Ryme Bouyakoub

Leber congenital amaurosis (LCA) encompasses the earliest and most severe retinal dystrophies and can occur as a non-syndromic or a syndromic disease. Molecular diagnosis in LCA is of particular importance in clinical decision-making and patient care since it can provide ocular and extraocular prognostics and identify patients eligible to develop gene-specific therapies. Routine high-throughput molecular testing in LCA yields 70%–80% of genetic diagnosis. In this study, we aimed to investigate the non-coding regions of one non-syndromic LCA gene, RPGRIP1, in a series of six families displaying one single disease allele after a gene-panel screening of 722 LCA families which identified 26 biallelic RPGRIP1 families. Using trio-based high-throughput whole locus sequencing (WLS) for second disease alleles, we identified a founder deep intronic mutation (NM_020366.3:c.1468-128T>G) in 3/6 families. We employed Sanger sequencing to search for the pathologic variant in unresolved LCA cases (106/722) and identified three additional families (two homozygous and one compound heterozygous with the NM_020366.3:c.930+77A>G deep intronic change). This makes the c.1468-128T>G the most frequent RPGRIP1 disease allele (8/60, 13%) in our cohort. Studying patient lymphoblasts, we show that the pathologic variant creates a donor splice-site and leads to the insertion of the pseudo-exon in the mRNA, which we were able to hamper using splice-switching antisense oligonucleotides (AONs), paving the way to therapies.

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.

Abstract Functional precision medicine (FPM) aims to personalize cancer treatment through ex vivo drug testing on patient tumor cells. Classical biochemical tests (like ATP assay) provide limited insight into how patient cells respond to treatment. Phenotypic markers based on imaging features called "radiomics" offer a promising approach to understanding tumor response. Combining FPM with phenotypic screening (PS), we evaluate the predictive power of biomarkers derived from brightfield time-lapse imaging of patient-derived organoids (PDO) from digestive cancer patients. We screened 24 patients, including 5 with colorectal (CRC) and 19 with pancreatic (PDAC) cancers, each with varying treatment histories (1 to 8 treatments prior to PDO biopsy and 12 different treatments). PDOs were exposed to the corresponding clinical treatments, and 96-hour post-treatment videos allowed for individual tracking of PDO responses. A total of 103 image features (18 intensity, 75 texture, 10 morphology) were extracted from each PDO. Predictive analysis was conducted at 24h, 48h, 72h, and 96h post-treatment, using single timepoints and time-averaging methods. PDO responses were assessed at 3 treatment doses, and area dose-response (ADR) was calculated using trapezoid estimation. Univariate and multivariate Cox models were applied to assess the association of image features with progression-free survival (PFS) and develop an image-based signature for PFS prediction. We compared image features extracted from entire movie sequences (49 frames), 24h-averaging (13 frames), 4h-averaging (3 frames), and single timepoints (24h, 48h, 72h, 96h). We found similar results from univariate Cox analyses and focused on single timepoint method, which identified 24 features significantly associated with PFS as early as 48 hours. These included intensity (7), texture (11), and shape (6) features (median C-index=0.63, 95% CI [0.60-0.67]). When combining each feature with clinical factors (disease and treatment), 7 features remained significantly associated with PFS. A subset of 3 non-correlated interpretable features was selected (one per category). A signature built from a texture feature measuring pixel intensity homogeneity, combined with disease and treatment best predicted PFS (C-index=0.72, HR=27.43 95% CI [1.89-399.28], p-value=0.046). Integrating image biomarkers with clinical variables enhances the predictive power of PDO-based functional drug screening. Extracting features from individual timepoints provides earlier insights than biochemical assays, which typically require 5 days post-treatment. This study encourages further exploration of how PS-based FPM could be implemented in a clinical setting. Future work will focus on validating the image-based signature in an independent cohort and across diverse experimental setups. Citation Format: Emilie Gontran, Leo Fillioux, Jerome Cartry, Ryme Bouyakoub, Sabrina Bedja, Alice Boileve, Ali Mouawia, Anna-Rose Gryspeert, Mia Cabantous, Gizem Altay, Maria Vakalopoulou, Stergios Christodoulidis, Jacques RR Mathieu, Paul-Henry Cournede, Fanny Jaulin. Phenotypic screening from patient-derived organoids to predict therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7154.

<p>EWS–WT1 is a determinant of DSRCT cells’ sensitivity to PARPi and ATRi. <b>A,</b> 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>B–E,</b> Dose–response survival curves of JN1 or R cells exposed to PARPi talazoparib (<b>B</b> and <b>C</b>) or ATRi M4344 (<b>D</b> and <b>E</b>) for 7 days in the presence or absence of siRNA-mediated silencing of EWS–WT1. Mean ± SD; <i>n</i> = 3. <b>F</b> and <b>G,</b> 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 <i>post hoc</i> Dunn test. <b>H</b> and <b>I,</b> Western blots of pCHK1, CHK1, pRPA2, RPA2, γH2AX, H2AX, and EWS–WT1 in JN1 (<b>H</b>) or R (<b>I</b>) 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. ****, <i>P</i> < 0.0001; ns, not significant. </p>