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 (ASNaseWT) and its glutaminase-deficient Q59L mutant (ASNaseQ59L) 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.

Escherichia coliL-asparaginase (ASNase) is a standard agent for treatment of acute lymphoblastic leukemia (ALL) and is being tested against other cancer types. As early as 1978, glutaminase activity was reported to contribute positively to the drug's anticancer activity (1) but also to toxic side effects (2). Because side effects often prevent patients from completing the full treatment regimen necessary to achieve durable remission (3) the research community is actively pursuing development of glutaminase-deficient ASNase variants (4–6). We previously reported that one such mutational variant, ASNaseQ59L, retains anticancer activity against asparagine synthetase (ASNS)-negative leukemia cell types in vitro (4). However, we and others have also noted that glutaminase activity appears to be a major determinant of anticancer activity against ASNS-positive cell types (1, 4, 7, 8) prompting the question: Do glutaminase-deficient ASNase variants retain anticancer activity in vivo?

The expression of asparagine synthetase (ASNS) in most cells in the body poses a serious challenge to therapy with ASNase. Although asparagine levels were not found in one report to be increased in the bone marrow during asparaginase therapy (9), others have suggested that production of asparagine by the liver (10) and cells of the tumor microenvironment (e.g., mesenchymal stem cells, refs. 11, 12; and adipocytes, ref. 13) may contribute significantly to ASNase resistance in vivo. Here, we asked whether glutaminase-deficient ASNase can exert anticancer activity against an ASNS-negative human leukemia cell line, Sup-B15, in an ASNS-positive environment in NSG mice. Accordingly, we examined the in vivo anticancer activity associated with the asparaginase and glutaminase activities of ASNase by comparing wild-type enzyme (ASNaseWT) with a glutaminase-deficient mutant, ASNaseQ59L, which we designed through molecular dynamics calculations, generated by saturation mutagenesis, and characterized in kinetic and pharmacologic assays (4).

ASNase variants

Escherichia coliL-asparaginase II (ASNase) recombinant proteins (wild-type (WT) and mutant Q59L) were produced as described previously (4).

Determination of asparaginase and glutaminase enzymatic activity

Asparaginase and glutaminase activities of ASNase were measured as described previously (4). One unit (U) of asparaginase or glutaminase activity is defined as the amount of enzyme required to generate 1 μmol of aspartate or glutamate per minute from 100 μmol/L asparagine or 2 mmol/L glutamine, respectively, in 23 mmol/L Tris buffer at pH 8.5 and 37°C. Specific activity is defined as units/mg of enzyme.

Mouse leukemia tumor model

Mouse studies were performed in a pathogen-free vivarium at The University of Texas MD Anderson Cancer Center under an approved Institutional Animal Care and Use Committee study protocol (ACUF #00001658-RN00). We injected 0.5 × 106 luciferase-engineered Sup-B15 cells in 100 μL phosphate-buffered saline (PBS) into each NSG mouse [NOD.Cg-PRKDC(scid) IL2RG(tm1Wjl); The Jackson Laboratory stock #005557] via the tail vein. After 2 weeks, leukemia burden was monitored using bioluminescence imaging (IVIS Imaging System, PerkinElmer), which was recently reported to be superior to peripheral blood monitoring of leukemia burden (14). We administered 100 μL 40 mg/mL D-potassium luciferin in PBS (Gold Biotechnology) i.p. and measured leukemia burden 10 minutes later. After confirmation of engraftment (signal intensity ∼1.0 × 105 p/s/cm2/sr), mice were randomized into 3 treatment groups (N = 5/group): (i) ASNase WT (ASNaseWT), (ii) ASNaseQ59L, and (iii) PBS as a negative control. Treatments were administered intraperitoneally at 20,000 U/kg/day in 100 μL PBS for 2 weeks. Leukemia burden was assessed weekly using bioluminescent imaging (∼9 weeks). Survival was monitored until termination of the study at day 234.

ASNase pharmacokinetics/pharmacodynamics mouse study

Asparagine, aspartic acid, glutamine, and glutamic acid in mouse whole blood were measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS). After administering 20,000 U/kg of either ASNaseWT or ASNaseQ59L (or a matched volume of PBS lacking ASNase) to healthy NSG mice by i.p. injection, we serially sampled whole blood from individual mice to permit longitudinal analysis. Blood samples were collected immediately prior to treatment (t = 0) and at 1, 2, 4, 12, and 23 hours after treatment. Whole blood was centrifuged at 3,000 × g for 3 minutes to separate plasma and red blood cells, and a 2 μL aliquot of the plasma layer was immediately quenched with formic acid, combined with stable isotope-labeled internal standards, and analyzed by LC-MS/MS. ASNase enzymatic activity in mouse whole blood were determined by a colorimetric activity assay described previously (4). Paired t test analysis was performed on all pharmacokinetics/pharmacodynamics (PK/PD) data (Supplementary Table S1).

We performed a series of pilot experiments to optimize the dosage of ASNaseWT and ASNaseQ59L for PK/PD studies and in vivo anticancer activity against a preclinical model of ASNS-negative leukemia using the luciferase-engineered Sup-B15 cell line, which we previously showed to lack detectable ASNS protein expression before and after treatment with ASNase (5). We found that 14 daily treatments of ASNaseWT and ASNaseQ59L at 5,000 U/kg administered i.p. failed to suppress leukemia progression (Supplementary Fig. S1A and S1B). At the dosage of 10,000 U/kg, ASNaseWT was capable of inhibiting leukemia cell growth while ASNaseQ59L was still unable to stop leukemia progression (Supplementary Fig. S1A and S1B). Mice tolerated the treatment well; we did not observe more than 10% weight loss (Supplementary Figs. S1C), but one mouse in the ASNaseWT group died on day 28 after the first treatment (Supplementary Figs. S1A). The corresponding PD study indicated that daily administration of 20,000 U/kg of ASNaseQ59L was able to deplete plasma asparagine, whereas the dosage of 10,000 U/kg was not (Supplementary Fig. S2). Those studies led to the selection of 20,000 U asparaginase activity per kg body weight (U/kg) as the dosage for further experiments.

Pharmacodynamics and pharmacokinetics of ASNase variants in healthy NSG mice

We then compared the pharmacodynamics of ASNaseWT and ASNaseQ59L in healthy NSG mice. At a dose of 20,000 U/kg, both ASNaseWT and ASNaseQ59L depleted asparagine very quickly and maintained that depletion (P < 0.05 compared with PBS treatment; Supplementary Table S1) after 2 hours and for at least 12 hours after treatment (Fig. 1A; Supplementary Fig. S3A). Even at 23 hours after treatment, plasma asparagine concentration was below the limit of quantitation (< 1 μmol/L) in mice treated with ASNaseWT and very low (∼5 μmol/L) in those treated with ASNaseQ59L. The greater extent of asparagine depletion by ASNaseWT was presumably due to the additional depletion of glutamine, which is a substrate needed for the synthesis of asparagine by ASNS in cells throughout the body. As expected, ASNaseWT and ASNaseQ59L treatment led to elevation of plasma aspartate concentration (from ∼10 to ∼25 μmol/L), presumably due to conversion of asparagine to aspartate (Fig. 1B; Supplementary Fig. S3B). We did not observe a significant difference between asparagine and aspartate profiles following ASNaseWT treatment and ASNaseQ59L treatment (P > 0.05, Supplementary Table S1).

Figure 1.

Pharmacokinetics and pharmacodynamics of ASNaseWT and ASNaseQ59Lin vivo. The pharmacodynamics of ASNaseWT and ASNaseQ59L in the plasma of non-tumor–bearing NSG mice were determined by LC-MS/MS-based analysis of the amino acids asparagine (A), aspartate (B), glutamine (C), and glutamate (D). NSG mice (3 per group) were treated with PBS (negative control), 20,000 U/kg ASNaseWT, or 20,000 U/kg ASNaseQ59L by intraperitoneal injection. Time 0 measurements were made on samples collected immediately before injection. E, The pharmacokinetics of ASNaseWT and ASNaseQ59L in plasma were determined by colorimetric assay of asparaginase activity. Error bars represent standard error of the mean.

Figure 1.

Pharmacokinetics and pharmacodynamics of ASNaseWT and ASNaseQ59Lin vivo. The pharmacodynamics of ASNaseWT and ASNaseQ59L in the plasma of non-tumor–bearing NSG mice were determined by LC-MS/MS-based analysis of the amino acids asparagine (A), aspartate (B), glutamine (C), and glutamate (D). NSG mice (3 per group) were treated with PBS (negative control), 20,000 U/kg ASNaseWT, or 20,000 U/kg ASNaseQ59L by intraperitoneal injection. Time 0 measurements were made on samples collected immediately before injection. E, The pharmacokinetics of ASNaseWT and ASNaseQ59L in plasma were determined by colorimetric assay of asparaginase activity. Error bars represent standard error of the mean.

Close modal

As also expected, ASNaseQ59L treatment did not decrease plasma glutamine concentration compared with PBS treatment (P > 0.2; Supplementary Table S1; Fig. 1C; Supplementary Figs. S3C). ASNaseWT, in contrast, decreased glutamine concentration significantly from ∼600 μmol/L (pretreatment) to ∼200 μmol/L at 2 hours after treatment (P < 0.05, Supplementary Table S1; Fig. 3C; Supplementary Fig. S3C), but that effect was short-lived; glutamine rapidly returned to baseline (or perhaps slightly elevated) concentration. The slightly increased glutamine level following ASNaseQ59L treatment was not statistically significant (P > 0.05; Supplementary Table S1).

Glutamate accumulated (ASNaseWT vs. PBS, P < 0.05; Supplementary Table S1) commensurately with the decrease of glutamine within the first 4 hours but regressed to baseline concentration within 8 hours (Fig. 1D; Supplementary Fig. S3D). Notably, no glutamate accumulation was detected following ASNaseQ59L treatment (ASNaseQ59L vs. PBS, P > 0.1; Supplementary Table S1), illustrating the absence of compensatory reaction and confirming its glutaminase deficiency.

Next, to analyze ASNase pharmacokinetics, we measured asparaginase activity in the same plasma samples as were collected for the analysis of ASNase pharmacodynamics. The pharmacokinetics of ASNaseWT and ASNaseQ59L were not statistically different (ASNaseWT vs. ASNaseQ59L, P > 0.05; Supplementary Table S1), but the maximal enzymatic activity (Cmax) of ASNaseWT was slightly lower than that of ASNaseQ59L; the Cmax of ASNaseWT and ASNaseQ59L occurred at 2 hours after administration and remained at 25% of maximum at 12 hours (Fig. 1E). Notably, the ASNaseWT pharmacokinetic profile (Fig. 1E) matched the glutamate pharmacodynamic profile (Fig. 1D) and inversely matched that of glutamine (Fig. 1C); rapid clearance of ASNase enzyme activity was associated with a rapid return of plasma glutamine concentration to baseline level. In contrast, the ASNaseQ59L pharmacokinetic profile matched the asparagine depletion profile (Fig. 1A). Overall, the pharmacokinetic and pharmacodynamic results suggested that administration of 20,000 U/kg i.p. daily should provide adequate conditions for the comparison of ASNaseWT and ASNaseQ59L anticancer activity in vivo.

Asparaginase activity alone does not produce cytotoxic anticancer activity in vivo

We next tested the in vivo anticancer activity of ASNaseWT and ASNaseQ59L against the Sup-B15 leukemia model (4). Following 14 daily treatments at 20,000 U/kg administered i.p., ASNaseWT and ASNaseQ59L suppressed leukemia progression, whereas the PBS-treated group exhibited rapid progression (Fig. 2A and B; Supplementary Fig. S4). We continued to monitor leukemia burden and survival rate after the cessation of treatment. ASNaseQ59L provided a growth delay of about 20 days (approximately 3 to 4 doubling times of the leukemia in mice) over the control group, whereas ASNaseWT yielded undetectable (background signal levels of) leukemia burden through the 66 days of imaging assessment (Fig. 2A and B; Supplementary Figs. S4). Strikingly, ASNaseWT extended survival to >100 days, with 2 of the mice showing no regrowth of this hard-to-cure leukemia through the time of sacrifice on day 234. A third mouse responded well but showed a small region of bioluminescence in the late stages of imaging, suggesting recurrence of the leukemia, and it survived until day 101 (Fig. 2C). ASNaseWT and ASNaseQ59L were both toxic as reflected by the loss of body weight following drug treatment (Fig. 2D; Supplementary Fig. S5). That side effect was more severe in the ASNaseWT group (up to ∼20% loss) than in the ASNaseQ59L group (up to ∼10% loss). Overall, the data in Fig. 2 indicate that ASNaseQ59L exhibited less toxic, but also less anticancer, activity than did ASNaseWT, which achieved results that approximate cure. It was unclear whether the early deaths of 2 mice from the ASNaseWT-treated group were caused by drug toxicity, but one of the 2 showed the largest early weight loss.

