Raajit Rampal M.D. Ph.D.Leukemia Service
Memorial Sloan-Kettering Cancer Center
Genetics and targeted therapy of MPNs
Scientific Questions/Advances
• Are mutations which activate JAK2 a hallmark of all MPN patients?
• What do mutations which occur in concert with JAK2/MPL mutations do?
• What have we learned about JAK2 inhibitors?
• What novel therapies are of potential benefit for MPN patients?
JAK2V617F mutations in myeloid malignancies
Prevalence of JAK2V617F :- PV: 95%- ET: 45-50%- MF: 45-50%- CMML: 5%- MDS: RARS-T (>50%)- AML: 2%
James et al. Nature 2005Levine et al. Cancer Cell 2005Baxter et al. Lancet 2005Kralovics et al. NEJM 2005Zhao et al. JBC 2005
JAK2V617F negative MPN
• JAK2V617F-negative PV- JAK2 exon 12 mutations- loss of function mutations in LNK, negative regulator of
JAK2 (Oh et al Blood 2010)
• JAK2V617F-negative ET/PMF- MPL mutations in 10%- LNK mutations in <5%
• Somatic mutations had not been identified in 30-40% of MPN patients
Identification of CALR Mutations in JAK2 wildtype MPN
Nangalia et al NEJM 2013Klampfl et al NEJM 2013
• CALR Mutations exclusive of JAK2/MPL mutations
• Seen exclusively in ET/MF
• Small set (<10%) of ET/MF patients JAK2/MPL/CALR-negative->unknown mutant disease allele
Gene expression – can we measure gene expression and learn something about pathogenesis of MPN?
• Determine if there is a common genetic signature associated with MPN or with JAK2V617F mutations
• Identify genes which segregate with clinical phenotype
• Identify candidate genes in JAK2/MPL/CALR-negative MPN
Ben Ebert/Todd Golub
Gene Expression Profiling of MPN Samples distinguishes MPN patients from controls but does not distinguish based on disease or JAK2 status
HomozygousHeterozygousJAK2 negativeControl
PVPMFET
CONTROLJAK2 shRNA
Is there a JAK2 signature in heterozygous/WT MPN patients?
JAK2 shRNA in HEL cells to generate JAK2 signature
Similar data with JAK inhibitor
PV MMM ETCONTROL
MMM MMMETHOMOZYGOUS WT HETEROZYGOUS
JAK2 shRNA signature in MPN and control samples
Seen in all MPN patients, not in controls
JAK2 is activated in all MPN patients regardless of specific mutation
JAK2 Signature Enriched in CALR-mutant MPN Patients
FDR qvalue= 0.012
NormalCALR mutant
0.70.60.50.40.30.20.10.0
Enric
hmen
t Sco
re (E
S)
JAK2 shRNA down expression signature Homozygous vs Normal UP signature
FDR qvalue= 0.321P-value= 0.035
NormalCALR mutant
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Enric
hmen
t Sco
re (E
S)
• Consistent with activated JAK2-signaling in CALR-mutant patients
Gene Expression Signature of CALR Mutation
• CALR Signature is Enriched in JAK2-mutant MPN patients
• Consistent with JAK2 and CALR mutations having same role in MPN pathogenesis – all leads to JAK2 activation
Cooperating Mutations in MPN Patients
• Recent studies have identified somatic disease alleles which occur in concert with JAK2/MPL mutations- TET2 loss of function mutations in 10% of MPN patients- ASXL1 mutations in 8-10% of MPN patients- IDH1/2 mutations in 3-5% of MPN patients- EZH2 mutations in 10-15% of patients
• Same mutations are seen in MDS and AML patients->they do not explain the PV/ET/MF concondrum
• In some cases (TET2, IDH1/2) these mutations occur most commonly at progression to AML
