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Autophagy and Glutamine Synthetase Induction Sustain ALL Blast Resistance to L‐Asparaginase in Human Bone Marrow Mesenchymal Stromal Cells
Author(s) -
Chiu Martina,
Bardelli Donatella,
Taurino Giuseppe,
Dander Erica,
Bianchi Massimiliano G,
Mirandola Prisco,
Carubbi Cecilia,
D'Amico Giovanna,
Rizzari Carmelo,
Bussolati Ovidio
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.809.8
Subject(s) - glutamine , asparagine synthetase , asparagine , asparaginase , mesenchymal stem cell , bone marrow , glutamine synthetase , autophagy , chemistry , flow cytometry , biochemistry , microbiology and biotechnology , enzyme , biology , immunology , leukemia , amino acid , lymphoblastic leukemia , apoptosis
L‐Asparaginase (ASNase) is one of the cornerstones of acute lymphoblastic leukaemia (ALL) treatment. Its antileukemic effect mainly relies on its ability to hydrolyse blood asparagine and glutamine. Asparagine depletion causes a severe nutritional stress in ALL blasts exhibiting low expression of asparagine synthetase (ASNS), which cannot, therefore, synthesize asparagine de novo . Conversely, given the role of glutamine as obliged nitrogen donor for asparagine synthesis, the glutaminolytic activity of ASNase is of relevance in the therapy of ASNS‐positive ALL blasts. It has been proposed that bone marrow mesenchymal stromal cells (MSCs) may support the resistance of ALL blasts to ASNase through the release of asparagine and glutamine. Given that no data are yet available on ASNase effects on MSCs themselves, aim of this work was to ascertain whether and how MSCs adapt to ASNase and if the inhibition of this adaptation reverts nutritional support to ALL blasts. MSCs isolated from healthy donors and the ALL cell lines RS4;11 have been cultured in DMEM and RPMI 1640 medium, respectively, supplemented with 10% FBS, 2 mM glutamine and antibiotics. Co‐cultures were established by adding RS4;11 cells to confluent monolayers of MSCs. ASNase from E. chrysanthemi was used at 1 U/ml, while glutamine synthetase (GS) was inhibited by 1 mM methionine‐L‐sulfoximine (MSO). Viability was measured through resazurin assay or flow cytometry. Autophagy was assessed by LC3 II detection in Western Blot. MSCs were poorly sensitive to ASNase (IC 50 > 10 U/ml) and, once treated with the enzyme, recovered their growth after a transient proliferative arrest. In MSCs, ASNase increased GS expression and activated a nutritional stress response marked by the phosphorylation of eIF2α. Moreover, ASNase inhibited mTORC1 activity, detected as the phosphorylation of p70S6K1, and, consequently, triggered autophagy. When GS was inhibited by MSO, the autophagic response was delayed, while CHOP was steadily induced and MSC growth was not rescued. When co‐cultured with MSCs, ALL blasts were effectively protected from ASNase‐induced cytotoxicity. The protective effect was markedly hindered when MSCs had been pre‐treated with ASNase and MSO. Our data show that the adaptation of MSCs to ASNase treatment through autophagy and GS induction is needed for ALL blast survival and may constitute an important therapeutic target.