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Purpose: Conventional anticancer treatments are often impaired by the presence of hypoxia. TH-302 selectively targets hypoxic tumor regions, where it is converted into a cytotoxic agent. This study assessed the efficacy of the combination treatment of TH-302 and radiotherapy in two preclinical tumor models. The effect of oxygen modification on the combination treatment was evaluated and the effect of TH-302 on the hypoxic fraction (HF) was monitored using [18F]HX4-PET imaging and pimonidazole IHC stainings.Experimental Design: Rhabdomyosarcoma R1 and H460 NSCLC tumor-bearing animals were treated with TH-302 and radiotherapy (8 Gy, single dose). The tumor oxygenation status was altered by exposing animals to carbogen (95% oxygen) and nicotinamide, 21% or 7% oxygen breathing during the course of the treatment. Tumor growth and treatment toxicity were monitored until the tumor reached four times its start volume (T4×SV).Results: Both tumor models showed a growth delay after TH-302 treatment, which further increased when combined with radiotherapy (enhancement ratio rhabdomyosarcoma 1.23; H460 1.49). TH-302 decreases the HF in both models, consistent with its hypoxia-targeting mechanism of action. Treatment efficacy was dependent on tumor oxygenation; increasing the tumor oxygen status abolished the effect of TH-302, whereas enhancing the HF enlarged TH-302′s therapeutic effect. An association was observed in rhabdomyosarcoma tumors between the pretreatment HF as measured by [18F]HX4-PET imaging and the T4×SV.Conclusions: The combination of TH-302 and radiotherapy is promising and warrants clinical testing, preferably guided by the companion biomarker [18F]HX4 hypoxia PET imaging for patient selection. Clin Cancer Res; 21(13); 2984–92. ©2015 AACR.

Supplementary figure 4. Body weight was monitored during and after treatment for both rhabdomyosarcoma (A) and H460 (B) tumor-bearing animals. The change in body weight is represented in percentage. Data represent mean {plus minus} SEM.

Supplementary Table 2. Time to reach 4 times start volume per treatment group for H460 bearing animals. Data represent mean {plus minus} SD, p-value and sensitization enhancement ratio.

Supplementary figure 3. A) Tumor growth of rhabdomyosarcoma bearing animals (n=8) treated with TH-302 (25mg/kg) in combination with 4, 8 or 12Gy of RT. Data represent mean {plus minus} SEM. B) T4xSV for rhabdomyosarcoma bearing animals treated with TH-302 in combination with RT 0, 4, 8 or 12Gy. C) T4xSV for rhabdomyosarcoma bearing animals exposed to different oxygen concentrations in combination with TH-302 and RT. D) T4xSV for H460 bearing animals exposed to different oxygen concentrations in combination with TH-302 and RT.

Supplementary figure 1. A) Treatment schedule for the rhabdomyosarcoma and H460 models. All animals were randomized over either the treatment or the control group. These groups were both treated with either NaCl or TH-302 (rhabdomysarcoma 25mg/kg, H460 50mg/kg). Rhabdomyosarcoma bearing animals started with oxygen modification at day 0 and a [18F]HX4 hypoxia PET scan. For histological controls only, this scan was repeated on day 4. Animals from the treatment group were exposed to radiotherapy (RT) at the end of treatment day 3 and their tumor volume was monitored until 4 times start volume was reached. H460 tumor bearing animals from the histological control group were injected with pimonidazole and Hoechst on the last treatment day and sacrificed. Animals from the treatment group were exposed to radiotherapy at day 4 and monitored until a tumor volume of 4 times start treatment volume was reached.

Supplementary Table 1. Time to reach 4 times start volume per treatment group for rhabdomyosarcoma bearing animals. Data represent mean {plus minus} SD, p-value and sensitization enhancement ratio.

Supplementary figure 5. Quantification of H460 histological staining for A) necrotic fraction (NF), B) relative vessel area (RVA), C) perfusion fraction (PF) and D) relative perfusion area (RPA). Data represent mean {plus minus} SEM.

