The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl(+)) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl(+) leukemias.     

Bcr-Abl-mediated resistance to apoptosis is independent of constant tyrosine-kinase activity

Bcr-Abl is one of the most potent antiapoptotic molecules and is the tyrosine-kinase implicated in Philadelphia (Ph) chromosome-positive leukemia. It is still obscure how Bcr-Abl provides the leukemic cell a strong resistance to chemotherapeutic drugs. A rational drug development produced a specific inhibitor of the catalytic activity of Bcr-Abl called STI571. This drug was shown to eliminate Bcr-Abl-positive cells both in vitro and in vivo, although resistant cells may appear in culture and relapse occurs in some patients. In the study described here, Bcr-Abl-positive cells treated with tyrosine-kinase inhibitors such as herbimycin A, genistein or STI571 lost their phosphotyrosine-containing proteins, but were still extremely resistant to apoptosis. Therefore, in the absence of tyrosine-kinase activity, Bcr-Abl-positive cells continue to signal biochemically to prevent apoptosis induced by chemotherapeutic drugs. We propose that secondary antiapoptotic signals are entirely responsible for the resistance of Bcr-Abl-positive cells. Precise determination of such signals and rational drug development against them should improve the means to combat Ph chromosome-positive leukemia.     

MPT0B169, a New Antitubulin Agent,Inhibits Bcr-Abl Expression and Induces Mitochondrion-Mediated Apoptosis in Nonresistant and Imatinib-Resistant Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) is a clonal disorder of hematopoietic stem/progenitor cells that is caused by the Bcr-Abl oncoprotein. Clinical resistance to the Bcr-Abl inhibitor imatinib is a critical problem in treating CML. This study investigated the antitumor effect and mechanism of MPT0B169, a new antitubulin agent, in K562 CML cells and their derived imatinib-resistant cells, IMR2 and IMR3. IMR2 and IMR3 cells showed complete resistance to imatinib-induced growth inhibition and apoptosis. Resistance involved ERK1/2 overactivation and MDR1 overexpression. MPT0B169 inhibited the growth of K562, IMR2, and IMR3 cells in a dose- and time-dependent manner. MPT0B169 substantially inhibited the mRNA and protein levels of Bcr-Abl, followed by its downstream pathways including Akt, ERK1/2, and STAT3 in these cells. MPT0B169 treatment resulted in a decrease in the polymer form of tubulin according to Western blot analysis. It triggered cell cycle arrest at the G2/M phase before apoptosis, which was related to the upregulation of the mitotic marker MPM2 and the cyclin B1 level, and a change in the phosphorylation of Cdk1. MPT0B169 induced apoptosis in nonresistant and imatinib-resistant cells via a mitochondrion-mediated caspase pathway. Further study showed that the agent led to a decrease in the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 and an increase in the apoptotic protein Bax. Taken together, our results suggest that MPT0B169 might be a promising agent for overcoming imatinib resistance in CML cells.     

Identification of chelerythrine as an inhibitor of BclXL function

The identification of small molecule inhibitors of antiapoptotic Bcl-2 family members has opened up new therapeutic opportunities, while the vast diversity of chemical structures and biological activities of natural products are yet to be systematically exploited. Here we report the identification of chelerythrine as an inhibitor of BclXL-Bak Bcl-2 homology 3 (BH3) domain binding through a high throughput screening of 107,423 extracts derived from natural products. Chelerythrine inhibited the BclXL-Bak BH3 peptide binding with IC50 of 1.5 micro m and displaced Bax, a BH3-containing protein, from BclXL. Mammalian cells treated with chelerythrine underwent apoptosis with characteristic features that suggest involvement of the mitochondrial pathway. While staurosporine, H7, etoposide, and chelerythrine released cytochrome c from mitochondria in intact cells, only chelerythrine released cytochrome c from isolated mitochondria. Furthermore BclXL-overexpressing cells that were completely resistant to apoptotic stimuli used in this study remained sensitive to chelerythrine. Although chelerythrine is widely known as a protein kinase C inhibitor, the mechanism by which it mediates apoptosis remain controversial. Our data suggest that chelerythrine triggers apoptosis through a mechanism that involves direct targeting of Bcl-2 family proteins.     

