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Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, occurs in people with neurofibromatosis type 1 (NF1) and sporadically. Whole-genome and multiregional exome sequencing, transcriptomic, and methylation profiling of 95 tumor samples revealed the order of genomic events in tumor evolution. Following biallelic inactivation of NF1, loss of CDKN2A or TP53 with or without inactivation of polycomb repressive complex 2 (PRC2) leads to extensive somatic copy-number aberrations (SCNA). Distinct pathways of tumor evolution are associated with inactivation of PRC2 genes and H3K27 trimethylation (H3K27me3) status. Tumors with H3K27me3 loss evolve through extensive chromosomal losses followed by whole-genome doubling and chromosome 8 amplification, and show lower levels of immune cell infiltration. Retention of H3K27me3 leads to extensive genomic instability, but an immune cell-rich phenotype. Specific SCNAs detected in both tumor samples and cell-free DNA (cfDNA) act as a surrogate for H3K27me3 loss and immune infiltration, and predict prognosis.Significance:MPNST is the most common cause of death and morbidity for individuals with NF1, a relatively common tumor predisposition syndrome. Our results suggest that somatic copy-number and methylation profiling of tumor or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups.This article is highlighted in the In This Issue feature, p. 517

Brown tumors are rare and generally self-limiting mass lesions of bone occurring in the context of hyperparathyroidism. Although commonly regarded as endocrine-driven tumor-like lesions, we detected pathogenic hotspot KRAS mutations in 10/16 brown tumors (62%) with similar frequencies found in cases affecting the peripheral and axial skeleton. Pathogenic mutations in other driver genes of the RAS-MAPK pathway were not identified. Our findings suggest brown tumors to represent true neoplasms driven by the activation of the RAS-MAPK signaling pathway. The frequent regression of brown tumors after normalization of hyperparathyroidism points to a second hit mediated by endocrine stimulation to be required for tumor development. Our findings underline the pathogenic relation of brown tumors to nonossifying fibroma and giant cell granuloma of the jaws which both appear histologically similar to brown tumors and are also driven by RAS-MAPK signaling pathway activation.

Oncohistones represent compelling evidence for a causative role of epigenetic perturbations in cancer. Giant cell tumours of bone (GCTs) are characterised by a mutated histone H3.3 as the sole genetic driver present in bone-forming osteoprogenitor cells but absent from abnormally large bone-resorbing osteoclasts which represent the hallmark of these neoplasms. While these striking features imply a pathogenic interaction between mesenchymal and myelomonocytic lineages during GCT development, the underlying mechanisms remain unknown. We show that the changes in the transcriptome and epigenome in the mesenchymal cells caused by the H3.3-G34W mutation contribute to increase osteoclast recruitment in part via reduced expression of the TGFβ-like soluble factor, SCUBE3. Transcriptional changes in SCUBE3 are associated with altered histone marks and H3.3G34W enrichment at its enhancer regions. In turn, osteoclasts secrete unregulated amounts of SEMA4D which enhances proliferation of mutated osteoprogenitors arresting their maturation. These findings provide a mechanism by which GCTs undergo differentiation in response to denosumab, a drug that depletes the tumour of osteoclasts. In contrast, hTERT alterations, commonly found in malignant GCT, result in the histone-mutated neoplastic cells being independent of osteoclasts for their proliferation, predicting unresponsiveness to denosumab. We provide a mechanism for the initiation of GCT, the basis of which is dysfunctional cross-talk between bone-forming and bone-resorbing cells. The findings highlight the role of tumour/microenvironment bidirectional interactions in tumorigenesis and how this is exploited in the treatment of GCT.

Supplementary Table 1 includes the metadata on all subjects, including basic demographics and medical history. Supplementary Table 2 provides statistics on chrom 8 gene expression among tumors with H3K27me3 loss. Note: no changes have been made to this file since our last resubmission.

Supplementary figures provide additional details on the genomic landscape of MPNST and interesting cases within the cohort.

Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, occurs in people with neurofibromatosis type 1 (NF1) and sporadically. Whole-genome and multiregional exome sequencing, transcriptomic, and methylation profiling of 95 tumor samples revealed the order of genomic events in tumor evolution. Following biallelic inactivation of NF1, loss of CDKN2A or TP53 with or without inactivation of polycomb repressive complex 2 (PRC2) leads to extensive somatic copy-number aberrations (SCNA). Distinct pathways of tumor evolution are associated with inactivation of PRC2 genes and H3K27 trimethylation (H3K27me3) status. Tumors with H3K27me3 loss evolve through extensive chromosomal losses followed by whole-genome doubling and chromosome 8 amplification, and show lower levels of immune cell infiltration. Retention of H3K27me3 leads to extensive genomic instability, but an immune cell-rich phenotype. Specific SCNAs detected in both tumor samples and cell-free DNA (cfDNA) act as a surrogate for H3K27me3 loss and immune infiltration, and predict prognosis.Significance:MPNST is the most common cause of death and morbidity for individuals with NF1, a relatively common tumor predisposition syndrome. Our results suggest that somatic copy-number and methylation profiling of tumor or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups.This article is highlighted in the In This Issue feature, p. 517

Top 30 loci associated with metastasis at diagnosis in the discovery set of 541 European osteosarcoma cases.

