Journal: Cancer Research Communications

Article Title: Combination AURKA and WEE1 Inhibition Exhibits Efficacy in EGFR or Pan-ERBB Inhibitor–Resistant Head and Neck Squamous Cell Carcinoma

doi: 10.1158/2767-9764.CRC-26-0368

Figure Lengend Snippet: Characterization of ErlR and AfaR HNSCC cell models. A and B, Dose response curves for ( A ) Fadu and ( B ) Cal27 parental, ErlR, or AfaR cell lines treated with the indicated dose range of afatinib or erlotinib for 72 hours. C, Representative Western blot images for active RTKs EGFR, MET, and HER2. D, Quantification of Western blots shown in C , with values normalized to those in parental cell lines. Graphs display baseline activation relative to total levels of each protein, based on n = 3. Note that for some proteins analyzed (e.g., MET), there was a significant intensity difference between FaDu and Cal27 models. For these cases, multiple exposures were taken for the relevant data, and quantification was performed based on primary Western blot data within a linear signal range. E, GSEA comparing Fadu and Cal27 AfaR or ErlR vs. Fadu and Cal27 parental cells, as indicated. RNA-seq analysis shown is averaged from two biological replicates. F and G, Representative Western blot images ( F ) and quantification ( G ) for indicated biomarkers of epithelial identity or EMT, based on n = 3 biological repeats. Bar graphs display data normalized for each parental cell line. P values are based on one-way ANOVA followed by Tukey multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.001; ****, P ≤ 0.0001. Data are shown as mean ± SEM of three biological replicates. ES, enrichment score.

Article Snippet: For drug treatments, cells were treated with vehicle (0.01% DMSO), VIC-1911 (VITRAC), adavosertib (MedChemExpress, HY-10993), erlotinib (MedChemExpress, E-4997), or afatinib (MedChemExpress, HY-10261).

Techniques: Western Blot, Activation Assay, RNA Sequencing, Comparison

Journal: Cancer Research Communications

Article Title: Combination AURKA and WEE1 Inhibition Exhibits Efficacy in EGFR or Pan-ERBB Inhibitor–Resistant Head and Neck Squamous Cell Carcinoma

doi: 10.1158/2767-9764.CRC-26-0368

Figure Lengend Snippet: Response of parental, AfaR, and ErlR models to AURKAi combinations with erlotinib or afatinib. A, Loewe synergy plot showing synergistic interaction between VIC-1911 and afatinib or erlotinib in FaDu and Cal27 parental, ErlR, and AfaR cell lines treated for 72 hours, n = 3. B, Quantification of dead cells from the models indicated treated for 72 hours with drugs indicated. FaDu and derived models were treated with vehicle, VIC-1911 (0.6 μmol/L), afatinib (0.2 μmol/L), erlotinib (1 μmol/L), or combination. Cal27 and derived models were treated with vehicle, VIC-1911 (0.6 μmol/L), afatinib (0.8 μmol/L), erlotinib (2 μmol/L), or combination for 24 hours. C and D, Clonogenic survival assay primary data ( C ) and corresponding ( D ) quantification. Cells were treated with the drug concentration indicated in C for 11 days. E, Quantification of annexin-positive cells for Fadu parental and Fadu AfaR treated for 3 days (left) and 5 days (right) with VIC-1911 (600 nmol/L). P values are based on one-way ANOVA followed by the Tukey multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Data are shown as mean ± SEM of three biological replicates.

Article Snippet: For drug treatments, cells were treated with vehicle (0.01% DMSO), VIC-1911 (VITRAC), adavosertib (MedChemExpress, HY-10993), erlotinib (MedChemExpress, E-4997), or afatinib (MedChemExpress, HY-10261).

