L-Asparaginase Strongly Affects Bioenergetics in Leukemic Cells

Abstract 779

L-asparaginase (L-asp) is an important component of childhood acute lymphoblastic leukemia (ALL) therapy. Its cytotoxic effect is based on the depletion of extracellular asparagine and glutamine. Leukemic cells are sensitive to this depletion due to the lower activity of asparagine synthetase compared to healthy cells. However, the mechanism of resistance development remains unclear. The aim of this study was to obtain further insights into the mechanisms underlying the cytotoxic effect of L-asp.

We used two models: resistant preB ALL leukemic cells derived from the REH (TEL/AML1[+]; very sensitive) and NALM6 (TEL/PDGFRB[+]; medium sensitive) cell lines by long-term incubation with L-asp. As a second model we used REH and NALM6 incubated with L-asp for 24 hrs, mimicking acute phase of treatment.

We performed GEP of REH, res-REH, NALM6 and res-NALM6. There was an overlap of 30 genes changed in both cases. Applying the pathway analysis we found that L-asp influences the regulation of lipid metabolism and apoptosis regulated by mitochondrial proteins. Next we merged our data with GEP data published by Holleman et al. (NEJM, 2004) of ALL patients′ samples sensitive/resistant to L-Asp. Two pathways, one regulating protein translation, the other metabolism, scored very highly. Next to glucose, glutamine is the other major cellular energy source, it is also important for activation of PI3K/Akt/mTOR pathway - the key regulator of translation. Since L-asp also depletes glutamine, we focused on the effect of L-asp treatment on bioenergetics and translation in leukemic cells. To confirm the effect of L-asp on PI3K/Akt/mTOR pathway we measured the amount of Akt protein and P70S6K/p-P70S6K (downstream target of mTOR) in REH and NALM6 treated for 24 hours with L-asp. Akt and p-P70S6K expression fell in both cell lines. Next we were interested if PI3K/mTOR inhibition affects the sensitivity of resistant cell lines. We measured the cytostatic effect of co-treatment: L-asp with mTORC1 inhibitor (rapamycin); with dual inhibitor of mTORC1 and PI3K (LY294002); and with specific inhibitor of PI3K (PX866) on REH, res-REH, NALM6 and res-NALM6. The MTS assay proved the L-asp/rapamycin combination the most effective. It strongly diminished viability of all cell lines, even in those resistant to L-asp.

c-Myc as an activator of glutamine catabolism showed a significant decrease in REH and NALM6 after incubation with L-asp. In addition, c-Myc was also decreased in res-REH compared with REH. Importantly, c-Myc activates glycolysis which is a main energy generator in cancer cells rather than oxidative phosphorylation (OXPHOS). We showed diminishement of Glucose transporter type 1 protein expression and 2.5-times reduced level of lactate, the product of cancer cell glycolysis, in media after L-asp exposure. Next we measured cell respiration that mirrors oxidative fosforylation (OXPHOS). REH and NALM6 showed increased capacity of respiratory chain after L-asp exposure, which was observed as an increase of endogenous respiration in uncoupled state. REH boosted the capacity of the respiratory chain from 163 to 236 pmol O₂/s/mg (p<0.01). NALM6 have increased the capacity of the respiratory chain from 78 to 155 pmol O₂/s/mg (p<0.01). Interestingly, REH has a significantly higher capacity of respiratory chain compared with NALM6 and a lower ability to regulate it. Moreover, res-REH had elevated the level of the endogenous oxygen consumption whereas res-NALM6 levels did not change. Our results indicate that leukemic cells, which normally depend on glycolysis for energy generation rather than OXPHOS, are able to swap these two mechanisms of energy generation under amino acid deprivation stress.

The data show that L-asp inhibits mTORC1 and strongly affects bioenergetics pathways of leukemic cells. It decreased c-Myc-regulated glycolysis and increased OXPHOS. We believe that these metabolic changes are mainly caused by the effort of the cells to mobilize another mitochondrial pathway as Krebs cycle and β-oxidation, with the aim to supply depleted amino acids. We conclude from these data that resistance of the cells is caused by better biochemical adaptability to the nutrient deprived environment.

Support: GAUK 92710, IGA NT1249, UNCE204012