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Rationale: Pancreatic cancer (PC) is associated with advanced presentation and poor survival. Currently approved therapies, including EGFR inhibitors, have minimal effect on patient survival. PCs have a high incidence of activated Ras, which may bypass inhibition of EGFR, resulting in resistance to EGFR inhibitors. Mixed lineage kinases (MLKs) are members of a large family of mitogen-activated protein kinase (MAPK) kinase kinases (MAPKKKs) that activate MAPK pathways, involving JNK, ERK 1/2 and p38 cell signaling pathways.1 MLK3 is required for proliferation of tumor cells with oncogenic kras mutations.2 The role of MLK3 in the pathophysiology and resistance to therapy of PC is not well known. Methods: The requirement of MLK3 for cell proliferation and survival of PC cell lines PANC 1 (gift of Dr. J. McCormick, Michigan State University) and MiaPaCa2 (ATCC), both of which have activating ras mutations, was investigated using RNA interference (siRNA) and MLK3 inhibitor K252a to inhibit the expression and activity of MLK3. Cells were also exposed to EGFR inhibitor Compound 56 at various concentrations. Proliferation was assessed with MTT after 48-72 h exposure. The effect of MLK3 inhibition on relevant cell signaling pathways was assessed by immunoblotting with activation-specific phosphoantibodies directed against MAPKs and the prosurvival kinase Akt. Results: MLK3 expression was significantly higher in PC cell lines than in a normal pancreatic epithelial cell line (gift of Dr. M. Tsao, Princess Margaret Hosp, Toronto). siRNA decreased MLK3 expression in both PC cell lines. K252a also significantly decreased MLK3 expression. Inhibition of MLK3 by siRNA or K252a was associated with decreased cell proliferation in both PC cell lines, with concurrent decrease in pERK and pAkt in PANC-1 cells and decreased pERK and pJNK in MiaPaCa2 cells. Concomitant exposure to MLK3 inhibitor and Compound 56 further inhibited cell proliferation and increased caspase 3 activity, compared to either agent alone. These results suggest that MLK3 plays an important role in survival and proliferation in PC cells with kras mutations and inhibition of MLK3 may enhance the effects of EGFR inhibitors in PC.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA