Abstract
The classical myeloproliferative neoplasms (MPNs) polycythemia vera, essential thrombocythemia, and primary myelofibrosis (MF) are driven by aberrant JAK2 signaling induced by mutations in JAK2, MPL, or CALR. MPN patients exhibit constitutional symptoms that impair quality of life, and they live with an elevated risk of deadly cardiovascular complications, bone marrow failure, and developing an incurable acute leukemia. While FDA-approved JAK2 inhibitors can improve patient quality of life they are unable to readily reduce driver allele burden/induce remission. However, MPN mouse model studies have demonstrated that MPN phenotypes require continued aberrant JAK2 signaling, indicating MPN patients may benefit from improved JAK2 inhibitors or approaches to antagonize this signaling. As suggested by numerous studies, the RAS/MEK/ERK pathway may play a role in the persistent survival of MPN-driving cells during JAK2 inhibitor therapy. Indeed, ruxolitinib combined with MEK, ERK, or SHP2 inhibitors can improve the efficacy of JAK2 inhibitor monotherapy in preclinical studies. MEK and ERK kinase inhibitors have presented significant challenges in clinical oncology, including inefficacy, toxicity, and the development of drug resistance. Recent advances in the development of small molecule RAS inhibitors have been translated to clinical studies in solid tumors but the potential of such anti-RAS targeted therapies in blood cancers remains under explored. Given aberrant JAK2 signaling in MPN signals in part through RAS activation, which activates multiple downstream pathways, direct RAS inhibition and subsequent suppression of multiple RAS controlled pathways could provide a significant advantage over RAS pathway selective kinase inhibitors such as MEK or ERK inhibitors. We hypothesized that direct inhibition of RAS may antagonize the growth and viability of MPN cells and enhance the effectiveness of anti-JAK2 therapy. We employed the recently developed RAS(ON) multi-selective inhibitor, RMC-7977, which inhibits signaling mediated by the active GTP-bound forms of wildtype H-RAS, K-RAS, and N-RAS, alone and in combination with the JAK2 inhibitor ruxolitinib in preclinical MPN models. We found that RMC-7977 suppressed levels of active ERK (pERK) in JAK2-driven MPN model SET2 and UKE1 cells at concentrations as low as 10 nM, with RMC-7977 exhibiting GI50 concentrations of 0.4 and 0.1 μM in these cells, respectively. While the MEK inhibitor binimetinib readily suppressed pERK levels, its GI50 was > 5 μM in these cells, suggesting direct inhibition of RAS may more efficiently suppress signaling requisite for JAK2-driven growth. Combining RMC-7977 with ruxolitinib synergistically compromised growth and survival of MPN model cells, mediated in part through enhanced apoptosis. Combined RMC-7977 and ruxolitinib treatment resulted in enhanced inhibition of pERK, pRSK3, and DUSP6 levels compared to monotherapies. These signaling and growth effects of RMC-7977 and ruxolitinib were not evident in non-MPN model cells, suggesting potential selectivity of this combination. Notably, RMC-7977 effectively suppressed pERK levels in, and growth of, ruxolitinib persistent MPN model cells suggesting in vitro mechanisms of JAK2 inhibitor persistence do not provide cross-resistance to direct inhibition of RAS. RMC-7977 suppressed the levels of pERK in primary MF patient cells, as well as the neoplastic growth of primary MPN progenitor cells, and could synergistically enhance growth inhibitory responses in combination with ruxolitinib. In an MPN mouse model driven by MPL-W515L, oral RMC-7977 treatment suppressed MPN phenotypes including leukocytosis, hepatosplenomegaly, and extramedullary hematopoiesis, and enhanced survival. RMC-7977 in combination with ruxolitinib further suppressed MPN phenotypes beyond the inhibitory effects of monotherapies, and significantly enhanced survival beyond ruxolitinib and RMC-7977 treatment alone. RMC-7977 treatment suppressed inflammatory cytokine levels including TNFα and IL-6, two cytokines implicated in MPN pathogenesis, among others. Importantly, treatment of healthy mice concurrently with RMC-7977 and ruxolitinib was well tolerated as evidenced by the absence of impact on hematologic parameters and body weight. Our results reveal that direct inhibition of RAS and JAK2 may represent a promising therapeutic strategy for MPN patients and provide evidence RAS inhibition has potential beyond solid tumor indications.
