Intrinsic Resistance to JAK2 Inhibition in Myeloﬁbrosisclincancerres.aacrjournals.org/content/clincanres/19/7/1729.full.pdf · Intrinsic Resistance to JAK2 Inhibition in Myeloﬁbrosis

Cancer Therapy: Preclinical

Intrinsic Resistance to JAK2 Inhibition in Myelofibrosis

Anna Kalota1, Grace R. Jeschke1, Martin Carroll1,2, and Elizabeth O. Hexner1,2

AbstractPurpose: Recent results have shown that myeloproliferative neoplasms (MPN) are strongly associated

with constitutive activation of the Janus-activated kinase (JAK)2 tyrosine kinase. However, JAK2 inhibitors

currently approved or under development for treating myeloproliferative neoplasms do not selectively

deplete the malignant clone, and the inhibition of activity of the drug target (JAK2) has not been rigorously

evaluated in the clinical studies. Therefore, in this study we developed an in vitro assay to gain insight into

how effectively JAK2 activity is inhibited in the samples of patients.

Experimental Design: We treated primary cells from normal donors and patients with MPN with

JAK2 inhibitors and measured phosphorylation of downstream targets STAT5 and STAT3 by flow

cytometry. Obtained results were next correlated with JAK2 V617F allele burden and plasma cytokine

level.

Results:We observed a dose-dependent decrease in pSTAT5 and pSTAT3 in ex vivo treated granulocytes.

However, phosphorylation of STAT3 and STAT5 in cells from patients with myelofibrosis was significantly

less inhibited when compared with cells from patients with polycythemia vera, essential thrombocythe-

mia, and normal donors. Sensitivity to inhibition did not correlate with JAK2 V617F clonal burden.

Mixing studies using plasma from patients with myelofibrosis did not transfer resistance to sensitive cells.

Likewise, no single cytokine measured seemed to account for the observed pattern of resistance.

Conclusions: Taken together, these observations suggest that there are cell intrinsic mechanisms

that define a priori resistance to JAK2 inhibition in myelofibrosis, and the lesion is localized upstream

of STAT3 and STAT5. Clin Cancer Res; 19(7); 1729–39. �2013 AACR.

IntroductionPolycythemia vera, essential thrombocythemia, and

myelofibrosis are myeloproliferative neoplasms (MPN)unified by the constitutive activation of the Janus-acti-vated kinase (JAK)2 pathway, conferred most commonlyby a point mutation in the pseudokinase domain of JAK2(JAK2V617F; refs. 1–4). The identification of this muta-tion has prompted wide interest in targeting JAK2 fortherapeutic benefit. Myelofibrosis, whether primary orevolved from polycythemia vera or essential thrombo-cythemia, has a variable but overall poor prognosis.Clinical investigation of JAK2 inhibitors has focused onmyelofibrosis with some unexpected results. Althoughreduction in spleen size and amelioration of constitu-tional symptoms are frequent and can be dramatic, onlymodest changes in mutant clonal burden have been

observed and complete responses by any criteria areexceedingly rare (5–7).

Notably, validation of inhibition of activity of the drugtarget (JAK2) has never been rigorously evaluated in clinicalstudies. JAK family proteins are nonreceptor tyrosinekinases that are associated with cytokine receptors. Cyto-kine engagement with receptor leads to the phosphoryla-tion of JAK2 and the recruitment and phosphorylation ofcytoplasmic signal transducers and activators of transcrip-tion (STAT3 and 5). Upon activation, phosphorylatedSTAT3 and STAT5 (pSTAT3 andpSTAT5) formhomodimersand translocate to the nucleus to activate transcription. InMPN, JAK2with aV617F substitution is constitutively activeand confers cytokine independence/hypersensitivity tohematopoietic tissues. Small-molecule tyrosine kinase inhi-bitors have been shown to be active in both preclinical andlate-phase clinical testing, with analyses of cytokine arraysand measurement of JAK2V617F clonal burden used aspotential biomarkers for response (6, 5). To study theefficacy of JAK2 inhibitors to inhibit JAK signaling in pri-mary patient cells, we usedphospho-specific flow cytometryon whole peripheral blood from patients with MPN andnormal controls to measure phosphorylation of STAT3 andSTAT5, the canonical targets of JAK2. We hypothesized thatthis measurement would be a feasible and informativepharmacodynamic assay for measuring JAK2 signaling inpatients on clinical trials and that differences in signalingmight be observed across the spectrum of disease.

