Abstract 5549: Identification of genes associated with drug persistence/resistance using CRISPR screening and scRNA-seq of ALK rearranged lung cancer

Abstract ALK-rearrangement is found in 3-5% of non-small cell lung cancer (NSCLC), and currently five ALK tyrosine kinase inhibitors (TKIs) have been approved, and several TKIs are in development. These ALK TKIs have contributed to a significant improvement in patient prognosis, such that the median survival of patients with advanced ALK-positive lung cancer has exceeded 5 years. However, it has been a problem that cancers eventually relapse due to drug resistance. To date, many investigators have reported the mechanism of ALK-TKI resistance; secondary mutation(s) in the ALK kinase domain that disrupt ALK-TKI binding, or the activation of bypass signaling pathways that promote cell proliferation even under ALK fusion protein-mediated signaling that is suppressed by ALK-TKIs. However, in a significant number of cases, the mechanism of resistance is still unknown. Therefore, in this study, we first performed a genome-wide CRISPR screen using two ALK-positive NSCLC cell lines derived from patients with ALK-rearranged cancer prior to ALK-TKI treatment. Cas9 and genome-wide sgRNA-introduced cells were cultured with alectinib or lorlatinib for 9 days, and sgRNAs in surviving drug-tolerant cells were analyzed by NGS. Several candidate genes were identified, such as MED12, PTEN, NF2 or MIG6, and confirmed whether knockout of these genes induced drug tolerance/resistance in the ALK-TKI-naive cells. For example, loss of MIG6, an EGFR-binding negative regulator protein, activates EGFR at very low concentrations of EGF (less than 1 ng/mL, which is similar to the healthy human plasma concentration) and induces marked resistance to ALK inhibitors. In contrast, NF2 knockout itself induces drug resistance rather than drug persistence, and we found that brigatinib may be able to overcome the resistance induced by NF2 loss, because brigatinib inhibited ALK as well as several other kinases at single digit nM. As recently published in the NF2 loss Schwannomatosis, NF2 loss induce multiple kinase activation not only mTOR pathway and MAPK pathways, and brigatinib was shown to be an effective treatment option for NF2 loss Schwannomatosis. In addition, from the analysis of alectinib-resistant pleural effusion-derived cells, we found AXL overexpression but not Gas6, a ligand of Axl, in the resistant tumor. Therefore, we performed an in vivo experiment by inoculating Axl-overexpressing ALK-positive cancer cells and analyzed the tumor tissue with or without alectinib treatment. Single-cell RNA sequencing revealed that MMP11-positive cancer-associated fibroblasts (CAFs) and a subset of tumor-associated macrophages (TAMs) were the major sources of Gas6 in the tumor microenvironment. This study uncovered some of the unidentified resistance mechanisms and potential therapeutic strategies to overcome the resistance. Citation Format: Ryohei Katayama, Takahiro Utsumi, Nobuyuki Kondo, Ken Uchibori, Makoto Nishio. Identification of genes associated with drug persistence/resistance using CRISPR screening and scRNA-seq of ALK rearranged lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5549.