Purpose: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a preactivated nucleoside released. 3′-Deoxyadenosine (3′-dA) is a naturally occurring adenosine analogue with established anticancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells, and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3′-dA was chemically modified to create the novel ProTide NUC-7738. Experimental Design: We describe the synthesis of NUC-7738. We determine the IC50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in patients with cancer. Results: We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3′-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide-binding protein 1. PK and tumor samples obtained from the ongoing first-in-human phase I clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective proapoptotic agent in cancer cells with effects on the NF-κB pathway. Conclusions: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and supports its further clinical evaluation as a novel cancer treatment within the growing pantheon of anticancer ProTides.

Anti-VEGF antibody bevacizumab has prolonged progression-free survival in several cancer types, however acquired resistance is common. Adaption has been observed pre-clinically, but no human study has shown timing and genes involved, enabling formulation of new clinical paradigms. In a window-of-opportunity study in 35 ductal breast cancer patients for 2 weeks prior to neoadjuvant chemotherapy, we monitored bevacizumab response by Dynamic Contrast-Enhanced Magnetic Resonance [DCE-MRI], transcriptomic and pathology. Initial treatment response showed significant overall decrease in DCE-MRI median Ktrans, angiogenic factors such ESM1 and FLT1, and proliferation. However, it also revealed great heterogeneity, spanning from downregulation of blood vessel density and central necrosis to continued growth with new vasculature. Crucially, significantly upregulated pathways leading to resistance included glycolysis and pH adaptation, PI3K-Akt and immune checkpoint signaling, for which inhibitors exist, making a strong case to investigate such combinations. These findings support that anti-angiogenesis trials should incorporate initial enrichment of patients with high Ktrans, and a range of targeted therapeutic options to meet potential early resistance pathways. Multi-arm adaptive trials are ongoing using molecular markers for targeted agents, but our results suggest this needs to be further modified by much earlier adaptation when using drugs affecting the tumor microenvironment., Highlights • DCE-MRI two weeks post-treatment of primary breast cancer shows heterogeneity of initial response to bevacizumab. • Gene expression shows heterogeneous changes but an overall decrease in angiogenic factors ESM1 and FLT1, and proliferation. • Increased PI3K-Akt and immune checkpoint signaling suggests combination of Bevacizumab with inhibitors of these pathways. • Increased PI3K-Akt and immune checkpoint signaling suggests combination of Bevacizumab with inhibitors of these pathways. Available evidence is that the anti-angiogenic treatment Bevacizumab currently has a limited range of indications in breast cancer compared to other cancers. Here, we show that multimodality dynamic monitoring of treatment response allow classification of patient's response as early as two weeks after treatment, and reveals new mechanisms associated with resistance. The implications, are that the role of bevacizumab in neoadjuvant breast cancer needs to be re-evaluated using early classification of resistant patients, but more generally that trials need to be designed prospectively, taking into account dynamic monitoring to be able to rapidly adapt available new agents for combination.

