Design of Novel BH3 Mimetics for the Treatment of Chronic Lymphocytic Leukemia

CONCISE REVIEW Design of novel BH3 mimetics for the treatment of chronic lymphocytic leukemia

C Billard1,2

Impaired programmed cell death is an important factor in the pathogenesis of chronic lymphocytic leukemia (CLL) and in the development of resistance to chemoimmunotherapy. Hence, the reactivation of apoptotic processes is likely to be a pertinent strategy for circumventing this resistance. Proteins from the Bcl-2 family are critical elements in defective apoptosis. Some compounds induce the apoptosis of CLL cells ex vivo by downregulation of prosurvival members of this family (for example, Bcl-2 and Mcl-1), whereas others act by upregulation of proapoptotic Bcl-2 homology (BH) 3-only members (for example, Noxa and Bim). The concept of BH3 mimetics was prompted by the fact that BH3-only proteins are speciﬁc antagonistic ligands of prosurvival Bcl-2 family members. This led to the design of small molecules capable of inhibiting the activity of prosurvival Bcl-2 proteins and inducing apoptosis in leukemia cells in vitro and antileukemic effects in animal models. Several putative or actual BH3 mimetics are currently being trialed in the clinic. Two novel BH3 mimetics that can speciﬁcally bind to and antagonize Mcl-1 (a crucial antiapoptotic factor in CLL) have recently been discovered. The evaluation of this type of compound’s clinical impact in CLL can now be considered.

Leukemia (2012) 26, 2032–2038; doi:10.1038/leu.2012.88 Keywords: CLL therapy; apoptosis induction; Bcl-2 family; Mcl-1; Noxa; BH3 mimetics

RECENT ADVANCES IN THE TREATMENT OF CHRONIC However, proliferative pools have been described in the bone LYMPHOCYTIC LEUKEMIA marrow and lymph nodes that feed the blood compartment, Although therapies for chronic lymphocytic leukemia (CLL) induce indicating that CLL is in fact a dynamic disease with a higher 8 remissions and palliate symptoms, it was only in the last few years leukemic cell turnover than previously assumed. Therefore, CLL that clear effects on patient survival have been noted. Prolonged may be due to an imbalance between proliferation and apoptosis. remissions and extended survival have been made possible by The impaired cell death program is thought to result from the marked progress in the development of chemoimmunotherapy complex interplay between several factors, including both protocols combining alkylating agents, purine analogs and mono- inherent defects in the apoptotic machinery of the leukemic cells clonal antibodies. On the basis of comparisons of randomized and an excess of survival signals delivered by their extrinsic 4,6,7,9 4–7,9 trials, the combination of fludarabine and cyclophosphamide (FC) microenvironment. As described in several reviews, was selected as the most effective chemotherapy regimen. The chemokines and cytokines (for example, BAFF, APRIL, CD40 addition of rituximab (to yield the FCR regimen) significantly ligand and IL-4) may provide a survival advantage through, for increases both the proportion and duration of complete remis- instance, NF-kB or PI3K/AKT pathways that are constitutively sions and prolongs overall survival (for a review, see Tam and activated in CLL cells and are known to stimulate the transcription Keating1; Gribben and O’Brien2). One of the major challenges in of antiapoptotic proteins. Indeed, CLL cells do overexpress some the field relates to how chemoimmunotherapy can be tailored antiapoptotic proteins, such as prosurvival members of the Bcl-2 according to each patient.3 However, the proportion of patients family (Bcl-2 and Mcl-1), survivin, X-inhibitor of apoptosis protein kip1 that are refractory to treatment with FCR (or allied agents) remains (IAP), p27 or inducible nitric oxide synthase (for a review, see 5 6 7 high and nearly all patients having achieved remission subse- Kolb et al. ; Chen et al. ; Packham and Stevenson ). Alterations quently relapse. Thus, CLL remains an incurable disease. A further, in other apoptosis regulators such as p53 or ATM (frequently 4–7,9 major challenge is to eliminate residual disease,1,4 and this requires observed) and in BCR signaling also have important roles. the development of novel strategies for combination therapy.2,4 As impaired susceptibility to apoptosis may be involved in not only leukemogenesis but also in CLL’s resistance to therapy, strategies for overcoming impairments in apoptotic cell death CLL, A DISEASE OF FAILED APOPTOSIS have attracted much attention.4–7 CLL arises from the oncogenic transformation and clonal expansion of a CD5-positive subpopulation of B lymphocytes. The leukemic cells that progressively accumulate in the blood are IMPACT OF BCL-2 FAMILY PROTEINS ON IMPAIRED APOPTOSIS quiescent but unable to develop their cell death program.5–7 IN CLL Hence, CLL was considered as a disease of accumulation due Members of the Bcl-2 family are essential factors in the control of to defective apoptosis rather than dysregulated proliferation. the intrinsic apoptosis pathway.10 They govern the mitochondrial

