Emerging evidence indicates that m
6A modification is involved in the progression of various cancers. Therefore, we speculate that BP may inhibit leukaemic cell proliferation and induce apoptosis, leading to suppression of leukemia tumorigenesis by regulating mRNA m
6A enrichment and expression. Using m
6A sequencing (m
6A-seq), we mapped the m
6A methylomes of AML cells under different treatment conditions. We found that the GGACC motif was highly enriched within m
6A sites of BP-treated AML cells (Fig.
3a). In total, we identified 10,478 m
6A peaks representing the transcripts of 7684 genes in control cells, 15,474 m
6A peaks representing the transcripts of 10,369 genes in BP-treated cells, 9403 m
6A peaks representing the transcripts of 6901 genes in cytarabine-treated cells, and 16,508 m
6A peaks representing the transcripts of 10,829 genes in combination-treated cells (Fig.
3b; Fig. S1a). Furthermore, m
6A-seq revealed that the expression of regulators related to RNA methylation changed under different treatment conditions compared with the control samples. Compared to those in the control group, methyltransferases, such as METTL3, METTL14, METTL16, WTAP, RBM15 and ZC3H13, in the BP treatment group were markedly upregulated, while the demethylase ALKBH5 was significantly downregulated. The combination treatment group shared similar changes, while there was no significant difference in the cytarabine group (Fig.
3c; Table S1). Given the significant changes in m
6A modification, we further explored the target genes affected by BP treatment. A total of 399 genes showed differential methylation modification in the BP group, in which methylation modification of 273 genes was upregulated, while that in 126 genes was downregulated. Similar to the BP group, 261 genes showed upregulated methylation modifications, and 139 genes showed downregulated methylation modifications in the BP and cytarabine combination group. Of the 139 genes with changes in methylation modification in the cytarabine group, 64 exhibited upregulated changes, while 75 were linked with downregulated changes (Fig.
3d). Four quadrant graph analysis revealed that the mRNA m
6A level increased in the BP treatment group and indicated that genes with upregulated methylation are expected to encompass genuine targets of BP treatment (Fig. S1b). Among genes with upregulated methylation in the BP group, we found that the expression of 5 genes was increased and that of 36 genes was decreased (FC > 2 &
p < 0.05). We employed the Molecular Signatures Database (MSigDB) to analyse the genes filtered above and found that 26 out of 36 downregulated genes and 3 out of 5 upregulated genes were included in the leukemia-related gene set (Fig.
3e; Table S2). We next investigated the pathways of the differentially expressed genes that were shared by ClueGO (plugin for Cytoscape software) and found that the significantly enriched pathways included mTORC1-mediated signalling, mTOR signalling and TOR signalling (Fig.
3f). Therapeutic targeting of the mTOR pathway as an anticancer strategy for leukemia has been under extensive investigation. mTOR signalling plays a central role in the proliferation and survival of AML and is aberrantly activated in 60% of AML patients [
32,
33]. mTOR, which is a serine/threonine kinase, is a major component of different protein complexes, including mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [
34]. mTORC1 controls mRNA translation and protein synthesis through phosphorylation of S6K and 4E-BP [
35], whereas mTORC2 mainly mediates signal transduction by regulating AKT kinase [
36,
37]. In the BP group, the expression of EIF4EBP1 and MLST8 was significantly decreased. Next, we focused on these two potential ALKBH5 targets for further study.