Effects of the JAK2 mutation on the hematopoietic stem and progenitor compartment in human myeloproliferative neoplasms

The human myeloproliferative neoplasm (MPN), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis are associated with mutations of signaling molecules, including the JAK2 V617F mutation, present in the majority of MPN patients.^1-4 JAK2 mutations are present in the hematopoietic stem cells (HSCs) from which the MPNs arise and are sufficient to produce MPNs in animal models.^2,5-9  Recently, several groups have reported murine models of JAK2 V617F MPN.^10-14  However, the effects of JAK2 V617F expression on hematopoietic stem and progenitor cell (HSPC) homeostasis were not consistent, with the reports of Mullaly et al¹⁴  and Li et al¹²  suggesting a neutral or detrimental effect on HSPC compartment size and/or function, whereas Akada et al reported HSPC expansion.¹¹  In MPN patients, the consequences of JAK2 V617F expression for HSPC compartment homeostasis and later myeloid differentiation are largely unknown. An understanding of the consequences of JAK2 V617F expression is important, as it will not only inform the biology of the disease but may also predict for the ability of JAK2 inhibitors to simply control or eradicate disease. In this paper, we assess the effects of JAK2 mutations on the homeostasis of the human HSPCs and later myeloid differentiation compartments, using a large cohort of MPN patients.

Institutional Ethics Committee approval from the Cambridge Institute of Medical Research was obtained in writing for recruited patients. Diagnosis was defined according to World Health Organiztion criteria. All material analyzed was bone marrow apart from 5 myelofibrosis (MF) patients where this was unavailable and peripheral blood (PB) was analyzed (supplemental Table 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article).

Staining and sorting for HSPC compartments were performed as previously described.¹⁵ 

JAK2 V617F genotyping and allele quantification were performed on individual colonies derived from HSPC compartments by pyrosequencing as previously described.¹⁶ 

Quantitative real-time PCR was performed with a Stratagene Mx3000P, using the ΔΔC_t formula with ABL1 and GUSB as the housekeeping control genes.

In patients with homozygous JAK2 V617F colonies, analysis was carried out using SNPs close to the JAK2 locus. Regions around the single nucleotide polymorphisms (SNPs) were amplified by PCR (primers available on request) to determine loss of heterozygosity and the proportion of hematopoiesis with a homozygous JAK2 V617F allele. In informative patients, the allele proportion for JAK2 V617F and for each SNP haplotype was quantitated by pyrosequencing. Comparison of the JAK2 V617F allele and SNP proportions was then used to calculate the proportion of JAK2 V617F homozygous and heterozygous cells (proportion of homozygous cells x = 2 [a − 0.5] where a is the proportion of the dominant SNP haplotype; and proportion of heterozygous cells y = 2 [% JAK2 V617F − x] (see Figure 2; supplemental Figure 2A).

Graphs and the indicated statistical analyses were generated in Prism Version 5.01 (GraphPad software).

We first assessed the size of the stem cell, individual myeloid progenitor, and later myeloid differentiation compartments in 41 samples from patients with JAK2-mutated PV, ET, or MF and 5 normal control bone marrow samples. No differences were detected between the sizes of the HSC-enriched (Lin⁻/CD34⁺/CD38⁻), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor, and granulocyte-monocyte progenitor (GMP) compartments between normal and ET or PV patients (Figure 1A; supplemental Figure 1A-B). In contrast, MF patients demonstrated enlarged HSC and GMP compartments compared with normal controls, with this increase reaching statistical significance compared with ET and PV patients (Figure 1A; supplemental Figure 1A). This increase was apparent for both PB and bone marrow from MF patients (supplemental Figure 1C). We also assessed the ability of purified HSC, CMP, megakaryocyte-erythroid progenitor, and GMP to proliferate and differentiate in vitro, measuring colony number, size, and composition from MPNs and normal HSPC populations. Only minimal differences between PV, ET, and MF patients and controls were demonstrated (Figure 1B-C; supplemental Figure 1D). Our data therefore suggest that the JAK2 V617F mutation has minimal effects on the size or in vitro behavior of the HSPC compartments in PV and ET patients.

In contrast, when we assessed the size of the later, maturing erythroid CD34⁻/CD71⁺/GPA⁺ progenitor compartment (Figure 1D), we found a significant expansion for both PV and MF patients compared with controls. Similar expansion was documented in the CD41⁺ megakaryocytic progenitor compartment between normal and MPN patients (Figure 1D; supplemental Figure 1E), which reached statistical significance for PV patients. These data demonstrate an in vivo expansion of the maturing erythroid and megakaryocyte compartments in PV and suggest an expansion of the megakaryocyte compartment in ET patients.

To correlate total compartment size with the size of the JAK2 V617F clone, we next assessed the JAK2 V617F-allele burden upon myeloid differentiation (Figure 2A). ET patients demonstrated a relatively stable, low allele burden, whereas in PV and MF patients the allele burden was generally higher (Figure 2A). Allele burden increased commensurate with the terminal myeloid amplification evident in PV patients (P = .02). In contrast, in MF patients, the average mutant allele burden was already high in the HSC-enriched fraction and remained stable throughout differentiation. This finding is also in keeping with alternate regulation of the HSPC compartment in V617F-positive MF.

