Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism

Although the immunomodulatory and cytoprotective properties of itaconate have been studied extensively, it is not known whether its naturally occurring isomers mesaconate and citraconate have similar properties. Here, we show that itaconate is partially converted to mesaconate intracellularly and that mesaconate accumulation in macrophage activation depends on prior itaconate synthesis. When added to human cells in supraphysiological concentrations, all three isomers reduce lactate levels, whereas itaconate is the strongest succinate dehydrogenase (SDH) inhibitor. In cells infected with influenza A virus (IAV), all three isomers profoundly alter amino acid metabolism, modulate cytokine/chemokine release and reduce interferon signalling, oxidative stress and the release of viral particles. Of the three isomers, citraconate is the strongest electrophile and nuclear factor-erythroid 2-related factor 2 (NRF2) agonist. Only citraconate inhibits catalysis of itaconate by cis-aconitate decarboxylase (ACOD1), probably by competitive binding to the substrate-binding site. These results reveal mesaconate and citraconate as immunomodulatory, anti-oxidative and antiviral compounds, and citraconate as the first naturally occurring ACOD1 inhibitor

Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism

Although the immunomodulatory and cytoprotective properties of itaconate have been studied extensively, it is not known whether its naturally occurring isomers mesaconate and citraconate have similar properties. Here, we show that itaconate is partially converted to mesaconate intracellularly and that mesaconate accumulation in macrophage activation depends on prior itaconate synthesis. When added to human cells in supraphysiological concentrations, all three isomers reduce lactate levels, whereas itaconate is the strongest succinate dehydrogenase (SDH) inhibitor. In cells infected with influenza A virus (IAV), all three isomers profoundly alter amino acid metabolism, modulate cytokine/chemokine release and reduce interferon signalling, oxidative stress and the release of viral particles. Of the three isomers, citraconate is the strongest electrophile and nuclear factor-erythroid 2-related factor 2 (NRF2) agonist. Only citraconate inhibits catalysis of itaconate by cis-aconitate decarboxylase (ACOD1), probably by competitive binding to the substrate-binding site. These results reveal mesaconate and citraconate as immunomodulatory, anti-oxidative and antiviral compounds, and citraconate as the first naturally occurring ACOD1 inhibitor. [Image: see text

Fine-mapping of the HNF1B multicancer locus identifies candidate variants that mediate endometrial cancer risk.

Common variants in the hepatocyte nuclear factor 1 homeobox B (HNF1B) gene are associated with the risk of Type II diabetes and multiple cancers. Evidence to date indicates that cancer risk may be mediated via genetic or epigenetic effects on HNF1B gene expression. We previously found single-nucleotide polymorphisms (SNPs) at the HNF1B locus to be associated with endometrial cancer, and now report extensive fine-mapping and in silico and laboratory analyses of this locus. Analysis of 1184 genotyped and imputed SNPs in 6608 Caucasian cases and 37 925 controls, and 895 Asian cases and 1968 controls, revealed the best signal of association for SNP rs11263763 (P = 8.4 × 10(-14), odds ratio = 0.86, 95% confidence interval = 0.82-0.89), located within HNF1B intron 1. Haplotype analysis and conditional analyses provide no evidence of further independent endometrial cancer risk variants at this locus. SNP rs11263763 genotype was associated with HNF1B mRNA expression but not with HNF1B methylation in endometrial tumor samples from The Cancer Genome Atlas. Genetic analyses prioritized rs11263763 and four other SNPs in high-to-moderate linkage disequilibrium as the most likely causal SNPs. Three of these SNPs map to the extended HNF1B promoter based on chromatin marks extending from the minimal promoter region. Reporter assays demonstrated that this extended region reduces activity in combination with the minimal HNF1B promoter, and that the minor alleles of rs11263763 or rs8064454 are associated with decreased HNF1B promoter activity. Our findings provide evidence for a single signal associated with endometrial cancer risk at the HNF1B locus, and that risk is likely mediated via altered HNF1B gene expression

Pre-Absorbed Immunoproteomics: A Novel Method for the Detection of Streptococcus suis Surface Proteins

