Journal: Computational and Structural Biotechnology Journal

Article Title: Computational design of an apoptogenic protein that binds BCL-xL and MCL-1 simultaneously and potently

doi: 10.1016/j.csbj.2022.06.021

Figure Lengend Snippet: Cell viability assay. (A) Viability assay. Leukemia (K562), colorectal cancer (HCT-116, SW620), and melanoma (MEWO, A375) cell lines were transduced with lentiviruses that encode each of the indicated constructs with a 3xFLAG tag. Then, cell viability was assayed 12 h after induction by 2 µg/ml doxycycline (Dox) (mean ± SD, n = 3). (B) Doxycycline-induced expression of the indicated proteins in transduced A375 cells as detected by western blotting using an anti-FLAG antibody. Cells were treated with the pan-caspase inhibitor Z-VAD-FMK prior to doxycycline induction to block BIM cleavage. A 10-fold serial dilution of doxycycline from 2 µg/ml was tested to modulate BIM expression. (C) Western blot analysis of transduced A375 cells harvested 12 h after induction by 2 µg/ml doxycycline. The expression of endogenous BCL-xL and MCL-1 along with γ-H2A.X and PARP cleavage were detected.

Article Snippet: For BIM detection, the pan-caspase inhibitor Z-VAD-FMK (MedChemExpress) was added 30 min prior to the addition of doxycycline.

Techniques: Viability Assay, Transduction, Construct, Expressing, Western Blot, Blocking Assay, Serial Dilution

Journal: Cancers

Article Title: Development of a Novel Biomarker Platform for Profiling Key Protein–Protein Interactions to Predict the Efficacy of BH3-Mimetic Drugs

doi: 10.3390/cancers17111852

Figure Lengend Snippet: PRIMAB production. ( A ) Supernatants collected from hybridomas from mice immunized with BCL-XL:BIM, BCL-2:BIM, or MCL-1:BIM covalent complexes were screened for selective binding to non-covalent immunogen complexes. ELISA readouts showing selective binding of mAb raised against the BCL-2:BIM covalent complex. Of 950 clones screened, 14 were highly selective; shown here is clone 2H3 that is a Heterodimer specific BCL-2: BIM (HSB2B). HSB2B not interact with BCL-2 or BIM protein alone but does bind to the BCL-2:BIM complex in a concentration-dependent manner. ( B ) The selective BCL-2 BH3-mimetic ABT199 (Venetoclax) was used to disrupt the BCL2:BIM complex. The PRIMAB signal aligned with Venetoclax treatment in a dose-dependent manner as determined by ELISA. ( C ) Supernatant from Hybridoma clone D32 raised against the BCL-XL:BIM complex was applied to the GST-BCL-XL:BIM complex and negative controls as indicated. The HSBXB D32 clone does not bind BCL-XL alone or to the other anti-apoptotic protein:BIM complexes. ( D ) The HSBXB clone was treated with the BCL2 selective BH3-mimetic Venetoclax or with the BCL-XL selective BH3-mimetic A1155463 at the indicated concentrations for 1 h. As indicated by ELISA, the HSBXB is not displaced by Venetoclax but is displaced by A1155463. ( E ) ELISA readouts showing selective binding of mAbs raised against the MCL-1:BIM complex. The HSMCB interacts with the MCL-1:BIM complex but neither protein alone. ( F ) To explore possible crossover binding of the various PRIMABs, biotinylated BIM BH3-domain containing peptides complexed with GST-MCL-1, GST-BCL-2, and GST-BCL-XL were incubated with each PRIMAB. Zero cross reactivity between any one of the PRIMABs and complexes used to make the others was observed.

Article Snippet: No signal was detected when BIM was deleted ( ) or complexes were disrupted with BH3-mimetics ( ).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Concentration Assay, Incubation

Journal: Cancers

Article Title: Development of a Novel Biomarker Platform for Profiling Key Protein–Protein Interactions to Predict the Efficacy of BH3-Mimetic Drugs

doi: 10.3390/cancers17111852

Figure Lengend Snippet: Various BCL-2 PPI disruptions by BH3-mimetics are detected in cells using PRIMBS. ( A ) Cell lines shown to have high levels MCL-1:BIM, BCL-2:BIM, or BCL-XL:BIM complexes were treated with the BH3-mimetics AZD5591, ABT-199, or A115463 respectively. Following treatments cells, were prepared and assessed by flow cytometry for complex signal. Error bars are the standard deviation (SD) for 3 wells. ( B ) Displacement of BIM from BCL-XL is detected by the HXBXB clone D32 in the megakaryoblast cell line SET-2 following treatment with A115463. This disruption of the BCL-XL:BIM complex is accompanied by a compensatory increase in the MCL-1:BIM complex as detected by the HSMCB PRIMAB. ( C ) The IC 50 s of cell lines U2932, H929, and U266B1 were determined for each of the BH3-mimetics ABT-199, AZD5591, and A1155463, respectively. PRIMAB readings were made at baseline and following 1 h of treatment at the determined IC 50 doses. Baseline readings were set at 1 and the post treatment % fold change in the BCL-2:BIM complex measurements was plotted against the IC 50 . R 2 values: 0.4, 0.87, 0.74, for HSB2B, HSMCB, HSBXB, respectively, indicate strong dependence of BH3-mimetic sensitivity on priming.

Article Snippet: No signal was detected when BIM was deleted ( ) or complexes were disrupted with BH3-mimetics ( ).

