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: Science Advances

Article Title: Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry

doi: 10.1126/sciadv.adi7840

Figure Lengend Snippet: ( A and B ) CD cells were G 2 -synchronized using RO-3306, and lysates were collected at the indicated time points after RO-3306 washout (“G 2 release”). Lysates were immunoblotted for pH3 to monitor mitotic entry. Three repeat experiments were quantified in (B). Fold change values ± SD. Arrows highlight the first pH3 peak of the respective groups indicating mitotic entry. ( C and D ) G 2 -synchronized CD cells were immunoblotted for cleaved caspase 3 (cl-Casp3) after G 2 release and quantified in (D) as fold change values ± SD ( n = 3). Asterisk (*) denotes between-group significance at the corresponding time point. ( E and F ) CD cells were G 2 -synchronized, and lysates were obtained immediately upon G 2 washout (G 2 , T0) and immunoblotted in biological duplicates. p-Cdk1 Y15: phosphorylated Cdk1 tyrosine-15. Three repeat experiments were quantified in (F). Fold change values ± SD. ( G and H ) Asynchronous Rac1 f/f (+DMSO) and Rac1 −/− [+DMSO or blebbistatin (5 μM)] CD cells were treated with cycloheximide (CHX; 100 μg/ml), and lysates were obtained at the indicated time points and immunoblotted for Wee1. Three repeat experiments are quantified in (H) as fold change ± SD. Asterisk (*) denotes significance between Rac1 −/− and Rac1 f/f or blebbistatin-treated Rac1 −/− at the corresponding time point. ( I and J ) Rac1 f/f and Rac1 −/− CD cells were G 2 -synchronized and treated with vehicle (DMSO) or 5 μM blebbistatin upon G 2 release. Lysates were collected at the indicated time points and immunoblotted for pH3 to monitor mitotic entry. Three independent repeat experiments are quantified (for Rac1 f/f , only the vehicle control is shown) in (J) as fold change values ± SD. Asterisk (*) denotes significance between Rac1 −/− and Rac1 f/f or blebbistatin-treated Rac1 −/− at the corresponding time point. * P < 0.05.

Article Snippet: Other cell culture treatments included nocodazole (Tocris Bioscience, #1228) at 100 ng/ml or the Wee1 inhibitor Adavosertib (MK-1775, Tocris Bioscience, #7589) at 1 μM, blebbistatin (Sigma-Aldrich, #203391) at 5 μM, or the ROCK inhibitor Y-27632 at 1 μM.

Techniques: Control

Journal: Science Advances

Article Title: Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry

doi: 10.1126/sciadv.adi7840

Figure Lengend Snippet: ( A to D ) Rac1 f/f and Rac1 −/− CD cells were G 2 -synchronized using RO-3306 and treated with the Wee1-specific inhibitor MK-1775 (1 μM) upon G 2 release. Lysates were collected at the indicated time points and immunoblotted for pH3 to monitor mitotic entry or cleaved caspase 3 to monitor cell death. Densitometry was used to quantify fold changes ± SD of three repeat experiments in (B) and (D). Arrows in (B) highlight the first pH3 peak indicating mitotic entry. ( E ) F-actin (white)– and DNA (blue)–labeled Rac1 f/f and Rac1 −/− (+MK-1775; 1 μM) CD cell monolayers analyzed by confocal microscopy with a mitotic metaphase cell shown in the center (scale bars, 10 μm). The top row column depicts metaphase F-actin (scale bars, 5 μm) as outlined by a red continuous box in the bottom row. Images are representative of at least three experiments. ( F ) Circularity quantification of mitotic metaphase F-actin as shown in (E) with a minimum of 10 measurements shown per group. Bars are means ± SD. ( G ) Live confocal mitosis imaging of SPY650-DNA (blue)– and SPY555-actin (white)–labeled vehicle (DMSO)– or MK-1775 (1 μM)–treated Rac1 f/f CD cells in vitro. The mitotic cell was manually segmented and colored in green. Scale bars, 10 μm. Sequences are representative of three repeat experiments with at least three to four mitoses analyzed per experiment. ( H ) Relative distribution of mitotic defects in vitro during live imaging cell division sequences with at least 10 mitoses analyzed per group. * P < 0.05.

Article Snippet: Other cell culture treatments included nocodazole (Tocris Bioscience, #1228) at 100 ng/ml or the Wee1 inhibitor Adavosertib (MK-1775, Tocris Bioscience, #7589) at 1 μM, blebbistatin (Sigma-Aldrich, #203391) at 5 μM, or the ROCK inhibitor Y-27632 at 1 μM.

Techniques: Labeling, Confocal Microscopy, Imaging, In Vitro

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Phase I Clinical Trial of the Wee1 inhibitor Adavosertib (AZD1775) with Irinotecan in Children with Relapsed Solid Tumors. A COG Phase 1 Consortium Report (ADVL1312)

doi: 10.1158/1078-0432.CCR-19-3470

Figure Lengend Snippet: Dose Escalation and DLT summary

Article Snippet: Adavosertib (10, 25 or 100 mg capsules) was supplied by Astra Zeneca and distributed by CTEP, NCI.

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Phase I Clinical Trial of the Wee1 inhibitor Adavosertib (AZD1775) with Irinotecan in Children with Relapsed Solid Tumors. A COG Phase 1 Consortium Report (ADVL1312)

doi: 10.1158/1078-0432.CCR-19-3470

Figure Lengend Snippet: Adavosertib pharmacokinetic (PK) parameters, median (range)

Article Snippet: Adavosertib (10, 25 or 100 mg capsules) was supplied by Astra Zeneca and distributed by CTEP, NCI.

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Phase I Clinical Trial of the Wee1 inhibitor Adavosertib (AZD1775) with Irinotecan in Children with Relapsed Solid Tumors. A COG Phase 1 Consortium Report (ADVL1312)

doi: 10.1158/1078-0432.CCR-19-3470

Figure Lengend Snippet: Median plasma concentration curves of adavosertib on day 1 and 5 (N = 12) at the recommended phase 2 dose of Irinotecan 90 mg/m2 and adavosertib 85 mg/m2. Blood samples were collected pre-dose and during cycle 1 at 1, 2, 4, 6 and 8 hrs after the first dose on day 1 and last dose on day 5. The adavosertib concentrations met the goal Wee1 target engagement concentration of 240 nM (red line).

Article Snippet: Adavosertib (10, 25 or 100 mg capsules) was supplied by Astra Zeneca and distributed by CTEP, NCI.

Techniques: Clinical Proteomics, Concentration Assay, Drug discovery

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Phase I Clinical Trial of the Wee1 inhibitor Adavosertib (AZD1775) with Irinotecan in Children with Relapsed Solid Tumors. A COG Phase 1 Consortium Report (ADVL1312)

doi: 10.1158/1078-0432.CCR-19-3470

Figure Lengend Snippet: Peripheral blood cell γH2AX fold induction on day 1, one hour after Irinotecan (column 4), and 4 hours after Irinotecan and adavosertib (column 5) and day 2, approximately 24 hours after chemotherapy (column 6). Bolded numbers indicate fold induction > 1 over baseline.

Article Snippet: Adavosertib (10, 25 or 100 mg capsules) was supplied by Astra Zeneca and distributed by CTEP, NCI.

Techniques:

Journal: Lancet (London, England)

Article Title: Gemcitabine plus adavosertib for platinum-resistant/refractory recurrent ovarian cancer: A randomised placebo-controlled trial

doi: 10.1016/S0140-6736(20)32554-X

Figure Lengend Snippet: Baseline characteristics (treated patients)

Article Snippet: Plasma adavosertib concentrations were profiled on day 1 (0, 1, 2, 4, 8, and 24 h) and day 2 (2, 4, and 8 h) using a validated liquid chromatography and tandem-mass spectrometry method (Covance Laboratories Inc., Madison, WI, USA).

Techniques: Mutagenesis, Staining

Journal: Lancet (London, England)

Article Title: Gemcitabine plus adavosertib for platinum-resistant/refractory recurrent ovarian cancer: A randomised placebo-controlled trial

doi: 10.1016/S0140-6736(20)32554-X

Figure Lengend Snippet: Most common adverse events by treatment arm and grade

Article Snippet: Plasma adavosertib concentrations were profiled on day 1 (0, 1, 2, 4, 8, and 24 h) and day 2 (2, 4, and 8 h) using a validated liquid chromatography and tandem-mass spectrometry method (Covance Laboratories Inc., Madison, WI, USA).

Techniques: