JAK2 TYROSINE KINASE AS A POTENTIAL NEW...

JAK2 TYROSINE KINASE AS A POTENTIAL NEW TARGET IN THE TREATMENT OF

CANCER AND CARDIOVASCULAR DISEASE

By

ANNET KIRABO

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2011

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© 2011 Annet Kirabo

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To my parents for their unfailing love, encouragement, wisdom and support

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ACKNOWLEDGMENTS

My deepest appreciation goes to my mentor Dr. Peter Sayeski. His excellent

guidance, advice and support have made my research successful. I would also like to

thank members of my Ph.D. supervisory committee; Drs. Chris Baylis, Hideko

Kasahara, and William Ogle. Their support, guidance and critical analysis of my

research will have a long-lasting impact on the success of my career.

Special thanks go to the various people who provided me with technical

assistance in various aspects of my research including Mr. Steve McClellan, Dr. Ann

Fu, Ms. Naime Fliess, Mr. Marcus Moore, Mr. Patrick Kerns, Mr. Bruce Cunningham,

Mr. Harold Snellen, Dr. Alvaro Gurovich, Dr. Jennifer Sasser, Dr. Yagna Jarajapu, Dr.

Jennifer Embury, Dr. Mary Reinhard, Dr. Heather Wamsley and Dr. Debra Ely. I thank

my friends Natasha Moningka, Lakeshia Murphy, Lisa Morrison and Dr. Chastity

Bradford for their support, advice and guidance. I also thank my lab mates Dr. Sung

Park, Anurima Majumder, Kavitha Gnanasambandan and Rebekah Baskin for their

friendship, and for providing a conducive research environment.

Lastly, I would like to thank members of my family. I thank my father Rev. Josiah

Ddembe and my mother Mrs. Proscovia Ddembe. Their strict and loving upbringing has

shaped me into who I am. Their constant assurance that they trust my decisions, and

that they are praying for me has enabled me to ‘fly out of the nest’ and be independent

in a responsible way. I thank my brothers Robert Ddembe, Nelson Wandira and James

Izimba Ddembe. They have always ‘had my back’ and around them, I have nothing to

fear. I thank my sisters Sarah Mirembe, Gertrude Tumwebaze, Martha Alitubera, Prossy

Musasizi and Naomi Mwesigwa for their love, prayers and moral support.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS.................................................................................................. 4

LIST OF TABLES............................................................................................................ 9

LIST OF FIGURES........................................................................................................ 10

ABSTRACT ................................................................................................................... 12

CHAPTER

1 INTRODUCTION .................................................................................................... 14

Jak2 Tyrosine Kinase: A Potential Therapeutic Target for AT1 Receptor Mediated Cardiovascular Disease ....................................................................... 14

The Janus Kinase Family of Proteins ............................................................... 16 Jak2 in Angiotensin II-Induced Cardiovascular Disease................................... 17 Pharmacological Jak2 Inhibition: An emerging Therapeutic Strategy in Jak2-mediated Diseases .................................................................................. 21

2 THE STILBENOID TYROSINE KINASE INHIBITOR, G6, SUPPRESSES JAK2-V617F MEDIATED HUMAN PATHOLOGICAL CELL GROWTH IN VITRO AND IN VIVO................................................................................................................... 27

Materials and Methods............................................................................................ 28 Cell Culture....................................................................................................... 28 Phospho-STAT Analysis .................................................................................. 28 In vivo Animal Model ........................................................................................ 28 Analysis of Peripheral Blood Cells.................................................................... 29 Histo-pathological Analysis............................................................................... 30 Bone Marrow Flow Cytometry .......................................................................... 30 Bone Marrow Immunohistochemistry ............................................................... 30 Statistical Analysis............................................................................................ 31

Results.................................................................................................................... 31 G6 Inhibits Jak2-V617F Dependent Human Erythroleukemia Cell

Proliferation ................................................................................................... 31 G6 Suppresses HEL Cell Growth by Inducing G1 Phase Cell Cycle Arrest

and Apoptosis ............................................................................................... 33 G6 Inhibits Jak2-V617F-Dependent Constitutive Activation of STAT5 ............. 34 G6 Reduces Blast Cells in the Peripheral Blood and the Spleen Weight to

Body Weight Ratio in a Murine Model of Jak2-Dependent, Human Erythroleukemia ............................................................................................ 35

G6 Corrects a Pathologically Low Myeloid to Erythroid Ratio by Reducing the Number of Human Erythroleukemia Cells in the Bone Marrow of Mice... 35

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G6 Reduces the Levels of phospho-STAT5 and Induces Cellular Apoptosis, in vivo ............................................................................................................ 37

G6 Treatment Results in Leukemic Regression and Normalization of Hematopoiesis in the Spleen......................................................................... 38

Discussion .............................................................................................................. 40

3 THE JAK2 INHIBITOR, G6, ALLEVIATES JAK2-V617F MEDIATED MYELOPROLIFERATIVE NEOPLASIA BY PROVIDING SIGNIFICANT THERAPEUTIC EFFICACY TO THE BONE MARROW......................................... 55

Materials and Methods............................................................................................ 56 Animals............................................................................................................. 56 Analysis of Peripheral Blood Cells.................................................................... 57 Interleukin-6 Analysis ....................................................................................... 57 Histological Analysis......................................................................................... 58 Immunohistochemistry...................................................................................... 58 Determination of Jak2-V617F Allele Burden in the Bone Marrow..................... 59 Clonogenic Assay............................................................................................. 59 Statistical Analysis............................................................................................ 59

Results.................................................................................................................... 60 G6 Provides Therapeutic Benefit in Peripheral Blood of Jak2-V617F MPN

Mice .............................................................................................................. 60 G6 Reduces Extramedullary Hematopoiesis in Jak2-V617F MPN Mice .......... 61 G6 Provides Therapeutic Benefit to the Spleen of Jak2-V617F MPN Mice...... 62 G6 Provides Therapeutic Benefit to the Bone Marrow of Jak2-V617F MPN

Mice by Alleviating Megakaryocytic and Myeloid Hyperplasia....................... 63 G6 Provides Therapeutic Benefit to the Bone Marrow in Jak2-V617F MPN

Mice by Reducing the Pathological Levels of Phospho-Jak2 and Phospho-STAT5............................................................................................ 64

G6 Provides Therapeutic Benefit to the Bone Marrow in Jak2-V617F MPN Mice by Significantly Reducing the Mutant Jak2 Allelic Burden .................... 65

G6 Prevents Jak2-V617F Mediated Clonogenic Growth .................................. 66 Discussion .............................................................................................................. 66

4 VASCULAR SMOOTH MUSCLE JAK2 MEDIATES ANGIOTENSIN II-INDUCED HYPERTENSION VIA INCREASED LEVELS OF REACTIVE OXYGEN SPECIES................................................................................................ 81

Materials and Methods............................................................................................ 82 Animals............................................................................................................. 82 Blood Pressure Measurements ........................................................................ 83 Aortic Contraction/Relaxation ........................................................................... 83 Histology........................................................................................................... 83 NO Measurements ........................................................................................... 83 H2O2 Detection ................................................................................................. 84 Rho Kinase Activity .......................................................................................... 84 Calcium Imaging............................................................................................... 84

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Statistical Analysis............................................................................................ 84 Results.................................................................................................................... 84

Generation of Mice with VSMC Deletion of Jak2.............................................. 84 Deletion of VSMC Jak2 Attenuates Ang II-Induced Hypertension .................... 85 VSMC Jak2 Null Mice were Protected from Ang II-Induced Aortic Wall

Thickening..................................................................................................... 86 Deletion of VSMC Jak2 Correlates with Reduced Ang II-Induced

Contraction of Aortic Rings and Increased Endothelium-derived Nitric Oxide............................................................................................................. 87

Deletion of VSMC Jak2 Enhances Endothelium Dependent Aortic Relaxation due to Reduced ROS and Increased NO Availability .................. 88

Deletion of VSMC Jak2 Results in Reduced Rho-Kinase Activity and Intracellular Ca2+ Levels in Response to Ang II............................................. 90

Deletion of VSMC Jak2 Prevents Angiotensin II-Induced Kidney Damage...... 91 Discussion .............................................................................................................. 92

5 VASCULAR SMOOTH MUSCLE JAK2 DELETION PREVENTS ANGIOTENSIN II-MEDIATED NEOINTIMA FORMATION FOLLOWING INJURY IN MICE.......... 105

Materials and Methods.......................................................................................... 107 Animals........................................................................................................... 107 Generation of Knockout Mice ......................................................................... 107 Vascular Injury Model ..................................................................................... 108 Histology......................................................................................................... 108 Immunohistochemistry.................................................................................... 109 Immunoblotting............................................................................................... 109 Cell Proliferation ............................................................................................. 110 Cell Migration ................................................................................................. 110 Statistical Analysis.......................................................................................... 111

Results.................................................................................................................. 111 Deletion of VSMC Jak2 Prevents Ang II-Mediated Neointima Formation and

Narrowing of the Vascular Lumen Following Injury ..................................... 111 Deletion of VSMC Jak2 Prevents Ang II-Mediated Vascular Fibrosis

Following Injury ........................................................................................... 112 Deletion of VSMC Jak2 Prevents the Loss of Smooth Muscle α-actin in

Response to Ang II-Mediated Vascular Injury ............................................. 113 Deletion of VSMC Jak2 Prevents Neointima Formation by Inhibiting Cell

Proliferation and Inducing Apoptosis........................................................... 113 VSMC Jak2 Induces Neointima Formation by Increasing Phosphorylation of

Jak2 and STAT5 ......................................................................................... 114 VSMC Jak2 Mediates Ang II-Induced Cell Proliferation and Migration........... 115 VSMC Jak2 deletion is associated with reduced Ang II-mediated activation

of STAT3 and STAT5.................................................................................. 116 Discussion ............................................................................................................ 117

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6 CONCLUSIONS AND PERSPECTIVES............................................................... 128

Jak2 is Important in Mammalian Biology............................................................... 128 Jak2 plays a Critical Role in the Pathogenesis of Hematological Malignancies.... 129 Therapeutic Efficacy of Jak2 Inhibitors in Hematological Malignancies ................ 130 G6 has Exceptional Bone Marrow Therapeutic Efficacy ....................................... 130 Conditional Deletion of Vascular Smooth Muscle Cell Jak2 is Protective against

Cardiovascular Disease Pathogenesis .............................................................. 132 Jak2 Mediates Cardiovascular Disease Pathogenesis via Multiple Non-

Redundant Mechanisms.................................................................................... 133 Jak2 Contributes to Increased Presence of ROS........................................... 133 VSMC Jak2 Expression Correlates with Reduced NO Availability ................. 135 VSMC Jak2 Expression Correlates with Increased Rho-kinase Activity ......... 135 VSMC Jak2 Expression Correlates with Increased Intracellular Calcium....... 136 VSMC Jak2 Mediates Ang II-Induced Growth Factor Effects and Local

Tissue Damage ........................................................................................... 137 Jak2 Inhibitors and Their Potential for Cardiovascular Disease Therapy.............. 138

Conclusions.................................................................................................... 139

LIST OF REFERENCES ............................................................................................. 142

BIOGRAPHICAL SKETCH.......................................................................................... 160

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LIST OF TABLES

Table page 2-1 Mass spectrometry results showing plasma and tissue concentrations of G6

at euthanasia. ..................................................................................................... 54

3-1 Summary of peripheral blood analyses showing erythrocyte and platelet indices of non-transgenic, and vehicle or G6 treated Jak2-V617F transgenic mice.................................................................................................................... 78

3-2 Summary of peripheral blood analyses showing leukocyte indices of non-transgenic, and vehicle or G6 treated Jak2-V617F transgenic mice................... 79

3-3 Mass spectrometry results showing plasma concentrations of G6 at euthanasia of Jak2-V617F transgenic mice........................................................ 80

6-1 Comparison of in vivo dosages of Jak2 inhibitors in murine models................. 141

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LIST OF FIGURES

Figure page 1-1 The Classical Jak/STAT Signaling Pathway. ........................................................ 23

1-2 Activation of the Jak2 signaling cascade via the AT1-receptor results in mitogenic growth responses. .............................................................................. 24

1-3 The mechanism by which Ang II mediates vasoconstriction............................... 25

1-4 Proposed mechanisms through which Jak2 mediates Ang II-dependent vasoconstriction.................................................................................................. 26

2-1 Model of Jak2-V617F mediated, human erythroleukemia. ................................. 44

2-2 G6 inhibits Jak2-V617F dependent HEL cell proliferation, in vitro ...................... 45

2-3 G6 suppresses HEL cell growth by inducing G1 phase cell cycle arrest. ........... 46

2-4 G6 induces the intrinsic apoptotic pathway in HEL cells..................................... 47

2-5 G6 preferentially inhibits Jak2-V617F dependent constitutive activation of STAT5. ............................................................................................................... 48

2-6 G6 decreases the percentage of blast cells in the peripheral blood and reduces the spleen weight to body weight ratio in a mouse model of Jak2-V617F mediated human erythroleukemia........................................................... 49

2-7 G6 improves the M:E ratio in a mouse model of Jak2-V617F mediated human erythroleukemia by reducing HEL cell engraftment in the bone marrow. .............................................................................................................. 50

2-8 G6 reduces the levels of phospho-STAT5 and induces cellular apoptosis in the bone marrow................................................................................................. 51

3-1 G6 provides therapeutic benefit in peripheral blood of Jak2-V617F transgenic mice.................................................................................................................... 71

3-2 G6 reduces extramedullary hematopoiesis in Jak2-V617F transgenic mice. ..... 72

3-3 G6 provides therapeutic benefit to the spleen of Jak2-V617F transgenic mice.................................................................................................................... 73

3-4 G6 provides therapeutic benefit to the bone marrow of Jak2-V617F transgenic mice by alleviating megakaryocytic and myeloid hyperplasia ........... 74

3-5 G6 reduces activation of Jak2 and STAT5 in Jak2-V617F transgenic mice ....... 75

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3-6 G6 reduces mutant allelic burden in bone marrow of Jak2-V617F transgenic mice.................................................................................................................... 76

3-7 G6 prevents Jak2-V617F-mediated cytokine-independent colony Formation..... 77

4-1 Generation of mice with vascular smooth muscle cell specific deletion of Jak2.................................................................................................................... 96

4-2 The Jak2 protein is absent in vascular smooth muscle cells of mutant mice. ..... 97

4-3 Deletion of vascular smooth muscle cell Jak2 attenuates Ang II-induced hypertension....................................................................................................... 98

4-4 VSMC Jak2 null mice are protected from Ang II induced aortic wall thickening. .......................................................................................................... 99

4-5 Deletion of vascular smooth muscle cell Jak2 correlates with reduced Ang II induced contraction of aortic rings .................................................................... 100

4-6 Deletion of vascular smooth muscle cell Jak2 correlates with increased levels of nitric oxide .................................................................................................... 101

4-7 Deletion of vascular smooth muscle cell Jak2 enhances endothelium dependent vascular relaxation due to reduced reactive oxygen species and increased nitric oxide availability ...................................................................... 102

4-8 Deletion of vascular smooth muscle cell Jak2 results in reduced Rho-kinase activity and intracellular Ca2+ levels in response to Ang II ................................ 103

4-9 VSMC Jak2 null mice are protected from Ang II-induced renal damage .......... 104

5-1 Deletion of VSMC Jak2 prevents Ang II-mediated neointima formation and narrowing of the vascular lumen following injury .............................................. 121

5-2 Deletion of VSMC Jak2 prevents Ang II-mediated fibrosis and loss of SMA following injury .................................................................................................. 122

5-3 Deletion of VSMC Jak2 inhibits cell proliferation and induces apoptosis.......... 123

5-4 VSMC Jak2 induces neointima formation by increasing phosphorylation of Jak2 and STAT5............................................................................................... 124

5-5 VSMC Jak2 mediates Ang II-induced cell proliferation and migration .............. 125

5-6 VSMC Jak2 deletion reduces Ang II-mediated activation of STAT3 and STAT5 .............................................................................................................. 127

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

JAK2 TYROSINE KINASE AS A POTENTIAL NEW TARGET IN THE TREATMENT OF

CANCER AND CARDIOVASCULAR DISEASE

By

Annet Kirabo

August 2011

Chair: Peter P. Sayeski Major: Medical Sciences -- Physiology and Pharmacology

Cardiovascular diseases including hypertension, stroke and heart attack are one

of the leading causes of death in the world. These diseases often result from multiple

risk factors including genetic background, environmental conditions and hematological

disorders. Most of the available treatment approaches for cardiovascular diseases are

targeted towards the renin-angiotensin-aldosterone system (RAAS) including direct

renin inhibition, ACE inhibition, Angiotensin II type 1 receptor (AT1-R) blockade, and

aldosterone receptor antagonism. However, many patients are non-responsive to the

available treatments, and there is still need to identify new therapeutic targets for

cardiovascular disease. The Jak2 signaling pathway is intricately coupled to the AT1-R

signaling processes involved in hypertension. In addition, hyper-activation of the Jak2

signaling pathway is central to the pathogenesis of hematological malignancies, which

also present an important predisposing factor for cardiovascular diseases such as

stroke and heart attack. In this dissertation, we investigated the involvement of Jak2 in

the pathogenesis of cardiovascular disease, and its potential as a therapeutic target.

We report here that G6, a novel stilbenoid based inhibitor of Jak2 tyrosine kinase,

has exceptional therapeutic efficacy in two different mouse models of Jak2-mediated

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hematological disease pathogenesis. In addition, we used the Cre-loxP system to

conditionally eliminate Jak2 tyrosine kinase expression within the vascular smooth

muscle cells (VSMC) of mice, followed by chronic infusion of Angiotensin II (Ang II). We

found that mice lacking Jak2 in their VSMC are largely protected from Ang II-induced

cardiovascular disease pathogenesis including hypertension and neointima formation

following vascular injury. These studies suggest that Jak2 plays a critical role in the

pathogenesis of cardiovascular disease via multiple, non-redundant mechanisms. As

such, Jak2 may provide a rational therapeutic approach for patients with various forms

of cardiovascular diseases.

CHAPTER 1 INTRODUCTION

1Jak2 Tyrosine Kinase: A Potential Therapeutic Target for AT1 Receptor Mediated Cardiovascular Disease

Cardiovascular diseases are among the leading causes of death in the United

States and other developed countries and hypertension is one of the major contributors

to cardiovascular disease, end-organ damage, and death in the Western world [1]. The

consequences of hypertension include myocardial ischemia, hypertensive heart

disease, renal failure, peripheral atherosclerosis, and stroke. Central to these processes

is the renin-angiotensin-aldosterone system (RAAS), which plays a major role in the

pathophysiological processes leading to hypertension.

Angiotensin II (Ang II) is the primary effecter hormone of the RAAS. There are two

G protein-coupled receptor subtypes through which Ang II mediates its actions; the Ang

II type 1 receptor (AT1-R) and Ang II type 2 receptor (AT2-R) [2,3]. Most of the

physiological and pathophysiological cardiovascular actions of Ang II are mediated

through the AT1-R [4,5]. The AT2-R is expressed at very high levels in the developing

fetus, but its expression is very low in the cardiovascular system of adults [6]. Under

normal physiological conditions, Ang II mediates responses that maintain electrolyte

and blood pressure homeostasis. It affects glomerular blood flow via arteriolar

vasoconstriction in the kidney and increases renal tubular sodium and water

reabsorption by stimulating synthesis and secretion of aldosterone. In addition, Ang II

1Reproduced with permission from Kirabo A, Sayeski PP (2010) Jak2 tyrosine kinase: a potential therapeutic target for AT1 receptor mediated cardiovascular disease. Pharmaceuticals 3: 3478-3493.

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stimulates release of vasopressin from the brain resulting in increased water retention. It

also drives the thirst response. Finally, Ang II acts directly on vascular smooth muscle

cells (VSMC) resulting in vasoconstriction and blood pressure regulation.

In addition to its hemodynamic effects, Ang II has direct antinatriuretic effects in

the kidney. In the proximal tubules, Ang II-mediated reabsorption of sodium is coupled

to reabsorption of bicarbonate via activation of apical Na+/H+ exchange, basolateral

Na+-HCO3- cotransport, and basolateral Na+/K+-ATPase and via insertion of H+-ATPase

into the apical membrane. In the distal tubules, Ang II increases amiloride-sensitive

sodium reabsorption via ENaC on sodium channels. It also stimulates Na+/H+ exchange

and the vacuolar H+-ATPase. In the collecting duct, Ang II also plays an important role

in regulation of the epithelial sodium channel. Ang II also regulates reabsorption of

sodium in the collecting duct via aldosterone. It stimulates production of aldosterone in

the zona glomerulosa of the adrenal cortex. Aldosterone then stimulates ion transport in

the principal cells of the collecting duct by opening sodium and potassium channels in

the luminal membrane, and increasing the activity of Na+/K+-ATPase pump in the

basolateral membrane. The antinatriuretic effects of Ang II on the various segments of

the nephron have been reviewed [7].

Perturbation of the RAAS is associated with the pathogenesis of a number of

cardiovascular diseases. Ang II action via the AT1-R is particularly vital in the

pathogenesis of cardiovascular disease resulting from hypertension. This is mainly due

to its vasoconstrictive actions on VSMCs resulting in increased peripheral resistance

and hypertension [6]. Ang II also acts on its receptors and mediates increased VSMC

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hyperplasia and hypertrophy, leading to increased peripheral vascular resistance. Most

of the pathophysiologic effects result from chronic Ang II stimulation which elicits growth

promoting effects leading to vascular disease [8]. Ang II infusion exacerbates neointima

formation in animals with vascular balloon catheter injury [9], which is inhibited by RAAS

blockers [10]. In addition, Ang II increases protein synthesis in VSMCs [11] and it

stimulates growth in a number of cell types including VSMC, fibroblasts, adrenal cortical

cells, cardiac myocytes, renal proximal tubular cells and tumor cells [12]. In cultured

VSMCs, Ang II promotes hyperplasia, hypertrophy and migration [13,14,15,16]. It has

also been implicated in inflammation, endothelial dysfunction, atherosclerosis,

hypertension and renal fibrosis [17]. Chronic Ang II infusion in rodents induces VSMC

proliferation in normal and injured vessels in vivo [9,18]. Interestingly, the growth factor-

like Ang II-dependent responses are largely independent of its hemodynamic effects

[19]. These studies suggest that Ang II acts as a growth factor under chronic exposure.

However, the mechanisms that mediate the growth promoting effects of Ang II are still

under scientific investigation. This dissertation is aimed at analyzing the involvement of

the tyrosine kinase, Jak2, in AT1-R mediated cardiovascular disease, and its potential

as a treatment option for cardiovascular disease.

The Janus Kinase Family of Proteins

There are four mammalian genes encoding the non-receptor Janus kinase (Jak)

family of proteins; Jak1, Jak2, Jak3 and Tyk2 [20]. They contain seven regions with

significant sequence homology and collectively, these regions are referred to as the Jak

homology domains (JH1-JH7) [21]. The JH1 domain contains the tyrosine kinase

domain, and is located within the carboxyl terminus of the protein. This domain binds

ATP and harbors the phospho-transferase activity of the protein. The JH2 domain

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shows close homology to the JH1 domain, but lacks tyrosine kinase activity. It is

therefore termed the pseudokinase domain. Acting via a cis mechanism, the JH2

domain negatively regulates the kinase activity of the JH1 domain [21,22]. The JH3 and

half of the JH4 domain encode an SH2 like motif whose function is not well understood

[23]. Finally, the remaining half of the JH4 domain, along with the entirety of the JH5,

JH6, and JH7 domains, collectively encode the FERM domain. The FERM domain

directly mediates the interaction of the Jak kinases with other cellular proteins such as

cytokine receptors [24,25,26].

The Jak kinases play a critical role in cytokine signaling. They transduce signals

from the cell surface to the nucleus via the tyrosine phosphorylation of the Signal

Transducers and Activators of Transcription (STAT) proteins. Phosphorylated STATs

translocate into the nucleus where they bind to cis-inducible promoter elements and

stimulate gene transcription (Figure 1-1). Insight into the in vivo function of each of the

Jaks was gained via the generation of specific Jak kinase family knockout mice. Among

the gene deletion models of the Jak family members, Jak2 deficient mice exhibited the

most severe phenotype. Jak2 null mice die embryonically around day E12.5 of gestation

due to impaired erythropoiesis and profound anemia [27,28]. These studies

demonstrate that Jak2 is important in mouse development via erythropoietin receptor-

dependent signaling. However, given the wide expression pattern of Jak2 in the body,

there is still need to investigate its other biologically relevant functions as a mediator for

cellular signaling in adult tissues.

Jak2 in Angiotensin II-Induced Cardiovascular Disease

Studies have shown that Ang II binding to the AT1-R triggers activation of Jak2,

leading to intracellular signaling cascades in VSMCs and cardiac myocytes

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[29,30,31,32,33,34,35,36,37]. Ang II stimulates Jak2 co-association to the AT1-R in

VSMCs leading to phosphorylation of Jak2 at Tyr 1007/Tyr 1008, phosphorylation of the

STATs, and translocation of the STATs into the nucleus [30,38,39,40], resulting in cell

growth/proliferative responses (Figure 1-2). Blockade of the RAAS by either angiotensin-

converting enzyme (ACE) inhibitors or AT1-R specific antagonists prevents injury-induced

neointima formation [10,41], and Ang II infusion exacerbates VSMC proliferation in

arterial walls [9]. In addition, the genes of the RAAS are up regulated in neointima

formation following vascular injury [42,43,44,45]. Interestingly, Jak2 has also been shown

to play a role in other cardiovascular signaling processes [46]. For example, in VSMC,

Jak2 plays a critical role in reactive oxygen species (ROS) dependent VSMC proliferation

[47]. It is also involved in the pathogenesis of atherosclerosis via its interaction with

cytokines such as interleukin 8 [48]. In addition, Jak2 activation has been linked to

neointima formation and vascular occlusion in rat carotid arteries subjected to balloon

injury, which is exacerbated by Ang II infusion [49]. Although it is well established that

Jak2 interacts with the AT1-R resulting in cell growth and hypertrophy, there is no in vitro

or in vivo evidence suggesting that the AT1-R mediated growth effects are exclusively

through Jak2 activation. Further studies need to be done to establish the relative

involvement of Jak2 activation in comparison to other pathways such as the mitogen-

activated protein (MAP) kinase or pp60c-src kinase in AT1-R mediated cardiovascular

remodeling.

Jak2 not only mediates Ang II-dependent growth promoting effects, but is also

involved in Ang II-induced contractile responses, increased vascular tone and

hypertension. The established mechanism by which Ang II mediates vasoconstriction

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involves the heterotrimeric G protein-mediated pathway [50]. In VSMCs, the binding of

Ang II to the AT1-R results in the activation of Gq [51] which leads to phospholipase C

(PLC) activation. This releases inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG)

from plasma membrane derived phosphatidylinositol 4,5-bisphosphate [52].

Diacylglycerol stimulates protein kinase C (PKC) while IP3 binds to its receptor on the

sarcoplasmic reticulum, allowing calcium efflux into the cytoplasm. Ang II also mediates

an influx of external Ca2+ via calcium release activated calcium (CRAC) channels

[53,54]. Ca2+ binds to calmodulin and activates myosin light chain kinase (MLCK), which

phosphorylates the myosin light chain and enhances the interaction between actin and

myosin, resulting in vasoconstriction [55]. The classical Ang II mediated signal

transduction leading to vasoconstriction is summarized in Figure 1-3.

Recently, Guilluy and colleagues demonstrated a role of Jak2 in the pathogenesis

of hypertension [56]. The authors showed that Jak2 is involved in the Ang II-mediated

activation of the Rho exchange factor, Arhgf1, resulting in enhanced vasoconstriction. It

is not known whether the phosphorylation of Arhgef1 by Jak2 involves the Jak2 pool

which is physically associated to AT1-R, or via an indirect mechanism.

ROS mediate signaling pathways involved in hypertension and vascular pathology

[57,58] and Ang II is involved in mediating oxidative stress and oxidant signaling

[55,59,60,61]. Many of the pathologic effects of Ang II in blood vessels are mediated by

the generation of ROS via activation of NAD(P)H oxidases [57]. Ang II stimulates the

activity of membrane-bound NAD(P)H oxidase in VSMCs and endothelial cells to

produce ROS in the form of superoxide and hydrogen peroxide. Generation of such

molecules causes vascular inflammation, fibrosis and endothelial dysfunction

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[17,57,62,63,64,65,66]. The Ang II-induced formation of ROS is not related to its

hemodynamic effects as it does not occur in norepinephrine-induced hypertension

[62,67]. Specifically, endothelial dysfunction was observed in rats made hypertensive by

Ang II infusion, but not norepinephrine infusion. Furthermore, the endothelial

dysfunction correlated positively with increased superoxide production in the arteries

[62,67,68].

ROS have been shown to mediate RhoA/Rho kinase-induced Ca2+ sensitization in

pulmonary vascular smooth muscle following chronic hypoxia [69]. Superoxide

generated by Ang II inactivates nitric oxide (NO) in endothelial cells and VSMCs

[70,71,72]. In addition, previous studies have shown that Rho kinase can be activated

by increased ROS [69,73]. However, the mechanisms by which Ang II activates

NAD(P)H oxidases to induce oxidative stress are still not well understood. A number of

tyrosine kinases and phosphatases are known to be regulated by oxidative stress

resulting in expression of inflammatory genes, endothelial dysfunction, VSMC growth,

and extracellular matrix formation [57,59,62,63,74].

There is evidence that Jak2 plays a critical role in mediating ROS dependent

VSMC proliferation [47]. Activation of Jak2 results in higher levels of ROS and Jak2

inhibition leads to a dramatic reduction in oxidative stress [75]. Mutations which cause

constitutive activation of Jak2, such as Jak2-V617F, increase the levels of ROS within

cells, and inhibition of Jak2 leads to reduction of ROS in these same cells [75,76].

