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Molecular targeted therapies are the standard of care for front‐line treatment of metastatic non‐small‐cell lung cancers (NSCLCs) harboring driver gene mutations. However, despite the initial dramatic responses, the emergence of acquired resistance is inevitable. Acquisition of secondary mutations in the target gene (on‐target resistance) is one of the major mechanisms of resistance. The mouse pro‐B cell line Ba/F3 is dependent on interleukin‐3 for survival and proliferation. Upon transduction of a driver gene, Ba/F3 cells become independent of interleukin‐3 but dependent on the transduced driver gene. Therefore, the Ba/F3 cell line has been a popular system to generate models with oncogene dependence and vulnerability to specific targeted therapies. These models have been used to estimate oncogenicity of driver mutations or efficacies of molecularly targeted drugs. In addition, Ba/F3 models, together with N‐ethyl‐N‐nitrosourea mutagenesis, have been used to derive acquired resistant cells to investigate on‐target resistance mechanisms. Here, we reviewed studies that used Ba/F3 models with EGFR mutations, ALK/ROS1/NTRK/RET fusions, MET exon 14 skipping mutations, or KRAS G12C mutations to investigate secondary/tertiary drug resistant mutations. We determined that 68% of resistance mutations reproducibly detected in clinical cases were also found in Ba/F3 models. In addition, sensitivity data generated with Ba/F3 models correlated well with clinical responses to each drug. Ba/F3 models are useful to comprehensively identify potential mutations that induce resistance to molecularly targeted drugs and to explore drugs to overcome the resistance. [ABSTRACT FROM AUTHOR]

Background: Approximately 10% of non‐small cell lung cancers (NSCLCs) that harbor epidermal growth factor receptor (EGFR) gene mutations have in‐frame insertions in exon 20 of the EGFR gene. These tumors do not usually respond to currently available EGFR‐tyrosine kinase inhibitors (TKIs). Tarloxotinib is a novel hypoxia‐activated prodrug that releases a potent, irreversible pan‐ERBB TKI (tarloxotinib‐E) under solid tumor hypoxia. Methods: We examined the efficacy of tarloxotinib‐E against several types of Ba/F3 cells with introduced EGFR exon 20 mutations (EGFR A763insFQEA, V769insASV, D770insSVD, H773insH and H773insNPH mutations). We assayed growth inhibition for tarloxotinib (prodrug), tarloxotinib‐E (active form), poziotinib, afatinib, and osimertinib in Ba/F3 cells with each EGFR exon 20 mutation. We also explored acquired resistance mechanisms to tarloxotinib‐E by establishing cells with resistance to tarloxotinib‐E via chronic drug exposure after N‐ethyl‐N‐nitrosourea mutagenesis treatment. Results: Among all tested Ba/F3 cell lines, IC50 was ≥72.1 times higher for tarloxotinib than for tarloxotinib‐E, which implies a wide therapeutic window with this prodrug strategy. Tarloxotinib‐E was efficacious against all tested Ba/F3 cells except for H773insH, which was less sensitive to all tested EGFR‐TKIs. As acquired resistance mechanisms to tarloxotinib‐E, we identified either T790M or C797S secondary mutations, depending on the original EGFR exon 20 mutation. Conclusions: These findings indicate that tarloxotinib‐E could be effective for NSCLC with EGFR exon 20 mutations. Our results also show that T790M or C797S mutations can confer acquired resistance to tarloxotinib‐E; and suggest that resistance mechanisms are influenced by the baseline EGFR exon 20 mutations. [ABSTRACT FROM AUTHOR]

Highlights • The treatment strategy for NSCLC with HER2 exon 20 insertion remains unclear. • Poziotinib showed potent activity against HER2 exon 20 insertions in vitro model. • C805S was identified as a mechanism underlying acquired resistance to poziotinib. • HSP90 inhibitors were able to overcome this resistant clone. Abstract Objectives Oncogenic HER2 mutations are present in 2–4% of lung adenocarcinomas, but the relevant clinical trials are unsatisfactory. The novel HER2 inhibitor poziotinib was recently developed and clinical trials are ongoing. We compared poziotinib with nine tyrosine kinase inhibitors (TKIs), and derived poziotinib-resistant clones to investigate the resistant mechanism. Materials and methods We introduced three common HER2 mutations A775_G776insYVMA (YVMA), G776delinsVC (VC) and P780_Y781insGSP (GSP), which account for 94% of HER2 exon 20 insertions in the literature, into Ba/F3 cells. We then compared the activity of poziotinib with that of nine TKIs (erlotinib, afatinib, dacomitinib, neratinib, osimertinib, AZ5104, pyrotinib, lapatinib, and irbinitinib), determined the 90% inhibitory concentration (IC 90) through a growth inhibition assay, and defined a sensitivity index (SI) as IC 90 divided by the trough concentration at the recommended dose as a surrogate for drug activity in humans. We also generated resistant clones by exposure to poziotinib in the presence of N-ethyl -N- nitrosourea, and HER2 secondary mutations that might serve as a resistance mechanism were searched. Results YVMA showed resistance to all tested drugs except neratinib, poziotinib and pyrotinib. Poziotinib was the only drug with an SI less than 10 for YVMA, the most common HER2 exon 20 insertion. We established 62 poziotinib-resistant clones, and among these, only C805S of HER2, which is homologous to C797S of the EGFR, was identified as a secondary mutation in 19 clones. We also revealed that heat shock protein (HSP) 90 inhibitors show potent anti-growth activity to the C805S secondary mutant clone. Conclusions Poziotinib showed the most potent activity against HER2 exon 20 mutations. We identified the secondary C805S at the covalent binding site of HER2 to poziotinib as a potential mechanism of acquired resistance. HSP90 inhibitors might be a therapeutic strategy for the C805S secondary mutation. [ABSTRACT FROM AUTHOR]

