Voltage-dependent anion channel 2 (VDAC2) drives rituximab resistance via inhibiting ferroptosis in diffuse large B cell lymphoma

To the Editor: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphoma, and exhibits significant morphologies and biological heterogeneity, which can be classified into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes based on cellular origin and gene expression profiles.^[1] Although rituximab-based regimens have markedly improved patient survival, 30–40% of patients still develop treatment resistance and relapse. Rituximab exerts its anti-lymphoma activity through CD20 binding, mediating complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and direct apoptosis induction.^[2] However, primary and acquired resistance significantly constrain its therapeutic efficacy in DLBCL, with the underlying mechanisms remaining not fully elucidated. Emerging evidence highlights metabolic reprogramming, particularly aerobic glycolysis, whereas lactate is the main metabolic product, and it is related to the resistance of tumor cells.^[3] Therefore, understanding the mechanism of this metabolic shift could contribute to the development of strategies for overcoming rituximab resistance in DLBCL. Detailed methods are provided in the Supplementary Materials, http://links.lww.com/CM9/C974.

To investigate the molecular mechanisms underlying rituximab resistance in DLBCL, we performed transcriptomic and proteomic profiling of resistant (ly1R) and wild-type (ly1) cells. Transcriptomic analysis identified 1143 genes upregulated and 2306 genes downregulated, while proteomic revealed 359 proteins increased and 392 decreased [Supplementary Figure 1A, B, http://links.lww.com/CM9/C974]. Integrated multi-omics showed 143 genes with consistent variation trends and displayed strong expression correlations, among which voltage-dependent anion channel 2 (VDAC2) markedly upregulated in resistant cells [Supplementary Figure 1C–E, http://links.lww.com/CM9/C974].

VDAC2 is a mitochondrial membrane pore-forming protein, which mediates ion and metabolite transport across mitochondrial membranes.^[4] It plays a crucial role in cancer progression by modulating the metabolic and apoptotic pathways. To further assess the prognostic significance of VDAC2 expression in DLBCL patients, we analyzed the Gene Expression Omnibus (GEO) datasets, and showed that patients with lower VDAC2 expression exhibited longer survival times within the entire cohort [Supplementary Figure 1F, http://links.lww.com/CM9/C974]. Subgroup analysis showed that low VDAC2 expression predicted better outcomes specifically in patients receiving R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), but not effect of patients treated with CHOP [Supplementary Figure 1G, H, http://links.lww.com/CM9/C974]. Additionally, patients with lower expression of VDAC2 derived greater benefit from the R-CHOP regimen [Supplementary Figure 1I, J, http://links.lww.com/CM9/C974]. Collectively, these findings suggest that VDAC2 is a prognostic biomarker, predicting the OS of patients treated with the R-CHOP regimen.

To investigate the biological function of VDAC2 in DLBCL, we established stable VDAC2-knockdown DLBCL cell lines [Figure 1A and Supplementary Figure 2A, http://links.lww.com/CM9/C974]. The cell counting kit-8 (CCK-8) assay revealed that VDAC2 depletion significantly suppressed cell proliferation [Figure 1B and Supplementary Figure 2B, http://links.lww.com/CM9/C974], and induced G0/G1 cell cycle arrest [Supplementary Figure 2C, http://links.lww.com/CM9/C974]. In vivo, VDAC2-knockdown significantly reduced tumor growth rates and volumes in mice [Figure 1C]. Furthermore, to explore the potential role of VDAC2 in rituximab resistance in DLBCL, cells with VDAC2 knockdown were exposed to varying concentrations of rituximab. VDAC2-deficient cells exhibited enhanced rituximab sensitivity, as evidenced by the significantly reduced half-maximal inhibitory concentration (IC50) values [Figure 1D and Supplementary Figure 2D, http://links.lww.com/CM9/C974]. Collectively, these results indicate that VDAC2 deficiency can inhibit cell proliferation and enhance the sensitivity of DLBCL cells to rituximab, highlighting its potential as a therapeutic target.

Figure 1.

