The dependency of childhood acute lymphoblastic leukaemia (cALL) cells on exogenous asparagine has made L-Asparaginase (ASNase) one of the key therapeutics in this haematological malignancy. Besides the relatively frequent immune-mediated inactivation of the enzyme, a number of molecular mechanisms of resistance to ASNase have been described. The complex interplay between the activation of amino acid response, autophagy, MEK/ERK, mTORC1, Wnt signaling and other pathways, implicated in the response to asparagine (and glutamine) deamination and subsequent metabolic adaptation and survival, has raised interest not only in cALL, but in a number of other malignancies. Therefore, in order to tackle ASNase-resistant refractory or relapsed cALL, new therapeutic approaches addressing the bioenergetic plasticity of lymphoblasts need to be investigated. The green tea polyphenol flavonoid EGCG is a well-established inhibitor of glutamate dehydrogenase 1 and 2 (GLUD1/2), but also of IDH1 and other cell metabolism related targets making it an attractive pleiotropic therapeutic. In this study we aimed to characterize the response to ASNase and to EGCG and to investigate the potential of EGCG to concomitantly block potential rescue metabolic mechanisms in cALL cells and thus augment the anti-leukaemic effect of ASNase.

REH, Sup-B15, and SEM B-cell cALL cell lines were examined after treatment with L-asparaginase and the epigallocatechin gallate (EGCG). Metabolomics analysis of cells, MTT assays, glutamine/glutamate luminescent assay, Annexin V/PI apoptosis test were applied, as well as assessment of mitochondrial function and ATP production by Seahorse XFp Analyzer (Agilent).

We found that L-Asparaginase alters a number of amino acid biosynthesis pathways as well as purine and pyrimidine synthesis. These metabolic changes are accompanied by a significant reduction of mitochondrial function and ATP production. Furthermore, there is a distinct cellular response to the minimal clinically recommended concentrations of 0.1 IU/ml of ASNase, and a relatively wide range of IC50 values for the three cell lines for EGCG – from ~8, 18 and 35uM. Upon following the progression of cell proliferation and cell death on days 1, 3 and 7 after treatment, the three lines exhibited diverse dynamics spanning from apoptosis as early as on day 1 to delayed additive effects of the combination that were observed only beyond 72h.

We observed distinct metabolomic alteration patterns and different dynamics in the response to L-Asparaginase in all three cell lines. However, targeting cALL metabolism with EGCG holds potential to enhance the cytostatic and cytotoxic effects of this key chemotherapeutic. ACKNOWLEDGEMENTS: National University Complex for Biomedical and Applied Research with participation in BBMRI-ERIC, NUCBPI-BBMRI.BG contract no. DО1-371/15.12.2023