Journal: bioRxiv

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

doi: 10.1101/2025.03.12.642754

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 Suppl. Fig. 3A). Circle size represents gene set size. (B, C) SILAC-based proteomics analysis of RPE1 TP53 KO control and EIF2A KO cells, showing differential protein expression at baseline (panel B) and upon AZD1775 treatment for 6, 24 and 48 h (panel C). (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 panel). Quantification of immunoblots is indicated (right panel). 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 ± SD (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 ± SD, 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 #1 and #2 cells (corresponding images provided in Suppl. Fig. 3H). Mean ± SD, n=3 for all conditions. (J, K) Representative histograms (panel J) and quantification (panel 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). (L) Parental RPE1 TP53 KO PAC KO , ATF4 KO #1 and GCN2 KO #2 cells were treated with AZD1775 (0, 250 or 500 nM) for 24 h, and indicated proteins were immunoblotted. Statistical analysis of panels D, K: unpaired t-test with p ≤ 0.05 considered significant.

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

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

Journal: bioRxiv

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

doi: 10.1101/2025.03.12.642754

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. Median ATF4 intensity from n=2 experiments is shown. (B, C) Experimental set-up (panel B). RPE1 TP53 KO ATF4-mScarlet-NLS reporter 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 (Panel B). mScarlet levels in RPE1 TP53 KO ATF4-mScarlet reporter cells were measured by flow cytometry. Data represent mean ± SD (n=3)(panel C). (D, E) Flow cytometry gating strategy (panel D) and analysis of γH2AX in RPE1 TP53 KO cells after treatment with AZD1775 (1 µM) and/or A92 (1 µM) in the absence (panel E, left) or presence (panel 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 #2 or GCN2 KO #2 cells after treatment with nocodazole, doxorubicin and AZD1775 (1 µM). Data represent mean ± SD (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. (I) RPE1 TP53 KO PAC KO and ATF4 KO #2 or GCN2 KO #2 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 was performed using unpaired t-tests, with p ≤ 0.05 considered significant.

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

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

Journal: bioRxiv

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

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) Relative gene and protein expression upon treatment with AZD1775 (24 h, 1 µM) measured by Ribo-seq and RNA-seq. (B) Differential protein expression upon treatment with AZD1775 (24 h, 1 µM) measured by Ribo-seq. (C) Pathway enrichment analysis of Ribo-seq data shown in (M). (D) Gene expression of ATF4-target genes DDIT4, DDIT3, ATF3 and PPP1R15A upon treatment with AZD1775 (24 h, 1 µM). (E) Codon usage in RPE1 TP53 KO cells upon treatment with DMSO or AZD1775 (24 h, 1 µM).

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques: Expressing, RNA Sequencing, Gene Expression

Journal: bioRxiv

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

doi: 10.1101/2025.03.12.642754

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 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. (C, D) NanoBRET kinase target engagement assay performed with HEK293T cells transfected with WEE1-NanoLuc (panel C) and NanoLuc-GCN2 (panel D) and incubated with K-10 tracer (0.5 μM) and indicated doses of CC1, AZD1775, Debio 0123 and ZNL-02-096 for 2 h prior to substrate addition and BRET signal detection.

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques: Western Blot, Transfection, Incubation

Journal: bioRxiv

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

doi: 10.1101/2025.03.12.642754

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 and . Data represent mean ± SD (n=5). (D) Synergy plots with ZIP synergy scores of heatmaps shown in . (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).

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2025.03.12.642754

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 Suppl. Fig. 3A). Circle size represents gene set size. (B, C) SILAC-based proteomics analysis of RPE1 TP53 KO control and EIF2A KO cells, showing differential protein expression at baseline (panel B) and upon AZD1775 treatment for 6, 24 and 48 h (panel C). (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 panel). Quantification of immunoblots is indicated (right panel). 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 ± SD (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 ± SD, 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 #1 and #2 cells (corresponding images provided in Suppl. Fig. 3H). Mean ± SD, n=3 for all conditions. (J, K) Representative histograms (panel J) and quantification (panel 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). (L) Parental RPE1 TP53 KO PAC KO , ATF4 KO #1 and GCN2 KO #2 cells were treated with AZD1775 (0, 250 or 500 nM) for 24 h, and indicated proteins were immunoblotted. Statistical analysis of panels D, K: unpaired t-test with p ≤ 0.05 considered significant.

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

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

Journal: bioRxiv

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

doi: 10.1101/2025.03.12.642754

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. Median ATF4 intensity from n=2 experiments is shown. (B, C) Experimental set-up (panel B). RPE1 TP53 KO ATF4-mScarlet-NLS reporter 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 (Panel B). mScarlet levels in RPE1 TP53 KO ATF4-mScarlet reporter cells were measured by flow cytometry. Data represent mean ± SD (n=3)(panel C). (D, E) Flow cytometry gating strategy (panel D) and analysis of γH2AX in RPE1 TP53 KO cells after treatment with AZD1775 (1 µM) and/or A92 (1 µM) in the absence (panel E, left) or presence (panel 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 #2 or GCN2 KO #2 cells after treatment with nocodazole, doxorubicin and AZD1775 (1 µM). Data represent mean ± SD (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. (I) RPE1 TP53 KO PAC KO and ATF4 KO #2 or GCN2 KO #2 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 was performed using unpaired t-tests, with p ≤ 0.05 considered significant.

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

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

Journal: bioRxiv

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

doi: 10.1101/2025.03.12.642754

Figure Lengend Snippet: (A) Relative gene and protein expression upon treatment with AZD1775 (24 h, 1 µM) measured by Ribo-seq and RNA-seq. (B) Differential protein expression upon treatment with AZD1775 (24 h, 1 µM) measured by Ribo-seq. (C) Pathway enrichment analysis of Ribo-seq data shown in (M). (D) Gene expression of ATF4-target genes DDIT4, DDIT3, ATF3 and PPP1R15A upon treatment with AZD1775 (24 h, 1 µM). (E) Codon usage in RPE1 TP53 KO cells upon treatment with DMSO or AZD1775 (24 h, 1 µM).

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques: Expressing, RNA Sequencing, Gene Expression

Journal: bioRxiv

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

doi: 10.1101/2025.03.12.642754

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 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. (C, D) NanoBRET kinase target engagement assay performed with HEK293T cells transfected with WEE1-NanoLuc (panel C) and NanoLuc-GCN2 (panel D) and incubated with K-10 tracer (0.5 μM) and indicated doses of CC1, AZD1775, Debio 0123 and ZNL-02-096 for 2 h prior to substrate addition and BRET signal detection.

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques: Western Blot, Transfection, Incubation

Journal: bioRxiv

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

doi: 10.1101/2025.03.12.642754

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 and . Data represent mean ± SD (n=5). (D) Synergy plots with ZIP synergy scores of heatmaps shown in . (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).

Article Snippet: The ATF4-mScarlet-NLS insert of pLHCX-ATF4 mScarlet NLS, which was a gift from David Andrews (Addgene, #115970) was obtained by PCR (Suppl.

Techniques: