NSUN2/YBX1-mediated m5C modification enhances phgdh expression to drive immune evasion via PD-L1 and CCL2 axis in high-grade b-cell lymphoma

Abstract High-grade B-cell lymphoma with double-hit (HGBL-DH), distinguished by simultaneous MYC and BCL-2 rearrangements, constitutes a notably aggressive lymphoma subtype with a dismal prognosis and scant treatment options. This investigation delves into the epitranscriptomic and immunometabolic mechanisms that drive HGBL-DH progression and immune evasion, concentrating on the m5C methylation pathway. We reveal that NSUN2, an essential m5C RNA methyltransferase, is markedly upregulated in HGBL-DH tissues compared to non-high-grade DLBCL, correlating with heightened global m5C levels. Through m5C-MeRIP-seq and transcriptomic analysis, we pinpoint PHGDH mRNA as a direct downstream target of NSUN2-mediated m5C modification, particularly in the 3'-UTR of PHGDH transcripts. Functional assays indicate that NSUN2 knockdown results in diminished m5C levels and a decrease in PHGDH mRNA and protein expression in SU-DHL-4 and SU-DHL-6 cells. Further research identified YBX1 as a potential m5C reader that aids PHGDH mRNA stabilization. RIP-PCR confirmed YBX1's binding to PHGDH mRNA, and site-directed mutagenesis validated the interaction domain. YBX1 knockdown led to a reduction in PHGDH mRNA half-life and protein levels, suggesting a YBX1-dependent post-transcriptional regulatory mechanism. Mechanistically, PHGDH fosters immune evasion by upregulating PD-L1 via transcriptional modulation involving PAX5. PHGDH knockdown resulted in reduced PD-L1 expression and hindered PAX5 nuclear translocation. Co-immunoprecipitation and pulldown assays demonstrated that PHGDH competes with PAX5 for binding to XPO1, a nuclear export protein. This competition promotes PAX5 nuclear retention, allowing it to activate PD-L1 transcription. Functionally, PHGDH suppression heightened CD8+ T-cell proliferation and cytotoxicity in co-culture systems, underscoring its critical role in tumor immune escape modulation. Moreover, we discovered that PAX5 transcriptionally activates CCL2, a chemokine that encourages tumor-associated macrophage (TAM) recruitment and M2 polarization. HGBL-DH tissues exhibited increased CCL2 expression and augmented CD206+ TAM infiltration. ELISA and luciferase reporter assays corroborated PAX5's regulation of CCL2 expression. Conditioned media from PHGDH- or PAX5-depleted cells diminished M2 TAM marker expression (IL-10, Arg-1) and enhanced CD8+ T-cell cytotoxic responses, implicating the PHGDH/PAX5/CCL2 axis in shaping the immunosuppressive microenvironment. Our proposed model indicates that NSUN2 and YBX1 collaborate to epigenetically upregulate PHGDH through m5C modification. PHGDH subsequently orchestrates tumor immune evasion along two converging pathways: (1) boosting PD-L1 expression via the PAX5-XPO1 axis to suppress T-cell activation; and (2) promoting M2 TAM polarization by driving CCL2 expression and secretion. These findings illuminate a new immunometabolic regulatory network in HGBL-DH and present promising targets for therapeutic intervention. In vivo validation utilizing patient-derived xenograft (PDX) models and humanized mice is ongoing to evaluate the synergistic effects of PHGDH inhibitors (e.g., NCT-503) alongside PD-1 blockade. This research not only clarifies the biological functions of m5C RNA methylation in hematologic malignancies but also provides a scientific basis for combinatorial immuno-metabolic therapies targeting high-risk lymphoma subtypes.