Total JAK2 V617F-allele burden does not distinguish between the effects of heterozygous and homozygous JAK2 V617F clones on myeloproliferation. To assess the role of JAK2 gene dosage in human MPNs, we devised a PCR-based pyrosequencing assay, which combined analysis of chromosome 9p SNPs with V617F allele burden, allowing us to quantify the contributions to hematopoiesis of the homozygous and heterozygous clones (supplemental Figure 2). In paired analysis of 6 individual PV patients with both homozygous and heterozygous clones (Figure 2B), the homozygous JAK2 V617F clone demonstrated a marked expansion from the HSC-enriched compartment to both the differentiated erythrocytic (P = .005) and granulocytic compartments (P = .01). However, no obvious expansion was demonstrated for the heterozygous clone in the same patients. Moreover, another 6 PV patients with only a heterozygous clone similarly lacked expansion of the later compartments, suggesting that abnormalities other than JAK2 V617F contribute to the terminal expansion in patients who lack a homozygous clone (Figure 2C).

Finally, to assess potential mechanisms explaining the terminal myeloid expansion associated with JAK2 V617F mutations, we assessed the expression pattern of total JAK2 upon myeloid differentiation. In normal and JAK2 V617F–positive patients, total JAK2 expression levels increased on myeloid maturation (Figure 2D). Therefore, although JAK2 is expressed in the HSPC compartment, increasing expression on differentiation may, at least in part, explain the greater phenotypic effect in later compartments that we have demonstrated. In addition, despite the small numbers analyzed, there were statistically significant differences in JAK2 levels between PV and MF patients and normal controls in certain compartments.

Our data suggest that JAK2 mutations do not significantly alter HSPC compartment size or in vitro behavior in PV and ET patients but predominantly exert their effects in later myeloid differentiation compartments. These data contrast with those of Jamieson et al, who demonstrated an increase in the size of the HSC and CMP compartments as well as differentiation skewed toward erythroid development in PV patients.¹⁵  However, the flow strategies differed slightly between these studies: our CD34⁺CD38⁻Lin⁻ fraction is HSC enriched, but less so than that of Jamieson et al,¹⁵  who used the same markers in combination with CD90.¹⁵  In addition, our larger study was conducted using samples obtained mainly from untreated patients at diagnosis and used predominantly bone marrow rather than PB samples. Moreover, our comparator was normal bone marrow and not normal PB. Indeed, PB from MPN patients has previously been demonstrated to contain an increased CD34⁺ fraction compared with normal PB.¹⁷  In contrast to PV and ET, the alterations in the size of the HSC and GMP population and the stable allele burden in JAK2 V617F–positive MF suggest an alteration in the regulation of the HSPC compartment through additional mechanisms, such as the presence of other mutations, microenvironment effects, and/or genetic modifiers. These findings are also in consonance with functional differences between human PV and MF V617F mutant HSCs as detected in xenotransplant experiments.¹⁸  Furthermore, the in vivo terminal expansion we demonstrate entirely corroborates the results in murine models^12-14  and human MPNs in vitro, where PV and ET patients demonstrated erythroid expansion compared with normal controls after long-term CD34⁺ cell culture experiments.¹⁹ 

We also demonstrate, using a novel PCR-based assay, that in individual patients the JAK2 V617F homozygous clone has a competitive advantage over the heterozygous clone and that the heterozygous clone does not significantly expand in vivo. Recent animal models have suggested that gene dosage of JAK2 V617F determines disease phenotype, with higher expression associated with PV and lower expression with an ET phenotype.^7,10,11  Our data link the presence or absence of homozygous clones²⁰  and the proliferative advantage associated with the homozygous clone with the eventual phenotype in human MPN. We also propose a possible explanation, at least in part, for these findings by the expression pattern of JAK2, which increases on myeloid differentiation. Moreover, our data raise the intriguing possibility that JAK2 expression is higher for MPN patients compared with normal controls, perhaps related to the allele burden of the JAK2 V617F mutation. Taken together, our findings inform the biology of the MPN and also have clinical relevance, as they predict that JAK2 inhibitors may have limited efficacy in eradicating the leukemic stem cells that sustain human MPNs.

The authors thank Simon McCallum and Anna Petrunkina-Harrison for expert flow sorting.

This work was supported by the Medical Research Council (United Kingdom), Senior Clinical Research Fellowship (B.J.P.H.), the Kay Kendall Leukemia Fund, Cancer Research United Kingdom, and the Leukaemia and Lymphoma Research. B.J.P.H. and A.R.G. were supported by the Leukemia & Lymphoma Society (SCOR grant) and the National Institute for Health Research Cambridge Biomedical Research Center. C.A.O. was supported by the Deutsche Forschungsgemeinschaft (fellowship).

Contribution: B.J.P.H. devised and oversaw the project; B.J.P.H. and S.A. designed the experiments and wrote the paper; S.A. and F.S. performed experiments; E.G., P.B., and C.A.O. provided clinical data; W.E. and A.B. provided some patient material; and A.R.G. provided patient material and valuable advice.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Brian J. P. Huntly, Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Hills Road, Cambridge, CB2 0XY, United Kingdom; e-mail: bjph2@cam.ac.uk.