Systematic profiling of AGO2-associated circRNAs and functional investigation reveal circN4BP2L2 as a survival-promoting regulator in T-ALL

Circular RNAs constitute an emerging area of intensive research in cancer. In T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematologic malignancy characterized by clonal proliferation of T-cell precursors, the function of most aberrantly expressed circRNAs is not yet understood. To identify circRNAs acting as miRNA sponges, we performed AGO2 immunoprecipitation and RNA sequencing (AGO2-RIP-seq) in four T-ALL cell lines. Our analysis revealed circRNAs consistently enriched in the AGO2-bound fraction, highlighting a new resource for T-ALL research. Notably, circRNAs were more enriched (7.3%) compared to linear RNAs (3%). For functional investigation, we focused on the most abundant AGO2-bound circRNAs that were also found to be dysregulated in T-ALL patients. Knockdown of circN4BP2L2 revealed its ability to promote cell proliferation and confer resistance to apoptosis in T-ALL cell lines, in addition to modulating key T-ALL pathogenic pathways. Analysis of gene expression of T-ALL patients disclosed peculiar features of T-ALL with high circN4BP2L2 expression. A marked overlap was observed between differentially expressed genes upon knockdown of circN4BP2L2 in vitro and the profiles of circN4BP2L2 stratified T-ALL cases. This work provides new insights into the circRNA-mediated regulatory networks driving this malignancy and presents circN4BP2L2 as a key RNA shaping oncogenic phenotype in T-ALL.