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XLS file 3927K, STK33 enzymatic activity and cellular activities for molecules a-l from Figure 6E

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XLS file - 175K, Effects of 515 small molecules on STK33 enzymatic activity vs. phosphorylation of RPS6 in cell lines

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Tumors harboring KRAS mutations remain an elusive target for oncology therapeutics. A recent publication (Scholl et al 2009) described a high throughput cellular RNAi screen which suggested a synthetic lethal relationship between STK33 and mutant KRAS. STK33 is a member of the calcium/calmodulin kinase family with a poorly characterized function. Although no genetic alterations in the STK33 gene have been reported in cancer, inhibiting STK33 kinase activity could offer a novel therapeutic approach to target mutant KRAS tumors. Thus, we sought to validate whether the survival of cancer cell lines with mutant KRAS is dependent on STK33, in parallel with a program to identify small molecule inhibitors of STK33 (Zhang Y et al AACR 2011). We focused on mutant KRAS leukemia cells because of their reported dependence on STK33. Using a panel of siRNA, 50-80% knockdown of STK33 at the RNA and protein level was achieved in NOMO-1KRASG13D and SKM-1KRASK117N cells. However we did not observe any alteration in cell viability. In addition, putative STK33 downstream signaling, (phosphorylation of p70 S6K thr389 and RPS6 ser235/236) was unaltered by modulation of STK33 expression. In contrast, knockdown of KRAS caused a significant reduction in both viability and signaling in these cell lines, confirming their dependence on KRAS. A panel of 27 cancer cell lines was screened with an siRNA library representing 1500 druggable genes. STK33 siRNA had no significant effect on viability regardless of KRAS mutational status. As expected, knockdown of KRAS significantly reduced cell viability, especially in mutant KRAS cells. A kinase dead mutant STK33 was used to test the dependence on STK33 kinase activity in mutant KRAS cell lines. Transient over-expression of kinase dead STK33 in PANC-1KRASG12D and DLD-1KRASG13D cells had no effect on viability, nor on the phosphorylation of p70 S6K thr389 and RPS6 ser235/236. In summary we have been unable to confirm a synthetic lethal relationship between STK33 and KRAS. Our data do not support inhibition of STK33 as a promising therapeutic approach for targeting mutant KRAS tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2011-252