The JAK2 V617F mutation occurs in hematopoieticstem cells in polycythemia vera and predisposestoward erythroid differentiationCatriona H. M. Jamieson*, Jason Gotlib†, Jeffrey A. Durocher‡, Mark P. Chao†, M. Rajan Mariappan§, Marla Lay§,Carol Jones§, James L. Zehnder†§, Stan L. Lilleberg‡, and Irving L. Weissman§¶�

*Department of Medicine and Moores Cancer Center, University of California at San Diego, La Jolla, CA 92093; Departments of †Medicineand §Pathology, and ¶Institute for Stem Cell Biology and Regenerative Medicine and Comprehensive Cancer Center, Stanford UniversitySchool of Medicine, Stanford, CA 94305; and ‡Transgenomic, Inc., Gaithersburg, MD 20878

Contributed by Irving L. Weissman, February 27, 2006

Although a large proportion of patients with polycythemia vera(PV) harbor a valine-to-phenylalanine mutation at amino acid 617(V617F) in the JAK2 signaling molecule, the stage of hematopoiesisat which the mutation arises is unknown. Here we isolated andcharacterized hematopoietic stem cells (HSC) and myeloid progen-itors from 16 PV patient samples and 14 normal individuals, testingwhether the JAK2 mutation could be found at the level of stemor progenitor cells and whether the JAK2 V617F-positive cellshad altered differentiation potential. In all PV samples analyzed,there were increased numbers of cells with a HSC phenotype(CD34�CD38�CD90�Lin�) compared with normal samples. Hema-topoietic progenitor assays demonstrated that the differentiationpotential of PV was already skewed toward the erythroid lineageat the HSC level. The JAK2 V617F mutation was detectable withinHSC and their progeny in PV. Moreover, the aberrant erythroidpotential of PV HSC was potently inhibited with a JAK2 inhibitor,AG490.

JAK � signaling � progenitors � cell fate � mutant allele

Myeloproliferative disorders (MPDs), such as polycythemiavera (PV), are clonal hematopoietic disorders that share

several characteristics (1–6), including the propensity to transforminto myelofibrosis or acute myelogenous leukemia (7).

Clonal derivation of PV from a primitive hematopoietic progen-itor was first suggested by X chromosome linkage analysis thatidentified the same glucose-6-phosphate dehydrogenase allele inerythrocytes, granulocytes, and platelets of females with PV whowere heterozygous for glucose-6-phosphate dehydrogenase alleles(8–10). PCR of the X-linked phosphoglycerate kinase gene wassubsequently used to demonstrate the heterogeneity of clonalinvolvement of the myeloid and erythroid lineages in female PVpatients (11). Subsequently, long-term PV marrow culture studiesdemonstrated unregulated proliferation of neoplastic progenitors,suggesting that primitive PV progenitors had an intrinsic defect thatallowed them to bypass negative regulatory signals (12). However,the disease could have arisen in self-renewing HSC, in non-self-renewing downstream multipotent progenitors, or even in myelo-erythroid progenitors.

Several recent reports provided critical insight into the geneticlesions involved in the development of PV by identifying a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the Januskinase 2 (JAK2) tyrosine kinase gene in a substantial proportion ofpatients with PV as well as other MPDs, including essentialthrombocythemia and idiopathic myelofibrosis (refs. 13–17 andreviewed in ref. 18). This recurrent somatic mutation results inconstitutive activation of the JAK2 tyrosine kinase (15, 16). Ex-pression of the mutant JAK2 gene in the Ba�F3 factor-dependentcell line led to erythropoietin hypersensitivity and growth factor-independent survival (15). The in vivo relevance of the JAK2 V617Fmutation to PV was tested in a mouse bone marrow transplantmodel (15). Marrow cells transduced with the JAK2 mutant allele

led to erythrocytosis after transplantation into lethally irradiatedrecipients (15). However, the role of the JAK2 V617F mutation inhuman PV pathogenesis and the stage of hematopoiesis at which itis expressed remained to be determined.

In this study, we performed a targeted molecular analysis ofcells with a hematopoietic stem cell (HSC) phenotype(CD34�CD38�CD90�Lin�) (19–22) and myeloid progenitors (23,24) in PV patient samples to identify the hematopoietic progenitorcompartment that harbors the JAK2 V617F mutation and theirresponses to a JAK2 inhibitor, AG490 (25, 26).