1Centre de Recherche des Cordeliers, UMRS 872 (Equipe 18), Paris, France and 2Universite´ Pierre et Marie Curie and Universite´ Paris-Descartes, UMRS 872, Paris, France. Correspondence: Dr C Billard, Centre de Recherche des Cordeliers, UMRS 872 (Equipe 18), 15 rue de l’Ecole de Me´decine, 75006 Paris, France. E-mail: [email protected] Received 3 January 2012; revised 27 February 2012; accepted 21 March 2012; accepted article preview online 28 March 2012; advance online publication, 27 April 2012 Novel BH3 mimetics for CLL therapy C Billard 2033 Extrinsic pathway Intrinsic pathway (death receptor) (mitochondrial)

Stimulus

TRAIL BH3 Ligands Fas L only TNF BH3 only Bax Bcl-2 Bax Bcl-2 Bax Ligands Bak Mcl-1 Bak Mcl-1 Bak Mitochondrial Death Death outer membrane receptors receptors permeabilization Activation Oligomerization DR4 Plasma Fas membrane Apaf-1 TNF-R Cyt C Release from mitochondria FADD Bid Pro-caspase-9 cleavage

Caspase-8 Effector caspase Caspase-9 activation activation activation

Apoptosis

Figure 1. The intrinsic and extrinsic pathways of caspase-dependent apoptosis. The intrinsic pathway is under the control of Bcl-2 family proteins. Prosurvival proteins (e.g., Bcl-2 and Mcl-1) sequester the proapoptotic proteins Bax and Bak. Upon activation, BH3-only proteins bind the prosurvival molecules and thus displace Bax and Bak from their prosurvival ligands. This leads to the release and activation (via a conformational change) of Bax and Bak that form homodimers responsible for pore formation at the outer membrane of mitochondria (and at other cellular membranes). Cytochrome c (Cyt c) is subsequently released from mitochondria, then interacts with the adapter Apaf-1 resulting in caspase-9 activation. There is also a hypothetical model in which some BH3-only proteins activate Bax and Bak directly. The extrinsic pathway is triggered by ligation of cell surface death receptors with their ligands and recruitment of adapters (e.g., FADD) enabling the activation of caspase-8. Under some circumstances, the latter can cleave the BH3-only Bid which interacts with prosurvival Bcl-2 proteins, thus creating a connection between the two apoptosis pathways. membrane permeabilization that enables the release of apopto- Bim/tBid BH3-only Bad Noxa genic factors (cytochrome c notably) and the subsequent caspase Puma activation cascade (Figure 1). The Bcl-2 family comprises three classes of proteins: (i) the prosurvival members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1, so on), (ii) the proapoptotic members with several Prosurvival Bcl-2 Bcl-w Bcl-xL Mcl-1 A1 Bcl-2 homology (BH) domains named BH1 to 4 (Bax, Bak, so on) and (iii) the BH3-only members (Bad, Bid, Puma, Bim, Noxa, so on), as presented in Figure 2. The functional activity of the prosurvival proteins is to sequester the proapoptotic Bax and Bak proteins. Multi-BH Bax Bak All the prosurvival members have the ability to sequester Bax, proapoptotic whereas only Mcl-1 and Bcl-xL can bind Bak. When activated, BH3- only proteins bind to prosurvival members, thus displacing Bax Figure 2. The three classes of Bcl-2 family members and their selective interactions. The prosurvival proteins (in red) sequester the and Bak from their prosurvival ligands (Figure 1). The release of proapototic multi-BH proteins Bax and Bak (in green). All prosurvival Bax and Bak enables their functional activation (that is, pore proteins have the ability to sequester Bax, whereas only Bcl-xL and formation and permeabilization of the mitochondrial membrane). Mcl-1 can bind to Bak. Upon apoptotic stimulation, the BH3-only The interactions between BH3-only and prosurvival proteins are proteins (in orange) specifically interact with the prosurvival selective: for example, Noxa binds to only Mcl-1 and A1, and Bad proteins. Bad interacts only with Bcl-2, Bcl-w and Bcl-xL; Noxa binds only to Bcl-2, Bcl-xL and Bcl-w, whereas Bim and Puma bind interacts only with Mcl-1 and A1; whereas Bim, Puma and tBid are to all five prosurvival proteins (Figure 2).10 able to bind to all prosurvival proteins. Most of the prosurvival Bcl-2 family members are overexpressed in CLL lymphocytes (Bcl-2, Bcl-xL, Mcl-1). The overexpression of Bcl-2 and perhaps more importantly the expression ratio relative STRATEGIES BASED ON APOPTOSIS INDUCTION IN CLL to its proapoptotic ligand Bax are undoubtedly involved in the Various strategies to reactivate the machinery of programmed cell survival advantage of leukemia cells.7 The roles of Bcl-xL and A1 death in CLL have been developed over the last decade or so. The are also probable.11 However, it has become clear that Mcl-1 is first and most extensively studied approach concerns identifying crucial for impaired apoptosis in CLL.6,7 In a first report, high agents capable of triggering the caspase-dependent mitochon- expression levels of Mcl-1 in the leukemic lymphocytes were drial apoptosis pathway in primary leukemia cells ex vivo. This led inversely correlated with in vitro and in vivo responses to to the discovery of many apoptosis inducers and, for some of fludarabine.12 Mcl-1 levels were further correlated with prog- them, the mechanisms of action that mainly involve effects on nostic markers (disease stage, IgVH mutation status, ZAP-70 Bcl-2 family proteins (as detailed in the next paragraph). Other positivity and CD38 expression)13 and, importantly, were found strategies have been described in a number of reviews.2,4–7,9 to be predictive of the CLL patients’ clinical outcomes.14 Examples include inhibition of the NF-kB or PI3K/AKT pathways In addition, inhibition of Mcl-1 expression is enough to trigger (using CAL-101), enhancement of p53/ATM-dependent apoptosis apoptosis in vitro in primary CLL cells.15 (with nutlin 3a or inhibitors of poly(ADP-ribose) polymerase-1,