Introduction: Cordycepin or 3′-deoxyadenosine is an extract from the fungus Cordyceps sinensis, a popular traditional medicine in the Far East. Although its active metabolite 3′-deoxydenosine 5′-triphosphate (3’dATP) has potent in vitro anti-cancer activity, it has shown limited efficacy in clinical trials. This is due to its poor bioavailability and rapid degradation by the ubiquitous catabolic enzyme adenosine deaminase (ADA). Here, using genetic and cellular cancer model systems, we investigated the biological impact of cordycepin in cancer cells. Experimental Procedures: Cell proliferation was measured in the presence of cordycepin and antagonists to determine its IC50. A haploid leukaemia-derived cell line, Hap1, was mutagenized using retroviral gene trapping and the mutant pool was selected for clones resistant to cordycepin which were sequenced by NGS. CRISP/Cas9 knock out combined with pharmacokinetic determination of the IC50 enabled us to validate the top hits. RNA deep sequencing-based transcriptomic analysis and gene set enrichment were used to identify the signalling pathways most affected by cordycepin and to define possible mechanisms of action. Results: We found 106 significant gene hits using the haploid genetic screen. The intracellular antiproliferative activity of cordycepin depended on both its uptake by human equilibrative nucleoside transporter 1 (hENT1) and its rate-limiting phosphorylation by the intracellular enzyme adenosine kinase (ADK). CRISPR/Cas9 knock out or selective inhibition of hENT1 or ADK led to a greater than 10-fold reduction in the activity of cordycepin. When extracellular degradation by ADA was inhibited, cordycepin showed up to 78x greater anti-proliferative potency in cancer cells. Furthermore, after 6h of treatment with cordycepin, cells showed significant differential expression of more 1000 genes. Integrating both datasets, we demonstrated by gene set enrichment analysis that, once successfully activated within the cell, 3’dATP induces cell death by a dual mechanism; by both activating the intrinsic apoptosis pathway and suppressing the TNFa-NFkB pro-survival signalling axis. Conclusions: We demonstrate here that, in the absence of ADA and in the presence of abundant hENT1 and ADK, the active metabolite of cordycepin, 3’ dATP, works as a dual driver of apoptosis in cancer cells. This suggests that a strategy of bypassing ADA and hENT1 while administering cordycepin or a chemically-modified version of cordycepin (that overcomes these resistance mechanisms) is essential to elicit any clinical anti-cancer effect. We are currently conducting a phase I trial of NUC-7738, a ProTide form of cordycepin designed to overcome these resistance mechanisms. Agents like NUC-7738 can enhance the stability and bioavailability of cordycepin, enabling it to effectively deliver its dualistic anticancer effect in patients. Citation Format: Hagen Schwenzer, Erica De Zan, Ruud van Stiphout, Cornelia H de Moor, Sebastian Nijman, Gareth Bond, Sarah P Blagden. Preclinical study of the fungal derivative cordycepin as an anti-cancer agent [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A062. doi:10.1158/1535-7163.TARG-19-A062

Purpose: Novel molecular stratification of the breast cancer population based on genomic aberrations has been recently reported.[1] The main focus has been on deriving prognostic classifiers by integrating gene expression and copy number aberrations (CNAs) data, but the role of other events, such as loss-of-heterozygosity (LOH), has not been extensively characterized in this integrated landscape. Here we consider the respective role of CNAs and LOH in breast cancer, in the context of an integrated genomic analysis, and their impact on clinical outcome. Methods: Currently, retrospective data with gene expression, CNA and LOH were used from 219 patients with early primary breast cancer who were resected in the period 1989-2003.[2] CNA and LOH calls were made using OncoSNP v1.4, a Bayesian model driven genomic analysis tool to call genomic aberrations and characterize LOH status, and statistical analyses were performed in R v3.0.1. The evaluated clinical outcome was distant-recurrence free survival (DRFS). Two gene sets were tested: A) general cancer drivers from the COSMIC cancer gene census and B) all genes across the genome. Significantly amplified (increased DNA copy number with respect to normal tissue), copy neutral (no change in copy number) and deleted cancer drivers were identified by deriving null distributions from permutation analysis. Survival analysis was done using both LogRank and Cox Proportional Hazards methods. The effect of LOH on the gene expression was tested using Mann-Whitney. Benjamini and Hochberg method was used to adjust for multiple testing. Results: A higher than expected LOH occurrence was detected in 168 out of 523 COSMIC defined cancer drivers. Of these, 10 were significantly amplified, 94 were copy neutral and 64 deleted. Within these three copy number groups, patients with LOH vs no LOH were compared for DRFS. LOH was a prognostic event in 14 out of 94 copy neutral group of genes, while 4 and 2 genes were found in the amplified and deleted genes respectively. Furthermore, tumors showing copy neutral LOH events had worse outcome than tumors where these genes are amplified (no LOH) or deleted (LOH). A whole genome analysis showed that a higher global LOH frequency, across the entire genome, is associated with poor prognosis as measured by a decreased DRFS, irrespective of the global level of CNAs in the tumour. Furthermore, 386 of the 5184 genes that showed a higher than expected LOH occurrence, were significantly associated with DRFS with respect to LOH status, and 51 of these were so independently of CNAs and global LOH. Conclusions: LOH in copy neutral patients has an effect on clinical outcome in a subset of cancer drivers and overall genes, irrespective of the global genomic alteration status. This finding supports an approach of measuring and integrating multiple genomic data information, which expectantly improves the accuracy of identifying subgroups of patients, resulting in improved treatment selection. Ongoing work, which will be presented at the meeting, is focused on characterizing these events, and confirming these analyses in the large TCGA dataset[3] with multiple cancer types. References: [1] Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature. 2012; 486(7403): 346-52. [2] Buffa FM, Camps C, Winchester L, Snell CE, Gee HE, Sheldon H, et al. microRNA-associated progression pathways and potential therapeutic targets identified by integrated mRNA and microRNA expression profiling in breast cancer. Cancer Res. 2011; 71(17): 5635-45. [3] Network TCGA. Comprehensive molecular portraits of human breast tumours. Nature. 2012; 490(7418): 61-70. Citation Format: Ruud GPM van Stiphout, Antoine deWeck, Laura Winchester, Syed A. Haider, Jiannis Ragoussis, Adrian L. Harris, Chris Holmes, Francesca M. Buffa. Distinct roles of copy number and loss-of-heterozygosity in predicting prognosis for breast cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-56.

