Figure 6 from Replication Stress Is an Actionable Genetic Vulnerability in Desmoplastic Small Round Cell Tumors
Authors: Kawai-Kawachi Asuka , Lenormand Madison M. , Astier Clémence , Herbel Noé , Cutrona Meritxell B. , Ngo Carine , Garrido Marlène , Eychenne Thomas , Dorvault Nicolas , Bordelet Laetitia , Song Feifei , Bouyakoub Ryme , Loktev Anastasia , Romo-Morales Antonio , Henon Clémence , Colmet-Daage Léo , Vibert Julien , Drac Marjorie , Brough Rachel , Schwob Etienne , Martella Oliviano , Pinna Guillaume , Shipley Janet M. , Mittnacht Sibylle , Zimmermann Astrid , Gulati Aditi , Mir Olivier , Le Cesne Axel , Faron Matthieu , Honoré Charles , Lord Christopher J. , Chabanon Roman M. , Postel-Vinay Sophie 
Published: January 2025

<p>The combination of PARPi and ATRi elicits a cGAS–STING–mediated cell-autonomous immune response. <b>A,</b> 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>B</b> and <b>C,</b> 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>B</b>) and CXCL10 (<b>C</b>) 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; <i>n</i> = 4; two-way ANOVA and <i>post hoc</i> Dunnett test, relative to the DMSO condition. <b>D,</b> 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; <i>n</i> = 3. Kruskal–Wallis test and <i>post hoc</i> Dunnett test, relative to the DMSO condition. <b>E,</b> 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 <a href="#fig4" target="_blank">Fig. 4H</a>. <b>F</b> and <b>G,</b> 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 (<b>F</b>) and CXCL10 (<b>G</b>) 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; <i>n</i> = 4; two-way ANOVA and <i>post hoc</i> Dunnett test, relative to the siCNTRL DMSO condition. <b>H,</b> 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; <i>n</i> = 3. Kruskal–Wallis test and <i>post hoc</i> Dunnett test, relative to the siCNTRL DMSO condition. <b>I,</b> Model of EWS–WT1–driven DSRCT sensitivity to PARPi and ATRi. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; ****, <i>P</i> < 0.0001; ns, not significant. ISG, IFN-stimulated genes.</p>