& 2012 Macmillan Publishers Limited Leukemia (2012) 2032 – 2038 Novel BH3 mimetics for CLL therapy C Billard 2034 PARP-1), inhibition of specific kinases (with dasatinib for example) and glucocorticoids.32,33 The fact that BH3-only proteins are and stimulation of the extrinsic apoptosis pathway through death modulated by a number of CLL cell apoptosis inducers suggested receptors (with TRAIL). Strategies trying to interfere with BCR that they might be relevant targets in this leukemia. Noxa appears signaling (fostanatimib) and with survival signals delivered by the to be of particular interest because it is a specific antagonistic leukemic microenvironment (particularly lenalidomide or CXCR4 ligand of the crucial survival factor Mcl-1.10 targeting) are currently under intense investigation. Lastly, Accordingly, studies on the various apoptosis inducers’ mecha- other approaches aim at evaluating novel chemotherapeutics nisms of action have indicated that the Noxa/Mcl-1 axis is an (bendamustine) and monoclonal antibodies (for example, fully attractive target for apoptosis-based therapeutic strategies in CLL. humanized anti-CD20 ofatumumab) with cell death properties. Some of these strategies are already tested in clinical trials (for a review, see Kolb et al.5; Chen et al.6; Packham and Stevenson7; and THERAPEUTIC USE OF APOPTOSIS INDUCERS IN CLL Burger et al.9). Only a few apoptosis inducers have been tested in clinical trials with CLL patients. Flavopiridol has been investigated with different administration schedules (24–72 h i.v. infusions) but APOPTOSIS INDUCERS AND THEIR MECHANISMS OF ACTION the trials failed to produce consistent therapeutic responses.34 A large number of structurally diverse inducers of CLL cell apoptosis More successful results were recorded using a pharmacologically have been identified. They comprise various synthetic compounds based dosing schedule (30 min i.v. bolus followed with a 4-h such as proteasome inhibitors, histone deacetylase inhibitors, Bcl-2 continuous i.v. infusion) on refractory and genetically high-risk inhibitors and a variety of mainly plant-derived natural products such patients, providing improvement in progression-free survival.35 as flavonoids (whose prototype is flavopiridol),16 other polyphenols However, it remains to establish predictive markers of toxicity (for example, resveratrol, gossypol and catechins), triterpenoids, required for dose escalation.35 Gossypol was one of the first xanthones, combretastatin analogs and the phloroglucinol plant-derived compounds to be clinically tested in solid tumors hyperforin.5,17–22 When known, the molecular mechanisms of but preliminary data with its isomer AT-101 seem to indicate action involve either inhibition of antiapoptotic molecules or only limited therapeutic efficacy in CLL.36 Regarding histone stimulation of proapoptotic proteins. Most targets of apoptosis deacetylase inhibitors, strong adverse effects were observed inducers are Bcl-2 family members. with depsipeptide in a Phase I study37 and weak activity of MGD0103 was reported in a recent Phase II study.38 Although the Downregulation of antiapoptotic proteins proteasome inhibitor bortezomib is now used for the treatment of multiple myeloma and mantle cell lymphoma, and has other Numerous natural and synthetic inducers of apoptosis in CLL cells potential clinical indications, the sole published Phase II trial in CLL are known to inhibit the expression of prosurvival Bcl-2 family failed to produce objective responses.39 proteins and/or other antiapoptotic molecules (for example, One possible explanation for these disappointing clinical data is inducible nitric oxide synthase and IAP).5 Regarding the Bcl-2 that the molecular targets of apoptosis inducers are not that protein itself, chemical inhibitors including antisense oligo- specific. For example, the inhibitory effect of flavopiridol on nucleotides are able to trigger apoptosis.5,23 Furthermore, transcription affects not only Mcl-1 and XIAP but also the many downregulation of Mcl-1 expression is a mechanism shared by proteins with an inherently rapid turnover rate due to the several compounds. Flavopiridol (the first known and most potent presence of an 50-AUUUA-30 sequence in the 30-UTR region and/or apoptosis inducer in CLL cells)16 downregulates Mcl-1 mRNA a PEST sequence.24 Furthermore, it is clearly demonstrated that transcription through its ability to inhibit cyclin-dependent kinases both proteasome and histone deacetylase inhibitors enhance the required for RNA polymerase II activation (thus decreasing expression of a large number of proteins other than Noxa, transcription of short-lived proteins such as Mcl-1 and XIAP).24 including the crucial antiapoptotic Mcl-1.26,40 To counteract this Other cyclin-dependent kinases inhibitors (roscovitin/seliciclib, major drawback for therapeutic purposes, the combination with SNS-032) and certain translational inhibitors of short-lived proteins 26,28,40 Mcl-1 downregulators was proposed. (silvestrol, homoharringtonine) are known to trigger CLL cell Many apoptosis inducers have not yet been evaluated in the apoptosis through Mcl-1 downregulation.24 Moreover, various clinic. Plant-derived compounds might be considered such as plant-derived apoptosis inducers (the gossypol isomer AT-101, for certain polyphenols as well as terpenoids and xanthones which example)25 were also found to decrease Mcl-1 protein levels. were reported to display therapeutic activity in xenograft These and many other literature data indicate that inhibition models.19,41 Hyperforin may be of particular interest because it of prosurvival Bcl-2 proteins in general and Mcl-1 in particular induces Noxa expression without significantly modulating Mcl-1 is a pertinent strategy for developing CLL therapies based on protein levels.29 Several derivatives (such as aristoforin) have apoptosis induction. greater stability and solubility, and are more potent in vivo in animal models than hyperforin.42 Upregulation of proapoptotic proteins Other inducers of CLL cell apoptosis act through upregulation of BH3-only proteins that are key molecules in triggering the BCL-2 FAMILY INHIBITORS mitochondrial apoptosis pathway. Inhibitors of histone deacety- On the basis of preclinical data, the Bcl-2 antisense strategy, in lase upregulate the expression of Bim and Noxa proteins26 which Bcl-2 mRNA is targeted for degradation, has been one of through epigenetic de-repression. Acadesine increases Bim, Noxa the first to be clinically evaluated in CLL. The Bcl-2 antisense and Puma mRNA levels.27 Inhibitors of proteosomal degradation oligonucleotide oblimersen displayed only modest activity as a (with bortezomib as a prototype) enhance the stability and single agent in a PhaseI/II trial.43 Though it showed a survival accumulation of various proteins, including Noxa.28 Upregulation benefit when combined with FC (particularly for patients in of Noxa is a mechanism by which hyperforin induces CLL cell relapse),44 oblimersen was not approved by the US Food and Drug apoptosis—possibly through inhibition of proteasomal activity.29 Administration.45 It is noticeable that oligonucleotides 4625 In connection with this, some natural proteasome inhibitors (targeting both Bcl-2 and Bcl-xL) and, interestingly, ISIS 20408 capable of increasing Noxa expression (such as polyphenols and (targeting Mcl-1) were found to exert in vivo effects in xenogratft triterpenoids) are also apoptosis inducers.19,30,31 Moreover, models.46,47 upregulation of BH3-only proteins (Puma, Bim and Noxa) is The strategy aiming to inhibit the activity of the prosurvival Bcl- associated with the cell death activity of drugs such as fludarabine 2 family proteins has also been developed. Several synthetic and

