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Supplementary Figures S1-S8. S1. Glucose concentration and pH in spheroid culture medium; S2. Dose-response and time-course experiment; S3. GFP-based and TOX8 assay comparison; S4. HT-29 spheroid-based clonogenic assay; S5. Final hit compounds' effects in spheroids formed with or without medium change S6. Nitazoxanide and tizoxanide effects comparison; S7. Nitazoxanide and irinotecan combination in clonogenic assay; S8. CD44 IHC staining.

Supplementary materials, Tables S1-S2. Supplementary Table S1. List of the 12 3D-selective screening hits, Supplementary Table S2. Structures and properties of the five final hit compounds.

Because dormant cancer cells in hypoxic and nutrient-deprived regions of solid tumors provide a major obstacle to treatment, compounds targeting those cells might have clinical benefits. Here, we describe a high-throughput drug screening approach, using glucose-deprived multicellular tumor spheroids (MCTS) with inner hypoxia, to identify compounds that specifically target this cell population. We used a concept of drug repositioning—using known molecules for new indications. This is a promising strategy to identify molecules for rapid clinical advancement. By screening 1,600 compounds with documented clinical history, we aimed to identify candidates with unforeseen potential for repositioning as anticancer drugs. Our screen identified five molecules with pronounced MCTS-selective activity: nitazoxanide, niclosamide, closantel, pyrvinium pamoate, and salinomycin. Herein, we show that all five compounds inhibit mitochondrial respiration. This suggests that cancer cells in low glucose concentrations depend on oxidative phosphorylation rather than solely glycolysis. Importantly, continuous exposure to the compounds was required to achieve effective treatment. Nitazoxanide, an FDA-approved antiprotozoal drug with excellent pharmacokinetic and safety profile, is the only molecule among the screening hits that reaches high plasma concentrations persisting for up to a few hours after single oral dose. Nitazoxanide activated the AMPK pathway and downregulated c-Myc, mTOR, and Wnt signaling at clinically achievable concentrations. Nitazoxanide combined with the cytotoxic drug irinotecan showed anticancer activity in vivo. We here report that the FDA-approved anthelmintic drug nitazoxanide could be a potential candidate for advancement into cancer clinical trials. Mol Cancer Ther; 14(6); 1504–16. ©2015 AACR.

A. Tumors were undetectable at 4 weeks by IVIS imaging when b-AP15 was administered from day 1 after injecting A673 cells expressing GFP-luciferase. NSG mice bearing A673 xenografts were treated with DMSO, bortezomib or b-AP15 after they reached 80-100mm3 and weekly tumor size measurements were taken. B. b-AP15 slowed the growth of the established A673 xenografts and C. prolonged survival of A673 tumor bearing mice compared to bortezomib. D. VLX-1570 and b-AP15 treatment did not cause significant weight loss in xenograft models. (two way Anova with Tukey's multiple comparison test).

Ewing sarcoma is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of Ewing sarcoma cells to provide the basis for the development of novel therapies and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds that selectively inhibit the growth of Ewing sarcoma cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in Ewing sarcoma cells also identified the proteasome as a node of vulnerability in Ewing sarcoma cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in Ewing sarcoma cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of Ewing sarcoma with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for Ewing sarcoma. Cancer Res; 76(15); 4525–34. ©2016 AACR.

Cell lines were treated with the indicated compounds for 96 h and then growth determined. Results represent the mean {plus minus} SD of 2-4 experiments in which each condition was assayed in triplicate.

Results and analysis of a genome-wide functional genomic screen using the Sigma-Aldrich shRNA Human Genome Library consisting of 63,093 shRNAs targeting 11,748 human genes.

A. Pair�wise expression profile comparisons of EWS cell lines treated with the known proteasome inhibitors bortezomib and MG�262 demonstrated highly significant overlap with profiles treated with compounds EWS-P and EWS-W. B. Overlap included 13 core genes established through published studies as being upregulated by 20S inhibitors such as bortezomib and MG-262. C. EWS cell lines TC-71 and CHP100 show demonstrate an increase in polyubiquitinated proteins following treatment with proteasome inhibitors MG-262, bortezomib, EWS-P, and EWS-W.

General Procedures for synthesis of 2,6-Bis-arylidenepiperidone scaffold

Ingenuity Pathway Analysis of positive hits identifies enrichment of genes involved in the ubiquitin-proteasome pathway.

72 hour IC50 growth inhibition levels (μM) of 23 compounds (labeled EWS-A through EWS-X) against five EWS cell lines and 11 non-EWS cell lines

Panel upper left: CHP100 cells exposed 3 hours to different concentrations of b-AP15. Extracts were labeled with HA-Ub-VS and subjected to western blot. Filters probed with HA antibody. Note the lack of inhibition of any of the DUBs labeled by HA-Ub-VS. Panel upper right: CHP100 cells exposed 3 hours to 1 uM b-AP15 Extracts were labeled with Ub-VS and subjected to western blot. Filters probed with USP14, UCHL5 or UCHL1 antibodies. b-AP15 treated extracts demonstrate inhibition of USP14, weak inhibition of UCHL5, and no inhibition of UCHL1. Middle Panels: Purified 26S proteasomes (5 nM) were treated with indicate concentrations for b-AP15 and DUB activity was determined by cleavage of Ub-AMC. Note the reduction in 26S associated proteasome activity following treatment with b-AP15. Lower Panels: Recombinant UCHL1 or UCHL3 was treated with b-AP15 and DUB activity was monitored using Ub-AMC cleavage as above. No inhibition of either UCHL1 or UCHL3 was observed.

The biological responses to dienone compounds with a 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore have been studied extensively. Despite their expected general thiol reactivity, these compounds display considerable degrees of tumor cell selectivity. Here we review in vitro and preclinical studies of dienone compounds including b-AP15, VLX1570, RA-9, RA-190, EF24, HO-3867, and MCB-613. A common property of these compounds is their targeting of the ubiquitin-proteasome system (UPS), known to be essential for the viability of tumor cells. Gene expression profiling experiments have shown induction of responses characteristic of UPS inhibition, and experiments using cellular reporter proteins have shown that proteasome inhibition is associated with cell death. Other mechanisms of action such as reactivation of mutant p53, stimulation of steroid receptor coactivators, and induction of protein cross-linking have also been described. Although unsuitable as biological probes due to widespread reactivity, dienone compounds are cytotoxic to apoptosis-resistant tumor cells and show activity in animal tumor models. Funding Agencies|Department of Defense Ovarian Cancer Research ProgramUnited States Department of Defense [OC160377]; Minnesota Ovarian Cancer Alliance; Randy Shaver Cancer Research Funds; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [NIGMS R01-GM130800]

Auranofin is a gold (1)-containing compound used for the treatment of rheumatic arthritis. Auranofin has anticancer activity in animal models and is approved for clinical trials for lung and ovarian carcinomas. Both the cytosolic and mitochondrial forms of the selenoprotein thioredoxin reductase (TrxR) are well documented targets of auranofin. Auranofin was recently reported to also inhibit proteasome activity at the level of the proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14. We here set out to re-examine the molecular mechanism underlying auranofin cytotoxicity towards cultured cancer cells. The effects of auranofin on the proteasome were examined in cells and in vitro, effects on DUB activity were assessed using different substrates. The cellular response to auranofin was compared to that of the 20S proteasome inhibitor bortezomib and the 19S DUB inhibitor b-AP15 using proteomics. Auranofin was found to inhibit mitochondrial activity and to an induce oxidative stress response at IC50 doses. At 2-3-fold higher doses, auranofin inhibits proteasome processing in cells. At such supra-pharmacological concentrations USP14 activity was inhibited. Analysis of protein expression profiles in drug-exposed tumor cells showed that auranofin induces a response distinct from that of the 20S proteasome inhibitor bortezomib and the DUB inhibitor b-AP15, both of which induced similar responses. Our results support the notion that the primary mechanism of action of auranofin is TrxR inhibition and suggest that proteasome DUB inhibition is an off-target effect. Whether proteasome inhibition will contribute to the antineoplastic effect of auranofin in treated patients is unclear but remains a possibility. (C) 2019 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved. Funding Agencies|Swedish Cancer Society; Radiumhemmets Forskningsfonder; Vetenskapsradet; Barncancerfonden; Knut and Alice Wallenbergs Foundation

Proteasome inhibitors have been shown to induce cell death in cancer cells by triggering an acute proteotoxic stress response characterized by accumulation of poly-ubiquitinated proteins, ER stress and the production of reactive oxygen species. The aggresome pathway has been described as an escape mechanism from proteotoxicity by sequestering toxic cellular aggregates. Here we show that b-AP15, a small-molecule inhibitor of proteasomal deubiquitinase activity, induces poly-ubiquitin accumulation in absence of aggresome formation. b-AP15 was found to affect organelle transport in treated cells, raising the possibility that microtubule-transport of toxic protein aggregates is inhibited, leading to enhanced cytotoxicity. In contrast to the antiproliferative effects of the clinically used proteasome inhibitor bortezomib, the effects of b-AP15 are not further enhanced by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Our results suggest an inhibitory effect of b-AP15 on the transport of misfolded proteins, resulting in a lack of aggresome formation, and a strong proteotoxic stress response.

