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Inflammatory bowel disease (IBD) is characterized by multiple alterations in cytokine expression and is a risk factor for colon cancer. The Omega class glutathione transferase GSTO1-1 regulates the release of the pro-inflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18) by deglutathionylating NEK7 in the NLRP3 inflammasome. When treated with azoxymethane and dextran sodium sulphate (AOM/DSS) as a model of IBD, Gsto1−/− mice were highly sensitive to colitis and showed a significant increase in the size and number of colon tumours compared with wild-type (WT) mice. Gsto1−/− mice treated with AOM/DSS had significantly lower serum IL-1β and IL-18 levels as well as significantly decreased interferon (IFN)-γ, decreased pSTAT1 and increased pSTAT3 levels in the distal colon compared with similarly treated WT mice. Histologically, AOM/DSS treated Gsto1−/− mice showed increased active chronic inflammation with macrophage infiltration, epithelial dysplasia and invasive adenocarcinoma compared with AOM/DSS treated WT mice. Thus, this study shows that GSTO1-1 regulates IL-1β and IL-18 activation and protects against colorectal cancer formation in the AOM/DSS model of IBD. The data suggest that while GSTO1-1 is a new target for the regulation of the NLRP3 inflammasome-associated cytokines IL-1β and IL-18 by small molecule inhibitors, there is a possibility that anti-inflammatory drugs targeting these cytokines may potentiate colon cancer in some situations.

Glutathione transferase omega-1 (GSTO1-1) is an enzyme whose function supports the activation of interleukin (IL)-1β and IL-18 that are implicated in a variety of inflammatory disease states for which small-molecule inhibitors are sought. The potent reactivity of the active-site cysteine has resulted in reported inhibitors that act by covalent labeling. In this study, structure-activity relationship (SAR) elaboration of the reported GSTO1-1 inhibitor C1-27 was undertaken. Compounds were evaluated for inhibitory activity toward purified recombinant GSTO1-1 and for indicators of target engagement in cell-based assays. As covalent inhibitors, the kinact/KI values of selected compounds were determined, as well as in vivo pharmacokinetics analysis. Cocrystal structures of key novel compounds in complex with GSTO1-1 were also solved. This study represents the first application of a biochemical assay for GSTO1-1 to determine kinact/KI values for tested inhibitors and the most extensive set of cell-based data for a GSTO1-1 inhibitor SAR series reported to date. Our research culminated in the discovery of 25, which we propose as the preferred biochemical tool to interrogate cellular responses to GSTO1-1 inhibition.

Cisplatin is an effective anticancer drug; however, cisplatin use often leads to nephrotoxicity, which limits its clinical effectiveness. In this study, we determined the effect of dichloroacetate, a novel anticancer agent, in a mouse model of cisplatin-induced AKI. Pretreatment with dichloroacetate significantly attenuated the cisplatin-induced increase in BUN and serum creatinine levels, renal tubular apoptosis, and oxidative stress. Additionally, pretreatment with dichloroacetate accelerated tubular regeneration after cisplatin-induced renal damage. Whole transcriptome sequencing revealed that dichloroacetate prevented mitochondrial dysfunction and preserved the energy-generating capacity of the kidneys by preventing the cisplatin-induced downregulation of fatty acid and glucose oxidation, and of genes involved in the Krebs cycle and oxidative phosphorylation. Notably, dichloroacetate did not interfere with the anticancer activity of cisplatin in vivo. These data provide strong evidence that dichloroacetate preserves renal function when used in conjunction with cisplatin.

Glutathione transferase Omega 1 (GSTO1-1) is an atypical GST reported to play a pro-inflammatory role in response to LPS. Here we show that genetic knockout of Gsto1 alters the response of mice to three distinct inflammatory disease models. GSTO1-1 deficiency ameliorates the inflammatory response stimulated by LPS and attenuates the inflammatory impact of a high fat diet on glucose tolerance and insulin resistance. In contrast, GSTO1-1 deficient mice show a more severe inflammatory response and increased escape of bacteria from the colon into the lymphatic system in a dextran sodium sulfate mediated model of inflammatory bowel disease. These responses are similar to those of TLR4 and MyD88 deficient mice in these models and confirm that GSTO1-1 is critical for a TLR4-like pro-inflammatory response in vivo. In wild-type mice, we show that a small molecule inhibitor that covalently binds in the active site of GSTO1-1 can be used to ameliorate the inflammatory response to LPS. Our findings demonstrate the potential therapeutic utility of GSTO1-1 inhibitors in the modulation of inflammation and suggest their possible application in the treatment of a range of inflammatory conditions.

