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Life, Death, BH3 Profiles, and the Salmon Mousse

2007; Cell Press; Volume: 12; Issue: 2 Linguagem: Inglês

10.1016/j.ccr.2007.07.011

1878-3686

Douglas R. Green,

Mitochondrial Function and Pathology

New drugs that neutralize the antiapoptotic members of the Bcl-2 family hold promise for rational cancer therapies, both alone and in combination with other agents. An understanding of how and why such agents may trigger apoptosis on their own, and how resistance to these drugs can occur, depends on the complexity of the Bcl-2 family interactions that control mitochondrial outer membrane permeabilization (MOMP). By extracting mitochondria from tumor cells and exposing them to peptides corresponding to the regulatory BH3-only proteins, MOMP predicts not only which cells will undergo apoptosis in response to Bcl-2 antagonists, but also why other cells may be resistant. New drugs that neutralize the antiapoptotic members of the Bcl-2 family hold promise for rational cancer therapies, both alone and in combination with other agents. An understanding of how and why such agents may trigger apoptosis on their own, and how resistance to these drugs can occur, depends on the complexity of the Bcl-2 family interactions that control mitochondrial outer membrane permeabilization (MOMP). By extracting mitochondria from tumor cells and exposing them to peptides corresponding to the regulatory BH3-only proteins, MOMP predicts not only which cells will undergo apoptosis in response to Bcl-2 antagonists, but also why other cells may be resistant. As any poet or philosopher can tell us, it is in the contemplation of death that we gain insight into life. Indeed, it was in their seminal The Meaning of Life that Monty Python had a dinner guest challenge the figure of Death to explain how their entire party had somehow all died at the same time, to which Death ominously replied, "The salmon mousse." In much the same way, if perhaps less ethereally, the study of the principles of cell death has long held the promise to preserve life, by, among other things, providing new insights for the treatment of cancer. In this issue of Cancer Cell, Deng and colleagues (2007) probe the mechanisms of sensitivity and resistance of tumor cells to the Bcl-2 antagonist ABT-737 and, in so doing, offer compelling evidence for an emerging view of the functions of the Bcl-2 family proteins in the control of apoptosis. This is a view that may, ultimately, give us the equivalent of a "salmon mousse" for cancer. Our story concerns the intrinsic, or mitochondrial pathway of apoptosis, in which mitochondrial outer membrane permeabilization (MOMP) results in diffusion of proteins from the intermembrane space to the cytosol. Holocytochrome c thus gains access to APAF-1, leading to caspase activation and death. Even without caspase activation, MOMP generally results in cell death. The decision, MOMP or no MOMP, translating into death or survival of the cell, is made by the interactions of the Bcl-2 family proteins, both pro- and antiapoptotic. Of these, Bax and Bak are the proapoptotic "effectors," which are likely to be directly responsible for the permeabilization of the outer membrane. These are antagonized by the actions of the antiapoptotic Bcl-2 proteins, including Bcl-2, Bcl-xL, Mcl-1, and Bfl/A1. A third class of Bcl-2 proteins, the BH3-only proteins, appear to make the "thumbs up or thumbs down" decision by regulating the other family members. Recently, several drugs that act as BH3 mimetics have been identified, including ABT-737, which antagonizes the functions of Bcl-2, Bcl-xL, and Bcl-w and has shown potent single-agent proapoptotic activity in some experimental tumors, but not others (Oltersdorf et al., 2005Oltersdorf T. Elmore S.W. Shoemaker A.R. Armstrong R.C. Augeri D.J. Belli B.A. Bruncko M. Deckwerth T.L. Dinges J. Hajduk P.J. et al.Nature. 2005; 435: 677-681Crossref PubMed Scopus (2927) Google Scholar). Importantly, ABT-737 is generally inactive in triggering apoptosis in normal cells, with an interesting exception being platelets. To understand how this works, and to further delineate mechanisms of resistance to ABT-737, Deng et al., 2007Deng J. Carlson N. Takeyama K. Dal Cin P. Shipp M. Letai A. Cancer Cell. 2007; (this issue)Google Scholar examined a number of ABT-737-sensitive or -resistant diffuse large B cell lymphoma lines (DLBCL) by their method of "BH3 profiling" (Certo et al., 2006Certo M. Del Gaizo Moore V. Nishino M. Wei G. Korsmeyer S. Armstrong S.A. Letai A. Cancer Cell. 2006; 9: 351-365Abstract Full Text Full Text PDF PubMed Scopus (998) Google Scholar). They isolated mitochondria from the different lines and exposed them to a panel of peptides corresponding to the BH3 regions of several BH3-only proteins and examined MOMP. In so doing, they identified three classes of resistance to ABT-737 and provided explanations for how they work. This approach depends on an understanding of the functions of the different BH3-only proteins (Figure 1). There is general agreement that these proteins act to neutralize the antiapoptotic Bcl-2 proteins, and they show distinct preferences in this function (Certo et al., 2006Certo M. Del Gaizo Moore V. Nishino M. Wei G. Korsmeyer S. Armstrong S.A. Letai A. Cancer Cell. 2006; 9: 351-365Abstract Full Text Full Text PDF PubMed Scopus (998) Google Scholar, Chen et al., 2005Chen L. Willis S.N. Wei A. Smith B.J. Fletcher J.I. Hinds M.G. Colman P.M. Day C.L. Adams J.M. Huang D.C. Mol. Cell. 2005; 17: 393-403Abstract Full Text Full Text PDF PubMed Scopus (1513) Google Scholar, Kuwana et al., 2005Kuwana T. Bouchier-Hayes L. Chipuk J.E. Bonzon C. Sullivan B.A. Green D.R. Newmeyer D.D. Mol. Cell. 2005; 17: 525-535Abstract Full Text Full Text PDF PubMed Scopus (988) Google Scholar), a function termed "sensitizer" or "derepressor" activity. The BH3-only protein BAD, for example, neutralizes Bcl-2, Bcl-xL, and Bcl-2, but neither Mcl-1 nor Bfl/A1, while another BH3-only protein, NOXA, neutralizes Mcl-1 (and to some extent Bfl/A1) but none of the others. BIM, PUMA, and probably BMF effectively bind and neutralize all of the different antiapoptotic Bcl-2 proteins. One prevalent view is that this neutralization of the antiapoptotic Bcl-2 proteins is not only necessary but also sufficient to cause MOMP via Bax and Bak (Willis et al., 2007Willis S.N. Fletcher J.I. Kaufmann T. van Delft M.F. Chen L. Czabotar P.E. Ierino H. Lee E.F. Fairlie W.D. Bouillet P. et al.Science. 2007; 315: 856-859Crossref PubMed Scopus (930) Google Scholar). Letai et al., 2002Letai A. Bassik M.C. Walensky L.D. Sorcinelli M.D. Weiler S. Korsmeyer S.J. Cancer Cell. 2002; 2: 183-192Abstract Full Text Full Text PDF PubMed Scopus (1351) Google Scholar and Kuwana et al., 2005Kuwana T. Bouchier-Hayes L. Chipuk J.E. Bonzon C. Sullivan B.A. Green D.R. Newmeyer D.D. Mol. Cell. 2005; 17: 525-535Abstract Full Text Full Text PDF PubMed Scopus (988) Google Scholar proposed an additional activity of some BH3-only proteins that is also necessary for MOMP: the activation of Bax and/or Bak. BH3-only proteins that have this "direct activator" function include BIM and BID. However, a recent study has shown that Bcl-2-inhibitable apoptosis proceeds in BID-BIM double knockout cells (Willis et al., 2007Willis S.N. Fletcher J.I. Kaufmann T. van Delft M.F. Chen L. Czabotar P.E. Ierino H. Lee E.F. Fairlie W.D. Bouillet P. et al.Science. 