Deciphering the m6A Epitranscriptomic Landscape of mRNAs in Breast Cancer Cells

N6-methyladenosine (m6A), the most prevalent modification in mRNAs, influences mRNA stability, splicing, and translation. Dysregulation of m6A patterns has been linked to various diseases, including cancer, highlighting its significance in cellular homeostasis. However, accurate detection and precise quantification of m6A sites within individual transcripts remains challenging. In this study, we employed nanopore sequencing to achieve transcriptome-wide, base-resolution map of the m6A methylome in human breast cancer cells. By investigating m6A distribution across breast cancer cell lines and implementing a CRISPR/Cas9-based knockout of the major m6A eraser ALKBH5, we provide insights into the differential methylation levels and motif-specific characteristics of m6A transcriptomic sites. We elucidated the m6A epitranscriptome in five well-established breast cancer cell lines derived from distinct molecular subtypes of the disease and confirmed a DRACH-dependent activity of ALKBH5. Comparative methylation analysis with the non-cancerous MCF-10A cell line revealed that MCF-7 and BT-474 breast cancer cells are primarily hypomethylated, while BT-20, MDA-MB-231 and SK-BR-3 cells show widespread hypermethylation. These cell line-based patterns highlight the potential regulatory role of m6A in breast cancer heterogeneity. Overall, our findings enhance the understanding of m6A dynamics in breast cancer.