Authors' Affiliations: 1Division of Hematology andOncology and 2Abram-son Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author:ElizabethO. Hexner, University of Pennsylvania, 2West Pavilion, Perelman Center for AdvancedMedicine, 3400 Civic CenterBlvd, Philadelphia, PA 19104. Phone: 215-614-1847; Fax: 215-573-7049;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-1907

�2013 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 1729

on February 3, 2019. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 5, 2013; DOI: 10.1158/1078-0432.CCR-12-1907

http://clincancerres.aacrjournals.org/

Materials and MethodsPeripheral blood samples

Peripheral blood samples from patients and normaldonors were collected, annotated, and stored through anInstitutional review board–approved hematologic diseasestissue bank. Previously collected, anonymous samples wereobtained from the Stem Cell and Xenograft Core facility(University of Pennsylvania, Philadelphia, PA).

ReagentsJAK2 inhibitors: CEP701 (lestaurtinib) was provided by

Cephalon Oncology or purchased from LC laboratories.CYT387 and INCB018424 were synthesized by ChemieTekand kindly provided by Dr. Ross Levine (Memorial Sloan-Kettering Cancer Center, New York, NY). Antibodies werepurchased from BD Bioscience and R&D Systems.

Flow cytometryWholeblood sampleswerefixed andpermeabilizedusing

a formaldehyde/triton/methanol method (8, 9) and ana-lyzed by flow cytometry. Briefly, 100 mL of whole bloodwasexposed to varying concentrations of the inhibitor for 15minutes at 37�Cand then stimulated for 20minutes at 37�Cwith10ng/mLgranulocytemacrophage colony-stimulatingfactor (GM-CSF; for pSTAT5 measurement) or 100 ng/mLgranulocyte colony-stimulating factor (G-CSF; for pSTAT3measurement). After stimulation, the samples were fixedwith 4% formaldehyde for 10minutes at room temperatureand permeabilized with 0.1% Triton X-100 for 15 minutesat 37�C. Next, samples were washed twice in cold PBSsupplemented with 4% bovine serum albumin (BSA), trea-ted with cold 100% methanol to enhance epitope avail-

ability, and stored at �20�C. Before analysis, samples werewashed twice in cold PBS supplemented with 4% BSA andthen incubated with directly labeled antibodies at roomtemperature for 30minutes in the dark. Data were acquiredon a BD FACSCalibur using CellQuest Pro software andanalyzed using FlowJo version 9.3.1. For sample of eachpatient, untreated mean fluorescence intensity (MFI) ofpSTAT3 and pSTAT5 was subtracted from treated samplesand MFI for cytokine-stimulated samples was set at 100%.Persistence of pSTAT3 and pSTAT5 in the presence ofinhibitor (X) was calculated as the ratio of mean fluores-cence values of gated events relative to cytokine-stimulatedsample; inhibition is described as 100�X. Statistical anal-ysis was conducted using GraphPad Prism 5.

JAK2 V617F allele burdenMononuclear cells and granulocytes were separated by

density using Ficoll-Paque (Amersham Bioscience). Thegenomic DNA from granulocytes was isolated from cellsusing Gentra Purgene Blood Kit from Qiagen. JAK2 V617Fallele burden was measured using a quantitative real-timePCR (qRT-PCR) assay adapted from Nussenzveig and col-leagues (10) to detect the G!T substitution at position1849 in exon 14 that uses an allele-specific (wild-type vs.mutant) primer with specificity enhanced bymismatch anda locked nucleic acid modification. Allele frequency wascalculated as described by Germer and colleagues (11).

Determination of cytokine level in plasma frompatients and normal donors

Plasma sampleswere obtainedby centrifugationofwholeperipheral blood at 600 � g for 10 minutes and stored at�80�C. Batched frozen samples were analyzed by theHuman Immunology Core (University of Pennsylvania)using a Luminex instrument and the Milliplex MAP Humancytokine 9-plex from Millipore.

Statistical analysisOne-way ANOVA and Student t test were used to analyze

differences between patient and normal donor samples.Correlations were calculated using the Spearman correla-tion coefficient. All calculations weremade using GraphPadPrism 5.

ResultsPhosphorylationof STAT5andSTAT3 isdetectablewithcytokine stimulation and is inhibited in the presence ofCEP701

To more precisely validate target and to define biologicresponse to treatment with JAK2 inhibitors in patientswith MPN, we investigated the level of target inhibition inperipheral blood samples collected from 29 patients withMPN (Table 1) and 6 normal donors. We measured phos-phorylated STAT3 and 5 (pSTAT3 and pSTAT5) as bio-markers for JAK2 inhibition. In our initial studies wedid not observe constitutive basal phosphorylation ofSTAT3 or STAT5 in any samples evaluated (Fig. 1A and