• Limited functional data suggest these mutations affect the epigenetic state of MPN cells->affect the way DNA is packaged and which parts of it are used in MPN cells
Leukemic Transformation of MPN
• Patients with PV, ET, and PMF are at high risk for transformation to AML- associated with a dismal prognosis
• Genetic/Epigenetic events which contribute to leukemic transformation are not known
• Approximately 50% of JAK2+ MPN patients transform to a JAK2-negative MPN*
*Campbell et al. Blood 2006 Theocarides et al. Blood 2007
Mutational Studies in post-MPN AML
Denotes frequently mutated in de novo AML
No mutations found in c-KIT, EZH2, or WT-1
Splicesome mutations
Model of MPN Pathogenesis
JAK2 rs10974944Other alleles
EZH2, TET2, ASXL1
AML
TET2, IKZF, IDH1/2Other Alleles
• Mutations which activate JAK2 are the most common lesion->best therapeutic target
• Possible other mutations affect response to JAK inhibitors
JAK Inhibitors in Current Clinical Development
Agent Company Activity Status
Ruxolitinib (INCB18424) Novartis/Incyte JAK1/JAK2 FDA approved, Phase II (PV)
TG101348/SAR302503 Sanofi Aventis JAK2, FLT3 Phase III
CYT387 Cytopia JAK1/JAK2/TYK2 Phase II
Pacritinib (SB1518) SBio JAK2, FLT3 Phase III
Lestaurtinib (CEP701) Cephalon JAK2/Flt3 Phase I
BMS-911453 Bristol-Myers Squibb JAK2 Phase I
NS-018 Nippon-Shinyaku JAK2/Src Phase I
AZD1480 Astra Zeneca JAK1/JAK2 Phase I
LY2784544 Eli Lily JAK2V617F Phase I
Tofacitinib (CP-690550) Pfizer JAK3 FDA approved (RA)
• Although structurally divergent, these inhibitors are all Type I (ATP competitive) JAK Inhibitors
Courtesy of Priya Koppikar
Resistance to JAK inhibitors in the treatment of MPNs
Phase II/III clinical trials with INCB18424 and other JAK inhibitors• Improvement in splenomegaly, constitutional symptoms, reduced progression to leukemia, survival benefit.• No decrease in allele burden in the majority of MPN patients • May be due, at least in part to presence of other (disease-initiating) alleles
JAK2-driven preclinical models argue other factors contribute• JAK2V617F knockin model: Disease initiating cells are resistant to JAK2 inhibitor treatment (Mullally et al. 2010)
MPN mutant clone persists in the presence of chronic JAK2 kinase inhibition
We have not identified second-site mutations in patients treated with INCB18424
The lack of an initial response argues for inherent insensitivity to JAK inhibitors: persistence Reactivation of JAK2/JAK-STAT signaling Activation of a redundant/collateral pathway
STAT
RASPI3K
AKTSTATSTAT
P
P
P
P
P
P
P
Nucleus
JAK2
MAPK
JAK2
JAK-STAT signaling in naïve MPN cells
RASPI3K
AKTSTAT
Nucleus
JAK2
MAPK
JAK2
JAK inhibitors block homodimeric JAK2 activation and downstream signaling in naïve cells
JAK-STAT Signaling is Intact in Patients Treated with Ruxolitinib
Koppikar et al. Nature 2012
STAT
RASPI3K
AKTSTATSTAT
P
P
P
P
P
P
P
Nucleus
JAK2
MAPK
JAK1/ TYK2
JAK inhibitors cannot inhibit heterodimeric JAK2 activation and downstream signaling in persistent cells
pJAK2 Associates with JAK1 and TYK2 in MPN Patients treated with Ruxolitinib, but Not in Inhibitor Naïve Patients
Koppikar et al. Nature 2012
• Do persistent cells remain JAK2 dependent?• Can persistence be targeted with agents which degrade
JAK2 or block JAK2 transactivation?
• Loss of JAK2 inhibits growth and signaling in persistent cells
Scr shJ2 81 shJ2 1 Scr shJ2 81 shJ2 1
SET-2 SET-2 INPer
pJAK2
JAK2
pSTAT3
pSTAT5
Tubulin
STAT3
STAT5
Persistent Cells are JAK2 dependent
Can we improve our ability to target JAK2
• Can we develop better therapies which improve the therapeutic window and target the malignant cell?
- additional therapies- alternate dosing strategies for JAK2
inhibitors
• Can novel compounds be used in a synergistic manner to inhibit JAK2 dependent proliferation?• HSP90 inhibition
Sachie Marubyashi, Priya Koppikar
PU-H71(HSP90 Inhibitor
JAK2 is an HSP90 Client and is Degraded by the Purine Scaffold HSP90 Inhibitor PU-H71
Marubayashi et al. JCI, 201025
Options: Click-Drag image to reposition | View smaller image | Expand to full image width | Reset image
JAK2V617F
JAK2V617FPU-H71 nM
pJAK2
JAK2
pSTAT3
STAT3
pSTAT5
STAT5
pMAPK
MAPK
HSP90
Raf1
Actin
• Similar results with W515L cells: JAK2 is an HSP90 client
PU-H71 Demonstrates Efficacy in vivo in JAK2V617F and MPLW515L Transplant Models
Survival
Spleen weights
HSP90 Inhibition Degrades JAK2/Inhibits JAK-STAT signaling in 1° MPN Samples
AUY922 (MSKCC IRB 12-076)
• Drug: AUY922 (HSP90 inhibitor)
• Study Design: Phase II single-arm
• Endpoint: Efficacy of AUY922 assessed by IWG criteria
• Patient Population/Eligibility: -PMF -Post-PV/ET MF-Refractory PV/ET (HU, phlebotomy, anagrelide)
p=0.02p=0.03
p=0.05
p=0.057p=0.057
p=0.05
29
Combination Treatment with JAK and HSP90 Inhibitor Shows Improved Efficacy Compared to Either Therapy Alone
Combination Treatment with JAK and HSP90 Inhibitor Shows Improved Efficacy Compared to Either Therapy
Alone
pJAK2
JAK2
pSTAT3
pMAPK
Actin
STAT3
MAPK
Veh INC30 PU+INC30INC90
Vehicle INC90 INC30 with PU
Reduced Reticulin Fibrosis with Combination Therapy
PU-H71 + Ruxolitinib Clinical Trial
• Drugs: PU-H71, Ruxolitinib
• Study Design: Phase I
• Endpoint: Efficacy assessed by IWG criteria
• Patient Population/Eligibility: -PMF on Ruxolitinib with persistent disease -Post-PV/ET MF on Ruxolitinib with persistent disease
Summary
• Mutations which activate JAK-STAT signaling are seen in almost all MPN patients->but there are additional genetic lesions seen in MPN patients which contribute to stem cell survival
• Additional novel therapeutic approaches targeted at JAK2 and at other oncogenic signaling pathways might offer benefit alone or in conjunction with JAK2 inhibitors
• Genetic studies of myeloid malignancies will likely identify novel mutations with pathogenetic and therapeutic relevance
Acknowledgements
• NHLBI, NCI, HHMI, LLS, Starr Cancer Consortium, Geoffrey Beene Foundation, Gabrielle’s Angel Foundation, MPN Foundation
MSKCC• Gabriela Chiosis• Nick Socci• Marty Tallman• Omar Abdel-Wahab
MDACC• Serge Verstovsek• Miloslav Beran• Taghi Mansouri
Harvard/Broad• Gary Gilliland• Ben Ebert• Todd Golub• Ann Mullally
Cornell• Dick Silver• Ari Melnick• Gail Roboz
Mayo• Reuben Mesa
Levine Lab• Priya Koppikar• Neha Bhagwat• Outi Kilpivaara• Jay Patel• Franck Rappaport• Alan Shih• Olga Guryanova• Lindsay Saunders• Ria Kleppe• Todd Hricik• Sophie McKenney
Northwestern• John Crispino• Jon Licht