Abstract

Cyclin dependent kinases (Cdks) not only drive cell cycle progression, but also control transcription. For example, Cdk7 and Cdk9 phosphorylate specific sites on the C-terminal domain of RNA polymerase II (pol II) to promote transcription initiation and elongation. Our previous experience with flavopiridol, a non-selective pan-Cdk inhibitor, in primary chronic lymphocytic leukemia (CLL) cells demonstrated inhibition of RNA pol II-driven mRNA synthesis, reduction of the expression of short lived anti-apoptotic proteins, and induction of apoptosis in CLL cells in vitro (

Blood
106
:
2513
–19,
2005
). Here we studied a novel Cdk inhibitor SNS-032, a 2-aminothiazole derivative with potent and selective inhibitory activity against Cdk2, Cdk7 and Cdk9. We hypothesized that similar to flavopiridol, SNS-032 would induce apoptosis in CLL cells through transcriptional inhibition of anti-apoptotic proteins. To test this hypothesis, we first studied toxicity of SNS-032 in primary CLL samples. At the clinically relevant concentration of 0.3 μM, SNS-032 induced 61 ± 13% apoptosis after 24 hr (mean ± S.D., n=19). This occurred in a time- and concentration-dependent manner that plateaued after 10–12 hr. Additional studies demonstrated this was not due to inactivation of SNS-032. A similar amount of apoptosis was induced in samples with a deletion of p53 gene, indicating a p53-independent cell death. This is important since clinical resistance to fludarabine in CLL is associated with loss of p53 function. Peripheral blood mononuclear cells from healthy donors were less sensitive to SNS-032, suggesting SNS-032 is selective against CLL cells. In direct comparisons SNS-032 was more potent than flavopiridol; 0.1 μM SNS-032 induced a similar amount of apoptosis as did 3 μM flavopiridol. Secondly, we investigated the inhibition of transcription by SNS-032. SNS-032 strongly inhibited the phosphorylation of pol II at both the Ser2 and Ser5 sites of its C-terminal domain, which are the substrates of Cdk9 and Cdk7, respectively. Inhibition of pol II function was associated with a decrease in RNA synthesis, measured by the incorporation of [3H]uridine. After 6 hr incubation, 0.1 μM SNS-032 inhibited 80% of RNA synthesis. Again, SNS-032 was about 30-fold more potent than flavopiridol at inhibiting transcription. As the most sensitive targets of transcription inhibitors are mRNAs and proteins with short intrinsic half-lives, we measured the mRNA levels of short-lived anti-apoptotic proteins by real-time RT-PCR. SNS-032 significantly reduced the mRNA levels of Mcl-1, XIAP and Bcl-2. The protein levels of Mcl-1 and XIAP were also reduced accordingly, whereas Bcl-2 protein remained stable, consistent with a longer half-life of the protein. As CLL cells exemplify a biological context of a tumor that is dependent upon the continued expression of anti-apoptotic proteins for survival, we conclude that inhibition of their expression led to the SNS-032-induced cell death. Thus, while the inhibition of transcription induced by both SNS-032 and flavopiridol is associated with apoptosis in CLL cells, SNS-032 is about 30-fold more potent. This may be due to differences in potency against Cdks and/or differential protein binding. These in vitro results provide rationale for a phase 1 trial of SNS-032 administered to patients with advanced B-lymphoid malignancies that is currently ongoing in multiple centers.

Author notes

Disclosure:Employment: Ute Hoch, Rachael E. Hawtin and Judith A. Fox are employees of Sunesis Pharmaceuticals. Research Funding: William G. Wierda and William Plunkett are recipients of research grants from Sunesis Pharceuticals.