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Autophagy is a survival mechanism that allows recycling of cellular breakdown products, particularly in stressed cells. Here we evaluate the hypotheses that up-regulation of autophagy is a common mechanism of resistance to chemotherapy, and that drug resistance can be reversed by inhibiting autophagy with a proton pump inhibitor. We exposed human PC3, LNCaP and MCF7 cells to seven clinically-used chemotherapy drugs ± pantoprazole, examined the up-regulation of autophagy and the effect on cellular proliferation by Western Blots, MTS assay and colony-forming assay. The distribution of drug effects and of autophagy was quantified in LNCaP tumor sections in relation to blood vessels and hypoxia by immunohistochemistry using γH2AX, cleaved caspase-3 and p62. All anticancer drugs led to up-regulation of autophagy in cultured tumor cells. Pantoprazole inhibited the induction of autophagy in a time- and dose-dependent manner, and sensitized cancer cells to the seven anti-cancer drugs. Treatment of LNCaP xenografts with paclitaxel induced both DNA damage and autophagy; autophagy was inhibited and markers of toxicity were increased by pantoprazole. Induction of autophagy is a general mechanism associated with resistance to anticancer drugs and that its inhibition is a promising therapeutic strategy to enhance the effects of chemotherapy and improve clinical outcomes.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer, they can be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary HNSCC tumors and their respective metastatic sites/recurrences. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. To complement the data from the functional screens and to further characterize our cell line collection, we utilized several high through put approaches including mutational analysis, proteomics and gene expression profiling. Moreover, we performed chemical screens using libraries of over 4000 FDA approved drugs with the aim to combine our functional genomic data with the results from the drug screens to discover drug/gene “hit” combinations that would provide a basis for development of novel anti-cancer therapies.While HNSCC derived cell lines represent a valuable tool to study this disease, their biology might not always accurately represent the biology of the tumors they were derived from. Therefore, we looked at the expression of the components of several key pathways that we discovered using the in vitro characterization in a patient material from over HNSCC surgical samples constructed into a TMA. Five of the hits belonged to the Notch signalling pathway and 4 others came from the proteomic analysis as they showed higher levels of surface expression in metastatic cells and/or under hypoxic conditions. Interestingly, higher proportion of patients with late stages of HNSCC belonged to the high Jag2 and Hey1 populations as compared to patients with low stages of HNSCC that mostly expressed low or intermediate levels of these proteins. This data is in line with our observations that Jag2 and Hey1 expression is elevated in cells derived from metastatic sites and is low in cells derived from primary tumors. Proportion of cells with high CD66 and TRAIL proteins was also higher among the late stage tumors suggesting that these biomarkers are of interest for further investigation. We discovered several molecular pathways that are important in metastatic/hypoxic HNSCC. We are investigating our ability to target these pathways using FDA approved drugs in order to achieve durable cures. Citation Format: Maria Kondratyev, Aleksandra Pesic, Anna Dvorkin-Sheva, Troy Ketela, Natalie Stickle, Laurie Aiiles, Reidar Grenman, Marianne Koritzinsky, Brad Wouters. Identification of therapeutic targets in HNSCC [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 2311.