ResultsPV Peripheral Blood Samples Have Increased Numbers of Cells with aHSC Phenotype. Phenotypic analysis of cells with a HSC phenotype(CD34�CD38�CD90�Lin�) and progenitor populations was per-formed with the aid of FACS in PV and normal peripheral bloodsamples (19–24). Analysis of early-phase PV patient samples (Table1) revealed increased numbers of cells with a HSC phenotype (Fig.1A). When compared with PV patients with no leukocytosis (PVwith normal WBC counts), patients who had PV with progressivemyeloproliferation characterized by increasing leukocytosis (PVwith high WBC counts) and splenomegaly had greater expansion ofthe HSC pool as well as an increase in the number of commonmyeloid progenitors (CMP) (Fig. 1A) and the appearance ofdistinctive IL-3 receptor �-high cells (Fig. 1 A and B).

PV Cells with a HSC Phenotype Exhibit Enhanced Erythroid Differen-tiation Potential. Hematopoietic progenitor assays revealed analteration in cell fate at the stem cell level in PV compared withnormal controls. There were both quantitative (Fig. 2A) andqualitative (Fig. 2B) differences in the colony types derived fromPV HSC compared with normal samples. PV HSC gave rise to apreponderance of large, abnormally shaped erythroid coloniescompared with normal controls (Fig. 2B). Direct sequencing anal-ysis of colonies derived from PV CD34�CD38�CD90�Lin� (HSC)

Conflict of interest statement: C.H.M.J. and I.L.W. have applied for U.S. patents entitled‘‘Methods of Identifying and Isolating Stem Cells and Cancer Stem Cells’’ and ‘‘Methods ofDiagnosing and Evaluating Blood Disorders’’ through the Stanford University Office ofTechnology and Licensing. I.L.W. receives consulting fees from and has equity ownership inCellerant Therapeutics (San Carlos, CA).

Freely available online through the PNAS open access option.

Abbreviations: HSC, hematopoietic stem cell; CMP, common myeloid progenitor; GMP,granulocyte�macrophage progenitor; MEP, megakaryocyte�erythrocyte progenitor; PV,polycythemia vera; MPD, myeloproliferative disorder; JAK2 V617F, Janus kinase 2 valine-to-phenylalanine mutation at amino acid 617; CFU, colony-forming unit; BFU, burst-forming unit; BFU-E, BFU-erythroid; CFU-G, CFU-granulocyte; CFU-M, CFU-macrophage;CFU-Mix, mixed colonies composed of granulocytes, erythrocytes, megakaryocytes, andmacrophages; CFU-Mega, CFU-megakaryocyte; CFU-GM, CFU-granulocyte�macrophage.

�To whom correspondence should be addressed at: Department of Pathology, 279 CampusDrive West, B257 Beckman Center, Stanford University School of Medicine, Stanford,CA 94305-5323. E-mail: irv@stanford.edu.

© 2006 by The National Academy of Sciences of the USA

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cells revealed that they harbored the JAK2 V617F mutation,suggesting that the JAK2 V617F mutation skewed differentiation ofHSC toward an erythroid fate (Fig. 2B).

The JAK2 V617F Mutation Occurs in PV Cells with a HSC Phenotype andTheir Progeny. Of the 16 PV peripheral blood and bone marrowmononuclear samples tested, 14 had the G 3 T mutation atnucleotide 1849 in exon 12 of the JAK2 gene, resulting in the V617Fmutation (Table 1). In six of six patients identified as having theJAK2 V617F mutation in the mononuclear cell fraction, a moretargeted molecular analysis demonstrated that the JAK2 V617Fmutation was also detectable in purified HSC (Fig. 2C) that weresorted in sufficient numbers for accurate mutation detection. Inpatients with HSC involvement by JAK2 V617F, the mutation couldalso be detected in the CMP in four of five patients analyzed, in the

granulocyte�macrophage progenitor (GMP) fractions in four offour patients analyzed, and in the megakaryocyte�erythrocyteprogenitor (MEP) fraction in four of four patients analyzed (Fig.2C). In addition, the JAK2 V617F mutation was found in the uniqueIL-3 receptor �-high fraction of a PV sample but was absent in allHSC and progenitor populations derived from normal samples(n � 5). The mutant allele frequency was similar between HSC andmore differentiated progeny, including the CMP, GMP, and MEPfractions (Fig. 2C), suggesting that the JAK2 V617F mutation isclonally transmitted by HSC to their progeny as had been suggestedby PCR and glucose-6-phosphate dehydrogenase analysis but notpreviously established by using FACS-purified HSC and myeloidprogenitors (8–11). Mutation analysis of colonies derived from PVHSC and committed progenitors revealed that not all coloniesderived from these progenitors contained the JAK2 V617F muta-

