Journal: British Journal of Haematology

Article Title: BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT IN HUMAN AML AND ALL CELLS IN ASSOCIATION WITH PERTURBATIONS IN NF-?B AND BIM

doi: 10.1111/j.1365-2141.2011.08591.x

Figure Lengend Snippet: (A-E) Logarithmically-growing U937 (A), HL-60 (B), MV-4-11 (C), Jurkat (D), and SEM (E) cells were exposed (24 hr) to the indicated concentrations of belinostat (PXD) ± bortezomib (SEM, 3 nM; other lines, 5 nM), after which the percentage of apoptotic cells was determined by annexin V/PI staining and flow cytometry. The results represent the means ± S.D. for experiments performed in triplicate on three separate occasions. AML cell lines were exposed (24 hr) to PTL (U937 and HL-60, 5 μM and 7.5 μM; MV-4-11, 5 μM; NB4, 3 μM) ± vorinostat (V, U937, 1.5 μM; HL-60 and NB4, 1 μM; MV-4-11, 0.5μM) or LBH589 (L, U937, 15 nM; NB4, 7.5 nM; HL-60, 5 nM; MV-4-11, 3 nM). After drug treatment, apoptosis was monitored by annexin V staining and flow cytometry. (F) Median Dose Effect analysis was employed to characterize interactions between belinostat and bortezomib in U937 cells, administered over a range of concentrations at a fixed ratio (40:1) for 24 hr, reflected by apoptosis induction (annexin V staining). Combination Index (CI) values < 1.0 correspond to a synergistic interaction.

Article Snippet: The proteasome inhibitor bortezomib and the pan-HDAC inhibitor belinostat were provided by Millennium (Cambridge, MA) and TopoTarget (Copenhagen, Denmark), respectively.

Techniques: Staining, Flow Cytometry

Journal: British Journal of Haematology

Article Title: BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT IN HUMAN AML AND ALL CELLS IN ASSOCIATION WITH PERTURBATIONS IN NF-?B AND BIM

doi: 10.1111/j.1365-2141.2011.08591.x

Figure Lengend Snippet: (A-C) U937, HL-60, Jurkat, and SEM cells were exposed (24 hr) to belinostat (U937, 200 nM and 300 nM; HL-60 and Jurkat, 300 nM; SEM, 100 nM) ± bortezomib (U937, 4 nM and 5 nM; HL-60 and Jurkat, 5 nM; SEM, 4 nM), after which cells were lysed and subjected to Western blot analysis to assess total and lysine 310 acetylated RelA/p65, serine 32/36 phosphorylated form of IκBα (A), total and lysine 40 acetylated α-tubulin (B), and expression of the precursor p100 and its active form p52 (C). Each lane was loaded with 30 μg of protein; blots were subsequently stripped and reprobed for expression β-actin to ensure equivalent loading and transfer of protein. Results of a representative experiment are shown; two additional studies yielded equivalent results. (D) U937 cells were stably transfected with NF-κB luciferase reporter as described in “Materials and Methods”. Cells were exposed to 200 nM or 300 nM belinostat with or without 5 nM bortezomib for 24 hr, after which cells were lysed and subjected to luciferase activity analysis. Relative luciferase activity was determined following normalization of values to total protein. NF-κB activity (reflected by RLU, relative light unit) was expressed as the fold-increase relative to values for untreated controls. (E) U937 cells were exposed to 200 nM or 300 nM belinostat in the presence or absence of 5 nM bortezomib, after which nuclear extracts were prepared and subjected to a RelA/p65-specific NF-κB-DNA binding assay as described in “Materials and Methods”. Wild-type (wt) and mutated (mt) consensus oligonucleotides were used as competitors for RelA/p65-DNA binding in order to demonstrate assay specificity. RelA/p65-DNA binding activity (reflected by relative light unit/RLU) was expressed as fold increase relative to untreated controls. For panels 2D and 2E, results represent the means ± S.D. for triplicate determinations performed on three separate occasions. Values shown represent P values for the significance of differences between cells exposed to belinostat alone versus those exposed to belinostat and bortezomib.