Summary: The NLRP3 inflammasome is a cytosolic complex sensing phagocytosed material and various damage-associated molecular patterns, triggering production of the pro-inflammatory cytokines interleukin-1 beta (IL)-1β and IL-18 and promoting pyroptosis. Here, we characterize glutathione transferase omega 1-1 (GSTO1-1), a constitutive deglutathionylating enzyme, as a regulator of the NLRP3 inflammasome. Using a small molecule inhibitor of GSTO1-1 termed C1-27, endogenous GSTO1-1 knockdown, and GSTO1-1−/− mice, we report that GSTO1-1 is involved in NLRP3 inflammasome activation. Mechanistically, GSTO1-1 deglutathionylates cysteine 253 in NIMA related kinase 7 (NEK7) to promote NLRP3 activation. We therefore identify GSTO1-1 as an NLRP3 inflammasome regulator, which has potential as a drug target to limit NLRP3-mediated inflammation. : NLRP3 inflammasome activation contributes to chronic inflammation associated with autoinflammatory disease, yet understanding of NLRP3 inflammasome regulation is incomplete. Hughes et al. show that the deglutathionylating enzyme GSTO1-1 promotes NLRP3 inflammasome activation through deglutathionylation of NEK7. Furthermore, the GSTO1-1 inhibitor C1-27 reduces NLRP3 inflammasome activation in vitro and in vivo. Keywords: NLRP3 inflammasome, GSTO1-1, glutathione, NEK7, IL-1β, deglutathionylation, pyroptosis

Purpose Glutathione-S-transferase omega 1-1 (GSTO1-1) is a cytosolic glutathione transferase enzyme, involved in glutathionylation, toll-like receptor signaling, and calcium channel regulation. GSTO1-1 dysregulation has been implicated in oxidative stress and inflammation, and contributes to the pathogenesis of several diseases and neurological disorders; however, its role in retinal degenerations is unknown. The aim of this study was to investigate the role of GSTO1-1 in modulating oxidative stress and consequent inflammation in the normal and degenerating retina. Methods The role of GSTO1-1 in retinal degenerations was explored by using Gsto1-/- mice in a model of retinal degeneration. The expression and localization of GSTO1-1 were investigated with immunohistochemistry and Western blot. Changes in the expression of inflammatory (Ccl2, Il-1β, and C3) and oxidative stress (Nox1, Sod2, Gpx3, Hmox1, Nrf2, and Nqo1) genes were investigated via quantitative real-time polymerase chain reaction. Retinal function in Gsto1-/- mice was investigated by using electroretinography. Results GSTO1-1 was localized to the inner segment of cone photoreceptors in the retina. Gsto1-/- photo-oxidative damage (PD) mice had decreased photoreceptor cell death as well as decreased expression of inflammatory (Ccl2, Il-1β, and C3) markers and oxidative stress marker Nqo1. Further, retinal function in the Gsto1-/- PD mice was increased as compared to wild-type PD mice. Conclusions These results indicate that GSTO1-1 is required for inflammatory-mediated photoreceptor death in retinal degenerations. Targeting GSTO1-1 may be a useful strategy to reduce oxidative stress and inflammation and ameliorate photoreceptor loss, slowing the progression of retinal degenerations.

Glutathione transferase kappa (GSTK1-1) is a highly conserved, mitochondrial enzyme potentially involved in redox reactions. GSTK1-1-deficient mice were generated to further study the enzyme's biological role. Reduced and total glutathione levels in liver and kidney were unchanged by GSTK1-1 deficiency and NADPH quinone oxidoreductase 1 expression was not elevated indicating that there is no general underlying oxidative stress in Gstk1(-/-) mice. Electron microscopy of liver and kidney showed no changes in mitochondrial morphology with GSTK1-1 deficiency. The death of a number of Gstk1(-/-) males with urinary tract problems prompted close examination of the kidneys. Electron microscopy revealed glomerular basement membrane changes at 3 months, accompanied by detectable microalbuminuria in male mice (albumin:creatinine ratio of 2.66±0.83 vs 1.13±0.20 mg/mmol for Gstk1(-/-) and wild-type (WT), respectively, P=0.001). This was followed by significant foot process effacement (40-55% vs 10% for Gstk1(-/-) and WT, respectively) at 6 months of age in all Gstk1(-/-) mice examined. Kidney tubules were ultrastructurally normal. Compared with human disease, the Gstk1(-/-) kidneys show changes seen in glomerulopathies causing nephrotic syndrome. Gstk1(-/-) mice may offer insights into the early development of glomerular nephropathies.

