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CD8+ T cell proliferation and differentiation into effector and memory states are high-energy processes associated with changes in cellular metabolism. CD28-mediated costimulation of T cells activates the PI3K/AKT/mammalian target of rapamycin signaling pathway and induces eukaryotic translation initiation factor 4E–dependent translation through the derepression by 4E-BP1 and 4E-BP2. In this study, we demonstrate that 4E-BP1/2 proteins are required for optimum proliferation of mouse CD8+ T cells and the development of an antiviral effector function. We show that translation of genes encoding mitochondrial biogenesis is impaired in T cells derived from 4E-BP1/2–deficient mice. Our findings demonstrate an unanticipated role for 4E-BPs in regulating a metabolic program that is required for cell growth and biosynthesis during the early stages of CD8+ T cell expansion.

Supplementary Tables 1 and 2 show respectively the fold change of lysosomal function and size in treated sensitive/resistant cells obtained by mixed effect modelling. Supplementary Table 3 shows patients enrolled per dose-level (DL) and dose limiting toxicities (DLTs). Supplementary Tables 4 and 5 show the summary of all and treatment related adverse effects.

CRISPR screen TOV1946. Results of the Itra sensitizing CRISPR screen on the TOV1946 cell line using drugZ algorithm.

List of cell lines- Lentiviral constructs, lentivirus generation and infection- Apoptosis Assay- FILIPIN staining- Lysosomal assay and immunofluorescence

CRISPR screen OVCAR5. Results of the Itra sensitizing CRISPR screen on the OVCAR5 cell line using drugZ algorithm.

Supplementary Figure 1: Itra dose response in ovarian cancer cell lines. - Supplementary Figure 2: Analysis on ovarian cancer cell lines used in the Itra-sensitizing CRISPR screen (OVCAR5 and TOV1946). - Supplementary Figure 3: c18orf8 and VPS54 Knockout effects on the TOV1946 cell line. - Supplementary Figure 4: Itra+CQ dose responses. - Supplementary Figure 5: 5/10 uM CQ response and apoptosis assay results. - Supplementary Figure 6: Itra+CQ dose response in c18orf8 and VPS54 knockout cells and calculation of synergy scores. - Supplementary Figure 7: Intratumoural Itra/CQ detection and Ki-67 analysis in patient samples.

Drug repurposing is an attractive option for oncology drug development. Itraconazole is an antifungal ergosterol synthesis inhibitor that has pleiotropic actions including cholesterol antagonism, inhibition of Hedgehog and mTOR pathways. We tested a panel of 28 epithelial ovarian cancer (EOC) cell lines with itraconazole to define its spectrum of activity. To identify synthetic lethality in combination with itraconazole, a whole-genome drop-out genome-scale clustered regularly interspaced short palindromic repeats sensitivity screen in two cell lines (TOV1946 and OVCAR5) was performed. On this basis, we conducted a phase I dose-escalation study assessing the combination of itraconazole and hydroxychloroquine in patients with platinum refractory EOC (NCT03081702). We identified a wide spectrum of sensitivity to itraconazole across the EOC cell lines. Pathway analysis showed significant involvement of lysosomal compartments, the trans-golgi network and late endosomes/lysosomes; similar pathways are phenocopied by the autophagy inhibitor, chloroquine. We then demonstrated that the combination of itraconazole and chloroquine displayed Bliss defined synergy in EOC cancer cell lines. Furthermore, there was an association of cytotoxic synergy with the ability to induce functional lysosome dysfunction, by chloroquine. Within the clinical trial, 11 patients received at least one cycle of itraconazole and hydroxychloroquine. Treatment was safe and feasible with the recommended phase II dose of 300 and 600 mg twice daily, respectively. No objective responses were detected. Pharmacodynamic measurements on serial biopsies demonstrated limited pharmacodynamic impact. In vitro, itraconazole and chloroquine have synergistic activity and exert a potent antitumor effect by affecting lysosomal function. The drug combination had no clinical antitumor activity in dose escalation.Significance:The combination of the antifungal drug itraconazole with antimalarial drug hydroxychloroquine leads to a cytotoxic lysosomal dysfunction, supporting the rational for further research on lysosomal targeting in ovarian cancer.

