PROJECT SUMMARY/ABSTRACT Genetic alterations in the tumor suppressor p53 gene (TP53) are found in most colorectal adenocarcinomas (CRC) and contributes to poor prognosis. The p53 protein encoded by TP53 is a key element of DNA damage checkpoints that are activated by DNA damage response and control genome integrity. Although decades of research generated immense information on the functional consequences of p53 mutations, therapeutic efforts targeted to mutant p53 have proven largely unfruitful. Existing therapeutic options for p53-deficient CRC are ineffective and cause toxic side effects stressing the need for better therapeutics. We developed a conceptually novel treatment strategy for selectively targeting p53-deficient cancer cells that takes advantage of their unique DNA repair deficiencies. Our preclinical research revealed that p53-deficient tumors accumulate DNA damage upon incorporation into DNA of a thymidine analogue (i.e., trifluorothymidine, a component of FDA-approved drug called TAS102). The thymidine analogue does not interrupt DNA replication but rather prompts DNA repair that requires p53-dependent checkpoint. We found that p53-deficient cells lacking the p53-dependent checkpoint selectively accumulate DNA breaks. Importantly, this DNA damage is strongly enhanced by inhibitors of poly (ADP) ribose polymerase (PARP) leading to cell death. This novel inducer- amplifier strategy was extensively validated in multiple preclinical models. Our preclinical data demonstrated a superior anti-tumor activity of TAS102 in combination with PARP inhibitor (PARPi) compared to either drug alone in p53-deficient cancer models. Based on our preclinical data, we developed a first-in-human Phase I clinical trial for advanced CRC with two FDA-approved drugs TAS102 and PARPi talazoparib. Thus, we hypothesize that the combination of TAS102 with PARPi talazoparib is an effective biomarker-driven treatment for patients with p53-deficient CRC. The current proposal is aimed to define the efficacy of our combination therapy strategy for the first time in humans with advanced CRC in a collaborative effort of basic, translational, and clinical scientists. The study will generate the biomarkers to guide clinical implementation and further development of our inducer-amplifier strategy by using patient-derived material from our ongoing clinical trial. The study will employ state-of-the art next-generation sequencing approaches to define genome-wide changes in response to the TAS102-PARPi combination in CRC models. Importantly, we will examine the antitumor efficacy of our novel two-drug therapy in p53-deficient CRC patients. Together, this work will provide mechanistic insights in the action of our two-drug therapy and will serve as a platform for development of better treatments. Our study matters for thousands of patients with aggressive p53-deficient CRC.