The m5C-binding protein YB-1 modulates proteasome inhibitors resistance in multiple myeloma via epigenetic modification

Abstract Multiple myeloma (MM) is a malignant proliferative disorder of plasma cells, and resistance to proteasome inhibitors (PIs) represents a major obstacle in MM treatment. Epigenetic reprogramming drives dynamic transcriptional heterogeneity in MM. As a novel epigenetic modification, RNA 5-methylcytosine (m5C) has emerged as a prognostic marker in tumors. Notably, Y-box binding protein 1 (YB-1) has been well-characterized as a cytoplasmic m5C reader that modulates RNA metabolism. However, the role and mechanisms of m5C in MM remain elusive. Here, we found that YB-1 is highly expressed in CD138+ cells isolated from MM patients compared to healthy donors and is associated with poor prognosis. In vitro experiments suggested that YB-1 knockdown could greatly trigger the apoptosis of MM cells and induce G2 phase arrest under the treatment of PIs. We also generated xenograft models using MM cell lines with stable YB-1 knockdown (KD) in immune-deficient NOD CRISPR Prkdc Il2r Gamma (NCG) mice. The results showed that the YB-1 KD group had a significant reduction in tumor burden and prolonged survival in response to bortezomib compared with the control group. In addition, we restored YB-1 expression by overexpressing wild-type (WT) YB-1 or the binding deficient mutant YB-1-Mut(W65F) on the basis of YB-1 knockdown. Subsequently, we observed that the restoration of WT-YB-1, but not YB-1-Mut(W65F), substantially rescued the survival of MM cells under PIs treatment, indicating that YB-1 regulates PIs resistance in MM in a m5C-dependent manner. To identify YB-1-mediated downstream effectors modulating MM PIs resistance, we conducted multiple high-throughput sequencing analyses (m5C-Bis-seq, RIP-seq, and RNA-seq) and obtained three potential downstream targets (SESN2, ZFP36, TTYH3). Further analysis of MM patient gene expression profiles suggested a stronger correlation between YB-1 and SESN2. We performed an RNA decay assay in MM cells and our data showed that YB-1 maintains SESN2 mRNA stability by binding to its m5C methylation sites. SESN2 is a highly conserved stress-induced protein that protects cells from external factors such as stress and autophagy. Based on survival analysis, we found that high expression of SESN2 is associated with poor prognosis of MM patients. Furthermore, in vitro phenotypic assays showed that the depletion of SESN2 significantly enhanced the killing effect of PIs on MM cells, while the supplementation of SESN2 substantially rescued the PIs sensitivity of MM cells resulting from YB-1 deficiency. In summary, our study reveals that YB-1 confers PIs resistance by stabilizing SESN2 mRNA via m5C modification in multiple myeloma. This discovery unveils a novel epigenetic mechanism underlying drug resistance and identifies YB-1 as a potential therapeutic target for overcoming chemoresistance in MM treatment. This mechanistic insight into YB-1's role in m5C-mediated drug resistance opens new therapeutic avenues.