Virma epitranscriptomically regulates cell proliferation, apoptosis, and mitochondrial function via m6A in multiple myeloma Free

Multiple Myeloma (MM) is a hematological neoplasm characterized by the clonal expansion of malignant plasma cells (PCs) within the bone marrow. Despite clinical advances, the majority of patients eventually experience disease relapse. Therefore, understanding the molecular basis of MM is essential for developing new therapeutic strategies. Epitranscriptomic alterations, such as post-transcriptional RNA modifications, have emerged as key mechanisms in tumor development. However, their role in MM remains poorly understood. In this study, we investigated the role of N6-methyladenosine (m6A), the most prevalent RNA modification, and its regulatory genes in the prognosis and development of MM.

We first measured global m6A levels in purified PCs from the bone marrow samples of individuals with premalignant stages such as MGUS and SMM, MM patients and healthy donors using ELISA. Interestingly, m6A levels were significantly increased from early disease stages through to MM. This increase correlated with the overexpression of 18 out of 20 m6A regulatory genes in MM samples, as observed in RNA-seq data from PCs of MM patients compared to healthy donors. Epigenomic analyses (ATAC-seq and H3K27ac ChIP-seq) confirmed increased chromatin accessibility and activation in the promoter region of m6A regulatory genes in MM PCs, demonstrating that these genes were epigenetically deregulated in MM. To assess the clinical relevance of m6A regulators we analyzed RNA-seq and clinical data from 642 MM patients from the CoMMpass cohort. Patients were stratified into high or low expression groups for each m6A gene individually, and Kaplan-Meier survival analyses were performed. High expression of most m6A genes was significantly associated with shorter progression-free survival (PFS) and overall survival (OS). Given the strong coexpression among m6A-related genes, we developed a composite expression signature referred to as the Epitranscriptomic Signature of m6A Epigenetic Genes (ESMEG) to better stratify MM patients. MM patients with high ESMEG scores exhibited significantly worse PFS and OS, independently of the presence or absence of high-risk genetic alterations in MM. Furthermore, gene ontology (GO) and gene set enrichment analysis (GSEA) revealed that high ESMEG scores were associated with enrichment of proliferation-related pathways, supporting a functional role for m6A regulator genes in promoting MM aggressiveness.

Among m6A regulators, VIRMA was identified as essential in MM based on data from the DepMap-Achilles' project. CRISPR-Cas9-mediated VIRMA inhibition in MM.1S-Cas9 and KMS-11-Cas9 cells reduced cell viability and global m6A levels in vitro. Notably, in vivo studies revealed a marked impairment of MM tumor growth, demonstrating its essentiality in MM. Direct RNA nanopore sequencing revealed a general reduction of m6A marks in VIRMA-knockdown conditions, as well as a reduced m6A stoichiometry. Importantly, levels of other RNA modifications, such as pseudouridine, 5-methylcytosine, inosine and 2'-O-methylated nucleotides, remained unchanged, supporting the specificity of VIRMA's effect on m6A modification. From all detected m6A sites, 609 of them had a significantly reduced m6A representativity across 475 genes after VIRMA knockdown. GO analysis showed that these genes were related to cell cycle, apoptosis, and mitochondrial functions. Specifically, functional studies validated that VIRMA inhibition led to G1 cell cycle arrest (flow cytometry), reduced NF-κB signaling (Western blot, ELISA, flow cytometry), and impaired mitochondrial respiration (Seahorse), supporting its key role in MM cell survival. Finally, to evaluate the potential of VIRMA as a therapeutic target, we developed lipid nanoparticles (LNPs) encapsulating a VIRMA-targeting siRNA (LNP-siVIRMA) using microfluidic technology. Treatment of MM cell lines with LNP-siVIRMA significantly inhibited MM cell proliferation in vitro. Importantly, in vivo administration led to a pronounced reduction in tumor growth, highlighting the therapeutic potential of targeting VIRMA in MM.In summary, our findings indicate that increased m6A and upregulation of its regulatory genes are associated with poor prognosis in MM. VIRMA is critical for MM cell survival through regulation of the cell cycle, apoptosis, and mitochondrial function. Its inhibition via LNP-siVIRMA represents a promising epitranscriptomic-based therapeutic strategy to improve the response of MM patients.