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Macrophage migration inhibitory factor (MIF1) is a pleiotropic cytokine involved in inflammation and cancer. Genetic knockout of Mif1 in the validated N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) model of bladder cancer (BCa) resulted in stage arrest at non-muscle invasive disease in prior studies. Small molecule inhibition of MIF1 reduced cancer-associated outcomes however it did not fully recapitulate genetic models. D-dopachrome tautomerase (gene name DDT - commonly referred to as MIF2) is a functional homologue of MIF1, and both MIF1 and MIF2 can bind the cell surface receptor CD74 on multiple cell types to initiate a signaling cascade. This interaction has been proposed to mediate part of the pro-tumorigenic effects of MIF1 and MIF2 and may explain the discordance in prior studies. We hypothesized MIF2 functions redundantly with MIF1 in BCa development and progression. TCGA analysis indicated MIF and DDT expression were increased in BCa patients compared to control. 4-IPP, a combined MIF1/MIF2 inhibitor, was more efficacious than ISO-1, a MIF1 only inhibitor, in preventing cellular proliferation in BCa cell lines. To evaluate these findings in vivo, WT and Mif1(−/−) animals were exposed to 0.05% BBN in drinking water for 16 weeks to initiate tumorigenesis, then evaluated over the subsequent 4 weeks for tumor formation and progression in the presence or absence of 4-IPP. 4-IPP reduced bladder weights in WT animals and bladder weights/tumor stage in Mif1(−/−) animals. To determine if MIF1/MIF2 function through CD74 in BCa, WT or Cd74(−/−) animals were used in the same BBN model. While these animals were partially protected against BBN induced BCa, 4-IPP did not enhance this effect. In conclusion, our data suggest MIF2 mechanistically functions in a similar pro-tumorigenic manner to MIF1, and this is at least partially through CD74. Dual inhibition of MIF homologues is more efficacious at reducing tumor burden in this model of BCa.

Acetaminophen (APAP) overdose causes liver injury in animals and humans. Although well-studied in animals, limited longitudinal data exist on cytokine release after APAP overdose in patients. The purpose of this study was to quantify concentrations of cytokines in APAP overdose patients to determine if early cytokine or complement measurements can distinguish between surviving and non-surviving patients. Plasma was obtained from healthy controls, APAP overdose patients with no increase in liver transaminases, and surviving and non-surviving APAP overdose patients with severe liver injury. Interleukin-10 (IL-10), and CC chemokine ligand-2 (CCL2, MCP-1) were substantially elevated in surviving and non-surviving patients, whereas IL-6 and CXC chemokine ligand-8 (CXCL8, IL-8) had early elevations in a subset of patients only with liver injury. Day 1 IL-10 and IL-6 levels, and Day 2 CCL2, levels correlated positively with survival. There was no significant increase in IL-1α, IL-1β or TNF-α in any patient during the first week after APAP. Monitoring cytokines such as CCL2 may be a good indicator of patient prognosis; furthermore, these data indicate the inflammatory response after APAP overdose in patients is not mediated by a second phase of inflammation driven by the inflammasome.

Advanced bladder cancer remains a major source of mortality, with poor treatment options. Cisplatin-based chemotherapy is the standard treatment, however many patients are or become resistant. One potential cause of chemoresistance is the Warburg effect, a metabolic switch to aerobic glycolysis that occurs in many cancers. Upregulation of the pyruvate dehydrogenase kinase family (PDK1–PDK4) is associated with aerobic glycolysis and chemoresistance through inhibition of the pyruvate dehydrogenase complex (PDH). We have previously observed upregulation of PDK4 in high-grade compared with low-grade bladder cancers. We initiated this study to determine if inhibition of PDK4 could reduce tumor growth rates or sensitize bladder cancer cells to cisplatin. Upregulation of PDK4 in malignant bladder cancer cell lines as compared with benign transformed urothelial cells was confirmed using qPCR. Inhibition of PDK4 with dichloroacetate (DCA) resulted in increased PDH activity, reduced cell growth, and G0–G1 phase arrest in bladder cancer cells. Similarly, siRNA knockdown of PDK4 inhibited bladder cancer cell proliferation. Cotreatment of bladder cancer cells with cisplatin and DCA did not increase caspase-3 activity but did enhance overall cell death in vitro. Although daily treatment with 200 mg/kg DCA alone did not reduce tumor volumes in a xenograft model, combination treatment with cisplatin resulted in dramatically reduced tumor volumes as compared with either DCA or cisplatin alone. This was attributed to substantial intratumoral necrosis. These findings indicate inhibition of PDK4 may potentiate cisplatin-induced cell death and warrant further studies investigating the mechanism through which this occurs. Mol Cancer Ther; 17(9); 2004–12. ©2018 AACR.

Relative expression of HIF-1a and PPARa

Combination Index

Pyruvate metabolism is critical for all mammalian cells. The pyruvate dehydrogenase complex couples the pyruvate formed as the primary product of glycolysis to the formation of acetyl-CoA required as the primary substrate of the citric acid cycle. Dysregulation of this coupling contributes to alterations in metabolic flexibility in obesity, diabetes, cancer, and more. The pyruvate dehydrogenase kinase family of isozymes phosphorylate and inactive the pyruvate dehydrogenase complex in the mitochondria. This function makes them critical mediators of mitochondrial metabolism and drug targets in a number of disease states. The liver expresses multiple PDKs, predominantly PDK1 and PDK2 in the fed state and PDK1, PDK2, and PDK4 in the starved and diabetic states. PDK4 undergoes substantial transcriptional regulation in response to a diverse array of stimuli in most tissues. PDK2 has received less attention than PDK4 potentially due to the dramatic changes in transcriptional gene regulation. However, PDK2 is more responsive than the other PDKs to feedforward and feedback regulation by substrates and products of the pyruvate dehydrogenase complex. Although underappreciated, this makes PDK2 particularly important for the minute-to-minute fine control of the pyruvate dehydrogenase complex and a major contributor to metabolic flexibility. The purpose of this review is to characterize the underappreciated role of PDK2 in liver metabolism. We will focus on known biological actions and physiological roles as well as what roles PDK2 may play in disease states. We will also define current inhibitors and address their potential as therapeutic agents in the future.

Ciclopirox (CPX) is an FDA-approved topical antifungal agent that has demonstrated preclinical anticancer activity in a number of solid and hematologic malignancies. Its clinical utility as an oral anticancer agent, however, is limited by poor oral bioavailability and gastrointestinal toxicity. Fosciclopirox, the phosphoryloxymethyl ester of CPX (Ciclopirox Prodrug, CPX-POM), selectively delivers the active metabolite, CPX, to the entire urinary tract following parenteral administration. We characterized the activity of CPX-POM and its major metabolites in in vitro and in vivo preclinical models of high-grade urothelial cancer. CPX inhibited cell proliferation, clonogenicity and spheroid formation, and increased cell cycle arrest at S and G0/G1 phases. Mechanistically, CPX suppressed activation of Notch signaling. Molecular modeling and cellular thermal shift assays demonstrated CPX binding to γ-secretase complex proteins Presenilin 1 and Nicastrin, which are essential for Notch activation. To establish in vivo preclinical proof of principle, we tested fosciclopirox in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) mouse bladder cancer model. Once-daily intraperitoneal administration of CPX-POM for four weeks at doses of 235 mg/kg and 470 mg/kg significantly decreased bladder weight, a surrogate for tumor volume, and resulted in a migration to lower stage tumors in CPX-POM treated animals. This was coupled with a reduction in the proliferation index. Additionally, there was a reduction in Presenilin 1 and Hes-1 expression in the bladder tissues of CPX-POM treated animals. Following the completion of the first-in-human Phase 1 trial (NCT03348514), the pharmacologic activity of fosciclopirox is currently being characterized in a Phase 1 expansion cohort study of muscle-invasive bladder cancer patients scheduled for cystectomy (NCT04608045) as well as a Phase 2 trial of newly diagnosed and recurrent urothelial cancer patients scheduled for transurethral resection of bladder tumors (NCT04525131).

