Generation of osimertinib-resistant cells from epidermal growth factor receptor L858R/T790M mutant non-small cell lung carcinoma cell line

Abstract

Background: Lung cancer contributes to high cancer mortality worldwide with 80% of total cases diagnosed as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain serves as a druggable target in NSCLC patients with exon 19 deletion and L858R mutation. However, patients eventually succumbed to resistance to first- and second-generation EGFR-TK inhibitors through activation of T790M mutation. Third-generation EGFR-TKI, Osimertinib exhibits high efficacy in patients with exon 19 deletion/L858R/T790M mutation but they experienced acquired resistance thereafter. Available treatment options in NSCLC patients remains a challenge due to unknown molecular heterogeneity responsible for acquired resistance to EGFR-TKI. In this study, we aim to generate Osimertinib-resistant (OR) cells from H1975 carrying L858R/T790M double mutation which can be used as a model to elucidate mechanism of resistance. Methods: OR cells were established via stepwise-dose escalation and limiting single-cell dilution method. We then evaluated Osimertinib resistance potential via cell viability assay. Proteins expression related to EGFR-signalling, epithelial to mesenchymal transition (EMT), and autophagy were analyzed via western blot. Results: OR cell lines exhibited increased drug resistance potential compared to H1975. Distinguishable mesenchymal-like features were observed in OR cells. Protein expression analysis revealed EGFR-independent signaling involved in the derived OR cells as well as EMT and autophagy activity. Conclusion: We generated OR cell lines in-vitro as evidenced by increased drug resistance potential, increased mesenchymal features, and enhanced autophagy activity. Development of Osimertinib resistance cells may serve as in-vitro model facilitating discovery of molecular aberration present during acquired mechanism of resistance.

Cell Line Models for Acquired Resistance to First-Line Osimertinib in Lung Cancers—Applications and Limitations

“…The authors reported that osimertinib sensitivity was restored through the knockdown of IRE1α or treatment with an IRE1α inhibitor (STF-083010) [38]. The potential role of enhanced autophagy in AR against first-line osimertinib has been reported by other groups that used H1975 or PC9 models [42,53]. One study suggested that combining osimertinib with an autophagy inhibitor (CQ) could improve osimertinib cytotoxicity [42].…”

“…The authors also reported that combining osimertinib with a TGFβR1 inhibitor (SB-431542) could overcome the osimertinib resistance mediated through this pathway [40]. Other studies that reported the involvement of EMT in acquiring resistance to first-line osimertinib used H1975 cells [13,46,53,54,[58][59][60], which were destined to develop EMT as an acquired resistance mechanism as described in our previous study [94]. Among mechanisms of EMT induction, increased ZEB1 expression [58], or decreased microRNA-200c in combination with increased ZEB1 expression [46] have been reported.…”

“…We classified the AR mechanisms into five categories: (a) aberration of EGFR itself, (b) activation of bypass signaling, (c) suppression of apoptosis, (d) EMT, and (e) other mechanisms. Mechanisms of acquired resistance to first-line osimertinib that were identified in cell line models [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46]48,[50][51][52][53][54][55][56][57][58][59][60]. This graph is color-coded by cell line models and mechanisms of resistance to first-line osimertinib.…”

OPALS: A New Osimertinib Adjunctive Treatment of Lung Adenocarcinoma or Glioblastoma Using Five Repurposed Drugs

“…In principle, when osimertinib is used to treat EGFR amplified or EGFR mutated NSCLC, such resistance evolves via: Multiple further EGFR mutations in EGFR T790M-positive NSCLC during osimertinib that increase the IC50 of osimertinib [ 15 ]; NSCLC that survives initial EGFR inhibitor treatment with either gefitinib or osimertinib, commonly do so through development of EGFR-independent activation of signal transducer and activator of transcription 3 (STAT3) and Src- YES-associated protein 1 (YAP1) signaling [ 16 , 17 ]; Transformation to squamous or small-cell lung cancer that is not dependent on EGFR for growth [ 18 ]; Evolution of parallel, growth-driving RTKs, including AXL, EGFR family members, and insulin growth factor 1 receptor, MET amplification, BRAF fusions, ALK fusions, Kras mutations and RET fusions [ 18 , 19 , 20 ]; Amplification of EGFR wild-type alleles conferring resistance to osimertinib [ 21 ], schematically depicted in Figure 1 . …”

“…Evolution of parallel, growth-driving RTKs, including AXL, EGFR family members, and insulin growth factor 1 receptor, MET amplification, BRAF fusions, ALK fusions, Kras mutations and RET fusions [ 18 , 19 , 20 ];…”