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Extracranial rhabdoid tumours (ECRTs) are an aggressive malignancy of infancy and early childhood. The vast majority of cases demonstrate inactivation of SMARCB1 (ECRTSMARCB1 ) on a background of a remarkably stable genome, a low mutational burden, and no other recurrent mutations. Rarely, ECRTs can harbour the alternative inactivation of SMARCA4 (ECRTSMARCA4 ) instead of SMARCB1. However, very few ECRTSMARCA4 cases have been published to date, and a systematic characterization of ECRTSMARCA4 is missing from the literature. In this study, we report the clinical, pathological, and genomic features of additional cases of ECRTSMARCA4 and show that they are comparable to those of ECRTSMARCB1. We also assess whether ECRTSMARCB1 , ECRTSMARCA4 , and small cell carcinomas of the ovary, hypercalcaemic type (SCCOHT) represent distinct or overlapping entities at a molecular level. Using DNA methylation and transcriptomics-based tumour classification approaches, we demonstrate that ECRTSMARCA4 display molecular features intermediate between SCCOHT and ECRTSMARCB1 ; however, ECRTSMARCA4 appear to be more closely related to SCCOHT by DNA methylation. Conversely, both transcriptomics and DNA methylation show a larger gap between SCCOHT and ECRTSMARCB1 , potentially supporting their continuous separate classification. Lastly, we show that ECRTSMARCA4 display concomitant lack of SMARCA4 (BRG1) and SMARCA2 (BRM) expression at the protein level, similar to what is seen in SCCOHT. Overall, these results expand our knowledge on this rare tumour type and explore the similarities and differences among entities from the 'rhabdoid tumour' spectrum. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Atypical teratoid/rhabdoid tumors (ATRTs) are very aggressive childhood malignancies of the central nervous system. The underlying genetic cause are inactivating bi-allelic mutations in SMARCB1 or (rarely) in SMARCA4. ATRT-SMARCA4 have been associated with a higher frequency of germline mutations, younger age, and an inferior prognosis in comparison to SMARCB1 mutated cases. Based on their DNA methylation profiles and transcriptomics, SMARCB1 mutated ATRTs have been divided into three distinct molecular subgroups: ATRT-TYR, ATRT-SHH, and ATRT-MYC. These subgroups differ in terms of age at diagnosis, tumor location, type of SMARCB1 alterations, and overall survival. ATRT-SMARCA4 are, however, less well understood, and it remains unknown, whether they belong to one of the described ATRT subgroups. Here, we examined 14 ATRT-SMARCA4 by global DNA methylation analyses. We show that they form a separate group segregating from SMARCB1 mutated ATRTs and from other SMARCA4-deficient tumors like small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) or SMARCA4 mutated extra-cranial malignant rhabdoid tumors. In contrast, medulloblastoma (MB) samples with heterozygous SMARCA4 mutations do not group separately, but with established MB subgroups. RNA sequencing of ATRT-SMARCA4 confirmed the clustering results based on DNA methylation profiling and displayed an absence of typical signature genes upregulated in SMARCB1 deleted ATRT. In summary, our results suggest that, in line with previous clinical observations, ATRT-SMARCA4 should be regarded as a distinct molecular subgroup.

International audience; Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.

