Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define...

R E S EARCH ART I C L E

CANCER

Exploiting selective BCL-2 family inhibitors to dissectcell survival dependencies and define improvedstrategies for cancer therapyJoel D. Leverson,1* Darren C. Phillips,1 Michael J. Mitten,1 Erwin R. Boghaert,1 Dolores Diaz,2

Stephen K. Tahir,1 Lisa D. Belmont,2 Paul Nimmer,1 Yu Xiao,1 Xiaoju Max Ma,2† Kym N. Lowes,3,4

Peter Kovar,1 Jun Chen,1 Sha Jin,1 Morey Smith,1 John Xue,1 Haichao Zhang,1 Anatol Oleksijew,1

Terrance J. Magoc,1 Kedar S. Vaidya,1 Daniel H. Albert,1 Jacqueline M. Tarrant,2 Nghi La,2

Le Wang,1 Zhi-Fu Tao,1 Michael D. Wendt,1 Deepak Sampath,2 Saul H. Rosenberg,1 Chris Tse,1

David C. S. Huang,3,4 Wayne J. Fairbrother,2 Steven W. Elmore,1 Andrew J. Souers1

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The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignan-cies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopeniacaused by BCL-XL inhibition. This prompted the generation of the BCL-2–selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown toenhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has beenlimited by neutropenia. We used venetoclax and the BCL-XL–selective inhibitors A-1155463 and A-1331852 toassess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxelcombination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting forthe exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selec-tively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxelwhen tested against a range of solid tumors. Therefore, BCL-XL–selective inhibitors have the potential to enhancethe efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. Thesestudies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight theirpotential as improved cancer therapeutics.

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INTRODUCTION

Multicellular organisms have evolved molecular mechanisms to elim-inate cells that are no longer required or that have become compro-mised through environmental insults. This active process of programmedcell death, or apoptosis, is especially important for eliminating cells withthe potential for malignant transformation. The ability to suppressapoptosis under conditions of cell stress has been defined as one ofthe hallmarks of a cancer cell (1), and thus, triggering apoptosis in can-cer cells represents a powerful therapeutic approach.

Apoptosis is regulated by a family of closely related proteins exem-plified by BCL-2 (B cell lymphoma protein 2), the first family memberdiscovered. BCL-2 family proteins are defined by one to four BCL-2homology motifs (BH1 to BH4) and can be subdivided into pro- andantiapoptotic subsets. Proapoptotic proteins include the BH3-only pro-teins such as BIM, BAD, BID, and NOXA, and the BH1 to BH4 pro-teins BAK and BAX, which serve as the ultimate effectors of apoptosisby oligomerizing to form pores in the mitochondrial outer membrane.BCL-2 and the closely related antiapoptotic proteins BCL-XL andMCL-1 can sequester their proapoptotic counterparts by binding to theirBH3 motifs and thereby inhibit the initiating steps of programmed celldeath (2–4).

1AbbVie Inc., North Chicago, IL 60064, USA. 2Genentech Inc., South San Francisco, CA 94080,USA. 3Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.4Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia.*Corresponding author. E-mail: [email protected]†Present address: Department of Genomics and Oncology, Roche Molecular Systems Inc.,4300 Hacienda Drive, Pleasanton, CA 94588, USA.

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Small molecules that mimic the BH3 motif have been developedwith the aim of binding antiapoptotic proteins, liberating proapoptoticproteins, and triggering apoptosis in cancer cells, many of which arethought to be “primed for death” due to the expression of high levels ofpro- and antiapoptotic protein complexes (5). ABT-737 and the re-lated orally bioavailable compound ABT-263 (navitoclax) are BH3 mi-metics that bind with high affinity to BCL-2, BCL-XL, and BCL-W,but not to MCL-1 (6, 7). Preclinically, navitoclax inhibits tumor growthas a single agent (8) and in combination with standard-of-care therapeu-tics such as gemcitabine, vincristine, and docetaxel (9, 10). In clinical studies,navitoclax exhibited objective antitumor activity in lymphoid malig-nancies but also induced rapid, reversible, and dose-dependent throm-bocytopenia that was dose-limiting in this setting (11, 12). Navitoclax-inducedthrombocytopenia was found to be a consequence of inhibiting BCL-XL

(13, 14), and this prompted the development of ABT-199/GDC-0199(venetoclax), a BCL-2–selective inhibitor that maintains efficacy in hema-tologic tumor models but spares platelets (15). Venetoclax has shownpromising signs of clinical activity in hematologic malignancies, demon-strating objective responses in chronic lymphocytic leukemia (CLL)(16) and non-Hodgkin’s lymphoma (17).

Navitoclax has also been evaluated for the treatment of solid tumorsin combination with chemotherapeutics. When combined with docetax-el, febrile neutropenia was the most commonly observed dose-limitingtoxicity for navitoclax (18). However, it has remained unclear whetherthis effect is driven by the inhibition of BCL-2, BCL-XL, or both proteins.We reasoned that, if the contributions of BCL-XL and BCL-2 inhibitioncould be defined, an improved strategy to minimize toxicity and thus

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maximize antitumor activity might be identified. To this end, we gen-erated the BCL-XL–selective inhibitors A-1155463 (19) and A-1331852to complement the BCL-2–selective agent venetoclax and its close an-alog A-1211212. Equipped with this toolkit, we now had the opportu-nity to chemically dissect the biological activities of navitoclax andattribute them to the inhibition of BCL-2, BCL-XL, or both targets.

In the studies described here, we use these inhibitors to define theroles of BCL-2 and BCL-XL in maintaining the survival of multiple hema-tologic and solid tumor cell lines. Using a variety of in vitro, ex vivo,and in vivo model systems, we also parse the contributions of BCL-2and BCL-XL inhibition to the antitumor efficacy and the myelotoxicityobserved when navitoclax is combined with docetaxel. Our data indicatethat BCL-XL inhibition is sufficient to recapitulate the efficacy ofnavitoclax in combination with docetaxel, whereas BCL-2 inhibitionlikely accounts for navitoclax-related neutropenia. Just as venetoclax rep-resented an improvement over the efficacy/toxicity profile of navitoclaxfor hematologic malignancies, so BCL-XL–selective inhibitors may bebetter tolerated, and thus enable greater efficacy, when combined withchemotherapeutic agents for the treatment of solid tumors.

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RESULTS

Selective BCL-2 family inhibitors parse the activity ofnavitoclax into BCL-2– and BCL-XL–dependent componentsNavitoclax most closely mimics the BH3-only protein BAD, bindingto BCL-2 (Ki = 0.044 nM) and BCL-XL (Ki = 0.055 nM) with highaffinity and more weakly to BCL-W (Ki = 21 nM; Table 1). Becauseits affinity for BCL-W is >300-fold weaker, the biological effects ofnavitoclax are most likely due to the inhibition of BCL-2, BCL-XL, orboth of these targets (Fig. 1A). Whereas venetoclax enables one to probespecifically for BCL-2–dependent phenomena, a BCL-XL–selective coun-terpart was needed to parse the effects of navitoclax more complete-ly. We used the recently described BCL-XL–selective inhibitor WEHI-539(20) as an effective lead structure to generate a more potent and chemi-cally stable molecule, A-1155463 (19) (Fig. 1A). A-1155463 binds toBCL-XL with high affinity (Ki < 0.010 nM) but has much weakeraffinity for BCL-2 (Ki = 74 nM), BCL-W (Ki = 8 nM), and MCL-1 (Ki >444 nM) (Table 1). As predicted by this binding profile, A-1155463disrupts BCL-XL–BIM but not BCL-2–BIM complexes in cells (Fig. 1B).A-1155463 kills BCL-XL–dependent Molt-4 cells (EC50 = 70 nM) but

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has no measurable cytotoxicity against BCL-2–dependent RS4;11 cells(EC50 > 5 mM) (Table 1). A-1155463 induces the hallmarks of apopto-sis, as evidenced by the release of cytochrome c from mitochondria,caspase activation, and the accumulation of caspase-dependent sub–G0-G1 DNA content in BCL-XL–dependent H146 cells (8, 15) (Fig. 1,C to E). In the absence of the essential apoptosis effector proteins BAKand BAX, no toxicity was observed with A-1155463 (fig. S1), thus exclud-ing any major off-target cytotoxicity. As anticipated (20–22), potentkilling of murine embryonic fibroblasts (MEFs) was observed only whenMcl-1 was deleted (fig. S1), demonstrating that this compound acted in ahighly specific manner. Together, venetoclax and A-1155463 provide theability to separate the activity of navitoclax into its respective BCL-2–and BCL-XL–dependent components.

As a first implementation of this chemical toolkit, we investigatedthe roles of BCL-2 and BCL-XL in mediating the survival of cancer celllines with known sensitivity to navitoclax. Small cell lung cancer (SCLC)cell lines have shown sensitivity to navitoclax that is inversely corre-lated with the expression of MCL-1 (23). Because BCL2 copy numbergains were observed in many of these cell lines (24), it was reasonable toinfer that this activity was due to the inhibition of BCL-2. Indeed,among a panel of 11 SCLC cell lines, two with BCL2 amplification,NCI-H889 and NCI-H211, were sensitive (EC50≤ 1.0 mM) to venetoclaxbut not to A-1155463 (Fig. 2A and Table 2), indicating that these celllines were BCL-2–dependent. However, most navitoclax-sensitive SCLClines either were more sensitive to the BCL-XL–selective inhibitor alone(NCI-H446, NCI-H847, NCI-H1417, and NCI-H1836) or required in-hibition of both BCL-2 and BCL-XL. For example, navitoclax killedNCI-H345, NCI-H69, DMS79, and NCI-H1048 more potently than dideither selective inhibitor alone. Notably, the combination of venetoclaxand A-1155463 was able to recapitulate the effect of navitoclax in thissetting, exhibiting synergistic killing of NCI-H69 (Bliss sum = 730 ± 26)and NCI-H345 (Bliss sum = 603 ± 89) cells (Fig. 2B). NCI-H187 cellswere susceptible to all three inhibitors, indicating that inhibition of eitherBCL-2 or BCL-XL alone was sufficient to trigger their killing. These datademonstrate the utility of venetoclax and A-1155463 for determiningwhether BCL-2 or BCL-XL is necessary and sufficient for the survivalof a given cell line. Furthermore, they indicate that BCL-2 and BCL-XL

do not function redundantly in all cellular contexts.We performed similar parsing studies in a panel of 24 acute myeloid

leukemia (AML) cell lines, 9 of which were sensitive to venetoclax(EC50 ≤ 0.1 mM) (Fig. 2C and Table 2), consistent with a recent report

Table 1. Biochemical and cellular activity of small-molecule BCL-2 familyinhibitors. For each compound listed, binding affinities (Ki) for BCL-2 familyproteins were calculated in time-resolved fluorescence resonance energy

transfer (TR-FRET) assays, and the effects on cell viability [median effectiveconcentration (EC50)] were assessed in BCL-2–dependent (RS4;11) or BCL-XL–dependent (Molt-4) cancer cell lines. All values are in nanomolar units.

BCL-XL/BCL-2
BCL-2–selective
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BCL-XL–selective

Navitoclax
A-874009 Venetoclax A-1211212 A-1155463
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A-1331852

BCL-2 TR-FRET
0.044 0.048 <0.010 <0.010 74 6
BCL-XL TR-FRET
0.055 0.463 48 14 <0.010 <0.010
MCL-1 TR-FRET
>224 >3900 >444 >444 >444 142
BCL-W TR-FRET
21 2 245 852 8 4
Molt-4 viability
303 134 >5000 >5000 70 6
RS4;11 viability
112 28 8 6 >5000 >5000
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(25). Most AML cell lines (14 of 24) were more sensitive to venetoclaxthan to a BCL-XL–selective inhibitor. Cell lines bearing the JAK2V617F mutation (UKE-1, SET-2, and HEL) were a notable exception,showing sensitivity to the BCL-XL–selective inhibitor but not to venetoclax.Again, the inhibition of both BCL-2 and BCL-XL was required for theeffective killing of some cell lines, including KG-1 and SKM-1. Like

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BCL-2/BCL-XL co-dependent SCLC cell lines,KG-1 (Bliss sum = 892 ± 22) and SKM-1(Bliss sum = 997 ± 154) showed synergisticsensitivity to the combination of venetoclaxand A-1155463 (Fig. 2D). OCI-AML3,NOMO-1, and ME-1 cells were resistant toall three inhibitors, potentially implicatingother BCL-2 family members in mediatingtheir survival. Indeed, MCL-1 was shown tomaintain the survival of OCI-AML3 whentreated with ABT-737 or venetoclax (25).

Synergistic killing of cancer celllines by the navitoclax-docetaxelcombination is driven byBCL-XL inhibitionABT-737 and navitoclax have been shownto synergize with taxanes in killing a vari-ety of cancer cell lines (10, 26–28). To dissectthe contributions of BCL-2 and BCL-XL

inhibition to the activity of this combination,we tested panels of breast cancer, non–smallcell lung cancer (NSCLC), and ovarian can-cer cell lines with combinations of docetaxeland navitoclax, venetoclax, or A-1155463.To assess potential combination activity, weused the Bliss independence model (28–30),according to which negative integers indi-cate antagonism, a value of zero indicatesadditive activity, and positive integers in-dicate synergy. Bliss scores were calculatedfor each combination in the dose matrixand then totaled to give a “Bliss sum”(Table 3). For the purposes of these studies,Bliss sum values >150 were considered in-dicative of synergy. The navitoclax-docetaxelcombination demonstrated synergistic kill-ing of several breast cancer cell lines (fig.S2) and enhanced the induction of pro-grammed cell death, as indicated by ele-vated caspase cleavage, poly(ADP-ribose)polymerase cleavage, and the accumulationof cells with sub–G0-G1 DNA content (fig.S3). The Bliss sum values for the A-1155463–docetaxel combinations showed a significantcorrelation with those of the navitoclax-docetaxel combinations when comparedamong breast cancer (r = 0.75, P < 0.0001,n = 28) (Fig. 3A) or NSCLC (r = 0.94, P <0.0001, n = 15) (Fig. 3B) cell lines. However,venetoclax-docetaxel Bliss values showed nocorrelation to those calculated for navitoclax-

docetaxel. In addition, the navitoclax-docetaxel and A-1155463–docetaxelcombinations exhibited synergistic killing of four of six ovarian cancer celllines tested (Bliss sum > 150), but no synergy was observed with thevenetoclax-docetaxel combination (fig. S4). Collectively, these data in-dicate that BCL-XL is the key target of navitoclax for inducing the syn-ergy observed with docetaxel in these solid tumor models.

Fig. 1. Selective BCL-2 family inhibitors enable the functional dissection of the effects of navitoclax.(A) Chemical structures and selectivity profiles of BH3mimetics used for in vitro studies. ABT-263 (navitoclax)
inhibits both BCL-2 and BCL-XL, whereas ABT-199 (venetoclax) selectively inhibits BCL-2 and A-1155463 se-lectively inhibits BCL-XL. (B) Quantitative measurement of BCL-XL–BIM and BCL-2–BIM complexes in Molt-4cells after 4-hour treatments with increasing concentrations of A-1155463. Data represent the average oftriplicate experiments, with error bars indicating the SD. (C) Cytochrome c levels present in NCI-H146 mito-chondrial and cytosolic fractions as determined by immunoblotting after treatments with increasing con-centrations of A-1155463, venetoclax, or navitoclax for 4 hours. (D) Caspase-3/7 activation in NCI-H146 cellsafter incubation with increasing concentrations of A-1155463 for 4 hours. Data represent the average oftriplicate experiments, with error bars indicating the SD. (E) Apoptosis (sub–G0-G1 accumulation) as assessedby fluorescence-activated cell sorting in NCI-H146 cells after 1-hour preincubation plus orminus the caspaseinhibitor Z-VAD-fmk (75 mM) and an additional 24 hours of incubation with increasing concentrations ofA-1155463. Data represent the average of duplicate experiments, with error bars indicating the SD.
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Orally bioavailable BCL-XL–selective inhibitor A-1331852enhances the efficacy of docetaxel in vivoWe next assessed the ability of a selective BCL-XL inhibitor to enhancethe efficacy of docetaxel in vivo. To this end, we used structure-based


design to generate A-1331852 (Fig. 4A), aBCL-XL–selective inhibitor with oral bio-availability. A-1331852 is a potent BCL-XL

inhibitor, binding BCL-XL with a Ki valueof <0.010 nM and demonstrating cellularactivity 10- to 50-fold more potent thanA-1155463 and navitoclax, respectively(Table 1). This molecule selectively disruptsBCL-XL–BIM complexes and induces thehallmarks of apoptosis in BCL-XL–dependentMolt-4 cells with median inhibitory con-centration (IC50) values in the low nano-molar range (Fig. 4, B to E, and Table 1)but does not affect MEF cells lacking BAKor BAX (fig. S5).Moreover, A-1331852 dem-onstrates antitumor efficacy in the Molt-4xenograft model, inducing tumor regres-sions as a single agent (Fig. 4F). Addition-ally, A-1331852 combines with venetoclaxto recapitulate the efficacy of navitoclaxin the NCI-H1963.FP5 xenograft modelof SCLC (Fig. 4G), thus providing in vivoconfirmation of the combination studiesshown in Fig. 2 (B and D).

Inhibition of tumor growth by A-1331852combined with docetaxel was determinedin seven subcutaneous xenograft modelsof solid tumors, including breast cancer,NSCLC, and ovarian cancer. Given as asingle agent, A-1331852 significantly (P <0.05) inhibited tumor growth in all sevenmodels (Table 4). Although its single-agentactivity was modest (TGImax < 60% in fiveof seven models), A-1331852 increased theefficacy of docetaxel in all seven models.As shown in Table 4, the maximum tumorgrowth inhibition (TGImax) for A-1331852as a single agent ranged between 34%(OVCAR-5) and 67% (A549-FP3). Themostdurable response to A-1331852 was a tumorgrowth delay (TGD) of 108%, observed inthe A549-FP3 model. This indicates thatthe median time required for the tumorsto reach a volume of 1 cm3 is about twiceas long when treated with A-1331852 ascompared to a sham-treated control. Whencomparing the combination to the most ef-fective single-agent treatment, the increase inamplitude and durability of the responsewas statistically significant (P < 0.05) infive of seven models. The effect was mostpronounced in the MDA-MB-231 LC3me-tastatic breast cancer model (Fig. 5A) andthe NSCLC models NCI-H1650 (Fig. 5B)

and NCI-H358 (Table 4). Overall, the single-agent and combination treat-ments were well tolerated by mice, without overt signs of toxicity or weightloss of >9%. These data demonstrate that BCL-XL inhibition alone canenhance the efficacy of docetaxel in a variety of solid tumor models.

Fig. 2. Venetoclax and A-1155463 define the BCL-2 family dependence profile of cancer cell lines.(A) SCLC cell lines were incubated with increasing concentrations of navitoclax (BCL-2/BCL-XL), venetoclax
(BCL-2–selective), or A-1155463 (BCL-XL–selective) for 48 hours before assessing cell viability. Cell killingEC50 values are plotted for each compound against the cell lines examined. (B) NCI-H69 and NCI-H345cells were incubated with increasing concentrations of A-1155463 in the presence or absence of venetoclaxfor 48 hours before assessing cell viability. (C) AML cell lines were treated as in (A) before assessing cellviability. Cell killing EC50 values are plotted for each compound against the cell lines examined. (D) KG-1 andSKM-1 cells were incubated with increasing concentrations of A-1155463 in the presence or absence ofvenetoclax for 48 hours before assessing cell viability.
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BCL-2 inhibition suppresses granulocyte colony formationand decreases circulating neutrophil countsAlthough the navitoclax-docetaxel combination is efficacious in pre-clinical studies, neutropenia has limited the dosing of this combination

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in the clinic (18). Using the toolkit of dual and selective inhibitors, wenext asked if BCL-2 or BCL-XL inhibition alone might be sufficient tosuppress granulopoiesis in colony-forming assays. Each compound wasincubated with isolated human bone marrow cells cultured in semisolidmedium over a period of 2 weeks. Venetoclax replicated the dose-dependentreduction in granulocyte colony formation caused by navitoclax, butthe BCL-XL–selective inhibitor A-1155463 had little or no effect (Fig. 6A).Likewise, navitoclax and venetoclax showed greater inhibition of gran-ulocyte colony formation than did A-1155463 when combined withdocetaxel (Fig. 6B).

To examine these effects in vivo, we evaluated orally bioavail-able BCL-2 family inhibitors, including A-874009 (a close analog ofnavitoclax), A-1211212 (a close analog of venetoclax), and A-1331852(Fig. 4A). Male Sprague-Dawley rats were dosed daily with each com-pound for 5 days, either alone or in combination with a single doseof docetaxel (5 mg/kg), before assessing their circulating platelet andneutrophil counts. Docetaxel decreased neutrophil counts significantly,with reductions between 61% (P < 0.01) and 71% (P < 0.01) relative tothe mean of the vehicle control groups (Fig. 6C). Rats treated with thedual BCL-2/BCL-XL inhibitor A-874009 or the BCL-2–selective in-hibitor A-1211212 had reduced neutrophils as well, in both casesshowing a reduction of 41% relative to the respective vehicle-treatedgroups (P < 0.01). However, rats dosed with the BCL-XL–selective in-hibitor A-1331852 showed no inhibition of granulopoiesis and ex-hibited increased neutrophil counts. When combinations of docetaxelwith A-874009, A-1211212, or A-1331852 were compared to docetaxelalone, the differences in neutrophil inhibition did not reach statis-tical significance (P = 0.383, 0.981, and 0.773, respectively). Thedual BCL-2/BCL-XL and BCL-XL–selective inhibitors both inducedsignificant reductions in circulating platelets (P < 0.01), though theBCL-2–selective compound A-1211212 did not (Fig. 6C). This is con-sistent with BCL-XL maintaining platelet survival and suggests thatthe lack of neutrophil inhibition was not due to insufficient expo-sure to A-1331852.

These studies indicate that BCL-2 inhibition accounts for the ex-acerbation of docetaxel-induced neutropenia caused by navitoclax (18)and support the hypothesis that BCL-XL–selective inhibitors will avoiddose-limiting neutropenia in this setting. However, because BCL-XL–selective inhibitors will still affect platelets, a question remained whetherefficacious levels of BCL-XL inhibition can be achieved in combinationwith docetaxel before thrombocytopenia becomes dose-limiting. Wetherefore analyzed data from phase 1 clinical trials of navitoclax, in-cluding single-agent studies in subjects with lymphoid malignancies(11) or solid tumors (31) and a navitoclax-docetaxel combination studyin subjects with advanced solid tumors (18). No apparent pharmaco-kinetic (PK) interactions were observed between navitoclax and docetaxelin the latter study (18). Moreover, coadministration with docetaxel didnot lead to substantial increases in navitoclax-induced thrombocytopeniarelative to navitoclax treatment alone (fig. S6). However, the maximumtolerated dose (MTD) of navitoclax when given in combination withdocetaxel was limited to 150 mg/day because of febrile neutropenia,which was observed at navitoclax exposures as low as 50.7 mg*hour/ml(AUC0–inf) (18) (table S1). This is in contrast to the single-agent setting,where MTDs of 315 or 325 mg/day were achieved before thrombocy-topenia became dose-limiting (11, 31). These doses correspond to meanexposures of 80.5 ± 44.3 mg*hour/ml and 91.0 ± 33.5 mg*hour/ml(AUC0–inf), which overlap with highly efficacious exposures of navitoclaxdetermined in preclinical studies (7) (Table 4). Because the PK of

Table 2. Cellkillingactivityof small-moleculeBCL-2 family inhibitors.SCLCand AML cell lines were incubatedwith increasing concentrations of navitoclax,venetoclax, or A-1155463 for 48 hours before assessing cell viability. Cell killingEC50 values are listed for each compound against the cell lines examined.

