L-Asparaginase (L-ASP) is a key component of acute lymphoblastic leukemia therapy. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. In the present study, we tested the hypothesis that L-ASP glutaminase activity is required for anticancer activity. We first used molecular dynamics simulations of the clinically used E. coli L-ASP enzyme to guide engineering of mutants that lack glutaminase activity. Dynamic mapping of enzyme-substrate contacts identified the backbone amine of residue Q59 as having frequent contact with glutamine but not asparagine substrate. That difference identified Q59 as a promising mutagenesis target for modifying substrate selectivity. Saturation mutagenesis and screening of the resulting Q59 mutants identified Q59L as retaining asparaginase activity yet exhibiting undetectable glutaminase activity. Using Q59L to test the glutaminase-anticancer hypothesis, we observed no anticancer activity by Q59L against cell lines that do express asparagine synthetase (ASNS), including six leukemia lines—CCRF-CEM, SR, MOLT-4, K562, NALM-6, and REH—and two ovarian cancer lines—OVCAR-8 and SK-OV-3. Wild-type (WT) L-ASP, on the other hand, effected a dose-response in all of those cell lines, suggesting that glutaminase activity is required to kill cancer cells that express ASNS. Unexpectedly, Q59L exhibited potent anticancer activity against cell lines that do not express detectable ASNS, including the leukemia cell lines Sup-B15 and RS4;11 and ASNS siRNA-treated OVCAR-8 cells. We conclude that the glutaminase activity of L-ASP is not necessary for anticancer activity against cell types that do not express ASNS. Since Q59L is expected to exhibit reduced toxicity relative to wild-type L-ASP because of its reduced glutaminase activity, these findings provide rationale for clinical assessment of Q59L L-ASP for the treatment of ASNS-deficient cancers.
Lorenzi:ERYtech Pharma: Consultancy, Membership on an entity’s Board of Directors or advisory committees, US 7985548, US 7985548 Patents & Royalties. Off Label Use: L-asparaginase is an enzyme-drug approved for treatment of acute lymphoblastic leukemia.
Asterisk with author names denotes non-ASH members.