Techniques: Derivative Assay, Clonogenic Cell Survival Assay, Concentration Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition of WEE1 Potentiates the Antitumoral Effect of the dl922-947 Oncolytic Virus in Malignant Mesothelioma Cell Lines

doi: 10.3390/ijms21197333

Figure Lengend Snippet: Synergistic effect of dl922-947 -AZD1775 combination on malignant mesothelioma (MM) cell lines. ( A ) Dose–response curves for dl922-947 alone, AZD1775 alone, and dl922-947 -AZD1775 combination in NCI-H28 and MSTO-211H cell lines 5 days after treatment. Results represent the means with standard deviation of 2 independent experiments, each conducted in triplicate, and are expressed as percentages of cell viability calculated with respect to control cells treated with DMSO alone. ( B ) Isobologram analysis to evaluate synergism between dl922-947 and AZD1775. Isobolograms are derived from the mean values of the dose–response experiments reported in (A) , through the CompuSyn software 1.0 (ComboSyn, Inc., Paramus, NJ, USA), at effect levels (Fa, fraction affected) of 25, 50, and 75%. Data points on the line indicate additivity; points below the line indicate synergy; points above the line indicate antagonism. The combination indexes (CIs) at 25, 50, and 75% of cell killing (CI25, CI50, CI75, respectively) and r values are also reported. Combination index (CI) values < 1 indicate synergism. ( C ) Histogram representing MET-5A cell viability analyzed 5 days after dl922-947 -AZD1775 co-treatment. Cell viability was calculated as a percentage with respect to control cells treated with DMSO alone. Results represent the means ± standard deviation of 3 experiments, each conducted in triplicate. The absorbance values of treated and control cells were subjected to paired Student’s t-test and showed no significant difference (ns, not significant).

Article Snippet: MM cells were seeded in 96-well plates 24 h before treatment with dl 922-947 and AZD1775 (MK1775, purchased from Axon Medchem), both alone and in combination at various concentrations in a constant ratio.

Techniques: Standard Deviation, Control, Derivative Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition of WEE1 Potentiates the Antitumoral Effect of the dl922-947 Oncolytic Virus in Malignant Mesothelioma Cell Lines

doi: 10.3390/ijms21197333

Figure Lengend Snippet: Cell death induction in malignant mesothelioma (MM) cells treated with dl922-947 and/or AZD1775. ( A ) Histograms report the means with standard deviations of at least three independent experiments representing the percentage of positive cells stained with annexinV–FITC and propidium iodide (PI) 96 h after treatment with dl922-947 and/or AZD1775 or DMSO, as a control. Statistically significant differences were evaluated by one-way repeated measures ANOVA with Tukey post-test and indicated as follows: * p < 0.05, significant; ** p < 0.01, very significant; and *** p < 0.001, extremely significant. ( B ) Caspase-3 and BCL-XL protein levels were analyzed by Western blotting in NCI-H28 and MSTO-211H cells treated as reported above. The antibody against caspase-3 detects both the full-length protein and the active cleaved form, which are shown separately at different exposure times (the full-length protein bands are shown at a shorter exposure time because they become overexposed at the time necessary for cleaved caspase-3 bands to appear). GAPDH was used as a loading control. Full-length caspase-3 band densities were quantified by densitometric analysis and normalized with the GAPDH band densities. Data are presented as relative values with respect to control values, set at 1. ( C ) Histograms reporting NCI-H28 and MSTO-211H cell viability 96 h after treatment with dl922-947 alone and in combination with Z-VAD-FMK. Results represent the means with standard deviations of at least 2 independent experiments, each conducted in triplicate, and are expressed as percentages of cell viability calculated with respect to untreated control cells. Statistically significant differences were evaluated by one-way repeated measures ANOVA with Tukey post-test and indicated as follows: * p < 0.05, significant; ** p < 0.01, very significant. ( D ) Representative fluorescence micrographs of DAPI-stained NCI-H28 and MSTO-211H cells treated with dl922-947 and/or AZD1775 as reported above. Arrows indicate some of the nuclei with clumped or condensed chromatin; arrowheads indicate some multilobed nuclei or multinucleation.

