Bim escapes displacement by BH3-mimetic anti-cancer drugs by double-bolt locking both Bcl-XL and Bcl-2

  1. Qian Liu
  2. Elizabeth J Osterlund
  3. Xiaoke Chi
  4. Justin Pogmore
  5. Brian Leber
  6. David William Andrews  Is a corresponding author
  1. Sunnybrook Research Institute, Canada
  2. University of Toronto, Canada
  3. McMaster University, Canada
10 figures, 2 tables and 1 additional file

Figures

ABT-263 does not displace Bim from binding to Bcl-XL.

(A) Live cell images of mCer3 and Venus intensities and intensity weighted FLIM images from FLIM-FRET measurements for the interaction between CBcl-XL and VBad. Higher magnification views are shown …

https://doi.org/10.7554/eLife.37689.003
Figure 1—source data 1

Source data fitted to Hill equations demonstrating that ABT-263 displaced tBid and Bad but does not displace Bim from binding to Bcl-XL.

https://doi.org/10.7554/eLife.37689.004
Figure 2 with 4 supplements
with four supplements: The impact of small-molecule inhibitors and BH3-sequence mutations on the interactions between anti-apoptotic proteins and BH3-only pro-apoptotic proteins.

(A) Values of R can be used to define binding interactions from FLIM-FRET data. Sample calculation of RABT-263 for CBcl-XL:VBad binding in MCF-7 cells. To maximize the accuracy and dynamic range of …

https://doi.org/10.7554/eLife.37689.005
Figure 2—source data 1

Source data for the impact of small-molecule inhibitors and BH3-sequence mutations on the interactions between anti-apoptotic proteins and BH3-only pro-apoptotic proteins.

https://doi.org/10.7554/eLife.37689.006
Figure 2—figure supplement 1
FLIM-FRET binding curves for Bcl-2 binding to BH3 proteins in MCF-7 cells.

(A–C) Both BH3-2A mutation and ABT-263 displace (A) VBad, (B) VtBid but not (C) VBimEL from CBcl-2 in MCF-7 cells. FLIM FRET binding curves are shown above representative images. Scale bar 10 μm. …

https://doi.org/10.7554/eLife.37689.007
Figure 2—figure supplement 1—source data 1

Source data fitted to a Hill equation to generate FLIM-FRET binding curves for Bcl-2 binding to BH3 proteins in MCF-7 cells.

https://doi.org/10.7554/eLife.37689.008
Figure 2—figure supplement 2
FLIM-FRET binding curves for Bcl-XL binding to BH3 proteins in BMK DKO cells.

(A–C) Both BH3-2A mutation and ABT-263 displace (A) VBad, (B) VtBid but not (C) VBimEL from CBcl-XL in BMK cells in which the genes for Bax and Bak have been deleted (DKO). FLIM FRET binding curves …

https://doi.org/10.7554/eLife.37689.009
Figure 2—figure supplement 2—source data 1

Source data fitted to a Hill equation to generate FLIM-FRET binding curves for Bcl-XL binding to BH3 proteins in BMK DKO cells.

https://doi.org/10.7554/eLife.37689.010
Figure 2—figure supplement 3
FLIM-FRET measurements for CBcl-XL binding to VBimEL-Bad in alive and dying MCF-7 cells.

Representative micrographs selected showing mCer3 and Venus intensities, Intensity weighted FLIM and selected regions of interest (ROIs) for (A) alive and (B) dying cells. Lifetime determinations …

https://doi.org/10.7554/eLife.37689.011
Figure 2—figure supplement 4
FLIM-FRET binding curves for VBad (left) and VBimEL (right) to CBcl-XL in MCF-7 cells in the presence of different concentrations of BH3-mimetics.

The curves illustrate the extent of binding for DMSO treated controls (black), or cells treated with 1.25 μM (pink), 2.5 μM (light blue), 5 μM (green), 10 μM (red), and 20 μM (dark blue) …

https://doi.org/10.7554/eLife.37689.012
Figure 2—figure supplement 4—source data 1

Source data fitted to a Hill equation to generate FLIM-FRET binding curves for Bad and BimEL to Bcl-XL in MCF-7 cells.

https://doi.org/10.7554/eLife.37689.013
MCF-7 cells are killed by transient expression of BimEL or Bid, are protected by stably expressed Bcl-XL or Bcl-2 and depend on MCL-1 for survival.

(A) Transient expression of exogenous VtBid (blue) and VBimEL (red) killed MCF-7 cells. MCF-7 cells were largely resistant to expression of VBad (green) as the resulting cell death was similar to …

https://doi.org/10.7554/eLife.37689.015
Figure 3—source data 1

Multiparametric source data for MCF-7 cell death in response to transient expression of BimEL or Bid and the protection afforded by stably expressed Bcl-XL or Bcl-2 and the dependence of MCF-7 cells on MCL-1 for survival.

https://doi.org/10.7554/eLife.37689.016
Figure 4 with 1 supplement
Resistance of CBcl-XL:VBimEL and CBcl-2:VBimEL complexes to ABT-263 is dependent on both the Bim BH3 and the Bim CTS.

(A) Stick diagrams for the different VBimEL constructs expressed in cells. (B) RABT-263 for CBcl-XL:VBH3-protein complexes (black) and CBcl-2:VBH3-protein complexes (blue). (C) RBH3-2A of CBcl-XL:VBH…

https://doi.org/10.7554/eLife.37689.017
Figure 4—source data 1

Source data for the calculation of R values for resistance of Bcl-XL:BimEL and Bcl-2:BimEL complexes to ABT-263 for the various mutant BH3 proteins.

https://doi.org/10.7554/eLife.37689.018
Figure 4—figure supplement 1
Mutations in the BH3 region and the Bim CTS impair Bim binding to Bcl-XL and Bcl-2.

FLIM-FRET binding curves for the proteins indicated to the left of each row and (A–F) CBcl-XL or (G–L) CBcl-2 in MCF-7 cells illustrate the extent of binding for untreated (black) or DMSO treated …

https://doi.org/10.7554/eLife.37689.019
Figure 4—figure supplement 1—source data 1

Source data fit to a Hill equation to determine how mutations in the BH3 region and the Bim CTS impair Bim binding to Bcl-XL and Bcl-2.

https://doi.org/10.7554/eLife.37689.020
The CTS contributes to Bim mediated inhibition of Bcl-XL and Bcl-2.

The BH3-proteins indicated to the right of the panels were expressed by transient transfection in MCF-7 cells and MCF-7 cells stably expressing either CBcl-XL or CBcl-2, as indicated. Commitment to …

https://doi.org/10.7554/eLife.37689.021
Figure 5—source data 1

Multiparametric cell death data for the mutants demonstrating that the Bim CTS contributes to Bim mediated inhibition of Bcl-XL and Bcl-2.

https://doi.org/10.7554/eLife.37689.022
Figure 6 with 1 supplement
h0 and h1 residues in the Bim BH3 contribute to the resistance of Bcl-XL:Bim complexes to ABT-263.

(A) Sequence alignment of the BH3 regions of Bad and Bim and the sequences of VBad-Bim mutants, residues from Bad (red), Bim (blue). (B) Identification of conserved hydrophobic residues in the Bim …

https://doi.org/10.7554/eLife.37689.023
Figure 6—source data 1

Source data for the calculation of R values for mutants demonstrating that the h0 and h1 residues in the Bim BH3 contribute to the resistance of Bcl-XL:Bim complexes to ABT-263.

https://doi.org/10.7554/eLife.37689.024
Figure 6—figure supplement 1
The h0 and h1 residues in the Bim BH3 region contribute to the resistance of Bcl-XL:Bim and Bcl-2:Bim complexes to ABT-263.