Article Snippet: The proteasome inhibitor bortezomib and the pan-HDAC inhibitor belinostat were provided by Millennium (Cambridge, MA) and TopoTarget (Copenhagen, Denmark), respectively.

Techniques: Western Blot, Expressing, Stable Transfection, Transfection, Luciferase, Activity Assay, DNA Binding Assay, Binding Assay

Journal: British Journal of Haematology

Article Title: BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT IN HUMAN AML AND ALL CELLS IN ASSOCIATION WITH PERTURBATIONS IN NF-?B AND BIM

doi: 10.1111/j.1365-2141.2011.08591.x

Figure Lengend Snippet: (A-B) U937, HL-60, Jurkat, and SEM cells were exposed (24 hr) to belinostat ± bortezomib administered at the same concentrations as those described in Fig 2. Cells were then lysed and subjected to Western blot analysis to monitor expression of the anti-apoptotic proteins XIAP, Bcl-xL, and survivin (A), as well as the pro-apoptotic Bcl-2 family protein Bim, including three isoforms (BimEL, BimL, and BimS). Each lane was loaded with 30 μg of protein; blots were subsequently stripped and re-probed for expression of β-actin to ensure equivalent loading and transfer. Representative results are shown; two additional experiments yielded equivalent findings. (C) U937 and Jurkat cells were stably transfected with shRNA directed against Bim (shBim) or scrambled sequence controls (shNC). Western blot analysis demonstrates downregulation of the three Bim isoforms. Each lane was loaded with 30 μg of protein. In parallel, the same membrane was blotted for α-tubulin as a loading control. (D-E) U937 (D) and Jurkat (E) cells transfected with shBim or shNC were exposed (24 hr) to 300 nM belinostat + 5 nM bortezomib, after which cell death was monitored by annexin V/PI analysis by flow cytometry. Values shown represent the percentage of cells in the right quadrants (lower, annexin V+/PI−, and upper, annexin V+/PI+). Two additional experiments yielded equivalent results.

Article Snippet: The proteasome inhibitor bortezomib and the pan-HDAC inhibitor belinostat were provided by Millennium (Cambridge, MA) and TopoTarget (Copenhagen, Denmark), respectively.

Techniques: Western Blot, Expressing, Stable Transfection, Transfection, shRNA, Sequencing, Membrane, Control, Flow Cytometry

Journal: British Journal of Haematology

Article Title: BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT IN HUMAN AML AND ALL CELLS IN ASSOCIATION WITH PERTURBATIONS IN NF-?B AND BIM

doi: 10.1111/j.1365-2141.2011.08591.x

Figure Lengend Snippet: (A-B) Primary blasts from a patient (#1) with AML (sub-type M2) were exposed (24 hr) to 100–500 nM belinostat in the presence or absence of 5 nM bortezomib, after which uptake of 7-AAD/DiOC6 (A) or annexin V/PI (B) were monitored by flow cytometry. For panel 4A, values refer to the percentage of cells in the upper (high 7-AAD uptake) or left (low DiOC6 uptake, reflecting loss of mitochondrial membrane potential or Δψm) quadrants. For panel 4B, values refer to the percentage of annexin V-positive (right quadrant) or PI-positive (upper quadrant) cells. (C) Blast samples from three additional AML patients were exposed to 300 nM belinostat ± bortezomib (#2 and #4, 5 nM; #3, 8 nM), after which 7-AAD uptake was monitored as described in panel 4A. Values represent the means ± S.D. for triplicate determinations. (D) Following treatment as described in panel C, blasts from two patients (#2 and #3) were lysed, and Western blot analysis performed to monitor PARP cleavage. CF = cleaved fragment. Each lane was loaded with 30 μg of protein; blots were subsequently stripped and re-probed for expression of β-actin to ensure equivalent loading and transfer.

Article Snippet: The proteasome inhibitor bortezomib and the pan-HDAC inhibitor belinostat were provided by Millennium (Cambridge, MA) and TopoTarget (Copenhagen, Denmark), respectively.

