Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: Effects of Wee1 knockdown and Wee1 kinase inhibitor AZD1775 on ACC cells. (A) Proliferation assay in Wee1‐silenced MUC‐1 cells. Cells were transfected with Wee1 siRNA or negative control (C‐) siRNA for 72 h. Cells were incubated with BrdU for 2 h, and its incorporation into newly synthetized DNA was measured (median and IQR of at least 3 independent experiments). Representative immunoblots of Wee1 silencing are shown. *** p < 0.001 vs C‐ siRNA. Mann–Whitney test. (B) Detection of MUC‐1 cell apoptosis by Pacific Blue Annexin V/SYTOX AAdvanced Apoptosis kit. Unlabelled cells were used as negative control. After 72 h of transfection, the percentage of Annexin V+ and SYTOX+ cell fractions were measured. Representative flow cytometric plots are shown. Early apoptotic, late apoptotic, and necrotic cells are plotted graphically and are expressed as fold vs C‐ siRNA (median and IQR of at least 3 independent experiments). ** p < 0.01 vs C‐ siRNA. Mann–Whitney test. (C) Cell proliferation assay in MUC‐1, NCI‐H295R, and TVBF‐7 cell lines. Subconfluent cells were stimulated with increasing concentrations of AZD1775 for 72 h. After 2 h of incubation with BrdU, its incorporation into newly synthetized DNA was measured (median and IQR of at least 3 independent experiments, respectively). * p < 0.05; ** p < 0.01; *** p < 0.001 vs basal condition. Friedman test with Dunn's post‐hoc test. (D) Cell viability assay in MUC‐1, NCI‐H295R, and TVBF‐7 cell lines. Subconfluent cells were stimulated with increasing concentrations of AZD1775 for 72 h. MTT assay was used to verify cell viability in response to AZD1775 treatment (median and IQR of at least 3 independent experiments, respectively). * p < 0.05; ** p < 0.01; *** p < 0.001 vs basal condition. Friedman test with Dunn's post‐hoc test. (E) Cell proliferation assay in two different patient‐derived primary cultures of ACC (ACC #1 and ACC #2). Subconfluent cells were stimulated with increasing concentrations of AZD1775 for 72 h. After 24 h of incubation with BrdU, its incorporation into newly synthetized DNA was measured (median and IQR of 5 replicates for each condition). * p < 0.05; ** p < 0.01; *** p < 0.001 vs basal absorbance value. Kruskal–Wallis test with Dunn's post‐hoc test. (F) Detection of MUC‐1 cell apoptosis by Pacific Blue Annexin V/SYTOX AAdvanced Apoptosis kit. Unlabelled cells were used as negative control. After 72 h of treatment with AZD1775, the percentage of Annexin V+ and SYTOX+ cell fractions were measured. Representative flow cytometric plots are shown. Early apoptotic, late apoptotic, and necrotic cells are plotted graphically and are expressed as fold vs basal condition (median and IQR of at least 3 independent experiments). * p < 0.05; ** p < 0.01 vs basal condition. Friedman test with Dunn's post‐hoc test.

Article Snippet: AZD1775 was purchased from TargetMol Chemicals Inc. (Boston, MA).

Techniques: Knockdown, Proliferation Assay, Transfection, Negative Control, Incubation, Western Blot, MANN-WHITNEY, Viability Assay, MTT Assay, Derivative Assay

Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: AZD1775 induced cell cycle dysregulation and DNA damage in MUC‐1 cells. (A) AZD1775 induced S phase accumulation, and G1/S and G2/M reduction in MUC‐1 cell line. Subconfluent MUC‐1 cells were treated with AZD1775 500 nM for 72 h, and DNA content was then evaluated by flow cytometry after propidium iodide (PI) staining. The graph shows the % of cells in each stage of the cell cycle (median and IQR of at least 3 independent experiments). * p < 0.05 vs basal condition. Wilcoxon test. (B) γ‐H2AX immunofluorescence staining on MUC‐1 cells untreated and treated with AZD1775 500 nM for 6 h. Nuclei were counterstained with DAPI to allow visualization. Representative images are shown. γ‐H2AX were quantified as foci/nucleus and at least 100 nuclei were scored for each experiment (median and IQR of at least 3 independent experiments). *** p < 0.001 vs basal condition. Wilcoxon test.

Article Snippet: AZD1775 was purchased from TargetMol Chemicals Inc. (Boston, MA).

