JAK2^V617F Promotes Stem Cell Amplification Driving MPN Clonal Dominance in Mice and Treatment by IFNa Prevents This Effect

Abstract 616

JAK2^V617F is the major mutation involved in classic myeloproliferative neoplasm (MPN). It promotes growth factor independent cell growth and is able to recapitulate MPN features in retroviral, transgenic (TG) or knock-in (KI) mouse models. Several mutations implicated in epigenetic modifications or leukemic transformations have been also identified in MPN and several reports have questioned the particular role of JAK2^V617F on hematopoietic stem cells (HSC) proliferation thus as a driver of MPN emergence. Therefore, we investigated the in vivo effect of an endogenous expression of JAK2^V617F on early stages of differentiation and their ability to compete for normal cells in a repopulation assay.

For this study, we develop a novel mouse conditional JAK2^V617F KI model based on the “FLEX switch” strategy. These KI mice were crossed with TG mice expressing the Cre recombinase under the control of the vav promoter in order to restrict JAK2^V617F expression to hematopoietic and some endothelial tissues. VavCre/JAK2^+/V617F KI mice developed high hematocrit (70 ± 2 %, control values 49 ± 1 % n=13), platelet (2.3 ± 0.1 × 10⁹ / mL, control values 0.84 ± 0.04 × 10⁹ / mL n=20) and white blood cell (20-40 × 10⁶/mL, control values between 6–10 × 10⁶ / mL) values and a splenomegaly at 2–3 months of age but after 6 months of age an anemia and a thrombocytopenia appeared. This model mimics human polycythemia vera with secondary myelofibrosis. At 2–3 months of age, cumulative numbers in bone marrow (BM) and spleen of CFU-E, BFU-E and GM-CFC were increased 15-, 3-, 1.2–fold, respectively, compared to control. Most CFU-E grew without the addition of erythropoietin. A 6-fold amplification of total early progenitors LSK and a tendency toward SLAM (LSK/CD48−/CD150+) cell amplification, mainly due to a significant 9-fold increase in the spleen, were also observed.

Competitive repopulation assays using 30% KI and 70% WT bone marrow cells demonstrated 17 weeks after BM transplantation (BMT) a rapid and strong amplification, from 30% to > 80%, of blood myeloid cells (Gr-1+/Mac1+) from KI origin. Late after transplantation (35 weeks), Lin-, LSK and SLAM cell compartments from KI origin raised from the initial 30% to almost 100% in the BM and even KI blood lymphoid cells (B220+ and CD3+) demonstrated a significant amplification compared to control. This shows that endogenous expression of JAK2^V617F gives an advantage to HSC, promoting clonal dominance in mice.

Then, we analyzed at which levels of differentiation acts IFNα, a drug promoting cycling of dormant cells and proven efficacious in PV treatment in human. In a chimeric model, we demonstrated that IFNα could prevent the development of MPN induced in vavCre/JAK2^+/V617F KI recipient mice by inhibiting the amplification of KI cells. Secondary BMT from treated animals demonstrated the eradication of disease-initiating cells after long-term treatment.

This study shows that IFNα acts at the level of the disease-initating cell by reverting the HSC promoting clonal dominance induced by JAK2^V617F.