JAK2 Mutations in MPN: A Worthy Therapeutic Target?

JAK2 Mutations in MPN: A
Worthy Therapeutic Target
Olatoyosi Odenike M.D
University of Chicago
Chicago, IL

Conflict of Interest Disclosure
Company
Gloucester
Pharmaceuticals
MGI Pharma/Eisai
Curagen/Topotarget
Celgene, MGI
Pharma, Cephalon,
Genzyme,
Amgen
Relationship
UC Tech Patent
Research Support
Advisory
Board/Consultancy

Phenotypic Mimicry in MPN
• Involvement of pluripotent HSC
• Increase in one or more peripheral
blood elements
• Marrow hypercellularity
• Extramedullary hematopoiesis
• Thrombo-hemorrhagic complications
• Predisposition to marrow fibrosis and
leukemic transformation

Recurring Cytogenetic Abnormalities
in MPN at Diagnosis
Disease
Specific
Non-specific
%
CML, CP
t(9;22)
>95
CML, AP/BP
t(9;22)
+8, +Ph, +19, i(17q), t(3;21)
80
CNL
none
+8, +9, del (20q), del (11q14)
10
CEL/HES
none
+8, t(5;12), dic(1;7), 8p11
60
PV
none
+8, +9, del (20q), del(13q),
del(9p)
15
PMF
+8, del(20q),-7/del(7q), del(11q),
del(13q), 12pnone
30
ET
none
+8, del(20q)
5

Activating Mutations of Tyrosine Kinases in MPN
Levine RL, et al., Nature Reviews Cancer, 2007

JAK2 Mutations in MPN
Exon 12
Mutations
V617F
JAK2
FERM Pseudokinase Kinase
COOH
• JAK2 V617F mutation
– Single nucleotide substitution in JAK2 exon 14 sequence
– Mutation occurs in the pseudokinase domain of the protein
• JAK2 exon 12 abnormalities
– Mutations, deletions, insertions
• Result in constitutive activation of JAK2 TK

JAK-2 mediated Signal Transduction Pathways
Ligand Bound Receptor
(EPOR, MPL, GCSF)
AKT
mTOR
P
P13K
P
P
JAK2
P
P
P
STAT
STAT
P
JAK2
STAT
STAT
P
P
P
RAS
MAPK
Target Gene
Activation
Survival
Differentiation
Proliferation

MPL mutations in MPN
• Detected in PMF and ET
– 5 to 10% frequency
• Two most common are W515L and
W515K
• Result in constitutive activation of JAK-
STAT signaling
• Cause PMF phenotype in mice
• Coexistence with JAK2V617F has been
described

JAK2V617F Mutational Frequency in
Myeloid Neoplasms
Disease
**Polycythemia Vera
*Essential Thrombocytosis
*Primary Myelofibrosis
CMMOL
RARS-T
Acute Myeloid Leukemia
Frequency (%)
>95%
≈50%
≈50%
3-9
≈40%

Question #1: The JAK2 V617F allele
frequency is:
a) Greater than 95% in Polycythemia
vera
b) Approximately 50% in Essential
thrombocythemia
c) Approximately 50% in Primary
Myelofibrosis
d) Relatively rare in AML
e) All of the above

Diagnostic algorithm for MPN in JAK2 Era
JAK2V617F genotyping when a
MPN is suspected
Positive
Negative
PV, ET, PMF
Likely
Use additional WHO criteria
PV: unlikely; test for
JAK2 ex 12
ET or PMF: possible;
test for MPL
Use additional WHO criteria
Adapted from Vannucchi AM et al, CA Cancer J Clin 2009

Question #2: The presence of the
JAK2V617F is sufficient to:
a) Establish a diagnosis of Polycythemia
vera
b) Establish a diagnosis of MDS-RARS-T
c) Establish a diagnosis of Primary
Myelofibrosis
d) Rule out a reactive cause of
thrombocytosis
e) Establish a diagnosis of Essential
thrombocythemia

