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Merck KGaA gartisertib

O ptimizing activation and inhibition of ATR . A , U-2 OS cells were incubated with thymidine (2 mM) for 24 h and released for 3 h at which point nocodazole (100 ng/ml) was added for a further 12 h. Cells were released from nocodazole into fresh medium for the times indicated. Cells were fixed, stained with propidium iodide (PI) and analysed by FACS. B , U-2 OS cells synchronized in S-phase (11 h after release from nocodazole) were pre-incubated for 1 h with the indicated concentrations of berzosertib or <t>gartisertib</t> before addition of HU (1 mM) for 1 h. Cells were lysed, and extracts were subjected to Western blotting with the antibodies indicated. C , same as ( B ) except that S-phase cells were pre-incubated with berzosertib or gartisertib (1 μM for 1 h) before addition of HU (1 mM) for the times indicated. D , optimized cell treatment workflow.

Gartisertib, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews

gartisertib - by Bioz Stars, 2026-03

90/100 stars

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1) Product Images from "Chemo-Phosphoproteomic Profiling with ATR Inhibitors Berzosertib and Gartisertib Uncovers New Biomarkers and DNA Damage Response Regulators"

Article Title: Chemo-Phosphoproteomic Profiling with ATR Inhibitors Berzosertib and Gartisertib Uncovers New Biomarkers and DNA Damage Response Regulators

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1016/j.mcpro.2024.100802

Figure Legend Snippet: O ptimizing activation and inhibition of ATR . A , U-2 OS cells were incubated with thymidine (2 mM) for 24 h and released for 3 h at which point nocodazole (100 ng/ml) was added for a further 12 h. Cells were released from nocodazole into fresh medium for the times indicated. Cells were fixed, stained with propidium iodide (PI) and analysed by FACS. B , U-2 OS cells synchronized in S-phase (11 h after release from nocodazole) were pre-incubated for 1 h with the indicated concentrations of berzosertib or gartisertib before addition of HU (1 mM) for 1 h. Cells were lysed, and extracts were subjected to Western blotting with the antibodies indicated. C , same as ( B ) except that S-phase cells were pre-incubated with berzosertib or gartisertib (1 μM for 1 h) before addition of HU (1 mM) for the times indicated. D , optimized cell treatment workflow.

Techniques Used: Activation Assay, Inhibition, Incubation, Staining, Western Blot

Phosphoproteomic screening of phosphorylation sites sensitive to berzosertib or gartisertib . A and B , Volcano plot showing phosphorylation sites affected by berzosertib ( A ) or gartisertib ( B ). The horizontal cut-off lines represent an adjusted p -value of 0.05. Phosphopeptides lower in abundance after inhibitor treatment are in the negative logFC region of the plots. The mass spectrometry proteomics raw data for this figure have been deposited to the ProteomeXchange Consortium via the jPOSTrepo partner repository with the dataset identifier PXD040469 . Data analysis scripts and annotated spectra are available via Zenodo under https://doi.org/10.5281/zenodo.10581948 . C , overlap of proteins and phosphorylation sites affected by berzosertib or gartisertib. D and E , Phosphomotif analysis for berzosertib ( D ) and gartisertib ( E ). F , the five proteins whose phosphorylation is most strongly affected by berzosertib (“Class 1” hits). The phosphorylated residue is highlighted in red in the “Motif” column. Fold change refers to the difference between HU±ATRi. G , schematic diagram showing the location of the ATR-dependent phosphorylation sites in the DNA helicase FANCJ and the protein kinase TLK2.

Figure Legend Snippet: Phosphoproteomic screening of phosphorylation sites sensitive to berzosertib or gartisertib . A and B , Volcano plot showing phosphorylation sites affected by berzosertib ( A ) or gartisertib ( B ). The horizontal cut-off lines represent an adjusted p -value of 0.05. Phosphopeptides lower in abundance after inhibitor treatment are in the negative logFC region of the plots. The mass spectrometry proteomics raw data for this figure have been deposited to the ProteomeXchange Consortium via the jPOSTrepo partner repository with the dataset identifier PXD040469 . Data analysis scripts and annotated spectra are available via Zenodo under https://doi.org/10.5281/zenodo.10581948 . C , overlap of proteins and phosphorylation sites affected by berzosertib or gartisertib. D and E , Phosphomotif analysis for berzosertib ( D ) and gartisertib ( E ). F , the five proteins whose phosphorylation is most strongly affected by berzosertib (“Class 1” hits). The phosphorylated residue is highlighted in red in the “Motif” column. Fold change refers to the difference between HU±ATRi. G , schematic diagram showing the location of the ATR-dependent phosphorylation sites in the DNA helicase FANCJ and the protein kinase TLK2.

Techniques Used: Phospho-proteomics, Mass Spectrometry, Residue

Image Search Results

( A ) Molecular structures of the four clinical ATR inhibitors Elimusertib, Gartisertib, Berzosertib, and Ceralasertib. ( B ) Heatmap displaying the binding affinities (p K D app ) for all target kinases identified with the Kinobeads technology. The designated target kinase ATR is highlighted with a red rectangle. ( C ) Residual binding of the three kinases ATR, PI3KCB, and PIK3C2A on Kinobeads upon increasing doses of Elimusertib. Binding affinities for each target kinase are given in the legend. .

Journal: Molecular Systems Biology

Article Title: Gemcitabine and ATR inhibitors synergize to kill PDAC cells by blocking DNA damage response

doi: 10.1038/s44320-025-00085-6

Figure Lengend Snippet: ( A ) Molecular structures of the four clinical ATR inhibitors Elimusertib, Gartisertib, Berzosertib, and Ceralasertib. ( B ) Heatmap displaying the binding affinities (p K D app ) for all target kinases identified with the Kinobeads technology. The designated target kinase ATR is highlighted with a red rectangle. ( C ) Residual binding of the three kinases ATR, PI3KCB, and PIK3C2A on Kinobeads upon increasing doses of Elimusertib. Binding affinities for each target kinase are given in the legend. .

Article Snippet: Gartisertib , Selleckchem , S9639.

Techniques: Binding Assay

( A ) Residual binding of ATR on Kinobeads upon increasing doses of Elimusertib and Ceralasertib (top), and Berzosertib and Gartisertib (bottom), based on label-free quantification (LFQ) intensities. Curve fit (R 2 ) and apparent affinity constants ( K D app ) are given in the legend. ( B ) Dose-dependent reduction in MS/MS spectra and unique peptides of ATR kinase in pulldown experiment with increasing concentrations of Elimusertib and Ceralasertib (top), and Berzosertib and Gartisertib (bottom).

Journal: Molecular Systems Biology

Article Title: Gemcitabine and ATR inhibitors synergize to kill PDAC cells by blocking DNA damage response

doi: 10.1038/s44320-025-00085-6

Figure Lengend Snippet: ( A ) Residual binding of ATR on Kinobeads upon increasing doses of Elimusertib and Ceralasertib (top), and Berzosertib and Gartisertib (bottom), based on label-free quantification (LFQ) intensities. Curve fit (R 2 ) and apparent affinity constants ( K D app ) are given in the legend. ( B ) Dose-dependent reduction in MS/MS spectra and unique peptides of ATR kinase in pulldown experiment with increasing concentrations of Elimusertib and Ceralasertib (top), and Berzosertib and Gartisertib (bottom).

Article Snippet: Gartisertib , Selleckchem , S9639.

Techniques: Binding Assay, Quantitative Proteomics, Tandem Mass Spectroscopy

( A ) Potencies (pEC 50 ) and curve fold changes (log 2 ) from phosphoproteomic decryptM experiments with Elimusertib and Gartisertib (top) and Berzosertib and Ceralasertib (bottom) in DNA-damaged AsPC-1 cells acquired by LC-MS/MS. Each dot represents one dose–response curve of a regulated p-peptide (red: pSQ/pTQ, gray: non-pSQ/pTQ). Known direct substrates of ATR are annotated by text. ( B ) Dose-dependent inhibition of CHEK1-pS317 by four ATR inhibitors in DNA-damaged AsPC-1 cells. PTM response was normalized to 1 µM GEM. ( C ) Density plot summarizing the drug potencies (pEC 50 ) by which the four ATRi regulate the phosphorylation of pSQ/pTQ-peptides in DNA-damaged AsPC-1 cells. The number of pSQ/pTQ-peptides regulated per drug is given in the legend. .

Journal: Molecular Systems Biology

Article Title: Gemcitabine and ATR inhibitors synergize to kill PDAC cells by blocking DNA damage response

doi: 10.1038/s44320-025-00085-6

Figure Lengend Snippet: ( A ) Potencies (pEC 50 ) and curve fold changes (log 2 ) from phosphoproteomic decryptM experiments with Elimusertib and Gartisertib (top) and Berzosertib and Ceralasertib (bottom) in DNA-damaged AsPC-1 cells acquired by LC-MS/MS. Each dot represents one dose–response curve of a regulated p-peptide (red: pSQ/pTQ, gray: non-pSQ/pTQ). Known direct substrates of ATR are annotated by text. ( B ) Dose-dependent inhibition of CHEK1-pS317 by four ATR inhibitors in DNA-damaged AsPC-1 cells. PTM response was normalized to 1 µM GEM. ( C ) Density plot summarizing the drug potencies (pEC 50 ) by which the four ATRi regulate the phosphorylation of pSQ/pTQ-peptides in DNA-damaged AsPC-1 cells. The number of pSQ/pTQ-peptides regulated per drug is given in the legend. .

Article Snippet: Gartisertib , Selleckchem , S9639.

Techniques: Liquid Chromatography with Mass Spectroscopy, Inhibition, Phospho-proteomics

Journal: Molecular Systems Biology

Article Title: Gemcitabine and ATR inhibitors synergize to kill PDAC cells by blocking DNA damage response

doi: 10.1038/s44320-025-00085-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Gartisertib , Selleckchem , S9639.

Techniques: Labeling, Recombinant, Luminescence Assay, Cell Viability Assay, Software, Mass Spectrometry

A small-molecule inhibitor and drug screen identifies PARPi and ATRi as candidate therapies for DSRCT. A, Schematic illustration of the workflow of small-molecule inhibitor and drug screen performed on the JN1 cell line. B, Waterfall plot displaying the difference in AUC between the JN1 cell line (AUC JN1 ) and the panel of 92 cell lines used for comparison (AUC median ) for the 79 evaluated small-molecule inhibitors or drugs. Red, PARPi; blue, ATRi; green, conventional cytotoxic. C–F, Dose–response survival curves of the DSRCT cell lines JN1 and R, and the A673 (Ewing sarcoma) and SaOS-2 (osteosarcoma) cell lines exposed to talazoparib ( C ), olaparib ( D ), M4344 ( E ), or AZD6738 ( F ) for 7 days. Mean ± SD; n = 3. G and H, Violin plots showing the relative sensitivity (log 2 -fold change of cell viability) of cell lines exposed to the PARPi talazoparib ( G ) or olaparib ( H ) after a single-dose exposure at 2.5 μmol/L for 5 days in the DepMap database (PRISM Repurposing 23Q2), in comparison with that of the JN1 and R cell lines. JN1 and R cell line sensitivities were extrapolated from the survival assays presented in C and E ; SFs were calculated at 2.5 μmol/L and log 2 transformed. Ewing sarcoma cell lines ( n = 16): RDES, A673, SKES1, CADOES1, EWS502, MHHES1, EW8, A673STAG2KO16, A673STAG2KO45, A673STAG2NT14, A673STAG2NT23, CBAGPN, CHLA10, SKNEP1, SKPNDW, and TC32; osteosarcoma cell lines ( n = 5): G292CLONEA141B1, MG63, U2OS, HOS, and SJSA1; soft-tissue sarcoma cell lines ( n = 7): S117, TE617T, HT1080, HS729, RD, RKN, and RH30, including rhabdomyosarcoma ( n = 4), leiomyosarcoma ( n = 1), fibrosarcoma ( n = 1), and NOS sarcoma cell lines ( n = 1), respectively. The BRCA1/2 -mutant IGROV1 ovarian cancer cell line and BRCA1 -mutant MDA-MB-436 breast cancer cell line were used as positive controls for sensitivity to PARPi. **, P < 0.01; ****, P < 0.0001; ns, not significant.

Journal: Cancer Research

Article Title: Replication Stress Is an Actionable Genetic Vulnerability in Desmoplastic Small Round Cell Tumors

doi: 10.1158/0008-5472.CAN-23-3603

Figure Lengend Snippet: A small-molecule inhibitor and drug screen identifies PARPi and ATRi as candidate therapies for DSRCT. A, Schematic illustration of the workflow of small-molecule inhibitor and drug screen performed on the JN1 cell line. B, Waterfall plot displaying the difference in AUC between the JN1 cell line (AUC JN1 ) and the panel of 92 cell lines used for comparison (AUC median ) for the 79 evaluated small-molecule inhibitors or drugs. Red, PARPi; blue, ATRi; green, conventional cytotoxic. C–F, Dose–response survival curves of the DSRCT cell lines JN1 and R, and the A673 (Ewing sarcoma) and SaOS-2 (osteosarcoma) cell lines exposed to talazoparib ( C ), olaparib ( D ), M4344 ( E ), or AZD6738 ( F ) for 7 days. Mean ± SD; n = 3. G and H, Violin plots showing the relative sensitivity (log 2 -fold change of cell viability) of cell lines exposed to the PARPi talazoparib ( G ) or olaparib ( H ) after a single-dose exposure at 2.5 μmol/L for 5 days in the DepMap database (PRISM Repurposing 23Q2), in comparison with that of the JN1 and R cell lines. JN1 and R cell line sensitivities were extrapolated from the survival assays presented in C and E ; SFs were calculated at 2.5 μmol/L and log 2 transformed. Ewing sarcoma cell lines ( n = 16): RDES, A673, SKES1, CADOES1, EWS502, MHHES1, EW8, A673STAG2KO16, A673STAG2KO45, A673STAG2NT14, A673STAG2NT23, CBAGPN, CHLA10, SKNEP1, SKPNDW, and TC32; osteosarcoma cell lines ( n = 5): G292CLONEA141B1, MG63, U2OS, HOS, and SJSA1; soft-tissue sarcoma cell lines ( n = 7): S117, TE617T, HT1080, HS729, RD, RKN, and RH30, including rhabdomyosarcoma ( n = 4), leiomyosarcoma ( n = 1), fibrosarcoma ( n = 1), and NOS sarcoma cell lines ( n = 1), respectively. The BRCA1/2 -mutant IGROV1 ovarian cancer cell line and BRCA1 -mutant MDA-MB-436 breast cancer cell line were used as positive controls for sensitivity to PARPi. **, P < 0.01; ****, P < 0.0001; ns, not significant.

Article Snippet: PARPi olaparib (AZD2281), talazoparib (BMN-673), and veliparib (ABT-888); the ATRi gartisertib (M4344), ceralasertib (AZD6738), and berzosertib (M6620); and the CHK1 inhibitors (CHK1i) prexasertib (LY2606368) and SRA-737, as well as cisplatin, topotecan, and SN-38, were purchased from Selleck Chemicals.

Techniques: Comparison, Transformation Assay, Mutagenesis

PARPi and ATRi have synergistic cytotoxic effects in models of DSRCT with high PARP1 expression. A and B, PARP1 expression ( A ) and PARylation levels ( B ) as assessed by IHC in a cohort of 16 DSRCT samples, compared with those of the JN1 and R cell lines (PARP1 and PAR expression levels are shown as H-scores). Representative cases (PARP1-high vs. PARP1-low tumors; PAR-high vs. PAR-low tumors) are shown to the right, compared with JN1 and R cells. C and D, Surface plots of Bliss independence scores calculated for the talazoparib–M4344 combination in JN1 ( C ) and R ( D ) cell lines at 7 days. E, The GR_13-PDX-O model was established from the primary peritoneal tumor of a patient with DSRCT, with confirmation of EWSR1::WT1 fusion by FISH and WT1-Cter IHC (Supplementary Fig. S8). F, Surface plot of Bliss independence scores calculated for the talazoparib–M4344 combination in the GR_13 PDX-O at 7 days. Mean ± SD; n = 3. Surface plots: the x -axis and y -axis values indicate drug concentrations, and the z -axis values indicate the associated synergy score; score < −10, antagonistic interaction; score = 0, absence of interaction; score > 10, synergistic interaction. G, Schematic illustration of an in vivo therapeutic experiment performed to evaluate the antitumor effect of PARPi talazoparib and ATRi M1774 in NSG mice engrafted with JN1 xenografts. H, Therapeutic responses to drug treatment in mice harboring JN1 xenografts. Mean tumor volume ± SD; two-way ANOVA and post hoc Dunnett test. I, Tumor volume at the time of mice sacrifice. Mean ± SD; one-way ANOVA and post hoc Šídák test. *, P < 0.01; ns, not significant. Tala, talazoparib.

Journal: Cancer Research

Article Title: Replication Stress Is an Actionable Genetic Vulnerability in Desmoplastic Small Round Cell Tumors

doi: 10.1158/0008-5472.CAN-23-3603

Figure Lengend Snippet: PARPi and ATRi have synergistic cytotoxic effects in models of DSRCT with high PARP1 expression. A and B, PARP1 expression ( A ) and PARylation levels ( B ) as assessed by IHC in a cohort of 16 DSRCT samples, compared with those of the JN1 and R cell lines (PARP1 and PAR expression levels are shown as H-scores). Representative cases (PARP1-high vs. PARP1-low tumors; PAR-high vs. PAR-low tumors) are shown to the right, compared with JN1 and R cells. C and D, Surface plots of Bliss independence scores calculated for the talazoparib–M4344 combination in JN1 ( C ) and R ( D ) cell lines at 7 days. E, The GR_13-PDX-O model was established from the primary peritoneal tumor of a patient with DSRCT, with confirmation of EWSR1::WT1 fusion by FISH and WT1-Cter IHC (Supplementary Fig. S8). F, Surface plot of Bliss independence scores calculated for the talazoparib–M4344 combination in the GR_13 PDX-O at 7 days. Mean ± SD; n = 3. Surface plots: the x -axis and y -axis values indicate drug concentrations, and the z -axis values indicate the associated synergy score; score < −10, antagonistic interaction; score = 0, absence of interaction; score > 10, synergistic interaction. G, Schematic illustration of an in vivo therapeutic experiment performed to evaluate the antitumor effect of PARPi talazoparib and ATRi M1774 in NSG mice engrafted with JN1 xenografts. H, Therapeutic responses to drug treatment in mice harboring JN1 xenografts. Mean tumor volume ± SD; two-way ANOVA and post hoc Dunnett test. I, Tumor volume at the time of mice sacrifice. Mean ± SD; one-way ANOVA and post hoc Šídák test. *, P < 0.01; ns, not significant. Tala, talazoparib.

Techniques: Expressing, In Vivo

PARPi and ATRi combination elicits DNA damage, replication stress, and genomic instability in DSRCT cells. A–D, Quantification of γH2AX ( A and B ) or RAD51 foci ( C and D ) in JN1 ( A and C ) or R ( B and D ) cells exposed to DMSO control, PARPi talazoparib (Tala), ATRi M4344, or a combination of both for 72 hours. Cisplatin was used as the positive control. A minimum of 500 nuclei was analyzed per condition. Violin plots show the absolute number of foci per nucleus. Thick line, median; thin lines, bottom and top quartiles; two-way ANOVA and post hoc Dunn test. E and F, Western blots of pCHK1, CHK1, pRPA2, RPA2, γH2AX, H2AX, and cleaved-PARP1 (cPARP) in JN1 ( E ) or R ( F ) cells exposed to DMSO control, PARPi talazoparib or olaparib, ATRi M4344 or AZD6738, or a combination of both for 48 hours. G and H, Representative immunofluorescence images ( G ) and quantification ( H ) of micronuclei-positive cells in JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours. A minimum of 500 cells was analyzed per condition. Mean ± SD; n = 3; one-way ANOVA and post hoc Dunn test. Arrows, micronuclei. Scale bar, 20 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Journal: Cancer Research

Article Title: Replication Stress Is an Actionable Genetic Vulnerability in Desmoplastic Small Round Cell Tumors

doi: 10.1158/0008-5472.CAN-23-3603

Figure Lengend Snippet: PARPi and ATRi combination elicits DNA damage, replication stress, and genomic instability in DSRCT cells. A–D, Quantification of γH2AX ( A and B ) or RAD51 foci ( C and D ) in JN1 ( A and C ) or R ( B and D ) cells exposed to DMSO control, PARPi talazoparib (Tala), ATRi M4344, or a combination of both for 72 hours. Cisplatin was used as the positive control. A minimum of 500 nuclei was analyzed per condition. Violin plots show the absolute number of foci per nucleus. Thick line, median; thin lines, bottom and top quartiles; two-way ANOVA and post hoc Dunn test. E and F, Western blots of pCHK1, CHK1, pRPA2, RPA2, γH2AX, H2AX, and cleaved-PARP1 (cPARP) in JN1 ( E ) or R ( F ) cells exposed to DMSO control, PARPi talazoparib or olaparib, ATRi M4344 or AZD6738, or a combination of both for 48 hours. G and H, Representative immunofluorescence images ( G ) and quantification ( H ) of micronuclei-positive cells in JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours. A minimum of 500 cells was analyzed per condition. Mean ± SD; n = 3; one-way ANOVA and post hoc Dunn test. Arrows, micronuclei. Scale bar, 20 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Techniques: Control, Positive Control, Western Blot, Immunofluorescence

EWS–WT1 is a determinant of DSRCT cells’ sensitivity to PARPi and ATRi. A, Western blot of EWS–WT1 in JN1 and R cells transfected with either siCNTRL or siEWS–WT1. Whole-cell lysates were generated 48 hours after transfection. B–E, Dose–response survival curves of JN1 or R cells exposed to PARPi talazoparib ( B and C ) or ATRi M4344 ( D and E ) for 7 days in the presence or absence of siRNA-mediated silencing of EWS–WT1. Mean ± SD; n = 3. F and G, Quantification of γH2AX in JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. Cisplatin was used as the positive control. A minimum of 500 nuclei was analyzed per condition. Violin plots show the absolute number of foci per nucleus. Thick line, median; thin lines, bottom and top quartiles; two-way ANOVA and post hoc Dunn test. H and I, Western blots of pCHK1, CHK1, pRPA2, RPA2, γH2AX, H2AX, and EWS–WT1 in JN1 ( H ) or R ( I ) cells exposed to DMSO control, PARPi talazoparib (Tala), ATRi M4344, or a combination of both for 48 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. ****, P < 0.0001; ns, not significant.

