Functional regulatory layers shaping the expression of flow cytometry markers in Acute Myeloid Leukemia

Abstract

Abstract Introduction: Flow cytometry is a central tool in the diagnosis, classification, and monitoring of acute myeloid leukemia (AML), allowing the identification of cell subpopulations based on the expression of surface markers. In recent years, interest has grown in understanding the genetic mechanisms underlying this heterogeneity, highlighting the role of non-coding RNAs, such as lncRNAs and miRNAs, which act as key regulators of gene expression by influencing the transcription, stability, and translation of mRNAs, including those associated with the coding of surface markers commonly analyzed in flow cytometry. Objectives: This work proposes to integrate functional, positional, and interaction data involving lncRNAs and miRNAs, with the aim of investigating the regulatory mechanisms that shape the immunophenotypic signature observed in AML, contributing to its more refined characterization in the context of precision medicine. Methodology: Surface markers for AML and their associated genes obtained via NCBI were selected, followed by functional enrichment. LncRNA and miRNA data were integrated from public databases (RNAcentral, LncBook, HMDD). Genomic positions were mapped to the GRCh38 genome and GTF annotations. R/RStudio with the GenomicRanges, biomaRt, and TxDb packages was used for genomic overlap analysis (FindOverlaps), highlighting pairs of miRNAs and lncRNAs with potential joint regulation. Results and discussion: Functional analysis of genes associated with surface markers used in flow cytometry in AML revealed enrichment in processes essential for hematopoiesis and immune response, such as cell differentiation, inflammatory signaling, and regulation of leukocyte adhesion, directly reflecting the biology of the disease (Table 1). The integration of public ncRNA data allowed the identification of lncRNAs and miRNAs associated with AML, considering the strength of evidence, methods, and causality, highlighting the growth and deepening of the literature in the field. These ncRNAs were mapped to the human genome GRCh38 for positional analysis, followed by investigation of genomic overlap between miRNAs and lncRNAs, which revealed specific pairs with potential regulatory interactions, particularly MIR196B/HOXA10-AS and MIR612/NEAT1. MIR196B is associated with oncogenic processes, while the antisense lncRNA HOXA10-AS exhibits significant expression in stem cells and has a functional relationship described in leukemias. MIR612 also has implications for oncogenic mechanisms, and the nuclear lncRNA NEAT1 is involved in the organization of nuclear bodies and is described as an immunoregulator, modulating cell lineage differentiation. The genomic overlap of these pairs suggests complex regulatory mechanisms, such as competition for miRNAs (“sponging”) or post-transcriptional modulation, that may influence the expression of genes encoding the markers identified in cytometry. These findings highlight the importance of considering additional layers of regulation in the immunophenotypic characterization of AML, going beyond the classic protein profile to incorporate non-coding regulation, as indicated by recent advances in the literature and guidelines. These results enrich the understanding of AML heterogeneity from the perspective of precision medicine and indicate directions for future research. Conclusion: This study deepens the understanding of biological heterogeneity in AML by evidencing regulatory mechanisms mediated by miRNAs and lncRNAs, which go beyond the classic regulation of DNA and proteins. It highlights the importance of integrating these molecular layers for a more precise and personalized approach to the disease. These findings seek to enrich the knowledge of AML pathogenesis and point to new research strategies that, aligned with advances in precision medicine, have the potential to impact diagnosis and treatment in this context.