Translational RelevanceThe identification of Janus-activated kinase (JAK)2

mutations in classical, Philadelphia-chromosome neg-ative myeloproliferative neoplasms (MPN) suggested atarget for small-molecule therapy for MPN, with thehope of responses akin to those seen in chronic myeloidleukemia treated with imatinib. Although subsequentstudies of JAK2 inhibitors have proven that they aretherapeutic, responses have been unexpected, and donot in fact meaningfully deplete the clonal burden ofdisease. The mechanism(s) through which JAK2 inhibi-tors exert activity are incompletely understood includinghow effectively they inhibit JAK2 kinase activity in bloodcells. We directly measured downstream inhibition ofSTAT3 and STAT5 phosphorylation in whole blood andfound that across disease subtypes, the response toinhibition varied with myelofibrosis overall resistant.Our findings identify a pattern of resistance that is not inthe classical "escapemutation" paradigm andmay beginto explain the attenuated clinical responses that havebeen observed in the clinic.

Kalota et al.

Clin Cancer Res; 19(7) April 1, 2013 Clinical Cancer Research1730




Tab

le1.

Patient

charac

teris

tics

ID#

Reg

istryID

Diagno

sis

Age

Sex

Rac

eWBC

(THO/mL)

%Blasts

HGB

(g/dL)

PLT

(THO/mL)

Cytogen

etics

Treatmen

thistory

V61

7F(%

)Yea

rsfrom

diagno

sis

2211

R12

67Pos

t-PVMye

lofibrosis

74M

W9.8

09.5(t)

4946

,XY[16]

PB,E

SA

890.9

2212

R06

02Polyc

ythe

mia

vera

56F

W4.6

014

.635

646

,XX[26]

PB,H

U,A

SA

6719

.222

22R12

43Prim

arymye

lofibrosis

68F

W5.2

08.5(t)

222

46XX[22]

THAL/PRED

740.7

2223

R10

92Ess

entia

ltho

mboc

ythe

mia

82F

W8.7

012

.860

046

,XX[20]

HU,A

NAG,A

SA,C

HLR

753.4

2273

R06

82Ess

entia

ltho

mboc

ythe

mia

78M

W6.5

012

.640

246

,XY[8]

HU,A

SA

634.3

2283

R06

33Prim

arymye

lofibrosis

46F

W4.1

09.1

245

46,XX[20]

ANAG,A

SA

011

.622

94R13

00Prim

arymye

lofibrosis

65F

W5.5

08.9

129

46,XX[20]

ESA

ND

0.4

2310

R09

73RARS-T

67F

AA

12.8

18.4

1961

46,XX[20]

HU,A

NAG,IFN

,ESA

700.8

2316

R12

31Prim

arymye

lofibrosis

67M

W13

.75

10.7

123

46,XY[20]

ESA

91.5

2317

R13

12Polyc

ythe

mia

vera

78F

W13

.10

14.8

346

ND

PB,H

U15

9.4

2327

R13

19Polyc

ythe

mia

vera

41M

W4.2

014

.417

1ND

PB,A

SA,IF

N79

12.7

2328

R13

20Ess

entia

ltho

mboc

ythe

mia

46M

W5

013

.432

046

,XY[20]

HU

7015

.823

40R12

31Prim

arymye

lofibrosis

67M

W11

.63

9.3

9346

,XY[20]

ESA

111.6

2341

R13

25Prim

arymye

lofibrosis

66M

W18

.10

10.1

1183

46,XY[21]

ESA,H

U,A

SA

730.1

2343

R13

22Polyc

ythe

mia

vera

56M

W12

.10

18.2

443

ND

PB,A

SA

480.1

2359

R11

82Ess

entia

ltho

mboc

ythe

mia

73F

AA

4.9

013

.372

146

,XX[20]

HU,A

SA

791.1

2369

R06

33Prim

arymye

lofibrosis

47F

W4.1

29.7

217

ND

ANAG,A

SA

08.4

2370

R12

69Prim

arymye

lofibrosis

58F

W9.5

013

.489

ND

none

570.6

2371

R13

32Ess

entia

ltho

mboc

ythe

mia

40M

W13

.70

17.1

608

46,X

Y[20]

ASA

911.9

2469

R06

82Ess

entia

ltho

mboc

ythe

mia

78M

W9.4

013

.255

046

,XY[20]

HU,A

SA

544.8

2470

R13

62Polyc

ythe

mia

vera

45F

W4.4

012

.836

846

,XX[25]

ANAG,A

SA,IFN

ND

17.2

2479

R13

08AML(from

pos

t-PVMF)

62M

AA

121.9

35ND(t)

91Com

plex

aPB,H

U,A

SA,IFN

580.8

2487

R06

70Pos

t-PVmye

lofibrosis

57F

W4.7

29.6

133

Com

plex

bPB,H

U,A

SA,IFN

,HSCT

521.3

2517

R13

78Prim

arymye

lofibrosis

63.1

MW

7.6

012

.311

046

,XY[20]

