Mechanisms Underlying Superior Efficacy of Co-Targeting BET Proteins and Anti-Apoptotic BCL2 or MCL1 Protein Against AML Blast Progenitor Cells
Authors: Mill Christopher Peter , Cai Tianyu , Fiskus Warren , Borthakur Gautam , Kornblau Steven M. , Kadia Tapan M. , DiNardo Courtney D. , Nowak Agnieszka J , Saenz Dyana T. , Saenz David N , Sun Baohua , Ganesh Thurika , Jin Wendy , Rao Vaishnavi , Khoury Joseph David , Shen Yu , Konopleva Marina Y. , Bhalla Kapil N. 
Published: October 2019

Abstract
Bromodomain extra-terminal (BET) protein (BETP) family of chromatin reader proteins includes BRD4 that binds to acetylated lysine in the active chromatin and transcription factors (TFs) at enhancers and promoters, as well as increases RNA pol II (RNAP2) mediated mRNA transcription of active oncogenes in AML. Disruption of binding and eviction of BRD4 from chromatin results in transcriptional attenuation of pro-growth and pro-survival oncoproteins. These include c-Myc, BCL2, MCL1, CDK6, while inducing p21, p27 and HEXIM1, thereby causing growth inhibition and apoptosis of AML blast progenitor cells (BPCs). Consistent with this, first generation BETP inhibitors (BETi) (e.g., OTX015) have been shown to reduce AML burden and induce clinical remissions, albeit in a minority of patients with AML. Recently, more potent BETis such as ABBV-075 (AbbVie Inc.) have been developed and are being investigated for their clinical efficacy in AML. Venetoclax (ABT-199, AbbVie) and A-1210477 bind and inhibit the antiapoptotic activity of BCL2 and MCL1, respectively, lowering the threshold for apoptosis in AML BPCs. Treatment with venetoclax alone, and in combinations with other anti-AML agents, is effective in inducing clinical remissions in AML. Here, we interrogated the epigenetic mechanisms underlying transcriptional repression due to BETi treatment, as well as determined the anti-AML activity of co-treatment with BETi and venetoclax or A-1210477 against AML BPCs. ATAC-Seq analysis showed that, in BETi-treated (over untreated control) AML BPCs, BETi treatment induced significantly greater perturbations in the accessible chromatin (number of peaks gained or lost), which were especially enriched for TF-binding sites for RUNX1, c-Myc, GATA2, PU.1 and ERG, especially in the DNA of BCL2, Bcl-xL, MCL1, MYC, BIM, PIM1, CDK6, BRD2/4, HEXIM1, CDKN1A, CEBPA and ITGAM. RNA-Seq analyses, followed by confirmation with QPCR, demonstrated that BETi treatment attenuated expression of MYC, BCL2, Bcl-xL and CDK6, while inducing mRNA expression of HEXIM1 and CDKN1A. BETi (ABBV-075) treatment also dose-dependently reduced protein levels of c-Myc, CDK6, MCL1 and BCL2, while inducing BIM, HEXIM1, CDKN1A and cleaved PARP levels in AML BPCs. This was associated with dose-dependent (10 to 250 nM) ABBV-075-induced apoptosis of cultured AML cell lines and AML BPCs. Whereas treatment with venetoclax (20 to 200 nM) or A-1210477 (1 to 10 µM) alone also induced apoptosis, co-treatment with ABBV-075 and venetoclax or A-1210477 synergistically induced apoptosis of AML BPCs, including CD34+ patient-derived AML BPCs (combination indices < 1.0). Notably, treatment with venetoclax significantly increased protein expression of MCL1 (p < 0.05), which has been recently reported as a potential mechanism of acquired-adaptive resistance to venetoclax. However, co-treatment with ABBV-075 significantly abrogated venetoclax-induced MCL-1 levels (p < 0.01), which likely contributed to the synergistic anti-AML activity of co-treatment with ABBV-075 and venetoclax against AML BPCs. We next determined the in vivo anti-AML efficacy of ABBV-075 and venetoclax in luciferase-transduced, AML BPCs (MOLM13) and patient-derived xenograft (PDX) models of AML BPCs engrafted in immune depleted (NSG) mice. Compared to treatment with either agent alone, co-treatment with ABBV-075 and venetoclax was significantly more effective in reducing AML cell burden (p < 0.001), without inducing toxicity, in AML BPC-engrafted NSG mice. Co-treatment with ABBV-075 and venetoclax also improved the median and overall survival of NSG mice engrafted with MOLM13 AML BPCs (p < 0.0001). Collectively, these pre-clinical findings elucidate the mechanistic rationale, and support evaluation of the clinical efficacy and safety of targeted co-treatment with BETi and BCL2 or MCL1 inhibitor in AML.

Disclosures
Kadia: Jazz: Consultancy, Research Funding; BMS: Research Funding; Novartis: Consultancy; Abbvie: Consultancy; Celgene: Research Funding; Celgene: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Abbvie: Consultancy; Amgen: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; BMS: Research Funding; Takeda: Consultancy; Pfizer: Consultancy, Research Funding. DiNardo:Karyopharm: Other: Advisory role; Medimmune: Other: Advisory role; Celgene: Other: Advisory role; Bayer: Other: Advisory role; Agios: Consultancy, Other: Advisory role; AbbVie: Consultancy, Other: Advisory role. Khoury:Stemline Therapeutics: Research Funding. Shen:AbbVie Inc: Employment. Konopleva:Stemline Therapeutics: Research Funding.