Effect of miR-15b and miR-16-2 overexpression on gene expression profiles in the human T-cell acute lymphoblastic leukemia (T-ALL) cell line CUTLL1.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy arising from the neoplastic transformation of immature T cells during their development in the thymus. Deciphering the developmental programs whose dysregulation leads to T-ALL pathogenesis is critical for the development of novel targeted therapies, which remain an urgent unmet need for the treatment of this disease. MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of numerous physiological processes and cancer. However, the specific role of miRNAs in human T- cell development and T-ALL pathogenesis remains largely unexplored. In this study, we comprehensively evaluated miRNA expression profiles across human T-cell development by microarray analysis and identified a dynamic expression pattern of miR-16-2, which is upregulated across early pre-T cell proliferative stages up to the resting stage of immature thymocytes immediately preceding TCRab expression, and decreased thereafter. We confirmed the coordinated regulation of miR-15b expression, consistent with the reported clustered genomic location of both miRNAs. Notably, functional studies identified the miR-15b/16-2 cluster as a negative regulator of early thymocyte proliferation, and showed that overexpression of miR-15b/16-2 in T-ALL cells impaired leukemic growth in vitro and tumor progression in patient-derived xenotransplantation assays. Mechanistically, miR-15b/16-2 expression represses the genes encoding BCL-2 and CYCLIN D3, leading to T-ALL apoptosis and cell cycle dysregulation, with an accumulation of G0-phase cells and a defective transition to the G2/M phase. Moreover, RNA-seq analysis revealed that miR-15b/16-2 expression in the human T-ALL cell line CUTLL1 represses cell cycle-associated pathways (i.e. G1-to-S transition, G2-to-M transition), upregulates apoptotic and programmed cell death pathways, and modulates genes associated to DNA replication and DNA damage repair. Overall, these findings support a novel function for miR-15b/16-2 as tumor suppressors in T-ALL, highlighting their role as promising targets for T-ALL therapy. Overall design: RNA-seq profiling of human T-ALL cell line CUTLL1 transduced with lentiviral vectors encoding either GFP alone (mock control vector, GFP) or the miR-15b/16-2 genomic cluster together with GFP (MIRS). GFP-positive transduced cells were enriched by flow cytometry sorting 4 days after transduction. The experiment was performed in triplicate.