Quiescent cell populations respond poorly to conventional cancer therapeutic drugs and tend to resume proliferation, resulting in tumor remission. Targeting such nonproliferating cell populations will require novel therapeutic concepts. Quiescent cell populations are generally present in poorly vascularized tumor areas characterized by both hypoxia and reduced nutrient availability. Recent studies have shown that metabolically compromised tumor cells are sensitive to targeting of mitochondrial bioenergetics. In addition to vulnerabilities due to microenvironmental factors, genetic alterations may result in increased dependence of tumor cells on mitochondrial energy production. This chapter discusses mitochondrial inhibitors that are considered as cancer therapeutics, with emphasis on desired pharmacological properties. Successful development of drugs that are effective on quiescent and metabolically compromised cell populations in solid tumors may enable tumor eradication.

Cancer cells are characterized by a higher rate of protein turnover and greater demand for protein homeostasis compared to normal cells. In this scenario, the ubiquitin–proteasome system (UPS), which is responsible for the degradation of over 80% of cellular proteins within mammalian cells, becomes vital to cancer cells, making the UPS a critical target for the discovery of novel cancer therapeutics. This review systematically categorizes all current reported small molecule inhibitors of the various essential components of the UPS, including ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), the 20S proteasome catalytic core particle (20S CP) and the 19S proteasome regulatory particles (19S RP), as well as their mechanism/s of action and limitations. We also discuss the immunoproteasome which is considered as a prospective therapeutic target of the next generation of proteasome inhibitors in cancer therapies.

Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27

An increasing number of manuscripts focus on the in vitro evaluation of established and novel anti-tumor agents in experimental models. Whilst the design of such in vitro assays is inherently flexible, some of these studies lack the minimum information necessary to critically evaluate their relevance or have been carried out under unsuitable conditions. The use of appropriate and robust methods and experimental design has important implications for generating results that are reliable, relevant, and reproducible. The Pharmacology and Molecular Mechanisms (PAMM) group of the European Organization for Research and Treatment of Cancer (EORTC) is the largest group of academic scientists working on drug development and bundle decades of expertise in this field. This position paper addresses all researchers with an interest in the preclinical and cellular pharmacology of anti-tumor agents and aims at generating basic recommendations for the correct use of compounds to be tested for anti-tumor activity using a range of preclinical cellular models of cancer.

Maintenance of protein homeostasis is a crucial process for the normal functioning of the cell. The regulated degradation of proteins is primarily facilitated by the ubiquitin proteasome system (UPS), a system of selective tagging of proteins with ubiquitin followed by proteasome-mediated proteolysis. The UPS is highly dynamic consisting of both ubiquitination and deubiquitination steps that modulate protein stabilization and degradation. Deregulation of protein stability is a common feature in the development and progression of numerous cancer types. Simultaneously, the elevated protein synthesis rate of cancer cells and consequential accumulation of misfolded proteins drives UPS addiction, thus sensitizing them to UPS inhibitors. This sensitivity along with the potential of stabilizing pro-apoptotic signaling pathways makes the proteasome an attractive clinical target for the development of novel therapies. Targeting of the catalytic 20S subunit of the proteasome is already a clinically validated strategy in multiple myeloma and other cancers. Spurred on by this success, promising novel inhibitors of the UPS have entered development, targeting the 20S as well as regulatory 19S subunit and inhibitors of deubiquitinating and ubiquitin ligase enzymes. In this review, we outline the manner in which deregulation of the UPS can cause cancer to develop, current clinical application of proteasome inhibitors, and the (pre-)clinical development of novel inhibitors of the UPS. Funding Agencies|Cancerfonden; Vetenskapsradet; Radiumhemmets forskningsfonder; Barncancerfonden; Knut and Alice Wallenberg Foundation; International Myeloma Foundation

Inhibitors of the 20S proteasome such as bortezomib (Velcade((R))) and carfilzomib (Kypriolis((R))) are in clinical use for the treatment of patients with multiple myeloma and mantle cell lymphoma. In an attempt to identify novel inhibitors of the ubiquitin-proteasome system (UPS) we used the connectivity map (CMap) resource, based on alterations of gene expression profiles by perturbagens, and performed COMPARE analyses of drug sensitivity patterns in the NCI60 panel. Cmap analysis identified a large number of small molecules with strong connectivity to proteasome inhibition, including both well characterized inhibitors of the 20S proteasome and molecules previously not described to inhibit the UPS. A number of these compounds have been reported to be cytotoxic to tumor cells and were tested for their ability to decrease processing of proteasome substrates. The antibiotic thiostrepton and the natural products celastrol and curcumin induced strong accumulation of polyubiquitinated proteasome substrates in exposed cells. Other compounds elicited modest increases of proteasome substrates, including the protein phosphatase inhibitor BCI-Cl and the farnesyltransferase inhibitor manumycin A, suggesting that these compounds inhibit proteasome function. Induction of chaperone expression in the absence of proteasome inhibition was observed by a number of compounds, suggesting other effects on the UPS. We conclude that the combination of bioinformatic analyses and cellular assays resulted in the identification of compounds with potential to inhibit the UPS. Funding Agencies|CancerfondenSwedish Cancer Society; VetenskapsradetSwedish Research Council; Radiumhemmets forskningsfonder; Knut och Alice Wallenbergs StiftelseKnut & Alice Wallenberg Foundation