Table 1. Characteristics of patients with PV

Patientno. Age�sex

Diseaseduration,

yearsWBCs per

mm3 Hb (g�dL)�Hct (%)Plateletsper mm3

Disease-specifictreatment at time ofsample evaluation

Samplesource

JAK2 V617Fmutation status

(PB)

1 43�M 7 19,200 14.7�44.8 687,000 PHB, HU PB �

2 59�F 6 14,400 13.3�41.8 248,000 PHB PB �

3 62�M 11 56,300 12.4�41.7 903,000 PHB, HU PB�BM �

4 68�F 0.5 9,200 16.4�51.6 511,000 HU PB �

5 47�F 3 7,300 13.4�42.8 567,000 PHB PB �

6 63�M 2 7,400 20.3�60.6 194,000 PHB PB �

7 68�F 1 12,600 16.2�48.9 734,000 PHB PB �

8 30�M 2 7,300 13.8�40.8 244,000 PHB PB �

9 65�F 1.5 15,000 15.5�49.5 666,000 PHB PB �

10 48�M 0.75 19,300 16.8�51.5 230,000 PHB PB �

11 61�F 0.5 10,800 16.1�49.4 1,302,000 PHB, HU PB �

12 46�M 15 4,100 14.6�43.9 308,000 PHB, HU PB �

13 68�M 1 3,000 18.7�57.6 461,000 PHB PB �

14 80�M 15 15,000 12.3�40.4 853,000 PHB, HU PB �

15 70�F 5.5 3,700 12.2�36.5 558,000 PHB, HU PB �

16 76�F 6.5 4,500 11.2�32.0 24,000 BUS* PB �

M, male; F, female; WBC, white blood cells; Hb, hemoglobin; Hct, hematocrit; PB, peripheral blood; BM, bone marrow; PHB, phlebotomy; HU, hydroxyurea;BUS, Busulfan.*Administered up to 2 months before sample evaluation.

Fig. 1. FACS-based progenitor profiling analysis demonstrated an increase in HSC in PV as well as a distinctive cell population with high IL-3 receptor �

expression. (A) Quantitative hematopoietic progenitor analysis. FACS analysis of primitive progenitors such as HSC and more committed progenitors includingCMP, GMP, MEP, IL-3R���CD45RA�, and IL-3R���CD45RA� cells revealed a statistically significant increase in the number of HSC per 105 mononuclear cells (P �0.011, two-tailed Student’s t test, early PV versus Normal) in PV patients with normal WBC counts (PV normal WBC counts; n � 7) compared with normal peripheralblood samples (Normal; n � 4). PV associated with leukocytosis and�or splenomegaly (PV high WBC counts; n � 3) was characterized by further expansion ofthe stem cell compartment (P � 0.006, statistically significant by unpaired two-tailed Student’s t test, advanced PV versus Normal) as well as an increase in CMP(P � 0.006, statistically significant by unpaired two-tailed Student’s t test, advanced PV versus Normal), IL-3 receptor �-high CD45RA� (P � 0.04, statisticallysignificant by unpaired two-tailed Student’s t test, advanced PV versus Normal), and IL-3 receptor �-high CD45RA� (P � 0.02, statistically significant by unpairedtwo-tailed Student’s t test, advanced PV versus Normal) populations compared with normal peripheral blood samples (Normal). (B) PV versus normal peripheralblood progenitor profiles. Representative FACS progenitor profiles, obtained with the aid of a modified FACSVantage and FLOJO software, demonstrating thatthe percentage of the CD34�CD38� lineage� fraction composed of myeloid progenitors, including CMP, GMP, MEP, and IL-3 receptor �-high (IL-3-high) cells,in PV peripheral blood (Left) versus normal peripheral blood (Right).

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tion (Fig. 2D), and therefore these mutant lineage cells cohabitatewith normal stem and progenitor cells in PV bone marrow andperipheral blood. Alternatively, it is possible that erythropoietin-independent marrow cells without the JAK2 V617F mutationcoexist with JAK2 V617F-positive cells in PV.