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that can cause infections in pigs and humans. Bacterial surface proteins are often investigated as potential vaccine candidates and biomarkers of virulence. In this study, a novel method for identifying bacterial surface proteins is presented, which combines immunoproteomic and immunoserologic techniques. Critical to the success of this new method is an improved procedure for generating two-dimensional electrophoresis gel profiles of S. suis proteins. The S. suis surface proteins identified in this study include muramidase-released protein precursor (MRP) and an ABC transporter protein, while MRP is thought to be one of the main virulence factors in SS2 located on the bacterial surface. Herein, we demonstrate that the ABC transporter protein can bind to HEp-2 cells, which strongly suggests that this protein is located on the bacterial cell surface and may be involved in pathogenesis. An immunofluorescence assay confirmed that the ABC transporter is localized to the bacterial outer surface. This new method may prove to be a useful tool for identifying surface proteins, and aid in the development of new vaccine subunits and disease diagnostics

Activity of the multikinase inhibitor dasatinib against ovarian cancer cells

BackgroundHere, we explore the therapeutic potential of dasatinib, a small-molecule inhibitor that targets multiple cytosolic and membrane-bound tyrosine kinases, including members of the Src kinase family, EphA2, and focal adhesion kinase for the treatment of ovarian cancer.MethodsWe examined the effects of dasatinib on proliferation, invasion, apoptosis, cell-cycle arrest, and kinase activity using a panel of 34 established human ovarian cancer cell lines. Molecular markers for response prediction were studied using gene expression profiling. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions with chemotherapeutic drugs.ResultsConcentration-dependent anti-proliferative effects of dasatinib were seen in all ovarian cancer cell lines tested, but varied significantly between individual cell lines with up to a 3 log-fold difference in the IC(50) values (IC(50) range: 0.001-11.3 micromol l(-1)). Dasatinib significantly inhibited invasion, and induced cell apoptosis, but less cell-cycle arrest. At a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for dasatinib plus carboplatin (mean CI values, range: 0.73-1.11) or paclitaxel (mean CI values, range: 0.76-1.05). In this study, 24 out of 34 (71%) representative ovarian cancer cell lines were highly sensitive to dasatinib, compared with only 8 out of 39 (21%) representative breast cancer cell lines previously reported. Cell lines with high expression of Yes, Lyn, Eph2A, caveolin-1 and 2, moesin, annexin-1, and uPA were particularly sensitive to dasatinib.ConclusionsThese data provide a clear biological rationale to test dasatinib as a single agent or in combination with chemotherapy in patients with ovarian cancer

Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations

Candidate locus analysis of the TERT-CLPTM1L cancer risk region on chromosome 5p15 identifies multiple independent variants associated with endometrial cancer risk.

Several studies have reported associations between multiple cancer types and single-nucleotide polymorphisms (SNPs) on chromosome 5p15, which harbours TERT and CLPTM1L, but no such association has been reported with endometrial cancer. To evaluate the role of genetic variants at the TERT-CLPTM1L region in endometrial cancer risk, we carried out comprehensive fine-mapping analyses of genotyped and imputed SNPs using a custom Illumina iSelect array which includes dense SNP coverage of this region. We examined 396 SNPs (113 genotyped, 283 imputed) in 4,401 endometrial cancer cases and 28,758 controls. Single-SNP and forward/backward logistic regression models suggested evidence for three variants independently associated with endometrial cancer risk (P = 4.9 × 10(-6) to P = 7.7 × 10(-5)). Only one falls into a haplotype previously associated with other cancer types (rs7705526, in TERT intron 1), and this SNP has been shown to alter TERT promoter activity. One of the novel associations (rs13174814) maps to a second region in the TERT promoter and the other (rs62329728) is in the promoter region of CLPTM1L; neither are correlated with previously reported cancer-associated SNPs. Using TCGA RNASeq data, we found significantly increased expression of both TERT and CLPTM1L in endometrial cancer tissue compared with normal tissue (TERT P = 1.5 × 10(-18), CLPTM1L P = 1.5 × 10(-19)). Our study thus reports a novel endometrial cancer risk locus and expands the spectrum of cancer types associated with genetic variation at 5p15, further highlighting the importance of this region for cancer susceptibility.This work was supported by the NHMRC Project Grant (ID#1031333). This work was also supported by Cancer Research UK (C1287/A10118, C1287/A 10710, C12292/A11174, C1281/A12014, C5047/A8384, C5047/A15007, C5047/A10692)This is the published version. It first appeared at http://link.springer.com/article/10.1007%2Fs00439-014-1515-4