Whereas transcriptional silencing of genes due to epigenetic mechanisms is one of the most important alterations in acute lymphoblastic leukemia (ALL), some recent studies indicate that DNA methylation contributes to down-regulation of miRNAs during tumorigenesis. To explore the epigenetic alterations of miRNAs in ALL, we analyzed the methylation and chromatin status of the miR-124a loci in ALL. Expression of miR-124a was down-regulated in ALL by hypermethylation of the promoter and histone modifications including decreased levels of 3mk4H3 and AcH3 and increased levels of 2mK9H3, 3mK9H3, and 3mK27H3. Epigenetic down-regulation of miR-124a induced an up-regulation of its target, CDK6, and phosphorylation of retinoblastoma (Rb) and contributed to the abnormal proliferation of ALL cells both in vitro and in vivo. Cyclin-dependent kinase 6 (CDK6) inhibition by sodium butyrate or PD-0332991 decreased ALL cell growth in vitro, whereas overexpression of pre-miR124a led to decreased tumorigenicity in a xenogeneic in vivo Rag2 were analyzed in a group of 353 patients diagnosed with ALL. Methylation of hsa-miR-124a was observed in 59% of the patients, which correlated with down-regulation of miR-124a (P < 0.001). Furthermore, hypermethylation of hsa-miR-124a was associated with higher relapse rate (P = 0.001) and mortality rate (P < 0.001), being an independent prognostic factor for disease-free survival (P < 0.001) and overall survival (P = 0.005) in the multivariate analysis. These results provide the grounds for new therapeutic strategies in ALL either targeting the epigenetic regulation of microRNAs and/or directly targeting the CDK6-Rb pathway.

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.

MicroRNAs (miRNA) are small noncoding, single-stranded RNAs that inhibit gene expression at a posttranscriptional level, whose abnormal expression has been described in different tumors. The aim of our study was to identify miRNAs potentially implicated in chronic myeloid leukemia (CML). We detected an abnormal miRNA expression profile in mononuclear and CD34 + cells from patients with CML compared with healthy controls. Of 157 miRNAs tested, hsa-miR-10a, hsa-miR-150, and hsa-miR-151 were down-regulated, whereas hsa-miR-96 was up-regulated in CML cells. Down-regulation of hsa-miR-10a was not dependent on BCR-ABL1 activity and contributed to the increased cell growth of CML cells. We identified the upstream stimulatory factor 2 (USF2) as a potential target of hsa-miR-10a and showed that overexpression of USF2 also increases cell growth. The clinical relevance of these findings was shown in a group of 85 newly diagnosed patients with CML in which expression of hsa-miR-10a was down-regulated in 71% of the patients, whereas expression of USF2 was up-regulated in 60% of the CML patients, with overexpression of USF2 being significantly associated with decreased expression of hsa-miR-10a (P = 0.004). Our results indicate that down-regulation of hsa-miR-10a may increase USF2 and contribute to the increase in cell proliferation of CML implicating a miRNA in the abnormal behavior of CML.

Activation of the Wnt/␤-catenin signaling pathway is a hallmark of a number of solid tumors. We analyzed the regulation of the Wnt/␤-catenin pathway in acute lymphoblastic leukemia (ALL) and its role in the pathogenesis of the disease. We found that expression of the Wnt inhibitors sFRP1, sFRP2, sFRP4, sFRP5, WIF1, Dkk3, and Hdpr1 was down-regulated due to abnormal promoter methylation in ALL IntroductionThe evolutionarily conserved Wnt-signaling pathway has pivotal roles during the development of many organ systems. Central to this pathway is a multiprotein scaffold consisting of adenomatous polyposis coli (APC), glycogen synthase kinase (GSK)-3␤, axin, and the transcriptional cofactor ␤-catenin. In the absence of Wnt ligands, ␤-catenin levels are kept low through constitutive phosphorylation by GSK-3␤, which leads to the ubiquitination and degradation of ␤-catenin. 1-3 Binding of the Wnt ligands to their receptors, Frizzled (Fz), leads to activation of the adaptor protein, Disheveled (Dvl), and the inhibition of GSK-3␤ activity, reducing phosphorylation and subsequent degradation of ␤-catenin. ␤-Catenin is stabilized and translocates to the nucleus where it binds members of the T-cell factor (Tcf)/lymphoid-enhancing factor (Lef) family of transcription factors and induces the expression of target gene such as CMYC and Cyclin D1. 4 This signaling pathway is controlled by a number of natural Wnt antagonists that interfere with ligand-receptor interactions or Dvl protein, including members of the Dickkopf (DKK) family, the secreted Frizzled-related protein (sFRP) family, the Wnt inhibitory factor-1 (WIF1), and the human Dapper protein-1 (HDPR1).Recent studies have demonstrated a role for Wnt signal transduction at several stages of lymphocyte development and in the self-renewal of hematopoietic stem cells. 5,6 All of them indicate a crucial role for the Wnt-signaling pathway in the proliferation of thymocytes and also of pro-B cells, suggesting that Wnt proteins can function as growth factors for progenitor cells of both the Band T-cell lineages. 4,7,8 Abnormal Wnt signaling has become a hallmark of some types of solid tumors, most notably colon and hepatocellular carcinomas, 9,10 and has been commonly associated with mutations in the amino-terminal region of ␤-catenin that make the molecule resistant to processing through the degradation pathway or in other proteins in this pathway, like the APC gene. 11 In addition, the functional loss of Wnt antagonists can contribute to activation of the Wnt pathway and result in carcinogenesis through deregulation of cell proliferation and differentiation. Recent studies have shown that impaired regulation of Wnt-antagonists such as sFRP, WIF1, HDPR1, and DKK3 by promoter hypermethylation is present in several human malignancies. [12][13][14][15][16][17] Given that Wnt signals are important for the survival and expansion of lymphocyte progenitors, it has been suggested that dysregulated Wnt signaling could be a mechanism underlying lymphoid leukemogenesis. Recent studies in...

While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cellspecific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.