Abstract A002: Genotype-directed synthetic cytotoxicity of ATR inhibition with radiotherapy
Authors: Ng Victor , Sinha Sonali , Novaj Ardijana , Ma Jennifer , McDermott Niamh , Longhini Ana , Gardner Rui , Rosen Ezra , Powell Simon , Mandelker Diana , Khan Atif , Setton Jeremy , Roulston Anne , Morris Stephen , Koehler Maria , Lee Nancy , Reis-Filho Jorge , Riaz Nadeem 
Published: January 2024

Abstract
The DNA damage response is crucial for the efficacy of radiotherapy (RT), prompting efforts to target this pathway with radiosensitizers to improve outcomes. Early clinical trials evaluating this approach, however, have failed to yield evidence of benefit in unselected populations. Here we demonstrate that a genotype-directed combination of DNA repair inhibition and RT can unleash the significant therapeutic benefit of this approach in pre-clinical models and present first experience from a Phase I/II trial (NCT05566574) testing this concept. Using CRISPR/Cas9, we created isogenic murine models (Atm-/- and WT) in 4T1 breast and CT26 colorectal cancer models, and then studied the synergy between camonsertib and RT across genotypes. In vitro, camonsertib radio-sensitized both Atm-/- and WT models, but was more potent and synergistic in Atm-/- models. In the orthotopically implanted 4T1 model, the combination of camonsertib (30 mg/kg, 3 days on; 4 days off) with RT (20Gy x 1) significantly delayed tumor growth in Atm-/- tumors compared to WT (p &lt; 0.001; n=10 mice per group). Overall survival (OS) also improved in mice with Atm-/- tumors (20% alive at 150 days) vs. WT (0% alive; p &lt; 0.001; log-rank test). Similar results were observed using other RT dose-fractionations (4Gy x 5 daily, 6Gy x 3, 8Gy x 2) with camonsertib. In the flank-implanted CT26 model, Atm-/- tumors had markedly improved response to combination therapy with 7/8 mice achieving a complete response (CR) compared to only 1/8 WT tumors (p&lt;0.001). OS was also significantly improved with 87% of mice with CT26 Atm-/- tumors cured compared to 0% of CT26 WT tumors (p &lt; 0.001). To evaluate the genotype-specificity for synergy from combination therapy, we performed similar experiments in a 4T1 Brca1-/- model, a key mediator of homologous recombination, and observed an absence of synergy, consistent with our hypothesis. Analysis of the microenvironment of 4T1 Atm-/- treated with combination therapy by flow cytometry did not reveal significant increases in CD4 or CD8 T-cells compared to vehicle nor increases in activated CD8 T-cells (Ki67+, PD1+, or GzmB+) suggesting the therapeutic benefit was primarily mediated by cell intrinsic effects. Similarly, re-challenge experiments in cured mice did not identify strong evidence of immunologic memory, again suggesting a modest immunologic role for efficacy of combination therapy in this setting. Based on these results, we initiated a Phase I/II study of camonsertib with RT (4Gy x 5) in patients with metastatic cancer that require palliative RT and have mutations in ATM (pathogenic or variant of unknown significance). The first two patients with pathogenic ATM mutant tumors enrolled in the Phase I portion of the study have demonstrated metabolic CR to combination therapy on PET scan. Overall, our findings suggest that genotype-directed concurrent radio-sensitization could represent a novel approach to develop more effective combinatorial synthetic cytotoxic RT-based treatments. Further research to test this hypothesis is ongoing.
Citation Format: Victor Ng, Sonali Sinha, Ardijana Novaj, Jennifer Ma, Niamh McDermott, Ana Longhini, Rui Gardner, Ezra Rosen, Simon Powell, Diana Mandelker, Atif Khan, Jeremy Setton, Anne Roulston, Stephen Morris, Maria Koehler, Nancy Lee, Jorge Reis-Filho, Nadeem Riaz. Genotype-directed synthetic cytotoxicity of ATR inhibition with radiotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A002.