Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance
Laura Bonapace, Beat C. Bornhauser, Maike Schmitz, Gunnar Cario, Urs Ziegler, Felix K. Niggli, Beat W. Schäfer, Martin Schrappe, Martin Stanulla, Jean-Pierre Bourquin
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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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 4

Obatoclax and rapamycin induce autophagic cell death in Bax–/–Bak–/– MEFs.

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Obatoclax and rapamycin induce autophagic cell death in Bax–/–Bak–/– MEF...
(A) Bax–/–Bak–/– MEFs transiently expressing the autophagy marker GFP-LC3 were treated with vehicle, obatoclax (100 nM), or rapamycin (10 nM) for 4 hours. Autophagosome formation was monitored by confocal microscopy. Scale bar: 20 μm. (B) Quantitation of autophagosome-positive cells. The data represent mean ± SD of 2 independent experiments, counting 200 cells each. (C) Treatment of Bax–/–Bak–/– MEFs with obatoclax induced generation of endogenous LC3-II as detected by Western analysis, an effect which was blocked by 3-MA treatment. (D) WT or Bax–/–Bak–/– (DKO) MEFs were incubated for 48 hours with obatoclax or ABT-737, and cell viability assessed by the MTT assay. (E) Inhibition of autophagy by 3-MA rescued Bax–/–Bak–/– MEFs but not WT MEFs from cell death induced by 48 hour exposure to obatoclax or rapamycin. (F) Downregulation of beclin-1 rescued Bax–/–Bak–/– MEFs but not WT MEFs from cell death induced by obatoclax as evaluated by the MTT assay. Efficiency of downregulation was determined by Western blot analysis. (G) Downregulation of ATG7 rendered Bax–/–Bak–/– MEFs but not WT MEFs resistant to obatoclax treatment. Efficiency of downregulation was assessed by Western blotting.