Targeting autophagy potentiates tyrosine kinase inhibitor–induced cell death in Philadelphia chromosome–positive cells, including primary CML stem cells
Cristian Bellodi, … , Paolo Salomoni, Bruno Calabretta
Cristian Bellodi, … , Paolo Salomoni, Bruno Calabretta
Published April 13, 2009
Citation Information: J Clin Invest. 2009;119(5):1109-1123. https://doi.org/10.1172/JCI35660.
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Targeting autophagy potentiates tyrosine kinase inhibitor–induced cell death in Philadelphia chromosome–positive cells, including primary CML stem cells

Abstract

Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210BCR/ABL-expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca2+, suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.

Authors

Cristian Bellodi, Maria Rosa Lidonnici, Ashley Hamilton, G. Vignir Helgason, Angela Rachele Soliera, Mattia Ronchetti, Sara Galavotti, Kenneth W. Young, Tommaso Selmi, Rinat Yacobi, Richard A. Van Etten, Nick Donato, Ann Hunter, David Dinsdale, Elena Tirrò, Paolo Vigneri, Pierluigi Nicotera, Martin J. Dyer, Tessa Holyoake, Paolo Salomoni, Bruno Calabretta

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Figure 4

Inhibition of autophagy potentiates IM-induced cell death in 32D-p210BCR/ABL cells.

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Inhibition of autophagy potentiates IM-induced cell death in 32D-p210BCR...
(A) 32D-p210BCR/ABL cells were cultured for 12 hours in the presence or absence of 1 μM IM, alone, or in combination with 5 μM CQ. Cell death was measured by annexin V staining. Values represent the mean ± SEM of 3 independent experiments. Data were analyzed by unpaired Student’s t test. (B) 32D-p210BCR/ABL cells were treated with 1 μM IM (12 hours), alone or in combination with 20 nM Ba. Cell death was measured as above. Values represent the mean ± SEM of 2 independent experiments. (C) CQ has no effects in 32D cells expressing the IM-resistant T315I p210BCR/ABL mutant. 32D cells expressing the IM-resistant T315I p210BCR/ABL mutant were treated with IM, with or without CQ, and analyzed for cell death induction as in A. (D) CQ-induced cell death is caspase independent. 32D-p210BCR/ABL cells were cultured for 6 hours in the presence or absence of different combination of 1 μM IM, 50 μM zVAD, and 5 μM CQ. The percentage of cell death was determined as above. Caspase activity was measured in extracts using a DVED fluorescent substrate. (E) Bcl-2 does not block CQ-mediated sensitization to IM. 32D-p210BCR/ABL cells ectopically expressing Bcl-2 (Bcl-2 32D-p210BCR/ABL) were cultured with 1 μM IM alone or in combination with 5 μM CQ. Cell death was measured as above. Values represent mean ± SEM (C–E). (F) Bcl-2 does not block IM-induced LC3-II accumulation. Western blot shows LC3-I/LC3-II levels in extracts of 32D-p210BCR/ABL cells ectopically expressing Bcl-2 treated for 6 or 12 hours with the indicated concentrations of IM. β-actin was detected as loading control.