Inhibition Assays with the JAK2 Inhibitor AG490. To ascertainwhether mutant JAK2 signaling played a role in the skeweddifferentiation potential of PV versus normal samples, hematopoi-etic progenitor assays were performed in the presence or absenceof AG490, a well characterized JAK2 inhibitor, at a dose (50 �M)that was reported to preferentially induce apoptosis in leukemiccompared with normal cells (25, 26). When purified normal HSC,CMP, and MEP were exposed to AG490 in hematopoietic pro-genitor assays, erythroid and mixed colony formation were partiallyinhibited, whereas myeloid colony formation was not affected (Fig.3A). In PV samples, however, there was a significant decrease in thenumber of mixed colonies and a diminution in erythroid coloniesderived from HSC (Fig. 3 B and C) after exposure to AG490,whereas the effects were not as dramatic for CMP and MEP (Fig.3B), suggesting an increased sensitivity of PV to JAK2 inhibition atthe stem cell level. Mutation analysis of HSC-derived colonies from

four patients with PV revealed that the JAK2 V617F mutationpersisted in some colonies that survived treatment with AG490(Fig. 3D). Further JAK2 V617F mutation analysis of individualHSC-derived colonies from two separate patients revealed differ-ences in individual patient sensitivity to JAK2 inhibition at the stemcell level, indicating that in some patients, HSC clones harboring theJAK2 V617F mutation are less sensitive to inhibition with AG490(Fig. 3E). These studies suggest that initially, PV patients have aproliferation of cells with a HSC phenotype, in part as a conse-quence of JAK2 V617F mutation expression, resulting in an in-crease in terminally differentiated progeny, whereas patients withevidence of progressive disease such as increasing WBC counts andsplenomegaly also have an expansion of the CMP compartment andthe production of a unique IL-3 receptor �-high population.

DiscussionWe have previously proposed (19, 27–30), and in some cases shown(23, 31), that, in the progression of myelogenous leukemias (and byanalogy many cancers), the self-renewing stem cell (here HSC)must be the first cell to sustain the genetic or epigenetic event thatinitiates the MPD, and in the myeloid lineage, only HSC self-renew(32, 33). However, it is possible that the first event, in some cases,

Fig. 2. The JAK2 V617F mutation occurs in PV HSC, is transmitted without alteration in mutant allele frequency to committed progenitors, and predisposes PV HSCtoward erythroid differentiation. (A) Quantitative analysis of differentiation potential of normal versus PV HSC in vitro. HSC (CD34�CD38�CD90�Lin�) derived fromnormal peripheral blood, bone marrow, or cord blood (Normal; n � 11) or PV (PV; n � 11) bone marrow or peripheral blood were FACS sorted with the aid of a modifiedFACSVantage onto 35-mm plates containing methylcellulose supplemented with recombinant human cytokines (Methocult GF� HH35; StemCell Technologies).Colonies including CFU-Mega, CFU-GM, CFU-G, CFU-M, BFU-E, and CFU-E as well as CFU-Mix colonies were scored with the aid of a Nikon Eclipse TS100 invertedmicroscope on day 14. Results are expressed as the number of colonies per 100 cells plated. (B) Qualitative analysis of altered in vitro differentiation potential of PVHSC. (Upper) Representative phase-contrast photomicrographs of colonies derived from FACS-sorted HSC revealed increased erythroid differentiation potential of PVHSC compared with normal HSC. (Magnification: �100.) HSC derived from normal peripheral blood, bone marrow, or cord blood (Normal; n � 11) or PV (PV; n � 11)bone marrow or peripheral blood were FACS-sorted onto methylcellulose supplemented with cytokines. (Lower) Mutation analysis performed on individual HSCcolonies derived from normal (n � 4) or PV samples (n � 4) demonstrated that normal HSC colonies lacked the G3 T mutation at nucleotide 1849 (black), whereas PVHSC colonies frequently harbored this JAK2 mutation (red), resulting in the valine-to-phenlyalanine substitution at position 617 (V617F). (C) PV HSC and their progenyharbor the JAK2 V617F mutation. Sequencing analysis revealed that PV peripheral blood and bone marrow samples derived from six of six patients with the JAK2 V617Fmutation in their peripheral blood mononuclear cells harbored the same G3 T mutation (black arrow) at nucleotide 1849 of JAK2 (red) in HSC as well as their progenyincluding CMP in four of five patients, GMP in four of four patients, and MEP in four of four patients analyzed, indicating clonal transmission of the mutation. (D)Expression of JAK2 V617F in normal versus PV colonies. Sequencing analysis was performed to detect the presence of the JAK2 V617F mutation (JAK2�) versus theabsence of JAK2 V617F mutation (JAK2�) in normal (Normal; n � 3) and PV (PV; n � 4) HSC, CMP, GMP, or MEP colonies. This analysis revealed that not all PVprogenitor-derived colonies from patients with the JAK2 mutation in peripheral blood mononuclear cells harbored the JAK2 V617F mutation. Results are expressedas the number of JAK2� and JAK2� colonies out of the total number of colonies analyzed that were derived from HSC, CMP, GMP, and MEP.

