Molecular Insights into Long Non-Coding RNAs in T-Cell Large Granular Lymphocyte Leukemia (T-LGLL)

T-cell Large Granular Lymphocyte Leukemia (T-LGLL) is the most frequent variant of LGLL, a rare chronic lymphoproliferative disorder characterized by the clonal expansion of cytotoxic T-cells, often driven by persistent antigenic stimulation and dysregulated apoptotic pathways. The disease exhibits significant clinical and biological heterogeneity, with two main immunophenotypic subsets: CD8+ T-LGLL and CD4+ T-LGLL. The JAK/STAT signaling pathways plays a pivotal role in disease pathogenesis, constitutive STAT3 phosphorylation contributing to disease progression, in particular in the cases characterized by the gain-of-function mutation on STAT3 gene. However, current therapies based on low-dose immunosuppressive and chemotherapeutic agents often yield unsatisfactory responses, with many patients exhibiting treatment resistance or relapse. Therefore, the identification of alternative therapeutic strategies and new therapeutic targets is crucial. While the genetic and immunological landscape of T-LGLL is well studied, the role of non-coding RNAs, particularly long non-coding RNAs (lncRNAs), remain unexplored. This study aims to investigate the role of lncRNAs in T-LGLL pathogenesis, analyzing their expression profiles and potential involvement in leukemic cell survival and immune dysregulation. Through RNA-sequencing analysis on 20 T-LGLL patients and 5 healthy donors (HD) we revealed 1,182 differentially expressed lncRNAs, identifying deregulated lncRNAs expression patterns in T-LGLL patients compared to healthy donors. Interestingly, symptomatic patients, corresponding to CD8+ T-LGLL STAT3 mutated cases, showed a lncRNA transcriptional profile distinguishable from all the other disease subgroups characterized by asymptomatic disease course, i.e. CD8+ STAT3 wild-type, CD4+ STAT5B mutated and CD4+ STAT5B wild-type T-LGLL, from now on defined as other groups, OTH. RT-qPCR validation of selected lncRNAs were performed in an independent cohort (20 T-LGLL and 5 HD). The collected data confirmed LINC00461, PVT1 and LINC02422 over-expression in CD8+ T-LGLL STAT3 mutated patients compared to OTH cases and HD; and HOTAIRM1 over-expression and FIRRE down-expression in T-LGLL patients compared to HD (p<0.05). Notably, an oncogenic role of these lncRNAs has already been reported in hematological neoplasms. Further, functional analysis revealed that the inhibition of STAT3 causes a reduction of PVT1 expression. Considering that PVT1 is over-expressed in CD8+ T-LGLL STAT3 mutated patients, these findings link its deregulation to STAT3 activation. Finally, we conducted a screening on cell lines to identify those suitable to perform functional studies of lncRNAs, identifying different hematological cell lines showing high expression of the lncRNAs selected in this study. Ongoing research aims to evaluate STAT3-dependent expression of all the other lncRNAs considered in this work and their role in disease pathogenesis, through analyses on both primary and line cells. In conclusion, this study provides the first report of lncRNAs dysregulation in T-LGLL, particularly in CD8+ STAT3 mutated patients, underscoring their involvement as novel players in the disease pathogenesis and clinical manifestations, particularly in treatment-requiring cases.