Page 198 - Haematologica May 2020

B. Mariotti et al.
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Figure 6. HuR is the endogenous miR-146b target gene in CD8 T large granular lymphocytes. (A) CD8 T large granular lymphocytes (T-LGL) were transfected with 200 pmol miR-scr or miR-146b. 24 hours (h) after transfection cells were processed and miR-146b, HuR mRNA, FasL mRNA and primary transcript (PT) expression were analyzed by real-time quatitative PCR (RT-qPCR). Data are expressed as MNE relative to U6 (miR-146b) and RPL32 (HuR, FasL mRNA and PT). Mean±standard error of the mean (SEM) of two independent experiments is shown. (B) HuR protein level was analyzed by Western blot as described in the Methods in CD8 (n=5) and CD4 (n=5) whole-cell extracts (15 mg). Normalized HuR protein levels are reported as arbitrary units (au) below the Western blot. *P<0.01 by unpaired t-test. Patients analyzed in each panel are specified in the Online Supplementary Table S5.
role for STAT3 mutations in this disease.6,7 However these mutations, mostly of the activating type, involve a vari- able percentage of pathological clones and in many cases are present in a very low percentage of LGL.32 Our results point to the role of STAT3 activation as the dominant fac- tor in the pathogenesis of the disease and in the induction of a specific miRNA profile. These findings are consistent with recent data from our lab indicating a correlation between STAT3 activation, phenotypic pattern of prolifer- ating LGLs and the presence of symptomatic disease, mostly characterized by neutropenia.5
The mechanism sustaining neutropenia in LGLL patients still remains poorly clarified. Since infiltration of pathological LGLs usually play only a marginal role in the pathogenesis of neutropenia, soluble factors have been reported to be the more relevant players in this feature. Among them, FasL has been detected at very high concen- trations in LGLL patients.9,12,28 In particular, a significant increase in FasL mRNA and protein expression was reported in patients with CD8+CD16+CD56– phenotype.5,9- 12 Consistently, we also found that the CD8 T-LGL popu- lation under investigation is characterized by higher levels of FasL expression. Comparative analysis of the differen- tially expressed miRNAs within different T-LGL subsets allowed us to identify miR-146b as a unique miRNA. In fact, miR-146b expression is decreased in CD8 T-LGLL, that is distinguished from the CD4 T-LGLL phenotype by high levels of constitutive STAT3 activation. Moreover, miR-146b expression inversely correlates with the levels of STAT3 tyrosine phosphorylation, with neutropenia and concurrently with FasL expression, suggesting the exis- tence of a STAT3-miR146b-FasL axis in T-LGL leukemia. Inhibition of constitutively activated STAT3 by STATTIC in CD8 T-LGLL patients increased miR-146b expression,
thus providing experimental evidence for a mechanistic link between constitutively activated STAT3 and inhibi- tion of miR-146b expression in CD8 T-LGLs. This finding is in line with data showing that induction of miR-146b expression by STAT3 occurs under normal physiological conditions only in non-transformed cells, but is lost in malignancy, mostly has a consequence miR-146b promot- er methylation, that prevents miR-146b expression even in the presence of constitutively activated STAT3.26,27 Here we provide the first evidence of miR-146b promoter methylation at the expected sites, thus pointing that a similar mechanism might also take place in CD8 T-LGLL. Inhibition of this process using DAC restored miR146b levels. Moreover, a direct role of activated STAT3 in induc- ing miR-146b promoter methylation, through regulating expression of DNA methyltransferase 1 (DNMT1) has been demonstrated in solid tumors27 and in malignant T lymphocytes.33 Similarly, we show that inhibition of constitutively active STAT3-YP reduces the expression of DNMT1, thereby providing a functional and mechanistic link between activation of STAT3 signaling pathway and its epigenetic control. Interestingly, a role for epigenetic mechanisms taking place in chronic LGL proliferations has been already reported by Caligiuri et al. in T-LGLL34 and by our group in CLPD-NK,35 and our data contribute to unravel the machineries differently activated in each sub- sets of patients (i.e. CD8+ vs. CD4+ LGLL) with relevant clinical impact.
Restoration of miR-146b expression in Jurkat cells and, most importantly, in patients CD8 T-LGLL, resulted in a significant reduction of the FasL mRNA expression level, which occurs in the absence of modification of the FasL primary transcript expression. Collectively, these data indicate that miR-146b affects FasL expression at a post-
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