Abstract
Background: Cancer immunotherapy relies on presentation of immunogenic peptides on major histocompatibility complex class-I (MHC-I) by the cancer cells that are recognized by cytotoxic CD8+ T cells. We have shown that radiation therapy (RT) enhances the presentation of immunogenic mutations in a lung cancer patient with complete response to RT and ipilimumab.1 In addition to the cancer mutanome, neoantigens generated by post-translational modifications like phosphorylation have been shown to be the targets of tumor-specific T cells.2,3 RT elicits changes in many signaling pathways involved in the response to DNA damage. Thus, we tested the hypothesis that the RT-modulated cellular phosphoproteome will result in qualitative and quantitative changes in the phosphopeptides (pPeptides) presented by MHC-I of cancer cells. Methods: Human breast adenocarcinoma MDA-MB-231 cells were irradiated daily with 8 Gy in three consecutive days (8 Gyx3) or left untreated. At 24 hours following the last RT dose, cells were harvested and MHC-I bound peptides were affinity-purified using the human MHC-I (A, B and C allele) specific W-6/32 antibody. High resolution LC-MS/MS was used to analyze the immunopeptidome according to established protocols.4 Pathway analysis of peptides was carried out in Qiagen Ingenuity Pathway Analysis. Results: Radiation increased the expression of MHC-I on the surface of MDA-MB-231 cells and the number of unique MHC-I bound peptides presented by the cells (mean 1918 in control vs 6180 in RT, n=3, p=0.0008). The total number of phosphorylated MHC-I ligands was increased 11-fold, representing, on average, 0.17% of ligands in untreated cells and 0.61% in irradiated cells (p=0.005). Phosphorylation usually occurred on serine or threonine residues and was found predominantly in position 4 or 5 of the peptide. The phosphorylated amino acid was often followed by proline, and stabilized by positively charged amino acids at either the +3 or -3 position relative to the phosphorylation site. The pPeptides presented only by the irradiated cells (and identified in at least 2 out of 3 samples) were derived from proteins belonging to pathways activated by radiation, including p53 and hypoxia signaling (HIF1a, JUN), regulation of cell proliferation (TGFl1, BCAR3) and senescence (CDC25B, JUN). Conclusions: Our results show, for the first time, that RT enhances the presentation of pPeptides by MHC-I. RT enhanced both the number and repertoire of pPeptides bound to surface MHC-I of cancer cells. Given the ability of pPeptides to serve as cancer antigens that are recognized by T cells, our data suggests that one way RT increases tumor immunogenicity may be through inducing the presentation of novel pPeptides. This hypothesis is being tested in ongoing studies.
References: 1.Formenti SC, et al. Nat Med 2018;24. 2.Engelhard VH, et al. J Immunother Cancer 2020;8. 3.Zarling AL, et al. J Exp Med 2000;192:1755-62. 4.Klatt MG, et al. J Proteomics 2020;228:103921.
Citation Format: Samantha J. Van Nest, Tomer M. Yaron, Martin G. Klatt, Noah E. Dephoure, Gad Getz, Laura Santambrogio, Olivier Elemento, David Scheinberg, Sandra Demaria, Lewis C. Cantley. Radiation therapy enhances the presentation of phosphopeptides by MHC-I on cancer cells [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-051.
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