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Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells


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#1 valiantchong

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Posted 13 October 2011 - 09:43 AM

Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells

Cristian Bellodi1, Maria Rosa Lidonnici2, Ashley Hamilton3, G. Vignir Helgason3, Angela Rachele Soliera2, Mattia Ronchetti2, Sara Galavotti1, Kenneth W. Young1, Tommaso Selmi1, Rinat Yacobi4, Richard A. Van Etten4, Nick Donato5, Ann Hunter6, David Dinsdale1, Elena Tirr├▓7, Paolo Vigneri7, Pierluigi Nicotera1, Martin J. Dyer1, Tessa Holyoake3, Paolo Salomoni1 and Bruno Calabretta2

1MRC Toxicology Unit, University of Leicester, Leicester, United Kingdom.
2Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
3Paul O'Gorman Leukaemia Research Centre, University of Glasgow, and Gartnavel General Hospital, Glasgow, United Kingdom.
4Molecular Oncology Research Institute and Division of Hematology/Oncology, Tufts-New England Medical Center, Boston, Massachusetts, USA.
5Division of Hematology/Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan, USA.
6Department of Haematology, Royal Infirmary Hospital, Leicester, United Kingdom.
7Department of Biomedical Sciences, University of Catania, Catania, Italy.


Address correspondence to: Paolo Salomoni, MRC Toxicology Unit, University of Leicester, Lancaster Road, Box 138, Leicester LE1 9HN, United Kingdom. Phone: 44-116-2525568; Fax: 44-116-2525616;

E-mail: ps90@le.ac.uk. Or to: Bruno Calabretta, Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania 19107, USA. Phone: (215) 503-4522; Fax: (215) 923-0249;

E-mail: bruno.calabretta@mail.jci.tju.edu.

First published April 13, 2009
Received for publication March 18, 2008, and accepted in revised form February 11, 2009.


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.


Introduction

Chronic myeloid leukemia (CML) is a malignancy arising from transformation of the hematopoietic stem cell, which typically evolves through 3 distinct disease stages: an indolent chronic phase (CP), characterized by the accumulation of mature granulocytes and myeloid precursors in the bone marrow and the peripheral blood; an accelerated phase, characterized by an increase in disease burden and in the frequency of progenitor/precursor cells; and an acute phase called blast crisis (BC), marked by increasing numbers of differentiation-arrested blast cells (1-3). The hallmark of all phases is the Philadelphia chromosome (Ph1), a reciprocal translocation of chromosomes 9 and 22, which generates the BCR/ABL fusion gene encoding a constitutively active tyrosine kinase (4). p210BCR/ABL exerts its oncogenic function by activating a cascade of intracellular signalling pathways, which leads to increased survival and proliferation and limited dependence on growth factors (5, 6). Two of the major pathways activated by BCR/ABL are the class I PI3K and the Ras pathways (7, 8), which are deregulated in most human cancers (9, 10). In normal hematopoietic cells, these 2 pathways are activated by stimulation of growth factor receptors with intrinsic or JAK-associated tyrosine kinase activity, suggesting that p210BCR/ABL effectively mimics growth factor-dependent signalling.

The generation of the BCR/ABL kinase, ATP-competitive inhibitor imatinib mesylate (IM) has revolutionized the therapy of CML, since this drug is highly effective in the CP of the disease (11). However, there are 3 major problems with IM-based therapy: (a) the limited response of CML-BC or Ph1 B cell acute lymphoblastic leukemia (ALL) patients to IM (11-13); (b) the development of resistance caused in approximately 40% of cases by mutations in the BCR/ABL kinase domain, which impair the ability of IM to interact with the protein (14-18); and (c) the relative insensitivity of Ph1 CML stem cells to IM (19). For these reasons, more potent BCR/ABL inhibitors, also targeting IM-resistant mutants, are being developed and tested (20, 21). However, at least one common BCR/ABL mutant (carrying the T315I mutation) is resistant to all tyrosine kinase inhibitors (TKIs) developed so far (22). A further limitation is that primitive Ph1 stem cells overexpress wild-type p210BCR/ABL and appear to be intrinsically resistant not only to treatment with IM but also to second generation (dasatinib [Das], nilotinib, and bosutinib) TKIs (19, 23-27). Therefore, there is the need to develop new therapeutic approaches that, in combination with TKIs, might be more effective in preventing the outgrowth of TKI-resistant CML/Ph1 ALL cells and target the stem cell population.

Macroautophagy (hereafter referred to as autophagy) is a degradative process in eukaryotic cells that results in the breakdown of intracellular material within lysosomes under homeostatic conditions or in response to stress signals (28, 29), allowing cells to adapt to environmental and/or developmental signals. Autophagy is a genetically controlled process, which progresses through definite steps, leading to the engulfment of long-lived proteins and whole organelles into multi-membraned vacuoles called autophagosomes (28, 29). Autophagosomes then fuse with lysosomes for final destruction and recycling (28, 29). While in certain cellular contexts autophagy can serve as an alternative cell death mechanism named type II cell death (30-32), it is becoming increasingly clear that this process can also act as a cell survival mechanism. In fact, autophagy is a process by which cells can adapt their metabolism to starvation caused by a decrease in metabolite concentrations or extracellular nutrients, a typical consequence of loss of growth factor signalling, allowing cells to evade programmed cell death (32, 33). Accordingly, inhibition of autophagy by knockdown of autophagy genes or by use of pharmacological inhibitors, such as chloroquine (CQ, an inhibitor of lysosomal acidification; ref. 34), results in cell death of growth factor-starved cells in which apoptosis has been genetically ablated (33, 35). In tumors displaying defective apoptosis, inhibition of autophagy causes caspase-independent necrotic cell death, which, in turn, augments inflammation, leading to enhanced tumor burden (36). In 2 recent studies, treatment of Myc-induced lymphomas with the autophagy inhibitor CQ, resulted in reduced tumor growth in vivo (34, 37), suggesting that induction of autophagy provides a protective mechanism in tumor cells. Thus, the consequences of autophagy inhibition are double-faced, as it can either promote or suppress tumorigenesis, depending on the tumor type or inflammation status.