Techniques: Flow Cytometry, Staining, Immunofluorescence

Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: Effects of FLNA knockdown on cell proliferation of MUC‐1 and one patient‐derived primary cultured ACC cells, and on MUC‐1 apoptosis. (A) Cell proliferation assay in MUC‐1 silenced for FLNA. Subconfluent cells were silenced with FLNA siRNA for 6 days, and then stimulated with increasing concentrations of AZD1775 for 72 h. After 2 h of incubation with BrdU, its incorporation into newly synthetized DNA was measured (median and IQR of at least 3 independent experiments, respectively). ** p < 0.01; *** p < 0.001 of treated C‐ siRNA vs basal C‐ siRNA. Friedman test with Dunn's post‐hoc test. ## p < 0.01; ### p < 0.001 of treated FLNA siRNA vs basal FLNA siRNA. Friedman test with Dunn's post‐hoc test.° p < 0.05;°° p < 0.01 of FLNA siRNA vs C‐ siRNA at the same drug concentration. Mann–Whitney test. (B) Cell proliferation assay in one patient‐derived primary culture of ACC silenced for FLNA. Subconfluent cells were silenced with FLNA siRNA for 6 days, and then stimulated with increasing concentrations of AZD1775 for 72 h. After 24 h of incubation with BrdU, its incorporation into newly synthetized DNA was measured (median and IQR of 5 replicates for each condition). * p < 0.05; ** p < 0.01 of treated C‐ siRNA vs basal C‐ siRNA. Kruskal–Wallis test with Dunn's post‐hoc test. # p < 0.05; ## p < 0.01 of treated FLNA siRNA vs basal FLNA siRNA. Kruskal–Wallis test with Dunn's post‐hoc test.° p < 0.05;°° p < 0.01 of FLNA siRNA vs C‐ siRNA at the same drug concentration. Mann–Whitney test. (C) MUC‐1 cell apoptosis was detected by Pacific Blue Annexin V/SYTOX AAdvanced Apoptosis Kit. Unlabelled cells were used as negative control. MUC‐1 were transfected with the FLNA‐specific siRNA for 6 days. After treatment with AZD1775, the percentage of Annexin V+ and SYTOX+ cell fractions were measured. Representative flow cytometric plots are shown. Early apoptotic, late apoptotic, and necrotic cells are plotted graphically and are expressed as fold vs C‐ siRNA (median and IQR of at least 3 independent experiments). * p < 0.05 of C‐ siRNA vs FLNA siRNA at the same drug concentration. Mann–Whitney test.

Article Snippet: AZD1775 was purchased from TargetMol Chemicals Inc. (Boston, MA).

Techniques: Knockdown, Derivative Assay, Cell Culture, Proliferation Assay, Incubation, Concentration Assay, MANN-WHITNEY, Negative Control, Transfection

Journal: ACS Pharmacology & Translational Science

Article Title: Differential Sensitivity to CDK2 Inhibition Discriminates the Molecular Mechanisms of CHK1 Inhibitors as Monotherapy or in Combination with the Topoisomerase I Inhibitor SN38

doi: 10.1021/acsptsci.9b00001

Figure Lengend Snippet: Abrogation of S phase arrest by CHK1i or WEE1i is insensitive to inhibition of CDK1/2 but requires CDC7 and CDC25 activity. (a) MDA-MB-231 cells were incubated with SN38 for 24 h and harvested as indicated. Cells were stained for DNA with propidium iodide and analyzed by flow cytometry. (b) Following removal of SN38 at 24 h, cells were incubated with 1 μM MK-8776 (CHK1i) or 1 μM AZD1775 (WEE1i) concurrently with 0–10 μM CVT-313 (CDK1/2i). (c) Same as panel b, except 0–10 μM XL413 (CDC7i) was added concurrently with CHKi or WEE1i. (d) MDA-MB-231 cells were incubated with SN38 for 0–24 h, and then CHK1i or WEE1i were added from 24 to 30 h, concurrently with 0–20 μM NSC663284 (CDC25i). Cells were immunostained with a fluorescent anti-pHH3 antibody, and DNA was stained with propidium iodide and then analyzed by flow cytometry. The gates and percentages shown represent the proportion of cells positive for pHH3.

Article Snippet: 3 Small Molecule Inhibitors SN38 (Pfizer, New York, NY), CVT-313 (Sigma-Aldrich), MK-8776 (Merck, Kenilworth, NJ), AZD1775 (Merck), and NSC663284 (Cayman Chemical, Ann Arbor, MI) were dissolved in dimethyl sulfoxide.

