Chronic lymphocytic leukemia requires BCL2 to sequester prodeath BIM, explaining sensitivity to BCL2 antagonist ABT-737
Victoria Del Gaizo Moore, … , Carl D. Novina, Anthony Letai
Victoria Del Gaizo Moore, … , Carl D. Novina, Anthony Letai
Published January 2, 2007
Citation Information: J Clin Invest. 2007;117(1):112-121. https://doi.org/10.1172/JCI28281.
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Research Article Oncology

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.

Authors

Victoria Del Gaizo Moore, Jennifer R. Brown, Michael Certo, Tara M. Love, Carl D. Novina, Anthony Letai

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Figure 5

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

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BIM is sequestered by BCL2 and displaced by antagonism of BCL2. (A) Anti...
(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.