(A–L) Binding of VBad-Bim chimeric proteins to CBcl-XL (left) and CBcl-2 (right) in MCF-7 cells. The VBad-Bim chimeric proteins are comprised of VBad with the BH3 region replaced with the …

https://doi.org/10.7554/eLife.37689.025
Figure 6—figure supplement 1—source data 1

Source data fitted to a Hill equation to determine the extent to which residues in the Bim BH3 region contribute to the resistance of Bcl-XL:Bimand Bcl-2:Bim complexes to ABT-263.

https://doi.org/10.7554/eLife.37689.026
Figure 7 with 1 supplement
The Bim CTS binds to Bcl-XL and Bcl-2 independent of binding to membranes (A) Sequence alignment for the Bim CTS and mutants.

Bim h1-h4, red; arginine residues within the CTS, green; substitutions, blue; numbering is for BimEL. (B) Selected images showing sub-cellular localization of VBimEL-mutants compared to MitoTracker. …

https://doi.org/10.7554/eLife.37689.027
Figure 7—source data 1

Source data for the experiments demonstrating that the Bim CTS binds to Bcl-XL and Bcl-2 independent of binding to membranes.

https://doi.org/10.7554/eLife.37689.028
Figure 7—figure supplement 1
Mutations in the Bim CTS reduce the affinity of binding to Bcl-XL and Bcl-2.

(A–L) FLIM-FRET data for CBcl-XL (left) and CBcl-2 (right) binding to the VBimEL mutants indicated to the left of the panels. Untreated and DMSO treated controls, black and green, respectively. …

https://doi.org/10.7554/eLife.37689.029
Figure 7—figure supplement 1—source data 1

Source data fitted to a Hill equation to quantify the effects of the indicated mutations in the BimCTS on binding affinities for Bcl-XL and Bcl-2.

https://doi.org/10.7554/eLife.37689.030
Bim CTS binds with non-transmembrane topology to cellular membranes.

(A) Selected images for sub-cellular localization of VBimEL and BimELV in BMK-DKO cells compared to MitoTracker. The scale bar represents 10 µm. (B) In live cells CBcl-XL binding to BimELV is …

https://doi.org/10.7554/eLife.37689.031
Figure 8—source data 1

Source data fitted to a Hill equation demonstrating that BimEL-venus undergoes FRET with mCer3-Bcl-XL.

https://doi.org/10.7554/eLife.37689.032
Figure 9 with 2 supplements
The Bim CTS binds to the cytoplasmic surface of membranes enabling concomitant binding to Bcl-XL (A–I) Interaction of the Bim CTS with liposomes and Bcl-XL measured using purified recombinant BimL protein.

The amino-acids and positions above the panels indicate the residues in BimL exchanged for single-cysteines for labeling with NBD (numbering for BimEL, subtract 56 for BimL numbering). Top row, (A,D,…

https://doi.org/10.7554/eLife.37689.033
Figure 9—source data 1

Source data for Interaction of the Bim CTS with liposomes and Bcl-XL measured using purified recombinant full length proteins.

https://doi.org/10.7554/eLife.37689.034
Figure 9—figure supplement 1
Residues in the Bim CTS are protected from iodide when bound to membranes and Bcl-XL.

(A–D) Representative Stern-Volmer quenching plots for specific residues in Bim. The fluorescence of BimL mutants labeled on the single cysteine with NBD (indicated at the top of each panel) was …

https://doi.org/10.7554/eLife.37689.035
Figure 9—figure supplement 1—source data 1

Source data for Stern-Volmer quwnching plots for representative mutants of Bim.

https://doi.org/10.7554/eLife.37689.036
Figure 9—figure supplement 2
The Bim CTS adopts a non-conventional conformation that allows binding to both membranes and Bcl-XL through hydrophobic interactions.

(A–K) FLIM-FRET binding curves for VBimEL (black) and VBimEL-BH3-2A (red) with the indicated amino acid substitutions for L185 indicate that non-charged amino acids are preferred for binding to CBcl-…

https://doi.org/10.7554/eLife.37689.037
Figure 9—figure supplement 2—source data 1

Source data fitted to a Hill equation for the mutants illustrating that the Bim-CTS binds both to membranes and to Bcl-XL.

https://doi.org/10.7554/eLife.37689.038
Author response image 1
ABT199 significantly affects the lifetime of CBcl2.

