Distinct roles for long-term hematopoietic stem cells and erythroid precursor cells in a murine model of Jak2V617F-mediated polycythemia vera

Abstract

In the current model of the pathogenesis of polycythemia vera (PV), the JAK2V617F mutation arises in hematopoietic stem cells (HSCs) that maintain the disease, while erythroid precursor populations expand, resulting in excessive red blood cell production. We examined the role of these specific cell populations using a conditional Jak2V617F knockin murine model. We demonstrate that the most immature long-term (LT) HSCs are solely responsible for initiating and maintaining the disease in vivo and that Jak2V617F mutant LT-HSCs dominate hematopoiesis over time. When we induced Jak2V617F expression in erythropoietin receptor expressing precursor cells, the mice developed elevated hematocrit, expanded erythroid precursors, and suppressed erythropoietin levels. However, the disease phenotype was significantly attenuated compared with mice expressing Jak2V617F in LT-HSCs. In addition to developing a PV phenotype, all mice transplanted with Jak2V617F LT-HSCs underwent myelofibrotic transformation over time. These findings recapitulate the development of post-PV myelofibrosis in human myeloproliferative neoplasms. In aggregate, these results demonstrate the distinct roles of LT-HSCs and erythroid precursors in the pathogenesis of PV.

Figure 1

Jak2V617F disease-initiating cells are contained exclusively in the LT-HSC compartment. (A) The percentage of donor chimerism assessed in peripheral blood of lethally irradiated WT recipients of Jak2^+/VFE2Acre⁺ LT-HSCs or a combination of Jak2^+/VFE2Acre⁺ ST-HSCs and MPPs (mean ± SEM, n = 3-5 in each group). (B) Hematocrit of recipients of Jak2^+/VFE2Acre⁺ LT-HSCs or a combination of Jak2^+/VFE2Acre⁺ ST-HSCs and MPPs (mean ± SEM, n = 3-5 in each group). (C) WBC count of recipients of Jak2^+/VFE2Acre⁺ LT-HSCs or a combination of Jak2^+/VFE2Acre⁺ ST-HSCs and MPPs (mean ± SEM, n = 3-5 in each group). (D) Platelet count of recipients of Jak2^+/VFE2Acre⁺ LT-HSCs or a combination of Jak2^+/VFE2Acre⁺ ST-HSCs and MPPs (mean ± SEM, n = 3-5 in each group). (E) The percentage of Jak2^+/VF(VF) donor chimerism assessed in CD150⁺CD48⁻ LSK cells (LT-HSCs) from mice transplanted with Jak2^+/VF LT-HSC cells or Jak2^+/VF ST-HSC + MPP cells (mean ± SEM, n = 3-4 in each group).

Figure 2

Jak2V617F MPN-initiating cells dominate hematopoiesis over time. (A) The percentage of Jak2^+/VF (VF) donor myeloid chimerism assessed in peripheral blood (PB) from lethally irradiated secondary recipients of Jak2^+/VFE2Acre⁺ and Jak2^+/+E2Acre⁺ LSK cells in 75:25, 50:50, and 25:75 ratios, respectively, measured 5-65 weeks posttransplantation (mean ± SEM; n = 2-6 in each group). (B) Jak2^+/VF (VF) to Jak2^+/+ (WT) chimerism ratios assessed in BM SLAM LSK, LSK, and LK compartments from lethally irradiated WT recipients of Jak2^+/VFE2Acre⁺ and Jak2^+/+E2Acre⁺ LSK cells in a 25:75 ratio, measured 65 weeks after transplantation (mean ± SEM; n = 2 in each group). (C) Histopathologic (HE) sections of BM and spleen (SPL) from representative mice transplanted with Jak2^+/VFE2Acre⁺ and Jak2^+/+E2Acre⁺ LSK cells in 50:50 or 25:75 ratios, demonstrating erythroid and megakaryocytic hyperplasia in both groups.

