ABSTRACT Asparaginase, an antileukemic enzyme that depletes the nonessential amino acid asparagine, is a core component of front-line therapy for acute lymphoblastic leukemias. However, the development of resistance to asparaginase-based therapy is a common problem with a poor prognosis, and the biology underlying asparaginase response and resistance is not well understood. Using a genome-wide genetic screen, we recently found that Wnt pathway activation profoundly sensitizes drug-resistant leukemias to asparaginase. This effect occurs in distinct treatment-resistant subtypes of acute leukemia, including T-ALL, hypodiploid B-ALL, and acute myeloid leukemia, but not in normal hematopoietic progenitors. Asparaginase sensitization is independent of the canonical β-catenin branch of Wnt signaling. Instead, this effect is mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), an understudied branch of Wnt signaling that inhibits GSK3-dependent protein ubiquitination and proteasomal degradation. Inhibiting the alpha isoform of GSK3 is sufficient to phenocopy this effect. In vivo, the combination of GSK3α-selective inhibition and asparaginase has profound therapeutic activity against patient-derived xenografts that are completely resistant to monotherapy with either agent. Thus, asparaginase-resistant leukemias survive treatment with this enzyme by relying on protein degradation, a catabolic source of amino acids, to replenish the pool of intracellular asparagine. This adaptive response is blocked by Wnt pathway activation, or by selective inhibition of GSK3α. However, the molecular determinants of response and resistance to the combination of GSK3α inhibition and asparaginase are unknown. These knowledge gaps are major obstacles to the rational application of this potent therapeutic combination for maximal clinical benefit, which will be addressed by investigating the following Specific Aims: 1) Define the molecular basis for the therapeutic index of GSK3α inhibition and asparaginase, and 2) Investigate why asparaginase resistance is solely dependent on GSK3α, when its closely related paralog GSK3β is redundant for regulation of canonical Wnt/β-catenin signaling. Successful completion of this proposal is expected to reveal major insights into the cellular response to amino acid starvation and its interaction with Wnt signaling. These processes are fundamental to metazoan biology, yet their molecular regulation and therapeutic exploitation remain poorly understood. We are uniquely poised to translate these advances into highly innovative and effective therapeutic strategies to maximize the therapeutic index of asparaginase in cancer therapy.

Project Narrative The antileukemic drug asparaginase is a central component of curative therapy for several acute leukemias, but the development of resistance is a major clinical problem with a poor prognosis. We found that inhibition of the kinase GSK3α induces asparaginase sensitization in several drug-resistant subtypes of acute leukemia. We propose to investigate the biology underlying this potent therapeutic interaction, which is expected to provide fundamental insights into the amino acid starvation response, and allow its rational therapeutic exploitation for maximal clinical benefit.