Transcription factor expression repertoire basis for epigenetic and transcriptional subtypes of colorectal cancers.

Colorectal cancers (CRCs) form a heterogenous group classified into epigenetic and transcriptional subtypes. The basis for the epigenetic subtypes, exemplified by varying degrees of promoter DNA hypermethylation, and its relation to the transcriptional subtypes is not well understood. We link cancer-specific transcription factor (TF) expression alterations to methylation alterations near TF-binding sites at promoter and enhancer regions in CRCs and their premalignant precursor lesions to provide mechanistic insights into the origins and evolution of the CRC molecular subtypes. A gradient of TF expression changes forms a basis for the subtypes of abnormal DNA methylation, termed CpG-island promoter DNA methylation phenotypes (CIMPs), in CRCs and other cancers. CIMP is tightly correlated with cancer-specific hypermethylation at enhancers, which we term CpG-enhancer methylation phenotype (CEMP). Coordinated promoter and enhancer methylation appears to be driven by downregulation of TFs with common binding sites at the hypermethylated enhancers and promoters. The altered expression of TFs related to hypermethylator subtypes occurs early during CRC development, detectable in premalignant adenomas. TF-based profiling further identifies patients with worse overall survival. Importantly, altered expression of these TFs discriminates the transcriptome-based consensus molecular subtypes (CMS), thus providing a common basis for CIMP and CMS subtypes.

Integration of the Transcriptome and Genome-Wide Landscape of BRD2 and BRD4 Binding Motifs Identifies Key Superenhancer Genes and Reveals the Mechanism of Bet Inhibitor Action in Rheumatoid Arthritis Synovial Fibroblasts.

Fibroblast-like synoviocytes (FLS), one of the main cell types of the rheumatoid arthritis (RA) synovium, possess phenotypic and molecular characteristics of transformed cells. JQ1, an inhibitor of the bromodomain and extra terminal domain family that includes BRD2, BRD3, BRD4, and BRDt, has shown efficacy in models of arthritis. We demonstrate that the active isomer of JQ1 but not its inactive isomer inhibits IL-1ß-induced RA-FLS activation and proliferation. To understand the mechanism of JQ1 action, we subjected JQ1-treated RA-FLS to transcriptional profiling and determined BRD2 and BRD4 cistromes by identifying their global chromatin binding sites. In addition, assay for transposable accessible chromatin by high throughput sequencing was employed to identify open and closed regions of chromatin in JQ1-treated RA-FLS. Through an integrated analysis of expression profiling, Brd2/Brd4 cistrome data, and changes in chromatin accessibility, we found that JQ1 inhibited key BRD2/BRD4 superenhancer genes, downregulated multiple crucial inflammatory pathways, and altered the genome-wide occupancy of critical transcription factors involved in inflammatory signaling. Our results suggest a pleiotropic effect of JQ1 on pathways that have shown to be individually efficacious in RA (in vitro, in vivo, and/or in humans) and provide a strong rationale for targeting BRD2/BRD4 for disease treatment and interception.

Epigenetic therapy activates type I interferon signaling in murine ovarian cancer to reduce immunosuppression and tumor burden.

Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.

TIM-3 Engagement Promotes Effector Memory T Cell Differentiation of Human Antigen-Specific CD8 T Cells by Activating mTORC1.

T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture system to specifically address the impact of anti-TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti-TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti-TIM-3 Ab. In these conditions, anti-TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti-TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and rhebl1 transcript. Altogether these findings suggest that anti-TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1.

Subtype and pathway specific responses to anticancer compounds in breast cancer.

Breast cancers are comprised of molecularly distinct subtypes that may respond differently to pathway-targeted therapies now under development. Collections of breast cancer cell lines mirror many of the molecular subtypes and pathways found in tumors, suggesting that treatment of cell lines with candidate therapeutic compounds can guide identification of associations between molecular subtypes, pathways, and drug response. In a test of 77 therapeutic compounds, nearly all drugs showed differential responses across these cell lines, and approximately one third showed subtype-, pathway-, and/or genomic aberration-specific responses. These observations suggest mechanisms of response and resistance and may inform efforts to develop molecular assays that predict clinical response.

Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells.

Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.

Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene.

We attempted to answer two central questions about epigenetic silencing of the tumor suppressor gene p16(INK4a) in this study: (1) whether the maintenance of associated histone modifications is dependent on DNA methylation and (2) whether such histone modifications can occur prior to DNA methylation. By coupling chromatin immunoprecipitation with gene targeting and the analysis of specific alleles, we found that elimination of DNA methylation from a p16(INK4a) allele resulted in profound changes in surrounding histones. After continued passage of such cells, methylation of histone H3 lysine-9 occurred in conjunction with re-silencing in the absence of DNA methylation. These results have important implications for understanding the biochemical events underlying the silencing of tumor suppressor genes and the resultant growth suppression.

Knockin of mutant PIK3CA activates multiple oncogenic pathways.

The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to "knock in" PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3beta phosphorylation. Paradoxically, the GSK3beta inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3beta target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3beta is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.

Mutation and copy number detection in human cancers using a custom genotyping assay.

Identification of biomarkers for positive and negative predictors of response to cancer therapeutics can help direct clinical strategies. However, challenges with tissue availability and costs are significant limiting factors for diagnostic assays. To address these challenges, we have customized a high-throughput single nucleotide polymorphism genotyping assay with the objective of simultaneously surveying known somatic mutations and copy number alterations for translational studies in cancer. As constructed, this assay can interrogate 376 known somatic mutations and quantify copy number alterations of genes commonly implicated in tumorigenesis or progression. Validation of this assay on a panel of 321 cell lines demonstrates sensitivity to accurately detect mutations, robust accuracy in the presence of infiltrating normal tissue, and the ability to detect both DNA copy number amplifications and deletions. This technology, with its high sensitivity, small DNA requirements, and low costs is an attractive platform for biomarker exploration in cancer.

Combined phosphatase and tensin homolog (PTEN) loss and fatty acid synthase (FAS) overexpression worsens the prognosis of Chinese patients with hepatocellular carcinoma.

We aimed to investigate the expression pattern of phosphatase and tensin homolog (PTEN), to evaluate the relationship between PTEN expression and clinicopathological characteristics, including fatty acid synthase (FAS) expression, and to determine the correlations of PTEN and FAS expression with survival in Chinese patients with hepatocellular carcinoma (HCC). The expression patterns of PTEN and FAS were determined using tissue microarrays and immunohistochemistry. The expression of PTEN was compared with the clinicopathological characteristics of HCC, including FAS expression. Receiver operator characteristic curves were used to calculate the clinical sensitivity and specificity of PTEN expression. Kaplan-Meier survival curves were constructed to evaluate the correlations of PTEN loss and FAS overexpression with overall survival. We found that the loss of PTEN expression occurred predominantly in the cytoplasm, while FAS was mainly localized to the cytoplasm. Cytoplasmic and total PTEN expression levels were significantly decreased in HCC compared with adjacent non-neoplastic tissue (both, p < 0.0001). Decreased cytoplasmic and total PTEN expression showed significant clinical sensitivity and specificity for HCC (both, p < 0.0001). Downregulation of PTEN in HCC relative to non-neoplastic tissue was significantly correlated with histological grade (p = 0.043 for histological grades I-II versus grade III). Loss of total PTEN was significantly correlated with FAS overexpression (p = 0.014). Loss of PTEN was also associated with poor prognosis of patients with poorly differentiated HCC (p = 0.049). Moreover, loss of PTEN combined with FAS overexpression was associated with significantly worse prognosis compared with other HCC cases (p = 0.011). Our data indicate that PTEN may serve as a potential diagnostic and prognostic marker of HCC. Upregulating PTEN expression and inhibiting FAS expression may offer a novel therapeutic approach for HCC.

High chromosome number in hematological cancer cell lines is a negative predictor of response to the inhibition of Aurora B and C by GSK1070916.

BACKGROUND: Aurora kinases play critical roles in mitosis and are being evaluated as therapeutic targets in cancer. GSK1070916 is a potent, selective, ATP competitive inhibitor of Aurora kinase B and C. Translation of predictive biomarkers to the clinic can benefit patients by identifying the tumors that are more likely to respond to therapies, especially novel inhibitors such as GSK1070916. METHODS: 59 Hematological cancer-derived cell lines were used as models for response where in vitro sensitivity to GSK1070916 was based on both time and degree of cell death. The response data was analyzed along with karyotype, transcriptomics and somatic mutation profiles to determine predictors of response. RESULTS: 20 cell lines were sensitive and 39 were resistant to treatment with GSK1070916. High chromosome number was more prevalent in resistant cell lines (p-value = 0.0098, Fisher Exact Test). Greater resistance was also found in cell lines harboring polyploid subpopulations (p-value = 0.00014, Unpaired t-test). A review of NOTCH1 mutations in T-ALL cell lines showed an association between NOTCH1 mutation status and chromosome number (p-value = 0.0066, Fisher Exact Test). CONCLUSIONS: High chromosome number associated with resistance to the inhibition of Aurora B and C suggests cells with a mechanism to bypass the high ploidy checkpoint are resistant to GSK1070916. High chromosome number, a hallmark trait of many late stage hematological malignancies, varies in prevalence among hematological malignancy subtypes. The high frequency and relative ease of measurement make high chromosome number a viable negative predictive marker for GSK1070916.

Tamoxifen-stimulated growth of breast cancer due to p21 loss.

Tamoxifen is widely used for the treatment of hormonally responsive breast cancers. However, some resistant breast cancers develop a growth proliferative response to this drug, as evidenced by tumor regression upon its withdrawal. To elucidate the molecular mediators of this paradox, tissue samples from a patient with tamoxifen-stimulated breast cancer were analyzed. These studies revealed that loss of the cyclin-dependent kinase inhibitor p21 was associated with a tamoxifen growth-inducing phenotype. Immortalized human breast epithelial cells with somatic deletion of the p21 gene were then generated and displayed a growth proliferative response to tamoxifen, whereas p21 wild-type cells demonstrated growth inhibition upon tamoxifen exposure. Mutational and biochemical analyses revealed that loss of p21's cyclin-dependent kinase inhibitory property results in hyperphosphorylation of estrogen receptor-alpha, with subsequent increased gene expression of estrogen receptor-regulated genes. These data reveal a previously uncharacterized molecular mechanism of tamoxifen resistance and have potential clinical implications for the management of tamoxifen-resistant breast cancers.

Gut-Restricted Selective Cyclooxygenase-2 (COX-2) Inhibitors for Chemoprevention of Colorectal Cancer.

Selective cyclooxygenase (COX)-2 inhibitors have been extensively studied for colorectal cancer (CRC) chemoprevention. Celecoxib has been reported to reduce the incidence of colorectal adenomas and CRC but is also associated with an increased risk of cardiovascular events. Here, we report a series of gut-restricted, selective COX-2 inhibitors characterized by high colonic exposure and minimized systemic exposure. By establishing acute ex vivo 18F-FDG uptake attenuation as an efficacy proxy, we identified a subset of analogues that demonstrated statistically significant in vivo dose-dependent inhibition of adenoma progression and survival extension in an APCmin/+ mouse model. However, in vitro-in vivo correlation analysis showed their chemoprotective effects were driven by residual systemic COX-2 inhibition, rationalizing their less than expected efficacies and highlighting the challenges associated with COX-2-mediated CRC disease chemoprevention.

High-throughput screening identifies novel agents eliciting hypersensitivity in Fanconi pathway-deficient cancer cells.

Inactivation of the Fanconi anemia (FA) pathway occurs in diverse human tumors among the general population and renders those tumors hypersensitive to DNA interstrand-cross-linking (ICL) agents. The identification of novel agents to which FA pathway-deficient cells were hypersensitive could provide new therapeutic opportunities and improve our molecular understanding of the FA genes. Using high-throughput screening, we assessed the growth of isogenic human cancer cells that differed only in the presence or absence of single FA genes upon treatment with 880 active drugs and 40,000 diverse compounds. We identified several compounds to which FA pathway-deficient cells were more sensitive than FA pathway-proficient cells, including two groups of structurally related compounds. We further investigated the compound eliciting the strongest effect, termed 80136342. Its mechanism of action was distinct from that of ICL agents; 80136342 did not cause increased chromosomal aberrations, enhanced FANCD2 monoubiquitination, H2AX phosphorylation, p53 activation, or ICL induction. Similar to ICL agents, however, 80136342 caused a pronounced G(2) arrest in FA pathway-deficient cells. When applied in combination with ICL agents, 80136342 had at least additive toxic effects, excluding interferences on ICL-induced toxicity and facilitating a combinational application. Finally, we identified one particular methyl group necessary for the effects of 80136342 on FA-deficient cells. In conclusion, using high-throughput screening in an isogenic human FA cancer model, we explored a novel approach to identify agents eliciting hypersensitivity in FA pathway-deficient cells. We discovered several attractive candidates to serve as lead compounds for evaluating structure-activity relationships and developing therapeutics selectively targeting FA pathway-deficient tumors.

Knock-in of mutant K-ras in nontumorigenic human epithelial cells as a new model for studying K-ras mediated transformation.

