A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2–interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence–specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.

The BCL-2 family interaction network lies at the crossroads of the cell’s life-death decision (1, 2). Among the oncogenic signals that drive the development and maintenance of cancer, the pathologic reprogramming of the BCL-2 family interaction network has emerged as a formidable barrier to modern day anticancer treatment. Antiapoptotic members of the BCL-2 family contain a surface hydrophobic groove that can bind, sequester, and neutralize the critical BH3 death domains of proapoptotic members (3). Small molecules, such as ABT-737 and ABT-263, that selectively inhibit the antiapoptotic proteins BCL-2 and BCL-X_L induce apoptosis of tumors that are especially dependent upon this subset of survival proteins (4, 5). However, the compounds are rendered ineffective by cellular expression of alternate antiapoptotic proteins, such as MCL-1 and BFL1/A1, which lie outside the molecules’ range of binding specificity or by the relative or absolute absence of proapoptotic effectors, such as BAX and BAK (6, 7). Thus, a pharmacologic quest is underway to develop next-generation agents that simulate broader spectrum BH3-dependent killing activities and effectively deactivate the deflector shields of relapsed and refractory cancers.

Chief among the killer BH3-only proteins, Bcl-2–interacting mediator of cell death (BIM) exhibits the most broad-ranging BCL-2 protein interactions, engaging all of the antiapoptotic proteins with high affinity (8, 9) and also directly triggering death effectors, such as BAX (10, 11). A recent genetic analysis elegantly demonstrated that only BIM BH3, but not other BH3 sequences inserted into the BIM protein, could rescue the leukocyte accumulation and autoimmune phenotype of Bim^–/– mice (12). These data emphasize the unique killing potency of BIM BH3, which may derive from its higher-affinity interactions with antiapoptotics, dual capacity to block antiapoptotics and directly activate proapoptotics, and/or engagement of yet uncharacterized proteins that contribute to induction of cell death. From a clinical translational standpoint, BIM induction appears essential for cell death responses to a host of chemotherapeutic agents, and BIM suppression by genetic deletion, miRNA-based suppression, or protein interaction-based neutralization or degradation accounts for the emergence of drug resistance (13–15). Therefore, harnessing the BIM BH3 helix to promote a death response and, in particular, chemical replacement of this death functionality, in the context of drug resistance, are logical pharmacologic goals (16–18).

We previously developed stabilized α helix of BCL-2 domains (SAHBs), which are structurally stable, protease-resistant, and cell-permeable compounds capable of targeting BCL-2 family proteins with high affinity (19, 20). Here, we examined the pharmacologic potential of BIM SAHB_A, a stapled BIM BH3 helix that contains an i, i+4 all-hydrocarbon crosslink spanning positions 154 and 158 (designated “A”), to restore cell death in the context of formidable and clinically relevant apoptotic resistance. We found that BIM SAHB_A reactivates cell death in a BH3 sequence–dependent fashion in vitro and in vivo and, importantly, preferentially affects cells driven by a deregulated apoptotic pathway.

BIM SAHB_A binds with high affinity to a broad range of BCL-2 family proteins. To explore the breadth of BIM BH3 activity, we generated a hydrocarbon-stapled BIM BH3 helix (amino acids 146–166) (19) and a negative control peptide based on an R153D reverse polarity mutation within the core BH3 sequence (ref. 10, Figure 1A, and Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI46231DS1). Circular dichroism analysis demonstrated that BIM SAHB_A and BIM SAHB_A(R153D) exhibit enhanced α-helical structure in solution compared with that of the corresponding unmodified BIM BH3 peptide (ref. 19 and Supplemental Figure 1), effectively recapitulating the native structure of this domain when bound to apoptotic targets (21–23). Equilibrium fluorescence polarization (FP) binding analysis using FITC-βAla-BIM SAHB_A peptides and recombinant and tagless BCL-X_LΔC, MCL-1ΔNΔC, BCL-wΔC, and BFL1/A1ΔC revealed 1–11 nM K_d values for BIM SAHB_A across antiapoptotic targets, whereas R153D mutagenesis consistently reduced binding activity (Figure 1, B–E). We previously demonstrated, both by FP (19) and NMR (10), that BIM SAHB_A and an analog optimized for structural analysis (amino acids 145–164), respectively, also bind to proapoptotic BAX. In contrast to the static interaction between BH3 domains and their canonical binding pocket on antiapoptotic proteins, the BIM BH3 helix engages a geographically distinct BH3-binding site on BAX (10), triggering a major structural reorganization that underlies the dynamic BAX activation process (24). Indeed, combining BIM SAHB_A with recombinant BAX triggered dose-responsive BAX-mediated liposomal release, linking the direct binding interaction to BAX’s transformation from an inactive monomer to a functional pore (Figure 1F). Importantly, the single R153D point mutation at the binding interface abrogated the BAX-triggering activity of BIM SAHB_A in this assay (Figure 1G). As a further measure of specificity, BIM SAHB_A–triggered and BAX-mediated liposomal release was blocked in the presence of antiapoptotic proteins BCL-X_LΔC, MCL-1ΔNΔC, BCL-wΔC, and BFL-1/A1ΔC (Supplemental Figure 2). Thus, BIM SAHB_A engages a diversity of multidomain BCL-2 family proteins, consistent with the established broad-spectrum interaction profile of native BIM protein (8–10, 19).

