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.
View: Text | PDF | Corrigendum
Research Article Hematology Article has an altmetric score of 16

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

×

Figure 6

Inhibition of autophagy increases IM-induced cell death in K562 cells.

Options: View larger image (or click on image) Download as PowerPoint
Inhibition of autophagy increases IM-induced cell death in K562 cells. (...
(A and B) Effects of CQ (A) and Ba (B) in IM-treated K562 cells. Cells were cultured for 48 hours in the presence or absence of 5 μM CQ and 2 μM IM (A) or Ba and IM as indicated (B), and cell death was measured by annexin V immunostaining. Values represent the mean ± SEM of 3 independent experiments. Data were analyzed by unpaired Student’s t test. (C and D) CQ potentiates the effect of IM in clonogenic assays of parental (C) and IM-resistant (D) K562 cells. Cells were plated in methylcellulose in the absence or in the presence of IM and CQ, and colonies were counted 10 days later. Values (expressed as percentage of control) represent the mean ± SEM of 3 (C) or 2 (D) independent experiments. (E–J) ATG5 or ATG7 downregulation enhances IM-induced cell death. K562 cells were transfected with control or ATG5 (E and F) or ATG7 (G and H) siRNAs and analyzed for ATG5 (E, upper panel) and ATG7 (G, upper panel) expression 48 hours after transfection. Levels of actin were measured as loading control. The asterisk indicates that bands are not specific for LC3. Forty-eight hours after transfection, cells were treated with IM and analyzed for LC3 and actin expression (E and G, lower panels). Cell death was measured by annexin V staining 24 (white bars) and 40 hours (black bars) after IM treatment (F and H). K562 cells were transduced with scrambled (SO) or ATG7 shRNA pGIPZ lentiviral vectors, and levels of ATG7, actin, and LC3 (I) and IM-induced cell death (annexin V staining) were measured at 24 (white bars) and 40 hours (black bars) (J). Values represent the mean ± SEM of 3 independent experiments.