Separate Roles of Asparagine and Glutamine in Cytostatic Effect of L-Asparaginase - Stable Isotope Tracing Approach
Authors: Hlozkova Katerina , Thakker Alpesh , Alquézar-Artieda Natividad , Zaliova Marketa , Trka Jan , Tennant Daniel A , Starkova Julia 
Published: November 2019

L-asparaginase (ASNase) is one of the crucial components of acute lymphoblastic leukemia (ALL) therapy. Although we have previously shown that ASNase triggers metabolic reprogramming of leukemic cells, the significance and interconnections of the changes have not yet been elucidated. Metabolic reprogramming is an accompanying feature in therapy response and is also triggered by commonly used cytostatic drugs. ASNase hydrolyzes two non-essential amino acids asparagine (Asn) and glutamine (Gln). Therapeutic ASNase concentration used in vitro transforms all Asn and Gln to aspartate (Asp) and glutamate (Glu), respectively.
We employed stable isotope tracing to understand the complexity and interconnection of metabolic processes altered in leukemia cells exposed to ASNase. Stable isotope tracing is a unique method enabling the quantification of metabolic flux. When substrate with 13C is metabolized by cells, enzymatic reactions rearrange 13C atoms resulting in specific labeling patterns in downstream metabolites. Therefore, this method allows us to determine exact pathways which are used to metabolize labeled nutrient. Glucose and Gln media concentrations were adjusted to 8mM and 1mM, respectively, to more resemble physiological conditions. All experiments were done with 0.8 IU/ml ASNase which is close to the plasma ASNase level in the induction phase of ALL treatment.
We studied the effect of ASNase on two B-ALL cell lines with different sensitivity to this cytostatic drug: REH (IC50=0.000231 IU/ml) and NALM6 (IC50=0.328894 IU/ml). U13C glucose tracing revealed that although pyruvate and lactate intracellular concentrations are not changed after ASNase treatment, the relative flux from glucose to these metabolites (expressed as M+3 which represents 3 13C atoms in 1 molecule) is lowered (e.g. lactate (M+3): NALM6 - (0.5898±0.0026) vs (0.4473±0.0031), p<0.0001); REH - (0.3417±0.0023) vs (0.2411±0.0024), p<0.0001). Unlike glycolytic intermediates, the levels of tricarboxylic acid (TCA) cycle intermediates are lowered after ASNase administration (e.g. relative quantification of malate: NALM6 - (1.6120±0.1202) vs (0.4358±0.0247), p<0.0001; REH - (0.8231±0.0689) vs (0.2484±0.0573), p<0.0001). Interestingly, the relative flux from glucose to TCA cycle (expressed as e.g. malate (M+1-M+4) which represents at least 1 13C atom in a malate molecule) is increased in NALM6 ((0.0947±0.0021) vs (0.3605±0.0070), p<0.0001) and lowered in REH cells ((0.3109±0.0100) vs (0.1449±0.0185), p<0.0001) after ASNase.
Since in our setting ASNase depletes both Asn and Gln, we tested what are the separate roles of Asn and Gln depletion in ASNase treatment. We discovered that omitting Gln from the media caused that TCA cycle intermediates are lowered in both cell lines to the same extent as after ASNase. Moreover, the flux from glucose to TCA cycle resembles the one after ASNase in both cell lines. In contrast the results of Asn withdrawal showed that level of TCA cycle intermediates are unchanged. The relative flux from glucose to TCA cycle is mildly downregulated without Asn in the media in both cell lines (e.g. malate (M+1-M+4): NALM6 - (0.1485±0.0014) vs (0.1158±0.0004), p<0.0001; REH - (0.2662±0.0031) vs (0.2024±0.0015), p<0.0001).
Many studies emerged that highlight TCA cycle importance in Asp production and hence in sustaining cell viability and proliferation. Although TCA cycle is diminished after ASNase treatment, our results showed that both NALM6 and REH cells are able to maintain Asp and Glu levels. Using U13C Asp and U13C Glu, we discovered that, unexpectedly, both cell lines are able to import Asp and Glu from the media in a dose-dependent manner.
Altogether, our results demonstrate different consequences of Asn and Gln depletion during ASNase treatment. According previous studies it is probably only Asn that is completely depleted after ASNase administration in ALL therapy with unchanged or lowered Gln concentration. Therefore, this fact should be considered in in vitro studies where Gln depletion could blur the effect of Asn depletion. Above that, we showed that leukemia cells are able to uptake Asp and Glu. This indicates the possible way how leukemic cells could cope with ASNase treatment.
This work is supported by NV18-07-00129 and Charles University Grant agency 79421.

Disclosures
No relevant conflicts of interest to declare.