ANAG,H

U,ASA

POS

9.0

2521

R12

36Prim

arymye

lofibrosis

81.8

FW

5.1

2210

.687

46,XX[20]

HU,E

SA

05.0

3013

R15

50Polyc

ythe

mia

vera

43M

W4.3

012

.722

746

,XY[20]

HU,IFN

,PB,A

SA

POS

1.7

3014

R06

39Ess

entia

ltho

mboc

ythe

mia

60.5

FW

70

11.5

1040

ND

ANAG,H

U,A

SA

013

.030

16R15

89Polyc

ythe

mia

vera

59M

AA

6.8

015

304

46,XY[20]

ANAG,H

U,A

SA

6.0

2518

R13

19Polyc

ythe

mia

vera

42M

W3.1

012

.612

2ND

ASA,P

B,IFN

POS

13.0

Abbreviations

:ANAG,a

nagrelide;

ASA,a

spirin;

CHLR

,chloram

bucil;ESA,e

rythropoietin/darbop

oietin;H

SCT,

hematop

oietic

stem

celltran

splantation;

HU,h

ydroxy

urea

;ND,n

oda

ta;P

B,p

hleb

otom

y;POS,p

ositive

but

unkn

ownperce

ntag

e.a47

,XY,þ

der(9

)t(1;9)(q21

;q21

)[1]/4

7,idem

,del(5)(q

21q34

)[19].b

46,XXþd

er(9)t(1;9)(q12

;q21

)[20].


www.aacrjournals.org Clin Cancer Res; 19(7) April 1, 2013 1731




Supplementary Fig. S1A; P ¼ 0.48). Therefore, we incorpo-rated cytokine activation into the assay. Unmanipulatedwhole blood from normal donors or patients was stimu-lated with GM-CSF or G-CSF known to activate phosphor-ylation of STAT5 or STAT3, respectively, in humanmyeloidcells (12, 13). Activation was measured in granulocytes bygating on CD15þ cells. There were no differences in cyto-kine responsiveness across disease andnormal samples. Thetotal level of intracellular STAT5 was measured to ensureadequate permeabilization of cells and did not change withstimulation or inhibition (Supplementary Fig. S1B andS1C).

We next used this assay to study the efficacy of JAK2inhibitors (Fig. 1B and C). For these studies, blood fromnormal donors, patients with essential thrombocythemia,polycythemia vera, and patients with the more advanceddisease, myelofibrosis (either primary or evolved frompolycythemia vera or essential thrombocythemia) was firstexposed to inhibitor, primarily lestaurtinib (CEP701) at aconcentration of 10 to 50 mmol/L and then stimulated withGM-CSF or G-CSF to activate phosphorylation of STAT5(Fig. 1B) or STAT3 (Fig. 1C), respectively. The range ofinhibitor concentration was based on mean steady-stateplasma concentrations of 7.7 to 12 mmol/L (1–40 mmol/L)previously described in treated patients for CEP701 (14),and from published pharmacokinetic information onachievable concentrations, when available, forINCB018424 andCYT387 (15).Weobserved adose-depen-dent decrease in cytokine-activated pSTAT3 and pSTAT5 inCD15þ granulocytes from normal donors when treated exvivo with CEP701 (Fig. 1B and C).

Neutrophils from patients with myelofibrosis areintrinsically resistant to JAK2 inhibitors

Next, we sought to measure response to treatment acrossMPN phenotypes. We found that the response to JAK2inhibitor differs among patients withMPN. Although phos-phorylation of STAT5 (Fig. 2A) and STAT3 (Fig. 2B) in bothessential thrombocythemia and polycythemia vera wassimilar and nearly completely abrogated in the presence of20 mmol/L CEP701, pSTAT3 and pSTAT5 in myelofibrosissamples were minimally inhibited (Fig. 2A and B). Inaggregate across samples, the mean inhibition at 20mmol/L in essential thrombocythemia/polycythemia verasamples as measured by pSTAT5 (69%), was significantlymore than that in myelofibrosis samples (36%, P ¼0.015; Fig. 3A and Supplementary Table S1). The responseto inhibition in normal donors (76% inhibited) was com-parable with that observed in essential thrombocythemiaand polycythemia vera samples (pSTAT5, 68.9% inhibited;P¼ 0.90). For pSTAT3, polycythemia vera/essential throm-bocythemia samples were more sensitive (73% inhibited)when compared with myelofibrosis samples (60% inhib-ited) although this was not significant (P ¼ 0.43). Thissuggests that either downstream signaling through STAT3 inmyelofibrosis samples is trulymore responsive to inhibitionthan STAT5 or may reflect a more subtle difference notcaptured in this number of samples. We also noted a