Question # 3:
In a 60 year old smoker with a
hemoglobin of 19g/dL and a low serum
erythropoietin level:
a) A diagnosis of Polycythemia vera can be made
unequivocally
b) It is necessary to perform a bone marrow
biopsy to diagnose Polycythemia vera
c) The finding of a JAK2V617F mutation in a
peripheral blood sample is diagnostic of
Polycythemia vera
d) Oxygen saturation should be measured to
diagnose the cause of the erythrocytosis
e) A red cell mass should be performed to make a
conclusive diagnosis of Polycythemia vera

The Ideal Target for MPN Therapy
• Present in patients with the disease
• Determined to be the causative
abnormality
• Has a unique activity that is
– Required for disease induction
– Dispensable for normal cellular function

Is JAK2V617F the initiating event in
MPN
YES
• Confers cytokine independent
growth and growth factor
hypersensitivity in vitro
• Causes PV phenotype in
murine models
NO
• JAK2 positive MPN can
transform into JAK2 negative
AML
• Pre-JAK2 mutations possible

Pre-JAK2 mutations
HSC
MPP
Acquisition of
JAK2 and similar
mutations
TET2
del 20q
Predisposing host genetic
Factors Epigenetic events

One mutation: Multiple phenotypes: Gene dosage effect
Vannucchi AM, Leukemia 2008

JAK2 mutations and Clinical
relevance

JAK2V617F and Thrombosis
• In ET, V617F mutation is associated with
– Higher Hb and leucocyte count
– Higher incidence of thrombosis and cardiovascular
events in homozygous patients
• Several studies have demonstrated an
association with thrombosis in MPN
– Potential confounding variables in virtually all
studies
• Significant association with splanchnic vein
thrombosis
– Odds ratio; 53.98 (13-222)
Austin SK et al, BJH 2008
Dentali F et al, Blood 2009

JAK2V617F and Thrombosis
Carrobio et al, Exp Hematol, 2009

Question #4
You are asked to see a 55 year old woman with
no significant past medical or surgical history
who develops Budd Chiari Syndrome. The
diagnostic work up for hypercoagulability
should definitely include
a) Bone marrow biopsy
b) Liver biopsy
c) CBC
d) Molecular testing of the peripheral
blood for JAK2V617F
e) c and d

JAK2V617F allele burden over time
Carrobio et al, Exp Hematol, 2009

JAK2V617F and Leukemic
Transformation in PMF
Proportion of Patients without Progression to
Leukemic Transformation
1,0
0,8
0,6
0,4
0,2
P=0.02
0 50 100 150 200
Time (months)
V617FC
- V617F -
V617F +
V617F +
Barosi G, Blood 2007
Guglielmelli P, Blood 2009

JAK2V617F and Overall Survival
Author
Campbell P, 2005
in PMF
N V617F+
(%)
152 55
Association
with Survival
Yes: HR 3.3
(95%CI 1.26-8.68)
Barosi G, 2007
304
63
No
Tefferi A, 2008
199
58
No
Gugliemelli P, 2009
186
68
No

JAK2V617F and Overall Survival
in PMF
V617F -
V617F -
V617F +
P = .34
V617F +
P = .73
Tefferi A, Leukemia 2008
Guglielmelli P, Blood 2009

Question #5
The presence of the JAK2V617F
mutation has been shown to:
a) Initiate myeloproliferative neoplasms in
humans
b) Contribute to the disease phenotype in
humans with essential thrombocythemia
c) Cause venous thrombosis
d) Cause leukemic transformation in humans
in prospective studies
e) Significantly shorten overall survival in
patients with myeloproliferative neoplasms

JAK2V617F allele burden and
survival in PMF
P= 0.0008
1 st 2 nd 3 rd
P=0.0001
4 th
Tefferi A, Leukemia 2008 Guglielmelli P, Blood, 2009