International audience; Ageing is correlated with elevated bladder cancer incidence, morbidity and mortality. Advanced age is also associated with elevated markers of chronic inflammation and perturbations in gut and urinary tract microbiota. One reason for the increased incidence and mortality of bladder cancer in the elderly might be that age-associated changes in multiple microbiomes induce systemic metabolic changes that contribute to immune dysregulation with potentially tumorigenic effects. The gut and urinary microbiomes could be dysregulated in bladder cancer, although the effect of these changes is poorly understood. Each of these domains - the immune system, gut microbiome and urinary microbiome - might also influence the response of patients with bladder cancer to treatment. Improved understanding of age-related alterations to the immune system and gut and urinary microbiomes could provide possible insight into the risk of bladder cancer development and progression in the elderly. In patients with bladder cancer, improved understanding of microbiota might also provide potential targets for therapeutic intervention.

Neoadjuvant cisplatin based chemotherapy is considered standard of care for patients with locally advanced bladder cancer (BCa). However, upwards of 50% of patients are ineligible due to poor performance status, underlying kidney disease, peripheral neuropathy, hearing loss and/or cardiac disease. There are currently no accepted alternative regimens with significant activity, and many cisplatin ineligible patients usually proceed directly to surgery. Prior research in other cancers has indicated intravenous vitamin C (IVC), or ascorbate, may serve as a beneficial adjuvant to less toxic therapeutic regimens. We initiated a single arm, Simon 2-stage, Window of Opportunity trial using high dose IVC with single cycle gemcitabine/carboplatin (GCa) (NCT04046094). The primary outcome was pathologic downstaging. Twelve patients with newly diagnosed CI-MIBC were enrolled. Patients received single cycle GCa and IVC titrated to 350-400 mg/dL for ~21 days followed by cystectomy after ~4-6 weeks. As reported, pathological downstaging ( Citation Format: Benjamin L. Woolbright, Ganeshkumar Rajendran, Erika Abbott, Zeb Zacharias, Jon Houtman, Rahul Parikh, Qi Chen, Ameer Hamza, Elizabeth Wulff-Burchfield, Jeffrey M. Holzbeierlein, Jeanne Drisko, Michael D. Henry, John A. Taylor. Intravenous vitamin C therapy for cisplatin ineligible bladder cancer patients (CI-MIBC): Early investigation into potential pharmacodynamic biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2119.

Cholestasis results in blockage of bile flow whether the point of obstruction occurs extrahepatically or intrahepatically. Bile acids are a primary constituent of bile, and thus one of the primary outcomes is acute retention of bile acids in hepatocytes. Bile acids are normally secreted into the biliary tracts and then released into the small bowel before recirculating back to the liver. Retention of bile acids has long been hypothesized to be a primary cause of the associated liver injury that occurs during acute or chronic cholestasis. Despite this, a surge of papers in the last decade have reported a primary role for inflammation in the pathophysiology of cholestatic liver injury. Furthermore, it has increasingly been recognized that both the constituency of individual bile acids that make up the greater pool, as well as their conjugation status, is intimately involved in their toxicity, and this varies between species. Finally, the role of bile acids in drug-induced cholestatic liver injury remains an area of increasing interest. The purpose of this review is to critically evaluate current proposed mechanisms of cholestatic liver injury, with a focus on the evolving role of bile acids in cell death and inflammation.

Cancer cells use alterations of normal metabolic processes to sustain proliferation indefinitely. Transcriptional and posttranscriptional control of the pyruvate dehydrogenase kinase (PDK) family is one way in which cancer cells alter normal pyruvate metabolism to fuel proliferation. PDKs can phosphorylate and inactivate the pyruvate dehydrogenase complex (PDHC), which blocks oxidative metabolism of pyruvate by the mitochondria. This process is thought to enhance cancer cell growth by promoting anabolic pathways. Inhibition of PDKs induces cell death through increased PDH activity and subsequent increases in ROS production. The use of PDK inhibitors has seen widespread success as a potential therapeutic in laboratory models of multiple cancers; however, gaps still exist in our understanding of the biology of PDK regulation and function, especially in the context of individual PDKs. Efforts are currently underway to generate PDK-specific inhibitors and delineate the roles of individual PDK isozymes in specific cancers. The goal of this review is to understand the regulation of the PDK isozyme family, their role in cancer proliferation, and how to target this pathway therapeutically to specifically and effectively reduce cancer growth.

Trisomy 21, Down Syndrome, results in a constellation of symptoms including increased risk for leukemia. In contrast, mounting evidence indicates that people with Down Syndrome have lower rates of numerous solid tumors, particularly carcinogen driven cancers, even when adjusted for age. The mechanisms that dictate why this occurs are not well understood but could potentially yield actionable targets for treating or preventing cancer formation. We hypothesized Ts65Dn mice, a murine model of Down Syndrome, would be protected against BCa formation and could be evaluated as a genetic basis for the discovery of novel therapeutic targets in bladder cancer. Using the well characterized N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) chemical carcinogen bladder cancer model we initiated tumors in Ts65Dn and WT mice. After 16 weeks BBN exposure, TS65Dn mice were found to have smaller bladder tumors by weight with a trend toward lower stages as compared to WT animals. RNA-sequencing was used to assess broad genomic changes in the tumors. Reactome analysis of the RNA-sequencing data indicated pathways associated with mitosis and the cell cycle were most affected in Ts65Dn mice, including downregulation of pathways associated with Aurora Kinase B (AURKB). AURKB is a pro-tumorigenic kinase known to enhance cell survival and proliferation. Bioinformatics analysis indicates that while AURKB is broadly expressed in most tissues, it is potently upregulated in bladder cancers, offering a potential therapeutic target. Using the AURKB specific inhibitor barasertib-HQPA we evaluated drug sensitivity in a set of BCa cell lines. Cell lines associated with a basal subtype gene signature (5637, HT-1376) were largely susceptible to barasertib with IC50 values in nM concentrations, whereas other BCa cell lines (HTB-5, T24) had IC50 values nearly 1,000X higher. We next assessed histone H3 phosphorylation, a known substrate of AURKB, to validate drug efficacy. Interestingly, inhibition of histone H3 phosphorylation was noted in all cell lines at nM concentrations, indicating that while barasertib is functionally active in both sets of cells, the anti-proliferative activity is not dependent upon phosphorylation of histone H3, but rather other factors that we have yet to determine. In conclusion, we have found Ts65Dn mice have reduced tumor formation when exposed to the BCa carcinogen BBN and furthermore, have identified AURKB as a potential therapeutic target. Use of Ts65Dn mice may be a relevant model for identifying potentially undefined tumorigenic agents in multiple cancers including bladder. Citation Format: Binh Vo, Erika Abbott, Carolyn J. Vivian, Xena Moore, Ganeshkumar Rajendran, Katie Dennis, Benjamin L. Woolbright, Scott J. Weir, John A. Taylor. Ts65Dn Down syndrome mice reveal a potential therapeutic vulnerability in a BBN bladder cancer model and subset of bladder cancer cell lines [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 3991.

Agent Blue and Agent Orange were defoliants used in high quantities during Vietnam era conflicts in Southeast Asia. The US Department of Veterans Affairs has recently recognized that exposure to both Agent Blue and Agent Orange can potentially increase risk of bladder cancer (BCa) and has added BCa to the list of diseases for which Veterans can seek military benefits. Agent Blue contained significant quantities of organic arsenic and although arsenic is a recognized carcinogen, the understanding of how arsenic affects BCa is still unclear in the literature. Moreover, the effects of the Agent Orange contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on BCa remains largely unstudied, especially in populations that were exposed during this period. We hypothesized chronic exposure to carcinogens TCDD or organic arsenic in the form of cacodylic acid (CA) would enhance cancer associated phenotypes. We treated benign urothelial cells (UROtsa and SV-HUC-1 cells) with low doses of cacodylic acid (0.05-5µM) or TCDD (0.03 - 3nM) for up to 6 months. While we found minor differences in proliferation rates there was no consistent trend across samples in either cell line nor was cellular migration affected. Because benign urothelial cells represent a transformed cell line and not true urothelial cells, we next wanted to assess how CA affected BCa formation in mice using the validated N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced BCa model. TCDD was omitted from this step as its carcinogenicity in mice is understood to dramatically exceed human levels and thus is not relevant to human data. To approximate early-life exposure akin to what Vietnam Era Veterans may have experienced, we treated C57Bl/6J mice with BBN for 16 weeks or 20 weeks with or without increasing concentrations of cacodylic acid (5, 10, 50ppm) for 4 weeks during BBN treatment, starting at week 4 and ending at week 8, and evaluated outcomes associated with BCa formation and progression. While previous reports have found CA promotes BBN induced BCa, we found co-treatment with CA had no effect at 16 weeks post BBN, but reduced both bladder weights (a surrogate for tumor volume) and tumor stage after 20 weeks. RNA-sequencing was used to determine how CA affected the transcriptome of BBN induced tumors. Differential gene expression and Reactome analysis indicated the predominant changes were associated with alterations in RNA processing and translation, EGFR signaling and more. Our work thus far indicates that the ability of TCDD/CA exposure to promote BCa formation is likely context dependent. Future studies will enhance our understanding of how CA interacts with BBN acutely, and attempt to define mechanisms of how arsenic may protect against BBN induced BCa in experimental models, and whether this is clinically relevant for Vietnam Era Veterans. Citation Format: Ganeshkumar Rajendron, Erika Abbott, Jeffery Thompson, Shachi Patel, Aaron Barchowsky, Katie Dennis, Benjamin L. Woolbright, John A. Taylor. Effect of Agent Orange and Agent Blue derived carcinogens on bladder cancer cell lines and a murine bladder cancer model [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 224.

