Hakai links m6A RNA methylation to immune regulation in colorectal cancer

To define the functional role of Hakai within the m 6 A writer complex in colorectal cancer (CRC) and to determine how its modulation influences RNA methylation dynamics and tumour-immune interactions. Hakai was knockdown in CRC cell models, followed by RNA sequencing to assess transcriptomic changes and MeRIP-seq to evaluate m 6 A methylation patterns. Protein expression and immune-related markers were validated using Western blot, flow cytometry, ELISA, and RT-qPCR. Protein-protein interactions were analysed by co-immunoprecipitation, and subcellular localisation was assessed by cell fractionation. Functional immune assays were performed by exposing peripheral blood mononuclear cells from healthy donors and lamina propria mononuclear cells from CRC patients to conditioned media from Hakai-knockdown cells. Hakai knockdown induced phenotypic alterations in both 2D and 3D CRC models, despite minimal global transcriptomic changes. Notably, significant modifications in m 6 A methylation were observed, particularly in immune-related transcripts, including cytokines TNF, IFNB1, CXCL1, and CXCL8, accompanied by an overall reduction in m 6 A levels. Mechanistically, Hakai interacted with the writer-associated protein VIRMA and regulated METTL3 subcellular localisation. Functionally, conditioned media from Hakai-knockdown cells altered immune marker expression in patient-derived immune cells, indicating modulation of tumour-immune crosstalk. Hakai functions as a regulatory component of the m⁶A writer complex in CRC, influencing RNA methylation and immune-related gene expression. Our findings support a previously unrecognised role for Hakai in tumour-immune crosstalk and suggest its potential relevance in the modulation of the CRC tumour microenvironment.