Hence, productions of ROS by the AT1-R, and Jak2 activation have been

experimentally demonstrated. However, it is still not known whether Jak2 mediates Ang

II-induced production of ROS via the AT1-R. There is still a need to elucidate the

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specific mechanisms by which Jak2 contributes to production of ROS, and whether it

plays a role in regulating NO availability. Furthermore, it is not known whether Jak2 can

activate Rho kinase via ROS-dependent mechanisms. The proposed mechanisms

through which Jak2 mediates vasoconstriction are represented in Figure 1-4.

Pharmacological Jak2 Inhibition: An emerging Therapeutic Strategy in Jak2-mediated Diseases

Jak2 kinase function is critical for normal hematopoietic growth factor signaling

[77]. On the other hand, hyper-kinetic Jak2 tyrosine kinase signaling causes several

hematologic diseases including some forms of leukemia, lymphoma, and myeloma.

Gain-of-function somatic mutations in the Jak2 allele are also known to be a causative

agent in the pathogenesis of the myeloproliferative neoplasms (MPN) [78]. MPNs are

clonal disorders of multipotent hematopoietic progenitors characterized by increased

hematopoiesis. They include polycythemia vera (PV), essential thrombocythaemia (ET)

and primary myelofibrosis (PMF). MPNs have a relatively high prevalence with the

number of cases ranging from about 130,000 to 150,000 in the United States [79].

The clinical symptoms of MPNs include bleeding, thrombosis, splenomegaly, and a

propensity for malignant transformation in the form of acute myeloid leukemia. One Jak2

mutation which causes MPNs is a valine to phenylalanine substitution at residue 617

(Jak2-V617F) within the pseudokinase domain. This mutation relieves the inhibitory

potential that the JH2 domain normally exerts on the JH1 kinase domain and the

consequence of this lost inhibitory potential is constitutive activation of the Jak2

signaling pathway [80,81,82,83,84]. The Jak2-V617F mutation has also been implicated

in other Jak2 mediated human diseases such as chronic myelomonocytic leukemia,

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myelodysplastic syndrome, systemic mastocytosis, chronic neutrophilic leukemia, and

acute myeloid leukemia [85,86,87].

Based on the identification of activating Jak2 mutations in MPNs, great effort has

been aimed at developing inhibitors that target Jak2 [88]. Accordingly, a number of small

molecule Jak2 inhibitors, which have potential therapeutic efficacy against Jak2-mediated

disorders, have been developed [89,90,91,92,93,94,95,96,97]. These compounds inhibit

the pathologic cell growth and signaling in cell lines transformed by Jak2 mutations in

vitro, in murine models in vivo, and in bone marrow samples obtained from MPN

patients and cultured ex vivo [89,97,98,99]. While some of these compounds are in pre-

clinical stages of development [100], others are currently in clinical trials for the treatment

of MPNs [89,101,102]. Early reports from these studies indicate that direct inhibition of

Jak2 with small molecule inhibitor therapy improved some clinical measures such as

spleen size and certain blood counts [101]. Side effects associated with Jak2 inhibitor

therapy included fatigue, neurotoxicity, and gastrointestinal disturbances [101]. However,

given the existing correlation between Jak2 kinase activity and cardiovascular disease,

perhaps changes in blood pressure or other cardiovascular readouts should be followed in

these patients. In these studies, we hypothesized that G6, a novel stilbenoid based

inhibitor of Jak2 tyrosine kinase, has therapeutic efficacy in Jak2-mediated

hematological disease pathogenesis. We also hypothesized that Jak2 plays a central

role in the causation of Ang II-induced cardiovascular disease including hypertension

and neointima formation.

22

ST

AT

ST

ATP

P

ST

AT S

TA

T

P

P

Transcription of genes involved in cell survival, migration and proliferation

JakJak

ST

AT

ST

AT

P P

Cell Membrane

Nuclear Membrane

Ligand binding to a cytokine receptor

ST

AT

ST

ATP

P

ST

AT S

TA

T

P

P


JakJak

ST

AT

ST

AT

P P

Cell Membrane

Nuclear Membrane

Ligand binding to a cytokine receptor

Figure 1-1. The Classical Jak/STAT Signaling Pathway. Ligand binding causes cytokine

receptors to dimerize which results in Jak phosphorylation, recruitment of the Signal Transducer and Activator of Transcription (STAT) signaling proteins, which are then tyrosine phosphorylated by the Jaks. The phosphorylated STATs dimerize, and translocate into the nucleus where they bind to cis-inducible promoter elements to stimulate gene transcription.

23

Ang II activation of the AT1-R

Jak2 P

ST

AT

ST

ATP

P

ST

AT

ST

ATP

P


Cell Membrane

Nuclear Membrane


Jak2 P

ST

AT

ST

ATP

P

ST

AT

ST

ATP

P


Cell Membrane

Nuclear Membrane

Figure 1-2. Activation of the Jak2 signaling cascade via the AT1-receptor results in

mitogenic growth responses. Angiotensin II binding results in phosphorylation of Jak2. Active Jak2 recruits and phosphorylates STATs, which then dimerize, translocate into the nucleus, and mediate the transcription of genes involved in cell survival, migration, and proliferation.

24

Ca2+

MLCK

MLCPMyosin p-Myosin

Actin

Contraction

Gα

PIP2

CaM

DAG IP3+

PLCGβγ


Cell Membrane

Ca2+

MLCK

MLCPMyosin p-Myosin

Actin

Contraction

Gα

PIP2

CaM

DAG IP3+

PLCGβγ


Cell Membrane

Figure 1-3. The mechanism by which Ang II mediates vasoconstriction. The binding of

Ang II to the AT1-R activates the heterotrimeric G protein signaling pathway which leads to phospholipase C (PLC) activation. This releases inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP2). IP3 binds to its receptor on the sarcoplasmic reticulum, allowing for Ca2+ efflux. Ang II also promotes an influx of external Ca2+ via calcium release activated calcium (CRAC) channels. Ca2+ binds to calmodulin and activates myosin light chain kinase (MLCK), which phosphorylates the myosin light chain and enhances the interaction between actin and myosin, resulting in enhanced vasoconstriction.

25

26

Ca2+

MLCK

MLCPMyosin p-Myosin

Actin

Rho Kinase

Jak2

NOENDOTHELIAL

CELL

VASCULAR SMOOTH MUSCLE CELL

Relaxation

Contraction

Gα


ROS Ca2+

MLCK

MLCPMyosin p-Myosin

Actin

Rho Kinase

Jak2

NOENDOTHELIAL

CELL


Relaxation

Contraction

Gα


ROS Ca2+

MLCK

MLCPMyosin p-Myosin

Actin

Rho Kinase

Jak2

NOENDOTHELIAL

CELL


Relaxation

Contraction

Gα


ROS Ca2+

MLCK

MLCPMyosin p-Myosin

Actin

Rho Kinase

Jak2

NOENDOTHELIAL

CELL


Relaxation

Contraction

Gα


ROS

Figure 1-4. Proposed mechanisms through which Jak2 mediates Ang II-dependent

vasoconstriction. Ang II binding to the AT1-R activates Jak2 (it is not known whether this involves the Jak2 pool physically associated with the AT1-R). Activated Jak2 phosphorylates Arhgf1 resulting in enhanced contraction via a Rho Kinase dependent mechanism. Jak2 is also believed to mediate intracellular increases in ROS. Higher levels of ROS increase intracellular Ca2+ sensitization, activate Rho Kinase, and scavenge endothelial nitric oxide all of which lead to enhanced VSMC contraction.

CHAPTER 2 2THE STILBENOID TYROSINE KINASE INHIBITOR, G6, SUPPRESSES JAK2-V617F

MEDIATED HUMAN PATHOLOGICAL CELL GROWTH IN VITRO AND IN VIVO

Hyper-kinetic Jak2 tyrosine kinase signaling is a contributor to several human

diseases including specific forms of leukemia, lymphoma, myeloma, and the

myeloproliferative neoplasms (MPNs). MPNs are clonal disorders of multipotent

hematopoietic progenitors characterized by increased hematopoiesis. The classic

MPNs include polycythemia vera (PV), essential thrombocythaemia (ET) and primary

myelofibrosis (PMF). A mutation resulting in a within the pseudokinase domain of Jak2

(Jak2-V617F) was identified in a large number of PV, ET, and PMF patients

[80,81,82,83,84]. This mutation has also been reported in chronic myelomonocytic

leukemia, myelodysplastic syndrome, systemic mastocytosis, chronic neutrophilic

leukemia, acute myeloid leukemia, and erythroleukemia [85,86,87]. The mutation

causes constitutive activation of the Jak2 signaling pathway when expressed in cells

[81,82,83,84]. Furthermore, its expression in murine bone marrow results in a neoplastic

phenotype [89,103,104].

Because of its pathogenicity in human disease, the Jak2-V617F mutation is a

target for therapeutic drug development. A number of laboratories have designed

and/or identified small molecule inhibitors that have potential Jak2 therapeutic efficacy

[89,90,91,92,93,94]. Additionally, our laboratory previously identified small molecule

compounds with anti-Jak2 tyrosine kinase activity [95,96]. In our most recent work,

2Reproduced from Kirabo A, Embury J, Kiss R, Polgar T, Gali M, et al. (2011) The stilbenoid tyrosine kinase inhibitor, G6, suppresses Jak2-V617F-mediated human pathological cell growth in vitro and in vivo. J Biol Chem 286: 4280-4291.

27

structure-based virtual screening was employed to identify a novel Jak2 inhibitor named

G6 [97]. Using cell free systems, we found that G6 demonstrated good potency and

specificity at suppressing Jak2-V617F kinase activity [97]. Based on that work, we

hypothesized that G6 would have therapeutic efficacy against Jak2-V617F mediated

pathogenesis. Here, we tested this hypothesis and found that G6 did indeed suppress

Jak2-V617F mediated, human pathological cell growth in vitro and in vivo.

Materials and Methods

Cell Culture

Human erythroleukemia 92.1.7 (HEL) cells were purchased from ATCC (Rockville,

MD). The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum

at 37°C in a 5% CO2 humidified atmosphere. Cell proliferation assays, DNA cell cycle

analysis, and annexin V/propidium iodide apoptotic levels were measured as we have

described previously [96].

Phospho-STAT Analysis

Phospho- STAT1 [pY701], STAT3 [pY705], and STAT5a/b [pY694/699] ([pY694]

for STAT5a and [pY699] for STAT5b) were similarly measured using the STAT1, 3, 5a/b

Phospho 3-Plex assay kit, a solid phase sandwich immunoassay, following the

manufacturer’s instructions (Invitrogen). The spectral properties of the 3 bead regions

specific for each analyte were monitored with a Luminex® 100TM instrument.

In vivo Animal Model

The in vivo efficacy of G6 was determined using a mouse model of Jak2-V617F

mediated, human erythroleukemia. All experimental protocols were performed

according to NIH standards established in the Guide for the Care and Use of Laboratory

Animals and approved by the Institutional Animal Care Use Committee at the University

28

of Florida. This approach is outlined in Figure 2-1. Thirty six male adult (3 months old)

NOD/SCID mice were purchased from Jackson Laboratory. After arrival and

acclimation, baseline body weights and peripheral blood samples were obtained and the

mice were subsequently randomized into 6 groups (n=6 per group). Mice were

inoculated intravenously via the lateral tail vein with 2 × 106 HEL cells expressing the

Jak2-V617F mutation. Body weights and blood samples were obtained each week to

monitor disease progression. Three weeks after HEL cell injection, the mice received

daily intra-peritoneal injections of G6 at dosage rates of 0.1, 1 and 10 mg/kg/day,

respectively, for 21 days. Three separate control groups were also included. The first

received HEL cells and subsequent daily injections of vehicle alone (DMSO). The

second group never received HEL cells, but received G6 at the 10 mg/kg/day dosage

over the same three week period of drug administration. The third group was completely

naïve to any treatment. After the three week period of drug or vehicle administration, all

groups were euthanized by CO2 asphyxiation and cervical dislocation. Spleen weight to

body weight ratios were obtained. A bone marrow aspirate from one femur was

obtained for flow cytometry analysis and determination of HEL cell engraftment. Tissue

samples (brain, lung, liver, kidney, spleen, and bone marrow) were fixed in 10% neutral-

buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and

eosin for histological analysis.

Analysis of Peripheral Blood Cells

A blood sample was obtained each week (~25 μl) via sub-mandibular bleeding into

a capillary tube. The samples were then smeared onto glass slides and stained using

DipQuick (Jorgensen Laboratories). Total white blood cell (WBC) counts, percentages

of immature granular leukocytes, monocytes and nucleated red blood cells (RBC)

29

among the peripheral blood cells were counted via a light microscope by a veterinary

pathologist.

Histo-pathological Analysis

Hematoxylin and eosin stained sections (liver, kidney, lung, brain, spleen, and

bone marrow) were examined for normal histological appearance as well as any lesions

via standard light microscopy.

Bone Marrow Flow Cytometry

At the time of euthanasia, marrow was harvested from one femur and teased apart

into single cell suspension in staining buffer by filtering it through a 50-μm nylon mesh

following the manufacturer’s protocol (eBioscience). Cell suspensions were incubated

on ice with APC conjugated anti-human CD45 antibody (BD Biosciences), washed, and

subjected to flow cytometry.

Bone Marrow Immunohistochemistry

Immunochemistry was carried out on tissue fixed in 10% neutral-buffered formalin

and paraffin-embedded. For detection of active STAT5, mouse monoclonal anti–

phospho-STAT5a/b (Y694/99; Advantex BioReagents LLP) was diluted 1:500 and

incubated on sections overnight at 4°C. Detection of the antigen–antibody complexes

was done by biotinylated secondary antibodies and streptavidin-peroxidase complex

(DAKO). Hematoxylin was used for counterstaining. Antigen retrieval was done by

heating (95°C, 20 min) with the BioGenex AR10 retrieval buffer. The staining intensity

was quantified using the NIS-Element D software. Apoptotic cells in the tissue were

identified via TUNEL. All TUNEL reagents were part of the ApopTag Kit (Millipore).

TUNEL-positive cells appeared as highly stained, brown nuclei against the methyl green

counterstain.

30

Pharmacokinetic and Pharmacodynamic Analysis of G6 in Mice

Baseline body weights and peripheral blood samples were obtained from three-

month-old male NOD SCID mice. The mice (n=12) were then injected in the tail vein

with 2x106 HEL cells. Three weeks later, peripheral blood samples were again obtained

in order to confirm that the animals were in blast crisis. Once this was validated, the

animals began receiving either vehicle control (DMSO) or G6 (1 mg/kg/day) via single,

daily IP injections for the next 14 days (n=6 mice per group). The mice were

subsequently euthanized and tissues (plasma, marrow, and spleen) were prepared. The

concentration of G6 was determined via liquid chromatography-mass spectrometry

using a quadratic standard curve (r=0.9902).

Statistical Analysis

Results are expressed as mean +/- SEM. Statistical comparisons were performed

by Student’s t test or the Mann-Whitney Rank Sum Test. Changes in peripheral blood

cell counts and bone marrow cellularity following HEL cell and drug treatment were

analyzed by a repeated-measures ANOVA followed by Bonferroni and Student-

Newman-Keuls post hoc test for multiple comparisons. p values of less than 0.05 were

considered statistically significant.

Results

G6 Inhibits Jak2-V617F Dependent Human Erythroleukemia Cell Proliferation

Using structure-based virtual screening, we recently identified a Jak2 tyrosine

kinase inhibitor called G6 [97]. This stilbenoid compound demonstrated good potency

and specificity for Jak2 tyrosine kinase as it inhibited Jak2-V617F enzymatic activity

(IC50 = 60 nM) while having no effect on c-Src tyrosine kinase activity at concentrations

as high as 25 µM [97]. Furthermore, it significantly inhibited the growth of cells whose

31

proliferation was driven by the Jak2-V617F mutation while having little to no effect on

cells whose proliferation was driven by other mechanisms including a JAK3-A572V

activating mutation, a c-Myc gene translocation, or immortalization via the SV-40 large T

antigen [97]. As such, we hypothesized that G6 would suppress Jak2-V617F mediated

pathological cell growth.

As a first estimate as to how well G6 could inhibit Jak2-V617F dependent

pathologic cell growth, we utilized the HEL cell line in vitro. This cell line is homozygous

for the V617F mutation which induces constitutive Jak2 phosphorylation and drives HEL

cell proliferation. Here, 5 x 104 HEL cells were treated with either DMSO or increasing

concentrations of G6 for 72 hours. The number of viable cells was then determined.

We found that G6 inhibited HEL cell growth in a dose-dependent manner with an IC50 of

~4.0 µM (Figure 2-2A). To determine if G6 could suppress HEL cell growth in a time

dependent manner, cells were treated with 25 µM of G6 for increasing periods of time.

We found that G6 inhibited HEL cell growth in a time dependent manner with 50%

inhibition being achieved after ~12 hours of treatment (Figure 2-2B). We next wanted to

determine whether the effects of G6 on HEL cell growth were reversible. For this, cells

were exposed to 25 µM of G6 for 0, 6, 24, 48 and 72 hours. At the end of each time

point, the cells were collected, washed extensively, and allowed to grow for an

additional 72 hours in the absence of any inhibitor. Viable cell numbers were then

determined. We found that for cells that were exposed to G6 for only 6 hours, nearly all

of them were able to proliferate after drug removal (Figure 2-2C). Analysis of the

recovery curve suggested that ~16 hours exposure to G6 prevented 50% of the cells

from recovering. For those cells that were exposed to G6 for 48 hours, virtually none

32

were able to subsequently grow after the drug was removed from the media. This

suggested that a 48 hour exposure of the cells to G6 committed them to a fate from

which they could not recover. Collectively, these data indicate that G6 inhibits HEL cell

growth in both a dose- and time-dependent manner and exposure of HEL cells to 25 µM

G6 for 48 hours is sufficient to prevent subsequent Jak2-V617F mediated, pathologic

cell growth.

G6 Suppresses HEL Cell Growth by Inducing G1 Phase Cell Cycle Arrest and Apoptosis

To determine the mechanism by which G6 reduces HEL cell growth, we first

measured cell cycle properties as a function of G6 treatment. Specifically, 5 x 105 HEL

cells were treated with G6 as a function of either dose or time. Three independent

experiments, each measured in triplicate, were averaged and the aggregate data were

graphed as a function of G6 concentration or G6 exposure time. We found that G6

dose-dependently increased the percentage of cells in G1 phase (Figure 2-3A),

decreased cells in S phase (Figure 2-3B), and increased cells in apoptosis (Figure 2-

3C). With respect to the time course study, we found that G6 promoted a time-

dependent increase in G1 phase (Figure 2-3D), a decrease in S phase (Figure 2-3E),

and an increase in apoptosis (Figure 2-3F) when compared to DMSO control treated

cells.

The apoptosis measurements in Figures 2-3C and 2-3F represent fragmented

DNA, which is only suggestive of late stage apoptosis. Therefore, to confirm this via

alternate means, we used annexin V/propidium iodide double staining. The values from

three independent dose- and time-course experiments were tabulated and graphed.

We found that G6 significantly induced apoptosis in both a dose- (Figure 2-4A) and

33

time- (Figure 2-4B) dependent manner as measured by the number of cells that were

annexin V positive and propidium iodide negative. Collectively, these data indicate that

the mechanism whereby G6 reduces HEL cell growth is via marked cell cycle arrest and

induction apoptosis.

G6 Inhibits Jak2-V617F-Dependent Constitutive Activation of STAT5

We previously showed that G6 mediated reductions in HEL cell numbers directly

correlate with suppression of Jak2 kinase activity [97]. We now wanted to determine

whether treatment with G6 and subsequent HEL cell growth inhibition also correlated

with reduced STAT signaling. This is important as it is possible that G6 could work

through mechanisms that are independent of the Jak/STAT signaling pathway. Here,

HEL cells were treated with either increasing concentrations of G6 or with 25 µM G6 for

increasing periods of time. The levels of phospho-STAT1 (pY701), phospho-STAT3

(pY705) and phospho-STAT5a/b (pY694/699) were then simultaneously measured. We

found that the dose- and time-dependent inhibition of phospho-STAT1 had little to no

correlation with the reduced levels of HEL cell growth (Figure 2-5A). The dose- and

time-dependent inhibition of phospho-STAT3 exhibited only a modest correlation with

the reduced levels of HEL cell growth (Figure 2-5B). Finally, we observed that the

dose- and time-dependent inhibition of phospho-STAT5a/b correlated very well with the

G6-dependent inhibition of HEL cell growth (Figure 2-5C). Thus, these results suggest

that STAT1, STAT3 and STAT5a/b are differentially affected by G6 treatment, with

STAT5 being the most sensitive. Furthermore, the reductions in phospho-STAT5

correlate very well with G6 mediated reductions in HEL cell growth and inhibition of

Jak2 kinase activity [97]. As such, our data suggest that G6 inhibits HEL cell growth via

a Jak2/STAT5 dependent mechanism.

34

G6 Reduces Blast Cells in the Peripheral Blood and the Spleen Weight to Body Weight Ratio in a Murine Model of Jak2-Dependent, Human Erythroleukemia

To test the in vivo efficacy of G6 at inhibiting Jak2-V617F pathologic cell growth,

we generated a mouse model of human erythroleukemia by injecting 2 × 106 HEL cells

expressing the Jak2-V617F mutation into the tail vein of immuno-deficient NOD/SCID

mice. We then treated the mice with increasing concentrations of G6 in order to

determine the therapeutic efficacy of the stilbenoid compound. Mice injected with HEL

cells followed by vehicle control injections rapidly developed a fully penetrant

hematopoietic disease. Specifically, we found that injection of HEL cells resulted in the

pathological appearance of blast cells in the peripheral blood and G6 treatment

significantly reduced this pathological effect, in a dose-dependent manner (Figure 2-

6A).

To determine the efficacy of G6 at inhibiting Jak2-dependent erythroleukemia via

alternate means, spleen weight to body weight ratios were determined. We found that

HEL cell injection and subsequent administration of vehicle control solution resulted in

an increased spleen weight to body weight ratio and this deleterious effect was

abrogated with G6 treatment (Figure 2-6B). These data indicate that G6 suppresses

Jak2-V617F mediated pathologic cell growth in vivo, as evidenced by reduced blast

cells in the peripheral blood and reduced spleen weight to body weight ratios.

G6 Corrects a Pathologically Low Myeloid to Erythroid Ratio by Reducing the Number of Human Erythroleukemia Cells in the Bone Marrow of Mice

The in vivo anti-tumor activity of G6 was further investigated using histo-

pathological analysis. Tissue sections from brain, liver, lungs and kidney appeared

histologically normal and indistinguishable across all six treatment groups suggesting

that G6 is not globally cytotoxic even at the 10 mg/kg dosage (data not shown).

35

However, quantification of cellular elements of the bone marrow revealed marked

changes; representative histological sections for each treatment group are shown

(Figure 2-7A). In mice that received HEL cells followed by vehicle control, two distinct

populations of neoplastic cells were observed. The first were consistent with

erythroblast morphology. These cells were approximately 15 µm with a dark gray blue

round nucleus and moderate, bluish cytoplasm (black arrows). The second population

of cells was myeloid blasts (monoblasts or myeloblasts). These atypical myeloid cells

were large (15-20 µm) with irregular nuclei that were frequently clefted. The chromatin

pattern was finely stippled and lacy. Nucleoli were prominent and occasionally multiple.

The nuclear to cytoplasmic ratio was high and the cytoplasm was pink (white arrows).

The absolute numbers of myeloid and erythroid cells for each treatment group

were determined (Figure 2-7B) and subsequently plotted as the myeloid to erythroid

(M:E) ratio (Figure 2-7C). For the naïve group of animals, the M:E ratio was ~1.4

(Figure 2-7C). Injection of HEL cells and subsequent treatment with vehicle control

caused a significant reduction of the M:E ratio that was driven by myeloid suppression

and increased numbers of erythroid cells. The 0.1 mg/kg dosage of G6 was without

effect on the cellular composition of the bone marrow as evidenced by the unchanged

M:E ratio. However, for the 1 and 10 mg/kg doses, there was a significant correction of

the M:E ratio that was driven by restoration of myeloid cells and suppression of

erythroid cells. With respect to the mice that received G6 alone, we observed a small

reduction in erythroid cell numbers and a moderate reduction in myeloid cells. However,

the M:E ratio of this group was not significantly different from that of the naïve mice.

36

We reasoned that the therapeutic correction of the M:E ratio by G6 was due to

reductions in the engraftment of the Jak2-V617F expressing, human erythroleukemia

cells in the bone marrow. To validate this, we carried out flow cytometry analysis of

bone marrow aspirates from the various treatment groups in order to identify the

percentage of cells that were human CD45 positive; a marker found on HEL cells, but

not any mouse cells (data not shown). The aggregate data for all animals were graphed

as a function of treatment group (Figure 2-7D). We found that HEL cell injection alone

resulted in robust bone marrow engraftment as evidenced by the appearance of human

CD45+ cells in the aspirates. The 0.1 mg/kg dose appeared to be without effect.

Starting at the 1 mg/kg dosage however, there was an observable decrease in the

percentage of human CD45+ cells among bone marrow mononuclear cells.

Overall, the data in Fig. 2-7 demonstrate that intravenous injection of HEL cells

into NOD/SCID mice results in marked Jak2-V617F mediated pathogenesis as

evidenced by a skewing of the M:E ratio and the engraftment of the human leukemic

cells in the bone marrow. However, G6 corrected these pathologies as evidenced by

reduced numbers of leukemic cells in the bone marrow and subsequent correction of

the M:E ratio.

G6 Reduces the Levels of phospho-STAT5 and Induces Cellular Apoptosis, in vivo

The data in Figures 2-2 to 2-5 demonstrate that in vitro, G6 suppresses pathologic

HEL cell growth via a mechanism that involves inhibition of STAT5 phosphorylation and

induction of apoptosis. We hypothesized that this also occurs in vivo. To confirm this,

we performed anti–phospho STAT5a/b immuno-histochemistry on bone marrow

sections of animals in all the treatment groups. Representative sections from all

37

treatment groups are shown (Figure 2-8A). The relative phospho-STAT5 signal (brown

colored stain) was then quantitated and graphed as a function of treatment group

(Figure 2-8B). We found that HEL cell injection alone resulted in a significant increase

in phospho-STAT5 staining, suggestive of an increased proliferative state. However,

G6 treatment at the 1 and 10 mg/kg doses significantly reduced this effect, suggesting

that G6 suppresses STAT5 phosphorylation in vivo.

To determine whether G6 induces apoptosis in vivo, bone marrow sections were

analyzed via TUNEL staining. Representative sections for the six groups are shown

(Figure 2-8C). The number of TUNEL positive cells per grid were then counted and

plotted as a function of treatment group (Figure 2-8D). We found that G6 induced

apoptosis in a dose-dependent manner. Overall, the data in Figure 2-8 demonstrate that

in vivo, G6 inhibits STAT5 phosphorylation and induces cellular apoptosis, two events

that are essential for suppressing Jak2-V617F mediated pathologic cell growth.

G6 Treatment Results in Leukemic Regression and Normalization of Hematopoiesis in the Spleen

To determine the effect of G6 on the spleen, histological sections were prepared

and viewed at 10X (Figure 2-9A) and 100X (Figure 2-9B) magnifications. Mice that

received HEL cells + DMSO displayed neoplastic erythroid morphology when compared

to naïve animals. Specifically, we observed large cells with lacy, vesicular chromatin

(white arrows). Mice that received HEL cells and the 0.1 mg/kg/day dosage exhibited

fewer neoplastic cells. Mice receiving HEL cells and the 1 and 10 mg/kg/day dosages

of G6 had even fewer neoplastic cells as well as increasing megakaryopoietic activity,

signs of leukemic regression and normalization of hematopoiesis. Finally, in the cohort

38

of mice that received G6 alone, four of the spleens were histologically normal, but two

displayed some necrosis and edema.

To obtain a quantitative measure for the efficacy of G6 in the spleen, the number

of pathological erythroblast foci was determined by computer assisted morphometric

analysis using a standard microscope and then plotted as a function of treatment group

(Figure 2-9C). We found that G6 treatment provided significant therapeutic benefit as

evidenced by a significant reduction in the number of erythroblast foci. Furthermore,

these results had a positive correlation with spleen size; namely, the reduction in splenic

erythroblast numbers directly correlated with decreased spleen size. Thus, the data in

Figure 2-9 suggest that G6 provides therapeutic benefit to the spleen in a mouse model

of Jak2-V617F mediated human erythroleukemia.

The Presence of G6 in the Plasma, Marrow and Spleen Correlates with Indicators of Therapeutic Efficacy

Finally, we wanted correlate the presence of G6 in hematopoietic tissues with

indicators of therapeutic efficacy. For this, baseline body weights and peripheral blood

samples were obtained from twelve NOD-SCID mice. The mice were subsequently

injected, intravenously, with 2x106 HEL cells. Three weeks later, peripheral blood

samples were again obtained to confirm that the animals were in blast crisis, at which

time the animals began receiving either vehicle control (DMSO) or G6 (1 mg/kg/day) via

single, daily IP injections. After 2 weeks of injection, analysis of peripheral blood

samples indicated that the G6-treated mice had significantly fewer blast cells in the

peripheral blood when compared with the DMSO-injected mice (Figure 2-10A). The

mice were euthanized the following day, and the spleen weight to body weight ratios

were determined for both treatment groups (Figure 2-10B). We found that for the mice

39

that received G6, the spleen weight to body weight ratio was reduced by ~40% when

compared with the mice that received vehicle control. To correlate these efficacious

indicators to the presence of G6, the concentration of G6 in the plasma, marrow and

spleen was determined. Analysis of the plasma samples that were collected at baseline

and after HEL cell injection, but prior to any vehicle/drug injection, completely lacked G6

(data not shown). For the terminal plasma samples that were collected at euthanasia

along with the marrow and spleen, G6 was completely absent in the samples that came

from vehicle control-injected mice, but present in the samples that came from G6-

treated mice (Table 2-1). Overall, the data in Figure 2-10 and Table 2-1 correlate

therapeutic efficacy in the form of decreased blast cells in the peripheral blood and

reduced spleen weight to body weight ratios with the presence of G6 in the plasma,

marrow and spleen.

Discussion

The main finding of this work is that G6 suppresses Jak2-V617F mediated

hyperplasia, in vitro and in vivo. Chemically, G6 is classified as a stilbenoid. Stilbenoids

are diarylethenes, that is, a hydrocarbon consisting of an ethene double bond

substituted with a phenyl group on both carbon atoms of the double bond. Stilbenoids

are known to have beneficial properties including anti-oxidative, anti-proliferative, and

tumor suppressive effects [105,106,107]. Resveratrol, a naturally occurring stilbenoid

found in the skin of red grapes, reduces the incidence of cardiovascular disease [108].

Piceatannol, a naturally occurring phenolic stilbenoid, exhibits anti-tyrosine kinase

activity. Specifically, it inhibits LMP2A, a tyrosine kinase associated with Epstein-Barr

virus infections [109,110]. The significance of these reports is that there is marked

40

precedent for stilbene compounds such as G6 possessing beneficial biological activity

in general, and anti tyrosine kinase activity in particular.

Evaluation of the in vivo data suggests that we have identified dosages of G6

that range from sub-therapeutic through toxic. Specifically, the 0.1 mg/kg/day dosage

provided some observable benefit. This low dosage reduced the percentage of blast

cells in the peripheral blood and leukemic cells in the spleen. However, it was unable to

alleviate the splenomegly or significantly reduce the numbers of HEL cells in the

marrow. The 1 mg/kg/day dosage was highly therapeutic as evidenced by reductions of

blast cells in the peripheral blood, reduced splenomegaly, elimination of HEL cells from

the marrow with correction of the M:E ratio, leukemic regression in the spleen, and

signs of return of normal hematopoiesis. Additionally, animals treated at this dose

displayed absolutely no signs of histological toxicity. Finally, while the 10 mg/kg/day

dosage clearly provided leukemic therapeutic benefit, one animal receiving this dose

exhibited some splenic necrosis. However, the brains, lungs, kidneys, and livers from

all these animals were histologically normal, indicating the G6-mediated cytotoxicity

might be targeted to hematopoietic organs at this dose. For the mice that received the

high dose of G6 alone, the marrow was hypo-cellular with two of six animals exhibiting

some bone marrow necrosis. Overall, the spleen weight to body weight ratio for this

cohort was increased (Figure 2-6B). Two of the six spleens from this group were

necrotic and edematous. However, the brains, lungs, kidneys, and livers from these

mice were histologically normal. As such, the data suggest that when given alone at the

10 mg/kg/day dosage, G6 can be cytotoxic to hematopoietic tissues.

41

An important consideration is understanding the precise linkage between G6-

mediated Jak2 inhibition, suppression of STAT5 phosphorylation and apoptosis within

HEL cells as it relates to our xenograft model. Jak2/STAT signaling is known to

positively regulate cell growth by directly increasing expression of the anti-apoptotic

marker, Bcl-xL, via STAT-binding elements located in its proximal promoter region

[111,112]. In a recently published work, we reported that G6 inhibits HEL cell growth via

the down regulation of Bcl-xL and this correlates with significantly reduced phospho-

STAT5 levels [113]. Furthermore, we showed that G6 treatment of HEL cells results in

upregulation of pro-apoptotic Bim, and cleavage of pro-apoptotic Bid, from its inactive

precursor to its active form [113]. In our work here, we show that G6 treatment results in

reduced STAT5 phosphorylation within HEL cells (Figure 2-5C) and within the bone

marrow (Figure 2-8A and 2-8B). Additionally, we show here that treatment of HEL cells

with G6 results in increased apoptosis (Figure 2-4A and 2-4B), and a dose-dependent

increase of apoptosis levels within the bone marrow of treated mice (Figure 2-8C and 2-

8D). As such, we believe that the underlying mechanism that allows G6 to provide

therapeutic efficacy involves the direct inhibition of Jak2, the corresponding suppression

of STAT5 phosphorylation, and apoptosis.

Recent works have reported paradoxical effects regarding the efficacy of Jak2

inhibitors when tested in vitro versus in vivo. For example, while the Jak2 inhibitor CEP-

701 exhibited good Jak2 efficacy in vitro, Santos et al. reported it failed to improve the

marrow fibrosis or alleviate the burden of marrow derived Jak2-V617F mutant clones in

humans suffering from PMF [114]. Similarly, while the Jak2 inhibitor INCB16562

exhibited good Jak2 efficacy in vitro, Koppikar et al. reported that it was unable to

42

reduce the number of malignant clones in the bone marrow using a mouse model of

MPLW515L-induced thrombocytosis and myelofibrosis [115]. Finally, while the Jak2

inhibitor compound CYT387 exhibited good Jak2 efficacy in vitro [93], work by Tyner et

al. showed that it was unable to eliminate Jak2-V617F mutant clones in vivo using a

murine myeloproliferative neoplasm model [100]. Our work here is significant in that we

show that G6 not only exhibits excellent therapeutic efficacy in vitro, but also in vivo as

measured by the critical elimination of mutant clones from the marrow of mice (Figure 2-

8D) and a corresponding correction of the M:E ratio (Figure 2-7C). As such, this work

suggests that stilbenoid based compounds such as G6 may possess unique Jak2

inhibitory properties that previous pyrimidine based compounds lack. In summary, we

show that the stilbenoid compound, G6, has therapeutic efficacy against Jak2-V617F

mediated human pathogenesis in vitro and in vivo. As such, this compound may have

practical applications in Jak2-related research and as a potential therapeutic agent.

43

-1 0 1 2 3 4 5 6

HEL cells, IV2x106 cells

NOD/SCID, N = 366 groups (n=6)Blood sampleBody weight

G6/DMSO – IP1. No treatment2. HEL+DMSO3. HEL+0.1mg/kg G64. HEL+1.0mg/kg G65. HEL+10mg/kg G66. 10mg/kg G6

Daily

Euthanized

Weeks

Weight + Blood Sample weekly

Figure 2-1. Model of Jak2-V617F mediated, human erythroleukemia: Four of the six groups (2, 3, 4, and 5) received 2 x 106 HEL cells via a single tail vein injection at Day 0. The disease was allowed to progress for 3 weeks at which time the mice began receiving daily injections of either vehicle control (DMSO) or G6 at the dosages of 0.1, 1, and 10 mg/kg/day for the next 21 days. 24 hours after the last injection, the mice were euthanized and prepared for analysis.

44

Num

ber

of v

iabl

e ce

lls (

% C

ontr

ol)

Time (Hours)

Initial Exposure Time to G6 (Hours)

B

C

A

G6 (μM)

0 20 40 60 80

0

40

80

120

0.01 0.1 1 10 100

0

40

80

120N

umbe

r of

via

ble

cells

(%

Con

trol

)

Nu

mbe

r of

via

ble

cells

(%

Con

trol

)

0 20 40 60 80

0

40

80

120

Figure 2-2. G6 inhibits Jak2-V617F dependent HEL cell proliferation, in vitro. A) HEL cells were treated with increasing doses of G6 for 72 hours and the number of viable cells was determined. B) HEL cells were treated 25 μM of G6 for 0, 6, 24, 48 and 72 hours and cell numbers were determined. C) HEL cells were treated with 25 μM of G6 for 0, 6, 12, 24, 48 and 72 hours. At the end of each time point, the cells were placed in media lacking inhibitor for an additional 72 hours. The number of viable cells was then determined. Shown are mean +/- SEM for three independent experiments, each run in triplicate.

45

0 5 10 15 20 25 3040

45

50

55

60

65

70

75

80

0 5 10 15 20 25 3015

20

25

30

35

40

0 5 10 15 20 25 305

10

15

20

25

30

35

0 20 40 60 8020

30

40

50

60

70

80G6 DMSO

0 20 40 60 8015

20

25

30

35

40

45 G6 DMSO

0 20 40 60 805

10

15

20

25

30

35

40

G6 DMSO

Time (hours) Time (hours) Time (hours)

G6 (µM) G6 (µM)G6 (µM)

G1

Pha

se (

%)

G1

Ph

ase

(%)

S P

hase

(%

)S

Pha

se (

%)

Apo

ptos

is (

%)

Ap

opto

sis

(%)

A

D

B C

FE

Figure 2-3. G6 suppresses HEL cell growth by inducing G1 phase cell cycle arrest. HEL cells were treated with increasing doses of G6 for 72 hours or with 25 μM G6 for increasing times. Cellular DNA contents were then determined by flow cytometry. Three independent experiments, each measured in triplicate, were averaged and the aggregate cell cycle data were graphed as a function of A-C) G6 concentration or D-F) G6 exposure time. Shown are the percentages of cells in G1 phase (A,D), S phase (B, E) and apoptosis (C, F). Shown are mean +/- SEM.

46

0 10 20 30 40 500

5

10

15

20

25

Apo

ptos

is (

%)

Time (Hours)

B

0

5

10

15

20

25

30

35

0.01 0.1 1 10 100

Apo

ptos

is (

%)

G6 (μM)

A

Figure 2-4. G6 induces the intrinsic apoptotic pathway in HEL cells. For apoptotic measurements, Annexin V/propidium iodide double staining was employed. The values from three independent A) dose response or B) time course experiments were graphed.

47

0

1

2

3

4

5

6

7

0.5

1.5

2.5

0 hr 12 hr 24 hr 48 hr 72 hr

G6 μM

P-S

TAT1

(uni

ts/m

l)P

-STAT1

(uni

ts/m

l)

*

**

0 3.125 6.25 9.375 12.5 25 0

5

10

15

20

25

30

0

5

10

15

20

25

30

35

P-S

TA

T3

(uni

ts/m

l)P

-STA

T3

(uni

ts/m

l)

0 3.125 6.25 9.375 12.5 25

G6 μM

0 hr 12 hr 24 hr 48 hr 72 hr

* *

**

0

10

20

30

40

50

60

70

80

0

20

40

60

80

100

120

P-S

TA

T5

(uni

ts/m

l)

0 3.125 6.25 9.375 12.5 25

G6 μM

P-S

TA

T5

(uni

ts/m

l)

0 hr 12 hr 24 hr 48 hr 72 hr

*

*

** **

****

**

C

BA

Figure 2-5. G6 preferentially inhibits Jak2-V617F dependent constitutive activation of STAT5. HEL cells were treated with either increasing concentrations of G6 or with 25 µM G6 for increasing periods of time. The levels of phospho-STAT1 (pY701), phospho-STAT3 (pY705) and phospho-STAT5a/b (pY694/699) were then simultaneously measured. Dose- and time-dependent inhibition of A) phospho-STAT1, B) phospho-STAT3, and C) phospho-STAT5a/b are shown. *, p < 0.05 versus DMSO control.

48

0 5 10 15 200

5

10

15

20

HEL+DMSOHEL+0.1 mg/kg G6HEL+1 mg/kg G6HEL+10 mg/kg G6

% B

last

Cel

l Coun

ts

Days of G6 Treatment

*

*

*

*

*

0.002

0.0022

0.0024

0.0026

0.0028

0.003

0.0032

Naïve

SCID

HEL+DM

SO

HEL+0.

1 mg/

kg G

6

HEL+1

mg/kg

G6

HEL+10

mg/

kg G

6

10 m

g/kg

G6

Sple

en W

t : B

ody

Wt R

atio # #

#

* *

B

A

Figure 2-6. G6 decreases the percentage of blast cells in the peripheral blood and reduces the spleen weight to body weight ratio in a mouse model of Jak2-V617F mediated human erythroleukemia. A) Percentages of blast cells in the peripheral blood plotted as a function of both treatment group and days of treatment. *, p < 1.0 x10-4 vs. DMSO treated mice. B) The spleen to body weight ratio was obtained and plotted as a function of treatment group. #, p < 0.05 vs. naïve mice; *, p < 0.05 versus. HEL+DMSO.

49

Naïve NOD-SCID HEL + DMSO HEL + 0.1mg/kg G6

HEL + 1mg/kg G6 HEL + 10mg/kg G6 10mg/kg G6

406080

100120140160180

Naïve S

CID

HEL+DMSO

HEL+0.1 m

g/kg G

6

HEL+1 m

g/kg G

6

HEL+10

mg/kg

G6

10 mg/k

g G6

Nu

mb

er o

f C

ells

Myeloids Erythroids

0.20.40.60.8

11.21.41.6

Naïve S

CID

HEL+DMSO

HEL+0.1 m

g/kg G6

HEL+1 mg/kg G

6

HEL+10 m

g/kg G

6

10 mg/kg

G6

M:E

Rat

io

#

#

#

#

* * * * * *

# #

* * *

0

5

10

15

20

25

HEL+DMSO

HEL+0.1 mg/kg G

6

HEL+1 mg/kg G

6

HEL+10 mg/kg

G6

10 mg/kg

G6

Hu

man

CD

45+

Cel

ls (

%) A D

B

C

Figure 2-7. G6 improves the M:E ratio in a mouse model of Jak2-V617F mediated human erythroleukemia by reducing HEL cell engraftment in the bone marrow. After a three week period of drug or vehicle administration, all groups were euthanized and histological sections of the femurs were prepared. A) Representative H&E stained bone marrow sections for each treatment group. B) The number of mature myeloid and erythroid cells was determined. #, p < 0.05 relative to naïve; *, p < 0.05 relative to HEL+DMSO. C) The myeloid and erythroid cell numbers plotted as the M:E ratio. #, p < 0.05 relative to naïve; *, p < 0.05 relative to HEL+DMSO. D) The average percentages of human CD45+ cells present in the bone marrow were plotted as a function of treatment group.

50



0.006

0.008

0.01

0.012

0.014

Naïve SCID

HEL+DMSO

HEL+0.1 mg /kg G

6

HEL+1 m

g/kg G

6

HEL+10 m

g/kg G

6

10 m

g/kg G

6

Ph

osp

ho

-ST

AT

5

*

*

# #

#



0

20

40

60

80

Naïve S

CID

HEL+DMSO

HEL+0.1

mg/kg

G6

HEL+1 m

g/kg G

6

HEL+10

mg/k

g G6

10 m

g/kg G

6

# o

f T

UN

EL

Po

siti

ve C

ells

* **

A B

C D

Figure 2-8. G6 reduces the levels of phospho-STAT5 and induces cellular apoptosis in the bone marrow. A) Representative anti phospho-STAT5 immuno-histochemistry bone marrow sections from the indicated treatment groups. B) Anti phospho-STAT5 staining was quantified and plotted as a function of treatment group. *p < 0.05 versus naïve; #p < 0.05 versus HEL+DMSO. C) Representative TUNEL stained bone marrow sections from each treatment group. D) Bone marrow TUNEL positive cells were counted and plotted as a function of treatment group. *p < 0.05 versus HEL+DMSO.

51

Naïve NOD-SCID HEL + DMSO HEL + 0.1 mg/kg G6

HEL + 1 mg/kg G6 HEL + 10 mg/kg G6 10 mg/kg G6



Ery

thro

blas

t Fo

ci (μ

M2 )

*

#

# # #

0

1000

2000

3000

4000

5000

6000

7000

Naïve S

CID

HEL+DMSO

HEL+0.1

mg/kg

G6

HEL+1 m

g/kg G

6

HEL+10

mg/k

g G6

10 m

g/kg G

6

A C

B

Figure 2-9. G6 treatment results in leukemic regression and normalization of hematopoiesis in the spleen. Histological sections of the spleen were prepared from each treatment group and viewed at A) 10X magnification and B) 100X magnification. Injection of HEL cells resulted in the appearance of neoplastic cells in the spleen (arrowheads in panel B) and this was reduced with G6 treatment. C) The number of erythroblast foci were counted and plotted as a function of treatment group. *p < 0.05 versus naïve; #p < 0.05 versus HEL+DMSO.

52

Figure 2-10. Therapeutic indicators of G6 efficacy correlate with the present of G6 in

the plasma, marrow, and spleen. A) Percentages of blast cells in the peripheral blood plotted as a function of both treatment group and time. *, p _ 0.05 versus DMSO-treated mice. B) Spleen to body weight ratio was obtained and plotted as a function of treatment group.

53

54

Table 2-1. Mass spectrometry results showing plasma and tissue concentrations of G6 at euthanasia.

a Average of replicate injections. b Calculations based on average of four replicate injections back-calculated using analyte concentrations (mg/ml) divided by prepared tissue concentration (g/ml) resulting in mg/g of tissue concentrations. c <0 indicates peak quantitates were below the 0 value of the standard curve. No Peak indicates no peak was observed in raw chromatography.

CHAPTER 3 THE JAK2 INHIBITOR, G6, ALLEVIATES JAK2-V617F MEDIATED

MYELOPROLIFERATIVE NEOPLASIA BY PROVIDING SIGNIFICANT THERAPEUTIC EFFICACY TO THE BONE MARROW

Polycythemia vera (PV), essential thrombocythemia (ET), and primary

myelofibrosis (PMF) are myeloproliferative neoplasms (MPN) lacking the Philadelphia

chromosome. They are caused by the transformation of an early hematopoietic stem

cell resulting in abnormal hematopoiesis [116,117]. These disorders are categorized

according to the syndromes caused by the terminally differentiated hematopoietic cells

such as increased production of red blood cells (PV), platelets (ET), and neutrophils

with concomitant fibrosis of the bone marrow tissue (PMF). Clinically, these diseases

are characterized by pathological peripheral blood syndromes such as leukocytosis,

erythrocytosis, and thrombocytosis. These syndromes predispose patients to vascular

diseases such as thrombosis, atherosclerosis, coronary heart disease and cerebral

ischemia [118,119,120,121,122]. In addition, patients with MPNs often have high levels

of circulating inflammatory cytokines, such as interleukin-6 (IL-6) which have been

associated with symptoms such as cachexia and listlessness [123,124,125,126,127].

Moreover, MPNs can often transform to acute myeloid leukemia (AML) [127]. Although

these disorders can be fatal with a life expectancy that can be as few as 5 years [128],

currently available treatments are limited.

The discovery of the Jak2-V617F mutation in most patients with MPN spurred the

development of small molecule Jak2 inhibitors via molecularly targeted drug discovery.

In preclinical experiments, many of these small molecules exhibited potent inhibition of

Jak2-mediated pathological cell growth. Some have subsequently progressed to clinical

trials where they exhibited some benefit by reducing clinical symptomologies associated

55

with the MPN phenotype [129]. However, none of these inhibitors have been reported to

be curative as they have little to no efficacy in the bone marrow and there is often a

relapse of the clinical disease manifestations after withdrawal of treatment. Thus,

current Jak2 inhibitors are largely palliative as they are unable to eradicate the Jak2

mutant burden in the bone marrow, which is the primary predilection site of the MPN

disease pathogenesis.

Recently, we developed a stilbenoid small molecule Jak2 inhibitor, G6, which

exhibits potent inhibition of Jak2-V617F mediated pathological cell growth in vitro and

ex vivo [96,112]. We subsequently reported that G6 has therapeutic potential in a NOD-

SCID mouse model of Jak2-V617F mediated hyperplasia as it eliminated the burden of

tumorigenic Jak2-V617F cells from the host bone marrow [131]. Therefore, we

hypothesized here, that G6 would be efficacious against Jak2-V617F-mediated

myeloproliferative neoplasia by providing significant efficacy to a number of tissues

including the bone marrow. To test this, we utilized a transgenic mouse model of Jak2-

V617F mediated myeloproliferative neoplasia and found that G6 treatment greatly

alleviated the MPN phenotype by providing significant therapeutic benefit to the

peripheral blood, liver, spleen and most notably, the bone marrow. As such, G6

appears to alter the natural history of Jak2-V617F mediated myeloproliferative

neoplasia by providing significant efficacy to the bone marrow where other Jak2

inhibitors have not.


Animals

Transgenic male mice expressing the Jak2-V617F mutated enzyme in the

hematopoietic system driven by the vav promoter and generated on a C57BL/6

56

background strain were used in these experiments [103]. All animal procedures were

approved by the Institutional Animal Care and Use Committee at the University of

Florida. Animals were maintained in accordance with NIH standards established in the

Guidelines for the Care and Use of Experimental Animals. The transgenic mice were

identified by PCR using primers TACAACCTCAGTGGGACAAAGAAGAAC and

CCATGCCAACTGTTTAGCAACTTCA which cover a 594 base-pair region in the coding

sequence of Jak2-V617F [103]. At 3 months of age, a baseline peripheral blood sample

was obtained from each mouse via sub-mandibular bleeding. The mice were then

injected with either vehicle (n=6) or 10 mg/kg/day of G6 (n=6) for 28 days. Other blood

samples were subsequently obtained after 14 and 28 days of vehicle or G6 treatment.

Analysis of Peripheral Blood Cells

Blood samples were obtained (~50 μl) via sub-mandibular bleeding into tubes

containing potassium salt of ethylenediamine tetraacetic acid. Complete blood counts

were obtained using a HESKA Vet ABC-Diff Hematology analyzer. Blood samples were

then smeared onto glass slides and stained using DipQuick (Jorgensen Laboratories

Inc. Loveland, CO).

Interleukin-6 Analysis

Blood samples were obtained (~50 µl) via sub-mandibular bleeding into tubes

containing potassium salt of ethylenediamine tetraacetic acid. Plasma was centrifuged

at 10 000g for 10 minutes and then stored at -80°C for subsequent analysis. Plasma

levels of IL-6 were measured using a commercially available mouse ELISA kit (Ray

Biotech) according to the manufacturer’s instructions.

57

Histological Analysis

Tissues (liver, spleen, and bone marrow) were fixed overnight in formaldehyde.

Femurs were decalcified for 16 hours. The tissues were subsequently dehydrated

through a graded ethanol series, paraffin embedded and sectioned (4 µm). The tissues

were then stained with hematoxylin and eosin (H&E), and they were examined for

normal histological appearance as well as any lesions via standard light microscopy by

two independent veterinary pathologist blinded to treatment groups. Bone marrow

analysis was done according to established guidelines [130]. The bone marrow was

evaluated for necrosis, fibrosis, hemorrhage, overall cellularity, M: E ratios and

megakaryocytic counts. All cell counts were made at 600X HPF. Spleen sections were

examined for evidence of extramedullary hematopoiesis (EMH) and a quantitative analysis

of megakaryocytes was made. Liver sections were examined for evidence of EMH at 40X.

Immunohistochemistry

Immunohistochemistry on bone marrow samples was carried out as previously

described [131]. Briefly, 5 µm sections mounted on gelatin-coated slides were dewaxed

in ethanol, rehydrated, then blocked in 3% H2O2 followed by 5% normal goat serum.

Sections were exposed to the primary antibody including anti-Jak2 (ab39636 Abcam),

anti-phospho-Jak2 (Ab32101 Abcam), or anti-phospho-STAT5 (Ab32364 Abcam)

overnight at 4oC. The sections were washed, and then treated with the biotinylated

secondary antibody. After secondary antibody incubation, the samples were washed,

exposed to the avidin-peroxidase reagent (Vectastain Elite, Vector Laboratories,

Burlingame, CA), and reacted with diaminobenzidine to produce a brown reaction

product. The sections were dehydrated in ethanol, mounted with Permount, and

observed by light microscopy.

58

Determination of Jak2-V617F Allele Burden in the Bone Marrow

RNA was isolated from bone marrow using the RNeasy Mini Kit (QIAGEN) and

DNA contamination was removed using the RNase-free DNase set (QIAGEN). cDNA

was synthesized using the high capacity cDNA Reverse Transcription Kit (Applied

Biosystems) on 2 µg RNA, at 60˚C. Real-time PCR was performed in a Multicolor Real-

Time PCR Detection System using TaqMan gene expression assays (Applied

Biosystems). PCR amplifications were performed in duplicate for human Jak2

(Hs01078124_m1) and mouse Jak2 (Mm00434577_m1) along with parallel

measurements of mouse β-actin cDNA as an internal control. The copy number of the

human Jak2 sequence relative to β- actin was calculated using a standard curve

technique. The allele burden was computed by calculating the ratio of the human Jak2

to the mouse Jak2 in the transgenic mice.

Clonogenic Assay

To determine the effectiveness of G6 in preventing the cytokine-independent

survival and proliferation of bone marrow cells obtained from the transgenic mice, bone

marrow cells from Jak2-V617F transgenic mice were harvested, cultured ex vivo, and

exposed to 25 µM of G6 for the indicated periods of time. The drug was then washed

away from the cells and they were plated in MethoCult media lacking EPO and TPO.

Five days later, the number of CFU-GM and CFU-E were counted and plotted as a

function of treatment group.


All results were expressed as mean +/- SEM. Statistical comparisons were

performed by Student’s t test. Changes in peripheral blood cell counts were analyzed by

a repeated-measures ANOVA followed by Bonferroni and Student-Newman-Keuls post

59

hoc test for multiple comparisons. p values of less than 0.05 (2-tailed) were considered

statistically significant.

Results

G6 Provides Therapeutic Benefit in Peripheral Blood of Jak2-V617F MPN Mice

Here, we employed a previously established transgenic mouse model of Jak2-

V617F mediated myeloproliferative neoplasia [133]. These mice express the human

Jak2-V617F cDNA under the control of the marrow stem cell promoter, vav. They

exhibit a number of phenotypes that recapitulate those observed in human MPN

including constitutive Jak/STAT signaling, myeloid neoplasia, leukocytosis,

thrombocytosis, erythrocytosis, and splenomegaly. Complete blood counts (CBC) were

first performed on three month old male mice to confirm the MPN phenotype. Mice fully

manifesting the MPN phenotype were randomly assigned to one of two groups (n=6 per

group) and then began receiving either 10 mg/kg/day of G6 or vehicle control solution.

CBCs were subsequently collected on days 14 and 28 of treatment via mandibular vein

bleeding and after 28 days of treatment, all the mice were euthanized and prepared for

analysis.

The CBC values were first examined by a repeated-measures analysis of

variance to determine whether there were any significant differences between the two

treatment groups (Table 3-1 and 3-2). Values from non-transgenic control mice are also

shown for comparison. We found that there were significant therapeutic improvements

in the erythrocyte and platelet indices including the red blood cell count (RBC),

hematocrit (HCT), mean corpuscular volume (MCV), red blood cell distribution width

(RDW), hemoglobin (HB), mean corpuscular hemoglobin (MCH), mean corpuscular

hemoglobin concentration (MCHC), platelet count (PLT), mean platelet volume (MPV)

60

and platelet distribution width (PDW) (Table 3-1). There were also significant

improvements in the leukocyte indices including the white blood cell count (WBC),

neutrophil count (NE), lymphocyte count (LO) and monocyte count (MO) (Table 3-2). To

confirm these observations via an alternate means, terminal peripheral blood smears

from all mice were examined in parallel (Figure 3-1A). In addition to validating many of

the CBC values, examination of the slides revealed an increased appearance of large

platelets in the vehicle treated MPN mice which were lacking in the G6 treated mice.

Previous work has shown that MPN patients have significantly elevated levels of

inflammatory cytokines, such as IL-6, in the peripheral blood [126]. To determine

whether this was also the case in our MPN mice and whether G6 could correct this,

terminal plasma IL-6 concentrations from both groups of mice were determined (Figure

3-1B). We found that the plasma concentrations of IL-6 were markedly elevated in the

vehicle-treated MPN mice; for reference, the values in non-transgenic mice are normally

35-43 pg/ml. However, we observed that G6 treatment completed normalized the

plasma levels of IL-6 in the MPN mice.

We next wanted to determine if the efficacious parameters observed in the

peripheral blood of G6 treated mice correlated with the presence of the drug in the

plasma. For this, mass spectrometry analysis was performed on the terminal plasma

samples and the levels of G6 were determined (Table 3-3). We found that G6 was

detectable in plasma samples from all the mice that received the drug, but not in the

plasma taken from mice that received vehicle control solution.

G6 Reduces Extramedullary Hematopoiesis in Jak2-V617F MPN Mice

Another pathology observed in the Jak2-V617F MPN mice is an abnormally high

degree of extramedullary hematopoiesis (EMH). Constitutive expression of the Jak2-

61

V617F transgene drives hematopoiesis in a number of tissues including the liver. In

order to determine whether G6 could reduce this Jak2-V617F mediated pathogenesis,

post mortem liver sections were examined by light microscopy and the levels of EMH

were quantified. Figure 3-2A shows representative liver sections from all conditions and

Figure 3-2B shows the quantitative values of EMH plotted as a function of condition.

We found that when compared to wild type mice, the MPN mice treated with vehicle

control solution exhibited an increased level of EMH. However, this was corrected with

G6 treatment. Overall, the data in Figure 3-2 indicate that G6 is efficacious in the liver

given its ability to normalize the levels of EMH in Jak2-V617F MPN mice.

G6 Provides Therapeutic Benefit to the Spleen of Jak2-V617F MPN Mice

The Jak2-V617F mouse recapitulates many of the spleen pathologies observed

in human MPN including splenomegaly and megakaryocytic hyperplasia. To determine

the efficacy of G6 in the spleen, several parameters were measured. First, at

euthanasia, spleens were immediately removed from the mice and gross spleen

weights were determined. Figure 3-3A shows representative spleens from each

condition and Figure 3-3B shows the quantitative spleen weight to body weight ratios.

We found that following 28 days of G6 treatment, the spleen size, which was

significantly increased in Jak2-V617F MPN mice, was significantly reduced with G6

treatment.

