SNS-032, a novel inhibitor of cyclin-dependent kinases 2, 7 and 9, blocks transcription of cyclin D1 and Mcl-1, causing cell death in mantle cell lymphoma cell lines

756 SNS-032 is a potent, selective inhibitor of cyclin-dependent kinases (Cdk) 2, 7 and 9 that inhibits both the cell cycle and transcription. Cdk7/cyclin H and Cdk9/cyclin T control RNA polymerase II (pol II)-dependent transcription. Phosphorylation of Ser5 (Cdk7) and Ser2 (Cdk9) of the pol II C-terminal domain (CTD) activates transcription initiation and elongation. Because lymphoid malignancies are dependent upon the continual expression of anti-apoptotic proteins for survival, we hypothesize that transient inhibition of transcription will cause a critical decrease in such proteins (e.g., Mcl-1, XIAP) and initiate apoptosis. Thus, Cdk7 and Cdk9 are attractive targets for cancer therapy in this context. Our studies in primary chronic lymphocytic leukemia (CLL) lymphocytes demonstrated that SNS-032 effectively decreases the phosphorylation of pol II CTD and inhibits transcription; this is associated with diminished levels of anti-apoptotic proteins and activation of apoptosis. The present study focuses on mantle cell lymphoma (MCL) cell lines (Granta 519, Jeko-1, Mino and SP-53) to evaluate this hypothesis in a growing cell model. The biology of MCL is characterized by the t(11:14)(q13;q32) translocation, which leads to the over-expression of Cyclin D1 which sustains the malignancy. Interestingly, the transcript and protein of Cyclin D1 are both short-lived, and thus may be targeted by SNS-032, providing further biological context to test this strategy. In addition, Cdk2, which is highly expressed in MCL cells, is also blocked by SNS-032. SNS-032 (0.3 to 1 µM) inhibited the phosphorylation of RNA pol II CTD at both the Ser2 and Ser5 sites; this is associated with inhibition of RNA synthesis in all MCL lines. As a result, the transcripts and protein levels of short lived protein decreased, including Cyclin D1 and Mcl-1, and apoptosis was induced within 24 hr in all cell lines except for Granta, shown by PARP cleavage and the emergence of a subG1 population. This is consistent with SNS-032 acting as a transcriptional inhibitor. Apoptosis was strongly induced by 0.3 µM SNS-032 in Jeko and Mino cells after a 24 hr exposure, despite their deficiency in p53 function, indicating that the SNS-032 induced apoptosis was independent of p53. Although apoptosis was not detected in Granta cells, clonogenic assays showed a strong, time-dependent inhibition of colony formation, with an IC50 of about 50 nM after 24 hr incubation, indicating that SNS-032 effectively jeopardized MCL repopulating ability. Thus, SNS-032 appears to block transcription by inhibition of Cdk7 and Cdk9, an action that leads to the decrease of survival proteins and Cyclin D1, and the death of MCL cell lines.