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Rhabdomyosarcoma (RMS) is a pediatric cancer with features of skeletal muscle; patients with unresectable or metastatic RMS fare poorly due to high rates of disease recurrence. Here, we use single cell and single nucleus RNA-sequencing to show that RMS tumors recapitulate the spectrum of embryonal myogenesis. Using matched patient samples from a clinical trial and orthotopic patient-derived xenografts (O-PDXs), we show chemotherapy eliminates the most proliferative component with features of myoblasts; after treatment, the quiescent immature population with features of paraxial mesoderm expands to reconstitute the developmental hierarchy of the original tumor. We discovered that this paraxial mesoderm population is dependent on EGFR signaling and is sensitive to EGFR inhibitors. Taken together, this data serves as a proof-of-concept that targeting each developmental state in RMS is an effective strategy for improving outcomes by preventing disease recurrence.

SummaryRhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.

Rhabdomyosarcoma (RMS) is a pediatric cancer of the developing skeletal muscle, and the mechanisms that drive this tumor are not completely understood. Despite the genetic alterations discovered in this disease, therapeutics targeting these mutations have not improved patient survival over the past 25 years. Since more than 60% of patients with metastatic disease do not survive, this creates an urgency to identify new vulnerabilities to exploit for therapeutic development. To do this we performed a comprehensive evaluation of the epigenomic landscape across 16 RMS orthotopic patient-derived xenograft tumors. We identified 41 super enhancers unique to RMS tumors. Within these, we discovered 9 novel lncRNAs of which 3 have potential cis-regulatory activity. We hypothesize that these lncRNAs are required for RMS tumorigenesis. Indeed, from preliminary knockdown analysis of one of these lncRNAs, lnc19_31, we observed rapid cell death in RMS cell lines and concomitant upregulation of apoptotic genes based on RNA-seq, such as TP53. Remarkably, we also observed more than 70 cancer consensus genes that were up- or downregulated, including genes that are frequently mutated in RMS patients such as BCOR and FGFR4. Within these we were excited to discover the loss of PAX3/FOX1 translocation, as this is a major driver of the alveolar subtype and often associated with poor prognosis. Taken together, we hypothesize that lncRNA deregulation is required for expression of key oncogenes and tumor suppressor and essential for RMS cell survival and tumorigenesis. These data will shed insight into a new underlying mechanism that drives this disease. These exciting findings present potential tumor vulnerabilities and, thus, new opportunities for future therapeutic discoveries that would otherwise be beyond our reach. Citation Format: Xiang Chen, Mireya Herrera-Herrera, Justina McEvoy. Novel lncRNA regulates PAX3/FOXO1 expression and is essential for RMS tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B26.

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss.

Summary Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G2/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G2/M pathway is the most effective approach in vivo for high-risk RMS.

In the 1970s, several human retinoblastoma cell lines were developed from cultures of primary tumors. As the human retinoblastoma cell lines were established in culture, growth properties and changes in cell adhesion were described. Those changes correlated with the ability of the human retinoblastoma cell lines to invade the optic nerve and metastasize in orthotopic xenograft studies. However, the mechanisms that underlie these changes were not determined. We used the recently developed knockout mouse models of retinoblastoma to begin to characterize the molecular, cellular, and genetic changes associated with retinoblastoma tumor progression and optic nerve invasion. Here we report the isolation and characterization of the first mouse retinoblastoma cell lines with targeted deletions of the Rb family. Our detailed analysis of these cells as they were propagated in culture from the primary tumor shows that changes in cadherin-mediated cell adhesion are associated with retinoblastoma invasion of the optic nerve prior to metastasis. In addition, the same changes in cadherin-mediated cell adhesion correlate with the invasive properties of the human retinoblastoma cell lines isolated decades ago, providing a molecular mechanism for these earlier observations. Most importantly, our studies are in agreement with genetic studies on human retinoblastomas, suggesting that changes in this pathway are involved in tumor progression.

Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf2 activity through the interaction with Nrf2 Neh2 domain. However, the mechanism(s) of Nrf2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule. Here, we report the generation of a new monoclonal antibody for the detection of endogenous Keap1 molecules. Immunocytochemical analysis of mouse embryonic fibroblasts with the antibody revealed that under normal, unstressed condition, Keap1 is localized primarily in the cytoplasm with minimal amount in the nucleus and endoplasmic reticulum. This subcellular localization profile of Keap1 appears unchanged after treatment of cells with diethyl maleate, an electrophile, and/or Leptomycin B, a nuclear export inhibitor. Subcellular fractionation analysis of mouse liver cells showed similar results. No substantial change in the subcellular distribution profile could be observed in cells isolated from butylated hydroxyanisole-treated mice. Analyses of sucrose density gradient centrifugation of mouse liver cells indicated that Keap1 appears to form multiprotein complexes in the cytoplasm. These results demonstrate that endogenous Keap1 remains mostly in the cytoplasm, and electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of Keap1.

