Summary A favorable target for radiation synthetic combinations would be a feature of cancer cells critically involved in growth, signaling, repair, or survival that can be blocked with an otherwise non-toxic drug, leaving tumors vulnerable to radiation without adverse effects on normal tissue. This project is directed at validating inhibition of telomerase reverse transcriptase (TERT) as a means to enhance the therapeutic index of radiation and achieving key progress toward translating this strategy to the clinic. While TERT is not expressed in most normal cells, approximately 90% of cancers display reactivation of TERT expression, supporting the catalytic activity of telomerase to maintain telomere integrity despite deregulated growth. While drugs targeting TERT have displayed sufficient safety in patients to evaluate effects of blocking telomere repeat synthesis, this has failed in solid tumors, as telomere erosion is too slow to affect tumor progression. Beyond its essential role in cancer cell immortality, TERT also contributes to pathways that support multiple cancer hallmarks. By limiting oxidative stress, accelerating double strand break repair and supporting cell survival, TERT expression in cancer cells may confer clinically significant resistance to radiation. This raises the question whether transiently targeting TERT during radiotherapy to enhance the toxicity of the resulting DNA damage to the cancer cells might significantly improve the therapeutic index of radiation. In recently published work, our groups described a novel class of TERT inhibitors inspired by the antibiotic chrolactomycin. Like the natural product, our streamlined natural product analogs react with an active site cysteine in the TERT reverse transcriptase active site. The optimized inhibitor, NU-1, is otherwise nontoxic in vitro or in vivo, but inhibits telomerase activity at low micromolar concentrations. NU-1 confers sensitivity to radiation to TERT-expressing cancer cells. Our data suggest that TERT may promote non-homologous end- joining repair, thereby affecting repair pathway choice. Finally, using a syngeneic tumor model in BALB/c mice, we have demonstrated marked sensitization to radiation in vivo, apparently mediated by persistent DNA damage and increased anti-tumor immune response. With these preliminary studies in hand, we propose to 1) Dissect the roles of TERT in double strand break repair and immune evasion, and 2) Improve the drug-like properties of NU-1 and use these novel compounds to understand how best to obtain radiation sensitization and an effective anti-tumor immune response.