Relationship of variables to metastasis at diagnosis in the discovery set.

SNPs in the NFIB locus associated with metastasis at diagnosis with P{less than or equal to}1x10-5 in the discovery stage, and functionally annotated with information from the ENCODE and 1000 Genomes Project data.

Associations of the top GWAS and imputed SNP with metastasis at diagnosis in the discovery stage (European ancestry) by inheritance model.

Top genotyped SNP associations with metastasis at diagnosis in the discovery and all replication studies.

Supplementary Figure 1. Manhattan plot of the Discovery stage. SNPs in the NFIB risk locus are highlighted in green. Supplementary Figure 2. LD structure of the NFIB risk locus. Plot was made using the CEU population of the 1000 Genomes Project data with LDlink (ref 54). Supplementary Figure 3. Significant relationship between the genotype of the metastasis associated SNP rs7034162, and expression of NFIB and IGFBP5 in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the distribution of expression of NFIBbetween the TT homozygous non-risk genotype (combined N=34) of rs7034162 genotypes to the heterozygous risk AT (combined N=10) and the homozygous risk AA genotypes (combined N=2). The green triangle represents the expression levels in the OSA cell line, the blue circle is expression levels in the HOS cell line, and the red square the U2OS cell line expression. These analyses were based on the publically available genotype and expression data from 17 osteosarcoma cell lines and 29 osteosarcoma tumors (ref 20). Supplementary Figure 4. No significant relationship between the genotype of the metastasis associated SNP, rs7034162, and expression of protein-encoding genes FREM1, ZDHHC1, and MPDZ in the neighborhood of NFIB in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the distribution of expression of FREM1, ZDHHC1, and MPDZ between the TT homozygous non-risk genotype (combined N=34) of rs7034162 genotypes to the heterozygous risk AT (combined N=10) and the homozygous risk AA genotypes (combined N=2). The green triangle represents the expression levels in the OSA cell line, the blue circle is expression levels in the HOS cell line, and the red square the U2OS cell line expression. These analyses were based on the publically available genotype and expression data from 17 osteosarcoma cell lines and 29 osteosarcoma tumors (ref 20). Supplementary Figure 5. The human osteosarcoma cell lines, U2OS and HOS, had higher NFIB protein levels then OSA cells. NFIB levels were determined using immunoblot analysis in the OSA, HOS and U2OS cells both basal (A) and after treatment with siRNA against NFIB (B). Supplementary Figure 6. NFIB expression significantly correlates with IGFBP5 expression in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the expression levels of NFIB with the corresponding IGFBP5 expression levels. Supplementary Figure 7. Expression levels of IGFBP5 decreases after NFIB suppression of human osteosarcoma cell lines. IGFBP5 expression of OSA, HOS and U2OS cells was determined 48 hours after transfection with control siRNA (si-NEG) or siRNA targeting NFIB (si-NFIB). Graph showing relative expression compared to control treated U2OS cells. Supplementary Figure 8. A model of how the NFIB risk locus leads to changes in NFIB expression and potentially may affect osteosarcoma metastatic potential. The risk allele (right panel) leads to lowered expression of NFIB and NFIB-mediated lower expression of IGFPB5, which then leads to less IGFBP5-mediated inhibition of IGF-1 (right panel, grey arrows), leading to an increase in proliferation, survival and metastasis of osteosarcoma cells (right panel, black arrows). The reference allele (left panel) leads to both higher expression of NFIB and higher expression of NFIB-mediated IGFPB5. In turn, higher IGFBP5 levels leads to increased inhibition of IGF-1 (left panel, black arrows), leading to a decrease in proliferation, survival and metastasis of osteosarcoma cells (left panel, grey arrows). Supplementary Figure 9. Plot of the PCA Eigenvectors of the Discovery stage. Supplementary Figure 10. QQ-plot of the Discovery stage.

Description of participating case studies.

Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage–fusion–bridge cycles, among others, which may lead to chromosomal instability and aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3–5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas6 revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.

Chordoma RNAseq_all cell lines_gene lists

Supplementary Information

Expression of the transcription factor brachyury (TBXT) is normally restricted to the embryo, and its silencing is epigenetically regulated. TBXT promotes mesenchymal transition in a subset of common carcinomas, and in chordoma, a rare cancer showing notochordal differentiation, TBXT acts as a putative oncogene. We hypothesized that TBXT expression is controlled through epigenetic inhibition to promote chordoma cell death. Screening of five human chordoma cell lines revealed that pharmacologic inhibition of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (JMJD3) leads to cell death. This effect was phenocopied by dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Inhibition of KDM6A/B with a novel compound KDOBA67 led to a genome-wide increase in repressive H3K27me3 marks with concomitant reduction in active H3K27ac, H3K9ac, and H3K4me3 marks. TBXT was a KDM6A/B target gene, and chromatin changes at TBXT following KDOBA67 treatment were associated with a reduction in TBXT protein levels in all models tested, including primary patient-derived cultures. In all models tested, KDOBA67 treatment downregulated expression of a network of transcription factors critical for chordoma survival and upregulated pathways dominated by ATF4-driven stress and proapoptotic responses. Blocking the AFT4 stress response did not prevent suppression of TBXT and induction of cell death, but ectopic overexpression of TBXT increased viability, therefore implicating TBXT as a potential therapeutic target of H3K27 demethylase inhibitors in chordoma. Our work highlights how knowledge of normal processes in fetal development can provide insight into tumorigenesis and identify novel therapeutic approaches.Significance:Pharmacologic inhibition of H3K27-demethylases in human chordoma cells promotes epigenetic silencing of oncogenic TBXT, alters gene networks critical to survival, and represents a potential novel therapy.

Overview ideogram of CGH data of all chordoma cell lines. Every cell line corresponds to one color. Lines on the left side are losses, and on the right side are gains of chromosomal material. Arrows mark the CDK6 region on 7q and p16 region on 9p.

Detailed CGH karyograms of the chordoma cell lines as shown in the ideogram in Supplementary Figure 1 (according to ISCN: International System for Human Cytogentic Nomenclature).

Results of the single tandem repeats analysis of the cell lines U-CH1, U-CH2, UCH3, UCH6, UCH7, U-CH10, U-CH11 and UCH12. The results of the sex typing marker AMEL and the detected alleles for the markers D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045 are given.

Supplementary Tables S1-S4. Identification of HEY1 regulated genes downstream of EWS-FLI1 silencing in Ewing sarcoma (S1); Combined immunohistochemical staining results for samples from patients with metastases at diagnosis on TMA1 and 2 according to site of metastasis (S2); Clinical characteristics and SIRT1 staining results of primary tumors from 43 ES patients treated at St. Jude Children´s Hospital between 1979 and 1987 using vincristine, doxorubicin, and cyclophosphamide-based regimens, with subsequent addition of ifosfamide and etoposide (S3); Materials used in the study (S4).

Expression data of mRNA as shown by box plot graphs for brachyury (A), collagen 2A1 (B) and CD24 (C).

Unsupervised cluster analysis of mRNA expression data of all chordoma cell lines. The T-gene (i.e., brachyury) is overexpressed (red). Cluster Analysis of chordoma cell lines with chondrocyte, liposarcoma and sarcoma expression data from GEO and own data from a cell line with cartilaginous differentiation and from tissue of a vertebral disc.

Effect of Abemaciclib (LY2835219) on the growth of chordoma lines U-CH1, U-CH6, CAL51 (negative control), and MCF7 (positive control). Cells were grown in 96-well-plates were incubated for 3 days at 37{degree sign}C with increasing concentrations of Abemaciclib (3, 6, 10, 30, 60, 100, 300, 600, 1000 nM) in the medium or left untreated for control. The amount of viable cells was determined by using the MTS cell viability test (GraphPad Prism software, Version 6).

Western blot analyses of 8 chordoma cell lines showing expression of brachyury at 47 kDa.

Clinical data of chordoma patients. Every sample was positive for brachyury. Asterisks (*) mark the parental chordomas of the established chordoma cell lines.

Proliferation assay using Alamar Blue assay for U-CH2, 3, 6 and 10. Increasing concentrations of Palbociclib lead to an increase of growth inhibition of chordoma cells.

Inversion microscopy views of the published chordoma cell lines U-CH1, U-CH2, U-CH 3, 6, 7, 10, 11, and 12. The cells showing the typical physaliphorous cytology; bars=100µm.

Supplementary Figure Legends. Legend for Supplementary Figures S1-S5.

Supplementary Figure Legend

Immunocytology of U-CH1 control vs inhibited cells with 1000 nM Palbociclib for 3 days. Ki-67 decreases from 20% down to

Data Supplement from Preclinical Characterization of Novel Chordoma Cell Systems and Their Targeting by Pharmocological Inhibitors of the CDK4/6 Cell-Cycle Pathway

Supplementary Figures S1-S5. MDM4 modulation by HEY1 occurs downstream of TP53 (S1); Modulation of SIRT1 and TP53 induction upon ectopic expression of Flag-tagged HEY1 in the B-cell malignancy cell lines "697" and NALM6, and in primary keratinocytes (S2); The degree of SIRT1 modulation depends on HEY1 levels (S3); Flow-diagram of samples analysed on tissue micro arrays (TMA1, TMA2, TMA3), and FFPE sections used in this study (S4); Kaplan-Meier survival estimates for patients analysed for SIRT1 expression on TMA3 (S5).

Expression data of mRNA as shown by box plot graphs for brachyury (A), collagen 2A1 (B) and CD24 (C).

List of up and down regulated genes on expression level with a p-value of 10-4 a FC of 10.

FISH results regarding the CDKN2A status in chordoma cell lines and the corresponding parental tumor.

Western blot analysis for cleaved PARP and cleaved caspase 3 of all chordoma cell lines as compared to HEK-293 cells (+/- stimulation with TNFα and Bortezomib for 18h). No corresponding bands are apparent in the cell lines after inhibition with 1000 nM Palbociclib for 3 days. U-CH 1-6 (A) and U-CH 7-12 (A).