The proteasome is a validated target of cancer therapeutics. Inhibition of proteasome activity results in the activation of the unfolded protein response (UPR) characterized by phosphorylation of eukaryotic initiation factor 2&alpha, (eIF2&alpha, ), global translational arrest, and increased expression of the proapoptotic CHOP (C/EBP homologous protein) protein. Defects in the UPR response has been reported to result in altered sensitivity of tumor cells to proteasome inhibitors. Here, we characterized the effects of the deubiquitinase (DUB) inhibitor VLX1570 on protein homeostasis, both at the level of the UPR and on protein translation, in acute lymphoblastic leukemia (ALL). Similar to the 20S inhibitor bortezomib, VLX1570 induced accumulation of polyubiquitinated proteins and increased expression of the chaperone Grp78/Bip in ALL cells. Both compounds induced cleavage of PARP (Poly (ADP-ribose) polymerase) in ALL cells, consistent with induction of apoptosis. However, and in contrast to bortezomib, VLX1570 treatment resulted in limited induction of the proapoptotic CHOP protein. Translational inhibition was observed by both bortezomib and VLX1570. We report that in distinction to bortezomib, suppression of translation by VXL1570 occurred at the level of elongation. Increased levels of Hsc70/Hsp70 proteins were observed on polysomes following exposure to VLX1570, possibly suggesting defects in nascent protein folding. Our findings demonstrate apoptosis induction in ALL cells that appears to be uncoupled from CHOP induction, and show that VLX1570 suppresses protein translation by a mechanism distinct from that of bortezomib.

This phase 1 study sought to characterize the safety, tolerability, and pharmacokinetic behavior of VLX1570, a small molecule inhibitor of the deubiquitinases (DUBs) that remove sterically bulky ubiquitin chains from proteins during processing in the19S regulatory subunit of the proteasome, in patients with relapsed and refractory multiple myeloma (MM). Fourteen patients were treated with escalating doses of VLX1570 ranging from 0.05 to 1.2 mg/kg as a brief intravenous (IV) infusion on Days 1, 2, 8, 9, 15, and 16 of a 28-day cycle. Due to its poor aqueous solubility, VLX1570 was formulated in polyethylene glycol, polyoxyethylated castor oil, and polysorbate 80 and administered as a brief intravenous (IV) infusion via a central venous catheter. Anti-myeloma effects were noted at doses at or above 0.6 mg/kg, however, two patients treated at the 1.2 mg/kg dose level experienced severe, abrupt, and progressive respiratory insufficiency, which was associated with diffuse pulmonary infiltrates on imaging studies, similar to those rarely noted with bortezomib and other inhibitors of the 20S proteasome, culminating in death. Although the contribution of VLX1570s formulation to the pulmonary toxicity could not be ruled out, the severity and precipitous nature of the toxicity and the steep relationship between dose and toxicity, the study was discontinued. Despite the severe pulmonary toxicity noted with VLX1570, efforts directed at identifying DUB inhibitors with greater therapeutic indices appear warranted based on the unique mechanism of action, robustness of preclinical antitumor activity, and activity of the DUB inhibitors in MM resistant to PIs targeting the 20S proteasome subunit. Funding Agencies|Uppsala University; Vivolux, AB

