Notable Efficacy of Co-Treatment with FHD-286, a Dual BRG1/BRM ATP-Ase Inhibitor, and Menin or BET Inhibitor, Decitabine or Venetoclax Against AML with MLL-r or Mutant NPM1

Abstract

BRG1 (SMARCA4) and BRM (SMARCA2) are the core ATPase within the multi-protein, ATP-dependent, chromatin remodeling BAF complexes that regulate gene transcription. FHD-286 is a potent, selective, orally administered, inhibitor of BRG1/BRM in early clinical development as a therapy for AML. In present studies, we determined that exposure to FHD-286 (10 to 100 nM) for 4 to 7 days induced CD11b and morphologic features of differentiation and loss of viability in AML cell lines (MOLM13, MV4-11 and OCI-AML3) and patient-derived (PD) AML cells with MLL1r or mtNPM1. Notably, FHD-286 also induced significant lethality in Menin inhibitor (MI) resistant AML cell lines and PD AML cells lacking Menin mutations. ChIP-Seq analysis showed that exposure to FHD-286 significantly reduced the occupancy of BRG1 and H3K27Ac on the chromatin and reduced the core transcriptional regulatory circuitry in MOLM13 cells. FHD-286 treatment concordantly reduced genome-wide ATAC-Seq and RNA-Seq peaks, with negative enrichment at the gene-sets of MYC and E2F targets, mTORC1 signaling, translation initiation/elongation and DNA replication. scRNA-Seq analysis in PD AML bone marrow cells with MLL1r or mtNPM1 showed significant decline in the myeloid/erythroid progenitor (MEP) cells. Mass spectrometry conducted on FHD-286-treated vs control cells revealed a significant decline in the protein levels of c-Myc and its targets, as well as of PLK1, BCL2, PU.1 and CDK4, but exhibited increased levels of HEXIM1, CDKN1A/1B, CEBPα and CD11b. Moreover, CyTOF analysis of PD AML cells with MLL1r or mtNPM1 showed significant decline in the protein levels of c-Myc, PU.1, BRG1, MEF2C, PBX3, BCL2 and MCL1, but exhibited increased levels of cleaved PARP and Caspase-3 in the AML stem/progenitor cells defined by high expression of CLEC12A, CD123, CD99 and CD33 but low expression of CD11b. A domain-specific CRISPR screen targeting epigenetic regulators revealed significant co-enrichments with FHD-286 treatment of the gRNAs against BRD4 and LSD1, suggesting them as the epigenetic mechanisms of FHD-286 resistance. Consistent with findings noted above, co-treatment with FHD-286 and venetoclax, BET inhibitor (OTX015), decitabine or MI (SNDX-50469) exerted synergistic lethality in AML cell lines (MOLM13, MV4-11 and OCI-AML3) and PD AML cells with MLL1r or mtNPM1 (delta synergy scores between 10 to 33 by the ZIP method). In a PD xenograft (PDX) model of MLL1r AML with FLT3 mutation, monotherapy with FHD-286 (oral gavage) for 4 to 6-weeks was significantly effective in reducing AML burden and improving overall survival. Separately, in vivo FHD-286 treatment vs vehicle control, also significantly inhibited the AML-initiating potential, by reducing the AML burden and improving survival of the re-engrafted mice with the FHD-286-treated PDX cells. Moreover, co-treatment with FHD-286 and venetoclax or decitabine or OTX015, vs each drug alone, or vehicle control, significantly reduced the AML burden and improved survival, without significant toxicity in the PDX model. Finally, co-treatment with FHD-286 and OTX015 or SNDX-5613 (oral gavage) was significantly more effective than each drug alone in reducing the AML burden and overall survival of mice engrafted with a separate PDX model of AML cells with mtNPM1 and FLT3-ITD, without significant toxicity. These findings demonstrate the pre-clinical efficacy of FHD-286-based rational combinations and underscore their promise against AML with MLL1r or mtNPM1.