L-Asparaginase Causes Metabolic Reprogramming in ALL Cells

Acute lymphoblastic leukemia (ALL) is the most frequent type of childhood cancer. The key component in the therapy, L-asparaginase (ASNase), hydrolyzes plasma asparagine and glutamine. Leukemic cells are sensitive to the depletion due to low activity of asparagine synthetase. Although the treatment is very effective, resistance and side effects remain a serious problem in some cases and its mechanism of action is not well understood. Our aim is to clarify the intracellular consequences of the amino acid depletion to define the reason of different patients´ response.

We have generated ASNase-resistant subclones through chronic exposure to the enzyme. Pathway analysis of gene expression profiles of the cell lines (REH;TEL/AML1-positive, NALM-6; TEL/PDGFRB1-positive and their resistant counterparts) and primary samples (sensitive and resistant to ASNase; Holleman et al. (NEJM, 2004)) revealed that ASNase affects the translation machinery and metabolism of leukemic cells. The key nutrient sensor positively regulating protein synthesis, pyrimidine synthesis, glycolysis and lipid synthesis is mTORC1. Since ASNase depletes glutamine that is essential for mTORC1 activity, we hypothesized that the effect of ASNase is driven through mTORC1 signaling. Main aim of the study was to explore the effect of ASNase on downstream mTORC1 targets in leukemic cells.

ASNase treatment inhibited protein synthesis, displayed by dephosphorylation of p-P70SK6 and p-S6. ASNase also decreased de-novo DNA synthesis as shown by dephosphorylation of p-CAD. This result was confirmed by analysis of de-novo pyrimidine synthesis intermediates, uridine monophosphate and uridine, measured by UPLC-ToF-MS. Both were decreased upon ASNase treatment. Except experiments done on ALL cell lines we also detected dephosphorylation of p-S6, p-CAD in primary samples.

Regarding the effect on glycolysis we observed inhibition of glucose uptake and decrease of lactate production in cells treated with ASNase. We also detected the decrease of protein levels of c-Myc, the activator of glucose and glutamine catabolism, and glucose transporter 1 (Glut-1). On the contrary, ASNase increased fatty acid oxidation (FAO) followed by the elevation of the capacity of cell respiration and NAD+/NADH ratio in both cell lines.

Next, we wanted to elucidate whether ASNase inhibits mTORC1 targets through RagB, the key protein mediating response to general amino acid deprivation. We established a RagB mutant leukemic cell line with constitutive activation of mTORC1 pathway despite deprivation of amino acids. ASNase treatment did not inhibit p-S6 and p-CAD in this cell line. Similarly, ASNase failed to increase FAO in cells with active mTORC1. These results suggest that the effect of ASNase on protein translation, de novo pyrimidine synthesis and FAO is mediated through RagB-mTORC1 pathway. By contrast, c-Myc expression was decreased in both RagB wild type and mutant cells, indicating that ASNase inhibits glycolysis in RagB-mTORC1 independent manner.

The activation of FAO has been suggested to have a pro-survival function in leukemic cells under nutrient stress conditions. We tested whether the increase of FAO in ALL cells treated with ASNase also serves to cope with the metabolic stress. Pharmacological inhibition of FAO significantly increased the sensitivity of ALL cells to ASNase. Moreover, cells with the inability to increase FAO (RagB mutant) were more sensitive to ASNase compared to RagB wild type cells.

Our results show that the inhibitory effect of ASNase on mTORC1 leads not only to apoptosis but also to metabolic reprogramming. We propose that inhibition of protein translation and pyrimidine synthesis are part of apoptotic processes whereas increased FAO and cell respiration represent pro-survival pathway. Altogether, our study suggests that targeting of FAO in leukemic cells resistant to ASNase is a promising new therapeutic strategy.

IGA-NT1249, GAUK-632513