narrower dynamic range for pSTAT3 measurements (Sup-plementary Fig. S1B). Thus, we observed that pSTAT5 andpSTAT3 can be measured in whole blood by phospho-flowcytometry in the presence of exogenous cytokine and thatpharmacologic inhibition is both dose- and MPN-subtypedependent. To extend this observation using 2 additionaltyrosine kinase inhibitors, we repeated these studies withCYT387 and INCB018424, agents currently being investi-gated/approved for the treatment of myelofibrosis. Wefound the samples of myelofibrosis to be similarly lesssensitive to inhibition with these compounds when com-pared with polycythemia vera samples (Fig. 3B). The sam-ples of myelofibrosis exposed to 10 and 20 mmol/L CYT387were significantly more resistant (28% and 36.4% inhibi-tion of pSTAT5, respectively) than polycythemia vera sam-ples (74% and 93% inhibition, P ¼ 0.003 for 10 mmol/L; P¼0.001 for 20mmol/L).Differences in response observed toINCB018424 were also significant (10 mmol/L; myelofibro-sis samples were 42% inhibited, whereas polycythemia verasamples were 72% inhibited, P ¼ 0.025). Taken together,these results suggest that peripheral blood neutrophils frompatients with myelofibrosis were intrinsically resistant toJAK2 inhibitors.

To begin to understand what, if any, coherence mightexist between terminally differentiated neutrophils andmore primitive progenitor compartments with respect tosignaling response, we measured pSTAT5 in CD15þ andCD34þ cells exposed to CYT387 and INCB018424 from apatient with acute myeloid leukemia that had evolved frompost-polycythemia vera myelofibrosis (Fig. 4). We foundthat CD34þ stimulation with GM-CSF results in a moreheterogeneous pSTAT5 signal, likely reflecting differences inGM-CSF receptor expression in this compartment (16), andthat the response to inhibition in CD34þ cells generallymirrors that in CD15þ cells.

The pattern of resistance is not correlated with JAK2V617F allele burden in myelofibrosis

To gain insight into a mechanism by which STAT3 andSTAT5 remain phosphorylated even in the presence ofupstream JAK2 inhibition, we investigated whether wecould detect a correlation between the frequency of JAK2V617F-mutant allele burden in samples from patients withthe mutation and response to drug. Overall, we found norelationship between allele frequency and level of targetinhibition in tested samples (Table 2 and SupplementaryFig. S2). Likewise, a similar (and near complete) pattern ofresistance was observed in a patient withmyelofibrosis whowas negative for the JAK2 V617F mutation. Thus, themutant to wild-type ratio of JAK2 does not seem to directlyaccount for the observed pattern of resistance. We did notethat in cells from patients without myelofibrosis, thereindeed seemed to be a significant direct correlation withhigher levels of JAK2 V617F and persistent signaling (P ¼0.04); although we interpret this cautiously, given thenumbers of samples (n¼ 2) from patients with lower alleleburdens.

Kalota et al.





Plasma components do not confer resistance to JAK2inhibitionWe next investigated the possibility that plasma compo-

nents might contribute to or confer resistance of myelofi-brosis samples to JAK2 inhibitors. We mixed cells from apatient with polycythemia vera (sample#: 2518) with plas-ma from patients with myelofibrosis (samples#: 2517 and2521) and evaluated inhibition of pSTAT5 (Fig. 5A–F). For agiven patient, response to CYT387 or INCB018424 as

measured by mean pSTAT5 fluorescence followed a similartrend regardless of plasma source, which suggested thatneither resistance to inhibition nor sensitivity to treatmentwas conferred by plasma components. We also analyzedbasal cytokine levels in plasma samples fromMPN samplesand normal donors. Plasma cytokines that signal throughJAK2 and/or are known to be altered in myelofibrosis weremeasured. No meaningful differences that could directlyexplain the differences in response were observed. For most

Side scatter

CD

15

Total STAT5

phospho S

tat5

Normal Donor 1 A

STAT5 (total)

STAT

5 (

pY

69

4)

10 µmol/L 20 µmol/L 50 µmol/L

GM-CSF (10 ng/mL)

CEP701 –+ + +

Unstimulated GM-CSF (10 ng/mL)

STAT3 (total)

STAT

3 (

pY

70

5)

G-CSF (100 ng/mL)

CEP701 –

– + + +

– 20 µmol/L 50 µmol/L

B

C

0 200 400 600 800 1,000

0

101

102

103

42.1

0 101

102

103

0

101

102

103

0.02 1.07

96.82.13

0 101

102

103

0

101

102

103

1.39 98.1

0.460.036

0 101

102

103

0

101

102

103

1.39 98.1

0.460.04

0 101

102

103

0

101

102

103

0.8 55

431.21

0 101

102

103

0

101

102

103

0 3.4

96.20.3

0 101

102

103

0

101

102

103

0 1.2

98.40.45

0 10 1 10 2 10 3

0

101

102

103

0.3 2.23

87.310.2

0 10 1 10 2 10 3

0

101

102

103

3.55 36.6

53.46.5

0 10 1 10 2 10 3

0

101

102

103

0.1 1.15

899.73

0 10 1 10 2 10 3

0

101

102

103

0.01 1.18

92.76.08

0 101

102

103

0

101

102

103

0.25 0.15

3.995.7

+

Unstained

Figure 1. Measurement of STAT5 and STAT3 phosphorylation in normal peripheral blood granulocytes. A, the gating strategy for isolating granulocytes basedon forward/side scatter properties and expression of CD15 antigen. Total (x-axis) and phospho- (y-axis) STAT5 (B) and STAT3 (C); detection of total STAT3 orSTAT5 served as an internal control for permeabilization. B and C, target inhibition in response to exposure to CEP701 at 10, 20, and 50 mmol/L doses.






of the 9 evaluated cytokines (MIP-1b, TNF-a, IL-6, GM-CSF,IFN-g , G-CSF, IFN-a, and Rantes), the observed concentra-tion in patients with MPN was higher than that in normaldonors, consistent with other reports (refs. 6, 17; Supple-mentary Fig. S3). In particular, the basal plasma levels of the2 cytokines also used exogenously in our experiments (G-and GM-CSF) were plotted against response to inhibitor,and there was no significant correlation between basal GM-CSF levels and response to inhibitor (P¼ 0.18, Supplemen-

tary Fig. S3B–S3D). These observations thus fail to show asoluble cell extrinsic factor that explains JAK2 inhibitorresistance in granulocytes from patients withmyelofibrosis.

DiscussionOur findings show that in myelofibrosis, cell intrinsic

properties bestow resistance to JAK2 inhibition, althoughthe mechanism remains unclear. We found that overall

A STAT5

Polycythemia vera (patient# 2317)

100

101

102

103

104

0

101

102

103

0 0.62

96.92.52

100

101

102

103

104

0

101

102

103

1.99 95.6

20.4

100

101

102

103

104

0

101

102

103

0.65 78.8

19.80.768

100

101

102

103

104

0

101

102

103

0.02 4.2

94.91

STAT5 (total)

STAT

5 (

pY

694)

GM-CSF (10 ng/mL)

CEP701+ + +-

- - 10 µmol/L 20 µmol/L

B STAT3

Primary myleofibrosis (patient# 2316)GM-CSF (10 ng/mL)

CEP701

+ + +- - - 10 µmol/L 20 µmol/L

STAT5 (total)

STAT

5 (

pY

694)

G-CSF (100 ng/mL)

CEP701

+ + +- - - 20 µmol/L 50 µmol/L

STAT3 (total)

STAT

3 (

pY

705)

100

101

102

103

104

0

101

102

103

9.92 84.1

4.231.86

100

101

102

103

104

0

101

102

103

0 1.7

94.53.85

100

101

102

103

104

0

101

102

103

6.49 80.1

11.12.39

100

101

102

103

104

0

101

102

103

7.25 60.8

27.14.88

100

101

102

103

104

0

101

102

103

0 0.87

954.13

100

101

102

103

104

0

101

102

103

2.29 56.8

391.94

100

101

102

103

104

0

101

102

103

0.6 34

64.40.9

100

101

102

103

104

0

101

102

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96.91.15

Polycythemia vera (patient# 2317)

Primary myleofibrosis (patient# 2316)

STAT3 (total)

STAT

3 (

pY

705)

G-CSF (100 ng/mL)

CEP701

+ + +- - - 20 µmol/L 50 µmol/L

10 0 10 1 10 2 10 3 10 4

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Figure 2. Relative resistance toinhibition of STAT5 and STAT3phosphorylation in myelofibrosis.Representative flow cytometryplots show level of inhibition ofSTAT5 (A) and STAT3 (B)phosphorylation after exposure toCEP701 for patients respectivelydiagnosed with polycythemia vera(top) and primary myelofibrosis(bottom).

