Glutaminase Activity of L-Asparaginase Contributes to Durable Preclinical Activity against Acute Lymphoblastic Leukemia

Abstract

We and others have reported that the anticancer activity of L-asparaginase (ASNase) against asparagine synthetase (ASNS)-positive cell types requires ASNase glutaminase activity, whereas anticancer activity against ASNS-negative cell types does not. Here, we attempted to disentangle the relationship between asparagine metabolism, glutamine metabolism, and downstream pathways that modulate cell viability by testing the hypothesis that ASNase anticancer activity is based on asparagine depletion rather than glutamine depletion per se. We tested ASNase wild-type (ASNase^WT) and its glutaminase-deficient Q59L mutant (ASNase^Q59L) and found that ASNase glutaminase activity contributed to durable anticancer activity against xenografts of the ASNS-negative Sup-B15 leukemia cell line in NOD/SCID gamma mice, whereas asparaginase activity alone yielded a mere growth delay. Our findings suggest that ASNase glutaminase activity is necessary for durable, single-agent anticancer activity in vivo, even against ASNS-negative cancer types.

Figure 1.. Pharmacokinetics and pharmacodynamics of ASNase^WT and ASNase^Q59L in vivo.

The pharmacodynamics of ASNase^WT and ASNase^Q59L in the plasma of non-tumor bearing NSG mice were determined by LC-MS/MS-based analysis of the amino acids (A) asparagine, (B) aspartate, (C) glutamine, and (D) glutamate. NSG mice (3 per group) were treated with PBS (negative control), 20,000 U/kg ASNase^WT, or 20,000 U/kg ASNase^Q59L by intraperitoneal injection. Time 0 measurements were made on samples collected immediately before injection. (E) The pharmacokinetics of ASNase^WT and ASNase^Q59L in plasma were determined by colorimetric assay of asparaginase activity. Error bars represent standard error.

Figure 2.. Anticancer activity of ASNase^WT and ASNase^Q59L in vivo.

(A) NSG mice (5 per group) xenografted with the luciferase-engineered leukemia cell line Sup-B15 were treated daily with the PBS negative control, 20,000 U/kg ASNase^WT, or 20,000 U/kg ASNase^Q59L for two weeks by intraperitoneal injection. Leukemia burden was monitored using bioluminescence imaging at the indicated time points. Day 0 is defined as one day before the first treatment. (B) Average bioluminescent signal in each treatment group as described for panel A. (C) Kaplan-Meier survival analysis of the mice in panel A. (D) The average of daily body weight loss of each group in panel A. Mice had unrestricted access to food and water. *Two mice died early, but it was not clear whether they died from ASNase^WT-related toxicity. Mean and SEM are shown.