Abstract B169: Characterization of in vitro activity and predictive biomarkers of the novel p21-activated kinase 4 (PAK4) inhibitor, PF-3758309, in colorectal cancer cell lines

Abstract

Abstract Background: The p21-activated kinase (PAK) family of serine/threonine kinases is overexpressed in multiple cancer types including breast, ovarian, colorectal, thyroid, and pancreatic. The two groups of PAK proteins, Group I (PAK 1–3) and Group II (PAK 4–6), are critical mediators of cell survival, motility, mitosis, transcription, and translation. The goal of this study was to assess the efficacy of a PAK4 inhibitor, PF-3758309, against a panel of colorectal cell lines and to identify potential predictive biomarkers of sensitivity or resistance that could be used to individualize therapy. Methods: A panel of 27 colorectal cancer (CRC) cell lines were exposed to increasing doses of PF-3758309 (0.015–1 µM) for 72 hours and analyzed for inhibition of proliferation using the sulforhodamine B (SRB) assay. Cell lines were designated sensitive (S), intermediate (I), or resistant (R) based on IC50's 0.125 and 1 µM, respectively. Assessment of induction of apoptosis was carried out using the caspase 3/7 assay at 24, 48, and 72 hours, whereas a soft agar colony assay was conducted on one sensitive and one resistant cell line to demonstrate anchorage- independent growth. To evaluate the effects on known target proteins of PAK-4, immunoblotting was performed for phosphorylated and total PAK-4, RhoA, GEF-H1, as well as proteins involved in apoptosis and the cell cycle. Lastly, to identify predictive genetic markers, five each of the most sensitive (IC50 1 mM) cell lines were subjected to gene array analysis using the Affymetrix U133 plus 2.0 gene chip. Results: Analysis of inhibition of proliferation in the 27 CRC cell lines demonstrated a greater than 10-fold (IC50 < 0.015 to > 1 mM) range of responsiveness to PF-3758309 that was accompanied by similarly diverse ranges of apoptosis induction. A sensitive cell line (HCT-116) demonstrated inhibition of anchorage-independent growth upon exposure to PF-3758309 that was not observed in the resistant (GEO) cell line. Overall, there did not appear to be an obvious relationship between effector protein expression and responsiveness to the PAK-4 inhibitor. Interestingly, gene array analysis indicated vimentin and ZEB1 expression to be increased, while E-cadherin was decreased, in the sensitive CRC cell lines. Other genes involved in cell cycle, MAPK pathway, and cytoskeleton dynamics were also differentially expressed. Conclusions: These data suggest that PAK4 inhibition by PF-3758309 results in efficacy against CRC cell lines as demonstrated by decreases in proliferation, anchorage-independent growth, and induction of apoptosis. The range of responsiveness to PF-3758309 and differential gene expression is being utilized to develop patient selective biomarkers for treatment with this novel compound. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B169.