Title: M1A regulator-mediated methylation modification and their prognostic value in multiple myeloma

Abstract

Background: N1‑methyladenosine (m1A), a dynamic modification of RNA with strong enrichment in the 5′-UTR, is gaining attention for its role across diverse biological and pathological processes such as cell differentiation, stress response and tumorigenesis1,2.However, the m1A regulatormediated methylation modification in multiple myeloma (MM) remains unclear. Aims: To investigate the transcriptomic m1A landscape and identify potential prognostic regulators in MM progression. Methods: We analyzed the expression of 10 m1A regulators and evaluated their prognostic value in three GEO datasets. Then, unsupervised clustering analysis was performed to evaluate the m1A modification patterns in MM and the m1A score was constructed to quantify m1A modification patterns of individual tumors using the PCA algorithm. Additionally, the potential role of YTHDF2 in MM pathogenesis was investigated in U266 cells. Results: Compared with healthy donors, the expression of three m1A readers (YTHDF1, YTHDF2, YTHDF3) was significantly upregulated in MM patients (Figure A). Multivariate cox analysis revealed that YTHDF2, YTHDF3 and TRMT6 could be independent prognostic factors for MM (Figure B). Based on the expression of ten m1A regulators, three distinct modification patterns were identified in MM patients, which were termed by clusters A-C respectively. There was a worse outcome in cluster B than in luster A and C, characterized by the increased expression of four regulators (YTHDF2, TRMT6/10C/61B) and remarkably deficient in innate immune cell infiltration (Figure C-E). Then, the K-M survival curve revealed that patients with low m1Ascores had a prominent survival probability (Figure F). Coincidentally, cluster B with a worse outcome exhibited the highest median score (Figure G). The reader protein YTHDF2 is associated with poor survival of MM patients and showed great superiority as a prognostic factor. Subsequent cell experiments demonstrated that YTHDF2 could promote the proliferation and inhibit apoptosis of U266 cells (Figure H, I). Notably, an evidently increased m1A level was observed in m1A dot-blot assay when YTHDF2 was over-expressed (Figure J). Summary/conclusion: This study identified the modification patterns and their superior prognostic value of m1A regulators and demonstrated that the reader protein YTHDF2 is a potentially crucial target for MM.