EP31670, a dual-BET + p300 inhibitor, demonstrates pre-clinical efficacy, including in combination with JAK inhibition, in the hMPLW515L adoptive transfer model of myelofibrosis (MF)

Abstract

Abstract Background: Bromodomain and Extra-Terminal (BET) motif inhibitor therapy, alone or in combination with JAK inhibition (JAKi), is emerging as a promising and potentially disease modifying therapy for the treatment of myeloproliferative neoplasms (MPNs), specifically myelofibrosis (MF). While these agents promote spleen volume and bone marrow (BM) fibrosis reduction, their impact on symptom burden is less pronounced, perhaps due to ineffective pro-inflammatory cytokine modulation. Alternative epigenetic modifying therapies that further quell NFkB-mediated inflammation might enhance clinical outcomes in MF. EP300 is a histone acetyltransferase increasingly implicated in MF and other myeloid diseases, and recent studies suggest p300 inhibition, like BRD4 inhibition, can suppress NFkB-mediated inflammation and fibrosis progression in vivo. The expression of many genes is synergistically controlled by BRD4 and p300, leading to the development of EP31670 (aka NEO2734), a dual BRD4 (BET) + p300 inhibitor. EP31670 has shown pre-clinical activity across multiple leukemia cell lines and in myeloid leukemia xenograft models, and a phase-I clinical trial of EP31670 is on-going for treatment-refractory hematologic malignancies, including MF (NCT05488548). Given encouraging pre-clinical activity of combined BRD4/p300 inhibition and the emerging role of both BRD4 and p300 in fibrosis, we sought to investigate efficacy of EP31670 in MF animal models. Methods The hMPLW515L adoptive transfer model was chosen for analysis. Lethally-irradiated BalbC mice were transplanted with lineage-negative BM cells retrovirally transfected with hMPLW515L-IRES-GFP plasmid and monitored for the development of MPN. Approximately 3 weeks post-transplant, mice were bled and cohorted into respective vehicle (VEH), EP31670 (3.5mg/kg BID), ruxolitinib (RUX; 60mg/kg BID), or combination (Combo) therapy arms. At the conclusion of the 3-week trial, mice were sacrificed and BM/spleen harvested for flow cytometric and histopathologic analysis. Serum cytokine profiling was performed using the Millipore 32-plex cytokine array kit on a Luminex platform. Results hMPLW515L mice treated with EP31670 demonstrated significant reductions in total white blood cell (K/uL: VEH 219, RUX 77, EP31670 36, Combo 15, p<0.005) and platelet counts (K/uL: VEH 2097, RUX 3029, EP31670 1286, Combo 799, p<0.05) compared to VEH and RUX treated arms, with distinct combinatorial activity observed in mice treated with EP31670/RUX together. Spleen volume reductions were observed across all treatment arms but reached statistical significance with combination therapy primarily (mg: VEH 472, RUX 377, EP31670 372, Combo 175, p<0.05). By flow analysis, significant reductions in total peripheral blood mutant cell fraction by GFP% were observed with EP31670, both alone and in combination with RUX (GFP: VEH 85%, RUX 84%, EP31670 71%, Combo 65%, P<0.005)—an effect not observed with RUX alone—consistent with selective loss of mutant cells in this compartment. Total BM Mac1+Gr1+ mature myeloid cell fractions were also significantly reduced with RUX/EP31670 combination (VEH 75%, RUX 76%, EP31670 68%, Combo 56%, p<0.05); however, this reduction did not extend to more primitive myeloid progenitor fractions. Improvements in BM reticulin fibrosis, along with reductions in total megakaryocyte number, were also observed with EP31670—an effect again further enhanced with combined RUX/EP31670. Finally, serum cytokine analysis revealed significant reductions in multiple inflammatory cytokines of EPI31670 +/- RUX treated mice not observed with RUX alone, including NFkB-mediated cytokines LIF, IL13, IP-10, MIG, and RANTES suggesting enhanced anti-inflammatory effects with EP31670, particularly in combination with JAKi. Conclusions In line with early results from ongoing human clinical trials, a low dose of EP31670 demonstrates significant pre-clinical efficacy in MF models—with reduction of pro-inflammatory cytokines, spleen volume, hematologic parameters, and BM fibrosis—both alone and in combination with JAKi. Notably, the efficacious dose of EP31670 in these mouse studies, in combination with JAKi, was lower than what is usually required for EP31670 efficacy when applied as monotherapy in other mouse tumor models. Dual BET+p300 inhibition suppresses pro-inflammatory cytokine production in vivo and represents a potential alternative to other emerging epigenetic modifying therapies for MF treatment.