Abstract Myeloproliferative Neoplasms (MPN) is a group of phenotypically defined blood cancers that affect thousands of people around the world. A driving mutation in JAK2 kinase (most commonly Jak2 V617F) is frequently found in MPN patients, occurring in over 99% of polycythemia vera cases as well as over 50% of essential thrombocythemia and primary myelofibrosis patients. JAK2 inhibitors have been clinically used to reduce disease burden, but they are not curative, highlighting the need for new therapeutic strategies. The mouse model endogenously expressing Jak2 V617F mutation develops lethal MPN with 100% penetrance, and is characterized by erythrocythemia, elevated hematocrit, and splenomegaly (Mullally et. al. Cancer Cell 2010). Previously, we found that phosphatidylinositol transfer proteins (PITP) indirectly modulate hematopoietic stem cell (HSC) function in mice. Megakaryocytes deficient in both PITPα and PITPβ oversecrete TGF-β1, leading to suppression of HSC proliferation. (Capitano & Zhao et. al. Blood 2018). Besides PITP primary function to shuttle lipids between submembranes in a cell (Carvou et. al. J. Cell Sci. 2010), they also play a role as a cofactor in phosphoinositide synthesis (Zhao et. al. Blood Adv. 2023). Given their impact on hematopoiesis, we hypothesized that PITPs might influence signaling and disease progression in Jak2 V617F-driven MPN. Following a pan-hematopoietic vavCre knockout of either PITPα or PITPβ in Jak2 V617F mutant mice, we found that PITPβ deficiency dramatically improved survival: 85% of double transgenic mice survived beyond 50 weeks, compared to only 10% of Jak2 V617F mice surviving past 25 weeks. Furthermore, Jak2 V617F mice with PITPβ KO exhibited normalized red blood cell count, hematocrit, reticulocyte count, and alleviated splenomegaly. This was specific to PITPβ loss and was not observed with deletion of the paralog PITPα. We evaluated these mice for erythroid lineage expansion associated with the Jak2 V617F mutation and found that PITPβ deficiency reduced the number proerythroblasts (Ery I) in the bone marrow and spleens of JAK2 V617F mice to the wildtype level. Furthermore, the level of early erythroid progenitors (CFU-e and BFU-e), as well as myeloid- (MPP3) and erythroid-biased (MPP2) multipotent progenitors was reduced by the PITPβ KO. In contrast, PITPα deficiency did not rescue erythroid expansion in Jak2 V617F mice. To evaluate the effect of PITPβ deficiency on stem cell function in Jak2 V617F MPN, we utilized methylcellulose colony forming assay for erythroid progenitors. We found that PITPβ KO reduced EPO hypersensitivity of Jak2 V617F bone marrow cells, reducing CFU-e colony formation to the wildtype level. Furthermore, preliminary data from the bone marrow transplantation suggests that PITPβ KO improved engraftment of transplanted stem and progenitor cells (Lineage- cKit+ Sca-1+) from JAK2 V617F bone marrow and mildly reduced myeloid reconstitution bias. To investigate the mechanism behind the rescue, we adapted flow cytometry panels to study signaling in rare hematopoietic progenitor populations at a single cell level. We observed JAK2 V617F driven hyperactivation of pSTAT5, pAKT, and pERK throughout the hematopoietic lineage in spleen, from early multipotent progenitors to more differentiated erythroid and myeloid progenitors in response to erythropoietin and stem cell factor. Importantly, we found that PITPβ deficiency selectively reduced pAKT across all populations tested, while leaving pSTAT5 or pERK unaffected. These findings suggest that reducing pAKT alone is sufficient to alleviate MPN symptoms and improve survival in mice. To validate these finding, we are currently testing if pharmacological AKT inhibition is sufficient to attenuate MPN in Jak2 V617F mice. In parallel, we are investigating if PITPβ deficiency impairs the production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a key secondary messenger in the AKT pathway. The results of both experiments will be ready by the ASH meeting. While these experiments are ongoing, our work has already revealed an unrecognized role for PITPβ in promoting Jak2 V617F-driven MPN through dysregulation of malignant pAKT activation. This discovery not only highlights a new therapeutic target for MPN, but also underscores the critical role of AKT signaling in MPN progression.
Vantsev et al. (Mon,) studied this question.