Figure 2.

Anticancer activity of ASNaseWT and ASNaseQ59Lin 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 ASNaseWT, or 20,000 U/kg ASNaseQ59L for 2 weeks by intraperitoneal injection. Leukemia burden was monitored using bioluminescence imaging at the indicated time points. Day 0 is defined as 1 day before the first treatment. B, Average bioluminescent signal in each treatment group as described for A. C, Kaplan–Meier survival analysis of the mice in A. D, The average of daily body weight loss of each group in A. Mice had unrestricted access to food and water. Two mice died early, but it was not clear whether they died from ASNaseWT-related toxicity. Mean and SEM are shown.

Figure 2.

Anticancer activity of ASNaseWT and ASNaseQ59Lin 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 ASNaseWT, or 20,000 U/kg ASNaseQ59L for 2 weeks by intraperitoneal injection. Leukemia burden was monitored using bioluminescence imaging at the indicated time points. Day 0 is defined as 1 day before the first treatment. B, Average bioluminescent signal in each treatment group as described for A. C, Kaplan–Meier survival analysis of the mice in A. D, The average of daily body weight loss of each group in A. Mice had unrestricted access to food and water. Two mice died early, but it was not clear whether they died from ASNaseWT-related toxicity. Mean and SEM are shown.

Close modal

We previously found that asparaginase activity played a role in the anticancer activity of ASNase in vitro, but glutaminase activity was dominant, especially in the case of ASNS-positive cell types (4). In the present study, we asked similar questions in vivo, intentionally biasing the case in favor of asparaginase activity by using xenografts of the ASNS-negative Sup-B15 line in NSG mice. In that model system, ASNaseQ59L exerted anticancer activity, but comparison with ASNaseWT showed that glutaminase activity was again dominant; ASNaseWT elicited a durable response (consistent with a cytotoxic mode of action) whereas ASNaseQ59L achieved only a fraction of the effect produced by ASNaseWT.

We previously found that ASNS expression (pretreatment and posttreatment) was a key mediator of resistance to ASNaseQ59Lin vitro (4). It was, therefore, unexpected in this study to find that ASNaseQ59L lacked significant anticancer activity in vivo. That is, depletion of asparagine alone in vivo elicited growth inhibition but not a durable response. One hypothesis to explain the discrepancy between in vitro and in vivo results is that the leukemia cell microenvironment consists of ASNS-positive cell types capable of synthesizing and transporting asparagine to the extracellular environment for consumption by leukemia cells (15, 16), thereby fueling resistance to ASNaseQ59L. In contrast, the in vitro cell culture models lack such additional sources of asparagine. Our results suggest that better in vitro models and/or a focus on in vivo data are critical for assessing the ability of glutaminase-deficient ASNase variants to overpower the supportive microenvironment.

Although our data support the conclusion that glutaminase activity is necessary for ASNase's in vivo anticancer activity, significant side effects, including pancreatitis, thrombosis, immunosuppression, and impaired functions of liver, kidney, or central nervous system, have been attributed to its glutaminase activity. Indeed, we observed greater weight loss in the ASNaseWT-treated group compared with the ASNaseQ59L-treated group during the 2-week treatment. In addition, 2 out of 5 mice in the ASNaseWT-treated group died, but none died in the ASNaseQ59L-treated group during the first 3 weeks. Aside from weight loss, we did not observe any obvious differences in the physical appearance or behavior of mice between 2 treatment groups, prompting a need for further studies to identify the causes of early death in the ASNaseWT-treated group. Additionally, there is a need to probe the metabolic pathways that are modulated downstream of the short-term glutamine decrease achieved by ASNaseWTin vivo. To overcome toxicities associated with ASNase glutaminase activity, combinations of ASNaseWT and glutaminase-deficient mutants such as ASNaseQ59L may be customized to achieve durable anticancer activity with minimal toxicity. In addition, monitoring glutamine concentration may be as important as monitoring asparagine concentration in patients during the course of ASNase therapy.