2007; 315: 856-859Crossref PubMed Scopus (930) Google Scholar), leading to two possible conclusions; either the "direct activator" concept is wrong, or other mechanisms exist to activate Bax and Bak. I favor the latter and have proposed that such alternative mechanisms exist (Green, 2006Green D.R. Cancer Cell. 2006; 9: 328-330Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Cells in which direct activators of Bax and/or Bak are present are therefore "primed for death" such that disruption of the antiapoptotic Bcl-2 functions results in death (Certo et al., 2006Certo M. Del Gaizo Moore V. Nishino M. Wei G. Korsmeyer S. Armstrong S.A. Letai A. Cancer Cell. 2006; 9: 351-365Abstract Full Text Full Text PDF PubMed Scopus (998) Google Scholar). It is in this context that Deng et al., 2007Deng J. Carlson N. Takeyama K. Dal Cin P. Shipp M. Letai A. Cancer Cell. 2007; (this issue)Google Scholar characterize the DLBCL lines in their study and describe three classes of resistance to ABT-737. In the first (class A), Bax and Bak are functional, but no signal is present to activate these effectors, and therefore no death occurs when antiapoptotic Bcl-2 proteins are neutralized. Mitochondria from such cells undergo MOMP in response to the direct activator BH3 sequences from BID or BIM, but not in response to the other BH3s. In this setting, neutralizing the antiapoptotic proteins with ABT-737 does not induce MOMP or death, because Bax or Bak is not engaged. In support of this idea, Deng et al., 2007Deng J. Carlson N. Takeyama K. Dal Cin P. Shipp M. Letai A. Cancer Cell. 2007; (this issue)Google Scholar show that a cell line that is normally sensitive to apoptosis induction by ABT-737 is rendered resistant by knocking down one of the direct activator BH3-only proteins, BIM. In their second class of resistance (class B), low or absent Bax and Bak appear to be responsible, and mitochondria from these cells do not undergo MOMP in response to any of the BH3 peptides. In such cells, neutralization of antiapoptotic proteins would not be expected to cause death unless some way to induce expression of Bax or Bak could be managed. The third resistance class (class C) is one that they (Del Gaizo Moore et al., 2007Del Gaizo Moore V. Brown J.R. Certo M. Love T.M. Novina C.D. Letai A. J. Clin. Invest. 2007; 117: 112-121Crossref PubMed Scopus (511) Google Scholar) and others (Konopleva et al., 2006Konopleva M. Contractor R. Tsao T. Samudio I. Ruvolo P.P. Kitada S. Deng X. Zhai D. Shi Y.X. Sneed T. et al.Cancer Cell. 2006; 10: 375-388Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar) had previously described for other types of lymphoma. In these cells, direct activators and Bax and/or Bak are present, and the cells are "primed for death," but the antiapoptotic Bcl-2 proteins sustaining survival are not affected by the drug. In AML and CLL, the presence of Mcl-1 correlated with such resistance, and mitochondria from such cells underwent MOMP in response to NOXA BH3. In the current study, one such resistant line was found to overexpress Bfl/A1. Is this emerging, complex view important? The simpler view, discussed above, that neutralization of all available antiapoptotic Bcl-2 family proteins is both necessary and sufficient for apoptosis does not account for the data presented by Deng et al., 2007Deng J. Carlson N. Takeyama K. Dal Cin P. Shipp M. Letai A. Cancer Cell. 2007; (this issue)Google Scholar. For example, while both BIM and PUMA interact with all of the antiapoptotic Bcl-2 proteins with equivalent affinity (e.g., Chen et al., 2005Chen L. Willis S.N. Wei A. Smith B.J. Fletcher J.I. Hinds M.G. Colman P.M. Day C.L. Adams J.M. Huang D.C. Mol. Cell. 2005; 17: 393-403Abstract Full Text Full Text PDF PubMed Scopus (1513) Google Scholar), BIM but not PUMA was observed to induce MOMP in mitochondria from class A cells. One recent report (Kim et al., 2006Kim H. Rafiuddin-Shah M. Tu H.C. Jeffers J.R. Zambetti G.P. Hsieh J.J. Cheng E.H. Nat. Cell Biol. 2006; 8: 1348-1358Crossref PubMed Scopus (701) Google Scholar) suggested that PUMA, like BIM and BID, is a direct activator BH3; however, this appears to be incompatible with