Abstract 5839: Identification of low-dose ligand dependent drug-resistance in ALK/ROS1 positive NSCLC via CRISPR library screening
Authors: Kondo Nobuyuki , Utsumi Takahiro , Uchibori Ken , Miyazaki Yasunari , Katayama Ryohei 
Published: March 2024

Abstract
Tyrosine kinase inhibitor (TKI) therapy has displayed significant effectiveness in treating patients with anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1)-positive non-small cell lung cancer (NSCLC). Nonetheless, the emergence of acquired resistance in most patients presents a major challenge. Drug-tolerant persister (DTP) cells have recently gained attention as potential sources of resistance, highlighting the need for a deeper understanding of drug resistance mechanisms. To address this issue, a comprehensive genome-wide CRISPR-Cas9 knockout screening was conducted using an ALK-positive NSCLC cell line derived from pleural effusion in a patient who had not been exposed to ALK-TKI treatment. Following a 9-day ALK-TKI treatment, sequencing analysis was performed to identify sgRNAs in DTP cells. Notably, this analysis detected the involvement of tumor suppressor genes, including NF2, BAX, and ERRFI1.NF2 depletion was found to induce a substantial resistance to ALK-TKIs, and a combination therapy involving mTOR inhibitors showed promise in partially overcoming this resistance. Moreover, knockout of ERRFI1 or BAX led to an increase in DTP cells, drawing particular attention to ERRFI1, also known as MIG6, which is known for negatively regulating EGFR signaling. One intriguing discovery was that the loss of MIG6 induced resistance to ALK-TKIs when exposed to a relatively low dose of EGF, comparable to concentrations in plasma of healthy adults. This resistance was attributed to the upregulation of the MAPK and PI3K/Akt/mTOR pathways. Similar to ALK-rearranged NSCLC, ROS1 fusion-positive NSCLC cell lines also showed resistance by MIG6 knockout. Interestingly, some cell lines with reduced MIG6 expression tended to develop resistance with low-dose EGF, even without prior treatment, and overexpression of MIG6 restored sensitivity to TKIs. We further explored the potential of combination therapy, combining ALK-TKIs with anti-EGFR antibodies, and this approach effectively addressed acquired resistance in both in vivo and in vitro models. Furthermore, an analysis of clinical samples revealed that individuals resistant to ALK-TKIs displayed decreased mRNA MIG6 levels in comparison to their sensitive counterparts. Investigating the mechanisms behind MIG6 expression, we confirmed that ALK-TKI therapy suppressed MIG6 expression levels, while factors such as corticosteroid usage or exposure to hypoxia induced the upregulation of MIG6, potentially influencing the formation of DTP cells. In conclusion, this research has unveiled a novel factor contributing to resistance in ALK and ROS1-TKI therapies by activating the EGFR pathway through exposure to low-dose ligands, shedding light on potential therapeutic strategies to combat acquired resistance.
Citation Format: Nobuyuki Kondo, Takahiro Utsumi, Ken Uchibori, Yasunari Miyazaki, Ryohei Katayama. Identification of low-dose ligand dependent drug-resistance in ALK/ROS1 positive NSCLC via CRISPR library screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5839.