Cell type
Cell line
CellTiter-Glo cell viability assay

NavitoclaxEC50 (mM)

VenetoclaxEC50 (mM)

A-1155463EC50 (mM)

SCLC
NCI-H889 0.053 0.015 6.310
NCI-H211
0.100 0.330 1.350
NCI-H187
0.037 0.850 0.180
NCI-H345
0.300 1.200 >10.000
NCI-H446
0.850 2.400 0.200
NCI-H1048
0.900 2.700 >10.000
NCI-H69
0.180 3.860 3.200
NCI-H847
0.140 3.900 0.003
NCI-H1417
0.100 4.100 0.007
DMS79
0.440 4.620 1.440
NCI-H1836
0.260 5.000 0.015
AML
GDM-1 0.011 0.100 1.170
EOL-1
0.011 0.100 2.250
MOLM-13
0.030 0.100 >5.000
HL-60
0.040 0.100 >5.000
MV4-11
0.040 0.100 >5.000
ML-2
0.050 0.100 >5.000
SIG-M5
0.011 0.100 >5.000
OCI-AML2
0.011 0.100 >5.000
MOLM-16
0.120 0.040 >5.000
OCI-AML5
0.180 0.110 >5.000
THP-1
0.450 0.820 >5.000
Kasumi-1
0.030 1.000 0.011
KG-1
0.110 1.200 >5.000
HNT-34
0.030 1.700 0.680
PL-21
0.190 1.810 >5.000
SKM-1
0.240 2.530 >5.000
UKE-1
0.050 3.060 0.490
SET-2
0.060 3.330 0.080
HEL
0.070 3.370 0.120
OCI-M2
0.160 4.050 0.100
OCI-M1
0.410 >5.000 0.230
OCI-AML3
>5.000 >5.000 >5.000
NOMO-1
>5.000 >5.000 >5.000
ME-1
>5.000 >5.000 >5.000



Table 3. Bliss synergy assessment of cell killing by BCL-2 family in-hibitors combined with docetaxel. Breast cancer, NSCLC, and ovariancancer cell lines were cultured in the presence of navitoclax, A-1155463,or venetoclax plus or minus docetaxel for 72 hours before assessing cellviability. The sum of Bliss scores across the combination dose matrix islisted for each cell line.

Celltype

continue

Cellline

d

Bliss sums

Navitoclax +docetaxel

A-1155463 +docetaxel

Venetoclax +docetaxel

Breastcancer(9 × 3dosematrix)

MDA-MB-231
264 277 −159
MDA-MB-231 (LC3)
253 305 −62
MDA-MB-175 (VII)
−19 290 −130
HCC1806
269 224 −46
MX-1
−43 53 −10
15
HCC1395 80 −31 −109
, 20

HCC1428
153 −203 −204
19
HCC38 67 42 −201
rch
ZR-75-1 271 103 −312
a
HCC1569 193 185 −102
on M

HCC1500
165 146 15
rg
MCF-7 (Luc) 100 63 −133
g.o
MDA-MB-361 66 110 −40
ma
SK-BR-3 83 101 −78
ce
UAC812 −105 −269 −210
ien

BT-474
5 −5 −43
.sc

HCC1187
99 0 −173
stm
HCC1937 220 332 −106
m

DU4475
242 229 168
fro

Hs578T
99 62 −161
ed
T-47D 289 250 −219
load
ZR-75-30 −290 −265 −418
wn
BT-20 126 260 −106
Do
HCC2218 −134 −250 −135
HCC1590
40 −105 −245
BT-549
41 117 −76
HCC1143
45 195 −215
MDA-MB-436
−110 −96 −386
NSCLC(5 × 5dosematrix)

LXFL529
656 734 236
SW1573
354 461 0
SKMES-1
174 152 80
A549
144 137 −19
H1650
335 312 146
H1975
69 6 −23
H1299
280 302 67
H23
95 97 6
H522
195 242 −19
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Bliss sums

Navitoclax +docetaxel

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A-1155463 +docetaxel

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Venetoclax +docetaxel

H661
109 94 −54
H460
128 105 15
H1568
−27 27 −29
H838
63 128 −6
EBC-1
−107 −103 18
H1703
1 29 6
Ovariancancer(9 × 3dosematrix)

OVCAR-3
635 683 −334
OVCAR-4
−33 −129 −231
OVCAR-5
511 706 92
OVCAR-8
247 159 −67
SKOV3
487 747 −53
IGROV1
33 −49 −191
Fig. 3. BCL-XL inhibition drives synergistic killing of solid tumor celllines in combination with docetaxel. (A and B) Panels of (A) breast
cancer (n = 28) and (B) NSCLC (n = 15) cell lines were cultured in the pres-ence of navitoclax, venetoclax, or A-1155463 plus or minus docetaxel for72 hours before assessing cell viability. The sum of Bliss scores across thecombination dose matrix was calculated for each cell line. Bliss sums wereplotted for navitoclax-docetaxel combinations versus the venetoclax-docetaxelcombinations or the A-1155463–docetaxel combinations. A significant correla-tion was observed between navitoclax-docetaxel and A-1155463–docetaxelBliss sums for both the breast cancer (Spearman: 0.75, P < 0.0001) and NSCLC(Spearman: 0.94, P < 0.0001) cell line panels.
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navitoclax is linear over the 150- to 325-mg dose range (11), these dataindicate that large increases in navitoclax exposure could be obtainedin combination with docetaxel if neutropenia were avoided. The dis-

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covery of BCL-XL–selective inhibitors like A-1331852 thus representsan opportunity to maximize BCL-XL inhibition and improve efficacywhen dosed in combination with docetaxel.

