Although mechanisms of acquired resistance of EGFR mutant non-small cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here, we observe that acquired resistance caused by the T790M gatekeeper mutation can occur either by selection of pre-existing T790M clones or via genetic evolution of initially T790M-negative drug tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug tolerant cells had a diminished apoptotic response to third generation EGFR inhibitors that target T790M EGFR; treatment with navitoclax, an inhibitor of BCL-XL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug resistant cancer cells can both pre-exist and evolve from drug tolerant cells, and point to therapeutic opportunities to prevent or overcome resistance in the clinic.

Tyrosine kinase inhibitors (TKIs) are effective treatments for non-small cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, relapse typically occurs after an average of one year of continuous treatment. A fundamental histological transformation from NSCLC to small cell lung cancer (SCLC) is observed in a subset of the resistant cancers, but the molecular changes associated with this transformation remain unknown. Analysis of tumor samples and cell lines derived from resistant EGFR mutant patients revealed that RB is lost in 100% of these SCLC transformed cases, but rarely in those that remain NSCLC. Further, increased neuroendocrine marker and decreased EGFR expression as well as greater sensitivity to BCL2 family inhibition are observed in resistant SCLC transformed cancers compared to resistant NSCLCs. Together, these findings suggest that this subset of resistant cancers ultimately adopt many of the molecular and phenotypic characteristics of classical SCLC.

Purpose A secondary epidermal growth factor receptor (EGFR) mutation, T790M, is the most common resistance mechanism in EGFR mutant adenocarcinomas that have progressed on erlotinib. Third generation EGFR inhibitors capable of inhibiting mutant EGFR with T790M produce responses in nearly two thirds of patients. However, acquired resistance mechanisms in patients treated with these drugs are yet to be described. Experimental Design To study acquired resistance to third generation EGFR inhibitors, T790M-positive cells derived from an erlotinib-resistant cancer were made resistant to a third generation TKI and then characterized using cell and molecular analyses. Results Cells resistant to a third generation TKI acquired an additional EGFR mutation, C797S, which prevented suppression of EGFR. Our results demonstrate that the allelic context in which C797S was acquired may predict responsiveness to alternative treatments. If the C797S and T790M mutations are in trans, cells will be resistant to third generation EGFR TKIs, but will be sensitive to a combination of first and third generation TKIs. If the mutations are in cis, no EGFR TKIs alone or in combination can suppress activity. If C797S develops in cells wild type for T790 (when third generation TKIs are administered in the first line setting), the cells are resistant to third generation TKIs, but retain sensitivity to first generation TKIs. Conclusions Mutation of C797S in EGFR is a novel mechanism of acquired resistance to third generation TKIs. The context in which the C797S develops with respect to the other EGFR alleles impacts the efficacy of subsequent treatments.