Inhibition of SHP2 Ameliorates JAK2V617F-Induced Myeloproliferative Neoplasms

Abstract

The oncogenic JAK2V617F mutation has been found in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF and advanced PV. Although ruxolitinib treatment can reduce splenomegaly and alleviate constitutional symptoms, it does not induce disease remission or cure bone marrow (BM) fibrosis in patients with MPN/MF. Thus, there is a critical need to identify new therapeutic target and develop novel targeted therapies for MPN/MF. We found that SHP2, a protein tyrosine phosphatase that positively regulates growth factor and cytokine signaling, is constitutively hyperphosphorylated in mouse and human MPN hematopoietic cells expressing JAK2V617F. We found that genetic ablation of SHP2 markedly inhibited the development of PV disease and BM fibrosis in Jak2V617F knock-in mouse models of MPN. So, we hypothesize that targeting of SHP2 alone or in combination with JAK2 inhibition might be useful for treatment of JAK2V617F-evoked MPN. In this study, we investigated the efficacy of SHP2 inhibition using an allosteric SHP2 inhibitor, SHP099, in hematopoietic cells and progenitors expressing JAK2V617F and a Jak2V617F knock-in mouse model of MPN.Treatment of SHP099 alone significantly reduced proliferation of murine BA/F3 cells expressing JAK2V617F. SHP099 treatment also significantly inhibited human JAK2V617F-positive UKE-1 cells. However, SHP099 only modestly affected proliferation of wild type JAK2-expressing BA/F3 cells at higher concentrations. SHP099 treatment also resulted in marked apoptosis in BA/F3-EpoR-JAK2V617F and UKE-1 cells but not in wild-type JAK2 expressing BA/F3-EpoR cells. We also found that SHP099 and ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Furthermore, SHP099 significantly inhibited hematopoietic progenitor colony growth in MPN/MF patient CD34+ cells.We previously generated conditional Jak2V617F knock-in mice. Whereas expression of heterozygous Jak2V617F (Jak2 VF/+) induces a PV-like disease, homozygous Jak2V617F (Jak2 VF/VF) expression promotes rapid progression to myelofibrosis. We utilized heterozygous Jak2V617F mice to test the in vivo efficacy of SHP099 against PV. Treatment of SHP099 significantly reduced the increase in WBC, neutrophil and RBC counts and markedly reduced splenomegaly in heterozygous Jak2V617F (Jak2 VF/+) mice. SHP099 treatment also significantly reduced hematopoietic stem/progenitors (HSPC) and myeloid precursors (Gr-1 +/Mac1 +) in the BM and spleens of Jak2V617F mice. Erythropoietin (Epo)-independent erythroid colony formation in the BM and spleens, a hall-mark feature of PV disease, was observed in Jak2V617F mice. Treatment of SHP099 markedly inhibited the Epo-independent CFU-E colonies in the BM and spleens of heterozygous Jak2V617F mice.We also tested the efficacy of SHP099 alone or in combination with ruxolitinib against myelofibrosis using our homozygous Jak2V617F (Jak2 VF/VF) mice. SHP099 treatment alone or in combination with ruxolitinib significantly reduced WBC and neutrophil counts and markedly reduced splenomegaly in Jak2 VF/VF mice. Treatment of SHP099 or SHP099/ruxolitinib combination also significantly reduced the HSPC and myeloid precursor cells in the BM and spleens of Jak2 VF/VF mice. Histopathologic analyzes revealed extensive BM fibrosis in vehicle-treated Jak2 VF/VFmice. SHP099 treatment alone caused marked reduction of BM fibrosis in Jak2 VF/VF mice whereas combined treatment of SHP099 and ruxolitinib almost completely inhibited BM fibrosis in Jak2 VF/VF mice. Additionally, we performed competitive BM transplantation assays using Mx1Cre; Jak2 VF/VF GFP+ and WT mice BM cells followed by drug treatments. We observed that combined treatment of SHP099 and ruxolitinib significantly reduced the percentage of Jak2V617F mutant GFP+ LSK and myeloid cells in the BM of chimeric mice, suggesting that SHP099 and ruxolitinib combination preferentially inhibits oncogenic Jak2V617F mutant hematopoietic progenitors. Overall, our results suggest that SHP099 alone or in combination with ruxolitinib may have therapeutic potential against PV and MF and support the clinical investigation of SHP2 inhibitor in patients with PV/MF. DisclosuresNo relevant conflicts of interest to declare.