Abstract #3265: RGB-286638, a multi-targeted protein kinase inhibitor, induces apoptosis involving the inhibition of RNA polymerase II carboxyl-terminal domain phosphorylation and the loss of the anti-apoptotic BCL2 family member Mcl-1.

The indeno-pyrazole compound RGB-286638 is a multi-targeted inhibitor with targets comprising the family of cyclin-dependent kinases (CDKs) and a range of other cancer-relevant Tyrosine- and Serine/Threonine kinases. The compound was also shown to have anti-proliferative activity against several tumor cell lines. In this study we further explored the broad cellular activity of RGB-286638 and investigated the kinetics and mechanism of its cell killing activity in more detail. We performed a comparative analysis of the anti-proliferative activity of RGB-286638 with a number of anticancer agents using a panel of 50 cancer cell lines representing 14 different solid tumor types. Our analysis confirmed the broad cellular activity of RGB-286638 (mean GI₅₀=25nM, mean GI₉₀=49 nM, mean TGI=73 nM). Furthermore, the comparison of cellular activity profiles revealed that the highest level of similarity was found to be with RGB-344064, a pre-clinical stage selective inhibitor of the CDKs (R=0.76). Next in similarity were etoposide (R=0.60) and SN-38 (R=0.49), an active metabolite of irinotecan. The lowest levels of similarity were found with gemcitabine (R=0.19) and staurosporine (R=0.17). Among the most sensitive tumor cell lines were A2780, CAKI1, MDA-MB231, ACHN and HCT116 (GI₅₀<10nM). Insensitive cell lines represented those known for the expression of multidrug resistant (MDR) transporter genes. The kinetics of cell killing by RGB-286638 was investigated on HCT-116 colon cancer cells using a clonal survival assay. At a dose of 0.10 µM, RGB-286638 inhibited 50% of colony formation within 1 hour of exposure and 90% within 6 hours. The results from a TUNEL assay of treated cells suggested that the mechanism of cell killing involves apoptosis. The rapid onset of apoptosis plus the known potent activity of RGB-286638 against CDK9 prompted us to investigate if the short-lived anti-apoptotic protein Mcl-1 might be one of the downstream targets of RGB-286638. It was found that inhibition of RNA pol II CTD phosphorylation occurred very rapidly, within 1 hour of compound addition, and preceded the disappearance of Mcl-1 protein and induction of PARP cleavage. The loss of Mcl-1 protein correlated with the transcriptional down-regulation of Mcl-1 mRNA, as demonstrated by quantitative RT-PCR. In conclusion, RGB-286638 is shown to act as a rapid inducer of apoptosis. The induction of apoptosis is likely to occur through a mechanism involving inhibition of RNA pol II CTD phosphorylation, which in turn results in the rapid disappearance of the anti-apoptotic BCL2 family member, Mcl-1.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3265.