Another interesting aspect of autophagy is its involvement in the response to chemotherapy. Autophagy inhibition has been shown to sensitize tumor cells to cell death induced by irradiation (38-40), alkylating agents (41), or arsenic trioxide (42), suggesting that cancer cells can react to chemotherapy by inducing autophagy as a self-defence mechanism. How an increase in intracellular lysosome-mediated catabolism results in reduced sensitivity to cell death is still unclear. Perhaps, clearance of mitochondria through autophagy (mitophagy) might cause a depletion of proapoptotic signals such as the release of cytochrome c (43, 44).

As mentioned above, p210BCR/ABL has the ability to activate several survival pathways, effectively mimicking growth factor signaling (5-8). This, in turn, causes transformed cells to acquire resistance to apoptosis induced by growth factor deprivation (5-8). Inhibition of BCR/ABL tyrosine kinase activity by IM results in induction of cell death, which is caused by a sudden decrease of intracellular survival signals and a relative increase in proapoptotic signals (45, 46). We show here that apoptosis is not the sole consequence of BCR/ABL inhibition, as IM treatment rapidly activates an autophagic process, which follows the induction of ER stress and relies on intracellular Ca2+. Of greater importance, inhibition of autophagy by pharmacological inhibitors potentiates IM-induced cell death in CML cell lines and primary CML cells, including those carrying partially IM-resistant BCR/ABL mutants. Colony formation of candidate CML stem cells, defined as CD34+CD38- and long-term culture-initiating cells (LTC-ICs), was also markedly suppressed by combination treatment. Knockdown of the autophagy genes ATG5 autophagy related 5 homolog (S. cerevisiae) (ATG5) and ATG7 autophagy related 7 homolog (S. cerevisiae) (ATG7) had comparable effects, supporting the specificity of the pharmacological inhibitors. These findings indicate that induction of autophagy provides a survival mechanism to IM-treated BCR/ABL-expressing cells, including the stem cell population, and strongly suggest that inhibition of autophagy may improve the therapeutic efficacy of TKIs in the treatment of CML.


Results

IM induces autophagy in p210BCR/ABL-expressing CML cell lines. IM induces death of CML cells, but additional effects have not been studied in detail. Therefore, we designed experiments to study cellular changes induced by IM in CML cells. To this end, the effects of IM treatment were initially analyzed in the erythro-megakaryocytic CML-BC line K562. To exclude effects due to apoptosis-related changes, cells were treated with N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD), a pan-caspase inhibitor. Morphological analysis of K562 cells cultured for 36 hours in the presence of 2 ?M IM showed a consistent decrease in cell size as compared with cells treated with zVAD alone (Figure 1A). A closer examination showed that the cytoplasm of IM/zVAD-treated cells was severely reduced and almost entirely filled with several translucent vacuoles (Figure 1A). Flow cytometry of physical parameters of IM/zVAD-treated cells revealed the appearance of a distinct population of smaller and denser cells, compared with those treated with zVAD only (forward light scatter-height [FSC-H] and side light scatter-height [SSC-H] parameters; Figure 1B, yellow arrow). This population was clearly distinct from apoptotic cells, which are smaller and less dense, as shown in cultures treated with IM only (Figure 1B). Mean FSC-H was also decreased in IM/zVAD-treated cells (Figure 1C). Similar changes were observed at earlier time points in cells treated with IM alone (data not shown). These morphological changes were reminiscent of those induced by growth factor deprivation in hematopoietic cells (33). A previous study showed that growth factor withdrawal activated autophagy in cells lacking the intrinsic apoptotic pathway and that this process was critical for maintaining cell survival (35). Thus, we investigated whether inhibition of BCR/ABL-dependent survival signals by IM treatment could also trigger autophagy. Autophagy is invariably associated with conversion of the microtubule-associated protein 1 light chain 3 (LC3), also known as ATG8, from the cytosolic LC3-I to the autophagosome-associated LC3-II form (47). Increased levels of LC3-II were clearly detected in extracts of IM-treated K562 cells (Figure 1D, upper panel). Immunofluorescence/confocal microscopy (IF/confocal microscopy) showed an increase in LC3-positive vacuoles in IM-treated K562 cells (data not shown). In cells transduced with a retrovirus encoding LC3 fused to the EGFP (EGFP-LC3), Western blotting showed a clear accumulation of EGFP-LC3-II in protein extracts as early as 6 hours after treatment with 2 ?M IM (Figure 1D, lower panel). Nevertheless, LC3-II accumulation could be due to inhibition, rather than induction, of autophagy. To exclude this possibility, we analyzed endogenous LC3-II levels in IM-treated K562 cells in the presence of CQ, an autophagy blocker. If IM blocked autophagy, one would expect equal levels of LC3-II in CQ- and CQ/IM-treated cells. Instead, LC3-II expression was higher in CQ/IM-treated cells (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI35660DS1), supporting an autophagy-inducing function of IM.






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