17040 Background: Erlotinib (E) and pemetrexed (P) have proven single agent activity in advanced NSCLC after first-line chemotherapy. The presence of K-Ras or EGFR T790M mutations in NSCLC tumors has been associated with primary or secondary resistance to erlotinib. We have previously reported that sequential administration of P followed by E results in synergistic cytotoxicity in NSCLC cells that contain the wild-type EGFR gene. In this study, we evaluate the cytotoxic effects of E and P in E-resistant human NSCLC cell lines that have either EGFR or K-Ras mutations and validate predictive biomarkers for drug sensitivity. METHODS E-resistant NSCLC cell lines [H1650 (EGFRdel E746-A750), H1975 (EGFRL858R/T790M), and A549 (K-RasG12S)] were exposed to E and P continuously for 72 hrs. Combinations of E and P were compared with E or P alone. Cellular effects assayed include cell survival by cell count and MTT assay, cell cycle distribution and apoptosis by flow cytometry after propidium iodide staining, and expression of different elements of the EGFR axis by immunoblots. The degree of cytotoxic synergism was expressed by the combination index (C.I.). RESULTS P and E have significant cytotoxic synergism (C.I. <1) in these E-resistant NSCLC cell lines. The cytotoxic synergism is, at least in part, mediated by the inhibitory effect of E on the P-induced activation of EGFR-mediated proliferation and/or survival responses (mainly via the PI3K/AKT pathway). Sensitivity of NSCLC cells to P and E might be predicted by the ratio of (p-EGFR:EGFR) to (p-AKT:AKT), reflecting tumor dependency on the EGFR axis for growth and survival. CONCLUSIONS Together with our previous data, we have demonstrated that the combination of P and E is synergistic in E-resistant NSCLC cell lines that have either a wild-type or mutant EGFR gene, or a mutant K-Ras gene. This combination deserves further evaluation in a clinical trial for the treatment of advanced NSCLC. No significant financial relationships to disclose.