Article Snippet: MM cells were seeded in 96-well plates 24 h before treatment with dl 922-947 and AZD1775 (MK1775, purchased from Axon Medchem), both alone and in combination at various concentrations in a constant ratio.

Techniques: Staining, Control, Western Blot, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition of WEE1 Potentiates the Antitumoral Effect of the dl922-947 Oncolytic Virus in Malignant Mesothelioma Cell Lines

doi: 10.3390/ijms21197333

Figure Lengend Snippet: Effect of dl922-947 and AZD1775 on the activation of cyclin-dependent kinase 1 (CDK1) and DNA damage signaling factors in malignant mesothelioma (MM) cell lines. NCI-H28 and MSTO-211H cell lines were treated with dl922-947 and AZD1775, both alone and in combination, and analyzed through Western blotting for the following factors: phosphorylated histone H2AX (γ-H2AX); phospho-replication protein A (RPA)32 Ser 4/Ser 8 and total RPA32; phospho-checkpoint kinase 1 (CHK1) Ser 345 and total CHK1; phospho-CDK1 Tyr 15 and total CDK1. DMSO was added to untreated control cells. An anti-GAPDH antibody was used for loading control.

Article Snippet: MM cells were seeded in 96-well plates 24 h before treatment with dl 922-947 and AZD1775 (MK1775, purchased from Axon Medchem), both alone and in combination at various concentrations in a constant ratio.

Techniques: Activation Assay, Western Blot, 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 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:

Journal: Nature Communications

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1038/s41467-025-66514-0

Figure Lengend Snippet: a Experimental setup of the viral insertional mutagenesis screen in ( b ). b Sense/total insertion ratios from mutagenesis screens performed in HAP1 cells treated with AZD1775 or DMSO. Genes with significantly less sense insertions in AZD1775-treated cells are indicated. c Schematic representation of high-ranking gene mutations causing sensitization to WEE1i. d Pathway enrichment analysis, depicting the most highly enriched pathways involved in sensitization to WEE1i. e , f Dose-response curve of AZD1775 in control RPE1 TP53 KO and RPE1 TP53 KO EIF2A KO cells ( e ) and OVCAR3, OVCAR3 EIF2A KO #1 and OVCAR3 EIF2A KO #2 cells ( f ). Cells were treated for 5 days and cellular viability was measured by MTT conversion. Data represent mean ± standard deviation (SD) (n = 3). g Immunofluorescence microscopy analysis of γH2AX in control RPE1 TP53 KO and EIF2A KO cells treated with AZD1775 (500 nM) for 24 h. Mean foci intensity per cell is plotted. Medians of n = 218, 212, 228 or 206 cells, respectively, measured across 3 independent experiments. Paired t-test (two-sided) of medians, p ≤ 0.05 was considered significant. h scEdU-seq maximum normalized log counts are plotted for control RPE1 TP53 KO and RPE1 TP53 KO EIF2A KO cells, ranked according to S-phase progression (y-axis) and binned per 400 kb (x-axis). A 40 megabase region of chromosome 2 is shown. All replicates are biological replicates unless indicated otherwise. Source data are provided as a file.

Article Snippet: To assess full-length GCN2 and WEE1 kinase activity upon treatment with WEE1i in vitro, AZD1775, Debio 0123, GCN2iB, and neratinib were sent to Eurofins for analysis with KinaseProfiler (items 14-934KP and 14-925KP, Eurofins).

