Genetic Deletion or Pharmacologic Inhibition of PTPN11 Impedes the Development and Progression of Myeloproliferative Neoplasms Induced By JAK2V617F and MPLW515L Mutants

Myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are clonal myeloid malignancies derived from mutated hematopoietic stem cells. The JAK2V617F mutation was found in ~95% cases of PV and 50-60% patients with ET and MF. Mutations in MPL and CALR were also detected in ET and MF. The JAK inhibitors, Ruxolitinib and Fedratinib, can reduce splenomegaly and alleviate constitutional symptoms but they are not sufficient to induce remission of MPN. So, there is an unmet need to identify new therapeutic targets and therapies for MPN/MF. The non-receptor protein tyrosine phosphatase 11 (PTPN11), which positively regulates hematopoietic cell signaling, is constitutively hyperphosphorylated in MPN patient cells and in hematopoietic cell lines expressing MPN driver mutants. So, we hypothesize that PTPN11 plays an important role in MPNs.

To examine the role of PTPN11 in JAK2V617F-induced MPN, we generated inducible PTPN11-deficient heterozygous JAK2V617F mice by crossing PTPN11 floxed mouse with Mx1Cre and JAK2V617F knock-in mice. While expression of heterozygous JAK2V617F (JAK2 ^VF/+) induced a PV-like MPN characterized by increased WBC, neutrophil, RBC, hemoglobin and platelet counts in the peripheral blood and enlargement of spleen size, deletion of PTPN11 normalized the blood counts and spleen size in JAK2 ^VF/+ mice. PTPN11 deletion significantly reduced the expansion of hematopoietic stem/progenitors, erythroid, megakaryocytic, and granulocytic cells in the bone marrow (BM) and spleens of JAK2 ^VF/+ mice. Furthermore, deletion of PTPN11 abrogated Epo-independent CFU-colonies, a hallmark feature of PV disease, in the BM and spleens of JAK2 ^VF/+ mice.

We also investigated the role of PTPN11 in the development of MF using homozygous JAK2V617F and MPLW515L mouse models. We found that deletion of PTPN11 significantly reduced WBC, neutrophil and platelet counts, normalized spleen size and abrogated BM fibrosis in JAK2V617F and MPLW515L mouse models. These results suggest that PTPN11 plays an important role in the development and progression of PV and MF.

Data from our PTPN11 genetic deletion study led us to investigate the effects of PTPN11 inhibition against MPN/MF. We tested the efficacy of an allosteric PTPN11 inhibitor SHP099 in MPN hematopoietic cell lines and mouse models of MPN/MF. We observed that treatment of SHP099 significantly reduced proliferation of hematopoietic cells expressing JAK2V617F and MPLW515L. SHP099 treatment also significantly inhibited hematopoietic progenitor colony growth in PV and MF patient CD34+ cells.

Next, we tested the in vivo efficacy of SHP099 against PV using heterozygous JAK2V617F mice. Treatment of SHP099 significantly inhibited the increase in WBC, neutrophil and RBC counts and markedly reduced splenomegaly in JAK2 ^VF/+ mice. SHP099 treatment also significantly reduced hematopoietic stem/progenitors and myeloid precursors in the BM and spleens of JAK2 ^VF/+ mice. Furthermore, SHP099 treatment abrogated Epo-independent CFU-E colony formation in the BM and spleens of JAK2 ^VF/+ mice.

We also tested the efficacy of SHP099 alone or in combination with ruxolitinib against myelofibrosis using homozygous JAK2V617F (JAK2 ^VF/VF) and MPLW515L mouse models. SHP099 treatment alone significantly reduced WBC and neutrophil counts and markedly reduced splenomegaly in JAK2 ^VF/VF and MPLW515L mice. Combined treatment of SHP099 and ruxolitinib resulted in greater reduction of blood cell counts and spleen size in JAK2 ^VF/VF and MPLW515Lmice. Histopathologic analyses showed that SHP099 treatment significantly reduced BM fibrosis in JAK2 ^VF/VF and MPLW515L mice while combined treatment of SHP099and ruxolitinib resulted in greater inhibition of BM fibrosis in these mice.

To gain insights into the mechanism of inhibition of MPN/MF by PTPN11 deletion or inhibition, we performed RNA-seq analysis on purified LSK cells from JAK2 ^VF/+ and PTPN11deleted-JAK2 ^VF/+ mice as well as from JAK2 ^VF/VF mice treated with ruxolitinib and SHP099/ruxolitinib. RNA-seq data analysis revealed that genes related to MYC targets, ribosome biogenesis and translation were significantly down-regulated by PTPN11 deletion or SHP099/ruxolitinib combination treatment. Overall, our results suggest that inhibition of PTPN11 alone or in combination with JAK2 inhibition might be useful for treatment of PV and MF.