Abstract In the United States, colon cancer and rectal cancer are the third most common cancer diagnosed in both men and women. The standard of care for stage II-III rectal cancer is neoadjuvant chemoradiation or short-course radiation followed by total mesorectal excision. However, the response to neoadjuvant radiation varies across patients, with some having minimal response to 10-30% having a pathologic complete response. Pathologic complete response is associated with improved clinical outcomes including resectability, sphincter preservation, local control, and overall survival. Therefore, novel strategies that sensitize rectal cancers to radiation therapy will have great potential to increase pathologic response rates, improve clinical outcomes, and support the emerging total neoadjuvant therapy paradigm and the experimental watch-and-wait approach. The long-term goal of this project is to increase the efficacy of radiation therapy for rectal cancer by targeting calcium/calmodulin-dependent protein kinase kinase 2 (Camkk2). Camkk2 belongs to a family of multifunctional Ser/Thr kinases that participate in the calcium/ calmodulin (CaM) signaling pathway and play a crucial role in controlling energy balance, inflammation and tissue regeneration. Several studies have reported that blocking Camkk2 using genetic approaches or the small molecule inhibitor STO-609 suppresses the growth of breast, prostate and liver cancers in vitro and in vivo. Furthermore, in preliminary studies we found that STO-609 sensitizes mouse colorectal cancer organoids to radiation in vitro. To investigate this question in vivo, we pioneered novel methods to generate solitary, autochthonous tumors in the distal colon that can be monitored with colonoscopy and reproduce the histology of human disease. In preliminary studies, we found that focal pelvic radiation effectively inhibits growth of these tumors. These models represent a major advance over other commonly used colorectal cancer mouse models, which develop many tumors predominantly in the small intestine and are therefore poorly suited to studying radiation therapy of rectal cancer. Based on these findings, we hypothesize that blocking Camkk2 signaling will improve the response to radiation in our novel mouse models of rectal cancer.