Background Sarcomas are cancers that arise from tissues of mesenchymal origin and there has been limited improvement in treatments over the last 30 years. The Warburg effect is a widespread metabolic phenotype of cancer, where glycolysis is favoured despite the presence of oxygen. Dichloroacetate (DCA) is a pyruvate dehydrogenase kinase (PDK) inhibitor in clinical use that can reverse the Warburg effect, inhibiting growth and enhancing apoptosis in a range of cancers. We have investigated its effectiveness against sarcoma cells in vitro and in vivo. Materials and methods Three cell lines (mouse fibrosarcoma S180, mouse osteosarcoma K7M2 and human fibrosarcoma HT1080-luc2) were examined for cell viability after DCA treatment in vitro (neutral red uptake assay), alone and in combination with doxorubicin. In vivo, K7M2 cells were injected s.c. into Rag1 -/- mice (2 sites per mouse, 7-9 mice per group) and established tumours were treated with DCA in the drinking water (0, 0.5, 1.0 and 1.5 g/L, delivering 0, 70, 125 and 165 mg/kg/day, respectively). Tumour growth was monitored with callipers. Plasma was collected on d1 and d15 for measurement of DCA levels (LC-MS). Results DCA significantly reduced the total viable cell number after 48 h of treatment in the mouse sarcoma lines (~15% at 0.5 mM DCA, and 30-40% at 5 mM DCA), however HT1080-luc2 cells showed on ly a1 0% reduction in cell number with 5 mM DCA. There was no morphological indication of apoptosis, suggesting DCA was decreasing proliferation. Chronic treatment of the mouse cells (5 mM DCA for 2 weeks) resulted in significantly slower growth rates as measured over 48 h (7 and 13% total cell number compared to untreated S180 and K7M2 cells, respectively). DCA did not synergise with doxorubicin but was additive at lower concentrations of doxorubicin. In vivo ,1 .0 and 1.5 g/L DCA significantly reduced tumour growth (33.0 and 33.1% reduction in tumour size on d13, p=0.001 and 0.01 respectively). Plasma DCA was undetectable on d1 of treatment, but by d15, 1.0 and 1.5 g/L DCA delivered 2-44 uM. Tumour DCA concentrations were also measured and found to be in the range of 25-470 uM, much lower than those typically used in in vitro studies. Conclusions DCA was effective against an in vivo sarcoma model, with tumour DCA concentrations in the micromolar range. These concentrations are achievable clinically, thus DCA warrants further investigation for sarcoma treatment.

Supported by NHMRC Career Development Award, National Breast Cancer Foundation Novel Concept Award, and Cancer Australia.

Dichloroacetate (DCA) has recently been proposed as a novel non-toxic anticancer agent that can reverse the glycolytic phenotype in cancer cells through the inhibition of pyruvate dehydrogenase kinase. Aims and Methods Three mouse cancer cell lines (V14, 4T1 and B16) were used to examine the histological changes caused by DCA in vivo. V14 or 4T1 mammary adenocarcinoma cells were injected subcutaneously and B16 melanoma cells were injected intravenously. Mice were treated with 0.5–2.0 g/L DCA in the drinking water. Results Growth of established V14 tumours was halted by DCA treatment (28% reduction in size, 1.5 g/L for 9 days, n = 8 per group, p = 0.08). Treatment from the day of cell injection resulted in fewer tumours being established (5/8 sites in treated mice vs 16/16 untreated, p = 0.03) and complete regression of one tumour. Histological assessment found DCA treatment resulted in increased tumour-infiltrating lymphocytes (TILs) but no change in mitotic rate, fibrosis or necrosis. Tumours from the 4T1 or B16 cell lines showed no growth inhibition and no evidence of TILs, correlating with a lack of sensitivity to growth inhibition in vitro. Conclusions In V14 tumours DCA appears to prevent or delay breast cancer formation either through growth inhibition or enhanced immune function.