Cyanobacteria are known to produce many toxins and other secondary metabolites. The study of their specific mode of action may reveal the biotechnological potential of such compounds. Portoamides A and B (PAB) are cyclic peptides isolated from the cyanobacteria Phormidium sp. due to their growth repression effect on microalgae and were shown to be cytotoxic against certain cancer cell lines. In the present work, viability was assessed on HCT116 colon cancer cells grown as monolayer culture and as multicellular spheroids (MTS), non-carcinogenic cells and on zebrafish larvae. HCT116 cells and epithelial RPE-1hTERT cells showed very similar degrees of sensitivities to PAB. PAB were able to penetrate the MTS, showing a four-fold high IC50 compared to monolayer cultures. The toxicity of PAB was similar at 4 °C and 37 °C suggesting energy-independent uptake. PAB exposure decreased ATP production, mitochondrial maximal respiration rates and induced mitochondrial membrane hyperpolarization. PAB induced general organelle stress response, indicated by an increase of the mitochondrial damage sensor PINK-1, and of phosphorylation of eIF2α, characteristic for endoplasmic reticulum stress. In summary, these findings show general toxicity of PAB on immortalized cells, cancer cells and zebrafish embryos, likely due to mitochondrial toxicity. This research was supported by the project CYANCAN - Uncovering the cyanobacterial chemical diversity: the search for novel anticancer compounds (reference PTDC/MEDQUI/30944/2017) co-financed by NORTE 2020, Portugal 2020 and the European Union through the ERDF, and by Foundation for Science and Technology through national funds and strategic fund UID/Multi/04423/2019. Ralph Urbatzka was supported by the FCT postdoc grant SFRH/BPD/112287/2015, Maria Lígia Sousa by the FCT PhD grant SFRH/BD/108314/2015 and Tiago Ribeiro by SFRH/BD/139131/2018. SL was supported by Cancerfonden, Vetenskapsrådet, Radiumhemmets forskningsfonder and Knut och Alice Wallenbergs stiftelse.

There is a need for preclinical models that can enable identification of novel radiosensitizing drugs in clinically relevant high-throughput experiments. We used a new high-throughput compatible total cell kill spheroid assay to study the interaction between drugs and radiation in order to identify compounds with radiosensitizing activity. Experimental drugs were compared to known radiosensitizers and cytotoxic drugs clinically used in combination with radiotherapy. VLX600, a novel iron-chelating inhibitor of oxidative phosphorylation, potentiated the effect of radiation in tumor spheroids in a synergistic manner. This effect was specific to spheroids and not observed in monolayer cell cultures. In conclusion, the total cell kill spheroid assay is a feasible high-throughput method in the search for novel radiosensitizers. VLX600 shows encouraging characteristics for development as a novel radiosensitizer.

Precision medicine approaches based on DNA or RNA analyses, are often not identifying actionable mutations in the patients, and have not been established for predicting presonse to commonly used drugs such as platins and taxols. In epithelial ovarian cancer (EOC), where paclitaxel and carboplatin is the most commonly used first-line treatment, however, local practice and a host of clinical studies have/are evaluating combinations with other drugs, implying that first line treatment regimens may vary in different countries and regions. As EOC patients that respond to first-line treatment have much better prognosis that those who don't, there is an urgent need for methods to predict the outcome to various available first line therapies, prior to treatment onset, so that the optimal treatment can be identified and offered to every patient. Zebrafish tumor xenograft (ZTX) models have recently been used to predict treatment outcome to first line therapy in multiple myeloma, neuroendocrine cancer, colorectal cancer and gastric cancer, but these studies have been small case studies including only a few patients. Furthermore, EOC, and the main drugs used to treat them have, however, not been studied in ZTX models in the past. Here we present results from the first 81 patients in an ongoing clinical study to delineate the sensitivity and specificity of ZTX models to predict treatment outcome to paclitaxel and carboplatin. Of these 81 patients, 21 presented with a primary tumor only, whereas 60 had metastatic disease. To date, 25 patient-derived tumor xenograft models have been developed by random selection of samples from this patient cohort, of which 12 were from paired primary and metastatic lesions from 6 patients. We show that tumors generated from metastatic omental lesions exhibited similar growth and metastatic dissemination as those generated from samples taken from ovarian lesions. Among the 25 patient models generated, 15 were analyzed for efficacy of paclitaxel and carboplatin in the ZTX models. We found that 11 of 15 responded to paclitaxel, 12 of 15 to carboplatin and 10 of 15 to both drugs. In conclusion, ZTX EOC models established from surgical samples allow prediction of treatment outcomes to 1st line treatment and thereby may aid treatment planning in the future. Citation Format: Karthik Selvaraj, Malin Vildevall, Lina Wirestam, Zaheer Ali, Anna Erkstam, Annelie Abrahamsson, Åsa Rydmark Kersley, Preben Kjölhede, Stig Linder, Charlotta Dabrosin, Anna Fahlgren, Lasse Jensen. Zebrafish tumor-derived xenograft-models for improved diagnosis and treatment planning in ovarian cancer patients [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 3000.

Ovarian clear cell carcinoma (OCCC) is a rare but chemorefractory tumor. About 50% of all OCCC patients have inactivating mutations of ARID1A, a member of the SWI/SNF chromatin-remodeling complex. Members of the SWI/SNF remodeling have emerged as regulators of the energetic metabolism of mammalian cells, however, the role of ARID1A as a modulator of the mitochondrial metabolism in OCCCs is yet to be defined. Here, we show that ARID1A loss results in increased mitochondrial metabolism and renders ARID1A-mutated cells increasingly and selectively dependent on it. The increase in mitochondrial activity following ARID1A loss is associated with increase in c-Myc expression and increased mitochondrial number and reduction of their size consistent with a higher mitochondrial cristae/outer membrane ratio. Significantly, preclinical testing of the complex I mitochondrial inhibitor IACS-010759 showed it extends overall survival in a preclinical model of ARID1A-mutated OCCC. These findings provide for the targeting mitochondrial activity in ARID1A-mutated OCCCs.

