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The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Adoptive immunotherapy using chimeric antigen receptor (CAR) expressing T cells targeting the CD19 B lineage receptor has demonstrated marked success in relapsed pre-B-cell acute lymphoblastic leukaemia (ALL). Persisting CAR-T cells generate sustained pressure against CD19 that may drive unique mechanisms of resistance. Pre-B ALL originates from a committed pre-B cell or an earlier progenitor, with potential to reprogram into other hematopoietic lineages. Here we report changes in lineage markers including myeloid conversion in patients following CD19 CAR therapy. Using murine ALL models we study the long-term effects of CD19 CAR-T cells and demonstrate partial or complete lineage switch as a consistent mechanism of CAR resistance depending on the underlying genetic oncogenic driver. Deletion of Pax5 or Ebf1 recapitulates lineage reprogramming occurring during CD19 CAR pressure. Our findings establish lineage switch as a mechanism of CAR resistance exposing inherent plasticity in genetic subtypes of pre-B-cell ALL. CAR-T targeting CD19 have been successfully used in a variety of B-cell malignancies but patients may eventually relapse. Here, the authors show that CD19 CAR-T resistance in pre-B cell ALL can be due to the induction of a myeloid lineage switch through an epigenetic alterations in master regulators of B cell development.

Identity determining transcription factors (TFs), or core regulatory (CR) TFs, are governed by cell-type specific super enhancers (SEs). Drugs to selectively inhibit CR circuitry are of high interest for cancer treatment. In alveolar rhabdomyosarcoma, PAX3-FOXO1 activates SEs to induce the expression of other CR TFs, providing a model system for studying cancer cell addiction to CR transcription. Using chemical genetics, the systematic screening of chemical matter for a biological outcome, here we report on a screen for epigenetic chemical probes able to distinguish between SE-driven transcription and constitutive transcription. We find that chemical probes along the acetylation-axis, and not the methylation-axis, selectively disrupt CR transcription. Additionally, we find that histone deacetylases (HDACs) are essential for CR TF transcription. We further dissect the contribution of HDAC isoforms using selective inhibitors, including the newly developed selective HDAC3 inhibitor LW3. We show HDAC1/2/3 are the co-essential isoforms that when co-inhibited halt CR transcription, making CR TF sites hyper-accessible and disrupting chromatin looping. Core regulatory transcription factors are usually regulated by cell-type specific super enhancers (SEs). Here, the authors screen for chemical probes able to distinguish between SE-driven and promoter-driven transcription and find that histone deacetylases are selectively required for core regulatory transcription.

Core regulatory transcription factors (CR TFs) orchestrate the placement of super-enhancers (SEs) to activate transcription of cell-identity specifying gene networks, and are critical in promoting cancer. Here, we define the core regulatory circuitry of rhabdomyosarcoma and identify critical CR TF dependencies. These CR TFs build SEs that have the highest levels of histone acetylation, yet paradoxically the same SEs also harbor the greatest amounts of histone deacetylases. We find that hyperacetylation selectively halts CR TF transcription. To investigate the architectural determinants of this phenotype, we used absolute quantification of architecture (AQuA) HiChIP, which revealed erosion of native SE contacts, and aberrant spreading of contacts that involved histone acetylation. Hyperacetylation removes RNA polymerase II (RNA Pol II) from core regulatory genetic elements, and eliminates RNA Pol II but not BRD4 phase condensates. This study identifies an SE-specific requirement for balancing histone modification states to maintain SE architecture and CR TF transcription. High levels of histone acetylation at rhabdomyosarcoma SEs, including SOX8, are detrimental to transcription via exclusion of RNA Pol II, but not BRD4, from phase condensates.

