Chronic lymphocytic leukemia requires BCL2 to sequester prodeath BIM, explaining sensitivity to BCL2 antagonist ABT-737

Abstract

Antiapoptotic B cell leukemia/lymphoma 2 (BCL2) family proteins are expressed in many cancers, but the circumstances under which these proteins are necessary for tumor maintenance are poorly understood. We exploited a novel functional assay that uses BCL2 homology domain 3 (BH3) peptides to predict dependence on antiapoptotic proteins, a strategy we call BH3 profiling. BH3 profiling accurately predicts sensitivity to BCL2 antagonist ABT-737 in primary chronic lymphocytic leukemia (CLL) cells. BH3 profiling also accurately distinguishes myeloid cell leukemia sequence 1 (MCL1) from BCL2 dependence in myeloma cell lines. We show that the special sensitivity of CLL cells to BCL2 antagonism arises from the requirement that BCL2 tonically sequester proapoptotic BIM in CLL. ABT-737 displaced BIM from BCL2's BH3-binding pocket, allowing BIM to activate BAX, induce mitochondrial permeabilization, and rapidly commit the CLL cell to death. Our experiments demonstrate that BCL2 expression alone does not dictate sensitivity to ABT-737. Instead, BCL2 complexed to BIM is the critical target for ABT-737 in CLL. An important implication is that in cancer, BCL2 may not effectively buffer chemotherapy death signals if it is already sequestering proapoptotic BH3-only proteins. Indeed, activator BH3-only occupation of BCL2 may prime cancer cells for death, offering a potential explanation for the marked chemosensitivity of certain cancers that express abundant BCL2, such as CLL and follicular lymphoma.

Figure 1. Schema of the intrinsic or mitochondrial programmed cell death pathway.

In response to death signaling, activator BH3-only proteins are triggered to interact with BAX and BAK, inducing BAX and BAK oligomerization. This oligomerization is followed by MOMP, which releases proapoptotic factors such as cytochrome c to the cytosol. Cytosolic cytochrome c forms a complex with apoptosis protease–activating factor–1 (APAF-1) and caspase-9 to make the holoenzyme known as the apoptosome, which in turn activates effector caspase-3, leading to widespread proteolysis. This pathway can be interrupted by antiapoptotic members, such as BCL2, which can bind activator BH3-only proteins, preventing their interaction with BAX and BAK. This inhibitory interaction can itself be antagonized by sensitizer BH3-only domains, which compete for the binding site in BCL2, displacing activators bound by BCL2. BFL1, BCL2-related gene expressed in fetal liver.

Figure 2. BCL2 antagonist ABT-737 efficiently kills primary CLL cells.

(A) CLL cells from 24 patient samples (numbered at right) were cultured for 48 hours with different concentrations of ABT-737 or negative control enantiomer (enant). Death was quantitated by annexin V staining and normalized to controls treated with solvent (DMSO). The effective concentration at which 50% killing is observed (EC₅₀) is shown. (B) CLL death induced by ABT-737 is rapid. CLL cells harvested from 5 patient samples (numbered at right) were cultured for 4 hours with different concentrations of compounds. Death was quantitated as in A. (C) Normal PBMCs are not sensitive to ABT-737. Four normal PBMC samples (numbered at right) were cultured for 24 hours in the presence of the indicated concentrations of compounds. Death was quantitated as in A.

Figure 3. Protein expression in CLL cells.

(A) BCL2 and BIM levels were uniform across 15 CLL samples tested by immunoblot of 5 μg whole-cell lysates. Three isoforms of BIM (BIM_EL, BIM_L, and BIM_S) are shown. (B) BCL2 and BIM levels in PBMCs were lower than in CLL. Whole-cell lysates (5 μg) evaluated by immunoblot. Three normal PBMC lysates are depicted at left, CLL lysates at right. (C) Short-term culture does not affect BCL2 or BIM protein levels. Two independent CLL lysates generated before and 48 hours after culture were probed for BCL2 and BIM. (D) BCL2 protein levels in 6 primary CLL cells (a–f) and primary FL cells are similar as seen by indexing immunoblots to lysates from the t(14;18)-containing H2 human lymphoma cell line. (E) Purified glutathione-S-transferase–tagged BCL2 (GST-BCL2) and GST-MCL1 (10–300 ng) were run next to 5 independent CLL lysates (5 μg) to quantitate BCL2 and MCL1. MCL1 is detectable only upon long exposure (bottom panel). Relative amounts of BCL2 and MCL1 were calculated using densitometry and are depicted in Table 3. (F) Antibody detecting full-length and cleaved MCL1 does not demonstrate MCL1 cleavage products in CLL lysates. (G) CLL lysates (10 μg) reveal low levels of BID and no detectable cleaved BID. DHL4 (DHL) cell lysate (10 μg), known to express BID, and recombinant caspase-8–cleaved his-tagged BID (tB) (35 ng) were run as positive controls for antibody recognition. Note that the his-tag causes slower migration of uncleaved BID. (Numbers at top of gels correspond to patient sample numbers.)

