Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance
Laura Bonapace, … , Martin Stanulla, Jean-Pierre Bourquin
Laura Bonapace, … , Martin Stanulla, Jean-Pierre Bourquin
Published March 1, 2010
Citation Information: J Clin Invest. 2010;120(4):1310-1323. https://doi.org/10.1172/JCI39987.
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Research Article Oncology

Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance

Abstract

In vivo resistance to first-line chemotherapy, including to glucocorticoids, is a strong predictor of poor outcome in children with acute lymphoblastic leukemia (ALL). Modulation of cell death regulators represents an attractive strategy for subverting such drug resistance. Here we report complete resensitization of multidrug-resistant childhood ALL cells to glucocorticoids and other cytotoxic agents with subcytotoxic concentrations of obatoclax, a putative antagonist of BCL-2 family members. The reversal of glucocorticoid resistance occurred through rapid activation of autophagy-dependent necroptosis, which bypassed the block in mitochondrial apoptosis. This effect was associated with dissociation of the autophagy inducer beclin-1 from the antiapoptotic BCL-2 family member myeloid cell leukemia sequence 1 (MCL-1) and with a marked decrease in mammalian target of rapamycin (mTOR) activity. Consistent with a protective role for mTOR in glucocorticoid resistance in childhood ALL, combination of rapamycin with the glucocorticoid dexamethasone triggered autophagy-dependent cell death, with characteristic features of necroptosis. Execution of cell death, but not induction of autophagy, was strictly dependent on expression of receptor-interacting protein (RIP-1) kinase and cylindromatosis (turban tumor syndrome) (CYLD), two key regulators of necroptosis. Accordingly, both inhibition of RIP-1 and interference with CYLD restored glucocorticoid resistance completely. Together with evidence for a chemosensitizing activity of obatoclax in vivo, our data provide a compelling rationale for clinical translation of this pharmacological approach into treatments for patients with refractory ALL.

Authors

Laura Bonapace, Beat C. Bornhauser, Maike Schmitz, Gunnar Cario, Urs Ziegler, Felix K. Niggli, Beat W. Schäfer, Martin Schrappe, Martin Stanulla, Jean-Pierre Bourquin

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Figure 7

RIP-1 kinase activity is essential for obatoclax-mediated GC sensitization.

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RIP-1 kinase activity is essential for obatoclax-mediated GC sensitizati...
(A) Electron microscopy images reveal features of necroptotic cell death after treatment for 72 hours with obatoclax and dexamethasone. No condensed chromatin was detectable, while Trail treatment induces condensed apoptotic nuclei (top panel) (original magnification, ×7,100). Cells treated with obatoclax and dexamethasone exhibited disintegrated plasma membranes, which was recapitulated by Na-azide. Trail treatment leaved membranes intact (bottom panel) (original magnification, ×5,400). (B) A more detailed view of the same experiment. An autophagosome formation with characteristic double membrane structures was detected in the cytoplasm. The plasma membrane was disrupted in cells treated with obatoclax and dexamethasone. N, nucleus; C, cytoplasm; A, autophagosome; PM, plasma membrane (original magnification, ×15,000). (C) In steroid-resistant (CEM-C1 and Jurkat) and steroid-sensitive (697) cells treated for 72 hours with dexamethasone (1 μM) and obatoclax (10% IC50), with or without the necroptosis inhibitor nec-1 (300 nM), nec-1 restored steroid resistance as assessed by the MTT assay. (D) Jurkat RIP1–/– cells were less sensitive to the double treatment for 72 hours with dexamethasone (1 mM) and obatoclax (10% IC50) compared with WT Jurkat cells. Cell viability was assessed by the MTT assay. (E) Downregulation of CYLD rescued Jurkat cells from cell death induced by combination treatment for 72 hours with dexamethasone (1 μM) and obatoclax (10% IC50). Efficiency of downregulation was assessed by Western blot analysis after 48 hours. (F) LC3-II generation occurred in Jurkat RIP1–/– cells and in WT cells after 8 hours of treatment with dexamethasone and obatoclax. (G) Treatment with nec-1 did not inhibit LC3-II generation induced by treatment with obatoclax and dexamethasone in Jurkat cells. ***P < 0.05.