Figure 1

Broad-spectrum BCL-2 family binding activity of a stapled BIM BH3 helix. (A) Sequence composition of BIM SAHB_A and its R153D mutant. “X” denotes the position of the nonnatural amino acid within each peptide. (B–E) BIM SAHB_A binds to a broad range of antiapoptotic targets with high affinity. R153D mutagenesis shifts the isotherms to the right, reflecting the impairment of binding activity. Data are mean ± SEM for experiments performed at least in triplicate. mP, units of millipolarization. (F and G) BIM SAHB_A triggers dose-responsive BAX-mediated liposomal release, whereas the R153D mutant has no effect. The liposomal release assays were performed in triplicate with similar results.

BIM SAHB_A blocks antiapoptotic sequestration of the BAX/BAK BH3 helices and triggers BAX/BAK-dependent mitochondrial cytochrome c release. An established paradigm for overcoming the apoptotic blockade of refractory cancer involves pharmacologic derepression of sequestered proapoptotic BCL-2 family members by competitive binding to antiapoptotic targets (8, 25–27). To explore the capacity of BIM SAHB_A to function as a derepressor compound, we performed competition assays using FITC-βAla-BAX SAHB_A/BCL-X_LΔC (K_d, 58.6 nM) and FITC-βAla-BAK SAHB_A/MCL-1ΔNΔC (K_d, 12.3 nM) (Figure 2, A and B). In each case, BIM SAHB_A dose responsively competed with the death effector BH3 helix for antiapoptotic binding, whereas BIM SAHB_A(R153D) was a relatively weak competitor (Figure 2, C and D).

Figure 2

BIM SAHB_A dissociates inhibitory antiapoptotic interactions and directly triggers BAX/BAK-dependent mitochondrial cytochrome c release. (A and B) The antiapoptotic proteins BCL-X_LΔC and MCL-1ΔNΔC, respectively, sequester the proapoptotic BH3 helices of BAX and BAK in high-affinity complexes. (C and D) BIM SAHB_A effectively dissociates the inhibitory complexes, whereas R153D mutagenesis markedly impairs competitive binding. (E) BAX/BAK-deficient MLM (mouse liver mitochondria) undergo cytochrome c release in response to combination treatment with recombinant BAX (50 nM) and increasing doses of BIM SAHB_A. The specificity of action is highlighted by (a) blockade of the effect by added recombinant BCL-X_LΔC (2 μM), (b) loss of activity upon R153D mutagenesis, and (c) the inability of BAX and BIM SAHB_As to influence cytochrome c release when administered as single agents. (F) To monitor BAK-dependent cytochrome c release, WT MLM that contain native BAK were exposed to BIM SAHB_A and its R153D mutant. BIM SAHB_A triggered BH3 sequence–dependent and BAK-dependent cytochrome c release, with little to no effect on the corresponding BAX/BAK-deficient MLM in the 0–200 nM dosing range. Data are mean ± SEM for experiments performed at least in triplicate.

To examine the functional impact of the broad targeting activity of BIM SAHB_A in the mitochondrial context, we performed cytochrome c release assays in the presence of BAX (Alb-cre^posBax^fl/–Bak^–/– mouse liver mitochondria [MLM] with added recombinant BAX) or BAK (WT MLM) or without either effector protein (Alb-cre^posBax^fl/–Bak^–/– MLM). Applying a 0–200 nM dosing range, BIM SAHB_A selectively triggered recombinant BAX- and native BAK-dependent cytochrome c release, with little to no release observed with BIM SAHB_A(R153D) or in the absence of BAX and BAK (Figure 2, E and F). Thus, we demonstrate that low nanomolar BIM SAHB_A has the capacity to broadly engage BCL-2 family targets to selectively trigger BH3 sequence–dependent and BAX/BAK-dependent mitochondrial apoptosis.

BIM SAHB_A activates caspase-3/7 and induces cell death in resistant hematologic cancer cells. We examined the utility of BIM SAHB_A in overcoming the apoptotic blockades of refractory cancer by testing a series of hematologic cancer cell lines that are relatively resistant to ABT-737 (5, 6, 28, 29), a selective BCL-2/BCL-X_L inhibitor whose activity is dampened by cellular expression of antiapoptotic proteins that lie outside its binding spectrum (4, 6, 7, 30). We first documented that BIM SAHB_A and its mutant derivative exhibit dose-equivalent uptake by the panel of cancer cell lines, which included OCI-AML3 (acute myeloid leukemia [AML]), U937 (monocytic leukemia/lymphoma), OPM-2 (multiple myeloma), Pfeiffer (diffuse large B cell lymphoma), and K562 (chronic myelogenous leukemia) cells (Figure 3A). Cultured cells were treated with vehicle or FITC-derivatized BH3 peptides (1 μM) in serum-free media for 2 hours, followed by centrifugation, trypsinization (to digest surface proteins and thereby eliminate potential nonspecific surface binding), washing, lysate preparation, electrophoresis, and fluorescence scan detection. Importantly, FITC-BIM SAHB_A and FITC-BIM SAHB_A(R153D) were present at similar levels within the cellular lysates, whereas the vehicle- and unmodified FITC-BIM BH3–treated cells showed no signal (Figure 3A). Live cell confocal microscopy of FITC-SAHB–treated cells further confirmed cellular uptake of the stapled peptides (Figure 3B). Whereas FITC-BIM SAHB_A largely colocalized with mitochondria, the predominant site of BCL-2 family targets (31), the R153D point mutant that exhibits substantially impaired binding activity showed little to no such colocalization. We further confirmed that at the micromolar dosing levels used for cellular studies, BIM SAHB_A peptides were solubilized as uniform, monomeric species, as assessed by gel filtration chromatography (Supplemental Figure 3).

