The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.

Valproate (VPA)(1) has been used for decades in the treatment of epilepsy, and is also effective as a mood stabilizer and in migraine therapy. It has been shown that VPA is also a histone deacetylase (HDAC) inhibitor. We have previously shown that VPA could trigger active demethylation of ectopically methylated transiently transfected DNA in HEK 293 cells. We therefore tested whether VPA treatment could bring about stable changes in the epigenome by causing changes in the state of DNA methylation of genomic DNA. Using a microarray gene expression analysis we identified the genes whose expression is induced by VPA treatment in HEK 293 cells. We found that a subset of these genes could also be induced by the classical DNA methylation inhibitor 5-aza-2'-deoxy-cytidine (5-aza-CdR) suggesting that VPA can alter the state of expression of genes, which are stably suppressed by DNA methylation. We mapped the state of methylation of three of these genes, MELANOMA ANTIGEN B2 GENE (MAGEB2), METALLOPROTEINASE 2 (MMP2) and WIF1, which are involved in tumor growth and metastasis. A chromatin immunoprecipitation (ChIP) assay revealed that VPA treatment caused as expected a change in the state of acetylation of these genes. Our data supports the concept that chromatin acetylation and DNA methylation are found in a dynamic interrelation and that the consequences of HDAC inhibitors are not limited to changes in histone acetylation but that they also bring about a change in the state of modification of DNA. The implications of our results on the future therapeutic utilities of VPA in cancer will be discussed.

NOD1 {nucleotide-binding oligomerization domain 1; NLRC [NOD-LRR (leucine-rich repeat) family with CARD (caspase recruitment domain) 1]} and NOD2 (NLRC2) are among the most prominent members of the NLR (NOD-LRR) family –proteins that contain nucleotide-binding NACHT domains and receptor-like LRR domains. With over 20 members identified in humans, NLRs represent important components of the mammalian innate immune system, serving as intracellular receptors for pathogens and for endogenous molecules elaborated by tissue injury. NOD1 and NOD2 proteins operate as microbial sensors through the recognition of specific PG (peptidoglycan) constituents of bacteria. Upon activation, these NLR family members initiate signal transduction mechanisms that include stimulation of NF-κB (nuclear factor-κB), stress kinases, IRFs (interferon regulatory factors) and autophagy. Hereditary polymorphisms in the genes encoding NOD1 and NOD2 have been associated with an increasing number of chronic inflammatory diseases. In fact, potential roles for NOD1 and NOD2 in inflammatory disorders have been revealed by investigations using a series of animal models. In the present review, we describe recent experimental findings associating NOD1 and NOD2 with various autoimmune and chronic inflammatory disorders, and we discuss prospects for development of novel therapeutics targeting these NLR family proteins.

Objective The genetic basis of inflammatory bowel disease (IBD) is incompletely understood. The aim of this study was to identify rare genetic variants involved in the pathogenesis of IBD. Design Exome sequencing and immunological profiling were performed in a patient with early onset Crohn's disease (CD). The coding region of the gene encoding X-linked inhibitor of apoptosis protein (XIAP) was sequenced in samples of 275 paediatric IBD and 1047 adult-onset CD patients. XIAP genotyping was performed in samples of 2680 IBD patients and 2864 healthy controls. Functional effects of the variants identified were investigated in primary cells and cultured cell lines. Results Our results demonstrate the frequent occurrence of private variants in XIAP in about four percent of male patients with paediatric-onset CD. While XIAP mutations are known to be associated with the primary immunodeficiency (PID) X-linked lymphoproliferative disease type 2 (XLP2), CD patients described here exhibited intestinal inflammation in the absence of XLP2 and harboured a spectrum of mutations partially distinct from that observed in XLP2. The majority of XIAP variants identified was associated with a selective defect in NOD1/2 signalling, impaired NOD1/2-mediated activation of NF-κB, and altered NF-κB-dependent cytokine production. Conclusions This study reveals the unanticipated, frequent occurrence of XIAP variants in male paediatriconset CD. The link between XIAP and NOD1/2, and the association of XIAP variants with XLP2, support the concept of PID in a subset of IBD patients. Moreover, these studies provide a rationale for the implementation of XIAP sequencing in clinical diagnostics in male patients with severe CD.

Introduction: Despite the enthusiasm surrounding cancer immunotherapy, most SCLC patients show very modest response to immune checkpoint inhibitor monotherapy treatment. Therefore, there is growing interest in combining immune checkpoint blockade with chemotherapy and other treatments to enhance immune checkpoint blockade efficacy. Based on favorable clinical trial results, chemotherapy and immunotherapy combinations have been recently approved by the U.S. Food and Drug Administration for frontline treatment for SCLC. Methods and Results: Here, we show that combined treatment of SRA737, an oral CHK1 inhibitor, and antiprogrammed death ligand 1 (PD-L1) leads to an antitumor response in multiple cancer models, including SCLC. We further show that combining low, non-cytotoxic doses of gemcitabine with SRA737 þ anti-PD-L1/anti-PD-1 significantly increased antitumorigenic CD8þ cytotoxic T cells, dendritic cells, and M1 macrophage populations in an SCLC model. This regimen also led to a significant decrease in immunosuppressive M2 macrophage and myeloid-derived suppressor cell populations, as well as an increase in the expression of the type I interferon beta 1 gene, IFNb, and chemokines, CCL5 and CXCL10. Conclusions: Given that anti-PD-L1/anti-PD-1 drugs have recently been approved as monotherapy and in combination with chemotherapy for the treatment of SCLC, and that the SRA737 þ low dose gemcitabine regimen is currently in clinical trials for SCLC and other malignancies, our preclinical data provide a strong rational for combining this regimen with inhibitors of the PD-L1/PD-1 pathway.

Previous lines of evidence have shown that inhibition of DNA methyltransferase (MeTase) can arrest tumor cell growth; however, the mechanisms involved were not clear. In this manuscript we show that out of 16 known tumor suppressors and cell cycle regulators, the cyclin-dependent kinase inhibitor p21 is the only tumor suppressor induced in the human lung cancer cell line, A549, following inhibition of DNA MeTase by a novel DNA MeTase antagonist or antisense oligonucleotides. The rapid induction of p21 expression points to a mechanism that does not involve demethylation of p21 promoter. Consistent with this hypothesis, we show that part of the CpG island upstream of the endogenous p21 gene is unmethylated and that the expression of unmethylated p21 promoter luciferase reporter constructs is induced following inhibition of DNA MeTase. These results are consistent with the hypothesis that the level of DNA MeTase in a cell can control the expression of a nodal tumor suppressor by a mechanism that does not involve DNA methylation.DNA methylation of cytosine residues located at the dinucleotide sequence CpG can suppress expression of genes either by directly interfering with the binding of transcription factors (1) or by attracting methylated-DNA binding proteins such as MeCP2 (2-4). To ascertain that the epigenetic information encoded in the methylation pattern is faithfully maintained, the expression of the maintenance DNA MeTase 1 enzyme DNMT1, which catalyzes the transfer of a methyl group from S-adenosyl-methionine to the 5Ј position on cytosines residing at the dinucleotide CpG (5), is tightly coordinated with DNA replication (6) and the state of growth of the cell (7). Different protooncogenic pathways can up-regulate dnmt1 expression (8), and high levels of dnmt1 mRNA have been observed in many cancer cells (9, 10). It has been proposed that increased DNA MeTase levels are a downstream component of oncogenic programs (11) and that they play a causal role in cellular transformation (12, 13). This hypothesis has been supported by the observation that a reduction in DNA MeTase levels by either 5-aza-deoxycytidine (13, 14), DNA MeTase antisense mRNA or antisense oligonucleotides can reverse tumor growth in vivo and in vitro (13-15).The mechanisms responsible for cellular transformation by DNMT1 and the reversal of transformation by DNA MeTase inhibition are unknown. One attractive hypothesis is that high levels of DNA MeTase lead to ectopic methylation and inactivation of tumor suppressor genes such as p16 (16). Similarly, the inhibition of DNA MeTase could result in demethylation and lead to activation of p16 (17, 18). However, because both the increase in DNA MeTase levels and its inhibition by pharmacological inhibitors are global processes, it is difficult to understand how they could predictably result in a discrete change of the methylation state of specific sites. A more likely explanation is that ectopic methylation of tumor suppressors in tumor cells is a slow and stochastic process, but the aberrant meth...