The oncogenic function of mutant ras in mammalian cells has been extensively investigated using multiple human and animal models. These systems include overexpression of exogenous mutant ras transgenes, conditionally expressed knock-in mouse models, and somatic cell knockout of mutant and wild-type ras genes in human cancer cell lines. However, phenotypic discrepancies between knock-in mice and transgenic mutant ras overexpression prompted us to evaluate the consequences of targeted knock-in of an oncogenic K-ras mutation in the nontumorigenic human breast epithelial cell line MCF-10A and hTERT-immortalized human mammary epithelial cells. Our results show several significant differences between mutant K-ras knock-in cells versus their transgene counterparts, including limited phosphorylation of the downstream molecules extracellular signal-regulated kinase and AKT, minor proliferative capacity in the absence of an exogenous growth factor, and the inability to form colonies in semisolid medium. Analysis of 16 cancer cell lines carrying mutant K-ras genes indicated that 50% of cancer cells harbor nonoverexpressed heterozygous K-ras mutations similar to the expression seen in our knock-in cell lines. Thus, this system serves as a new model for elucidating the oncogenic contribution of mutant K-ras as expressed in a large fraction of human cancer cells.

DFMO and 5-Azacytidine Increase M1 Macrophages in the Tumor Microenvironment of Murine Ovarian Cancer.

Although ovarian cancer has a low incidence rate, it remains the most deadly gynecologic malignancy. Previous work has demonstrated that the DNMTi 5-Azacytidine (5AZA-C) activates type I interferon signaling to increase IFNγ+ T cells and natural killer (NK) cells and reduce the percentage of macrophages in the tumor microenvironment. To improve the efficacy of epigenetic therapy, we hypothesized that the addition of α-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, may further decrease immunosuppressive cell populations improving outcome. We tested this hypothesis in an immunocompetent mouse model for ovarian cancer and found that in vivo, 5AZA-C and DFMO, either alone or in combination, significantly increased survival, decreased tumor burden, and caused recruitment of activated (IFNγ+) CD4+ T cells, CD8+ T cells, and NK cells. The combination therapy had a striking increase in survival when compared with single-agent treatment, despite a smaller difference in recruited lymphocytes. Instead, combination therapy led to a significant decrease in immunosuppressive cells such as M2 polarized macrophages and an increase in tumor-killing M1 macrophages. In this model, depletion of macrophages with a CSF1R-blocking antibody reduced the efficacy of 5AZA-C + DFMO treatment and resulted in fewer M1 macrophages in the tumor microenvironment. These observations suggest our novel combination therapy modifies macrophage polarization in the tumor microenvironment, recruiting M1 macrophages and prolonging survival. SIGNIFICANCE: Combined epigenetic and polyamine-reducing therapy stimulates M1 macrophage polarization in the tumor microenvironment of an ovarian cancer mouse model, resulting in decreased tumor burden and prolonged survival.

MCT-1 protein interacts with the cap complex and modulates messenger RNA translational profiles.

MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis.

De novo CpG island methylation in human cancer cells.

A major obstacle toward understanding how patterns of abnormal mammalian cytosine DNA methylation are established is the difficulty in quantitating the de novo methylation activities of DNA methyltransferases (DNMT) thought to catalyze these reactions. Here, we describe a novel method, using native human CpG island substrates from genes that frequently become hypermethylated in cancer, which generates robust activity for measuring de novo CpG methylation. We then survey colon cancer cells with genetically engineered deficiencies in different DNMTs and find that the major activity against these substrates in extracts of these cells is DNMT1, with minor contribution from DNMT 3b and none from DNMT3a, the only known bona fide de novo methyltransferases. The activity of DNMT1 against unmethylated CpG rich DNA was further tested by introducing CpG island substrates and DNMT1 into Drosophila melanogaster cells. The exogenous DNMT1 methylates the integrated mammalian CpG islands but not the Drosophila DNA. Additionally, in human cancer cells lacking DNMT1 and DNMT3b and having nearly absent genomic methylation, gene-specific de novo methylation can be initiated by reintroduction of DNMT1. Our studies provide a new assay for de novo activity of DNMTs and data suggesting a potential role for DNMT1 in the initiation of promoter CpG island hypermethylation in human cancer cells.

A mechanism of T cell dependent selection of antigen engaged Germinal Center B cells.

A model of B cell affinity selection is proposed, and an explanation of peripheral tolerance mechanisms through antibody repertoire editing is presented. We show that affinity discrimination between B cells is driven by a competition between obtaining T cell help and removal of B cells from the light zone, either through apoptosis or by a return to the dark zone of germinal centers. We demonstrate that this mechanism also allows for the negative selection of self reactive B cells and maintenance of B cell tolerance during the Germinal Center reaction. Finally, we demonstrate that clonal expansion upon return to the Germinal Center dark zone amplifies differences in the antigen affinity of B cells that survive the light zone.