Gemcitabine therapeutically disrupts essential SIRT1-mediated p53 repression in atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumors (ATRTs) are highly malignant embryonal tumors of the central nervous system with a dismal prognosis. Using a newly developed and validated patient-derived ATRT culture and xenograft model, alongside a panel of primary ATRT models, we found that ATRTs are selectively sensitive to the nucleoside analog gemcitabine. Gene expression and protein analyses indicate that gemcitabine treatment causes the degradation of sirtuin 1 (SIRT1), resulting in cell death through activation of nuclear factor κB (NF-κB) and p53. Furthermore, we discovered that gemcitabine-induced loss of SIRT1 results in a nucleus-to-cytoplasm translocation of the sonic hedgehog (SHH) signaling activator GLI2, explaining the observed additional gemcitabine sensitivity in SHH-subtype ATRT. Treatment of ATRT xenograft-bearing mice with gemcitabine resulted in a >30% increase in median survival and yielded long-term survivors in two independent patient-derived xenograft models. These findings demonstrate that ATRTs are highly sensitive to gemcitabine treatment and may form part of a future multimodal treatment strategy for ATRTs.

PHF6 cooperates with SWI/SNF complexes to facilitate transcriptional progression.

Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are mutated in nearly 25% of cancers. To gain insight into the mechanisms by which SWI/SNF mutations drive cancer, we contributed ten rhabdoid tumor (RT) cell lines mutant for SWI/SNF subunit SMARCB1 to a genome-scale CRISPR-Cas9 depletion screen performed across 896 cell lines. We identify PHF6 as specifically essential for RT cell survival and demonstrate that dependency on Phf6 extends to Smarcb1-deficient cancers in vivo. As mutations in either SWI/SNF or PHF6 can cause the neurodevelopmental disorder Coffin-Siris syndrome, our findings of a dependency suggest a previously unrecognized functional link. We demonstrate that PHF6 co-localizes with SWI/SNF complexes at promoters, where it is essential for maintenance of an active chromatin state. We show that in the absence of SMARCB1, PHF6 loss disrupts the recruitment and stability of residual SWI/SNF complex members, collectively resulting in the loss of active chromatin at promoters and stalling of RNA Polymerase II progression. Our work establishes a mechanistic basis for the shared syndromic features of SWI/SNF and PHF6 mutations in CSS and the basis for selective dependency on PHF6 in SMARCB1-mutant cancers.

A kinome drug screen identifies multi-TKI synergies and ERBB2 signaling as a therapeutic vulnerability in MYC/TYR subgroup atypical teratoid rhabdoid tumors.

BACKGROUND: Atypical teratoid rhabdoid tumor (ATRT) is a rare, devastating, and largely incurable pediatric brain tumor. Although recent studies have uncovered 3 molecular subgroups of ATRTs with distinct disease patterns, and signaling features, the therapeutic profiles of ATRT subgroups remain incompletely elucidated. METHODS: We examined the effect of 465 kinase inhibitors on a panel of ATRT subgroup-specific cell lines. We then applied multiomics analyses to investigate the underlying molecular mechanism of kinase inhibitor efficacy in ATRT subgroups. RESULTS: We observed that ATRT cell lines are broadly sensitive to inhibitors of the PI3K and MAPK signaling pathways, as well as CDKs, AURKA/B kinases, and polo-like kinase 1. We identified 2 classes of multikinase inhibitors predominantly targeting receptor tyrosine kinases including PDGFR and EGFR/ERBB2 in MYC/TYR ATRT cells. The PDGFRB inhibitor, Dasatinib, synergistically affected MYC/TYR ATRT cell growth when combined with broad-acting PI3K and MAPK pathway inhibitors, including Rapamycin and Trametinib. We observed that MYC/TYR ATRT cells were also distinctly sensitive to various inhibitors of ERBB2 signaling. Transcriptional, H3K27Ac ChIPSeq, ATACSeq, and HiChIP analyses of primary MYC/TYR ATRTs revealed ERBB2 expression, which correlated with differential methylation and activation of a distinct enhancer element by DNA looping. Significantly, we show the brain penetrant EGFR/ERBB2 inhibitor, Afatinib, specifically inhibited in vitro and in vivo growth of MYC/TYR ATRT cells. CONCLUSIONS: Taken together, our studies suggest combined treatments with PDGFR and ERBB2-directed TKIs with inhibitors of the PI3K and MAPK pathways as an important new therapeutic strategy for the MYC/TYR subgroup of ATRTs.

Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers.

SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of CBP and p300 as a synthetic lethal target in SMARCB1-deficient cancers by using a dual siRNA screening method based on the "simultaneous inhibition of a paralog pair" concept. Treatment with CBP/p300 dual inhibitors suppresses growth of cell lines and tumor xenografts derived from SMARCB1-deficient cells but not from SMARCB1-proficient cells. SMARCB1-containing SWI/SNF complexes localize with H3K27me3 and its methyltransferase EZH2 at the promotor region of the KREMEN2 locus, resulting in transcriptional downregulation of KREMEN2. By contrast, SMARCB1 deficiency leads to localization of H3K27ac, and recruitment of its acetyltransferases CBP and p300, at the KREMEN2 locus, resulting in transcriptional upregulation of KREMEN2, which cooperates with the SMARCA1 chromatin remodeling complex. Simultaneous inhibition of CBP/p300 leads to transcriptional downregulation of KREMEN2, followed by apoptosis induction via monomerization of KREMEN1 due to a failure to interact with KREMEN2, which suppresses anti-apoptotic signaling pathways. Taken together, our findings indicate that simultaneous inhibitors of CBP/p300 could be promising therapeutic agents for SMARCB1-deficient cancers.

Aberrant DNA methylation distorts developmental trajectories in atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumors (AT/RTs) are pediatric brain tumors known for their aggressiveness and aberrant but still unresolved epigenetic regulation. To better understand their malignancy, we investigated how AT/RT-specific DNA hypermethylation was associated with gene expression and altered transcription factor binding and how it is linked to upstream regulation. Medulloblastomas, choroid plexus tumors, pluripotent stem cells, and fetal brain were used as references. A part of the genomic regions, which were hypermethylated in AT/RTs similarly as in pluripotent stem cells and demethylated in the fetal brain, were targeted by neural transcriptional regulators. AT/RT-unique DNA hypermethylation was associated with polycomb repressive complex 2 and linked to suppressed genes with a role in neural development and tumorigenesis. Activity of the several NEUROG/NEUROD pioneer factors, which are unable to bind to methylated DNA, was compromised via the suppressed expression or DNA hypermethylation of their target sites, which was also experimentally validated for NEUROD1 in medulloblastomas and AT/RT samples. These results highlight and characterize the role of DNA hypermethylation in AT/RT malignancy and halted neural cell differentiation.

Targeting of RRM2 suppresses DNA damage response and activates apoptosis in atypical teratoid rhabdoid tumor.

BACKGROUND: Atypical teratoid rhabdoid tumors (ATRT) is a rare but aggressive malignancy in the central nervous system, predominantly occurring in early childhood. Despite aggressive treatment, the prognosis of ATRT patients remains poor. RRM2, a subunit of ribonucleotide reductase, has been reported as a biomarker for aggressiveness and poor prognostic conditions in several cancers. However, little is known about the role of RRM2 in ATRT. Uncovering the role of RRM2 in ATRT will further promote the development of feasible strategies and effective drugs to treat ATRT. METHODS: Expression of RRM2 was evaluated by molecular profiling analysis and was confirmed by IHC in both ATRT patients and PDX tissues. Follow-up in vitro studies used shRNA knockdown RRM2 in three different ATRT cells to elucidate the oncogenic role of RRM2. The efficacy of COH29, an RRM2 inhibitor, was assessed in vitro and in vivo. Western blot and RNA-sequencing were used to determine the mechanisms of RRM2 transcriptional activation in ATRT. RESULTS: RRM2 was found to be significantly overexpressed in multiple independent ATRT clinical cohorts through comprehensive bioinformatics and clinical data analysis in this study. The expression level of RRM2 was strongly correlated with poor survival rates in patients. In addition, we employed shRNAs to silence RRM2, which led to significantly decrease in ATRT colony formation, cell proliferation, and migration. In vitro experiments showed that treatment with COH29 resulted in similar but more pronounced inhibitory effect. Therefore, ATRT orthotopic mouse model was utilized to validate this finding, and COH29 treatment showed significant tumor growth suppression and prolong overall survival. Moreover, we provide evidence that COH29 treatment led to genomic instability, suppressed homologous recombinant DNA damage repair, and subsequently induced ATRT cell death through apoptosis in ATRT cells. CONCLUSIONS: Collectively, our study uncovers the oncogenic functions of RRM2 in ATRT cell lines, and highlights the therapeutic potential of targeting RRM2 in ATRT. The promising effect of COH29 on ATRT suggests its potential suitability for clinical trials as a novel therapeutic approach for ATRT.