Leukemia (2012) 2032 – 2038 & 2012 Macmillan Publishers Limited Novel BH3 mimetics for CLL therapy C Billard 2035 Table 1. Antagonists of prosurvival Bcl-2 family proteins as putative or true BH3 mimetics

Compound Targets Binding Bax/Bak BH3 mimetic Other mechanisms Preclinical/ afﬁnitya apoptosisb clinical data (CLL)

HA14-1 Bcl-2 Low No No Noxa induction, caspase- No independent, ROS generation BH3-I Bcl-xL Lowc No No Unknown No Antimycin A3 Bcl-2, Bcl-xL Low No No Noxa induction No Chelerythrine Bcl-xL Low/nod No No p38/JNK activation, PKC inhibition? No SAHBA Bcl-2, Bcl-xL High Yes True Unknown No ABT-737/ Bcl-2, Bcl-xL, Bcl-w Highe Yes True Bad-like Autophagy Yes/yes ABT-263 GX15-070 Bcl-2, Bcl-xL, Bcl-w, Low Partial/nod Putative pan Noxa induction, caspase-independent, Yes/yes Mcl-1, A1 BH3-only autophagy Gossypol Bcl-2, Bcl-xL, Bcl-w, Modest Slight/nod Putative pan Noxa induction, caspase-independent, Yes/no Bcl-B, Mcl-1, A1 BH3-only Autophagy, ROS generation AT-101 (gossypol Bcl-2, Bcl-xL, Bcl-w, Modest ND Putative Caspase independent Yes/yes isomer) Bcl-B, Mcl-1, A1 ApoG2 (gossypol Bcl-2, Bcl-xL, Mcl-1 Modest Slight/nod Putative Noxa induction Yes/no derivative) BI-97C1 (ApoG2 Bcl-2, Bcl-xL, Modest Partial Putative Noxa and Bim induction, autophagy No derivative) Mcl-1, A1 TW37 (gossypol Bcl-2, Bcl-xL, Mcl-1 Modest ND Putative Unknown Yes/no analog) S1 Bcl-2, Mcl-1 Modest Yes Putative Noxa induction No 072RB Bcl-xL, Mcl-1 High NDf Putative Unknown Yes/no e g BimS2A Mcl-1 High ND True Noxa-like Unknown No Mcl-1 SAHB Mcl-1 Highe NDh True Noxa-like Unknown No

a b Abbreviations: CLL, chronic lymphocytic leukemia; ND, not determined. Affinity: low, mM; modest, sub mM; high, nM. Based on Bax/Bak double deficiency. cNo binding into the hydrophobic groove but to another site of Bcl-xL. dDepending on the study. eCrystallographic analysis confirms the binding into the hydrophobic groove of prosurvival proteins. fOnly one study showing Bak and Bax activation. gApotosis only in Bcl-xL-deficient or Bad-expressing cells. hBak-dependent apoptosis.

natural inhibitors were identified: HA14-1 (the first to be charac- degradation, attempts were made to stabilize the a-helix. To this terized by Croce’s group)48 BH3-I, antimycin A3 or chelerythrine end, Korsmeyer’s group generated hydrocarbon-stapled peptides (Table 1). However, these compounds were subsequently found to that were helical and protease resistant; one such small organic exert cytotoxic effects independently of their interaction with the molecule, SAHBA (Table 1) derived from the BH3 domain of Bid, prosurvival proteins.49,50 In parallel, the concept of BH3 mimetics was found to induce apoptosis in leukemia cell lines and inhibit has emerged, that is, the design of agents that mimic the action the growth of human myeloid leukemia in an animal model.53 No of BH3-only proteins and thus inhibit the functional activity of further preclinical or clinical study with this interesting compound prosurvival Bcl-2 family proteins.51 This concept is described in has yet been published. more detail in the following section. Using a method based on nuclear magnetic resonance screening and structure/activity relationship to identify compounds that bind to the BH3-binding groove of Bcl-xL, Olsterdorf et al.54 THE BH3 MIMETIC CONCEPT discovered ABT-737, a small organic molecule that inhibits Bcl-2, This concept is based on the discovery that the BH3-only proteins Bcl-xL and Bcl-w with a binding affinity (nM range) markedly are specific antagonistic ligands of prosurvival Bcl-2 members, higher than previously reported compounds—even in the whose functional activity is to sequester the proapoptotic presence of human serum. Another study has shown that of members Bax and Bak.10 Once activated, a BH3-only protein seven putative BH3 mimetics tested (including HA14-1, BH3I-1, inserts its a-helical BH3 domain into the hydrophobic groove of a antimycin A and chelerythrine), only ABT-737 induces Bax/Bak- prosurvival protein. The design of small molecules capable of mediated apoptosis, demonstrating that ABT-737 is a true BH3 mimicking the BH3 domain thus enables prosurvival proteins mimetic (Table 1).49 Furthermore, ABT-737 does not bind Mcl-1 to be targeted directly, with neutralization of their functional and A1, indicating that it is a Bad-like BH3 mimetic.54 ABT-737 activity. This strategy usefully attacks the initial molecular events induces apoptosis in malignant cell lines and primary tumor responsible for triggering the mitochondrial apoptosis pathway. cells, and has potential therapeutic efficacy in human tumors in animal models54—but only when Mcl-1 is absent or impaired.49 Malignant cells overexpressing Mcl-1 are resistant to ABT-737 and DEVELOPMENT OF BH3 MIMETICS so combination therapy with Mcl-1 downregulators has been The first implementation of this novel apoptosis-based strategy suggested.49 was to design peptidomimetics of the BH3 domain, as for example Screening a natural product library led to the design of a small peptide derived from Bad (BH3-only protein that binds to GX15-070 (also known as obatoclax), an indol bipyrrole compound Bcl-2, Bcl-xL and Bcl-w) with affinity for Bcl-xL; however, these derived from progidiosin. GX15-070 was reported to bind to compounds proved to be unable to permeate into cells.51 Croce’s Bcl-2, Bcl-xL, Bcl-w and Mcl-1 (as well as Bcl-B and A1) but 55 group designed a Bad-derived cell-permeable small peptide with with rather low affinities (mM range). In fact, this putative BH3 in vitro proapoptotic effects and in vivo antileukemic activity.52 mimetic with a pan BH3-like behavior was found to disrupt Given that this type of peptide is prone to rapid proteolytic Mcl-1/Bak interaction and thus overcome resistance to ABT-737.55