Histological sections through the spleen revealed a disorganized splenic

architecture in the Jak2-V617F MPN mice treated with vehicle control solution and this

was alleviated with G6 treatment (Figure 3-3C). Examination of the sections at higher

power revealed a marked megakaryocytic hyperplasia in the Jak2-V617F MPN mice

which was absent in the G6 treated mice (Figure 3-3D). To quantitate this hyperplasia,

62

the average numbers of megakaryocytes per high power field were plotted as a function

of condition (Figure 3-3E). We found that G6 treatment returned the number of

megakaryocytes to normal, non-transgenic levels. Collectively, the data in Figure 3-3

indicate that, in a mouse model of Jak2-V617F mediated myeloproliferative neoplasia,

G6 provides significant therapeutic benefit to the spleen as determined by a significantly

reduced spleen weight to body weight ratio, a restoration of normal splenic architecture,

and an elimination of megakaryocytic hyperplasia.

G6 Provides Therapeutic Benefit to the Bone Marrow of Jak2-V617F MPN Mice by Alleviating Megakaryocytic and Myeloid Hyperplasia

The ability of a drug to provide therapeutic benefit in the bone marrow of MPN

patients is critically important since this is the site of initiation of disease pathogenesis.

Additionally, this has been the point of failure for current generation Jak2 inhibitors. To

assess the efficacy of G6 in the bone marrow, we first examined marrow sections.

Figure 3-4A shows representative histological sections from each group. We found that

when compared to non-transgenic controls, the vehicle-treated Jak2-V617F MPN mice

had a hyper-cellular marrow due to myeloid and megakaryocytic hyperplasia and this

corresponded with the increased platelet counts observed in the peripheral blood (Table

3-1). However, G6 appeared to restore the marrow to non-diseased conditions. To

confirm this quantitatively, the average number of megakaryocytes per high power field

were determined from all animals and plotted as a function of treatment group (Figure 3-

4B). We found that in the Jak2-V617F MPN mice, G6 reduced the number of

megakaryocytes in the marrow to near WT levels.

It is well accepted that an altered M:E ratio is often one of the characteristic signs

of Jak2-V617F-mediated myeloproliferative neoplasia. In order to determine whether

63

G6 could correct the abnormally high M:E ratio in the bone marrow of the Jak2-V617F

MPN mice, we carried out a quantitative analysis of the myeloid and erythroid cells on

the marrow sections (Figure 3-4C). We found that when compared to the WT mice,

there was a robust increase in the M:E ratio in the vehicle treated Jak2-V617F MPN

mice that was driven by myeloid hyperplasia. However, G6 treatment returned the M:E

ratio to wild type levels. Altogether, the data in Figure 3-4 demonstrate that G6 has

marked therapeutic benefit in the bone marrow. Specifically, it reduced the pathologic

increase in megakaryocytic and myeloid hyperplasia in the marrow as a consequence of

which, the M:E ratio was completely normalized.

G6 Provides Therapeutic Benefit to the Bone Marrow in Jak2-V617F MPN Mice by Reducing the Pathological Levels of Phospho-Jak2 and Phospho-STAT5

In order to determine whether the therapeutic benefit observed in the marrow

with G6 treatment is a result of reduced Jak/STAT signaling, we carried out anti

phospho-Jak2 and anti phospho-STAT5 immuno-histochemistry staining of the bone

marrow sections. Figure 3-5A shows representative images of the anti phospho-Jak2

immuno-histochemistry at two magnifications. Qualitatively, we found that bone marrow

sections obtained from the Jak2-V617F MPN mice treated with vehicle control had a

robust increase in phospho-Jak2 levels when compared to the wild type mice. However,

the phospho-Jak2 staining was reduced to wild type levels in the Jak2-V617F MPN

mice that were treated with G6. These qualitative observations were supported

quantitatively when the numbers of anti phospho-Jak2 stained cells were counted and

plotted as a function of treatment group (Figure 3-5B).

The therapeutic effect within the bone marrow was further verified by the ability of

G6 to reduce the levels of the proliferative marker, phospho-STAT5. STAT5 is an

64

immediate downstream target of Jak2 and is hyper-phosphorylated in Jak2-V617F

expressing cells [113,131]. Figure 3-5C shows representative bone marrow pictures of

the anti phospho-STAT5 immuno-histochemistry stained sections and Figure 3-5D

shows the quantification of all sections plotted as a function of treatment group. We

similarly observed that when compared to wild type mice, the Jak2-V617F MPN mice

that were given vehicle control solution had pathologically high levels of phospho-

STAT5. Again however, G6 fully corrected this pathogenesis by returning the phospho-

STAT5 levels to non-transgenic levels.

In summary, the data in Figure 3-5 show that G6 has striking therapeutic efficacy

in the bone marrow. Specifically, the Jak2-V617F MPN mice have significantly elevated

levels of phospho-Jak2 and its proliferative downstream target, phospho-STAT5.

However, G6 treatment normalizes these values to non-diseased levels.

G6 Provides Therapeutic Benefit to the Bone Marrow in Jak2-V617F MPN Mice by Significantly Reducing the Mutant Jak2 Allelic Burden

The greatest obstacle to current Jak2 inhibitors is the inability of these drugs to

eliminate Jak2-V617F mutant clones from the bone marrow. To determine the efficacy

of this parameter in our MPN model, we measured the mRNA levels of both the human

Jak2-V617F mutant mRNA transcripts and endogenous mouse Jak2-WT transcripts.

We found that while G6 treatment significantly reduced the levels of the mutant V617F

transcripts (Figure 3-6A), endogenous wild type transcripts were only slightly reduced

by G6 treatment, and this change was not significant (Figure 3-6B). Furthermore, we

found that the ratio of these two parameters (i.e., the mutant burden within the marrow)

was reduced, on average, by ~67% with G6 treatment when compared to Jak2-V617F

MPN mice that received vehicle control injections (Figure 3-6C). In addition, one-third

65

of the G6 treated mice exhibited virtual elimination of all Jak2-V617F transcripts from

the marrow. Thus, the data in Figure 3-6 demonstrate that G6 significantly reduces the

burden of Jak2-V617F mutant cells from the bone marrow in a model of Jak2-V617F

mediated myeloproliferative neoplasia.

G6 Prevents Jak2-V617F Mediated Clonogenic Growth

Lastly, we wanted to assess whether G6 can stop the clonogenic growth potential

of Jak2-V617F transformed cells as this would be crucial to any therapeutic possibility.

For this, cells were harvested from the bone marrow of Jak2-V617F MPN mice and

cultured ex vivo in the presence of 25 M of G6 for either 0, 12, or 24 hours. The cells

were then washed extensively to remove drug and plated in medium lacking EPO and

TPO. Five days later, the number of granulocyte-macrophage colony forming units

(Figure 3-7A) and erythroid colony forming units (Figure 3-7B) were counted and plotted

as a function of time. We found that G6 significantly suppressed the clonogenic growth

potential of Jak2-V617F cells in a time dependent manner; 12 hours of drug exposure

resulted in ~50% growth inhibition and 24 hours of drug exposure virtually eliminated all

subsequent clonogenic growth. As such, these results indicate that brief exposures of

Jak2-V617F cells to G6 prevent subsequent clonogenic growth.

Discussion

Since the discovery of the Jak2-V617F mutation in most patients with MPN, a

number of molecularly targeted Jak2 inhibitors have been developed. However, the

clinical benefits provided by these inhibitors so far have largely been palliative due to

their inability to eliminate malignant clones from the bone marrow. As such, these drugs

have no ability to alter the natural history of the disease. Furthermore, those few drugs

that have exhibited some efficacy in the marrow have done so only after numerous

66

cycles of treatment [129]. The consequence of such frequent treatment regimens

includes a number of undesirable side effects including myelotoxicity. Worse, there is a

relapse of disease after cessation of treatment. Thus, the identification of Jak2 inhibitors

that can provide significant bone marrow efficacy in the absence of repeated drug

administration is highly desirable. We present here pre-clinical data demonstrating that

the Jak2 inhibitor, G6, provides exceptional therapeutic efficacy against Jak2-V617F

mediated myeloproliferative neoplasia. The drug significantly reduced the Jak2-V617F

allele burden in the bone marrow. This reduction of the mutant burden in the marrow

was concomitant with the elimination of myeloid hyperplasia, correction of the M:E ratio,

normalization of the levels of phospho-Jak2 and phospho-STAT5, and an elimination of

Jak2-V617F dependent clonogenic growth potential. Overall, these results indicate that

G6 is highly efficacious in the bone marrow.

In addition to providing exceptional bone marrow efficacy, G6 also corrected

virtually every pathological MPN indicator in the peripheral blood including the red blood

cell count, hematocrit, mean corpuscular volume, red blood cell distribution width,

hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin

concentration, platelet count, mean platelet volume, platelet distribution width, white

blood cell count, neutrophil count, lymphocyte count, monocyte count, and the levels of

IL-6 (Table 3-1, Table 3-2 and Figure 3-1). It also eliminated the extramedullary

hematopoiesis in the liver that was being driven by the Jak2-V617F transgene (Figure

3-2). Lastly, within the spleen, G6 alleviated splenomegaly, significantly reduced the

megakaryocytic hyperplasia, and restored the normal architecture to this tissue (Figures

67

3-3 and 3-4). As such, G6 significantly ameliorates or eliminates the pathogenesis of

nearly every indicator of the MPN phenotype.

We recently reported that G6 eliminates the Jak2-V617F mutant burden from the

bone marrow using a HEL cell xenograft model of Jak2-V617F mediated hyperplasia

[131]. This xenograft model has the advantage of closely replicating many aspects of

human disease including a low tumor burden in the context of the endogenous marrow

niche. One limitation of this model however, is the lack of the associated MPN

phenotype. Our work here is significant in that we show that G6 is also highly effective

in the bone marrow using a mouse model of Jak2-V617F mediated, human

myeloproliferative neoplasia. As such, the comprehensive elimination of the mixed

PV/ET phenotype from these Jak2-V617F mice suggests that G6 may have therapeutic

potential for the treatment of MPN.

Given the causative role of Jak2 kinase in human disorders, Jak2 small

molecules may have significant therapeutic potential. Accordingly, within the past

several years, a number of groups have developed Jak2 inhibitors. One problem with

virtually all these compounds however, is that while they demonstrated excellent

efficacy in vitro, they have little to no efficacy in vivo [93, 100, 114,115]. This critical

inability to reduce the mutant Jak2 burden in the bone marrow was the focus of a recent

and sobering review describing current obstacles and limitations in this area of research

[132]. Our work here is significant because in addition to having in vitro efficacy

[97,113], we now show that G6 has exceptional in vivo efficacy using a second,

independent model of Jak2-V617F mediated pathogenesis.

68

Perhaps the single greatest problem with current generation Jak2 inhibitors is

that they are merely palliative and not curative in any way [129,132]. In other words,

while they alleviate a number of MPN associated symptomologies, they do not alter the

burden of mutant Jak2 clones in the bone marrow and hence, cannot change the

natural progression of the disease. The clear efficacy observed in the bone marrow with

G6 treatment (Figures 3-4 to 3-6) suggests that the drug may in fact be curative rather

than merely palliative. Furthermore, our observation that brief exposures of Jak2-V617F

cells to G6 completely eliminate all subsequent Jak2-V617F dependent clonogenic

growth (Figure 3-7), suggests that the bone marrow efficacy may be permanent.

G6 was identified using structure based virtual screening [97]. It belongs to a

group of diarylethene compounds known as stilbenes. Previously, we demonstrated that

the stilbenoid core element of G6 is critically essential for it therapeutic potential [131].

Stilbenes have therapeutic efficacy in a wide variety of disease conditions including

cancer, stress, cardiovascular, and viral diseases [105,106,107,108,109,110]. Given

that stilbenes such as resveratrol and piceatannol are naturally occurring [108,109,110],

they are likely to have limited side effects in vivo. In the current study, we did not

observe any apparent side effects associated with G6 treatment, suggesting that it may

be suitable and safe for administration to humans with MPN.

In conclusion, the results in this study demonstrated that the small molecule Jak2

inhibitor, G6, provides unique and superior therapeutic benefit in the bone marrow using

a mouse model of Jak2-V617F mediated myeloproliferative neoplasia. The bone

marrow is the predilection site for MPN disease pathogenesis. Therefore, this work is

69

significant in that G6 may be a promising candidate for progression into clinical trials for

the treatment of MPN.

70

WT MPN + Vehicle MPN + G6

0

50

100

150

200

250

300

MPN + Vehicle MPN + G6

Pla

sma

IL-6

con

cent

ratio

n (p

g/m

l)

A

B

40x

100x

*


0

50

100

150

200

250

300

MPN + Vehicle MPN + G6

Pla

sma

IL-6

con

cent

ratio

n (p

g/m

l)

A

B

40x

100x

*

Figure 3-1. G6 provides therapeutic benefit in peripheral blood of Jak2-V617F transgenic mice. A) Representative blood smears showing giant platelets at the indicated magnifications. B) Plasma interleukin-6 concentrations. *p=6.37x10-8 versus vehicle treated.

71

0

1

2

3

4

5

6

# of

EM

H/f

ield


A B

10X

20X

100X


0

1

2

3

4

5

6

# of

EM

H/f

ield


A B

10X

20X

100X


Figure 3-2. G6 reduces extramedullary hematopoiesis in Jak2-V617F transgenic mice. A) Liver sections showing extramedullary hematopoiesis sites at the indicated magnifications. B) Number of extramedullary hematopoiesis sites plotted as a function of treatment group.

72

A WT MPN + Vehicle MPN + G6

0

5

10

15

20

Sp

ln/B

wt

(mg

/g)

**B**


10 mm10 mm10 mm

C WT MPN + Vehicle MPN + G6

02468

1012141618

# of

Meg

akar

yocy

tes/

HP

F **



D

E

Figure 3-3. G6 provides therapeutic benefit to the spleen of Jak2-V617F transgenic mice. A) Representative spleens. B) Spleen weight to body weight ratios graphed as a function of treatment group. C) Histological sections through the spleen at lower magnification showing splenic architecture. D) Histological sections through the spleen at higher magnification showing effect of G6 on megakaryocytic hyperplasia in transgenic mice. E) Number of megakaryocytes per high power field plotted as a function of treatment group. **p<0.001 and *p<0.05 versus vehicle treated.

73

# of

Meg

akar

yocy

tes/

HP

F

0

2

4

6

8

10

12

14 * *



A

0

2

4

6

8

10

12

M:E

Rat

io

C ****


B

Figure 3-4. G6 provides therapeutic benefit to the bone marrow of Jak2-V617F transgenic mice by alleviating megakaryocytic and myeloid hyperplasia. A) Bone marrow sections showing effect of G6 on megakaryocytic hyperplasia. B) Average number of megakaryocytes per high power field plotted as a function of treatment group. C) M:E ratios plotted as a function of treatment group. *p<0.05 and **p<0.001 versus vehicle treated.

74

phos

pho

-Jak

2

% p

hosh

o-Ja

k2 s

tain

ing



**

A B

0

20

40

60

80

100

120

% p

hosp

ho-S

TA

T5

stai

ning

phos

pho-

ST

AT

5

WT MPN + Vehicle MPN + G6C*

*

D


40x

100x

40x

100x

0102030405060708090

100

Figure 3-5. G6 reduces activation of Jak2 and STAT5 in Jak2-V617F transgenic mice. A) Representative anti-phospho-Jak2 immunohistochemistry in bone marrow sections from the indicated treatment groups. B) Quantification of the anti-phospho-Jak2 staining plotted as a function of treatment group. C) Representative anti-phospho-STAT5 immunohistochemistry in bone marrow sections. D) Quantification of the anti-phospho-STAT5 staining plotted as a function of treatment group. *p<0.05 versus vehicle treated.

75

0

0.5

1

1.5

2

2.5

Jak2

-V61

7F/β

-Act

in m

RN

A *A

B


0

0.5

1

1.5

2

2.5

WT

Jak

2/β

-Act

in m

RN

A


*

0

0.5

1

1.5

2


*

Jak2

-V61

7F//

WT

Jak

2C

Figure 3-6. G6 reduces mutant allelic burden in bone marrow of Jak2-V617F transgenic mice. A) Number of Jak2-V617F mutant transcripts from the bone marrow of indicated treatment groups. B) Number of endogenous mouse Jak2 transcripts. C) Allelic burden in Jak2-V617F in the indicated treatment groups. *p<0.05.

76

77

0

5

10

15

20

25

30

35

0 12 24

CF

U-E

Time of G6 Exposure (Hours)

*

*

B

0

5

10

15

20

25

30

35

0 12 24

CF

U-E


*

*

B

0

5

10

15

20

25

30

35

0 12 24

CF

U-G

M


*

*

A

Figure 3-7. G6 prevents Jak2-V617F-mediated cytokine-independent colony formation. A) Number of CFU-GM and B) number of CFU-E, plotted as a function of treatment group. *p<0.05.

Table 3-1. Summary of peripheral blood analyses showing erythrocyte and platelet indices of non-transgenic, and vehicle or G6 treated Jak2-V617F transgenic mice.

HB (g/dL) MCH (pg) MCHC (g/dL) Non Transgenic 11.9 ± 0.1 12.3 ± 0.2 28.1 ± 0.4

Jak2V617F Vehicle G6 Vehicle G6 Vehicle G6 Baseline 14.3 ± 0.3 14.4 ± 0.6 10.6 ± 0.1 10.6 ± 0.1 27.1 ± 0.2 26.9 ± 0.3

Week 2 15.5 ± 0.7 12.5 ± 1.4# 11.3 ± 0.1 11.8 ± 0.2* 29.8 ± 0.2* 29.8 ± 0.3*

Week 4 13.9 ± 0.4 12.1 ± 0.2 11.1 ± 0.2 11.9 ± 0.4#* 29.7 ± 0.2 28.2 ± 0.3#*

PLT (K/µL) MPV (fL) PDW (%) Non Transgenic 1198 ± 151 4.0 ± 0.4 30.2 ± 0.6

Jak2V617F Vehicle G6 Vehicle G6 Vehicle G6 Baseline 2879 ± 145 2802 ± 234 5.3 ± 0.1 5.3 ± 0.1 28.7 ± 0.4 28.9 ± 0.4

Week 2 2732 ± 465 1708 ± 259#* 5.4 ± 0.1 5.0 ± 0.2# 25.9 ± 1.2 28.1 ± 1.5 Week 4 3311 ± 313 1180 ± 253#* 5.4 ± 0.2 4.1 ± 0.3#* 27.2 ± 1.4 31.7 ± 1.9#

RBC (M/μL) HCT (%) MCV (fL) RDW (%) Non Transgenic 9.7 ± 0.1 42.4 ± 0.2 43.7 ± 0.3 18.3 ± 0.5

Jak2V617F Vehicle G6 Vehicle G6 Vehicle G6 Vehicle G6 Baseline 13.5 ± 0.4 13.7 ± 0.7 53.0 ± 1.3 53.5 ± 1.9 39.3 ± 0.3 39.3 ± 0.5 20.4 ± 0.3 20.5 ± 0.2

Week 2 13.7 ± 0.6 10.7 ± 1.3#* 52.2 ± 2.0 42.1 ± 4.8#* 38.3 ± 0.5 39.6 ± 0.9 20.5 ± 0.3 19.5 ± 0.5

Week 4 12.6 ± 0.5 10.3 ± 0.5#* 47.2 ± 1.4 43.0 ± 0.5* 37.2 ± 0.6 42.2 ± 1.4#* 20.2 ± 0.4 18.3 ± 0.4#*

RBC, red blood cells; HCT, hematocrit; MCV, mean corpuscular volume; RDW, red blood cell distribution width; HB, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelets; MPV, mean platelet volume; PDW, platelet distribution width. #p < 0.05 in reference to vehicle treated; *p < 0.05 in reference to baseline.

78

79

Table 3-2. Summary of peripheral blood analyses showing leukocyte indices of non-transgenic, and vehicle or G6 treated Jak2-V617F transgenic mice. WBC (K/µL) NE (K/µL) NE (%) Non Transgenic 9.1 ± 0.9 2.4 ± 0.3 23.4 ± 2.3

Jak2V617F Vehicle G6 Vehicle G6 Vehicle G6 Baseline 16.8 ± 1.6 14.8 ± 0.9 5.3 ± 0.5 5.8 ± 0.9 34.1 ± 2.6 34.9 ± 2.9

Week 2 19.8 ± 2.4 12.0 ± 3.1# 6.2 ± 0.9 4.3 ± 1.1 31.3 ± 2.2 30.0 ± 2.6

Week 4 21.3 ± 2.6 10.3 ± 0.9# 7.5 ± 1.1 2.7 ± 0.3#* 35.1 ± 1.1 27.3 ± 3.6

WBC, white blood cells; NE, neutrophils; LY, lymphocytes; MO, monocytes; EO, eosinophils; BA, basophils. #p < 0.05 in reference to vehicle treated; *p < 0.05 in reference to baseline.

LY (K/μL) LY (%) MO (K/µL) MO (%) Non Transgenic 6.4 ± 0.7 62.4 ± 1.5 0.8 ± 0.1 7.9 ± 0.9


Week 2 10.3 ± 1.3 6.7 ± 1.5# 52.1 ± 2.9 57.6 ± 2.8 2.4 ± 0.4 1.3 ± 0.3# 12.2 ± 1.2 9.3 ± 1.1#

Week 4 10.8 ± 1.3 6.1 ± 0.8# 50.7 ± 0.9 59.0 ± 3.8# 2.0 ± 0.3 0.8 ± 0.1# 9.13 ± 0.3 8.0 ± 0.8

EO (K/μL) EO (%) BA (K/µL) BA (%) Non Transgenic 0.6 ± 0.2 4.7 ± 0.6 0.1 ± 0.0 0.7 ± 0.4


Week 2 0.8 ± 0.1 0.5 ± 0.2 4.0 ± 0.6 2.6 ± 0.7 0.1 ± 0.0 0.1 ± 0.1 0.5 ± 0.2 0.6 ± 0.1

Week 4 0.9 ± 0.2 0.5 ± 0.1 4.5 ± 1.0 4.7 ± 0.6 0.1 ± 0.0 0.1 ± 0.0 0.6 ± 0.2 0.1 ± 0.0

Table 3-3. Mass spectrometry results showing plasma concentrations of G6 at euthanasia of Jak2-V617F transgenic mice.

Animal ID Plasma

(µg/mL)* Vehicle Treated

1 < 0 2 < 0 3 < 0 4 < 0 5 < 0 6 < 0

G6 Treated 1 48.6 2 0.155 3 0.036 4 0.025 5 0.030

Standard curve was quadratic (1/x2) r = 0.9902 and range from 0.005-5.00µg/mL. < 0 indicates, peak quantitates below 0 value of the standard carve, *Average of replicate injections - calculations by Analyst Software 1.4.2.

80

CHAPTER 4 3VASCULAR SMOOTH MUSCLE JAK2 MEDIATES ANGIOTENSIN II-INDUCED HYPERTENSION VIA INCREASED LEVELS OF REACTIVE OXYGEN SPECIES

Hypertension is a major risk factor for cardiovascular disease (CVD) and death [1].

Angiotensin II (Ang II) plays a major role in the regulation of normal physiological

responses of the cardiovascular system and the pathogenesis of hypertension. Previous

studies have shown that Ang II stimulation of the AT1 receptor leads to increased

activation of the Jak2 signaling pathway, and increased Jak2 activity correlates with

various Ang II-mediated cardiovascular diseases [29,30,31,32,33,34,35,36,49,133,134].

Recent work demonstrated that the Rho exchange factor, Arhgef1, mediates the effects

of Ang II on vascular tone and blood pressure [56]. It was demonstrated in this work that

Jak2 may be involved in this in vivo process; however, this correlation was made using

the Jak2 pharmacological inhibitor, AG490. Although AG490 is a potent inhibitor of

Jak2, it also inhibits other tyrosine kinases [135,136]. It inhibits EGFR phosphorylation

1,000 times more potently than it inhibits Jak2 [137]. Moreover, systemic administration

of a pharmacological inhibitor is unable to discriminate between potential Jak2 target

tissues such as brain, kidney, heart, or the vasculature.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of

hypertension and Ang II is involved in mediating ROS-dependent signaling

[55,59,60,61,138]. One mechanism by which ROS trigger hypertension is via

scavenging of nitric oxide (NO) [139]. ROS also increase intracellular free Ca2+ levels,

3Reproduced with permission from Kirabo A, Kearns PN, Jarajapu YP, Sasser JM, Oh SP, Grant MB et al. (2011) Vascular smooth muscle Jak2 mediates angiotensin II-induced hypertension via increased levels of reactive oxygen species. Cardiovasc Res. 2011 Mar 22. [Epub ahead of print]

81

which contribute to increased vascular tone [138]. In VSMC and endothelial cells, Ang II

stimulates the activity of membrane-bound NAD(P)H oxidase producing ROS such as

superoxide and hydrogen peroxide (H2O2) [17,57,62,63,64,65,66]. Superoxide

generation in response to Ang II inactivates NO in both these cell types [70,71,72].

Interestingly, the Ang II-induced formation of ROS is not dependent on its hemodynamic

effects, since this phenomenon is not observed in norepinephrine-induced hypertension

[62,67,68]. Furthermore, work in human leukemia cells demonstrated that Jak2 is

involved in ROS production as inhibition of Jak2 resulted in decreased ROS levels [75].

We hypothesized that the mechanism of Ang II-induced hypertension is by Jak2

tyrosine kinase activation within VSMC leading to increased ROS generation. Utilizing a

conditional knockout mouse approach in which Jak2 was deleted from VSMC, we

identified Jak2 as a key modulator of Ang II induced vascular contraction via a ROS-

dependent mechanism.


Animals

Animals were maintained according to NIH standards established in the

Guidelines for the Care and Use of Experimental Animals in a specific pathogen-free

facility at the Laboratory Animal Center of University of Florida. All protocols were

approved by the Institutional Animal Care and Use Committee at the University of

Florida. Mice harboring a floxed Jak2 allele were crossed with mice expressing Cre

recombinase under the control of the SM22α promoter and the Jak2 null allele was

identified using primers 5’-GTCTATACACCACCACTCCTG and 5’-

GAGCTGGAAAGATAGGTCAGC.

82

Blood Pressure Measurements

Mice were anesthetized by isoflurane (2%-5%, aerosolized) and surgically fitted

with telemetry probes placed in the left carotid artery. Buprenorphine was the post

operative analgesia (0.05 – 0.1 mg/kg every 6-12 hours for 48 hours, IP). After 10 days

of recovery, baseline blood pressure measurements were made. Mice were again

anesthetized with isoflurane and micro-osmotic pumps were placed subcutaneously for

infusion of 1,000 ng/kg/min Ang II. Radio telemetry recordings were then performed

over the ensuing four week period.

Aortic Contraction/Relaxation

Mice were euthanized via CO2 asphyxiation followed by cervical dislocation. 2-

mm abdominal aorta ring segments were then mounted on a wire myograph in Krebs-

bicarbonate buffer equilibrated with 95% O2, 5% CO2 at 37°C. The rings were allowed to

equilibrate for 45 minutes, stimulated with different pharmacological agents and

changes in contraction/relaxation were recorded. Following treatment with each

vasoactive agent, the rings were allowed to recover for 30 minutes, with 6 washes

during this time period.

Histology

Tissue samples were prepared and stained with hematoxylin and eosin or

Masson’s trichrome. Immunohistological detection of anti-smooth muscle α-actin was

carried out using the Rat on Mouse AP-Polymer Kit. The anti-Jak2 antibody was

purchased commercially (Abcam #ab39636).

NO Measurements

Aortic rings were incubated with 7 µM fluorescent dye 4-amino-5-methylamino-

2',7'-difluorescein (DAF-FM) aerated with 95% O2-5% CO2 at 37°C for 45 minutes.

83

Samples for basal accumulation of NO were taken. The rings were then treated with

Ang II (10–7 M) or Ach (10–6 M) for 30 minutes, removed, dried, and weighed.

Fluorescence was measured at an excitation wavelength of 495 nm and an emission

wavelength of 520 nm and normalized to tissue weight.

H2O2 Detection

H2O2 production was measured using the Amplex Red Hydrogen

Peroxide/Peroxidase Assay Kit and presented as the catalase-inhabitable signal

normalized to total cellular protein.

Rho Kinase Activity

Rho kinase activity was determined using the CycLex Rho-kinase Assay Kit or

directly measuring the phosphorylation levels of myosin phosphatase subunit 1

(MYPT1), a down stream target of Rho-kinase.

Calcium Imaging

Ca2+ levels within individual cells were determined via fura-2 loading and a cooled

charge-coupled device camera fitted to a fluorescence microscope.


Comparison of genotypes and treatments was performed by unpaired/paired

Student’s t-test, analysis of variance followed by the Bonferroni t-test, or Friedman’s

test.

Results

Generation of Mice with VSMC Deletion of Jak2

The Cre-loxP system was used to ablate Jak2 within VSMCs. The schematic

representation of the floxed Jak2 allele (Figure 4-1A) and the breeding strategy used to

generate such mice (Figure 4-1B) are shown. The genotypes of all offspring were

84

analyzed by PCR (Figure 4-1C). Mice homozygous for the floxed Jak2 allele were

identified by the presence of a 310 bp band as opposed to the 230 bp band of the WT

allele. Identification of SM22αCre was confirmed by the presence of a 201 bp Cre

specific amplicon. The site-specific recombination event to obtain the null Jak2 allele

was verified by a PCR assay of the abdominal aorta using primers 278 and 279; the 355

bp band could only be generated after Cre mediated excision of the Jak2 start codon. In

order to confirm that the SM22α promoter targets Cre expression to VSMC, our mice

were crossed with the ubiquitously expressed Rosa26 β-galactosidase reporter mouse

[144] and X-Gal staining was analyzed in tissue sections. VSMCs of renal arteries

showed no staining in the SM22αCre(-)Jak2fl/fl/Rosa26 control mice (Figure 4-1D, left).

However, there was efficient Cre expression and recombination within VSMC leading to

intensive X-Gal staining in the renal arteries of SM22αCre(+)Jak2fl/fl /Rosa26 mice

(Figure 4-1D). Overall, these data demonstrate a high degree of Cre activity in VSMC.

