Restored Biosynthetic Pathways Induced By MSCs Serve As Rescue Mechanism in Leukemia Cells after L-Asparaginase Therapy

The cause of relapse in childhood acute lymphoblastic leukemia (ALL) is often associated with resistance to the standard chemotherapy treatment. The primary goal of this project was to elucidate the resistance mechanism of L-asparaginase (ASNase), one of the crucial drugs used in ALL therapy. The cytotoxic effect of ASNase relies on the depletion of extracellular asparagine (Asn) and glutamine, which is disastrous for leukemic cells since they have minimal activity of de novo synthesis of these amino acids that paradoxically have essential roles in leukemic cells' metabolism.

We previously showed that ASNase caused metabolic reprogramming by which leukemic cells escaped the cytostatic effect of the treatment. In the present study, we investigated the role of the main aspects of the in vivo environment on the resistance mechanism of leukemic cells (BCP-ALL cell lines: NALM-6, REH, RS4-11 and SUP-B15 and primary ALL cells). By co-culturing them with mesenchymal stem cells (MSCs) and treating them with ASNase-pretreated culture media, we mimicked the bone marrow matrix and the in vivo half-life of the drug (1.28±0.35 days). The ASNase concentrations used in the ASNase-pretreated culture media were 0.04, 0.4 and 4 IU/mL.

In concordance with previous results, we showed that leukemic cell survival was increased in the co-culture model compared to the “classical” in vitro treatment after five days using flow cytometry (NALM-6 - 0.04IU/mL: 17.37±2.8% p<0.0001, 0.4IU/mL: 18±2.8% p<0.0001, 4IU/mL: 25.87±2.3% p<0.0001; REH - 0.04IU/mL: 27.51±3.3% p<0.0001, 0.4IU/mL: 22.06±3.3% p<0.0001, 4IU/mL: 23.96±3.3% p<0.0001; RS4:11 – 0.04IU/mL: 50.07±2.9% p<0.0001, 0.4UI/mL: 60.14±2.9% p<0.0001, 4IU/mL: 34.50±3% p<0.0001; SUP-B15 - 0.04IU/mL: 13.90±2% p<0.0001, 0.4UI/mL: 19.55±2.3% p<0.0001, 4IU/mL: 5.72±2% p<0.05). While ASNase-mediated metabolic rewiring of leukemic cells persisted in both mono and co-culture: reduced glycolysis and increased fatty acid oxidation, the activity of mTOR-regulated biosynthetic pathways differed. The latter pathway was assessed by western blot quantification of the downstream targets of mTOR, S6 and CAD, which are protein and nucleotide synthesis mediators, respectively. In both cultures, the phosphorylated forms of S6 and CAD were inhibited after ASNase treatment (4IU/mL). However, the effect was significantly less profound in the co-culture model (REH: p-S6 (1.826 log (2) fold change, p=0.0043); p-CAD (2.385 log (2) fold change, p=0.0152), NALM-6: p-S6 (1.380 log (2) fold change, p=0.0106); p-CAD (0.78 log (2) fold change, p=n.s)). Similar changes in phospo-S6 were observed in primary BCP-ALL cells isolated from pediatric patients treated with ASNase. As shown by stable isotope tracing, asparagine synthesized de novo and released from MSCs compensated for asparagine depletion (after ASNase administration) and induced resistance of leukemic cells. Asparagine was sufficient to restore protein and nucleotide synthesis and partially rescued the viability of leukemic cells.

In conclusion, the presence of MSCs sustains biosynthetic pathways, making leukemic cells more accessible to bioenergetic rewiring, which may counteract ASNase cytotoxicity. These findings present a potential therapeutical target for resistant patients. (Supported by GAČR GA20-27132S and GAUK 1262120)