& 2012 Macmillan Publishers Limited Leukemia (2012) 2032 – 2038 Novel BH3 mimetics for CLL therapy C Billard 2036 GX15-070 induces apoptosis, exerts antitumor effects in various GX15-070 and AT-101 have limited therapeutic activities when preclinical models and was the first putative BH3 mimetic to enter administered as single agents.36,68,69 It is not known whether clinical trials in oncology.56 However, it has been reported that these disappointing results are due to the fact that the GX15-070 acts rather by inducing Noxa activation, caspase- compounds are not bona fide BH3 mimetics. Regarding ABT-263, independent apoptosis and autophagy than through its putative somewhat promising data from ongoing trials in CLL were BH3 mimetic property (Table 1).57–59 reported.70,71 Although the final conclusions of these studies The natural polyphenol gossypol was already known for its have to be awaited, these preliminary data suggest that ABT-263 proapoptotic capacities and had already entered clinical trials may be of potential interest for CLL therapy—possibly resulting when it was found that this compound can displace the binding of from the ability of this true BH3 mimetic to neutralize not only Bcl- BH3 peptides to Bcl-xL and antagonize the antiapoptotic activity 2 but also Bcl-xL (Table 1). of the latter.60 In view of its ability to interact with all prosurvival Bcl-2 proteins (Bcl-2, Bcl-xL, Bcl-w, Bcl-B, Mcl-1 and A1) with a modest affinity (sub mM), gossypol resembles a pan-BH3 mimetic. BH3 MIMETICS AS THERAPEUTIC AGENTS FOR CLL It can induce apoptosis of primary CLL cells ex vivo17 and its Given that ABT-263 is a Bad-like but not a Noxa-like BH3 mimetic isomer AT-101 overcomes stroma-mediated Mcl-1 induction and with the ability to bind Mcl-1, a number of studies have proposed apoptosis protection.25 However, gossypol does not function the combination of this compound with agents capable of solely as a BH3 mimetic.49,57,59 The polyphenol was indeed found downregulating Mcl-1 expression for malignancies in which Mcl- to activate Noxa and trigger caspase-independent and autophagic 1 has a critical role in therapy resistance.49 However, the use of cell death (Table 1).17,57,61 Several more stable, less toxic pharmacological inhibitors of Mcl-1 would require highly complex derivatives of gossypol (notably TW37, ApoG2 and its derivative treatment schedules. An alternative, more direct strategy is to BI-97C1, see Table 1) are under investigation.62–64 develop small molecules that selectively mimic Noxa, that is, Other recently described putative BH3 mimetics include S1 having a BH3 domain capable of inserting into the groove of Mcl-1 (a synthetic heterocyclic compound) capable of inhibiting both to effectively antagonize its functional activity. This type of Noxa- Bcl-2 and Mcl-1, disrupting Bcl-2/Bax and Mcl-1/Bak interactions like BH3 mimetics is not presently available but might be of and inducing Bax/Bak-dependent apoptosis.65 A BH3 peptide considerable interest in CLL therapy, as (i) Mcl-1 is a crucial survival derived from Bim (a pan-BH3-only protein), named 072RB, elicites factor in this leukemia and (ii) the Mcl-1/ Noxa axis is an attractive primary CLL cell apoptosis and this effect is associated with target for apoptosis-based strategies. downregulation of both Bcl-xL and Mcl-1 and with stimulation of Bax and Bak activities (Table 1).66 NOVEL BH3 MIMETICS AS MCL-1 ANTAGONISTS Inspite of potential importance of this approach, attempts to NONE OF THE BH3 MIMETICS IS A DIRECT MCL-1 INHIBITOR design small BH3 peptides with the ability to engage and bind the The results of binding studies of various molecules designed to hydrophobic groove of Mcl-1 proved to be unsuccessful until a mimic BH3 domains indicate that only ABT-737 has a high binding few years ago. One possible explanation for this failure was affinity for prosurvival Bcl-2 ligands. In this connection, it