Lentiviral Insertional Mutagenesis Helps To Uncover The Mechanisms Of Resistance To Azd9291 And Co-1686 In Egfr-Mutant Lung Adenocarcinoma

The discovery of cancer-driver mutations, accounting for the growth and spreading of cancer cells, led to the development of anti-cancer targeted therapies, which hit in a specific manner cell pathways directly involved in tumor progression. This new class of therapeutic agents has been shown to be more effective and less toxic than conventional chemotherapy in advanced forms of cancer. However, the inevitable development of acquired resistance has limited their success. An example of this concept is the case of Epidermal Growth Factor Receptor (EGFR)-mutant lung cancer. The discovery of EGFR mutations that confer sensitivity to the Tyrosine Kinase Inhibitors (TKI) erlotinib and gefitinib have underlined the importance of defining molecular subgroups to design more efficacious targeted therapies. Unfortunately, on average ~1 year after starting treatment, resistance to these agents, caused by EGFR secondary mutation T790M, occurs at high frequency. This observation suggests that strategies to delay or prevent the emergence of this resistance mechanism would prolong the lives of many lung cancer patients with EGFR mutations. AZD9291 and CO-1686 are two novel third-generation EGFR TKIs designed to irreversibly and specifically target both the initial activating EGFR mutations and the resistance T790M. Phase I/II studies show compelling clinical activity of these compounds. Nevertheless the observed progression-free survival is about 12 months and the reasons of the relapse are under evaluation. We took advantage of a lentiviral vector (LV) -based insertional mutagenesis platform, developed by our lab, to screen genes that confer resistance to CO-1686. To this aim T790M+ (PC9BRc1) and T790M- (PC9) human lung adenocarcinoma cells were transduced with a genotoxic LV, harboring the Spleen Focus Forming Virus enhancer/promoter in the Long Terminal Repeats (LV-SF-LTR) or a non-genotoxic LV with self-inactivating LTR. After 2 weeks, transduced cells were divided in three groups receiving Erlotinib, CO-1686 or DMSO as control. In our rationale, the integration of the genotoxic LV in the cellular genome can potentially deregulate the expression of neighboring genes that contribute to confer resistance to these TKIs. Therefore, exposure to the treatment would result in the selection and expansion of the cell clones harboring those specific traceable mutations. Drug-resistant colonies were obtained after 4 weeks of erlotinib and 6 weeks of CO-1686 treatment. While results on PC9BRc1 are still pending, sequencing analysis performed on 100,000 LV integration sites retrieved by PCR-based technologies on PC9 drug-resistant colonies identified SOS1 as the principal LV-induced gene deregulation responsible for the resistance to both Erlotinib and CO-1686. A similar experiment aimed at identifying the AZD9291-induced resistance in lung cancer cells is on-going. The identification of biomarkers of resistance will allow the development of new therapies to overcome resistance, improving the life expectancy of lung adenocarcinoma patients.