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
View: Text | PDF
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

×

Figure 3

MC38 tumor growth and mouse survival in a syngeneic colon tumor model using FdU alone or in combination with UNG inhibition with UGI.

Options: View larger image (or click on image) Download as PowerPoint
MC38 tumor growth and mouse survival in a syngeneic colon tumor model us...
We introduced the cDNA sequence of UGI into a CW57.1 doxycycline-inducible lentiviral expression plasmid and introduced the expression construct into MC38 cells. After confirming successful inducible inhibition of UNG in the transduced cell line (see Figure 2B), 2.5 × 105 MC38 cells were injected into the right hind limb of female C57BL/6J mice. Overall, 60 mice were used in 2 independent experiments. In the first experiment, the mice were divided into 4 groups of 5 mice, and the second, 4 groups of 10 mice. The 4 experimental groups were (a) UNG+, empty vector with no UNG inhibition and no FdU treatment, (b) UNG–, UNG inhibition with no FdU treatment; 1, (c) FdU treatment with no UNG inhibition (UNG+/FdU) and, (d) combination of UNG inhibition and FdU treatment (UNG–/FdU). (A) FdU was administered by intraperitoneal injection using a 50 mg/kg daily dose on days 3 to 12 and UNG inhibition was induced with doxycycline starting on day 1. (B) Tumor volumes for the 4 experimental groups were measured on the indicated days. Measurements from the replicate experiments were pooled and plotted for statistical analysis (n = 15 for each group). Error bars for tumor growth are SEM. Tumor growth curves were compared using an unpaired 2-tailed t test between groups: UNG–/FdU vs. UNG+/FdU (P = 0.09) and UNG–/FdU vs. UNG– (P = 0.12). (C) Kaplan-Meier survival analysis for the 4 experimental groups (n = 15 per group). Statistical comparisons of survival between groups used the log-rank Mantel-Cox test: UNG–/FdU vs. UNG+/FdU (P value = 0.005 and UNG–/FdU vs. UNG– (P = 0.005).