The presence of quiescent cell populations in solid tumors represents a major challenge for disease eradication. Such cells are generally present in poorly vascularized tumor areas, show limited sensitivity to traditional chemotherapeutical drugs, and tend to resume proliferation, resulting in tumor reseeding and growth. There is growing recognition of the importance of developing therapies that target these quiescent cell populations to achieve long-lasting remission. Recent studies have shown that the combination of hypoxia and reduced nutrient availability in poorly vascularized areas results in limited tumor metabolic plasticity coupled with an increased sensitivity to perturbations in mitochondrial flux. Targeting of mitochondrial bioenergetics in these quiescent cell tumor populations may enable tumor eradication and improve the prognosis of patients with cancer.

A large number of natural products have been advocated as anticancer agents. Many of these compounds contain functional groups characterized by chemical reactivity. It is not clear whether distinct mechanisms of action can be attributed to such compounds. We used a chemical library screening approach to demonstrate that a substantial fraction (similar to 20%) of cytotoxic synthetic compounds containing Michael acceptor groups inhibit proteasome substrate processing and induce a cellular response characteristic of proteasome inhibition. Biochemical and structural analyses showed binding to and inhibition of proteasome-associated cysteine deubiquitinases, in particular ubiquitin specific peptidase 14 (USP14). The results suggested that compounds bind to a crevice close to the USP14 active site with modest affinity, followed by covalent binding. A subset of compounds was identified where cell death induction was closely associated with proteasome inhibition and that showed significant antineoplastic activity in a zebrafish embryo model. These findings suggest that proteasome inhibition is a relatively common mode of action by cytotoxic compounds containing Michael acceptor groups and help to explain previous reports on the antineoplastic effects of natural products containing such functional groups. Funding Agencies|Cancerfonden; Vetenskapsradet; Radiumhemmets forskningsfonder; Knut och Alice Wallenbergs Stiftelse; Barncancerfonden

Ovarian cancer is the leading cause of death from gynecologic malignancy in the Western world. This is due, in part, to the fact that despite standard treatment of surgery and platinum/paclitaxel most patients recur with ultimately chemoresistant disease. Ovarian cancer is a unique form of solid tumor that develops, metastasizes and recurs in the same space, the abdominal cavity, which becomes a unique microenvironment characterized by ascites, hypoxia and low glucose levels. It is under these conditions that cancer cells adapt and switch to mitochondrial respiration, which becomes crucial to their survival, and therefore an ideal metabolic target for chemoresistant ovarian cancer. Importantly, independent of microenvironmental factors, mitochondria spatial redistribution has been associated to both tumor metastasis and chemoresistance in ovarian cancer while specific sets of genetic mutations have been shown to cause aberrant dependence on mitochondrial pathways in the most aggressive ovarian cancer subtypes. In this review we summarize on targeting mitochondria for treatment of chemoresistant ovarian cancer and current state of understanding of the role of mitochondria respiration in ovarian cancer. We feel this is an important and timely topic given that ovarian cancer remains the deadliest of the gynecological diseases, and that the mitochondrial pathway has recently emerged as critical in sustaining solid tumor progression. Funding Agencies|Department of Defense Ovarian Cancer Research Program [OC160377]; Minnesota Ovarian Cancer Alliance; Randy Shaver Cancer Research Funds

Human cancers are characterized by intrinsic or acquired resistance to apoptosis and evasion of apoptosis has been proposed to contribute to treatment resistance. Bis-benzylidine piperidone compounds, containing α,β-unsaturated carbonyl functionalities, have been extensively documented as being effective in killing apoptosis-resistant cells and to display promising antineoplastic activities in a number of tumor models. We here explored the phenotypic response of colon cancer cells to b-AP15, a bis-benzylidine piperidone previously shown to inhibit the proteasome deubiquitinases (DUBs) USP14 and UCHL5. Whereas similar overall mRNA and protein expression profiles were induced by b-AP15 and the clinically available proteasome inhibitor bortezomib, b-AP15 induced stronger increases of chaperone expression. b-AP15 also induced a stronger accumulation of polyubiquitinated proteins in exposed cells. These proteins were found to partially colocalize with organelle structures, including mitochondria. Mitochondrial oxidative phosphorylation decreased in cells exposed to b-AP15, a phenomenon enhanced under conditions of severe proteotoxic stress caused by inhibition of the VCP/p97 ATPase and inhibition of protein translocation over the ER. We propose that mitochondrial damage caused by the association of misfolded proteins with mitochondrial membranes may contribute to the atypical cell death mode induced by b-AP15 and related compounds. The robust mode of cell death induction by this class of drugs holds promise for treatment of tumor cells characterized by apoptosis resistance.