Fig. 4. Orally bioavailable inhibitorA-1331852 enables the functional character-
ization of BCL-XL in vivo. (A) The chemicalstructures and selectivity profiles of BH3 mi-metics used for in vivo studies are depicted.A-874009 inhibits both BCL-2 and BCL-XL,whereas A-1211212 selectively inhibits BCL-2and A-1331852 selectively inhibits BCL-XL. (B)Quantitative measurement of BCL-XL–BIMand BCL-2–BIM complexes in Molt-4 cellsafter 4-hour treatments with increasingconcentrations of A-1331852. Data representthe average of triplicate experiments, witherror bars indicating the SD. (C) Cytochromec levels present in Molt-4 mitochondrial andcytosolic fractions as determined by immu-noblotting after 4-hour treatments withincreasing concentrations of A-1331852.(D) Caspase-3/7 activation in Molt-4 cells af-ter incubation with increasing concentrationsof A-1331852 for 4 hours. Data represent theaverage of triplicate experiments, with errorbars indicating the SD. (E) Exposure of phos-phatidylserine as determined by annexin Vstaining of Molt-4 cells after 1-hour preincu-bation plus or minus the caspase inhibitorZ-VAD-fmk (75 mM) and an additional 24 hoursof incubation with increasing concentra-tions of A-1331852. Data represent the av-erage of triplicate experiments, with errorbars indicating the SD. (F) Mice bearingMolt-4 T cell acute lymphocytic leukemia–xenografted tumors were treated with a ve-hicle control (solid gray circles), venetoclax(daily for 14 days) at 100 mg/kg (open bluecircles), or A-1331852 (twice a day for 14 days)at 25 mg/kg (open red circles). The pointsof each curve reflect the average volume of10 tumors. Error bars indicate the SD of themeans. (G) Mice bearing subcutaneous xe-nografts of NCI-H1963.FP5 were treated withvehicle control (solid gray circles), navitoclax(daily for 14 days) at 100 mg/kg (open greencircles), venetoclax (daily for 14 days) at50 mg/kg (open blue circles), A-1331852(twice a day for 14 days) at 25 mg/kg (openred circles), or a combination of the latter twocompounds (solid purple circles). The pointsof each curve reflect the average volume offive tumors. Error bars indicate the SD.



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DISCUSSION

The work that led to the discovery and functional characterization ofBCL-2 family proteins relied on a variety of cell and molecular biologyapproaches, genetically engineered mouse models, and RNA interference–based methods. With the synthesis of the first validated BH3 mimetics,


ABT-737 and navitoclax (6, 7), came theability to inhibit BCL-2 and BCL-XL di-rectly with cell-permeable small molecules,facilitating a host of new discoveries. Sinceits introduction, the BCL-2–selective in-hibitor ABT-199/GDC-0199 (venetoclax)has been used to define the role of BCL-2in specific hematopoietic lineages (32), a va-riety of hematologic cancers (25, 33–38),and even some solid tumors, including es-trogen receptor–positive breast cancer (39).In addition, certain roles for BCL-XL havebeen deduced using a subtractive parsingmethod that compares the effects of ABT-737 or navitoclax to those of venetoclax(38, 40–42). For example, Bah and co-workersused ABT-737 and venetoclax to demon-strate that BCL-XL inhibition is requiredfor the killing of breast cancer cell lines incombination with paclitaxel (41). Althoughthis approach is informative, these inhibi-tors alone cannot distinguish between celllines that are co-dependent on BCL-2 andBCL-XL for survival and those that relysolely on BCL-XL for survival. With thegeneration of the BCL-XL–selective inhib-itors described here, it is possible to inter-rogate the role of BCL-XL directly in vitroand in vivo and to determine whether itsselective inhibition is sufficient for a giv-

en effect. This is demonstrated by the studies in Fig. 2, which definethe contributions of BCL-2 and BCL-XL in maintaining the survival ofSCLC and AML cell lines. When used together, the small-moleculeBH3 mimetics described here represent a powerful toolkit for dissect-ing the roles of BCL-2 and BCL-XL and for defining more effectivetherapeutic strategies. As these molecules find broader use in the research

Table 4. Inhibition of tumor xenograft growth by administration ofA-1331852, docetaxel, or the combination. TGImax = 100 (1 − Tv/Cv), whereTv and Cv are the mean tumor volumes of the treated and control groups,respectively. TGD is the extended period of time that a treated tumor re-quires to reach a volume of 1 cm3 relative to the control group. TGD = 100

(T/C − 1), where T and C are the median time periods required for the treatedand control groups, respectively, to reach 1 cm3. Each treatment groupof NCI-H747, A549-FP3, EBC-1, and OVCAR-5 models consisted of five mice.Each treatment group of the NCI-H358 model consisted of eight mice. Eachtreatment group of NCI-H1650 and MDA-MB-231 LC3 consisted of ten mice.

Xenograft
A-1331852 Docetaxel
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TGImax
TGD TGImax TGD TGImax
Vol 7 Issue 279 279ra40

TGD

NCI-H1650
48* 33* 82* 52* 98*† 170*†
MDA-MB-231 LC3
55* 55* 73* 82* 93*† 141*†
NCI-H747
50* 33* 52* 113* 84* 167*†
A549-FP3
67* 108* 71* 108* 74* 131*
EBC-1
52* 22* 76* 44* 88*† 94*†
OVCAR-5
34* 54* 39* 54 57*† 85*†
NCI-H358
62* 80* 58* 47* 91*† 233*†
*Significantly different from sham-treated control group. †Significantly different from most effective component of the combination as a single agent.