Subclassification of epithelioid sarcoma with potential therapeutic impact.

Epithelioid sarcoma is a rare and aggressive mesenchymal tumour, the genetic hallmark of which is the loss of expression of SMARCB1, a key member of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodelling complex. Hampered by its rarity, epithelioid sarcoma has received little research attention and therapeutic options for this disease remain limited. SMARCB1-deficient tumours also include malignant rhabdoid tumour, atypical teratoid and rhabdoid tumour, epithelioid malignant peripheral nerve sheath tumour, and poorly differentiated chordoma. Histologically, it can be challenging to distinguish epithelioid sarcoma from malignant rhabdoid tumour and other SMARCB1-deficient tumours, whereas methylation profiling shows that they represent distinct entities and facilitates their classification. Methylation studies on SMARCB1-deficient tumours, although not including epithelioid sarcomas, reported methylation subgroups which resulted in new clinical stratification and therapeutic approaches. In addition, emerging evidence indicates that immunotherapy, including immune checkpoint inhibitors, represents a promising therapeutic strategy for SMARCB1-deficient tumours. Here, we show that some epithelioid sarcomas share methylation patterns of malignant rhabdoid tumours indicating that this could help to distinguish these entities and guide treatment. Using gene expression data, we also showed that the immune environment of epithelioid sarcoma is characterised by a predominance of CD8+ lymphocytes and M2 macrophages. These findings have potential implications for the management of patients with epithelioid sarcoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Atypical teratoid/rhabdoid tumoroids reveal subgroup-specific drug vulnerabilities.

Atypical teratoid/rhabdoid tumors (ATRTs) represent a rare, but aggressive pediatric brain tumor entity. They are genetically defined by alterations in the SWI/SNF chromatin remodeling complex members SMARCB1 or SMARCA4. ATRTs can be further classified in different molecular subgroups based on their epigenetic profiles. Although recent studies suggest that the different subgroups have distinct clinical features, subgroup-specific treatment regimens have not been developed thus far. This is hampered by the lack of pre-clinical in vitro models representative of the different molecular subgroups. Here, we describe the establishment of ATRT tumoroid models from the ATRT-MYC and ATRT-SHH subgroups. We demonstrate that ATRT tumoroids retain subgroup-specific epigenetic and gene expression profiles. High throughput drug screens on our ATRT tumoroids revealed distinct drug sensitivities between and within ATRT-MYC and ATRT-SHH subgroups. Whereas ATRT-MYC universally displayed high sensitivity to multi-targeted tyrosine kinase inhibitors, ATRT-SHH showed a more heterogeneous response with a subset showing high sensitivity to NOTCH inhibitors, which corresponded to high expression of NOTCH receptors. Our ATRT tumoroids represent the first pediatric brain tumor organoid model, providing a representative pre-clinical model which enables the development of subgroup-specific therapies.

Primary cilia contribute to the aggressiveness of atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant brain tumor in infants that is characterized by loss of nuclear expression of SMARCB1 or SMARCA4 proteins. Recent studies show that AT/RTs comprise three molecular subgroups, namely AT/RT-TYR, AT/RT-MYC and AT/RT-SHH. The subgroups show distinct expression patterns of genes involved in ciliogenesis, however, little is known about the functional roles of primary cilia in the biology of AT/RT. Here, we show that primary cilia are present across all AT/RT subgroups with specific enrichment in AT/RT-TYR patient samples. Furthermore, we demonstrate that primary ciliogenesis contributes to AT/RT biology in vitro and in vivo. Specifically, we observed a significant decrease in proliferation and clonogenicity following disruption of primary ciliogenesis in AT/RT cell line models. Additionally, apoptosis was significantly increased via the induction of STAT1 and DR5 signaling, as detected by proteogenomic profiling. In a Drosophila model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with more than 20% of flies reaching adulthood. We also found significantly extended survival in an orthotopic xenograft mouse model of AT/RT upon disruption of primary ciliogenesis. Taken together, our findings indicate that primary ciliogenesis or its downstream signaling contributes to the aggressiveness of AT/RT and, therefore, may constitute a novel therapeutic target.

Evaluation of targeting autophagy for the treatment of malignant rhabdoid tumours.

