Differential Requirements for the RON Ligand Binding and Kinase Domains in Myeloproliferative Neoplasms

The BCR-ABL-negative myeloproliferative neoplasms (MPNs) polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are clonal disorders involving hyper-activation of signal transduction pathways (primarily JAK/STAT) in hematopoietic stem cells. This results in excess production of terminally differentiated blood cells. JAK inhibitors are part of the standard of care for patients with PV and PMF, but are associated with dose-limiting cytopenias, are unable to fully eliminate the MPN-initiating cell populations, and have minimal effects on bone marrow fibrosis in PMF. RON tyrosine kinase has been implicated in MPN pathogenesis, especially in contributing to the loss of sensitivity to JAK inhibitors (PMID:36537918). We show that several RON isoforms are over-expressed in the JAK2-mutated cell lines HEL, SET2, and UKE1. This includes the short isoform (sfRON) that lacks an extracellular ligand binding domain and has been reported in other tumors to be ligand-independent and constitutively active. However, our preliminary data suggests that MPN patient cells also have increased expression of the full-length RON isoform with an intact ligand binding domain, suggesting that RON could be a cell-surface therapeutic target in MPN patients. The goal of this study is to investigate the relative contributions of the ligand binding or kinase domains of RON to MPN pathogenesis in preclinical models. To elucidate the importance of the ligand binding domain to RON pathogenicity in PV, we mated JAK2-lox-STOP-lox-V617F mice (PMID:20541703) to Stk-/- mice, which have homozygous deletion of the RON extracellular ligand binding domain (PMID:9680329) to generate JAK2VF/+;Stk-/-;Vav1-Cre mice. Deletion of this domain significantly extended median survival in the context of JAK2-mutated MPN (229 days for JAK2VF/+;Stk-/-;Vav1-Cre mice and 167 days for JAK2VF/+;Vav1-Cre mice (p<0.05)). Given that truncated, intracellular RON isoforms are still capable of downstream signaling, we also developed a JAK2-mutated model with conditional homozygous deletion of the RON kinase domain by mating JAK2-lox-STOP-lox-V617F mice to Mst1r-TK-/- mice (PMID:11518730) to generate JAK2VF/+;Mst1r-TK-/-;Vav1-Cre mice. The analysis of JAK2VF/+;Mst1r-TK-/-;Vav1-Cre mice is still ongoing, but there is a trend in decreased white blood cell counts in JAK2VF/+;Mst1r-TK-/-;Vav1-Cre mice compared with JAK2VF/+;Vav1-Cre controls. This data suggests that both cell surface and kinase domains of RON likely contribute to PV disease progression. To determine if the distinction between the different RON domains is more critical in myelofibrosis, we also assessed survival and disease burden of the two RON inactivation models with retroviral overexpression of the MPLW515Lmutation, which drives a PMF-like phenotype in mice. Contrasting with our findings in JAK2-mutated MPN, we found that RON ligand domain deletion did not alter disease outcomes in the MPLW515L PMF model, and that only kinase domain deletion was associated with improved median survival: 94 days for MPLW515L mice while 9/10 MPLW515L;Mst1r-TK-/- mice remained alive until termination of the experiment at 150 days (p<0.05). Consistent with these results, pharmacological inhibition of RON with the MET/RON inhibitor crizotinib significantly decreased disease burden in MPLW515L mice. Bone marrow fibrosis and cytokine levels in MPLW515L;Mst1r-TK-/- mice are under evaluation. Together, this supports an important role for ligand-independent RON activation in PMF. Deletion of the ligand binding domain in Stk-/- mice is associated with minimal effects on healthy hematopoiesis (PMID:36537918). Similarly, Mst1r-TK-/- mice have normal blood counts and Mst1r-TK-/- bone marrow cells have normal long-term repopulating activity, suggesting that RON activity is not critical for normal hematopoietic stem cell function. This predicts that targeting RON would be safe with a reasonable therapeutic window. This work highlights an important role for RON activity in MPN disease progression, which appears to be specifically related to ligand-independent signaling in the setting of PMF driven by MPLW515L. The promising survival benefits and minimal hematopoietic toxicity associated with genetic inactivation of RON kinase activity in vivo support further investigation of RON inhibitors in PMF.