DNA-PK inhibition enhances neoantigen diversity and increases T cell responses to immunoresistant tumors
Allison J. Nielsen, Gabriella K. Albert, Amelia Sanchez, Jiangli Chen, Jing Liu, Andres S. Davalos, Degui Geng, Xander Bradeen, Jennifer D. Hintzsche, William Robinson, Martin McCarter, Carol Amato, Richard Tobin, Kasey Couts, Breelyn A. Wilky, Eduardo Davila
Allison J. Nielsen, Gabriella K. Albert, Amelia Sanchez, Jiangli Chen, Jing Liu, Andres S. Davalos, Degui Geng, Xander Bradeen, Jennifer D. Hintzsche, William Robinson, Martin McCarter, Carol Amato, Richard Tobin, Kasey Couts, Breelyn A. Wilky, Eduardo Davila
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Research Article Immunology Oncology

DNA-PK inhibition enhances neoantigen diversity and increases T cell responses to immunoresistant tumors

Abstract

Effective antitumor T cell activity relies on the expression and MHC presentation of tumor neoantigens. Tumor cells can evade T cell detection by silencing the transcription of antigens or by altering MHC machinery, resulting in inadequate neoantigen-specific T cell activation. We identified the DNA–protein kinase inhibitor (DNA-PKi) NU7441 as a promising immunomodulator that reduced immunosuppressive proteins, while increasing MHC-I expression in a panel of human melanoma cell lines. In tumor-bearing mice, combination therapy using NU7441 and the immune adjuvants stimulator of IFN genes (STING) ligand and the CD40 agonist NU-SL40 substantially increased and diversified the neoantigen landscape, antigen-presenting machinery, and, consequently, substantially increased both the number and repertoire of neoantigen-reactive, tumor-infiltrating lymphocytes (TILs). DNA-PK inhibition or KO promoted transcription and protein expression of various neoantigens in human and mouse melanomas and induced sensitivity to immune checkpoint blockade (ICB) in resistant tumors. In patients, protein kinase, DNA-activated catalytic subunit (PRKDC) transcript levels were inversely correlated with MHC-I expression and CD8+ TILs but positively correlated with increased neoantigen loads and improved responses to ICB. These studies suggest that inhibition of DNA-PK activity can restore tumor immunogenicity by increasing neoantigen expression and presentation and broadening the neoantigen-reactive T cell population.

Authors

Allison J. Nielsen, Gabriella K. Albert, Amelia Sanchez, Jiangli Chen, Jing Liu, Andres S. Davalos, Degui Geng, Xander Bradeen, Jennifer D. Hintzsche, William Robinson, Martin McCarter, Carol Amato, Richard Tobin, Kasey Couts, Breelyn A. Wilky, Eduardo Davila

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Figure 3

DNA-PK inhibition drives TCRvβ diversity of highly functional tumor-reactive CD8+ T cells.

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DNA-PK inhibition drives TCRvβ diversity of highly functional tumor-reac...
(A) Schematic of drug treatment and tissue processing with representative flow cytometric analysis of TCRvβ on CD8+ TILs. SSC-A, side scatter area. (B) UMAP distribution of the absolute number of CD8+ TILs clustered by TCRvβ chain (number labels and color scale for differentiation) (untreated: n = 15; SL40: n = 9; NU: n = 5; NU-SL40: n = 8). (C) Number of CD8+ TILs by TCRvβ chain per 1 million single-cell events. A rout outlier test was performed. Blue and red bars represent significant decreases or increases in TCRvβ counts in treatment conditions compared with no treatment. Each dot represents a single mouse (untreated: n = 15; SL40: n = 9; NU: n = 5; NU-SL40: n = 8). (D) Schematic of C57BL/6 B16-F10 tumor model and tumor collection for TIL isolation via magnetic bead–positive selection followed by ex vivo culturing with or without IFN-γ–pretreated (100 U/mL for 24 hours) B16-F10 melanoma cells. (E) Representative flow plot with adjunct MFI histograms representing the number of isolated CD8+ TILs expressing GzmB and PD-1 obtained from control and NU-SL40–treated mice (16-hour coculture). (F) Heatmap of TCRvβ distribution of CD8+ TILs that expressed PD-1 and produced GzmB. TILs were pooled from tumors (untreated: n = 4; NU-SL40: n = 5), and counts were normalized to 2 × 105 CD3+ cells. The sum of the TCRvβ chain in each condition is represented above the columns, the sum of total TCRvβ in each condition is indicated to the right of each row. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by multiple unpaired, 2-tailed t test.