The glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

Abstract

• We used molecular dynamics, saturation mutagenesis, and enzymologic screening to develop a glutaminase-free mutant (Q59L) L-ASP.• We then used Q59L to show that glutaminase activity is not required for L-ASP activity against ASNS-negative cancer cells.L-Asparaginase (L-ASP) is a key component of therapy for acute lymphoblastic leukemia. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. Here, we show that L-ASP's glutaminase activity is not always required for the enzyme's anticancer effect. We first used molecular dynamics simulations of the clinically standard Escherichia coli L-ASP to predict what mutated forms could be engineered to retain activity against asparagine but not glutamine. Dynamic mapping of enzyme substrate contacts identified Q59 as a promising mutagenesis target for that purpose. Saturation mutagenesis followed by enzymatic screening identified Q59L as a variant that retains asparaginase activity but shows undetectable glutaminase activity. Unlike wild-type L-ASP, Q59L is inactive against cancer cells that express measurable asparagine synthetase (ASNS). Q59L is potently active, however, against ASNS-negative cells. Those observations indicate that the glutaminase activity of L-ASP is necessary for anticancer activity against ASNS-positive cell types but not ASNS-negative cell types.Because the clinical toxicity of L-ASP is thought to stem from its glutaminase activity, these findings suggest the hypothesis that glutaminase-negative variants of L-ASP would provide larger therapeutic indices than wild-type L-ASP for ASNS-negative cancers. (Blood. 2014;123(23):3596-3606) Introduction L-Asparaginase (L-ASP) is an enzyme drug used in combination with vincristine and a glucocorticoid (eg, dexamethasone) to treat acute lymphoblastic leukemia (ALL).1,2 We 3-6 and others 7 have reported a rationale for testing L-ASP against low-asparagine synthetase (ASNS) solid tumors as well. L-ASP's primary known enzymatic activity is deamidation of asparagine to aspartic acid and ammonia, but it also deamidates glutamine to glutamic acid and ammonia, although with lower affinity and lower maximal rate. L-ASP therapy is often limited by toxic side effects that are generally attributed to the glutaminase activity. 8,9 Those side effects often preclude completion of the full treatment regimen, resulting in poor outcome. 10 The question that arises, however, is whether the therapeutic index of L-ASP could be increased by decreasing its glutaminase activity 8,9 or whether that would also decrease the anticancer effect commensurately.One side of the debate hypothesizes that L-ASP's therapeutic index can be improved by increasing glutaminase activity. In support of that hypothesis, data collected over the last decade have suggested that glutaminase activity generally increases the efficacy of L-ASP and is sometimes required to achieve an anticancer effect. Those studies have reported asparaginase activity to be expendable. [11][12][13][...

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity

“…This choice of mutation was reinforced by the fact that the counterpart of Glu-63 in the low L-glutaminase H. pylori and E. coli L-asparaginases is indeed a glutamine. In addition to the E63Q mutant we also created the E63L mutation based on the recent study of the E. coli enzyme conducted by Chan et al (17) reporting Q59L (analogues position to Glu-63 in ErA) as a highly L-asparaginase-specific variant.…”

Effect of Premedications in a Murine Model of Asparaginase Hypersensitivity

“…The mechanism of action of asparaginase is not completely understood, but the active enzyme depletes asparagine and possibly glutamine systemically (Wu et al, 1978;Asselin et al, 1989;Chan et al, 2014). Common adverse events to asparaginase include allergic reactions, often accompanied by the development of antiasparaginase IgG antibodies (Pieters et al, 2011).…”

Is glutamine depletion needed in ALL disease?

“…The Q59L variant ASNase has undetectable glutaminase coactivity, while retaining its ASNase activity. 1 …”

“…Chan et al have clearly demonstrated this biochemical effect. 1 The authors next explored the antileukemia activity of this Q59L protein, demonstrating it to be "potentially active" against Asn synthetase (ASNS)-negative cells, but inactive against cells expressing measurable ASNS. The authors further propose that in ASNSnegative ALL cells, the Q59L variant ASNase protein may be less toxic to patients, while providing a large therapeutic index.…”