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Children with treatment-refractory or relapsed (R/R) tumors face poor prognoses. As the genomic underpinnings driving R/R disease are not well defined, we describe here the genomic and transcriptomic landscapes of R/R solid tumors from 202 patients enrolled in Beat Childhood Cancer Consortium clinical trials. Tumor mutational burden (TMB) was elevated relative to untreated tumors at diagnosis, with one-third of tumors classified as having a pediatric high TMB. Prior chemotherapy exposure influenced the mutational landscape of these R/R tumors, with more than 40% of tumors demonstrating mutational signatures associated with platinum or temozolomide chemotherapy and two tumors showing treatment-associated hypermutation. Immunogenomic profiling found a heterogenous pattern of neoantigen and MHC class I expression and a general absence of immune infiltration. Transcriptional analysis and functional gene set enrichment analysis identified cross-pathology clusters associated with development, immune signaling, and cellular signaling pathways. While the landscapes of these R/R tumors reflected those of their corresponding untreated tumors at diagnosis, important exceptions were observed, suggestive of tumor evolution, treatment resistance mechanisms, and mutagenic etiologies of treatment. Significance: Tumor heterogeneity, chemotherapy exposure, and tumor evolution contribute to the molecular profiles and increased mutational burden that occur in treatment-refractory and relapsed childhood solid tumors.

Despite decades of multi-module therapies, RMS remains incurable once it has metastasized, thus new therapeutic strategies are warranted. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase, overexpressed in virtually all, mutationally activated in about 10% of RMS, and directly activated by PAX3-FOXO1 fusion protein which makes it a tractable target for immunotherapy. We generated fifteen binders against FGFR4 and characterized them further as candidate molecules for immune therapy. We found that 3A11, a mouse IgG antibody, bound to FGFR4 positive cell lines. The VL and VH domain of 3A11 was cloned and made into scFvFc format (mouse Fv and Human IgG1 Fc). The chimeric form of 3A11 antibody was successfully produced in vitro and retained its FGFR4 specificity with an observed binding affinity at nanomolar range. By ELISA using the extra cellular domain of human FGFR1, FGFR2, FGFR3 or FGFR4, 3A11 scFvFc showed dose dependent binding to FGFR4 only. We then made 3A11 into a second generation CAR. Human T cells transduced with 3A11 CAR construct were found to be highly potent at inducing IFN-γ, TNF-α, IL-2 and cytotoxicity when the FGFR4-CART was co-cultured with RMS cells, but not with RMS cells with FGFR4 knocked out and FGFR4 negative cells. Our in vivo testing also found that 3A11 CART was able to eliminate RMS cells in murine xenograft metastatic models. Here we report the successful generation of binders specific to human FGFR4. FGFR4 CAR developed from 3A11 was able to kill FGFR4 positive target cells both in-vivo and in-vitro. Thus, 3A11 CAR T cells targeting FGFR4 may provide effective immune therapies for rhabdomyosarcoma and other FGFR4 expressing cancers and clinical trials are planned. Citation Format: Adam Cheuk, Meijie Tian, Jeetendra Kumar, Peter Azorsa, Nityashree Shivaprasad, Dina Schneider, Berkley Gryder, Jun Wei, Young Song, Xinyu Wen, Sivasish Sindiri, Joon-Yong Chung, Zhongyu Zhu, Dimiter Dimitrov, Stephen Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Potent tumoricidal activity of a FGFR4 CART in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1885.