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occurs in a progenitor that cannot self-renew. In that view, succes-sive clonal progeny from the altered HSC clone could accumulateprogression events, so that at the end of the process, a multiplyaltered clone could enable poorly regulated self-renewal by turningon the hematopoietic self-renewal pathway(s) (19). A corollary ofthat hypothesis is that the initiating events in these MPDs that can

progress to acute leukemias sustain the events in HSC. Previousstudies have demonstrated that PV arises from an oligopotent ormultipotent hematopoietic progenitor (8–12). Consistent with thisobservation, a previously unrecognized mutation at position 617 ofthe JAK2 signaling molecule (JAK2 V617F mutation) was detectedin individual colony-forming unit (CFU)-granulocyte�macrophage

Fig. 3. Aberrant PV HSC erythroid differentiation potential is inhibited but JAK2 V617F-positive colonies are not completely eliminated by AG490, a JAK2 inhibitor.(A) Effect of JAK2 inhibition on normal HSC differentiation potential in vitro. HSC, CMP, or MEP derived from normal bone marrow, peripheral blood, or cord blood(n � 7) were FACS sorted onto methylcellulose supplemented with or without AG490 in addition to cytokines. Colonies of CFU-G, CFU-M, BFU-E, and CFU-E, as well asCFU-Mix, were scored with the aid of a Nikon Eclipse TS100 inverted microscope on day 14. Results are expressed as the number of colonies per 100 cells plated. Atwo-tailed unpaired Student’s t test performed with EXCEL software revealed that there was no statistically significant difference in BFU-E colony formation by normalperipheralbloodHSCbeforeorafterAG490treatment(P�0.67),whereasnormalbonemarrowandcordbloodderivedHSCappearedtobemoresensitivetotheeffectsof AG490 with regard to erythroid colony-forming potential (P � 0.054), as was the mixed-colony-forming potential of normal samples (P � 0.063). There were nostatisticallysignificantdifferences intheproportionsofHSC-derivedCFU-GM,CFU-G,CFU-Mega,orCFU-MbeforeorafterAG490treatment. (B)EffectofJAK2inhibitionon PV HSC differentiation potential in vitro. Hematopoietic progenitor assays were performed on HSC, CMP, or MEP derived from PV bone marrow or peripheral bloodsamples (n � 9) that were FACS-sorted onto methylcellulose supplemented with or without AG490 in addition to cytokines. Colonies including CFU-Mega, CFU-GM,CFU-G,CFU-M,BFU-E,andCFU-EaswellasCFU-MixwerescoredwiththeaidofaNikonEclipseTS100 invertedmicroscopeonday14.Resultsareexpressedasthenumberof colonies per 100 cells plated. A two-tailed unpaired Student’s t test performed with EXCEL software revealed that there was a statistically significant difference in thenumber CFU-Mix derived from PV HSC before and after AG490 treatment (n � 0.027). Although there was a trend toward a reduction in PV HSC-derived BFU-E afterthe addition of AG490, it was not statistically significantly different (P � 0.17) from untreated controls. There was no reduction in other colony types derived from HSCor other progenitor populations before or after AG490 treatment. (C) Qualitative assessment of JAK2 inhibition in normal versus PV HSC. The effect of JAK2 inhibitionwith AG490 (50 �M) on normal versus PV HSC in vitro differentiation potential was assessed. Representative phase-contrast photomicrographs were obtained with aNikon Eclipse TS100 microscope and SPOT software. (Magnification: �100.) Normal (Upper) or PV (Lower) HSC were FACS-sorted onto methylcellulose supplementedwith or without AG490 (50 �M) in addition to cytokines. (D) Analysis of JAK2 V617F expression by normal versus PV HSC colonies before and after JAK2 inhibition.Sequencing analysis of JAK2 V617F mutation (JAK2�) expression was performed on HSC colonies derived from three normal individuals (Normal 1–3) versus fourpatients with PV (PV1–4) before and after in vitro treatment with AG490 (50 �M), a JAK2 inhibitor. This analysis revealed that the proportion of HSC colonies harboringthe JAK2 V617F mutation (JAK2�) as opposed to those without the JAK2 V617F mutation (JAK2�) varies among PV patients and that the JAK2 V617F mutation persistsdespite AG490 treatment in the HSC of three of four PV patients. Results are expressed as the number of JAK2� and JAK2� colonies of the total number of coloniesderived from HSC from each individual before and after in vitro AG490 treatment. (E) PV HSC and their progeny harbor the JAK2 V617F mutation. Sequencing analysisrevealed that PV samples from patients with the JAK2 V617F mutation in their peripheral blood mononuclear cells harbored the same G3 T mutation (black arrow)at nucleotide 1849 of JAK2 (red) in their HSC but that their HSC-derived colonies had different sensitivities to in vitro JAK2 inhibition with AG490 (50 �M).