Journal: Cancer Research

Article Title: Replication Stress Is an Actionable Genetic Vulnerability in Desmoplastic Small Round Cell Tumors

doi: 10.1158/0008-5472.CAN-23-3603

Figure Lengend Snippet: EWS–WT1 is a determinant of DSRCT cells’ sensitivity to PARPi and ATRi. A, Western blot of EWS–WT1 in JN1 and R cells transfected with either siCNTRL or siEWS–WT1. Whole-cell lysates were generated 48 hours after transfection. B–E, Dose–response survival curves of JN1 or R cells exposed to PARPi talazoparib ( B and C ) or ATRi M4344 ( D and E ) for 7 days in the presence or absence of siRNA-mediated silencing of EWS–WT1. Mean ± SD; n = 3. F and G, Quantification of γH2AX in JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. Cisplatin was used as the positive control. A minimum of 500 nuclei was analyzed per condition. Violin plots show the absolute number of foci per nucleus. Thick line, median; thin lines, bottom and top quartiles; two-way ANOVA and post hoc Dunn test. H and I, Western blots of pCHK1, CHK1, pRPA2, RPA2, γH2AX, H2AX, and EWS–WT1 in JN1 ( H ) or R ( I ) cells exposed to DMSO control, PARPi talazoparib (Tala), ATRi M4344, or a combination of both for 48 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. ****, P < 0.0001; ns, not significant.

Techniques: Western Blot, Transfection, Generated, Control, Positive Control

EWS–WT1 drives enhanced DNA replication stress and R-loops, which contribute to DSRCT cells’ sensitivity to PARPi and ATRi. A, Assessment of replication fork speed (kb/minute) in JN1 cells subjected to siRNA-mediated silencing of EWS–WT1 or CCND1. A minimum of 50 forks was analyzed per condition. Mean ± SD; each dot represents a single replication fork; n = 2, one-way ANOVA and post hoc Dunnett test. B, Assessment of replication fork speed (kb/minute) in JN1 cells exposed to DMSO control, or a combination of PARPi talazoparib (Tala) and ATRi M4344 for 6 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. A minimum of 50 forks was analyzed per condition. Mean ± SD; each dot represents a single replication fork; n = 2; two-way ANOVA and post hoc Šídák test. C and D, DNA:RNA hybrid dot blot of genomic DNA extracted from JN1 ( C ) or R ( D ) cells exposed to PARPi talazoparib, ATRi M4344, or a combination of both in the presence or absence of siRNA-mediated silencing of EWS–WT1 as in B . S9.6, RNA:DNA hybrids; ssDNA, loading control. E, Assessment of replication fork speed (kb/minute) in RNase H1–overexpressing JN1 cells subjected to siRNA-mediated silencing of EWS–WT1. Synchronized cells were collected 14 hours after transfection. A minimum of 50 forks was analyzed per condition. Mean ± SD; each dot represents a single replication fork; n = 2; unpaired t test. E, Dose–response survival curves of JN1 cells exposed to PARPi talazoparib ( F ) or olaparib ( G ), and ATRi M4344 ( H ) or AZD6738 ( I ) for 7 days in the presence or absence of siRNA-mediated silencing of EWS–WT1 and/or RNase H1 overexpression. Mean ± SD; n = 3; two-way ANOVA. *, P < 0.05; ****, P < 0.0001; ns, not significant.

Journal: Cancer Research

Article Title: Replication Stress Is an Actionable Genetic Vulnerability in Desmoplastic Small Round Cell Tumors

doi: 10.1158/0008-5472.CAN-23-3603

Figure Lengend Snippet: EWS–WT1 drives enhanced DNA replication stress and R-loops, which contribute to DSRCT cells’ sensitivity to PARPi and ATRi. A, Assessment of replication fork speed (kb/minute) in JN1 cells subjected to siRNA-mediated silencing of EWS–WT1 or CCND1. A minimum of 50 forks was analyzed per condition. Mean ± SD; each dot represents a single replication fork; n = 2, one-way ANOVA and post hoc Dunnett test. B, Assessment of replication fork speed (kb/minute) in JN1 cells exposed to DMSO control, or a combination of PARPi talazoparib (Tala) and ATRi M4344 for 6 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. A minimum of 50 forks was analyzed per condition. Mean ± SD; each dot represents a single replication fork; n = 2; two-way ANOVA and post hoc Šídák test. C and D, DNA:RNA hybrid dot blot of genomic DNA extracted from JN1 ( C ) or R ( D ) cells exposed to PARPi talazoparib, ATRi M4344, or a combination of both in the presence or absence of siRNA-mediated silencing of EWS–WT1 as in B . S9.6, RNA:DNA hybrids; ssDNA, loading control. E, Assessment of replication fork speed (kb/minute) in RNase H1–overexpressing JN1 cells subjected to siRNA-mediated silencing of EWS–WT1. Synchronized cells were collected 14 hours after transfection. A minimum of 50 forks was analyzed per condition. Mean ± SD; each dot represents a single replication fork; n = 2; unpaired t test. E, Dose–response survival curves of JN1 cells exposed to PARPi talazoparib ( F ) or olaparib ( G ), and ATRi M4344 ( H ) or AZD6738 ( I ) for 7 days in the presence or absence of siRNA-mediated silencing of EWS–WT1 and/or RNase H1 overexpression. Mean ± SD; n = 3; two-way ANOVA. *, P < 0.05; ****, P < 0.0001; ns, not significant.

