PURPOSE:The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. METHODS:Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). RESULTS:A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/β-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. CONCLUSIONS:High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.

Supplementary Figure from Epigenome-Wide Study Identifies Epigenetic Outliers in Normal Mucosa of Patients with Colorectal Cancer

AbstractNongenetic predisposition to colorectal cancer continues to be difficult to measure precisely, hampering efforts in targeted prevention and screening. Epigenetic changes in the normal mucosa of patients with colorectal cancer can serve as a tool in predicting colorectal cancer outcomes. We identified epigenetic changes affecting the normal mucosa of patients with colorectal cancer. DNA methylation profiling on normal colon mucosa from 77 patients with colorectal cancer and 68 controls identified a distinct subgroup of normally-appearing mucosa with markedly disrupted DNA methylation at a large number of CpGs, termed as “Outlier Methylation Phenotype” (OMP) and are present in 15 of 77 patients with cancer versus 0 of 68 controls (P < 0.001). Similar findings were also seen in publicly available datasets. Comparison of normal colon mucosa transcription profiles of patients with OMP cancer with those of patients with non-OMP cancer indicates genes whose promoters are hypermethylated in the OMP patients are also transcriptionally downregulated, and that many of the genes most affected are involved in interactions between epithelial cells, the mucus layer, and the microbiome. Analysis of 16S rRNA profiles suggests that normal colon mucosa of OMPs are enriched in bacterial genera associated with colorectal cancer risk, advanced tumor stage, chronic intestinal inflammation, malignant transformation, nosocomial infections, and KRAS mutations. In conclusion, our study identifies an epigenetically distinct OMP group in the normal mucosa of patients with colorectal cancer that is characterized by a disrupted methylome, altered gene expression, and microbial dysbiosis. Prospective studies are needed to determine whether OMP could serve as a biomarker for an elevated epigenetic risk for colorectal cancer development.Prevention Relevance:Our study identifies an epigenetically distinct OMP group in the normal mucosa of patients with colorectal cancer that is characterized by a disrupted methylome, altered gene expression, and microbial dysbiosis. Identification of OMPs in healthy controls and patients with colorectal cancer will lead to prevention and better prognosis, respectively.

African Americans (AA) have a higher incidence and mortality rate for colorectal cancer (CRC) compared to Caucasians. DNA methylation profiling on normal colon mucosa from 77 CRC patients and 68 patients without cancer (controls) of AA and Caucasian descent identified a distinct subgroup of CRC patients (11AA and 4 Caucasians) who are statistical “outliers” at a large number of CpGs. Such highly epigenetically disrupted CRC patients are termed “Outlier Methylation Phenotype” (OMP). Comparison of methylome profiles of CRC patients and controls revealed that AA CRC patients have greater disruption of the normal colon methylome than do Caucasian patients. A large fraction of the overall methylation differences found between CRC patients and controls is attributable to OMPs. Estimation of epigenetic ages shows that OMPs have lower epigenetic ages compared to their chronological ages. In other words, OMPs undergo epigenetic age deceleration. Comparison of normal colon mucosa transcription profiles of OMP cancer patients with those of non-OMP cancer patients indicates genes whose promoters are hypermethylated in the OMP patients are also transcriptionally down-regulated, and that many of the genes most affected are involved in interactions between epithelial cells, the mucus layer, and the microbiome. Analysis of 16S RDNA profiles suggests that normal colon mucosa of OMPs are enriched in bacterial genera associated with CRC risk, advanced tumor stage, chronic intestinal inflammation, malignant transformation, nosocomial infections and KRAS mutations. In conclusion, our study suggests a distinct epigenetically disrupted OMP group in CRC that are responsible for disrupted methylome, altered gene expression and microbial dysbiosis. Furthermore, a higher frequency of OMPs among African Americans (11/42) than among Caucasians (5/35) may contribute to observed racial disparities in CRC incidence and outcome. Citation Format: Jayashri Ghosh, Bryant M. Schultz, Joe Chan, Claudia Wultsch, Imad Shureiqi, Stephanie Chow, Ahmet Doymaz, Sophia Varriano, Melissa Driscoll, Jen Muse, Frida E. Kleiman, Konstantinos Krampis, Jean-Pierre J. Issa, Carmen Sapienza. Do epigenetic outliers impact racial disparities in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6265.

Background Alterations in the epigenome are a risk factor in multiple disease states. We have demonstrated in the past that disruption of the epigenome during early pregnancy or periconception, as demonstrated by altered methylation, may be associated with both assisted reproductive technology and undesirable clinical outcomes at birth, such as low birth weight. We have previously defined this altered methylation, calculated based on statistical upper and lower limits of outlier CpGs compared to the population, as an ‘outlier methylation phenotype’ (OMP). Our aim in this study was to determine whether children thus identified as possessing an OMP at birth by DNA methylation in cord blood persist as outliers in early childhood based on salivary DNA methylation. Results A total of 31 children were included in the analysis. Among 24 children for whom both cord blood DNA and salivary DNA were available, DNA methylation patterns, analyzed using the Illumina Infinium MethylationEPIC BeadChip (850 K), between cord blood at birth and saliva in childhood at age 6–12 years remain stable ( R 2 range 0.89–0.97). At birth, three out of 28 children demonstrated an OMP in multiple cord blood datasets and hierarchical clustering. Overall DNA methylation among all three OMP children identified as outliers at birth was remarkably stable (individual R 2 0.908, 0.92, 0.915), even when only outlier CpG sites were considered ( R 2 0.694, 0.738, 0.828). Conclusions DNA methylation signatures in cord blood remain stable over time as demonstrated by a strong correlation with epigenetic salivary signatures in childhood. Future work is planned to identify whether a clinical phenotype is associated with OMP and, if so, could undesirable clinical outcomes in childhood and adulthood be predicted at birth.

In vitro fertilization (IVF) is associated with DNA methylation abnormalities and a higher incidence of adverse pregnancy outcomes. However, which exposure(s), among the many IVF interventions, contributes to these outcomes remains unknown. Frozen embryo transfer (ET) is increasingly utilized as an alternative to fresh ET, but reports suggest a higher incidence of pre-eclampsia and large for gestational age infants. This study examines DNA methylation in human placentas using the 850K Infinium MethylationEPIC BeadChip array obtained after 65 programmed frozen ET cycles, 82 fresh ET cycles and 45 unassisted conceptions. Nine patients provided placentas following frozen and fresh ET from consecutive pregnancies for a paired subgroup analysis. In parallel, eight mouse placentas from fresh and frozen ET were analyzed using the Infinium Mouse Methylation BeadChip array. Human and mouse placentas were significantly hypermethylated after frozen ET compared with fresh. Paired analysis showed similar trends. Sex-specific analysis revealed that these changes were driven by male placentas in humans and mice. Frozen and fresh ET placentas were significantly different from controls, with frozen samples hypermethylated compared with controls driven by males and fresh samples being hypomethylated compared with controls, driven by females. Sexually dimorphic epigenetic changes could indicate differential susceptibility to IVF-associated perturbations, which highlights the importance of sex-specific evaluation of adverse outcomes. Similarities between changes in mice and humans underscore the suitability of the mouse model in evaluating how IVF impacts the epigenetic landscape, which is valuable given limited access to human tissue and the ability to isolate specific interventions in mice.

While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T-cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical "brake" on T-cell activation. Hence, TCR engagement of EGR4(-/-) T cells leads to enhanced Ca2+ responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFN gamma production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca2+ entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti-tumor immunity in EGR4(-/-) mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T-cell differentiation and function.

Ca2 + is a ubiquitous and dynamic second messenger molecule that is induced by many factors including receptor activation, environmental factors, and voltage, leading to pleiotropic effects on cell function including changes in migration, metabolism and transcription. As such, it is not surprising that aberrant regulation of Ca2 + signals can lead to pathological phenotypes, including cancer progression. However, given the highly context-specific nature of Ca2 +-dependent changes in cell function, delineation of its role in cancer has been a challenge. Herein, we discuss the distinct roles of Ca2 + signaling within and between each type of cancer, including consideration of the potential of therapeutic strategies targeting these signaling pathways.