Proapoptotic Bcl-2 family member Bim is involved in the control of mast cell survival and is induced together with Bcl-XL upon IgE-receptor activation

Mast cells play critical roles in the regulation of acute and chronic inflammations. Apoptosis is one of the mechanisms that limit and resolve inflammatory responses. Mast cell survival can be controlled by growth factors and activation of the IgE-receptor FcvarepsilonRI. Members of the Bcl-2 protein family are critical regulators of apoptosis and our study provides evidence that the proapoptotic BH3-only family member Bim is essential for growth factor deprivation-induced mast cell apoptosis and that Bim levels increase upon FcvarepsilonRI activation. Bim deficiency or Bcl-2 overexpression delayed or even prevented cytokine withdrawal-induced mast cell apoptosis in culture. The prosurvival protein Bcl-XL and the proapoptotic Bim were both induced upon FcvarepsilonRI activation. These results suggest that Bim and possibly also other BH3-only proteins control growth factor withdrawal-induced mast cell apoptosis and that the fate of mast cells upon FcvarepsilonRI activation depends on the relative levels of pro- and antiapoptotic Bcl-2 family members.     

Targeting the Bcl-2-regulated apoptosis pathway by BH3 mimetics: a breakthrough in anticancer therapy?

Induction of apoptosis in tumor cells by direct activation of the Bcl-2-regulated apoptosis pathway by small molecule drugs carries high hopes to overcome the shortcomings of current anticancer therapies. This novel therapy concept builds on emerging insights into how Bcl-2-like molecules maintain mitochondrial integrity and how pro-apoptotic BH3-only proteins lead to its disruption. Means to unleash the pro-apoptotic potential of BH3-only proteins in tumor cells, or to bypass the need for BH3-only proteins by directly blocking possible interactions of Bcl-2-like pro-survival molecules with Bax and/or Bak, constitute interesting options for the design of novel anticancer therapies. For the optimization and clinical implementation of these novel anticancer strategies, a detailed understanding of the role of individual BH3-only proteins in cell death signaling in healthy cells and during tumor suppression is required. In this review, we will touch on the latest findings on BH3-only protein function and attempts to define the molecular properties of the so-called 'BH3 mimetics,' a novel class of anticancer agents, able to prompt apoptosis in tumor cells, regardless of their p53 or Bcl-2 status.     

Direct Interaction of Bax and Bak Proteins with Bcl-2 Homology Domain 3 (BH3)-only Proteins in Living Cells Revealed by Fluorescence Complementation

The key event in the mitochondrial pathway of apoptosis is the activation of Bax and Bak by BH3-only proteins through a molecular mechanism that is still a matter of debate. Here we studied interactions among anti- and proapoptotic proteins of the Bcl-2 family in living cells by using bimolecular fluorescence complementation analysis. Our results indicate that the antiapoptotic proteins Mcl-1 and Bcl-x_L bind preferably to the BH3-only proteins Bim, PUMA, and Noxa but can also bind to Bak and Bax. We also found a direct interaction between Bim, PUMA, or Noxa with either Bax or Bak during apoptosis induction. In HeLa cells, interaction of Bim with Bax occurs in cytosol, and then Bim-Bax complexes translocate to mitochondria. Complexes of either PUMA or Noxa with Bax or Bak were always detected at mitochondria. Overexpression of Bcl-x_L or Mcl-1 delayed Bim/Bax translocation to mitochondria. These results reveal the ability of main BH3-only proteins to directly activate Bax and Bak in living cells and suggest that a complex network of interactions regulate the function of Bcl-2 family members during apoptosis.     

BH3-only proteins in cell death initiation, malignant disease and anticancer therapy

Induction of apoptosis in tumour cells, either by direct activation of the death receptor pathway using agonistic antibodies or recombinant ligands, or direct triggering of the Bcl-2-regulated intrinsic apoptosis pathway by small molecule drugs, carries high hopes to overcome the shortcomings of current anticancer therapies. The latter therapy concept builds on a more detailed understanding of how Bcl-2-like molecules maintain mitochondrial integrity and how BH3-only proteins and Bax/Bak-like molecules can undermine it. Means to unleash the apoptotic potential of BH3-only proteins in tumour cells, or bypass the need for BH3-only proteins by blocking possible interactions of Bcl-2-like prosurvival molecules with Bax and/or Bak allowing their direct activation, constitute interesting options for the design of novel anticancer therapies.     

