Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab

Abstract

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux®) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, sprycel®) a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src Family Kinases. We analyzed 16 CRC lines for: 1) KRAS mutation status, 2) dependence on mutant KRAS signaling, 3) expression level of EGFR and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo, and HCT116) cell lines, and two KRAS wild type cell lines (SW48 and CaCo2). In vitro, using Poly-D-Lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab whereas parental controls showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater anti-proliferative effect on KRAS mutant line as compared to either agent alone both in vitro and in vivo. To investigate potential mechanisms for this anti-proliferative response in the combinatorial therapy we performed Human Phospho-kinase Antibody Array analysis measuring the relative phosphorylation levels of phosphorylation of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the classical MAPK pathway, AKT/mTOR pathway and the family of STAT transcription factors when compared to the untreated control or monotherapy treatments. Next we analyzed tumor growth with cetuximab, dasatinib or the combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an anti-tumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation as measured by Ki67 and higher rates of apoptosis as measured by TUNEL. The data presented herein indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key-signaling pathways. Further, these results suggest that signaling via the EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. This data strengthen the rationale for clinical trials in this genetic setting combining cetuximab and dasatinib.