Single nucleotide changes were introduced into the non-structural (NS) coding sequence of the H-1 parvovirus (PV) infectious molecular clone and the corresponding virus stocks produced, thereby generating H1-PM-I, H1-PM-II, H1-PM-III, and H1-DM. The effects of the mutations on viral fitness were analyzed. Because of the overlapping sequences of NS1 and NS2, the mutations affected either NS2 (H1-PM-II, -III) or both NS1 and NS2 proteins (H1-PM-I, H1-DM). Our results show key benefits of PM-I, PM-II, and DM mutations with regard to the fitness of the virus stocks produced. Indeed, these mutants displayed a higher production of infectious virus in different cell cultures and better spreading capacity than the wild-type virus. This correlated with a decreased particle-to-infectivity (P/I) ratio and stimulation of an early step(s) of the viral cycle prior to viral DNA replication, namely, cell binding and internalization. These mutations also enhance the transduction efficiency of H-1PV-based vectors. In contrast, the PM-III mutation, which affects NS2 at a position downstream of the sequence deleted in Del H-1PV, impaired virus replication and spreading. We hypothesize that the NS2 protein—modified in H1-PM-I, H1-PM-II, and H1-DM—may result in the stimulation of some maturation step(s) of the capsid and facilitate virus entry into subsequently infected cells.

Hepatoblastoma (HB) is the most common pediatric liver cancer. While standard cisplatin-based therapy cures ∼80% of patients, resistance remains a major challenge, with no standard second-line treatment. This study aimed to identify new therapeutic options for cisplatin-resistant HB. Fourteen HB cell lines and one pediatric hepatocellular carcinoma cell line were molecularly profiled. Eight cell lines were screened with 101 compounds and 58 combinations in monolayer cultures. The most synergistic combination was further validated in two low-passage patient-derived primary HB cultures and 12 HB spheroid models. In vivo efficacy was evaluated in cisplatin-resistant subcutaneous (n = 2) and orthotopic (n = 1) HB xenografts, and a liver-specific MYC-driven HB-like model. Mechanistic analyses were performed using transcriptomics, DNA damage, and apoptosis assays. Cell lines recapitulated key HB genomic alterations and mainly represented the cisplatin-resistant liver progenitor transcriptomic subtype, characterized by upregulated DNA repair pathways, potentially contributing to chemoresistance. ATR was identified as a critical target for overcoming resistance. The potent ATR inhibitor elimusertib demonstrated strong synergy with cisplatin in 2D and 3D cultures. In vivo, the elimusertib/cisplatin combination significantly suppressed tumor growth in xenografts, with manageable toxicity, inducing tumor cell death through enhanced DNA damage and p53 activation. The combination also showed antitumor efficacy in the MYC-driven HB-like model, which mirrors the cisplatin resistance and DNA repair features of the human liver progenitor HB subtype. Additional in vitro studies suggested that elimusertib synergizes with other standard HB chemotherapies and inhibits mTOR. ATR mediates cisplatin resistance in HB. The combination of elimusertib and cisplatin demonstrated preclinical efficacy across patient-derived cultures and multiple in vivo HB models, supporting its potential as a promising therapy for cisplatin-resistant HB. Chemoresistance significantly reduces survival in patients with hepatoblastoma, highlighting the need for new therapies. In this study, we found that blocking ATR with the drug elimusertib synergizes with cisplatin, strongly reducing tumor growth in diverse preclinical models, including cell cultures and mouse models of cisplatin-resistant hepatoblastoma. These results highlight a promising new approach to improve outcomes for children with cisplatin-resistant hepatoblastoma and support further clinical evaluation.

EWSR1::FLI1, the oncogenic chimeric transcription factor driving Ewing sarcoma (EwS) induces expression of exquisitely EwS-specific neogenes (Ew_NGs) through neomorphic binding and transcription activation at GGAA microsatellites in genomic regions that are silent in normal tissues. We show that peptides encoded by Ew_NGs are presented on HLA-I complexes on EwS cells. The cytokine secretion of CD8+ T cells specific for Ew_NG-encoded HLA-I-bound peptides is activated by all HLA-I-matched EwS cells but not by non-EwS cells. These T cells kill EwS cells in an HLA-I restricted manner. This cytotoxicity is dependent on the expression of EWSR1::FLI1 and of the corresponding Ew_NG. It can be reproduced by transduction of the TCR into donor T cells (TCR-T) which kill EwS cells in vivo. Moreover, we show that neither off target nor allogeneic activation are observed with TCR-T thus paving the way for cell therapy in relapsed/resistant EwS patients for which therapeutic options are very limited.

Les tumeurs pédiatriques du foie (PLCs) sont rares et représentent 1 à 2% des néoplasmes pédiatriques. Parmi elles, la tumeur la plus fréquente est l'hépatoblastome (HB) qui survient majoritairement chez les jeunes enfants (< 5 ans). Bien que l'HB soit le plus souvent sporadique, il peut se développer dans le cadre de maladies génétiques telles que le syndrome de Beckwith-Wiedemann, caractérisé par une croissance excessive et des altérations génétiques de la région chromosomique 11p15.5. L'HB peut être également associé à une polypose adénomateuse familiale associée à des mutations du gène APC. Bien que les traitements à base de cisplatine et anthracyclines soient très efficaces pour les HB de risque standard, les HB de haut risque présentent un taux de survie nettement plus faible en raison de la résistance à la chimiothérapie. Les carcinomes hépatocellulaires (CHC) pédiatriques et les carcinomes hépatocellulaires fibrolamellaires (FLC) surviennent en général chez des enfants plus agés avec une survie globale d'environ 30% à 5 ans. Des tumeurs bénignes comme les adénomes hépatocellulaires (HCA) peuvent également survenir, et sont généralement liées à des maladies génétiques métaboliques qui peuvent dans de rares cas évoluer en carcinomes hépatocellulaires. Au cours de ce travail, nous avons identifié de nouveaux gènes impliqués dans la tumorigénèse des PLCs. Parmi eux, nous avons montré que, même en l'absence de BWS, la plupart des HBs et les CHC pédiatriques étaient porteurs d'altérations génétiques et/ou épigénétiques au niveau du locus 11p15.5 conduisant à la surexpression de l'oncogène IGF2. Nos résultats ont également montré que les anomalies du 11p15.5 se présentaient sous forme de mosaïques et d'expansions clonales pré-malignes dans 10% des tissus hépatiques non-tumoraux. L'analyse des hépatoblastomes mutés APC a démontré la présence d'infiltrats immunitaires sous forme de structures lymphoides tertiaires induites par la chimiothérapie par cisplatine. Une classification transcriptomique a permis de mettre en évidence 4 groupes d'HB «hepatocytic hot», «hepatocytic cold», «liver progenitor» et «mesenchymal» en lien avec l'état de différenciation, l'infiltrat immunitaire et fortement corrélés au contingent histologique de l'échantillon tumoral. Nous avons montré que les cellules d'hépatoblastomes étaient capables d'une plasticité importante entre les différents sous-groupes moléculaires. De plus, nous avons montré que les cellules du groupe «liver progenitor» étaient capables d'accumuler des charges massives de mutations induites par le cisplatine conduisant à une signature mutationnelle spécifique SBS35 et entraînant le développement de récidives et métastases fortement mutées. Le criblage de molécules thérapeutiques dans des lignées cellulaires dérivées de PLCs nous a permis d'identifier des cibles prometteuses pour le traitement des cellules « Liver progenitor » résistantes au cisplatine : CHEK1, BIRC5 et PLK1. Enfin, les lignées cellulaires qui comportaient des altérations génétiques de la voie Ras/MAPK étaient particulièrement sensibles au Trametinib, un inhibiteur de MEK1/2.

How tumors generate a cryptic “dark” proteome absent from healthy tissues, and whether it can be reversibly activated, remains unclear. In Ewing sarcoma, all tumors are driven by EWSR1::ETS fusions, making it a tractable model to study dark proteome activation. Integrating matched transcriptomes, translatomes and proteomes from 48 patient tumors with long-read RNA sequencing, single-cell Ribo-seq, proteomics and immunopeptidomics in cell line models, we show that EWSR1::FLI1 acts as a reversible switch recurrently inducing hundreds of cancer-specific neoproteins. Many arise from canonical coding regions via intragenic transcription start sites, generating truncated or out-of-frame neoproteins. We uncover a fusion-dependent increase in ribosomal readthrough into poly(A) tails, generating a stable 80-amino-acid TRPM4 neoprotein that, despite lacking a stop codon, is the most abundant tumor-specific microprotein across patients. Proteomic, immunopeptidomic, and immunofluorescence analyses validate neoproteins as tumor-restricted antigens, revealing a therapeutically actionable cancer dark proteome controlled by one oncogenic fusion. Highlights: Fusion proteins activate in- and out-of-frame neoproteins through intragenic transcription. Neoproteins exhibit distinct protein structures and localizations compared to their host genes. Mass spectrometry detects 911 cryptic proteins, including 29 EwS neoproteins, 15 HLA-I presented. TRPM4-dORF is a stop-less 80-aa neoprotein with abundant peptide evidence exclusively in EwS.