Erlotinib Resistance Via A Persister Bottleneck Results In Mixed Expression Of Egfr T790m And Emt-Like State With High Axl Expression

Abstract Background: Recent studies have suggested that EGFR mutant cells exposed to high concentrations of EGFR TKI (erlotinib) develop resistance through a persister bottleneck and reflect a spectrum of resistance mechanisms. Our goal was to exploit this approach to further understand mechanisms of EGFR TKI resistance and develop new therapeutic targets. Methods: To generate erlotinib resistant “persister” cell clones (EPs), PC9 cells were seeded at low density and exposed to 2.5 µM of erlotinib for ~6 weeks. In this time approximately 99.9% of cells died, while a small surviving fraction entered a quiescent state defined as “persisters” followed by proliferation of clones. Over 20 clonal subpopulations of resistant clones were obtained, of which four have been characterized via proliferation assays, immunoblotting, DNA sequencing and RNA-Seq analysis. Results: DNA sequencing and subsequent phenotypical analysis revealed varying EGFR T790M allele frequency and osimertinib sensitivity. Two clones were sensitive to osimertinib, with their T790M allele frequencies being 3.7% and 7.0%. The other two clones were resistant to osimertinib with allele frequencies being 0% and 8.9%. Common mechanisms of EGFR TKI resistance (MET amplification, KRAS mutations, BRAF/MEK mutations) were not observed in any of the EP clones. DNA sequencing also identified 13 unique mutations across the clones compared to the parental PC9 and none of these were known oncogenes or associated with TKI resistance. Maintenance of downstream signaling despite loss of EGFR phosphorylation in the presence of erlotinib was observed in all the clones. Analysis of the RNA-seq data revealed all four clones show an increase in vimentin, a marker of EMT, with one clone showing a 25-fold increase. Three of the four clones had greater than two-fold expression of SLUG mRNA, a known transcriptional factor of EMT resistance, while no changes in other EMT transcription factors (Snail, Twist, Zeb1) were identified. Interesting, AXL, a receptor tyrosine kinase known to be associated with EMT, was the only up-regulated gene across all four clones. Treatment with AXL TKI alone (RXDX-106 and R428) did not affect cell viability, but one EP clone demonstrated sensitivity to erlotinib when combined with R428 but not RXDX-106. Downstream signaling also shows that this combination results in strong reduction in ERK phosphorylation compared to either single agent. Conclusions: Our data suggests that EPs have developed resistance through a combination of T790M and EMT-like state, with AXL upregulation as a common theme. One clone, harboring some T790M and osimertinib sensitivity, was also sensitive to erlotinib when combined with R428. Citation Format: Sina Hosseinian, Anurima Majumder, Matthew Smith, Fumi Kinose, Eric Haura. Erlotinib resistance via a persister bottleneck results in mixed expression of EGFR T790M and EMT-like state with high AXL expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1883.