P980: JAK2V617F-DEPENDENT DOWN REGULATION OF SHP1 EXPRESSION PARTICIPATES IN THE SELECTION OF MPN CELLS IN THE PRESENCE OF TGFΒ

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Background: Myeloproliferative neoplasms (MPNs) are characterized by an increased production of blood cells, due to acquisition in HSC of mutations in the JAK2, CALR or MPL genes. The JAK2V617F mutation, leading to a constitutively active form of JAK2, is the most frequent mutation. During disease evolution the clonal selection of mutated cells is dependent on cell-intrinsic and extrinsic factors present in the niche microenvironment such as cytokines. TGFβ, whose local secretion is increased in MPNs, and more particularly in myelofibrosis is known to negatively regulate normal hematopoietic stem cells (HSC) proliferation. However, its mode of action is not clearly established. We hypothesized that JAK2V617F mutant HSC are resistant to TGFβ antiproliferative effect explaining the pathological stem cell invasion in myelofibrosis. Using multiomics approaches to study the intracellular signaling pathways in MPNs we recently identified the down regulation of SHP-1 expression in JAK2V617F cells compared to wild type cells. Of note, the phosphatase SHP-1 has recently been shown to be required for the TGFβ-induced quiescence of HSCs. Aims: We hypothesized a relationship between SHP-1 down regulation and the resistance of JAK2V617F mutated cells to the antiproliferative effect of TGFβ. The work presented herein aimed at verifying this hypothesis. Methods: We used the HEL erythroleukemia cell line which carries several copies of the JAK2V617F mutated gene. We also used an isogenic model obtained by transducing either the JAK2 wild type (JAK2wt) of JAK2V617F allele in the UT-7 megakaryoblastic cell line. Proliferation was measured using the CCK8. Protein expression was measured using western blot while gene expression was measured using RT-qPCR. Results: The expression of SHP-1 was reduced by 30% at the RNA level and 50% at the protein level in UT-7 cells transduced with JAK2V617F compared to JAK2wt -transduced cells. In bone marrow cells taken from JAK2V617F knock-in mice the protein level of SHP1 was reduced by 70% compared to wild-type mice. EPO treatment of UT-7 cells (which express the EPO-R) showed a down regulation of SHP-1 expression which was abrogated in the presence of the JAK2 inhibitor ruxolitinib. These results suggest that SHP-1 expression is modulated by JAK2 signaling leading to its down regulation in JAK2V617F cells. Although TGFβ receptor and SMAD2/3 expression was similar in both cell type, JAK2V617F UT-7 cells were less responsive to TGFβ signaling compared to JAK2wt cells (reduced phosphorylation of SMAD2/3). The proliferation of JAK2wt UT-7 cells was significantly reduced in the presence of TGFβ 10ng/mL while JAK2V617F UT-7 cells were not affected. To confirm the involvement of SHP-1 we transduced HEL cells with the cDNA coding for SHP-1 in order to restore higher levels of the protein in this JAK2V617F mutated cell line and observed a significant reduction in the proliferation when treated with TGFβ compared to cells transduced with the empty vector. Finally, we performed an in vitro competitive assay where JAK2V617F UT-7 cells transduced by an m-cherry fluorescent vector were mixed 50/50 with JAK2wt UT-7 and cultured with TGFβ. After 14 days of culture with TGFβ 10ng/mL we observed a significant positive selection of the JAK2 mutant cells over JAK2wt cells. Summary/Conclusion: In conclusion, we found a JAK2-dependent down-regulation of SHP1 expression which is related to the resistance of JAK2V617F cells to the antiproliferative effect of TGFβ explaining clonal selection of mutant stem cells in myelofibrosis