The "CpG Island Methylator Phenotype" (CIMP) has been found to be a useful concept in stratifying several types of human cancer into molecularly and clinically distinguishable subgroups. We have identified an additional epigenetic stratification category, the "Outlier Methylation Phenotype" (OMP). Whereas CIMP is defined on the basis of hyper-methylation in tumor genomes, OMP is defined on the basis of highly variant (either or both hyper- and hypo-methylation) methylation at many sites in normal tissues. OMP was identified and defined, originally, as being more common among low birth weight individuals conceived in vitro but we have also identified OMP individuals among colon cancer patients profiled by us, as well as multiple types of cancer patients in the TCGA database. The cause(s) of OMP are unknown, as is whether these individuals identify a clinically useful subgroup of patients, but both the causes of, and potential consequences to, this epigenetically distinct group are of great interest.

Despite pharmacological prophylaxis using calcineurin (CN) inhibitors (i.e., cyclosporin A and Tacrolimus), graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Upon antigen stimulation, activated T cell receptor (TCR) signaling triggers rapid Ca2+ influx. This induces both CN-mediated NFAT activation, and increase of mitochondrial Ca2+ content, a major driver of metabolic activity. However, mitochondrial Ca2+ overload triggers opening of the mitochondrial permeability transition pore and cell death. We hypothesize that pharmacologically increasing mitochondrial Ca2+ load may decrease T cell survival capability, thereby reducing the GVH reaction. If this can be accomplished, it may lead to new strategies for inhibition of GVHD, which is conceptually different from the use of CN inhibitors. To test this hypothesis, we employed a high throughput drug screening system with the proliferation and cytotoxicity of TCR-activated human T cells as the readout, and screened the NPL-800 library (https://www.timtec.net) composed of 800 pure natural compounds. With a stringent criterion in consideration of dose-dependent effect, 26 compounds stood out for reducing the count of activated human T cells with a reduction rate of at least 30% at both 1.0uM and 10.0uM. Positive hits included inhibitors of DNA synthesis, the Na-K-ATPase and mitochondrial metabolism. We were particularly interested in artesunate (ART), which is a derivative of artemisinin that has been used for treating malaria in patients. While artemisinin acts by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in P. falciparum malaria, which causes passive endoplasmic reticulum (ER) Ca2+ depletion and the subsequent cytosolic Ca2+ influx, ART did not inhibit SERCA in T cells. Ex vivo culture assays showed that ART dose-dependently reduced the survival of TCR-activated murine T cells. This inhibitory effect of ART was abrogated by inhibiting Ca2+ influx using BTP2, a potent inhibitor of store-operated Ca2+ channels. Furthermore, treatment of murine CD8 T cells with ART induced significant increases in mitochondrial Ca2+ loading upon TCR activation. These data suggest that inhibition of T cell survival by ART was dependent on TCR activation-induced Ca2+ influx and associated with enhanced mitochondrial Ca2+ uptake. We examined the impact of ART on GVHD in Balb/c mice receiving C57BL/6 (B6) mouse T cell-depleted bone marrow (TCD-BM) and CD4+ T cells. Intraperitoneal injection of ART (10 mg/kg, every other day) from day 1 to day 28 after transplantation reduced clinical signs of GVHD in these recipients and significantly improved their overall survival. Similar inhibition effects of ART on GVHD were observed in miHA-mismatched B6 anti-Balb/b and haplo-identical B6 anti-BDF1 mouse models of GVHD. Further investigations showed that in vivo administration of ART caused significant decreases in the number of host-reactive donor T cells in the spleen and liver of Balb/c mice 7 days after transfer of B6 TCD-BM plus CD4+ T cells. ART treatment did not affect the capacity of donor T cells to produce effector cytokines (e.g., IFN-g and TNF-α) in individual cells. Importantly, in vivo administration of ART preserved anti-leukemia activity of donor T cells and did not impair the reconstitution of hematopoiesis and lymphocytes. Collectively, our findings indicate that pharmacologically increasing mitochondrial Ca2+ loading may have significant implications in the development of novel strategies to prevent GVHD and other T cell-mediated inflammatory disorders in a broad context. Since ART therapy has been clinically approved, this work could be immediately translated into patients. Disclosures No relevant conflicts of interest to declare.