The Warburg effect occurs in 90% of tumours, where there is a high rate of glycolysis even in the presence of oxygen. This results in increased lactate production and reduced pyruvate oxidation. Dichloroacetate (DCA) is used clinically for congenital lactic acidosis and can reverse the Warburg effect by inhibiting the pyruvate dehydrogenase kinases (PDKs), activating pyruvate dehydrogenase thus promoting oxidative metabolism of pyruvate. The PDKs have different sensitivities towards DCA inhibition (PDK2>PDK4>PDK1>>PDK3). We have investigated the effects of DCA on cancer growth in vivo and in vitro, and examined the expression of the PDK isoforms to explain variation of cancer cell responses to DCA. DCA halted the growth of established V14 mouse mammary tumours in vivo, but was ineffective against 4T1 mouse mammary tumours. In 8 human cancer cell lines of breast, colon, pancreatic and prostate origin, 5 mM DCA (48 hr) inhibited growth by 5-40% in vitro. Western blotting for expression of PDK isoforms revealed that the most sensitive T-47D cells expressed PDK2 and low levels of the other PDKs, whereas less sensitive cells expressed high levels of PDK1 and/or PDK3. In siRNA knockdown (kd) experiments, PDK3-kd in MCF7 cells (low DCA sensitivity, high PDK3 levels) increased sensitivity to DCA by 30%, while DCA did not further inhibit growth of PDK2-kd T-47D cells, confirming that sensitivity to DCA growth inhibition is determined by the PDK profiles. PDK1 & 3 are up-regulated in hypoxia, which may reduce the effectiveness of DCA. To the contrary, DCA increased apoptosis in hypoxia in MCF7 cells, suggesting that DCA can enhance effects of other stressors/drugs, an area of ongoing investigation. Use of the PDK profiles to target DCA sensitive tumours will improve the outcomes of clinical trials using DCA that are currently underway Citation Format: Bevan P. Gang, Melissa Rooke, Sheenu Mishra, Jane E. Dahlstrom, Anneke C. Blackburn, Cancer Metabolism and Genetics Group. Sensitivity to dichloroacetate is determined by PDK expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5421. doi:10.1158/1538-7445.AM2013-5421 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

While genetic testing for familial cancer susceptibility factors has excelled in recent years, the prevention options for those carrying high risk alleles have not. Altered bioenergetics has now been acknowledged as an emerging hallmark of cancer, and is believed to be a critical characteristic acquired during tumorigenesis. Several very safe drugs are available that can modify bioenergetics. Dichloroacetate (DCA) inactivates pyruvate dehydrogenase kinase, resulting in activation of pyruvate dehydrogenase, reduced lactic acid production and increased mitochondrial activity. Metformin, a type 2 diabetes treatment which activates AMPK, thereby inhibiting mTOR, has unambiguously been demonstrated to reduce the risk of many cancer types in diabetics where insulin treatments do not. We have tested these drugs as chemopreventive agents against the mammary tumours that occur in the spontaneous BALB/c-Trp53+/- mouse tumour model. In vitro studies on breast cancer cell lines indicate that DCA (1-5 mM), metformin (30-300 uM) or the combination of the two can significantly inhibit breast cancer cell growth over 4 days of treatment. These results support the possibility that DCA and metformin could prevent or delay breast cancer formation in BALB/c-Trp53+/- mice. To examine this, four groups of female BALB/c-Trp53+/- mice were given distilled water (n=75), DCA (1.5 g/L in drinking water, ~180 mg/kg/day, n=53), metformin (0.25 g/L in drinking water, ~30 mg/kg/day, n=61) or DCA + metformin (n=51) from 8 weeks of age, and monitored for tumour development over 78 weeks. The overall tumour-free survival curves were not significantly different (Kaplan-Meier analysis) between the treatment groups, suggesting that metformin does not reduce cancer risk in non-diabetics. However, the occurrence of mammary tumours in the four groups was altered. DCA reduced the number and increased the latency of mammary tumours (20.8% of DCA treated mice with mean latency of 63.8 weeks compared to 28.0% of untreated mice with mean latency of 55.0 weeks), whereas metformin had no effect (26.2% of mice, average latency 54.7 weeks). Examining the mammary tumour-free survival curves, DCA appeared to eliminate the early onset mammary tumours (latency