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Undifferentiated pleomorphic sarcoma (UPS) and malignant peripheral nerve sheath tumor (MPNST) are aggressive soft tissue sarcomas that do not respond well to current treatment modalities. The limited availability of UPS and MPNST cell lines makes it challenging to identify potential therapeutic targets in a laboratory setting. Understanding the urgent need for improved treatments for these tumors and the limited cellular models available, we generated additional cell lines to study these rare cancers. Patient-derived tumors were used to establish 4 new UPS models, including one radiation-associated UPS-UPS271.1, UPS511, UPS0103, and RIS620, one unclassified spindle cell sarcoma-USC060.1, and 3 new models of MPNST-MPNST007, MPNST3813E, and MPNST4970. This study examined the utility of the new cell lines as sarcoma models by assessing their tumorigenic potential and mutation status for known sarcoma-related genes. All the cell lines formed colonies and migrated in vitro. The in vivo tumorigenic potential of the cell lines and corresponding xenografts was determined by subcutaneous injection or xenograft re-passaging into immunocompromised mice. USC060.1 and UPS511 cells formed tumors in mice upon subcutaneous injection. UPS0103 and RIS620 tumor implants formed tumors in vivo, as did MPNST007 and MPNST3813E tumor implants. Targeted sequencing analysis of a panel of genes frequently mutated in sarcomas identified TP53, RB1, and ATRX mutations in a subset of the cell lines. These new cellular models provide the scientific community with powerful tools for detailed studies of tumorigenesis and for investigating novel therapies for UPS and MPNST., (© 2022. The Author(s), under exclusive licence to United States and Canadian Academy of Pathology.)

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that frequently harbor genetic alterations in polycomb repressor complex 2 (PRC2) components-SUZ12 and EED. Here, we show that PRC2 loss confers a dedifferentiated early neural-crest phenotype which is exclusive to PRC2-mutant MPNSTs and not a feature of neurofibromas. Neural crest phenotype in PRC2 mutant MPNSTs was validated via cross-species comparative analysis using spontaneous and transgenic MPNST models. Systematic chromatin state profiling of the MPNST cells showed extensive epigenomic reprogramming or chromatin states associated with PRC2 loss and identified gains of active enhancer states/super-enhancers on early neural crest regulators in PRC2-mutant conditions around genomic loci that harbored repressed/poised states in PRC2-WT MPNST cells. Consistently, inverse correlation between H3K27me3 loss and H3K27Ac gain was noted in MPNSTs. Epigenetic editing experiments established functional roles for enhancer gains on DLX5-a key regulator of neural crest phenotype. Consistently, blockade of enhancer activity by bromodomain inhibitors specifically suppressed this neural crest phenotype and tumor burden in PRC2-mutant PDXs. Together, these findings reveal accumulation of dedifferentiated neural crest like state in PRC2-mutant MPNSTs that can be targeted by enhancer blockade., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Importance: Neurofibromatosis type 1 (NF1) is a complex genetic disorder that is associated with not only neurofibromas, but also an increased susceptibility to other neoplasms., Objective: To evaluate the prevalence of neoplasia and outcomes among patients with NF1., Design, Setting, and Participants: This cohort study was conducted among patients with NF1 at a single academic cancer center from 1985 to 2020 with median (range) follow-up of 2.9 years (36 days to 30.5 years). Of 2427 patients evaluated for NF1, 1607 patients who met the National Institutes of Health consensus criteria for NF1 were included. This group was compared with estimates from Surveillance, Epidemiology, and End Results (SEER) Cancer Statistics Review 1975 to 2015 and SEER participants database unless otherwise specified. Data were analyzed from August 2018 to March 2020., Main Outcomes and Measures: Disease-specific survival (DSS) was measured from diagnosis date to date of neoplasm-specific death or censorship and calculated using the Kaplan-Meier method. Survival curves were compared using the log-rank test. Deaths from disease were considered a DSS end point; other deaths were considered censored observations. Secondary outcome measures were comparisons of (1) overall survival of patients with NF1 with neurofibroma neoplasms vs those without nonneurofibroma neoplasms, (2) neoplasm prevalence in the NF1 group vs general population estimates, and (3) age at diagnosis in the NF1 group vs general population estimates for the most common neoplasms in the NF1 group., Results: Among 1607 patients with NF1, the median (range) age at initial visit was 19 years (1 month to 83 years) and 840 (52.3%) were female patients. Among 666 patients who developed other neoplasms in addition to neurofibromas (41.4%), 295 patients (18.4%) developed glioma and 243 patients (15.1%) developed malignant peripheral nerve sheath tumor (MPNST), the most common neoplasms. Patients with NF1, compared with the general population, developed several neoplasms at a younger mean (SD) age (low-grade glioma: 12.98 [11.09] years vs 37.76 [24.53] years; P < .0001; high-grade glioma [HGG]: 27.31 [15.59] years vs 58.42 [19.09] years; P < .0001; MPNST: 33.88 [14.80] years vs 47.06 [20.76] years; P < .0001; breast cancer: 46.61 [9.94] years vs 61.71 [13.85] years; P < .0001). Patients with NF1 developed neoplasms more frequently compared with the general population (odds ratio, 9.5; 95% CI, 8.5-10.5; P < .0001). Among patients with NF1, significantly lower 5-year DSS rates were found among those with undifferentiated pleomorphic sarcoma (1 of 5 patients [20.0%]), HGG (8 of 34 patients [23.1%]), MPNST (72 of 228 patients [31.6%]), ovarian carcinoma (4 of 7 patients [57.1%]), and melanoma (8 of 12 patients [66.7%]) compared with those who had neoplasms classified as other (110 of 119 patients [92.4%]) (all P < .001) ., Conclusions and Relevance: This cohort study found that among patients with NF1, those who developed undifferentiated pleomorphic sarcoma, HGG, MPNST, ovarian carcinoma, or melanoma had significantly lower DSS rates compared with those who developed other neoplasms. This study also found that patients with NF1 developed some neoplasms more frequently and at a younger age compared with individuals without NF1. HGGs and MPNST were noteworthy causes of death among patients NF1. This information may be useful for NF1 patient counseling and follow-up.

