Presenter(s)
H. Askarifirouzjaei1,2, J. H. Oh1, R. Elkin1, D. Han3, H. Qi3, M. Vermeulen3, H. Lin1,3, C. B. Simone II3, M. Baltaci2, R. James1, S. N. Powell2, J. Humm1, A. Haimovitz-Friedman2, and J. O. Deasy1; 1Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, NY, 2Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, 3New York Proton Center, New York, NY
Purpose/Objective(s): Proton and photon irradiation generate distinct patterns of DNA damage, yet whether these differences produce biologically distinct and therapeutically exploitable cellular states remains unclear. We hypothesized that proton irradiation induces a checkpoint-governed repair architecture that is selectively vulnerable to XPB destabilization by the FDA-approved drug spironolactone.
Materials/Methods: Using (DLD1 parental, DLD1 BRCA2(-), HCT116, KPC) tumor cells, single-cell RNA sequencing, differential differential gene cooperation via geometric network analysis, multiplex phospho-imaging, and cell-cycle–restricted profiling at 24 hours after 8 Gy Bragg peak irradiation using a 100 MeV proton beam, we compared photon and proton responses and assessed the impact of spironolactone (20 µM), a known destabilizer of the TFIIH helicase XPB (ERCC3).
Results: Cell survival assays demonstrated robust synthetic lethality between spironolactone and proton irradiation, reducing the dose required for 90% cell kill from ~9.0 Gy (no drug) to ~5.1 Gy with spironolactone. In contrast, photon irradiation showed no comparable enhancement. In the absence of drug, photon irradiation favored coordinated replication-coupled repair characterized by PCNA–MCM–CDC6 co-engagement and sustained transcriptional robustness. In contrast, proton irradiation without drug induced a cohesive ATR–CHK1–WEE1–CDK1 S/G2 checkpoint governance state with enhanced RAD51-BRCA1 coupling. S-phase–restricted analyses of proton irradiation plus spironolactone cells demonstrated disrupted proton-favored architecture, tightening PCNA-FEN1-EXO1-MSH2-RPA2-RFC2-POLD3 modules and increasing integrated stress response signaling (ATF4-DDIT3-AP-1) without early apoptotic caspase synchronization. G2/M–restricted analyses of proton irradiation plus drug demonstrated destabilization of mitotic progression modules (PLK1-CDK1-CCNB1-BIRC5) alongside strengthened cohesion and structural surveillance coupling (SMC1A-SMC3-RAD21-BLM), consistent with inheritance of unresolved replication blocks.
Conclusion: Collectively, these data support a two-component interaction model: spironolactone collapses the proton-favored checkpoint governance state, forcing cells into a replication-coupled configuration; however, the structural complexity of proton-induced lesions renders this forced state non-resolving under impaired transcription restart, apparently producing persistent transcription–replication conflicts, fork congestion, and progressive replication stress. Thus, spironolactone apparently does not simply increase DNA damage, but induces a maladaptive state transition that converts proton lesions into mechanically difficult substrates. These findings establish an unexpected synthetic lethality between protons and the FDA-approved drug spironolactone and support further translational evaluations of spironolactone with proton therapy.