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.
View: Text | PDF
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

×

Figure 3

Obatoclax induces autophagy in GC-resistant ALL cells.

Options: View larger image (or click on image) Download as PowerPoint
Obatoclax induces autophagy in GC-resistant ALL cells. (A) After transie...
(A) After transient transfection with GFP-LC3, Jurkat cells were treated for 4 hours as indicated. The characteristic punctuate staining pattern indicative of autophagosome formation was detected by confocal microscopy in cells treated with dexamethasone and obatoclax or rapamycin (rapa). Scale bar: 10 μm. (B) Quantitation of autophagosome-positive cells. The data represent mean ± SD of 2 independent experiments, counting 200 cells each. (C) Detection of endogenous LC3-II by Western blot analysis. Jurkat cells were treated with obatoclax and dexamethasone for the indicated time points in the presence or absence of 3-MA and after 24 hours of treatment as indicated. (D) Inhibition of autophagy by 3-MA or bafilomycin impaired sensitization to dexamethasone by 10 nM rapamycin or obatoclax (10% IC50). Cell viability was assessed by MTT. (E) Treatment with obatoclax and dexamethasone inhibited clonogenic survival of Jurkat cells. 3-MA rescued GC-resistant cells from cell death induced by combination treatment. Cells were treated for 72 hours with compounds, and clonogenic survival in methylcellulose was assessed after washing and incubation for 7 days. (F) Downregulation of beclin-1 or ATG7 using siRNA (si) impaired the resensitization of Jurkat cells to dexamethasone by obatoclax or rapamycin compared with scrambled (scr) controls. Cell viability was assessed by MTT. Efficiency of downregulation at 48 hours was analyzed by Western analysis. (G) Downregulation of beclin-1 and ATG7 in Jurkat cells protected cells from obatoclax- and dexamethasone-induced cell death in the clonogenic assay. ***P < 0.05.