e16540 Background: Neo-adjuvant cisplatin-based chemotherapy (NAC) is considered standard of care for patients with locally advanced disease. However, ̃40% of patients are cisplatin ineligible (CI) due to renal insufficiency, hearing loss or poor performance status. Gemcitabine and carboplatin (GCa) has limited success in this setting. Patients usually proceed directly to cystectomy without realizing the potential survival benefit afforded by NAC. Intravenous ascorbate (vitamin C) administration (IVC) has been shown to improve the efficacy of carboplatin and gemcitabine-based therapy in other models. This single-arm, Simon 2-stage, window of opportunity trial included IVC with single cycle GCa to evaluate pathologic downstaging. We report on the interim first stage analysis of 12 patients. Methods: Patients with newly diagnosed CI-MIBC were enrolled and received single cycle GCa and IVC titrated to peak plasma concentration of 350 to 400 mg/dL (̃20 mM) for 21 days followed by cystectomy at 4-6 weeks from initiation of treatment. The primary outcome is pathological stage at cystectomy. Patients are then followed per NCCN guidelines with standard of care bloodwork, physical exam and imaging studies until progression and/or death. QOL is being evaluated by Functional Assessment of Cancer Therapy-Bladder (FACT-Bl). Results: All 12 patients completed GCa/IVC with 11 having had a cystectomy and 1 pending surgery. Pathological downstaging (yp < T2) was noted in 4 patients with 3 CRs (ypT0N0Mx) and 1 with residual ypTisN0Mx only. Of note, 1 CR was seen in a patient with locally advanced plasmacytoid variant. Participants tolerated treatment well with minimal treatment related AE/SAEs. Conclusions: Interim analysis of GCa-IVC NAC shows good tolerability with > 36% rate of downstaging. Of those with a pathological response, 75% achieved a CR. Continuation criteria has been met for stage 2. FACT-BI analysis and clinical follow up is ongoing and will be reported at study completion. Clinical trial information: NCT04046094.

e16557 Background: Fosciclopirox (F) is being developed for the treatment of non-muscle invasive (NMIBC) and muscle invasive (MIBC) bladder cancer. F is a prodrug which is rapidly and completely metabolized in blood to its active metabolite, ciclopirox (CPX). In preclinical models of bladder cancer, CPX acts in part as a γ-secretase inhibitor by binding to γ-secretase complex proteins Presenilin 1 and Nicastrin, resulting in Notch and Wnt inhibition. The F Recommended Phase 2 Dose (RP2D), 900 mg/m2 administered IV over 20 minutes, was identified in the Phase 1 dose escalation trial (NCT03348514) in advanced solid tumor patients. Methods: The F RP2D was investigated in two early phase NMIBC and MIBC clinical proof of concept trials. In NCT04608045, neoadjuvant F was administered as monotherapy in cisplatin-ineligible (C-I) MIBC patients and in combination with gemcitabine + cisplatin in chemotherapy-eligible (C-E) MIBC patients. Clinical stage was assessed in pre-treatment (TURBT/CT) and post-treatment pathological state determined at radical cystectomy, (RC). The steady-state plasma and urine pharmacokinetics of F were also characterized. In NCT04525131, F was administered once daily for five days prior to TURBT. Pre- and post-treatment (at TURBT) bladder tumor samples underwent single cell sequencing to identify treatment effects on gene expression. Plasma, urine, and bladder tumor concentrations of F and its metabolites were determined in samples collected at TURBT. Results: Five C-E and 4 C-I MIBC patients received neoadjuvant F prior to RC. Twelve NMIBC patients received F prior to TURBT. There were no treatment-related serious adverse events observed in either study. Each patient experienced at least one treatment-emergent adverse event (TEAE), none of which resulted in study discontinuation. The most common TEAEs were nausea, fatigue, and constipation. Pathologic downstaging (< ypT2) of bladder tumors was observed in 3 C-E MIBC patients with 2 CRs (ypT0). Two of 4 C-I patients had evident clinical response by CT scan with only microscopic residual ypT2 disease. Treatment-related changes in expression of Notch 1, Notch 2, Hes 1, Hey-1, c-Myc, ß-catenin and survivin were observed in the majority of NMIBC patients. F disappeared from plasma within 2 hours of administration. The mean CPX elimination half-life of CPX, apparent systemic clearance, and volume of distribution values were 8.8 hours, 46 L/hr and 549 L, respectively. Mean plasma, tumor and urine concentrations of CPX at TURBT were approximately 27, 9 and 100 µM, respectively. Conclusions: To date, fosciclopirox is well tolerated and achieves sufficient systemic, tumor, and urine CPX exposure at the RP2D. Evidence of target inhibition was demonstrated in NMIBC tumors and preliminary signs of clinical activity observed in MIBC patients. Safety and efficacy trials are planned to confirm and expand findings in NMIBC and MIBC patients. Clinical trial information: NCT04608045; NCT04525131.

Cisplatin remains an integral part of the treatment for muscle invasive bladder cancer. A large number of patients do not respond to cisplatin-based chemotherapy and efficacious salvage regimens are limited. Immunotherapy has offered a second line of treatment; however, only approximately 20% of patients respond, and molecular subtyping of tumors indicates there may be significant overlap in those patients that respond to cisplatin and those patients that respond to immunotherapy. As such, restoring sensitivity to cisplatin remains a major hurdle to improving patient care. One potential source of compounds for enhancing cisplatin is naturally derived bioactive products such as phytochemicals, flavonoids and others. These compounds can activate a diverse array of different pathways, many of which can directly promote or inhibit cisplatin sensitivity. The purpose of this review is to understand current drug development in the area of natural products and to assess how these compounds may enhance cisplatin treatment in bladder cancer patients.

Acetaminophen (APAP) overdose-induced acute liver failure is an important clinical problem in the United States and the current antidote N-acetylcysteine, has a short early therapeutic window. Since most patients present late to the clinic, there is need for novel late-acting therapeutic options. Though the neuronal guidance cue netrin-1, has been shown to promote hepatic repair and regeneration during liver ischemia/reperfusion injury, its effect in APAP-induced hepatotoxicity is unknown. In the quest for a late-acting therapeutic intervention in APAP-induced liver injury, we examined the role of netrin-1 in a mouse model of APAP overdose. Male C57BL/6J mice were cotreated with exogenous netrin-1 or vehicle control, along with 300 mg/kg APAP and euthanized at 6, 12, and 24 h. Significant elevations in alanine aminotransferase indicative of liver injury were seen in control mice at 6 h and this was not affected by netrin-1 administration. Also, netrin-1 treatment did not influence mitochondrial translocation of phospho-JNK, or peroxynitrite formation indicating that there was no interference with APAP-induced injury processes. Interestingly however, netrin-1 administration attenuated liver injury at 24 h, as seen by alanine aminotransferase levels and histology, at which time significant elevations in the netrin-1 receptor, adenosine A2B receptor (A2BAR) as well as macrophage infiltration was evident. Removal of resident macrophages with clodronate liposomes or treatment with the A2BAR antagonist PSB1115 blocked the protective effects of netrin-1. Thus, our data indicate a previously unrecognized role for netrin-1 in attenuation of APAP hepatotoxicity by enhancing recovery and regeneration, which is mediated through the A2BAR and involves resident liver macrophages.

