Both gains and losses of DNA methylation are common in cancer, but the factors controlling this balance of methylation remain unclear. Triple-negative breast cancer (TNBC), a subtype that does not overexpress hormone receptors or HER2/NEU, is one of the most hypomethylated cancers observed. Here, we discovered that the TET1 DNA demethylase is specifically overexpressed in about 40% of patients with TNBC, where it is associated with hypomethylation of up to 10% of queried CpG sites and a worse overall survival. Through bioinformatic analyses in both breast and ovarian cancer cell line panels, we uncovered an intricate network connecting TET1 to hypomethylation and activation of cancer-specific oncogenic pathways, including PI3K, EGFR, and PDGF. TET1 expression correlated with sensitivity to drugs targeting the PI3K-mTOR pathway, and CRISPR-mediated deletion of TET1 in two independent TNBC cell lines resulted in reduced expression of PI3K pathway genes, upregulation of immune response genes, and substantially reduced cellular proliferation, suggesting dependence of oncogenic pathways on TET1 overexpression. Our work establishes TET1 as a potential oncogene that contributes to aberrant hypomethylation in cancer and suggests that TET1 could serve as a druggable target for therapeutic intervention. This study addresses a critical gap in knowledge of how and why methylation is prognostic in breast cancer and shows how this information can be used to stratify patients with TNBC for targeted therapy. .

The c-abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes, and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as in the regulation of the actin cytoskeleton. Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia. Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in chronic myelogenous leukemia and revolutionized the field of targeted cancer therapy. In this minireview, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity. Structural Overview of the c-Abl Kinase CoreThe kinase core of the c-Abl protein has a domain organization similar to that of the Src family kinases, with sequential Src homology (SH) 3 3 and SH2 domains, an SH2/kinase linker, and a bilobed kinase domain (Fig. 1). This core is flanked by an N-terminal "cap" (N-cap) region with a signal sequence for myristoylation, which serves dual roles in regulation of kinase activity and in membrane localization. C-terminal to the kinase domain is a long region of Ͼ600 amino acids encoded by a single exon, which controls interaction of Abl with other SH3-containing proteins and the actin cytoskeleton. This region also regulates nuclear-cytoplasmic shuttling of the kinase (1-4). These key structural and regulatory features are discussed in detail below. The Myristoylated N-cap Is Critical for Down-regulation of AblThe N-cap is ϳ80 amino acids in length and is myristoylated in the 1b splice variant of Abl (5). The first crystal structure of the Abl core (residues 1-531) revealed that this N-terminal myristic acid group binds a deep hydrophobic pocket in the C-terminal lobe (C-lobe) of the kinase domain ( Fig. 1) (6). Binding of the myristoyl group into this pocket induces a bend in C-lobe helix ␣I, allowing the SH2 domain to dock onto the C-lobe of the kinase domain (Fig. 2). Interaction of the myristoylated N-cap with the C-lobe is critical to maintenance of the autoinhibited state, as mutation of the myristoylation signal sequence results in a highly active kinase (7). Interestingly, small molecules that bind to this site also modulate kinase activity, supporting an allosteric connection between this regulatory pocket and the kinase active site (8 -11).In addition to binding the C-lobe of the kinase domain, the N-cap also influences kinase regulation via the SH3 and SH2 domains. Although the N-cap region was disordered in the first crystal structure of the c-Abl core, a more recent structure with a modified N-cap revealed that Ser 69 (numbered according to Protein Data Bank (PDB) code 2FO0) 4 is phosphorylated and contacts the short connector joining the SH3 and SH2 domains. Mutation of Ser 69 increased Abl activity, identifying this site as a potential input for regulatory kinases (12). Additional contacts were observed between N...

BackgroundEpithelial-mesenchymal transition (EMT) is known to impart metastasis and stemness characteristics in breast cancer. To characterize the epigenetic reprogramming following Twist1-induced EMT, we characterized the epigenetic and transcriptome landscapes using whole-genome transcriptome analysis by RNA-seq, DNA methylation by digital restriction enzyme analysis of methylation (DREAM) and histone modifications by CHIP-seq of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells relative to cells induced to undergo EMT by Twist1.ResultsEMT is accompanied by focal hypermethylation and widespread global DNA hypomethylation, predominantly within transcriptionally repressed gene bodies. At the chromatin level, the number of gene promoters marked by H3K4me3 increases by more than one fifth; H3K27me3 undergoes dynamic genomic redistribution characterized by loss at half of gene promoters and overall reduction of peak size by almost half. This is paralleled by increased phosphorylation of EZH2 at serine 21. Among genes with highly altered mRNA expression, 23.1% switch between H3K4me3 and H3K27me3 marks, and those point to the master EMT targets and regulators CDH1, PDGFRα and ESRP1. Strikingly, Twist1 increases the number of bivalent genes by more than two fold. Inhibition of the H3K27 methyltransferases EZH2 and EZH1, which form part of the Polycomb repressive complex 2 (PRC2), blocks EMT and stemness properties.ConclusionsOur findings demonstrate that the EMT program requires epigenetic remodeling by the Polycomb and Trithorax complexes leading to increased cellular plasticity. This suggests that inhibiting epigenetic remodeling and thus decrease plasticity will prevent EMT, and the associated breast cancer metastasis.

Background: Abl kinases are regulated by noncatalytic domains that allosterically impact kinase domain structure and inhibitor sensitivity. Results: Enhanced SH3/linker interaction suppresses c-Abl core protein dynamics and sensitizes Bcr-Abl to kinase domain inhibitors. Conclusion: SH3/linker interaction influences kinase dynamics in the context of Bcr-Abl. Significance: Stabilizers of SH3/linker interaction may sensitize Bcr-Abl to kinase domain inhibitors, providing a new route to allosteric kinase control.

A central challenge in the development of epigenetic cancer therapy is the ability to direct selectivity in modulating gene expression for disease-selective efficacy. To address this issue, we characterized by RNA-seq, DNA methylation and ChIP-seq analyses the epigenetic response of a set of colon, breast and leukemia cancer cell lines to small molecule inhibitors against DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343). We also characterized the effects of DAC as combined with the other compounds. Averaged over the cancer cell models employed, we found that DAC affected 8.6% of the transcriptome and that 95.4% of the genes affected were upregulated. DAC preferentially regulated genes that were silenced in cancer and that were methylated at their promoters. In contrast, Depsi affected the expression of 30.4% of the transcriptome but showed little selectivity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 affected only 2% of the transcriptome, with UNC0638 and GSK343 preferentially targeting genes marked with H3K9me2 or H3K27me3, respectively. When combined with histone methylase inhibitors, the extent of gene upregulation by DAC was extended while still maintaining selectivity for DNA methylated genes and silenced genes. However, the genes upregulated by combination treatment exhibited limited overlap, indicating the possibility of targeting distinct sets of genes based on different epigenetic therapy combinations. Overall, our results demonstrated that DNA methyltransferase inhibitors preferentially target cancer-relevant genes and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining selectivity.