Techniques: Control, Dot Blot, Transfection, Over Expression

The combination of PARPi and ATRi elicits a cGAS–STING–mediated cell-autonomous immune response. A, Western blots of pTBK1, TBK, pIRF3, and IRF3 in JN1 cells exposed to DMSO control, PARPi talazoparib (Tala), ATRi M4344, or a combination of both for 72 hours. B and C, RT-qPCR analysis of RNA isolated from JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours. CCL5 ( B ) and CXCL10 ( C ) mRNA were analyzed separately relative to RPLP0. Box and whisker plots show arbitrary units of gene expression, normalized to the DMSO condition. Boxes, median and lower and upper quartiles; whiskers, the 5th to 95th percentile range; n = 4; two-way ANOVA and post hoc Dunnett test, relative to the DMSO condition. D, Quantification of PD-L1 cell-surface expression by flow cytometry in JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours. Scatter plot shows the percentage of PD-L1–positive cells within the DAPI-negative population, normalized to the DMSO condition. Mean ± SD; n = 3. Kruskal–Wallis test and post hoc Dunnett test, relative to the DMSO condition. E, Western blots of pTBK1, TBK, pIRF3, and IRF3 in JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. Appropriate silencing of EWS–WT1 was verified as shown in . F and G, RT-qPCR analysis of RNA isolated from JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. CCL5 ( F ) and CXCL10 ( G ) mRNA were analyzed separately relative to RPLP0. Box and whisker plots show arbitrary units of gene expression, normalized to the siCNTRL DMSO condition. Boxes, median and lower and upper quartiles; whiskers, the 5th to 95th percentile range; n = 4; two-way ANOVA and post hoc Dunnett test, relative to the siCNTRL DMSO condition. H, Quantification of PD-L1 cell-surface expression by flow cytometry in JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. Scatter plot shows the percentage of PD-L1–positive cells within the DAPI-negative population, normalized to the siCNTRL DMSO condition. Mean ± SD; n = 3. Kruskal–Wallis test and post hoc Dunnett test, relative to the siCNTRL DMSO condition. I, Model of EWS–WT1–driven DSRCT sensitivity to PARPi and ATRi. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. ISG, IFN-stimulated genes.

Journal: Cancer Research

Article Title: Replication Stress Is an Actionable Genetic Vulnerability in Desmoplastic Small Round Cell Tumors

doi: 10.1158/0008-5472.CAN-23-3603

Figure Lengend Snippet: The combination of PARPi and ATRi elicits a cGAS–STING–mediated cell-autonomous immune response. A, Western blots of pTBK1, TBK, pIRF3, and IRF3 in JN1 cells exposed to DMSO control, PARPi talazoparib (Tala), ATRi M4344, or a combination of both for 72 hours. B and C, RT-qPCR analysis of RNA isolated from JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours. CCL5 ( B ) and CXCL10 ( C ) mRNA were analyzed separately relative to RPLP0. Box and whisker plots show arbitrary units of gene expression, normalized to the DMSO condition. Boxes, median and lower and upper quartiles; whiskers, the 5th to 95th percentile range; n = 4; two-way ANOVA and post hoc Dunnett test, relative to the DMSO condition. D, Quantification of PD-L1 cell-surface expression by flow cytometry in JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours. Scatter plot shows the percentage of PD-L1–positive cells within the DAPI-negative population, normalized to the DMSO condition. Mean ± SD; n = 3. Kruskal–Wallis test and post hoc Dunnett test, relative to the DMSO condition. E, Western blots of pTBK1, TBK, pIRF3, and IRF3 in JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. Appropriate silencing of EWS–WT1 was verified as shown in . F and G, RT-qPCR analysis of RNA isolated from JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. CCL5 ( F ) and CXCL10 ( G ) mRNA were analyzed separately relative to RPLP0. Box and whisker plots show arbitrary units of gene expression, normalized to the siCNTRL DMSO condition. Boxes, median and lower and upper quartiles; whiskers, the 5th to 95th percentile range; n = 4; two-way ANOVA and post hoc Dunnett test, relative to the siCNTRL DMSO condition. H, Quantification of PD-L1 cell-surface expression by flow cytometry in JN1 cells exposed to DMSO control, PARPi talazoparib, ATRi M4344, or a combination of both for 72 hours, in the presence or absence of siRNA-mediated silencing of EWS–WT1. Scatter plot shows the percentage of PD-L1–positive cells within the DAPI-negative population, normalized to the siCNTRL DMSO condition. Mean ± SD; n = 3. Kruskal–Wallis test and post hoc Dunnett test, relative to the siCNTRL DMSO condition. I, Model of EWS–WT1–driven DSRCT sensitivity to PARPi and ATRi. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. ISG, IFN-stimulated genes.