Techniques: Flow Cytometry, Membrane, Western Blot, Expressing

Journal: British Journal of Haematology

Article Title: BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT IN HUMAN AML AND ALL CELLS IN ASSOCIATION WITH PERTURBATIONS IN NF-?B AND BIM

doi: 10.1111/j.1365-2141.2011.08591.x

Figure Lengend Snippet: (A-B) Blasts from patients with B-cell ALL (A) or T-cell ALL (B) were exposed (24 hr) to the indicated concentrations of belinostat ± 5 nM bortezomib, after which uptake of 7-AAD/DiOC6 (A) or annexin V/PI (B) were determined by flow cytometry as described in Fig 4. (C) Three primary ALL (B-cell ALL = 2, and T-cell ALL = 1) samples and four cord blood (CB) CD34+ cell samples were exposed (24 hr) to belinostat (B-ALL, 400 nM; T-ALL, 300 nM; CB CD34+, 500 nM) ± 5 nM bortezomib, after which cell death (7AAD+ cells) was determined by flow cytometry as described in above. Values represent the means ± S.D. for triplicate determinations. (D) Representative photomicrographs of Wright-Giemsa stained cytospin slides for primary B- and one T-cell ALL specimens viewed under oil at 60x magnification. (E) Following treatment as described in panel 5A and 5B, blasts from B-cell and T-cell ALL patients were lysed, and Western blot analysis performed to monitor PARP cleavage. Each lane was loaded with 30 μg of protein; blots were subsequently stripped and re-probed for expression of β-actin to ensure equivalent loading and transfer. (F) Normal cord blood (CB) CD34+ cells were exposed to 500 nM belinostat ± 5 nM bortezomib, after which annexin V/PI uptake was monitored as in panel 5B.

Article Snippet: The proteasome inhibitor bortezomib and the pan-HDAC inhibitor belinostat were provided by Millennium (Cambridge, MA) and TopoTarget (Copenhagen, Denmark), respectively.

Techniques: Flow Cytometry, Staining, Western Blot, Expressing

Journal: British Journal of Haematology

Article Title: BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT IN HUMAN AML AND ALL CELLS IN ASSOCIATION WITH PERTURBATIONS IN NF-?B AND BIM

doi: 10.1111/j.1365-2141.2011.08591.x

Figure Lengend Snippet: (A-B) Blasts from patients with AML, B-cell ALL, or T-cell ALL were exposed (24 hr) to belinostat (AML, 100, 300 and 500 nM; B-ALL, 200 and 400 nM; T-ALL, 100 and 300 nM) ± bortezomib (AML, 8 and 10nM; B-ALL and T-ALL, 5 nM), after which cells were lysed and subjected to Western blot analysis to assess acetylation of α-tubulin (lysine 40, A) and RelA/p65 (lysine 310), phosphorylation of IκBα (serine 32/36), and processing of p100 to p52 (B). Levels of total p65 and α-tubulin are shown as controls. Each lane was loaded with 30 μg of protein; blots were subsequently stripped and reprobed for β-actin to ensure equivalent loading and transfer of protein. Duplicate experiments yielded equivalent results.

Article Snippet: The proteasome inhibitor bortezomib and the pan-HDAC inhibitor belinostat were provided by Millennium (Cambridge, MA) and TopoTarget (Copenhagen, Denmark), respectively.

Techniques: Western Blot, Phospho-proteomics

Journal: British Journal of Haematology

Article Title: BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT IN HUMAN AML AND ALL CELLS IN ASSOCIATION WITH PERTURBATIONS IN NF-?B AND BIM

doi: 10.1111/j.1365-2141.2011.08591.x

Figure Lengend Snippet: (A-B) Primary AML, B-ALL, and T-ALL blasts were treated (24 hr) with the indicated concentrations of belinostat ± bortezomib (AML, 8 nM; B-ALL and T-ALL, 5 nM) as described in Fig 6. Following treatment, Western blot analysis was performed to monitor expression levels of the pro-apoptotic proteins XIAP, Bcl-xL, and survivin, as well as the proapoptotic protein Bim (BimEL, BimL, and BimS). Each lane was loaded with 30 μg of protein; blots were subsequently stripped and re-probed for expression of β-actin to ensure equivalent loading and transfer. Duplicate experiments yielded equivalent results.

