Studiando il Pathway di mTorStudiando il Pathway di mTor

Funzionalità, Meccanismi di Escape,, p ,Potenziali Targets di Sinergismo Inibitorio

Federica Recine, MDDepartment of Medical Oncology,

San Camillo and Forlanini Hospitals, Rome, Italy

mTOR protein kinase(Mammalian Target of Rapamycin

Macmillan Publishers Ltd, Nature Reviews Clinical Oncology 2010; 7(4)209‐219.

mTOR: The Central Regulator of Growth and Metabolism

• mTOR is a central regulator

Nutrients Growth Factors

Normal Cell • mTOR is a central regulator that senses changes in:

– Availability of growthGrowth Factors Availability of growth factors

– Availability of nutrientsmTOR

Availability of nutrients

– Availability of fuel/energy

Protein Synthesis• mTOR regulation can affect:

Protein Synthesis

Cell Growth

– Angiogenesis

– Cell growthCell Growth             

& Proliferation Bioenergetics

Angiogenesis  

– Nutrient uptake

– MetabolismWullschleger et al. Cell. 2006;124(3):471‐484.Humar et al. FASEB J. 2002;16(8):771‐780.Edinger and Thompson.Mol Biol Cell. 2002;13(7):2276‐2288.

mTOR signaling pathwaysg g p y

• mTOR activity is linked to PI3K/AKT signaling

• PTEN regulates negatively PI3K• PI3K AKT mTOR is a downstream• PI3K‐AKT‐mTOR is a downstream 

component of several growth factor signaling pathways

l ( )• Upstream signals (ex IGF‐1)  activate PI3K/AKT pathway 

• Activated mTOR phosphorylates p p ydownstream effector molecules S6K1 and 4EBP1 

Translation

S. Faivre  et al. Nature 2006; 16 (9): 674

Cyclin D1, c‐Myc, HIF1α, and others

mTOR pathwaysmTOR pathwaysAKT‐dipendent mechanisms

• AKT is a substrate of mTORC2 (but (indirectly activates mTORC1 by phosphorylating and inhibiting TSC2)

• The TSC complex (TSC1 /TSC2) are key components in the upstream regulation of mTORof mTOR

• AKT phosphorylation of TSC2 leads to TSC inactivation activation of the small GTPase Rheb, which switchesmTORC1 signaling

Memmott M. et al., Cell Segnal, 2009

mTOR signaling in cancermTOR signaling in cancer

• Aberrant activation of the PI3K/AKT/mTOR pathway leads to cell‐cycle progression, tumor 

i i d lif tiangiogenesis, and proliferation

Deregulation of mTOR can result in• Deregulation of mTOR can result in loss of growth control and metabolism

• Mutations in the mTOR pathway have been linked to specific cancersp

J Zha et al. Clinical Cancer Research 2010; 16 (9): 2512‐2517Huang J et al. Science 2007; 318 (5857): 1744‐1748Engelman JA et al. Nat Rev Cancer 2009;9 (8):550‐562

mTOR signaling in cancermTOR signaling in cancer

• Upstream aberrant activation:– overexpression of RTKs (HER1‐4, p ( ,

PDGFR/KIT, IGFR) and Ras– activating mutations of PIK3CA 

p110 subunit of PI3K– PTEN inactivation or loss – mutation of NF1– Amplifications of AKTp– inactivation or mutations of 

TSC1/2

• Downstream deregulation:– S6K1 and IF4E hyperactivation– p53 loss– p53 loss 

J Zha et al. Clinical Cancer Research 2010; 16 (9): 2512‐2517Huang J et al. Science 2007; 318 (5857): 1744‐1748Engelman JA et al. Nat Rev Cancer 2009;9 (8):550‐562

mTOR inhibitor classesmTOR inhibitor classes

Direct active site inhibitorsRapalogsRapalogs

• Everolimus • Temsirolimus

• BEZ235• Perifosina

Janes et al, Nat Med 2010• Temsirolimus • Perifosina

• BKM120

Why mTor inhibitors in NET?Comprehensive analyses have definitely shown activation of mTor pathway in 

Why mTor inhibitors in NET?p y y p y

both experimental, sporadic and Hereditary NETs

LL ff LL ff LessonsLessons fromfrom clinicalclinicalLessonsLessons fromfromHereditaryHereditarySyndromesSyndromes

LessonsLessons fromfrompreclinicalpreclinical studiesstudies

LessonsLessons fromfrom clinicalclinicalstudystudyRADIANTRADIANT SAGASAGAyy

‐‐MENMEN11 genegeneVHLVHL

‐‐PIPI33KK‐‐AKTAKTPTENPTEN

TranslationalTranslationalmedicinemedicine‐‐VHLVHL genegene

‐‐TSCTSC complexcomplex‐‐NFNF‐‐11 genegene

‐‐PTENPTEN‐‐TSCTSC22‐‐miRmiR2121

medicinemedicine

gg‐‐DAXX/ATRXDAXX/ATRX ‐‐mTormTor (S(S66KK11‐‐44EBPEBP11))

P li i l T i l’Preclinical Trial’s messages

•High expression rates of pmTOR have been demonstrated in poorlydifferentiated NETs, suggesting a potential role of mTOR inhibitors in NETdifferentiated NETs, suggesting a potential role of mTOR inhibitors in NETtreatment

•Everolimus blocks IGF production and consequence PI3K activation

•Everolimus treatment led to NET cell growth inhibition and high expressionf h l l ( O NA d h h l d O l l )of the molecular targets (mTOR mRNA and phosphorylated mTOR levels)

predicted response to mTOR inhibition

Zatelli et al., Endocr Related Cancer, 2010Zitzmann et al., Neuroendocrinology and Cancer Lett, 2007, 2010Svejda et al., Cancer, 2011Meric-Bernstam et al., Clin Cancer Res, 2012

Clinical Trial’s messages

Dose‐ and time‐dependent rise in lactate dehydrogenase with everolimus treatment has

Yao et al., JCO, 2008

Dose‐ and time‐dependent rise in lactate dehydrogenase with everolimus treatment hasbeen associated with better PFS. This is postulated to be related to tumor hypoxia frommTOR inhibition

Yao et al., JCO, 2010Yao et al., NEJM, 2011Duran et al., Br J Cancer, 2006Yao et al., JCO, 2010

h h b ?Why mTor inhibitor in Breast Cancer?• Preclinical studies have demonstrated activation of the PI3K/mTOR pathway 

after long term estrogen deprivation

• Growing evidence supports a close interaction between the mTOR pathway d ER i liand ER signaling

• Everolimus synergizes with letrozole in preclinical models• Everolimus synergizes with letrozole in preclinical models

• mTOR activation is a mechanism of escape to long term• mTOR activation  is  a mechanism  of  escape  to  long  term estrogen  deprivation 

Do Rapalogs reverse resistance to endocrine therapy?

Martin LA et al. J Biol Chem 2003

Mill TW l J Cli I 2010

Do  Rapalogs reverse  resistance  to  endocrine  therapy? 

Miller TW et al. J Clin Invest 2010

deGraffenried  LA Clin  Cancer  Res  2004) Boulay et al.  Clin Canc Res 20004

BOLERO‐2: Study Design

• Primary endpoint: PFS (investigator assessment)

S d d i t OS ORR li i l b fit t f t

Treatment until disease progression Randomized 2:1; stratified by sensitivity to 

i h l th f

• Secondary endpoints: OS, ORR, clinical benefit rate, safety

p gor unacceptable toxicity

previous hormonal therapy, presence of visceral metastases

Postmenopausal women with ER‐positive advanced breast cancer who progressed on

Exemestane 25 mg/day +Everolimus 10 mg/day

(n = 485)cancer who progressed on previous nonsteroidal AI 

therapy*Exemestane 25 mg/day +

(N = 724)g/ y

Placebo(n = 239)

Baselga J et al. N Engl J Med 2012;366:520‐9.

*> 50% of patients in each arm with ≥ 3 previous therapies

BOLERO‐2: PFSBOLERO‐2: PFS  

Local CentralHR: 0.44 (95% CI: 0.36-0.53;Log rank P value: < 1 x 10-16)100

HR: 0.36 (95% CI: 0.28-0.45;Log rank P value: < 1 x 10-16)100

EVE + EXE (E/N = 267/485)PBO + EXE (E/N = 190/239)

EVE + EXE: 7.4 mosPBO + EXE: 3.2 mos

nts

(%)

100

80

60EVE + EXE (E/N = 155/485)PBO + EXE (E/N = 127/239)

EVE + EXE: 11.0 mosPBO + EXE: 4.1 mos

100

80

60

Patie

n

40

20

0

40

20

0

Patients at Risk, n

00 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96

Wks

485 436 365 303 246 188 136 96 64 45 34 21 13 9 2 2Everolimus 0

00 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96

Wks

485 422 351 284 224 176 119 86 57 38 32 22 12 7 2 2Everolimus 0

• Significant PFS benefit with everolimus observed in all patient subgroups 

239 190 131 95 63 45 29 19 12 8 6 6 4 2 0 0Placebo 0 239 179 112 74 56 36 23 18 8 5 4 4 3 1 0 0Placebo 0

g p g pincluding: age, hormone sensitivity, visceral metastasis, ECOG PS, previous chemotherapy, number of previous therapies, and PR status

Baselga J et al. N Engl J Med 2012;366:520‐9.

BOLERO-2 (12-Mo Follow-up): SafetyBOLERO-2 (12-Mo Follow-up): Safety

Adverse Events, % Exemestane + Everolimus Exemestane + Placebo (n = 482) (n = 238)

All Grades Grade 3/4 All Grades Grade 3/4

Stomatitis 59 8 11 1

Rash 39 1 6 0

F ti 36 5 27 1Fatigue 36 5 27 1

Diarrhea 33 3 19 < 1

Appetite decreased 30 1 12 < 1Appetite decreased 30 1 12 < 1

Nausea 29 2 28 1

Noninfectious 15 3 0 0Noninfectious pneumonitis

15 3 0 0

Hyperglycemia 14 6 2 < 1

Baselga J et al. N Engl J Med 2012;366:520‐9.

Why mTor inhibitor in Kidney Cancer?

• PI3K/Akt/mTOR pathway activation is common in RCC*

• Inhibition of mTOR with rapalogs has clinical value in advanced/metastatic RCC:advanced/metastatic RCC:– Temsirolimus increases overall survival of pts with multiple poor 

prognostic factors (compared with IFN)p g ( p )– Everolimus increases PFS of pts who progress on VEGFR TKIs (compared 

with placebo)

• Consistent toxicity profile of rapamycin analogs 

*Lin F et al Ann Clin Lab Sci 2006Lin F, et al, Ann Clin Lab Sci 2006 Pantuck AJ et al, Cancer 2007Robb V et al, J Urol 2006

Late‐Breaking Abstract # 5026g

RECORD 1 RAD001 (Everolimus) + BestRECORD‐1:  RAD001 (Everolimus) + Best Supportive Care (BSC) vs BSC + Placebo in Pts With Metastatic RCC After Progression on 

VEGFr TKI TherapyVEGFr‐TKI Therapy

R. Motzer, B. Escudier, S. Oudard, C. Porta,T. Hutson, S. Bracarda, R. Figlin, J. Thompson, 

V. Grünwald, N. Hollaender, G. Urbanowitz, A. Kay, A. Ravaud, for the RECORD‐, , , y, ,1 Study Group

Study Design

RR

TargetN = 362TargetN = 362 Everolimus + BSC

AANNDDN 362

Stratification

N 362

Stratification

Everolimus BSC

U

DDOOMM

• Prior VEGFrTKI: 1 or 2

• Prior VEGFrTKI: 1 or 2

Upon Disease

Progression

IIZZAA• MSKCC risk

group1: favorable, intermediate

• MSKCC risk group1: favorable, intermediate

Placebo + BSC

AATTII

Final analysis

intermediate, or poorintermediate, or poor

Interim analysis

Interim analysis

OONN

analysisanalysis analysis2:12:1

• Interim analyses planned after ≈ 30% and 60% of targeted 290 events1 Motzer et al. J Clin Oncol. 2004;22:454-463.

Progression-Free Survival by Treatment C t l R di l R iCentral Radiology Review

100

80

Hazard ratio = 0.3095% CI [0.22, 0.40]

80

60%

Median PFSEverolimus: 4.0 mo60

40babi

lity,

% Placebo: 1.9 mo40

20

Prob P value < 0.001

Everolimus (n = 272)Placebo (n = 138)

0

( )

0 2 4 6 8 10 12Months

Patients at RiskEverolimus 272 132 47 8 2 0 0Placebo 138 32 4 1 0 0 0

Rapalogs and resistance mechanisms 

• Incomplete and substrate‐selective mTORC‐1 inhibitionselective mTORC 1 inhibition

• Feedback loop circuits activated by mTORC1 inhibition:by mTORC1 inhibition:

– feedback activation of the PI3K pathwaypathway

– mTORC‐2 mediated AKT iperactivationiperactivation

– feedback activation of MAPK

( lk)(crosstalk)

O’Reilly K E et al. Cancer Res. 2006 February 1; 66(3): 1500 1508.Seth A W et al. J Clin Invest 2011; 121(4):1231‐124q1Yatscoff RW et al. Ther Drug Monit 1993 ; 15: 478‐482

PI3K/mTOR pathway inhibitorsPI3K/mTOR pathway inhibitors

• Dual PI3K/mTOR inhibitors• ‘Pure’ PI3K inhibitors XL147• AKT inhibitors• Dual mTORC1/2 inhibitors

GDC0941BKM120

RKT

CAL101BYL719PI3K

XL765PerifosineMK2206XL765

BEZ235GDC0980

MK2206GDC‐0068AKT

GDC0980OSI‐027AZD8055INK 128mTOR INK‐128mTOR

Clinical Benefits for Perifosine/Capecitabine Combination Not Realized in Phase III Study forCombination Not Realized in Phase III Study for 

Metastatic Colorectal Cancer

• X‐PECT was a phase III study that randomly assigned 468 patients with p y y g prefractory colorectal cancer (CRC) to receive perifosine/capecitabine (P‐CAP; 234 patients) or capecitabine (CAP; 234 patients)

• The median OS was 6.4 months and 6.9 months for patients receiving P‐CAP and CAP, respectively (p = 0.315). The difference in OS also was not significant when analyzed based on K RAS statussignificant when analyzed based on K‐RAS status. 

• In addition, the difference in PFS was not significant for patients in the P‐CAP arm of the study (10 9 weeks for P CAP and 11 4 weeks for CAP; p =CAP arm of the study (10.9 weeks for P‐CAP and 11.4 weeks for CAP; p = 0.752).

O’Reilly K E et al. ASCO 2012

BEZ235: D l PI3K d TOR I hibitDual PI3K and mTOR Inhibitor

• BEZ235 is a potent oral inhibitor of PI3K and theBEZ235 is a potent oral inhibitor of PI3K  and the downstream effectors, mTORC1/2

• Significantly inhibits growth and induces apoptosis in i t f t ll lia variety of tumor cells lines

• BEZ235 suppresses estrogen‐independent growth in ER+ breast cancer cells

• BEZ235 has reversed resistance to other anti‐cancer therapies in a variety of tumor cell lineslines

• In vivo studies have confirmed the anti‐growth and anti‐angiogenic potential of BEZ235

• BEZ235 exhibited dose‐ and day‐dependent PI3K inhibition in a y pPhase I study and is now entering Phase II development

Maira SM, et al. Mol Cancer Ther 2008;7:1851–1863Serra V, et al. Cancer Res 2008;68:8022–8030Engelman JA, et al. Nat Med 2008;14:1315–1316Brachmann S, et al. Proc Natl Acad Sci USA 2009;106:22299–22304Eichhorn PJ, et al. Cancer Res 2008;68:9221–9230Brünner‐Kubath C, et al. Breast Cancer Res Treat 2011;129:387–400Bhende PM, et al. Leukemia 2010;24:1781–1784;

ConclusionsConclusions

• Overall, molecular clues are the proof of the concept and havefurnished the rationale for the use of mTOR inhibitors in thetreatment of disregulated mTor cancers

• Prognostic and predictive biomarkers for mTOR‐targetedtherapies are neededtherapies are needed

I t i i d i t d d• Improvements in overcoming drug resistance are needed