Figure 3

Erythroid lineage–restricted Jak2V617F expression results in an attenuated MPN phenotype. (A) Jak2 allele-specific PCR performed on Jak2^+/+ E2Acre⁺, Jak2^+/VFE2Acre⁺, and Jak2^+/VFEpoRCre⁺ LSK, MEP, or GMP cells purified from primary mice. Jak2WT and Jak2V617F refer to forward primers (a common reverse primer was used). NTC indicates no template control. Results demonstrate absent Jak2V617F expression in Jak2^+/+ cells, Jak2V617F expression in all Jak2^+/VFE2Acre⁺ cell populations and Jak2V617F expression in Jak2^+/VFEpoRCre⁺ MEP cells only. (B) WBC count, hematocrit, and platelet counts of age-matched Jak2^+/+E2Acre⁺, Jak2^+/+EpoRcre⁺, Jak2^+/VFEpoRcre⁺, and Jak2^+/VFE2Acre⁺ mice aged 8-12 weeks (mean ± SEM; n = 4 in each group);*P < .05, **P < .0005. (C) Frequency of CD71⁺, Ter119⁺ erythroid precursors in spleen from age-matched Jak2^+/+E2Acre⁺, Jak2^+/+EpoRcre⁺, Jak2^+/VFEpoRcre⁺, and Jak2^+/VFE2Acre⁺ mice (mean ± SEM; n = 4 in each group); **P < .0005. (D) Serum EPO levels from age-matched Jak2^+/+E2Acre⁺, Jak2^+/VFEpoRcre⁺, and Jak2^+/VFE2Acre⁺ mice (mean ± SEM; n = 8 in each group); **P < .001. (E) Frequency of LSK and LK cells in BM from age-matched Jak2^+/+E2Acre⁺, Jak2^+/+EpoRcre⁺, Jak2^+/VFEpoRcre⁺, and Jak2^+/VFE2Acre⁺ mice (mean ± SEM; n = 4 in each group); *P < .05. (F) Frequency of CMP, GMP, and MEP cells in BM from age-matched Jak2^+/+E2Acre⁺, Jak2^+/+EpoRcre⁺, Jak2^+/VFEpoRcre⁺, and Jak2^+/VFE2Acre⁺ mice (mean ± SEM; n = 4 in each group); *P < .05. (G) The percentage of donor chimerism assessed in peripheral blood of recipients of Jak2^+/VFE2Acre⁺ or Jak2^+/VFEpoRcre⁺ unfractionated BM (mean ± SEM, n = 4 in each group). (H) Hematocrit of recipients of Jak2^+/VFE2Acre⁺ or Jak2^+/VFEpoRcre⁺ unfractionated BM (mean ± SEM, n = 4 in each group).

Figure 4

Mice transplanted with Jak2V617F mutant LT-HSCs develop MPN and myelofibrosis. (A) Histopathologic (HE) sections of BM from a representative mouse transplanted with Jak2^+/VF ST-HSC + MPP cells (n = 4 in group) or Jak2^+/VF LT-HSC cells (n = 3 in group), demonstrating diffuse erythroid hyperplasia and megakaryocyte hyperplasia in the Jak2^+/VF LT-HSC recipient. The Jak2^+/VF LT-HSC recipient also shows evidence of megakaryocytic clustering, enlarged forms with bizarre, hypernucleated nuclei, marked emperipolesis, and abnormal megakaryocyte localization in marrow sinuses or in association with trabecular bone. These abnormalities are not seen in the Jak2^+/VF ST-HSC + MPP recipient. (B) Reticulin sections of BM from a representative mouse transplanted with Jak2^+/VF ST-HSC + MPP cells (n = 4 in group) or Jak2^+/VF LT-HSC cells (n = 3 in group), demonstrating a fine reticulin fiber network with crossing fibers deposition consistent with grade 1-2 reticulin fibrosis in the Jak2^+/VF LT-HSC recipient. These abnormalities are not seen in the Jak2^+/VF ST-HSC + MPP recipient. (C) Histopathologic (HE) sections of spleen from a representative mouse transplanted with Jak2^+/VF ST-HSC + MPP cells (n = 4 in group) or Jak2^+/VF LT-HSC cells (n = 3 in group), demonstrating erythroid hyperplasia and megakaryocyte hyperplasia with clustering and atypical nuclear features in the Jak2^+/VF LT-HSC recipient. These abnormalities are not seen in the Jak2^+/VF ST-HSC + MPP recipient.