Deoxyuridine Contamination and Innate Immune Signaling

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

Investigators

Abstract

ABSTRACT Most chemotherapies target DNA replication, and their efficacy depends on the DNA damage response that integrates DNA repair with the cell cycle. Widely used standard-of-care antimetabolites inhibit thymidylate synthase and increase the incorporation of deoxyuridine (dU) into DNA by polymerases. dU contamination in DNA is limited by the DDR kinase ATR that induces cell cycle checkpoints and reduces the rate of DNA replication. This project investigates the impact of dU contamination on innate immune responses. ATR inhibitors (ATRi) induce origin firing across active replicons and cause ribonuclease reductase degradation in otherwise unperturbed cells. This increases both the amount of DNA replication and the amount of free dUTP in cells. Thus, ATRi increase the incorporation of dU into DNA by polymerases. Since ATRi also inhibit cell cycle checkpoints, ATRi induce more dU contamination than antimetabolites. Our preliminary data show that ATRi-induced IFN-β is dependent on uracil DNA glycosylase (UNG) which removes dU from DNA. Our data are consistent with a model in which UNG-dependent base excision repair (BER) removes dU from DNA and generates cytoplasmic dsDNA that induces IFN-α/β. Unexpectedly, however, we did not observe a difference in the amount of cytoplasmic dsDNA in wild-type (WT) and UNG knockout (∆UNG) cells after treatment with ATRi. We reasoned that cytoplasmic DNA in ∆UNG cells may not be recognized by pattern recognition receptors because it is dA:dU-rich. Indeed, replacing just 20 thymidine bases (T) with dU in a 200 bp dsDNA fragment significantly decreased IFN-β induction in cells. Our data suggest that the integrity of dA:T base pairs is critical for the IFN-α/β response in cells treated with chemotherapy and ionizing radiation (XRT). cGAS recognizes DNA in a sequence-independent manner. However, dsDNA fragments are transcribed in the cytoplasm by RNA Polymerase III (Pol III) to generate RNA that is recognized by RIG1. It was recently shown that dA:dU-rich DNA is inhibitory for Pol II transcription. We hypothesize that dA:dU-rich fragments cannot be transcribed by Pol III and that dA:dU-rich fragments antagonize innate immune responses. An important corollary to this hypothesis is that the efficacy of dU repair in tumors may impact immune responses in patients treated with ATRi, antimetabolites, and checkpoint inhibitors. Three Specific Aims will address this hypothesis. Aim 1: Identify pattern recognition receptors (PRRs) that do not recognize DNA fragments when thymidine is replaced with dU. Aim 2: Determine whether dU disrupts Pol III-dependent innate immune receptor signaling. Aim 3: Investigate the impact of dU contamination in mouse models of cancer treated with XRT. These experiments are highly significant and impactful for the 100,000’s of cancer patients treated with antimetabolites and checkpoint inhibitors every year.