Abstract Book

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ESTRO 37

4 University Medical Center Hamburg - Eppendorf UKE, Biochemistry, Hamburg, Germany 5 University Medical Center Hamburg - Eppendorf UKE, Radiotherapy, Hamburg, Germany Purpose or Objective Breast cancer comprises a heterogeneous group of tumors of whom 20% are categorized as triple-negative (TNBC). Important biological characteristics and potential therapeutic targets of TNBC include high proliferation, a basal-like and mesenchymal phenotype and a defect in the DNA repair pathway Homologous Recombination (HR), which feeds the observed elevated chromosomal instability in these tumors. TNBCs show an enrichment of cancer stem cells and therapy resistance. This project aims to develop treatment intensification strategies based on the simultaneous exploitation of the HR- deficiency and the stem-like phenotype, using specific inhibitors for RAD51, CHK1 and PARP1 in combination with irradiation. Material and Methods Expression of HR-related (RAD51, BRCA1, PTEN, CHK1, MRE11, ATR, ATM) and stem-like factors (ZEB1, E- Cadherin, Vimentin, ALDH1) as well as HR functionality (via RAD51 foci, MMC-sensitivity and plasmid reporter assay) were determined in the TNBC line MDA-231 WT, its two sublines preferentially metastasizing to brain (BR) or bone (SA) and in the luminal BC line MCF7. DNA replication (fiber assay) and migration assay were also tested. Radiosensitivity and the radiosensitizing effect of different inhibitors was analyzed by colony assay and correlated to the CIN in the METABRIC database. Results Distinct differences in the expression of HR-related proteins were observed, with an elevated expression of CHK1, MRE11 and ATM in BR and SA relative to WT and MCF7. BR and SA showed a typical stem-cell like protein expression profile, together with a higher migration capacity, increased HR-capacity, resistance against MMC and less DNA damage, in line with an HR-proficient phenotype. After irradiation no advantage in survival for the BR and SA cell lines was observed, suggesting that not HR, but superordinate CHK1 mediated DDR promotes radioresistance. This was confirmed by a distinct radiosensitization after CHK1i; the most radioresistant WT cell line was most strongly sensitized by an EF=3. That effect also showed up in replication-processes, the higher the EF the stronger the inhibitory effect on replication. The effect of other inhibitors on radiosensitivity is currently being investigated. A second promising target is RAD51, because a METABRIC analysis (952 TNBCs) showed that in TNBC with high CIN RAD51 and CHK1 are significantly stronger expressed than in TNBC with low CIN. Conclusion In conclusion the results presented here show that DNA repair and a stem-like phenotype are closely intertwined in determining resistance to tumor therapy of TNBCs with high CIN. OC-0380 Cell cycle checkpoint modulates radiotherapy fraction size sensitivity in normal and malignant cells N. Somaiah 1 , S. Anbalagan 2 , C. Strom 2 , J. Downs 3 , P. Jeggo 4 , A. Wilkins 1 , S. Boyle 1 , K. Rothkamm 5 , J. Yarnold 1 1 The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, Division of Cancer Biology and Division of Radiotherapy and Imaging, Sutton, United Kingdom 2 The Institute of Cancer Research, Division of Radiotherapy and Imaging, London, United Kingdom 3 The Institute of Cancer Research, Division of Cancer Biology, London, United Kingdom 4 University of Sussex, Genome Damage and Stability Centre, Sussex, United Kingdom 5 University Medical Centre Hamburg-Eppendorf,

Department of Radiotherapy and Radiation Oncology, Hamburg, Germany Purpose or Objective Cancers are on average less sensitive to fraction size than the surrounding dose limiting healthy tissues; however recent randomised clinical trials show that breast and prostate cancers are more sensitive to fraction size than previously thought. The implication is that small fractions spare breast and prostate cancers as much as the healthy tissues, so 2 Gy fractions lose their advantages. Understanding cell and molecular mechanisms determining fraction size sensitivity is essential if this therapeutic variable is to be fully exploited for patient benefit. The hypothesis under test in this study is that fraction size sensitivity is modulated by the G1/S checkpoint, reflecting error-prone non-homologous end joining (NHEJ) of radiation induced DNA double-strand A panel of normal and tumour cell lines were used: 1. Normal wild type (WT) fibroblasts (primary skin culture S009 & 1BR hTERT), 2. p53-mutant transformed Li- Fraumeni fibroblasts (MDAH087 & MDAH041), 3. DNA ligase IV deficient fibroblasts (411BR hTERT), 4. p53- mutant glioma tumour cells with defective DNA-PKcs (M059J) and WT control (M059K), 5. prostate tumour cells LNCaP (WT p53) and PC3 (defective p53) and 6. ovarian tumour cells A2780 (WT p53) and A2780/E6 (defective p53). Cells were exposed to single acute X-ray doses (0 – 8 Gy) or 2 smaller fractions of either 2, 3 or 4Gy (8h apart) or 1 Gy daily fractions, plated for colony survival assay and harvested for FACS analysis. Recovery factor (RF) was calculated as the ratio of split dose to single dose survival as a measure of sensitivity to fraction size. Results Both normal and tumour cells with WT p53 demonstrated significant split dose recovery (S009, 1BR hTERT, LNCaP, A2780 WT), whereas Li-Fraumeni fibroblasts (MDAH041 and MDAH087) and tumour cells with defective G1/S checkpoint (M059K, M059J, PC3 and A2780/E6) lost the sparing effect of smaller fractions (Table 1a & b). p53- competent cells were predominantly in G1 phase after split dose IR in contrast to p53-mutant cells in S/G2. There was a lack of significant split dose recovery in NHEJ-deficient fibroblasts (411BR hTERT) and in p53- mutant DNA-PKcs defective cells. Furthermore, siRNA knockdown of p53 in 1BR hTERT cells and DNA-PKcs inhibitor (KU006468) treatment of M059K cells reduced split dose recovery. breaks in G0/1 phase. Material and Methods

Conclusion Our data support the hypothesis that cells defective in p53 are less sensitive to RT fraction size than cells with an intact G1/S checkpoint, consistent with higher fidelity repair by HR &/or NHEJ in S/G2. The loss of split dose recovery observed in DNA ligase IV and DNA-PKcs mutant cells is interpreted as the dependence of fraction size sensitivity on intact NHEJ.

OC-0381 Lessons from isogenic models of radioresistant prostate cancer cells