Evaluating Clonal Dominance in a Murine Knock-in Model of Jak2V617F MPN

Abstract

Abstract 614Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoiesis but the mechanisms of clonal dominance in these diseases are poorly understood. The JAK2V617F mutation is found in the majority of patients with MPN and is sufficient to confer the MPN phenotype. We recently described a Jak2V617F knock-in MPN model in which the mutation was expressed from the endogenous murine Jak2 promoter and the disease phenotype closely recapitulated human polycythemia vera (PV). In the model we found that the MPN-initiating population is contained within the CD150+CD48− LineagelowSca1+cKithigh(LSK) long-term hematopoietic stem cell (HSC) compartment, and showed that the MPN is cell autonomous and serially transplantable. In long-term competitive transplantation experiments we found that the Jak2V617F CD150+CD48− LSK population demonstrates gradual clonal expansion over time and we are now investigating the regulation of this primitive HSC population in vivo.Erythropoietin (EPO) signaling is reported to be the fundamental defect in polycythemia vera (PV). The JAK2V617F mutation is present in 95% PV patients and can be detected in the HSC compartment. We evaluated the role of EPO signaling in the JAK2V617F mutant HSC compartment using a conditional Jak2V617F knock-in murine model. Floxed Jak2+/VF mice were crossed with Vav Cre or erythropoietin receptor GFP Cre (ErGFPcre) mice resulting in Jak2V617F expression in all hematopoietic lineages or in erythroid restricted Jak2V617F expression respectively. Jak2V617F-ErGFPcre mice demonstrated elevated hematocrit, expanded committed erythroid progenitors and suppressed EPO levels but had an attenuated MPN phenotype as compared with Jak2V617F-Vavcre mice. Notably, the hematopoietic stem and progenitor cell (HSPC) compartment was not expanded in Jak2V617F-ErGFPcre mice and HSCs from both Jak2V617F-Vavcre and Jak2V617F-ErGFPcre mice did not activate phosphoStat5 signaling in response to EPO stimulation. These results indicate that expression of Jak2V617F in the HSC compartment is required for development of a full MPN phenotype and suggest that cytokine receptors other than the EPO receptor are important in mediating clonal dominance within the HSC compartment in JAK2V617F mediated MPN.TET2 is one of three TET gene family members that appear to play a role in DNA demethylation. Acquired TET2 deletions and loss-of-function mutations have been found across a broad spectrum of myeloid malignancies indicating that TET2 may drive a common pathogenic step in myeloid cancers, such as the establishment and/or enhancement of clonal dominance. Tet2 null HSCs have recently been shown to have a competitive repopulating advantage over wild-type HSCs in murine transplantation assays. TET2 loss-of-function mutations are found in approximately 12% of MPN patients, are often found co-mutated with JAK2V617F and have been associated with leukemic transformation of MPN. To evaluate the effects of loss of Tet2 function on the self-renewal and differentiation of Jak2V617F mutant HSCs, we crossed Jak2V617F knock-in mice with Tet2 conditional knockout mice. At 6 weeks of age, Jak2V617F/Tet2 +/− Vav Cre mice do not demonstrate significant differences in MPN phenotype as compared with Jak2V617F/Tet2+/+ Vav Cre mice, in terms of peripheral blood counts, hematopoietic stem and progenitor cell numbers or in colony formation. Additional studies using older mice and Jak2V617F/Tet2−/− Vav Cre animals are underway to further investigate the impact of loss of Tet2 function on Jak2V617F mutant HSCs.Understanding the mechanisms that contribute to clonal dominance in MPN will help facilitate the development of strategies to selectively target MPN stem cells therapeutically, and thereby advance the treatment of MPN patients. Disclosures:No relevant conflicts of interest to declare.