Histological and molecular analyses of urothelial carcinoma often reveal intratumoural and intertumoural heterogeneity at the genomic, transcriptional and cellular levels. Despite the clonal initiation of the tumour, progression and metastasis often arise from subclones that can develop naturally or during therapy, resulting in molecular alterations with a heterogeneous distribution. Variant histologies in tumour tissues that have developed distinct morphological characteristics divergent from urothelial carcinoma are extreme examples of tumour heterogeneity. Ultimately, heterogeneity contributes to drug resistance and relapse after therapy, resulting in poor survival outcomes. Mutation profile differences between patients with muscle-invasive and metastatic urothelial cancer (interpatient heterogeneity) probably contribute to variability in response to chemotherapy and immunotherapy as first-line treatments. Heterogeneity can occur on multiple levels and averaging or normalizing these alterations is crucial for clinical trial and drug design to enable appropriate therapeutic targeting. Identification of the extent of heterogeneity might shape the choice of monotherapy or additional combination treatments to target different drivers and genetic events. Identification of the lethal tumour cell clones is required to improve survival of patients with urothelial carcinoma.

Though mitochondrial oxidant stress plays a critical role in the progression of acetaminophen (APAP) overdose-induced liver damage, the influence of mitochondrial bioenergetics on this is not well characterized. This is important, since lifestyle and diet alter hepatic mitochondrial bioenergetics and an understanding of its effects on APAP-induced liver injury is clinically relevant. Pyruvate dehydrogenase (PDH) is critical to mitochondrial bioenergetics, since it controls the rate of generation of reducing equivalents driving respiration, and pyruvate dehydrogenase kinase 4 (PDK4) regulates (inhibits) PDH by phosphorylation. We examined APAP-induced liver injury in PDK4-deficient (PDK4(−/−)) mice, which would have constitutively active PDH and hence elevated flux through the mitochondrial electron transport chain. PDK4(−/−) mice showed significant protection against APAP-induced liver injury when compared to wild type (WT) mice as measured by ALT levels and histology. Deficiency of PDK4 did not alter APAP metabolism, with similar APAP-adduct levels in PDK4(−/−) and WT mice, and no difference in JNK activation and translocation to mitochondria. However, subsequent amplification of mitochondrial dysfunction with release of mitochondrial AIF, peroxynitrite formation and DNA fragmentation were prevented. Interestingly, APAP induced a rapid decline in UCP2 protein levels in PDK4-deficient mice. These data suggest that adaptive changes in mitochondrial bioenergetics induced by enhanced respiratory chain flux in PDK4(−/−) mice render them highly efficient in handling APAP-induced oxidant stress, probably through modulation of UCP2 levels. Further investigation of these specific adaptive mechanisms would provide better insight into the control exerted by mitochondrial bioenergetics on cellular responses to an APAP overdose.

Introduction and Objective Bladder cancer is a common solid tumor. Outcomes are poor in advanced disease, with few novel clinical therapeutics introduced over the previous several decades. Otto Warburg's original hypothesis that cancer cells use aerobic glycolysis to produce ATP instead of traditional oxidative phosphorylation in the mitochondria was a landmark discovery in its time. Recent studies indicate metabolic changes in cancer are far more complex than originally anticipated though. The purpose of this review is to understand metabolic changes that occur in bladder cancer, how targeting these changes could potentially be used therapeutically, and the current treatments that target these metabolic changes Methods A literature review on recent advances in cancer metabolism with an emphasis on bladder cancer was performed. Results Significant metabolic change occurs in bladder cancer; however, these changes associated are not yet well understood. Therapeutic development in this area is growing and a diverse array of actionable targets such as mitochondrial DNA, mitochondrial metabolic enzymes and cellular signaling proteins have been identified. Many of these proteins may also be involved in chemoresistance. Conclusion Metabolism is a growing area of therapeutic interest in bladder cancer, but more studies are required to advance therapeutic development in this area.

Alcoholic hepatitis is the most severe and acute form of alcoholic liver disease. The mortality rate associated with alcoholic hepatitis is high, largely due to the lack of suitable pharmacological interventions. While there has been substantial research in the area, generating pharmacological interventions has been plagued by the lack of a robust mouse model both for testing and for understanding the underlying pathology. A number of major notable advances have been made in this area recently, with the goal of generating a mouse model of alcoholic hepatitis. The purpose of this article is to review recent advances in modeling alcoholic liver disease both in vitro and in vivo in the mouse, and place them in the context of the greater spectrum of alcoholic liver disease, with a focus on how we can translate current advances into a high-fidelity model of alcoholic hepatitis. In addition, we will review the basic mechanisms of alcoholic hepatitis as it is currently understood, focusing on recent advancements in diagnosis, prognosis and current pathophysiology, especially as it relates to the profound immune dysfunction present during alcoholic hepatitis.

Bladder cancer (BCa) is a disease strongly related to aging and remains a major source of morbidity and mortality in the elderly. Older patients are at greater risk of developing BCa and treatment outcomes remain poor. Interventions aimed at delaying recurrence and enhancing therapy would be highly beneficial given the aging US population and association with BCa. Aging is associated with baseline immune dysfunction, increasing risk for chronic diseases such as cancers. Compounding this issue, dysregulated inflammation in cancer results in a switch in the inflammatory milieu toward a pro-tumorigenic phenotype. Recent studies have indicated immune dysregulation in multiple cancers is also likely related to the changes in the underlying microbiome. The impact of age-related changes in the microbiome and inflammatory status, and how this relates to progression of tumorigenesis in the bladder have not yet been defined though. We hypothesized aging would enhance inflammation and carcinogenesis in a mouse model of carcinogen induced BCa. To determine how aging affects BCa, we exposed middle aged mice (14 months old at study start), and young mice (2 months old at study start) to the urothelial carcinogen N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) 0.05% in drinking water for 6-20 weeks. Pathological analysis indicated older mice had higher indices of cellular inflammation at baseline and after BBN. Similarly, older mice had significantly more upregulation of pro-inflammatory mediators after 6 weeks exposure to BBN. While inflammatory metrics were increased, this did not extend to tumor size or stage. Middle aged mice (14 months) do not express prototypical biomarkers of aging yet though, and represent a very mild phenotype. To test the idea that truly older mice would be more susceptible to carcinogenesis, we treated young mice (8 weeks at start) or truly old mice (18 months at start) with 0.05% BBN for 16 weeks. We found older animals had larger tumors indicative of increased tumorigenesis after 16 weeks BBN. Moreover, multiple older mice muscle invasive tumors which were absent in the young group and increased recruitment of neutrophils indicative of increased inflammation. We subsequently analyzed the fecal microbiome using 16S sequencing of bacterial DNA. ß-diversity indices indicated substantial differences in microbiota constituency between young and older mice with cancer, although these differences were not observed at baseline. Future goals are to determine if aging-related alterations in the microbiome are responsible for the observed changes in tumor formation and inflammation. In conclusion, aged animals experience more inflammation, major changes in gut microbial content, and develop larger and more aggressive tumors after exposure to carcinogens implicating a pathological role for aging in BCa. Citation Format: Benjamin L. Woolbright, Erika Abbott, Ganeshkumar Rajendran, Cuncong Zhong, Hao Xuan, Shahid Umar, John Arthur Taylor. Intersection of aging, the microbiome and inflammation in a mouse model of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2911.

Mitochondrial biogenesis (MB) is an adaptive response to maintain metabolic homeostasis after mitochondrial dysfunction. Induction of MB during APAP hepatotoxicity has not been studied. To investigate this, mice were treated with toxic doses of APAP and euthanized between 0 and 96 h. At early time points, APAP caused both mitochondrial dysfunction and reduction of mitochondrial mass, indicated by reduced activity of electron transport chain (ETC) complexes I and IV and depletion of mitochondrial DNA (mtDNA), respectively. Both ETC activity and mtDNA gradually recovered after 12 h, suggesting that MB occurs at late time points after APAP overdose. Immunofluorescent staining of mitochondria with mitochondrial outer membrane protein Tom20 further demonstrated that MB occurs selectively in hepatocytes surrounding necrotic areas. MB signaling mediators including PPARγ co-activator 1-α (Pgc-1α), nuclear respiratory factor-1 (Nrf-1) and mitochondrial fission protein dynamin-related protein-1 (Drp-1) were induced. Pgc-1α was selectively increased in hepatocytes surrounding necrotic areas. In addition, the time course of MB induction coincides with increased liver regeneration. Post-treatment with the known MB inducer SRT1720 increased Pgc-1α expression and liver regeneration, resulting in protection against late liver injury after APAP overdose. Thus, induction of MB is an important feature during APAP hepatotoxicity and liver regeneration.

