Abstract 3036: BRD4 degrader and inhibitor of beta catenin-TCF7L2 are synergistically active against human AML cells resistant to BET inhibitor
Authors: Saenz Dyana T. , Fiskus Warren C. , Manshouri Taghi , Mill Christopher P. , Horrigan Steve , Soldi Raffaella , Khoury Joseph D. , Sharma Sunil , Verstovesek Srdan , Bhalla Kapil N. 
Published: August 2019

Abstract
Treatment with bromodomain and extra-terminal protein inhibitor (BETi) reduces in vivo AML cell burden, inducing clinical remissions in AML. Yet, resistance to BETi treatment develops uniformly. Here, following at least 10 exposures of secondary (s) AML control (parental) SET2 and HEL92.1.7 cells to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, we generated BETi persister-resistant (BETi-P/R) SET2-P/R and HEL-P/R cells. These cells showed &gt; 10-fold resistance to OTX015 and cross-resistance to other BETis. Compared to the controls, SET2-P/R and HEL-P/R cells lacked additional genetic alterations or altered levels of TRIM33, SPOP, DUB3 or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). However, SET2-P/R and HEL-P/R cells demonstrated significantly higher nuclear levels of β-catenin, the transcription factor TCF7L2 and adaptor protein TBL1X (TBL1), associated with increased expression of TCF7L2 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, with enrichment of STAT5, MYC, PU.1 and GATA2 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 induction of gene-sets involving MYC/MAX, STAT5, NFkB and TCF7L2 targets. QPCR and Western analyses confirmed increase in the mRNA and protein levels of TCF7L2, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Also, confocal microscopy demonstrated increased binding of β-catenin with TBL1 and TCF7L2 in the nucleus in BETi-P/R sAML cells. β-catenin inhibitor BC2059, which disrupts binding of nuclear β-catenin with TBL1 and TCF7L2 and depletes β-catenin levels, exerted similar lethality in BETi-P/R sAML and control sAML cells. Both shRNA-mediated knockdown of BRD4 and BRD4-PROTAC (proteolysis-targeting chimera) ARV-771 (Arvinas, Inc.), which degrades BRD4/3/2, induced similar level of apoptosis in BETi-P/R and control sAML cells. Co-treatment with ARV-771 and BC2059 synergistically induced lethality in BETi-P/R sAML cells as well as in patient-derived, CD34+ sAML BPCs (combination indices &lt; 1.0). This was associated with marked attenuation of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL levels. Also, compared to each agent alone, 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 sAML burden and improved survival of NSG mice engrafted with HEL-P/R cells (p &lt; 0.01). Thus, increased levels and activity of β-catenin-TCF7L2-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BRD4 degrader and β-catenin-TCF7L2 inhibitor is a promising therapeutic strategy against BETi-P/R sAML BPCs.
Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Christopher P. Mill, Steve Horrigan, Raffaella Soldi, Joseph D. Khoury, Sunil Sharma, Srdan Verstovesek, Kapil N. Bhalla. BRD4 degrader and inhibitor of beta catenin-TCF7L2 are synergistically active against human AML cells resistant to BET inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3036.