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(CFU-GM), burst-forming unit (BFU)-erythroid (BFU-E), andendogenous erythroid colonies derived from a patient with essen-tial thrombocythemia. In three patients with MPDs, the JAK2mutation was identified in CD34� progenitor cells, with the mutantform predominating in a mixed clonality pattern with wild-typeJAK2 (34). However, in preliminary studies, JAK2 V7617F was notdetected in B or T lymphocyte fractions, allowing the possibilitythat the target population was not multipotent but myeloid-committed (13, 15, 34). In this study, direct sequencing analysis ofthe JAK2 V617F mutation in primitive hematopoietic progenitors,including HSC and their progeny, revealed PV to be a HSC disease,wherein the JAK2 V617F mutation alters the differentiation po-tential of HSC.

Our previous progenitor profiling studies involving chronic my-elogenous leukemia, a clonal disorder wherein the initial BCR-ABLtranslocation occurs at the level of HSC, showed that as the diseaseprogresses from a chronic MPD to blast crisis, there was anexpansion of the GMP pool (23). Progression was attributed, inpart, to aberrant acquisition of self-renewal potential as a result of�-catenin activation within the committed GMP population, as wellas increased proliferative capacity secondary to BCR-ABL ampli-fication (23). This study demonstrates that progenitor profilingcould provide critical insights into the stage of hematopoiesis atwhich key events involved in progression of MPDs, such as chronicmyelogenous leukemia, occur.

In this study, phenotypic and functional progenitor profilingtogether with targeted JAK2 sequencing analysis revealed fivepreviously unrecognized findings: (i) in PV, there is an increase inthe number of cells with a HSC phenotype, expansion of the CMPpool, and emergence of an IL-3 receptor �-high progenitor popu-lation with disease progression; (ii) there is a quantitative andqualitative alteration in differentiation potential of PV HSC; (iii)cells with a HSC phenotype, from the majority of PV samplestested, harbored the JAK2 V617F mutation which (iv) was trans-mitted in a clonal manner to more committed progenitors; and (v)the multilineage differentiation potential of PV HSC was moresusceptible to inhibition with a JAK2 antagonist, AG490, thannormal HSC.

Many scientific and clinical reports have demonstrated thathuman HSC, regardless of the tissue of origin, share the samephenotype (CD34�CD38�CD90�Lin�) and can be purified byFACS (19–22). In this study, we observed that the number of cellswith a HSC phenotype was increased in the peripheral blood ofpatients with JAK2 V617F-positive PV compared with normalsamples and that the in vitro differentiation capacity of PV wasprofoundly altered. These findings suggest that, in addition toenhanced proliferative capacity, one of the primary defects in PVmay be altered differentiation potential at the stem cell level.However, additional mutations are likely responsible for propaga-tion of JAK2 V617F-positive clones and expansion of the CMPcompartment, such as changes in survival, self-renewal, or replica-tive capacity that may hasten the progression of PV and evolutionto acute leukemia as has been demonstrated with other MPDs (23,28–30, 35–38).

We identified an IL-3 receptor �-high progenitor population thatwas unique to PV patients. Prior reports showed that PV marrowBFU-E, CFU-GM, and CFU-megakaryocyte (CFU-Mega) exhibitmarked hypersensitivity to recombinant IL-3. The mechanism wasnot found to caused by enhanced binding of recombinant IL-3 to itsreceptor (3, 39). The growth factors to which hematopoietic pro-genitors are hypersensitive in MPDs (e.g., IL-3, granulocyte�macrophage colony stimulating factor, erythropoietin, stem cellfactor, thrombopoietin, and insulin-like growth factor-1) all employJAK2 for signaling, and therefore, constitutive activation of theJAK2 signaling pathway may partly explain this observation (18,40). Functional analysis of IL-3 receptor signaling in the IL-3receptor �-high population should be undertaken to evaluate its

relevance to increased JAK2 tyrosine kinase activity, and morebroadly, to the pathogenesis of PV and other MPDs.