Detailed protocol of the HYDRA-1 (NCT03081702) Trial. TITLE: A phase I/II trial investigating the tolerability, toxicity and efficacy of hydroxychloroquine and itraconazole in patients with advanced platinum-resistant epithelial ovarian cancer (EOC) (HYDRA-1 study).

Supplementary Figure 1. (A) There is no effect on cell proliferation in RKO cells. (B) There is no effect on S-phase fraction in RKO and paired HCT116 cells. (C) 50-65% Clonogenic cell survival in RKO and paired HCT116 cells. (D) BER mRNA expression is unchanged in RKO cells. qRT-PCR analyses show no significant effects of prolonged hypoxia (72 h x 0.2% O2) on the mRNA levels of selected BER genes in RKO cells. Carbonic anhydrase IX (CAIX) is shown as a positive control for hypoxia.

Genotype analysis.

The mTOR signaling pathway is a central regulator of protein synthesis and cellular metabolism in response to the availability of energy, nutrients, oxygen, and growth factors. mTOR activation leads to phosphorylation of multiple downstream targets including the eukaryotic initiation factor 4E (eIF4E) binding proteins-1 and -2 (EIF4EBP1/4E-BP1 and EIF4EBP2/4E-BP2). These binding proteins inhibit protein synthesis, but are inactivated by mTOR to stimulate cell growth and metabolism. However, the role of these proteins in the context of aberrant activation of mTOR, which occurs frequently in cancers through loss of PTEN or mutational activation of the PI3K/AKT pathway, is unclear. Here, even under conditions of aberrant mTOR activation, hypoxia causes dephosphorylation of 4E-BP1/4E-BP2 and increases their association with eIF4E to suppress translation. This is essential for hypoxia tolerance as knockdown of 4E-BP1 and 4E-BP2 decreases proliferation under hypoxia and increases hypoxia-induced cell death. In addition, genetic deletion of 4E-BP1 and 4E-BP2 significantly accelerates all phases of cancer development in the context of PTEN loss–driven prostate cancer in mice despite potent PI3K/AKT and mTOR activation. However, even with a more rapid onset, tumors that establish in the absence of 4E-BP1 and 4E-BP2 have reduced levels of tumor hypoxia and show increased cell death within hypoxic tumor regions. Together, these data demonstrate that 4E-BP1 and 4E-BP2 act as essential metabolic breaks even in the context of aberrant mTOR activation and that they are essential for the creation of hypoxia-tolerant cells in prostate cancer. Mol Cancer Res; 16(4); 682–95. ©2018 AACR.

Supplementary Data from A Phase II Randomized Trial of Chemoradiation with or without Metformin in Locally Advanced Cervical Cancer

Purpose:Tumor hypoxia is associated with poor response to radiation (RT). We previously discovered a novel mechanism of metformin: enhancing tumor RT response by decreasing tumor hypoxia. We hypothesized that metformin would decrease tumor hypoxia and improve cervical cancer response to RT.Patients and Methods:A window-of-opportunity, phase II randomized trial was performed in stage IB–IVA cervical cancer. Patients underwent screening positron emission tomography (PET) imaging with hypoxia tracer fluoroazomycin arabinoside (FAZA). Only patients with FAZA uptake (hypoxic tumor) were included and randomized 2:1 to receive metformin in combination with chemoRT or chemoRT alone. A second FAZA-PET/CT scan was performed after 1 week of metformin or no intervention (control). The primary endpoint was a change in fractional hypoxic volume (FHV) between FAZA-PET scans, compared using the Wilcoxon signed-rank test. The study was closed early due to FAZA availability and the COVID-19 pandemic.Results:Of the 20 consented patients, 6 were excluded due to no FAZA uptake and 1 withdrew. FHV of 10 patients in the metformin arm decreased by an average of 10.2% (44.4%–34.2%) ± SD 16.9% after 1 week of metformin, compared with an average increase of 4.7% (29.1%–33.8%) ± 11.5% for the 3 controls (P = 0.027). Those with FHV reduction after metformin had significantly lower MATE2 expression. With a median follow-up of 2.8 years, the 2-year disease-free survival was 67% for the metformin arm versus 33% for controls (P = 0.09).Conclusions:Metformin decreased cervical tumor hypoxia in this trial that selected for patients with hypoxic tumor.See related commentary by Lyng et al., p. 5233

Characterization of ENZ-resistant cell lines

Clinical characteristics of prostate cancer patients with matched diagnostic (archival) castration-sensitive and metastatic castration-resistant tissue biopsies

Supplementary File 2 shows transcriptional pathways associated with alpha or beta from the linear-quadratic formalism.

