Identification of a miR-146b-FasL axis in the development of neutropenia in T-LGL leukemia

T Large Granular Lymphocytes Leukemia (T-LGLL) is a rare disease characterized by LGLs proliferation. The main clinical manifestation of T-LGLL is a severe neutropenia that is also the most common indication for treatment in this disease. From the molecular point of view, the high levels of soluble Fas ligand (FasL) found in these patients are the main cause of neutropenia. We recently described that the increased FasL expression was induced by the high STAT3 activation that specifically characterizes T-LGLs of neutropenic patients. Although STAT3 is a transcriptional activator of a huge number of oncogenes, so far its relationship with microRNAs has not been evaluated in T-LGLL. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. Hierarchical Clustering Analysis of miRNA expression profile of T-LGLs identified two groups of samples: in the first cluster, patients are characterised by CD8+ T-LGLs, neutropenia and high levels of activated STAT3, while CD4+ TLGLs, normal neutrophil counts and low levels of STAT3 activation distinguish remaining patients. Remarkably, we demonstrate a CD8-specific and STAT3 activation-dependent inhibition of miR-146b expression, this latter being related to miR-146b promoter methylation. We also provide experimental evidence that the STAT3-dependent reduction of miR-146b expression allows increased HuR-mediated stabilization of FasL mRNA, leading to increased FasL production, which in turn has been shown to play a role in neutropenia. Restoring miR-146b expression in CD8 T-LGLs leads to a reduction of HuR protein and, in turn, of FasL mRNA expression. Finally, we demonstrated that treatment with Bortezomib, a proteasome inhibitor, lead to a 70% reduction of STAT3 activation and 67% reduction of DNMT1 expression in T-LGL. Finally, in line with our previous results, reduction of HuR protein expression (-44%) and FasL mRNA (-43%) was observed in Bortezomib-treated T-LGL, consistent with the restoration of miR-146b expression. All together these data provide mechanistic insights for the link between STAT3 activation, miR-146b expression and neutropenia in CD8 T-LGLs and indicate a possible use of Bortezomib in the treatment of neutropenia in T-LGLL.