Synthetic Lethality of Wnt Pathway Activation and Asparaginase in Drug-Resistant Acute Leukemias

Asparaginase, a bacterial enzyme that depletes the nonessential amino acid asparagine, is an integral component of acute leukemia therapy. However, asparaginase resistance is a common clinical problem whose biologic basis is poorly understood. We hypothesized, based on the concept of synthetic lethality, that gain-of-fitness alterations in the drug-resistant cells had conferred a survival advantage that could be exploited therapeutically. To identify molecular pathways that promote fitness of leukemic cells upon treatment with asparaginase, we performed a genome-wide CRISPR/Cas9 loss-of-function screen in the asparaginase-resistant T-ALL cell line CCRF-CEM. Cas9-expressing CCRF-CEM cells were transduced with a genome-wide guide RNA library (Shalem et al. Science343, 84-87, 2014), treated with either vehicle or asparaginase (10 U/L), and guide RNA representation was assessed. Our internal positive control, asparagine synthetase, was the gene most significantly depleted in asparaginase-treated cells (RRA significance score = 1.56 x 10-7), followed closely by two regulators of Wnt signaling, NKD2 and LGR6 (RRA score = 6 x 10-6and 2.19 x 10-5, respectively). To test how these genes regulate Wnt signaling in T-ALL, we transduced CCRF-CEM cells with shRNAs targeting NKD2 or LGR6, or with an shLuciferase control. Knockdown of NKD2 or LGR6 increased levels of active β-catenin, as well as the activity of a TopFLASH reporter of canonical Wnt/β-catenin transcriptional activity (P < 0.0001), indicating that NKD2 and LGR6 are negative regulators of Wnt signaling in these cells. We then validated the screen results using shRNA knockdown of NKD2 or LGR6, which profoundly sensitized these cells to asparaginase (P< 0.0001) and potentiated asparaginase-induced apoptosis (P < 0.0001). Inhibition of glycogen synthase kinase 3 (GSK3) is a key event in Wnt-induced signal transduction. Thus, we tested whether CHIR99021, an ATP-competitive inhibitor of both GSK3 isoforms (GSK3α and GSK3β), could phenocopy the effect of Wnt pathway activation. Pharmacologic inhibition of GSK3 induced significant sensitization to asparaginase across a panel of cell lines representing distinct subtypes of treatment-resistant acute leukemia, including T-ALL, AML and hypodiploid B-ALL (Fig. 1a, b). Importantly, GSK3 inhibition did not sensitize normal hematopoietic progenitors to asparaginase, suggesting a leukemia-specific effect. Wnt-induced sensitization to asparaginase was independent of β-catenin