Autophagy Inhibition Sensitizes Acute Lymphoblastic Leukemia Cells to L-Asparaginase
Authors: Takahashi Hiroyoshi , Inoue Jun , Sakaguchi Kimiyoshi , Takagi Masatoshi , Mizutani Shuki , Inazawa Johji 
Published: October 2019

Abstract
Autophagy is an intracellular protein and organelle degradation system, and is upregulated in response to cellular stress such as amino acid starvation. On the other hand, L-asparaginase (L-asp) plays an essential role in acute lymphoblastic leukemia (ALL) therapy by reducing intracellular asparagine and glutamine in ALL cells. However, the relationship of L-asp and autophagy is largely unknown.
Here we show that L-asp induced cytoprotective autophagy. Three ALL cell lines of varied genetic background were used for in vitro experiments (REH, ETV6-RUNX1+ B-cell precursor (BCP) ALL; 697, E2A-PBX1+ BCP-ALL; TS-2, MEF2D-DAZAP1+ BCP-ALL). The cells were exposed to chroloquine (CQ) or bafilomycin A1 as autophagy inhibitors for 3 hours. LC3B-II, autophagy flux marker, was significantly increased under L-asp treatment with CQ as compared to only CQ condition, which was confirmed in independent experiments at immunofluorescence staining. Transmission electron microscopy showed that both the number and the area of autophagic vesicles per cell were markedly increased in L-asp with CQ condition. Thus, autophagy was induced by L-asp increasing turnover and clearance of autophagosomes in ALL cells. The toxic effect of 4 groups (control, CQ, L-asp, and L-asp plus CQ) by flow cytometry using Annexin-V staining indicated that combination treatment with L-asp and CQ for 48 hours induced significant cell death in the three ALL cell lines. Furthermore, inhibition of autophagy by CQ comparably sensitize REH cells to L-asp as ATG7 silencing by short interfering RNA. Cell growth assays for 6-9 days showed that L-asp monotreatment suppressed cell growth but did not increase the percentage of dead cells. In contrast, combination treatment with L-asp and CQ decreased the number of living cells and significantly increased the percentage of dead cells in time-dependent manner. Cell cycle analysis showed that cell cycle arrest at G1 phase was induced and the percentage of cells in sub-G1 phase remained a small increase by L-asp monotreatment, indicating leukemia cells endured amino acid deficiency by G1 arrest. In contrast, addition of CQ to L-asp significantly increased the sub-G1 population instead of decreasing G1 population. The apoptosis-related protein expressions using western blot analysis showed that combination treatment with L-asp and CQ induced cleavage of caspase 3 and PERP. In addition, a pan-caspase inhibitor benyloxycarbonyl Val-Ala-Asp (O-methyl)-fluoro-methylketone (z-VAD) significantly reduced the percentage of Annexin-V positive cells in the combination treatment with L-asp and CQ, which suggested that the autophagy inhibition upon L-asp treatment induced apoptotic cell death. We next transduced REH cell line with a luciferase-expressing viral vector. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were transplanted with these cells via tail vein and 6,000 U/kg L-asp and/or 50 mg/kg CQ were injected intraperitoneally once per day for survival analysis. The combination treatment with L-asp and CQ clearly reduced the leukemia burden as detected by luciferase intensity and improved outcome (L-asp plus CQ vs L-asp at day 28 after administration: 82% vs 0%, respectively. p <0.001). Of note, LC3B dots detected by immunofluorescence staining were apparently increased by the combination treatment with L-asp and CQ in the ALL cells derived from peripheral blood, bone marrow, and central nervous system at day 14 after transplantation.
Taken together, these data suggest that autophagy plays cytoprotective role in L-asp-treated ALL cells both in vitro and in vivo, and autophagy inhibition upon L-asp treatment may be a useful approach for ALL.

Disclosures
No relevant conflicts of interest to declare.