BMF deficiency promotes drug-tolerant persister cell formation in ALK/ROS1-positive lung cancer

Abstract Non-small cell lung cancer (NSCLC) patients with anaplastic lymphoma kinase (ALK)-fusion oncogene respond remarkably well to ALK-tyrosine kinase inhibitor (ALK-TKI) therapies. However, the emergence of resistance remains a major challenge in long-term ALK-TKI treatment. The aim of this study was to elucidate the factors contributing to the formation of drug-tolerant persister cells (DTPs) in ALK-positive lung cancer and to explore therapeutic strategies for targeting these cells. To this end, we performed genome-wide CRISPR-Cas9 knockout screening using patient-derived ALK-positive NSCLC cells. This screening identified the apoptosis-associated factor BMF as a key regulator of DTP formation. BMF expression increased within 24 h of ALK-TKI treatment or dual ERK and PI3K pathway inhibition. BMF-knockout cells exhibited impaired apoptosis and an increased DTP population following ALK-TKI treatment. This suggests that BMF plays a pivotal role in ALK-TKI-induced apoptosis induction. Pharmacological inhibition or siRNA-mediated knockdown of MCL-1 with ALK-TKI treatment effectively induced apoptosis and eliminated BMF knocked-out cells. Furthermore, siRNA-mediated knockdown of the transcription factor FOXO1 increased BMF expression, suggesting that FOXO1-mediated signaling contributes to the suppression of BMF. Thus, FOXO1 inhibition could enhance the efficacy of ALK-targeted therapies. Notably, the role of BMF observed in ALK-positive lung cancer models was recapitulated in ROS1-positive cell lines. Overall, BMF plays a critical role in apoptosis induction and is upregulated during targeted therapy for ALK/ROS1-positive lung cancer. BMF loss promotes DTP formation and contributes to therapeutic resistance. Targeting MCL-1 or FOXO1 may be a promising strategy for eliminating DTP cells and enhancing the efficacy of ALK/ROS1-TKIs.