David C. S. Huang

The Bcl-2 family: roles in cell survival and oncogenesis

Apoptosis, the cell-suicide programme executed by caspases, is critical for maintaining tissue homeostasis, and impaired apoptosis is now recognized to be a key step in tumorigenesis. Whether a cell should live or die is largely determined by the Bcl-2 family of anti- and proapoptotic regulators. These proteins respond to cues from various forms of intracellular stress, such as DNA damage or cytokine deprivation, and interact with opposing family members to determine whether or not the caspase proteolytic cascade should be unleashed. This review summarizes current views of how these proteins sense stress, interact with their relatives, perturb organelles such as the mitochondrion and endoplasmic reticulum and govern pathways to caspase activation. It briefly explores how family members influence cell-cycle entry and outlines the evidence for their involvement in tumour development, both as oncoproteins and tumour suppressors. Finally, it discusses the promise of novel anticancer therapeutics that target these vital regulators.

ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets

Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2–like 1 (BCL-XL), which has shown clinical efficacy in some BCL-2–dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-XL inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2–selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2–dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2–dependent hematological cancers.

Bim: a novel member of the Bcl‐2 family that promotes apoptosis

Certain members of the Bcl‐2 family inhibit apoptosis while others facilitate this physiological process of cell death. An expression screen for proteins that bind to Bcl‐2 yielded a small novel protein, denoted Bim, whose only similarity to any known protein is the short (nine amino acid) BH3 motif shared by most Bcl‐2 homologues. Bim provokes apoptosis, and the BH3 region is required for Bcl‐2 binding and for most of its cytotoxicity. Like Bcl‐2, Bim possesses a hydrophobic C‐terminus and localizes to intracytoplasmic membranes. Three Bim isoforms, probably generated by alternative splicing, all induce apoptosis, the shortest being the most potent. Wild‐type Bcl‐2 associates with Bim in vivo and modulates its death function, whereas Bcl‐2 mutants that lack survival function do neither. Significantly, Bcl‐xL and Bcl‐w, the two closest homologues of Bcl‐2, also bind to Bim and inhibit its activity, but more distant viral homologues, adenovirus E1B19K and Epstein–Barr virus BHRF‐1, can do neither. Hence, Bim appears to act as a ‘death ligand’ which can only neutralize certain members of the pro‐survival Bcl‐2 sub‐family.

The Proapoptotic Activity of the Bcl-2 Family Member Bim Is Regulated by Interaction with the Dynein Motor Complex

Bcl-2 family members that have only a single Bcl-2 homology domain, BH3, are potent inducers of apoptosis, and some appear to play a critical role in developmentally programmed cell death. We examined the regulation of the proapoptotic activity of the BH3-only protein Bim. In healthy cells, most Bim molecules were bound to LC8 cytoplasmic dynein light chain and thereby sequestered to the microtubule-associated dynein motor complex. Certain apoptotic stimuli disrupted the interaction between LC8 and the dynein motor complex. This freed Bim to translocate together with LC8 to Bcl-2 and to neutralize its antiapoptotic activity. This process did not require caspase activity and therefore constitutes an initiating event in apoptosis signaling.

BH3-Only proteins-essential initiators of apoptotic cell death.

Overexpression of Bcl-2 can promote cancer or autoimmunity and affect sensitivity of tumor cells to chemotherapeutic drugs. Mutations in BH3-only proteins or their regulators may therefore also be pathogenic. Indeed, Bim deficiency causes a systemic lupus erythematosus (SLE)-like autoimmune disease (Bouillet et al. 1999xBouillet, P, Metcalf, D, Huang, D.C.S, Tarlinton, D.M, Kay, T.W.H, Kontgen, F, Adams, J.M, and Strasser, A. Science. 1999; 286: 1735–1738Crossref | PubMed | Scopus (1075)See all ReferencesBouillet et al. 1999) and may predispose to lymphomagenesis. It will be interesting to see what diseases develop in mice lacking other BH3-only proteins and to investigate whether abnormalities in BH3-only proteins cause disease in humans. Since these proteins are essential initiators of cell death pathways, understanding their regulation may provide novel therapeutic targets to prevent pathological cell death. Inhibitors of BH3-only proteins may alleviate degenerative disorders, whereas small molecule mimetics may be used to kill cancer cells or self-antigen-specific lymphocytes in autoimmune disease (Wang et al. 2000xWang, J.L, Liu, D, Zhang, Z.J, Shan, S, Han, X, Srinivasula, S.M, Croce, C.M, Alnemri, E.S, and Huang, Z. Proc. Natl. Acad. Sci. USA. 2000; 97: 7124–7129Crossref | PubMed | Scopus (790)See all ReferencesWang et al. 2000).*E-mail: huang_d@wehi.edu.au or strasser@wehi.edu.au

Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.

