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 4

Measurement of the tumor microenvironment with CyTOF demonstrates inhibiting UNG catalytic activity with UGI in combination with FdU treatment alters the immune microenvironment.

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Measurement of the tumor microenvironment with CyTOF demonstrates inhibi...
To determine the influence of UNG inhibition via UGI on the MC38 tumor microenvironment, 2.5 × 105 MC38 cells containing the CW57.1 UGI guide were injected into the right hind limb of female C57BL/6J mice (20 total). Mice were divided into 4 groups of 10 mice; (a) Control, (b) UNG inhibition via doxycycline-induced UGI expression starting on day one, (c) floxuridine (FdU) treatment at 50 mg/kg/dose daily via intraperitoneal injection days 3 to 12, and (d) UNG inhibition combined with FdU treatment. On day 20, mice were sacrificed and tumors excised. Tumors were then homogenized and stained with a panel of 40 antibodies with the Fc region complexed with unique heavy metal isotopes. This demonstrated a numerical increase in CD4+ T lymphocytes and B cells in the combination treatment group. Selected cell subtypes are shown in the figure with an entire list and heatmap provided in Supplemental Figure 8. Data are shown as box-and-whisker plots, with the mean indicated by a horizontal line, middle quartiles represented by the box bounds, and range denoted by the whiskers. Significance calculated using pairwise 2-tailed t tests. P values were adjusted for multiple comparisons within each cell type using Benjamini-Hochberg. Tc cells, CD8+ T lymphocytes; Th cells, CD4+ T lymphocytes; Treg cells, regulatory T cells; Mac_III, CD206–CD11b+Ki67+ macrophages; Mac_VII, CD206–CD11b+Ki67+IAIE+ macrophages; Mac_IX, CD206+CD11b+Ki67+PD-L1– macrophages; Monocyte_I, CD11b+Ly6G+ monocytes; Monocyte_II, CD11b+Ly6G+PD-L1+IAIE+ monocytes.