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posted on 2025-01-02, 08:21 authored by Asuka Kawai-Kawachi, Madison M. Lenormand, Clémence Astier, Noé Herbel, Meritxell B. Cutrona, Carine Ngo, Marlène Garrido, Thomas Eychenne, Nicolas Dorvault, Laetitia Bordelet, Feifei Song, Ryme Bouyakoub, Anastasia Loktev, Antonio Romo-Morales, Clémence Henon, Léo Colmet-Daage, Julien Vibert, Marjorie Drac, Rachel Brough, Etienne Schwob, Oliviano Martella, Guillaume Pinna, Janet M. Shipley, Sibylle Mittnacht, Astrid Zimmermann, Aditi Gulati, Olivier Mir, Axel Le Cesne, Matthieu Faron, Charles Honoré, Christopher J. Lord, Roman M. Chabanon, Sophie Postel-Vinay 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.
Funding
Fondation pour la Recherche Médicale (FRM)
European Society for Medical Oncology (ESMO)
Canceropôle PACA (Canceropole PACA)
Fondation Bettencourt Schueller (Bettencourt Schueller Foundation)
Institut Servier (Servier Institute)
Fondation des Treilles
HORIZON EUROPE European Research Council (ERC)
Fondation ARC pour la Recherche sur le Cancer (ARC)
Institut National de la Santé et de la Recherche Médicale (Inserm)
Institut National Du Cancer (INCa)
History
ARTICLE ABSTRACT
Desmoplastic small round cell tumor (DSRCT) is an aggressive sarcoma subtype that is driven by the EWS–WT1 chimeric transcription factor. The prognosis for DSRCT is poor, and major advances in treating DSRCT have not occurred for over two decades. To identify effective therapeutic approaches to target DSRCT, we conducted a high-throughput drug sensitivity screen in a DSRCT cell line assessing chemosensitivity profiles for 79 small-molecule inhibitors. DSRCT cells were sensitive to PARP inhibitors (PARPi) and ataxia–telangiectasia and Rad3–related inhibitors (ATRi), as monotherapies and in combination. These effects were recapitulated using multiple clinical PARPi and ATRi in three biologically distinct, clinically relevant models of DSRCT, including cell lines, a patient-derived xenograft–derived organoid model, and a cell line–derived xenograft mouse model. Mechanistically, exposure to a combination of PARPi and ATRi caused increased DNA damage, G2–M checkpoint activation, micronuclei accumulation, replication stress, and R-loop formation. EWS–WT1 silencing abrogated these phenotypes and was epistatic with exogenous expression of the R-loop resolution enzyme RNase H1 in reversing sensitivity to PARPi and ATRi monotherapies. The combination of PARPi and ATRi also induced EWS–WT1–dependent cell-autonomous activation of the cyclic GMP–AMP synthase–stimulator of IFN genes innate immune pathway and cell-surface expression of PD-L1. Taken together, these findings point toward a role for EWS–WT1 in generating R-loop–dependent replication stress that leads to a targetable vulnerability, providing a rationale for the clinical assessment of PARPi and ATRi in DSRCT.Significance: EWS–WT1, the unique oncogenic driver of desmoplastic small round cell tumors, confers sensitivity to PARP and ATR inhibitors, supporting the potential of these drugs in treating patients with this aggressive sarcoma subtype.