Techniques: Mutagenesis, Control, Standard Deviation, Immunofluorescence, Microscopy

Journal: Nature Communications

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1038/s41467-025-66514-0

Figure Lengend Snippet: a Gene set enrichment analysis of RNA-seq data of RPE1 TP53 KO control and EIF2A KO cells (more detailed analysis in Supplementary Fig. ). Circle size represents gene set size (control: n = 3, EIF2A KO : n = 1). b , c SILAC-based proteomics analysis of RPE1 TP53 KO control and EIF2A KO cells, showing differential protein expression at baseline ( b ) and upon AZD1775 treatment for 6, 24 and 48 h ( c ) (n = 2). Red datapoint indicates eIF2A, green datapoints indicate other screen hits. d RPE1 TP53 KO control and EIF2A KO cells were treated with AZD1775 or Debio 0123 (1 µM) for 24 h, followed by a 10’ puromycin pulse. Puromycin incorporation was visualized by immunoblotting (left) and quantified (right). Data represent mean ± SD; RPE1 TP53 KO EIF2A KO treated with Debio 0123 (n = 4); control RPE1 TP53 KO treated with DMSO or AZD1775 (n = 7); Other conditions (n = 5). e RPE1 TP53 KO PAC KO cells were treated with AZD1775 (250 nM) and ISRIB for 24 h and subsequently labeled with L-azidohomoalanine (AHA). Immunoblot of AHA-labeled proteins and quantification is shown. Data represent mean and individual datapoints (n = 2). f Schematic overview of the CRISPR/Cas9 genome-wide AZD1775 resistance screen. g MAGeCK scores for individual genes in the RPE1 TP53 KO AZD1775 positive selection screen (day 12 vs day 0). h Cell confluency analysis of parental RPE1 TP53 KO PAC KO or GCN2 KO cells. Mean ± range, n = 3 for control and GCN2 KO #1, n = 2 for GCN2 KO #2. i Quantification of clonogenic survival assays of parental RPE1 TP53 KO PAC KO and ATF4 KO cells (corresponding images provided in Supplementary Fig. ). Mean ± SD, n = 3 for all conditions. j , k Representative histograms ( j ) and quantification ( k ) of ATF4-mScarlet flow cytometry measurements in RPE1 TP53 KO ATF4-mScarlet reporter cells treated with thapsigargin (1 µM), AZD1775 (1 µM), Debio 0123 (1 µM) and/or A92 (1 µM) for 24 h. Data represent mean ± SD (n = 3). Statistical analysis of ( d , k ): unpaired t-test (two-sided) with p ≤ 0.05 considered significant. All replicates are biological replicates unless indicated otherwise. Source data are provided as a file.

Article Snippet: To assess full-length GCN2 and WEE1 kinase activity upon treatment with WEE1i in vitro, AZD1775, Debio 0123, GCN2iB, and neratinib were sent to Eurofins for analysis with KinaseProfiler (items 14-934KP and 14-925KP, Eurofins).

Techniques: RNA Sequencing, Control, Multiplex sample analysis, Expressing, Western Blot, Labeling, CRISPR, Genome Wide, Selection, Flow Cytometry

Journal: Nature Communications

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1038/s41467-025-66514-0

Figure Lengend Snippet: a RPE1 TP53 KO PAC KO cells were treated with AZD1775 (250 nM) for 6 h in the presence or absence of ISRIB (1 μM), pulse labeled with EdU, and processed for quantitative image-based cytometry. Cell cycle stage was defined by DNA content and EdU positivity. Each point represents a cell. Two independent replicates are defined by two different colors. Bars represent means of each replicate. Experimental set-up ( b ) and flow cytometry analysis ( c ) of RPE1 TP53 KO ATF4-mScarlet-NLS reporter cells. Cells were treated overnight with nocodazole. Mitotic cells were isolated and replated in the presence or absence of palbociclib. After 2 h, AZD1775 (1 µM) and/or A92 (1 µM) was added for 20 h. Data represent mean ± SD (n = 3). Flow cytometry gating strategy ( d ) and analysis of γH2AX in RPE1 TP53 KO cells after treatment with AZD1775 (1 µM) and/or A92 (1 µM) in the absence ( e , left) or presence ( e , right) of nocodazole. Data represent mean ± SD (n = 4). f Flow cytometry analysis of MPM2-positivity in RPE1 TP53 KO cells after treatment with nocodazole, doxorubicin, AZD1775 (1 µM) and/or A92 (1 µM). Data represent mean ± SD (n = 3). g Flow cytometry analysis of MPM2-positivity in control RPE1 TP53 KO PAC KO and ATF4 KO #1 or GCN2 KO #1 cells after treatment with nocodazole, doxorubicin and AZD1775 (1 µM). Data represent mean ± SD (parental conditions n = 4, KO conditions n = 3). h Immunoblot of resting PBMCs or PBMCs stimulated with anti-CD3/CD28 beads. PBMCs were treated with DMSO, AZD1775 (500 nM) or thapsigargin (500 nM) for 24 h. Representative blot of n = 3 independent experiments. i RPE1 TP53 KO PAC KO and GCN2 KO cells were treated with AZD1775 and/or cisplatin for 5 days. Cell survival was analyzed by MTT conversion. Data represent mean ± SD (n = 4). Statistical analysis for c , e , f , g was performed using unpaired t-tests (two-sided), with p ≤ 0.05 considered significant. All replicates are biological replicates unless indicated otherwise. Source data are provided as a file.

