Cancer Letters 332 (2013) 202–205

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Cancer Letters

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Mini-review

BH3 profiling – Measuring integrated function of the mitochondrial apoptoticpathway to predict cell fate decisions

Victoria Del Gaizo Moore a, Anthony Letai b,⇑a Chemistry Department, Elon University, McMichael Science Building 311, Elon, NC 27244, United Statesb Harvard Medical School, Mayer 430, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, United States

a r t i c l e i n f o

Keywords:ApoptosisMitochondriaBH3-onlyBH3BCL-2

0304-3835/$ - see front matter � 2012 Published bydoi:10.1016/j.canlet.2011.12.021

⇑ Corresponding author. Tel.: +1 617 632 2348; faxE-mail address: anthony_letai@dfci.harvard.edu (A

a b s t r a c t

Apoptosis is a form of programmed cell death that is controlled at the mitochondrion by the BCL-2 familyof proteins. While much has been learned about the structure and function of these proteins over the pasttwo decades, the important goal of predicting cell fate decisions in response to toxic stimuli is largelyunrealized. BH3 profiling is a functional approach that can be used to predict cellular responses to stimulibased on measuring the response of mitochondria to perturbation by a panel of BH3 domain peptides.BH3 profiling has proven useful in identifying and understanding cellular dependence on individualanti-apoptotic proteins like BCL-2 or MCL-1. Consequently, it can also be used to predict cellular responseto chemotherapy agents such as ABT-737 that target these individual proteins.

� 2012 Published by Elsevier Ireland Ltd.

1. Background on BCL-2 proteins

The B cell leukemia/lymphoma-2 (BCL-2) family of proteinsinteract at the crux of the mitochondrial pathway of apoptosis. Ini-tially identified as a participant in the t(14;18) translocation of fol-licular lymphomas, BCL-2 was the first oncogene described thatinhibited cell death [1–3]. Over the two decades since its discovery,multiple proteins with structural and functional similarities havebeen identified to comprise what is called the BCL-2 family of pro-teins. BCL-2 proteins are categorized broadly as anti- or pro-apop-totic. Homo- and heterodimerization of these proteins regulatesapoptosis at the mitochondrion [4–8]. In very simplified terms,when the abundance of active pro-apoptotic proteins exceeds thebinding capacity of anti-apoptotic proteins, apoptosis proceeds topermeabilization of the mitochondrial outer membrane, the hall-mark of the mitochondrial pathway of apoptosis.

All family members share homology in one or more BCL-2 homol-ogy domains (called BH domains, numbered BH1–H4) that areessential for their function [6,9]. Anti-apoptotic members such asBCL-2 and BCL-XL share homology in all four BH regions. A sur-face-exposed hydrophobic cleft formed by BH1–3 participates inhetero- and homodimerization with the BH3 domain of other familymembers [10]. Multi-domain pro-apoptotic members BAX and BAKhave conserved homology in BH1–BH3 as well as a C-terminal mem-brane anchor and are the effectors of mitochondrial outer membranepermeabilization (MOMP) [11,12]. While they are monomers in

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healthy cells, BAX and BAK can be triggered to homo-oligomerizeand participate in pores that cause MOMP, allowing the release ofcytochrome c and other pro-apoptotic molecules that commit a cellto apoptosis [12,13]. Either multidomain pro-apoptotic protein issufficient for release of cytochrome c, but at least one of the two isrequired for this function. The anti-apoptotic proteins, includingBCL-2, MCL-1, BCL-w, BCL-XL and BFL1/A1, can inhibit apoptosisby sequestering monomeric activated BAX and BAK before theycan oligomerize [14] (Fig. 1).

