RON kinase as a novel therapeutic target in myeloproliferative neoplasms

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Abstract The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineage compartments. The driver mutations of MPNs, namely activating mutations in either Janus Kinase 2 (JAK2), thrombopoietin receptor (MPL), or calreticulin (CALR), function to upregulate JAK/STAT signaling. During disease progression, MPN patients experience increased pro-inflammatory cytokine secretion, leading to remodeling of the bone marrow microenvironment and subsequent fibrosis. Currently, the only curative treatment for MPNs is stem cell transplantation, but most patients are poor candidates due to age or comorbidities. The JAK inhibitor ruxolitinib is an approved targeted therapy for MPN patients and has shown promise in its ability to reduce splenomegaly and the cytokine storm observed in patients. However, JAK inhibitors alone are not sufficient to reduce bone marrow fibrosis or to eliminate the JAK2-mutated clone. Furthermore, JAK inhibitor persistence, or reactivation of JAK/STAT signaling upon chronic JAK inhibitor treatment, has been observed in both MPN mouse models and MPN patients. Therefore, there is an urgent need for new treatment options in MPN. The tyrosine kinase RON is a member of the MET kinase family, and signaling through RON promotes activation of downstream signals, including pAkt, pErk, and pSTAT3. RON signaling has well characterized roles in erythroblast proliferation and pro-inflammatory cytokine production. Specifically, the constitutively active short-form RON (sfRON) isoform is necessary for Friend virus-induced erythroleukemia in mice via signaling through STAT3. In addition, it was shown that the canonical full-length RON (flRON) isoform is phosphorylated by JAK2 to stimulate erythroblast proliferation. However, the role of RON in MPN pathogenesis is currently unknown. Preliminary data from our lab has shown that pharmacological inhibition of RON with the ALK/MET/RON/ROS1 inhibitor crizotinib inhibits colony formation and JAK/STAT signaling in both patient MPN cells and JAK2-mutated cell lines. Furthermore, we demonstrated that shRNA knockdown of both flRON and sfRON isoforms in JAK2-mutated cell lines phenocopies the inhibitory effects of crizotinib. We also found that RON isoform phosphorylation is enhanced in JAK inhibitor persistent cells, suggesting that RON may potentiate the JAK2 persistence phenotype in response to JAK inhibitors. Therefore, we hypothesize that RON is a novel mediator of JAK/STAT signaling in MPNs, and that inhibiting signaling through RON will have therapeutic value in MPN patients. We propose to examine the roles of RON signaling in MPN disease progression and initiation using MPN mouse models. We also aim to determine the therapeutic window of targeting RON kinase in MPN by examining its role in the normal hematopoietic system. Lastly, we propose to delineate the mechanism by which RON potentiates JAK/STAT signaling in both JAK inhibitor naïve and JAK inhibitor persistent MPN cells.