Article Snippet: To assess full-length GCN2 and WEE1 kinase activity upon treatment with WEE1i in vitro, AZD1775, Debio 0123, GCN2iB, and neratinib were sent to Eurofins for analysis with KinaseProfiler (items 14-934KP and 14-925KP, Eurofins).

Techniques: Labeling, Cytometry, Flow Cytometry, Isolation, Control, Western Blot

Journal: Nature Communications

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1038/s41467-025-66514-0

Figure Lengend Snippet: a Schematic overview of the CRISPR screen in ( b ). b NormZ scores of individual genes in an RPE1 TP53 KO PAC KO Cas9 negative selection AZD1775 CRISPR screen (day 15 vs. day 0). Positive normZ scores indicate suppression, and negative normZ scores indicate synergy. Genes are color-coded by category; Orange: cell cycle checkpoint kinases, Green: ribosome quality control, Blue: ISR kinases GCN1L1 ( GCN1 ) and EIF2AK4 (GCN2). c Schematic representation of ribosome quality control pathway members with high-ranking screen hits sensitizing to WEE1i. d Schematic representation of single ribosome mRNA translation kinetics measured by socRNA-mediated GFP translation. e Representative image of socRNA-mediated GFP translation as analyzed in ( f ). f GFP foci intensity over time (n = 1, tracking of 67 (no drug) or 73 (AZD1775) socRNAs). Lines and shaded areas indicate mean ± SD. g Elongation speed of a single ribosome on a socRNA in control or AZD1775-treated cells. Data represent mean ± SD (no drug: n = 58, AZD1775: n = 54, from 2 experiments), unpaired t-test (two-sided) with p ≤ 0.05 considered significant. h Schematic representation of a socRNA containing an Xbp1 pause sequence. Puromycin addition triggers the release of ribosomes and nascent peptide chains from a socRNA, allowing quantification of ribosome copies per socRNA. i Visual representation of ribosome release upon puromycin addition to socRNA, as quantified in ( j ). j Fraction of analyzed socRNAs containing indicated numbers of translating ribosomes per socRNA after AZD1775 treatment (300 nM, 24 h) with or without GCN2i A92 (1 µM). Mean ± range (No drug n = 3; AZD1775 n = 4, AZD1775 + A92 n = 2). k Immunoblot of RPE1 TP53 KO cells after 30 min treatment with emetine (0.5 µg/ml) and puromycin pulse (n = 1). l Immunoblot of RPE1 TP53 KO cells co-treated with AZD1775 (250 nM) and indicated doses of emetine according to the indicated timeline. Representative experiment of n = 2. m Immunoblot of RPE1 TP53 KO cells treated with AZD1775 (250 nM) for 4.5 h, and during the last 30 min co-treatment with emetine (0.5 µg/ml). Representative experiment of n = 2. All replicates are biological replicates unless indicated otherwise. Source data are provided as a file.

