Paper Details

ABSTRACT

The JAK2V617F mutation is the predominant oncogenic driver in myeloproliferative neoplasms (MPNs), occurring in nearly all patients with polycythemia vera (PV) and approximately half of those with essential thrombocythemia and myelofibrosis. JAK2 inhibitors approved for the treatment of MPNs inhibit both mutant and wild-type (WT) JAK-STAT signaling, limiting their therapeutic window and disease-modifying potential. We previously reported the first-generation JAK2V617F-selective pseudokinase inhibitor INCB160058 (Stubbs MC, et al. Blood; 2023;142[suppl1]:860). Here we describe INCB203954, a next-generation JAK2V617F-selective inhibitor with enhanced potency and selectivity.

INCB203954 bound the pseudokinase domain of JAK2V617F with picomolar affinity and demonstrated a 7-fold improvement in whole-blood potency (IC₅₀ 650 nM vs 4500 nM) and a 4.4-fold increase in cellular selectivity for JAK2V617F over WT JAK2 (22-fold vs 5-fold) compared with INCB160058. Consistent with its mutant-selective mechanism, INCB203954 disrupted ligand-independent thrombopoietin receptor (TPO-R) dimerization induced by JAK2V617F at concentrations as low as 20 nM while preserving physiologic ligand-dependent WT TPO-R dimerization up to micromolar concentrations.

These potency and selectivity improvements translated into physiologically relevant assays using primary patient-derived CD34⁺ cells harboring JAK2V617F. In colony-forming unit assays, INCB203954 selectively and effectively inhibited JAK2V617F-driven erythroid and myeloid colony formation at concentrations as low as 50 nM while sparing colonies derived from WT donor CD34⁺ cells up to micromolar concentrations. Similarly, in liquid culture assays, INCB203954 selectively suppressed spontaneous megakaryocyte differentiation from JAK2V617F⁺ CD34⁺ cells with minimal effects on normal megakaryopoiesis. In contrast, the JAK1/JAK2 inhibitor ruxolitinib non-selectively inhibited clonogenic growth and megakaryocyte differentiation in both mutant and WT CD34⁺ hematopoietic cells.

The disease-modifying potential of selective JAK2V617F inhibition was evaluated in a competitive transplantation murine model of JAK2V617F-driven PV using INCB196616, an analogue of INCB203954 whose comparable biochemical potency, cellular activity, and pharmacokinetic properties provide a predictive model for the expected in vivo activity of INCB203954. Congenic recipients were transplanted with 40% Jak2V617F knock-in and 60% WT competitor bone marrow (BM) cells ubiquitously expressing GFP. Four weeks post-transplant, following hematopoietic reconstitution and PV establishment, mice received INCB196616 treatment for 10 weeks. Treatment substantially improved the PV phenotype (with marked reductions in hematocrit, hemoglobin, and red blood cell counts) and selectively reduced Jak2V617F-derived erythroid, myeloid, and platelet populations while preserving WT hematopoiesis in peripheral blood. At sacrifice, INCB196616 markedly reduced Jak2V617F⁺ BM hematopoietic stem cells (HSCs) and progenitor cells populations, while preserving overall BM cellularity including at the highest dose tested (60 mg/kg). Importantly, depletion of mutated long-term (LT) HSCs was accompanied by progressive repopulation of the BM by WT LT-HSCs, indicating selective elimination of disease-driving cells while preserving normal hematopoiesis. Consistent with these findings, treatment with INCB196616 also resolved erythroid and megakaryocytic hyperplasia in the BM, normalized splenomegaly, and reduced inflammatory cytokine levels. Furthermore, secondary transplantation studies demonstrated loss of disease-propagating capacity, confirming effective targeting of disease-initiating stem cells.

Collectively, these findings demonstrate that selective JAK2V617F inhibition with INCB203954 results in preferential elimination of JAK2V617F hematopoiesis, including disease-propagating stem and progenitor cells, while preserving normal hematopoiesis. The combination of enhanced potency and JAK2V617F selectivity in physiologically relevant ex vivo and in vivo assays supports the clinical development of INCB203954 as a next-generation targeted therapy for patients with JAK2V617F⁺ MPNs, with a phase 1 study planned to initiate in early 2027.