was provided by a comparative crystallographic analysis of the reported that among six putative inhibitors of Bcl-2 family proteins hydrophobic grooves of Mcl-1 and Bcl-xL.72 The groove of Bcl-xL (including GX15-070, gossypol, ApoG2 and chelerythrine), only shows an extreme flexibility (with movements of helical segments ABT-737 induces Bax/Bak-mediated apoptosis.57 Another study in the binding site) enabling interaction with various ligands. In revealed that of seven putative BH3 mimetics, only ABT-737 contrast, the pocket of Mcl-1 is conformationally rigid—a feature inhibits Bcl-2 in a cell-based system.59 In contrast, the other that probably prevents first-generation BH3 ligands from entering.72 six compounds (gossypol, ApoG2, GX15-070, S1, HA14-1 and Another explanation relates to the fact that these ligands were antimycin A3) do not use the BH3 mimetic property to induce screened against soluble fragments of the target proteins, cell death, but employ another mechanism: they induce Noxa whereas it is now clear that Bcl-2 proteins change their confor- which can then bind to and antagonize Mcl-1.59 This explains the mation after insertion into the cell membranes (as reviewed in disruption of the Mcl-1/Bak complex detected in previous studies Shamas-Din et al.73). with for example S1 or GX15-070.55,65 These data (i) confirm that Nevertheless, two novel BH3 peptides capable of specifically ABT-737 is a bona fide BH3 mimetic in contrast to the other binding to Mcl-1 have recently been discovered. Firstly, a compounds and (ii) demonstrate that none of the putative pan- structure/function study performed to identify the functional BH3 mimetics GX15-070, gossypol, ApoG2 and S1 are direct Mcl-1 epitopes on the binding BH3 domain from Bim led to the inhibitors. characterization of a novel Bim BH3 variant (BimS2A) that is highly 74 Inasmuch as ABT-737 is a Bad-like BH3 mimetic unable to bind specific for Mcl-1. Like Noxa, BimS2A promotes Bak-dependent Mcl-1, it appears that there is no BH3 mimetic targeting cell death, but only when Bcl-xL is absent or neutralized (due to specifically Mcl-1 among the currently characterized compounds. the fact that Bak is sequestered by not only Mcl-1 but also Bcl-xL This does not mean that these compounds and their eventual as shown in Figure 2). Moreover, crystallographic experiments derivatives will be devoid of clinical interest. Besides, future confirmed that BimS2A engages within the groove of Mcl-1 studies will be able to determine whether some compounds (such (Table 1). In addition, the hydrophobic residues of the BH3 domain as 072RB or gossypol derivatives) can bind directly to Mcl-1 with a responsible for the interaction with the binding site of Mcl-1 have high affinity or induce Bax/Bak-dependent apoptosis in accor- been identified. This might enable the design of Noxa-like dance with the criteria defining a true antagonist of Bcl-2 family compounds with the ability to kill cells whose survival depends proteins suggested by Lessene et al.67 on Mcl-1.74,75 By screening a library of stabilized alpha-helix of Bcl-2 domains (SAHB), another group found that the BH3 helix of Mcl-1 (i) is itself BH3 MIMETICS IN THE CLINIC an exclusive and potent Mcl-1 inhibitor, (ii) dissociates the Mcl-1/ A few of the putative or actual BH3 mimetics have entered clinical Bak complex and (iii) sensitizes malignant cells to Bak and trials. Only three—GX15-070, AT-101 (gossypol isomer) and ABT- caspase-dependent apoptosis.76 A structural analysis also revealed 263 (navitoclax, the oral version of ABT-737)—are currently under key peptidic determinants allowing the generated compound to clinical evaluation in hematologic malignancies (including CLL).68 bind to Mcl-1. The characterization of this Mcl-1 SAHB (Table 1) According to published Phase I/II studies with CLL patients, both thus defines the structural and functional features of targeted

