Abstract Book

S193

ESTRO 37

Purpose or Objective A decade ago it was proposed that tumour recurrence in glioblastoma (GBM) was associated with radioresistance of GBM stem cells (GSC), which demonstrate enhanced DNA double strand break (DSB) repair secondary to preferential activation of DNA damage response (DDR). The underlying reason for upregulated DDR and consequent radiation resistance of GSCs is enigmatic, despite having broad implications for our understanding of the cancer stem cell phenotype, gliomagenesis and treatments for GBM. We investigated DNA replication stress (RS) as a determinant of GSC radioresistance and as a potential therapeutic target. Material and Methods A panel of primary GBM cell cultures and human GBM specimens were utilised. The DNA fibre assay was used to compare RS in CD133+ and CD133- cell sorted populations. Confocal immunofluorescence visualised expression of GSC markers and RS response proteins in human GBMs and murine intracranial xenografts. RNA sequencing examined expression of long genes (>800kbp) 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

Purpose or Objective Patients with breast cancer are often treated by ionizing radiation (IR) and PARP inhibitors; however, the effectiveness of these treatments is often limited by lack of response and/or resistance. Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxyribonucleotide triphosphate (dNTP) triphosphohydrolase with a well-established role in HIV-1 restriction by depleting dNTPs required for reverse transcription and replication. SAMHD1 is also overexpressed in breast and dysregulated in autoimmune disease. In addition to its well-established dNTPase activity, our lab has recently defined a novel role for SAMHD1 in DNA end resection to facilitate DNA double strand break (DSB) repair by homologous recombination (HR), independent of its dNTPase activity. Because of its role in DNA DSB repair and its link to breast cancer, we hypothesized that SAMHD1 could be targeted to improve Using genetic, biochemical, cell biological, and molecular biological approaches combined with structural insight, we characterized the role of SAMHD1 in DSB repair. We further queried the TCGA database for patients with breast cancer who were treated with or without IR to determine if there was an association of SAMHD1 expression level with clinical outcome. We also determined if targeting SAMHD1 for proteasomal degradation with novel virus like particles (VLPs) containing Vpx, a viral accessory protein, can sensitize breast cancer cells and tumors to DNA damaging agents. Results We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. In patients with breast cancer treated with IR, low SAMHD1 expression was associated with a statistically significant improvement in overall survival, while for those not treated with IR, no significant difference in association of SAMHD1 expression with outcome was observed. Breast cancer cells and tumors treated with VLPs containing Vpx were hypersensitive to IR and PARP inhibitor. Conclusion Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity. In addition,our results identify SAMHD1 as a novel biomarker for stratifying breast cancer patients for treatment with IR. Finally, our results establish proof of concept for targeting SAMHD1 with VLPs containing Vpx as a novel therapeutic strategy for sensitizing breast cancer to IR, PARP inhibitors, and other agents that induce DNA DSBs. R. Carruthers 1 , S. Ahmed 2 , K. Strathdee 1 , S. Ramachandran 3 , E. Hammond 3 , A. Chalmers 1 1 University of Glasgow, Institute of Cancer Sciences, Glasgow, United Kingdom 2 University of Sunderland, School of pharmacy, Sunderland, United Kingdom 3 University of Oxford, Department of radiation oncology, Oxford, United Kingdom breast cancer control. Material and Methods OC-0378 DNA replication stress due to long gene expression causes radioresistance in GBM stem cells