Pitpβ Loss Ameliorates Jak2 V617F-Induced Myeloproliferative Neoplasms (MPN) in Mice
Authors: Vantsev Nikita , Zhao Liang , Abrams Charles S. , Tong Wei 
Published: December 2024

Myeloproliferative Neoplasms (MPN) is a blood disorder that affects ~300,000 people in the US. Mutations in the JAK/STAT pathway, which is responsible for essential cytokine signaling in blood development, are a common cause of MPN. One of the most common mutations, JAK2 V617F is present in 50-60% of all MPN cases. Although JAK inhibitors are shown to improve patient outcomes, they are not curative, suggesting the need for new therapeutic avenues. The mouse model bearing the Jak2 V617F mutation at the endogenous locus develops lethal MPN with 100% penetrance. MPN is characterized by elevated hematocrit, splenomegaly, and erythroid expansion in the spleen and bone marrow (Mullally et. al. Cancer Cell 2010).

Previously, we showed that phosphatidylinositol transfer protein α (PITPα) is essential for platelet-mediated tumor metastasis (Zhao et. al. Nat Commun 2017). Furthermore, Pf4-Cre conditional knockout (CKO) in mice of PITPα, or both PITPα and PITPβ, results in defective megakaryocytes with abnormal α-granule morphology that indirectly affects hematopoiesis. These mice exhibit moderately reduced hematopoietic stem cell (HSC) numbers with reduced competitive reconstitution ability, attributed to over-secretion of TGF-β1 (Capitano & Zhao et. al. Blood 2018). PITPs are known to regulate phosphoinositide synthesis by transferring phosphatidylinositol and phosphatidylcholine from the ER to other membrane compartments in the cell (Carvou et. al. J. Cell Sci. 2010). They have also been implicated in vesicular synthesis and exocytosis, and involved in cellular signaling as a carrier of metabolic precursors to inositol triphosphates (IP3) and diacylglycerol (Ile et. al. Nat Chem Biol 2006). However, their role in MPN or JAK2 signaling has not been explored.

Due to the effect of PITP family proteins on hematopoiesis, we set out to determine the role of PITP in Jak2 V617F-mediated MPN. We generated pan-hematopoietic Jak2 V617F knock-in mice combined with PITPα or PITPβ KO using vavCre. We found that PITPβ KO, but not PITPα KO, significantly improved survival of Jak2 V617F mutant mice. Importantly, PITPβ deficiency normalized red blood cell count, hematocrit, reticulocyte count, and alleviated splenomegaly of Jak2 V617F MPN mice.

We further evaluated the effect of PITP KO on erythroid populations in Jak2 V617F mice using flow cytometry. Jak2V617F markedly elevated the level of proerythroblasts (Ery I) and orthochromatic erythroblasts (Ery IV) in the bone marrow. In the spleen, Jak2 V617F elevated proerythroblasts (Ery I), and basophilic and polychromatic erythroblasts (Ery II). PITPβ deficiency significantly reduced all affected populations, whereas PITPα deficiency did not have an effect. Furthermore, PITPβ deficiency reduced erythroid precursor expansion in Jak2 V617F mutant mice. The Jak2 V617F mice exhibited a markedly expanded population of colony-forming progenitors (CFU-e) in the bone marrow and spleen. PITPβ, but not PITPα, deficiency completely normalized CFU-e population in Jak2 V617F mice to WT level.

We next analyzed the HSPC populations in these mice. Jak2 V617F mice have a 2-fold increase in Lineage-Sca-1+cKit+ (LSK) compartment in their bone marrow. We found that LSK expansion is largely due to an increase in erythroid/megakaryocyte-biased multipotent progenitors (MPP2) and myeloid-biased MPP3. LT-HSC as well as lymphoid-biased MPP4 were not affected. PITPβ deficiency restored both MPP2 and MPP3 populations to WT level.

Furthermore, we analyzed the effect of PITPβ knockout on erythropoietin (EPO)-mediated progenitor cell growth. PITPβ deficiency significantly reduced EPO-dependent and cytokine-independent erythroid colony formation (CFU-E) of Jak2 V617F bone marrow. We then examined EPO-mediated JAK/STAT signaling in primary erythroid progenitors. Jak2 V617F conferred cytokine-independent phosphorylation of Stat5 in proerythroblasts (Ery I) and early basophilic erythroblasts (EryA), which was abrogated by PITPβ deficiency.

Our findings revealed a previously unappreciated interaction between JAK2 V617F and the phosphatidylinositol transfer protein β. PITPβ KO mice have a normal survival rate and display normal hematopoiesis and erythropoiesis, indicating that PITPβ does not impact wildtype JAK2 signaling. Hence, our work points to a potential novel therapeutic avenue for the treatment of JAK2 V617F-mediated polycythemia vera or MPN in general.