Fig. 5. BCL-XL–selective inhibitor A-1331852 enhances the efficacy of docetaxel in vivo. The growthinhibition of established tumors in SCID-bg mice is illustrated. Each graph describes the change of tumor
volume (mm3, ordinate) as a function of the time after initiation of treatment (days, abscissa). Each pointon the curves represents the mean volume of 10 tumors. Error bars depict the SD. A-1331852 wasadministered orally and docetaxel was administered intravenously for all studies. (A) MDA-MB-231 LC3metastatic breast cancer xenograft. Vehicle control (solid gray circles), docetaxel (DTX) (once) at 7.5 mg/kg (openblue circles), A-1331852 (daily for 14 days) at 25 mg/kg (open red circles), and combination of docetaxel(once) at 7.5 mg/kg and A-1331852 (daily for 14 days) at 25 mg/kg (solid purple circles). (B) NCI-H1650NSCLC xenograft. Vehicle control (solid gray circles), docetaxel (once) at 7.5 mg/kg (open blue circles),A-1331852 (daily for 14 days) at 25 mg/kg (open red circles), and combination of docetaxel (once)at 7.5 mg/kg and A-1331852 (daily for 14 days) at 25 mg/kg (solid purple circles).
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community, they should facilitate a more complete understanding ofBCL-2 family biology and the roles these proteins play in normal tissuesand disease states.

Antiapoptotic BCL-2 family proteins have been widely studied incancer cells, where they are often overexpressed and maintain survivalby sequestering large amounts of their proapoptotic counterparts, astate that has been referred to as being primed for death (5). An ele-gant peptide-based technique referred to as “BH3 profiling” has beenused extensively to probe these interactions and to determine the pro-file of BCL-2 family dependence for a host of tumor cell types (43).One limitation of this method is the lack of highly selective BH3 pep-tides for targeting certain BCL-2 family members, which can make itdifficult to fully define their roles. The discovery of selective BH3 mi-metics like venetoclax, A-1155463, A-1331852, and cell-active MCL-1–selective inhibitors (44) will now enable a form of chemical BH3profiling, whereby one can determine precisely which BCL-2 familymembers are required for the survival of any given cell population.The ability of these molecules to penetrate live cells also obviatesthe need for detergent-based permeabilization associated with peptide-based methods and enables one to probe BCL-2 family dependencyby conducting simple cell viability assays. Furthermore, navitoclax,venetoclax, and A-1331852 are orally bioavailable and sufficiently po-tent to interrogate BCL-2 family member dependence in vivo, makingthem especially attractive translational tools.

Despite major investments into the discovery and development ofsmall-molecule therapeutics, many compounds still fail in the clinic be-cause of a lack of efficacy (45). In some cases, this may be due tooff-target activities causing dose-limiting toxicities that preclude theachievement of efficacious exposures. This emphasizes the need tocarefully dissect a molecule’s target inhibition profile and to better un-derstand the roles each target plays in efficacy and toxicity. As demon-strated here, selective BCL-XL inhibitors can offer important advantagesover their less selective predecessor, navitoclax, for the treatment of

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solid tumors. BCL-XL–selective inhibition enhanced the efficacy ofdocetaxel in breast cancer, NSCLC, and ovarian cancer models, butit did not inhibit granulopoiesis assessed ex vivo or in vivo. In con-trast, BCL-2–selective inhibition suppressed granulopoiesis to a similarextent as was observed with dual BCL-2/BCL-XL inhibitors. AlthoughBcl-2 knockout mice show no overt signs of defective granulopoiesisbefore succumbing to polycystic kidney disease (46, 47), bone marrowfrom Bcl-2−/− reconstituted mice shows a reduced capacity for granu-locyte colony formation, especially when cultured in the absence ofcytokines such as interleukin-3 and stem cell factor (48). Moreover,neutropenia has been observed in a portion of CLL subjects receivingvenetoclax (16).

Fig. 6. BCL-2–selective inhibition suppresses granulopoiesis ex vivoand reduces circulating neutrophil counts in vivo. (A) Isolated human
bone marrow cells were used to perform granulocyte colony-forming as-says in the presence of increasing concentrations of navitoclax (BCL-2/BCL-XL),venetoclax (BCL-2–selective), or A-1155463 (BCL-XL–selective). Neutrophilcolonies were enumerated by light microscopy after 15 to 16 days of cul-ture. (B) Neutrophil colony formation was assessed as in (A) after treatmentof human bonemarrow samples with increasing concentrations of navitoclax,venetoclax, or A-1155463 ± 5 nM docetaxel. Colonies were enumeratedafter 14 days of growth in culture. All data in (A) and (B) represent themeans of triplicate experiments, with error bars indicating the SD. Student’st tests compared test compound single-agent effects to solvent control sam-ples. For combination experiments, test compounds in combination withdocetaxel were compared to samples treated with docetaxel alone. (C) Groups(n = 10 per group) of male Sprague-Dawley rats were dosed with docetaxel(5 mg/kg, intravenously, once), the BCL-2/BCL-XL inhibitor A-874009 (30 mg/kg,orally, daily for 5 days), the BCL-2–selective inhibitor A-1211212 (50 mg/kg,orally, daily for 5 days), the BCL-XL–selective inhibitor A-1331852 (7 mg/kg, oral-ly, daily for 5 days), or their respective vehicles. Groups were also dosedwith combinations of docetaxel and the various BCL-2 family inhibitors.Box and whisker plots depict the means (n = 10) and ranges for total neu-trophil and platelet counts assessed in blood collected on day 6. Tukey-Kramer tests compared vehicle- and compound-treated groups at a 5%significance level. Significant reductions in neutrophils or platelets relativeto vehicle controls (P < 0.01) are denoted by asterisks.



Navitoclax clinical data indicated that, if exacerbated neutropeniain combination with docetaxel could be avoided, higher exposures andmore complete inhibition of BCL-XL might be achieved before throm-bocytopenia becomes dose-limiting. However, these analyses werelimited by the small number of subjects (n = 7) with both exposureand hematology data available. In addition, our in vivo studies of BCL-XL–selective inhibitors were limited to rodent models, and so the possibil-ity of encountering dose-limiting toxicities other than thrombocytopeniain the clinic cannot be ruled out. Nevertheless, the data presented hereindicate that BCL-XL–selective inhibitors have the potential for im-proved safety and efficacy profiles, and provide further impetus for ex-ploring this concept in the clinic.

