Characterizing the Disease-Initiating Cell Population In a Murine Model of Jak2V617F+ Myeloproliferative Neoplasm

Abstract 317

The JAK2V617F mutation is the most common somatic mutation in BCR-ABL negative myeloproliferative neoplasms (MPN) and small molecule JAK2 kinase inhibitors are currently being evaluated in clinical trials in MPN patients. We recently reported a Jak2V617F conditional knockin mouse MPN model in which expression of Jak2V617F was under the control of the endogenous Jak2 promoter and the phenotype closely recapitulated the features of human polycythemia vera (PV) (Mullally and Lane et al, Cancer Cell 2010). In this model the MPN phenotype developed as a result of expansion and erythroid skewing of committed myeloid progenitor populations, while the hematopoietic stem cell (HSC) compartment (enriched within Lineage^lowSca1⁺cKit^high, LSK cells) was not expanded. In contrast to the expanded progenitor population, only LSK cells had the unique capacity to initiate the MPN. Notably, treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. In light of these findings, we wished to further characterize MPN-initiating LSK cells in this model, with a view to gaining understanding of the differential molecular circuitry of JAK2V617F mutant and normal HSC. The LSK compartment, although enriched for HSC, may be further defined by immunophenotype into long-term HSC (LT-HSC, LSKCD150+CD48-), short-term HSC (ST–HSC, LSKCD150-CD48-), or multipotent progenitor cells (MPP, LSKCD150-CD48+). Gene expression analysis of total LSK cells revealed enrichment for markers of myeloid progenitor differentiation in Jak2V617F LSK cells compared to Jak2 wild-type (WT) controls, although there were no quantitative differences in the LSK sub-populations between the two groups. We therefore sought to evaluate whether the MPN-initiating cell population was contained within the LT-HSC compartment. Employing single cell sorting multi-parameter flow cytometry and limiting dilution bone marrow transplantation assays, we provide further functional characterization of the MPN-initiating population in this model. We demonstrate that Jak2V617F LT-HSCs are highly enriched for MPN-initiating activity in vivo and are able to maintain the MPN through multiple rounds of serial transplantation for periods greater than one year. The MPN phenotype remains stable during this time without evolution to myelofibrosis or leukemia. These findings provide important biological insights into MPN initiation and maintenance and have implications for the development of curative therapeutic strategies for MPN patients.