Background: Radiotherapy is a mainstay of curative treatment for squamous cell carcinomas (SCC) and is frequently combined with radiosensitizing drugs to improve efficacy. Outcomes for HPV-negative SCCs remain heterogeneous, in part due to variable resistance to ionizing radiation (IR) and modest benefit of existing radiosensitizing drugs. Hypothesis driven testing of novel radiosensitizing drugs has previously been limited due to a lack of robust, efficient, and reproducible methods to assess drug radiation combinations. Aims: To improve the throughput of preclinical studies on radiosensitizers, we have developed an in vitro system for assessing drug/radiation combinations, validated using the known radiosensitizing chemotherapeutic drug cisplatin. We extended the assay to assess the effect of ML-385, a NRF2 inhibitor that increases cellular reactive oxygen species (ROS), and abemaciclib and palbociclib, two CDK4/6 inhibitors predicted to radiosensitize cancer cells by inhibiting entry into more resistant phases of the cell cycle. Experimental Methods: We adapted the 9-day viability assay of Abazeed et al. (Cancer Res., 2013) to enable drug/radiation efficacy assessments. We first validated the assay’s ability to recapitulate clonogenic survival among 19 HPV-negative SCC cell lines. Drug/radiation experiments were performed in the 6 most radioresistant SCC cell lines. ROS levels after 4Gy IR was used to confirm NRF2 suppression. Flow cytometry was used to evaluate cell cycle phase. AKT inhibitor GSK690693 was used in combination with a NRF2 shRNA knockdown cell line to investigate the mechanism of sensitivity. Western blot was used to detect p16. Cellular response to multiple IR doses was summarized using an area-under-the-curve (AUC) metric. The delta AUC across drug doses was used to evaluate radiosensitization. Results: Among the 19 cell lines, AUCs with the clonogenic and 9-day viability assays were strongly correlated (Pearson r=0.74, p=3.00 × 10–4). Six (32%) of the cell lines were reproducibly radioresistant (AUC >3.5) using both assays. All of these cell lines saw sensitization with cisplatin. None of these 6 cell lines harboured mutations in the canonical NRF2 pathway, whereas all 6 harboured either CCND1 amplification, CDKN2A mutation, or both. Only one cell line showed radiosensitization with the NRF2 inhibitor ML-385, an effect that was abrogated by PIK3CA or AKT pathway inhibition. In contrast, 5 of the 6 cell lines showed reproducible radiosensitization following CDK4/6 inhibition; with robust p16 expression detected in the lone unaffected cell line. Conclusion: The 9-day viability assay allows efficient evaluation of putative radiosensitizers. Our tests of putative radiosensitizing drugs in a cohort of radioresistant cell lines have identified CDK4/6 inhibitors to be of interest for further investigation in biomarker-selected populations.

Citation Format: Meghan Lambie, Rita Gill, Laurie Ailles, Scott V. Bratman. Selective radiosensitization in preclinical models of HPV-negative squamous cell carcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-086.