Abstract 4485: Resistance to ferroptosis in high grade diffuse large B-cell lymphoma is mediated through the Nrf2 signaling pathway

Abstract Introduction: High-grade B-cell lymphomas (HGBCL) with C-MYC and BCL-2 rearrangements are aggressive and resistant to standard treatment. Some B cell lymphomas, including HGBCLs and diffuse large B cell lymphomas (DLBCL), are susceptible to ferroptosis, a cell death pathway marked by toxic lipid peroxides. We have shown that some DLBCLs are sensitive to ferroptosis induced by gold high-density lipoprotein (HDL)-like nanoparticles (Au-HDL NPs), while others, including some HGBCLs, are more resistant. Differential gene expression (DEG) analysis of RNAseq data suggests that resistance to Au-HDL NP-induced ferroptosis correlates with increased expression of Nrf2 associated genes. We hypothesized that Nrf2 protects HGBCL cells against ferroptosis induced by Au-HDL NPs and other reactive oxygen species (ROS)-inducing chemotherapy agents and tested this by generating Nrf2 knockout OCI-ly1 cells (Nrf2-/-). Methods: DLBCL (SUDHL-4) and HGBCL (RC, OCI-ly1, OCI-ly8, OCI-ly18) cell lines were studied. Au-HDL NPs were synthesized as previously described. RNA from RC and SUDHL4 cells treated with Au-HDL NPs or vehicle were subjected to RNA-seq. DEGs were validated by RT-qPCR and Western blot. Nrf2 -/- OCI-ly1 cells were generated using CRISPR/Cas9. Viability was assessed by MTS and Annexin V-FITC/PI staining, while lipid peroxidation was quantified by C11 BODIPY. Results: HGBCL cell lines (RC, OCI-ly1, OCI-ly8, OCI-ly18) were less sensitive to Au-HDL NP-induced ferroptosis (IC50 = 31.4, 27.2, 58.2, and 26.0 nM, respectively) compared with the DLBCL cell line SUDHL4 (8.1 nM). RNAseq, RT-qPCR and western blot revealed higher expression of Nrf2 regulated genes, particularly NQO1 in Au-HDL NP-treated RC (19.9-fold), OCI-ly1 (9.0-fold), and OCI-ly8 (36.5-fold) cells compared with SUDHL4 (4.2-fold). Nrf2 -/- OCI-ly1 cells were less viable after treatment with Au-HDL NPs than SUDHL4 (30%, 14%, 6% vs. 104%, 75%, and 41% at doses of 10, 20, and 40 nM Au-HDL NPs, respectively). Annexin V+/PI+ was increased in Nrf2 -/- OCI-ly1 cells (58%) compared to OCI-ly1wt cells (32%), and lipid peroxidation levels were also higher in Nrf2 -/- cells, suggesting that Nrf2 mediates resistance to Au-HDL NPs by reducing oxidative burden. Compared to OCI-ly1wt cells, Nrf2 -/- cells increased sensitivity to other ferroptosis inducers, such as erastin (IC50 1.1 vs 5.7 μM) and RSL3 (IC50 26.5 vs 57.3 nM). Nrf2 -/- cells exhibited increased sensitivity to ROS-related chemotherapy agents compared to OCI-ly1wt cells, including doxorubicin (IC50 1.2 vs 2.6 nM) and venetoclax (IC50 21.5 vs 52.3 μM). Conclusion: Nrf2 signaling plays a crucial role in conferring resistance of aggressive lymphomas to ROS-dependent cell death mechanisms, including ferroptosis. Targeting Nrf2 could be a promising strategy to overcome resistance to ferroptosis and enhance the efficacy of ROS-inducing chemotherapy agents. Citation Format: Meiying Yang, Jonathan S. Rink, Adam Y. Lin, Shuo Yang, Colby S. Thaxton, Leo I. Gordon. Resistance to ferroptosis in high grade diffuse large B-cell lymphoma is mediated through the Nrf2 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4485.