Abstract 2623: Molecular mechanism of synergy between BET-protein bromodomain antagonist (BA) and pTEFb kinase inhibitor against human AML blast progenitor cells

Abstract

Abstract We have previously reported that treatment with BET-protein bromodomain antagonist (BA) reduces the enhancer/promoter occupancy of BRD4 and induces the expression of HEXIM1, which sequesters and inhibits pTEFb (a complex of CDK9 and cyclin T). pTEFb phosphorylates serine 2 in the C-terminal heptad repeats in the stalled RNA pol II (RNAP2), causing its pause-release and the mRNA transcript elongation. While relatively sparing normal bone marrow progenitor cells, BA treatment attenuates the expression of cMYC, BCL2 and CDK4/6 and induces apoptosis of cultured (OCI-AML3, MOLM13 and MV4-11) and primary AML blast progenitor cells (BPCs), including those expressing FLT3-ITD. Because of its ability to inhibit CDK9 and pTEFb, we determined whether co-treatment with flavopiridol (FP), a known inhibitor of CDK9, would enhance the activity of BA against the cultured and primary AML BPCs. Our findings demonstrate that co-treatment with BA and FP synergistically induced apoptosis of the cultured and primary AML BPCs (CI of < 1.0). Combination of BA and FP, versus each drug alone, was associated with greater attenuation of the levels of p-RNAP2, cMYC, BCL-2 and CDK4/6 but greater induction of HEXIM1 and p21 levels. Notably, co-treatment with FP and BA markedly reduced the levels of the anti-apoptotic protein MCL1, which is known to be transcriptionally regulated by pTEFb-mediated phosphorylation of p-RNAP2. However, treatment with BA and FP did not affect the levels of cyclin T or CDK9. Co-treatment with BA and FP also synergistically induced apoptosis of MOLM13-TKIR cells (expressing FLT3-ITD) that exhibit > 100-fold in vitro resistance to FLT3 tyrosine kinase inhibitors. We next determined whether HEXIM1 levels regulate the activity of BA and FP in the AML BPCs. While shRNA-mediated stable knockdown of HEXIM1 significantly reduced the lethal activity of BA in OCI-AML3/HKD and MOLM13/HKD cells, tetracycline-inducible ectopic overexpression of HEXIM1 significantly enhanced BA-induced apoptosis of MOLM13 and OCI-AML3 cells. Notably, the significant improvement in the median survival of NOD/SCID mice treated with JQ1 (50 mg/kg/5 days per week/for 3 weeks) was not observed in the similarly treated mice engrafted with MOLM13/HKD cells (p < 0.01). The lethality induced by co-treatment with JQ1 and FP was significantly reduced in MOLM13/HKD versus MOLM13 cells; conversely, it was significantly increased in MOLM13 cells with ectopic overexpression of HEXIM1 (p < 0.01). These findings indicate that co-treatment with BA and FP exerts synergistic lethality, and BA-mediated induction of HEXIM1 is mechanistically involved in mediating the lethal effects of BA and FP against human AML BPCs. Citation Format: Santhana G. T. Devaraj, Bhavin Shah, Warren Fiskus, Baohua Sun, Saikat Saha, Sai Ravi Pingali, Swaminathan P. Iyer, Sunil Sharma, James E. Bradner, Kapil N. Bhalla. Molecular mechanism of synergy between BET-protein bromodomain antagonist (BA) and pTEFb kinase inhibitor against human AML blast progenitor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2623. doi:10.1158/1538-7445.AM2015-2623