We used targeted JAK2 mutation screening of cells with a HSCphenotype and committed progenitors to confirm the clonal HSCorigin of PV. The JAK2 V617F mutation was identified in HSC aswell as their progeny including CMP, GMP, MEP, and the IL-3receptor �-high population. JAK2 V617F appears to be propagatedwithout diminution in mutant allele frequency from HSC to morecommitted hematopoietic progenitors. Analysis of a larger cohortof samples will be required to assess whether JAK2 mutant allelefrequency increases preferentially in more terminally differentiatedprogenitor populations and whether this contributes to the prolif-eration of particular lineages in different MPDs. Although inhibi-tion of JAK2 with AG490 decreased the aberrant erythroid po-tential of PV HSC, it did not eradicate all JAK2 V617F-positivecolonies and also exhibited inhibitory effects on the erythroidpotential of normal HSC but to a lesser extent than PV HSC. Thesedata suggest that a tyrosine kinase inhibitor with more specificityfor the JAK2 V617F protein will likely be required to preferentiallytarget JAK2 mutation-positive clones and to avoid inhibitory effectson normal erythropoiesis.

Materials and MethodsSamples. Peripheral blood and bone marrow samples (n � 16) weredonated by patients with PV. Normal bone marrow (n � 14) or cordblood (n � 2) and peripheral blood samples (n � 4) were providedby healthy volunteers. Samples were obtained with informed con-sent according to Stanford University Institutional Review Board-approved protocols. Normal bone marrow and cord blood sampleswere also purchased from All Cells (Berkeley, CA).

Huma HSC and Myeloid Progenitor Flow-Cytometric Analysis and CellSorting. Mononuclear fractions were extracted from peripheralblood or bone marrow after Ficoll density centrifugation accordingto standard methods (23, 24). Samples were analyzed fresh orsubsequent to rapid thawing of samples previously frozen in 90%FBS and 10% DMSO in liquid nitrogen. In some cases, CD34� cellswere enriched from mononuclear fractions with the aid of immu-nomagnetic beads (CD34� Progenitor Isolation Kit; MiltenyiBiotec, Auburn, CA).

Before HSC and myeloid progenitor FACS analysis and sorting,CD34-enriched cell populations or mononuclear cells were stainedwith lineage marker-specific phycoerythrin-Cy5-conjugated anti-bodies including CD2 (RPA-2.10), CD11b (ICRF44), CD20 (2H7),CD56 (B159), and GPA (GA-R2) from Becton Dickinson Pharm-ingen; CD3 (S4.1), CD4 (S3.5), CD7 (CD7-6B7), CD8 (3B5), CD10(5-1B4), CD14 (TUK4), and CD19 (SJ25-C1) from Caltag (SouthSan Francisco, CA); APC-conjugated anti-CD34 (HPCA-2; BectonDickinson Pharmingen); and biotinylated anti-CD38 (HIT2;Caltag) in addition to phycoerythrin-conjugated anti-IL-3 receptor� (9F5; Becton Dickinson Pharmingen) and FITC-conjugatedanti-CD45RA (MEM56; Caltag) followed by staining with strepta-vidin-Alexa Fluor 594 (Invitrogen) to visualize CD38-biotin-stained cells and resuspension in propidium iodide to exclude deadcells. The methodology was the same for HSC staining except thata phycoerythrin-conjugated anti-human CD90 antibody (BectonDickinson Pharmingen) was used instead of an anti-IL-3 receptor� antibody. Unstained samples and isotype controls were includedto assess background fluorescence. After staining, cells were ana-lyzed and sorted by using a modified FACSVantage (BectonDickinson Immunocytometry Systems) equipped with a 599-nmdye laser and a 488-nm argon laser. Double-sorted HSC wereidentified as CD34�CD38�CD90� and lineage-negative. CMPwere identified based on CD34�CD38�IL-3R��CD45RA�Lin�

staining, and their progeny including GMP were CD34�CD38�IL-3R��CD45RA�Lin�, whereas MEP were identified based onCD34�CD38�IL-3R��CD45RA�Lin� staining (16, 17, 23, 24).

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Hematopoietic Progenitor Assays. Normal and PV HSC(CD34�CD38�CD90�Lin�), CMP (CD34�CD38�IL-3R��-CD45RA�Lin�), GMP (CD34�CD38�IL-3R��CD45RA�Lin�),and MEP (CD34�CD38�IL-3R��CD45RA�Lin�) were sortedwith the aid of a FACSVantage directly onto 35-mm plates con-taining complete methylcellulose (GF � H4435; StemCell Tech-nologies, Vancouver) according to the manufacturer’s specifica-tions, with or without a 50 �M concentration of the JAK2 inhibitorAG490 (Tyrphostin B42; Calbiochem) (23–26). Colonies wereincubated in a 37°C 7% CO2 humidified incubator and scored onday 14 as CFU-Mix (mixed colonies composed of granulocytes,erythrocytes, megakaryocytes, and macrophages), BFU-E or CFU-erythroid (CFU-E), CFU-granulocyte (CFU-G), CFU-macro-phage (CFU-M), CFU-Mega, or CFU-GM (24). Phase-contrastphotomicrographs of colonies were obtained on day 14 with aNikon phase-contrast inverted microscope at �100 magnificationwith the aid of SPOT software.

