Development of a putative Zn2+-chelating but highly selective MMP-13 inhibitor.

Starting from an already known MMP-13 inhibitor, 1, we pursued an SAR-approach focusing on optimizing interactions close to the Zn2+ binding site of the enzyme. We found the oxetane containing compound 32 (MMP-13 IC50 = 42 nM), which exhibited complete inhibition of collagenolysis in in vitro studies and an excellent selectivity profile among the MMP family. Interestingly, docking studies propose that the oxetane ring in 32 is oriented towards the Zn2+ ion for chelating the metal ion. Chelating properties of MMP13-inhibitors are often connected with non-selectivity within the enzyme family. Compound 32 demonstrates a rare example where the selectivity can be explained via combinatorial effects of interactions within the S1' loop and a chelating effect of the oxetane moiety. Furthermore, in vivo pharmacokinetic studies were performed demonstrating a concentration of 1.97 µM of 32 within the synovial fluid of the rat knee joint, which makes the compound a promising lead compound for further optimization and development for osteoarthritis.

Transcriptional Behavior of Regulatory T Cells Predicts IBD Patient Responses to Vedolizumab Therapy.

BACKGROUND: Inflammatory bowel disease (IBD) involves chronic T cell-mediated inflammatory responses. Vedolizumab (VDZ), a monoclonal antibody against α4ß7 integrin, inhibits lymphocyte extravasation into intestinal mucosae and is effective in ulcerative colitis (UC) and Crohn's disease (CD). AIM: We sought to identify immune cell phenotypic and gene expression signatures that related to response to VDZ. METHODS: Peripheral blood (PBMC) and lamina propria mononuclear cells (LPMCs) were analyzed by flow cytometry and Cytofkit. Sorted CD4 + memory (Tmem) or regulatory T (Treg) cells from PBMC and LPMC were analyzed by RNA sequencing (RNA-seq). Clinical response (≥2-point drop in partial Mayo scores [UC] or Harvey-Bradshaw index [CD]) was assessed 14 to 22 weeks after VDZ initiation. Machine-learning models were used to infer combinatorial traits that predicted response to VDZ. RESULTS: Seventy-one patients were enrolled: 37 received VDZ and 21 patients remained on VDZ >2 years. Fourteen of 37 patients (38%; 8 UC, 6 CD) responded to VDZ. Immune cell phenotypes and CD4 + Tmem and Treg transcriptional behaviors were most divergent between the ileum and colon, irrespective of IBD subtype or inflammation status. Vedolizumab treatment had the greatest impact on Treg metabolic pathways, and response was associated with increased expression of genes involved in oxidative phosphorylation. The strongest clinical predictor of VDZ efficacy was concurrent use of thiopurines. Mucosal tissues offered the greatest number of response-predictive biomarkers, whereas PBMC Treg-expressed genes were the best predictors in combinatorial models of response. CONCLUSIONS: Mucosal and peripheral blood immune cell phenotypes and transcriptional profiles can inform VDZ efficacy and inform new opportunities for combination therapies.

Vedolizumab (VDZ) is effective in the treatment of IBD. Immunophenotyping and RNAseq of T cells were used to inform its mechanism of action. Changes in T regulatory cells in the periphery and mucosa have the greatest relationship to VDZ response.

The Multiple Sclerosis Data Alliance Catalogue: Enabling Web-Based Discovery of Metadata from Real-World Multiple Sclerosis Data Sources.

