A novel Menin-MLL1 inhibitor, DS-1594a, prevents the progression of acute leukemia with rearranged MLL1 or mutated NPM1.

BACKGROUND: Mixed lineage leukemia 1-rearranged (MLL1-r) acute leukemia patients respond poorly to currently available treatments and there is a need to develop more effective therapies directly disrupting the Menin‒MLL1 complex. Small-molecule-mediated inhibition of the protein‒protein interaction between Menin and MLL1 fusion proteins is a potential therapeutic strategy for patients with MLL1-r or mutated-nucleophosmin 1 (NPM1c) acute leukemia. In this study, we preclinically evaluated the new compound DS-1594a and its salts. METHODS: We evaluated the preclinical efficacy of DS-1594a as well as DS-1594a·HCl (the HCl salt of DS-1594a) and DS-1594a·succinate (the succinic acid salt of DS-1594a, DS-1594b) in vitro and in vivo using acute myeloid leukemia (AML)/acute lymphoblastic leukemia (ALL) models. RESULTS: Our results showed that MLL1-r or NPM1c human leukemic cell lines were selectively and highly sensitive to DS-1594a·HCl, with 50% growth inhibition values < 30 nM. Compared with cytrabine, the standard chemotherapy drug as AML therapy, both DS-1594a·HCl and DS-1594a·succinate mediated the eradication of potential leukemia-initiating cells by enhancing differentiation and reducing serial colony-forming potential in MLL1-r AML cells in vitro. The results were confirmed by flow cytometry, RNA sequencing, RT‒qPCR and chromatin immunoprecipitation sequencing analyses. DS-1594a·HCl and DS-1594a·succinate exhibited significant antitumor efficacy and survival benefit in MOLM-13 cell and patient-derived xenograft models of MLL1-r or NPM1c acute leukemia in vivo. CONCLUSION: We have generated a novel, potent, orally available small-molecule inhibitor of the Menin-MLL1 interaction, DS-1594a. Our results suggest that DS-1594a has medicinal properties distinct from those of cytarabine and that DS-1594a has the potential to be a new anticancer therapy and support oral dosing regimen for clinical studies (NCT04752163).

Novel orally bioavailable EZH1/2 dual inhibitors with greater antitumor efficacy than an EZH2 selective inhibitor.

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 functions as a catalytic subunit of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Tumorigenic functions of EZH2 and its synthetic lethality with some subunits of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes have been observed. However, little is known about the function of EZH1 in tumorigenesis. Herein, we developed novel, orally bioavailable EZH1/2 dual inhibitors that strongly and selectively inhibited methyltransferase activity of both EZH2 and EZH1. EZH1/2 dual inhibitors suppressed trimethylation of histone H3 lysine 27 in cells more than EZH2 selective inhibitors. They also showed greater antitumor efficacy than EZH2 selective inhibitor in vitro and in vivo against diffuse large B-cell lymphoma cells harboring gain-of-function mutation in EZH2. A hematological cancer panel assay indicated that EZH1/2 dual inhibitor has efficacy against some lymphomas, multiple myeloma, and leukemia with fusion genes such as MLL-AF9, MLL-AF4, and AML1-ETO. A solid cancer panel assay demonstrated that some cancer cell lines are sensitive to EZH1/2 dual inhibitor in vitro and in vivo. No clear correlation was detected between sensitivity to EZH1/2 dual inhibitor and SWI/SNF mutations, with a few exceptions. Severe toxicity was not seen in rats treated with EZH1/2 dual inhibitor for 14 days at drug levels higher than those used in the antitumor study. Our results indicate the possibility of EZH1/2 dual inhibitors for clinical applications.

Rapid receptor-proximal signaling assays for FcR gamma-containing receptors.

Novel, cell-based assays, based on bioluminescence resonance energy transfer, have been developed for FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling at receptor-proximal steps. In a stable transfectant of the HEK-293 cell line expressing human FcepsilonRIalpha, FcepsilonRIbeta, and FcRgamma-GFP2 and Syk(1-265)-Rluc fusion proteins, FcepsilonRI cross-linking markedly increased BRET2 ratios, which are the ratios of GFP2 emission to Rluc emission. These ratios reflect the FcRgamma-GFP2-Syk(1-265)-Rluc interaction in living cells. The signals are specifically inhibited by the Src-family kinase inhibitor PP2. Separately, in transient transfectants expressing GPVI, FcRgamma-GFP2, and Syk(1-265)-Rluc, the GPVI-specific ligand convulxin induced a two-fold increase in the BRET2 ratio and this increase was also inhibited by PP2. Finally, a differential assay was developed which permits the measurement of FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling in the same cell. These assays provide useful methods for monitoring FcRgamma-Syk interaction in real time in living cells and may contribute to the understanding of signal regulation through FcRgamma-containing receptors.