Figure 3

Cellular uptake and localization of BIM SAHB_As. (A) Cultured cancer cells were exposed to FITC derivatives of BIM BH3 peptides (1 μM) for 2 hours, followed by centrifugation, trypsinization, washing, lysate preparation, electrophoresis, and fluorescence detection. FITC-BIM SAHB_A and its R153D point mutant were present at similar levels in the cellular lysates, whereas no uptake was observed for the unmodified FITC-BIM BH3 peptide. (B) Live cell confocal microscopy of U937 cancer cells treated with FITC-BIM SAHB_As (1 μM, 2 hours) demonstrated the uptake and intracellular distribution of the peptides. FITC-BIM SAHB_A, but not the impaired R153D mutant, largely colocalizes with mitochondria, the site of BCL-2 family targets. Nuclei (Hoechst), SAHBs (FITC), mitochondria (MitoTracker), and the BIM SAHB_A/mitochondria colocalization (FITC/MitoTracker) are shown in blue, green, red, and yellow, respectively. An example of the BIM SAHB_A/mitochondria colocalization is highlighted by the white arrow. Original magnification, ×100.

Cancer cells were then exposed to serial dilutions of vehicle, BIM SAHB_A, and BIM SAHB_A(R153D), and the effect of treatment was monitored by cell viability assay, annexin V binding FACS analysis, and caspase-3/7 activation assay (Figure 4). In each case, BIM SAHB_A impaired cancer cell viability in a dose-responsive and sequence-dependent manner (IC₅₀, 1.8–3.4 μM), an effect that correlated with both annexin V positivity and caspase-3/7 activation. When applied to nonmalignant cells that also demonstrated efficient uptake of BIM SAHB_A (Supplemental Figure 4), such as the human fibroblast cell line WS1 and WT and Bax^–/–Bak^–/– (DKO) mouse embryo fibroblasts (MEFs), neither BIM SAHB_A nor BIM SAHB_A(R153D) had any cytotoxic or caspase-3/7 activation effects in the same 0–10 μM dosing range (Figure 5). Only when increasing the dose to 20 μM do we begin to observe a decrease in WS1 cell viability, which coincides with caspase-3/7 activation (Supplemental Figure 5A); in adherent MEFs, this dose escalation does not impact cell viability, but a mild elevation in caspase-3/7 activation is observed in WT MEFs but not DKO MEFs, consistent with a BAX/BAK-dependent proapoptotic mechanism (Supplemental Figure 5, B and C). To confirm that the relative lack of toxicity of human BIM SAHB_A in murine fibroblasts did not derive from cross-species variation, we repeated the experiment with the corresponding murine BIM SAHB_As (Supplemental Table 1) and obtained similar results (data not shown). Thus, these data indicate that at low micromolar dosing, BIM SAHB_A activates the death pathway in a variety of hematologic cancer cells that are otherwise resistant to selective BCL-2/BCL-X_L inhibition (6, 7). Importantly, the relative resistance of nonmalignant fibroblasts to BIM SAHB_A treatment suggested that a therapeutic window may exist for BIM BH3 replacement in vivo, with preferential toxicity to cells driven by deregulated apoptosis.

Figure 4

Sequence-dependent cytotoxicity of BIM SAHB_A in hematologic cancer cells resistant to selective BCL-2/BCL-X_L inhibition. BIM SAHB_A dose responsively reduces the viability of (A) OCI-AML3, (B) U937, (C) OPM-2, (D) Pfeiffer, and (E) K562 cancer cells, as measured at 24 hours by CellTiter-Glo assay (left column). R153D mutagenesis markedly impairs the cytotoxic activity of BIM SAHB_A. Cells exposed to BIM SAHB_A, but not its R153D mutant, exhibit dose-dependent annexin V binding at 6 hours after treatment, as monitored by FACS analysis (middle column). The negative effect of BIM SAHB_A on cancer cell viability correlates with dose-responsive activation of caspase-3/7, as assessed by monitoring the cleavage of proluminescent caspase-3/7 substrate at 6 hours after treatment (right column). Data are mean ± SEM for experiments performed at least in triplicate. RLU, relative luminescence units.

Figure 5

Viability of human and mouse fibroblasts exposed to BIM SAHB_A peptides. WS1 human fibroblasts showed no response to BIM SAHB_A peptides (0–10 μM), as assessed by (A) viability and (B) caspase-3/7 activation assays performed at 24 and 6 hours after treatment, respectively. Staurosporine (100 nM), which impaired viability and induced caspase-3/7 activation, served as a positive control. Adherent WT and DKO MEFs likewise showed no response to BIM SAHB_A treatment in this dose range, as assessed by (C and E) viability and (D and F) caspase-3/7 activation assays. As a positive control, (C and D) staurosporine (100 nM) triggered caspase-3/7 activation and reduced viability of WT MEFs, (E and F) whereas DKO MEFs exhibited relative resistance to staurosporine with no caspase-3/7 activation, as reported previously (50). Data are mean ± SEM for experiments performed at least in triplicate.

Mechanism of BIM SAHB_A–induced cytotoxicity. To further probe the protein interaction-based mechanism of BIM SAHB_A activity, we treated OCI-AML3 cells with BIM SAHBs (0–10 μM) and monitored for disruption of the inhibitory MCL-1/BAK complex (32) and conversion of BAX to its conformationally activated form (10) by immunoprecipitation with antibodies to MCL-1 and the BAX activation epitope (6A7) (33), respectively. Cellular treatment with BIM SAHB_A, using the same dose range that induced cytotoxicity, annexin V binding, and caspase-3/7 activation (Figure 4), triggered both dose-responsive dissociation of MCL-1/BAK (Figure 6A) and exposure of the BAX activation epitope (Figure 6B), consistent with pharmacologic induction of the mitochondrial apoptotic pathway. Importantly, BIM SAHB_A(R153D) showed no such effects. In accordance with the observed BIM SAHB_A–induced biochemical changes, OCI-AML3 cells likewise manifested the morphologic features of apoptosis, as reflected by cellular contraction, chromatin condensation and marginalization, and nuclear fragmentation of Wright-Giemsa–stained cytospun cells (Figure 6C).

Figure 6

BIM SAHB_A treatment induces biochemical and morphological changes that reflect a proapoptotic mechanism of action. (A) OCI-AML3 cells treated with BIM SAHB_A (0–10 μM) manifest dose-responsive dissociation of the native BAK/MCL-1 complex, as assessed by MCL-1 immunoprecipitation and anti-BAK Western analysis. No effect is observed upon treatment with BIM SAHB_A(R153D). (B) BIM SAHB_A (0–10 μM) likewise triggers BAX activation, as monitored by BAX immunoprecipitation using the 6A7 antibody, which selectively recognizes BAX’s N-terminal activation epitope. R153D mutagenesis abrogates the effect. (C) A Wright-Giemsa stain of BIM SAHB_A–treated OCI-AML3 cells (5 μM) revealed cellular shrinkage, chromatin condensation and marginalization, and nuclear fragmentation, all hallmark morphological features of apoptosis induction (arrowheads). Vehicle- and BIM SAHB_A(R153D)–treated cells showed no such effects. Original magnification, ×100; third column, additional ×15 zoom. (D) BIM SAHB_A (25 mg/kg/d i.v.) suppressed tumor growth compared with vehicle (n = 7 per arm; 2-way ANOVA, P = 0.018), as assessed by in vivo bioluminescence imaging of a subcutaneous OCI-AML3 tumor xenograft model. Data are mean ± SEM. *P < 0.05.

Tumor suppressive activity of BIM SAHB_A as a single agent and in combination. To extend these findings to an in vivo context, we tested the efficacy of BIM SAHB_A in an OCI-AML3 human xenograft model (34). OCI-AML3-LucNeo cells (5 × 10⁶ cells) were implanted subcutaneously with 30% Matrigel into the right hind flank of 6-week-old female NOD scid gamma (NSG) mice. Mice with equivalent disease, as determined by bioluminescent imaging on day 8 after inoculation, were divided into 2 groups (n = 7 per arm), treated daily with either i.v. vehicle (5% DMSO/D5W) or BIM SAHB_A (25 mg/kg), and imaged on days 0, 4, 7, and 10. Throughout the treatment period, mice receiving BIM SAHB_A manifested a statistically significant suppression of tumor growth compared with that of vehicle-treated animals, as reflected by an approximately 50% decrease in relative bioluminescence (2-way ANOVA, P = 0.018) (Figure 6D).

We next explored whether the single agent activity of BIM SAHB_A could be combined with other proapoptotic agents to enhance the antitumor response. BIM SAHB_A was combined with either TRAIL, an extrinsic apoptotic pathway stimulus, or ABT-737, an intrinsic apoptotic pathway stimulus, in synergy analyses (35) performed on OCI-AML3 cells. The cancer cells were treated with serial dilutions of BIM SAHB_A, TRAIL, or ABT-737 or the combination of BIM SAHB_A and TRAIL or BIM SAHB_A and ABT-737, followed by viability assay at 24 hours (Figure 7, A and B). In each case, synergy was observed, as reflected by combination indices of less than 1 at the 50%, 75%, and 90% effective doses. To determine whether a drug combination could enhance the antitumor activity of BIM SAHB_A in vivo, we repeated the OCI-AML3 xenograft study (n = 8 per arm), examining the impact of adding the orally bioavailable BCL-2/BCL-X_L inhibitor ABT-263 (5, 36) (100 mg/kg/d per orem [PO]) to either BIM SAHB_A or its point-mutant control BIM SAHB_A(R153D) (25 mg/kg/d i.v.). Mice were treated for 15 days with vehicle, the single agent SAHB, or the combination of SAHB with ABT-263, and bioluminescence was measured on days 0, 3, 7, 10, 13, and 16. As previously observed (Figure 6D), single agent BIM SAHB_A suppressed tumor growth throughout the treatment period (P = 0.0299) (Figure 7C). Single R153D point mutagenesis abrogated this effect, with BIM SAHB_A(R153D) treatment showing no statistically significant tumor suppressive activity (P = 0.3934). The addition of ABT-263 to BIM SAHB_A yielded the most significant antitumor response (P = 0.0009), whereas combining ABT-263 with BIM SAHB_A(R153D) did not achieve a significant antitumor effect (P = 0.1982) (Figure 7C). Importantly, neither BIM SAHB_A nor BIM SAHB_A/ABT-263 combination treatment adversely affected peripheral white blood cell counts (Supplemental Figure 6), and no weight loss was observed among the treatment groups (Figure 7D). Taken together, the in vivo efficacy data are consistent with BIM BH3 sequence–dependent antitumor activity, both as a single agent and in combination with ABT-263, in an otherwise aggressive and relatively resistant xenograft model of human AML.

Figure 7

Antitumor activity of combination treatment with BIM SAHB_A. Treatment of OCI-AML3 cells with the combination of (A) BIM SAHB_A/TRAIL or (B) BIM SAHB_A/ABT-737 caused a synergistic decrease in cell viability, as reflected by CalcuSyn analysis and the plotted dose-effect curves (combination index [CI] <1). EC₅₀, 50% effective concentration; ED₇₅, 75% effective dose; ED₉₀, 90% effective dose. (C) The combination of BIM SAHB_A (25 mg/kg/d i.v.) and ABT-263 (100 mg/kg/d PO) caused the most significant suppression of OCI-AML3 tumor growth in vivo, as assessed by in vivo bioluminescence imaging. Two-way ANOVA, overall P = 0.0052. Vehicle versus: BIM SAHB_A, P = 0.0299; BIM SAHB_A(R153D), P = 0.3934; BIM SAHB_A and ABT-263, P = 0.0009; and BIM SAHB_A(R153D) and ABT-263, P = 0.1982. Data are mean ± SEM (n = 8 per arm). *P < 0.05; **P < 0.01 (D) No weight loss manifested among the 5 treatment cohorts over the 16-day observation period. Data are mean ± SEM (n = 8 per arm).

Therapeutic potential of BIM BH3 replacement. To explore the in vivo capacity of BIM SAHB_A to selectively reactivate the death pathway in cells driven by a formidable genetic apoptotic blockade, we transplanted the bone marrow (2 × 10⁶ cells) from Bim^–/– mice (37) into Rag2^–/–gc^–/– recipients, yielding uniform cohorts of mice manifesting the immune cell accumulation of native Bim^–/– mice (38), as evidenced by organ infiltrates (Figure 8A), hypergammaglobulinemia (Supplemental Figure 7A), and splenomegaly (Supplemental Figure 7B) evaluated at 19, 15, and 12 weeks post transplant, respectively. Histologic analysis of solid organs, such as the kidney and liver by H&E stain and anti-B220 immunohistochemistry, documented the presence of pathologic B cell infiltrates throughout the tissues (Figure 8A). Post transplant day–matched mouse cohorts (n = 4) were treated with daily i.v. injections of vehicle (5% DMSO/D5W) or 10 mg/kg of murine BIM SAHB_As for 5 days, followed by necropsy and histologic analysis. The relative degree of apoptosis induction was measured by TUNEL staining, which revealed a statistically significant increase in TUNEL positivity in the liver and kidney infiltrates of BIM SAHB_A–treated mice compared with that in vehicle- and BIM SAHB_A(R153D)–treated animals (Figure 8, B–D). Importantly, no increase in TUNEL staining was observed in the surrounding normal liver and kidney parenchyma of BIM SAHB_A–treated mice (Figure 8, B, C, and E). Of note, Bim^–/– bone marrow–reconstituted Rag2^–/–gc^–/– mice treated with BIM SAHB_A for 5 days also manifested a trend toward lower spleen weight and spleen weight per body weight compared with that of vehicle- and BIM SAHB_A(R153D)–treated animals (Supplemental Figure 8, A and B). An analogous study performed with WT B6/129 mice showed equivalent spleen weights across the vehicle, BIM SAHB_A, and BIM SAHB_A(R153D) treatment groups (Supplemental Figure 8, C and D). In addition, no suppressive effects on peripheral white blood cell counts or peripheral or splenic lymphocyte subsets were detected in the WT mice (Supplemental Figure 9). Thus, we find that BIM SAHB_A triggered cell death of deregulated Bim^–/– immune cells in a BH3 sequence–specific manner and spared normal host tissues, highlighting the potential of this BIM BH3 replacement strategy to surmount pathologic apoptotic blockades in vivo.

Figure 8

Pharmacologic replacement of BIM BH3 restores the death pathway in Bim^–/– immune cell infiltrates. (A) The infiltrative B cell disease of Bim^–/– mice was recapitulated in irradiated Rag2^–/–gc^–/– mice transplanted with the bone marrow of Bim^–/– mice. Histologic analysis by H&E stain and anti-B220 immunohistochemistry at 19 weeks post transplant revealed extensive B cell infiltration of solid organs. Original magnification: ×2 (whole organs); ×40 (H&E), ×20 (B220 immunohistochemistry). (B and C) Increased TUNEL staining was evident in the aberrant infiltrates of BIM SAHB_A–treated mice as compared with that in vehicle- and BIM SAHB_A(R153D)–treated animals. Importantly, the observed increase in TUNEL positivity in BIM SAHB_A–treated mice appeared restricted to the deregulated B cells and was not evident in the normal parenchymal tissue. Original magnification, ×100. (D) The percentage TUNEL positivity within the immune cell infiltrates was calculated by counting approximately 6,000 cells per 5-μm tissue section from each experimental animal (n = 4 per cohort) in a blinded fashion. A statistically significant increase in percentage of TUNEL positivity was observed in the kidney (P = 0.007) and liver (P = 0.0007) infiltrates of BIM SAHB_A–treated mice, an effect that was abrogated by R153D point mutagenesis. Data are mean ± SD. **P < 0.01. (E) Importantly, no increase in the number of TUNEL-positive cells was found in kidney and liver parenchymal tissue devoid of the infiltrates. Data are mean ± SD.