A novel Bim-BH3-derived Bcl-XL inhibitor: Biochemical characterization,in vitro,in vivo and ex-vivo anti-leukemic activity

BH3-only members of the Bcl-2 family exert a fundamental role in apoptosis induction. This work focuses on the development of a novel peptidic molecule based on the BH3 domain of Bim. The antiapoptotic molecule Bcl-XL, involved in cancer development/progression and tumour resistance to cytotoxic drugs, is a target for Bim. According to a rational study of the structural interactions between wt Bim-BH3 and Bcl-XL, we replaced specific residues of Bim-BH3 with natural and non-natural aminoacids and added an internalizing sequence, thus increasing dramatically the inhibitory activity of our modified Bim-BH3 peptide, called 072RB. Confocal microscopy and flow cytometry demonstrated cellular uptake and internalization of 072RB, followed by co-localization with mitochondria. Multiparameter flow cytometry demonstrated that the 072RB dose-dependent growth inhibition of leukaemia cell lines was due to apoptotic cell death. No effect was observed when cells were treated with the internalizing vector alone or a mutated control peptide (single aminoacid substitution L94A). Ex-vivo derived leukemic cells from acute myeloid leukaemia (AML) patients underwent cell death when cultured in vitro in the presence of 072RB. Conversely, no significant cytotoxic effect was observed when 072RB was administered to cultures of peripheral blood mononuclear cells, either resting or PHA-stimulated, and bone marrow cells of normal donors. Xenografts of human AML cells in NOD/SCID mice displayed a significant delay of leukemic cell growth upon treatment with 072RB administered intravenously (15 mg/Kg three times, 48 hours after tumour cell injection). Altogether, these observations support the therapeutic potentials of this novel BH3 mimetic.     

Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax^-/-Bak^-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.     

Development of Noxa-like BH3 mimetics for apoptosis-based therapeutic strategy in chronic lymphocytic leukemia

Despite real advances made in chemoimmunotherapy, chronic lymphocytic leukemia (CLL) is still an incurable disease. New therapeutic strategies based on the restoration of the cell death program seemed relevant. Some members of the Bcl-2 family are critical players in the defective apoptotic program in CLL cells and/or targets of apoptosis inducers in vitro. The concept of BH3 mimetics has led to the characterization of small molecules mimicking proapoptotic BH3-only members of the Bcl-2 family by their ability to bind and antagonize the prosurvival members. Some putative or actual BH3 mimetics are already being tested in clinical trials with somewhat promising results. However, none of them has a high enough interaction affinity with Mcl-1, a crucial antiapoptotic factor in CLL. It has been suggested that resistance to BH3 mimetics can be overcome by using inhibitors of Mcl-1 expression. An alternative and more direct strategy is to design mimetics of the Noxa BH3 domain, which is a specific antagonistic Mcl-1 ligand. The development of such Noxa-like BH3 mimetics, capable of directly interacting with Mcl-1 and efficiently neutralizing its antiapoptotic activity, is extremely important to evaluate their impact on the clinical outcome of patients with CLL.     

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.     

C. elegans ced-13 can promote apoptosis and is induced in response to DNA damage

The p53 tumor suppressor promotes apoptosis in response to DNA damage. Here we describe the Caenorhabditis elegans gene ced-13, which encodes a conserved BH3-only protein. We show that ced-13 mRNA accumulates following DNA damage, and that this accumulation is dependent on an intact C. elegans cep-1/p53 gene. We demonstrate that CED-13 protein physically interacts with the antiapoptotic Bcl-2-related protein CED-9. Furthermore, overexpression of ced-13 in somatic cells leads to the death of cells that normally survive, and this death requires the core apoptotic pathway of C. elegans. Recent studies have implicated two BH3-only proteins, Noxa and PUMA, in p53-induced apoptosis in mammals. Our studies suggest that in addition to the BH3-only protein EGL-1, CED-13 might also promote apoptosis in the C. elegans germ line in response to p53 activation. We propose that an evolutionarily conserved pathway exists in which p53 promotes cell death by inducing expression of two BH3-only genes.     

Degenerative disorders caused by Bcl-2 deficiency prevented by loss of its BH3-only antagonist Bim.