Estrogen receptor alpha (ESR1) is 1 of the 2 intracellular receptors for estrogen and is expressed by hepatocytes in the liver. The role of ESR1 in the regulation of toxicant-induced liver injury and compensatory regeneration is not completely clear. We investigated the role of ESR1 in liver regeneration after carbon tetrachloride (CCl4)-induced liver injury using wild type (WT) and ESR1 knockout (ESR1-KO) rats. Adult female WT and ESR1-KO rats were treated with 1 mL/kg CCl4and euthanized over a time course of 0 to 48 hours. Liver injury measured by serum alanine amino transaminase, and histopathological analysis showed significantly higher liver injury in ESR1-KO as compared to WT rats. Hematoxylin and eosin staining revealed 2-fold higher necrosis and significant inflammatory cell infiltration in ESR1-KO rats. Chloracetate esterase staining revealed higher neutrophil infiltration in ESR1-KO rat livers. Interestingly, proliferating cell nuclear antigen immunohistochemistry showed that in spite of 2-fold higher liver injury, the ESR1-KO rats had equal liver regeneration as compared to WT rats. Western blot analysis of cyclin D1 and phosphorylated Rb, proteins involved in the initiation of the cell cycle, was significantly higher at all time points in ESR1-KO rats. Further analysis revealed faster activation of canonical Wnt/β-catenin and NF-κB signaling in ESR1-KO rats characterized by higher activated β-catenin and phosphorylated p65 at 12 hours after CCl4treatment. Taken together, these data indicate that ESR1-mediated signaling inhibits liver regeneration by downregulation of Wnt signaling resulting in lower cyclin D1 activation after chemical-induced liver injury.

Microcystins are a group of toxins produced by freshwater cyanobacteria. Uptake of microcystin-leucine arginine (MC-LR) by organic anion transporting polypeptide 1B2 in hepatocytes results in inhibition of protein phosphatase 1A and 2A, and subsequent cell death. Studies performed in primary rat hepatocytes demonstrate prototypical apoptosis after MC-LR exposure; however, no study has directly tested whether apoptosis is critically involved in vivo in the mouse, or in human hepatocytes. MC-LR (120 μg/kg) was administered to C57BL/6J mice and cell death was evaluated by alanine aminotransferase (ALT) release, caspase-3 activity in the liver, and histology. Mice exposed to MC-LR had increases in plasma ALT values, and hemorrhage in the liver, but no increase in capase-3 activity in the liver. Pre-treatment with the pan-caspase inhibitor z-VAD-fmk failed to protect against cell death measured by ALT, glutathione depletion, or hemorrhage. Administration of MC-LR to primary human hepatocytes resulted in significant toxicity at concentrations between 5 nM and 1 μM. There were no elevated caspase-3 activities and pretreatment with z-VAD-fmk failed to protect against cell death in human hepatocytes. MC-LR treated human hepatocytes stained positive for propidium iodide, indicating membrane instability, a marker of necrosis. Of note, both increases in PI positive cells, and increases in lactate dehydrogenase release, occurred before the onset of complete actin filament collapse. In conclusion, apoptosis does not contribute to MC-LR-induced cell death in the in vivo mouse model or in primary human hepatocytes in vitro. Thus, targeting necrotic cell death mechanisms will be critical for preventing microcystin-induced liver injury.

Cholestasis is a result of obstruction of the biliary tracts. It is a common cause of liver pathology after exposure to toxic xenobiotics and during numerous other liver diseases. Accumulation of bile acids in the liver is thought to be a major driver of liver injury during cholestasis and can lead to eventual liver fibrosis and cirrhosis. As such, current therapy in the field of chronic liver diseases with prominent cholestasis relies heavily on increasing choleresis to limit accumulation of bile acids. Many of these same diseases also present with autoimmunity before the onset of cholestasis though, indicating the inflammation may be an initiating component of the pathology. Moreover, cytotoxic inflammatory mediators accumulate during cholestasis and can propagate liver injury. Anti-inflammatory biologics and small molecules have largely failed clinical trials in these diseases though and as such, targeting inflammation as a means to address cholestatic liver injury remains debatable. The purpose of this review is to understand the different roles that inflammation can play during cholestatic liver injury and attempt to define how new therapeutic targets that limit or control inflammation may be beneficial for patients with chronic cholestatic liver disease.

Bladder cancer (BCa) is a common solid tumor marked by high rates of recurrence, especially in non-muscle invasive disease. Prostaglandin E2 (PGE2) is a ubiquitously present lipid mediator responsible for numerous physiological actions. Inhibition of cyclooxygenase (COX) enzymes by the non-steroidal anti-inflammatory (NSAID) class of drugs results in reduced PGE2 levels. NSAID usage has been associated with reductions in cancers such as BCa. Clinical trials using NSAIDs to prevent recurrence have had mixed results, but largely converge on issues with cardiotoxicity. The purpose of this review is to understand the basic science behind how and why inhibitors of PGE2 may be effective against BCa, and to explore alternate therapeutic modalities for addressing the role of PGE2 without the associated cardiotoxicity. We will address the role of PGE2 in a diverse array of cancer-related functions including stemness, immunosuppression, proliferation, cellular signaling and more.

Objective: Regulation of TF (tissue factor):FVIIa (coagulation factor VIIa) complex procoagulant activity is especially critical in tissues where plasma can contact TF-expressing cells. One example is the liver, where hepatocytes are routinely exposed to plasma because of the fenestrated sinusoidal endothelium. Although liver-associated TF contributes to coagulation, the mechanisms controlling the TF:FVIIa complex activity in this tissue are not known. Approach and Results: Common bile duct ligation in mice triggered rapid hepatocyte TF-dependent intrahepatic coagulation coincident with increased plasma bile acids, which occurred at a time before observable liver damage. Similarly, plasma TAT (thrombin-antithrombin) levels increased in cholestatic patients without concurrent hepatocellular injury. Pathologically relevant concentrations of the bile acid glycochenodeoxycholic acid rapidly increased hepatocyte TF-dependent procoagulant activity in vitro, independent of de novo TF synthesis and necrotic or apoptotic cell death. Glycochenodeoxycholic acid increased hepatocyte TF activity even in the presence of the phosphatidylserine-blocking protein lactadherin. Interestingly, glycochenodeoxycholic acid and taurochenodeoxycholic acid increased the procoagulant activity of the TF:FVIIa complex relipidated in unilamellar phosphatidylcholine vesicles, which was linked to an apparent decrease in the K m for FX (coagulation factor X). Notably, the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a bile acid structural analog, did not increase relipidated TF:FVIIa activity. Bile acids directly enhanced factor X activation by recombinant soluble TF:FVIIa complex but had no effect on FVIIa alone. Conclusions: The results indicate that bile acids directly accelerate TF:FVIIa–driven coagulation reactions, suggesting a novel mechanism whereby elevation in a physiological mediator can directly increase TF:FVIIa procoagulant activity.

Cholestasis can be induced by obstruction of bile ducts or intrahepatic toxicity of drugs and chemicals. However, the mode of cell death during cholestasis, i.e., apoptosis or necrosis, has been controversial. There are fundamental reasons for the controversies, both of which are discussed here, namely the design of experiments and the use of parameters with limited specificity for a certain mode of cell death. Based on the assumption that cholestatic liver injury is caused by accumulation of bile acids, rodent (mainly rat) hepatocytes have been exposed to hydrophobic, glycine-conjugated bile acids, which resulted in apoptotic cell death. The problems with this experimental design are that in rodents bile acids are predominantly taurine conjugated and rodent hepatocytes are never exposed to these levels of glycine-conjugated bile acids. In contrast, taurine-conjugated bile acids trigger inflammatory gene activation in rodent hepatocytes and a necro-inflammatory injury in vivo. On the other hand, human hepatocytes are more resistant to glycine-conjugated bile acids and die by necrosis when exposed to high biliary levels of these bile acids. In this chapter, we describe multiple assays including the caspase activity assay, which is specific for apoptosis, and the general cell death assays alanine aminotransferase or lactate dehydrogenase activities in cell culture medium or plasma. An increase in these enzyme activities without caspase activity indicates necrotic cell death. Thus, both the experimental design and the selection of cell death parameters are critical for the relevance of the experiments for the human pathophysiology.