Leukemia (2012) 2032 – 2038 & 2012 Macmillan Publishers Limited Novel BH3 mimetics for CLL therapy C Billard 2037 76 Mcl-1 inhibition. Accordingly, the novel BH3 peptides, BimS2A epigallocatechin-3-gallate (EGCG), in B-cell chronic lymphocytic leukemia. Blood and Mcl-1 SAHB, are Mcl-1-specific antagonists capable of 2004; 104: 788–794. inducing apoptosis through a Noxa-like mechanism. 19 Kress CL, Konopleva M, Martinez-Garcia V, Krajewsla M, Lefebvre S, Hyer M et al. Triterpenoids display single agent anti-tumor activity in a transgenic mouse model of chronic lymphocytic leukemia and small B cell lymphoma. PLoS One CONCLUSION AND FUTURE DIRECTIONS 2007; 2:e559. 20 Menasria F, Azebaze AG, Billard C, Faussat AM, Nkengfack AE, Meyer M et al. Both BimS2A and Mcl-1 SAHB turn out to be BH3 mimetics targeting selectively Mcl-1. Their identification constitutes proof Apoptotic effects on B-cell chronic lymphocytic leukemia (B-CLL) cells of heterocyclic compounds isolated from Guttiferaes. Leuk Res 2008; 32: 1914–1926. of concept that this type of compound can be designed and 21 Billard C, Menasria F, Quiney C, Faussat AM, Finet JP, Combes S et al. provides the molecular basis for developing the novel therapeutic 4-arylcoumarin analogues of combretastatins stimulate the apoptosis of agents for cancers in which Mcl-1 has a crucial role. Eagerly leukemic cells from chronic lymphocytic leukemia patients. Exp Hematol 2008; awaited additional studies will have to establish whether these 36: 1625–1633. compounds may trigger apoptosis in primary CLL cells and exert 22 Quiney C, Billard C, Faussat AM, Salanoubat C, Ensaf A, Naı¨t-Si Y et al. Pro- antileukemic effects in animal models of this leukemia. Lastly, apoptotic properties of hyperforin in leukemic cells from patients with chronic clinical evaluation of the impact of Mcl-1-specific BH3 mimetics on lymphocytic leukemia. 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