Apoptosis is triggered when proapoptotic members of the Bcl-2 protein family bearing only the BH3 association domain bind to Bcl-2 or its homologs and block their antiapoptotic activity. To test whether loss of the BH3-only protein Bim could prevent the cellular attrition caused by Bcl-2 deficiency, we generated mice lacking both genes. Mice without Bcl-2 have a fragile lymphoid system, become runted, turn gray, and succumb to polycystic kidney disease. Concomitant absence of Bim prevented all these disorders. Indeed, loss of even one bim allele restored normal kidney development, growth, and health. These results demonstrate that Bim levels set the threshold for initiation of apoptosis in several tissues and suggest that degenerative diseases might be alleviated by blocking BH3-only proteins.     

BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly

Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.     

The orf virus inhibitor of apoptosis functions in a Bcl-2-like manner, binding and neutralizing a set of BH3-only proteins and active Bax

We have previously shown that the Orf virus protein, ORFV125, is a potent inhibitor of the mitochondrial pathway of apoptosis and displays rudimentary sequence similarities to cellular anti-apoptotic Bcl-2 proteins. Here we investigate the proposal that ORFV125 acts in a Bcl-2-like manner to inhibit apoptosis. We show that the viral protein interacted with a range of BH3-only proteins (Bik, Puma, DP5, Noxa and all 3 isoforms of Bim) and neutralized their pro-apoptotic activity. In addition, ORFV125 bound to the active, but not the inactive, form of Bax, and reduced the formation of Bax dimers. Mutation of specific amino acids in ORFV125 that are conserved and functionally important in mammalian Bcl-2 family proteins led to loss of both binding and inhibitory functions. We conclude that ORFV125’s mechanism of action is Bcl-2-like and propose that the viral protein’s combined ability to bind to a range of BH3-only proteins as well as the active form of Bax provides significant protection against apoptosis. Furthermore, we demonstrate that the binding profile of ORFV125 is distinct to that of other poxviral Bcl-2-like proteins.     

Roles of Bim in apoptosis of normal and Bcr-Abl-expressing hematopoietic progenitors

Bcr-Abl kinase is known to reverse apoptosis of cytokine-dependent cells due to cytokine deprivation, although it has been controversial whether chronic myeloid leukemia (CML) progenitors have the potential to survive under conditions in which there are limited amounts of cytokines. Here we demonstrate that early hematopoietic progenitors (Sca-1(+) c-Kit(+) Lin(-)) isolated from normal mice rapidly undergo apoptosis in the absence of cytokines. In these cells, the expression of Bim, a proapoptotic relative of Bcl-2 which plays a key role in the cytokine-mediated survival system, is induced. In contrast, those cells isolated from our previously established CML model mice resist apoptosis in cytokine-free medium without the induction of Bim expression, and these effects are reversed by the Abl-specific kinase inhibitor imatinib mesylate. In addition, the expression levels of Bim are uniformly low in cell lines established from patients in the blast crisis phase of CML, and imatinib induced Bim in these cells. Moreover, small interfering RNA that reduces the expression level of Bim effectively rescues CML cells from apoptosis caused by imatinib. These findings suggest that Bim plays an important role in the apoptosis of early hematopoietic progenitors and that Bcr-Abl supports cell survival in part through downregulation of this cell death activator.     

BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax

During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.     

Regulation of pro-apoptotic BH3-only proteins and its contribution to cancer progression and chemoresistance

The BH3-only members of the Bcl-2 protein family function as damage sensors in the cell and initiate the apoptotic cascade. Apoptosis is the primary mechanism by which the body gets rid of genetically defective cells and is critical for preventing the accumulation of cells with tumorigenic potential. BH3-only proteins have evolved to respond to distinct forms of cellular stress or DNA damage by inactivating the protective function of the prosurvival members of the Bcl-2 family. Therefore, a downregulated expression or activity of these proteins may favour tumor development. Moreover, the pro-apoptotic proteins are required for the success of most cancer treatments, including chemotherapy. Resistance to chemotherapy, a common feature of cancer, often reflects an inability of tumor cells to undergo apoptosis. Deciphering the regulation and activity of the BH3-only proteins may provide the basis for novel therapeutic strategies aimed at promoting tumor cell death or enhancing susceptibility to chemotherapeutic agents. This review summarizes the current knowledge of BH3-only proteins and their contribution to tumorigenesis and chemoresistance.