JAK2 Mutation Screening. Mononuclear cells. JAK2 V617F mutationgenotyping was performed on peripheral blood or bone marrowmononuclear cells derived from patients with PV, as well as normalperipheral blood, bone marrow, and cord blood. Red blood cellswere lysed, and DNA was extracted with the QIAamp DNA BloodMini kit according to the manufacturer’s directions (Qiagen, Va-lencia, CA) and then stored at �80°C until amplification-basedtesting. Extracted DNA was prepared for JAK2 mutation analysis byLightCycler (Roche Applied Science, Indianapolis) methodology(see section B of Supporting Methods, which is published as sup-porting information on the PNAS web site).HSC- and progenitor-targeted JAK2 mutation analysis. Targeted JAK2V617F sequencing analysis was performed on cDNA derived fromFACS-sorted HSC, CMP, GMP, and MEP from normal peripheralblood, bone marrow, or cord blood versus PV peripheral blood orbone marrow. In some experiments, methylcellulose-containingcolonies (whole-plate) derived from individual progenitor popula-tions were resuspended in 1 ml of TRIzol (Invitrogen), RNA wasextracted, and cDNA was made and sequenced for the JAK2 V617Fmutation (see section C of Supporting Methods for information onPCR and JAK2 primers).Clonal JAK2 mutation analysis. To further investigate whether JAK2V617F occurred as a clonal mutation, sequencing analysis of JAK2was performed on individual colonies derived from HSC, CMP,GMP, and MEP populations with or without in vitro inhibition of

JAK2 with AG490. Individual colonies were plucked and resus-pended in 200 �l of RLT buffer supplemented with �-mercapto-ethanol (RNeasy kit; Qiagen) and frozen immediately at �80°C.Samples were thawed and RNA was extracted, followed by cDNApreparation and PCR amplification with JAK2-specific primers (seesection C of Supporting Methods).Mutation scanning and DNA sequencing. Mutation analysis of the JAK2cDNA PCR product was conducted by using fluorescent denaturinghigh-performance liquid chromatography (DHPLC) technologyand Surveyor mismatch cleavage analysis, both with the WAVE-HSSystem (Transgenomic). Aliquots of PCR product (3–15 �l) fromall samples were scanned for mutations by DHPLC, confirmed bySurveyor mismatch cleavage, and identified with bidirectionalsequence analysis on an ABI 3100 sequencer using BigDye V3.1terminator chemistry (Applied Biosystems). In addition, for semi-quantitative determination of mutant and normal allele frequen-cies, relative peak areas of denaturing high performance liquidchromatography elution profiles, and Surveyor mismatch cleavageproducts were determined after normalization and comparisonwith reference controls by using the WAVE NAVIGATOR software.

Statistical Analysis. Standard deviation, standard error of the mean,and numbers of HSC and progenitors per 105 mononuclear cellswere measured by using FLOJO (Treestar, San Carlos, CA) andMicrosoft EXCEL software. Two-tailed Student’s t test (EXCEL) wasused to analyze statistical differences in the number of differentprogenitors between PV patient samples and normal controls.

We thank Lenn Fechter and Drs. Steven Coutre, Stanley Schrier,Caroline Berube, and Lawrence Leung (all of Stanford University) forproviding samples from patients with PV; Drs. William Maloney, DerrickRossi, and David Bryder (all of Stanford University) for their assistancein obtaining and processing normal age-matched bone marrow samples;Libuse Jerabek for excellent laboratory management; the StanfordUniversity FACS facility for expert assistance; members of the StanfordUniversity Division of Hematology for their support; and the patientswho made this research possible. This work was supported by a StanfordCenter for Clinical Immunology Yu–Bechmann fellowship for Genomicsand Oncology and an Aplastic Anemia and Myelodysplastic SyndromeInternational Foundation Investigator Award (both to C.H.M.J.); theWalter and Beth Weissman Fund; the Smith Family Fund; NationalInstitutes of Health Grants CA086065 and CA86017 (to I.L.W.), K23HL04409 (to J.G.), and 2P01CA49605 (to C.J. and J.L.Z.); and a deVilliers grant from the Leukemia Society (to I.L.W.).

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