MiR-146b restoration in T-large granular lymphocyte leukemia: Molecular insights for a new therapeutic strategy

Abstract

Abstract INTRODUCTION T-Large Granular Lymphocyte Leukemia (T-LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of cytotoxic LGLs. The disease exhibits significant heterogeneity, with clinical manifestations ranging from indolent to life-threatening conditions. T-LGL lymphoproliferation is driven by both somatic gain-of-function mutations, mostly in STAT3 and STAT5B genes, and by a pro-inflammatory microenvironment. STAT3 mutations characterize the CD8+ T-LGLL subtype and are strongly associated with symptomatic disease, especially neutropenia, which negatively impacts patient outcome. Currently, immunosuppressive treatments have limited efficacy with a high rate of relapsed/refractory cases. Therefore, identifying novel molecular targets is paramount to improve therapeutic strategies. We recently demonstrated that miR-146b downregulation induces a high Fas ligand (FasL) production and subsequent neutrophil death, through STAT3-miR-146b-ELAVL1-FasL axis. Building on these data, we aimed to investigate the molecular consequences of miR-146b restoration in patient-derived and normal LGLs, as the first step to proceed towards developing a possible innovative class of RNA-based therapies for STAT3-mutated T-LGLL. METHODS Six CD8+ STAT3-mutated T-LGLL patients were selected alongside 6 healthy donors (HD) matched for sex and age. LGLs were isolated from patients based on LGL-specific marker CD57 expression, while CD8+ cytotoxic T lymphocytes (CTLs) were purified from HD. Primary cells were transfected with either a miR-146b mimic or a negative control (scramble). After 24 hours, cell viability and miR-146b transfection efficiency were assessed, and total RNA was extracted to perform RNA sequencing. Gene expression was quantified using CircComPara. Differentially expressed gene (DEG) analysis was conducted using DESeq2 v1.46.0 (adj-P≤0.05) and gene set enrichment analysis using clusterProfiler v4.14.6 and enrichplot v1.28.0. RESULTS MiR-146b uptake at 24 hours post-transfection compared to scramble control was confirmed (p<0.01), maintaining a cell viability of at least 60%. According to transcriptomic data and descriptive principal component analysis, the changes between scramble and miRNA-transfected conditions are marked in patient-derived cells (2,404 DEGs) whereas are mild in HD samples (154 DEGs). Our data demonstrated that miR-146b restoration was able to successfully normalise the expression of its known targets, most importantly ELAVL1, leading to FasL reduction, thus reverting the pathogenic axis associated with neutropenia in T-LGLL.Interestingly, miR-146b is found to exert the desired effect not only on ELAVL1, but also on another target, IRAK1, a kinase that is crucial for the response against foreign pathogens and has been associated with STAT3 activation. In addition, several genes including epigenetic modifiers (e.g., HDAC1), oncogenic regulators (e.g., JUNB), and inflammatory mediators (e.g., IL32 and IL2RB), were downregulated following miR-146b restoration, reaching expression levels comparable to those in HD. Interestingly, CCL5, a chemokine implicated in LGL proliferation via IL-6 production by monocytes, and SBNO2, required in STAT3-dependent hematopoietic malignancies,were significantly reduced in miR-146b-transfected LGLs (p<0.01). Additional analyses disclosed that several signaling pathways, including TNFα/NFkB, IL6/JAK/STAT3 signaling, inflammatory responses, and viral carcinogenesis, were enriched in genes downregulated upon miR-146b restoration in leukemic LGLs, whereas miR-146b transfection does not elicit significant pathway perturbations in normal CTLs. Finally, we demonstrated that the expression level of nearly half of the genes altered in the disease was completely restored or positively affected by the miR-146b restoration, while only a small proportion of genes not altered in the disease were affected, mostly very mildly, by the treatment. CONCLUSIONS Our results demonstrate that miR-146b restoration selectively affects leukemic cells with negligible effect on healthy CTLs. Moreover, robust data support the ability of miR-146b restoration to correct key targets implicated in T-LGLL-associated neutropenia. The miR-146b restoration also modulates the expression of genes and the activation of pathways aberrantly regulated in leukemic cells from the most symptomatic disease subtype, suggesting a broader anti-leukemic effect.