PDF file 97K, Table S1 shows the clinical demographics of the patient cohort treated with radiotherapy for prostate cancer

The Supplementary Data file contains 10 supplementary figures and 3 supplementary tables.

Purpose: Tumor hypoxia is a negative prognostic factor in multiple cancers, due in part to its role in causing resistance to radiotherapy. Hypoxia arises in tumor regions distal to blood vessels as oxygen is consumed by more proximal tumor cells. Reducing the rate of oxygen consumption is therefore a potential strategy to reduce tumor hypoxia. We hypothesized that the anti-diabetic drug metformin, which reduces oxygen consumption through inhibition of mitochondrial complex I, would improve radiation response by increasing tumor oxygenation.Experimental Design: Tumor hypoxia was measured in xenografts before and after metformin treatment using 2-nitroimidazole hypoxia markers quantified by immunohistochemistry (IHC), flow cytometry, and positron emission tomography (PET) imaging. Radiation response was determined by tumor growth delay and clonogenic survival in xenografts with and without administration of metformin. The impact of metformin use on outcome was assessed in 504 patients with localized prostate cancer treated with curative-intent, image-guided radiotherapy (IGRT) from 1996 to 2012. Three-year biochemical relapse-free rates were assessed using the Kaplan–Meier method.Results: Metformin treatment significantly improved tumor oxygenation in two xenograft models as measured by IHC, flow cytometry, and PET imaging. Metformin also led to improved radiotherapy responses when mice were administered metformin immediately before irradiation. Clinically, metformin use was associated with an independent and significant decrease in early biochemical relapse rates (P = 0.0106).Conclusion: Our data demonstrate that metformin can improve tumor oxygenation and response to radiotherapy. Our study suggests that metformin may represent an effective and inexpensive means to improve radiotherapy outcome with an optimal therapeutic ratio. Clin Cancer Res; 19(24); 6741–50. ©2013 AACR.

PDF file 209K, Figure S1 illustrates the method for IHC analysis. It also shows a reduction in hypoxic fraction after metformin exposure in POP-92S and LNCaP xenografts

PDF file 81K, Supplementary Legends

Supplementary Data - PDF file 113K, This file contains supplementary materials and methods, and the legends of the 14 supplementary figures. Suppl. Table 1 shows the patients characteristic from the clinical study described in main Fig 1. Suppl. Table 2 shows the relationship between LAMP3 protein expression and continuous clinical parameters, incl. HP5. Suppl. Table 3 shows the relationship between LAMP3 protein expression and categorical clinical parameters, incl. FIGO stage. Suppl. Fig 1 shows the association between LAMP3 gene copy number and LAMP3 gene expression in human cervix tumors described in main Fig 1, as well as the LAMP3 gene copy numbers in normal human cervix tissues. Suppl. Fig 2 provides in vitro evidence for the inducibility of the ME180 cancer cell line. Suppl. Fig 3 provides in vivo evidence for the metastatic capacity of the isogenic ME180 cell line, as well as its inducibility. Suppl. Fig 4 shows in vitro validation of the ME180-GADD34-C cells. Suppl. Fig 5 contains the raw data that belongs to main Fig 3C, which shows that Inhibition of UPR signaling suppresses hypoxia-induced lymph node metastasis. Suppl. Fig 6 shows the in vitro validation of ME180 LAMP3-knockdown cells. Suppl. Fig 7 shows representative images of LAMP3 expression in LAMP3 knockdown and empty vector control tumors described in main Fig 4B. Suppl. Fig 8 contains the raw data that belongs to main Fig 4C, which shows that knockdown of LAMP3 inhibits hypoxia-induced lymph node metastasis. Suppl. Fig 9 shows the human VEGF mRNA expression in ME180-GADD34-C orthotopic xenografts described in main Fig 3B. Suppl. Fig 10 shows the quantification of the microvessel density and hVEGF expression in shLAMP3 orthotopic xenografts described in main Fig 4B. Suppl. Fig 11 shows the hypoxic fraction shLAMP3 orthotopic xenografts described in main Fig 4B. Suppl. Fig 12 contains data from the soft agar colony formation assay that shows that knockdown of LAMP3 in HeLa cells inhibits colony formation. Suppl. Fig 13 is supplementary to main Fig 6B and shows that knockdown of LAMP3 in HeLa cells, just like in ME180 cells, inhibits transwell migration. Suppl. Fig 14 is supplementary to main Fig 6D and shows that knockdown of LAMP3 in Caski cells, just like in ME180 cells, inhibits wound closure in the scratch wound healing assay. This figure also shows that overexpression of LAMP3 accelerates wound closure