Malignant rhabdoid tumour (MRT) is a rare and aggressive paediatric tumour that typically arises in the kidneys or central nervous system (CNS). The malignancy often affects patients under the age of three and is associated with an extremely poor survival rate, with most deaths occurring within the first year of presentation. Thus, there is an unmet and urgent medical need for novel therapeutic strategies for this malignancy. One of the major issues when treating MRT patients is the emergence of chemoresistance. Autophagy has become an area of focus in the study of chemoresistance due to its reported dual role as both a pro-survival and pro-death mechanism. The role of autophagy in the chemotherapeutic response of MRT remains largely unknown. A greater understanding of the role of autophagy may lead to the development of therapeutic strategies to enhance chemotherapeutic effect and improve the clinical outcome of MRT patients. This study evaluated the cellular response to cisplatin, a representative chemotherapeutic agent used in the treatment of MRT, and the role of autophagy in mediating cisplatin resistance. Our results demonstrated that cisplatin induced apoptosis and autophagy concomitantly in a panel of MRT cell lines. Furthermore, inhibition of caspase-induced apoptosis with Z-VAD-FMK also inhibited autophagy levels demonstrating a complex interplay between these two pathways. In addition, blocking autophagy at the early stages of the autophagic process using the pharmacological inhibitor SAR405 or through the genetic knockdown of critical autophagic protein ATG5 by siRNA did not sensitise cells to cisplatin-induced apoptosis. Collectively, these results suggest that induction of autophagy does not appear to elicit a pro-survival effect in the chemotherapeutic response of MRT cells.

Meningioma with rhabdoid features: Pathologic findings in dogs.

Rhabdoid meningioma is a rare type of meningeal neoplasm in humans. This study reports the clinical, pathological, and ultrastructural features of 4 cases of canine meningioma with rhabdoid features. The cases were female and 8 to 12 years of age. Biopsies from complete surgical resections were examined for all cases. The whole brain with tumor recurrence was collected at necropsy in 2 dogs. Histologically, the tumors consisted of discohesive sheets of oval-polygonal cells with abundant eosinophilic cytoplasm and occasional paranuclear hyaline-like inclusions. Cells were intensely immunopositive for vimentin, negative for melan A and S100 protein in all cases, and showed variable immunolabeling for cytokeratin in 2 cases. Focal glial fibrillary acidic protein (GFAP)-immunopositive cells were present in 1 case. Ultrastructurally, the rhabdoid cells in case 1 contained prominent cytoplasmic whorls of intermediate filaments, recapitulating the ultrastructural features of rhabdoid meningioma in humans. In cases 2 and 3, the meningioma cells contained interdigitating cell processes folded in a maze-like fashion resembling rhabdoid-like meningioma in humans. In case 4, the voluminous cytoplasm contained many round-to-flattened mitochondria admixed with rough endoplasmic reticulum, indicating a predominant oncocytic differentiation and not the rhabdoid differentiation suggested by light microscopy. Thus, rhabdoid morphology occurs in different types of meningiomas, and ultrastructural findings are essential for a correct diagnosis.

NSD1 mediates antagonism between SWI/SNF and polycomb complexes and is required for transcriptional activation upon EZH2 inhibition.

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.

The Impact of Surgical Resection and Adjuvant Therapy on Survival in Pediatric Patients with Atypical Teratoid/Rhabdoid Tumor: Systematic Review and Pooled Survival Analysis.

BACKGROUND: Atypical teratoid/rhabdoid tumor (ATRT) is a rare malignant neoplasm in the pediatric population. ATRT is characterized by rhabdoid cells combined with the loss of either the INI1 (integrase interactor 1) or BRG1 (Brahma-related gene-1) protein. OBJECTIVE: To systematically review and analyze patient and tumor characteristics, prognosis, and impact of treatment on survival in pediatric patients with ATRT confirmed by alterations in INI1 or BRG1. This systematic review is the first to include only pediatric cases of ATRT confirmed with either INI1 or BRG1 alterations. METHODS: MEDLINE was searched using the terms "atypical teratoid/rhabdoid tumor" AND "paediatric/pediatric." Cases were included if confirmed by loss of INI1 or BRG1. The extracted dataset was analyzed using descriptive statistics, log-rank test, and Kaplan-Meier survival analysis via SPSS. RESULTS: A total of 38 articles were included in this study. The average age at diagnosis was 3 years. The most common locations reported are the supratentorial region and cerebral hemispheres. Ninety-three patients were reported to show evidence of dissemination. The average overall survival was 29 months. A significant difference in survival was noted between the tumor location groups, particularly worse outcomes for patients with spinal ATRT (P < 0.001). Extent of resection and adjuvant therapy were significant for survival (χ2 = 10.107, P = 0.018 and χ2 = 20.38, P < 0.0001, respectively). CONCLUSIONS: ATRT of the central nervous system in pediatric populations is a rare neoplasm associated with a poor prognosis in most patients. Future studies should be directed to find a standardized treatment protocol.