the above results as well, at least under these experimental conditions. Perhaps more importantly, the emerging view laid out by Deng et al., 2007Deng J. Carlson N. Takeyama K. Dal Cin P. Shipp M. Letai A. Cancer Cell. 2007; (this issue)Google Scholar is compatible with the idea that a therapeutic window may exist for BH3-mimetic drugs, such as ABT-737, to preferentially kill tumors rather than normal tissues. In any simpler view, no such window should logically exist, since tumors should be expected to have equal or higher levels of antiapoptotic proteins as compared to normal tissues, and thus be more resistant than normal cells to their neutralization. However if tumors are preferentially "primed for death" by direct activators of Bax and/or Bak, then they may have increased sensitivity to such neutralizers, unless they belong to one of the classes of resistance described by Deng et al., 2007Deng J. Carlson N. Takeyama K. Dal Cin P. Shipp M. Letai A. Cancer Cell. 2007; (this issue)Google Scholar. Such priming may occur, in part, because oncogenes that drive proliferation can also engage direct activators of Bax and Bak. For example, c-Myc drives the function of BIM (Egle et al., 2004Egle A. Harris A.W. Bouillet P. Cory S. Proc. Natl. Acad. Sci. USA. 2004; 101: 6164-6169Crossref PubMed Scopus (417) Google Scholar). In keeping with these notions, one recent study explores another drug, TW-37, which neutralizes Bcl-2 and Bcl-xL with affinities similar to those of ABT-737, but also effectively blocks Mcl-1 (with similar affinity to the other antiapoptotic proteins) (Verhaegen et al., 2006Verhaegen M. Bauer J.A. Martin de la Vega C. Wang G. Wolter K.G. Brenner J.C. Nikolovska-Coleska Z. Bengtson A. Nair R. Elder J.T. et al.Cancer Res. 2006; 66: 11348-11359Crossref PubMed Scopus (124) Google Scholar). A therapeutic window was found for melanoma versus primary cells in mice, a finding that is difficult to reconcile with simple "neutralization models" of Bcl-2 family function. However, if melanoma, like the lymphomas studied by Letai and colleagues, are "primed for death" by the engagement of direct activators of Bax and Bak, then the specificity of this drug for transformed cells can make sense. We shall see if the patterns of resistance defined by Deng et al., 2007Deng J. Carlson N. Takeyama K. Dal Cin P. Shipp M. Letai A. Cancer Cell. 2007; (this issue)Google Scholar apply similarly to this agent. Ultimately, the functional mechanisms controlling the Bcl-2 family and MOMP may turn out to be very much a matter of life and death. Fully effective tumor therapy requires that, like the guests at Monty Python's ill-fated dinner party, every targeted cell dies. Combinations of therapies to engage the mechanisms of apoptosis, for example, by conventional radiation or chcmotherapies, while inhibiting antiapoptotic mechanisms using BH3 mimetics, hold promise for new avenues of cancer therapy. But it is easy to kill tumor cells—the trick is leaving the healthy tissues intact. The BH3 profiling approach that Letai and colleagues have introduced represents a step toward teasing out such conditions. BH3 Profiling Identifies Three Distinct Classes of Apoptotic Blocks to Predict Response to ABT-737 and Conventional Chemotherapeutic AgentsDeng et al.Cancer CellAugust 14, 2007In BriefCancer cells exhibit many abnormal phenotypes that induce apoptotic signaling via the intrinsic, or mitochondrial, pathway. That cancer cells nonetheless survive implies that they select for blocks in apoptosis. Identifying cancer-specific apoptotic blocks is necessary to rationally target them. Using a panel of 18 lymphoma cell lines, we show that a strategy we have developed, BH3 profiling, can identify apoptotic defects in cancer cells and separate them into three main classes based on position in the apoptotic pathway. Full-Text PDF Open Archive