Background: Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in the overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both antileukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs. Methods: Altogether, 19 leukemia cell lines, primary leukemia cells from 26 patients and 2 healthy controls were used. Glycolytic function and mitochondrial respiration were measured using Seahorse Bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence. Results: Using cell lines and primary patient samples we characterized the basal metabolic state of cells derived from different leukemia subtypes and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile. Lymphoid leukemia cell lines and patients sensitive to L-asparaginase clustered into the low glycolytic cluster. While lymphoid leukemia cells with lower sensitivity to L-asparaginase together with resistant normal mononuclear blood cells gathered into the high glycolytic cluster. Furthermore, we observed a correlation of specific metabolic parameters with the sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in the other cytostatic drugs tested by us.
Childhood T-cell acute lymphoblastic leukemia (T-ALL) still remains a therapeutic challenge due to relapses which are resistant to further treatment. l-asparaginase (ASNase) is a key therapy component in pediatric T-ALL and lower sensitivity of leukemia cells to this drug negatively influences overall treatment efficacy and outcome. PTEN protein deletion and/or activation of the PI3K/Akt signaling pathway leading to altered cell growth and metabolism are emerging as a common feature in T-ALL. We herein investigated the relationship amongst PTEN deletion, ASNase sensitivity and glucose metabolism in T-ALL cells. First, we found significant differences in the sensitivity to ASNase amongst T-ALL cell lines. While cell lines more sensitive to ASNase were PTEN wild type (WT) and had no detectable level of phosphorylated Akt (P-Akt), cell lines less sensitive to ASNase were PTEN-null with high P-Akt levels. Pharmacological inhibition of Akt in the PTEN-null cells rendered them more sensitive to ASNase and lowered their glycolytic function which then resembled PTEN WT cells. In primary T-ALL cells, although P-Akt level was not dependent exclusively on PTEN expression, their sensitivity to ASNase could also be increased by pharmacological inhibition of Akt. In summary, we highlight a promising therapeutic option for T-ALL patients with aberrant PTEN/PI3K/Akt signaling.
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.
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