HOXA9/METTL3/BMI1 axis is required for promoting anthracycline resistance in acute myeloid leukemia cells by modulating BMI1 expression through  mRNA m6A modification Free

Background Acute myeloid leukemia (AML) is the most common adult acute leukemia. The traditional “3+7” induction regimen with anthracycline and cytarabine remains a standard therapy, but 30%-40% of patients develop drug resistance to anthracyclines, leading to relapse. Refractory/relapsed AML has dismal survival rates, posing a major challenge. Elucidating mechanisms of therapy resistance and relapse is crucial for improving outcomes in high-risk AML.

Methods ATAC-seq analyzed chromatin accessibility of anthracycline-sensitive and -resistant AML cells, focusing on METTL3 locus. RNA-seq, qRT-PCR and Western blot measured METTL3 expression in cell lines and primary cells. METTL3 was regulated by small-molecule inhibition, shRNA knockdown and overexpression to study its effect on m6A modification and phenotypes in resistant cells. MeRIP-seq and RNA-seq identified METTL3 target genes in shMETTL3-transfected resistant cells. MeRIP-qPCR validated m6A-modified mRNAs and actinomycin D assays tested RNA stability. BMI1 and METTL3 inhibitors were used to explore their functional relationship in drug resistance.

Results

In anthracycline-sensitive AML patients, over 50% of the global chromatin accessible regions are enriched in promoter regions. Conversely, in anthracycline-resistant AML patients, promoter-accessible regions account for less than 25% of the total. Treatment with anthracyclines induces changes in promoter chromatin accessibility in only approximately 5% of the genome in sensitive patients, whereas this proportion reaches approximately 25% in resistant patients. Notably, METTL3 exhibits significantly elevated chromatin accessibility in anthracycline-resistant AML cells. Following anthracycline exposure, METTL3 accessibility is further enhanced in resistant cells but decreased in sensitive cells. m6A modification was significantly upregulated in AML drug-resistant cells, accompanied by consistently higher METTL3 expression. Integrative multi-omics analyses identified HOXA9 as a key transcriptional regulator of METTL3, with HOXA9 positively modulating METTL3 expression. Clinical datasets (TCGA and GSE6891) revealed that elevated HOXA9 expression was significantly associated with poor prognosis in AML patients. HOXA9 levels were markedly higher in drug-resistant and relapsed/refractory (R/R) AML cells. HOXA9 knockdown in resistant cells suppressed proliferation and induced apoptosis. Dual-luciferase reporter assays confirmed that HOXA9 specifically bound to the “TAACGTG” motif within CpG island 3 of the METTL3 promoter.

Mechanistically, reducing METTL3 expression in drug-resistant cells decreased m⁶A modification, inhibited cell proliferation, and triggered apoptosis. Conversely, METTL3 overexpression in sensitive cells enhanced m⁶A modification, promoted proliferation, suppressed apoptosis, and augmented doxorubicin resistance. METTL3 promoted m⁶A modification of BMI1 mRNA, stabilizing BMI1 transcripts and elevating BMI1 expression in anthracycline-resistant cells.

BMI1 was overexpressed in anthracycline-resistant cells and correlated with poor prognosis in AML patients. Inhibiting BMI1 in resistant cells induced dose-dependent proliferation arrest and apoptosis, while restoring sensitivity to anthracyclines.

Conclusion Our study demonstrates that chromatin accessibility in the promoter regions of anthracycline-resistant AML cells is significantly decreased compared to anthracycline-sensitive counterparts. Anthracycline elicits more pronounced changes in promoter chromatin accessibility in resistant cells than in sensitive cells, underscoring a heightened involvement of promoter-level epigenetic regulation in anthracycline-resistant AML. METTL3 exhibits both upregulated expression and enhanced chromatin accessibility in anthracycline-resistant AML cells relative to sensitive cells. Furthermore, anthracycline exposure leads to a further increase in chromatin accessibility at the METTL3 promoter in resistant cells, suggesting dynamic epigenetic modulation of this key m⁶A regulator during drug resistance. Our study demonstrated that The HOXA9/METTL3/BMI1 axis promotes anthracycline resistance in acute myeloid leukemia through mRNA m6A modification and suggested the therapeutic potential of targeting the METTL3/BMI1 axis in the treatment of anthracycline-resistant AML.