Abstract Book

S1284

ESTRO 37

Results In all 16 cell lines IC 50 were lower in anoxia than normoxia resulting in cytotoxicity ratios ranging from >2.3 to >39.9. CP-506 caused a concentration-dependent decrease in survival of clonogens from hypoxic spheroids but had no effect on those from non-hypoxic spheroids. In vivo , CP-506 (800 mg/kg QD5) caused tumor growth inhibition (TGI) in several xenograft models, e.g. 99.6% (MDA-MB-468), 81% (A2780) and 41% (SiHa). In an independent validation experiment in mice bearing MDA- MB-468 tumors, 800, 600, 400 and 200 mg/kg CP-506 QD5 caused a TGI of 99.8%, 97.6%, 63.5% and 24.7%, respectively. Similar results were obtained in other xenograft models, where CP-506 increased mean T4×SV 1.5- (H460) and 2.4-fold (MDA-MB-231) and mean T3×SV 1.7-fold (HCT116). In MDA-MB-468 xenografts HF was severely decreased by CP-506 400 mg/kg QD3 (HF CCI 3- fold, HF pimo 15-fold) and QD5 (HF CCI 11.8-fold, HF pimo 150- fold) compared to vehicle treatment. In CT26 xenografts, RT + CP-506 significantly (p≤0.01) increased mean T4×SV (saline: 5.5 d, CP-506: 7.3 d, RT + saline: 7.5 d, RT + CP- 506: 13.3 d). Conclusion In vitro , CP-506 preferentially kills hypoxic tumor cells and reduces clonogenic cell survival of hypoxic spheroids only. In vivo, CP-506 monotherapy strongly inhibits tumor growth and reduces HF. In combination with RT it also strongly inhibits tumor growth. Further studies to determine the antitumor effects of CP-506 as monotherapy and in combination with RT in several preclinical tumor models are ongoing. EP-2328 AT13387 in combination with radiotherapy in clinically relevant models of glioblastoma S. Chahal 1 , A. Chalmers 1 , S. Short 2 1 Institute of Cancer Sciences, University of Glasgow- College of Medical- Veterinary & Life Sciences, Glasgow, United Kingdom 2 Leeds Institute of Cancer and Pathology, Wellcome Trust Brenner Building- St James's University Hospital, Leeds, United Kingdom Purpose or Objective Glioblastoma is incurable and tumour recurrence occurs within the high radiation dose volume in 90% of cases. Treatment failure in glioblastoma has been attributed to intrinsic radioresistance of glioblastoma stem-like cells which exhibit upregulation of the DNA damage response (DDR) with particular dependence on homologous recombination (HR) repair. Pre-clinical data show that first generation Hsp90 inhibitors downregulate HR repair and increase radiation sensitivity of glioblastoma cells, stem-like cells and xenografts. AT13387 is a second generation Hsp90 inhibitor with demonstrable pre-clinical single agent and combination activity in multiple tumour types, a favourable toxicity profile and encouraging CNS penetration. It is currently undergoing phase II evaluation in prostate and non-small cell lung cancer. The aims of the study is to measure in vitro efficacy of the Hsp90 inhibitor AT13387 in combination with radiotherapy and elucidate underlying mechanism to support a phase I clinical trial of this novel combination in glioblastoma cancer patients. Material and Methods E2 and G7 primary GBM cell lines were cultured in cancer stem cell(serum free)medium. For clonogenic assays cells were seeded in Matrigel coated 6well dishes for 4hrs to allow attachment before drugging with Hsp90 inhibitor AT13387 (Astex Pharmaceuticals). For drug-radiation

combination studies, cells were exposed to the inhibitor or DMSO control for 24 hour prior to irradiation with either sham or irradiated at 1 to 6Gy in tissue culture vessels using an XStrahl RS225 cabinet at room temperature with 195kV/15mA X rays. Visible colonies stained with crystal violet were manually counted. Clonogenic survival data were fitted using a linear quadratic model and SER0.37 and SF4Gy values were calculated from the fitted curve. For western blotting whole cell lysates were prepared and processed in SDS buffer, blotted onto membranes and probed with primary antibodies overnight followed by appropriate secondary antibodies for 1-3 hours. Bound antibodies were visualised using chemiluminescence (Thermo Scientific) and bands quantified by Image Studio Lite. Results Dose dependent effects of AT13387 on important MMR proteins (MSH2, MLH1) , GBM oncoproteins (EGFR, PDGFR, Akt, CDK4) and key DDR proteins (Rad51, DNA-PK) in E2 and G7 cell line was documented by western blot. Hsp70 was shown to be effectively upregulated in a dose dependent manner. MTT cell viability showed IC50 of 140nM in G7, and 230nM in E2. Clonogenic survival assays measuring sensitivity effects of E2, G7 cell lines to (i) radiation alone and (ii) radiation plus AT13387, exhibited marked radiosensitizing activity at 100nM with 24hour timepoint. In vivo pharmacodynamic effects of AT13387 G7 intracrancial GBM xenograft models by immuno- histochemical detection of Hsp70 and client oncoproteins Our studies suggest a novel approach to GBM cancer therapy in which AT13387, a second generation Hsp90 inhibitor is combined with radiotherapy. EP-2329 Mitochondrial dysfunction inhibits HIF-1α stabilization and expression of downstream targets M.W. Van Gisbergen 1 , K. Offermans 1 , A.M. Voets 2 , N.G. Lieuwes 1 , R. Biemans 1 , R.F. Hoffmann 3 , L.J. Dubois 1 , P. Lambin 1 1 Dept of Radiotherapy, GROW - School for Oncology and Developmental Biology- Maastricht Comprehensive Cancer Centre- Maastricht University Medical Centre- the Netherlands, Maastricht, The Netherlands 2 Dept of Clinical Genomics, GROW - School for Oncology and Developmental Biology- Maastricht Comprehensive Cancer Centre- Maastricht University Medical Centre- the Netherlands, Maastricht, The Netherlands 3 Dept of Pathology and Medical Biology, University Medical Centre Groningen- the Netherlands, Groningen, The Netherlands Purpose or Objective Hypoxia inducible factor (HIF-1) is involved in the regulation of tumor cell metabolism during hypoxic stress, as HIF-1 is able to reprogram metabolism towards the low yield-energy glycolytic pathway. In hypoxic tumors, expression of carbonic anhydrase 9 (CAIX), a HIF- 1 target, is found to be upregulated and is an important player in the cellular pH homeostasis. Furthermore, a defective oxidative phosphorylation (OXPHOS) machinery could also contribute to the tumoral metabolic reprogramming. Mitochondrial DNA (mtDNA) variations often result in a bioenergetic dysfunction and a metabolic switch towards glycolysis resulting in an unbalanced pH homeostasis. Therefore, we hypothesized that cells with dysfunctional OXPHOS display elevated CAIX expression levels. is on-going. Conclusion