Ablating UNG activity in a mouse model inhibits colorectal cancer growth by increasing tumor immunogenicity
Eric S. Christenson, Brandon E. Smith, Thanh J. Nguyen, Alens Valentin, Soren Charmsaz, Nicole E. Gross, Sarah M. Shin, Alexei Hernandez, Won Jin Ho, Srinivasan Yegnasubramanian, James T. Stivers
Eric S. Christenson, Brandon E. Smith, Thanh J. Nguyen, Alens Valentin, Soren Charmsaz, Nicole E. Gross, Sarah M. Shin, Alexei Hernandez, Won Jin Ho, Srinivasan Yegnasubramanian, James T. Stivers
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Research Article Genetics Immunology Oncology

Ablating UNG activity in a mouse model inhibits colorectal cancer growth by increasing tumor immunogenicity

Abstract

Uracil DNA glycosylase (UNG) excises uracil and 5-fluorouracil bases from DNA and is implicated in fluorodeoxyuridine (FdU) resistance. Here we explore the effects of inhibiting UNG activity, or depleting the UNG protein, in 2 mouse syngeneic models for colorectal cancer. Overexpressing the small UNG inhibitor protein (UGI) in mismatch repair–deficient (MMR-deficient) MC38 cells injected into C57BL/6J mice delayed tumor growth and prolonged survival when combined with FdU. Combining UNG inhibition with FdU numerically increased CD4+ T lymphocytes and B cells compared with FdU or UNG inhibition alone, suggesting an immune component to the effects. In contrast, shRNA depletion of UNG in the absence of FdU treatment resulted in 70% of mice clearing their tumors, and a 3-fold increase in overall survival compared with FdU. Analysis of MC38 tumor–infiltrating immune cells showed UNG depletion increased monocyte and dendritic cell populations, with CD8+ T cells also numerically increased. shRNA depletion of UNG in MMR-proficient CT-26 cells injected into BALB/c mice produced minimal benefit; the addition of anti–PD-1 antibody synergized with UNG depletion to increase survival. Cytotoxic T cell depletion abolished the benefits of UNG depletion in both models. These findings suggest UNG inhibition and/or depletion could enhance antitumor immune responses in humans.

Authors

Eric S. Christenson, Brandon E. Smith, Thanh J. Nguyen, Alens Valentin, Soren Charmsaz, Nicole E. Gross, Sarah M. Shin, Alexei Hernandez, Won Jin Ho, Srinivasan Yegnasubramanian, James T. Stivers

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Figure 8

shRNAUNG knockdown in BALB/c mice inoculated with CT-26 tumors.

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shRNAUNG knockdown in BALB/c mice inoculated with CT-26 tumors. The anti...
The antitumor effects of UNG protein shRNA depletion in the presence and absence of FdU treatment was investigated in the BALB/c MMR-competent mouse strain. CT-26 cells (5 × 105) were injected into the right hind limb of female BALB/c mice. The experiment was performed twice, with 5 mice and 10 mice per group. (A) The 4 groups in each experiment were (a) shRNActrl without FdU treatment, (b) shRNActrl with FdU treatment, (c) shRNAUNG without FdU treatment, and (d) shRNAUNG with FdU treatment. The FdU treatment was performed in an identical fashion as described in the legend to Figure 3. (B) Tumor volumes for the 4 experimental groups were measured on the indicated days. Tumor volumes from the replicate experiments were combined and plotted for statistical analysis (n = 15 for each group). Error bars are SEM, and differences in tumor growth were compared using an unpaired 2-tailed t test. A significant reduction in tumor growth was observed for shRNActrl + FdU vs. shRNActrl (P = 0.0005), but depletion of UNG had no statistically significant benefit in this syngeneic model. (C) Kaplan-Meier survival analysis for the 4 experimental groups. Survival comparisons between groups used the log-rank Mantel-Cox test, which showed improved survival with shRNActrl + FdU versus shRNActrl (P = 0.0002). Depletion of UNG had no statistically significant benefit on survival in this syngeneic model.