The miR-200c/141-ZEB2-TGFß axis is aberrant in human T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small- to medium-sized prolymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, and no clear T-cell receptor a or ß CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of microRNA (miRNA) is aberrant and often heterogeneous in T-PLL. We identified 35 miRNA that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR- 200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFßR3 and aberrant TGFß1- induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFß pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNA in T-PLL and pave the way for new therapeutic targets in this disease.

miR-181a is a novel player in the STAT3-mediated survival network of TCRαß+ CD8+ T large granular lymphocyte leukemia.

T-LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of constitutive activation of the STAT3 and ERK pathway. Notably, in 40% of patients, constitutive STAT3 activation is due to STAT3 activating mutations, whereas in 60% this is unknown. As miRNAs are amongst the most potent regulators in health and disease, we hypothesized that aberrant miRNA expression could contribute to dysregulation of these pathways. miRNA sequencing in T-LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miR-181a. Furthermore, geneset enrichment analysis (GSEA) of downregulated targets of miR-181a implicated involvement in regulating STAT3 and ERK1/2 pathways. Flow cytometric analyses showed increased SOCS3+ and DUSP6+ T-LGL cells upon miR-181a inhibition. In addition, miR-181a-transfected human CD8+ T cells showed increased basal STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line, we could show that miR-181a is an actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition and ERK1/2 phosphorylation by DUSP6 inhibition and verified this mechanism in an independent cell line. In addition, miR-181a inhibition resulted in a higher sensitivity to FAS-mediated apoptosis. Collectively, our data show that miR-181a could be the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

Dysregulated signaling, proliferation and apoptosis impact on the pathogenesis of TCRγδ+ T cell large granular lymphocyte leukemia.

TCRγδ+ T-LGL leukemia is a rare form of chronic mature T cell disorders in elderly, which is generally characterized by a persistently enlarged CD3+CD57+TCRγδ+ large granular lymphocyte population in the peripheral blood with a monoclonal phenotype. Clinically, the disease is heterogeneous, most patients being largely asymptomatic, although neutropenia, fatigue and B symptoms and underlying diseases such as autoimmune diseases or malignancies are also often observed. The etiology of TCRγδ+ T-LGL proliferations is largely unknown. Here, we aimed to investigate underlying molecular mechanisms of these rare proliferations by performing gene expression profiling of TCRγδ+ T-LGL versus normal TCRγδ+ T cell subsets. From our initial microarray dataset we observed that TCRγδ+ T-LGL leukemia forms a separate group when compared with different healthy control TCRγδ+ T cell subsets, correlating best with the healthy TemRA subset. The lowest correlation was seen with the naive subset. Based on specific comparison between healthy control cells and TCRγδ+ T-LGL leukemia cells we observed up-regulation of survival, proliferation and hematopoietic system related genes, with a remarkable down-regulation of apoptotic pathway genes. RQ-PCR validation of important genes representative for the dataset, including apoptosis (XIAP, CASP1, BCLAF1 and CFLAR), proliferation/development (ID3) and inflammation (CD28, CCR7, CX3CR1 and IFNG) processes largely confirmed the dysregulation in proliferation and apoptosis. Based on these expression data we conclude that TCRγδ+ T-LGL leukemia is likely the result of an underlying aberrant molecular mechanisms leading to increased proliferation and reduced apoptosis.