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 5

shRNA knockdown of UNG leads to clearance of MC38 tumors in C57BL/6J mice.

Options: View larger image (or click on image) Download as PowerPoint
shRNA knockdown of UNG leads to clearance of MC38 tumors in C57BL/6J mic...
To determine the antitumor effects of UNG protein depletion in the presence and absence of FdU treatment, we used 2 doxycycline-inducible shRNA constructs that targeted UNG (shRNAUNG seq A, shRNAUNG seq B), and a control shRNA (shRNActrl) that did not target any genomic mRNA sequence. After confirming successful delivery of the shRNAUNG and shRNActrl constructs, 2.5 × 105 MC38 cells were injected into the right hind limb of female C57BL/6J mice. The experiment, which was performed twice, with each experiment consisting of 4 groups of 10 mice: (a) shRNActrl without FdU treatment, (b) shRNActrl with FdU treatment, (c) shRNAUNG without FdU treatment, and (d) shRNAUNG with FdU treatment. FdU treatment was performed in an identical fashion as described in the legend to Figure 3. (A) Tumor volumes for the 4 experimental groups were measured on the indicated days using shRNAUNG sequence A. (B) Tumor volumes for 10 individual mice inoculated with shRNAUNG sequence A and shRNActrl in the presence and absence of FdU. The asterisk indicates the overlapping data for 7 mice receiving shRNAUNG sequence A that had no palpable tumors. One mouse receiving the shRNActrl also had no palpable tumors. Error bars are SEM. Tumor growth was compared using an unpaired 2-tailed t test between the experimental groups: shRNActrl vs. shRNAUNG sequence A (P < 0.0001), shRNActrl + FdU treatment vs. shRNAUNG (P = 0.003), and shRNActrl + FdU vs. shRNAUNG + FdU (P = 0.003). (C) Kaplan-Meier survival analysis for the 4 experimental groups. Seven out of 10 mice in both the shRNAUNG (sequence A) and shRNAUNG/FdU treatment groups had cleared their tumors 7 days after inoculation. Survival comparisons were made the log-rank Mantel-Cox test: shRNAUNG vs. shRNActrl (P = 0.0009), shRNActrl + FdU treatment vs. shRNAUNG (P = 0.013), and shRNActrl + FdU vs. shRNAUNG + FdU (P = 0.012). (D and E) Given the absence of an effect of FdU in the experiments in A and B, shRNA depletion of UNG was repeated in an additional experiment with the alternative shRNA guide shRNAUNG sequence B in the absence of FdU (5 mice per group), showing similar results.