• The inducibility of drug tolerant cells depended on the type of cell line. • Acquisition of DTC phenotype was a cellular response to high dose of EGFR-TKI. • High expression of RYK was a molecular mechanism of the drug-tolerant state. Emergence of acquired resistance is almost inevitable during EGFR-tyrosine kinase inhibitor therapy for non-small-cell lung cancer (NSCLC) harboring EGFR mutations. Drug tolerance, a reversible state of drug insensitivity in the early phases of tyrosine kinase inhibitor therapy, is considered to serve as the basis of recurrent disease. Therefore, it is important to elucidate the molecular mechanisms of drug tolerance. Five EGFR -mutated NSCLC cell lines were used in this study. We established drug-tolerant cells (DTCs) via 72 h treatment with osimertinib (600 nM) or afatinib (60 nM). Acquisition of drug tolerance was evaluated by growth inhibitory assay, and the molecular mechanisms of drug tolerance were analyzed by phospho-RTK array. DTCs were successfully induced in PC9, HCC4006, and H1975 cells against osimertinib and in PC9 cells against afatinib. We observed that a high drug concentration was required to induce DTCs, and HCC4006 cells become tolerant when a higher dose of afatinib (>180 nM) was used. In the analysis of HCC4006 DTCs against osimertinib, we observed increased receptor-like tyrosine kinase (RYK) expression, and siRNA-mediated RYK knockdown inhibited the proliferation of DTCs. These results suggest that induction of DTCs is dose-dependent, and increased RYK expression was the mechanism of drug tolerance in HCC4006 cells against osimertinib. [ABSTRACT FROM AUTHOR]

Highlights • Possible secondary resistant mutations against front-line osimertinib were examined. • Together with Del 19, only C797S conferred significant resistance to osimertinib. • Together with L858R, C797 and L718 mutations conferred resistance to osimertinib. • TKI sensitivities depended on combinations of secondary and activating mutations. Abstract Objectives Non-small cell lung cancers (NSCLCs) that harbor activating mutations for epidermal growth factor receptor (EGFR) show remarkable initial response to EGFR-tyrosine kinase inhibitors (TKIs), but inevitably acquire resistance, half of which are due to a T790 M secondary mutation when first-generation (1 G) or 2 G EGFR-TKIs are used. Osimertinib, a 3 G EGFR-TKI, is a standard of care in this situation, but eventually also evokes resistance, reportedly due to some tertiary EGFR mutations. However, the FLAURA trial showed the superiority of osimertinib over 1 G EGFR-TKIs in treatment-naïve patients, thus providing an option of first-line osimertinib treatment. Resistance in this setting is also inevitable, but its mechanism is unclear. We investigated whether resistance mutations that emerged with T790 M were responsible for the osimertinib resistance in the first-line setting; i.e. without T790 M, and if so, what treatment option was available. Materials and Methods We used literature search to identify EGFR mutations at codons L718, G724, L792, G796, and C797 as mechanisms of osimertinib resistance in the presence of T790 M. These mutations were introduced into Ba/F3 cells in cis with activating EGFR mutations but not with T790 M; inhibitory effects of five EGFR-TKIs were evaluated. Results Only C797S conferred significant resistance against osimertinib when exon 19 deletion was the activating mutation. However, co-existence of L858R with C797S, C797 G, L718Q, or L718 V mutations all conferred resistance to osimertinib. Erlotinib showed the greatest activity for C797S-mediated resistance. However, 2 G EGFR-TKIs (afatinib or dacomitinib) were effective for other resistance mutations. Conclusion After first-line osimertinib failure, 1 G or 2 G EGFR-TKIs are effective, depending on combinations of secondary and activating mutations. [ABSTRACT FROM AUTHOR]