The Role of PTPN1 in the Progression of JAK2V617F-induced Myeloproliferative Neoplasms

The deletion of chromosome 20q (del20q) is a common karyotypic abnormality associated with myeloid malignancies, including 15% of myeloproliferative neoplasms (MPN), 10% of myelodysplastic syndrome (MDS), 5% of MDS/MPN overlapping diseases, and 5% of acute myeloid leukemia (AML). Myelofibrosis (MF), the deadliest among MPNs, exhibits the most frequent association with del20q. In MPN, the oncogenic JAK2V617F mutation was detected in approximately 97% of patients with polycythemia vera (PV), and 50-60% of patients with essential thrombocythemia (ET) and myelofibrosis. We have observed a significant co-occurrence of the JAK2V617F mutation and del20q abnormality involving PTPN1 deletion in patients with MF. Therefore, we hypothesize that PTPN1 deficiency may cooperate with JAK2V617F in the progression to MF.
In this study, we investigated the effects of PTPN1 deficiency in JAK2V617F-induced MPN using a using a conditional knockout allele for PTPN1 and a mouse model of MPN with knock-in JAK2V617F mutation. Mice expressing heterozygous JAK2V617F (JAK2V617F/+) exhibit a PV-like MPN characterized by an increase in red blood cells (RBC), white blood cells (WBC), neutrophils, and platelets in the peripheral blood. Heterozygous deletion of PTPN1 significantly increased WBC, neutrophil, and platelet counts but reduced RBC and hemoglobin levels in JAK2V617F/+ mice. Flow cytometric analysis showed significantly increased myeloid (Gr-1+Mac-1+) and megakaryocytic (CD41+CD61+) precursors in the BM and spleens of PTPN1-deficient JAK2V617F/+ mice compared with JAK2V617F/+ mice. Additionally, deletion of PTPN1 significantly increased hematopoietic stem cells and myeloid progenitors in the BM and spleens of PTPN1-deficient JAK2V617F/+ mice compared to JAK2V617F/+ mice. Spleen weight was significantly increased in JAK2V617F/+ mice, while deletion of PTPN1 further enhanced splenomegaly in PTPN1-deficient JAK2V617F/+ mice. Moreover, PTPN1 deletion significantly enhanced bone marrow fibrosis in JAK2V617F/+ mice. Bone marrow transplantation assays confirmed that the phenotypes observed in PTPN1-deficient JAK2V617F/+ mice are cell intrinsic. Furthermore, the hematopoietic stem cells (HSC) from PTPN1-deficient JAK2V617F/+ mice display greater clonal advantage than the HSC of JAK2V617F/+ mice. Deletion of both alleles of PTPN1 resulted in more robust and accelerated development of myelofibrosis in JAK2V617F/+ mice. The cooperative effects of JAK2V617F and PTPN1 deficiency in myelofibrotic transformation are associated with increased production of proinflammatory cytokines, including IL-1β, IL-6, and TGF-β.
Biochemical analyses revealed greater activation of STAT1, STAT3, STAT5, ERK1/2, and NF-kB signaling in bone marrow of PTPN1-deficient JAK2V617F/+ mice compared with bone marrow of JAK2V617F/+ or WT mice. CRISPR/Cas9-mediated deletion of PTPN1 also resulted in increased proliferation and enhanced activation of these signaling molecules in JAK2V617F-positive hematopoietic cells. RNA-sequencing analysis showed enrichment of genes related to hematopoietic stem cells, MAPK cascade, cell cycle, and WNT signaling pathways in PTPN1-deficient JAK2V617F/+ LSK cells, which have significant overlap with genes enriched in hematopoietic cells of patients with myelofibrosis. Furthermore, deletion of PTPN1 significantly reduced ruxolitinib sensitivity against JAK2V617F-positive hematopoietic cells, suggesting that del20q involving PTPN1 deletion may alter the ruxolitinib response in MPN. Overall, this study demonstrates that PTPN1 is an important target in del20q-associated MPN, and deficiency of PTPN1 collaborates with JAK2V617F in the progression to myelofibrosis.