BACKGROUND: One of the major objectives of the Multiple Sclerosis Data Alliance (MSDA) is to enable better discovery of multiple sclerosis (MS) real-world data (RWD). METHODS: We implemented the MSDA Catalogue, which is available worldwide. The current version of the MSDA Catalogue collects descriptive information on governance, purpose, inclusion criteria, procedures for data quality control, and how and which data are collected, including the use of e-health technologies and data on collection of COVID-19 variables. The current cataloguing procedure is performed in several manual steps, securing an effective catalogue. RESULTS: Herein we summarize the status of the MSDA Catalogue as of January 6, 2021. To date, 38 data sources across five continents are included in the MSDA Catalogue. These data sources differ in purpose, maturity, and variables collected, but this landscaping effort shows that there is substantial alignment on some domains. The MSDA Catalogue shows that personal data and basic disease data are the most collected categories of variables, whereas data on fatigue measurements and cognition scales are the least collected in MS registries/cohorts. CONCLUSIONS: The Web-based MSDA Catalogue provides strategic overview and allows authorized end users to browse metadata profiles of data cohorts and data sources. There are many existing and arising RWD sources in MS. Detailed cataloguing of MS RWD is a first and useful step toward reducing the time needed to discover MS RWD sets and promoting collaboration.

The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Human Myeloid-derived Suppressor Cells are Associated With Chronic Immune Suppression After Severe Sepsis/Septic Shock.

OBJECTIVE: We hypothesized that after sepsis in humans, MDSCs will be persistently increased, functionally immunosuppressive, and associated with adverse clinical outcomes. BACKGROUND: Cancer and sepsis have surprisingly similar immunologic responses and equally dismal long term consequences. In cancer, increased myeloid-derived suppressor cells (MDSCs) induce detrimental immunosuppression, but little is known about the role of MDSCs after sepsis. METHODS: Blood was obtained from 74 patients within 12 hours of severe sepsis/septic shock (SS/SS), and at set intervals out to 28 days, and also in 18 healthy controls. MDSCs were phenotyped for cell surface receptor expression and enriched by cell sorting. Functional and genome-wide expression analyses were performed. Multiple logistic regression analysis was conducted to determine if increased MDSC appearance was associated with in-hospital and long-term outcomes. RESULTS: After SS/SS, CD33CD11bHLA-DR MDSCs were dramatically increased out to 28 days (P < 0.05). When co-cultured with MDSCs from SS/SS patients, antigen-driven T-cell proliferation and TH1/TH2 cytokine production were suppressed (P < 0.05). Additionally, septic MDSCs had suppressed HLA gene expression and up-regulated ARG1 expression (P < 0.05). Finally, SS/SS patients with persistent increased percentages of blood MDSCs had increased nosocomial infections, prolonged intensive care unit stays, and poor functional status at discharge (P < 0.05). CONCLUSIONS: After SS/SS in humans, circulating MDSCs are persistently increased, functionally immunosuppressive, and associated with adverse outcomes. This novel observation warrants further studies. As observed in cancer immunotherapy, MDSCs could be a novel component in multimodality immunotherapy targeting detrimental inflammation and immunosuppression after SS/SS to improve currently observed dismal long-term outcomes.

Informatics-Based Discovery of Disease-Associated Immune Profiles.

Advances in flow and mass cytometry are enabling ultra-high resolution immune profiling in mice and humans on an unprecedented scale. However, the resulting high-content datasets challenge traditional views of cytometry data, which are both limited in scope and biased by pre-existing hypotheses. Computational solutions are now emerging (e.g., Citrus, AutoGate, SPADE) that automate cell gating or enable visualization of relative subset abundance within healthy versus diseased mice or humans. Yet these tools require significant computational fluency and fail to show quantitative relationships between discrete immune phenotypes and continuous disease variables. Here we describe a simple informatics platform that uses hierarchical clustering and nearest neighbor algorithms to associate manually gated immune phenotypes with clinical or pre-clinical disease endpoints of interest in a rapid and unbiased manner. Using this approach, we identify discrete immune profiles that correspond with either weight loss or histologic colitis in a T cell transfer model of inflammatory bowel disease (IBD), and show distinct nodes of immune dysregulation in the IBDs, Crohn's disease and ulcerative colitis. This streamlined informatics approach for cytometry data analysis leverages publicly available software, can be applied to manually or computationally gated cytometry data, is suitable for any clinical or pre-clinical setting, and embraces ultra-high content flow and mass cytometry as a discovery engine.

