N6-methyladenosine demethylase ALKBH5 promotes the development of multiple myeloma

Objective: This study aimed at investigating the role of alkylation repair homolog protein 5 (ALKBH5), a N6-methyladenosine (m6A) demethylase, in the development of multiple myeloma (MM) as well as the underlying mechanisms.Methods: The m6A level of total RNA and ALKBH5 expression in MM patients were investigated. Moreover, ALKBH5 expression in MM cells was determined, followed by investigating the effects of ALKBH5 knockdown on MM cell proliferation and m6A levels. Furthermore, m6A sequencing (m6A-seq) of the shALKBH5-transfected and shNC-transfected RPMI 8226 cells was preformed, and bioinformatic analyses, such as identification of differentially methylated peaks and functional enrichment analyses were then conducted. The expression of key target genes of up-regulated methylated peaks was validated by quantitative PCR (qPCR).Results: The m6A level of total RNA was significantly decreased in MM patients, while ALKBH5 expression was remarkably elevated. Knockdown of ALKBH5 inhibited MM cell proliferation and upregulated m6A levels. Bioinformatics analyses of m6A-seq data identified 2421 differentially methylated peaks (619 up- and 1802 down-regulated) between shALKBH5-transfected and shNC-transfected RPMI 8226 cells. The target genes of up-regulated methylated peaks were remarkably enriched in several pathways such as hsa04330: Notch signaling pathway (EP300 and MAML2) and hsa04068: FoxO signaling pathway (ARAF, ATM, EP300, and FOXO3). qPCR further revealed that ALKBH5 knockdown reduced the expression of EP300, ARAF, ATM and FOXO3, and elevated MAML2 expression.Conclusion: Our findings indicate that ALKBH5 facilitates MM development by through m6A demethylation. Notch signaling pathway and FoxO signaling pathway may be downstream mechanisms underlying ALKBH5 demethylation in MM.