Retinoblastoma is a rare pediatric cancer of the retina. Nearly all retinoblastomas are initiated through the biallelic inactivation of the retinoblastoma tumor susceptibility gene (RB1). Whole-genome sequencing has made it possible to identify secondary genetic lesions following RB1 inactivation. One of the major discoveries from retinoblastoma sequencing studies is that some retinoblastoma tumors have stable genomes. Subsequent epigenetic studies showed that changes in the epigenome contribute to the rapid progression of retinoblastoma following RB1 gene inactivation. In addition, gene amplification and elevated expression of p53 antagonists, MDM2 and MDM4, may also play an important role in retinoblastoma tumorigenesis. The knowledge gained from these recent molecular, cellular, genomic, and epigenomic analyses are now being integrated to identify new therapeutic approaches that can help save lives and vision in children with retinoblastoma, with fewer long-term side effects.

Most pediatric solid tumors originate from perturbations during development, however the mechanisms that drive these disruptions and promote tumor progression are still poorly understood. We believe that epigenetic deregulation of cancer genes and developmental pathways is a common driver in pediatric cancers, especially those with a low mutation rate. To test this novel idea, we are focusing on rhabdomyosarcoma (RMS), a developing muscle tumor with striking genetic differences between two of the major subtypes. We hypothesize that differential epigenetic deregulation of differentiation programs and cancer genes between the major subtypes are essential for tumorigenesis. Indeed, our preliminary integrative analysis reveal distinct epigenetic landscapes between the two subtypes particularly at the loci of HOX cluster genes A-C, including an oncogenic HOXC long non-coding RNA (lncRNA) called HOTAIR. Knockdown of HOTAIR led to derepression of HOXD10, a candidate tumor suppressor, and a decrease in RMS proliferation, migration, and invasion. Thus, we believe HOX gene deregulation is required for tumorigenesis, and their implication in several adult cancers suggests this currently unappreciated mechanism is likely to be more broadly relevant. In addition, we identified a group of RMS specific novel lncRNAs, of which one demonstrates an essential role in cell survival. Our innovative approach to integrate epigenetic data sets from tumor types with varying genetic differences shed light on the mechanisms that drive tumorigenesis, which may lead to identifying previously unknown potential therapeutic targets. Since current therapeutic options have not changed over the last twenty-five years, these data will especially provide a strong translational impact for children with RMS, whose survival outcomes are less than 20% in the case of metastatic disease. Note: This abstract was not presented at the meeting. Citation Format: Justina McEvoy, Michael Dyer, Xiang Chen. Novel non-genetic mechanisms drive rhabdomyosarcoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3873. doi:10.1158/1538-7445.AM2017-3873

Genetically engineered mouse models (GEMMs) of human cancer are important for advancing our understanding of tumor initiation and progression as well as for testing novel therapeutics. Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. GEMMs faithfully recapitulate the histopathology, molecular, cellular, morphometric, neuroanatomical and neurochemical features of human retinoblastoma. In this study, we analyzed the genomic and epigenomic landscape of murine retinoblastoma and compared them to human retinoblastomas to gain insight into shared mechanisms of tumor progression across species. Similar to human retinoblastoma, mouse tumors have low rates of single nucleotide variations. However, mouse retinoblastomas have higher rates of aneuploidy and regional and focal copy number changes that vary depending on the genetic lesions that initiate tumorigenesis in the developing murine retina. Furthermore, the epigenetic landscape in mouse retinoblastoma was significantly different from human tumors and some pathways that are candidates for molecular targeted therapy for human retinoblastoma such as SYK or MCL1 are not deregulated in GEMMs. Taken together, these data suggest there are important differences between mouse and human retinoblastomas with respect to the mechanism of tumor progression and those differences can have significant implications for translational research to test the efficacy of novel therapies for this devastating childhood cancer.

Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX) and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191) was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

Two distinct pathways for the degradation of mammalian cyclin E have previously been described. One pathway is induced by cyclin E phosphorylation and is dependent on the Cul1/Fbw7-based E3 ligase. The other pathway is dependent on the Cul3-based E3 ligase, but the mechanistic details of this pathway have yet to be elucidated. To establish the role of Cul3 in the degradation of cyclin E in vivo, we created a conditional knockout of the Cul3 gene in mice. Interestingly, the biallelic loss of Cul3 in primary fibroblasts derived from these mice results in increased cyclin E expression and reduced cell viability, paralleling the loss of Cul3 protein expression. Cell cycle analysis of viable, Cul3 hypomorphic cells shows that decreasing the levels of Cul3 increases both cyclin E protein levels and the number of cells in S phase. In order to examine the role of Cul3 in an in vivo setting, we determined the effect of deletion of the Cul3 gene in liver. This gene deletion resulted in a dramatic increase in cyclin E levels as well as an increase in cell size and ploidy. The results we report here show that the constitutive degradation pathway for cyclin E that is regulated by the Cul3-based E3 ligase is essential to maintain quiescence in mammalian cells.

10036 Background: The genomic landscape of pediatric solid tumors is actively being investigated and to date, few recurrent actionable mutations have been identified. Using orthotopic xenografts, w...

SummaryIt is widely believed that the molecular and cellular features of a tumor reflect its cell of origin and can thus provide clues about treatment targets. The retinoblastoma cell of origin has been debated for over a century. Here, we report that human and mouse retinoblastomas have molecular, cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type-specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Furthermore, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro.

// Justina McEvoy 1,* , Panduka Nagahawatte 2,* , David Finkelstein 2 , Jennifer Richards-Yutz 6 , Marcus Valentine 13 , Jing Ma 14 , Charles Mullighan 14 , Guangchun Song 14 , Xiang Chen 2 , Matthew Wilson 4 , Rachel Brennan 12 , Stanley Pounds 3 , Jared Becksfort 2 , Robert Huether 2 , Charles Lu 7 , Robert S. Fulton 7,8 , Lucinda L. Fulton 7,8 , Xin Hong 7,8 , David J. Dooling 7,8 , Kerri Ochoa 7,8 , Elaine R. Mardis 7,8,9 , Richard K.Wilson 7,8,10 , John Easton 2 , Jinghui Zhang 2 , James R. Downing 14 , Arupa Ganguly 5,6,* and Michael A. Dyer 1,4,11 for the St. Jude Children’s Research Hospital – Washington University Pediatric Cancer Genome Project 1 Departments of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA. 2 Computational Biology and Bioinformatics, St. Jude Children’s Research Hospital, Memphis, TN, USA. 3 Biostatistics, St. Jude Children’s Research Hospital, Memphis, TN, USA. 4 Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN 5 Department of Genetics, School of Medicine, Philadelphia, PA, USA 6 Genetic Diagnostic Laboratory at University of Pennsylvania, School of Medicine, Philadelphia, PA, USA. 7 The Genome Institute, Washington University School of Medicine in St Louis, St Louis, Missouri, USA. 8 Department of Genetics, Washington University School of Medicine in St Louis, St Louis, Missouri, USA. 9 Siteman Cancer Center, Washington University School of Medicine in St Louis, St Louis, Missouri, USA. 10 Department of Medicine, Washington University School of Medicine in St Louis, St Louis, Missouri, USA 11 Howard Hughes Medical Institute, Chevy Chase, MD 12 Oncology, St. Jude Children’s Research Hospital, Memphis, TN, USA. 13 Cytogenetics, St. Jude Children’s Research Hospital, Memphis, TN, USA. 14 Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA. * These authors contributed equally to this work. Correspondence: Michael A. Dyer , email: // Keywords : chromothripsis, retinoblastoma, RB1, MYCN Received : December 13, 2013 Accepted : January 7, 2014 Published : January 11, 2014 Abstract Retinoblastoma is a rare childhood cancer of the developing retina. Most retinoblastomas initiate with biallelic inactivation of the RB1 gene through diverse mechanisms including point mutations, nucleotide insertions, deletions, loss of heterozygosity and promoter hypermethylation. Recently, a novel mechanism of retinoblastoma initiation was proposed. Gallie and colleagues discovered that a small proportion of retinoblastomas lack RB1 mutations and had MYCN amplification [ 1 ]. In this study, we identified recurrent chromosomal, regional and focal genomic lesions in 94 primary retinoblastomas with their matched normal DNA using SNP 6.0 chips. We also analyzed the RB1 gene mutations and compared the mechanism of RB1 inactivation to the recurrent copy number variations in the retinoblastoma genome. In addition to the previously described focal amplification of MYCN and deletions in RB1 and BCOR , we also identified recurrent focal amplification of OTX2 , a transcription factor required for retinal photoreceptor development. We identified 10 retinoblastomas in our cohort that lacked RB1 point mutations or indels. We performed whole genome sequencing on those 10 tumors and their corresponding germline DNA. In one of the tumors, the RB1 gene was unaltered, the MYCN gene was amplified and RB1 protein was expressed in the nuclei of the tumor cells. In addition, several tumors had complex patterns of structural variations and we identified 3 tumors with chromothripsis at the RB1 locus. This is the first report of chromothripsis as a mechanism for RB1 gene inactivation in cancer.