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 4

DNA-PK inhibition increases tumor-associated antigen expression levels, induces a unique neoantigen expression profile in melanoma, and represents better targets for human TILs.

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DNA-PK inhibition increases tumor-associated antigen expression levels, ...
(A and B) B16-F10 melanoma cells were treated with 2 μM NU7441 or DMSO control for 72 hours, at which point gradual darkening was observed and the OD405 recorded. (C) Bar graph comparing the levels of RNA per FPKM of known melanogenesis-associated antigens 48 hours after treatment with 2 μM NU7441 or DMSO control. The fold change between DMSO and NU7441 treatments is noted above the bars. (D) B16-F10 melanoma cells were treated with 2 μM NU7441 or DMSO control for 48 hours, and the levels of the indicated proteins were determined by Western blotting. The fold change between groups is shown to the right of each band. (E and F) B16-F10 melanoma cells were treated as described in A, and the neoantigens and FPKM were determined as described in Methods. (E) Venn diagram representing the number of uniquely expressed or shared B16-F10 neoantigens present in control-treated melanomas and those induced by NU7441. (F) The gene name and amino acid mutation expressed following DNA-PKi treatment are shown on the left. The matched bar graph shows the levels of RNA per FPKM of neoantigen-producing genes exposed to NU7441 treatment, and the binding affinity of these epitopes for H2-Db and H2-Kb was determined using the MHC binding prediction algorithms from the Immune Epitope Database and Tools (IEDB) (iedb.org) site. (G) Schematic showing the generation of melanoma cell lines and TILs from a patient melanoma tumors and the experiments performed in H–J. (H) The MB3429 melanoma cell line was treated with 2 μM NU7441 or DMSO control for 48 hours, and the levels of the indicated transcripts were determined by RT-PCR and are shown as ΔCt. (I) MB3429 melanoma cells were treated with 2 μM NU7441 or DMSO control for 48 hours, and the levels of the indicated proteins were determined by Western blotting (fold change between groups is indicated to the right of each band). (J) Matched TILs and tumors were derived from the same tumor fragment. The tumor cell line was treated with DMSO or DNA-PKi (2 μM NU7441) for 48 hours, at which point the drug was washed off prior to coculturing with TILs at a 1:1 ratio for 18 hours. Cytotoxicity was determined by annexin V staining with flow cytometric gating on tumor cells (based on light scatter and CD3–) and viability dye.