ESTRO 37 Abstract book

S190

ESTRO 37

Material and Methods 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. OC-0378 DNA replication stress due to long gene expression causes radioresistance in GBM stem cells 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 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)

models were used: the highly proliferative LLC lung cancer and the moderately proliferative PC3 prostate cancer. Tumour hypoxia (pimonidazole), blood perfusion (hoechst perfusion), microvascular density (CD31) and pericyte coverage (αSMA and Desmin) were assessed by immunohistochemistry. Chemotherapy diffusion was assessed by doxorubicin intravenous injections. Results After 2 weeks, all RT schedules induced a tumour volume reduction in the PC3 model and a tumour growth slowdown in the LLC model. In both models, RT substantially improved vascular pericyte coverage regardless of the fractionation schedule and chemotherapy diffusion was majored. Unlike vascular morphologic changes, hypoxia and perfusion modifications occurred differently according to the dose per fraction. In the slowly proliferative PC3 model, despite microvascular density was unchanged, hypoxia was reduced from -26% to -85% and perfusion was majored from +28% to +203% compare to control, in a dose per fraction-dependent manner. In the highly proliferative LLC model, microvascular density was affected but perfusion was surprisingly improved with the highest doses per fraction. Hypoxia was reduced in all the hypofractionated schedules. In this model, hypoxia, perfusion and microvascular density changes were observed within 3 days after last RT session and compensated after 7 days whereas the changes were more stable in the PC3 model. Conclusion In our tumour models, hypofractionated RT improves tumour perfusion, reduces hypoxia and increases pericyte coverage. The differences between the 2 models suggest that the duration of radio-induced hypoxia and perfusion improvement could be balanced by the tumor growth rate. These finding will help better understanding the radiobiological effects of high doses per fraction and modeling hypofractionated RT schedules. OC-0377 Targeting a Novel Function for SAMHD1 in DNA Repair for Radiation Therapy and PARP Inhibition D. Yu 1 , W. Daddacha 1 , A. Koyen 1 , A. Bastien 1 , P. Head 1 , V. Dhere 1 , G. Nabeta 1 , E. Connolly 1 , E. Werner 1 , M. Madden 1 , M. Daly 1 , E. Minten 1 , D. Whelan 2 , H. Zhang 1 , R. Anand 3 , C. Shepard 4 , R. Sundaram 5 , X. Deng 1 , W. Dynan 1 , Y. Wang 1 , R. Bindra 5 , P. Cejka 3 , E. Rothenberg 2 , P. Doetsch 1 , B. Kim 4 1 Emory University, Radiation Oncology, Atlanta GA, USA 2 NYU, Biochemistry and Molecular Pharmacology, New Yoprk, USA 3 Università della Svizzera italiana, Institute for Research in Biomedicin, Bellinzona, Switzerland 4 Emory University, Pediatrics, Atlanta GA, USA 5 Yale University School of Medicine, Department of Radiation Oncology, New Haven, USA 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 breast cancer control.