Ca2+ signals, which facilitate pluripotent changes in cell fate, reflect the balance between cation entry and export. We found that overexpression of either isoform of the Ca2+ -extruding plasma membrane calcium ATPase 4 (PMCA4) pump in Jurkat T cells unexpectedly increased activation of the Ca2+-dependent transcription factor nuclear factor of activated T cells (NFAT). Coexpression of the endoplasmic reticulum-resident Ca2+ sensor stromal interaction molecule 1 (STIM1) with the PMCA4b splice variant further enhanced NFAT activity; however, coexpression with PMCA4a depressed NFAT. No PMCA4 splice variant dependence in STIM1 association was observed, whereas partner of STIM1 (POST) preferentially associated with PMCA4b over PMCA4a, which enhanced, rather than inhibited, PMCA4 function. A comparison of global and near-membrane cytosolic Ca2+ abundances during store-operated Ca2+ entry revealed that PMCA4 markedly depressed near-membrane Ca2+ concentrations, particularly when PMCA4b was coexpressed with STIM1. PMCA4b closely associated with both POST and the store-operated Ca2+ channel Orai1. Furthermore, POST knockdown increased the near-membrane Ca2+ concentration, inhibiting the global cytosolic Ca2+ increase. These observations reveal an unexpected role for POST in coupling PMCA4 to Orai1 to promote Ca2+ entry during T cell activation through Ca2+ disinhibition.

Background Pre-implantation embryos exhibit sexual dimorphisms in both primates and rodents. To determine whether these differences reflected sex-biased expression patterns, we generated transcriptome profiles for six 40,XX, six 40,XY, and two 39,X mouse embryonic stem (ES) cells by RNA sequencing. Results We found hundreds of coding and non-coding RNAs that were differentially expressed between male and female cells. Surprisingly, the majority of these were autosomal and included RNA encoding transcription and epigenetic and chromatin remodeling factors. We showed differential Prdm14-responsive enhancer activity in male and female cells, correlating with the sex-specific levels of Prdm14 expression. This is the first time sex-specific enhancer activity in ES cells has been reported. Evaluation of X-linked gene expression patterns between our XX and XY lines revealed four distinct categories: (1) genes showing 2-fold greater expression in the female cells; (2) a set of genes with expression levels well above 2-fold in female cells; (3) genes with equivalent RNA levels in male and female cells; and strikingly, (4) a small number of genes with higher expression in the XY lines. Further evaluation of autosomal gene expression revealed differential expression of imprinted loci, despite appropriate parent-of-origin patterns. The 39,X lines aligned closely with the XY cells and provided insights into potential regulation of genes associated with Turner syndrome in humans. Moreover, inclusion of the 39,X lines permitted three-way comparisons, delineating X and Y chromosome-dependent patterns. Conclusions Overall, our results support the role of the sex chromosomes in establishing sex-specific networks early in embryonic development and provide insights into effects of sex chromosome aneuploidies originating at those stages.

The imprinted Kcnq1 domain contains a differentially methylated region (KvDMR) in intron 11 of Kcnq1. The Kcnq1ot1 non-coding RNA emerges from the unmethylated paternal KvDMR in antisense direction, resulting in cis-repression of neighboring genes. The KvDMR encompasses the Kcnq1ot1 promoter, CTCF sites and other DNA elements, whose individual contribution to regulation of the endogenous domain is unknown. We find that paternal inheritance of a deletion of the minimal Kcnq1ot1 promoter derepresses the upstream Cdkn1c gene. Surprisingly, Kcnq1ot1 transcripts continue to emerge from alternative sites, evidence that silencing depends, not on the ncRNA, but on the promoter sequence. Detailed analyses of Kcnq1ot during cardiogenesis show substantial chromatin reorganization coinciding with discontinuous RNA production in both wild-type and mutant mice, with loss of imprinting. We show that CTCF binds to both methylated and unmethylated alleles of the KvDMR. Furthermore, we report a multitude of enhancers within the Kcnq1ot1 region, and present conformational dynamics of a novel heart enhancer engaged in Kcnq1 expression. Our results have important implications on tissue-specific imprinting patterns and how transcriptional mechanisms compete to maximize the expression of vital genes, in addition to shifting our perception on the role of the long ncRNA in regulating this imprinted domain.