To achieve a survival advantage, cancer cells overexpress antiapoptotic BCL-2 family proteins (39) and neutralize the proapoptotic members by interaction-mediated sequestration (40, 41) and/or genetic deletion (14, 15, 42, 43). Targeted pharmacologic strategies to reactivate apoptosis in cancer have focused on inhibiting individual (32) or subsets (4, 5, 44, 45) of BCL-2 family antiapoptotic proteins. Relapsed and refractory cancer, however, is often characterized by the emergence of apoptotic blockade reinforcements that derive from (a) a surge in expression of alternate antiapoptotic proteins or (b) genetic silencing/deletion of proapoptotic proteins, such as the BH3-only subclass. In this context, multimodal targeting of the BCL-2 family pathway with agents designed to block all antiapoptotic proteins, directly trigger the multidomain death proteins, and even pharmacologically replace the death functionality of deleted BH3-only proteins, may be ideally suited to confront chemoresistant disease.

The BH3-only protein BIM has emerged as the most potent death sentinel, engaging all antiapoptotic targets (8, 9, 12) and directly triggering the multidomain proapoptotic proteins (10, 11, 19, 46). Thus, we generated a structurally reinforced and cell permeable stapled BIM BH3 helix to maximally reactivate the BCL-2 family death pathway in resistant cancer. We found that BIM SAHB_A binds to a diversity of antiapoptotic and proapoptotic targets with high affinity and potently triggers BAX- and BAK-dependent mitochondrial apoptosis in vitro. In a cellular context, BIM SAHB_A dissociates inhibitory heterodimeric interactions between antiapoptotic and proapoptotic proteins, such as MCL-1/BAK, and triggers conformational activation of the proapoptotic effector protein BAX. Consistent with these biochemical changes induced by low micromolar dosing, BIM SAHB_A dose responsively impairs the viability of hematologic cancer cells, promoting annexin V binding, caspase-3/7 activation, and the morphologic features of apoptosis. Combining BIM SAHB_A with an extrinsic or intrinsic apoptotic pathway stimulant caused synergistic cancer cell death, highlighting the potential utility of BIM SAHB_A in lowering the apoptotic threshold to enhance the cytotoxicity of chemotherapeutic agents. Importantly, BIM SAHB_A imposes preferential toxicity upon cancer cells, as no significant changes in viability were observed for human and murine fibroblasts subjected to BIM SAHB_A treatment at the same micromolar dosing levels.

In the in vivo context, as a single agent and in combination with ABT-263, BIM SAHB_A suppressed tumor growth in a xenograft model of human AML. In addition, BIM SAHB_A selectively restored the death pathway in tissue-infiltrating Bim^–/– immune cells. Single point mutagenesis of the BIM SAHB_A helical binding surface abrogated death-inducing activity, and no acute toxicity was observed in normal tissues, highlighting the specificity of action of BIM SAHB_A. Interestingly, the therapeutic window observed for this and other BCL-2 family modulators (4, 20, 44) appears to derive from the antiapoptotic reserve of unstressed cells, which sustain targeted activation of the BCL-2 family pathway by deploying their natural reservoir of antiapoptotics to bind and sequester activated proapoptotic proteins. In contrast, diseased cells whose antiapoptotic reserve is taxed by tonic death signaling are unable to withstand further BAX/BAK-mediated mitochondrial assault, making them preferentially susceptible to the apoptotic trigger (8). Thus, multimodal targeting of the BCL-2 family pathway through combined antiapoptotic inhibition, direct proapoptotic activation, and pharmacologic replacement of genetically deleted BH3 functionalities may unmask a critical opportunity to overturn the apoptotic blockades of relapsed and refractory cancer and achieve a durable clinical remission.

View Supplemental data

We thank E.D. Smith, A.L. Christie, and L. Cameron for assistance with graphics, animal treatment and imaging, and confocal microscopy, respectively. This work was supported by a Lauri Strauss Leukemia Foundation Discovery Grant, a Leukemia and Lymphoma Society Special Fellow Award, and NIH grant 1K08CA151450 to J.L. LaBelle and NIH grants 5P01CA92625 and 5R01CA050239, a Leukemia and Lymphoma Society SCOR grant, the Todd J. Schwartz Pediatric Oncology Fund, a grant from the Wolpoff Family Foundation, and a Burroughs Wellcome Fund Career Award to L.D. Walensky. We thank the LaTorre family for their generous support of C. Lawrence.

Address correspondence to: Loren D. Walensky, Dana-Farber Cancer Institute, 450 Brookline Avenue, Mayer 664, Boston, Massachusetts 02215, USA. Phone: 617.632.6307; Fax: 617.582.8240; E-mail: loren_walensky@dfci.harvard.edu.

Conflict of interest: Loren D. Walensky is a scientific advisory board member and consultant for Aileron Therapeutics.