Other pro-apoptotic family members that share homology onlyin the BH3 domain, called BH3-only proteins, are subdivided aseither activators or sensitizers. ‘‘Activators’’, such as BIM, BID andpossibly PUMA, exert their pro-death function by interactingdirectly with BAX and BAK and induce the allosteric changes thatlead to BAX and BAK homo-oligomerization [15–23]. Another waythat anti-apoptotic proteins inhibit apoptosis is by binding andsequestering activators, preventing their activation of BAX andBAK. BH3-only proteins that lack activator function, called‘‘sensitizers’’, exert their pro-death function by competitive dis-placement of activator BH3-only proteins or activated BAX or BAKmonomers from inhibitor anti-apoptotic proteins like BCL-2 [16](Fig. 1). These binding interactions involve the binding of the hydro-phobic face of the amphipathic alpha-helical BH3 domain to thehydrophobic BH3-binding cleft in anti-apoptotic proteins. It is clearthat the ability of an anti-apoptotic protein like BCL-2 to inhibit sub-sequent death signaling in a cellular context will depend entirely onits not already being occupied by any one of many pro-apoptoticmolecules. This probably explains why measurements of levels ofsingle anti-apoptotic proteins are generally insufficient to predict

Fig. 1. Model of apoptotic control by BCL-2 family proteins. Cellular damageinduces upregulation of activator and/or sensitizer proteins. BAX and BAK oligo-merization is then induced by direct binding of activators like BID and BIM leadingto MOMP then cytochrome c release and subsequent cell death. Anti-apoptoticproteins inhibit apoptosis by binding and sequestering activators or activated BAXor BAK. Sensitizers can bind to anti-apoptotic proteins and displace pre-boundactivators, or previously activated BAX or BAK, inducing BAX/BAK oligomerizationand mitochondrial permeabilization (adapted from Certo et al. [15]).

Fig. 2. The binding code. Summary of the selective binding between BH3-onlypeptides and different anti-apoptotic BCL-2 family members. + indicates moderatebinding, ++ tight binding, and – no binding interaction based on fluorescentpolarization binding studies (adapted from Certo et al. [15]).

V. Del Gaizo Moore, A. Letai / Cancer Letters 332 (2013) 202–205 203

response to toxic stimuli. As a result, some have employed systemsbiology approaches to understand cell fate decisions based on initialconditions of BCL-2 family proteins [24].

Early experiments examining the interactions among BCL-2 pro-teins revealed differential binding patterns. In the course of studyingwhether the BH3 domain of BH3-only proteins separated from thecontext of the whole protein could interact with multi-domainanti- and pro-apoptotic proteins, it was found that individual BH3domains interacted differently [25]. For example BAD was foundto bind to BCL-2 but not MCL-1. NOXA displayed an opposite bindingpattern and BID, BIM or PUMA interacted with all anti-apoptoticproteins as well as BAX and BAK [15,21,26]. Experiments utilizingrecombinant anti-apoptotic proteins and fluoroscein-tagged BH3domain peptides in fluorescence polarization assays and gave riseto the ‘‘binding code’’ [15] (Fig. 2). The binding code revealed a selec-tivity of interaction among BCL-2 proteins. It may be that cellsexploit this selectivity of interaction to finely tune individual cellsto select stimuli.

2. BCL-2 proteins in cancer

A common observation that is nevertheless often overlooked isthat many cancer cells that express large amounts of anti-apopto-tic proteins are exquisitely chemosensitive [17–19]. Examples ofthis apparent paradox include acute lymphoblastic leukemia(ALL) and chronic lymphocytic leukemia (CLL), both of which con-sistently express large amounts of BCL-2 [16,17]. While cure is fre-quently obtained only in the case of ALL, both diseases show a veryhigh rate of initial response to chemotherapy. If BCL-2 acts to inhi-bit cell death, how can this be? The answer lies in the understand-ing that commitment to apoptotic death depends up on levels of

many BCL-2 family proteins, both pro- and anti-apoptotic. Onecan imagine, therefore, that if the BCL-2 in these cancers is largelyoccupied by pro-apoptotic proteins, there would be a negligibleamount of protection against apoptosis afforded by the BCL-2 pres-ent. In the case of ALL and CLL, it is clear that these cancers simul-taneously express large amounts of the pro-apoptotic activatorBH3-only protein BIM which is sequestered by BCL-2 [16,17].

During oncogenesis, oncogene activation, metastasis and geno-mic instability are examples of common phenotypes which caninduce pro-apoptotic signaling, including up-regulation of activatorBH3-only proteins [15,27,28]. In cancer cells such as ALL and CLL,which concomitantly overexpress anti-apoptotic proteins, apopto-sis can be avoided despite because the highly expressed BCL-2 bindsand sequesters the excess BIM thereby preventing BAX/BAK activa-tion [16,17,28]. These cells therefore depend on continued functionof their anti-apoptotic BCL-2 to avoid apoptosis. Such cancer cellscould thus be described as addicted to BCL-2. When substantial acti-vators, or perhaps activated BAX or BAK, are present in cells but theyare bound to anti-apoptotic proteins such as BCL-2 or MCL-1, andaddiction to BCL-2 or MCL-1 ensues, we refer to these cells as being‘‘primed for death’’ [4,15,18,29–31]. If activators could be displacedfrom anti-apoptotic proteins in these primed cells, they would bepredicted to cause apoptotic cell death. Indeed, as predicted by thismodel, the primed cancer cells in ALL and CLL very readily die viaapoptosis when treated with a BCL-2 antagonist ABT-737 [16,17].

3. BH3 profiling

BH3 profiling was designed as a tool to predict cellular fate deci-sions and understand addiction to BCL-2 family proteins. The basisof BH3 profiling is to contact mitochondria with known concentra-tions of BH3 peptides and measure the resulting permeabilizationof the outer mitochondrial membrane. As long as conditions arewell-controlled, comparisons can then be made between themitochondria of different cell lines, different tissues or subject to dif-ferent perturbations. Initially, BH3 profiling was conducted with iso-lated mitochondria [15–18]. The read-out of results was cytochromec release (Fig. 3A). In intact mitochondria, cytochrome c is containedin the intermembrane space. When BAX/BAK are engaged and oligo-merize, MOMP occurs and cytochrome c is released, which can bedetected by ELISA or Western blot. Understanding the binding codeprovided the opportunity to selectively identify dependence on indi-vidual BCL-2 family members. For instance, a mitochondrion thatwas sensitive to the NOXA BH3 peptide would indicate a depen-dence on MCL-1, while one that was sensitive to BAD BH3 wouldbe dependent on BCL-2 (or perhaps BCL-XL or BCL-w, which alsointeract with BAD BH3).

An initial test venue for BH3 profiling was in cell lines over-expressing BCL-2 or MCL-1. We found that the technique could accu-rately identify when the cell was dependent on either individualprotein [15]. Protein analysis showed that cellular dependence oc-curred only when upregulated BIM was sequestered by the relevantanti-apoptotic protein. Extending BH3 profiling to in vivo systems, a

Fig. 3. BH3 Profiling Assay Protocols. Flow charts representing the various steps for BH3 profiling using cytochrome c ELISA (A), JC-1 in a plate reader format (B), and JC-1 withflow cytometry (C) (adapted from Ryan et al. [33]).

Fig. 4. BH3 profiling using isolated mitochondria and whole cell assays produce comparable results. BCL-2 1863, MCL-1 1780 and MDA-MB-231 cell lines were used to eitherisolate mitochondria via a heavy membrane preparation and profiled with 100 lM peptides either using a cytochrome c ELISA (HM-CC) (A) or whole cells used in the JC-1(WC-JC-1) profiling assay (B). Two Upper panels in A represent data from a single experiment while all the rest triplicates; means shown with bars for SD (adapted from Ryanet al. [33]).

204 V. Del Gaizo Moore, A. Letai / Cancer Letters 332 (2013) 202–205

murine leukemia model that was demonstrated to be BCL-2 depen-dent in vivo via a conditional BCL-2 allele was subjected to BH3 pro-filing, revealing a BCL-2 dependent BH3 profile [15,28]. The BCL-2 inthis case was found to be in complex with BIM and PUMA BH3 only-proteins. A related MCL-1 dependent leukemia model demonstratesa distinct BH3 profile, with high NOXA activity and low BAD activity,consistent with MCL-1 dependence [29].

