PRMT5 Inhibition Sensitizes B-Cell Lymphoma to Ferroptosis via ATF5-SLC7A11 Axis
核心洞察
PRMT5 inhibition enhances dimethyl fumarate (DMF)-mediated ferroptosis in DLBCL and MCL cell lines and patient samples by promoting lipid peroxidation.
PRMT5 regulates SLC7A11 (搜索) expression at the transcriptional level through the ATF5 (搜索) transcription factor, which also controls ATF4 (搜索) expression, reducing glutathione synthesis.
The PRMT5-AKT-MYC (搜索) signaling axis drives ATF5 (搜索) expression, and combined high expression of MYC and ATF5 correlates with worse survival in DLBCL and MCL patients.
Researchers have uncovered a novel mechanism by which PRMT5 inhibition sensitizes B-cell lymphoma cells to ferroptosis, a form of regulated cell death driven by lipid peroxidation. The findings, published in Leukemia, demonstrate that targeting PRMT5 in combination with ferroptosis inducers such as dimethyl fumarate (DMF) produces synergistic anti-tumor effects in diffuse large B-cell lymphoma (搜索) (DLBCL) and mantle cell lymphoma (搜索) (MCL), offering a potential new therapeutic strategy for these aggressive malignancies.
PRMT5 regulates lipid peroxidation and ferroptosis sensitivity
Reanalysis of RNA-seq data from DLBCL cell lines OCI-Ly7 and TMD8 revealed that PRMT5 knockout enriches gene signatures related to fatty acid metabolism, cholesterol homeostasis, glycolysis, and lipid peroxidation, in addition to the expected PI3K-AKT-mTOR and MYC (搜索) target pathways. This prompted the investigators to explore whether PRMT5 plays a role in ferroptosis regulation.
Flow cytometric analysis using the oxidation-sensitive probe BODIPY C11 demonstrated that PRMT5 knockdown alone did not induce apparent lipid peroxidation but significantly enhanced DMF-mediated ferroptosis across all tested cell lines, including one MCL cell line, two activated B-cell (ABC) DLBCL lines, and one germinal center B-cell (GCB) DLBCL line. This effect was dose-dependent. Similar results were observed with two specific PRMT5 inhibitors, which also enhanced DMF-mediated lipid peroxidation in two MCL patient samples (MCL-4 and MCL-9). Notably, a three-hour DMF treatment following five-day pretreatment with PRMT5 inhibitors did not induce apoptosis, confirming that the observed cell death was ferroptosis-specific.
PRMT5 controls SLC7A11 (搜索) expression at the transcriptional level
Immunoblotting revealed that PRMT5 knockout reduced protein levels of SLC7A11 (搜索) and GPX4 (搜索), two major ferroptosis-inhibitory proteins, while AIFM2 remained unchanged. Treatment with PRMT5 inhibitors led to a significant decrease in SLC7A11 expression across two DLBCL cell lines, two MCL cell lines, and one MCL patient sample, whereas GPX4 expression was either unchanged or only slightly reduced.
A rescue experiment confirmed that restoring PRMT5 expression via retroviral delivery of PRMT5 cDNA significantly increased SLC7A11 (搜索) expression, but not GPX4 (搜索) or AIFM2. Quantitative PCR analysis further demonstrated reduced SLC7A11 mRNA following PRMT5 knockout or inhibition. The researchers found no physical interaction between PRMT5 and SLC7A11, indicating that PRMT5 regulates SLC7A11 at the transcriptional level rather than through posttranslational modification.
Functionally, PRMT5 inhibition reduced intracellular glutathione (GSH) levels and decreased the ratio of reduced GSH to its oxidized form (GSSG), consistent with impaired cystine import through SLC7A11 (搜索) and diminished GSH synthesis.
