Abstract 4600: P-TEFb is a therapeutic target in human bortezomib-resistant multiple myeloma cells
Authors: Yun Dai, Shuang Chen, Liang Zhou, Yu Zhang, Yun Leng, Xin-Yan Pei, Hui Lin, Robert Z. Orlowski, Steven Grant
Journal: Cancer Research (2014)

Abstract Despite proteasome inhibitor (e.g., bortezomib/btz and carfilzomib/cfz) success in multiple myeloma (MM), drug resistance remains a key problem. MM cells commonly over-express Mcl-1, a protein implicated in survival and drug-resistance. Mcl-1 exhibits rapid protein turnover (half-life &amp;lt; 1h), suggesting that efficient transcriptional machinery for de novo synthesis may be required for its constitutive expression in MM. Eukaryotic protein-coding gene transcription is controlled by RNA polymerase II (RNAPII), which is regulated by negative (N-TEF) and positive elongation factors (P-TEF). P-TEFb, a holoenzyme Cdk9/cyclin T complex, activates RNAPII by phosphorylating the S2 site within the carboxy-terminal domain (CTD). Here, the role of P-TEFb in Mcl-1 maintenance was investigated in MM cells. Anti-apoptotic Bcl-2 family members profiling revealed high Mcl-1 protein levels in virtually all MM lines tested and up-regulation in drug-resistant lines (e.g., btz-resistant PS-R cells). Similar patterns occurred in human MM xenograft mouse models and primary CD138+ MM cells. Ectopic Mcl-1 over-expression strikingly diminished btz lethality, arguing that Mcl-1 is a critical determinant of btz-resistance. All tested MM cell lines, primary MM specimens, and murine xenograft samples displayed constitutive P-TEFb activation i.e., high CTD (S2) phosphorylation, which correlated with a) up-regulation of P-TEFb subunits i.e., Cdk9 (42 and 55kD isoforms) and its partner cyclin T1 (but not T2a/b or K); b) marked 42kD T186 phosphorylation, while drug-resistant cells also displayed sharply increased 55kD T186 phosphorylation; and c) constitutive P-TEFb complex assembly and RNAPII interactions, but little inhibitory association with HEXIM which binds to P-TEFb via 7SK snRNP. The function of P-TEFb in Mcl-1 regulation was then validated by genetic and pharmacological strategies. First, Cdk9 or cyclin T1 shRNA knock-down dramatically inhibited CTD S2 phosphorylation and down-regulated Mcl-1. Second, pan-Cdk inhibitors e.g., dinaciclib rapidly inhibited P-TEFb and down-regulated Mcl-1, leading to apoptosis prior to cell cycle perturbations. These events primarily reflected P-TEFb complex disassembly, but not levels of P-TEFb subunits or T186 phosphorylation. Third, like Mcl-1 knock-down, Cdk9 shRNA or a specific Cdk9 inhibitor significantly restored susceptibility of btz-resistant cells to btz or cfz, and also sensitized them to the BH3-mimetic ABT-737 which does not target Mcl-1. Finally, pan-Cdk inhibitors reduced tumor burden and eliminated human CD138+ cells in the bone marrow of murine models bearing drug-naïve or btz-resistant MM cells. Collectively, these findings implicate constitutive activation of P-TEFb in maintenance of high Mcl-1 levels in MM, and argue that P-TEFb may serve as a rational target to overcome btz-resistance. This study was supported by the MD Anderson SPORE in Myeloma. Citation Format: Yun Dai, Shuang Chen, Liang Zhou, Yu Zhang, Yun Leng, Xin-Yan Pei, Hui Lin, Robert Z. Orlowski, Steven Grant. P-TEFb is a therapeutic target in human bortezomib-resistant multiple myeloma 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 4600. doi:10.1158/1538-7445.AM2014-4600