Having confirmed the appropriate genetic manipulations within these mice, we

next wanted to confirm the specific absence of Jak2 protein within VSMC. For this,

immuno-histochemistry (IHC) was carried out on renal arteries of several genotypes to

demonstrate differential Jak2 staining patterns (Figure 4-2). Collectively, these data

demonstrate that the Cre-mediated conditional deletion of the first coding exon of Jak2

within VSMC gives rise to mice that correspondingly lack Jak2 protein in VSMC.

Deletion of VSMC Jak2 Attenuates Ang II-Induced Hypertension

While it is well accepted that the Ang II type AT1 receptor couples to Jak2

signaling [29,30,31,32,33,34,133], the functional role of this interaction is not clearly

understood and little is known about the direct role of Jak2 in blood pressure regulation.

We hypothesized that Jak2 within VSMC plays a critical role in Ang II mediated

85

hypertension. For all subsequent studies, we used two genotypes; SM22αCre(-)Jak2fl/fl

herein designated as control and SM22αCre(+)Jak2fl/fl herein designated as the VSMC

Jak2 null. Using telemetry blood pressure recordings, we found that the basal mean

arterial pressure was slightly lower in VSMC Jak2 null mice, when compared to controls

(Figure 4-3A). After two baseline recordings, the mice were implanted with osmotic

minipumps infusing Ang II at a rate of 1,000 ng/kg/min for 28 days. Infusion of Ang II

resulted in an increase in MAP in both groups. However the increase in VSMC Jak2 null

mice was significantly lower (p < 0.001) compared to that observed in the control group,

both during the dark (Figure 4-3A) and the light (Figure 4-3B) periods. Examination of

the heart rate data indicated that the heart rates were not significantly different between

the two groups during these same periods (Figure 4-3C and 4-3D). These results

indicate that deletion of VSMC Jak2 attenuates Ang II induced hypertension, but has no

effect on heart rate.

VSMC Jak2 Null Mice were Protected from Ang II-Induced Aortic Wall Thickening

Chronically elevated Ang II levels promote pathological vascular wall remodeling in

animals [141,142]. To determine the affects of Ang II infusion on vascular remodeling in

our mice, aortas from both genotypes were prepared for analysis. Representative

sections are shown as Figure 4-4A while the aggregate data for all animals is shown in

Figure 4-4B. In the controls, Ang II infusion increased the aortic wall thickness relative

to untreated littermate controls. However, this Ang II-mediated pathological thickening

was significantly reduced in the null mice. Computer assisted morphometric analysis

indicated that the tunica intima thickness as a percentage of the total thickness, was

similar in VSMC Jak2 null mice and controls (Figure 4-4C). In contrast, the percent

tunica media thickness was significantly increased and the percent tunica adventitia

86

thickness was significantly reduced in aortic wall sections of controls when compared to

the null mice (Figure 4-4C). Collectively, these data indicate that VSMC Jak2 null mice

are protected from pathological vascular remodeling that occurs as a consequence of

chronic Ang II infusion.

Deletion of VSMC Jak2 Correlates with Reduced Ang II-Induced Contraction of Aortic Rings and Increased Endothelium-derived Nitric Oxide

Next, we wanted to determine whether VSMC Jak2 null mice have reduced Ang

II-mediated aortic contraction when compared to controls. For this, abdominal aortic

rings from control and null genotypes were isolated and their constrictive properties

were measured. In response to KCl, there was no significant difference in the absolute

contraction between the control and VSMC Jak2 null rings, suggesting that the

contractile machinery was similar in both genotypes (Figure 4-5A). In contrast, rings

constricted with Ang II showed a marked difference with Ang II inducing a forceful

contraction in the control rings, but not in the null rings; a representative response is

shown as Figure 4-5B while the aggregate data from all experiments are shown as

Figure 4-5C. To determine if this effect was specific for Ang II or common to vasoactive

molecules, aortic rings from both genotypes were constricted with the α1-adrenergic

receptor agonist, phenylephrine. No significant difference in the phenylephrine induced

contraction between the two genotypes was observed (Figure 4-5D). Additionally, pre-

treatment with the AT2 receptor blocker PD123319 or the Ang-(1–7) selective

antagonist A-779, had no significant effect on the percent change between the Ang II-

induced constriction in Jak2 null and the control rings, indicating that the reduced

contractile property of the VSMC Jak2 null rings in response to Ang II was independent

of AT2 and Mas receptor signaling (data not shown).

87

In order to determine if the endothelium played a role in the observed differences

in the Ang II-mediated contraction of the vessels, aortic rings from both genotypes were

either left intact or the endothelium was denuded prior to Ang II-mediated constriction.

Removal of the endothelium increased the Ang II-induced aortic contraction of the null

rings to levels that were similar to controls (Figure 4-6A). When the data were plotted as

the percent change in constriction after EC removal, we found that the percent change

between the endothelium intact and endothelium denuded aortic rings was significantly

greater in VSMC Jak2 null mice, when compared to controls (Figure 4-6B). These

results suggest that the inability of the VSMC Jak2 null rings to contract in response to

Ang II is likely due to endothelium derived inhibitory factors. In order to determine

whether NO was one such factor, rings were pre-treated with either vehicle or 1mM of

the nitric oxide synthase (NOS) inhibitor L-NAME, followed by Ang II treatment. Pre-

treatment with L-NAME lead to a partial increase of the Ang II-dependent contraction of

the null rings (Figure 4-6C). When the data were plotted as the percent change in

contraction after L-NAME treatment, we found that rings from the null mice had a

significantly greater increase in contraction when compared to controls (Figure 4-6D).

Overall, these results indicate that deletion of VSMC derived Jak2 correlates with

reduced Ang II-mediated contraction. Furthermore, the data support that there is

increased bio-available NO in the rings of the null mice, which is likely responsible for

part of the reduced Ang II-induced contraction in the aortic rings of these animals.

Deletion of VSMC Jak2 Enhances Endothelium Dependent Aortic Relaxation due to Reduced ROS and Increased NO Availability

To understand the relationship between NO and ROS in this process, aortic rings

were first constricted with phenylephrine (Phe) (10-6 M) to elicit an initial Phe-induced,

88

maximal contraction. Increasing concentrations of acetylcholine (Ach) were then added,

and the percent of Ach-mediated relaxation of the rings was determined. Ach caused a

concentration-dependent relaxation of the rings which was significantly enhanced in the

null rings when compared to the controls (Figure 4-7A). However, when the endothelium

was first denuded prior to Ang II stimulation, there was no relaxation in either set of

rings, indicating that the relaxation was entirely endothelium dependent. Accordingly,

these results support that deletion of VSMC Jak2 enhances endothelium dependent

vascular relaxation in response to Ach.

ROS have been implicated in the pathogenesis of hypertension via the

scavenging of NO [55,59,60,61,138,139]. In order to determine whether reduced levels

of ROS were responsible for the improved endothelium dependent relaxation observed

in the null mice, aortic rings were pre-treated with the anti-oxidant, superoxide

dismutase (SOD), followed by treatment with increasing concentrations of Ach. Pre-

incubation with SOD improved aortic relaxation of the control rings to levels that were

comparable to that observed with the Jak2 null rings, indicating that the control rings

had higher levels of ROS (Figure 4-7B). To determine whether there were inherent

differences in the ability of VSMC to relax, aortic rings were pre-treated with the NO

inhibitor L-NAME, and then treated with increasing concentrations of the exogenous NO

donor, DETA NONOate, a compound that will directly relax the VSMC (Figure 4-7C).

There was no significant difference in aortic relaxation between control and null rings

indicating that the impaired endothelium dependent aortic relaxation observed in the

control mice was not due to an impaired ability of the VSMC to relax, per se.

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We next measured NO levels directly and found that basal levels of NO were

significantly greater in the aortic rings taken from null mice when compared to controls

(Figure 4-7D). Similarly, Ang II (10–7) and Ach (10–6) caused greater increases in NO

production in aortic rings of VSMC Jak2 nulls when compared to the controls (Figure 4-

7D). Finally, levels of ROS were measured in cultured VSMC from both genotypes. Both

at baseline and in response to Ang II, more H2O2 was detected in VSMC obtained from

control mice than VSMC Jak2 nulls (Figure 4-7E). Collectively, the results in Figure 4-7

indicate that the aortic rings from VSMC Jak2 null mice have enhanced endothelium

dependent relaxation when compared to controls. Furthermore, this enhanced

relaxation correlates with decreased ROS and increased NO.

Deletion of VSMC Jak2 Results in Reduced Rho-Kinase Activity and Intracellular Ca2+ Levels in Response to Ang II

Rho kinase is a critical mediator of VSMC contraction [143,144,145]. As such,

Ang II-dependent Rho kinase activity was measured in cultured VSMC derived from

control and null mice. While Ang II treatment caused a time-dependent increase in Rho-

kinase activity in the control cells, it was completely lacking in the VSMC Jak2 null cells

(Figure 4-8A). To demonstrate this result another way, Western blot analysis was used

to analyze the activation of myosin phosphatase subunit 1 (MYPT1), a down stream

target of Rho kinase. As expected, Ang II treatment increased the phosphorylation of

MYPT1 in a time-dependent manner in the control cells (Figure 4-8B, top). However,

VSMC Jak2 null cells completely lacked this effect (Figure 4-8B, top). Densitometry was

then performed on all blots so that quantitative values could be compared between the

two genotypes (Figure 4-8B, bottom). Control cells exhibited a robust Ang II-dependent

phosphorylation of MYPT1 which was completely lacking in the VSMC Jak2 null cells.

90

Lastly, given that ROS can increase intracellular Ca2+ levels and this can

contribute to increased vascular tone [138], we hypothesized that the VSMC Jak2 null

cells would have decreased intracellular Ca2+ levels compared to controls. To test this,

control and VSMC Jak2 null cells were first depolarized with KCl and intracellular Ca2+

levels were determined. Both cells types generated a peak Ca2+ response that was

virtually identical, indicating that the Ca2+ signaling machinery is comparable in both cell

types (Figure 4-8C, top panels). However, when VSMC Jak2 null cells were treated with

Ang II, intracellular Ca2+ elevation was significantly blunted when compared to control

cells (Figure 4-8C, bottom panels). All responses were then graphed as a function of

both treatment and genotype (Figure 4-8D). We found that deletion of Jak2 within

VSMC significantly blunted, but did not prevent Ang II-mediated intracellular Ca2+

elevation. In summary, the data in Figure 4-8 demonstrate that deletion of Jak2 within

VSMC results in a less contractile phenotype when compared to control cells; namely,

in response to Ang II, the Jak2 nulls cells have reduced Rho kinase activity, reduced

MYPT1 phosphorylation, and reduced intracellular Ca2+ levels.

Deletion of VSMC Jak2 Prevents Angiotensin II-Induced Kidney Damage

End-organ damage is an important clinical sequel of hypertension and renal

failure. Since mice lacking VSMC Jak2 had attenuated Ang II-induced hypertension, we

hypothesized that they would be protected from kidney damage. Cross sections through

the kidneys were stained with either trichrome (Figure 4-9A) or Periodic Acid Schiff

(PAS) (Figure 4-9B). Trichrome staining revealed pronounced interstitial (Figure 4-9A

upper panel) and peri-glomerular fibrosis (Figure 4-9A lower panel) in the control when

compared to the VSMC Jak2 null mice. PAS staining revealed that glomeruli in the

control mice were enlarged and showed signs of degeneration when compared to the

91

VSMC Jak2 null mice (Figure 4-9B). There was also increased peri-vascular T-

lymphocyte infiltration in the control mice treated with Ang II, but not in the Jak2 null

mice (Figure 4-9C). Consistent with the protection from structural kidney damage, the

Jak2 null mice developed less albiminuria after 4 weeks of Ang II infusion when

compared to the control mice (Figure 4-9D). Urine albumin was undetectable in the

control and Jak2 null mice that did not receive Ang II infusion (data not shown).

Discussion

Ang II-induced actions via the AT1 receptor are considered to play a major role in

the pathogenesis of hypertension. However, the downstream signaling mechanisms

through which Ang II exerts its actions are not fully understood. We have investigated

the role of Jak2 in mediating Ang II-induced vasoconstriction and hypertension by using

mice whose VSMC are devoid of Jak2. This study identifies Jak2 as a mediator of Ang

II-induced vasoconstriction and hypertension through multiple non-redundant

mechanisms, by contributing to increased presence of ROS. To our knowledge, this is

the first study to report that Jak2 is involved in the production of ROS in VSMC, thereby

being centrally located to influence many of the singling molecules involved in

modulating the Ang II-induced vasoactive effects including NO, Ca2+ and Rho-kinase.

Recent studies have demonstrated that the Rho exchange factor, Arhgef1,

mediates the effects of Ang II on vascular tone and blood pressure, and that Jak2, by

phosphorylating Arhgef1 on Tyr 738, plays a role in this process [56]. Our data here

both confirm and extend those observations as we show that VSMC Jak2 null cells are

unable to activate Rho in response to Ang II (Figure 4-8A). In addition to

phosphorylation dependent Rho kinase activation, previous studies have shown that

Rho kinase can be activated by increased ROS [69,73]. The cartoon in Figure 1-4

92

integrates the signaling modalities involved in the Ang II and Jak2 mediated vascular

contraction. Our work here indicates that Jak2 generates ROS. The higher levels of

ROS reduce the levels of NO in the endothelium and increase the levels of Ca2+ in the

VSMC, both of which lead to enhanced contraction. Furthermore, Jak2 can activate Rho

kinase via both ROS-dependent [69,73] and phosphorylation-dependent mechanisms

[56]. Collectively, these data indicate that while Jak2 is not required for Ang II

contraction per se, it plays an absolutely critical role in modulating overall Ang II-

induced vascular tone via multiple, non-redundant mechanisms. We believe that by

contributing to the production of ROS, Jak2 is centrally located to mediate most of the

mechanisms which are known to be mediated by Ang II. This might explain the

dramatically reduced in vivo hypertensive response to Ang II in VSMC Jak2 null mice

when compared to the control group.

It is possible that the reduced vascular pathology observed in the null mice

(Figure 4-4) is due to the lower blood pressure in these animals. Previous studies

however have shown that Ang II has growth factor-like properties that act independent

of its hemodynamic effects. For example, Ang II induces ROS production leading to

endothelial dysfunction through mechanisms that are independent of its pressor actions

and norepinephrine-induced hypertension fails to elicit these same deleterious

outcomes [67,68]. Additionally, administration of renin-angiotensin system (RAS)

inhibitors provide numerous cardio protective effects that are independent of blood

pressure reduction [146]. Based on these observations, we hypothesize that the Ang II-

induced vascular pathology observed in the control mice occurs via a mechanism that is

independent of its pressor effect and instead mediated by its growth factor-like

93

properties acting through Jak2. Clearly however, this needs to be demonstrated

experimentally.

Studies have shown that in mice, Ang II is infused at a dose rate of 1,000

ng/kg/min to achieve the relevant patho-physiological effects [147,148,149]. Dose

response studies of Ang II revealed that while the infusion of a dose as low as 200

ng/min/kg into rats demonstrated increases in systolic pressure by approximately 40

mmHg, which was accompanied by development of cardiac hypertrophy and a

decrease in body weight [150], mice required a dose of 1,000 ng/min/kg of Ang II to

achieve discernible patho-physiological effects similar to those observed in humans with

Ang II mediated disease pathogenesis [151]. We recognize that the dosage of Ang II

used in this study is very high and superphysiological. As a result, the high levels of Ang

II used in these mice could lead to additional non-specific vaso-active effects

independent of Ang II receptors. Therefore, interpretation of these results in a

physiological setting in humans where the blood Ang II levels are significantly lower

needs to be done with caution. Thus, whether Jak2 mediates Ang-induced increase in

vascular tone via NO, ROS, Rho-kinase or Calcium at physiological Ang II levels in vivo

still needs to be elucidated. However, the slightly lower blood pressure at baseline in the

Jak2 null mice may suggest an effect of Jak2 even at normal circulating Ang II levels.

Gain-of-function, somatic mutations in the Jak2 allele are known to cause various

human diseases including the classical myeloproliferative neoplasms [78]. As a

consequence, great effort has been made to develop small molecule inhibitors that

target Jak2 and a limited number of these inhibitors are currently in clinical trials. While

changes in parameters such as spleen size, blood counts, cytokine levels, fatigue,

94

neurotoxicity, and gastrointestinal disturbances were monitored, our results here

suggest that changes in blood pressure may also serve as a key clinical parameter to

follow.

In conclusion, these studies identify a novel role of Jak2 tyrosine kinase in

regulating vascular tone by increasing ROS. Elevated ROS leads to increased

vasoconstriction and hypertension by scavenging endothelial NO, increasing

intracellular Ca2+, and increasing Rho kinase activity. Hence, this work strongly supports

the consideration of Jak2 as a new therapeutic target for the management of

hypertension.

95

XJak2fl/fl SM22αCre(+)

X

Jak2fl/flSM22αCre(+)Jak2fl/+

SM22αCre(+)Jak2fl/fl

XJak2fl/fl SM22αCre(+)

X

Jak2fl/flSM22αCre(+)Jak2fl/+

SM22αCre(+)Jak2fl/fl

BHR H R

R R

5’ 3’

PGK-neo

R R

Jak2 wildtype

Jak2 floxed

Jak2 null

LoxP site

SM22α-Cre mediated recombination

Exon 2

ATG

267 268

267 268

278 279

278 279

HR H R

R R

5’ 3’

PGK-neo

R R

Jak2 wildtype

Jak2 floxed

Jak2 null

LoxP site

SM22α-Cre mediated recombination

Exon 2

ATG

267 268

267 268

278 279

278 279

A

+/+-

fl/fl

-+/++

fl/fl

+Jak2 Allele:

310 bp230 bp

201 bp

355 bpJak2 null

Jak2 allele

+/+-

fl/fl

-+/++

fl/fl

+Jak2 Allele

310 bp230 bp

201 bp

355 bpJak2 null

C

SM22Cre

SM22Cre:

C

D

E

C

D

E

SM22Cre(-)Jak2fl/flRosa26βgal

SM22Cre(+)Jak2fl/flRosa26βgal

SM22Cre(-)Jak2fl/flRosa26βgal

SM22Cre(+)Jak2fl/flRosa26βgal

D

Figure 4-1. Generation of mice with vascular smooth muscle cell specific deletion of Jak2. A) Cre-mediated deletion of the Jak2 gene; arrows indicate location of primers. B) Breeding strategy to convert the floxed Jak2 allele into a vascular smooth muscle cell specific null Jak2 mutation. C) Results of a PCR assay to verify the presence of a floxed Jak2 allele (top), the SM22αCre specific amplicon (middle) and the null Jak2 allele in vascular smooth muscle cells (bottom). D) X-Gal staining of kidney tissue sections derived from Rosa26 β-gal mice with and without the SM22αCre transgene.

96

A B C

D E F

G H I

J K L

H&E Anti-SMA Anti-Jak2

SM22Cre(-)Jak2+/+

SM22Cre(-)Jak2FL/FL

SM22Cre(+)Jak2+/+

SM22Cre(+)Jak2FL/FL

A B C

D E F

G H I

J K L

H&E Anti-SMA Anti-Jak2

SM22Cre(-)Jak2+/+

SM22Cre(-)Jak2FL/FL

SM22Cre(+)Jak2+/+

SM22Cre(+)Jak2FL/FL

Figure 4-2. The Jak2 protein is absent in vascular smooth muscle cells of mutant mice. Representative renal artery pictures for hematoxylin and eosin staining, immuno-histochemistry of anti-smooth muscle actin (SMA), and immuno-histochemistry of anti-Jak2 for the indicated genotypes; SM22αCre(-)Jak2+/+ (A-C), SM22αCre(-)Jak2fl/fl (D-F), SM22αCre(+)Jak2+/+ (G-I) and SM22αCre(+)Jak2fl/fl (J-L).

97

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Figure 4-3. Deletion of vascular smooth muscle cell Jak2 attenuates Ang II-induced hypertension. Long-term radio-telemetric recording of mean arterial pressure was performed in VSMC Jak2 null (n=6) and age-matched controls (n=6). Two base line recordings were performed before the mice were implanted with Ang II mini pumps infusing 1,000 ng/kg/min of Ang II (day 0). Further recording was continued over the ensuing 28 days. The values shown represent daily average 12-hour mean arterial pressure recordings for the active dark period (A) and the resting light period (B). Heart rates were also plotted as a function of both genotype and time. The average 12-hour mean heart rate recordings for the active dark period (C) and the resting light period (D) are shown. Data represent means +/- SE (* p<0.05, ** p<0.01, ANOVA).

98

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Figure 4-4. VSMC Jak2 null mice are protected from Ang II induced aortic wall thickening. A) Aortic histological sections from control and VSMC Jak2 null mice were stained with trichrome. B) The total aortic wall thickness from each animal was measured and then plotted as a function of treatment and genotype. C) Aortic wall layers of the intima, media, and adventitia were defined, and their thickness is presented as a percentage of the total wall thickness. Data represent means ± SE; n = 6. *p < 0.05 vs. untreated control; # p <0.05 vs. Ang II treated control, paired Student's t-test.

99

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Figure 4-5. Deletion of vascular smooth muscle cell Jak2 correlates with reduced Ang II induced contraction of aortic rings. A) KCl-induced absolute contraction of aortic rings from both genotypes. B) Representative profiles showing Ang II (10-7 M) induced contraction in aortic rings obtained from Control and VSMC Jak2 null mice. C) Aggregate Ang II-induced contraction data plotted as a percent of KCl. D) Phenylephrine-induced contraction plotted as a percent of KCl. Data represent means +/- SE (* p <0.05; n=8, paired Student's t-test).

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Figure 4-6. Deletion of vascular smooth muscle cell Jak2 correlates with increased levels of nitric oxide. A) Ang II (10-7 M) induced contraction data plotted as a percent of KCl either in presence or absence of the endothelium. B) The percent change in Ang II-induced contraction between endothelial cell (EC) intact and EC denuded aortic rings. C) Ang II-induced contraction of aortic rings pretreated with vehicle or N(G)-nitro-L-arginine methyl ester (L-NAME) and plotted as a percent of KCl. D) The percent change in Ang II induced contraction between vehicle treated and L-NAME treated aortic rings for both genotypes. Data represent means +/- SE (* p <0.05; n=8, paired Student's t-test).

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Figure 4-7. Deletion of vascular smooth muscle cell Jak2 enhances endothelium dependent vascular relaxation due to reduced reactive oxygen species and increased nitric oxide availability. A) Dose-dependent acetylcholine induced relaxation, plotted as a percent of maximum phenylephrine (10-6 M) induced contraction with or without the endothelium. Data represent means +/- SE (n=8). B) Dose-dependent acetylcholine induced relaxation, plotted as a percent of maximum phenylephrine (10-6 M) induced contraction with superoxide dismutase pretreatment. Data represent means +/- SE (n=8). C) Percent DETA NONOate-induced relaxation in aortic rings pre-incubated with L-NAME. D) DAF fluorescence nitric oxide measurements in aortic rings at baseline, or following Ang II (10–7) and acetylcholine (10–6) treatment (* p < 0.05 vs. control, paired Student's t-test). E) Catalase inhibitable H2O2 in vascular smooth muscle cells obtained from control and Jack2 null mice at baseline, and in response to Ang II treatment (* p < 0.05 vs. vehicle control, # p <0.05 vs. control, unpaired Student's t-test).

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Figure 4-8. Deletion of vascular smooth muscle cell Jak2 results in reduced Rho-kinase activity and intracellular Ca2+ levels in response to Ang II. A) Time-dependent Ang II induced Rho kinase activity in vascular smooth muscle cells obtained for control and VSMC Jak2 null mice. B) Representative immunoprecipitation/western blot analysis of phospho-MYPT showing an Ang II-induced phosphorylation of MYPT by Rho-kinase (top). All blots were quantitated via densitometric analysis and graphed as a function of genotype and time (bottom) (* p <0.05 vs. untreated condition, Friedman’s test). C) KCl and Ang II-induced increase in intracellular Ca2+ as measured by fura-2 fluorescent imaging in individual cells. D) The average Ca2+ responses plotted as a function of treatment and genotype (p<0.05 vs. control, unpaired Student's t-test).

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Figure 4-9. VSMC Jak2 null mice are protected from Ang II-induced renal damage. A)

Trichrome blue staining showing fibrosis in the interstitial (upper panel) and around glomeruli (lower panel) in the control and Jak2 null mice treated with Ang II. B) Periodic Acid Schiff staining showing glomerular degeneration and enlargement in the control mice, but not in the Jak2 null mice treated with Ang II. C) Immunohistochemistry of CD3 showing increased T-lymphocyte infiltration in control mice, but not in the Jak2 null mice. D) Urine albumin excretion in the Ang II treated control and Jak2 null mice. *p<0.05 versus Ang II treated control.

CHAPTER 5 4VASCULAR SMOOTH MUSCLE JAK2 DELETION PREVENTS ANGIOTENSIN II-

MEDIATED NEOINTIMA FORMATION FOLLOWING INJURY IN MICE

Damage to the vascular endothelial cell (EC) layer often caused by stenting,

angioplasty, or bypass surgery is one of the initial key events in the pathogenesis of

various vascular diseases including neointima formation, atherosclerosis, restenosis,

and hypertension. It causes phenotypic switching of vascular smooth muscle cells

(VSMC) from a contractile to a synthetic phenotype characterized by increased cell

proliferation, migration, and production of extracellular matrix [152,153]. Neointima

formation resulting in vascular EC injury is also characterized by a general loss of

critical VSMC contractile markers including smooth muscle α-actin (SMA), myosin

heavy chain (SM-MHC), SM22α and calponin (CNN) [153,154]. However, the

mechanistic processes that trigger these phenotypic alterations are still not fully

understood.

Janus kinase 2 (Jak2) is well known for its role in hematopoiesis and cytokine

signaling. Mice that are completely Jak2 null die at embryonic day 12.5 due to impaired

hematopoiesis and profound anemia [27,28]. Conversely, mutations that lead to hyper-

kinetic Jak2 kinase activity result in various hematological diseases characterized

increased cell proliferation and hematopoiesis [78]. As a consequence, numerous Jak2

inhibitors are currently under pre-clinical and clinical investigation for their potential in

the treatment of Jak2 mediated hematological diseases, but to date, none have been

approved.

4Reproduced with permission from Kirabo A, Oh SP, Kasahara H, Wagner KU, Sayeski PP (2011) Vascular smooth muscle Jak2 deletion prevents angiotensin II-mediated neointima formation following injury in mice J Mol Cell Cardiol. 50(6):1026-34.

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It is well accepted that the Jak2 signaling pathway couples to the angiotensin II

type 1 receptor (AT1-R) [155]. However, the physiological and/or pathological

importance of this coupling is poorly understood in the in vivo setting. Angiotensin II

(Ang II) has been shown to exhibit growth promoting and migration properties in

cultured VSMCs [13,15]. In addition, chronic infusion of Ang II induces VSMC

proliferation in normal and injured vessels in vivo [9,18]. Unfortunately, there have been

conflicting reports as to which kinase signaling pathway(s) mediate(s) Ang II-induced

cell proliferation and migration. For instance, some studies have shown that Ang II

mediates its growth promoting effects via the Mitogen-Activated Protein (MAP) kinase

pathway in vitro [14]. On the other hand, studies have reported a correlative

involvement of the Jak/STAT signaling pathway in the growth factor-like signaling

properties of Ang II and subsequent vascular neointima formation [49,156]. Previous

attempts to determine the specific kinase pathway involved in Ang II-induced neointima

formation have been limited by the lack of specific kinase inhibitors and the lack of

conditional knockout animal models. For example, AG490 has been used in these types

of studies [49], but in addition to inhibiting Jak2, it also inhibits Jak3 and MAP kinase

[157]. Thus, it is still unclear which kinase signaling pathway acts downstream of the

AT1-R to mediate VSMC proliferation, migration, and neointima formation.

Previous studies have long shown that Ang II binding causes physical interaction

of Jak2 with the AT1-R resulting in the subsequent activation of the Signal Transducers

and Activators of Transcription (STAT) proteins [30]. Activated STATs in turn form

homo- and hetero-dimers which translocate into the nucleus and bind cis-inducible

elements resulting in the activation of growth promoting genes [158,159]. However,

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there is no evidence indicating that the pathological AT1-R-mediated growth effects

occur exclusively through Jak2. The current study was aimed at determining whether

Jak2 plays a central role in the pathogenesis of Ang II-mediated neointima formation

following injury. Using an approach whereby we conditionally deleted Jak2 from the

VSMC of mice, our data indicate for the first time that Jak2 plays a rate liming role in the

causation of Ang II-induced neointima formation following vascular injury in vivo.


Animals

Male mice generated on an FVB background strain were used in these

experiments. All procedures using laboratory animals were approved by the Institutional

Animal Care and Use Committee at the University of Florida. Animals were maintained

in accordance with NIH standards established in the Guidelines for the Care and Use of

Experimental Animals.

Generation of Knockout Mice

VSMC deletion of Jak2 was achieved by crossing mice carrying loxP sites around

the first coding exon of the Jak2 gene [160] with mice expressing Cre recombinase

under the control of the SM22α promoter [161]. Specifically, male mice of genotype

SM22αCreJak2fl/+ were crossed with Jak2fl/fl females, which resulted in mice whose

VSMCs are devoid of Jak2 (SM22αCre(+)Jak2fl/fl). Genotyping was done by PCR using

primers 5’-GCTAAACATGCTTCATCGTCGGTC and 5’-

CAGATTACGTATATCCTGGCAGCG in the Cre coding region, 5’-

ATTCTGAGATTCAGGTCTGAGC and 5’-CTCACAACCATCTGTATCTCAC in the Jak2

coding region, and 5’-GTCTATACACCACCACTCCTG and 5’-

GAGCTGGAAAGATAGGTCAGC to identify the null Jak2 allele. Expression of VSMC

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SM22αCre was confirmed by crossing mice expressing SM22αCre(+)Jak2fl/fl with

Rosa26 β-galactosidase reporter mice, and X-Gal (5-bromo-4-chloro-3-indolyl β-D-

galactoside) staining was analyzed as previously described [140].

