Cumerlato, M.; Maccagno, M.; Labrador, M.; Luciano, B.; Mereu, E.; Porro, M.; Bandini, C.; Maisano, D.; D'Agostino, M.; Larocca, A.; Gay, F. M.; Tamburrini, A.; Anselmi, F.; Porporato, P. E.; Oliviero, S.; Bruno, B.; Morelli, E.; Munshi, N.; PIVA, R.

Proteasome inhibitors (PIs) are cornerstone therapies for multiple myeloma (MM), yet resistance remains a major barrier to durable responses. To identify druggable vulnerabilities that enhance PI efficacy, we performed a small-molecule chemical screen in the presence of carfilzomib (CFZ). We identified bedaquiline (BDQ), an FDA-approved antimycobacterial agent, as a potent synergistic partner. BDQ and its fumarate salt (BDQ-F) significantly amplified CFZ-induced cytotoxicity in PI-sensitive and PI-resistant MM cells, in AL amyloidosis and other B-cell malignancies, with minimal toxicity toward normal cells. Mechanistic studies confirmed that BDQ specifically targets the ATP5F1C subunit of mitochondrial ATP synthase. BDQ-CFZ combination triggered extensive apoptosis, exacerbating proteotoxic stress and proteasome-associated pathways. BDQ specifically enhanced CFZ inhibition of the proteasome chymotrypsin-like activity. Importantly, BDQ synergized with multiple proteasome and ubiquitin-activating enzyme inhibitors, but not with other standard MM agents, underscoring its selective interaction with the UPS pathway. BDQ-CFZ co-treatment markedly reduced MM cell viability and tumor burden in patient-derived cells and zebrafish xenograft models. These findings support the therapeutic repurposing of BDQ to potentiate PI efficacy and overcome resistance in MM and related B-cell malignancies.