Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Counts of BCL2-related immunoassays (BCL2-BIM complex, BCL2-BAX complex, BCL2 total level) from the fixed numbers of HL60 cells ( n = 3). b , Counts of BCLxL-related immunoassays (BCLxL-BIM complex, BCLxL-BAX complex, BCLxL-BAD complex, BCLxL-BAK complex, BCLxL total level) from the fixed numbers of PC9 cells ( n = 3). c , Counts of MCL1-related immunoassays (MCL1-BIM complex, MCL1-BAK complex, MCL1 total level) from the fixed numbers of PC9 cells ( n = 3). The single-molecule counts were rescaled to account for the labeling efficiencies of the immunoassays calculated in Extended Data Fig. as well as the specific incubation conditions for direct comparison. All data were measured from independent inter-chip experiments. CVs obtained from independent inter-chip measurement for all the immunoassays and cell numbers ( n = 3). Individual data points shown for independent biological replicates.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Labeling, Incubation, Comparison

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a – d , Changes in endogenous BCL2-family PPI profiles of HL60 cells through apoptosis progression by 2 μM of staurosporine. Active BAX/BAK level ( a ), total levels of anti-apoptotic proteins ( b ), BIM complexes ( c ), BAX complexes ( n = 10 independent images) ( d ). e , Schematic of the PBA to measure the unoccupied populations of surface-immobilized anti-apoptotic proteins. f , Changes in BIM BH3 PBAs for anti-apoptotic proteins from HL60 cells through apoptosis progression by staurosporine ( n = 10 independent images). g , Schematic of the comparison of the BCL2 protein complex compositions in different AML cell lines (left). The density of surface-immobilized BCL2 was constantly maintained by optimizing the total protein concentration of crude cell extracts in all AML cell lines (right) ( n = 10 independent images). h – j , Compositions of the BCL2 PPI profiles in AML cell lines. BCL2 protein complexes ( h ), BCL2-BIM BH3 PBA ( n = 10 independent images) ( i ), integrated BCL2 complex compositions ( j ). k , Changes in BCL2-BIM BH3 PBA and BCL2-BAD PBA with increasing amounts of BAD probe ( n = 10 independent images). BIM BH3 probe was presented at 10 nM. The single-molecule counts were rescaled to account for the labelling efficiencies of the immunoassays calculated in Extended Data Fig. . Data represent means ± s.d.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Comparison, Protein Concentration

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a-d , PBA binding curves for each PPI probes (eGFP-labeled BIM BH3 , BIM EL , BAD, NOXA) to anti-apoptotic proteins (mCherry-labeled BCL2, BCLxL, MCL1) to calculate dissociation constant ( K d ). ( a ) BIM BH3 -PBA, ( b ) BIM EL -PBA, ( c ) BAD-PBA, ( d ) NOXA-PBA. e , Comparison of the binding affinities between each binding pairs. f , Correlations between the calculated K d values and the occupancy values at a fixed PPI probe concentration. g , Dissociation of BCL2-BIM complex after in vitro competition by BAD-eGFP PPI probes ( n = 10 independent images). Protein complexes were surface-immobilized by anti-BCL2 IP antibody and detected with anti-BIM detection antibody after the competition. h , Schematic of in vitro PBA competition between BIM BH3 -eGFP PPI probe and competitors (PPI probe or BH3 mimetics) on surface-immobilized BCL2 proteins. i , Remained BCL2-BIM BH3 PBA after in vitro binding competition with BH3 mimetics (ABT-199, AZD-5991, WEHI-539). BIM BH3 PPI probe was presented in 10 nM. j,k , BCL2 PBA counts with different PPI probes from four AML cell lines. ( j ) BCL2-BIM EL PBA, ( k ) BCL2-BAD PBA ( n = 10 independent images). l , Schematic for selective binding of PPI probes for unoccupied BCL2 proteins. Error bars represent means±s.d.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Binding Assay, Labeling, Comparison, Concentration Assay, In Vitro

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Comparison of BCL2 family PPI profiles from the healthy donor PBMC sample and HL60 cells with SMPC ( n = 10 independent images). The data were normalized to the HL60 cell. b , Correlation between ex vivo AUC for ABT-199 and combination of BCL2-related metrics (BCL2 total level, BCL2-BIM BH3 PBA, BCL2-BAD PBA, and BCL2-BAX CPX) (One-sided F -test, p -value = 1.3e-10). c , Linear correlations between two different BCL2 PBA metrics. d , Correlations between ex vivo AUC and IC 50 of primary AML samples for ABT-199 ( n = 32). e , Schematic of Lasso regression analysis for the selection of PPI metrics highly correlated with drug response. The training and test groups were randomly selected from the primary AML sample cohort. The Lasso regression models were generated using the training group and evaluated based on Pearson’s R as well as prediction outcomes for the test group. 67 models were selected from 10,000 different initial models. f , Identification of outliers in the ABT-199 drug efficacy prediction models. The residuals of each outlier in the model ( ex vivo AUC – estimated score) were indicated. g , Correlations between ex vivo AUC and IC 50 of primary AML samples for AZD-5991 ( n = 27). h , Correlation between ex vivo AUC for AZD-5991 and combination of MCL1-related metrics (MCL1 total level, MCL1-BIM BH3 PBA, MCL1-NOXA PBA, and MCL1-BAK CPX) (One-sided F -test, p -value = 2.5e-5).

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Comparison, Ex Vivo, Selection, Generated

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Schematic for BH3 profiling based on JC-1 staining, and measurement of BCL2 levels using flow cytometry or western blotting for primary AML samples. b,c , BH3 profiling on HL60 cells. ( b ) Relative fluorescence units (RFU) of 590 nm wavelength for HL60 cells through the treatment of BH3 peptides or BH3 mimetics, ( c ) Depolarization of HL60 cells through the BH3 profiling. Depolarization by 10 μM of BAD peptides was indicated. d,e , Correlations for ex vivo ABT-199 AUC with ( d ) depolarizations by BAD (10 μM) – HRK (10 μM) peptides, ( e ) combination of multiple PPI metrics (BCL2-BIM BH3 PBA, BCL2-BAX CPX, and BCLxL-BAK CPX). The outliers identified in model ( d ) were indicated ( n = 14) (One-sided F -test, p -value = 0.011, 8.1e-05). f,g , ROC curves for ex vivo ABT-199 responses with ( f ) depolarizations by BAD (10 μM) – HRK (10 μM) peptides, ( g ) Combination of multiple PPI metrics (Two-sided t -test, p -value = 0.07, 0.009). h , Correlations between BCL2 protein levels determined by SMPC and flow cytometry for primary AML samples (n = 14 ). i , Prediction models for ABT-199 ex vivo AUC with BCL2 protein levels determined by flow cytometry (One-sided F -test, p -value = 0.077). j , Correlations between BCL2 protein levels determined by SMPC and western blotting for primary AML samples ( n = 32). The raw blot image is provided as a Source Data. k , Prediction models for ABT-199 ex vivo AUC with BCL2 protein levels determined by western blotting (One-sided F -test, p -value = 0.0003).

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Staining, Flow Cytometry, Western Blot, Fluorescence, Ex Vivo