Article Snippet: The proteasome inhibitor bortezomib and the pan-HDAC inhibitor belinostat were provided by Millennium (Cambridge, MA) and TopoTarget (Copenhagen, Denmark), respectively.

Techniques: Western Blot, Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: The Histone Deacetylase 9 Stroke-Risk Variant Promotes Apoptosis and Inflammation in a Human iPSC-Derived Smooth Muscle Cells Model

doi: 10.3389/fcvm.2022.849664

Figure Lengend Snippet: Smooth muscle cells with the stroke risk variant show higher level of apoptosis and respond to HDAC inhibitors treatment. (A) Flow cytometric analysis of Annexin V (FITC) show percentage gated for in WT1 (2.9%), iHDAC9 (9.6%), HDAC9v-1 (27.64%), and HDAC9v-2 (25.74%) SMC and unstained (dotted lines) and (B) Annexin V staining quantification ( n = 3). (C) SMC flow cytometric analysis of Annexin V (FITC) show percentage gated for HDAC9v-1 untreated (DMSO; 25.92%) and upon treatment with BRD4354 (HDAC9/HDAC5 inhibitor; 7.2%) (10 μmol/L); AstraZeneca HDAC class II inhibitors AZ01 (3.52%) and AZ02 (4.30%) (10 μmol/L); sodium valproate [valproic acid (VA); 3%] (1 mmol/L); and HDAC class I inhibitor AZ03 (19.81%) (10 μmol/L); and (D) Annexin V positive staining quantification ( n = 3). The results are presented as means ± SD of three independent experiments. * P < 0.05; ** P < 0.01; ns, not significant. Statistical analysis was performed by 1-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Based on these observations, we set up a phenotypic drug screen, by comparing the efficacy of the HDAC pan-inhibitor sodium valproate (VA), BRD4354, a zinc chelating reversible inhibitor with selectivity for HDAC5 and HDAC9, two HDAC class II inhibitors (AZ01 and AZ02) and a HDAC class I inhibitor (AZ03) kindly provided by AstraZeneca ( ).

Techniques: Variant Assay, Staining, Comparison

Journal: Frontiers in Cardiovascular Medicine

Article Title: The Histone Deacetylase 9 Stroke-Risk Variant Promotes Apoptosis and Inflammation in a Human iPSC-Derived Smooth Muscle Cells Model

doi: 10.3389/fcvm.2022.849664

Figure Lengend Snippet: Valproate and AZ class II inhibitors rescue risk SMC proliferation and migration phenotypes. (A) Proliferation rate measured by Ki67 staining in HDAC9-v1 control (DMSO) and treated with BRD4354, AZ01, AZ02 HDAC class II inhibitors and sodium valproate (VA) and (B) quantification of Ki67 staining expressed relative to cells number (nuclei). (C) Representative images of scratch wound assays of iHDAC9 and HDAC9v-1 SMC treated with AZ01, AZ02, and VA at the indicated time points (0, 6, and 12 h) and (D) quantification of the wound areas. (E) Cropped blot of phospho-p53 (P-P53), total P53 and β-actin control for HDAC9v-1 control (DMSO) and upon treatment with AZ01, AZ02 and VA (a) and quantification (b). Nuclei were stained with DAPI. Scale bar = 100 μm. The results are presented as means ± SD of three independent experiments. ** P < 0.01; *** P < 0.001; ns, not significant. Statistical analysis was performed by 1-way (B) and 2-way (D) ANOVA with Tukey’s multiple comparison test.

Article Snippet: Based on these observations, we set up a phenotypic drug screen, by comparing the efficacy of the HDAC pan-inhibitor sodium valproate (VA), BRD4354, a zinc chelating reversible inhibitor with selectivity for HDAC5 and HDAC9, two HDAC class II inhibitors (AZ01 and AZ02) and a HDAC class I inhibitor (AZ03) kindly provided by AstraZeneca ( ).

Techniques: Migration, Staining, Control, Comparison