Figure 4. BH3 profiling of CLL mitochondria reveals BCL2 dependence.

Mitochondria were isolated from independent primary CLL patient samples and treated with BH3-only domain peptides as indicated (100 μM) or ABT-737 or negative control enantiomer (100 nM). DMSO (1%) is a solvent control. Cytochrome c release was analyzed via ELISA. Note that activators BID and BIM BH3 peptides interact with all antiapoptotic proteins tested and furthermore can directly activate BAX and BAK (8) so that they act as positive controls for cytochrome c release assays. BADmu, a point mutant of the BAD BH3-only domain, was used as a negative control. n = 7, except for BADmu, where n = 5, and ABT-737 and negative control enantiomer, where n = 3. Error bars represent SD.

Figure 5. BIM is sequestered by BCL2 and displaced by antagonism of BCL2.

(A) Anti-BCL2 immunoprecipitation of CLL whole-cell lysates (50 μg). (B and C) CLL whole-cell lysates (50 μg) immunoprecipitated with anti-BCL2 (B) or anti-BIM (C) antibodies. Immunoprecipitation pellet (p), supernatant (s), and 100% of total lysate used for immunoprecipitation were loaded (40, 38 [B]; 38 [C]). (D) Subcellular fractions of primary CLL cells (50 μg) were immunoblotted for BIM and BCL2. Fractionation verified with marker proteins glucose 6 phosphate dehydrogenase (G6P), ER-binding protein (BiP), and mitochondrial superoxide dismutase (MnSOD). HM, heavy membrane; LM, light membrane. (E) Anti-BCL2 immunoprecipitation from 50 μg CLL lysates (Triton X-100 used for 2 samples at left, CHAPS for 2 at right). Incubation with DMSO (1%), 1 μM negative control enantiomer, or 1 μM ABT-737 displaced BIM to the supernatant identified by anti-BIM immunoblot. (F) Lysates of CLL mitochondria (15 μg) in RIPA buffer were immunoblotted for BAK, BAX, and MnSOD (loading control). DHL4 lysate (15 μg) was used as a positive control for BAK. Patient sample numbers shown at top of blots in A–F. (G) CLL cells were incubated with DMSO (0.1%), ABT-737, or negative control enantiomer (10 nM, 100 nM, or 1 μM) for 4 hours. Percentage of cells that were dead was assessed by annexin V. Oligomerization of BAX and BAK evaluated by anti-BAX or anti-BAK immunoblot of chemically cross-linked whole-cell lysates. (H) Mitochondria isolated from CLL samples were incubated with DMSO (1%) or BAD BH3 peptide (100 μM). Samples were preincubated with antibodies directed against either the human BIM BH3 domain or irrelevant antigen (CD56). n = 5. Error bars show ± SD.

Figure 6. BH3 profiling correctly predicts ABT-737 sensitivity in myeloma cell lines.

(A) Mitochondria isolated from LP1 cells treated with 100 μM BH3 peptides show an MCL1-dependent pattern of cytochrome c release. Mitochondria isolated from L363 cells display a BCL2-dependent pattern. (B) Lysates (10 μg) of LP1 and L363 cell lines subjected to Western blot, comparing levels of MCL1, BCL2, and BIM. (C) L363 cells are sensitive and LP1 cells resistant to 48-hour treatment with ABT-737 and negative control enantiomer. n = 3. Error bars show ± SD. (D and E) Reduction of BIM confers resistance to ABT-737. BIM levels in L363 cells were reduced using lentiviral-delivered shRNA against exon 5 of BIM. (D) Western blot demonstrating knockdown of BIM in shRNA BIM–treated cells compared with negative control shRNA luciferase–treated (nc) cells. BCL2 levels are also shown at bottom. Quantification of protein bands by densitometry reveals 40% total BIM knockdown (white bars) with more than half of BIM_L and BIM_S isoforms reduced, as compared with shRNA luciferase (black bars). Samples were normalized to actin. (E) L363 cells infected with lentiviruses expressing shRNA BIM are less sensitive to 48-hour treatment with ABT-737 (1 μM) than L363 cells infected with lentiviruses expressing shRNA luciferase. These data indicate that the presence of BIM complexed with BCL2 is responsible for ABT-737 sensitivity. n = 3. Error bars show ± SD.

Figure 7. Model of ABT-737–induced death at the mitochondria.

Mitochondrial BCL2 sequesters BIM in CLL cells. Upon addition of ABT-737, BIM is displaced and BCL2 becomes occupied by ABT-737. Freed BIM then interacts with BAX or BAK, inducing oligomerization leading to cytochrome c release and irreversible commitment to programmed cell death. BCL2 primed with activator BH3-only proteins renders the cancer cell sensitive to treatment with ABT-737 and possibly other chemotherapeutic agents.