CRISPR screening identifies BMF loss as a drug-tolerant persister cell survival factor in ALK-rearranged lung cancer

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (ALK-TKIs) have significantly improved the clinical outcomes of patients with ALK-rearranged non-small cell lung cancer (NSCLC). However, the development of resistance remains a major obstacle to long-term therapeutic success. Drug resistance is believed to develop from drug-tolerant persister (DTP) cells that survive drug treatment. Therefore, understanding the molecular mechanisms underlying DTP formation and survival is critical for developing strategies to overcome resistance and extend patient survival.To identify the factors related to the survival of DTP cells, we conducted a genome-wide CRISPR-Cas9 knockout screening using patient-derived ALK-positive NSCLC cells. From the Crispr screening, we identified the pro-apoptotic factor BMF (Bcl-2 modifying factor) as a critical determinant of DTP formation. BMF expression was rapidly induced following ALK-TKI exposure or dual inhibition of ERK and PI3K signaling, suggesting its role as a mediator of apoptotic induction by ALK-TKIs. Strikingly, BMF knockout cells exhibited impaired apoptosis and an increased fraction of DTPs under ALK-TKI treatment, highlighting its essential function in limiting drug tolerance.Mechanistic studies revealed that pharmacologic or siRNA-mediated inhibition of MCL-1 restored apoptosis and eliminated BMF-deficient DTP cells, underscoring the therapeutic potential of targeting anti-apoptotic pathways. Furthermore, knockdown of FOXO1 resulted in the upregulation of BMF, indicating that FOXO1 acts as a transcriptional repressor of BMF. These findings suggest the existence of a regulatory axis in which FOXO1 suppresses BMF, thereby promoting the survival of DTP cells.In addition, patient tumor samples collected before and after ALK-TKI therapy, together with publicly available datasets, were analyzed to evaluate the clinical relevance of these findings. The clinical analyses revealed that low BMF expression was associated with poor treatment response and shorter overall survival in patients with ALK-positive non-small cell lung cancer (NSCLC). Moreover, BMF genomic loss was found relatively frequently across diverse cancer types, suggesting that BMF deficiency may be a common mechanism contributing to drug tolerance and resistance beyond ALK-driven lung cancer.In summary, our study demonstrates that BMF deficiency promotes the formation of DTP cells and contributes to therapeutic resistance in ALK-positive NSCLC. Importantly, targeting MCL-1 or FOXO1 may be a promising strategy for eradicating DTPs and enhancing the durability of ALK-TKI responses. These findings provide mechanistic insight into the regulation of persister cell apoptosis and highlight actionable vulnerabilities that could be exploited to improve patient outcomes.