JAK2 inhibitors in MPN

JAK2 inhibitors: Preclinical
• Bind to JAK2 kinase catalytic site at low
nanomolar concentrations
– Both wildtype and mutant allele inhibited
• Inhibit the growth of V617F + cell lines and
primary MPN cells
– Progenitors from MPN patients more
sensitive than healthy controls
• Activity demonstrated in murine models of
V617F+ MPN

JAK2 Inhibitors in the clinic
Agent
CEP701
XL019
TG01348
INCB018424
SB1518
AZD1480
Company
Cephalon
Exelixis
TargeGen
Incyte
S*Bio
AstraZeneca
Biological Target
JAK2 and FLT3
JAK2
JAK2
JAK1 and JAK2
JAK2 and FLT3
JAK2

A Phase I Study of XL019, a
Selective JAK2 Inhibitor, in
Patients with Primary
Myelofibrosis, Post-Polycythemia
Vera, or Post-Essential
Thrombocythemia Myelofibrosis
Shah et al. ASH 2008

Phase I Study XL019: Safety Data
Related Adverse Events
Non-
Hematologic
AEs
Hematologic
AEs
Formication
Peripheral
Neuropathy
Confusional state
Balance Disorder
Paresthesia
Dysgeusia
Fatigue
Nausea
Skin Rash
Anemia
Thrombocytopenia
Neutropenia
25 mg
(N=16)
n (%)
2(12)
1(6)
1(6)
1(6)
1(6)
1(6)
1(6)
1(6)
1(6)
2 (40)
1(6)
• Dose levels of 25 or 50 mg (N=21)
• No drug-related hematologic adverse events (AEs)
• Asymptomatic NCS changes were observed in 3 patients dosed at 25 mg
0
0
0
All Grades
50 mg
(N=5)
n (%)
2(40)
2(40)
1 (20)
0
0
0
0
0
0
0
0
25 mg
(N=16)
n (%)
0
0
0
0
0
0
0
0
0
0
0
Grade 3 or 4
50 mg
(N=5)
n (%)
0
0
0
0
0
0
0
0
0
0
0
0

Phase I/II Study of INCB18424
• Phase I dose escalation
study
– Reduction in
splenomegaly and
improvement in
constitutional symptoms
was observed
– Identified 25 mg 2x/day
as maximum tolerated
dose (MTD), with
reversible
thrombocytopenia as the
dose-limiting toxicity
• Expansion Cohort:
– Goals: Establish safety
and efficacy with longterm
dosing 25 mg
2x/day (BID)
- Evaluation of:
•Lower doses
– 10 and 15 mg BID
•Once daily dosing
regimen
Verstovsek et al. ASH 2007 and 2008

Demographics
N 152
Age: Median (Range) 65 (40 – 84)
Male:Female 95:57 (62.5%:37.5%)
Disease Subtype:
PMF
PPV-MF
PET-MF
79 (53.7%)
47 (32.0%)
21 (14.3%)
% JAK2 Mutation Positive 80.3%
% Transfusion Dependent 31.6%

Treatment-Emergent Laboratory Findings
15 mg BID
25 mg BID
Toxicity
Grade
Hgb*
(N=26)
Platelets
(N=32 )
Hgb*
(N=29)
Platelets
(N=47)
3
2 (8%)
0
6
(21%)
11
(23%)
4
0
0
1
(3.4%)
3
(6.4%)
*Includes subjects who were transfusion independent at entry with a > 2 gm Hgb decline

INCB018424: Safety Summary
• INCB018424 was generally well tolerated,
with few adverse events other than
mechanism-based effects on
hematopoiesis
– 115/154 patients (inclusive of all Phase
I and single daily dose patients) remain
on study with a median duration of 12
months
• Myelosuppression was readily reversible
with dose interruption and/or reduction

