Histone deacetylases mediate the silencing of miR-15a, miR-16, and miR-29b in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) demonstrates a global down-regulation of miR-15a and miR-16 and a selective silencing of the related miR-29b in aggressive disease. Deletions in chromosome 13 [del(13q14)] partially account for the loss of expression of miR-15a and miR-16, but the mechanisms by which miR-29b becomes silenced is unknown. In the present study, we show that the histone deacetylases (HDACs) are overexpressed in CLL and mediate the epigenetic silencing of miR-15a, miR-16, and miR-29b. HDAC inhibition triggered the accumulation of the transcriptionally activating chromatin modification H3K4me2 and restored the expression of miR-15a, miR-16, and miR-29b in approximately 35% of samples. Ectopic expression of miR-15a and miR-16 and HDAC inhibition-induced expression of miR-15a, miR-16, or miR-29b in primary CLL cells was associated with declines in the levels of Mcl-1, but not Bcl-2, mitochondrial dysfunction, and induction of cell death. Therefore, our results show that HDACs aberrantly silence the expression of the critical tumor suppressors miR-15a, miR-16, and miR-29b in CLL. Deacetylase inhibition may be a therapeutic strategy that restores the expression of these miRs to antagonize Mcl-1, an important survival protein in these cells. Consequently, CLL patients who exhibit such epigenetic silencing may benefit from HDAC inhibitor-based therapy.

HDAC Inhibition Induces MicroRNA-182, which Targets RAD51 and Impairs HR Repair to Sensitize Cells to Sapacitabine in Acute Myelogenous Leukemia.

PURPOSE: The double-strand breaks elicited by sapacitabine, a clinically active nucleoside analogue prodrug, are repaired by RAD51 and the homologous recombination repair (HR) pathway, which could potentially limit its toxicity. We investigated the mechanism by which histone deacetylase (HDAC) inhibitors targeted RAD51 and HR to sensitize acute myelogenous leukemia (AML) cells to sapacitabine. EXPERIMENTAL DESIGN: Chromatin immunoprecipitation identified the role of HDACs in silencing miR-182 in AML. Immunoblotting, gene expression, overexpression, or inhibition of miR-182 and luciferase assays established that miR-182 directly targeted RAD51. HR reporter assays, apoptotic assays, and colony-forming assays established that the miR-182, as well as the HDAC inhibition-mediated decreases in RAD51 inhibited HR repair and sensitized cells to sapacitabine. RESULTS: The gene repressors, HDAC1 and HDAC2, became recruited to the promoter of miR-182 to silence its expression in AML. HDAC inhibition induced miR-182 in AML cell lines and primary AML blasts. miR-182 targeted RAD51 protein both in luciferase assays and in AML cells. Overexpression of miR-182, as well as HDAC inhibition-mediated induction of miR-182 were linked to time- and dose-dependent decreases in the levels of RAD51, an inhibition of HR, increased levels of residual damage, and decreased survival after exposure to double-strand damage-inducing agents. CONCLUSIONS: Our findings define the mechanism by which HDAC inhibition induces miR-182 to target RAD51 and highlights a novel pharmacologic strategy that compromises the ability of AML cells to conduct HR, thereby sensitizing AML cells to DNA-damaging agents that activate HR as a repair and potential resistance mechanism. Clin Cancer Res; 22(14); 3537-49. ©2016 AACR.

Expression and prognostic assessment of dipeptidyl peptidase IV and related enzymes in B-cell chronic lymphocytic leukemia.

Recent observations of the deregulated expression of several dipeptidyl peptidase (DP) IV-like enzymes in human cancers have led to presumptions of their pathogenic role in cancer. To further explore this concept we have characterized the expression of all DPIV-like enzymes in chronic lymphocytic leukemia (CLL). We have demonstrated the constitutive expression of DPIV, DP8, DP9, DPII and PEP mRNA and DPIV, DP8 and DP9 protein in CLL. FAP mRNA was not detected in CLL or normal B-lymphocytes. This correlated with an absence of FAP protein on the cell surface. This study also shows that DP8 mRNA expression is significantly upregulated in CLL compared to normal tonsil B-lymphocytes (p < 0.05) which may suggest biological importance in this disease. DP expression could not be correlated with any molecular or clinical prognostic markers for CLL in this cohort including IgVH mutational status, CD38, ZAP-70 or CD49d expression (n = 58). However, the constitutive expression of the DPIV-like enzymes in CLL and their emergence as potent immune regulators makes them candidate therapeutic targets in this disease.

Dipeptidyl peptidase expression during experimental colitis in mice.

BACKGROUND: We have previously demonstrated that inhibition of dipeptidyl peptidase (DP) activity partially attenuates dextran sulfate sodium (DSS) colitis in mice. The aim of this study was to further investigate the mechanisms of this protection. MATERIALS AND METHODS: Wildtype (WT) and DPIV(-/-) mice consumed 2% DSS in drinking water for 6 days to induce colitis. Mice were treated with saline or the DP inhibitors Ile-Pyrr-(2-CN)*TFA or Ile-Thia. DP mRNA and enzyme levels were measured in the colon. Glucagon-like peptide (GLP)-2 and GLP-1 concentrations were determined by radioimmunoassay, regulatory T-cells (Tregs) by fluorescence activated cell sorting (FACS) on FOXp3+T cells in blood, and neutrophil infiltration assessed by myeloperoxidase (MPO) assay. RESULTS: DP8 and DP2 mRNA levels were increased (P < 0.05) in WT+saline mice compared to untreated WT mice with colitis. Cytoplasmic DP enzyme activity was increased (P < 0.05) in DPIV(-/-) mice at day 6 of DSS, while DP2 activity was increased (P < 0.05) in WT mice with colitis. GLP-1 (63%) and GLP-2 (50%) concentrations increased in WT+Ile-Pyrr-(2-CN)*TFA mice compared to day-0 controls. MPO activity was lower in WT+Ile-Thia and WT+Ile-Pyrr-(2-CN)*TFA treated mice compared to WT+saline (P < 0.001) at day 6 colitis. CONCLUSIONS: DP expression and activity are differentially regulated during DSS colitis, suggesting a pathophysiological role for these enzymes in human inflammatory bowel disease (IBD). DP inhibitors impaired neutrophil recruitment and maintenance of the Treg population during DSS-colitis, providing further preclinical evidence for the potential therapeutic use of these inhibitors in IBD. Finally, DPIV appears to play a critical role in mediating the protective effect of DP inhibitors.

Dipeptidyl peptidase 8 and 9--guilty by association?

Dipeptidyl peptidases (DP) 8 and 9 are members of the DPIV enzyme family. Other members include DPIV, fibroblast activation protein (FAP) and the non-enzymes DP6 and DP10. DPIV family members have diverse biological roles, and have been implicated in a range of diseases including diabetes, cancer, inflammatory bowel disease, multiple sclerosis (MS), arthritis and asthma. While DP8/9 biological functions are yet to be established, they have been predicted to have similar roles to the other DPs due to high sequence similarities within the active site of the enzymes. While there is mounting evidence towards the involvement of DP8 and/or DP9 in innate and acquired immunity, direct proof for the link between DP8 and DP9 and human disease is yet to be definitively shown, thus DP8 and 9 proteins remain guilty by association.