INCB160058 selectively targets JAK2V617F-driven hematopoiesis in diverse and drug-resistant models of myeloproliferative neoplasms

Abstract INCB160058 is a first-in-class, orally bioavailable Janus kinase 2 (JAK2) V617F mutant-selective inhibitor, currently being studied in phase 1 clinical trials (eg, NCT06313593) in patients with myeloproliferative neoplasms (MPNs) harboring JAK2V617F mutation. Previously, INCB160058 was shown to bind the pseudokinase domain (JH2) of JAK2. This unique binding results in a novel mechanism of action where INCB160058 selectively inhibits ligand-independent oncogenic JAK2V617F homodimers associated with class I cytokine receptors (eg, thrombopoietin receptor), while preserving physiological, cytokine-dependent signaling. Aberrant JAK2/STAT5 signaling and proliferation in JAK2V617F+ cells are inhibited with minimal impact on signaling and growth of wild-type counterparts (Stubbs. Blood 2023;142[Suppl 1]:860). This study evaluated the activity of INCB160058 in JAK2V617F+ cells possessing different secondary mutations or having previously been treated with MPN-directed therapy, simulating possible clinical scenarios. We evaluated the effect of INCB160058 on the clonogenic potential of JAK2V617F⁺ CD34⁺ hematopoietic stem and progenitor cells using colony-forming unit (CFU) assays and assessed JAK2V617F-dependent erythroid and megakaryocyte differentiation potential through liquid culture assays. In CFU assays, INCB160058 inhibited colony formation by JAK2V617F+ CD34+ cells, irrespective of additional mutations, including high-risk variants such as ASXL1 or EZH2, while exhibiting minimal impact on normal CD34+ cells. These findings demonstrate that INCB160058 effectively inhibits the aberrant proliferation of MPN CD34⁺ cells irrespective of additional mutations, highlighting that their oncogenic growth remains primarily dependent on the JAK2V617F mutation. In addition, in liquid culture assays, INCB160058 selectively impaired cytokine-independent JAK2V617F-driven erythroid and megakaryocytic maturation of CD34+ cells from JAK2V617F+ polycythemia vera, essential thrombocythemia, and myelofibrosis patient samples, while sparing erythroid and megakaryocyte maturation of CD34+ cells from healthy donors. Importantly, both acute and chronic INCB160058 treatment reduced oncogenic signaling pathways (pSTAT5, pERK, and pAKT), JAK2/STAT5 target gene expression (eg, PIM1, ID1, CISH) as well as cytokine production (eg, IL8, MCP1, and MIP-1B) in JAK2V617F+ CD34+ cells, with minimal effects on normal CD34+ cells. We also assessed the in vitro efficacy of INCB160058 on cells exhibiting persistence to ruxolitinib, which was established by exposing JAK2V617F-dependent SET2 cells to increasing concentrations of ruxolitinib, resulting in a population capable of proliferating despite high-dose ruxolitinib treatment. Notably, treatment with INCB160058 inhibited STAT5 phosphorylation and suppressed proliferation to a similar extent in both ruxolitinib-persistent and inhibitor-naive JAK2V617F-dependent SET2 cells, suggesting potential benefit in ruxolitinib-resistant or refractory MPNs. Furthermore, currently approved JAK2 inhibitors for MPNs induce paradoxical hyperphosphorylation of the JAK2 activation loop (Y1007/1008) associated with withdrawal syndrome; in contrast, INCB160058 reduced activation loop phosphorylation, consistent with its binding to the JH2 domain of JAK2V617F. These findings support the potential of INCB160058 to overcome ruxolitinib resistance and mitigate withdrawal-related signaling effects in clinical settings. In conclusion, our findings demonstrate that INCB160058 selectively targets JAK2V617F-mediated pathways and inhibits constitutive differentiation of CD34+ cells driven by the mutation, while preserving cytokine-dependent signaling for normal hematopoietic differentiation across numerous clinically relevant in vitro and ex vivo MPN models.