Smarcb1 Loss Results in a Deregulation of esBAF Binding and Impacts the Expression of Neurodevelopmental Genes.

The murine esBAF complex plays a major role in the regulation of gene expression during stem cell development and differentiation. As one of its core subunits, Smarcb1 is indispensable for its function and its loss is connected to neurodevelopmental disorders and participates in the carcinogenesis of entities such as rhabdoid tumours. We explored how Smarcb1 regulates gene programs in murine embryonic stem cells (mESC) and in this way orchestrates differentiation. Our data underline the importance of Smarcb1 expression and function for the development of the nervous system along with basic cellular functions, such as cell adhesion and cell organisation. Using ChIP-seq, we were able to portray the consequences of Smarcb1 knockdown (kd) for the binding of esBAF and PRC2 as well as its influence on histone marks H3K27me3, H3K4me3 and H3K27ac. Their signals are changed in gene and enhancer regions of genes connected to nervous system development and offers a plausible explanation for changes in gene expression. Further, we describe a group of genes that are, despite increased BAF binding, suppressed after Smarcb1 kd by mechanisms independent of PRC2 function.

Atypical teratoid/rhabdoid tumor in adults: a systematic review of the literature with meta-analysis and additional reports of 4 cases.

INTRODUCTION: Atypical teratoid/rhabdoid tumor (AT/RT) is a highly aggressive embryonal CNS neoplasm, characterized by inactivation of SMARCB1 (INI1) or rarely of SMARCA4 (BRG1). While it is predominantly a childhood tumor, AT/RT is rare in adults. METHODS: We provide a comprehensive systematic review of literature with meta-analysis; 92 adult cases were found from 74 articles. We additionally present 4 cases of adult AT/RTs (age ranging from 19 to 29 years), located to cerebellum in 2 cases, to ponto-cerebellar angle in 1 case and to spinal cord in the remaining case. RESULTS: Microscopic features of our 4 cases showed a highly cellular tumor with rhabdoid morphology and high mitotic activity. All tumor cells lacked nuclear SMARCB1/INI1 protein expression. In case no. 3 we also performed methylation profiling which clustered the tumor with pediatric AT/RT-MYC subgroup. Prognosis remains poor in both pediatric and adult population with a median overall survival of 11 months. Our review demonstrated median overall survival of 15 months among the adult populations. However, consistent with a recent review, adult AT/RT seems to have highly variable prognosis and some patients reach long term survival with 22.9% of 5-year survival without evidence of disease and mean follow up time of 35.9 months (SD = 36.5). 27.1% of dissemination was also reported among the adult population. CONCLUSIONS: Adult AT/RTs predominantly arise in female patients and in supratentorial location. Midline structures, including the sellar region, are the most affected sites, especially among females aged > 40 years. Male gender is more prevalent between the age of 18 and 40 years and more frequently associated with non-midline tumors. Factors significantly associated with better prognosis are patient's age (< 40 years), combined radio-chemotherapy adjuvant approach and Ki-67 score < 40%.

Diverse outcomes in extra-cranial rhabdoid tumors: A single institute experience.

Rhabdoid tumors (RTs) are a rare and aggressive pediatric cancer that commonly presents with alterations in the tumor suppressor gene SMARCB1. However, RT prognosis is still poor, with no standard treatment available. Moreover, no predictive biomarkers have been identified for determining its aggressiveness or chemo- and radio-sensitivities. Herein, four cases of extra-cranial RTs (ERTs) are described, two of whom are long-term survivors. These two surviving patients were positive for p16, whereas the other two were p16-negative. Our findings suggest that biologically distinct types of ERTs exist and that p16 expression may be a potential positive prognostic biomarker of ERTs. Nevertheless, further studies are required to confirm our findings.

Spatial molecular profiling of a central nervous system low-grade diffusely infiltrative tumour with INI1 deficiency featuring a high-grade atypical teratoid/rhabdoid tumour component.