VDAC2 promotes rituximab resistance via lactate/ferroptosis axis in DLBCL. (A) VDAC2 silencing efficiency in DLBCL cells was confirmed by qRT-PCR and Western blot. (B) Cell proliferation was analyzed in VDAC2-knockdown cells using the CCK-8 assay. (C) The volume and tissues of NOD/SCID mice xenograft tumors with VADC2-knockdown cells. (D) The IC50 values for sh-VDAC2 cells to rituximab for 24 h. (E) Lactate production was quantified using a Lactate Assay Kit (Jiancheng Corporation, Nanjing, China). (F) ECAR was measured in sh-VDAC2 cells. (G) The IC50 values for rituximab and lactate treatment were determined after 24 h. (H) KEGG enrichment analysis of differentially expressed genes in rituximab-resistant cells. (I) Lipid ROS levels in sh-VDAC2 cells were measured using C11-BODIPY staining followed by flow cytometry. (J) MDA and GSH levels were assesses in VDAC2-knockdown cells. (K) Lipid ROS levels were measured in sh-VDAC2 cells pre-treated with Ferrostatin-1 or ZVAD-FMK. (L) Ferroptosis-related gene expression was analyzed via qRT-PCR and Western blot. (M) Assessed therapeutic effect of rituximab (12.5 mg/kg) by xenograft mouse models with VDAC2-knockdown cells. (N) Ferroptosis-related gene expression was analyzed in lactate-treated sh-VDAC2 cells. Statistical significance: ^*P <0.05, ^†P <0.01, ^‡P <0.001 (Student’s t-test). 2-DG: 2-Deoxy-D-glucose; C11-BODIPY: C11-BODIPY 581/591; CCK-8: Cell Counting Kit-8; DLBCL: Diffuse large B-cell lymphoma; ECAR: Extracellular acidification rate; Fer-1: Ferrostatin-1; IC50: Half-maximal inhibitory concentration; GPX4: Glutathione peroxidase 4; GSH: Glutathione; KEGG: Kyoto Encyclopedia of Genes and Genomes; Lac: Lactate; MDA: Malondialdehyde; mRNA: Messenger RNA; NOD/SCID: Non-obese diabetic/severe combined immunodeficiency; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; Rtx: Rituximab; ROS: Reactive oxygen species; SLC7A11: Solute carrier family 7 member 11; VDAC2: Voltage-dependent anion channel 2; ZVAD-FMK: Z-Val-Ala-Asp(OMe)-fluoromethyl ketone.

Additionally, DLBCL cells with stable VDAC2 overexpression were established [Supplementary Figure 3A, http://links.lww.com/CM9/C974]. VDAC2 overexpression enhanced cell proliferation [Supplementary Figures 3B, C, http://links.lww.com/CM9/C974], and promoted cell cycle progression by reducing G1-phase populations while increasing S-phase [Supplementary Figures 3D, http://links.lww.com/CM9/C974]. Importantly, the sensitivity of cells to rituximab was diminished upon VDAC2 overexpression [Supplementary Figure 3E, http://links.lww.com/CM9/C974]. These findings underscore VDAC2 as a key regulator of DLBCL cell proliferation and rituximab sensitivity.

Previous studies have identified VDAC2 as a mitochondrial channel protein involved in energy metabolism. Notably, malignant cells often prioritize adenosine triphosphate (ATP) production through glycolysis during development and progression. We identified HexokiII (HKII) and Pyruvate Kinase M2 (PKM2) as crucial downstream effectors of VDAC2; their expression was reduced upon VDAC2 knockdown [Supplementary Figure 4A, http://links.lww.com/CM9/C974]. Consistently, VDAC2 knockdown substantially decreased lactate production [Figure 1E and Supplementary Figure 4B, http://links.lww.com/CM9/C974] and significantly reduced glycolytic rate and glycolytic capacity [Figure 1F and Supplementary Figure 4C, http://links.lww.com/CM9/C974]. These results indicated that VDAC2 modulates the metabolic shift toward glycolysis in DLBCL cells. Lactate is a key glycolytic metabolite with documented involvement in tumor progression and therapy resistance;^[5] we further investigated its contribution to VDAC2-mediated rituximab resistance. Exogenous lactate reversed the enhanced sensitivity to rituximab in VDAC2 knockdown cells [Figure 1G and Supplementary Figure 4D, http://links.lww.com/CM9/C974]. Collectively, these results suggest that VDAC2-mediated metabolic reprogramming toward glycolysis, with lactate serving as a critical mediator of the resulting rituximab resistance in DLBCL.