ATF5 (搜索) emerges as a critical transcription factor downstream of PRMT5
Reanalysis of RNA-seq data identified ATF5 (搜索) as the most significantly regulated and abundantly expressed gene after PRMT5 knockout. Analysis of the GOYA trial cohort (553 de novo DLBCL patients) revealed that high ATF5 expression, but not ATF4 (搜索), is associated with poor overall survival. Higher combined expression of both ATF5 and ATF4 correlated with worse survival compared to lower expression levels. A similar trend was observed in MCL based on reanalysis of Affymetrix data from 123 MCL samples.
Immunoblotting showed that ATF5 (搜索) and ATF4 (搜索) expression was increased in DLBCL and MCL cell lines compared to normal naive B cells. PRMT5 inhibition, either pharmacologically or genetically, reduced expression of both transcription factors. Overexpression of ATF5 or ATF4 reduced the sensitivity of lymphoma cells to DMF, confirming their involvement in ferroptosis regulation.
ATF5 (搜索) knockdown reduced SLC7A11 (搜索) protein and mRNA levels, and dual-luciferase reporter assays demonstrated that both ATF5 and ATF4 (搜索) exhibit transcriptional activity on the SLC7A11 promoter through the AARE1 and AARE2 binding regions. ChIP-qPCR confirmed ATF5 binding to these promoter regions. Co-immunoprecipitation revealed a physical interaction between ATF5 and ATF4, suggesting they may dimerize to regulate transcription.
The PRMT5-AKT-MYC (搜索) axis drives ATF5 (搜索) expression
The researchers found that ATF5 (搜索) regulation by PRMT5 does not involve altered splicing or direct methylation. Instead, ATF5 expression is controlled through the PRMT5-AKT-MYC (搜索) signaling axis. Treatment with two AKT inhibitors, AZD5356 and AKTi-V, reduced ATF5 and ATF4 (搜索) expression in both DLBCL and MCL cell lines, as well as in two MCL patient samples. MYC knockout reduced ATF5, ATF4, and SLC7A11 (搜索) expression, but not GPX4 (搜索) or AIFM2, and decreased ATF5 mRNA levels.
Dual-luciferase reporter assays and MYC (搜索) ChIP-qPCR confirmed that MYC binds to the F2 region of the ATF5 (搜索) promoter. Reanalysis of gene expression data from two datasets showed that higher combined expression of ATF5 and MYC correlates with worse survival in both DLBCL and MCL patients following immunochemotherapy, suggesting their potential as prognostic biomarkers.
In vivo validation of PRMT5 inhibition and ferroptosis synergy
Cell viability assays demonstrated that PRMT5 inhibition via shRNA or CRISPR/Cas9-mediated sgRNA significantly enhanced DMF-induced ferroptotic cell death, with the combination exhibiting a synergistic effect. Synergy was also observed between the PRMT5 inhibitor GSK3326595 and two other ferroptosis inducers: the GPX4 (搜索) inhibitor RSL3 and the SLC7A11 (搜索) inhibitor Erastin. The reduced cell viability was rescued by the ferroptosis inhibitor Ferrostatin-1 (搜索) and by N-acetylcysteine (NAC), a GSH precursor, but not by the pan-caspase inhibitor Q-VD-OPh, confirming ferroptosis-dependent cell death.
In cell line-derived xenograft (CDX) models using NSG mice, PRMT5 knockout via sgRNA alone resulted in modest tumor growth reduction but significantly enhanced DMF-mediated suppression of tumor progression, as evidenced by reductions in tumor volume, size, and weight. No changes in body weight were observed in recipient mice. Importantly, the combination of PRMT5 knockout and DMF significantly increased production of 4-hydroxynonenal (4-HNE), a key lipid peroxidation product, without altering cleaved caspase-3 levels, confirming that ferroptosis contributes to tumor growth inhibition in vivo.
These findings establish the PRMT5-ATF5 (搜索)-SLC7A11 (搜索) axis as a critical regulator of ferroptosis in B-cell lymphomas and provide a mechanistic rationale for combining PRMT5 inhibitors with ferroptosis inducers as a therapeutic strategy for DLBCL and MCL.