The leading cause of mortality for patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the development of malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors. This multi-institutional study from the National Cancer Institute and Washington University in St. Louis used fragment size analysis and ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Following in silico enrichment for short cfDNA fragments and copy number analysis to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier that differentiates MPNST from PN with 86% pretreatment accuracy (91% specificity, 75% sensitivity) and 89% accuracy on serial analysis (91% specificity, 83% sensitivity). Healthy controls without NF1 (participants = 16, plasma samples = 16), PN (participants = 23, plasma samples = 23), and MPNST (participants = 14, plasma samples = 46) cohorts showed significant differences in tumor fraction in plasma (P = 0.001) as well as cfDNA fragment length (P < 0.001) with MPNST samples harboring shorter fragments and being enriched for tumor-derived cfDNA relative to PN and healthy controls. No other covariates were significant on multivariate logistic regression. Mutational analysis demonstrated focal NF1 copy number loss in PN and MPNST patient plasma but not in healthy controls. Greater genomic instability including alterations associated with malignant transformation (focal copy number gains in chromosome arms 1q, 7p, 8q, 9q, and 17q; focal copy number losses in SUZ12, SMARCA2, CDKN2A/B, and chromosome arms 6p and 9p) was more prominently observed in MPNST plasma. Furthermore, the sum of longest tumor diameters (SLD) visualized by cross-sectional imaging correlated significantly with paired tumor fractions in plasma from MPNST patients (r = 0.39, P = 0.024). On serial analysis, tumor fraction levels in plasma dynamically correlated with treatment response to therapy and minimal residual disease (MRD) detection before relapse. Study limitations include a modest MPNST sample size despite accrual from 2 major referral centers for this rare malignancy, and lack of uniform treatment and imaging protocols representing a real-world cohort. Tumor fraction levels derived from cfDNA fragment size and copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, accurately distinguished MPNST from its benign PN precursor, and dynamically correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations.

MYCN activates canonical MYC targets involved in ribosome biogenesis, protein synthesis, and represses neuronal differentiation genes to drive oncogenesis in neuroblastoma (NB). How MYCN orchestrates global gene expression remains incompletely understood. Our study finds that MYCN binds promoters to up-regulate canonical MYC targets but binds to both enhancers and promoters to repress differentiation genes. MYCN binding also increases H3K4me3 and H3K27ac on canonical MYC target promoters and decreases H3K27ac on neuronal differentiation gene enhancers and promoters. WDR5 facilitates MYCN promoter binding to activate canonical MYC target genes, whereas MYCN recruits G9a to enhancers to repress neuronal differentiation genes. Targeting both MYCN’s active and repressive transcriptional activities using both WDR5 and G9a inhibitors synergistically suppresses NB growth. We demonstrate that MYCN cooperates with WDR5 and G9a to orchestrate global gene transcription. The targeting of both these cofactors is a novel therapeutic strategy to indirectly target the oncogenic activity of MYCN .

Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Here, we show that the zinc finger transcription factor CASZ1 up-regulates MYOD signature genes and induces skeletal muscle differentiation in normal myoblasts and ERMS. The oncogenic activation of the RAS-MEK pathway suppresses CASZ1 expression in ERMS. ChIP-seq, ATAC-seq and RNA-seq experiments reveal that CASZ1 directly up-regulates skeletal muscle genes and represses non-muscle genes through affecting regional epigenetic modifications, chromatin accessibility and super-enhancer establishment. Next generation sequencing of primary RMS tumors identified a single nucleotide variant in the CASZ1 coding region that potentially contributes to ERMS tumorigenesis. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis. Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer with impaired myogenic differentiation despite the presence of myogenic transcription factors. Here, the authors show that CASZ1 directly regulates these myogenic factors and the loss of CASZ1 is due to RAS-MEK signaling in ERMS.

To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

To define alterations early in tumor formation, we studied nerve tumors in neurofibromatosis 1 (NF1), a tumor predisposition syndrome. Affected individuals develop neurofibromas, benign tumors driven by NF1 loss in Schwann cells (SCs). By comparing normal nerve cells to plexiform neurofibroma (PN) cells using single-cell and bulk RNA sequencing, we identified changes in 5 SC populations, including a de novo SC progenitor–like (SCP-like) population. Long after Nf1 loss, SC populations developed PN-specific expression of Dcn , Postn , and Cd74 , with sustained expression of the injury response gene Postn and showed dramatic expansion of immune and stromal cell populations; in corresponding human PNs, the immune and stromal cells comprised 90% of cells. Comparisons between injury-related and tumor monocytes/macrophages support early monocyte recruitment and aberrant macrophage differentiation. Cross-species analysis verified each SC population and unique conserved patterns of predicted cell-cell communication in each SC population. This analysis identified PROS1-AXL, FGF-FGFR, and MIF-CD74 and its effector pathway NF-κB as deregulated in NF1 SC populations, including SCP-like cells predicted to influence other types of SCs, stromal cells, and/or immune cells in mouse and human. These findings highlight remarkable changes in multiple types of SCs and identify therapeutic targets for PN.