Proton Irradiation Sensitizes Cancer Cell Lines With Acquired Radioresistance By Exploiting Differentially Dysregulated Dna Damage Response (Ddr) Pathways.

Abstract 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 potential for PBT to overcome radioresistance in these models by targeting the DNA repair machinery. Due to the high cost of PBT, precise patient stratification is needed to ensure that PBT is applied only to patients who benefit significantly from this procedure over photon-RT. Our preliminary data represents a proof-of-concept for feasibility of patient stratification to PBT. Citation Format: Pek Lim Chu, Eugenia L.L. Yeo, Dennis J.J. Poon, Ce-belle Chen, Minqin Ren, Saumitra Vajandar, Dewi Susanti, Hong Qi Tan, Sung Yong Park, Thomas Osipowicz, Kwok Wai Lo, Stanley K. Liu, Khee Chee Soo, Melvin Lk Chua. Proton irradiation sensitizes cancer cell lines with acquired radioresistance by exploiting differentially dysregulated DNA damage response (DDR) pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3062.