We performed spatial epigenetic and transcriptomic analyses of a highly unusual low-grade diffusely infiltrative tumour with INI1 deficiency (CNS LGDIT-INI1), which harboured a high-grade component corresponding to an atypical teratoid/rhabdoid tumour (AT/RT). Methylation profiles of both low-grade and high-grade components yielded high similarity with AT/RTs of the MYC subgroup, whereas RNA expression analyses revealed increased translational activity and MYC pathway activation in the high-grade component. Close follow-up of patients harbouring CNS LGDIT-INI1 is warranted.

Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors.

BACKGROUND: Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear which epitopes T cells might recognize on AT/RT cells. METHODS: Here, we report a comprehensive mass spectrometry (MS)-based analysis of naturally presented human leukocyte antigen (HLA) class I and class II ligands on 23 AT/RTs. MS data were validated by matching with a human proteome dataset and exclusion of peptides that are part of the human benignome. Cryptic peptide ligands were identified using Peptide-PRISM. RESULTS: Comparative HLA ligandome analysis of the HLA ligandome revealed 55 class I and 139 class II tumor-exclusive peptides. No peptide originated from the SMARCB1 region. In addition, 61 HLA class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas, but no concordance was found with extracranial tumors. More than 80% of AT/RT exclusive peptides were able to successfully prime CD8+ T cells, whereas naturally occurring memory responses in AT/RT patients could only be detected for class II epitopes. Interestingly, >50% of AT/RT exclusive class II ligands were also recognized by T cells from glioblastoma patients but not from healthy donors. CONCLUSIONS: These findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA class I and class II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion.

Role of Cyclin D1 and BCOR Immunohistochemistry in Differentiating Clear Cell Sarcoma of Kidney From its Mimics.

BACKGROUND AND AIM: Clear cell sarcoma of kidney (CCSK) is the second most common pediatric renal malignancy, constituting ∼3% of renal tumors. Due to its morphologic diversity, the diagnosis of CCSK is often challenging. Recent studies have identified internal tandem duplication of BCL6 corepressor (BCOR) gene in CCSKs which coupled with cyclin D1 immunoreactivity, is helpful in differentiating it from its mimics, particularly blastema-rich Wilms tumor (WT), malignant rhabdoid tumor (MRT), and congenital mesoblastic nephroma (CMN). We aimed to evaluate the utility of cyclin D1 and BCOR immunohistochemistry in differentiating CCSK from its morphologic mimics. MATERIALS AND METHODS: Our cohort comprised of 38 pediatric renal tumors which included CCSK (n=18), WT (n=10), MRT (n=5), and CMN (n=5) cases. A detailed clinicopathologic analysis was performed, and tissue microarray were constructed for CCSK and WT, while MRT and CMN tumors were individually stained. RESULTS: The age ranged from 2 months to 16 years with male:female ratio of 3:1. Strong, diffuse nuclear immunoreactivity for cyclin D1 and BCOR was noted in 61% (n=11/18) and 83% (n=15/18) of CCSK, respectively, while it was significantly less in WT (n=3/10 for cyclin D1) (n=2/10 for BCOR). None of the MRT and CMN examples demonstrated any immunoreactivity. Interestingly, only the blastemal component of WTs showed distinct, rare nuclear immunoreactivity for cyclin D1 or BCOR and the combination of these was never positive in a given case. CONCLUSION: Our results provide evidence that concurrent immunopositivity with cyclin D1 and BCOR is helpful in distinguishing CCSK from its morphologic mimics.

Organoid-based drug screening reveals neddylation as therapeutic target for malignant rhabdoid tumors.

Malignant rhabdoid tumors (MRTs) represent one of the most aggressive childhood malignancies. No effective treatment options are available, and prognosis is, therefore, dismal. Previous studies have demonstrated that tumor organoids capture the heterogeneity of patient tumors and can be used to predict patient response to therapy. Here, we perform drug screening on patient-derived normal and tumor organoids to identify MRT-specific therapeutic vulnerabilities. We identify neddylation inhibitor MLN4924 as a potential therapeutic agent. Mechanistically, we find increased neddylation in MRT organoids and tissues and show that MLN4924 induces a cytotoxic response via upregulation of the unfolded protein response. Lastly, we demonstrate in vivo efficacy in an MRT PDX mouse model, in which single-agent MLN4924 treatment significantly extends survival. Our study demonstrates that organoids can be used to find drugs selectively targeting tumor cells while leaving healthy cells unharmed and proposes neddylation inhibition as a therapeutic strategy in MRT.