Background: Drug screening for the identification of compounds with anticancer activity is commonly performed using cell lines cultured under normal oxygen pressure and physiological pH. However, solid tumors are characterized by a microenvironment with limited access to nutrients, reduced oxygen supply and acidosis. Tumor hypoxia and acidosis have been identified as important drivers of malignant progression and contribute to multicellular resistance to different forms of therapy. Tumor acidosis represents an important mechanism mediating drug resistance thus the identification of drugs active on acid-adapted cells may improve the efficacy of cancer therapy. Methods: Here, we characterized human colon carcinoma cells (HCT116) chronically adapted to grow at pH 6.8 and used them to screen the Prestwick drug library for cytotoxic compounds. Analysis of gene expression profiles in parental and low pH-adapted cells showed several differences relating to cell cycle, metabolism and autophagy. Results: The screen led to the identification of several compounds which were further selected for their preferential cytotoxicity towards acid-adapted cells. Amongst 11 confirmed hits, we primarily focused our investigation on the benzoporphyrin derivative Verteporfin (VP). VP significantly reduced viability in low pH-adapted HCT116 cells as compared to parental HCT116 cells and normal immortalized epithelial cells. The cytotoxic activity of VP was enhanced by light activation and acidic pH culture conditions, likely via increased acid-dependent drug uptake. VP displayed the unique property to cause light-dependent cross-linking of proteins and resulted in accumulation of polyubiquitinated proteins without inducing inhibition of the proteasome. Conclusions: Our study provides an example and a tool to identify anticancer drugs targeting acid-adapted cancer cells. Funding Agencies|Swedish Cancer Society [CAN 2012/415]; Association for International Cancer Research [11-0522]; Robert Lundberg Foundation

The MYC proto-oncogene serves as a rheostat coupling mitogenic signaling with the activation of genes regulating growth, metabolism and mitochondrial biogenesis. Here we describe a novel link between mitochondria and MYC levels. Perturbation of mitochondrial function using a number of conventional and novel inhibitors resulted in the decreased expression of MYC mRNA. This decrease in MYC mRNA occurred concomitantly with an increase in the levels of tumor-suppressive miRNAs such as members of the let-7 family and miR-34a-5p. Knockdown of let-7 family or miR-34a-5p could partially restore MYC levels following mitochondria damage. We also identified let-7-dependent downregulation of the MYC mRNA chaperone, CRD-BP (coding region determinant-binding protein) as an additional control following mitochondria damage. Our data demonstrates the existence of a homeostasis mechanism whereby mitochondrial function controls MYC expression.

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Developing natural products as potential antineoplastic drugs is a meticulous process involving both compound isolation and biological testing. Many studies are based on primary screening using tumor cell viability as the readout followed by compound isolation. We here present an approach which utilizes both 2-D and 3-D cultured of tumor cells for screening and immortalized human non-transformed cells for counter screening. This procedure increases the precision of identifying tumor-specific cytotoxic compounds with interesting pharmacological properties. Using this straight-forward approach, we screened 500 plant extracts from the Egyptian flora for anticancer activity. The primary screen on 2-D cultured cells yielded 41 hits, 12 of which showed significant cytotoxicity on 3-D cultured cells. Of these, 4 extracts showed limited cytoxicity to normal cells. We conclude that only ~10% of the cytotoxic extracts showed desired properties with regard to tumor parenchyme penetration and tumor-specific activity. Extracts from Euphorbia dendroides L. herb, Ononis vaginalis Vahl. herb and Quercus robur L. branches were found to induce tumor apoptosis and were considered the most promising. These three extracts showed significant inhibition in the Ehrlich ascites carcinoma in vivo model and did not show severe toxicity on healthy animals.

Background: Breast cancer cells with tumor-initiating capabilities (BSCs) are considered to maintain tumor growth and govern metastasis. Hence, targeting BSCs will be crucial to achieve successful treatment of breast cancer. Methods: We characterized mammospheres derived from more than 40 cancer patients and two breast cancer cell lines for the expression of estrogen receptors (ERs) and stem cell markers. Mammosphere formation and proliferation assays were performed on cells from 19 cancer patients and five healthy individuals after incubation with ER-subtype selective ligands. Transcriptional analysis was performed to identify pathways activated in ER beta-stimulated mammospheres and verified using in vitro experiments. Xenograft models (n = 4 or 5 per group) were used to study the role of ERs during tumorigenesis. Results: We identified an absence of ERa but upregulation of ER beta in BSCs associated with phenotypic stem cell markers and responsible for the proliferative role of estrogens. Knockdown of ER beta caused a reduction of mammosphere formation in cell lines and in patient-derived cancer cells (40.7%, 26.8%, and 39.1%, respectively). Gene set enrichment analysis identified glycolysis-related pathways (false discovery rate amp;lt; 0.001) upregulated in ER beta-activated mammospheres. We observed that tamoxifen or fulvestrant alone was insufficient to block proliferation of patient-derived BSCs while this could be accomplished by a selective inhibitor of ER beta (PHTPP; 53.7% in luminal and 45.5% in triple-negative breast cancers). Furthermore, PHTPP reduced tumor initiation in two patient-derived xenografts (75.9% and 59.1% reduction in tumor volume, respectively) and potentiated tamoxifen-mediated inhibition of tumor growth in MCF7 xenografts. Conclusion: We identify ER beta as a mediator of estrogen action in BSCs and a novel target for endocrine therapy. Funding Agencies|Swedish Society of Medicine; Swedish Society for Medical Research (SSMF); Magnus Bergvalls Stiftelse; Karolinska Institutes Theme Center in Breast Cancer (BRECT); Linnecenter for Prevention of Breast and Prostate cancer (CRisP); Swedish Cancer Society; Stockholm Cancer Society; King Gustav V Jubilee Fund; Karolinska Institutet; Stockholm County Council Research Strategy Committee; Swedish Breast Cancer Association; Science for Life-Astra Zeneca; Marie Curie Actions via the VINNOVA program Mobility for Growth [291795]

// Magdalena Mazurkiewicz 1 , Ellin-Kristina Hillert 1 , Xin Wang 2 , Paola Pellegrini 2 , Maria Hagg Olofsson 1 , Karthik Selvaraju 2 , Padraig D’Arcy 2 , Stig Linder 1, 2 1 Cancer Center Karolinska, Department of Oncology and Pathology, Karolinska Institute, S-171 76 Stockholm, Sweden 2 Department of Medical Health Sciences (IMH), Linkoping University, S-751 85 Linkoping, Sweden Correspondence to: Stig Linder, email: Stig.Linder@liu.se Keywords: acute lymphoblastic leukemia, proteasome, deubiquitinase, ER stress, VLX1570 Received: December 02, 2016 Accepted: February 07, 2017 Published: February 18, 2017 ABSTRACT The non-genotoxic nature of proteasome inhibition makes it an attractive therapeutic option for the treatment of pediatric malignancies. We recently described the small molecule VLX1570 as an inhibitor of proteasome deubiquitinase (DUB) activity that induces proteotoxic stress and apoptosis in cancer cells. Here we show that acute lymphoblastic leukemia (ALL) cells are highly sensitive to treatment with VLX1570, resulting in the accumulation of polyubiquitinated proteasome substrates and loss of cell viability. VLX1570 treatment increased the levels of a number of proteins, including the chaperone HSP70B’, the oxidative stress marker heme oxygenase-1 (HO-1) and the cell cycle regulator p21 Cip1 . Unexpectedly, polyubiquitin accumulation was found to be uncoupled from ER stress in ALL cells. Thus, increased phosphorylation of eIF2α occurred only at supra-pharmacological VLX1570 concentrations and did not correlate with polyubiquitin accumulation. Total cellular protein synthesis was found to decrease in the absence of eIF2α phosphorylation. Furthermore, ISRIB (Integrated Stress Response inhibitor) did not overcome the inhibition of protein synthesis. We finally show that VLX1570 can be combined with L-asparaginase for additive or synergistic antiproliferative effects on ALL cells. We conclude that ALL cells are highly sensitive to the proteasome DUB inhibitor VLX1570 suggesting a novel therapeutic option for this disease.

Inhibition of deubiquitinase (DUB) activity is a promising strategy for cancer therapy. VLX1570 is an inhibitor of proteasome DUB activity currently in clinical trials for relapsed multiple myeloma. Here we show that VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). Exposure of multiple myeloma cells to VLX1570 resulted in thermostabilization of USP14 at therapeutically relevant concentrations. Transient knockdown of USP14 or UCHL5 expression by electroporation of siRNA reduced the viability of multiple myeloma cells. Treatment of multiple myeloma cells with VLX1570 induced the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and an apoptotic response. Sensitivity to VLX1570 was moderately affected by altered drug uptake, but was unaffected by overexpression of BCL2-family proteins or inhibitors of caspase activity. Finally, treatment with VLX1570 was found to lead to extended survival in xenograft models of multiple myeloma. Our findings demonstrate promising antiproliferative activity of VLX1570 in multiple myeloma, primarily associated with inhibition of USP14 activity.