Initial AXL and MCL-1 inhibition contributes to abolishing lazertinib tolerance in EGFR-mutant lung cancer cells

Abstract

Lazertinib, a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), demonstrates marked efficacy in EGFR-mutant lung cancer. However, resistance commonly develops, prompting consideration of therapeutic strategies to overcome initial drug resistance mechanisms. This study aimed to elucidate the adaptive resistance to lazertinib and advocate novel combination treatments that demonstrate efficacy in preventing resistance as a first-line treatment for EGFR mutation-positive NSCLC. We found that AXL knockdown significantly inhibited lung cancer cell viability in the presence of lazertinib, indicating that AXL activation contributes to lazertinib resistance. However, long-term culture with a combination of lazertinib and AXL inhibitors led to residual cell proliferation and increased the MCL-1 expression level, which was mediated by the nuclear translocation of the transcription factor YAP. Triple therapy with an MCL-1 or YAP inhibitor in combination with lazertinib and an AXL inhibitor significantly reduced cell viability and increased the apoptosis rate. These results demonstrate that AXL and YAP/MCL-1 signals contribute to adaptive lazertinib resistance in EGFR-mutant lung cancer cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be a highly effective strategy in eliminating lazertinib-resistant cells.

Keywords: AXL; EGFR; EGFR‐TKI; MCL‐1; YAP.

FIGURE 1

AXL plays a pivotal role in the viability of EGFR‐mutant NSCLC cells treated with lazertinib. (A) EGFR‐mutant NSCLC cell lines PC‐9, HCC4011, H1975, PC‐9GXR, KPP‐03, HCC827, and HCC4006 were incubated with indicated concentrations of lazertinib for 72 h. Cell growth was determined using MTT assays, and the IC₅₀ values of lazertinib for each cell line were determined. *p < 0.05 (B) PC‐9 cell viability after 7 days of treatment with indicated concentrations of lazertinib or gefitinib, replenished every 72 h. (C) MTT assays evaluating the effect of a combination of EGFR inhibitor lazertinib (100 nmol/L) and knockdown of 56 receptor tyrosine kinases from Silencer® Select human kinase siRNA library V4 on PC‐9 cell viability. The 56 RTKs are rank ordered from highest to lowest according to their inhibitory effect on the viability of PC‐9 cells relative to nonspecific control siRNA. (D) PC‐9, HCC4011, H1975, and KPP‐03 cells were treated with nonspecific control siRNA or AXL‐specific siRNAs and were incubated with or without lazertinib (100 nmol/L) for 72 h. Cell viability was detected using MTT assays. *p < 0.05.

FIGURE 2

The AXL inhibitor sensitized AXL‐overexpressing EGFR‐mutant NSCLC cells to lazertinib. (A) EGFR‐mutant NSCLC cell lines PC‐9, HCC4011, H1975, PC‐9GXR, KPP‐03, HCC827, and HCC4006 were lysed, and the indicated proteins were detected using western blotting. (B) EGFR‐mutant NSCLC cell lines PC‐9, HCC4011, H1975, and KPP‐03 were incubated with lazertinib (100 nmol/L) or a combination of lazertinib (100 nmol/L) and AXL inhibitor ONO7475 (100 nmol/L) for 72 h. Cell growth was determined using MTT assays. *p < 0.05. (C) Quantitative determination of the inhibition of cell viability of high‐AXL‐expressing and low‐AXL‐expressing EGFR‐mutant cells treated with the EGFR‐TKI lazertinib (100 nmol/L) in the presence or absence of ONO7475 (100 nmol/L) for 72 h. Paired Student's t‐tests were used for comparisons. (D) PC‐9 and HCC4011 cells were treated with lazertinib (100 nmol/L), ONO7475 (100 nmol/L), or a combination of lazertinib and ONO7475 for 4 h. (E) PC‐9 and HCC4011 cells were treated with lazertinib (100 nmol/L), ONO7475 (100 nmol/L), or a combination of lazertinib and ONO7475 for 72 h. (F) PC‐9, HCC4011, and H1975 cells were visualized using crystal violet staining following 9 days of treatment with lazertinib (100 nmol/L), ONO7475 (100 nmol/L), or a combination of lazertinib and ONO7475 where the drugs were replenished every 72 h. (G) PC‐9, HCC4011, and H1975 cells were treated with lazertinib (100 nmol/L), ONO7475 (100 nmol/L), or a combination of lazertinib and ONO7475 in which the drugs were replenished every 72 h. Absorbance was measured every 3 days. *p < 0.05.

FIGURE 3

MCL‐1 knockdown sensitized EGFR‐mutant NSCLC cells to a combination of lazertinib and ONO7475. (A) PC‐9, HCC4011, and H1975 cells were treated with lazertinib (100 nmol/L), ONO7475 (100 nmol/L), or a combination of lazertinib and ONO7475 for 48 h. The cells were lysed, and the indicated proteins were detected using western blotting. (B) PC‐9 and HCC4011 cells were treated with nonspecific control siRNA or MCL‐1‐specific siRNA and incubated with lazertinib (100 nmol/L) or a combination of lazertinib (100 nmol/L) and ONO7475 (100 nmol/L) for 48 h. The cells were lysed, and the indicated proteins were detected using western blotting. (C) PC‐9 and HCC4011 cells treated with nonspecific control siRNA or MCL‐1‐specific siRNA and incubated with lazertinib (100 nmol/L) or a combination of lazertinib and ONO7475 (100 nmol/L) for 72 h. Cell growth was determined using MTT assays. *p < 0.05. (D) Apoptotic cell percentages in PC‐9 and HCC4011 cells double stained with annexin V and propidium iodide were determined by flow cytometry following treatment with lazertinib (100 nmol/L) and ONO7475 (100 nmol/L) with or without siMCL‐1 for 48 h. *p < 0.05.

FIGURE 4

Co‐treatment with MCL‐1 inhibitor, AXL inhibitor, and lazertinib promoted apoptosis in EGFR‐mutant NSCLC cells. (A) PC‐9, HCC4011, and H1975 cells were treated with lazertinib (100 nmol/L), ONO7475 (100 nmol/L), MCL‐1 inhibitor S63845 (1 μmol/L), or combinations for 72 h. Cell growth was determined using MTT assays. *p < 0.05. (B) PC‐9, HCC4011, and H1975 cells were visualized using crystal violet staining following 9 days of treatment with lazertinib (100 nmol/L), ONO7475 (100 nmol/L), S63845 (1 μmol/L), or combinations where the drugs were replenished every 72 h. (C) PC‐9, HCC4011, and H1975 cells were treated with lazertinib (100 nmol/L) with or without ONO7475 (100 nmol/L), S63845 (1 μmol/L) for 48 h. The cells were lysed, and the indicated proteins were detected using western blotting. (D) Apoptotic cell percentages of PC‐9, HCC4011, and H1975 cells, which were double stained with annexin V and propidium iodide were detected using flow cytometry following treatment with lazertinib (100 nmol/L) with or without ONO7475 (100 nmol/L) and S63845 (1 μmol/L) for 48 h. *p < 0.05. (E) PC‐9 CDX tumors were treated with the vehicle control, 3 mg/kg lazertinib daily, 3 mg/kg lazertinib daily plus 10 mg/kg ONO‐7475 daily, and 3 mg/kg lazertinib daily plus 10 mg/kg ONO‐7475 daily plus 25 mg/kg S63845 twice a week (n = 6 per group). *Comparison of Lazertinib + ONO‐7475 with Lazertinib plus ONO‐7475 plus S63845 gave p < 0.05. (F) PC‐9 cells were treated with lazertinib (100 nmol/L) with or without ONO7475 (100 nmol/L) and S63845(1 μmol/L) for 48 h. Proteins were immunoprecipitated from the cell lysates and subjected to immunoblotting using anti‐MCL‐1, anti‐Bax, and anti‐Bak mAbs.

FIGURE 5

YAP–MCL‐1 axis‐induced tolerant cells with a combination of lazertinib and AXL inhibitor in EGFR‐mutant NSCLC cells. (A) PC‐9 and HCC4011 cells were treated with lazertinib (100 nmol/L), ONO7475 (100 nmol/L), or a combination of lazertinib and ONO7475 for 48 h. The cells were lysed, and the indicated proteins were detected using western blotting. (B) PC‐9 and HCC4011 cells were treated with nonspecific control siRNA or YAP‐specific siRNA and incubated with lazertinib (100 nmol/L) or a combination of lazertinib and ONO7475 (100 nmol/L) for 48 h. The cells were lysed, and the indicated proteins were detected using western blotting. (C) PC‐9 and HCC4011 cells were treated with nonspecific control siRNA or YAP‐specific siRNAs and incubated with lazertinib (100 nmol/L) or a combination of lazertinib and ONO7475 (100 nmol/L) for 72 h. Cell growth was determined using MTT assays. *p < 0.05. (D) PC‐9 and HCC4011 cells were treated with lazertinib (100 nmol/L) or a combination of lazertinib and ONO7475 (100 nmol/L) for 48 h. Proteins were fractured into the nucleus and cytoplasm using NE‐PER™ Nuclear and Cytoplasmic Extraction Reagents. (E) Immunofluorescence showing nuclear localization of YAP in EGFR‐mutant NSCLC cells exposed to lazertinib (100 nmol/L) or a combination of lazertinib and ONO7475 (100 nmol/L) for 48 h. Scale bars, 20 μm. (F) qPCR of MCL‐1 in PC‐9 and HCC4011 cells treated with nonspecific control siRNA or YAP‐specific siRNAs and incubated lazertinib (100 nmol/L) or a combination of lazertinib and ONO7475 (100 nmol/L) for 48 h. *p < 0.05.

FIGURE 6

Inhibition of YAP via downregulation of MCL‐1‐sensitized EGFR‐mutant NSCLC cells to lazertinib and AXL inhibitor. (A) PC‐9 and HCC4011 cells were treated with lazertinib (100 nmol/L) with or without ONO7475 (100 nmol/L) and YAP inhibitor verteporfin (1 μmol/L) for 48 h. The cells were lysed, and the indicated proteins were detected using western blotting. (B) qPCR of MCL‐1 in PC‐9 and HCC4011 cells treated with lazertinib (100 nmol/L) and ONO7475 (100 nmol/L) with or without verteporfin (1 μmol/L) for 48 h. *p < 0.05. (C) PC‐9 and HCC4011 cells were treated with lazertinib (100 nmol/L) with or without ONO7475 (100 nmol/L) and verteporfin (1 μmol/L) for 72 h. Cell growth was determined using MTT assays. *p < 0.05. (D) PC‐9 and HCC4011 cells were visualized using crystal violet staining following 9 days of treatment with lazertinib (100 nmol/L), ONO7475 (100 nmol/L), verteporfin (1 μmol/L), or combinations, with the drugs replenished every 72 h. (E) PC‐9 and HCC4011 cells were treated with lazertinib (100 nmol/L) with or without ONO7475 (100 nmol/L) and verteporfin (1 μmol/L) for 48 h. The cells were lysed, and the indicated proteins were detected using western blotting. (F) Schematic presentation of the mechanism by which a combination of lazertinib and ONO7475 promote the YAP–MCL‐1 axis and facilitate the synthesis of antiapoptotic proteins in EGFR‐mutant NSCLC cells.