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Leukemia Research 36 (2012) 808– 810

Contents lists available at SciVerse ScienceDirect

Leukemia Research

jou rna l h omepa g e: www.elsev ier .com/ locate / leukres

ditorial

role for caspase inhibitors in differentiation therapy of myeloid leukaemia

eywords:ifferentiation therapycute myeloid leukaemiaitamin D3aspase inhibitors

The paper by Chen-Deutch et al. 2012 [1] describes an interest-ng way forward to the possible use of 1�,25-dihydroxyvitamin D3D3) in differentiation therapy of acute myeloid leukaemia (AML)y the combined use of D3 and the pan-caspase inhibitor Q-VD-OPhQVD).

AML is a heterogeneous disease which accounts for 80% of adulteukaemias. Immature blast cells accumulate due to the diseaserising in a stem cell or primitive progenitor and the progeny failingo differentiate adequately. Differentiation therapy aims to restoreerminal maturation resulting in the leukaemia cells having a lim-ted lifespan, and dying via apoptosis. A patient benefit of this typef therapy is the prospect of a less-aggressive treatment by target-ng the cellular defect in the leukaemia cells and therefore sparingormal cells as much as is possible. Milder treatments for AML are

mportant for a number of reasons. Thirty-five percent of patientsre aged ≥75 years and the median age at diagnosis is 72 [2];hese patients cannot tolerate aggressive chemotherapy becausef co-existing age-related conditions such as declining bone mar-ow function and further ablation of normal haematopoiesis; whilstounger patients have benefited from more aggressive treatmentshe outcome for elderly AML patients has not really changed dur-ng the last 20 years and only around 5% survive long-term whenreated by conventional means [3–5]; and stem cell transplantations not an option for many elderly patients [6]. Importantly, devising

ilder treatments for very elderly patients with acute leukaemia,nd other cancers, poses a real challenge to the heath care service.y 2034, 23% of the UK population is projected to be aged ≥65 years,% will be ≥85 years old, and around 40% of older persons will beiagnosed with some form of cancer.

Differentiation therapy can be highly effective as exemplified byhe longstanding and successful use of all-trans retinoic acid (ATRA)o drive acute promyelocytic leukaemia (APL) cells to differenti-te towards granulocytes: cure rates when ATRA is combined with

nthracylin-based chemotherapy are up to 80% [7]. ATRA was firstsed in APL in 1987 but despite many efforts differentiation ther-py has not extended to other sub-types of AML. Is there a future for

DOI of original article: http://dx.doi.org/10.1016/j.leukres.2012.03.023.

145-2126/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.leukres.2012.03.030

differentiation therapy of AML? Findings from the Studzinski lab-oratory (UMDNJ-New Jersey Medical School, Newark, New Jersey,USA) have championed the use of D3. It has long been known that D3drives growth arrest and monocytic differentiation of cultured AMLcells. The problems with the therapeutic use of D3 are the unac-ceptable frequency of occurrence of hypercalcaemia, many AMLpatients’ cells respond poorly when treated in culture with D3, andthe need to find D3 analogues with more potent anti-cancer activ-ity. There are analogues with a lower calcaemic capacity; these areused in the Chen-Deutch study which also describes a novel meansof enhancing the differentiating effect of D3.

The Studzinski laboratory has been one of the leaders indescribing the molecular mechanisms of D3-driven monocytic dif-ferentiation of myeloid leukaemia cells. In recent years, they haveidentified cellular events that render some myeloid leukaemia cellsresistant to D3-mediated differentiation and developed ways ofsensitising cells to the effect of D3 by co-administrating a varietyof ‘potentiating’ agents, including cyclooxygenase inhibitors, theantioxidants carnosic acid and silibinin, and the kinase inhibitorSB202190 [reviewed in 8]. In some D3-refractory AML cell linesinhibition of the Cot1 oncogene, or of one of its downstream tar-gets (ERK5), restores hormone sensitivity [9]. Two recent papersfrom Studzinski’s group have focused attention on the role ofhaematopoietic progenitor kinase 1 (HPK1) in regulating D3-mediated differentiation in cell lines that typify AML and patients’cells. HPK1 is a mammalian yeast ste20-related serine/threoninekinase that belongs to the germinal centre kinase superfamily andis expressed in haematopoietic progenitor cells and mature leuko-cytes [10]. The N-terminal kinase domain (HPK1-N) is coupled to aC-terminal Citron homology domain (HPK1-C), via four proline-richmotifs, and HPK1 is cleaved into these two fragments by caspase-3[11].

HPK1 is known to play an important role in prolifera-tion/survival/death decisions and this kinase can be activatedby a number of stimuli, including epidermal growth factor,prostaglandin E2 (PGE2), transforming growth factor-� (TGF�), ery-thropoietin and stimulation of the antigen receptors of T and Blymphocytes. The downstream targets of HPK1 are members of theMAPK signalling pathway, especially the JNK signalling cassette andthe NF-�B pathway [11]. In lymphoid cells, HPK1 acts as a caspase-activated life/death switch. In non-stimulated T cells full lengthHPK1 (fl-HPK1) binds to the NF-�B regulatory proteins IKK� andIKK-� and phosphorylates IKK�, via its HPK1-N domain, to activate

signalling via NF-�B which confers resistance to activation-inducedcell death and negatively regulates T-cell signalling [12]. The func-tion of HPK1 appears to change once the T-cell has been stimulatedby antigen and is undergoing proliferation. In these cells HPK1 is

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Editorial / Leukemia R

leaved, by caspase-3, into the two fragments HPK1-N and HPK1-C.he non-catalytic HPK1-C domain still binds to IKK� and IKK-� butn the absence of the catalytic HPK1-N subunit cannot phosphory-ate the IKK complex. This leads to decreased NF-�B signalling andn increased sensitivity to activation-induced cell death [13,14].

Recently, HPK1 has been shown to play a role in regulating IL--mediated monocytic differentiation of normal murine myeloidrogenitor cells [15]. Arnold and co-workers have shown that IL-

activates caspase-3 which in turn cleaves HPK1. IL-3-stimulatedonocytic differentiation of mouse primary haematopoietic pro-

enitor cells requires a persistent increase in JNK activity, andPK1-N can act as a MAP4K to constitutively activate the JNK sig-alling pathway and generate a signal(s) that renders the cells lessensitive to apoptotic stimuli and essential for monocytic differen-iation [15]. Interestingly, HPK1 activators such as PGE2 and TGF-�an increase the sensitivity of the promyeloid cell line HL60 andther myeloid leukaemia cell lines to D3-stimulated monocytic dif-erentiation.

As above HPK1 plays a crucial role in JNK activation duringonocytic differentiation and of interest was whether HPK1 and its

leaved products have a similar function in D3-provoked monocyticifferentiation of human myeloid leukaemia cells. Earlier this yearhe Studzinski laboratory addressed this issue and demonstratedor human myeloid leukaemia cell lines that fl-HPK1 appears toave a dual role as a positive regulator of D3-stimulated growthrrest and monocytic differentiation and a mediator of resistanceo D3-stimulated differentiation [16]. Studzinski and co-workerstudied the expression of HPK1 in HL60-40AF cells, a sub-linef HL60 cells that fails to undergo growth arrest and monocyticifferentiation when treated with D3. mRNA and protein levelsf fl-HPK1 and HPK1-C were found to be massively increasedn HL60-40AF cells when compared with control HL60 cells, andtudzinski and co-workers concluded that the very high level ofPK1-C contributes to restraint of D3-stimulated differentiation.hese investigators also suggested that HPK1 can play a permissiveole in D3-stimulated monocytic differentiation. In D3-sensitiveyeloid leukaemia cell lines, the levels of fl-HPK1 and HPK1-C

re fairly low and D3-stimulation was associated with a moder-te increase in the expression of fl-HPK1 and a decrease in thexpression of HPK1-C. The level of HPK1 was further increasedy treatment of the cells with a differentiation sensitising cocktail.he rise in fl-HPK1 and a fall in HPK1-C expression correlated wellith the expression of differentiation markers and the activation

f JNK and its downstream targets, suggesting a role for fl-HPK1 inhe differentiation process that is mediated by enhanced JNK sig-alling. This was confirmed by the observation that knockdown of-HPK1 in D3-sensitive cell lines decreased their responsivenesso D3 and reduced D3-driven activation of the JNK signalling path-ay. Thus, by contrast to the pro-differentiation effect of HPK1-Nithin normal murine haematopoietic cells, it appears that full

ength HPK1 is required for maximal JNK signalling and optimal D3-nduced monocytic differentiation of myeloid leukaemia cell linesnd that HPK1-C inhibits differentiation. In keeping, D3-inducedifferentiation of the myeloid cell lines was augmented by co-reatment with a pan-caspase inhibitor to prevent cleavage ofPK1.

Of relevance to these observations is a recent study whichhowed that normal pancreatic cells express a high level of fl-HPK1nd expression of fl-HPK1 protein is lost in malignant pancreaticells. In human pancreatic cancer cell lines treatment with the pro-easomal inhibitor MG132 was sufficient to induce expression of-HPK1 which stabilised the expression of p21 and p27 leading

o cell cycle arrest [17]. Interestingly, higher than normal plasmaevels of proteasome and ubiquitin proteins and enzyme activi-ies have been reported in AML and correlated with poor survivalrospects [18]. So perhaps in malignant cells proteolytic cleavage

h 36 (2012) 808– 810 809

of HPK1 plays an important role in their escape from the normalcontrols on cell differentiation and proliferation.

It has been known for some time that AML patients’ cells showconsiderable variability in their responsiveness in culture to D3induction of cell differentiation. As seen for one of the patientsin the Chen-Deutsch study [1], and described elsewhere [19,20],some patients’ cells are quite sensitive to D3 and others completelyrefractory. The paper by Chen-Deutsch et al., examines whetherexcessive caspase-mediated cleavage of HPK1 contributes to thepoor responsiveness of some AML patients’ cells to D3 [1]. Rela-tively high levels of caspase-2 and caspase-3 have been reportedin AML blasts. In these cells the caspases are not provoking apo-ptosis because none of the expected pro-apoptotic products werefound in the viable cells and the rate of apoptosis was fairly low.There is growing evidence in the literature to support the notionthat caspases have functions that are unrelated to their role in theapoptotic process. This seems to be the case for AML blast cells sincethe Chen-Deutch paper has shown that pan-caspase inhibition (byQVD) has a profound enhancing effect on the sensitivity of AMLpatients’ cells in culture to D3 [1]. The samples investigated wererepresentative of various sub-types of AML. For example, imma-ture non-differentiated FAB-M1 AML cells that carry a mutationin C/EBP� (like 15% of all M1-AML cases) were poorly respon-sive to D3, and treatment of these cells with a caspase inhibitorresulted in complete responsiveness to D3. This was associatedwith an increased expression of fl-HPK1, increased D3-inducedJNK1 signalling, and an increased expression of C/EBP�, a transcrip-tion factor that is important to monocytic differentiation. Togetherthe studies using cell lines that typify AML cells and patients’cells have shown that caspase activity plays a role in resistanceto D3-mediated differentiation. Hence, the possible use of caspaseinhibitors to improve the efficacy of D3-based differentiation ther-apy is worthy of further investigation.

Beyond the success of ATRA in treating APL there has been some-what of a lull of interest in both D3- and ATRA-based differentiationtherapies for leukaemias and for other hard-to-treat malignancies.However, as seen in the Chen-Deutch study [1], a better under-standing of the molecular mechanisms by which differentiatingoccurs leading to agents that can enhance pro-differentiation cel-lular events, or remove ‘a molecular brake’, may provide newcombination differentiation therapies. The way forward for differ-entiation therapy of non-APL AML looks much healthier, includingregimens personalised in accordance with molecular defects seenin patients’ cells. In this regard, it is interesting to see that thereis also the prospect of a new ATRA-based therapy. Zelant andco-workers have provided insight to why ATRA fails to work innon-APL where upon target genes that are important to differ-entiation are not properly activated due to the H3K4me1/me2lysine-specific demethylase 1 (LSD1/KDMI) repressing target genepromoters. Accordingly, inhibitors of LSD1 restore responsivenessof non-APL AML cells to ATRA [21]. Without a doubt these, andother observations, open a quest for further preclinical studiesto arrive at efficacious differentiation therapies for non-APL AML,particularly as to the need to treat very elderly patients moreeffectively.

Funding source

None.

Conflict of interest

The authors declare that there are no conflicts of interest.

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cknowledgements

None.Contributions. G.B. and P.J.H. researched, wrote and approved

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eferences

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[2] Juliusson G, Antunovic P, Derolf A, Lehmann S, Mollgard L, Stockelberg D, et al.Age and acute myeloid leukemia: real world data on decision to treat and out-comes from the Swedish Acute Leukemia Registry. Blood 2009;113:4179–87.

[3] Kohrt HE, Coutre SE. Optimising therapy for acute myeloid leukemia. J NatCompr Cancer Netw 2008;6:1003–16.

[4] Dombret H, Raffoux E, Gardin C. Acute myeloid leukemia in the elderly. SeminOncol 2008;35:430–8.

[5] Farag SS, Archer KJ, Mrozek K, Ruppert AS, Carrol AJ, Vardiman JW, et al. Pre-treatment cytogenetics add to other prognostic factors predicting completeremission and long-term outcome in patients 60 years of age or older withacute myeloid leukemia: results from Cancer and Leukemia Group B 8461.Blood 2006;108:63–73.