Purpose:Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance.Experimental Design:We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer.Results:ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased.Conclusions:Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer.

List of antibodies used for Western Blot analyses

Radiotherapy is integral to the care of a majority of patients with cancer. Despite differences in tumor responses to radiation (radioresponse), dose prescriptions are not currently tailored to individual patients. Recent large-scale cancer cell line databases hold the promise of unravelling the complex molecular arrangements underlying cellular response to radiation, which is critical for novel predictive biomarker discovery. Here, we present RadioGx, a computational platform for integrative analyses of radioresponse using radiogenomic databases. We fit the dose–response data within RadioGx to the linear-quadratic model. The imputed survival across a range of dose levels (AUC) was a robust radioresponse indicator that correlated with biological processes known to underpin the cellular response to radiation. Using AUC as a metric for further investigations, we found that radiation sensitivity was significantly associated with disruptive mutations in genes related to nonhomologous end joining. Next, by simulating the effects of different oxygen levels, we identified putative genes that may influence radioresponse specifically under hypoxic conditions. Furthermore, using transcriptomic data, we found evidence for tissue-specific determinants of radioresponse, suggesting that tumor type could influence the validity of putative predictive biomarkers of radioresponse. Finally, integrating radioresponse with drug response data, we found that drug classes impacting the cytoskeleton, DNA replication, and mitosis display similar therapeutic effects to ionizing radiation on cancer cell lines. In summary, RadioGx provides a unique computational toolbox for hypothesis generation to advance preclinical research for radiation oncology and precision medicine.Significance:The RadioGx computational platform enables integrative analyses of cellular response to radiation with drug responses and genome-wide molecular data.

Supplementary File 1 shows transcriptional pathways associated with the radioresponse indicators SF2 or AUC.

Supplementary File 4 shows transcriptional pathways associated with radioresponse within distinct tissue types.

Purpose: Conditions of poor oxygenation (hypoxia) are present in many human tumors, including cervix cancer, and are associated with increased risk of metastasis and poor prognosis. Hypoxia is a potent activator of the PERK/eIF2α signaling pathway, a component of the unfolded protein response (UPR) and an important mediator of hypoxia tolerance and tumor growth. Here, the importance of this pathway in the metastasis of human cervix carcinoma was investigated.Experimental Design: Amplification and expression of LAMP3, a UPR metastasis-associated gene, was examined using FISH and immunofluorescence in a cohort of human cervix tumors from patients who had received oxygen needle electrode tumor oxygenation measurements. To evaluate the importance of this pathway in metastasis in vivo, we constructed a series of inducible cell lines to interfere with PERK signaling during hypoxia and used these in an orthotopic cervix cancer model of hypoxia-driven metastasis.Results: We show that LAMP3 expression in human cervix tumors is augmented both by gene copy number alterations and by hypoxia. Induced disruption of PERK signaling in established orthotopic xenografts resulted in complete inhibition of hypoxia-induced metastasis to the lymph nodes. This is due, in part, to a direct influence of the UPR pathway on hypoxia tolerance. However, we also find that LAMP3 is a key mediator of hypoxia-driven nodal metastasis, through its ability to promote metastatic properties including cell migration.Conclusion: These data suggest that the association between hypoxia, metastasis, and poor prognosis is due, in part, to hypoxic activation of the UPR and expression of LAMP3. Clin Cancer Res; 19(22); 6126–37. ©2013 AACR.

Compound full list of two libraries

Supplementary Tables and Figures - PDF file 274K, 34-C orthotopic xenografts; Supplementary figure 10: Microvessel density and hVEGF expression in shLAMP3 orthotopic xenografts; Supplementary figure 11: Hypoxic fraction shLAMP3 orthotopic xenografts; Supplementary figure 12: LAMP3 promotes colony formation in soft agar; Supplementary figure 13: Transwell migration LAMP3 deficient HeLa cells; Supplementary figure 14: Scratch wound healing assay

List of primers used for qPCR analyses

Description of additional methods and procedures used in the study. Also includes Supplementary References.

List of shRNAs on the custom reductase library.

Supplementary Figure Legends from Identification of P450 Oxidoreductase as a Major Determinant of Sensitivity to Hypoxia-Activated Prodrugs

Evaluation of SN30000-induced bystander cell killing in a co-culture system (S1); SN30000 clonogenic survival curves for HT-29, PANC-1 cells and HKO1 (S2); Plating efficiency in HT-29, PANC-1 and HKO2 reductase screens (S3); Density distributions and correlations of reductase screen sequencing data (S4); Log2 enrichment factors of shRNAs significantly selected in the reductase screens (S5); Depletion of POR protein by shRNAs in PANC-1 and HT-29 cells (S6); Replicate SN30000 clonogenic survival assays performed in stably transduced HT-29 and PANC-1 cells (S7); Measurement of plating efficiency in HKO1 and HKO2 whole-genome screens (S8); Coincidence of shRNAs significantly selected (Z > 1.96) in HT-29 and PANC-1 reductase screens and the HKO1 genome-wide screen (S9); Validation of POR shRNAs and clonogenic survival following SN30000 treatment of POR-knockout HCT116 cells (S10); Spatial distribution of POR and CA-IX expression in tongue SCC (S11); Quantitation of POR and CA-IX expression in HNSCC (S12); POR IHC staining in isogenic xenografts used for method development (S13); POR mRNA expression and sequence variation in the TCGA HNSCC dataset (S14).

The effectiveness of radiation treatment is impacted by spatially defined features of the tumor microenvironment including areas of tumor hypoxia. This is due in part to direct protection against radiation damage by hypoxia, as well as through the influence of hypoxia on tumor, stroma and immune cell phenotypes. Despite this understanding, our progress in advancing therapies to address tumor hypoxia in patients have been largely unsuccessful. This is driven in part to the incredible variability in tumor hypoxia across patients as well as to a limited understanding of the underlying causes and consequences of hypoxia on an individual patient basis. As a step towards more precision approaches for hypoxia directed therapies we are characterizing hypoxia and its causes in individual patients and in matched patient derived xenografts. Our data reveal a striking variation in hypoxia across patients, and a corresponding variation in both oxygen consumption and hypoxia tolerance in patient derived organoids. We hypothesize that levels of hypoxia within individual tumors are determined through separate processes that drive changes in oxygen demand coupled with those that influence adaptation and survival to hypoxic stress. We have also identified ULK1 as a key contributor to both of these processes. Loss or inhibition of ULK1, a gene activated downstream of the unfolded protein response during hypoxia, results in defects in both mitochondrial and ER turnover leading to increases in both oxygen consumption and ER stress. Consequently, knockdown of ULK1 in established pancreatic cancer xenografts results in development of more hypoxia, while at the same time promotes the death of cells that become hypoxic. This has a dramatic impact on tumor growth, leading to complete remission in some models and suggesting that ULK1 should be pursued as a tumor microenvironment therapeutic target. Citation Format: Ji Zhang, Dan Cojocari, Rob Cairns, Marianne Koritzinsky, Bradly G. Wouters. Determinants of hypoxia in pancreatic cancer and identification of ULK1 as a potential therapeutic target. [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 SY31-02.