BMKd3 cells expressing CBcl2 were incubated 24 hours in DMEM media alone (untreated) or media plus DMSO, ABT199 or ABT263. FLIM data was collected on the ISS-Alba, each point represents average …

Tables

Table 1
Values of RBH3-2A and RABT-263 for all reported interactions.
https://doi.org/10.7554/eLife.37689.014
Cell lines
Constructs
cBcl-XLcBcl-2
RBH3-2ARABT-263RBH3-2ARABT-263
Value95% CIValue95% CIValue95% CIValue95% CI
vBad2.442.5046.368.234.333.4818.633.24
vtBid33.702.6043.956.8425.333.9830.169.82
vBimEL81.434.6586.265.1066.854.7083.068.69
vBad-Bim70.194.0397.966.4554.913.9265.467.40
vBimEL-dN101.874.3790.817.0182.574.2790.4710.25
vBimEL-Bad26.122.7060.554.4332.745.8065.756.36
vBimEL-dCTS15.083.1769.054.5713.602.2160.026.73
vBimEL-Bad-dCTS10.614.2640.255.6711.954.0712.516.60
vBad-Bim-WAANDND78.515.81NDND50.157.22
vBad-Bim-I146Y46.563.2083.245.3426.784.2153.175.43
vBad-Bim-Q148R74.454.2489.826.2156.945.4653.467.33
vBad-Bim-I153M96.534.6092.786.00102.308.9468.517.51
vBad-Bim-I146Y-Q148R50.463.4690.187.5937.733.8861.777.54
vBad-Bim-I146Y-I153M56.803.2788.976.1242.594.3369.077.73
vBimEL-I146Y70.997.5888.6610.8554.173.4965.328.72
vBimEL-I146Y-dCTS12.292.8938.458.318.574.0352.866.02
vBimEL-L185E26.683.6241.8510.2416.093.8751.0212.29
vBimEL-I146Y-L185E19.444.1040.018.075.799.8136.858.81
vBimEL-CTS2A77.994.4388.796.2460.243.7780.6911.40
vBimEL-I181E49.1950.2363.866.1647.202.9673.2610.45
vBimEL-L185E26.683.6241.8510.2416.093.8751.0212.29
vBimEL-I188E67.175.1450.967.6647.473.3663.048.04
vBimEL-V192E52.5753.5970.147.6454.503.5664.849.70
vBimEL-L185A159.1014.88
vBimEL-L185D20.315.29
vBimEL-L185E26.163.57
vBimEL-L185F99.995.15
vBimEL-L185G86.827.91
vBimEL-L185H49.104.50
vBimEL-L185M89.984.42
vBimEL-L185P58.953.61
vBimEL-L185R48.083.33
vBimEL-L185S73.494.67
vBimEL-L185stop26.874.94
BimELv89.415.80
Key resources table
Reagent
type (species)
or resource
DesignationSource
or reference
IdentifiersAdditional
information
Antibodyantibody to Bcl-XL (Rabbit polyclonal)PMID: 14681679(1:10,000), human, mouse reactivity
Antibodyantibody to Beta-actin (Mouse monoclonal)AbgentCat. #: 8H10D10(1:5000), human, rat, mouse reactivity
AntibodyDonkey anti-rabbit (polyclonal)Jackson Immuno Research LaboratoriesCat. #: 711-035-150(1:10,000)
AntibodyDonkey anti-mouse (polyclonal)Jackson Immuno Research LaboratoriesCat. #: 711-035-152(1:10,000)
Cell line (H.sapiens)MCF-7PMID: 3790748Dr. Ronald N. Buick (University of Toronto)
Cell line (M. musculus)Baby Mouse Kidney(BMK)-DKO (Bax and Bak knockout) cellsPMID: 11836241Dr. Eileen White (Rutgers University)
Chemical compound, drugNavitoclax; ABT-263SelleckchemCat. #: S1001in DMSO
Chemical compound, drugA-1155463ChemietekCat #: CT-A115in DMSO
Chemical compound, drugA-1331852ChemietekCat. #: CT-A115in DMSO
Chemical compound, drugS-63845ChemietekCat. #: 1799633-27-4in DMSO
Chemical compound, drugVenetoclax; ABT-199ChemietekCat. #: CT-A199in DMSO
Chemical compound, drugTMREThermoFisher ScientificCat. #: T669
Chemical compound, drugDRAQ5Biostatus, UKCat. #: DR05500
Chemical compound, drugMitoTracker RedThermoFisher ScientificCat. #: M22425