Background Malignant mesothelioma (MM) is an aggressive cancer with limited treatment options and poor prognosis. Malignant pleural mesothelioma comprises 80% of the cases and has worse outcome than malignant peritoneal mesothelioma. An in-depth knowledge of genetic, transcriptomic and immunogenic events involved in MM is critical for successful development of prognostics and therapeutic modalities. Methods We performed whole-exome sequencing of germline and tumors of 122 patients with pleural (n=59), peritoneal (n=61) and tunica vaginalis (n=2) mesothelioma, and RNA-sequencing of 100 tumors to identify pathogenic variants, somatic mutational signatures, and prognostic gene expression signatures, predictive of patient survival and tumor response to therapies. We validated our findings using the TCGA and Bueno et al. mesothelioma datasets. Results The important findings from this study include: a) Key somatic mutational signatures are associated with DNA repair pathways and BRCA1 associated protein-1 (BAP1) is the most commonly mutated gene (~13% with germline mutation). b) We identified a set of 48 genes, a “mesothelioma prognostic signature”, whose high expression level is associated with poor survival (Cox regression, FDR < 0.1). These genes are enriched for genes related to cell cycle and DNA repair. This signature is highly predictive of patient survival in two other independent, pleural mesothelioma cohorts: TCGA (Hazard ratio (HR) = 2.6, P = 6.94e-10) and Bueno et al. mesothelioma dataset (HR = 1.49, P = 4.34e-07), after controlling for age and gender. c) Among the 48 genes, the expression of CCNB1 is highly predictive of patient survival suggesting its important role in MM, possibly via its involvement in the CDK1-CCNB1-CCNF complex (HR = 2.54, P = 1.89e-08 for TCGA; HR = 1.40, P = 1.65e-05 for Bueno et al. dataset). d) Using a synthetic lethality (SL) based precision-oncology computational framework for analyzing the patients' transcriptomic data, we were able to accurately predict response to an anti-PD1 immune checkpoint inhibitor and combination therapies with pemetrexed (chemotherapy) in mesothelioma patients. The SL profiles successfully predicted the overall patient-response observed across targeted, immuno- and chemotherapies in 11 independent mesothelioma clinical trials (Spearman's ρ = 0.64, P = 0.0348). This is the first analysis shown to successfully predict overall patient-response for various treatments within a cancer type. Conclusions By analyzing the tumor genomic and transcriptomics data of a large cohort of MM patients, we identify gene expression prognostic markers predictive of patient survival and response to therapy, both as independent signatures and via their SL interactions. These findings lay a basis for the future development of personalized therapy approaches for mesothelioma patients. Citation Format: Nishanth Ulhas Nair, Qun Jiang, Jun S. Wei, Vikram A. Misra, Betsy Morrow, Leandro C. Hermida, Joo Sang Lee, Idrees Mian, Jingli Zhang, Manjistha Sengupta, Javed Khan, Eytan Ruppin, Raffit Hassan. Genomic and transcriptomic profiling of malignant mesothelioma patients identifies gene signatures predictive of survival and response to immuno and chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 667.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that accounts for 12% of childhood cancer deaths. Like many childhood cancers, neuroblastoma exhibits a relative paucity of somatic single nucleotide variants (SNVs) and small insertions and deletions (indels) compared to adult cancers. Here, we assessed the contribution of somatic structural variation (SV) in neuroblastoma using a combination of whole genome sequencing (WGS; n=135) and single nucleotide polymorphism (SNP) genotyping (n=914) of matched tumor-normal pairs. Our study design allowed for orthogonal validation and replication across platforms. SV frequency, type, and localization varied significantly among high-risk tumors. MYCN non-amplified high-risk tumors harbored an increased SV burden overall, including a substantial excess of tandem-duplication events across the genome. Genes disrupted by SV breakpoints were enriched in neuronal lineages and autism spectrum disorder (ASD). The postsynaptic adapter protein-coding gene SHANK2, located on chromosome 11q13, was disrupted by SVs in 14% of MYCN non-amplified high-risk tumors based on WGS and 10% in the SNP array cohort. Expression of SHANK2 was low across human-derived neuroblastoma cell lines and high-risk neuroblastoma tumors. Forced expression of SHANK2 in neuroblastoma cell models resulted in significant growth inhibition (P=2.62×10-2 to 3.4×10-5) and accelerated neuronal differentiation following treatment with all-trans retinoic acid (P=3.08×10-13 to 2.38×10-30). These data further define the complex landscape of structural variation in neuroblastoma and suggest that events leading to deregulation of neurodevelopmental processes, such as inactivation of SHANK2, are key mediators of tumorigenesis in this childhood cancer.

Small cell neuroendocrine cancers (SCNCs) are recalcitrant cancers arising from diverse primary sites that lack effective treatments. Using chemical genetic screens, we identified inhibition of ataxia telangiectasia and rad3 related (ATR), the primary activator of the replication stress response, and topoisomerase I (TOP1), nuclear enzyme that suppresses genomic instability, as synergistically cytotoxic in small cell lung cancer (SCLC). In a proof-of-concept study, we combined M6620 (berzosertib), first-in-class ATR inhibitor, and TOP1 inhibitor topotecan in patients with relapsed SCNCs. Objective response rate among patients with SCLC was 36% (9/25), achieving the primary efficacy endpoint. Durable tumor regressions were observed in patients with platinum-resistant SCNCs, typically fatal within weeks of recurrence. SCNCs with high neuroendocrine differentiation, characterized by enhanced replication stress, were more likely to respond. These findings highlight replication stress as a potentially transformative vulnerability of SCNCs, paving the way for rational patient selection in these cancers, now treated as a single disease.