Purpose: Tumor hypoxia is associated with poor response to radiation (RT). We previously discovered a novel mechanism of metformin: enhancing tumor RT response by decreasing tumor hypoxia. We hypothesized that metformin would decrease tumor hypoxia and improve cervical cancer response to RT. Patients and Methods: A window-of-opportunity, phase II randomized trial was performed in stage IB–IVA cervical cancer. Patients underwent screening positron emission tomography (PET) imaging with hypoxia tracer fluoroazomycin arabinoside (FAZA). Only patients with FAZA uptake (hypoxic tumor) were included and randomized 2:1 to receive metformin in combination with chemoRT or chemoRT alone. A second FAZA-PET/CT scan was performed after 1 week of metformin or no intervention (control). The primary endpoint was a change in fractional hypoxic volume (FHV) between FAZA-PET scans, compared using the Wilcoxon signed-rank test. The study was closed early due to FAZA availability and the COVID-19 pandemic. Results: Of the 20 consented patients, 6 were excluded due to no FAZA uptake and 1 withdrew. FHV of 10 patients in the metformin arm decreased by an average of 10.2% (44.4%–34.2%) ± SD 16.9% after 1 week of metformin, compared with an average increase of 4.7% (29.1%–33.8%) ± 11.5% for the 3 controls (P = 0.027). Those with FHV reduction after metformin had significantly lower MATE2 expression. With a median follow-up of 2.8 years, the 2-year disease-free survival was 67% for the metformin arm versus 33% for controls (P = 0.09). Conclusions: Metformin decreased cervical tumor hypoxia in this trial that selected for patients with hypoxic tumor. See related commentary by Lyng et al., p. 5233

Cancer metabolism adapts the metabolic network of its tissue of origin. However, breast cancer is not a disease of a single origin. Multiple epithelial populations serve as the culprit cell of origin for specific breast cancer subtypes, yet our knowledge of the metabolic network of normal mammary epithelial cells is limited. Using a multi-omic approach, here we identify the diverse metabolic programmes operating in normal mammary populations. The proteomes of basal, luminal progenitor and mature luminal cell populations revealed enrichment of glycolysis in basal cells and of oxidative phosphorylation in luminal progenitors. Single-cell transcriptomes corroborated lineage-specific metabolic identities and additional intra-lineage heterogeneity. Mitochondrial form and function differed across lineages, with clonogenicity correlating with mitochondrial activity. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed lineage-rooted metabolic vulnerabilities of mammary progenitors. Bioinformatics indicated breast cancer subtypes retain metabolic features of their putative cell of origin. Thus, lineage-rooted metabolic identities of normal mammary cells may underlie breast cancer metabolic heterogeneity and targeting these vulnerabilities could advance breast cancer therapy.

2-Aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a direct role in both metabolism and protein stability. Oxidation of the metabolite cysteamine produces hypotaurine, while oxidation of N-terminal cysteines targets protein substrates for N-degron pathway-mediated degradation. Despite these known functions, the (patho)physiological roles of ADO remain obscure. By analyzing TCGA datasets we discovered that high ADO expression is associated with poor outcome for patients with hepatocellular carcinoma (HCC) (HR 1.2, p Citation Format: Sandy C-E Lee, Andrea H. Pyo, Helia Mohammadi, Ji Zhang, Anna Dvorkin-Gheva, Lucie Malbeteau, Stephen Chung, Shahbaz Khan, Thomas Kislinger, Julie A. Reisz, Courtney Jones, Marianne Koritzinsky. ADO is essential for redox homeostasis in liver cancer. [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 4783.

Purpose: Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance. Experimental Design: We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer. Results: ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased. Conclusions: Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer.

