ESTRO 37 Abstract book

S191

ESTRO 37

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 breaks in G0/1 phase.

in GSC enriched and deplete cell cultures. Finally we targeted GSC RS response with PARP (olaparib) and ATR (VE-821) inhibition in vitro in combination with radiation. Results GSC enriched cultures show increased expression of the RS markers pCHK1, pATR, gamma H2AX and RPA relative to GSC depleted cultures by Western blot and immunofluorescence. Gamma H2AX foci in GSCs colocalise with areas of intense BrDU incorporation in S phase cells. CD133 + GSC show reduced DNA replication velocity relative to CD133- populations by DNA fibre assay. CD133+ cells exhibit a prominent mid S phase accumulation relative to CD133 – cells by flow cytometry, consistent with elevated RS and difficulty replicating long genes in late S phase. We show that long neural genes are overexpressed in GSCs relative to non-GSCs, suggesting replication/transcription collisions as a mechanism of elevated RS in GSCs. DNA replication fork slowing by exposure to aphidocolin generates radiation resistance in GSC depleted cultures, linking RS to radioresistance. Confocal immunofluorescence of multiple human GBMs illustrates elevated RPA staining intensity in cell populations staining positively for putative GSC markers. Finally we demonstrate that targeting of RS response in GSCs via dual combined inhibition of PARP and ATR preferentially reduces cell viability in GSCs compared to non-GSCs and induces gamma H2AX foci in GSCs. This combination dramatically reduces neurosphere generation and potently radiosensitises GSC. Conclusion We propose RS as a novel determinant of radiation resistance and an underlying mechanism of preferential DDR activation and radioresistance in GSC. Our observations demonstrate that GSCs exhibit elevated RS due to constitutive expression of long neural genes generating activation of DDR via replication/transcription collisions and subsequent enhanced DNA repair. Our findings shed new light on GSC biology and identify novel therapeutics with potential to improve clinical outcomes. OC-0379 DNA repair and stemness determine the sensitizing effect of CHK1,RAD51 and PARP1 inhibition in TNBC F. Meyer 1 , S. Becker 1 , A. Niecke 1 , B. Riepen 1 , A. Zielinski 1 , S. Werner 2 , C. Peitzsch 3 , L. Hein 3 , A. Dubrovska 3 , H. Wikmann 2 , S. Windhorst 4 , Y. Goy 5 , A.C. Parplys 1 , C. Petersen 5 , K. Rothkamm 1 , K. Borgmann 1 1 University Medical Center Hamburg - Eppendorf UKE, Radiation Biology/Experimental Radiooncology, Hamburg, Germany 2 University Medical Center Hamburg - Eppendorf UKE, Tumorbiology, Hamburg, Germany 3 Onkoray, Instiute of Radiation Biology, Dresden, Germany 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.