L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine and glutamine. To evaluate the importance of glutaminase activity, we exploited a recently developed mutant of Helicobacter pylori ASNase (dm HpA), with amino acid substitutions M121C/T169M. The mutant form has the same asparaginase activity as wild-type but lacks glutaminase activity. Wild-type and dm HpA were compared with the clinically used ASNases from E. coli (L-ASP) and Erwinia chrysanthemi (ERWase). Asparaginase activity was similar for all isoforms, while glutaminase activity followed the rank order: ERWase > L-ASP > wild-type HpA > dm HpA. Cytotoxic efficacy of ASNases was tested on 11 human leukemia cell lines and two patient-derived ALL samples. Two cell lines which we had previously shown to be asparagine-dependent were equally sensitive to the asparaginase isoforms. The other nine lines and the two patient-derived samples were more sensitive to isoforms with higher glutaminase activities. ERWase was overall the most effective ASNase on all cell lines tested whereas dm HpA, having the lowest glutaminase activity, was the least effective. These data demonstrate that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full anti-leukemic efficacy.

L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine (Asn) and glutamine (Gln). We and others have shown that its cytotoxicity depends more on Gln than Asn depletion. Novel ASNases with different activities have been developed in an attempt to decrease side effects and allergic reaction. To evaluate the importance of glutaminase activity, we developed a novel form of Helicobacter pylori ASNase (dm HpA) using site-directed mutagenesis, with amino acid substitutions M121C/T169M. This mutant form was designed to have the same asparaginase activity as wild type (wt), but lack glutaminase activity. WT and dm HpA were compared with the clinically used ASNases from E. coli (EcA-II) and Erwinia chrysanthemi (ErA). Asparaginase and glutaminase activities of each enzyme were measured with Nessler's assay at pH 8.6. The ratio of glutaminase/asparaginase activity was 1.4% for dm HPase, 7.5% for wt HPase, 41% for EcA-II, and 99% for ErA (at 0.6 IU), respectively. L-Asparaginases (0.01 to 3 IU/ml over 72 hrs) were tested on 8 human leukemia cell lines: pre B ALL (BV173, Nalm-6, RCH-ACV, RS4;11, SEM, SupB15), T cell ALL (Molt-4), and acute promyelocytic leukemia (HL-60). Viable cells were determined by trypan blue exclusion. Two cell lines which we had previously shown to be Asn-dependent (RS4;11 and SupB15) were equally sensitive to the asparaginase isoforms (>90% kill at 0.03 IU/mL). The other 6 lines were more sensitive to isoforms with higher glutaminase activities (Table). ErA was the most effective on all three cell lines on which it was tested. These data show that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full efficacy. These sensitivities to ASNases match previous data on Asn- and Gln-dependent cell proliferation (Ehsanipour et al., Cancer Research, 2013). % cell growth inhibition at 3 IU/ml ASNase (n=1-2 for each cell line; nd: not done)Cell linedm HpAwt HpAEcA-IIErARS4;11919291ndSupB15919193ndRCH-ACV13362866SEM22484165BV17324326ndNalm-61621574HL-60204033ndMolt4325963nd Citation Format: Jean-Hugues Parmentier, Maristella Maggi, Erika Tarasco, Claudia Scotti, Vassilios Avramis, Steven D. Mittelman. Glutaminase activity determines cytotoxicity of L-asparaginases on leukemia cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3378. doi:10.1158/1538-7445.AM2014-3378