[6] Deeg HJ, Sandmaier BM. Who is fit for allogeneneic transplantation? Blood2010;116:4762–70.

[7] Sanz M, Grimwade D, Tallman M, Lowenberg B, Fenaux P, Estay E, et al.Guidelines on the management of acute promyelocytic leukemia: recommen-dations from an expert panel on behalf of the European LeukemiaNet. Blood2009;113:1875–91.

[8] Hughes PJ, Marcinkowska E, Gocek E, Studzinski GP, Brown G. Vitamin D3-driven signals for myeloid cell differentiation – implications for differentiationtherapy. Leukemia Res 2010;34:553–65.

[9] Wang X, Gocek E, Novik V, Harrison JS, Danilenko M, Studzinski GP. Inhi-bition of Cot1/Tip2 oncogene in AML cells reduces ERK5 activation andup-regulates p27Kip1 concomitant with enhancement of differentiation andcell cycle arrest induced by silibinin and 1,25-dihydroxyvitamin D(3). Cell Cycle2010;9:4542–51.

10] Kiefer F, Tibbles LA, Anafi M, Janssen A, Zanke BW, Lassam N, et al. HPK1,a hematopoietic protein kinase activating the SAPK/JNK pathway. EMBO J1996;15:7013–25.

11] Boomer JS, Tan TH. Functional interactions of HPK1 with adaptor proteins. J CellBiochem 2005;95:34–44.

12] Shui JW, Boomer JS, Han J, Xu J, Dement GA, Zhou G, et al. Hematopoietic progen-itor kinase 1 negatively regulates T cell receptor signaling and T cell-mediatedimmune responses. Nat Immunol 2007;8:84–91.

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14] Arnold R, Liou J, Drexler HCA, Weiss A, Kiefer F. Caspase-mediated cleavage ofhematopoietic progenitor kinase 1 (HPK1) converts an activator of NF kappa Binto an inhibitor of NF kappa B. J Biol Chem 2001;276:14675–84.

15] Arnold R, Frey CR, Müller W, Brenner D, Krammer PH, Kiefer F, et al. SustainedJNK signaling by proteolytically processed HPK1 mediates IL-3 independentsurvival during monocytic differentiation. Cell Death Differ 2007;14:568–75.

16] Chen-Deutsch X, Studzinski GP. Dual role of hematopoietic progenitor kinase1 (HPK1) mas a positive regulator of 1�,25-dihydroxyvitamin D-induced dif-ferentiation and cell cycle arrest of AML cells and as a mediator of vitamin Dresistance. Cell Cycle 2012;11:1364–73.

17] Wang H, Song X, Logsdon C, Zhou G, Evans DB, Abbruzzese JL, et al. Proteasomal-mediated degradation and functions of hematopoietic progenitor kinase 1 inpancreatic cancer. Cancer Res 2009;69:1063–70.

18] Ma W, Kantarijian H, Zhang X, Wang X, Estrov Z, O’Brien S, et al.Ubiquitin–proteasome system profiling in acute leukemias and its clinical rel-evance. Leukemia Res 2011;35:526–33.

19] Gocek E, Kiebinski M, Baurska H, Haus O, Kutner A, Marcinkowska E. Differentsusceptibilities to 1,25-dihydroxyvitamin D3-induced differentiation of AMLcells carrying various mutations. Leukemia Res 2010;34:649–57.

20] Zhang J, Harrison JS, Uskokovic M, Danilenko M, Studzinski GP. Silibinin caninduce differentiation as well as enhance vitamin D3-induced differentiationof human AML cells ex vivo and regulates the levels of differentiation-relatedtranscription factors. Hematol Oncol 2010;28:124–32.

21] Schenk T, Chen WC, Gollner S, Howell L, Jin L, Hebestreit K, et al. Inhi-bition of the LSD1 (KDM1) demethylase reactivates the all-trans-retinoicacid differentiation pathway in acute myeloid leukemia. Nat Med 2012,http://dx.doi.org/10.1038/nm.2661.

Geoffrey Brown ∗

Philip J. HughesSchool of Immunity and Infection, College of Medicaland Dental Sciences, The University of Birmingham,

Birmingham, UK

∗ Corresponding author at: School of Immunity andInfection, College of Medical and Dental Sciences,

The University of Birmingham, Vincent Drive,Edgbaston, Birmingham, West Midlands B15 2TT,

UK. Tel.: +44 0 121 414 4082;fax: +44 0 121 414 3599.

E-mail address: g.brown@bham.ac.uk (G. Brown)

30 March 2012Available online 23 April 2012