INTRODUCTION AND OBJECTIVES:PDK4 is a member of the PDK enzyme family which phosphorylate and inactivate the pyruvate dehydrogenase (PDH) complex, promoting aerobic glycolysis, known as the Warburg...

Genitourinary cancers encompass some of the most common solid tumours and have high rates of morbidity and mortality. Inflammation is associated with enhanced tumorigenesis, and a number of pro-inflammatory mediators, such as macrophage migration inhibitory factor (MIF), also promote tumorigenesis. Studies of the role of MIF (which largely functions via the type II transmembrane receptor CD74) in prostate, bladder and kidney cancers suggest that it is a pro-tumorigenic factor in genitourinary malignancy. Inhibiting MIF activity in cell culture and in preclinical animal models of genitourinary cancers reduces the phenotypic hallmarks of cancer, such as proliferation, angiogenesis and tumour aggressiveness, by downregulating signalling pathways such as those regulated by extracellular signal-regulated kinase (ERK), protein kinase B and p53, and MIF may also reverse immunosuppression. Progress has been made in our understanding of the role of MIF (and its family member d-dopachrome tautomerase (DDT)) in genitourinary cancers and how it can be therapeutically targeted. Macrophage migration inhibitory factor (MIF) can promote tumorigenesis by acting directly on cancer cells and by reversing immunosuppression. Advances have been made in our understanding of the role of MIF in the promotion of, and as a treatment target for, genitourinary cancers.

Personalized medicine portends a future where patients receive therapy based on mutational and gene expression profiles intrinsic to their tumor. Recent advances in molecular subtyping of tumors have pushed us closer to using patient-specific data to guide therapy. The purpose of this review is to understand how these advances may be used to understand tumor development and direct therapeutic regimens clinically.Multiple reports have identified specific molecular subtypes present in bladder cancer. A variety of classification schemes are currently being suggested based on different groups observations on gene expression, mutational profile, and histological variability. Notably, recent novel findings indicate standard of care with neoadjuvant platinum-based chemotherapy effectively removes the basal subtype specifically, indicating clinical data largely supports the use of molecular subtyping as a way to treat tumors.Although varied classifications are present in the field currently, more work is required to truly define which subtypes are responsive to specific therapies. Current data supports the idea that molecular subtyping will benefit patients; however, these data also argue in favor of combinatorial therapy and molecular profiling throughout treatment.

PurposeBladder cancer is a "Warburg-like" tumor characterized by a reliance on aerobic glycolysis and expression of pyruvate kinase M2 (PKM2). PKM2 oscillates between an active tetramer and an inactive dimer. We aim to further characterize PKM2, in particular PKM2 dimer, as a urinary biomarker of bladder cancer and a potential target for treatment.MethodsHTB-9, HTB-5, and UM-UC3 bladder cancer cells were assessed for proliferation under differential glucose levels using the hexosaminidase assay. Western blot and Blue-native analysis was performed for protein expression of PKM2. Shikonin, an herb that is known to bind and inhibit PKM2, was utilized to determine if PKM2 has a role in glucose usage and cellular proliferation in bladder cancer cells by caspase activity assay. Institutional review board approval was obtained to collect healthy control and bladder cancer patient urine samples. The ScheBo M2-PK EDTA Plasma Test was performed on urine samples to assess urine Tumor M2-PK values.ResultsThe three bladder cancer cell lines tested all demonstrate statistically significant increases in proliferation when exposed to higher level of glucose (200mg/dL). Similarly, low doses of glucose (25mg/dL) result in reduced proliferation. Increased cell growth in higher glucose concentration correlated with up-regulation of PKM2 protein expression. Shikonin, a PKM2 inhibitor, reduced cell proliferation and switched PKM2 isoforms from the dimer to tetramer. Lastly, dimer PKM2 (Tumor-M2PK) levels were assessed in the urine samples from bladder cancer (Bca) patients and healthy controls. Tumor M2-PK significantly correlated with the presence of BCa in our subjects.ConclusionsOur studies demonstrate the potential of PKM2, specifically the dimer (Tumor-M2PK) as a target of drug therapy and as a urinary marker for bladder cancer.

Cholestasis can be induced by obstruction of bile ducts or intrahepatic toxicity of drugs and chemicals. However, the mode of cell death during cholestasis, i.e., apoptosis or necrosis, has been controversial. There are fundamental reasons for the controversies, both of which are discussed here, namely the design of experiments and the use of parameters with limited specificity for a certain mode of cell death. Based on the assumption that cholestatic liver injury is caused by accumulation of bile acids, rodent (mainly rat) hepatocytes have been exposed to hydrophobic, glycine-conjugated bile acids, which resulted in apoptotic cell death. The problems with this experimental design are that in rodents bile acids are predominantly taurine conjugated and rodent hepatocytes are never exposed to these levels of glycine-conjugated bile acids. In contrast, taurine-conjugated bile acids trigger inflammatory gene activation in rodent hepatocytes and a necro-inflammatory injury in vivo. On the other hand, human hepatocytes are more resistant to glycine-conjugated bile acids and die by necrosis when exposed to high biliary levels of these bile acids. In this chapter, we describe multiple assays including the caspase activity assay, which is specific for apoptosis, and the general cell death assays alanine aminotransferase or lactate dehydrogenase activities in cell culture medium or plasma. An increase in these enzyme activities without caspase activity indicates necrotic cell death. Thus, both the experimental design and the selection of cell death parameters are critical for the relevance of the experiments for the human pathophysiology.

PURPOSE OF REVIEW: This article provides a brief overview of mechanisms of inflammatory liver injury and how this applies to drug hepatotoxicity with a particular emphasis on the role of inflammation in acetaminophen-induced liver injury. RECENT FINDINGS: Significant progress has been made in the last decade in our understanding of the initiation of sterile inflammation after necrotic cell death by the release of damage-associated molecular patterns and their recognition by toll-like receptors and others on macrophages. These events trigger the formation of cytokines and chemokines directly or with assistance of inflammasome activation thereby activating and recruiting leukocytes including neutrophils and monocyte-derived macrophages into the necrotic areas. Although this sterile inflammatory response is mainly geared towards the removal of necrotic cell debris and preparation of regeneration, there are conditions where these innate immune cells can aggravate the initial injury. The mechanisms and controversial findings of the innate immunity are being discussed in detail. In contrast, drug metabolism and formation of a reactive metabolite that binds to proteins in the absence of extensive cell death, can induce an adaptive immune response, which eventually also results in severe liver injury. However, the initiating event appears to be the formation of protein adducts, which act as haptens to activate an adaptive immune response. Overall, these mechanisms are less well understood. SUMMARY: The past decade has revolutionized our understanding of the mechanisms that control the interplay between cell death and innate or adaptive immune responses. This report provides an update on these mechanisms.

How different cell death modes and cell survival pathways cross talk remains elusive. We determined the interrelation of apoptosis, necrosis, and autophagy in tumor necrosis factor (TNF)-α/actinomycin D (ActD) and lipopolysaccharide/D-galactosamine (GalN)-induced hepatotoxicity in vitro and in vivo . We found that TNF-α/ActD-induced apoptosis was completely blocked by a general caspase inhibitor ZVAD-fmk at 24 hours but hepatocytes still died by necrosis at 48 hours. Inhibition of caspases also protected mice against lipopolysaccharide/GalN-induced apoptosis and liver injury at the early time point, but this protection was diminished after prolonged treatment by switching apoptosis to necrosis. Inhibition of receptor-interacting protein kinase (RIP)1 by necrostatin 1 partially inhibited TNF-α/ZVAD-induced necrosis in primary hepatocytes. Pharmacologic inhibition of autophagy or genetic deletion of Atg5 in hepatocytes did not protect against TNF-α/ActD/ZVAD-induced necrosis. Moreover, pharmacologic inhibition of RIP1 or genetic deletion of RIP3 failed to protect and even exacerbated liver injury after mice were treated with lipopolysaccharide/GalN and a pan-caspase inhibitor. In conclusion, our results suggest that different cell death mode and cell survival pathways are closely integrated during TNF-α–induced liver injury when both caspases and NF-κB are blocked. Moreover, results from our study also raised concerns about the safety of currently ongoing clinical trials that use caspase inhibitors.