114 Background: Preclinical data show hydroxychloroquine (HCQ) and suba-itraconazole (SI) together enhance prostate cancer cell death. The proposed mechanism is lysosome dysfunction including sequestration of cholesterol in endosomes and inhibition of gogi-lyosome trafficking. HCQ/SI could delay androgen deprivation therapy (ADT) and its associated morbidity in men with biochemical relapse of prostate cancer. In this phase I/II study, maximum tolerated dose (MTD), recommended phase II dose (RP2D), safety, pharmacokinetics (PK), and preliminary activity of HCQ/SI was investigated in such patients. Methods: Patients received escalating doses of HCQ with fixed SI 150mg BD in rolling 6 design. This will be followed by a planned phase II Simon 2-stage cohort expansion. Results: Eleven men were treated with HCQ/SI. Median age 73 (range 69-77), baseline PSA 4.4 µg/L (1.6-22.4) and doubling time 5.3 months (3.3-15.3). Two experienced dose-limiting toxicity: grade 3 diarrhoea and grade 3 alanine transferase elevation, both at HCQ 600mg BD. Most common treatment-related adverse events (AEs) were hypertension (91% all grade/18% grade 3), QTc prolongation (55%/0%), diarrhoea (36%/9%), and nausea (36%/0%). There were no grade 4 AEs or deaths. While there were no PSA responses (≥50% fall from baseline), PSA PFS by PCWG3 criteria was 5.5 months (2.0-9.0), and PSA doubling time was prolonged at 4 and 12 weeks in 82% and 45% respectively. ADT-free and metastasis-free survival are 14.3 months (95% CI 4.9-23.8) and 15.9 months (95% CI unevaluable) respectively. PK data will be presented. Conclusions: HCQ/SI demonstrated acceptable safety with MTD 600mg BD and RP2D 400mg BD. There is early signal of activity and phase II enrolment is to begin. Clinical trial information: NCT03513211.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary HNSCC tumors and their respective metastatic sites or recurrences. While extensive efforts are being made by both industry and academia to develop novel anti-cancer drugs, this process is still very slow and expensive. Drug repurposing is becoming increasingly popular due to a reduced risk to patients and lower costs of drug development compared to “de novo” discovery. We utilized 2 large chemical libraries (Selleck and Prestwick) that together contain about 4000 drugs approved by FDA and similar agencies worldwide. The libraries were screened against the HNSCC lines described above in order to discover new drugs targeting head and neck cancer including drugs that target selectively metastatic cells compared to their primary tumor counterparts. We accomplished screening of 30 HNSCC lines for all of which we have also obtained the functional genomic, mutational and gene expression data. We aim to combine our functional genomic data with the results from the drug screens to discover drug/gene “hit” combinations that would provide a basis for development of novel anti-cancer therapies. For this purpose, we are looking at correlations between the drugs and the shRNAs that have similar pattern of effects across the 30 cell lines. To complement the functional screens data, we performed targeted sequencing of the most commonly altered genes in HNSCC as reported by the TCGA. This data will help understand which pathways are affected by the drugs we select to investigate. Out of the 30 lines, we have 13 sets of “matched” lines that belong to the same patients, coming from primary tumors, metastatic sites or recurrences. As a first step of the analysis, we looked at drugs that affected cells derived from metastases but not those from the respective primary tumors. Interestingly, many of the metastasis-specific drugs were antibiotics. We selected 10 of the drugs for further validation; dose response-based viability assays and growth curves experiments confirmed that 5 of the drugs were selectively affecting survival and proliferation of the metastatic cell lines; the mechanism of this effect are currently being investigated. Citation Format: Maria Kondratyev, Aleksandra Pesic, Anna Dvorkin-Sheva, Troy Ketela, Jason Moffat, Marianne Koritzinsky, Brad Wouters. Repurposing of FDA approved drugs for treatment of metastatic HNSCC [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 507.

HNSCC is the 6th most common malignancy in the world. The prognosis is favorable during early stages; however, the disease is rarely diagnosed early due to the lack of symptoms. Most HNSCC patients present with metastatic disease for which the survival rates remain low. Hypoxia serves as a bad prognostic factor in HNSCC correlating with worse survival and resistance to radiotherapy. We aimed to discover novel targets in metastatic HNSCC utilizing a unique collection of matched sets of cell lines derived from primary tumors and their respective metastatic sites. We carried out expression profiling across the lines to reveal potential common changes in gene expression between the cells derived from primary and metastatic sites in each patient as well as between normoxia and hypoxia. This analysis revealed significant changes in gene expression between the described conditions. Interestingly, beta defensin 2 was one of the genes that came up as overexpressed in metastasis as well as in cells cultured in hypoxia. Beta defensins are small cationic peptides that belong to the innate immune system and exhibit antimicrobial and antiviral activities. Few studies reported their abnormal expression patterns in various cancers including HNSCC. While various novel anticancer therapies are currently being developed, early diagnosis remains one of the biggest challenges in cancer research. A discovery of soluble serum factors that can reliably detect the presence of primary or metastatic disease would serve as an extremely valuable tool in defining diagnosis and prognosis, predicting the response to treatment, and monitoring disease progression. We hypothesized the beta defensin 2 can serve as a serum biomarker of hypoxia and metastasis in HNSCC patients. Utilizing a commercial ELISA kit developed to detect and quantify levels of beta-defensin in human sera, we demonstrated that media collected from cell lines derived from metastases contained higher levels of beta-defensin compared to the cell lines derived from the primary tumors. Moreover, sera from HNSCC patients showed higher levels of beta defensin compared to the normal controls. As a next step, we tested sera samples from 40 HNSCC patients, of whom 20 had lymph node metastasis and 20 did not. While these data are still under analysis, preliminary results suggest that higher concentrations of beta-defensin correlate with the presence of lymph node metastasis in HNSCC patients. Utilizing two large chemical libraries that together contain about 4,000 FDA-approved drugs, we performed high-throughput screening of the HNSCC lines described above in order to discover new drugs targeting head and neck cancer, including drugs that target selectively metastatic cells compared to their primary tumor counterparts. Interestingly, many of the metastasis-specific drugs were antibiotics. Together with the findings described above, these data clearly suggest a connection between patient microbiome and the metastatic process in HNSCC. Citation Format: Maria Kondratyev, Aleksandra Pesic, Carl Virtanen, Marianne Koritzinsky, Brad Wouters. Antibacterial defense and metastatic progression—lessons from head and neck cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B10.

Background: Autophagy allows recycling of cellular components and may facilitate cell survival after chemotherapy. Pantoprazole inhibits proton pumps and is reported to inhibit autophagy. Here we evaluate the effects of pantoprazole to modify cytotoxicity of the anticancer drug docetaxel, and underlying mechanisms. Methods: Effects of docetaxel±pantoprazole were studied against wild-type and autophagy-deficient PC3 cells and against four human xenografts. Effects of pantoprazole on autophagy were evaluated by quantifying LC3-I, LC3-II and p62 proteins in western blots, and by fluorescent microscopy of cells transfected with RFP-GFP-LC3. The distribution of drug effects and of autophagy was quantified in tumour sections in relation to blood vessels and hypoxia by immunohistochemistry using γH2AX, cleaved caspase-3, Ki67 and LC3/ p62. Results: Pantoprazole increased the toxicity of docetaxel in vitro, increased docetaxel-induced expression of γH2AX and cleaved caspase-3, and decreased Ki67 in tumour sections. Pantoprazole increased growth delay of four human xenografts of low, moderate and high sensitivity to docetaxel, with minimal increase in toxicity. Docetaxel led to increased autophagy throughout tumour sections. Pantoprazole inhibited autophagy, and effects of pantoprazole were reduced against genetically modified cells with decreased ability to undergo autophagy. Conclusions: Autophagy is a mechanism of resistance to docetaxel chemotherapy that may be modified by pantoprazole to improve therapeutic index.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. We are particularly interested in the identification of contextual synthetic lethal oncogenes expressed on the cell surface, as those are easily targetable by therapeutic antibodies. To identify these targets, we performed high-throughput flow cytometry screening that enables evaluation of 370 validated cell surface antibodies. Cell surface targets differentially expressed in metastatic lines included CECAM and CCR6 that were previously reported to be implicated in metastasis and tumor progression as well as Thy1, a known marker of stem cells involved in regulation of cell adhesion. Cell surface targets induced under hypoxic conditions across the cell lines included CA9, an enzyme that is known to regulate pH in hypoxic cells and be associated with tumor progression, as well as CD338, CD264 and CD312, that were previously associated with stemness in a few models of cancer. Interestingly, the described proteins were also found to be differentially essential in the shRNA screens, highlighting their functional importance in tumor progression and hypoxia survival. We are currently investigating the role of these proteins in HNSCC metastasis utilizing our unique collection of matched pairs of HNSCC lines from multiple patients. Moreover, we are utilizing our pipeline of patient derived HNSCC xenografts to test the effect of knocking down the described genes in patient tumors. We are also testing the expression of selected hits in histological sections of patient tumorsthe 400-patient TMA by immunohistochemistry looking for correlation with tumor grade, aggressiveness, levels of hypoxia as well as presence/absence of metastasis in the patient. Citation Format: Maria Kondratyev, Aleksandra Pesic, Azin Sayad, Troy Ketela, Natalie Stickle, Carl Virtanen, Jason Moffat, Laurie Ailles, Marianne Koritzinsky, Brad Wouters. Cell surface targets in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3054.