Activation of the cell surface receptor Fas/APO‐1 (CD95) induces apoptosis in lymphocytes and regulates immune responses. The cytoplasmic membrane protein Bcl‐2 inhibits lymphocyte killing by diverse cytotoxic agents, but we found it provided little protection against Fas/APO‐1‐transduced apoptosis in B lymphoid cell lines, thymocytes and activated T cells. In contrast, the cowpox virus protease inhibitor CrmA blocked Fas/APO‐1‐transduced apoptosis, but did not affect cell death induced by gamma‐radiation or serum deprivation. Signalling through Fas/APO‐1 did not down‐regulate Bcl‐2 or induce its antagonists Bax and Bcl‐xS. In Fas/APO‐1‐deficient lpr mice, Bcl‐2 transgenes markedly augmented the survival of antigen‐activated T cells and the abnormal accumulation of lymphocytes (although they did not interfere with deletion of auto‐reactive cells in the thymus). These data raise the possibility that Bcl‐2 and Fas/APO‐1 regulate distinct pathways to lymphocyte apoptosis.

Programmed anuclear cell death delimits platelet life span.

Platelets are anuclear cytoplasmic fragments essential for blood clotting and wound healing. Despite much speculation, the factors determining their life span in the circulation are unknown. We show here that an intrinsic program for apoptosis controls platelet survival and dictates their life span. Pro-survival Bcl-x(L) constrains the pro-apoptotic activity of Bak to maintain platelet survival, but as Bcl-x(L) degrades, aged platelets are primed for cell death. Genetic ablation or pharmacological inactivation of Bcl-x(L) reduces platelet half-life and causes thrombocytopenia in a dose-dependent manner. Deletion of Bak corrects these defects, and platelets from Bak-deficient mice live longer than normal. Thus, platelets are, by default, genetically programmed to die by apoptosis. The antagonistic balance between Bcl-x(L) and Bak constitutes a molecular clock that determines platelet life span: this represents an important paradigm for cellular homeostasis, and has profound implications for the diagnosis and treatment of disorders that affect platelet number and function.

Substantial Susceptibility of Chronic Lymphocytic Leukemia to BCL2 Inhibition: Results of a Phase I Study of Navitoclax in Patients With Relapsed or Refractory Disease

PURPOSE BCL2 overexpression is a hallmark of chronic lymphocytic leukemia (CLL). The novel BH3 mimetic navitoclax (ABT-263) specifically inhibits BCL2 and related proteins BCL-x(l) and BCL-w, potently inducing apoptosis of CLL cells in vitro. A phase I trial in patients with CLL was conducted to evaluate the safety, pharmacokinetics, and biologic activity of oral navitoclax. PATIENTS AND METHODS Twenty-nine patients with relapsed or refractory CLL received daily navitoclax for 14 days (10, 110, 200, or 250 mg/d; n = 15) or 21 days (125, 200, 250, or 300 mg/d; n = 14) of each 21-day cycle. Dose escalation decisions were informed by continual reassessment methodology. RESULTS Lymphocytosis was reduced by more than 50% in 19 of 21 patients with baseline lymphocytosis. Among 26 patients treated with navitoclax ≥ 110 mg/d, nine (35%) achieved a partial response and seven maintained stable disease for more than 6 months. Median treatment duration was 7 months (range, 1 to ≥ 29 months). Median progression-free survival was 25 months. Activity was observed in patients with fludarabine-refractory disease, bulky adenopathy, and del(17p) CLL. Thrombocytopenia due to BCL-x(l) inhibition was the major dose-limiting toxicity and was dose-related. Low MCL1 expression and high BIM:MCL1 or BIM:BCL2 ratios in leukemic cells correlated with response. We determined that the navitoclax dose of 250 mg/d in a continuous dosing schedule was optimal for phase II studies. CONCLUSION BCL2 is a valid therapeutic target in CLL, and its inhibition by navitoclax warrants further evaluation as monotherapy and in combination in this disease.

Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome

Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 ‘apoptosome’, which seems to amplify rather than initiate the caspase cascade.

Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.