Background & aims Hypoxic hepatitis is a clinical condition precipitated by prolonged periods of oxygen deprivation to the liver. It can have several underlying causes. Despite its prevalence in critically ill patients, which can reach upwards of 10%, very little is known about the mechanisms of injury. Thus, we set out to measure previously identified circulating biomarkers in an attempt to describe mechanisms of injury following hypoxic hepatitis. Methods Plasma from patients diagnosed with hypoxic hepatitis was collected for this study. Biomarkers of hepatocellular injury, mitochondrial damage and cell death were measured. These results were compared against results obtained from well-characterized acetaminophen overdose patients. Results At peak injury, ALT measured 4082±606 U/L and gradually decreased over 5 days, corresponding to the clinically observed pattern of hypoxic hepatitis. Levels of GDH showed a similar pattern, but neither ALT nor GDH were significantly higher in these patients than in acetaminophen patients. Plasma levels of DNA fragments mimicked hepatocellular injury as measured by ALT and miRNA-122. Interestingly, we found a significant increase in caspase-cleaved cytokeratin-18; however, the full-length form greatly exceeded the cleaved form at the time of maximum injury (45837±12085 vs 2528±1074 U/L). We also found an increase in acHMGB1 at later time points indicating a possible role of inflammation, but cytokine levels at these times were actually decreased relative to early time points. Conclusions The mechanism of injury following hypoxic hepatitis involves mitochondrial damage and DNA fragmentation. Importantly, necrosis, rather than apoptosis, is the main mode of cell death.

Because of the liver’s function in the body as both a filter for toxins and an innate immune organ, the liver is predisposed to cellular turnover. Under healthy conditions, this occurs through the ...

Ciclopirox (CPX) is a FDA-approved topical antifungal agent that has demonstrated preclinical anticancer activity in solid and hematologic malignancies. It's clinical utility as an anticancer agent, however, is limited by poor oral bioavailability, gastrointestinal toxicity, and poor water solubility. Fosciclopirox, the phosphoryloxymethyl ester of CPX (Ciclopirox Prodrug, CPX-POM), is rapidly and completely metabolized to CPX, the active metabolite, which subsequently undergoes renal elimination resulting in urine concentrations of CPX that exceed in vitro IC50's several-fold. We characterized the activity of CPX-POM and its major metabolites in vitro utilizing authenticated human T24, HT-1376, and UM-UC-3 high-grade urothelial cancer cell lines. CPX inhibited cell proliferation, clonogenicity, and spheroid formation, and increased cell cycle arrest at S and G0/G1 phases. Mechanistically, CPX suppressed activation of Notch signaling, which was partially rescued by ectopic expression of the intracellular domain of Notch1. Molecular modeling and cellular thermal shift assays demonstrated CPX binding to γ-secretase complex proteins Presenilin1 and Nicastrin, which are essential for Notch activation. Interrogation of The Cancer Genome Atlas (TCGA) database demonstrated that both proteins were upregulated in bladder tumor tissue, and that higher levels of Presenilin1 and Nicastrin were significantly associated with lower overall survival in muscle invasive bladder cancer (MIBC) patients. To establish in vivo preclinical proof of principle, we tested fosciclopirox in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) mouse bladder cancer model in two separate studies. Intraperitoneal (IP) administration of CPX-POM once daily for four weeks at doses ranging from 25 to 200 mg/kg significantly decreased bladder weight and resulted in a migration to lower stage tumors in CPX-POM treated animals compared to untreated animals. This was coupled with a reduction in proliferation index, as well as reductions in Presenilin1 and Hey1 expression in bladder tumor tissues in CPX-POM treated animals. A similar anti-tumor response was observed following once daily versus three times weekly IP CPX-POM in this chemical carcinogen mouse model of bladder cancer. The safety, dose tolerance, pharmacokinetics and pharmacodynamics of intravenous (IV) CPX-POM were characterized in a US multi-center, First-in-Human, Phase 1, open-label, dose escalation study (NCT03348514). Eight cohorts of 19 patients received IV CPX-POM doses ranging from 30 to 1200 mg/m2 for as many as six 21-day treatment cycles. Adequate systemic and urinary tract CPX exposures were achieved at the maximum tolerated dose of 900 mg/m2 with evidence of Notch inhibition. An expansion cohort study in 12 cisplatin-ineligible MIBC patients receiving two treatment cycles of CPX-POM prior to radical cystectomy (RC) is underway. Evidence of pharmacologic activity is being characterized in bladder tumor tissues obtained at RC. Citation Format: Scott James Weir, Prasad Dandawate, Prabhu Ramamoorthy, Parthasarathy Ranjarajan, Robyn Wood, Amanda Brinker, Benjamin Woolbright, Mehmet Tanol, Tammy Ham, William McCulloch, Michael Dalton, Michael J. Baltezor, Roy A. Jensen, John A. Taylor, Shrikant Anant. Fosciclopirox suppresses growth of high-grade urothelial cancer by targeting Notch signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6405.

Macrophage migration inhibitory factor (MIF1) is a pro-tumorigenic cytokine overexpressed in multiple cancers. Our previous data in a carcinogen (BBN) induced murine bladder cancer (BCa) model indicates genetic depletion of MIF1 induces stage arrest. Pharmacological inhibition of MIF1 with the inhibitor CPSI-1306 reduces tumor stage and bladder weights. These data indicate MIF1 is a therapeutic target in BCa. However, a second protein with significant homology to MIF1 was recently identified and termed MIF2. Both MIF1 and MIF2 bind and activate their receptor CD74 yielding significant functional homology and thus, targeting both proteins may be more efficacious. We hypothesize MIF2 recapitulates the pro-tumorigenic effects of MIF1 in BCa in a CD74 dependent manner. We assessed MIF1 and MIF2 by ELISA in serum from human patients with BCa and MIF1/2 expression were evaluated in TCGA. WT mice (C57Bl/6 background), MIF-/-, and CD74-/- mice were treated with 0.05% BBN for 16 weeks to induce BCa, and then switched to water. Tumors were allowed to develop until 20 weeks post initiation of BBN, a time frame consistent with muscle invasive tumor development. Some animals were given either vehicle, or a dual MIF1/MIF2 inhibitor 4-IPP (80mg/kg i.p. daily) for 4 weeks after cessation of BBN. We assessed bladder weights as a surrogate for tumor weight, tumor stage and immunohistochemistry for proliferation, angiogenesis and inflammation. In vitro cell proliferation was measured using the hexosaminidase assay and tautomerase activity was measured using two separate enzymatic assays for MIF tautomerase activity. MIF1 and MIF2 mRNA levels were elevated in TCGA. Analysis of serum from BCa patients indicated increased MIF1 and MIF2 in BCa patients, although this didn't reach statistical significance. 4-IPP treated C57Bl/6 animals had reductions in bladder weight, although tumor staging was similar between groups. 4-IPP treatment had no effect on bladder weights or tumor stage in CD74-/- mice supporting a role for CD74 mediated MIF signaling. To more fully assess the role of MIF2 directly, MIF1-/- mice were treated with 4-IPP to solely block MIF2 4-IPP treatment lead to major reductions in tumor stage, tumor incidence and bladder weight indicating inhibition of MIF2 may be more effective when MIF1 is chronically absent. Inflammation and proliferation trended lower, but did not reach statistical significance. MIF1 depletion did not affect MIF2 mRNA levels in bladder, heart or liver. In vitro treatment with 4-IPP substantially reduced proliferation of two high grade BCa cell lines (HTB-5, HTB-9) at concentrations consistent with doses found to inhibit recombinant MIF activity. These data indicate MIF1/2 enhance BCa tumorigenesis through activation of CD74. Combined MIF1/MIF2 may improve therapeutic targeting of MIF, especially during chronic MIF1 inhibition. Citation Format: Ganeshkumar Rajendran, Benjamin L. Woolbright, Erika Abbott, Austin Martin, Katie Dennis, John Arthur Taylor. MIF-2 in bladder cancer: potential therapeutic target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5150.