Reference information: J Clin Invest. 2012;122(6):2018–2031. doi:10.1172/JCI46231.

See the related article at Therapeutic potential of a peptide targeting BCL-2 cell guardians in cancer.

1. Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell. 2004;116(2):205–219.
   View this article via: PubMed CrossRef Google Scholar
2. Tait SW, Green DR. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol. 2010;11(9):621–632.
   View this article via: PubMed CrossRef Google Scholar
3. Sattler M, et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science. 1997;275(5302):983–986.
   View this article via: PubMed CrossRef Google Scholar
4. Oltersdorf T, et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature. 2005;435(7042):677–681.
   View this article via: PubMed CrossRef Google Scholar
5. Tse C, et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res. 2008;68(9):3421–3428.
   View this article via: PubMed CrossRef Google Scholar
6. Konopleva M, et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell. 2006;10(5):375–388.
   View this article via: PubMed CrossRef Google Scholar
7. van Delft MF, et al. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell. 2006;10(5):389–399.
   View this article via: PubMed CrossRef Google Scholar
8. Certo M, et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell. 2006;9(5):351–365.
   View this article via: PubMed CrossRef Google Scholar
9. Chen L, et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell. 2005;17(3):393–403.
   View this article via: PubMed CrossRef Google Scholar
10. Gavathiotis E, et al. BAX activation is initiated at a novel interaction site. Nature. 2008;455(7216):1076–1081.
    View this article via: PubMed CrossRef Google Scholar
11. Kuwana T, et al. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell. 2005;17(4):525–535.
    View this article via: PubMed CrossRef Google Scholar
12. Merino D, et al. The role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins. J Cell Biol. 2009;186(3):355–362.
    View this article via: PubMed CrossRef Google Scholar
13. Inomata M, Tagawa H, Guo YM, Kameoka Y, Takahashi N, Sawada K. MicroRNA-17-92 down-regulates expression of distinct targets in different B-cell lymphoma subtypes. Blood. 2009;113(2):396–402.
    View this article via: PubMed CrossRef Google Scholar
14. Mestre-Escorihuela C, et al. Homozygous deletions localize novel tumor suppressor genes in B-cell lymphomas. Blood. 2007;109(1):271–280.
    View this article via: PubMed CrossRef Google Scholar
15. Tagawa H, et al. Genome-wide array-based CGH for mantle cell lymphoma: identification of homozygous deletions of the proapoptotic gene BIM. Oncogene. 2005;24(8):1348–1358.
    View this article via: PubMed CrossRef Google Scholar
16. Kashiwagi H, et al. TAT-Bim induces extensive apoptosis in cancer cells. Ann Surg Oncol. 2007;14(5):1763–1771.
    View this article via: PubMed CrossRef Google Scholar
17. Kazi A, et al. The BH3 alpha-helical mimic BH3-M6 disrupts BCL-XL, BCL-2 and MCL-1 protein-protein interactions with BAX, BAK, BAD or BIM and induces apoptosis in a BAX- and BIM-dependent manner. J Biol Chem. 2011;286(11):9382–9392.
    View this article via: PubMed CrossRef Google Scholar
18. Ponassi R, et al. A novel Bim-BH3-derived Bcl-XL inhibitor: biochemical characterization, in vitro, in vivo and ex-vivo anti-leukemic activity. Cell Cycle. 2008;7(20):3211–3224.
    View this article via: PubMed CrossRef Google Scholar
19. Walensky LD, et al. A stapled BID BH3 helix directly binds and activates BAX. Mol Cell. 2006;24(2):199–210.
    View this article via: PubMed CrossRef Google Scholar
20. Walensky LD, et al. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science. 2004;305(5689):1466–1470.
    View this article via: PubMed CrossRef Google Scholar
21. Fire E, Gulla SV, Grant RA, Keating AE. Mcl-1-Bim complexes accommodate surprising point mutations via minor structural changes. Protein Sci. 2010;19(3):507–519.
    View this article via: PubMed Google Scholar
22. Herman MD, et al. Completing the family portrait of the anti-apoptotic Bcl-2 proteins: crystal structure of human Bfl-1 in complex with Bim. FEBS Lett. 2008;582(25–26):3590–3594.
    View this article via: PubMed CrossRef Google Scholar
23. Liu X, Dai S, Zhu Y, Marrack P, Kappler JW. The structure of a Bcl-xL/Bim fragment complex: implications for Bim function. Immunity. 2003;19(3):341–352.
    View this article via: PubMed CrossRef Google Scholar
24. Gavathiotis E, Reyna DE, Davis ML, Bird GH, Walensky LD. BH3-triggered structural reorganization drives the activation of proapoptotic BAX. Mol Cell. 2010;40(3):481–492.
    View this article via: PubMed CrossRef Google Scholar
25. Chipuk JE, Fisher JC, Dillon CP, Kriwacki RW, Kuwana T, Green DR. Mechanism of apoptosis induction by inhibition of the anti-apoptotic BCL-2 proteins. Proc Natl Acad Sci U S A. 2008;105(51):20327–20332.
    View this article via: PubMed CrossRef Google Scholar
26. Kim H, et al. Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nat Cell Biol. 2006;8(12):1348–1358.
    View this article via: PubMed CrossRef Google Scholar