Dysregulation of the receptor tyrosine kinase AXL is known to promote cancer cell growth and survival in many sarcomas, including the rare subtype, malignant peripheral nerve sheath tumors (MPNST). MPNSTs are largely chemoresistant and carry a poor prognosis. AXL is an attractive potential therapeutic target, as it is aberrantly expressed, and its activation may be an early event in MPNST. However, the effect of AXL inhibition on MPNST development and progression is not known. Here, we investigated the role of AXL in MPNST development and the effects of AXL and MEK1/2 co-inhibition on MPNSTs. We used western blotting to examine AXL expression and activation in MPNST cell lines. We analyzed the effects of exogenous growth arrest-specific 6 (GAS6) expression on downstream signaling and the proliferation, migration, and invasion of MPNST cells. The effect of AXL knockdown with or without mitogen-activated protein kinase (MAPK) inhibition on downstream signal transduction and tumorigenesis was also examined in vivo and in vitro . We found that AXL knockdown increased MAPK pathway signaling. This compensation, in turn, abrogated the antitumorigenic effects linked to AXL knockdown in vivo . AXL knockdown, combined with pharmacological MEK inhibition, reduced the proliferation and increased the apoptosis of MPNST cells both in vitro and in vivo . The pharmacological co-inhibition of AXL and MEK1/2 reduced MPNST volumes. Together these findings suggest that AXL inhibition enhances the sensitivity of MPNST to other small molecule inhibitors. We conclude that combination therapy with AXL inhibitor may be a therapeutic option for MPNST.

Undifferentiated pleomorphic sarcomas (UPSs) are aggressive mesenchymal malignancies with no definitive cell of origin or specific recurrent genetic hallmarks. These tumors are largely chemoresistant; thus, identification of potential therapeutic targets is necessary to improve patient outcome. Previous studies demonstrated that high expression of activated protein kinase B (AKT) in patients with UPS corresponds to poor disease-specific survival. Here, we demonstrate that inhibiting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling using a small molecule inhibitor reduced UPS cell proliferation and motility and xenograft growth; however, increased phosphorylation of insulin-like growth factor 1 receptor (IGF1R) indicated the potential for adaptive resistance following treatment through compensatory receptor activation. Co-treatment with a dual PI3K/mTOR inhibitor and an anti-IGF1R kinase inhibitor reduced in vivo tumor growth rates despite a lack of antiproliferative effects in vitro. Moreover, this combination treatment significantly decreased UPS cell migration and invasion, which is linked to changes in p27 subcellular localization. Our results demonstrate that targeted inhibition of multiple components of the IGF1R/PI3K/mTOR pathway was more efficacious than single-agent therapy and suggest that co-targeting this pathway could be a beneficial therapeutic strategy for patients with UPS.

Friedreich's ataxia (FRDA) is caused by the expansion of GAA repeats located in the Frataxin (FXN) gene. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing to disease progression. The mechanism leading to repeat expansion and decreased FXN transcription remains unclear. Using single-molecule analysis of replicated DNA, we detected that expanded GAA repeats present a substantial obstacle for the replication machinery at the FXN locus in FRDA cells. Furthermore, aberrant origin activation and lack of a proper stress response to rescue the stalled forks in FRDA cells cause an increase in 3'-5' progressing forks, which could enhance repeat expansion and hinder FXN transcription by head-on collision with RNA polymerases. Treatment of FRDA cells with GAA-specific polyamides rescues DNA replication fork stalling and alleviates expansion of the GAA repeats, implicating DNA triplexes as a replication impediment and suggesting that fork stalling might be a therapeutic target for FRDA., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Friedreich's ataxia (FRDA) represents a rare neurodegenerative disease caused by expansion of GAA trinucleotide repeats in the first intron of the FXN gene. The number of GAA repeats in FRDA patients varies from approximately 60 to <1000 and is tightly correlated with age of onset and severity of the disease symptoms. The heterogeneity of Friedreich's ataxia stresses the need for a large cohort of patient samples to conduct studies addressing the mechanism of disease pathogenesis or evaluate novel therapeutic candidates. Herein, we report the establishment and characterization of an FRDA fibroblast repository, which currently includes 50 primary cell lines derived from FRDA patients and seven lines from mutation carriers. These cells are also a source for generating induced pluripotent stem cell (iPSC) lines by reprogramming, as well as disease-relevant neuronal, cardiac, and pancreatic cells that can then be differentiated from the iPSCs. All FRDA and carrier lines are derived using a standard operating procedure and characterized to confirm mutation status, as well as expression of FXN mRNA and protein. Consideration and significance of creating disease-focused cell line and tissue repositories, especially in the context of rare and heterogeneous disorders, are presented. Although the economic aspect of creating and maintaining such repositories is important, the benefits of easy access to a collection of well-characterized cell lines for the purpose of drug discovery or disease mechanism studies overshadow the associated costs. Importantly, all FRDA fibroblast cell lines collected in our repository are available to the scientific community.

Objective: Friedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat expansion disorder caused by epigenetic silencing of the frataxin gene (FXN). Current research suggests that damage and variation of mitochondrial DNA (mtDNA) contribute to the molecular pathogenesis of FRDA. We sought to establish the extent of the mutation burden across the mitochondrial genome in FRDA cells and investigate the molecular mechanisms connecting FXN downregulation and the acquisition of mtDNA damage., Methods: Damage and mutation load in mtDNA of a panel of FRDA and control fibroblasts were determined using qPCR and next-generation MiSeq sequencing, respectively. The capacity of FRDA and control cells to repair oxidative lesions in their mtDNA was measured using a quantitative DNA damage assay. Comprehensive RNA sequencing gene expression analyses were conducted to assess the status of DNA repair and metabolism genes in FRDA cells., Results: Acute or prolonged downregulation of FXN expression resulted in a significant increase in mtDNA damage that translated to a significant elevation of mutation load in mtDNA. The predominant mutations identified throughout the mtDNA were C>T, G>A transitions (P = 0.007). Low FXN expression reduced capacity to repair oxidative damage in mtDNA. Downregulation of FXN expression strongly correlated (r = 0.73) with decreased levels of base excision repair (BER) DNA glycosylase NTHL1., Interpretation: Downregulation of FXN expression in FRDA cells elevates mtDNA damage, increases mutation load of the mitochondrial genome, and diminishes DNA repair capacity. Progressive accumulation of mtDNA mutations in vulnerable FRDA patient cells reduces mitochondrial fitness ultimately leading to cell death.

Friedreich's ataxia (FRDA) is a severe neurodegenerative disease caused by homozygous expansion of the guanine-adenine-adenine (GAA) repeats in intron 1 of the FXN gene leading to transcriptional repression of frataxin expression. Post-translational histone modifications that typify heterochromatin are enriched in the vicinity of the repeats, whereas active chromatin marks in this region are underrepresented in FRDA samples. Yet, the immediate effect of the expanded repeats on transcription progression through FXN and their long-range effect on the surrounding genomic context are two critical questions that remain unanswered in the molecular pathogenesis of FRDA. To address these questions, we conducted next-generation RNA sequencing of a large cohort of FRDA and control primary fibroblasts. This comprehensive analysis revealed that the GAA-induced silencing effect does not influence expression of neighboring genes upstream or downstream of FXN. Furthermore, no long-range silencing effects were detected across a large portion of chromosome 9. Additionally, results of chromatin immunoprecipitation studies confirmed that histone modifications associated with repressed transcription are confined to the FXN locus. Finally, deep sequencing of FXN pre-mRNA molecules revealed a pronounced defect in the transcription elongation rate in FRDA cells when compared with controls. These results indicate that approaches aimed to reactivate frataxin expression should simultaneously address deficits in transcription initiation and elongation at the FXN locus., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Friedreich's ataxia (FRDA) is an autosomal recessive neurological disease caused by expansions of guanine-adenine-adenine (GAA) repeats in intron 1 of the frataxin (FXN) gene. The expansion results in significantly decreased frataxin expression. We report that human FRDA cells can be corrected by zinc finger nuclease-mediated excision of the expanded GAA repeats. Editing of a single expanded GAA allele created heterozygous, FRDA carrier-like cells and significantly increased frataxin expression. This correction persisted during reprogramming of zinc finger nuclease-edited fibroblasts to induced pluripotent stem cells and subsequent differentiation into neurons. The expression of FRDA biomarkers was normalized in corrected patient cells and disease-associated phenotypes, such as decreases in aconitase activity and intracellular ATP levels, were reversed in zinc finger nuclease corrected neuronal cells. Genetically and phenotypically corrected patient cells represent not only a preferred disease-relevant model system to study pathogenic mechanisms, but also a critical step towards development of cell replacement therapy.

Conotruncal and related heart defects (CTDs) are a group of serious and relatively common birth defects. Although both maternal and inherited genotypes are thought to play a role in the etiology of CTDs, few specific genetic risk factors have been identified. To determine whether common variants acting through the genotype of the mother (e.g. via an in utero effect) or the case are associated with CTDs, we conducted a genome-wide association study of 750 CTD case-parent triads, with follow-up analyses in 358 independent triads. Log-linear analyses were used to assess the association of CTDs with the genotypes of both the mother and case. No association achieved genomewide significance in either the discovery or combined (discovery+follow-up) samples. However, three loci with p-values suggestive of association (p<10-5) in the discovery sample had p-values <0.05 in the follow-up sample and p-values in the combined data that were lower than in the discovery sample. These included suggestive association with an inherited intergenic variant at 20p12.3 (rs6140038, combined p = 1.0 × 10(-5)) and an inherited intronic variant in KCNJ4 at 22q13.1 (rs2267386, combined p = 9.8 × 10(-6)), as well as with a maternal variant in SLC22A24 at 11q12.3 (rs11231379, combined p = 4.2 × 10(-6)). These observations suggest novel candidate loci for CTDs, including loci that appear to be associated with the risk of CTDs via the maternal genotype, but further studies are needed to confirm these associations.

Background: Based on studies in animals and humans, PAX3 and T (brachyury) are candidate genes for spina bifida. However, neither gene has been definitively identified as a risk factor for this condition., Methods: Sanger sequencing was used to identify variants in all PAX3 and T exons and promoter regions in 114 spina bifida cases. For known variants, allele frequencies in cases were compared with those from public databases using unadjusted odds ratios. Novel variants were genotyped in parents and assessed for predicted functional impact., Results: We identified common variants in PAX3 (n = 2) and T (n = 3) for which the allele frequencies in cases were significantly different from those reported in at least one public database. We also identified novel variants in both PAX3 (n = 11) and T (n = 1) in spina bifida cases. Several of the novel PAX3 variants are predicted to be highly conserved and/or impact gene function or expression., Conclusion: These studies provide some evidence that common variants of PAX3 and T are associated with spina bifida. Rare and novel variants in these genes were also identified in affected individuals. However, additional studies will be required to determine whether these variants influence the risk of spina bifida., (Copyright © 2013 Wiley Periodicals, Inc.)

Nonsense-mediated decay (NMD) is an RNA decay pathway that downregulates aberrant mRNAs and a subset of normal mRNAs. The regulation of NMD is poorly understood. Here we identify a regulatory mechanism acting on two related UPF (up-frameshift) factors crucial for NMD: UPF3A and UPF3B. This regulatory mechanism, which reduces the level of UPF3A in response to the presence of UPF3B, is relieved in individuals harboring UPF3B mutations, leading to strongly increased steady-state levels of UPF3A. UPF3A compensates for the loss of UPF3B by regulating several NMD target transcripts, but it can also impair NMD, as it competes with the stronger NMD activator UPF3B for binding to the essential NMD factor UPF2. This deleterious effect of UPF3A protein is prevented by its destabilization using a conserved UPF3B-dependent mechanism. Together, our results suggest that UPF3A levels are tightly regulated by a post-transcriptional switch to maintain appropriate levels of NMD substrates in cells containing different levels of UPF3B.

T-cell receptor-beta (TCRbeta) genes naturally acquire premature termination codons (PTCs) as a result of programmed gene rearrangements. PTC-bearing TCRbeta transcripts are dramatically down-regulated to protect T-cells from the deleterious effects of the truncated proteins that would otherwise be produced. Here we provide evidence that two responses collaborate to elicit this dramatic down-regulation. One is rapid mRNA decay triggered by the nonsense-mediated decay (NMD) RNA surveillance pathway. We demonstrate that this occurs in highly purified nuclei lacking detectable levels of three different cytoplasmic markers, but containing an outer nuclear membrane marker, suggesting that decay occurs either in the nucleoplasm or at the outer nuclear membrane. The second response is a dramatic partitioning shift in the nuclear fraction-to-cytoplasmic fraction mRNA ratio that results in few TCRbeta transcripts escaping to the cytoplasmic fraction of cells. Analysis of TCRbeta mRNA kinetics after either transcriptional repression or induction suggested that this nonsense codon-induced partitioning shift (NIPS) response is not the result of cytoplasmic NMD but instead reflects retention of PTC(+) TCRbeta mRNA in the nuclear fraction of cells. We identified TCRbeta sequences crucial for NIPS but found that NIPS is not exclusively a property of TCRbeta transcripts, and we identified non-TCRbeta sequences that elicit NIPS. RNA interference experiments indicated that NIPS depends on the NMD factors UPF1 and eIF4AIII but not the NMD factor UPF3B. We propose that NIPS collaborates with NMD to retain and degrade a subset of PTC(+) transcripts at the outer nuclear membrane and/or within the nucleoplasm.