518 Background: Fosciclopirox (CPX-POM) is being developed for the treatment of non-muscle invasive and muscle invasive bladder cancer. CPX-POM selectively delivers its active metabolite, ciclopirox (CPX), to the entire urinary tract following systemic administration. In a chemical carcinogen mouse model of bladder cancer, CPX-POM treatment resulted in significant decreases in bladder weight, migration to lower stage tumors, inhibition of cell proliferation as well as Notch 1 and Wnt signaling pathways. Methods: Study CPX-POM-001 (NCT03348514) is US multi-site, Phase I, open-label, dose escalation study characterizing the safety, dose tolerance, pharmacokinetics (PK) and pharmacodynamics of IV CPX-POM in advanced solid tumor patients. The PK of CPX-POM, CPX and ciclopirox glucuronide (CPX-G), were characterized in plasma and urine. Circulating biomarkers of Wnt and Notch, IL-6, IL-8 and VEGF were determined. Results: Nineteen patients were enrolled in the study. The starting dose of 30 mg/m2 was administered once daily on Days 1-5 of each 21-day treatment cycle. Doses were escalated to 1200 mg/m2. The MTD was determined to be 900 mg/m2. Overall, the number of treatment-related AE's tended to increase in frequency with dose, nausea and vomiting being the most common. Grade 3 confusion was observed in the 1200 mg/m2 cohort. Four AE's of Grade 1 confusion at 600 and 900 mg/m2. There was no evidence of QTc prolongation or other ECG abnormality. One patient in the 240 mg/m2 dose cohort, with a diagnosis of indolent primary fallopian tube tumor, achieved a partial response per RECIST 1.1. Metabolism of CPX-POM was rapid and complete. The clearance of CPX was dose proportional and time-independent. At MTD, steady-state 24-hour urine CPX concentrations of 215 µM were achieved. Evidence of Notch and Wnt inhibition was observed. Conclusions: IV CPX-POM was well tolerated with treatment-related AEs primarily CNS-related. At MTD, systemic and urinary CPX exposures exceeding in vitro IC50 values by several-fold. The 900 mg/m2 dose is currently being evaluated in an expansion cohort study in cisplatin-ineligible muscle invasive bladder cancer patients scheduled for cystectomy. Clinical trial information: NCT03348514.

TPS604 Background: Fosciclopirox (Ciclopirox Prodrug, CPX-POM) is being developed for the treatment of non-muscle invasive and muscle invasive (MIBC) bladder cancer. CPX-POM selectively delivers its active metabolite, ciclopirox (CPX), to the entire urinary tract following systemic administration. In a validated, chemical carcinogen mouse model of bladder cancer, CPX-POM treatment results in significant decreases in bladder weight, a clear migration to lower stage tumors, dose-dependent reductions in Ki67 and PCNA staining, and inhibition of Notch 1 and Wnt signaling. The safety, dose tolerance, pharmacokinetics and pharmacodynamics of IV CPX-POM have recently been characterized in 19 patients with advanced solid tumors (CPX-POM-001, NCT03348514). The safety and dose tolerance of IV CPX-POM was characterized across a dose range of 30 to 1200 mg/m2. The CPX-POM Recommended Phase 2 Dose (PR2D) of 900 mg/m2 administered IV over 20 minutes on Days 1-5 every 21 days was selected. Methods: Twelve cisplatin ineligible MIBC patients (Stage >T2, NO-N1, M0), scheduled for radical cystectomy (RC) will be enrolled in this window of opportunity study. Patients will receive two 21-day treatment cycles followed by RC within 14 days of completion of the second cycle. Safety and tolerability assessments will be made based on observed adverse and serious adverse events, physical examination, vital signs, electrocardiogram, clinical laboratory tests, and concomitant medications. Assessment of complete and partial pathologic response will be determined at RC. Ki67, Notch and Wnt signaling, and CD8+ lymphocyte tumor infiltration will be determined by immunohistochemistry. An unbiased approach to characterizing CPX-POM mechanisms of action will also be employed using RNAseq and ChIPseq. Serial blood (plasma) and complete urine specimens will be collected on Days 5-6 of Cycle 1 for determination of drug and metabolite concentrations by LC-MS/MS. Plasma and urine steady-state pharmacokinetics of CPX-POM, CPX and ciclopirox glucuronide will be characterized. Urine ß-glucuronidase activity is also being determined by ELISA. Clinical trial information: NCT03348514.

We previously reported that delayed treatment with Mito-tempo (MT), a mitochondria-targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300 mg/kg APAP followed by 20 mg/kg MT 1.5 h or 3 h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24 h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10 mg/kg) 2 h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine, the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.

Acute liver failure (ALF) due to a variety of causes has been well established clinically for a number of decades (1). An intense research focus has recently been generated in the realm of acute-on-chronic liver failure (ACLF), especially in light of the high number of cirrhotic patients that eventually present to the clinic (2). The pathology and clinical history of ACLF is poorly understood and due to its nebulous clinical nature, there are no current mainline treatments.

Advanced bladder cancer remains a major source of mortality, with poor treatment options. Cisplatin-based chemotherapy is the standard treatment, however many patients are or become resistant. One potential cause of chemoresistance is the Warburg effect, a metabolic switch to aerobic glycolysis that occurs in many cancers. Upregulation of the pyruvate dehydrogenase kinase family (PDK1–PDK4) is associated with aerobic glycolysis and chemoresistance through inhibition of the pyruvate dehydrogenase complex (PDH). We have previously observed upregulation of PDK4 in high-grade compared with low-grade bladder cancers. We initiated this study to determine if inhibition of PDK4 could reduce tumor growth rates or sensitize bladder cancer cells to cisplatin. Upregulation of PDK4 in malignant bladder cancer cell lines as compared with benign transformed urothelial cells was confirmed using qPCR. Inhibition of PDK4 with dichloroacetate (DCA) resulted in increased PDH activity, reduced cell growth, and G0–G1 phase arrest in bladder cancer cells. Similarly, siRNA knockdown of PDK4 inhibited bladder cancer cell proliferation. Cotreatment of bladder cancer cells with cisplatin and DCA did not increase caspase-3 activity but did enhance overall cell death in vitro. Although daily treatment with 200 mg/kg DCA alone did not reduce tumor volumes in a xenograft model, combination treatment with cisplatin resulted in dramatically reduced tumor volumes as compared with either DCA or cisplatin alone. This was attributed to substantial intratumoral necrosis. These findings indicate inhibition of PDK4 may potentiate cisplatin-induced cell death and warrant further studies investigating the mechanism through which this occurs. Mol Cancer Ther; 17(9); 2004–12. ©2018 AACR.

Cholestasis is a reduction in bile flow that occurs during numerous pathologies. Blockage of the biliary tracts results in hepatic accumulation of bile acids or their conjugate bile salts. The molecular mechanisms behind liver injury associated with cholestasis are extensively studied, but not well understood. Multiple models of obstructive cholestasis result in a significant inflammatory infiltrate at the sites of necrosis that characterize the injury.This review will focus on direct bile acid toxicity during cholestasis, bile acid signaling processes and on the development and continuation of inflammation during cholestasis, with a focus on novel proposed molecular mediators of neutrophil recruitment. While significant progress has been made on these molecular mechanisms, a continued focus on how cholestasis and the innate immune system interact is necessary to discover targetable therapeutics that might protect the liver while leaving global immunity intact.While bile acid toxicity likely occurs in humans and other mammals when toxic bile acids accumulate, persistent inflammation is likely responsible for continued liver injury during obstructive cholestasis. Targeting molecular mediators of inflammation may help prevent liver injury during acute cholestasis both in murine models and human patients.

Cholestatic liver injury is a pathological component of numerous disease states. Much of the current literature on cholestatic liver injury is derived from in vitro studies using rodent hepatocytes or cell lines transfected with bile acid (BA) uptake transporters. While these studies demonstrate BA-driven apoptosis, it is debatable whether these models reflect the human pathophysiology, as primary human hepatocytes undergo primarily necrosis. HepaRG cells are a bipotential, human hepatoma line that express apical and basolateral BA transporters. Thus, we sought to determine whether HepaRG cells could replicate the response of primary human hepatocytes to BA exposure in vitro. HepG2 cells, primary murine hepatocytes (PMH) or HepaRG cells, were exposed to taurocholic acid (TCA), or glycochenodeoxycholate (GCDC) and lactate dehydrogenase release were measured to determine cell death. Cell death occurred dose-responsively in HepaRG cells when exposed to GCDC; however, HepG2 cells died acutely only at very high concentrations of GCDC. In HepaRG cells, pre-treatment with the caspase inhibitor z-VD-FMK had no effect on cell death, indicating a lack of apoptotic cell death, and while c-jun N-terminal kinase (JNK) protein was activated by GCDC treatment in HepaRG cells, the inhibition of JNK did not protect. Although previous data indicate that TCA stimulates pro-inflammatory gene induction in PMH, there was no change in gene expression after TCA stimulation in HepaRG cells, which mimicked previous data found in primary human hepatocytes. These data provide evidence for HepaRG cells as a new model for the study of the effect of BA on human hepatocytes.