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 9

Anti–PD-1 therapy in the context of UNG depletion delays CT-26 tumor growth and increases BALB/c mouse survival in a CD8+ T cell–dependent manner.

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Anti–PD-1 therapy in the context of UNG depletion delays CT-26 tumor gro...
(A) BALB/c mice were treated with anti–PD-1 (aPD1) antibodies (0.1 mg twice weekly via intraperitoneal injection), with or without shRNA-mediated UNG depletion (shRNAUNG). Female mice were injected with 5 × 105 CT-26 cells expressing either shRNAUNG or shRNActrl, and the experiment was performed twice with 4 groups consisting of 5 mice each: (a) shRNActrl without aPD1 antibodies (shRNActrl/aPD1–), (b) shRNActrl + aPD1 antibodies (shRNActrl/aPD1+), (c) shRNAUNG without aPD1 antibodies (shRNAUNG/aPD1–), and (d) shRNAUNG + aPD1 antibodies (shRNAUNG/aPD1+). (B) Tumor growth for the 4 experimental groups was measured on the indicated days (colors correspond to A). Error bars for tumor growth are SEM, and the significance between groups was evaluated using an unpaired 2-tailed t test. A reduced growth rate for shRNAUNG/aPD1+ vs. shRNActrl/aPD1+ was observed (P = 0.026), while tumor growth in the mice receiving the 2 monotherapy regimes did not differ from the no-treatment control. (C) Tumor growth kinetics for individual CD8proficient mice receiving the shRNActrl/aPD1+ (tan square) and shRNAUNG/aPD1+ (red triangle) treatments. (D) Survival of the 4 experimental treatment groups (colors correspond to A). Significance was tested using the log-rank Mantel-Cox test, which showed a significantly improved survival in CD8proficient mice treated with shRNAUNG/aPD1+ vs. shRNActrl/aPD1+ (P = 0.005). (E) Tumor growth kinetics for CD8depleted mice using the 4 treatments: shRNActrl/aPD1– (black circle), shRNActrl/aPD1+ (tan square), shRNAUNG/aPD1– (pink triangle), and shRNAUNG/aPD1+ (red triangle). Tumors volumes were measured on the indicated days and the error bars are SEM. No significant differences in the tumor growth curves for the 4 groups of CD8depleted mice were observed as determined by an unpaired 2-tailed t test (P > 0.05). (F) Survival of CD8depleted mice using the 4 treatment modalities [shRNActrl/aPD1– (black circle), shRNActrl/aPD1+ (tan square), shRNAUNG/aPD1– (pink triangle), and shRNAUNG/aPD1+ (red triangle)] showed no significant differences as determined by an unpaired 2-sided t test (P > 0.05).