Critical Role of SHP2 in the Initiation and Maintenance of Myeloproliferative Neoplasms Evoked By JAK2V617F

The JAK2V617F mutation has been found in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of the JAK2 tyrosine kinase and its downstream signaling pathways. JAK2 inhibitor therapy can reduce splenomegaly and constitutional symptoms but is not sufficient to cure or produce complete remission in patients with MPNs. Identification of critical signaling pathways/molecules required for the initiation and maintenance of MPNs will facilitate the development of more effective targeted therapies.

To determine the role of JAK2V617F mutation in MPNs, we previously generated a conditional Jak2V617F knock-in mouse. We observed that expression of heterozygous Jak2V617F in mice hematopoietic compartments is sufficient to induce a PV-like MPN, whereas homozygous Jak2V617F expression accelerates the development of myelofibrosis. We have found that SHP2, a protein tyrosine phosphatase that positively regulates hematopoietic signaling, is constitutively phosphorylated in mouse and human hematopoietic cells expressing JAK2V617F. However, the contribution of SHP2 in the pathogenesis of MPNs induced by JAK2V617F remains elusive. Here, we sought to determine the role of SHP2 in JAK2V617F-evoked MPNs using conditional SHP2 knockout (SHP2 floxed) and Jak2V617F knock-in mice and MxCre line.

Whereas expression of heterozygous Jak2V617F induced a PV-like MPN characterized by increase in red blood cells (RBC), white blood cells (WBC), neutrophils and platelets in the peripheral blood and splenomegaly, deletion of SHP2 normalized the blood parameters and spleen size in Jak2V617F knock-in mice. Flow cytometric analysis showed that deletion of SHP2 inhibited the expansion of erythroid, megakaryocytic, and granulocytic precursors in the bone marrow (BM) and spleens of Jak2V617F mice. Deletion of SHP2 also inhibited the expansion of hematopoietic stem cells and megakaryocyte-erythroid progenitors in the BM and spleens of Jak2V617F knock-in mice. Furthermore, deletion of SHP2 markedly inhibited the erythropoietin (Epo)-independent CFU-E colonies in the BM and spleens of Jak2V617F mice.

In order to determine whether the effects of SHP2 deletion in Jak2V617F mice were cell autonomous, BM cells from uninduced control, MxCre;V617F/+ and MxCre;V617F/+;SHP2fl/fl mice were transplanted into lethally irradiated syngeneic recipient mice. Four weeks after transplantation, mice were injected with pI-pC to induce deletion of SHP2 and expression of Jak2V617F simultaneously. Transplanted animals receiving MxCre;V617F/+ BM developed a PV-like disease within 6 weeks after pI-pC induction. However, recipients of MxCre;V617F/+;SHP2fl/fl BM failed to induce the PV disease due to deletion of SHP2. Together, these data suggest that SHP2 is required for the initiation of PV-like MPN mediated by Jak2V617F.

To determine if SHP2 is required for the maintenance of MPN evoked by Jak2V617F, uninduced wild type or MxCre;SHP2fl/fl mice BM cells were transduced with retroviruses expressing Jak2V617F and transplanted into lethally irradiated syngeneic recipient animals. Five weeks after transplantation, we assessed the peripheral blood counts to confirm the development of MPN disease. Transplanted animals receiving Jak2V617F-transduced wild type or MxCre;SHP2fl/fl mice BM were then divided into two groups- one group was injected with pI-pC (to induce deletion of SHP2 after establishment of the MPN disease); another group (control group) was injected with saline. We observed that deletion of SHP2 by pI-pC induction significantly inhibited the RBC, hematocrit, WBC and platelet counts in the peripheral blood and reduced the spleen size in transplanted animals expressing Jak2V617F. Moreover, deletion of SHP2 markedly inhibited the Epo-independent erythroid colonies in the BM and spleens of transplanted animals expressing Jak2V617F. Thus, SHP2 is not only required for the initiation of MPN but also required for the maintenance of MPN mediated by Jak2V617F. We have also observed that deletion of SHP2 blocks the development of myelofibrosis in Jak2V617F knock-in mice. In conclusion, our results suggest that SHP2 plays a critical role in the initiation and maintenance of MPNs evoked by Jak2V617F.