Bromodomain and extra-terminal protein (BETP) inhibitors (BETis) disrupt the chromatin binding and activity of the BETP BRD4 in facilitating RNA pol II-mediated mRNA transcription, thereby depleting levels of active oncoproteins including c-Myc, CDK6, BCL2, PIM1 and MCL1. BETi treatment also increases protein levels of p21, p27 and HEXIM1, thereby causing growth inhibition and apoptosis of AML blast progenitor cells (BPCs), including post-MPN, secondary AML (sAML) BPCs. Treatment with BETi (e.g., OTX015) has been shown to reduce AML burden and induce clinical remissions. However, BETi-refractory AML develops uniformly. Previous reports utilizing mouse AML models have highlighted that persister-resistance to BETi (BETi-P/R) in AML stem progenitor cells is observed despite BETi treatment and reduction of BRD4 occupancy on the chromatin. This is mediated by re-expression of c-Myc due to transcriptional activity of WNT-β-catenin. In the present studies, we developed human sAML models of BETi-P/R to elucidate the mechanisms and develop targeted therapies against BETi-P/R sAML BPCs. Utilizing human sAML control (parental) SET2 and HEL92.1.7 cells and subjecting them to at least 10 exposures to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, we first generated the BETi-P/R SET2-P/R and HEL-P/R cells. These cells were > 10-fold resistant to OTX015 and exhibited cross-resistance to other BETis, including JQ1 and ABBV-075. As compared to the control sAML cells, SET2-P/R and HEL-P/R cells neither exhibited additional genetic alterations by NextGen whole-exome sequencing, nor showed altered levels of TRIM33, SPOP or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). In contrast, compared to the control, SET2-P/R and HEL-P/R cells demonstrated significantly higher nuclear levels and binding of β-catenin to the transcription factor TCF7L2 (TCF4) and TBL1X (TBL1), associated with increased expression of TCF4 targets, including c-Myc, Cyclin D1, TERT and Survivin. ATAC-Seq and ChIP-Seq (H3K27Ac mark) analyses showed significant gain of peaks and active enhancers in HEL-P/R over HEL92.1.7 cells, including enrichment of the STAT5, MYC, PU.1, GATA2 and MYB transcription factor binding sites, as well as newly gained peaks in the enhancers of JAK1/2, RUNX1, PU.1, MYC, BCL2L1 and CTNNB1. RNA-Seq analysis showed significant increase/decrease in mRNA expressions (340/247), with increased expression of gene-sets involving MYC/MAX, STAT5, NFkB and TCF4 targets. QPCR and Western analyses confirmed significant perturbation in gene expressions, with increase in TCF4, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Consistent with the finding that shRNA-mediated knockdown of BRD4 exerted similar lethal effects in BETi-P/R versus control cells, we also discovered that BETP-PROTAC (proteolysis targeting chimera) ARV-771 (Arvinas, Inc.) that degraded BRD4/3/2 was equipotent in inducing apoptosis of BETi-P/R and control sAML cells. Also, consistent with increased nuclear levels and binding (utilizing confocal microscopy) of β-catenin with TBL1 and TCF4 in BETi-P/R sAML BPCs, β-catenin inhibitor BC2059 (Beta-Cat Pharma), which disrupts the binding of nuclear β-catenin with TBL1 and TCF4 and depletes β-catenin levels, exerted similar lethal effects in BETi-P/R sAML and control sAML cells. Consistent with these findings, we also determined that co-treatment with ARV-771 and BC2059 exerted synergistic in vitro lethality against BETi-P/R sAML BPCs (combination indices < 1.0), which was associated with greater reduction in levels of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL. Co-treatment with ARV-771 and BC2059 was also synergistically lethal against 12 patient-derived samples of CD34+ sAML BPCs. Notably, compared to treatment with each agent alone or vehicle control, in vivo treatment with ARV-771 (30 mg/kg SQ daily x 5, per week) and BC2059 (30 mg/kg IP BIW per week) for 3 weeks, significantly reduced the sAML burden and improved survival of the NSG mice engrafted with luciferase-transduced HEL-P/R cells (p < 0.01). These findings demonstrate that increased levels and activity of β-catenin-TCF7L2-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BETP degrader and β-catenin-TCF4 inhibitor is synergistically lethal against BETi-P/R sAML BPCs.
Soldi:Beta Cat Pharma: Employment. Bose:Astellas Pharmaceuticals: Research Funding; Celgene Corporation: Honoraria, Research Funding; Blueprint Medicines Corporation: Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding; CTI BioPharma: Research Funding; Incyte Corporation: Honoraria, Research Funding. Kadia:BMS: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Celgene: Research Funding; BMS: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy; Celgene: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Pfizer: Consultancy, Research Funding; Abbvie: Consultancy; Abbvie: Consultancy. DiNardo:Abbvie: Honoraria; Medimmune: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Bayer: Honoraria; Agios: Consultancy. Horrigan:Beta Cat Pharma: Employment. Khoury:Stemline Therapeutics: Research Funding. Verstovsek:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy.
Asterisk with author names denotes non-ASH members.