Abstract 253: Identification of STK33 kinase inhibitors for the validation of a synthetic lethal relationship between STK33 and mutant KRAS
Authors: Zhang Yihong , Kurzeja Robert , Munzli Anke , Zondlo James , Miguel Tisha San , Robinson John , McCarter John , Schwandner Ralf , Judd Ted , Fernando Manory , Babij Carol , Carnahan Josette , Dussault Isabelle 
Published: June 2012

Abstract
The kinase activity of the serine/threonine kinase STK33 was recently shown to be required for the survival of cancer cell lines dependent on the KRAS oncogene. This suggests that an STK33 kinase inhibitor could be useful to treat cancer patients whose tumors harbor KRAS mutations. Our goal was to identify STK33 kinase inhibitors to explore the role of this kinase in mutant KRAS cancer cell lines. First, full-length wild-type or kinase dead (KD) STK33 proteins were prepared using a baculovirus/insect expression system and purified by chromatography. Preliminary experiments identified two peptides, CREBtide and p70S6K peptides, as potential substrates for STK33. Both peptides were therefore used to track STK33 kinase activity during purification steps. We found that phosphorylation of the p70S6K peptide correlated well with STK33 presence during purification, but the peptide was not phosphorylated by the kinase dead STK33. In contrast, CREBtide phosphorylation showed a poor correlation with STK33 positive fractions and also occurred in the presence of a kinase dead STK33. The p70S6K peptide was thus chosen as the substrate for final optimization of an STK33 kinase assay. Over 400 000 compounds were screened using a high throughput STK33 kinase assay. Multiple hits were identified and IC50s were determined for the top 1043 compounds. Multiple potent STK33 kinase inhibitors were identified, including 69 compounds with IC50s &lt;100 nM and 6 with IC50s &lt;10 nM. Since p70S6K and RPS6 phosphorylation were previously shown to be dependent on STK33 in mutant KRAS AML cell lines, we tested our STK33 inhibitors for their ability to inhibit these signaling nodes in cells. In our hands, the levels of p70S6K phosphorylation were too low to be measured accurately, thus we tested the effect of the 510 most potent STK33 kinase inhibitors on RPS6 phosphorylation in NB4 and SKM-1 mutant KRAS cell lines. While many compounds inhibited RPS6 phosphorylation, there was no correlation between the potency of the compounds in the STK33 enzyme assay and on RPS6 phosphorylation in cells. To assess whether the kinase activity of STK33 is required for the survival of mutant KRAS cell lines, we tested the effect of the 145 most potent STK33 kinase inhibitors on the survival of 4 AML cell lines, 2 mutant for KRAS (NB4 and SKM-1) and 2 wild-type for KRAS (U-937 and OCI-AML3). There was no correlation between the effect of the compounds on STK33 kinase activity and their effects on cell survival. Many compounds inhibited cell growth, but not in a KRAS-dependent manner. In summary, we have identified STK33 kinase inhibitors using a high throughput screen. These compounds had no selective growth inhibitory effects on KRAS dependent cells. We conclude that the STK33 kinase activity does not contribute to the survival of KRAS-dependent cell lines.
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 253. doi:10.1158/1538-7445.AM2011-253