Article Snippet: To assess full-length GCN2 and WEE1 kinase activity upon treatment with WEE1i in vitro, AZD1775, Debio 0123, GCN2iB, and neratinib were sent to Eurofins for analysis with KinaseProfiler (items 14-934KP and 14-925KP, Eurofins).

Techniques: CRISPR, Selection, Control, Sequencing, Western Blot

Journal: Nature Communications

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1038/s41467-025-66514-0

Figure Lengend Snippet: a Relative ATF4-mScarlet MFI in RPE1 ATF4-mScarlet reporter cells after treatment with 125, 250, 500, or 1000 nM of AZD1775, RP-6306, VE-822 or AZD7762. Data represent mean ± SD (DMSO: n = 4; other conditions: n = 3). Statistical analysis was performed using a one-way ANOVA with Dunnett’s multiple comparisons with p ≤ 0.05 considered significant. b Immunoblot of RPE1 TP53 KO cells treated with siRNA targeting WEE1 for 72 h and AZD1775 (250 nM) for 2.5 or 5 h. Representative blot of n = 3 experiments. WEE1 and GCN2 engagement as measured by NanoBRET kinase target engagement assay. HEK293 cells were transfected with WEE1-NanoLuc ( c ) and NanoLuc-GCN2 ( d ) and incubated with K-10 tracer (0.5 μM), and indicated doses of CC1, GCN2iB, AZD1775, Debio 0123, ZNL-02-096, RP-6306 or neratinib for 2 h prior to substrate addition and BRET signal detection. Data represent mean ± SD (n = 3). Representative histograms ( e ) and quantification ( f ) of ATF4-mScarlet flow cytometry measurements in RPE1 TP53 KO ATF4-mScarlet reporter cells treated with siRNAs targeting GCN1 and AZD1775 or neratinib (1 µM) for 5 h. Data represent mean ± SD (n = 3), statistical analysis: two-way ANOVA with Tukey’s multiple comparisons test, with p ≤ 0.05 considered significant. g Model of WEE1 inhibitor mode of action and consequences. All replicates are biological replicates unless indicated otherwise. Source data are provided as a file.

Article Snippet: To assess full-length GCN2 and WEE1 kinase activity upon treatment with WEE1i in vitro, AZD1775, Debio 0123, GCN2iB, and neratinib were sent to Eurofins for analysis with KinaseProfiler (items 14-934KP and 14-925KP, Eurofins).

Techniques: Western Blot, Drug discovery, Transfection, Incubation, Flow Cytometry

Journal: Nature Communications

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1038/s41467-025-66514-0

Figure Lengend Snippet: a Relative ATF4-mScarlet MFI in RPE1 TP53 KO ATF4-mScarlet reporter cells after treatment with the indicated doses of Debio 0123. Data represent mean ± SD (n = 3). b Cell viability in parental RPE1 TP53 KO PAC KO , GCN2 KO #1 and GCN2 KO #2 cells after treatment with Debio 0123 and RP-6306 at the indicated doses (n = 5). c ZIP synergy scores from cell survival matrices shown in ( b , d ). Data represent mean ± SD (n = 5). d Synergy plots with ZIP synergy scores of heatmaps shown in ( b ). e Relative ATF4-mScarlet MFI in RPE1 TP53 KO ATF4-mScarlet reporter cells treated with the indicated doses of AZD1775, Debio 0123 and RP-6306. Data represent mean ± SD (n = 4). Statistical analysis ( a , c , e ) was performed using paired t-tests (two-sided), with p ≤ 0.05 considered significant. All replicates are biological replicates unless indicated otherwise. Source data are provided as a file.

Article Snippet: To assess full-length GCN2 and WEE1 kinase activity upon treatment with WEE1i in vitro, AZD1775, Debio 0123, GCN2iB, and neratinib were sent to Eurofins for analysis with KinaseProfiler (items 14-934KP and 14-925KP, Eurofins).

Techniques:

Journal: Nature Communications

Article Title: Harnessing free energy calculations for kinome-wide selectivity in drug discovery campaigns with a Wee1 case study

doi: 10.1038/s41467-025-62722-w

Figure Lengend Snippet: a Thermodynamic cycle leveraged by ligand-based relative binding free energy calculations (L-RB-FEP+) to estimate the binding affinity of a design idea (orange) (ΔG expt design ) relative to experimental binding affinity of reference compound (green) (ΔG expt reference ) in the crystallographic structure of AZD1775 (compound 1 ) within the Wee1 binding pocket (gray surface representation; PDB ID 5V5Y ). Arrows A and B represent the alchemical perturbation between the reference and design idea in solvent (ΔG FEP solvent ) and the binding pocket, (ΔG FEP complex ), respectively, while arrows 1 and 2 represent experimental binding affinities. b Sample schematic of an L-RB-FEP+ profiling cascade to predict potency and selectivity against three off-targets such that only the most promising designs need to be promoted to subsequent stages.

Article Snippet: AstraZeneca’s Wee1 inhibitor, AZD1775 (Compound 1 ), licensed from Merck & Co was studied in multiple Phase 2 clinical trials over a period of nine years before being discontinued due to a narrow therapeutic window mainly driven by combined myelosuppression and gastrointestinal toxicity.

Techniques: Binding Assay, Solvent

Journal: Nature Communications

Article Title: Harnessing free energy calculations for kinome-wide selectivity in drug discovery campaigns with a Wee1 case study

doi: 10.1038/s41467-025-62722-w

Figure Lengend Snippet: a Schematic of Wee1 in cell cycle regulation. The cell cycle is composed of four major phases, including G1 (gap 1), S (DNA synthesis), G2 (gap 2) and M (mitosis). Wee1 is a gatekeeper of the G2-M cell cycle checkpoint through phosphorylation of CDK1/2. Inhibition of Wee1 forces cells into unscheduled mitosis and culminates in premature cell death. b Binding pocket representation of AZD1775 bound to Wee1 (PDB ID: 5V5Y ; generated in Maestro (Schrödinger Release 2024-2: Maestro, Schrödinger, LLC, New York, NY, 2024.) c 2D schematic of AZD1775 scaffold with highlighted R1 group occupying the hydrophobic pocket around the gatekeeper (GK) residue, R2 group located under the p-loop and the R3 group protruding into the solvent-exposed region.

Article Snippet: AstraZeneca’s Wee1 inhibitor, AZD1775 (Compound 1 ), licensed from Merck & Co was studied in multiple Phase 2 clinical trials over a period of nine years before being discontinued due to a narrow therapeutic window mainly driven by combined myelosuppression and gastrointestinal toxicity.

Techniques: DNA Synthesis, Phospho-proteomics, Inhibition, Binding Assay, Generated, Residue, Solvent

Journal: Nature Communications

Article Title: Harnessing free energy calculations for kinome-wide selectivity in drug discovery campaigns with a Wee1 case study

doi: 10.1038/s41467-025-62722-w

Figure Lengend Snippet: a Compound 10 (PDB ID: 9D0R), b compound 11 (PDB ID: 9D0Q), c compound 14 (PDB ID: 9D0S), d PD-166285 (compound 15 ; PDB ID: 5VC5 ), and e overlay of compounds 10, 11 , and 14 (green) with AZD1775 (orange) in the Wee1 binding pocket (select residues for 5V5Y displayed in cyan ribbons and gray surface). All ligand interaction diagrams generated in Maestro (Schrödinger Release 2024-2: Maestro, Schrödinger, LLC, New York, NY, 2024).

Article Snippet: AstraZeneca’s Wee1 inhibitor, AZD1775 (Compound 1 ), licensed from Merck & Co was studied in multiple Phase 2 clinical trials over a period of nine years before being discontinued due to a narrow therapeutic window mainly driven by combined myelosuppression and gastrointestinal toxicity.

Techniques: Binding Assay, Generated