Malignancy remains the leading cause of disease-related death in children. To identify potential tumor-driving molecular targets and characterize immunogenomic profiles in pediatric cancers, we performed RNA-seq analysis on a cohort of 788 pediatric solid tumors across 14 different diagnoses in conjunction with additional 147 normal tissues for comparison. Sequencing data were analyzed for expressed mutations, fusion events, and expressional patterns, providing therapeutic targets and rich cancer biology for these childhood cancers. Furthermore, we describe a comprehensive and in-depth immunogenomic landscape of these solid tumors including immune cell infiltrate, neoepitope analysis from expressed mutations and fusions, expressional patterns of clinically relevant immune checkpoint genes, expression of tumor-specific genes as potential pharmacological or immunological targets, and T cell receptor repertoire. Across the cohort, we observed a striking correlation between the expressed neoepitope burden in tumors and enrichment of the effector immune signatures. Intriguingly, canonical fusions (e.g. EWS-FLI1) contribute a disproportionally large number of neoepitopes in these typically low mutational tumors. Histology-specific immunogenomic patterns are also apparent. Several of the pediatric cancers such as alveolar soft part sarcoma and osteosarcoma exhibit rich immune cell infiltration and evidence for activated T cell activities, whereas others such as Wilms tumors and synovial sarcoma generally have a very low T cell infiltration. In addition, we demonstrated a significant positive correlation between tumor-infiltrating CD8+ T cells and overall survival in patients with osteosarcoma, revealing the clinical importance of these tumor-infiltrating immune cells in these childhood cancers. Moreover, an orthogonal evaluation of immunopeptidome in osteosarcoma, a cancer type displaying high immune infiltrates, confirmed our transcriptomic findings on potential targetable tumor-specific genes. Finally, we took an adoptive cell therapy-based approach to target a tumor-specific gene PRAME identified by our transcriptomic and immunopeptidomic studies and showed significant in-vitro cytotoxicity using T cells expressing TCRs specifically targeting PRAME in osteosarcoma U2OS cells. Therefore, we demonstrate that RNA-seq is a powerful tool to identify clinically relevant and histology-specific genomic alterations and translationally relevant immunogenomic patterns for pediatric cancers. This study also represents one of the largest of its type to date and provides a framework for future translational efforts in pediatric cancer. Citation Format: Jun S. Wei, Andrew S. Brohl, Sivasish Sindiri, David Milewski, Young K. Song, Sushma Nagaraj, Vineela Gangalapudi, Xinyu Wen, Marc Ladanyi, Javed Khan. Immuno-transcriptomic profiling identifies actionable genomic alterations in pediatric solid malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3445.

