Chapter 080. Cancer Cell Biology and Angiogenesis (Part 9) Acetylation of the amino terminus of the core histones H3 and H4 induces an open chromatin conformation that promotes transcription initiation. Histone acetylases are components of coactivator complexes recruited to promoter/enhancer regions by sequence-specific transcription factors during the activation of genes (Fig. 80-4). Histone deacetylases (HDACs; at least 17 are encoded in the human genome) are recruited to genes by transcriptional repressors and prevent the initiation of gene transcription. Methylated cytosine residues in promoter regions become associated with methyl-cytosine–binding proteins that recruit protein complexes with HDAC activity. The balance between permissive and inhibitory chromatin structure is therefore largely determined by the activity of transcription factors in modulating the "histone code" and the methylation status of the genetic regulatory elements of genes. The pattern of gene transcription is aberrant in all human cancers, and in many cases, epigenetic events are responsible. Unlike genetic events that alter DNA primary structure (e.g., deletions), epigenetic changes are potentially reversible and appear amenable to therapeutic intervention. In many human cancers, including pancreatic cancer and multiple myeloma, the p16 Ink4a promoter is inactivated by methylation, thus permitting the unchecked activity of CDK4/cyclin D and rendering pRB nonfunctional. In sporadic forms of renal, breast, and colon cancer, the von Hippel–Lindau (VHL), breast cancer 1 (BRCA1), and serine/threonine kinase 11 (STK11) genes, respectively, are epigenetically silenced. Other targeted genes include the p15 Ink4b CDK inhibitor, glutathione-S- transferase (which detoxifies reactive oxygen species), and the E-cadherin molecule (important for junction formation between epithelial cells). Epigenetic silencing can occur in premalignant lesions and can affect genes involved in DNA repair, thus predisposing to further genetic damage. Examples include MLH1 (mut L homologue) in hereditary nonpolyposis colon cancer (HNPCC, also called Lynch syndrome), which is critical for repair of mismatched bases that occur during DNA synthesis, and 0 6 -methylguanine-DNA methyltransferase, which removes alkylated guanine adducts from DNA and is often silenced in colon, lung, and lymphoid tumors. Many human leukemias have chromosomal translocations that code for novel fusion proteins with enzymatic activities that alter chromatin structure. The PML-RAR fusion protein, generated by the t(15;17) observed in most cases of acute promyelocytic leukemia (APL), binds to promoters containing retinoic acid response elements and recruits HDAC to these promoters, effectively inhibiting gene expression. This arrests differentiation at the promyelocyte stage and promotes tumor cell proliferation and survival. Treatment with pharmacologic doses of all-trans retinoic acid (ATRA), the ligand for RARα, results in the release of HDAC activity and the recruitment of coactivators, which overcomes the differentiation block. This induced differentiation of APL cells has greatly improved treatment of these patients and has provided a treatment paradigm for the reversal of epigenetic changes in cancer. However, for other leukemia- associated fusion proteins, such as AML-ETO and the MLL fusion proteins seen in AML and ALL, no ligand is known. Therefore, efforts are ongoing to determine the structural basis for interactions between translocation fusion proteins and chromatin remodeling proteins, and to use this information to rationally design small molecules that will disrupt specific protein-protein associations. Drugs that block the enzymatic activity of HDAC are being developed. A number of different chemical classes of HDAC inhibitors have demonstrated antitumor activity in clinical studies against cutaneous T cell lymphoma (e.g., vorinostat) and some solid tumors. HDAC inhibitors may target cancer cells via a number of mechanisms including upregulation of death receptors (DR4/5, FAS, and their ligands) and p21 Cip1/Waf1 , as well as inhibition of cell cycle checkpoints. Major therapeutic efforts are also under way to reverse the hypermethylation of CpG islands that characterizes many solid tumors. Drugs that induce DNA demethylation, such as 5-aza-2'-deoxycytidine, can lead to reexpression of silenced genes in cancer cells with restoration of function. However, 5-aza-2'-deoxycytidine has limited aqueous solubility and is myelosuppressive. Other inhibitors of DNA methyltransferases are in development. In ongoing clinical trials, inhibitors of DNA methylation are being combined with HDAC inhibitors. The hope is that by reversing coexisting epigenetic changes, the deregulated patterns of gene transcription in cancer cells will be at least partially reversed. Aberrant signal transduction pathways activate a number of transcription factors that promote tumor cell proliferation and survival. These include signal transducer and activator of transcription (STAT)-3 and STAT5, NFκB, β-catenin (a component of the APC tumor-suppressor pathway), the heterodimer of c-Jun and Fos known as AP1, and c-Myc. The ability to target these transcription factors therapeutically does not currently exist. However, structural and molecular approaches may make it possible to identify small molecules that would inhibit protein-protein interactions needed for transcription factor dimerization or interaction with coactivator proteins. A small-molecule inhibitor has been developed that blocks the association of Myc with its partner Max, thereby inhibiting Myc-induced transformation. Many transcription factors are activated by phosphorylation, which can be prevented by tyrosine- or serine/threonine kinase inhibitors. The transcription factor NFκB is a heterodimer composed of p65 and p50 subunits that associate with an inhibitor, IκB, in the cell cytoplasm. In response to growth factor or cytokine signaling, a multi-subunit kinase called IKK (IκB-kinase) phosphorylates IκB and directs its degradation by the ubiquitin/proteasome system. NFκB, free of its inhibitor, translocates to the nucleus and activates target genes, many of which promote the survival of tumor cells. Novel drugs called proteasome inhibitors block the proteolysis of IκB, thereby preventing NFκB activation. For unexplained reasons, this is selectively toxic to tumor cells. Further studies have indicated that the antitumor effects of proteasome inhibitors are more complicated and involve the inhibition of the degradation of multiple cellular proteins. Proteasome inhibitors [bortezomib (Velcade)] have shown very significant activity in patients with multiple myeloma, including partial and complete remissions. Inhibitors of IKK are also in development, with the hope of more selectively blocking the degradation of IκB, thus "locking" NFκB in an inhibitory complex and rendering the cancer cell more susceptible to apoptosis-inducing agents. . Chapter 080. Cancer Cell Biology and Angiogenesis (Part 9) Acetylation of the amino terminus of the core histones H3 and H4 induces an open chromatin conformation. cutaneous T cell lymphoma (e.g., vorinostat) and some solid tumors. HDAC inhibitors may target cancer cells via a number of mechanisms including upregulation of death receptors (DR4/5, FAS, and their. rendering pRB nonfunctional. In sporadic forms of renal, breast, and colon cancer, the von Hippel–Lindau (VHL), breast cancer 1 (BRCA1), and serine/threonine kinase 11 (STK11) genes, respectively,