Quantitative profiling of N6-methyladenosine methylome at childhood Ph positive B-cell acute lymphoblastic leukemia reveals a potential mechanism for transcriptional regulation and differential methylation pattern Running title N6-methyladenosine methylome in the childhood Ph positive B-ALL

Abstract Background: N6-Methyladenosine (m6A) is the most common methylation modification of messenger RNA (mRNA). Recent researches have revealed potential regulatory functions in various fundamental bioprocesses, suggesting a critical role of m6A in tumorigenesis. Acute lymphoblastic leukemia (ALL) is the most common malignant tumor in children. Methods: The RNA-seq transcriptome data of childhood B-ALL were downloaded from The Oncomine database. The identification of differentially expressed m6A RNA methylation regulators between cancer samples and normal control samples were achieved using t-test. The key regulators of m6A RNA methylation are tested in the bone marrow of childhood B-ALL by qRT-PCR. Then, we profile next-generation sequencing (MeRIP-Seq and RNA-Seq) in the bone marrow of childhood B-ALL at diagnosis. The Transcriptomes of bone marrow are from four patients. Three patients are diagnosed with childhood Philadelphia chromosome–positive ALL. Another one is Ph-negative B-cell ALL without gene fusions expression profile. Results: The results showed that the expression levels of FTO, METTL14, YTHDF2 and YTHDF2 in ALL patients were significantly up-regulated (P<0.05) compared with control group. The qRT-PCR reveals that the bone marrow in childhood B-ALL had m6A RNA methylation genes mRNA expression. We found that m6A is a highly conserved modification of mRNA. The percentage of more than 2 m6A modifications in each mRNA is approximately 30%, only one is about 40%, and 2 is about 30%. The m6A modification sites have a typical consensus sequence RRACU. The m6A modification sites in B-ALL are enriched near the stop codons and within 30-untranslated regions (3’UTR). Gene ontology (GO) analysis showed that the major molecular functions in these transcripts were responsible for molecule binding, transferase, transcription regulatory, and transferase activity. The cellular components were responsible for intracellular, nucleus and membrane-bounded organelle. The biological processes were responsible for metabolic process. The KEGG indicates that virus infection and type I interferons pathway are crucial. The versatile roles of m6A in childhood B-ALL is significant in mediating mRNA decay. We also discover that highly expressed genes are relatively more methylated. There is a positive correlation between m6A peak enrichment and mRNA abundance, suggesting a regulatory role of m6A in gene expression. Collusions: Collectively, we could show that m6A landscape is altered in the bone marrow of childhood B-ALL. Our reports demonstrate the functional importance of the m6A methylation in B-ALL especially Ph+ ALL in childhood, and provide profound insights into leukemogenesis. Our studies also further enlarge our knowledge on the function of RNA m6A modification and modulation of transcriptomic processes such as m6A methylation might be a meaning target for therapeutic interventions.