Background and purpose: Hypoxia is a hallmark of solid cancers and associated with metastases and treatment failure. During tumor progression epithelial cells often acquire mesenchymal features, a phenomenon known as epithelial-to-mesenchymal transition (EMT). Intratumoral hypoxia has been linked to EMT induction. We hypothesized that signals from the tumor microenvironment such as growth factors and tumor oxygenation collaborate to promote EMT and thereby contribute to radioresistance. Materials and methods: Gene expression changes under hypoxia were analyzed using microarray and validated by qRT-PCR. Conversion of epithelial phenotype upon hypoxic exposure, TGF beta addition or oncogene activation was investigated by Western blot and immunofluorescence. Cell survival following ionizing radiation was assayed using clonogenic survival. Results: Upon hypoxia, TGF beta addition or EGFRvIII expression, MCF7, A549 and NMuMG epithelial cells acquired a spindle shape and lost cell-cell contacts. Expression of epithelial markers such as E-cadherin decreased, whereas mesenchymal markers such as vimentin and N-cadherin increased. Combining hypoxia with TGF beta or EGFRvIII expression, lead to more rapid and pronounced EMT-like phenotype. Interestingly, E-cadherin expression and the mesenchymal appearance were reversible upon reoxygenation. Mesenchymal conversion and E-cadherin loss were associated with radioresistance. Conclusions: Our findings describe a mechanism by which the tumor microenvironment may contribute to tumor radioresistance via E-cadherin loss and EMT.

Head and neck carcinoma (HNSCC) is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. The idea behind this selective targeting is based on discovering molecular pathways that became essential in metastatic cells, and then exploiting this vulnerability through targeted agents. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. To complement the functional screens data, we performed targeted sequencing of the most commonly altered genes in HNSCC as reported by the TCGA profiled gene expression in the HNSCC cell lines cultured under normoxia and hypoxia using Illumina microarrays. These analyses led to the discovery of genes that are differentially essential in metastatic cells as well as in cells exposed to hypoxic conditions. Utilizing CRISPR technology, we assembled a library of guide RNAs targeting the discovered hits and are currently validating the top identified genes using both in vitro and in vivo systems. To validate hits that are essential in metastatic cells in vivo, we engineered selected “matched” pairs of HNSCC cell lines to express CAS9 protein upon induction with doxycycline. The cells are then transduced with the library of guides and injected subcutaneously into mice; tumors from control and doxycycline treated animals are compared. The difference in genes that are essential for growth of tumors seeded by primary tumor derived and metastasis derived HNSCC cell lines is then assessed in a quantitative manner. Citation Format: Maria Kondratyev, Aleksandra Pesic, Troy Ketela, Azin Sayad, Stephano Marastoni, Jason Mofat, Carl Virtanen, Natalie Stickle, Reider Grenman, Brad Wouters. Novel targets in metastatic HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 407.

Purpose With this modeling study, we wanted to estimate the potential gain from incorporating fluorodeoxyglucose-positron emission tomography (FDG-PET) scanning in the radiotherapy treatment planning of CT Stage N2-N3M0 non–small-cell lung cancer (NSCLC) patients. Methods and materials Twenty-one consecutive patients with clinical CT Stage N2-N3M0 NSCLC were studied. For each patient, two three-dimensional conformal treatment plans were made: one with a CT-based planning target volume (PTV) and one with a PET-CT–based PTV, both to deliver 60 Gy in 30 fractions. From the dose–volume histograms and dose distributions on each plan, the dosimetric factors predicting esophageal and lung toxicity were analyzed and compared. For each patient, the maximal tolerable prescribed radiation dose for the CT PTV vs. PET-CT PTV was calculated according to the constraints for the lung, esophagus, and spinal cord. From these results, the tumor control probability (TCP) was estimated, assuming a clinical dose–response curve with a median toxic dose of 84.5 Gy and a γ 50 of 2.0. Dose–response curves were modeled, taking into account geographic misses according to the accuracy of CT and PET in our institutions. Results The gross tumor volume of the nodes decreased from 13.7 ± 3.8 cm 3 on the CT scan to 9.9 ± 4.0 cm 3 on the PET-CT scan ( p = 0.011). All dose–volume characteristics for the esophagus and lungs decreased in favor of PET-CT. The esophageal V 45 (the volume of the esophagus receiving 45 Gy) decreased from 45.2% ± 4.9% to 34.0% ± 5.8% ( p = 0.003), esophageal V 55 (the volume of the esophagus receiving 55 Gy) from 30.6% ± 3.2% to 21.9% ± 3.8% ( p = 0.004), mean esophageal dose from 29.8 ± 2.5 Gy to 23.7 ± 3.1 Gy ( p = 0.004), lung V 20 (the volume of the lungs minus the PTV receiving 20 Gy) from 24.9% ± 2.3% to 22.3% ± 2.2% ( p = 0.012), and mean lung dose from 14.7 ± 1.3 Gy to 13.6 ± 1.3 Gy ( p = 0.004). For the same toxicity levels of the lung, esophagus, and spinal cord, the dose could be increased from 56.0 ± 5.4 Gy with CT planning to 71.0 ± 13.7 Gy with PET planning ( p = 0.038). The TCP corresponding to these doses was estimated to be 14.2% ± 5.6% for CT and 22.8% ± 7.1% for PET-CT planning ( p = 0.026). Adjusting for geographic misses by PET-CT vs. CT planning yielded TCP estimates of 12.5% and 18.3% ( p = 0.009) for CT and PET-CT planning, respectively. Conclusion In this group of clinical CT Stage N2-N3 NSCLC patients, use of FDG-PET scanning information in radiotherapy planning reduced the radiation exposure of the esophagus and lung, and thus allowed significant radiation dose escalation while respecting all relevant normal tissue constraints. This, together with a reduced risk of geographic misses using PET-CT, led to an estimated increase in TCP from 13% to 18%. The results of this modeling study support clinical trials investigating incorporation of FDG-PET information in CT-based radiotherapy planning.

