Abstract P001: Discovery and validation of mTOR as an immune mediator of the FLASH effect
Authors: Johnson Erickson Danielle P. , Parkman Gennie L. , Morimoto Alec , Shaver Benjamin A. , Seitz Zacharias , Delonais-Dick Ava , Cui Sunan , Cao Ning , Zeng Jing , Sanders Justin , Noble William S. , Wu Christine C. , Rengan Ramesh , MacCoss Michael J. 
Published: January 2025

Abstract
An area of particular interest in FLASH is the potential influence of the immune microenvironment in the sparing of normal tissue. After finding loss of the FLASH effect via survival studies in immunocompromised SCID mice, we examined proteomic analyses performed upon the skin of BALB/c mice for classifiers of FLASH that may be involved in the immune response. Intriguingly, one of our top hits was a known immune regulator, mTOR (mammalian target of rapamycin). Here we have validated and further explored the role of mTOR in FLASH. For these studies we exposed the pelvic region of 6-7-week-old female BALB/c mice to proton radiation at two different dose rates: FLASH (∼80Gy/s) and conventional (∼0.5Gy/s), with doses of either 0, 12, or 14 Gy, and mice were irradiated in the entrance region of a 50 MeV proton beam. After 27 days, surviving animals were euthanized, and their pelts and colons were collected for analysis. Protein extracts from skin were prepared for Data Independent Acquisition-Mass Spectrometry (DIA-MS) with protein aggregation capture. Peptide and protein levels were quantified using the Skyline software platform, with normalization performed using the limma batch correction. Through recursive feature elimination using a support vector machine, we identified tryptic peptides whose abundance distinguished between FLASH and conventional radiation treatments. Our analysis revealed significant upregulation of mTOR and Raptor (the regulatory associated protein of mTOR) in response to FLASH radiation, suggesting a pivotal role for mTOR signaling in mediating the differential effects of FLASH versus conventional radiation. mTOR is known to promote PD-L1 expression on cancer cells and enhance the infiltration of immune-suppressive lymphocytes into tumors through increased signaling of interferon and TGF-β. Based on these results, we sought to explore whether combining FLASH radiation with immune checkpoint inhibitors could enhance therapeutic efficacy in melanoma treatment. We conducted experiments treating melanoma cell lines and mice with the combination of conventional or FLASH radiation, with and without anti-PD-1, and noted an increase in overall survival of mice treated with FLASH + anti-PD-1 suggesting a potential therapeutic benefit from this combination. In addition, lysates from in vitro and in vivo studies displayed increased levels of phospho-mTOR (Ser2448), as well as downstream phosphorylated substrates signifying the role of mTOR in mediation of this response. Furthermore, immunofluorescent analyses of the normal colons harvested from the MS cohort demonstrate increased mTOR signaling in FLASH via elevated downstream phospho-S6 ribosomal protein. mTOR is a known regulator of the immune response in cancer therapies; interestingly, it may also be a regulator of FLASH radiation in normal tissue. We have validated our MS hit by western blot and IF, but further studies are aimed at the elucidation of this mechanism.
Citation Format: Danielle P. Johnson Erickson, Gennie L. Parkman, Alec Morimoto, Benjamin A. Shaver, Zacharias Seitz, Ava Delonais-Dick, Sunan Cui, Ning Cao, Jing Zeng, Justin Sanders, William S. Noble, Christine C. Wu, Ramesh Rengan, Michael J. MacCoss. Discovery and validation of mTOR as an immune mediator of the FLASH effect. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P001