Dr. Raajit Rampal, MD, Ph.D

Report
Genetics and targeted therapy of
MPNs
Raajit Rampal M.D. Ph.D.
Leukemia Service
Memorial Sloan-Kettering Cancer Center
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 2005
Levine et al. Cancer Cell 2005
Baxter et al. Lancet 2005
Kralovics et al. NEJM 2005
Zhao 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
Klampfl et al NEJM 2013
Nangalia 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
Homozygous
Heterozygous
JAK2 negative
Control
PV
PMF
ET
Is there a JAK2 signature in heterozygous/WT MPN patients?
CONTROL
JAK2 shRNA in HEL cells to
generate JAK2 signature
JAK2 expression
1500
1000
500
0
LUC Control
GFP Control
shRNA1 JAK2
shRNA2 JAK2
Similar data with JAK inhibitor
JAK2
shRNA
JAK2 shRNA signature in MPN and control samples
HOMOZYGOUS
PV
MMM
WT
ET
HETEROZYGOUS
MMM
ET
CONTROL
MMM
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
JAK2 shRNA down expression signature
Homozygous vs Normal UP signature
0.5
Enrichment Score (ES)
Enrichment Score (ES)
0.6
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Normal
CALR mutant
FDR qvalue= 0.012
•
0.4
0.3
0.2
0.1
0.0
Normal
CALR mutant
FDR qvalue= 0.321
P-value= 0.035
Consistent with activated JAK2-signaling in CALR-mutant patients
Enrichment Score (ES)
Gene Expression Signature of CALR Mutation
• CALR Signature is
Enriched in JAK2-mutant
MPN patients
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Normal
Homozygous
FDR qvalue= 0.002
• 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
No mutations
found in c-KIT,
EZH2, or WT-1
Splicesome mutations
Denotes frequently
mutated in de novo AML
Model of MPN Pathogenesis
JAK2 rs10974944
Other alleles
EZH2, TET2, ASXL1
• Mutations which activate
JAK2 are the most
common lesion->best
therapeutic target
• Possible other mutations
affect response to JAK
inhibitors
TET2, IKZF, IDH1/2
Other Alleles
AML
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
JAK-STAT signaling in naïve MPN cells
JAK2
P
JAK2
P
STAT
P
P
P
RAS
PI3K
P
MAPK
AKT
Nucleus
STAT
STAT
P
JAK inhibitors block homodimeric JAK2
activation and downstream signaling in naïve cells
JAK2
RAS
JAK2
PI3K
STAT
MAPK
AKT
Nucleus
JAK-STAT Signaling is Intact in Patients
Treated with Ruxolitinib
INCB18424 Treated
Untreated
Pt-3
Pt-4
-
+
-
+
-
Pt-2
Pt-1
Pt-5
+
-
+
-
Pt-9
+
-
+
INCB18424
pSTAT3
STAT3
pSTAT5
STAT5
Koppikar et al. Nature 2012
JAK inhibitors cannot inhibit heterodimeric JAK2
activation and downstream signaling in persistent cells
JAK1/
TYK2
JAK2
P
P
STAT
P
P
P
RAS
PI3K
P
MAPK
AKT
Nucleus
STAT
STAT
P
pJAK2 Associates with JAK1 and TYK2 in MPN Patients
treated with Ruxolitinib, but Not in Inhibitor Naïve Patients
IN tx
Pt-1
Pt-2 Pt-3
Untreated
Pt-4 Pt-5
Pt-6
IN tx
Pt-7
Pt-8 Pt-9
pJAK2
IP: JAK1
JAK2
JAK1
pJAK2
IP: TYK2
JAK2
TYK2
•
•
Do persistent cells remain JAK2 dependent?
Can persistence be targeted with agents which degrade
JAK2 or block JAK2 transactivation?
Koppikar et al. Nature 2012
Persistent Cells are JAK2 dependent
SET-2
Scr
shJ2 81 shJ2 1
SET-2 INPer
Scr
shJ2 81
shJ2 1
pJAK2
JAK2
pSTAT3
STAT3
pSTAT5
STAT5
Tubulin
• Loss of JAK2 inhibits growth and signaling in persistent cells
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
PU-H71
(HSP90
Inhibitor
Sachie Marubyashi, Priya Koppikar
JAK2 is an HSP90 Client and is Degraded by
the Purine Scaffold HSP90 Inhibitor PU-H71
JAK2V617F
PU-H71 nM
JAK2V617F
pJAK2
JAK2
pSTAT3
STAT3
pSTAT5
STAT5
pMAPK
MAPK
HSP90
Raf1
Actin
• Similar results with W515L cells: JAK2 is an HSP90 client
25
Marubayashi et al. JCI, 2010
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)
Combination Treatment with JAK and HSP90 Inhibitor Shows
Improved Efficacy Compared to Either Therapy Alone
p=0.03
p=0.02
3000
p=0.05
Platelets (K/uL)
200
p=0.05
100
2000
1000
p=0.057
Spleen weights (g)
0.8
0.6
0.4
0.2
29
IN
C
90
+P
U
PU
IN
C
30
w
ith
C
90
IN
IN
C
30
0.0
IN
C
90
+P
U
PU
w
ith
C
90
IN
C
30
IN
IN
C9
0+
PU
PU
w
ith
IN
C
30
IN
C
90
p=0.057
IN
C
30
0
0
IN
C
30
WBC (K/ul)
300
Combination Treatment with JAK and HSP90 Inhibitor
Shows Improved Efficacy Compared to Either Therapy
Alone
Veh
INC30
INC90
PU+INC30
pJAK2
JAK2
pSTAT3
STAT3
pMAPK
MAPK
Actin
Reduced Reticulin Fibrosis with Combination
Therapy
Vehicle
INC90
INC30 with PU
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
Levine Lab
• Priya Koppikar
• Neha Bhagwat
• Outi Kilpivaara
• Jay Patel
• Franck Rappaport
• Alan Shih
• Olga Guryanova
• Lindsay Saunders
• Ria Kleppe
• Todd Hricik
• Sophie McKenney
Cornell
• Dick Silver
• Ari Melnick
• Gail Roboz
MDACC
• Serge Verstovsek
• Miloslav Beran
• Taghi Mansouri
Harvard/Broad
• Gary Gilliland
• Ben Ebert
• Todd Golub
• Ann Mullally
MSKCC
• Gabriela Chiosis
• Nick Socci
• Marty Tallman
• Omar Abdel-Wahab
Northwestern
• John Crispino
• Jon Licht
Mayo
• Reuben Mesa
•NHLBI, NCI,
HHMI, LLS, Starr
Cancer
Consortium,
Geoffrey Beene
Foundation,
Gabrielle’s Angel
Foundation, MPN
Foundation

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