Impact on Spleen Size Measured by Palpation
Spleen Size, cm
22.5
20.0
17.5
15.0
12.5
10.0
10 mg BID
N=23 to Start
15 mg BID
N=32 to Start
25 mg BID
N=39 to Start
N=11
7.5
5.0
2.5
N=19
N=15
N=9 N=5
0.0
0 56 112 168 224 280 336
Days on Therapy

Spleen Volume Decrease by MRI Parallels
Spleen Size Reduction by Palpation
Proportion Achieving Response
100
75
50
25
50% size reduction by palpation
(Clinical Response by IWG
Criteria) corresponds to 35%
volume reduction by MRI
56%
22/39
52%
12/23
50%
12/24
LIVER
LIVER
SPLEEN
BEFORE
THERAPY
AFTER
6 MONTHS
0
25 mg BID/Palpation
15 mg BID/Palpation
15 mg BID/MRI
SPLEEN
53% decrease
In spleen
volume, 32%
decrease in
liver volume

INCB018424 Improves Splenomegaly
Independent of JAK2 Mutation Status
Spleen length, cm
22.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
JAK mutation POSITIVE; N = 33
JAK mutation NEGATIVE; N = 6
0
0 56 112 168 224 280 336
Time on Therapy (days)
Note: 25mg BID cohort; data are censored after a dose change.

Decrease in JAK2V617F Allele Burden
100
N=19
P=0.0001
V617F (% Baseline)
90
80
70
100
90
80
70
N=20
P=0.0005
N=14
P=0.016
N=14
P=0.040
N=20
P=0.0007
15mg BID
25mg BID
N=17
P=0.023
N=20
P=0.047
N=16
P=0.041
N=3
N=16
P=0.039
0 84 168 252 336 420 504
Days on Therapy
N=17
P=0.024 N=15
P=0.017
N=12
P=0.033

Rapid Improvement in Constitutional Symptoms
Bone Pain
Pruritis
Night Sweats
Symptoms Associated
with Elevated Cytokines
Fatigue
Impaired Appetite
Impaired Ability to Bend
Impaired Ability to Move
Symptoms Associated
with Splenomegaly
Abdominal Discomfort
0
10
20
30
40
50
60
70
80
Proportion of Patients with ≥ 50% Improvement
• Myelofibrosis Symptom Assessment Form (MFSAF) used (N=55 respondents)
• Improvement in symptoms were seen as early as 2 weeks

Clonal megakaryocyte
Proliferation
Cytokines in MF
PDGF
bFGF
TGF-β
Clonal monocyte and
histiocyte proliferation
bFGF
Reactive fibroblast
proliferation and fibrosis
bFGF
TNF-α
bFGF
VEGF
Neoangiogenesis
VEGF
bFGF
TNF-α
TGF-β
TGF-β
Osteosclerosis
TGF-β
Ineffective erythropoiesis
TGF-β
Tefferi A. N Engl J Med 342:1255, 2000

Reduction in Pro-Inflammatory Cytokines
V617F+
V617F-
PPV
PMF
PET
PPV
PET
fold change
11
6
3
1.5
-1.5
-3
-6
-11
Baseline
Day 28 on
Therapy

Question # 6
At this current stage of clinical trial
development, JAK2 inhibitors
should be considered for:
a) All patients with JAK2 V617F + Essential
thrombocythemia
b) All patients with JAK2 V617F + Polycythemia
vera
c) All patients with JAK2 V617F+ Primary
Myelofibrosis
d) Patients with symptomatic Primary
myelofibrosis or post-ET or post-PV
myelofibrosis
e) None of the above

JAK2 inhibitors- hype versus hope
• Several compounds under current investigation
• Lead compounds so far have uniformly had
favorable effects on spleen
– but have been myelosuppressive or neurotoxic and
several are still in dose finding phase
• Since JAK2 is critical for normal hematopoietic
signaling
– therapeutic index may improve with inhibitors that
target mutant allele
• Identification of other key pathogenetic factors
will be essential for the development of optimal
targeted therapeutic approaches