Purpose An overview of the Rapid Learning methodology, its results, and the potential impact on radiotherapy. Material and results Rapid Learning methodology is divided into four phases. In the data phase, diverse data are collected about past patients, treatments used, and outcomes. Innovative information technologies that support semantic interoperability enable distributed learning and data sharing without additional burden on health care professionals and without the need for data to leave the hospital. In the knowledge phase, prediction models are developed for new data and treatment outcomes by applying machine learning methods to data. In the application phase, this knowledge is applied in clinical practice via novel decision support systems or via extensions of existing models such as Tumour Control Probability models. In the evaluation phase, the predictability of treatment outcomes allows the new knowledge to be evaluated by comparing predicted and actual outcomes. Conclusion Personalised or tailored cancer therapy ensures not only that patients receive an optimal treatment, but also that the right resources are being used for the right patients. Rapid Learning approaches combined with evidence based medicine are expected to improve the predictability of outcome and radiotherapy is the ideal field to study the value of Rapid Learning. The next step will be to include patient preferences in the decision making. ?? 2013 The Authors. Published by Elsevier Ireland Ltd.

Gemcitabine constitutes one of the backbones for chemotherapy treatment in pancreatic ductal adenocarcinoma (PDAC), but patients often respond poorly to this agent. Molecular markers downstream of gemcitabine treatment in preclinical models may provide an insight into resistance mechanisms. Using cytokine arrays, we identified potential secretory biomarkers of gemcitabine resistance (response) in the transgenic KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) mouse model of PDAC. We verified the oncogenic role of the cytokine tissue inhibitor of matrix metalloproteinases 1 (TIMP1) in primary pancreatic tumors and metastases using both in vitro techniques and animal models. We identified potential pathways affected downstream of TIMP1 using the Illumina Human H12 array. Our findings were validated in both primary and metastatic models of pancreatic cancer. Gemcitabine increased inflammatory cytokines including TIMP1 in the KPC mouse model. TIMP1 was upregulated in patients with pancreatic intraepithelial neoplasias grade 3 and PDAC lesions relative to matched normal pancreatic tissue. In addition, TIMP1 played a role in tumor clonogenic survival and vascular density, while TIMP1 inhibition resensitized tumors to gemcitabine and radiotherapy. We observed a linear relationship between TIMP-1 expression, liver metastatic burden, and infiltration by CD11b+Gr1+ myeloid cells and CD4+CD25+FOXP3+ Tregs, whereas the presence of tumor cells was required for immune cell infiltration. Overall, our results identify TIMP1 upregulation as a resistance mechanism to gemcitabine and provide a rationale for combining chemo/radiotherapy with TIMP1 inhibitors in PDAC. Cancer Res; 77(21); 5952–62. ©2017 AACR.