Faithful inheritance of the chromatin structure is essential for maintaining the gene expression integrity of a cell. Histone modification by acetylation and deacetylation is a critical control of chromatin structure. In this study, we test the hypothesis that histone deacetylase 1 (HDAC1) is physically associated with a basic component of the DNA replication machinery as a mechanism of coordinating histone deacetylation and DNA synthesis. Proliferating cell nuclear antigen (PCNA) is a sliding clamp that serves as a loading platform for many proteins involved in DNA replication and DNA repair. We show that PCNA interacts with HDAC1 in human cells and in vitro and that a considerable fraction of PCNA and HDAC1 colocalize in the cell nucleus. PCNA associates with histone deacetylase activity that is completely abolished in the presence of the HDAC inhibitor trichostatin A. Trichostatin A treatment arrests cells at the G 2 -M phase of the cell cycle, which is consistent with the hypothesis that the proper formation of the chromatin after DNA replication may be important in signaling the progression through the cell cycle. Our results strengthen the role of PCNA as a factor coordinating DNA replication and epigenetic inheritance.Epigenetic markings play an essential role in regulating the gene expression program of vertebrate cells. One of the fundamental challenges of cell division is therefore coordinating the processes of genetic and epigenetic inheritance. The cell must possess multiple mechanisms to coordinate these processes (1). For example, DNA methylation is coordinated with DNA replication (2) by physical association of maintenance DNA methyltransferase 1 with the DNA replication fork protein PCNA 1 (3). Inhibition of DNA methyltransferase 1 leads to inhibition of initiation of DNA replication (4).Similar to DNA methylation, DNA replication-coupled chromatin assembly is essential for the inheritance of the epigenetic code. The specific targeting of nucleosome assembly to the newly synthesized DNA is achieved by direct interaction of histone chaperone CAF-1 with PCNA (5). PCNA is a homotrimeric protein that forms a sliding clamp around DNA and functions as a DNA polymerase processivity factor during replication and nucleotide excision repair. Through its multiple protein-protein interactions, PCNA coordinates events in replication, epigenetic inheritance, repair, and cell cycle control (6). A recent study in Saccharomyces cerevisiae has shown that several mutations in PCNA decrease silencing at telomeres and at the mating-type HMR locus (7). Furthermore, mutations in the Drosophila PCNA gene mus209 suppress repression in the vicinity of heterochromatin (8). The disruption of epigenetic silencing has been attributed to the inability of some of these mutants to associate with CAF-1. However, synergism of several PCNA mutants with CAF-1 mutants suggested that PCNA may participate in silencing through another factor.During nucleosome assembly, histones H3 and H4 undergo transient acetylation before their deposi...

DNA methyltransferase 1 (DNMT1) catalyzes the postreplication methylation of DNA and is responsible for maintaining the DNA methylation pattern during cell division. A long list of data supports a role for DNMT1 in cellular transformation and inhibitors of DNMT1 were shown to have antitumorigenic effects. It was long believed that DNMT1 promoted tumorigenesis by maintaining the hypermethylated and silenced state of tumor suppressor genes. We have previously shown that DNMT1 knock down by either antisense oligonucleotides directed at DNMT1 or expressed antisense activates a number of genes involved in stress response and cell cycle arrest by a DNA methylation-independent mechanism. In this report we demonstrate that antisense knock down of DNMT1 in human lung carcinoma A549 and embryonal kidney HEK293 cells induces gene expression by a mechanism that does not involve either of the known epigenomic mechanisms, DNA methylation, histone acetylation, or histone methylation. The mechanism of activation of the cell cycle inhibitor p21 and apoptosis inducer BIK by DNMT1 inhibition is independent of the mechanism of activation of the same genes by histone deacetylase inhibition. We determine whether DNMT1 knock down activates one of the nodal transcription activation pathways in the cell and demonstrate that DNMT1 activates Sp1 response elements. This activation of Sp1 response does not involve an increase in either Sp1 or Sp3 protein levels in the cell or the occupancy of the Sp1 elements with these proteins. The methylation-independent regulation of Sp1 elements by DNMT1 unravels a novel function for DNMT1 in gene regulation. DNA methylation was believed to be a mechanism for suppression of CG-rich Sp1-bearing promoters. Our data suggest a fundamentally different and surprising role for DNMT1 regulation of CG-rich genes by a mechanism independent of DNA methylation and histone acetylation. The implications of our data on the biological roles of DNMT1 and the therapeutic potential of DNMT1 inhibitors as anticancer agents are discussed.DNA modification by methylation of cytosines residing at the dinucleotide sequence CG plays an important role in epigenomic programming of gene expression (1). Not all CGs are methylated, and the pattern of distribution of methylated and unmethylated CGs is cell type-specific (2). DNA methylation in regulatory regions of genes plays a role in silencing genes either by directly inhibiting the interaction of transcription factors with their regulatory sequences (3, 4) or by attracting methylated DNA-binding proteins, which in turn recruit histone deacetylases and histone methyltransferases, resulting in an inactive chromatin structure (5, 6). DNA methylation is catalyzed by DNA methyltransferases DNMTs, 1 which transfer the methyl moiety from the methyl donor S-adenosylmethionine to 5th position on the cytosine ring (7). DNMT1 is responsible for maintaining the DNA methylation pattern during embryonal development and cell division (8, 9). DNMT1 deregulation was proposed to play a critical ro...