The cell(s) of origin of AT/RTs remain(s) unknown. We previously developed a mouse model consisting of tamoxifen inducible system in a Smarcb1Flox/Flox;Rosa26-CreERT2 background. We obtained two molecular subgroups of intracranial tumors, one with neuronal and the other with non-neuronal features, consistent with the diversity observed in human AT/RTs. To investigate the potential cell(s) of origin of these various AT/RTs, we first explored whether different time points of Smarcb1 inactivation correlated with anatomic location and/or molecular subgroups. We observed that the neuronal group, primarily developing from the subventricular zone and the spinal cord, was almost exclusively obtained with the earliest inactivation time points (E6-E7). In contrast, the non-neuronal group emerged upon Smarcb1 inactivation at any time point (E6-E10) and showed intracranial but extra-parenchymal/meningeal origins. High-resolution analysis of anatomic distribution of 55 human AT/RT and molecular subgroups is in progress. In order to more specifically identify the cell(s) of origin for the neuronal group, we next generated developmental stage-specific conditional knockout mice carrying Smarcb1 inactivation by restricting Cre expression with promoters characteristic for various neural stem cells/progenitors. While Smarcb1Flox/Flox;Atoh1CreERT2 showed ataxia but failed to give rise to any tumor at any embryonal time point, Smarcb1Flox/Flox;Ascl1CreERT2 did not show any phenotype. Targeting Nestin-expressing cells led to tumors with morphologic rhabdoid features; these tumors again showed molecular diversity as observed in human AT/RTs. In conclusion, we show that deletion of Smarcb1 determines mouse AT/RT subtypes depending on spatial and temporal factors. Our new mouse models not only give insight into the cell(s) of origin, but also provide interesting preclinical models of AT/RTs. Citation Format: Zhi-Yan Han, Mamy Andrianteranagna, Maria Jesus-Lobon-Iglesias, Arnault Tauziède-Espariat, Kevin Beccaria, Paul Fréneaux, Joshua J. Waterfall, Julien Masliah-Planchon, Christine Bourneix, Gaelle Pierron, Amaury Leruste, Céline Chauvin, Didier Surdez, Pascale Varlet, Christelle Dufour, Olivier Delattre, Volodia Dangouloff-Ros, Franck Bourdeaut. Spatial and temporal conditions for Smarcb1 deletion determines mouse AT/RT (atypical teratoid/rhabdoid tumor) subtype [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B68.

The cell(s) of origin of AT/RTs remains unclear. We previously developed a mouse model consisting in tamoxifen inducible system in a Smarcb1(Flox/Flox);Rosa26-Cre(ERT2) background. We obtained two subgroups of intracranial tumors, one with neuronal and the other with non-neuronal features, in keeping with the diversity observed in human AT/RTs. To investigate the potential cell(s) of origin of those various AT/RTs, we first endeavored to specify whether different time points of Smarcb1 inactivation correlated with specific anatomic location and/or molecular subgroups. Thereby, we identified that the neuronal group, mostly developing from the sub-ventricular zone and the spinal cord, was almost exclusively obtained with the earliest inactivation time point(E6-E7). In contrary, the non-neuronal group emerged after Smarcb1 inactivation at any time point(E6-E10), and showed intracranial but extra-parenchyma/meningeal origins. In order to more specifically identify the cell(s) of origin for the neuronal group, we next generated developmental stage-specific conditional knockout mice carrying Smarcb1 inactivation by restricting Cre expression upon promoters characteristic for various neural stem cells/progenitors. While Smarcb1(Flox/Flox);Atoh1Cre(ERT2) showed ataxia but failed to give rise to any tumor whatever the embryonal time point, targeting Nestin-expressing cells led to tumors with morphological rhabdoid features; these again showed some molecular diversity as observed in human AT/RTs. The study of the impact of Smarcb1 inactivation in other promoter-restricted backgrounds is ongoing. In conclusion, we show that spatio-temporal deletion of Smarcb1 determines mouse AT/RT subtypes. Our new mouse models not only give insight on the cell(s) of origin, but also provide interesting preclinical models.

Summary: Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/β and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These results strongly support testing pazopanib/CfT combination therapy in future clinical trials for RTs. : Rhabdoid tumors (RTs) are aggressive pediatric tumors characterized by SMARCB1 inactivation. Chauvin et al. identify two SMARCB1-dependent targeted therapies for RT: pazopanib, which inhibits PDGFR and FGFR2, and the potassium channel inhibitor clofilium tosylate, which induces endoplasmic reticulum stress. Combining both drugs induces cell apoptosis and reduces PDX tumor growth. Keywords: rhabdoid tumors, SMARCB1, pazopanib, clofilium tosylate, high-throughput drug screening, tyrosine kinase inhibitors