Purpose: Conventional anticancer treatments are often impaired by the presence of hypoxia. TH-302 selectively targets hypoxic tumor regions, where it is converted into a cytotoxic agent. This study assessed the efficacy of the combination treatment of TH-302 and radiotherapy in two preclinical tumor models. The effect of oxygen modification on the combination treatment was evaluated and the effect of TH-302 on the hypoxic fraction (HF) was monitored using [18F]HX4-PET imaging and pimonidazole IHC stainings. Experimental Design: Rhabdomyosarcoma R1 and H460 NSCLC tumor-bearing animals were treated with TH-302 and radiotherapy (8 Gy, single dose). The tumor oxygenation status was altered by exposing animals to carbogen (95% oxygen) and nicotinamide, 21% or 7% oxygen breathing during the course of the treatment. Tumor growth and treatment toxicity were monitored until the tumor reached four times its start volume (T4×SV). Results: Both tumor models showed a growth delay after TH-302 treatment, which further increased when combined with radiotherapy (enhancement ratio rhabdomyosarcoma 1.23; H460 1.49). TH-302 decreases the HF in both models, consistent with its hypoxia-targeting mechanism of action. Treatment efficacy was dependent on tumor oxygenation; increasing the tumor oxygen status abolished the effect of TH-302, whereas enhancing the HF enlarged TH-302′s therapeutic effect. An association was observed in rhabdomyosarcoma tumors between the pretreatment HF as measured by [18F]HX4-PET imaging and the T4×SV. Conclusions: The combination of TH-302 and radiotherapy is promising and warrants clinical testing, preferably guided by the companion biomarker [18F]HX4 hypoxia PET imaging for patient selection. Clin Cancer Res; 21(13); 2984–92. ©2015 AACR.

Background Postoperative radiation therapy for stage I endometrial cancer improves locoregional control but is without survival benefit. To facilitate treatment decision support for individual patients, accurate statistical models to predict locoregional relapse (LRR), distant relapse (DR), overall survival (OS), and disease-free survival (DFS) are required. Methods and Materials Clinical trial data from the randomized Post Operative Radiation Therapy for Endometrial Cancer (PORTEC-1; N=714 patients) and PORTEC-2 (N=427 patients) trials and registered group (grade 3 and deep invasion, n=99) were pooled for analysis (N=1240). For most patients (86%) pathology review data were available; otherwise original pathology data were used. Trial variables which were clinically relevant and eligible according to data constraints were age, stage, given treatment (pelvic external beam radiation therapy (EBRT), vaginal brachytherapy (VBT), or no adjuvant treatment, FIGO histological grade, depth of invasion, and lymph-vascular invasion (LVSI). Multivariate analyses were based on Cox proportional hazards regression model. Predictors were selected based on a backward elimination scheme. Model results were expressed by the c-index (0.5-1.0; random to perfect prediction). Two validation sets (n=244 and 291 patients) were used. Results Accuracy of the developed models was good, with training accuracies between 0.71 and 0.78. The nomograms validated well for DR (0.73), DFS (0.69), and OS (0.70), but validation was only fair for LRR (0.59). Ranking of variables as to their predictive power showed that age, tumor grade, and LVSI were highly predictive for all outcomes, and given treatment for LRR and DFS. The nomograms were able to significantly distinguish low- from high-probability patients for these outcomes. Conclusions The nomograms are internally validated and able to accurately predict long-term outcome for endometrial cancer patients with observation, pelvic EBRT, or VBT after surgery. These models facilitate decision support in daily clinical practice and can be used for patient counseling and shared decision making, selecting patients who benefit most from adjuvant treatment, and generating new hypotheses.

The current increasing amount of digitalized medical data in health-care demands for solutions to store, share, mine, and analyze these data. Today, medical knowledge and evidence is based on outdated data. Tomorrow, we aim to have a rapid learning health medicine system in which evidence can be generated instantly, based on the most recent data available. The development of this system requires dedication and support of health-care providers, politicians, and patients on many levels. The aim of this system is improvement of health-care quality and support in clinical decision making. Full integration of data handling systems within the clinic and between institutes is inevitable in the near future.

Evidence is accumulating that radiotherapy of non-small cell lung cancer patients can be optimized by escalating the tumour dose until the normal tissue tolerances are met. To further improve the therapeutic ratio between tumour control probability and the risk of normal tissue complications, we firstly need to exploit inter patient variation. This variation arises, e.g. from differences in tumour shape and size, lung function and genetic factors. Secondly improvement is achieved by taking into account intra-tumour and intra-organ heterogeneity derived from molecular and functional imaging. Additional radiation dose must be delivered to those parts of the tumour that need it the most, e.g. because of increased radio-resistance or reduced therapeutic drug uptake, and away from regions inside the lung that are most prone to complication. As the delivery of these treatments plans is very sensitive for geometrical uncertainties, probabilistic treatment planning is needed to generate robust treatment plans. The administration of these complicated dose distributions requires a quality assurance procedure that can evaluate the treatment delivery and, if necessary, adapt the treatment plan during radiotherapy.

Background and purpose: The purpose of this study was to prospectively investigate metabolic changes of rectal tumors after 1 week of treatment of either radiochemotherapy (28 x 1.8 Gy + Capecitabine) (RCT) or hypofractionated radiotherapy (5 x 5 Gy) alone (RT). Materials and methods: Fourty-six rectal cancer patients, 25 RCT- and 21 RT-patients, were included in this study. Sequential FDG-PET-CT scans were performed for each of the included patients both prior to treatment and after the first week of treatment. Consecutively, the metabolic treatment response of the tumor was evaluated. Results: For the patients referred for pre-operative RCT, significant reductions of SUV(mean) (p

Background Hypoxia is a key physiological and microenvironmental difference between tumour and normal tissues. Hypoxia induces DNA damage, genomic aberrations and reprogramming of DNA repair mechanisms. A crucial mechanism in a highly dynamic hypoxic tumour microenvironment is metabolic reprogramming of tumour cells. Genome-wide multi-modal molecular profiles have been generated, however it remains unclear whether upregulation of metabolic genes is substantially enriched with somatically acquired alterations. To answer this, we performed a pan-cancer integrative analysis of genomic and transcriptomic profiles of 10 TCGA tumour types (n = 5,500). Methods 2,750 metabolic genes were extracted from KEGG pathways, and differentially upregulated genes were identified for each tumour type. High-ranking (10%) upregulated genes were assessed for correlation between mRNA abundance and DNA copy-number changes, and subsequently tested for metabolic-enrichment using genome-wide permutation analysis. For each tumour type, highly correlated genesets were subsequently examined for their potential as drug targets and biomarkers of disease relapse. Results Integrative analysis of mRNA and copy-number data revealed clusters of tumours that exhibit similar landscape of metabolic gene profiles, and those having unique metabolic copy number aberrations. Within these profiles, we identify a core set of genes that exhibit strong correlation (Spearman's ρ>0.3; q Conclusions We isolate tumour-type specific and core pan-cancer metabolic gene signatures revealing within and cross disease heterogeneity in metabolic profiles. We highlight a core set of pan-cancer metabolic candidate drivers which are recurrently over-expressed due to genomic amplifications induced by hypoxia. Citation Format: Syed Haider, Alan McIntyre, Ruud GPM van Stiphout, Laura M. Winchester, Simon Wigfield, Adrian L. Harris, Francesca M. Buffa. Genomic alterations underlie a pan-cancer metabolic transcriptome shift. [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 3750. doi:10.1158/1538-7445.AM2015-3750

The main objective of the present study was to determine alterations of calcium handling in the diabetic rat heart during the transition from adaptive to maladaptive phase of cardiomyopathy. By inhibiting the nuclear enzyme poly(ADP-ribose) polymerase (PARP), we also investigated the possible role of this enzyme in the sequence of pathological events. Six weeks after induction of type I diabetes by injection of streptozotocin in rats, the hearts were perfused according to Langendorff. Intracellular-free calcium (Ca(2+)(i)) levels were measured by surface fluorometry using Indo-1 AM. Cyclic changes in Ca(2+)(i) concentrations and hemodynamic parameters were measured simultaneously. The hearts were challenged by infusion of isoproterenol. Six weeks of diabetes resulted in reduced inotropy and lusitropy. The diabetic hearts (DM) expressed a significantly elevated end-diastolic Ca(2+)(i) level (control, 111-/+20 vs DM, 221-/+35 nM). The maximal transport capacity of SERCA2a and conductance of RyR2 were reduced. These changes were not accompanied by major alterations in the tissue content of SERCA2a, RyR2, phospholamban and Na(+)/Ca(2+) exchanger. In response to beta-adrenergic activation, SERCA2a transport capacity and RyR2 conductance were stunted in the DM hearts. Inhibition of PARP induced minor changes in the mechanical function and calcium handling of the DM hearts. In conclusion, the observed changes in contractility and in Ca(2+)(i) handling are most likely attributable to functional disturbances of SERCA2a and RyR2 in this transitional phase of diabetes. At this stage of diabetes, PARP does not appear to play a significant pathogenetic role in the alterations in contractile function and calcium handling.

// Alessandro Valli 1,2,7 , Miguel Rodriguez 3,4 , Loukas Moutsianas 5 , Roman Fischer 2 , Vita Fedele 2 , Hong-Lei Huang 2 , Ruud Van Stiphout 1 , Dylan Jones 1 , Michael Mccarthy 2 , Maria Vinaxia 3,4 , Kaori Igarashi 6 , Maya Sato 6 , Tomoyoshi Soga 6 , Francesca Buffa 1 , James Mccullagh 7 , Oscar Yanes 3,4 , Adrian Harris 1,* and Benedikt Kessler 2,* 1 Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK 2 Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK 3 Centre for Omic Sciences, Rovira i Virgili University, Reus, Spain 4 Biomedical Research Centre in Diabetes and Associated Metabolic Disorders, Madrid, Spain 5 The Wellcome Trust Centre for Human Genetics, Nuffield Department of Medicine, Oxford, UK 6 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan 7 Mass Spectrometry Research Facility CRL, Department of Chemistry, University of Oxford, Oxford, UK * equal senior authors Correspondence: Alessandro Valli, email: // Keywords : cancer metabolism, fatty acids, HIF1α, HIF2α, hypoxia, Kennedy pathway, lipidomics, PAF Received : June 01, 2014 Accepted : December 10, 2014 Published : December 11, 2014 Abstract The biochemistry of cancer cells diverges significantly from normal cells as a result of a comprehensive reprogramming of metabolic pathways. A major factor influencing cancer metabolism is hypoxia, which is mediated by HIF1α and HIF2α. HIF1α represents one of the principal regulators of metabolism and energetic balance in cancer cells through its regulation of glycolysis, glycogen synthesis, Krebs cycle and the pentose phosphate shunt. However, less is known about the role of HIF1α in modulating lipid metabolism. Lipids serve cancer cells to provide molecules acting as oncogenic signals, energetic reserve, precursors for new membrane synthesis and to balance redox biological reactions. To study the role of HIF1α in these processes, we used HCT116 colorectal cancer cells expressing endogenous HIF1α and cells in which the hif1α gene was deleted to characterize HIF1α-dependent and independent effects on hypoxia regulated lipid metabolites. Untargeted metabolomics integrated with proteomics revealed that hypoxia induced many changes in lipids metabolites. Enzymatic steps in fatty acid synthesis and the Kennedy pathway were modified in a HIF1α-dependent fashion. Palmitate, stearate, PLD3 and PAFC16 were regulated in a HIF-independent manner. Our results demonstrate the impact of hypoxia on lipid metabolites, of which a distinct subset is regulated by HIF1α.