ABSTRACT Colorectal cancer (CRC) is the second-leading cause of cancer deaths in the US, and unlike many other tumor types, there are no known effective therapies targeting dominant oncogenic drivers. Almost all CRCs have mutations that activate canonical Wnt/β-catenin signaling, but direct inhibition of β-catenin is difficult, and blocking Wnt ligand activity leads to significant on-target bone toxicity. Thus, while targeting Wnt directly is challenging, aberrant Wnt pathway activation may induce tumor-specific vulnerabilities that can be exploited for CRC therapy. Using a genome-wide genetic screen, we found that Wnt activation induces profound sensitization to therapeutic asparagine depletion using asparaginase in drug-resistant leukemias. This effect is dependent on Wnt-induced inhibition of GSK3, but is independent of APC or β-catenin. Instead, asparaginase sensitization is mediated by Wnt-induced inhibition of GSK3-dependent protein degradation, a catabolic source of amino acids required for asparaginase resistance. CRC provides a unique context in which to test predictions from our model, because these tumors almost all have mutations that activate Wnt/β-catenin, but these can function either upstream or downstream of GSK3. Using human CRC cell lines and genetically engineered mouse intestinal organoids, we found that asparaginase had little effect on CRCs with mutations of the downstream Wnt factor APC, but was profoundly toxic to cases with R-spondin translocations, which activate Wnt signaling via ligand-induced inhibition of GSK3, and thus inhibit GSK3-dependent protein degradation. Importantly, this approach has little detectable toxicity to normal intestinal or epithelial cells. This suggests that this approach has a potent therapeutic index that could transform clinical outcomes for the thousands of patients who die of CRC every year, and a clinical trial based on these data is under development. However, we do not understand key aspects of the biology underlying this RSPO/Wnt-induced therapeutic vulnerability. Defining the precise molecular events that dictate RSPO/Wnt induced asparaginase sensitivity is critical for prospectively identifying clinical responders, designing rational approaches to improve therapeutic response, and overcoming treatment resistance. These knowledge gaps will be addressed in the following Aims: 1) Determine how RSPO ligands induce sensitization to asparaginase. 2) Investigate the role of GSK3α body formation as a cellular response to asparagine starvation. 3) Determine the role of oncogenic KRAS and TP53 mutations in therapeutic response of RSPO fusion CRC to asparaginase. This proposal is expected to provide fundamental insights into the amino acid starvation response and its impairment by aberrant Wnt signaling, cellular processes fundamental to metazoan life whose molecular basis and therapeutic exploitation remain poorly understood. Given our highly complementary expertise in asparaginase biology and...