MIG6 loss confers resistance to ALK/ROS1 inhibitors in NSCLC through EGFR activation by low-dose EGF
Nobuyuki Kondo, … , Yasunari Miyazaki, Ryohei Katayama
Nobuyuki Kondo, … , Yasunari Miyazaki, Ryohei Katayama
Published November 2, 2023
Citation Information: JCI Insight. 2023;8(24):e173688. https://doi.org/10.1172/jci.insight.173688.
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Research Article Oncology

MIG6 loss confers resistance to ALK/ROS1 inhibitors in NSCLC through EGFR activation by low-dose EGF

Abstract

Although tyrosine kinase inhibitor (TKI) therapy shows marked clinical efficacy in patients with anaplastic lymphoma kinase–positive (ALK+) and ROS proto-oncogene 1–positive (ROS1+) non–small cell lung cancer (NSCLC), most of these patients eventually relapse with acquired resistance. Therefore, genome-wide CRISPR/Cas9 knockout screening was performed using an ALK+ NSCLC cell line established from pleural effusion without ALK-TKI treatment. After 9 days of ALK-TKI therapy, sequencing analysis was performed, which identified several tumor suppressor genes, such as NF2 or MED12, and multiple candidate genes. Among them, this study focused on ERRFI1, which is known as MIG6 and negatively regulates EGFR signaling. Interestingly, MIG6 loss induced resistance to ALK-TKIs by treatment with quite a low dose of EGF, which is equivalent to plasma concentration, through the upregulation of MAPK and PI3K/AKT/mTOR pathways. Combination therapy with ALK-TKIs and anti-EGFR antibodies could overcome the acquired resistance in both in vivo and in vitro models. In addition, this verified that MIG6 loss induces resistance to ROS1-TKIs in ROS1+ cell lines. This study found a potentially novel factor that plays a role in ALK and ROS1-TKI resistance by activating the EGFR pathway with low-dose ligands.

Authors

Nobuyuki Kondo, Takahiro Utsumi, Yuki Shimizu, Ai Takemoto, Tomoko Oh-hara, Ken Uchibori, Sophia Subat-Motoshi, Hironori Ninomiya, Kengo Takeuchi, Makoto Nishio, Yasunari Miyazaki, Ryohei Katayama

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Figure 1

CRISPR library screening identifies MIG6 depletion in ALK-TKI–resistant cells.

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CRISPR library screening identifies MIG6 depletion in ALK-TKI–resistant ...
(A) Schematic diagram of the workflow of genome-wide CRISPR/Cas9 library screening to identify critical genes related to alectinib and lorlatinib resistance in the JFCR-028-3 cell line. NGS, next-generation sequencing. (B) The abundance of sgRNA for each gene in CRISPR library screening was evaluated by the β-score using the MAGeCK algorithm. Positively selected genes after both alectinib and lorlatinib treatments (cutoff of –log10 β-score > 2.5) are indicated as blue dots. MIG6 (ERRFI1) is indicated as a red dot. (C) Immunoblot analysis of NF2 knocked out in JFCR-028-3 cells. (D) JFCR-028-3 cells were treated with the indicated concentrations of lorlatinib with or without 1 μmol/L of PP242 for 72 hours. Cell viability was measured using the CellTiter-Glo assay (n = 3). (E) Immunoblot analysis of MIG6 knocked out in JFCR-028-3 cells. (F and G) Colony formation assays were performed in JFCR-028-3 cells. JFCR-028-3 sg-control (Cntl) or sg-MIG6 cells were treated with 10 nmol/L of alectinib or 3 nmol/L of lorlatinib for 2 weeks. Surviving cells were stained with crystal violet. Representative images are shown in F. Relative cell viability was measured using a spectrophotometer after solubilizing the stained crystal violet with the acetic acid buffer from each well (G). (H) Quantitative reverse transcription PCR (RT-qPCR) of MIG6 mRNA was performed using JFCR-028-3 cells treated with 300 nmol/L of alectinib for the indicated hours. (C–H) Similar experiments were performed twice (C and E) or 3 times (D and F–H), and representative data are shown. Each point represents mean ± SD of 3 technical replicates; *P < 0.05, ***P < 0.001 (2-way ANOVA following Dunnett post hoc test).