Kalota et al.





polycythemia vera and essential thrombocythemia samplesare sensitive to inhibition by multiple investigational inhi-bitors, with responses overall comparable with those ofnormal donors. In contrast, in cells from patients withtreatment-na€�ve myelofibrosis, STAT5 remained phosphor-ylated despite treatment with inhibitors, suggesting eitherincomplete upstream inhibition of JAK2 and/or alternativeinputs to STAT5 (and possibly STAT3) that are specific tomyelofibrosis, typically a more advanced and geneticallycomplex disease. Our studies did not support a role forspecific cell extrinsic soluble factors in plasma as majormediators of resistance, nor could we correlate the clonalburden of JAK2 V617F with the resistance phenotype,suggesting that additional acquired features specific tomye-lofibrosis mediated the attenuated response.Our signaling studies focused largely on mature, termi-

nally differentiated neutrophils from patients, an experi-mental design conceived with the intent of developing afeasible peripheral blood-based pharmacodynamic assayfor measuring response to treatment. But whether ourobservation in neutrophils extends to signaling in more

primitive, disease-initiating compartments is also of greatinterest. Our exploratory studies of CD34þ blasts andneutrophils from a patient with acute leukemia evolvedfrom post-polycythemia vera myelofibrosis showed aresponse thatmirrors the response in granulocytes, support-ing the possibility that a signaling phenotype carriesthrough to terminal differentiation, but additional studiesare needed to draw major conclusions about signalingresponses throughout differentiation.Of note, other studieshave confirmed that granulocytes from patients with mye-lofibrosis are derived largely from themalignant clone (18).Although our studies cannot directly distinguish signalingwithin clonally derived granulocytes versus those derivedfrom the premalignant stem cell pool, the signaling patternsinmyelofibrosis tended to be relatively uniform, andwouldbe expected to represent, at least proportionally, clonallyderived cells. In contrast, clonal dominance is not thepredominant pattern in polycythemia vera and essentialthrombocythemia, and this may explain the overlappingsignaling phenotype among polycythemia vera, essentialthrombocythemia, and normal donors (18). Although we

B

0 20 40 600

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Dose of CEP701 (µmol/L)

Normal donors

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PV/ET

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Figure 3. A,CD15þcells frompatientswithmyelofibrosis are significantlymore resistant to JAK2 inhibition thancells frompatientswith polycythemia vera (PV),essential thrombocythemia (ET), and normal controls. Graphs showmolecular response to various doses of JAK2 inhibitor, CEP701, in normal donors whencompared with samples from patients with polycythemia vera/essential thrombocythemia and myelofibrosis (MF). Molecular response to the drug ispresented as a percentage of STAT5 (left) and STAT3 (right) phosphorylation remaining after exposure to CEP701. Mean fluorescence from cells stimulatedwith GM-CSF for pSTAT5 or G-CSF for pSTAT3 minus background (mean fluorescence of unstimulated cells) was set as a 100%. B, resistance to inhibitioncan be generalized to additional JAK2 inhibitors under clinical development. Response to CYT387 (right) and INCB018424 (left) are plotted as the percentageSTAT5 phosphorylation after exposure to JAK2 inhibitors. MFI from cells stimulated with GM-CSF for pSTAT5 minus background (mean fluorescence ofunstimulated cells) was set as a 100%.






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CD15+ cells (patient# 2479)

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+50 µmol/L

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Figure 4. Coherence between terminally differentiated and progenitor signaling response. Measurement of pSTAT3 and pSTAT5 in CD15þ andCD34þ cells exposed to INCB018424 (top) and CYT387 (bottom) from a patient with acute myeloid leukemia evolved from post-polycythemia veramyelofibrosis.

Kalota et al.





found no direct correlation between JAK2 V617F alleleburden and resistance in myelofibrosis, but the samplesfrom patients with essential thrombocythemia or polycy-themia vera with higher JAK2 V617F seemed to be less

sensitive to treatment than samples with lower allele bur-dens. Taken together, one wonders whether clonal domi-nance in a bulk population might influence probability ofindividual cell signaling.

Figure 5. Plasma components donot alter sensitivity to JAK2 inhibitor.Effect of plasma components onresponse to CYT387 (A–C) orINCB018424 (D–F) as measured bySTAT5 phosphorylation andpresented as MFI. Peripheral bloodcells from 2 patients withmyelofibrosis (2521, A and 2517, B)were treated ex vivowith GM-CSF inthe presence or absence of CYT387,as whole, unperturbed blood (WB),as plasma-depleted bloodreconstituted with native plasma(e.g., 2521 þ 2521), or withpolycythemia vera patient (2518)plasma of equivalent volume(e.g., 2521 þ 2518). Likewise,polycythemia vera peripheral bloodwas exposed to CYT387 (C) orINCB018424 (F) as whole blood,native reconstituted blood, or withplasma from either myelofibrosispatient (2518 þ 2517 and 2518 þ2521). Myelofibrosis blood cells(2517, D and 2521, E) were treatedwith GM-CSF with or withoutINCB018424 in native plasma orpolycythemia vera plasma.

