The ability of cells to tolerate amino acid starvation is fundamental to survival under stress. Some cancer cells are vulnerable to asparagine depletion, which is exploited therapeutically using asparaginase. We recently found that Wnt pathway activation induces a profound therapeutic vulnerability to asparaginase in acute leukemia and in colorectal cancer (Hinze et al., In press; Hinze et al., 2019). This effect is mediated by a β-catenin independent branch of Wnt signaling termed Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3-dependent protein degradation. Asparaginase resistance is uniquely dependent on GSK3α because its inhibition fully phenocopied Wnt-induced sensitization to asparaginase (p < 0.0001), whereas selective GSK3β inhibition had no effect (p=ns). This is surprising because GSK3α and GSK3β are closely related paralogs thought to be redundant for many of their biologic functions. Thus, we set out to define why asparaginase resistance is selectively dependent on GSK3α activity. To define the GSK3α domains responsible for asparaginase resistance, we tested whether resistance could be restored by expression of a series of GSK3 alleles in which the N-terminal, kinase, and C-terminal domains of GSK3α and GSK3β were swapped in various configurations. Fusing the N-terminus of GSK3α to the kinase and C-terminal domains of GSK3β fully restored asparaginase resistance in GSK3α depleted T-ALL (p<0.0001). By contrast, fusing the N-terminus of GSK3β to the kinase and C-terminus of GSK3α had no activity (p=ns). To investigate how the N-terminus of GSK3α regulates asparaginase response, we first applied structural prediction algorithms, which revealed that the N-terminal domain of GSK3α is a low-complexity domain, a feature lacking in its paralog GSK3β. Immunofluorescence confocal microscopy revealed that this domain mediates spatial sequestration of GSK3α into membrane-less cytoplasmic bodies in response to amino acid starvation. GSK3a bodies appeared to be distinct from known phase-separated compartments such as stress granules and aggresomes. However, GSK3α bodies colocalized with K48-linked ubiquitin and the proteasome, suggesting that they assemble the cellular machinery for protein ubiquitination and proteasomal degradation. This suggests that GSK3α body formation promotes efficient generation of amino acids via catabolic protein degradation, and promotes survival during starvation in normal and malignant cells. Importantly, GSK3α body formation was stimulated in response to starvation of several amino acids (p<0.0001), and promoted survival of normal cells in response to amino acid starvation. In human cancer, GSK3α body formation predicted response to asparaginase resistance (p<0.0001), and sensitivity to asparaginase combined with a GSK3a inhibitor (p<0.0001). We propose that GSK3α body formation provides a cellular mechanism to promote catalytic efficiency of protein degradation as an adaptive response to amino acid starvation.
Citation Format: Laura Hinze, Sabine Schreek, Andre Zeug, Evgeni Ponimaskin, Roxane Labrosse, James Degar, Beat Bornhauser, Jean-Pierre Bourquin, Martin Stanulla, Alejandro Gutierrez. Spatial sequestration of GSKα as a cellular response to amino acid starvation [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR06.