The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation

Abstract

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 hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had 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. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.

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 α expression. (A) Quantitative hematopoietic progenitor analysis. FACS analysis of primitive progenitors such as HSC and more committed progenitors including CMP, GMP, MEP, IL-3Rα⁺⁺CD45RA⁻, and IL-3Rα⁺⁺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 peripheral blood samples (Normal; n = 4). PV associated with leukocytosis and/or splenomegaly (PV high WBC counts; n = 3) was characterized by further expansion of the 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 α-high CD45RA⁻ (P = 0.04, statistically significant by unpaired two-tailed Student's t test, advanced PV versus Normal), and IL-3 receptor α-high CD45RA⁺ (P = 0.02, statistically significant by unpaired two-tailed Student's t test, advanced PV versus Normal) populations compared with normal peripheral blood samples (Normal). (B) PV versus normal peripheral blood progenitor profiles. Representative FACS progenitor profiles, obtained with the aid of a modified FACSVantage and flojo software, demonstrating that the percentage of the CD34⁺CD38⁺ lineage⁻ fraction composed of myeloid progenitors, including CMP, GMP, MEP, and IL-3 receptor α-high (IL-3-high) cells, in PV peripheral blood (Left) versus normal peripheral blood (Right).

Fig. 2.

The JAK2 V617F mutation occurs in PV HSC, is transmitted without alteration in mutant allele frequency to committed progenitors, and predisposes PV HSC toward erythroid differentiation. (A) Quantitative analysis of differentiation potential of normal versus PV HSC in vitro. HSC (CD34⁺CD38⁻CD90⁺Lin⁻) 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 with the aid of a modified FACSVantage 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 inverted microscope 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 PV HSC. (Upper) Representative phase-contrast photomicrographs of colonies derived from FACS-sorted HSC revealed increased erythroid differentiation potential of PV HSC 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 HSC colonies derived from normal (n = 4) or PV samples (n = 4) demonstrated that normal HSC colonies lacked the G → T mutation at nucleotide 1849 (black), whereas PV HSC colonies frequently harbored this JAK2 mutation (red), resulting in the valine-to-phenlyalanine substitution at position 617 (V617F). (C) PV HSC and their progeny harbor the JAK2 V617F mutation. Sequencing analysis revealed that PV peripheral blood and bone marrow samples derived from six of six patients with the JAK2 V617F mutation in their peripheral blood mononuclear cells harbored the same G → T mutation (black arrow) at nucleotide 1849 of JAK2 (red) in HSC as well as their progeny including 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 the absence 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 PV progenitor-derived colonies from patients with the JAK2 mutation in peripheral blood mononuclear cells harbored the JAK2 V617F mutation. Results are expressed as the number of JAK2− and JAK2+ colonies out of the total number of colonies analyzed that were derived from HSC, CMP, GMP, and MEP.

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 as CFU-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. A two-tailed unpaired Student's t test performed with excel software revealed that there was no statistically significant difference in BFU-E colony formation by normal peripheral blood HSC before or after AG490 treatment (P = 0.67), whereas normal bone marrow and cord blood derived HSC appeared to be more sensitive to the effects of 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 no statistically significant differences in the proportions of HSC-derived CFU-GM, CFU-G, CFU-Mega, or CFU-M before or after AG490 treatment. (B) Effect of JAK2 inhibition on PV HSC differentiation potential in vitro. Hematopoietic progenitor assays were performed on HSC, CMP, or MEP derived from PV bone marrow or peripheral blood samples (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, and CFU-E as well as CFU-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. A two-tailed unpaired Student's t test performed with excel software revealed that there was a statistically significant difference in the number 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 after the 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 HSC or other progenitor populations before or after AG490 treatment. (C) Qualitative assessment of JAK2 inhibition in normal versus PV HSC. The effect of JAK2 inhibition with AG490 (50 μM) on normal versus PV HSC in vitro differentiation potential was assessed. Representative phase-contrast photomicrographs were obtained with a Nikon Eclipse TS100 microscope and spot software. (Magnification: ×100.) Normal (Upper) or PV (Lower) HSC were FACS-sorted onto methylcellulose supplemented with or without AG490 (50 μM) 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 four patients with PV (PV1–4) before and after in vitro treatment with AG490 (50 μM), a JAK2 inhibitor. This analysis revealed that the proportion of HSC colonies harboring the JAK2 V617F mutation (JAK2+) as opposed to those without the JAK2 V617F mutation (JAK2−) varies among PV patients and that the JAK2 V617F mutation persists despite 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 colonies derived from HSC from each individual before and after in vitro AG490 treatment. (E) PV HSC and their progeny harbor the JAK2 V617F mutation. Sequencing analysis revealed that PV samples from patients with the JAK2 V617F mutation in their peripheral blood mononuclear cells harbored the same G → 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 μM).