The BET protein BRD4 binds to acetylated lysines on the histone proteins, recruits pTEFb kinase, a complex of cyclin T and CDK9, to gene promoters to phosphorylate serine 2 of the C-terminus of RNA pol (RNAP) II. This promotes the pause release of RNAP II, with the elongation and expression of the mRNA transcripts of several oncogenes as well as their target genes, which are essential for the growth and survival of AML cells. We have recently determined that the BET protein antagonist JQ1, which disrupts the binding of BRD4 to acetylated lysines, potently induces apoptosis in cultured (OCI-AML3 and MOLM13) and patient-derived primary AML blast progenitor cells. JQ1 treatment also improved the survival of the immune-depleted mice engrafted with MOLM13 or primary AML cells. The lethal activity of JQ1 is associated with marked induction of hexamethylene bisacetamide (HMBA)-inducible protein 1 (HEXIM1) and p21, while MYC, BCL2 and CDK4/6 expressions are concomitantly downregulated in the cultured and primary AML cells. HEXIM1 binds and sequesters pTEFb, thereby inhibiting its kinase activity for RNAP II. To determine the role of HEXIM1 induction on the lethal effects of JQ1, we achieved a stable lentivirus mediated knockdown (KD) of the mRNA and protein expressions of HEXIM1 by approximately 90% in OCI-AML3 and MOLM13 cells. There was no significant difference in the protein expressions of BRD4, MYC and BCL2, or of cyclin T and CDK9 levels in the nuclear fraction in the AML cells with HEXIM1 knockdown (HKD) versus the AML cells expressing non-targeted (NT) shRNA. However, in the HKD cells, higher levels of the complex of cyclin T with CDK9 as pTEFb were detected in the immunoprecipitates of cyclin T. As compared to the NT AML, JQ1 (250 to 2000 nM)-induced HEXIM1 and apoptosis was reduced in HKD AML cells. This was associated with abrogation of JQ1-mediated HEXIM1 and p21 induction in the HKD AML cells. Similarly, HMBA (5 mM)-induced HEXIM1 and apoptosis was also inhibited in the HKD versus NT AML cells. Co-treatment with JQ1 and pan-HDAC inhibitor panobinostat (PS) synergistically induced apoptosis of cultured (MOLM13 and OCI-AML3) and primary AML blast progenitor cells, as well as conferred superior in vivo survival on the mice engrafted with cultured and primary AML blast progenitor cells. Importantly, co-treatment with JQ1 and PS also synergistically induced apoptosis of the cultured HKD AML cells. These findings indicate that HEXIM1 induction is a biomarker and plays a mechanistic role in the lethal activity of BRD4 antagonist against human AML cells.

Citation Format: Santhana G. T. Devaraj, Warren Fiskus, Sunil Sharma, Jun Qi, Bhavin Shah, Leasha J. Schaub, Melissa Rodriguez, Ka Liu, Swaminathan P. Iyer, James E. Bradner, Kapil N. Bhalla. HEXIM1 induction exerts a mechanistic role and is a biomarker of lethal activity of BRD4 antagonist against human AML cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3686. doi:10.1158/1538-7445.AM2014-3686