P334: ultimate clarification of the relationship between asparagine synthetase activity and sensitivity of leukemia to l-asparaginase

Background: L-asparaginase (ASNase) is one of the crucial drugs used in treatment of childhood acute lymphoblastic leukemia (ALL). Leukemia cells are sensitive to ASNase because they have lower asparagine synthetase (ASNS) level compared to healthy cells and other tumors. However, correlations of ASNS gene expression or enzyme production with sensitivity of leukemia to ASNase are inconsistent. Notably, ASNase used in vitro depletes all asparagine (Asn) and glutamine (Gln) whereas it is probably only Asn that is completely depleted in vivo after ASNase administration in ALL treatment with unchanged or lowered Gln concentration. Aims: The main aim is to evaluate the role of ASNS enzymatic activity, rather than protein level, in predicting sensitivity of ALL cells to ASNase. To do that, ASNS enzymatic activity in ALL cells was measured together with sensitivity of these cells both to ASNase and Asn-depletion only. Methods: We employed stable isotope tracing to study the activity of ASNS under different nutrient conditions. Sensitivity of ALL cells to ASNase, Asn-depletion and chloroquine (CQ) was measured using MTS assay. Protein levels were detected using western blot (WB). Results: We measured the activity of ASNS together with ASNS protein levels in four B-ALL and five T-ALL cell lines. Six of them had ASNS detectable on WB (ASNS-plus). Surprisingly, none of the tested cell line synthesized Asn when it was present in the culture media, even in case when ASNS was clearly detectable on WB. On the other hand, when Asn was limited in the media, ASNS-plus cells were able to synthesize Asn whereas ASNS-null cells were not. We found out that all tested ASNS-plus cells used exclusively aspartate (Asp) synthesized from Gln via TCA cycle. Then, ASNS catalyzed the transfer of an amide group to Asp from the second Gln. Noteworthy, all of the tested ASNS-plus cell lines were able to import Asp in a dose-dependent manner. However, they did not use this imported Asp to synthesize Asn. These results indicate that ASNS prefers Asp produced via TCA cycle and that substrate channeling could play a role in ASNS activity. Then, we measured the sensitivity of all studied cell lines to ASNase and Asn-depletion. In B-ALL, all ASNS-plus cell lines (NALM6, REH and UOC-B6) were less sensitive to Asn-depletion compared to ASNS-null cell line RS4;11. Furthermore, in T-ALL, ASNS-plus cell lines (JURKAT, MOLT4 and CCRF-CEM) together with one ASNS-null cell line (ALL-SIL) were less sensitive to Asn-depletion compared to the second ASNS-null cell line (DND-41). Since autophagy could provide Asn when it is limited in the media, we looked into autophagy activity. Interestingly, ALL-SIL cells were more sensitive to autophagy inhibitor CQ and also had higher autophagic flux compared to DND-41 cells. In accordance with that, we detected higher mTOR activity in DND-41 than in ALL-SIL cells. Regarding sensitivity to ASNase in both B-ALL and T-ALL, ASNS protein positivity only correlated with sensitivity to ASNase in mimicked in vivo conditions (Asn-depletion). Summary/Conclusion: Altogether, our study for the first time determined ASNS activity in leukemia cells under different nutrient availability. Importantly, ASNS activity correlates with ASNS protein levels in Asn-deplete conditions. In general, ASNS-plus cells are less sensitive to Asn-depletion than ASNS-null cells. However, the second mentioned could overcome ASNase treatment by high autophagy state and therefore bias the correlation between ASNS protein level and sensitivity to ASNase. This work was supported by NU20J-03-00032 and NU22-07-00087. Keywords: Resistance, L-asparaginase, Acute lymphoblastic leukemia