27. Willis SN, et al. Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science. 2007;315(5813):856–859.
    View this article via: PubMed CrossRef Google Scholar
28. Deng J, Carlson N, Takeyama K, Dal Cin P, Shipp M, Letai A. BH3 profiling identifies three distinct classes of apoptotic blocks to predict response to ABT-737 and conventional chemotherapeutic agents. Cancer Cell. 2007;12(2):171–185.
    View this article via: PubMed CrossRef Google Scholar
29. Li G, et al. Effective targeting of STAT5-mediated survival in myeloproliferative neoplasms using ABT-737 combined with rapamycin. Leukemia. 2010;24(8):1397–1405.
    View this article via: PubMed CrossRef Google Scholar
30. Deng J, et al. Proapoptotic BH3-only BCL-2 family protein BIM connects death signaling from epidermal growth factor receptor inhibition to the mitochondrion. Cancer Res. 2007;67(24):11867–11875.
    View this article via: PubMed CrossRef Google Scholar
31. Martinou JC, Youle RJ. Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev Cell. 2011;21(1):92–101.
    View this article via: PubMed CrossRef Google Scholar
32. Stewart ML, Fire E, Keating AE, Walensky LD. The MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizer. Nat Chem Biol. 2010;6(8):595–601.
    View this article via: PubMed CrossRef Google Scholar
33. Hsu YT, Youle RJ. Nonionic detergents induce dimerization among members of the Bcl-2 family. J Biol Chem. 1997;272(21):13829–13834.
    View this article via: PubMed CrossRef Google Scholar
34. Callens C, et al. Targeting iron homeostasis induces cellular differentiation and synergizes with differentiating agents in acute myeloid leukemia. J Exp Med. 2010;207(4):731–750.
    View this article via: PubMed CrossRef Google Scholar
35. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharm Rev. 2006;58(3):621–681.
    View this article via: PubMed CrossRef Google Scholar
36. Gandhi L, et al. Phase I study of Navitoclax (ABT-263), a novel Bcl-2 family inhibitor, in patients with small-cell lung cancer and other solid tumors. J Clin Oncol. 2011;29(7):909–916.
    View this article via: PubMed CrossRef Google Scholar
37. Takeuchi O, Fisher J, Suh H, Harada H, Malynn BA, Korsmeyer SJ. Essential role of BAX,BAK in B cell homeostasis and prevention of autoimmune disease. Proc Natl Acad Sci U S A. 2005;102(32):11272–11277.
    View this article via: PubMed CrossRef Google Scholar
38. Bouillet P, et al. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science. 1999;286(5445):1735–1738.
    View this article via: PubMed CrossRef Google Scholar
39. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science. 1984;226(4678):1097–1099.
    View this article via: PubMed CrossRef Google Scholar
40. Brunelle JK, Ryan J, Yecies D, Opferman JT, Letai A. MCL-1-dependent leukemia cells are more sensitive to chemotherapy than BCL-2-dependent counterparts. J Cell Biol. 2009;187(3):429–442.
    View this article via: PubMed CrossRef Google Scholar
41. Letai A, Sorcinelli MD, Beard C, Korsmeyer SJ. Antiapoptotic BCL-2 is required for maintenance of a model leukemia. Cancer Cell. 2004;6(3):241–249.
    View this article via: PubMed CrossRef Google Scholar
42. Meijerink JP, et al. Hematopoietic malignancies demonstrate loss-of-function mutations of BAX. Blood. 1998;91(8):2991–2997.
    View this article via: PubMed Google Scholar
43. Egle A, Harris AW, Bouillet P, Cory S. Bim is a suppressor of Myc-induced mouse B cell leukemia. Proc Natl Acad Sci U S A. 2004;101(16):6164–6169.
    View this article via: PubMed CrossRef Google Scholar
44. Nguyen M, et al. Small molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes MCL-1-mediated resistance to apoptosis. Proc Natl Acad Sci U S A. 2007;104(49):19512–19517.
    View this article via: PubMed CrossRef Google Scholar
45. Paoluzzi L, et al. Targeting Bcl-2 family members with the BH3 mimetic AT-101 markedly enhances the therapeutic effects of chemotherapeutic agents in in vitro and in vivo models of B-cell lymphoma. Blood. 2008;111(11):5350–5358.
    View this article via: PubMed CrossRef Google Scholar
46. Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, Korsmeyer SJ. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell. 2002;2(3):183–192.
    View this article via: PubMed CrossRef Google Scholar
47. Bird GH, Bernal F, Pitter K, Walensky LD. Synthesis and biophysical characterization of stabilized alpha-helices of BCL-2 domains. Methods Enzymol. 2008;446:369–386.
    View this article via: PubMed Google Scholar
48. Pitter K, Bernal F, Labelle J, Walensky LD. Dissection of the BCL-2 family signaling network with stabilized alpha-helices of BCL-2 domains. Methods Enzymol. 2008;446:387–408.
    View this article via: PubMed Google Scholar
49. Billen LP, Kokoski CL, Lovell JF, Leber B, Andrews DW. Bcl-XL inhibits membrane permeabilization by competing with Bax. PLoS Biol. 2008;6(6):e147.
    View this article via: PubMed CrossRef Google Scholar
50. Zong WX, Ditsworth D, Bauer DE, Wang ZQ, Thompson CB. Alkylating DNA damage stimulates a regulated form of necrotic cell death. Genes Dev. 2004;18(11):1272–1282.
    View this article via: PubMed CrossRef Google Scholar