CYT387

MF cells in PV plasma

0

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60

80

2517 WB

2517+2517

2517+2518

Mean flu

ore

scence


Untreated GM-CSF 1 µmol/L 10 µmol/L

Untreated GM-CSF 1 µmol/L 10 µmol/L Untreated GM-CSF 1 µmol/L 10 µmol/L

Untreated GM-CSF 1 µmol/L 10 µmol/L Untreated GM-CSF 1 µmol/L 10 µmol/L

Untreated GM-CSF 1 µmol/L 10 µmol/L 0

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ore

scence

CYT387

Mean flu

ore

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2521+2521

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INCB018424

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B E


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scence

INCB018424

D


PV cells in MF plasma

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2518+2521

CYT387

Mean flu

ore

scence

INCB018424

Mean flu

ore

scence

PV cells in MF plasma

0

20

40

60

80

2518 WB

2518+2518

2518+2517

2518+2521

C F

Table 2. Level of mutant JAK2 V617F allele frequency in granulocytes does not correlate with response toinhibition of phosphoSTAT5

Stratified by JAK2 V617F allele freq Mean allele freq (%) Mean % pSTAT5 at 20 mmol/L n

All patients with JAK2 V617F mutation<50% 17 49.6 5>50% 74 48.6 11

Polycythemia vera/essential thrombocythemia<50% 31 4.05 2>50% 75 35.74 7

Myelofibrosis<50% 8 80.03 3>50% 73 71.02 4






Acquired resistance to targeted therapies has been welldescribed (19), although classical escape mutations do notseem to be a major mechanism of resistance to JAK2inhibitors. Perhaps unique features of JAK2 as a target leadto less selective pressure for outgrowthof resistant clones, sothat classical gatekeeper mutations as seen in chronic mye-loid leukemia would not be favored: JAK2 is absolutelyrequired for normal hematopoiesis, and consequentlydefines a more narrow therapeutic window between dis-eased, JAK2 dysregulated and normal cells. Elegant exper-imental engineered ex vivo systems of chronic drug exposurehave likewise shown that resistance mutations do notaccount for persistent (or reactivated) downstream signal-ing in JAK2 V617F cells that grow despite the presence ofinhibitor (20). Rather, signaling in trans of JAK2 by JAK1(and TYK2) seems to mediate a novel mechanism of druginsensitivity that seems to be epigenetically determined atthe JAK2 locus itself. Our data are distinguished from thesein that the cells have not been chronically exposed to drugbut instead are derived from treatment-na€�ve patients andyet our observationsmay have similar underpinnings. Mye-lofibrosis is increasingly recognized as an entity governed byepigenetic dysregulation that confers competitive advantageto the diseased stem cell compartment, an advantage withwhich JAK2 V617F itself may conspire (21, 22), but wherethe mutation alone cannot confer such an advantage (23).

We used pSTAT5 as a surrogate for activation of the JAK2pathway. One limitation of this strategy is that we cannotexclude the possibility that JAK2 was appropriately inhib-ited in myelofibrosis samples, whereas STAT5 remainedactivated in response to other signaling pathways. We werenot able to directly detect JAK2/phospho-JAK2 in primaryMPN samples. Future studies using other strategies willaddress whether novel heterodimers can be detected inprimary cells, and if present, whether they correlate with

persistent activationof STAT3andSTAT5.Ourmethods alsocould not specifically address microenvironmental factorsknown to play an important role in drug response andresistance, which might carry through in progeny of dis-eased stem/progenitor cells (24). Of particular interest iswhether terminally differentiated cell signaling in a clonalpopulation faithfully recapitulates that of more primitiveancestors within the stem/progenitor cell compartment.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A. Kalota, E.O. HexnerDevelopment of methodology: A. Kalota, M. Carroll, E. O. HexnerAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): A. Kalota, G.R. Jeschke, E.O. HexnerAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): A. Kalota, G.R. Jeschke, M. Carroll, E.O.HexnerWriting, review, and/or revision of the manuscript: A. Kalota, G.R.Jeschke, M. Carroll, E.O. HexnerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A. Kalota, E.O. HexnerStudy supervision: A. Kalota, E.O. Hexner

AcknowledgmentsThe authors thank Joy Cannon (StemCell and Xenograft Core, University

of Pennsylvania) for coordination of samples and Gerald Wertheim for helpwith qPCR calculations.

Grant SupportM. Carroll has previously received research funding from Cephalon

Oncology. E.O. Hexner is supported by an NIH grant (K23-HL-093366-01A1) and American Society of Hematology Scholar (fellow) in ClinicalTranslational Research.

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked advertise-ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 18, 2012; revised January 9, 2013; accepted January 30,2013; published OnlineFirst February 5, 2013.

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2013;19:1729-1739. Published OnlineFirst February 5, 2013.Clin Cancer Res Anna Kalota, Grace R. Jeschke, Martin Carroll, et al. Intrinsic Resistance to JAK2 Inhibition in Myelofibrosis

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