Chemical compound, drugMitoTracker GreenThermoFisher ScientificCat. #: M7514
Chemical compound, drugAlexa 647-maleimideThermoFisher Scientific, Molecular probesCat. #: A20347
Chemical compound, drugAlexa568-maleimideThermoFisher Scientific, Molecular probesCat. #. A20341
Chemical compound, drugNBDMolecular ProbesCat. #: D-2004
Chemical compound, drugPC (L-α-phosphatidylcholine)Avanti Polar LipidsCat. #:840051Cfor making liposomes, used 48% PC
Chemical compound, drugDOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine)Avanti Polar LipidsCat. #: 840035Cfor making liposomes, used 10% DOPS
Chemical compound, drugPI (L-α-phosphatidylinositol)Avanti Polar LipidsCat. #:840042Cfor making liposomes, used 10% PI
Chemical compound, drugPE (L-α-phosphatidylethanolamine)Avanti Polar LipidsCat. #: 841118Cfor making liposomes, used 28% PE
Chemical compound, drugTOCL, (18:1 Cardiolipin)Avanti Polar LipidsCat. #: 710335Cfor making liposomes, used 4% TOCL
Commercial assay or kitFugene HDPromegaCat. #: E2311
Commercial assay or kitTransIT-X2MirusCat. #: Mir 6003
Gene (H. sapiens)BaxPMID: 14522999,GI: L22473.1For recombinant protein
Gene (H. sapiens)Bcl-XLPMID: 18547146GI: Z23115.1For recombinant protein
Gene (H. sapiens)Bcl-2PMID: 22464442GI: M14745.1For expression of CBcl-2 in cells
Gene (H. sapiens)BadPMID: 22464442GI: AB451254.1For expression of VBad in cells
Gene (H. sapiens)Bcl-XLPMID: 22464442GI: NM_138578.3For expression of CBcl-XL in cells
Gene (M. musculus)BidPMID: 16642033,
PMID: 19062087
GI: NM_007544.4For recombinant protein
Gene (M. musculus)BimLthis paperGI: AAD26594.1This lab, plasmid # 2187, for recombinant BimL purification
Gene
(M. musculus)
tBidPMID: 22464442GI: NM_007544.4for expression of VtBid in cells
Gene (M. musculus)BimELPMID: 22464442GI: XM_006498614.3for expression of VBimEL in cells
OtherCell Carrier-384, UltraPerkinElmerCat. #: 6057300for live cell imaging
OtherNon-binding surface, 96-well plate, black with clear bottomCorningCat. #: 3881For recombinant protein and liposome assays critical to use non-binding plate
OtherOpera PhenixPerkinElmerCat. #: HH14000000
OtherISS-AlbaPMID: 25631031Custom built by ISS for DWA lab
Software, algorithmGraphPad PrismSan Diego, CaliforniaVersion 6Scientific graphing program, used to perform statistical analysis
Software, algorithmImageJPMID: 17936939MBF - ImageJ for microscopy, Dr. Tony Collins (McMaster University)FLIM-FRET analysis Macro, in this paper: https://github.com/DWALab/Liu_et_al_2018_eLife
Software, algorithmCellProfilerPMID: 17269487Colocalization analysis, in this paper: https://github.com/DWALab/Liu_et_al_2018_eLife
Transfected ConstructmVenus-pEGFP-C1otherGI: KU341334.1Dr. Ray Truant (McMaster University). Backbone EGFP-C1 (Clonetech)
Transfected ConstructmCerulean3-pEGFP-C1PMID: 21479270Dr. Mark A Rizzo (University of Maryland). Backbone EGFP-C1 (Clonetech)
Transfected ConstructpSPUTKStratagene Santa Clara CACat. #: CB4278654Cotransfected to reduce overexpression in live cells

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