The asns inhibitor asx-173 potentiates l-asparaginase anticancer activity

Abstract Background Cancer cells reprogram metabolic pathways to meet increased energy and biosynthetic demands. Among those pathways, elevated asparagine metabolism regulated by asparagine synthetase (ASNS) has been linked to progression of various hematological cancers, driving cell proliferation, chemoresistance, and metastasis. ASNS inhibition represents a promising therapeutic strategy, but inhibitors have shown limited efficacy due to poor specificity and cell permeability. Methods We performed an unbiased phenotypic screen for inhibitors of the Wnt/β-catenin pathway. Subsequent mechanistic characterization via biochemical and cellular assays revealed that the resulting inhibitors were specifically targeted to asparagine synthetase (ASNS). We evaluated the anti-cancer activity of the top inhibitor alone and in combination with L-asparaginase (ASNase), a key component of childhood acute lymphoblastic leukemia therapy. We also assessed anticancer efficacy against the OCI-AML2 xenograft preclinical model of acute myeloid leukemia (AML). Results Phenotypic screening coupled with direct binding studies using purified human ASNS protein unveiled ASX-173 as a cell-permeable small molecule that inhibits ASNS at nanomolar concentrations (ki, inhibition constant of 0.4 nM). Using a paired cell line model consisting of ASNS-deficient RS4;11 cells (RS4;11) and their ASNS-expressing counterpart (RS4;11_ASNS), we found that increasing concentrations of ASX-173 effectively restored sensitivity of the ASNase-resistant RS4;11_ASNS line to ASNase. Mechanistically, the combination treatment disrupted nucleotide synthesis and induced cell cycle arrest and apoptosis. In a mouse model of AML, the combination significantly delayed the growth of OCI-AML2 xenografts. Although the combination therapy induced mild toxicity, evidenced by body weight loss (<15%), mice fully recovered after treatment cessation. Pharmacodynamic analysis revealed decreased plasma asparagine concentrations from approximately 60 µM baseline to 25 µM after two weeks of either ASNase alone or ASNase in combination with ASX-173, whereas ASX-173 alone had no effect on plasma asparagine. Conclusions There is a long-running history of cell lines reported to be sensitive to ASNase in vitro but not in vivo—a phenomenon that can be explained by the lack of ASNS inhibition in vivo. ASX-173 now enables such inhibition. In combination with ASNase, the resulting bicompartmental blockade of asparagine metabolism results in potent anticancer activity, a clear mechanism of action, and in vivo efficacy. Future studies to optimize pharmacokinetics, test rational combinations, and examine additional cancer models will help to realize the full therapeutic potential of targeting asparagine metabolism for the treatment of hematological malignancies.