Drug repurposing is an attractive option for oncology drug development. Itraconazole is an antifungal ergosterol synthesis inhibitor that has pleiotropic actions including cholesterol antagonism, inhibition of Hedgehog and mTOR pathways. We tested a panel of 28 epithelial ovarian cancer (EOC) cell lines with itraconazole to define its spectrum of activity. To identify synthetic lethality in combination with itraconazole, a whole-genome drop-out genome-scale clustered regularly interspaced short palindromic repeats sensitivity screen in two cell lines (TOV1946 and OVCAR5) was performed. On this basis, we conducted a phase I dose-escalation study assessing the combination of itraconazole and hydroxychloroquine in patients with platinum refractory EOC (NCT03081702). We identified a wide spectrum of sensitivity to itraconazole across the EOC cell lines. Pathway analysis showed significant involvement of lysosomal compartments, the trans-golgi network and late endosomes/lysosomes; similar pathways are phenocopied by the autophagy inhibitor, chloroquine. We then demonstrated that the combination of itraconazole and chloroquine displayed Bliss defined synergy in EOC cancer cell lines. Furthermore, there was an association of cytotoxic synergy with the ability to induce functional lysosome dysfunction, by chloroquine. Within the clinical trial, 11 patients received at least one cycle of itraconazole and hydroxychloroquine. Treatment was safe and feasible with the recommended phase II dose of 300 and 600 mg twice daily, respectively. No objective responses were detected. Pharmacodynamic measurements on serial biopsies demonstrated limited pharmacodynamic impact. In vitro, itraconazole and chloroquine have synergistic activity and exert a potent antitumor effect by affecting lysosomal function. The drug combination had no clinical antitumor activity in dose escalation. Significance: The combination of the antifungal drug itraconazole with antimalarial drug hydroxychloroquine leads to a cytotoxic lysosomal dysfunction, supporting the rational for further research on lysosomal targeting in ovarian cancer.

CD8

The tumor suppressor p53 is an important regulator of cell fate response after DNA damage. Cell fate response following metabolic stresses has also been linked to p53-dependent pathways. In this study, we asked if 5′-adenosine monophosphate-activated protein kinase (AMPK), the master sensor of cellular energy balance, played a role in p53-dependent apoptosis of neural progenitor cells (NPCs) in the hippocampus after irradiation. Adult mice with targeted disruption of p53 or prkaa2 (gene that encodes AMPKα) in the brain were used to determine the role of p53 and AMPK, respectively, in radiation-induced apoptosis of NPCs in the hippocampus. The p53-dependent apoptosis of NPCs was associated with an increase in phospho-AMPK expression in the dentate gyrus at 8 hours after irradiation. Activation of AMPK was seen in granule neurons and subgranular NPCs. Compared with wildtype mice, apoptosis of NPCs was significantly attenuated in AMPK deficient (nestinCre: prkaa2fl/fl) mice after irradiation. AMPK deficiency did not however alter p53 activation in NPCs after irradiation. We conclude that AMPK may regulate apoptosis of hippocampal NPCs after irradiation. These findings suggest that cellular metabolism may play a role in determining cell fate response such as apoptosis after DNA damage in NPCs.

2-aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a role in both metabolism and protein stability. ADO directly metabolizes cysteamine to produce hypotaurine and taurine in mammals. ADO has also been recently identified to promote oxygen dependent stability of a subset of substrates involved in the N-degron pathway in mammals (IL32, RGS4 and RGS5). The ability of ADO to target protein stability of signaling molecules suggests that it may have the potential to transduce rapid responses to hypoxia and affect tumour initiation and progression phenotypes. Here, we have successfully knocked down and knocked out ADO using two independent siRNAs and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system, respectively. We have assessed proliferation and migration through the Incucyte® ZOOM system by imaging cell confluency over time. Survival was assessed through a clonogenic assay. siRNA mediated knockdown of ADO in cervical (HeLa and SiHa), pancreatic (Panc1 and Capan2) and liver (SNU499 and Huh6) cancer cell lines drastically reduced proliferation, survival, and migration in normoxia. These results were also replicated in hypoxia (0.2% O2) across all 6 cell lines. Out of the 6 cell lines, the liver cancer cell lines were most drastically affected by the knockdown of ADO. This phenotype was replicated in the ADO KO cell lines. Taken together, these data suggest that expression of ADO may contribute to phenotypes that induce aggressive tumour phenotypes by targeting the stability of specific proteins and altering cellular metabolism in mammals. Citation Format: Sandy Che-Eun Serena Lee, Andrea Hye An Pyo, Marianne Koritzinsky. ADO contributes to tumour initiating phenotypes [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 1431.