The new results presented here have potentially significant implications. First, attempts to achieve better clinical responses by increasing ASNase treatment intensity (and, therefore, glutaminase activity) are often overruled by evidence of increased toxicity (2, 17), but the new results here are consistent with arguments in favor of increasing the glutaminase dosage for both ASNS-negative and ASNS-positive leukemia cell types. The single-agent ASNaseWT treatment regimen used in the current study thus achieved results similar to those reported with optimized vincristine, deXamethasone, L-asparaginase (VXL) combination therapy in a patient-derived xenograft model of acute lymphoblastic leukemia, whereby weekly administration of vincristine (0.15 mg/kg) together with weekday treatment with dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) for 4 weeks yielded 34-week survival of 1 of 5 mice engrafted with the chemosensitive T-ALL xenograft ALL-16 (18). On that note, given the single-agent activity observed here for ASNaseQ59L, further in vivo studies to assess Q59L's potential in the VXL regimen are warranted.

Another recently published report indicated that a low-glutaminase mutant of Erwinia chrysanthemi ASNase (a different backbone than the E. coli ASNase used in the present studies) retained anticancer activity in vivo (6). Specifically, with 14-day and 29-day study designs, a low-glutaminase ASNase mutant yielded anticancer activity in the short term. The new results presented here using a glutaminase-deficient variant of ASNase with even lower relative glutaminase activity (<0.2% of WT) provide critical additional information. In our extended survival analysis, glutaminase-deficient ASNaseQ59L was insufficiently active to prevent recurrence of the leukemia. In contrast, ASNaseWT at the same dose in terms of asparaginase activity yielded a durable response, with 2 of the mice surviving until sacrifice at day 234. Overall, the results presented here indicate that ASNase glutaminase activity is a key component, although not the only component, of the mechanism of action of ASNase.

J.N. Weinstein has ownership interest (including stock, patents, etc.) in NIH. P.L. Lorenzi is a consultant/advisory board member for Erytech Pharma. No potential conflicts of interest were disclosed by the other authors.

Conception and design: W.-K. Chan, T.D. Horvath, S.B. Rempe, S. Sukharev, J.N. Weinstein, P.L. Lorenzi

Development of methodology: W.-K. Chan, T.D. Horvath, J.N. Weinstein, P.L. Lorenzi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W.-K. Chan, T.D. Horvath, L. Tan, T. Link, M.A. Pontikos, L.A. Martin, E. Yin, M. Konopleva, J.N. Weinstein, P.L. Lorenzi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W.-K. Chan, T.D. Horvath, A. Anishkin, D. Du, M. Konopleva, J.N. Weinstein, P.L. Lorenzi

Writing, review, and/or revision of the manuscript: W.-K. Chan, T.D. Horvath, M. Konopleva, J.N. Weinstein, P.L. Lorenzi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W.-K. Chan, M.A. Pontikos, J.N. Weinstein

Study supervision: W.-K. Chan, J.N. Weinstein, P.L. Lorenzi

This work was supported in part by NCI grant numbers CA143883 (J.N. Weinstein), CA083639 (J.N. Weinstein), CA235510 (J.N. Weinstein), and CA016672 (J.N. Weinstein and P.L. Lorenzi); Cancer Prevention and Research Institute of Texas grant number RP130397 (J.N. Weinstein and P.L. Lorenzi); NIH high-end instrument grant 1S10OD012304-01 (P.L. Lorenzi), the Chapman Foundation, and the Michael and Susan Dell Foundation (honoring Lorraine Dell). SNL is a multimission laboratory managed and operated by NTESS for the US DOE, NNSA, under contract DE-NA-0003525 (S.B. Rempe). Sup-B15-luc cells were a kind gift from Michael Jensen (Seattle Children's Research Hospital).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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