Abstract 3062: Proton irradiation sensitizes cancer cell lines with acquired radioresistance by exploiting differentially dysregulated DNA damage response (DDR) pathways

Radiotherapy (RT) is a primary treatment modality that is used in 50% of cancer patients. Clinically, tumor radioresistance poses a challenge to achieve complete anti-tumor response following conventional photon-RT. Diverse mechanisms have been proposed to underpin radioresistance. However, targeting these aberrant pathways to overcome tumor radioresistance remains a clinical challenge. Here, we investigate if proton beam therapy (PBT) invokes differential cellular responses compared to photon-RT in a broad panel of radioresistant (RR) cancer cell lines. We generated isogenic RR human cancer cell lines: 22Rv1 prostate cancer, FaDu hypopharyngeal cancer and C666-1 nasopharyngeal cancer, by exposure of respective wildtype (wt) to 90 Gy photon-RT (2 Gy x 45 fr). RR was confirmed by clonogenic survival, with surviving fraction ratio (SFRR/SFwt) at 1-4 Gy (range: 1.2-1.8). Whole exome sequencing (100x; Illumina NovaSeq) was performed to profile RR-associated mutational drivers. We performed transcriptomic and proteome profiling of cellular response such as non-homologous end-joining (NHEJ) and homologous recombination (HR) repair at 1-24 h after 4 Gy PBT (2.5 MeV, 2 Gy/min) and photon-RT (0.66 MeV, 0.716 Gy/min). At baseline, we found common mutations and differential activation of several pro-survival (Akt, mTOR), epithelial-mesenchymal transition (EMT) (PDGF, TGFb) and DNA repair pathways (ATM, BRCA1, NHEJ- and HR-associated) genes in RR- relative to wt-22Rv1. Post-photon-RT, we observed residual persistent DNA repair in wt-cell lines up to 48 h. In contrast, repair in RR-cell lines was more proficient, as shown by recovery of γH2AX, ATM-Chk2 within 6 h, with significant NHEJ activation (DNA-PKcs). Next, we observed that RR-cell lines were more sensitive to PBT than photon-RT (mean SF4Gy PBT/SF4Gy photon-RT = 0.61), with delayed DNA repair (γH2AX) observed up to 48 h, which may be attributed to the difficulty in repairing PBT-induced DNA damage. This phenomenon, seems to be primarily orchestrated by decreased NHEJ activation combined with diminished cell cycle checkpoint arrest (p21) and anti-apoptotic (bcl-2) signaling. Radiosensitization by PBT was however not observed in wt (mean SF4Gy proton/SF4Gy photon = 1.14), corroborated by gene and protein expression of DDR-related pathways. We observed dysregulation of multiple pathways relating to EMT and DNA repair in our panel of RR-cancer cell lines, compared to the wt-counterparts. Additionally, we show the po