Techniques: Flow Cytometry, Standard Deviation, Disruption

Journal: Cancers

Article Title: Development of a Novel Biomarker Platform for Profiling Key Protein–Protein Interactions to Predict the Efficacy of BH3-Mimetic Drugs

doi: 10.3390/cancers17111852

Figure Lengend Snippet: BH3-mimetic shifting of priming complexes in cells can be visualized with PRIMABs. ( A ) Cell blocks were made from ABT-199, A1155463, and AZD5591 treated and untreated U2932, U266B1, and H929 cells to generate TMA slides PRIMABs were used to stain cells as indicated. IHC imaging was performed. Scale bar: 0.020 mm. ( B ) Pixel intensity of IHC in ( A ) was quantified using ImageQuant 800. ( C ) Immunofluorescence imaging of HCC1937 breast cancer cells treated with the BCL-XL BH3-mimetic A1331852 was performed using HSBXB and BCL-XL antibody to measure the BCL-XL:BIM complex and BCL-XL protein alone, respectively. Magnification: 63×. BCL-XL signal (red) colocalizes with HSBXB (green) as indicated in the shift of the green to yellow in untreated cells.

Article Snippet: No signal was detected when BIM was deleted ( ) or complexes were disrupted with BH3-mimetics ( ).

Techniques: Staining, Imaging, Immunofluorescence

Journal: Cancers

Article Title: Development of a Novel Biomarker Platform for Profiling Key Protein–Protein Interactions to Predict the Efficacy of BH3-Mimetic Drugs

doi: 10.3390/cancers17111852

Figure Lengend Snippet: BIM KD/KO impacts PRIMAB staining and priming. ( A ) HCC1937 breast cancer cells were treated with siRNA targeting BIM. Following treatment, intracellular staining of the MCL-1:BIM complex and total MCL-1, including the unbound MCL-1, was performed. Flow cytometry analysis of the cell line samples are reported as MFI. Following treatment, MCL-1 levels remained consistent with levels in untreated cells, while the level of MCL-1:BIM complex was reduced. The level of reduction on the complex coincided with the KD of BIM protein. ( B ) HCC1937 breast cancer cells were treated with BCL-XL targeting siRNA or BCL-XL selective BH3-mimetic A-1331852 and prepared into cell blocks arrayed into TMAs following treatment. IHC was performed and images collected provide a visual assessment of the loss of the BCL-XL:BIM complex measured by HSBXB in the siRNA treated cells at a similar level as the A-1331852 treated cells. In addition, MEF-WT and MEF-BIM -/- cells were made into cell blocks and arrayed onto TMAs. The BSBXB-PRIMAB signal and the BCL-XL signals were reduced in the MEF-BIM -/- cells. ( C ) CRISPR/Cas9 was used to target exon 5 of Bim in a tetraploid human breast cancer cell line (MCF7). The MCF7 2E9 BIM knockout cell line has a two base pair deletion (c.451-452 delGA) in two alleles and a 1 bp insertion (c.452_453 insG) in the other two alleles as confirmed by NGS sequencing. Position of the guide RNA is shown in blue, PAM site in green, and the BH3 domain indicated in orange. ( D ) Western blot analysis showing absence of BIM in MCF7 2E9 compared to otherwise isogenic wild-type MCF7 line (1C5). ( E ) MCF7-WT and MCF7-BIM KO were stained with HSMCB. The flow readout (MFI) indicated a significant loss of signal in the permeabilized BIM KO cells compared to the permeabilized WT cells. The signal in non-permeabilized cells indicated nonspecific background staining. The MCF7-WT and MCF7-BIM KO cells were made into cell blocks and used to generate TMAs. IHC images on samples stained with HSMCB. Images of cytoplasmic segmented staining were analyzed using HALO. Student’s two-tailed t -test was used for analysis. Magnification: 20×. ( F ) BH3 profiling was performed on MCF7-WT and MCF7-BIM KO permeabilized and treated with BH3-mimetic peptides and controls as indicated.

Article Snippet: No signal was detected when BIM was deleted ( ) or complexes were disrupted with BH3-mimetics ( ).

Techniques: Staining, Flow Cytometry, CRISPR, Knock-Out, Sequencing, Western Blot, Two Tailed Test

Journal: Cancers

Article Title: Development of a Novel Biomarker Platform for Profiling Key Protein–Protein Interactions to Predict the Efficacy of BH3-Mimetic Drugs

doi: 10.3390/cancers17111852

Figure Lengend Snippet: Correlation of PRIMAB signals to AML patient response to treatment. ( A ) Archival AML Patient bone marrow and peripheral blood samples were analyzed with the PRIMABs or with antibodies to the constituent anti-apoptotic proteins and BIM. Testing was performed on ficoll purified peripheral blood biopsies collected immediately prior to treatment with cytarabine based therapy (7+3). These were fixed, permeabilized, and stained with antibodies to BCL-2, BCL-XL, BIM, and MCL-1 and PRIMABs HSB2B, HSMCB, or HSBXB prior to analysis by flow cytometry. Readouts were compared to the event free survival in patients (EFS). Neither the anti-apoptotic proteins nor BIM expression measurements showed significant correlation to EFS. Significant correlation was seen with both the HSB2B PRIMAB ( p = 0.026) and HSMCB PRIMAB ( p = 0.037). The HSBXB signal did not correlate to response. (See ). ( B ) BH3 profiling was also performed on the same patient samples. BIM priming did not significantly correlate to EFS ( p = 0.107), nor did the MS-1 peptide JC1 signal induced by the MS-1 peptide ( p = 0.958). Student’s two-tailed t -test was used for statistical analysis.

Article Snippet: No signal was detected when BIM was deleted ( ) or complexes were disrupted with BH3-mimetics ( ).

Techniques: Purification, Staining, Flow Cytometry, Expressing, Two Tailed Test