Quantitative DNA methylation analysis of candidate genes in cervical cancer.

Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

WIF1 is a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix.

Aberrant activation of the Wnt/ß-catenin signaling axis is a prominent oncogenic mechanism in numerous cancers including cervical cancer. Wnt inhibitory factor-1 (WIF1) is a secreted protein that binds Wnt and antagonizes Wnt activity. While the WIF1 gene is characterized as a target for epigenetic silencing in some tumor types, WIF1 expression has not been examined in human cervical tissue and cervical cancer. Here, we show that WIF1 is unmethylated and its gene product is expressed in normal cervical epithelium and some cultured cervical tumor lines. In contrast, several cervical cancer lines contained dense CpG methylation within the WIF1 gene, and expression of both WIF1 transcript and protein was restored by culturing cells in the presence of the global DNA demethylating agent 5-aza-2'-deoxycytidine. Using single-molecule MAPit methylation footprinting, we observed differences in chromatin structure within the WIF1 promoter region between cell lines that express and those that do not express WIF1, consistent with transcriptional activity and repression, respectively. The WIF1 promoter was aberrantly methylated in ∼60% (10 of 17) high-grade highly undifferentiated squamous cell cervical tumors examined, whereas paired normal tissue showed significantly lower levels of CpG methylation. WIF1 protein was not detectable by immunohistochemistry in tumors with quantitatively high levels of WIF1 methylation. Of note, WIF1 protein was not detectable in two of the seven unmethylated cervical tumors examined, suggesting other mechanisms may contribute WIF1 repression. Our findings establish the WIF1 gene as a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix.

Simultaneous single-molecule mapping of protein-DNA interactions and DNA methylation by MAPit.

Sites of protein binding to DNA are inferred from footprints or spans of protection against a probing reagent. In most protocols, sites of accessibility to a probe are detected by mapping breaks in DNA strands. As discussed in this unit, such methods obscure molecular heterogeneity by averaging cuts at a given site over all DNA strands in a sample population. The DNA methyltransferase accessibility protocol for individual templates (MAPit), an alternative method described in this unit, localizes protein-DNA interactions by probing with cytosine-modifying DNA methyltransferases followed by bisulfite sequencing. Sequencing individual DNA products after amplification of bisulfite-converted sequences permits assignment of the methylation status of every enzyme target site along a single DNA strand. Use of the GC-methylating enzyme M.CviPI allows simultaneous mapping of chromatin accessibility and endogenous CpG methylation. MAPit is therefore the only footprinting method that can detect subpopulations of molecules with distinct patterns of protein binding or chromatin architecture and correlate them directly with the occurrence of endogenous methylation. Additional advantages of MAPit methylation footprinting as well as considerations for experimental design and potential sources of error are discussed.

DNMT1 and DNMT3B modulate distinct polycomb-mediated histone modifications in colon cancer.

DNA methylation patterns are established and maintained by three DNA methyltransferases (DNMT): DNMT1, DNMT3A, and DNMT3B. Although essential for development, methylation patterns are frequently disrupted in cancer and contribute directly to carcinogenesis. Recent studies linking polycomb group repression complexes (PRC1 and PRC2) to the DNMTs have begun to shed light on how methylation is targeted. We identified previously a panel of genes regulated by DNMT3B. Here, we compare these with known polycomb group targets to show that approximately 47% of DNMT3B regulated genes are also bound by PRC1 or PRC2. We chose 44 genes coregulated by DNMT3B and PRC1/PRC2 to test whether these criteria would accurately identify novel targets of epigenetic silencing in colon cancer. Using reverse transcription-PCR, bisulfite genomic sequencing, and pyrosequencing, we show that the majority of these genes are frequently silenced in colorectal cancer cell lines and primary tumors. Some of these, including HAND1, HMX2, and SIX3, repressed cell growth. Finally, we analyzed the histone code, DNMT1, DNMT3B, and PRC2 binding by chromatin immunoprecipitation at epigenetically silenced genes to reveal a novel link between DNMT3B and the mark mediated by PRC1. Taken together, these studies suggest that patterns of epigenetic modifiers and the histone code influence the propensity of a gene to become hypermethylated in cancer and that DNMT3B plays an important role in regulating PRC1 function.

Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas.

DNA hypermethylation-mediated gene silencing is a frequent and early contributor to aberrant cell growth and invasion in cancer. Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Morbidity and mortality are high in glioma patients because tumors are resistant to treatment and are highly invasive into surrounding brain tissue rendering complete surgical resection impossible. Invasiveness is regulated by the interplay between secreted proteases (eg, cathepsins) and their endogenous inhibitors (cystatins). In our previous studies we identified cystatin E/M (CST6) as a frequent target of epigenetic silencing in glioma. Cystatin E/M is a potent inhibitor of cathepsin B, which is frequently overexpressed in glioma. Here, we study the expression of cystatin E/M in normal brain and show that it is highly and moderately expressed in oligodendrocytes and astrocytes, respectively, but not in neurons. Consistent with this, the CST6 promoter is hypomethylated in all normal samples using methylation-specific PCR, bisulfite genomic sequencing, and pyrosequencing. In contrast, 78% of 28 primary brain tumors demonstrated reduced/absent cystatin E/M expression using a tissue microarray and this reduced expression correlated with CST6 promoter hypermethylation. Interestingly, CST6 was expressed in neural stem cells (NSC) and markedly induced upon differentiation, whereas a glioma tumor initiating cell (TIC) line was completely blocked for CST6 expression by promoter methylation. Analysis of primary pediatric brain tumor-derived lines also showed CST6 downregulation and methylation in nearly 100% of 12 cases. Finally, ectopic expression of cystatin E/M in glioma lines reduced cell motility and invasion. These results demonstrate that epigenetic silencing of CST6 is frequent in adult and pediatric brain tumors and occurs in TICs, which are thought to give rise to the tumor. CST6 methylation may therefore represent a novel prognostic marker and therapeutic target specifically altered in TICs.

DNA methyltransferase 3B (DNMT3B) mutations in ICF syndrome lead to altered epigenetic modifications and aberrant expression of genes regulating development, neurogenesis and immune function.

Genome-wide DNA methylation patterns are established and maintained by the coordinated action of three DNA methyltransferases (DNMTs), DNMT1, DNMT3A and DNMT3B. DNMT3B hypomorphic germline mutations are responsible for two-thirds of immunodeficiency, centromere instability, facial anomalies (ICF) syndrome cases, a rare recessive disease characterized by immune defects, instability of pericentromeric satellite 2-containing heterochromatin, facial abnormalities and mental retardation. The molecular defects in transcription, DNA methylation and chromatin structure in ICF cells remain relatively uncharacterized. In the present study, we used global expression profiling to elucidate the role of DNMT3B in these processes using cell lines derived from ICF syndrome and normal individuals. We show that there are significant changes in the expression of genes critical for immune function, development and neurogenesis that are highly relevant to the ICF phenotype. Approximately half the upregulated genes we analyzed were marked with low-level DNA methylation in normal cells that was lost in ICF cells, concomitant with loss of repressive histone modifications, particularly H3K27 trimethylation, and gains in transcriptionally active H3K9 acetylation and H3K4 trimethylation marks. In addition, we consistently observed loss of binding of the SUZ12 component of the PRC2 polycomb repression complex and DNMT3B to derepressed genes, including a number of homeobox genes critical for immune system, brain and craniofacial development. We also observed altered global levels of certain histone modifications in ICF cells, particularly ubiquitinated H2AK119. Therefore, this study provides important new insights into the role of DNMT3B in modulating gene expression and chromatin structure and reveals new connections between DNMT3B and polycomb-mediated repression.