Abstract Approximatively 40% of diffuse large B-cell lymphoma (DLBCL) patients exhibit primary resistance to standard therapy. Moreover, relapse is a common event in first-line responders. Overexpression of BCL-2 is a major determinant of resistance to chemotherapy in several B-cell neoplasms, including DLBCL. While venetoclax, a specific BCL-2 inhibitor, has dramatically changed the therapeutic landscape of other B-cell malignancies, only 12% of DLBCL patients exhibit a complete response to venetoclax, with a mean progression-free survival of 1 month. In this scenario, new therapeutic strategies are urgently needed. We tested the possibility to overcome the resistance of DLBCL cells to venetoclax by rewiring their reactive oxygen species (ROS) set point, which is higher in cancer cells compared to their healthy counterparts. We employed a panel of 14 DLBCL cell lines of both germinal center B-cell (GCB) and activated B-cell (ABC) derivation. Cell death, ROS accumulation, and NADPH levels were evaluated upon treatment of DLBCL cells with three inhibitors of NADPH production, the central electron donor required to maintain ROS scavengers in their reduced form. The synergism between venetoclax and inhibitors of NADPH production was assessed through isobologram analysis. RNA-Seq, quantitative real-time PCR (qRT-PCR), and western blot techniques were used to dissect the mechanism of cooperation between venetoclax and inhibition of NADPH production. The Zebrafish model was used for in vivo validation of the therapeutic approach. We observed that inhibiting NADPH production dramatically improves the response to venetoclax by more than 500-fold, while no toxic effects were observed in normal B-cells. By generating isogenic cell lines with differing sensitivity to venetoclax, we demonstrated that the resistance to this drug positively correlates with the expression of the anti-apoptotic protein BCL-xL. Moreover, RNA-seq and protein analyses indicated that the increase of ROS levels engages the integrated stress response (ISR). Pharmacological inhibition and gene silencing of key effectors of the ISR rescued DLBCL cells from cell death induced by our therapeutic strategy. We also confirmed the ROS-dependency of BCL-xL downregulation using both ROS scavengers and by pulsing cells with exogenous hydrogen peroxide. Finally, in vivo studies confirmed the sensitization of DLBCL cells to venetoclax upon the inhibition of NADPH production. Taken together, these results provide the first proof-of-principle evidence for a ROS-based strategy to increase the therapeutic window of venetoclax in DLBCL, suggesting a viable therapeutic avenue to treat refractory patients. Citation Format: Francesco Ciccarese, Vittoria Raimondi, Alberto Corradin, Natascia Tiso, Giovanni Risato, Micol Silic-Benussi, Ilaria Cavallari, Donna Mia D'Agostino, Vincenzo Ciminale. A novel strategy to overcome resistance of diffuse large B-cell lymphoma to venetoclax [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-032.

Abstract PO-032: A novel strategy to overcome resistance of diffuse large B-cell lymphoma to venetoclax