Then, we performed an integrative analysis of transcriptomic and proteomic data from resistant DLBCL cells, which revealed significant enrichment of the ferroptosis-related pathway via Kyoto Encyclopedia of Genes and Genomes (KEGG) [Figure 1H]. Using the ferroptosis inducer erastin (which inhibits system Xc^– and depletion glutathione [GSH]) and ferroptosis inhibitor (Fer-1), we observed that VDAC2 knockdown significantly enhanced erastin-induced cell death, while VDAC2 overexpression provided partial protection. Importantly, erastin-induced cell death was specifically reversed by Fer-1 but not by the apoptosis inhibitor ZVAD-FMK (Z-Val-Ala-Asp(OMe)-fluoromethyl ketone) [Supplementary Figures 5A, B, http://links.lww.com/CM9/C974]. These findings suggest that VDAC2 modulates ferroptosis in DLBCL cells. Furthermore, we detected the characteristic ferroptosis features including lipid reactive oxygen species (ROS), lipid peroxides, and malondialdehyde (MDA). VDAC2 knockdown increased lipid ROS and MDA levels while decreasing GSH levels [Figures 1I, J and Supplementary Figures 5C, D, http://links.lww.com/CM9/C974]. Notably, the elevated lipid ROS levels was reversed by Fer-1 but not ZVAD-FMK [Figure 1K and Supplementary Figure 5E, http://links.lww.com/CM9/C974]. VDAC2 knockdown also significantly downregulated the key ferroptosis regulators, solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) [Figure 1L and Supplementary Figure 5F, http://links.lww.com/CM9/C974]. Moreover, erastin co-treatment synergistically enhanced VDAC2-knockdown cells to rituximab sensitivity [Supplementary Figure 5G, http://links.lww.com/CM9/C974]. Subsequently, in vivo assessment of the impact of VDAC2 knockdown on the sensitivity of cells to rituximab was done. VDAC2-knockdown xenograft tumors exhibited enhanced sensitivity to rituximab, demonstrating significantly reduced tumor volume and weight compared with the controls [Figure 1M]. In addition, VDAC2-knockdown xenograft tumors elevated levels of MDA and reduced intracellular GSH [Supplementary Figure 5H, http://links.lww.com/CM9/C974]; they also decreased expression of glycolytic regulators (HKII, PKM2) and ferroptosis-related proteins (SLC7A11, GPX4) [Supplementary Figure 5I, http://links.lww.com/CM9/C974].

Although our findings indicate that lactate production is enhanced by VDAC2 expression, the role of lactate in regulating ferroptosis remains to be elucidated. We observed that exogenous lactate could effectively rescue VDAC2 knockdown-mediated expression downregulation of key ferroptosis regulators SLC7A11 and GPX4 [Figure 1N and Supplementary Figure 6A, http://links.lww.com/CM9/C974]. It also abrogated lipid ROS accumulation, intracellular GSH depletion, and increased MDA levels [Supplementary Figures 6B–D, http://links.lww.com/CM9/C974], and attenuated the enhanced cell death induced by erastin treatment of VDAC2-knockdown cells [Supplementary Figure 6E, http://links.lww.com/CM9/C974]. Concurrently, lactate partially reversed the synergistic sensitization to rituximab caused by erastin and VDAC2 deficiency [Supplementary Figure 6F, http://links.lww.com/CM9/C974]. In summary, these results suggest that lactate is a crucial metabolic mediator in VDAC2-conferred rituximab resistance through ferroptosis suppression.

In conclusion, our findings propose VDAC2 as a potential prognostic biomarker and therapeutic target for DLBCL. Targeting the VDAC2–lactate–ferroptosis axis may represent a novel strategy to overcome rituximab resistance in DLBCL patients, providing a foundation for future clinical translation of VDAC2-targeted therapies and biomarker applications.

Funding

This work was supported by the National Natural Science Foundation (No. 82470191), National Natural Science Foundation of Chongqing (Nos. CSTB2024NSCQ-LZX0069 and CSTB2025NSCQ-GPX0588), Chongqing Science and Health Joint medical innovation project (Nos. 2022DBXM003, 2023QNXM047, and 2025ZDXM030), Clinical Research Special Project of Xinqiao Hospital (No. 2024F028), and Young Doctor Incubation Program of Xinqiao Hospital, Army Medical University (No. 2023YQB037).

Conflicts of interest

None.

Supplementary Material

cm9-139-2988-s001.docx ^{(1.9MB, docx)}