Mitochondrial Dysfunction Induces Asparaginase Resistance in R-Spondin Fusion Colorectal Cancer

Improved therapies are required for the thousands of patients who die of colorectal cancer (CRC) every year. We previously found that asparaginase, a cancer therapeutic that degrades asparagine, has efficacy in R-spondin fusion CRCs because these fusions block upregulation of proteasomal protein degradation, a source of intracellular amino acids, when asparagine is lacking from the environment (Hinze et al., Cancer Discovery, 2020). To anticipate mechanisms of treatment resistance that may emerge, we applied a CRISPR-based genetic screen to identify modifiers of asparaginase response in R-spondin fusion CRC. We found that guide RNAs targeting multiple mitochondria-localized proteins were enriched in asparaginase-treated cells, as these genes comprised 59% of the top 100 “hits” on the screen. This finding suggested that impairing mitochondrial function induces asparaginase resistance, which was surprising because mitochondrial function is required to produce intracellular aspartate, a key substrate for asparagine biosynthesis. Indeed, in R-spondin fusion CRC driven cells, we confirmed that shRNA knockdown of multiple mitochondrial factors, or a pharmacologic inhibitor of mitochondrial translation, each induced asparaginase resistance. Mitochondria contain >1100 proteins, 99% of which are nuclear-encoded, cytoplasmically-synthesized, and then imported into mitochondria. We found that induction of mitochondrial dysfunction led to a marked increase in total K48-linked polyubiquitinated proteins, along with cytoplasmic accumulation of multiple nuclear-encoded mitochondria proteins. Our findings support the model that triggering mitochondrial dysfunction impairs mitochondrial import of nuclear-encoded mitochondrial proteins, which are then degraded via the ubiquitin proteasome system. This provides an alternative mechanism through which these cells can increase protein degradation despite expression of the R-spondin fusion. Protein degradation releases free amino acids, providing these cells with a catabolic source of intracellular asparagine that allows them to survive during asparaginase-induced depletion of asparagine. Our findings provide a molecular understanding of how the failure of import of mitochondrial proteins may be an anticipated mechanism of resistance to asparaginase in patients. Citation Format: Su Hyun Lee, Yun-Cheol Chae, Alejandro Gutierrez. Mitochondrial dysfunction induces asparaginase resistance in R-spondin fusion colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1800.