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www.sciencetranslationalmedicine.org/cgi/content/full/7/279/279ra40/DC1Materials and MethodsFig. S1. BCL-XL–selective inhibitor A-1155463 kills Mcl-1−/− MEF cells but not Bak−/− Bax−/− MEFs.Fig. S2. Navitoclax synergizes with docetaxel to kill breast cancer cell lines.Fig. S3. Navitoclax-docetaxel combination kills breast cancer cell lines by inducing apoptosis.Fig. S4. Selective BCL-XL inhibition suffices for synergy with docetaxel in ovarian cancer cell lines.Fig. S5. BCL-XL–selective inhibitor A-1331852 kills Mcl-1−/− MEF cells but not Bak−/− Bax−/− MEFs.Fig. S6. Relationship between exposure and platelet reduction is similar for navitoclax with orwithout docetaxel.Table S1. Plasma exposures and platelet effects of navitoclax-docetaxel combinations.References (49–51)

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Acknowledgments: We thank J. Damen and M. Huber of StemCell Technologies for technicaladvice and performing granulocyte colony-forming assays. We thank D. Sheinson forperforming statistical analyses. We also thank M. Mabry for helpful discussions and coiningthe phrase “chemical parsing.” Funding: Work in the Huang Lab is supported by grants andfellowships from the Australian National Health and Medical Research Council (researchfellowships to D.C.S.H.; project grants to D.C.S.H.; program grants 461219, 461221, and1016701; and Independent Research Institutes Infrastructure Support Scheme grant361646), the Cancer Council Victoria (grant-in-aid to D.C.S.H.), the Leukemia and LymphomaSociety (Specialized Centers of Research grants), the Australian Cancer Research Foundation,and a Victorian State Government Operational Infrastructure Support grant. Author contribu-tions: J.D.L. designed experiments, oversaw biology experiments, and wrote the manuscript.D.C.P. designed and performed breast cancer experiments in vitro, oversaw biologyexperiments, and wrote the manuscript. M.J.M., A.O., and T.J.M. performed in vivo efficacy experiments.K.S.V., E.R.B., D.H.A., and D.S. designed, oversaw, and analyzed in vivo efficacy experiments. D.D. de-signed and oversaw rat studies. J.M.T. and N.L. performed rat studies. L.D.B. designed and oversawNSCLC experiments in vitro. K.N.L. performed ovarian cancer studies in vitro. P.K. performed bio-chemical affinity assessments of all compounds. Y.X. performed breast cancer experiments in vitro.X.M.M. performed AML experiments in vitro. P.N., S.J., J.X., J.C., and H.Z. performed biological characteriza-tions of BCL-XL–selective inhibitors in vitro. M.S. and S.K.T. designed colony-forming experiments andperformed SCLC experiments in vitro. L.W., Z.-F.T., and M.D.W. designed and synthesized compounds.C.T., D.C.S.H., andW.J.F. oversaw biology efforts. S.H.R. and S.W.E. oversaw chemistry and biology efforts.A.J.S. designed compounds, oversaw chemistry and biology efforts, and wrote the manuscript. Allauthors contributed to the interpretation of data and to the review and editing of the manuscript.Competing interests: J.D.L., D.C.P., M.J.M., E.R.B., J.C., P.N., S.K.T., J.X., P.K., M.S., A.O., T.J.M., K.S.V., D.H.A.,Y.X., H.Z., L.W., Z.T., M.D.W., C.T., S.H.R., S.W.E., and A.J.S. are employees of AbbVie and hold companystock. D.D., L.D.B., J.M.T., N.L., D.S., and W.J.F. are employees of Genentech and hold company stock.Financial support for this research was provided by AbbVie and Genentech. AbbVie, Genentech, andWalter and Eliza Hall Institute of Medical Research participated in the design and conduct of studies,interpretation of data, and review and approval of the publication. Patents cover all of the small mo-lecules described in this study. Data and materials availability: ABT-263 (navitoclax), ABT-199 (ve-netoclax), A-874009, A-1155463, A-1211212, and A-1331852 may be obtained through appropriatematerial transfer agreements. Please address compound requests to [email protected].

Submitted 9 December 2014Accepted 13 February 2015Published 18 March 201510.1126/scitranslmed.aaa4642

Citation: J. D. Leverson, D. C. Phillips, M. J. Mitten, E. R. Boghaert, D. Diaz, S. K. Tahir,L. D. Belmont, P. Nimmer, Y. Xiao, X. M. Ma, K. N. Lowes, P. Kovar, J. Chen, S. Jin, M. Smith,J. Xue, H. Zhang, A. Oleksijew, T. J. Magoc, K. S. Vaidya, D. H. Albert, J. M. Tarrant, N. La, L. Wang,Z.-F. Tao, M. D. Wendt, D. Sampath, S. H. Rosenberg, C. Tse, D. C. S. Huang, W. J. Fairbrother,S. W. Elmore, A. J. Souers, Exploiting selective BCL-2 family inhibitors to dissect cell survivaldependencies and define improved strategies for cancer therapy. Sci. Transl. Med. 7, 279ra40(2015).

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DOI: 10.1126/scitranslmed.aaa4642, 279ra40 (2015);7 Sci Transl Med et al.Joel D. Leverson

dependencies and define improved strategies for cancer therapyExploiting selective BCL-2 family inhibitors to dissect cell survival

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effects seen with less selective drugs. (one member of this protein family) is effective for killing tumors, but without the common sideLinhibiting BCL-X

. have used a toolkit of BCL-2 family inhibitors with different specificities to show that specificallyet alNow, Leverson neutropenia.Unfortunately, some of these drugs are also associated with dose-limiting hematologic toxicities, such as

a great deal of interest in developing drugs that can inhibit the antiapoptotic members of the BCL-2 pathway.anticancer treatments often hinges on the ability to induce cancer cell death by apoptosis. As a result, there has been

The BCL-2 family is a group of related proteins that regulate apoptosis in a variety of ways. The success of

A more refined antitumor strategy

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