Project Summary Despite exciting progress made recently in precision medicine, several common cancers remain difficult to treat, including lung, colorectal, and pancreatic cancer, which together account for over 200,000 deaths annually. One common molecular factor in these tumors is high levels of reactive oxygen species, which lead to oxidative damage in DNA – most notably, 8-oxoguanine (8-OG), which is both toxic and mutagenic. As a result, tumor cells evolve strategies to support rapid growth, and thus often misregulate the enzymes that combat this damage: namely MTH1 and OGG1, which remove 8-OG from the nucleotide pool and from DNA itself. We hypothesize that developing approaches to control the activities of these enzymes will provide new and promising strategies for controlling tumor growth. However, until very recently no one has been able to measure or modulate these enzymes' activities. In preliminary work leading up to this proposal, novel and sensitive chemical probes have been devised that are the only existing reporters that can measure the cellular activities of MTH1 and OGG1. In addition, these probes have been used to identify new small-molecule modulators of these pathways, including, excitingly, the only known activators of the two enzymes. Third, new hypotheses have been developed regarding how modulating the activities of these pathways via small molecules, singly or in combination, can provide biologically important, and potentially clinically useful, outcomes in cancer. The Kool/Ford collaborative team will develop and employ these molecular tools to investigate the promise of modulating these important repair pathways. The specific aims for the four-year term of the project are to develop new probes to quantify repair activities in tumor cells and tissues; to identify and develop new small-molecule inhibitors and activators of the enzymes; to test novel biological hypotheses regarding how targeted up- or down-regulation may suppress tumor growth; and to test a new hypothesis for preventing tumorigenesis in individuals who are genetically susceptible to developing cancer. This research is important because it addresses multiple common and deadly cancers that remain difficult to treat. In addition, the collaborative team will develop several molecular tools that are likely to be useful to the cancer research community as a whole. Moreover, if successful, this work may lead to new targeted strategies for cancer treatment, and practical methods for evaluating patients for these therapies. This research plan is innovative in several ways: it will develop and apply novel molecular tools for assessing damage repair pathways; it will lead to the development of the only known small-molecule activators of damage repair, and it presents new hypotheses regarding how modulating repair activities will be helpful in treatment - and even prevention - of these serious malignancies.