Vascular Injury Model

Iron chloride-induced vascular injury was carried out as previously described with

a few modifications [162]. Briefly, mice at 3 months of age were anaesthetized using

isoflurane. An incision was made directly on top of the right common carotid artery and

vascular injury was induced by applying a sterile Q-tip saturated with 10% ferric chloride

(Sigma, St. Louis, MO, USA) for 3 minutes. The left common carotid artery was

exposed by blunt dissection, but not injured and thus served as a contralateral control.

At the same time, the animals received an Alzet Model 1004 osmotic minipump (Alzet

Corp) for subcutaneous infusion of 1,000 ng/kg/min of Ang II [151]. The incision was

closed and the animals were allowed to recover. To prevent thrombosis, animals were

injected subcutaneously with 20 units of heparin prior to surgery. Animals were

euthanized at 7 days (n=6 for each genotype) or 14 days (n=6 for each genotype)

following vascular injury. The carotid arteries were fixed using 10% formalin and

processed for histological determination of neointima formation.

Histology

Tissue samples were prepared for histology as previously described [168]. Briefly,

tissues were fixed overnight at 4°C in 10% buffered formalin (Fisher Scientific,

Pittsburgh, PA). The tissues were subsequently dehydrated through a graded ethanol

series, paraffin embedded and sectioned. Five micrometer sections were stained with

hematoxylin and eosin (H&E) for morphological analysis. Transverse sections of the

carotid arteries were subjected to morphometry for assessing the intima/media ratio (I/M

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ratio). Other tissues were stained with Masson’s trichrome using a kit (87019 Richard-

Allan Scientific) for analysis of fibrosis.

Immunohistochemistry

5 micron sections mounted on gelatin-coated slides were dewaxed in ethanol,

rehydrated, then blocked in 3% H2O2 followed by 5% normal goat serum. Sections

were exposed to the primary antibody overnight at 4oC, washed, and then treated with

the biotinylated secondary antibody. After secondary antibody incubation, the samples

were washed, exposed to the avidin-peroxidase reagent (Vectastain Elite, Vector

Laboratories, Burlingame, CA), and reacted with diaminobenzidine to produce a brown

reaction product. The sections were dehydrated in ethanol, mounted with Permount,

and observed by light microscopy. Immunohistological detection of anti-smooth muscle

α-actin (CM001B) was carried out using the Rat on Mouse AP-Polymer Kit (Biocare

Medical) according to the manufacturer’s instructions. Anti-Jak2 (ab39636 Abcam), anti-

phospho-Jak2 (Ab32101 Abcam), anti-phospho-STAT5 (Ab32364 Abcam), and Ki-67

(M7249 DAKO) immunohistochemical detection was performed as previously described

[163]. Apoptosis detection was carried out using a TUNEL staining kit (S7100

Chemicon) according to the manufacturer’s instructions.

Immunoblotting

Protein sample was extracted from the homogenates of primary VSMC cultures

and immunoprecipitated using STAT3 antibody (SC-482, Santa Cruz), or STAT5

antibody (SC-28685, Santa Cruz), followed by western-blotting using phospho-STAT3

antibody (SC-8059, Santa Cruz), or phospho-STAT5 antibody (716900, Invitrogen)

respectively, as previously described [164].

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Cell Proliferation

VSMCs from each genotype were isolated and cultured as described [165]. Cell

proliferation was assessed on the basis of mitochondrial dehydrogenase activity of the

cells using the MTT dye–reduction assay [166]. In brief, after the addition of ligand or

vehicle, the cells were incubated with the MTT labeling solution (0.5 mg/mL) at 37°C for

4 hours. The cells were then solubilized in 100 µL of 0.1N HCl and isopropyl alcohol,

and shaken for 30 seconds on a plate rotator. Absorbance at 540 nm was measured

with the use of a microtiter plate reader with a reference wavelength of 690 nm. Cell

counts were also carried out to determine the number of viable cells using trypan blue

exclusion.

Cell Migration

The migration of VSMCs was determined using a QCMTM 24-well colorimetric cell

migration assay kit (Millipore) according to the manufacturer’s instructions. Briefly, the

cells were suspended in serum free media (DMEM) at a concentration of 1 x 106

cells/mL. 0.3 mL aliquots of the cell suspension were added to the top chambers of the

transwell membranes with 8-µm pores. The lower transwell compartments contained 0.5

mL of DMEM containing various migration factors including Ang II (10-7M), FBS (10%)

or platelet-derived growth factor (PDGF-BB) (20 ng/ml). Incubation was continued for 24

hours at 37°C in a CO2 incubator. The adherent cells were then stained, washed, and

allowed to air dry. The die was extracted from the cells, and optic density was measured

at 560 nm.

Apoptosis

Annexin V/propidium iodide staining was employed to determine early apoptosis in

the control and the Jak2 null VSMCs. VSMCs were serum-starved overnight and then

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treated with either vehicle, 10% FBS, 20 ng/ml PDGF or 10−7 M Ang II for 24 h. Cells

(105) were re-suspended in 100 μl of 1x binding buffer and apoptotic levels were

determined via the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen) following

the manufacturer's instructions and analyzed on a FACSCalibur flow cytometer

(BectonDickinson).Apoptotic cells were also identified using the terminal

deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method

which specifically labels the 3′-hydroxyl termini of DNA strand breaks. The TUNEL

assay was carried out using the ApopTag Fluorescein In Situ Apoptosis Detection Kit

(Millipore) according to the manufacturer's instructions.


All results were expressed as means +/- SEM. Statistical comparison of the

different genotypes were performed by unpaired Student’s t test. P values of less than

0.05 were considered statistically significant.

Results

Deletion of VSMC Jak2 Prevents Ang II-Mediated Neointima Formation and Narrowing of the Vascular Lumen Following Injury

Vascular remodeling is a pathologic response to vascular injury characterized by

VSMC proliferation, migration, neointima formation, and a narrowing of the vascular

lumen [167]. We wanted to determine whether VSMC Jak2-null mice are protected from

neointima formation and narrowing of the vascular lumen following vascular injury. For

this, the right carotid arteries of control (SM22αCre(-);Jak2fl/fl) and VSMC Jak2-null

(SM22αCre(+);Jak2fl/fl) mice were subjected to iron chloride-induced vascular injury

with simultaneous Ang II infusion. Left carotid arteries were exposed via blunt

dissection, but not subjected to iron chloride-induced injury and thus served as

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contralateral controls. Mice were euthanized 7 and 14 days later. H&E representative

sections in Figure 5-1A show a clear increase in thickness of the neointima in the

injured arteries of the control mice at day 7 and 14, but neither in the VSMC Jak2-null

mice nor any of the contralateral arteries. Using computer assisted quantitative

morphometric analysis, we found that compared to the non-injured contralateral carotid

arteries, vascular injury induced significant increases in the intima/media ratio in the

control mice at day 7 and 14, which was lacking in the VSMC Jak2 null mice (Figure 5-

1B). As a consequence, there was a significant narrowing on the carotid artery lumen in

the injured arteries from the control mice while the VSMC Jak2-null mice were protected

from this deleterious effect (Figure 5-1C). These collective results suggest that deletion

of VSMC Jak2 prevents neointima formation and the subsequent narrowing of the

vessel following vascular injury.

Deletion of VSMC Jak2 Prevents Ang II-Mediated Vascular Fibrosis Following Injury

Vascular fibrosis is an important component of vascular injury response [168].

Thus, we quantified extracellular matrix (ECM) deposition using trichrome-blue staining

of sections from iron chloride-injured control and Jak2 null mouse carotid arteries 7 and

14 days after injury. Representative sections indicated that the density of trichrome blue

staining was significantly reduced in the Jak2 null mice at both time points when

compared to the control group (Figure 5-2A) and quantitative analysis of all sections

found this difference to be significant (Figure 5-2B). Overall, these results suggest that

deletion of VSMC Jak2 reduces Ang II-induced ECM deposition following vascular

injury.

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Deletion of VSMC Jak2 Prevents the Loss of Smooth Muscle α-actin in Response to Ang II-Mediated Vascular Injury

Vascular injury is known to reduce the expression of contractile markers within

VSMC [152,153]. We hypothesized that the Jak2-null mice would be protected from the

injury-induced loss of smooth muscle α-actin (SMA). Immunohistochemistry of injured

carotid sections showed that there was a significant loss of SMA immunoreactivity (red

stain) at both day 7 and day 14 in the control mice when compared to the contralateral

carotid artery (Figure 5-2C). However, SMA staining was preserved in the injured

carotid arteries in the Jak2 null mice, and was comparable to that observed in their

contralateral arteries. Quantitative analysis of all sections again found this difference

between the two genotypes to be significant (Figure 5-2D). As such, these results

suggest that VSMC Jak2-null mice are protected from the loss of the contractile

phenotype that occurs following vascular injury.

Deletion of VSMC Jak2 Prevents Neointima Formation by Inhibiting Cell Proliferation and Inducing Apoptosis

We hypothesized that a mechanism whereby deletion of VSMC Jak2 prevents

neointima formation is via reduced cellular proliferation. To determine this, the levels of

the proliferative marker, Ki-67, were measured in sections from each condition.

Representative sections indicated an increase in the number of Ki-67 positive nuclei in

the injured arteries of the control mice at day 7 and 14, but not in the VSMC Jak2 null-

mice (Figure 5-3A). Quantification of Ki-67 positive nuclei across all sections found that

when compared to the non-injured contralateral arteries, injury caused significant

increases in the number of Ki-67 positive cells in the control mice at day 7 and 14 and

this was lacking in the Jak2-null mice (Figure 5-3B). These results suggest that deletion

of VSMC Jak2 prevents neointima formation by inhibiting cell proliferation.

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Previous studies have shown that induction of apoptosis is a possible mechanism

for the prevention of neointima formation [156,169]. Therefore, we wanted to determine

whether the reduced neointima formation observed in the VSMC Jak2-null mice

correlates with increased apoptosis. Shown are representative TUNEL stained sections

(Figure 5-3C; brown stained cells indicated by red arrows) and the average number of

TUNEL positive cells plotted as a function of treatment group (Figure 5-3D). There was

no significant difference in the number of TUNEL positive cells observed in injured

carotid arteries taken from the control group when compared to the non-injured,

contralateral arteries. In contrast, there was a significant increase in the number of

apoptotic cells 7 days after injury in the Jak2-null mice and this returned to near

baseline levels by day 14. Altogether, the results in Figure 5-3 suggest that VSMC Jak2

deletion prevents neointima formation by inhibiting cell proliferation and inducing

apoptosis in VSMCs.

VSMC Jak2 Induces Neointima Formation by Increasing Phosphorylation of Jak2 and STAT5

Immunohistochemistry was carried out in order to determine the relative levels of

Jak2, phospho-Jak2, and phospho-STAT5 (a downstream target of Jak2) in the injured

and non-injured carotid arteries of both the control and VSMC Jak2-null mice (Figure 5-

4). With respect to the control mice, we found that there were readily detectable levels

(brown stain) of Jak2, phospho-Jak2 and phospho-STAT5 in the contralateral arteries

and vascular injury increased this staining pattern both in the media and neointima. With

respect to the VSMC Jak2-null mice, there was little to no staining for Jak2, phospho-

Jak2, and phospho-STAT5 both in the contralateral control and the injured arteries.

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These results demonstrate that the reduced neointima formation observed in the VSMC

Jak2-null mice correlates with reduced phosphorylation of Jak2 and STAT5 in VSMC.

VSMC Jak2 Mediates Ang II-Induced Cell Proliferation and Migration

In order to determine whether the Jak2 dependent effects observed in vivo are

specific for Ang II per se, we isolated VSMC from control and VSMC Jak2-null mice and

cultured them ex vivo. The cells were subsequently treated with either vehicle, 10%

FBS, platelet derived growth factor (PDGF), or Ang II, and then evaluated for cell

proliferation and cell migration properties. For the control cells, FBS, PDGF, and Ang II

all induced robust cell proliferation (Figure 5-5A). For the VSMC Jak2 null cells, FBS

and PDGF induced robust cells proliferation while Ang II did not (Figure 5-5B), using

MTT dye–reduction assay. Similar results were obtained when we counted the number

of viable cells using trypan blue exclusion (Figure 5-5C and 5-5D). These results

suggest that Jak2 mediates Ang II-induced VSMC proliferation.

Next, we wanted to determine the role of Jak2 in VSMC migration in response to

these same stimuli. We found that there was no significant difference in cell migration

between the genotypes when the cells were treated with FBS or PDGF (Figure 5-5E).

However, when the cells were treated with Ang II, cell migration in the Jak2-null cells

was significantly attenuated when compared to the control cells (Figure 5-5E),

suggesting that Jak2 mediates Ang II induced cell migration. Collectively, the data in

Figure 5 correlate the specific loss of VSMC Jak2 with significantly impaired Ang II-

mediated VSMC proliferation and migration.

VSMC Jak2 is Required for Ang II-Mediated Cell Survival

The in vivo immunohistochemistry data indicate that Jak2 promotes cell survival as

cells that lack Jak2 are more prone to apoptosis after injury (Figure 5-3C and 5-3D).

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Here, we wanted to determine whether this Jak2-dependent event was specific for Ang

II. For this, primary cultures of VSMC from control and VSMC Jak2-null mice were

serum starved overnight. The following day, the cells were treated with the indicated

ligands and early apoptosis was measured 24 h later. We found that in the control cells,

~10% of all cells treated with vehicle control solution were in early apoptosis (Figure 5-

6A). Treatment with FBS, PDGF, and Ang II promoted cell survival as indicated by the

lower levels of apoptosis in these cells. When the same experiment was performed in

the VSMC Jak2-null cells, the ability of Ang II to decrease the levels of apoptosis was

lost, thereby indicating a critical role for Jak2 in Ang II-mediated cell survival. To

demonstrate this using an alternate approach, the cells were treated in the same way,

but this time apoptosis levels were measured via TUNEL stain, an indicator of both early

and late apoptosis (Figure 5-6B). Similarly, we found that Jak2 is essential for promoting

Ang II-mediated cell survival since the null cells were unable to decrease the levels of

apoptosis in response to Ang II. As such, these data correlate with our in vivo data

suggesting that the VSMC Jak2-null mice have reduced Ang II-mediated neointima

formation due to increased apoptosis.

VSMC Jak2 deletion is associated with reduced Ang II-mediated activation of STAT3 and STAT5

We next wanted to determine whether the activation of the Jak2/STAT signaling

pathway is also specific for Ang II. For this, we analyzed the relative abilities of FBS,

PDGF and Ang II to induce phosphorylation of STAT3 and STAT5; both of which are

mitogenic proteins and are downstream signaling targets of Jak2. With respect to

STAT3, ligand induced phosphorylation of STAT3 was significantly reduced in the null

cells when compared to the control cells for all conditions (Figure 5-7A and 5-7B).

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These results suggest that activation of STAT3 is primarily mediated by Jak2 since the

absence of Jak2 renders the cells incapable of phosphorylating STAT3 under any

condition. In addition, we found that within the control cells, the Ang II-induced

phosphorylation of STAT3 was significantly increased, when compared to the other

stimulating ligands. With respect to STAT5, the phospho-STAT5 levels were not

significantly different in the FBS and PDGF treated control cells when compared to

vehicle treated cells, but were significantly different in the Ang II treated cells (Figure 5-

7C and 5-7D). Furthermore, there was no significant difference in ligand induced

phosphorylation of STAT5 amongst the Jak2-null cells (Figure 5-7C and 5-7D). As such,

these results suggest that Jak2 is the primary mediator of Ang II-induced

phosphorylation of STAT5, and that activation of the Jak2/STAT5 pathway is specific for

Ang II.

Discussion

Neointima formation is a pathologic consequence of vascular endothelial damage

which often results from various risk factors including percutaneous vascular surgery

procedures. We have investigated the impact of VSMC deletion of Jak2 on the

prevention of Ang II-mediated neointima formation following vascular injury in mice. This

study identifies Jak2 as a critical mediator of the pathological processes involved in Ang

II-mediated neointima formation. To our knowledge, this is the first report to establish a

causal relationship between Jak2 and Ang II-mediated neointima formation following

vascular injury.

Following endothelial damage, the process of neointima formation starts with an

initiation of VSMC proliferation and migration into the intima, followed by a sustained

proliferation of VSMC in the neointima [167,170]. This compromises the vascular lumen,

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and limits blood supply to distal areas of the vessel. Ang II plays a key role in mediating

the processes involved in the pathogenesis of neointima formation following vascular

injury [171]. However, the specific mechanisms involved in Ang II-mediated neointima

formation remain largely unidentified. Ang II induces tyrosine-phosphorylation and

activation of Jak2 in rat VSMC resulting in activation of its downstream signaling

substrates, the STATs [30]. Subsequent studies showed that the Ang II-induced

activation of Jak2 results in proliferation of cultured VSMC [31]. We found that not only

did VSMC Jak2 deletion prevent Ang II-induced neointima formation, but it also

prevented the pathological phenotypic alterations induced by vascular injury including

vascular fibrosis and loss of the contractile marker, SMA. The mechanisms by which

VSMC Jak2 deletion prevents neointima formation include inhibition of cell proliferation

and migration. Furthermore, we found that Ang II-induced activation of the proliferative

proteins, STAT3 and STAT5, is impaired in the VSMC Jak2-null cells. The Jak2-

mediated growth promoting and migratory effects are specific for Ang II since other

ligands such as 10% FBS and PDGF did not lead to differential effects on cell

proliferation and migration in the control and the Jak2-null cells. From these data, we

conclude that Jak2 plays a rate liming role in the causation of Ang II-induced neointima

formation following vascular injury in vivo.

It is well established that Ang II plays a key role in mediating neointima formation

following vascular injury [171]. This provided us with an excellent model of examining

the role of VSMC Jak2 deletion in an environment in which neointima formation

following injury is exacerbated by chronic Ang II infusion. Although studies have shown

that the Jak2 signaling pathway couples to the AT1-R resulting in VSMC proliferation in

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vitro [155], the only study to date examining the relationship between Jak2 and

neointima formation in vivo reported that Jak2 is only transiently expressed during the

process of neointima formation [49]. The same study also reported that treatment of

vascular strips with Ang II ex vivo enhances phosphorylation of Jak2 [49]. Therefore,

until now, the pathophysiological link between Jak2 and Ang II-mediated neointima

formation has been only correlative and no study has implicated Jak2 in playing a

causative role in Ang II-mediated vascular remodeling in vivo.

Placing the Cre recombinase cDNA under the control of tissue specific promoters

has allowed for the conditional deletion of genes such as Jak2. However, previous work

has clearly demonstrated that the pattern of Cre expression in the targeted tissue is a

mosaicism [172,173]. In other words, most cells will express Cre, but some will not. The

reason(s) for this is not fully understood, but it is thought that it may be due to modifying

events such as epigenetic silencing of the transgene within a given cell [174,175].

Hence, the gene targeted for deletion, in this case Jak2, is not removed from the cell.

Quantitative examinations of our anti-Jak2 immuno-histochemistry (Figure 5-4) as well

as Rosa26/LacZ expression patterns (data not shown) indicate that Jak2 is deleted from

~95% of all VSMC in the carotid arteries of these mice. This degree of deletion is

clearly enough to impair Jak2 function as determined by the significantly reduced VSMC

migration, VSMC proliferation, neo-intimal formation, and preservation of contractile

markers, when compared to controls. Given that Jak2 has been implicated in a number

of cardiovascular diseases including hypertension, heart failure, and diabetes

[56,176,177,178], we now have an excellent model for the future examination of the role

of VSMC derived Jak2 in these and other disorders.

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In conclusion, the present study demonstrated for the first time that Jak2 plays a

central role in the causation of Ang II-induced neointima formation following vascular

injury in vivo. Therefore, inhibition of Jak2 may provide a potential prophylactic

therapeutic strategy for prevention of neointima formation. In addition, Jak2 inhibition

may prevent initiation and progression of neointima thickening following angioplasty

and/or vascular stenting.

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0

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Day 7 Day 14 Day 14Day 7

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Figure 5-1. Deletion of VSMC Jak2 prevents Ang II-mediated neointima formation and narrowing of the vascular lumen following injury. A) H&E stained sections showing neointima formation in control mice. The dotted lines separate the intima from media. B) Intima/media ratio. C) Lumen area. *p<0.05 vs. contralateral control, **p<0.01 vs. contralateral control.

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Figure 5-2. Deletion of VSMC Jak2 prevents Ang II-mediated fibrosis and loss of SMA following injury. A) Trichrome-blue stained sections showing increased collagen density in control mice. B) Quantification of trichrome-blue staining. C) Immunohistochemistry of SMA. D) Quantification of SMA staining. *p<0.05 vs. contralateral non-injured artery.

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Figure 5-3. Deletion of VSMC Jak2 inhibits cell proliferation and induces apoptosis. A) Representative sections showing immunohistochemistry of Ki-67. B) Quantification of the number of Ki-67-positive nuclei. C) Representative sections showing immunohistochemistry of TUNEL. D) Quantification of the number of TUNEL positive cells. *p<0.05 vs. contra-lateral non-injured control.

123

Contralateral Injured Contralateral Injured

Control Jak2 Null

Jak2

p-Jak2

p-STAT5

Figure 5-4. VSMC Jak2 induces neointima formation by increasing phosphorylation of Jak2 and STAT5. Immunohistochemistry of representative sections showing relative expression of Jak2, phospho-Jak2 and phospho-STAT5 in injured and non-injured carotid arteries of the control and the Jak2-null mice.

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Null

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C D

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*

Figure 5-5. VSMC Jak2 mediates Ang II-induced cell proliferation and migration. VSMC were treated with 10% FBS, 20 ng/ml PDGF or 10-7M Ang II as indicated. Resulting cell proliferation of the control cells A) and VSMC Jak2 null cells B) is shown using MTT dye–reduction assay. C) and D) cell proliferation using trypan blue exclusion. E) Cell migration in these same cells. *p<0.05 vs. control. Shown are mean +/- SEM for three independent experiments, each run in triplicate.

125

Figure 5-6. Jak2 is required for Ang II-mediated VSMC survival. After overnight serum starvation, VSMC were treated with vehicle control solution, 10% FBS, 20 ng/ml PDGF or 10−7 M Ang II as indicated. Twenty-four hours later, apoptosis levels were measured via annexin V/propidium iodide selection (A) or via TUNEL staining (B). *p≤0.05 versus vehicle treated, #p≤0.05 versus control genotype. Shown are mean±SEM for three independent experiments, each run in triplicate.

126

p-STAT5

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+/+ -/- +/+ -/- +/+ -/-+/+ -/-10% FBS PDGF Ang IIVehicle

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* * * *+/+ -/- +/+ -/- +/+ -/-+/+ -/-10% FBS PDGF Ang IIVehicle

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Vehicle 10% FBS PDGF Ang II

Control Null #

Figure 5-7. VSMC Jak2 deletion reduces Ang II-mediated activation of STAT3 and STAT5. A) Representative blots of phospho-STAT3 and STAT3. B) Phospho-STAT3 quantification. C) Representative blots of phospho-STAT5 and STAT5. D) Phospho-STAT5 quantification. *p<0.05 versus ligand-treated control cells, #p<0.05 vs. vehicle-treated control cells, &p<0.05 vs. Ang II treated control cells. Shown are mean +/- SEM for three independent experiments.

127

CHAPTER 6 CONCLUSIONS AND PERSPECTIVES

Jak2 tyrosine kinase is an emerging therapeutic target for various disorders

including hematological malignancies and cardiovascular diseases. In this dissertation,

we showed for the first time that G6, a small molecule Jak2 inhibitor developed in our

lab, has therapeutic efficacy against two independent mouse models of Jak2-mediated

hematological malignancies. We also demonstrated that mice lacking Jak2 within VSMC

are largely protected from cardiovascular diseases including hypertension, kidney

damage, and neointima formation following vascular injury. In addition to providing a

new superior inhibitor of Jak2-mediated disorders, the main significance of these

studies is that they reveal Jak2 as a potential new therapeutic target for treatment of

cardiovascular disease. A detailed account of the implications and perspectives of the

studies in this dissertation is discussed below.

Jak2 is Important in Mammalian Biology

Jak2 tyrosine kinase plays a critical role in cytokine signaling and hematopoiesis. It

is activated by many different growth factors and cytokines including erythropoietin

(EPO), growth hormone (GH), prolactin (PRL), interferon (IFN) and interleukins (IL)

[179,180]. Jak2 knockout mice die around embryonic day 12.5 due impaired

erythropoiesis [27,28]. These reports suggest that Jak2 is essential for mammalian

biology and development. Although Jak2 is ubiquitously expressed in all mammalian

tissue, the biological and physiological importance of its expression in the individual

tissue systems is largely unknown. Traditionally, Jak2 is known to be localized in the

cytoplasm associated with cytokine receptors, as well as the plasma membrane. Recent

reports have, however, shown that Jak2 can be localized in the nucleus, where it is

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kinetically active, playing a role in nuclear factor stabilization and histone

phosphorylation [181,182,183]. The physiological outcome of the nuclear actions of

Jak2 is not fully understood. Understanding the specific functioning of Jak2 would

require creating tissue specific conditional Jak2 deletion animal models. In addition,

there is need to carry out tamoxifen-induced deletion of Jak2 at various stages of

development in order to determine whether it is important at all stages of life. There is

also need to develop specific Jak2 inhibitors in order to gain insight into the in vivo roles

of Jak2.

Jak2 plays a Critical Role in the Pathogenesis of Hematological Malignancies

Despite its beneficial role in mammalian biology and development, aberrant Jak2

signaling has been linked to various human diseases. Constitutive Jak2 signaling via

either cytokine-autocrine signaling or formation of Jak2 fusion proteins has been

implicated in the causation of cancer [184,185,186]. Hyperkinetic Jak2 kinase activity

has also been linked to a number of hematological malignancies including acute

lymphoid leukemia and chronic myeloid leukemia [85,86]. Somatic mutations in Jak2

are also responsible for the causation of MPN, which have a high prevalence in the

United States. There are approximately 22 cases of PV, 24 cases of ET, and 1.46 cases

of PMF out of every 100,000 people, which amount to 65,243 patients with PV, 71,078

with ET, and 4,330 with PMF in the United States. Some cases of leukemia, lymphoma,

and myeloma are Jak2-mediated, and these have a combined incidence of 48 per

100,000 in the United States. Despite the high incidence of Jak2-mediated neoplastic

conditions, there are currently no effective treatments. The only available treatments for

MPN are only palliative involving platelet-lowering agents such as phlebotomy,

hydroxyurea, anagrelide and interferon-α. Although these treatment strategies provide

129

some temporary relief, they are not curative and they produce a wide range of

undesirable side effects. Thus, there is need to develop molecularly targeted treatment

options which are effective in eliminating the etiology of the disease, rather than

providing temporary symptomatic relief. The discovery of the specific Jak2 mutations

such as the Jak2-V617F mutation has brought us a step closer to achieving this goal.

The Jak2-V617F has been associated with most patients with MPNs; ~98% of all PV

cases, and ~50% of all ET and PMF cases [80,81,82,83,84]. As a consequence, this

somatic cell gain of function mutation has been an important target for drug

development.

Therapeutic Efficacy of Jak2 Inhibitors in Hematological Malignancies

Although numerous attempts have been made to develop small molecule Jak2

inhibitors, their therapeutic potential in treating hematological malignancies is still

questionable. Many of these compounds are very potent in inhibiting Jak2 in vitro with

reasonable specificity, but they are unable to alleviate the disease burden in vivo. For

example, small molecule Jak2 inhibitors CEP-701, INCB16562 and CYT387 exhibited

significant efficacy in inhibiting Jak2-V617F-mediated neoplasia in vitro and provided

only palliative improvements in vivo [93, 100, 114]. There was a general failure of these

compounds to alleviate the burden of bone marrow-derived Jak2-V617F mutant clones

in vivo [100, 114,116,132]. Thus, there is still need to develop small molecule Jak2

inhibitors that have targeted therapeutic efficacy in the bone marrow, which is the

specific site of hematological disease progression.

G6 has Exceptional Bone Marrow Therapeutic Efficacy

In this dissertation, we show that a small molecule Jak2 inhibitor, G6,

demonstrates excellent therapeutic efficacy using cell lines cultured in vitro, and in vivo

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using two independent mouse models of Jak2-V617F-mediated disease pathogenesis.

This work is specifically significant because in contrast to other previously reported Jak2

inhibitors, G6 has exceptional therapeutic efficacy in the bone marrow. Using both a

NOD-SCID xenograft and a transgenic mouse model of Jak2-V617F mediated

myeloproliferative neoplasia, we demonstrate that G6 not only alleviates the palliative

symptoms, but it is also able to significantly eliminate the Jak2-V617F mutant burden in

the bone marrow.

Specifically, G6 completely eliminated HEL cells from the bone marrow of recipient

NOD-SCID mice. In a mouse model of Jak2-V617F driven MPN, G6 provided

exceptional efficacy to both the plasma and spleen as determined by corrections of

nearly every cellular compartment in the blood and the elimination of myeloproliferative

neoplasia from the spleen. This in turn resulted in significant alleviation of

splenomegaly in the transgenic mice. Furthermore, within the marrow, G6 eliminated

the myeloproliferative neoplasia phenotype. It also markedly decreased the levels of

phospho-STAT5 and significantly reduced the levels of Jak2-V617F transcripts in the

bone marrow of these MPN mice; in fact, 33% of the treated mice exhibited complete

elimination of all Jak2-V617F transcripts from the marrow. These reductions collectively

allowed for a normalization of the bone marrow as determined by corrections in the M:E

ratios. Finally, brief exposures of Jak2-V61F bone marrow cells to G6 eliminated their

subsequent clonogenic ability thereby indicating a potent inhibitory effect of G6 on Jak2-

V617F expressing cells.

