PROJECT SUMMARY/ABSTRACT Defects in DNA repair are prevalent in human cancer and present an opportunity for targeted tumor treatment with agents that induce DNA damage only in cells with specific DNA repair defects. O6-methylguanine-DNA methyltransferase (MGMT), a direct reversal repair enzyme that removes O6-alkylguanine lesions deposited by alkylating agents such as temozolomide (TMZ), is the most frequently silenced DNA repair gene in cancer, with promoter hypermethylation observed in ~10% of all tumors. Loss of MGMT occurs in the majority of lower grade gliomas, ~50% of glioblastomas (GBM), and in a broad array of other cancer types, including ~20-40% of colon cancer and melanoma. MGMT is ubiquitously expressed in normal tissue, and reduced MGMT expression in tumors predicts for clinical benefit from TMZ, owing to the accumulation of DNA damage specifically in tumor cells. Importantly, the efficacy of TMZ in MGMT-deficient tumor cells is dependent upon intact mismatch repair (MMR) proteins, and resistance to TMZ arises due to MMR loss in ~50% of recurrent gliomas and ~20% of recurrent GBM. Strategies are needed to avoid or overcome this major resistance mechanism in order to therapeutically exploit MGMT silencing in cancer. In recent work, we identified a novel 2-fluoroethylating agent, KL-50, which overcomes TMZ resistance through the induction of DNA interstrand crosslinks (ICLs) specifically in tumor cells lacking MGMT, leading to MMR-independent activity in glioma models. We aim here to evaluate more broadly the therapeutic potential of this targeted DNA modifying agent. We hypothesize that the MGMT- dependent crosslinking activity of KL-50 can be harnessed to exploit unique combinations of DNA repair defects, combined with DDR inhibitors (DDRi’s) to enhance therapeutic potential, and utilized to overcome certain mechanisms of resistance to TMZ, independent of cancer histology. In Aim 1, we will determine the prevalence of cancers with combined MGMT and ICL repair defects and establish the therapeutic potential of KL-50 in this subset of cancer. We will characterize the overlap of MGMT and ICL repair defects in a large pan-cancer database and evaluate the activity of KL-50 in experimental in vitro and in vivo models representing this subset of tumors, alone and in combination with DDRi’s. In Aim 2, we will analyze resistance mechanisms to TMZ and KL-50 in MGMT-silenced tumors and determine the ability of KL-50 to avoid or overcome TMZ-induced resistance in non-glioma tumors. Using colon cancer, melanoma, and pancreatic neuroendocrine tumor cell models, we will perform focused CRISPR-Cas9 screening and develop drug-resistant cell lines to identify key resistance factors. Emergence of drug resistance and the ability of KL-50 to target TMZ-resistant tumors will further be analyzed in patient-derived primary cells and xenograft models. These studies will advance our understanding of targeted DNA modifying agents and may support the initiation...