ALKBH3 loss induces a gain of m¹A in the transcriptome of Hodgkin lymphoma cells and is associated with poor clinical outcome. (A) Western blot validation of the efficient shRNA-mediated depletion of ALKBH3 in HD-MY-Z cells upon the addition of doxycycline and workflow of the RNA high-throughput sequencing using an m¹A antibody to enrich m¹A-modified mRNA-fragment analysis developed to detect changes in m¹A peaks upon ALKBH3 depletion in HD-MY-Z cells. (B) Distribution of RNA location sites for the m¹A peaks undergoing changes in upon ALKBH3 depletion in HD-MY-Z cells. (C) GO analysis (GO) of the genes with differential m¹A content in HD-MY-Z cells upon ALKBH3 depletion (hypergeometric test with a false discovery rate [FDR] adjusted P < .05). (D) Western blot of COL1A2 and COL1A1 protein levels upon shRNA-mediated depletion of ALKBH3 in HD-MY-Z cells. (E) Kaplan-Meier analysis of OS in primary Hodgkin lymphomas according to ALKBH3-methylation status determined by pyrosequencing. The P value corresponds to the log-rank test. Results of the univariate Cox regression analysis are represented by the HR and 95% CI. (F) Multivariate Cox regression analysis of OS, represented by a forest plot, considering the clinical characteristics of the cohort of primary Hodgkin lymphoma patients. ALKBH3 promoter hypermethylation is an independent prognostic factor for OS. Values of P < .05 were considered statistically significant. *P < .05. EBV, Epstein-Barr virus; M, methylated; U, unmethylated.