The aim of this study was to evaluate the applicability of fluorine-19 magnetic resonance spectroscopy ((19)F MRS) for monitoring in vivo the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) after using an attenuated Salmonella Typhimurium strain recombinant to provide cytosine deaminase (TAPET-CD). The (19)F MRS measurements were done on mice bearing the human colon tumour xenograft (HCT116). The intratumoural conversion is greater when TAPET-CD/5-FC is delivered intratumourally (i.tu.) than when TAPET-CD is delivered intravenously (i.v.) and 5-FC intraperitoneally (i.p.). Repeat measurements of the same tumour also yielded important information on the tumour colonization by TAPET-CD through the correlated 5-FC to 5-FU conversion efficacy. The in vivo MRS spectra were confirmed by in vitro (19)F MRS of perchloric acid extracts of the tumour tissue. No 5-FU metabolites were detectable in vivo in the tumours. However, the in vitro measurements revealed, besides 5-FC and 5-FU, the presence of small amounts of catabolites. Finally, spectra obtained in vitro from liver extracts of tumour-bearing mice treated i.tu. with TAPET-CD/5-FC showed no 5-FU and only little amounts of catabolites. Our data illustrate most importantly the potential of (19)F MRS to monitor biologically-based treatments involving cytosine deaminase. ispartof: British Journal of Cancer vol:89 issue:9 pages:1796-801 ispartof: location:England status: published

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies, we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Utilizing CRISPR methodology, we established that “fixing” the mutation results in reversal of metastatic phenotype of the cells, making them Notch independent similar to their primary tumor counterparts. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Overall, our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies. Citation Format: Maria Kondratyev, Aleksandra Pesic, Troy Ketela, Azin Sayad, Stephano Marastoni, Carl Virtanen, Laurie Ailles, Soroush Samadian, Mikhail Bashkurov, Marianne Koritzinsky, Brad Wouters. Novel therapeutic targets in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3020. doi:10.1158/1538-7445.AM2017-3020

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that metastatic, but not primary tumor cells, undergo apoptosis upon knockdown of Notch3 and that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies. Citation Format: Maria Kondratyev, Aleksandra Pesic, Stephano Marastoni, Troy Ketela, Jason Moffat, Carl Virtanen, Azin Sayad, Mikhail Bashkurov, Alessandro Dati, Laurie Ailles, Reidar Grenman, Marianne Koritzinsky, Brad Wouters. Novel therapeutic targets in head and neck cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3794.

e16502Background: Enhancing the effectiveness of docetaxel for men with mCRPC is an unmet clinical need. Preclinical studies in our laboratory demonstrated that high dose pantoprazole can prevent o...

Stromal expression of hypoxia inducible factor 2 alpha (HIF-2 alpha) and carbonic anhydrase 9 (CA9) are associated with a poorer prognosis in colorectal cancer (CRC). Tumour cell death, regulated by a hypoxic stromal microenvironment, could be of importance in this respect. Therefore, we correlated apoptosis, TP53 mutational status and BNIP3 promoter hypermethylation of CRC cells with HIF-2 alpha- and CA9-related poor outcome. In a series of 195 CRCs, TP53 mutations in exons 5-8 were analysed by direct sequencing, and promoter hypermethylation of BNIP3 was determined by methylation-specific PCR. Expressions of HIF-2 alpha, CA9, p53, BNIP3 and M30 were analysed immunohistochemically. Poorer survival of HIF-2 alpha and CA9 stromal-positive CRCs was associated with wild-type TP53 (P=0.001 and P=0.0391), but not with BNIP3 methylation. Furthermore, apoptotic levels were independent of the TP53 status, but lower in unmethylated BNIP3 CRCs (P=0.004). It appears that wild-type TP53 in CRC cells favours the progression of tumours expressing markers for hypoxia in their stroma, rather than in the epithelial compartment. Preserved BNIP3 function in CRC cells lowers apoptosis, and may thus be involved in alternative cell death pathways, such as autophagic cell death. However, BNIP3 silencing in tumour cells does not impact on hypoxia-driven poorer prognosis. These results suggest that the biology of CRC cells can be modified by alterations in the tumour microenvironment under conditions of tumour hypoxia.

Introduction: Enzalutamide is a recently approved potent androgen receptor antagonist for prostate cancer both in the pre- and post-docetaxel settings. Its action relies upon 3 defined mechanisms of action: 1) blocking testosterone binding to the androgen receptor (AR), 2) preventing nuclear translocation of AR, and 3) inhibiting AR-DNA binding. However, despite its utility in prolonged overall survival in both these settings, disease relapse is inevitable, suggesting that resistance to the drug is somehow being acquired and is of vital concern. Methods: Using a combination of high-throughput functional genomic (kinome-shRNA) and chemical genomic (pharmacological inhibitor) screens, along with various in vitro / in vivo assays (proliferation, apoptosis, necrosis, soft agar growth, etc.) we have identified the IkB kinase (IKK) / IkB / NFkB signaling axis as important for acquisition of, and continued resistance to, Enzalutamide. Results: Using a panel of Enzalutamide-resistant and -sensitive cell lines, we show that targeting this signaling axis will selectively kill Enzalutamide-resistant cells while not affecting the proliferation of sensitive cells. Moreover, our data shows that the pharmacological inhibitors and shRNAs are effective even without Enzalutamide administration, negating any potential drug-drug interaction issues. Finally, we have found that our cells are resistant to Enzalutamide in an AR-V7-independent manner, suggesting that NFkB signaling is acting in a novel manner to bring about the resistance phenotype. Conclusions: Through in vitro and in vivo experiments, we provide evidence that targeting activators of NFκB signalling can be a strategy to help patients with Enzalutamide-resistant prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Sujeeve Jeganathan, Amina Zoubeidi, Martin Gleave, Brad G. Wouters, Anthony M. Joshua. Using functional and chemical genomics to identify mechanisms of Enzalutamide resistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 732. doi:10.1158/1538-7445.AM2015-732

One of the most fundamental aspects of gene therapy for cancer continues to be targeting of therapeutic gene expression specifically to the tumor. In order to exclusively target malignant cells while at the same time sparing normal tissue, cancer gene therapy needs to combine highly selective gene delivery with highly specific gene expression. Specific bacterial strains have been considered as gene delivery vehicles as an alternative to viral vectors. We and others have been investigating the potential use of non-pathogenic anaerobic bacteria such as Clostridium. Their tumor specificity is based upon the unique physiology of solid tumors which is often characterized by regions of hypoxia and necrosis. We have constructed Clostridium strain that expresses the prodrug converting enzyme, nitroreductase that converts the prodrug CB1954 into a toxic agent that can kill dividing and non-dividing cells. As the enzyme is specifically targeted, high therapeutic doses are achieved only in the tumor. In vivo experiments using combined treatment of prodrug and recombinant bacteria administration resulted in a significant anti-tumoral response. Besides to Clostridium, the ability of the attenuated Salmonella to replicate preferentially in solid tumors demonstrated its potential use as gene delivery vector. However, despite its preferential tumor accumulation, normal tissues are also colonized albeit to a lesser extent. Although bacteria are cleared rapidly from the normal tissues, it negatively affects the specificity of the salmonella mediated gene transfer system. One way to address this problem is to obtain controlled gene expression when using attenuated Salmonella by exploiting tumor hypoxia. In that context, introducing a therapeutic gene under the transcriptional control of a hypoxia regulatory element might be promising.