Techniques: Inhibition, Activity Assay, Incubation, Staining, Flow Cytometry

Journal: Cell chemical biology

Article Title: Development and Characterization of a Wee1 Kinase Degrader

doi: 10.1016/j.chembiol.2019.10.013

Figure Lengend Snippet: (A) Immunoblot analysis of MOLT4 parental or CRBN−/− cells treated with ZNL-02-096 for 5 hrs; (B) Immunoblot analysis of MOLT4 cells pre-treated with DMSO, Carfilzomib (Carf.), MLN4924 (MLN), pomalidomide (Pom.), or AZD1775 for 2 hrs, and then co-treated with ZNL-02-096 for 5 hrs; (C) Immunoblot analysis of MOLT4 cells treated with ZNL-02-096 for the indicated time points; (D) Immunoblot analysis of MOLT4 cells treated with ZNL-02-096 or ZNL-02-178 for the indicated time points; (E) Log2 fold-change in abundance of proteins as measured using multiplexed quantitative-mass spectrometry-based proteomics of MOLT4 cells treated with ZNL-02-096 (100 nM) for 2 hrs vs. p-value. n = 3 biological replicates.

Article Snippet: X-ray crystal structure model of Wee1 bound AZD1775 (pdb: 5v5y) was superimposed to each of the docked poses of Wee1 using Pymol ‘align’ command (The PyMOL Molecular Graphics System, Version 1.8.6.0 Schrodinger, LLC).

Techniques: Western Blot, Mass Spectrometry

Journal: Cell chemical biology

Article Title: Development and Characterization of a Wee1 Kinase Degrader

doi: 10.1016/j.chembiol.2019.10.013

Figure Lengend Snippet: (A) Chemical structure of AZD1775, with linker attachment point highlighted in blue (B) Chemical structures of Wee1 degraders and the negative control, ZNL-02-178 (C) Histogram of the shortest pairwise distances between lenalidomide and AZD1775, with the red arrow corresponding to the docking pose in panel D (Rosetta) (D) Shortest pairwise distance docking pose between lenalidomide and AZD1775 (Rosetta)

Article Snippet: X-ray crystal structure model of Wee1 bound AZD1775 (pdb: 5v5y) was superimposed to each of the docked poses of Wee1 using Pymol ‘align’ command (The PyMOL Molecular Graphics System, Version 1.8.6.0 Schrodinger, LLC).

Techniques: Negative Control

Journal: Cell chemical biology

Article Title: Development and Characterization of a Wee1 Kinase Degrader

doi: 10.1016/j.chembiol.2019.10.013

Figure Lengend Snippet: (A) Immunoblot analysis of MOLT4 cells treated with the ZNL-02-096, ZNL-02-178, AZD1775, or pomalidomide for 24 hrs. (B) MOLT4 cells treated with ZNL-02-096, ZNL-02-178, or AZD1775 for the indicated time points and stained with propidium iodide. Reported as mean ± SEM for n=3 biological replicates; (C) Parental or CRBN−/− MOLT4 cells treated with ZNL-02-096, ZNL-02-178, AZD1775, or Lenalidomide for 24 hrs and stained with propidium iodide. Reported as mean ± SEM for n=3 biological replicates.

Article Snippet: X-ray crystal structure model of Wee1 bound AZD1775 (pdb: 5v5y) was superimposed to each of the docked poses of Wee1 using Pymol ‘align’ command (The PyMOL Molecular Graphics System, Version 1.8.6.0 Schrodinger, LLC).

Techniques: Western Blot, Staining

Journal: Cell chemical biology

Article Title: Development and Characterization of a Wee1 Kinase Degrader

doi: 10.1016/j.chembiol.2019.10.013

Figure Lengend Snippet: (A) Cell viability of parental or CRBN−/− MOLT4 cells treated with AZD1775, ZNL-02-096, or ZNL-02-178 for 72 hrs, using CellTiter-Glo. Plotted as mean ± SEM for n=4 replicates; (B) Cell viability of OVCAR8 cells treated with AZD1775, ZNL-02-096, or ZNL-02-178 for 72 hrs, using CellTiter-Glo. Plotted as mean ± SEM for n=4 replicates; (C) Cell viability of OVCAR8 cells co-treated with ZNL-02-096 and Olaparib, or AZD1775 and Olaparib for 72 hrs, using CellTiter-Glo. Plotted as mean ± SEM for n=4 replicates.

Article Snippet: X-ray crystal structure model of Wee1 bound AZD1775 (pdb: 5v5y) was superimposed to each of the docked poses of Wee1 using Pymol ‘align’ command (The PyMOL Molecular Graphics System, Version 1.8.6.0 Schrodinger, LLC).

Techniques: