P-250: ALTERATION OF THE m6A REGULATORY GENES, WITH THE SPECIAL IMPLICATION OF VIRMA, IS CRUCIAL FOR MULTIPLE MYELOMA DEVELOPMENT

Introduction: Epitranscriptomics has emerged as a crucial field uncovering RNA modifications that play a vital role in human diseases, being the methylation of N⁶-adenosine (m6A) the one that suffers the most prevalent alterations in human tumors. Therefore, the study of the levels and the associated genes regulating m6A may provide a different perspective for a deeper understanding of the biology of multiple myeloma (MM) and the potential impact as a biomarker or therapeutic target in this disease. We aimed to elucidate the role of the m6A and its regulatory genes in the prognosis and pathogenesis of MM.

Methods: We analyzed the m6A levels and the status of its 20 regulatory genes based on RNA-seq, ATAC-seq, and ChIP-seq data, in plasma cells (PCs) from healthy donors and MM patients. RNA-seq data from 619 MM patients from the CoMMPass dataset (IA18) were used. VIRMA inhibition was carried out by CRISPR-Cas9 technology. VIRMA levels (western blot), global m6A levels (m6A-ELISA), cell proliferation (flow cytometry), and whole proteome (mass spectrometry) were analyzed.

Results: m6A global levels were significantly increased in plasma cells from patients with SMM and MM, which correlated with the overexpression of 18 of the 20 genes that regulate m6A. Interestingly, the expression of these genes was epigenetically regulated as demonstrated by greater accessibility and activation (H3K27Ac) of the chromatin in their promoter region. In terms of prognosis, MM patients with a higher number of m6A genes upregulated (ESMEG score: Expression Signature of m6A Epitranscriptomic Genes) showed significantly poorer progression-free (PFS) and overall survival (OS) in the univariate and multivariate study. Finally, we focused on VIRMA, as we identified that VIRMA inhibition was essential in virtually all MM cell lines included in the Dep-MAP-Achilles project. We demonstrated that the inhibition of VIRMA decreased significantly the m6A level and cell proliferation of MM cells. Inhibition of VIRMA was associated with proteome change in which IKBKB downregulation was highlighted. Lack of IKBKB was hypothesized to reduce NF-kb translocation to the cell nucleus, and consequently, to lower NF-kb signaling, decreasing cell survival, and anti-apoptotic effects. These results demonstrate the alteration of the m6A profile in the disease, where the m6A genes, especially VIRMA, have a critical role in MM pathophysiology.

Conclusions: Our results demonstrate that MM is characterized by an epigenetically deregulated epitranscriptome. Moreover, the expression score of genes that regulate the m6A emerges as a promising biomarker for refining the stratification of MM patients. Notably, within m6A genes, VIRMA plays an essential role in the pathogenesis of MM via reverting the overactivation of the NF-kb signaling pathway observed in MM plasma cells, indicating its potential for the development of targeted therapies to improve the treatment response of MM patients.