In addition, the in vivo efficacy of G6 was attained at reasonably low doses (1-10

mg/kg/day) when compared to other small molecule Jak2 inhibitors (Table 6-1). This is

131

particularly important because high in vivo dose requirement of a drug can limit its

clinical potential due to the high possibility of non-target actions. These results suggest

that G6 provides superior efficacy against Jak2-V617F mediated pathogenesis when

compared to other inhibitors. Unlike other known Jak2 inhibitors, G6 has a unique

chemical structure. It is classified among a group of compound known as stilbenes.

These compounds such as resveratrol, piceatannol and diethylstilbestrol, are reported

to have anti-proliferative, anti-oxidative, anti-neovascularization and tumor-suppressive

effects [105,106,107]. Thus, the exceptional efficacy of G6 as a Jak2 inhibitor could be

in part due to its unique chemical nature as a stilbenoid compound.

Further studies need to focus on the pharmacokinetic parameters of G6 in mice

following a single administration of the effective dose. This is important in order to

determine whether sufficient concentrations of G6 reach the important predilection sites

of disease, and how it is metabolized in vivo. Further studies to develop structural

variants of G6 with increased target specificity and solubility may enhance its potential

as a therapeutic agent. Since oral administration is often preferred compared to other

routes, there is need to determine bioavailability of G6 following oral dosage.

Conditional Deletion of Vascular Smooth Muscle Cell Jak2 is Protective against Cardiovascular Disease Pathogenesis

In addition to using a pharmacological approach to demonstrating the involvement

of Jak2 in human disease pathogenesis, we have also used a genetic strategy to

demonstrate that Jak2 plays a critical role in the pathogenesis of cardiovascular

disease. In this dissertation, we show that mice lacking Jak2 within VSMC are largely

protected from cardiovascular diseases including hypertension and neointima formation

132

following vascular injury. The mechanisms through which Jak2 mediates cardiovascular

disease pathogenesis are discussed below in details.

Jak2 Mediates Cardiovascular Disease Pathogenesis via Multiple Non-Redundant Mechanisms

Cardiovascular diseases are believed to be the number one causes of death in the

world. Although great effort and resources have been dedicated to understanding the

pathogenesis of these diseases, there is still an urgent need to investigate the signaling

mechanisms involved in the causation of cardiovascular disease. Since the discovery

that Jak2 couples to the AT1 receptor, there have been numerous reports implicating

Jak2 in the pathogenesis of cardiovascular disease. The mechanisms through which

Jak2 mediates cardiovascular disease are still not fully understood. In this dissertation,

we have identified Jak2 as a mediator of Ang II-induced hypertension via multiple non-

redundant mechanisms. We found that by contributing to the increased presence of

ROS, Jak2 is centrally located to mediate cardiovascular disease via multiple

mechanisms. These mechanisms include ROS mediated scavenging of endothelial NO,

increasing intracellular Ca2+, and increasing Rho-kinase activity.

Jak2 Contributes to Increased Presence of ROS

In this study, we found that compared to controls, the mice that lack Jak2 in their

VSMC have lower levels of ROS. Previous studies have also demonstrated that in

human leukemia cells, inhibition of Jak2 resulted in decreased ROS levels [75].

However, the mechanism(s) by which Jak2 contributes to the increased production of

ROS is/are not known. NAD(P)H oxidase is the primary source of ROS in eukaryotic

cells. There are 7 isoforms of NAD(P)H oxidases (Nox) including Nox1, Nox2, Nox3,

Nox4, Nox5, Duox1 and Duox2. The Nox family of enzymes transports electrons across

133

the plasma membrane leading to generation of ROS, which then mediate a wide variety

of physiological and pathophysiological processes including cardiovascular disease

[192]. The mechanisms for activation of Nox are not fully understood. We know that Ang

II stimulates the activity of membrane-bound Nox in VSMC and endothelial cells

resulting in increased production of ROS such as superoxide (•O2−) and hydrogen

peroxide (H2O2) [17,57,62,63,64,65,66]. It is possible that Jak2 may mediate Ang II-

induced production of ROS by direct activation of Nox but this still needs to be

investigated.

The ROS are comprised of three different reduction products of oxygen including

superoxide, H2O2, and the hydroxyl radical (•OH). The obvious differences in the

molecular chemical properties of these reduction products dictate differential signaling

functions [188]. Because superoxide has a negative charge, it is believed to be

incapable of crossing the cell membrane via anion channels. However, studies have

shown that superoxide can cross membrane lipid bi-layer via the chloride channel-3

(ClC-3) [189]. In this dissertation, we found that expression of Jak2 in VSMC resulted in

a partial impairment of the endothelium dependent vasorelaxation in aortic rings.

However, when the rings were pre-treated with the anti-oxidant superoxide dismutase

(SOD), their vasorelaxation was dramatically improved (Figure 4-7). These results

suggest that presence of Jak2 in VSMC results in increase presence of superoxide,

which scavenge endothelial NO, resulting in reduced vasorelaxation. It is not known

whether superoxide scavenged NO extracellularly before diffusing into the VSMC or

intracellularly. H2O2 is believed to be the main ROS involved in the pathogenesis of

hypertension because it is freely permeable and is able diffuse outside of the cell and

134

scavenge NO. Numerous studies have shown that free oxygen radicals including H2O2

and OH• can act directly on VSMC and modulate vascular tone. They can induce

vasoconstriction or vasodilatation depending on various factors such as concentration

and the specific vessel type. The vascular effects of free oxygen radicals have been

reviewed [190].

VSMC Jak2 Expression Correlates with Reduced NO Availability

NO is a vasoactive substance produced from the endothelium by endothelial NO

synthase (eNOS) [191,192]. It acts on VSMC by activating intracellular soluble

guanylate cyclase (sGC) enzyme which converts guanosine triphosphate to cGMP,

resulting in vasorelaxation [193]. Endothelial dysfunction and low NO bioavailability is

a major cause of cardiovascular diseases resulting from hypertension. One of the main

mechanisms resulting in low NO bioavailability is scavenging by ROS [139]. Another

mechanism is lack of BH4, which leads to eNOS dysfunction and a switch from NO

release to the formation of oxygen radicals [194]. Ang II action increases production of

ROS, which in turn inactivate NO in both VSMC and endothelial cells [70,71,72]. This

Ang II-induced formation of ROS is independent of its hemodynamic effects, since it is

not observed in norepinephrine-induced hypertension [62,67,68]. In this dissertation, we

report for the first time that increased Jak2 expression in VSMC results in reduced NO

availability via a ROS dependent mechanism.

VSMC Jak2 Expression Correlates with Increased Rho-kinase Activity

Rho-associated kinase (Rho-kinase) is an effector molecule of the small GTPase

Rho. It plays a pivotal role in vascular smooth muscle contraction, cell adhesion and cell

motility [195]. These processes are involved in the pathogenesis of cardiovascular

disease including hypertension and coronary artery spasm [196,197]. Rho-kinase

135

regulates myosin light chain (MLC) by either direct phosphorylation or by inactivation of

myosin phosphatase through the phosphorylation of myosin-binding subunit. As such,

increased Rho-kinase activity increases smooth muscle contraction via regulation of the

phosphorylation state of MLC. In addition, Rho-kinase increases intracellular Ca2+

sensitization in VSMC in response to agonist stimulation [198,199].

Ang II is known to activate Rho-kinase activity. Binding of Ang II to the AT1-R

results in increased Rho-kinase activity and vasoconstriction by acting via the Rho

exchange factor, Arhgef1 and Jak2 has been shown to mediate this process [56].

Interestingly, previous studies have shown that Rho-kinase can be activated by

increased ROS [69,73]. These results, and the data presented in this dissertation

suggest that Jak2 mediates Ang II-induced vasoconstriction by activating Rho-kinase

via both ROS- and phosphorylation-dependent mechanisms.

VSMC Jak2 Expression Correlates with Increased Intracellular Calcium

Increase in intracellular Ca2+ is one of the major triggers of contraction in VSMC.

In response to an agonist such as Ang II, there is increased influx of Ca2+ into the

cytoplasm. Ca2+ binds to calmodulin and activates myosin light chain kinase (MLCK),

which phosphorylates the myosin light chain and enhances the interaction between

actin and myosin, resulting in vasoconstriction [55]. ROS have been shown to mediate

RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle

following chronic hypoxia [69]. In addition, whole cell patch clamp studies have shown

that oxidative stress induced by H2O2 increases the activity of L-type Ca2+ channels

[200]. The ROS mediated increase in intracellular free Ca2+ contributes to increased

vascular tone [138]. These data suggest that increased presence of ROS result in

increased influx and sensitization of intracellular Ca2+. Here we found that presence of

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VSMC Jak2 resulted in increased presence of ROS, which in turn resulted in increased

intracellular Ca2+.

VSMC Jak2 Mediates Ang II-Induced Growth Factor Effects and Local Tissue Damage

Vascular remodeling and kidney damage are known to play an important role in

cardiovascular disease pathogenesis. In this investigation, we found that mice lacking

the Jak2 tyrosine kinase in their VSMC are resistant to Ang II-induced vascular

remodeling as manifested by increased thickness of the tunica media. We also found

that these mice are protected from kidney damage and neointima formation following

vascular injury. These local tissue effects of Ang II were associated with increased

activation of the Jak-STAT pathway. However, it is still not known whether the local

tissue effects of Ang II are independent of its hemodynamic effects. Further studies

need to be done in which control mice are infused with Ang II, but their blood pressure

reduced to levels observed in the VSMC Jak2 null mice. This can be achieved by

treating the mice with a drug combination comprising of hydralazine, reserpine and

thiazide, which are collectively referred to as the triple therapy. Hydralazine lowers

blood pressure by increasing guanosine monophosphate levels resulting in decreased

action of the second messenger IP3, and limiting calcium release from the sarcoplasmic

reticulum. Reserpine exerts its antihypertensive effects by acting as an antagonist to the

vesicular monoamine transporter (VMAT), there by preventing the sympathetic actions

of catecholamines. Thiazide is a diuretic and it lower blood pressure by blocking the

thiazide-sensitive Na+-Cl− symporter, preventing reabsorption of sodium and chloride

ions. The different actions of these drugs in hypertension have been reviewed [201].

137

http://en.wikipedia.org/wiki/Guanosine_monophosphate

http://en.wikipedia.org/wiki/Second_messenger

http://en.wikipedia.org/wiki/Inositol_triphosphate

http://en.wikipedia.org/wiki/Inositol_triphosphate

http://en.wikipedia.org/w/index.php?title=Calcium_release&action=edit&redlink=1

http://en.wikipedia.org/wiki/Sarcoplasmic_reticulum

http://en.wikipedia.org/wiki/Sarcoplasmic_reticulum

http://en.wikipedia.org/wiki/VMAT

http://en.wikipedia.org/wiki/Catecholamine

http://en.wikipedia.org/wiki/Sodium-chloride_symporter





The triple therapy drug combination will eliminate the pressor effects of Ang II while

allowing its local tissue actions.

Jak2 Inhibitors and Their Potential for Cardiovascular Disease Therapy

The pathogenesis of cardiovascular disease resulting from hypertension often

involves a complex combination of causes including genetic and environmental factors

[202]. The current pharmacological treatments for hypertension are mainly targeted

towards inhibition or prevention of action of vasoconstrictor hormones including Ang II.

Treatment of resistant hypertension currently entails choosing medications with

complementary mechanisms of action such as optimizing diuretic use, and/or

mineralocorticoid antagonism [203]. However, due to the multifactorial nature of the

disease pathogenesis, there are still subsets of patients in whom available treatments

are increasingly becoming ineffective. Treatment of resistant hypertension presents an

increasing dilemma in the clinical setting, and patients with resistant hypertension have

increased cardiovascular risk [203]. Therefore, there is still need to identify other genetic

targets, to provide more individualized treatments for such patients. In addition, a

number of patients are non-responsive to mono-antihypertensive therapy and there is

often need to use combination therapy [204].

Since Jak2 has been shown to regulate Ang II-mediated signaling downstream of

the AT1-R, it may represent a valuable new target for anti-hypertensive therapeutic

strategies. AG490, a tyrphostin well known for inhibiting Jak2 [162] has been shown to

prevent hypertension [56], and neointima formation [49] in animal models. As such,

these studies and the studies presented in this dissertation implicate Jak2 as an

important modulator of blood pressure and cardiovascular disease. Therefore,

therapeutic approaches using inhibition of Jak2 to regulate Ang II-mediated AT1-R

138

stimulation is an intriguing novel target for the treatment of hypertension. This implies a

potential new therapeutic target for multi-drug resistant hypertension. The realization of

therapeutic benefit from Jak2 inhibition in cardiovascular diseases may entail identifying

optimized inhibitors with unique profiles to maximize therapeutic potential. In addition,

given the potential side effects that may arise from Jak2 inhibition, there is need for a

risk/benefit assessment of using Jak2 as a target in treatment of cardiovascular

disease. Being a downstream signaling molecule of the AT1-R, Jak2 is well positioned,

and may offer a new, more specific target in the treatment of Ang II-mediated

cardiovascular diseases. Ang II acts on the AT1-R resulting in the stimulation of multiple

downstream signaling cascades, leading to various effects. Since Jak2 is a downstream

signaling molecule of the AT1-R, it may present a more specific target for treatment of

cardiovascular disease while avoiding side effects arising from inhibition of non diseased

signaling pathways.

Conclusions

Jak2 plays a critical role in the pathogenesis of various human diseases including

cancer and cardiovascular disease. A considerable amount of research has been

dedicated to developing drugs using Jak2 as a therapeutic target in hematological

malignancies. Although numerous in vitro studies have demonstrated involvement of

Jak2 in cardiovascular disease pathogenesis, there is a general lack on in vivo evidence

and very little attention has been given to this important signaling molecule as a

treatment strategy in cardiovascular disease. In this dissertation, we have not only

discovered a potential therapeutic agent in Jak2-mediated neoplasia, but we have also

found that Jak2 plays a causative role in Ang II-induced cardiovascular disease.

Cardiovascular disease has complex and multi-factorial etiologies and is unlikely to be

139

successfully treated with a single approach. Current standard therapies include

inhibition of the RAAS as well as enhancement of nitric oxide-mediated relaxation of

VSMC. Jak2 is centrally located as a downstream signaling molecule of the AT1-R and it

is involved in many of the signaling cascades regulated by Ang II including oxidative

stress. It appears that in a multi-factorial disease such as hypertension, Jak2 may

provide a more specific target as a therapeutic approach. Jak2 inhibition offers a

potential base for further study and development of therapeutic options to overcome

cardiovascular disease and ultimately, may be considered as an adjunct or alternative

to current therapeutic agents.

140

Table 6-1. Comparison of in vivo dosages of Jak2 inhibitors in murine models.

Inhibitor Name

Administered In Vivo Dosage (mg/kg/day)

Reference

TG101348

60-120 mg/kg twice daily (oral)

Wernig, G. et al. (2008)

TG101209

100 mg/kg twice daily (oral)

Pardanani, A. et al. (2007)

CEP701

30 mg/kg twice daily (oral)

Hexner, E. O. et al. (2008)

INCB018424

180 mg/kg/day (oral)

Quintas-Cardama, A. et al. (2010)

P1

100 mg/kg/day (oral)

Mathur, A. et al. (2009)

CYT387

25-50 mg/kg twice daily

(oral)

Tyner, J. W. et al. (2010)

WP1066

40 mg/kg every other day (ip)

Iwamaru, A. et al. (2006)

141

LIST OF REFERENCES

1. Hansson L, Zanchetti A, Carruthers SG, Dahlof B, Elmfeldt D, et al. (1998) Effects of intensive blood-pressure lowering and low-dose aspirin in patients with hypertension: principal results of the hypertension optimal treatment (hot) randomised trial. Hot study group. Lancet 351: 1755-1762.

2. Duncia JV, Carini DJ, Chiu AT, Johnson AL, Price WA, et al. (1992) The discovery of

dup 753, a potent, orally active nonpeptide angiotensin ii receptor antagonist. Med Res Rev 12: 149-191.

3. Chiu AT, Mccall DE, Nguyen TT, Carini DJ, Duncia JV, et al. (1989) Discrimination of

angiotensin ii receptor subtypes by dithiothreitol. Eur J Pharmacol 170: 117-118.

4. Chiu AT, Mccall DE, Price WA, Jr., Wong PC, Carini DJ, et al. (1991) In vitro pharmacology of dup 753. Am J Hypertens 4: 282s-287s.

5. Wong PC, Price WA, Jr., Chiu AT, Duncia JV, Carini DJ, et al. (1991) In vivo

pharmacology of dup 753. Am J Hypertens 4: 288s-298s.

6. Matsubara H (1998) Pathophysiological role of angiotensin ii type 2 receptor in cardiovascular and renal diseases. Circ Res 83: 1182-1191.

7. Kobori H, Nangaku M, Navar LG, Nishiyama A (2007) The intrarenal renin-

angiotensin system: from physiology to the pathobiology of hypertension and kidney disease. Pharmacol Rev 59: 251-287.

8. Katz AM (1992) Is angiotensin ii a growth factor masquerading as a vasopressor?

Heart Dis Stroke 1: 151-154.

9. Daemen MJ, Lombardi DM, Bosman FT, Schwartz SM (1991) Angiotensin II induces smooth muscle cell proliferation in the normal and injured rat arterial wall. Circ Res 68: 450-456.

10. Powell JS, Clozel JP, Muller RK, Kuhn H, Hefti F, et al. (1989) Inhibitors of

angiotensin-converting enzyme prevent myointimal proliferation after vascular injury. Science 245: 186-188.

11. Chiu AT, Roscoe WA, Mccall DE, Timmermans PB (1991) Angiotensin II-1

receptors mediate both vasoconstrictor and hypertrophic responses in rat aortic smooth muscle cells. Receptor 1: 133-140.

12. Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, et al. (1993)

Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 45: 205-251.

142

13. Geisterfer AA, Peach MJ, Owens GK (1988) Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res 62: 749-756.

14. Xi XP, Graf K, Goetze S, Fleck E, Hsueh WA, et al. (1999) Central role of the MAPK

pathway in ang II-mediated DNA synthesis and migration in rat vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 19: 73-82.

15. Berk BC, Vekshtein V, Gordon HM, Tsuda T (1989) Angiotensin II-stimulated protein

synthesis in cultured vascular smooth muscle cells. Hypertension 13: 305-314.

16. Fingerle J, Muller RM, Kuhn H, Pech M, Baumgartner HR (1995) Mechanism of inhibition of neointimal formation by the angiotensin-converting enzyme inhibitor cilazapril. A study in balloon catheter-injured rat carotid arteries. Arterioscler Thromb Vasc Biol 15: 1945-1950.

17. Mehta PK, Griendling KK (2007) Angiotensin II cell signaling: Physiological and

pathological effects in the cardiovascular system. Am J Physiol Cell Physiol 292: C82-97.

18. Su EJ, Lombardi DM, Wiener J, Daemen MJ, Reidy MA, et al. (1998) Mitogenic

effect of angiotensin II on rat carotid arteries and type II Or III mesenteric microvessels but not type I mesenteric microvessels is mediated by endogenous basic fibroblast growth factor. Circ Res 82: 321-327.

19. Griffin SA, Brown WC, Macpherson F, Mcgrath JC, Wilson VG, et al. (1991)

Angiotensin II causes vascular hypertrophy in part by a non-pressor mechanism. Hypertension 17: 626-635.

20. Yamaoka K, Saharinen P, Pesu M, Holt VE, 3rd, Silvennoinen O, et al. (2004) The

Janus Kinases (Jaks). Genome Biol 5: 253.

21. Saharinen P, Vihinen M, Silvennoinen O (2003) Autoinhibition of Jak2 Tyrosine Kinase is dependent on specific regions in its pseudokinase domain. Mol Biol Cell 14: 1448-1459.

22. Luo H, Rose P, Barber D, Hanratty WP, Lee S, et al. (1997) Mutation in the Jak

Kinase JH2 domain hyperactivates drosophila and mammalian Jak-Stat pathways. Mol Cell Biol 17: 1562-1571.

23. Kampa D, Burnside J (2000) Computational and functional analysis of the putative

SH2 domain in Janus Kinases. Biochem Biophys Res Commun 278: 175-182.

24. Girault JA, Labesse G, Mornon JP, Callebaut I (1998) Janus Kinases and focal adhesion kinases play in the 4.1 band: a superfamily of band 4.1 domains important for cell structure and signal transduction. Mol Med 4: 751-769.

143

25. Tanner JW, Chen W, Young RL, Longmore GD, Shaw AS (1995) The conserved box 1 motif of cytokine receptors is required for association with Jak Kinases. J Biol Chem 270: 6523-6530.

26. Zhao Y, Wagner F, Frank SJ, Kraft AS (1995) The amino-terminal portion of the

Jak2 protein kinase is necessary for binding and phosphorylation of the granulocyte-macrophage colony-stimulating factor receptor beta c chain. J Biol Chem 270: 13814-13818.

27. Neubauer H, Cumano A, Muller M, Wu H, Huffstadt U, et al. (1998) Jak2 deficiency

defines an essential developmental checkpoint in definitive hematopoiesis. Cell 93: 397-409.

28. Parganas E, Wang D, Stravopodis D, Topham DJ, Marine JC e a. (1998) Jak2 is

essential for signaling through a variety of cytokine receptors. Cell 93: 385-395.

29. Bhat GJ Thekkumkara TJ Thomas WG Conrad KM Baker KM (1994) Angiotensin II stimulates sis-inducing factor-like DNA binding activity. Evidence that the at1a receptor activates transcription factor-stat91 and/or a related protein. J Biol Chem 269: 31443-31449.

30. Marrero MB, Schieffer B, Paxton WG, Heerdt L, Berk BC, et al. (1995) Direct

stimulation of Jak/Stat pathway by the Angiotensin II AT1 receptor. Nature 375: 247-250.

31. Marrero MB, Schieffer B, Li B, Sun J, Harp JB, et al. (1997) Role of Janus

Kinase/Signal Transducer And Activator of Transcription and Mitogen-Activated Protein Kinase cascades in angiotensin II- and platelet-derived growth factor-induced vascular smooth muscle cell proliferation. J Biol Chem 272: 24684-24690.

32. Ali MS, Sayeski PP, Dirksen LB, Hayzer DJ, Marrero MB, et al. (1997) Dependence

on the motif yipp for the physical association of Jak2 kinase with the intracellular carboxyl tail of the angiotensin II AT1 receptor. J Biol Chem 272: 23382-23388.

33. Mcwhinney CD, Hunt RA, Conrad KM, Dostal DE, Baker KM (1997) The Type I

Angiotensin II receptor couples to Stat1 and Stat3 activation through Jak2 kinase in neonatal rat cardiac myocytes. J Mol Cell Cardiol 29: 2513-2524.

34. Dostal DE, Hunt RA, Kule CE, Bhat GJ, Karoor V, et al. (1997) Molecular

mechanisms of angiotensin II in modulating cardiac function: intracardiac effects and signal transduction pathways. J Mol Cell Cardiol 29: 2893-2902.

35. Pan J, Fukuda K, Kodama H, Makino S, Takahashi T, et al. (1997) Role of

angiotensin II in activation of the Jak/Stat pathway induced by acute pressure overload in the rat heart. Circ Res 81: 611-617.

144

36. Kodama H, Fukuda K, Pan J, Makino S, Sano M, et al. (1998) Biphasic activation of

the Jak/Stat pathway by angiotensin II in rat cardiomyocytes. Circ Res 82: 244-250.

37. Mcwhinney CD, Dostal D, Baker K (1998) Angiotensin II activates Stat5 through

Jak2 kinase in cardiac myocytes. J Mol Cell Cardiol 30: 751-761.

38. Frank GD, Saito S, Motley ED, Sasaki T, Ohba M, et al. (2002) Requirement of Ca(2+) and PKCdelta for Janus Kinase 2 activation by angiotensin II: involvement of pyk2. Mol Endocrinol 16: 367-377.

39. Marrero MB, Paxton WP, Schieffer B, Ling BN, Bernstein KE (1996) Angiotensin II

signalling events mediated by tyrosine phosphorylation. Cell Signal 8: 21-26.

40. Venema RC, Venema VJ, Eaton DC, Marrero MB (1998) Angiotensin II-induced tyrosine phosphorylation of Signal Transducers and Activators of Transcription 1 is regulated by Janus-Activated Kinase 2 and Fyn Kinases and Mitogen-Activated Protein Kinase Phosphatase 1. J Biol Chem 273: 30795-30800.

41. Prescott MF, Webb RL, Reidy MA (1991) Angiotensin-Converting Enzyme inhibitor

versus angiotensin II, AT1 receptor antagonist. Effects on smooth muscle cell migration and proliferation after balloon catheter injury. Am J Pathol 139: 1291-1296.

42. Rakugi H, Jacob HJ, Krieger JE, Ingelfinger JR, Pratt RE (1993) Vascular injury

induces angiotensinogen gene expression in the media and neointima. Circulation 87: 283-290.

43. Rakugi H, Kim DK, Krieger JE, Wang DS, Dzau VJ, et al. (1994) Induction of

angiotensin converting enzyme in the neointima after vascular injury. Possible role in restenosis. J Clin Invest 93: 339-346.

44. Iwai N, Izumi M, Inagami T, Kinoshita M (1997) Induction of renin in medial smooth

muscle cells by balloon injury. Hypertension 29: 1044-1050.

45. Viswanathan M, Stromberg C, Seltzer A, Saavedra JM (1992) Balloon angioplasty enhances the expression of angiotensin ii at1 receptors in neointima of rat aorta. J Clin Invest 90: 1707-1712.

46. Sandberg EM, Ma X, Vonderlinden D, Godeny MD, Sayeski PP (2004) Jak2

tyrosine kinase mediates angiotensin II-dependent inactivation of Erk2 via induction of Mitogen-Activated Protein Kinase Phosphatase 1. J Biol Chem 279: 1956-1967.

145

47. Madamanchi NR, Li S, Patterson C, Runge MS (2001) Reactive Oxygen Species regulate heat-shock protein 70 via the Jak/Stat pathway. Arterioscler Thromb Vasc Biol 21: 321-326.

48. Gharavi NM, Alva JA, Mouillesseaux KP, Lai C, Yeh M, et al. (2007) Role of the

Jak/Stat pathway in the regulation of interleukin-8 transcription by oxidized phospholipids in vitro and in atherosclerosis in vivo. J Biol Chem 282: 31460-31468.

49. Seki Y, Kai H, Shibata R, Nagata T, Yasukawa H, et al. (2000) Role of the Jak/Stat

pathway in rat carotid artery remodeling after vascular injury. Circ Res 87: 12-18.

50. Macrez-Lepretre N, Kalkbrenner F, Morel JL, Schultz G, Mironneau J (1997) G protein heterotrimer Galpha13beta1gamma3 couples the angiotensin AT1a receptor to increases in cytoplasmic Ca2+ in rat portal vein myocytes. J Biol Chem 272: 10095-10102.

51. Ushio-Fukai M, Griendling KK, Akers M, Lyons PR, Alexander RW (1998) Temporal

dispersion of activation of phospholipase c-beta1 and -gamma isoforms by angiotensin ii in vascular smooth muscle cells. Role of alphaq/11, alpha12, and beta gamma g protein subunits. J Biol Chem 273: 19772-19777.

52. Rossier MF, Capponi AM (2000) Angiotensin II and calcium channels. Vitam Horm

60: 229-284. 53. Penner R, Fasolato C, Hoth M (1993) Calcium influx and its control by calcium

release. Curr Opin Neurobiol 3: 368-374.

54. Parekh AB (2003) Store-operated Ca2+ entry: dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane. J Physiol 547: 333-348.

55. Yan C, Kim D, Aizawa T, Berk BC (2003) Functional interplay between angiotensin

II and nitric oxide: cyclic GMP as a key mediator. Arterioscler Thromb Vasc Biol 23: 26-36.

56. Guilluy C, Bregeon J, Toumaniantz G, Rolli-Derkinderen M, Retailleau K, et al.

(2010) The rho exchange factor arhgef1 mediates the effects of angiotensin II on vascular tone and blood pressure. Nat Med 16: 183-190.

57. Griendling KK, Sorescu D, Ushio-Fukai M (2000) NAD(P)H oxidase: Role in cardiovascular biology and disease. Circ Res 86: 494-501.

146

58. Ohtsu H, Frank GD, Utsunomiya H, Eguchi S (2005) Redox-dependent protein kinase regulation by angiotensin II: mechanistic insights and its pathophysiology. Antioxid Redox Signal 7: 1315-1326.

59. Touyz RM (2004) Reactive Oxygen Species and angiotensin II signaling in vascular

cells -- implications in cardiovascular disease. Braz J Med Biol Res 37: 1263-1273.

60. Taniyama Y, Griendling KK (2003) Reactive Oxygen Species in the vasculature:

molecular and cellular mechanisms. Hypertension 42: 1075-1081.

61. Ushio-Fukai M, Alexander RW, Akers M, Yin Q, Fujio Y, et al. (1999) Reactive Oxygen Species mediate the activation of akt/protein kinase b by angiotensin II in vascular smooth muscle cells. J Biol Chem 274: 22699-22704.

62. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, et al. (1996) Angiotensin

II-mediated hypertension in the rat increases vascular superoxide production via membrane nadh/nadph oxidase activation. Contribution to alterations of vasomotor tone. J Clin Invest 97: 1916-1923.

63. Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, et al. (1998) Role of

NADH/NADPH oxidase-derived H2O2 in angiotensin ii-induced vascular hypertrophy. Hypertension 32: 488-495.

64. Ushio-Fukai M, Zafari AM, Fukui T, Ishizaka N, Griendling KK (1996) P22phox is a

critical component of the superoxide-generating NADP/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. J Biol Chem 271: 23317-23321.

65. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW (1994) Angiotensin II

stimulates NADP and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74: 1141-1148.

66. Zhang H, Schmeisser A, Garlichs CD, Plotze K, Damme U, et al. (1999) Angiotensin

II-induced superoxide anion generation in human vascular endothelial cells: role of membrane-bound NADH-/NADPH-oxidases. Cardiovasc Res 44: 215-222.

67. Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA, et al. (1997) Role of

superoxide in angiotensin II-induced but not catecholamine-induced hypertension. Circulation 95: 588-593.

68. Harrison DG (1997) Endothelial function and oxidant stress. Clin Cardiol 20: Ii-11-

17.

147

69. Jernigan NL, Walker BR, Resta TC (2008) Reactive Oxygen Species mediate rhoa/rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 295: L515-529.

70. Gryglewski RJ, Palmer RM, Moncada S (1986) Superoxide anion is involved in the

breakdown of endothelium-derived vascular relaxing factor. Nature 320: 454-456.

71. Rubanyi GM, Vanhoutte PM (1986) Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol 250: H822-827.

72. Dzau VJ (2001) Theodore Cooper Lecture: Tissue Angiotensin and pathobiology of

vascular disease: A unifying hypothesis. Hypertension 37: 1047-1052.

73. Jin L, Ying Z, Webb RC (2004) Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta. Am J Physiol Heart Circ Physiol 287: H1495-1500.

74. Yasunari K, Kohno M, Kano H, Yokokawa K, Minami M, et al. (1999) Antioxidants

improve impaired insulin-mediated glucose uptake and prevent migration and proliferation of cultured rabbit coronary smooth muscle cells induced by high glucose. Circulation 99: 1370-1378.

75. Walz C, Crowley BJ, Hudon HE, Gramlich JL, Neuberg DS, et al. (2006) Activated

Jak2 with the V617F point mutation promotes g1/s phase transition. J Biol Chem 281: 18177-18183.

76. Sattler M, Verma S, Shrikhande G, Byrne CH, Pride YB, et al. (2000) The bcr/abl

tyrosine kinase induces production of reactive oxygen species in hematopoietic cells. J Biol Chem 275: 24273-24278.

77. Ihle JN (1995) Cytokine receptor signalling. Nature 377: 591-594.

78. Delhommeau F, Pisani DF, James C, Casadevall N, Constantinescu S, et al. (2006)

Oncogenic mechanisms in myeloproliferative disorders. Cell Mol Life Sci 63: 2939-2953.

79. Ma X, Vanasse G, Cartmel B, Wang Y, Selinger HA (2008) Prevalence of

polycythemia vera and essential thrombocythemia. Am J Hematol 83: 359-362.

80. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, et al. (2005) Acquired mutation of the tyrosine kinase Jak2 in human myeloproliferative disorders. Lancet 365: 1054-1061.

148

81. James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, et al. (2005) A unique clonal Jak2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434: 1144-1148.

82. Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, et al. (2005) Activating

mutation in the tyrosine kinase Jak2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 7: 387-397.

83. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, et al. (2005) A gain-of-

function mutation of Jak2 in myeloproliferative disorders. N Engl J Med 352: 1779-1790.

84. Zhao R, Xing S, Li Z, Fu X, Li Q, et al. (2005) Identification of an acquired Jak2

mutation in polycythemia vera. J Biol Chem 280: 22788-22792.

85. Jelinek J, Oki Y, Gharibyan V, Bueso-Ramos C, Prchal JT, et al. (2005) Jak2 mutation 1849G>T is rare in acute leukemias but can be found in cmml, philadelphia chromosome-negative cml, and megakaryocytic leukemia. Blood 106: 3370-3373.

86. Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, et al. (2005) Widespread occurrence

of the Jak2 V617F mutation in chronic myeloproliferative disorders. Blood 106: 2162-2168.

87. Levine RL, Loriaux M, Huntly BJ, Loh ML, Beran M, et al. (2005) The Jak2V617F

activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia. Blood 106: 3377-3379.

88. Levine RI, Pardanani A, Tefferi A, Gilliland DG (2007) Role of Jak2 in the

pathogenesis and therapy of myeloproliferative disorders. Nat Rev Cancer 7: 673-683.

89. Wernig G, Kharas MG, Okabe R, Moore SA, Leeman DS, et al. (2008) Efficacy of

TG101348, a selective Jak2 inhibitor, in treatment of a murine model of Jak2V617F-induced polycythemia vera. Cancer Cell 13: 311-320.

90. Lipka DB, Hoffmann LS, Heidel F, Markova B, Blum MC, et al. (2008) LS104, a non-

ATP-competitive small-molecule inhibitor of Jak2, is potently inducing apoptosis in jak2v617f-positive cells. Mol Cancer Ther 7: 1176-1184.

91. Ferrajoli A, Faderl S, Van Q, Koch P, Harris D, et al. (2007) WP1066 disrupts janus

kinase-2 and induces caspase-dependent apoptosis in acute myelogenous leukemia cells. Cancer Res 67: 11291-11299.

149

92. Gozgit JM, Bebernitz G, Patil P, Ye M, Parmentier J, et al. (2008) Effects of the Jak2 inhibitor, AZ960, on pim/bad/bcl-xl survival signaling in the human Jak2 V617F cell line set-2. J Biol Chem 283: 32334-32343.

93. Pardanani A, Lasho T, Smith G, Burns CJ, Fantino E, et al. (2009) CYT387, a

selective Jak1/Jak2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients. Leukemia 23: 1441-1445.

94. Antonysamy S, Hirst G, Park F, Sprengeler P, Stappenbeck F, et al. (2009)

fragment-based discovery of Jak-2 inhibitors. Bioorg Med Chem Lett 19: 279-282.

95. Sandberg EM, Ma X, He K, Frank SJ, Ostrov DA, et al. (2005) Identification of

1,2,3,4,5,6-hexabromocyclohexane as a small molecule inhibitor of Jak2 tyrosine kinase autophosphorylation [correction of autophophorylation]. J Med Chem 48: 2526-2533.

96. Sayyah J, Magis A, Ostrov DA, Allan RW, Braylan RC, et al. (2008) Z3, a Novel

Jak2 tyrosine kinase small-molecule inhibitor that suppresses Jak2-mediated pathologic cell growth. Mol Cancer Ther 7: 2308-2318.

97. Kiss R, Polgar T, Kirabo A, Sayyah J, Figueroa NC, et al. (2009) Identification of a

novel inhibitor of Jak2 tyrosine kinase by structure-based virtual screening. Bioorg Med Chem Lett 19: 3598-3601.

98. Pardanani A, Hood J, Lasho T, Levine RL, Martin MB, et al. (2007) TG101209, a

small molecule Jak2-selective kinase inhibitor potently inhibits myeloproliferative disorder-associated Jak2V617F and MPLW515l/k mutations. Leukemia 21: 1658-1668.

99. Hexner EO, Serdikoff C, Jan M, Swider CR, Robinson C, et al. (2008) Lestaurtinib

(Cep701) is a Jak2 inhibitor that suppresses Jak2/Stat5 signaling and the proliferation of primary erythroid cells from patients with myeloproliferative disorders. Blood 111: 5663-5671.

100. Tyner JW, Bumm TG, Deininger J, Wood L, Aichberger KJ, et al. (2010) Cyt387, a

novel Jak2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms. Blood 115: 5232-5240.

101. Verstovsek S (2009) Therapeutic potential of Jak2 inhibitors. Hematology Am Soc

Hematol Educ Program: 636-642.

150

102. Geron I, Abrahamsson AE, Barroga CF, Kavalerchik E, Gotlib J, et al. (2008) Selective inhibition of Jak2-driven erythroid differentiation of polycythemia vera progenitors. Cancer Cell 13: 321-330.

103. Xing S, Ho WTT, Zhao WM, Ma JM, Wang SF, et al. (2008) Transgenic expression

of Jak2(V617F) causes myeloproliferative disorders in mice. Blood 111: 5109-5117.

104. Lacout C, Pisani DF, Tulliez M, Gachelin FM, Vainchenker W, et al. (2006)

Jak2V617F expression in murine hematopoietic cells leads to mpd mimicking human PV with secondary myelofibrosis. Blood 108: 1652-1660.

105. Larrosa M, Tomas-Barberan FA, Espin JC (2003) Grape polyphenol resveratrol

and the related molecule 4-hydroxystilbene induce growth inhibition, apoptosis, S-phase arrest, and upregulation of cyclins A, E, and B1 in human sk-mel-28 melanoma cells. J Agric Food Chem 51: 4576-4584.

106. Le Corre L, Chalabi N, Delort L, Bignon YJ, Bernard-Gallon DJ (2005) Resveratrol

and breast cancer chemoprevention: molecular mechanisms. Mol Nutr Food Res 49: 462-471.

107. Kimura Y (2005) New anticancer agents: in vitro and in vivo evaluation of the

antitumor and antimetastatic actions of various compounds isolated from medicinal plants. In Vivo 19: 37-60.

108. Pace-Asciak CR, Hahn S, Diamandis EP, Soleas G, Goldberg DM (1995) The red

wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease. Clin Chim Acta 235: 207-219.

109. Geahlen RL, Mclaughlin JL (1989) Piceatannol (3,4,3',5'-tetrahydroxy-trans-

stilbene) is a naturally occurring protein-tyrosine kinase inhibitor. Biochem Biophys Res Commun 165: 241-245.

110. Swanson-Mungerson M, Ikeda M, Lev L, Longnecker R, Portis T (2003)

Identification of latent membrane protein 2a (lmp2a) specific targets for treatment and eradication of epstein-barr virus (ebv)-associated diseases. J Antimicrob Chemother 52: 152-154.

111. Baker SJ, Rane SG, Reddy EP (2007) Hematopoietic cytokine receptor signaling.

Oncogene 26: 6724-6737.

112. Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF (1999) Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- Mice: a direct role for Stat5 in BCL-X(L) induction. Cell 98: 181-191.

151

113. Majumder A, Govindasamy L, Magis A, Kiss R, Polgar T, et al. (2010) Structure-function correlation of G6, a novel small molecule inhibitor of Jak2: indispensability of the stilbenoid core. J Biol Chem 285: 31399-31407.

114. Santos FP, Kantarjian HM, Jain N, Manshouri T, Thomas DA, et al. (2010) Phase 2

study of CEP-701, an orally available Jak2 inhibitor, in patients with primary or post-polycythemia vera/essential thrombocythemia myelofibrosis. Blood 115: 1131-1136.

115. Koppikar P, Abdel-Wahab O, Hedvat C, Marubayashi S, Patel J, et al. (2010)

Efficacy of the Jak2 inhibitor INCB16562 in a murine model of MPLW515l-induced thrombocytosis and myelofibrosis. Blood 115: 2919-2927.

116. Talarico L, Wolf BC, Kumar A, Weintraub LR (1989) Reversal of bone marrow

fibrosis and subsequent development of polycythemia in patients with myeloproliferative disorders. Am J Hematol 30: 248-253.

117. Campbell PJ, Green AR (2006) The myeloproliferative disorders. N Engl J Med

355: 2452-2466.

118. Marchioli R, Finazzi G, Landolfi R, Kutti J, Gisslinger H, et al. (2005) Vascular and neoplastic risk in a large cohort of patients with polycythemia vera. J Clin Oncol 23: 2224-2232.

119. Harrison CN, Campbell PJ, Buck G, Wheatley K, East CL, et al. (2005)

Hydroxyurea compared with anagrelide in high-risk essential thrombocythemia. N Engl J Med 353: 33-45.

120. Cervantes F, Alvarez-Larran A, Arellano-Rodrigo E, Granell M, Domingo A, et al.

(2006) Frequency and risk factors for thrombosis in idiopathic myelofibrosis: analysis in a series of 155 patients from a single institution. Leukemia 20: 55-60.

121. De Stefano V, Za T, Rossi E, Vannucchi AM, Ruggeri M, et al. (2008) Recurrent

thrombosis in patients with polycythemia vera and essential thrombocythemia: incidence, risk factors, and effect of treatments. Haematologica 93: 372-380.

122. Kiladjian JJ, Cervantes F, Leebeek FW, Marzac C, Cassinat B, et al. (2008) The

impact of Jak2 and MPL mutations on diagnosis and prognosis of splanchnic vein thrombosis: a report on 241 cases. Blood 111: 4922-4929.

123. Wang JC, Chang TH, Goldberg A, Novetsky AD, Lichter S, et al. (2006)

Quantitative analysis of growth factor production in the mechanism of fibrosis in agnogenic myeloid metaplasia. Exp Hematol 34: 1617-1623.

152

124. Schmitt A, Jouault H, Guichard J, Wendling F, Drouin A, et al. (2000) Pathologic interaction between megakaryocytes and polymorphonuclear leukocytes in myelofibrosis. Blood 96: 1342-1347.

125. Xu M, Bruno E, Chao J, Huang S, Finazzi G, et al. (2005) Constitutive mobilization

of cd34+ cells into the peripheral blood in idiopathic myelofibrosis may be due to the action of a number of proteases. Blood 105: 4508-4515.

126. Panteli KE, Hatzimichael EC, Bouranta PK, Katsaraki A, Seferiadis K, et al. (2005)

Serum Interleukin (Il)-1, Il-2, Sil-2ra, Il-6 and thrombopoietin levels in patients with chronic myeloproliferative diseases. Br J Haematol 130: 709-715.

127. Tefferi A (2000) Myelofibrosis with myeloid metaplasia. N Engl J Med 342: 1255-

1265.

128. Mesa RA, Silverstein MN, Jacobsen SJ, Wollan PC, Tefferi A (1999) Population-based incidence and survival figures in essential thrombocythemia and agnogenic myeloid metaplasia: an olmsted county study, 1976-1995. Am J Hematol 61: 10-15.

129. Santos FP, Verstovsek S (2011) Jak2 inhibitors: What's the true therapeutic

potential? Blood Rev 25: 53-63.

130. Elmore SA (2006) Enhanced histopathology of the bone marrow. Toxicol Pathol 34: 666-686.

131. Kirabo A, Embury J, Kiss R, Polgar T, Gali M, et al. (2011) The stilbenoid tyrosine

kinase inhibitor, G6, suppresses Jak2-V617F-mediated human pathological cell growth in vitro and in vivo. J Biol Chem 286: 4280-4291.

132. Wadleigh M, Tefferi A (2010) Preclinical and clinical activity of ATP mimetic Jak2

inhibitors. Clin Adv Hematol Oncol 8: 557-563.

133. Bhat GJ, Thekkumkara TJ, Thomas WG, Conrad KM, Baker KM (1995) Activation of the Stat pathway by angiotensin II in T3cho/At1a cells. cross-talk between angiotensin II and interleukin-6 nuclear signaling. J Biol Chem 270: 19059-19065.

134. Pan J, Fukuda K, Saito M, Matsuzaki J, Kodama H, et al. (1999) Mechanical

stretch activates the jak/stat pathway in rat cardiomyocytes. Circ Res 84: 1127-1136.

135. Kleinberger-Doron N, Shelah N, Capone R, Gazit A, Levitzki A (1998) Inhibition of

cdk2 activation by selected tyrphostins causes cell cycle arrest at late G1 and S phase. Exp Cell Res 241: 340-351.

153

136. Oda Y, Renaux B, Bjorge J, Saifeddine M, Fujita DJ, et al. (1999) c-src is a major cytosolic tyrosine kinase in vascular tissue. Can J Physiol Pharmacol 77: 606-617.

137. Levitzki A (1990) Tyrphostins--potential antiproliferative agents and novel

molecular tools. Biochem Pharmacol 40: 913-918.

138. Paravicini TM, Touyz RM (2006) Redox signaling in hypertension. Cardiovasc Res 71: 247-258.

139. Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M (2000) Modulation of

protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 20: 2175-2183.

140. Mao X, Fujiwara Y, Orkin SH (1999) Improved reporter strain for monitoring Cre

recombinase-mediated DNA excisions in mice. Proc Natl Acad Sci U S A 96: 5037-5042.

141. Falkenhahn M, Gohlke P, Paul M, Stoll M, Unger T (1994) The Renin-Angiotensin

system in the heart and vascular wall: new therapeutic aspects. J Cardiovasc Pharmacol 24 Suppl 2: S6-13.

142. Tham DM, Martin-Mcnulty B, Wang YX, Da Cunha V, Wilson DW, et al. (2002)

Angiotensin II injures the arterial wall causing increased aortic stiffening in apolipoprotein e-deficient mice. Am J Physiol Regul Integr Comp Physiol 283: R1442-1449.

143. Muthalif MM, Parmentier JH, Benter IF, Karzoun N, Ahmed A, et al. (2000)

Ras/Mitogen-Activated Protein Kinase mediates norepinephrine-induced phospholipase D activation in rabbit aortic smooth muscle cells by a phosphorylation-dependent mechanism. J Pharmacol Exp Ther 293: 268-274.

144. Sawada N, Itoh H, Ueyama K, Yamashita J, Doi K, et al. (2000) Inhibition of rho-

associated kinase results in suppression of neointimal formation of balloon-injured arteries. Circulation 101: 2030-2033.

145. Begum N, Duddy N, Sandu O, Reinzie J, Ragolia L (2000) Regulation of myosin-

bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of rho kinase and phosphatidylinositol-3-kinase-dependent signaling pathways. Mol Endocrinol 14: 1365-1376.

146. Boos CJ, Dawes M (2004) ACE cardiovascular protection: europa versus hope.

Cardiovasc Drugs Ther 18: 179-180.

154

147. Huang F, Thompson JC, Wilson PG, Aung HH, Rutledge JC, et al. (2008) Angiotensin II increases vascular proteoglycan content preceding and contributing to atherosclerosis development. J Lipid Res 49: 521-530.

148. Inoue N, Muramatsu M, Jin D, Takai S, Hayashi T, et al. (2009) Involvement of

vascular angiotensin II-forming enzymes in the progression of aortic abdominal aneurysms in angiotensin II- infused apoe-deficient mice. J Atheroscler Thromb 16: 164-171.

149. Henriques TA, Huang J, D'souza SS, Daugherty A, Cassis LA (2004)

Orchidectomy, but not ovariectomy, regulates angiotensin II-induced vascular diseases in apolipoprotein e-deficient mice. Endocrinology 145: 3866-3872.

150. Cassis LA, Marshall DE, Fettinger MJ, Rosenbluth B, Lodder RA (1998)

Mechanisms contributing to angiotensin II regulation of body weight. Am J Physiol 274: E867-876.

151. Daugherty A, Manning MW, Cassis LA (2000) Angiotensin II promotes

atherosclerotic lesions and aneurysms in apolipoprotein e-deficient mice. J Clin Invest 105: 1605-1612.

152. Owens GK, Vernon SM, Madsen CS (1996) Molecular regulation of smooth muscle

cell differentiation. J Hypertens Suppl 14: S55-64.

153. Owens GK, Kumar MS, Wamhoff BR (2004) Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84: 767-801.

154. Sobue K, Hayashi K, Nishida W (1999) Expressional Regulation of smooth muscle

cell-specific genes in association with phenotypic modulation. Mol Cell Biochem 190: 105-118.

155. Kirabo A, Sayeski PP (2010) Jak2 tyrosine kinase: a potential therapeutic target for

AT1 receptor mediated cardiovascular disease. Pharmaceuticals 3: 3478-3493.

156. Shibata R, Kai H, Seki Y, Kato S, Wada Y, et al. (2003) Inhibition of Stat3 prevents neointima formation by inhibiting proliferation and promoting apoptosis of neointimal smooth muscle cells. Hum Gene Ther 14: 601-610.

157. Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, et al. (1996) Inhibition of

acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 379: 645-648.

158. Ihle JN (1996) STATs: Signal Transducers and Activators of Transcription. Cell 84: 331-334.

155

159. Schieffer B, Bernstein KE, Marrero MB (1996) The role of tyrosine phosphorylation in angiotensin II mediated intracellular signaling and cell growth. J Mol Med 74: 85-91.

160. Krempler A, Qi Y, Triplett AA, Zhu J, Rui H, et al. (2004) Generation of a

conditional knockout allele for the janus kinase 2 (Jak2) gene in mice. Genesis 40: 52-57.

161. Li L, Miano JM, Mercer B, Olson EN (1996) Expression Of the sm22alpha

promoter in transgenic mice provides evidence for distinct transcriptional regulatory programs in vascular and visceral smooth muscle cells. J Cell Biol 132: 849-859.

162. Wang X, Xu L (2005) An optimized murine model of ferric chloride-induced arterial

thrombosis for thrombosis research. Thromb Res 115: 95-100.

163. Chen G, Wu J, Sun C, Qi M, Hang C, et al. (2008) Potential role of Jak2 in cerebral vasospasm after experimental subarachnoid hemorrhage. Brain Res 1214: 136-144.

164. Sayeski PP, Ali MS, Harp JB, Marrero MB, Bernstein KE (1998) Phosphorylation of

P130cas by angiotensin II is dependent on c-src, intracellular Ca2+, and protein kinase C. Circ Res 82: 1279-1288.

165. Ray Jl, Leach R, Herbert JM, Benson M (2001) Isolation of vascular smooth

muscle cells from a single murine aorta. Methods Cell Sci 23: 185-188.

166. Denizot F, Lang R (1986) Rapid Colorimetric assay for cell growth and survival. modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89: 271-277.

167. Schwartz SM, Reidy MA, O'brien ER (1995) Assessment of factors important in

atherosclerotic occlusion and restenosis. Thromb Haemost 74: 541-551.

168. Wakabayashi K, Suzuki H, Sato T, Iso Y, Katagiri T, et al. (2006) Eplerenone suppresses neointimal formation after coronary stent implantation in swine. Int J Cardiol 107: 260-266.

169. Shibata R, Kai H, Seki Y, Kato S, Morimatsu M, et al. (2001) Role of rho-

associated kinase in neointima formation after vascular injury. Circulation 103: 284-289.

170. Davis C, Fischer J, Ley K, Sarembock IJ (2003) The Role of inflammation in

vascular injury and repair. J Thromb Haemost 1: 1699-1709.

156

171. Laporte S, Escher E (1992) Neointima formation after vascular injury is angiotensin ii mediated. Biochem Biophys Res Commun 187: 1510-1516.

172. Palmiter RD, Wilkie TM, Chen HY, Brinster RL (1984) Transmission distortion and

mosaicism in an unusual transgenic mouse pedigree. Cell 36: 869-877.

173. Wilkie TM, Brinster RL, Palmiter RD (1986) Germline and somatic mosaicism in transgenic mice. Dev Biol 118: 9-18.

174. Long MA, Rossi FM (2009) Silencing inhibits cre-mediated recombination of the

z/ap and z/eg reporters in adult cells. Plos One 4: E5435.

175. Calero-Nieto FJ, Bert AG, Cockerill PN Transcription-dependent silencing of inducible convergent transgenes in transgenic mice. Epigenetics Chromatin 3: 3.

176. Grote K, Luchtefeld M, Schieffer B (2005) Janus under stress--role of Jak/Stat

signaling pathway in vascular diseases. Vascul Pharmacol 43: 357-363.

177. Lata K, Madiraju N, Levitt L Jak2 mutations and coronary ischemia. N Engl J Med 363: 396-397.

178. Wang H, Li Y, Liu H, Liu S, Liu Q, et al. (2009) Peroxynitrite mediates glomerular

lesion of diabetic rat via Jak/Stat signaling pathway. J Endocrinol Invest 32: 844-851.

179. Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Silvennoinen O (1995) Signaling

through the hematopoietic cytokine receptors. Annu Rev Immunol 13: 369-398.

180. Valentino L, Pierre J (2006) Jak/Stat signal transduction: regulators and implication in hematological malignancies. Biochem Pharmacol 71: 713-721.

181. Nilsson J, Bjursell G, Kannius-Janson M (2006) Nuclear Jak2 and transcription

factor nf1-c2: a novel mechanism of prolactin signaling in mammary epithelial cells. Mol Cell Biol 26: 5663-5674.

182. Lobie Pe, Ronsin B, Silvennoinen O, Haldosen LA, Norstedt G, et al. (1996)

Constitutive nuclear localization of janus kinases 1 and 2. Endocrinology 137: 4037-4045.

183. Dawson MA, Bannister AJ, Gottgens B, Foster SD, Bartke T, et al. (2009) Jak2

phosphorylates histone h3y41 and excludes hp1alpha from chromatin. Nature 461: 819-822.

184. Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, et al. (1997) A Tel-Jak2

fusion protein with constitutive kinase activity in human leukemia. Science 278: 1309-1312.

157

185. Dagvadorj A, Collins S, Jomain JB, Abdulghani J, Karras J, et al. (2007) Autocrine

prolactin promotes prostate cancer cell growth via Janus Kinase-2-Signal Transducer And Activator of Transcription-5a/B signaling pathway. Endocrinology 148: 3089-3101.

186. Reiter A, Walz C, Watmore A, Schoch C, Blau I, et al. (2005) The T(8;9)(P22;P24)

is a recurrent abnormality in chronic and acute leukemia that fuses pcm1 to Jak2. Cancer Res 65: 2662-2667.

187. Bedard K, Krause KH (2007) The Nox family of ROS-generating NADPH

Oxidases: physiology and pathophysiology. Physiol Rev 87: 245-313.

188. Pryor WA (1986) Oxy-radicals and related species: their formation, lifetimes, and reactions. Annu Rev Physiol 48: 657-667.

189. Hawkins BJ, Madesh M, Kirkpatrick CJ, Fisher AB (2007) Superoxide flux in

endothelial cells via the chloride channel-3 mediates intracellular signaling. Mol Biol Cell 18: 2002-2012.

190. Rubanyi GM (1988) Vascular effects of oxygen-derived free radicals. Free Radic

Biol Med 4: 107-120.

191. Moncada S, Palmer RM, Higgs EA (1991) Nitric Oxide: Physiology, Pathophysiology, and Pharmacology. Pharmacol Rev 43: 109-142.

192. Moncada S, Higgs A (1993) The L-Arginine-Nitric Oxide pathway. N Engl J Med

329: 2002-2012.

193. Ignarro Lj (1989) Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res 65: 1-21.

194. Radi R, Beckman JS, Bush KM, Freeman BA (1991) Peroxynitrite oxidation of

sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J Biol Chem 266: 4244-4250.

195. Fukata Y, Amano M, Kaibuchi K (2001) Rho-Rho-Kinase pathway in smooth

muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol Sci 22: 32-39.

196. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, et al. (1997) Calcium

sensitization of smooth muscle mediated by a rho-associated protein kinase in hypertension. Nature 389: 990-994.

158

197. Shimokawa H, Seto M, Katsumata N, Amano M, Kozai T, et al. (1999) Rho-Kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm. Cardiovasc Res 43: 1029-1039.

198. Hirata K, Kikuchi A, Sasaki T, Kuroda S, Kaibuchi K, et al. (1992) Involvement of

rho p21 in the gtp-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem 267: 8719-8722.

199. Gong MC, Iizuka K, Nixon G, Browne JP, Hall A, et al. (1996) Role of Guanine

Nucleotide-Binding Proteins--Ras-family or trimeric proteins or both--In Ca2+ Sensitization of smooth muscle. Proc Natl Acad Sci U S A 93: 1340-1345.

200. Akaishi T, Nakazawa K, Sato K, Saito H, Ohno Y, et al. (2004) Hydrogen peroxide

modulates whole cell Ca2+ currents through L-type channels in cultured rat dentate granule cells. Neurosci Lett 356: 25-28.

201. Wright JM, Musini VM (2009) First-line drugs for hypertension. Cochrane Database

Syst Rev: Cd001841.

202. Staessen JA, Wang J, Bianchi G, Birkenhager WH (2003) Essential hypertension. Lancet 361: 1629-1641.

203. Acelajado MC, Calhoun DA, Oparil S (2009) Reduction of blood pressure in

patients with treatment-resistant hypertension. Expert Opin Pharmacother 10: 2959-2971.

204. Nesbitt SD Overcoming therapeutic inertia in patients with hypertension. Postgrad

Med 122: 118-124.

205. Anderson JE, Tefferi A, Craig F, Holmberg L, Chauncey T, et al. (2001) Myeloablation and autologous peripheral blood stem cell rescue results in hematologic and clinical responses in patients with myeloid metaplasia with myelofibrosis. Blood 98: 586-593.

159

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BIOGRAPHICAL SKETCH

Annet Kirabo grew up in a small village of Kigulu county in Iganga district, Uganda.

She attended various elementary schools including Kaliro demonstration school, Iganga

town council and Bishop Willis Demonstration School. Annet attained her ordinary level

high school diploma at Iganga Secondary School and her advanced level high school

diploma at Bukoyo Secondary School where she majored in physics, chemistry and

biology, with a minor in fine art. She graduated high school as a valedictorian of her

class in 1997 and she was selected as one of the top 2,000 high school graduates to

receive the Uganda government sponsored college education. Annet first developed

interest in medical-related research when she was a student in the School of Veterinary

Medicine, Makerere University, Uganda. Under the supervision of Professor Ojock

Lonzy, she carried out a gross and histo-pathological survey on the prevalence of

Johne’s disease in Uganda cattle. Annet graduated with a Bachelor of Veterinary

Medicine in 2002 as the top student in her class and on the deans list. She then moved

to the United States of America where she attained a Master of Science in Cell and

Molecular Biology at St. Cloud State University, Minnesota. Her research as a master’s

student focused on the mechanistic interaction between obesity and reproduction under

the supervision of Dr. Oladele Gazal. Annet performed her Ph.D. studies in the

laboratory of Dr. Peter Sayeski, Department of Physiology and Functional Genomics,

University of Florida College of Medicine, Gainesville, Florida. Her research mainly

focused on understanding involvement of Jak2 tyrosine kinase in human cardiovascular

disease and hematological malignancies and how it could be a potential therapeutic

target for these diseases. Annet graduated with a Ph.D. in August 2011.

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