Abstract Anaplastic lymphoma kinase (ALK)-rearrangements occur in approximately 2-5% of non-small cell lung cancers (NSCLC). Several ALK tyrosine kinase inhibitors (ALK-TKIs) have been developed, and six are currently approved for clinical use. Although these therapies have markedly improved clinical outcomes, most patients eventually relapse due to acquired drug resistance, which remains a significant clinical challenge and limits long-term survival. To date, numerous mechanisms of ALK-TKI resistance have been identified, including secondary mutations in the ALK kinase domain and activation of bypass signaling pathways, such as EGFR or MET. However, in a significant number of cases, the underlying mechanisms remain unknown, highlighting the need for unbiased approaches to identify novel drivers of ALK-TKI resistance. In this study, we first performed a genome-wide CRISPR activation (CRISPRa) gain-of-function screen in patient-derived ALK-positive NSCLC cell lines to identify genetic pathways conferring resistance to ALK-TKIs. The CRISPRa system enables sgRNA-directed activation of endogenous genes via dCas9-VP64, a nuclease-dead Cas9 fused to the transcriptional activator VP64. In JFCR-028-3 cells expressing dCas9-VP64, introduction of sgEGFR or sgMET led to upregulation of the targeted proteins and conferred resistance to ALK-TKIs. For the genome-wide CRISPRa screen, cells expressing dCas9-VP64 were transduced with a pooled sgRNA library targeting over 18,000 genes. The in vitro CRISPRa screen was performed by culturing the cells with alectinib, lorlatinib, or brigatinib for nine days, and sgRNAs enriched in the surviving drug-tolerant cells were analyzed by next-generation sequencing. The top-ranked hits in JFCR-028-3 included receptor tyrosine kinases (RTKs) such as EGFR, MET, and FGFR1, as well as the anti-apoptotic factor BCL2. Gene ontology analysis further revealed significant enrichment of pathways related to cell proliferation, RTK signaling, and regulation of apoptotic signaling among the hit genes. The screens with the three ALK-TKIs identified both shared and drug-specific hit genes, which may reflect differences in their off-target inhibitory profiles. Furthermore, the in vivo CRISPRa screen revealed top hits that differed from those in vitro, with enrichment of ligand-dependent factors, including EGFR and KIT, reflecting the influence of the physiological microenvironment. Together, these results reveal multiple novel factors responsible for ALK-TKI resistance, providing insight into previously unidentified mechanisms. Citation Format: Mai Nagasaka, Marie Kawahara, Ken Uchibori, Makoto Nishio, Ryohei Katayama. Identification of drug resistance factors in ALK-rearranged lung cancer using genome-wide CRISPR activation screening abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 395.
Nagasaka et al. (Fri,) studied this question.