Other work has tested a series of lymphoma cell lines, and dem-onstrated that BH3 profiling correctly identifies those cell lines thatare BCL-2 dependent based on correlation with response to theBCL-2 antagonist ABT-737 [18]. Throughout our work on many celltypes, we have found an excellent correlation between a BH3 profilethat indicates BCL-2 dependence and sensitivity to ABT-737. This isperhaps not surprising, since ABT-737, and its clinically-testedderivative, ABT-263, are essentially small molecule mimetics ofthe BAD BH3 domain, and the BAD BH3 is the key peptide indicatingBCL-2 dependence in the BH3 profiling assay. We have also per-formed BH3 profiling of primary cancer cells, including ALL andCLL, and similarly found that a BH3 profile indicative of BCL-2dependence was predictive of ABT-737 sensitivity [16,17]. Theseexperiments were the first instance that suggested that BH3 profil-ing could be a useful diagnostic for clinical treatment. Since BH3profiling is rapid, taking less than 4 h from blood draw to profiling

results, as well as accurate, it could potentially be used to predictclinical response to an antagonist of an anti-apoptotic protein.

4. Whole cell assay

BH3 profiling using isolated mitochondria can require between107 and 109 cells depending on the number of mitochondria per cell.To make BH3 profiling clinically useful in situations where fewercells are obtained, to improve upon the time it takes from patientbiopsy to results, and harness the capability of using mixed cellpopulations, a whole cell assay was developed. First, a readout ofcytochrome c release other than western blot or ELISA was sought.Testing revealed that the dual emission fluorescent probe JC-1would be ideal. Reading a JC-1 fluorescent signal at A595, healthymitochondria with an intact membrane potential and mitochondriawhere BAX/BAK have oligomerized causing the decline and eventualabolishment of the membrane potential could be distinguished.Since JC-1 is cell permeant and fluorescence is detectable using afluorimeter or by flow cytometry the next obstacle to attack wasdelivery of the activator and sensitizer peptides to the cells. Sincethe binding of peptides to anti-apoptotic proteins is dependent onstructure interactions attachment to a cell-penetrating signal, such

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as TAT or Antennapedia domain, could change binding or induce off-target effects [32]. Therefore an alternative way to introduce pep-tides into the cell was sought. Since long term cell viability wasnot a concern, we turned to detergents. After evaluating multipledetergents and concentrations, ultra low concentration of digitonin(0.001–0.005%) were found to permeabilize the plasma membranewithout affecting the mitochondrial membrane potential in bothcell lines and primary samples for up to 3 h (Fig. 3B) [33]. Further sta-bilization of the cells was then achieved by using a trehalose-basedbuffer for experiments [34] and the assay was then adapted to flowcytometry (Fig. 3C). Overall, BH3 profiling results using isolatedmitochondria and whole cells produce similar results, indicatingthat utilizing the trehelose and JC-1 does not affect the results(Fig. 4). By adapting the assay to flow cytometry not only was theprocedure time decreased but mixed populations of cells could thenbe used because cell surface markers could allow definition of sub-populations of interest. One example of its use is to distinguish theBH3 profiles of distinct T-cell subsets of the thymus, explainingthe great sensitivity of CD4+CD8+lymphocytes to apoptotic stimuli[33]. In the laboratory, we now find that we can perform BH3 profil-ing on any normal or malignant tissue once it is reduced to a singlecell suspension, and have found success in analyzing cell subsetseven when they represent a very small fraction of the total cells, aslong as we can identify cell surface markers to distinguish them.

5. Conclusions

Apoptosis transpires via the dynamic interaction of BCL-2 pro-teins at the mitochondrion. Dependence, or addiction, to an anti-apoptotic protein can lead to a cancer phenotype and priming ofthe mitochondria can allow initial chemosensitivity. Both situa-tions can be monitored conveniently by BH3 profiling, the noveltechnique that assesses the aggregate state of BCL-2 proteins atthe mitochondrion (Fig. 4). After multiple iterations, BH3 profilingis now conducted with whole cells and with heterogenous sam-ples. The data from this assay not only is informative from a basicscience standpoint but also is a step toward personalized medicinewhere response to a drug can be evaluated prior to administration.

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