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 6

Obatoclax induces disruption of a complex of MCL-1 and beclin-1.

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Obatoclax induces disruption of a complex of MCL-1 and beclin-1. (A) FLA...
(A) FLAG-tagged beclin-1 was overexpressed in 293T cells, and MCL-1 immunoprecipitates were analyzed for the presence of beclin-1, with or without obatoclax treatment for 3 hours. (B) Jurkat cells were treated with 100 nM obatoclax, 30 nM ABT-737, or 10 nM rapamycin for 3 hours, and MCL-1 or beclin-1 immunoprecipitates were analyzed for the presence of proteins as indicated. Low-dose obatoclax disrupted the interaction between beclin-1 and MCL-1, while interactions between BCL2 and beclin-1 were unaffected. Likewise, ABT-737 or rapamycin did not affect complexes between beclin-1 and MCL-1 or BCL2. (C) MCL-1 levels were assessed after treatment with vehicle or the combination of dexamethasone and obatoclax at indicated time points, with or without cycloheximide (CHX). Western blot analyses revealed that MCL-1 levels were unaffected by combination treatment. (D) BIM induction was assessed by Western blot in steroid-resistant (Jurkat, VHR-01, and VHR-02) and steroid-sensitive (RS4;11, 697, SR-01, and SR-02) cell lines and primary cells treated with DEX (1 μM), obatoclax (100 nM), or the combination for 24 hours.