Techniques: Western Blot, Control, Quantitative RT-PCR, Isolation, Whisker Assay, Gene Expression, Expressing, Flow Cytometry

Ataxia‐telangiectasia‐and‐Rad3‐related protein (ATR) inhibitors in clinical development.

Journal: MedComm

Article Title: Targeting the DNA damage response in cancer

doi: 10.1002/mco2.788

Figure Lengend Snippet: Ataxia‐telangiectasia‐and‐Rad3‐related protein (ATR) inhibitors in clinical development.

Article Snippet: Gartisertib (M4344) was developed by Vertex Pharmaceuticals.

Techniques: Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Chemo-Phosphoproteomic Profiling with ATR Inhibitors Berzosertib and Gartisertib Uncovers New Biomarkers and DNA Damage Response Regulators

doi: 10.1016/j.mcpro.2024.100802

Figure Lengend Snippet: O ptimizing activation and inhibition of ATR . A , U-2 OS cells were incubated with thymidine (2 mM) for 24 h and released for 3 h at which point nocodazole (100 ng/ml) was added for a further 12 h. Cells were released from nocodazole into fresh medium for the times indicated. Cells were fixed, stained with propidium iodide (PI) and analysed by FACS. B , U-2 OS cells synchronized in S-phase (11 h after release from nocodazole) were pre-incubated for 1 h with the indicated concentrations of berzosertib or gartisertib before addition of HU (1 mM) for 1 h. Cells were lysed, and extracts were subjected to Western blotting with the antibodies indicated. C , same as ( B ) except that S-phase cells were pre-incubated with berzosertib or gartisertib (1 μM for 1 h) before addition of HU (1 mM) for the times indicated. D , optimized cell treatment workflow.

Article Snippet: This work was supported by the 10.13039/501100000265 Medical Research Council (grant number MC_UU_12016/1 ; M. G., P. L., F. L., I. M., J. R.) and by the healthcare business of 10.13039/100009945 Merck KGaA , Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945 ) who provided berzosertib and gartisertib free of charge.

Techniques: Activation Assay, Inhibition, Incubation, Staining, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Chemo-Phosphoproteomic Profiling with ATR Inhibitors Berzosertib and Gartisertib Uncovers New Biomarkers and DNA Damage Response Regulators

doi: 10.1016/j.mcpro.2024.100802

Figure Lengend Snippet: Phosphoproteomic screening of phosphorylation sites sensitive to berzosertib or gartisertib . A and B , Volcano plot showing phosphorylation sites affected by berzosertib ( A ) or gartisertib ( B ). The horizontal cut-off lines represent an adjusted p -value of 0.05. Phosphopeptides lower in abundance after inhibitor treatment are in the negative logFC region of the plots. The mass spectrometry proteomics raw data for this figure have been deposited to the ProteomeXchange Consortium via the jPOSTrepo partner repository with the dataset identifier PXD040469 . Data analysis scripts and annotated spectra are available via Zenodo under https://doi.org/10.5281/zenodo.10581948 . C , overlap of proteins and phosphorylation sites affected by berzosertib or gartisertib. D and E , Phosphomotif analysis for berzosertib ( D ) and gartisertib ( E ). F , the five proteins whose phosphorylation is most strongly affected by berzosertib (“Class 1” hits). The phosphorylated residue is highlighted in red in the “Motif” column. Fold change refers to the difference between HU±ATRi. G , schematic diagram showing the location of the ATR-dependent phosphorylation sites in the DNA helicase FANCJ and the protein kinase TLK2.

Techniques: Phospho-proteomics, Mass Spectrometry, Residue

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