Background: Despite decades of multimodule therapies, RMS remains incurable once it has metastasized; thus, new therapeutic strategies are warranted. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase, overexpressed in virtually all, mutationally activated in about 7.5% of RMS, and directly activated by PAX3-FOXO1 fusion protein, which makes it a tractable target for immunotherapy. Material and Methods: Using monoclonal antibody technologies and a yeast display B-cell library, we generated 15 human or mouse binders specific to human FGFR4 and engineered into human scFvFc. All binders were successfully produced in vitro, and we further characterized them using FACS and ELISA for their specificity. Octet was used to measure the binding affinity against human FGFR4. For those lead hits, they were made into different formats of therapeutic including CAR and BiTE. We then performed in vitro killing assays and/or in vivo xenograft model to determine the efficacy of those therapeutics in killing RMS cells. Results: m410 and m412 were two lead hits and scFvFcs of these two binders were successfully produced in vitro and showed FGFR4 specificity with a binding affinity at nanomolar concentration. By ELISA, these binders showed dose-dependent binding to FGFR4 protein but not to other FGFR family members. We then made m410 and m412 into CAR and BiTE format, respectively. T cells transduced with m410 CAR construct were found highly potent in inducing gamma interferon, TNF alpha, and cytotoxicity when the FGFR4-CART are cocultured with RMS cells. Our in vivo testing found them to be effective in eliminating RMS cells in murine xenograft models. When T cells were cocultured with RMS cells in the presence of m412 BiTE in vitro, potent selective antitumor effect was observed, suggesting this can be another promising strategy for RMS immunotherapy. Conclusions: Here our data demonstrated that we had successfully generated binders specific to human FGFR4. The CAR and BiTE developed from these binders were able to kill FGFR4-positive target cells. Our data suggest that these FGFR4 CARs and FGFR4 BiTEs could provide effective immune therapies for rhabdomyosarcoma and other FGFR4-expressing cancers. Citation Format: Adam Cheuk, Nityashree Shivaprasad, Dina Schneider, Marielle Yohe, Meijie Tan, Peter Azorsa, Ronald Sams, Silvia Pomella, Berkley Gryder, Rossella Rota, Ben Stanton, Jun Wei, Young Song, Xinyu Wen, Sivasish Sindiri, Jeetendra Kumar, Robert Hawley, Joon-Yong Chung, Doncho Zhelev, Zhongyu Zhu, Dimiter Dimitrov, Stephen Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Development of FGFR4-specific chimeric antibody receptor (CAR) T cell and bispecific T cell engager (BiTE) for rhabdomyosarcoma (RMS) immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A08.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer with a peak incidence at 3-5 years of age. Despite the improved survival rate of 90% for newly diagnosed children with ALL, the outcome for patients with relapsed disease is poor with a less than 30% overall survival. CD19 CAR T-cell therapy has shown remarkable response rates in relapsed/refractory disease. Long-term survival analysis has shown that initial response rates exceed 80%. However, durable response rates at one year are closer to 40%. Little is known about factors predicting durable response to CAR T therapy. We hypothesize that patients with CD19 CAR T-cell resistant ALL have a distinct disease compared to responders to therapy that can be identified in pretreatment leukemia. Utilizing advanced genomic, epigenetic, proteomic, and single-cell (sc) techniques, we characterized patient bone marrow aspirates (BMA) to identify mechanisms of resistance. Patients enrolled in PLAT-02 at Seattle Children’s Hospital were categorized according to the durability of their response to CD19 CAR T therapy. To characterize the molecular and genomic alterations specific to the therapy-resistant ALLs, we performed comprehensive analyses on pre-treatment therapy-resistant and sensitive BMAs using whole-exome sequencing, RNA- BMAs seq, scRNA-seq, sc B cell receptor (BCR)-seq, methylation array, H3K27ac ChIP-seq, ATAC-seq, and CyTOF. Additionally, we developed murine patient-derived xenografts (PDXs) for future studies. Initial mutation analyses revealed 5 hotspot mutations (ABL1, 2 x KRAS, IKZF1, and EP300) and actionable fusion (2 ABL1, 2 ETV6, 2 ETV5, KMT2A). Interestingly, we identified a KMT2A-AFF1 fusion in a sensitive leukemia, which has been demonstrated to predispose patients to CD19 CAR T resistance through lineage switching. Additionally, we identified a novel CREBBP-fusion in leukemias resistant to CD19 CAR T-induced B-cell aplasia. Alterations of CREBBP have previously been associated with ALL that is refractory to conventional therapies. Integrated gene expression and epigenetic analyses are ongoing to identify genes or pathways associated with resistant disease. scRNA- and scBCR-seq data are being analyzed and integrated with CyTOF analyses to detect mixed lineage and gene expression-based heterogeneity that may predict clonal selection by CAR T pressure. Finally, we developed and genetically analyzed murine PDXs for 64% of the patient samples, establishing a valuable resource for future studies and developing novel therapies for resistant leukemias. This study is one of the most integrative and comprehensive genomic profiling approaches to identify the molecular traits of therapy-resistant ALL in patient samples. We hope to identify and develop crucial biomarkers predicting responsiveness to CAR T-cell therapy. Citation Format: Katherine E. Masih, Rebecca Gardner, Berkley E. Gryder, Abdalla Abdelmaksoud, Ashley Wilson, Serifat Adebola, Benjamin Z. Stanton, Young K. Song, Justin Lack, Chaoyu Wang, Xinyu Wen, Zachary Rae, Adam Cheuk, Gregoire Altan-Bonnet, Michael Kelly, Jun S. Wei, Michael C. Jensen, Rimas J. Orentas, Javed Khan. A comprehensive and integrative omic analysis of multiply relapsed refractory pediatric pre-B cell acute lymphoblastic leukemia predicts response to CD19 CAR T-cell therapy [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A11.

9054 Background: BRCA1 associated protein 1 ( BAP1), a nuclear deubiquitinase involved in DNA double-strand break repair is frequently mutated in MM. Because poly(ADP-ribose) polymerase inhibitors (PARPIs) induce synthetic lethality in BRCA1/2 mutant cancers, we sought to evaluate efficacy of olaparib in patients with MM and correlate it with pathogenic germline and somatic mutations in DNA repair genes. Methods: Phase II single-center study (NCT03531840) enrolled patients with advanced pleural or peritoneal mesothelioma who had progressed on prior therapies, age >18 years, ECOG performance status

The majority of neuroblastoma patients have tumors that initially respond to chemotherapy, but a large proportion of patients will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole genome sequencing of 23 paired diagnostic and relapsed neuroblastomas showed clonal evolution from the diagnostic tumor with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK signaling pathway. Seven events were detected only in the relapse tumor while the others showed clonal enrichment. In neuroblastoma cell lines we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18, 61%) and these lesions predicted for sensitivity to MEK inhibition in vitro and in vivo. Our findings provide the rationale for genetic characterization of relapse neuroblastoma and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.

Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs) is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19) or antibody-based therapy (anti-CD20) in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling’s T-squared test (Hotelling–Lawley multivariate analysis of variance) for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues). We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK) with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK1, GPR173, or with one another provided the most promising paired-hits. We propose that targeting these markers together would increase the specificity and thereby the safety of CAR-based therapy for neuroblastoma.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 affected individuals (cases) using a combination of whole-exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per Mb (0.48 nonsilent) and notably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, and an additional 7.1% had focal deletions), MYCN (1.7%, causing a recurrent p.Pro44Leu alteration) and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1 and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies that rely on frequently altered oncogenic drivers., National Human Genome Research Institute (U.S.) (Grant U54HG003067), National Cancer Institute (U.S.) (Contract HHSN261200800001E)

Using two MYCN transgenic mouse strains, we established 10 transplantable neuroblastoma cell lines via serial orthotopic passage in the adrenal gland. Tissue arrays demonstrate that by histochemistry, vascularity, immunohistochemical staining for neuroblastoma markers, catecholamine analysis, and concurrent cDNA microarray analysis, there is a close correspondence between the transplantable lines and the spontaneous tumors. Several genes closely associated with the pathobiology and immune evasion of neuroblastoma, novel targets that warrant evaluation, and decreased expression of tumor suppressor genes are demonstrated. These studies describe a unique and generalizable approach to expand the utility of transgenic models of spontaneous tumor, providing new tools for preclinical investigation.

MicroRNA 34a (miR-34a) is a potential tumor suppressor gene and has been identified as a miRNA component of the p53 network. To better understand the biological pathways involved in miR-34a action, a parallel global protein and mRNA expression profiling on miR-34a treated neuroblastoma cells (IMR32) was performed using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. Global profiling showed that miR-34a causes much smaller mRNA expression changes compared to changes at the protein level. A total of 1495 proteins represented by two or more peptides were identified from the quantitative ICAT analysis, of which 143 and 192 proteins are significantly up- or down-regulated by miR-34a, respectively. Pathway analysis of these differentially expressed proteins showed the enrichment of apoptosis and cell death processes in up-regulated proteins but DNA replication and cell cycle processes in the down-regulated proteins. Ribosomal proteins are the most significant set down-regulated by miR-34a. Additionally, biological network analysis to identify direct interactions among the differentially expressed proteins demonstrated that the expression of the ubiquitous transcription factor YY1, as well as its downstream proteins, is significantly reduced by miR-34a. We further demonstrated that miR-34a directly targets YY1 through a miR-34a-binding site within the 3' UTR of YY1 using a luciferase reporter system. YY1 is a negative regulator of p53, and it plays an essential role in cancer biology. Therefore, YY1 is another important direct target of miR-34a which closely regulates TP53 activities.

Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, representing 8% of all childhood tumors. The most aggressive subtype of RMS is associated with chromosomal translocations creating a fusion transcription factor, most commonly involving PAX3 and FOXO1 (fusion-positive (FP)-RMS). Our group has recently comprehensively characterized the genetic and epigenetic landscape of FP-RMS. We found that FP-RMS has a strikingly low somatic mutational burden and that the PAX3-FOXO1 fusion gene is the primary oncogenic driver. Specifically, we found that PAX3-FOXO1 reprograms the cis-regulatory epigenetic landscape by inducing de novo super enhancers in collaboration with the bromodomain and extra-terminal domain (BET) protein family member BRD4, and the master transcription factors MYOG, MYOD1, and MYCN. We therefore hypothesized that FP-RMS would be vulnerable to disruption of the PAX3-FOXO1 super enhancer oncogenic circuitry. Method and Results: To test this, we developed a novel readout assay to simultaneously monitor PAX3-FOXO1 super enhancer and general transcriptional activity as well as cell viability, and screened against a 62,643 small-molecule library at a concentration of 10 µM. We found a total of 573 compounds with selective ALK super enhancer inhibition, which were selected for dose response assay with four 10-fold dilutions ranging from 20 μM to 2 nM. Further prioritization using a systematic computational model reduced this number to 64. Among the compounds with known activities, the multi-kinase PKC-412/midostaurin was previously identified in screens for PAX3-FOXO1 inhibitors. Other known compounds included topoisomerase I and topoisomerase II inhibitors, where both enzymes have recently been reported to play important roles in BRD4-facilitated DNA dynamics involving super enhancers and the transcriptional machinery. Among this list we identified 33 novel compounds of unknown mechanism of action. We therefore performed RNA-seq analysis to determine the transcriptional impact of the effect of these compounds. Pathway, gene set enrichment and Connectivity Map analyses of RNA-seq expression signatures implicate several of the novel compounds as inhibitors of epigenetic regulators, including inhibitors of BRD4, histone deacetylases, histone demethylases, histone methyltransferases, and chromodomain proteins. Conclusions: Our systematic approach identified several small molecules that specifically disrupt the activity of the PAX3-FOXO1 fusion oncogene in RMS. Further characterization of the specificity and selectivity of the lead compounds is ongoing, and exact mechanism of action is currently being elucidated. Our ultimate goal is to develop a clinically effective precision therapeutic targeting the previously “undruggable” PAX3-FOXO1 oncogenic driver of aggressive FP-RMS. Citation Format: Robert G. Hawley, Girma M. Woldemichael, Berkley E. Gryder, Young Song, Sivasish Sindiri, Vineela Gangalapudi, Jun S. Wei, James B. McMahon, Jack F. Shern, Javed Khan. Identification of novel inhibitors of the PAX3-FOXO1 oncogenic driver in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5867.

To discover novel drugs for neuroblastoma treatment, we have previously screened a panel of drugs and identified 30 active agents against neuroblastoma cells. Here we performed microarray gene expression analysis to monitor the impact of these agents on a neuroblastoma cell line and used the connectivity map (cMAP) to explore putative mechanism of action of unknown drugs. We first compared the expression profiles of ten compounds shared in both our dataset and cMAP database and observed the high connectivity scores for 7 of 10 matched drugs regardless of the differences of cell lines utilized. The screen of cMAP for uncharacterized drugs indicated the signature of Epoxy anthraquinone derivative (EAD) matched the profiles of multiple known DNA targeted agents (topoisomerase I/II inhibitors, DNA intercalators, and DNA alkylation agents) as predicted by its structure. Similar result was obtained by querying against our internal NB-cMAP (http://pob.abcc.ncifcrf.gov/cgi-bin/cMAP), a database containing the profiles of 30 active drugs. These results suggest that Epoxy anthraquinone derivative may inhibit neuroblastoma cells by targeting DNA replication inhibition. Experimental data also demonstrate that Epoxy anthraquinone derivative indeed induces DNA double-strand breaks through DNA alkylation and inhibition of topoisomerase activity. Our study indicates that Epoxy anthraquinone derivative may be a novel DNA topoisomerase inhibitor that can be potentially used for treatment of neuroblastoma or other cancer patients.

Background: The completion of the Human Genome Project (HGP) has paved the way for new, more detailed and accurate molecular diagnostic classification of cancer. With the information from the HGP, cancers can be categorized not only on the morphology or limited immunohistological markers, but also according to their ‘molecular fingerprints’, such as gene expression profiles. Technologies detecting these signatures have been developed to measure simultaneously multiple genes or proteins in one assay with high sensitivity and specificity. Objective: To evaluate potential innovative new methods of diagnosis and prognosis in pediatric cancers. Methods: A variety of promising new diagnostic technologies was selected using molecular signatures that harness the results from HGP, including DNA microarray, bead-based detection system, multiplexed real-time polymerase chain reaction, MesoScale Discovery and isotope-coded affinity tag, as well as their applications in biomarker discovery for pediatric tumors. Label-...

Purpose: Amplified MYCN oncogene defines a subgroup of neuroblastomas with poor outcome. However, a substantial number of MYCN single-copy neuroblastomas exhibits an aggressive phenotype similar to that of MYCN-amplified neuroblastomas even in the absence of high MYCN mRNA and/or protein levels. Experimental Design: To identify shared molecular mechanisms that mediate the aggressive phenotype in MYCN-amplified and single-copy high-risk neuroblastomas, we defined genetic programs evoked by ectopically expressed MYCN in vitro and analyzed them in high-risk versus low-risk neuroblastoma tumors (n = 49) using cDNA microarrays. Candidate gene expression was validated in a separate cohort of 117 patients using quantitative PCR, and protein expression was analyzed in neuroblastoma tumors by immunoblotting and immunohistochemistry. Results: We identified a genetic signature characterized by a subset of MYCN/MYC and E2F targets, including Skp2, encoding the F-box protein of the SCFSkp2 E3-ligase, to be highly expressed in high-risk neuroblastomas independent of amplified MYCN. We validated the findings for Skp2 and analyzed its expression in relation to MYCN and E2F-1 expression in a separate cohort (n = 117) using quantitative PCR. High Skp2 expression proved to be a highly significant marker of dire prognosis independent of both MYCN status and disease stage, on the basis of multivariate analysis of event-free survival (hazard ratio, 3.54; 95% confidence interval, 1.56-8.00; P = 0.002). Skp2 protein expression was inversely correlated with expression of p27, the primary target of the SCFSkp2 E3-ligase, in neuroblastoma tumors. Conclusion: Skp2 may have a key role in the progression of neuroblastomas and should make an attractive target for therapeutic approaches.

The small round blue cell tumors of childhood, which include neuroblastoma, rhabdomyosarcoma, non-Hodgkin’s lymphoma, and the Ewing’s family of tumors, are so called because of their similar appearance on routine histology. Using cDNA microarray gene expression profiles and artificial neural networks (ANNs), we previously identified 93 genes capable of diagnosing these cancers. Using a subset of these, together with some additional genes (total 39), we developed a multiplex polymerase chain reaction (PCR) assay to diagnose these cancer types. Blinded testing of 96 new samples (26 Ewing’s family of tumors, 29 rhabdomyosarcomas, 24 neuroblastomas, and 17 lymphomas) using ANNs in a complete leave-one-out analysis demonstrated that all except one sample were accurately diagnosed as their respective category. Moreover, using an ANN-based gene minimization strategy in a separate analysis, we found that the top 31 genes could correctly diagnose all 96 tumors. Our results suggest that this molecular test based on a multiplex PCR reaction may assist the physician in the rapid confirmation of the diagnosis of these cancers.

Human tumor xenografts have been used extensively for rapid screening of the efficacy of anticancer drugs for the past 35 years. The selection of appropriate xenograft models for drug testing has been largely empirical and has not incorporated a similarity to the tumor type of origin at the molecular level. This study is the first comprehensive analysis of the transcriptome of a large set of pediatric xenografts, which are currently used for preclinical drug testing. Suitable models representing the tumor type of origin were identified. It was found that the characteristic expression patterns of the primary tumors were maintained in the corresponding xenografts for the majority of samples. Because a prerequisite for developing rationally designed drugs is that the target is expressed at the protein level, we developed tissue arrays from these xenografts and corroborated that high mRNA levels yielded high protein levels for two tested genes. The web database and availability of tissue arrays will allow for the rapid confirmation of the expression of potential targets at both the mRNA and the protein level for molecularly targeted agents. The database will facilitate the identification of tumor markers predictive of response to tested agents as well as the discovery of new molecular targets. [Cancer Res 2007;67(1):32–40]

Currently, patients with neuroblastoma are classified into risk groups (e.g., according to the Children’s Oncology Group risk-stratification) to guide physicians in the choice of the most appropriate therapy. Despite this careful stratification, the survival rate for patients with high-risk neuroblastoma remains

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., Author Summary To decipher the dynamic mutational process and identify the causative genomic events in rhabdomyosarcoma (RMS), we developed a systematic method that incorporates multiple types of genomic information to estimate normal cell contamination, tumor clonality, and a timeline of somatic events that occurred prior to the tumor presentation. Our results demonstrate two distinct evolutionary paths resulting in PAX-fusion-negative-rhabdomyosarcoma (PFN-RMS) and PAX-fusion-positive-rhabdomyosarcoma (PFP-RMS): (1) In PFN-RMS, genomic loss of heterozygosity on chromosome 11p15.5 and non-synonymous mutations in RAS pathway and cell cycle genes (FGFR4, KRAS, NRAS, HRAS and CCDN1), as well as several genes not previously known to be drivers of RMS, including PKN1, CUL2, and TTK, occurs early in the evolutionary history of tumor; (2) In contrast, the PAX gene fusion event in PFP-RMS tumors is an early detectable event which consistently occurs prior to a whole genome duplication event. These findings provide new insights into the biology and molecular events that initiate RMS tumorigenesis and may help identify actionable drivers for targeted therapies.

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., Author Summary The Ewing sarcoma family of tumors is a group of aggressive cancers that primarily affects the pediatric and young adult population. Increasingly, genomics are being used to better define the disease biology and to identify targets for therapy in many cancer types. Here, we report one of the first and largest genomic studies to date in the Ewing sarcoma family of tumors. Using a combination of modern sequencing techniques in >100 samples, we discover that Ewing sarcomas have a genome that is less complex compared to most cancer types previously surveyed. We find that this cancer is frequently affected by mutations in STAG2, a gene that has recently gained attention due to its importance in the biology of several cancer types. We show that Ewing sarcoma patients whose tumors are affected by STAG2 loss may have a worse prognosis. Additionally, we identify a subset of tumors that were diagnosed as Ewing sarcoma that appear to be distinct from the majority based on genetic and molecular characteristics. Our findings help to define the genetic landscape of Ewing sarcoma and provide a starting point for improving individualization of diagnosis, prognosis and treatment in this cancer.

Despite gains in survival, outcomes for patients with metastatic or recurrent rhabdomyosarcoma remain dismal. In a collaboration between the National Cancer Institute, Children's Oncology Group, and Broad Institute, we performed whole-genome, whole-exome, and transcriptome sequencing to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two genotypes are evident in rhabdomyosarcoma tumors: those characterized by the PAX3 or PAX7 fusion and those that lack these fusions but harbor mutations in key signaling pathways. The overall burden of somatic mutations in rhabdomyosarcoma is relatively low, especially in tumors that harbor a PAX3/7 gene fusion. In addition to previously reported mutations in NRAS, KRAS, HRAS, FGFR4, PIK3CA, and CTNNB1, we found novel recurrent mutations in FBXW7 and BCOR, providing potential new avenues for therapeutic intervention. Furthermore, alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of cases, providing a framework for genomics-directed therapies that might improve outcomes for patients with rhabdomyosarcoma. Significance: This is the most comprehensive genomic analysis of rhabdomyosarcoma to date. Despite a relatively low mutation rate, multiple genes were recurrently altered, including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, and BCOR. In addition, a majority of rhabdomyosarcoma tumors alter the receptor tyrosine kinase/RAS/PIK3CA axis, providing an opportunity for genomics-guided intervention. Cancer Discov; 4(2); 216–31. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 131

The complexity of the transcriptome has been appreciated in recent years in light of the Encyclopedia of DNA Elements (ENCODE) project. While less than 3% of the genome is annotated with protein-coding genes, 62% of the genome is long RNA molecules (>200 nucleotides). With an increasing sequencing throughput and a decreasing cost, RNA sequencing (RNA-seq) is becoming a frequently used technology in transcriptome research. This chapter summarizes the advantages of RNA-seq compared to hybridization-based microarrays, the RNA-seq experiment workflow and reviews current applications of RNA-seq in the field of cancer research. With advantages in expression analysis of non-coding genes, fusion genes, investigation of expressed allelic imbalance, pseudogenes, viral integrated genes, and post-transcriptional regulation analysis including splicing, polyadenylation as well as RNA editing, RNA-seq technology has a tremendous potential to elucidate the complexity of the transcriptome and its working mechanisms in the initiation and development of cancer. Along with expression and pathway analysis, RNA-seq data can clarify the functional consequences of any potential genomic (DNA) variances and thus help to distinguish “driver” from “passenger” genomic events. In summary, RNA-seq allows a genome-wide interrogation of complex transcriptomes and will help elucidate important biological mechanisms linked to cancer phenotypes. In addition, the non-protein-coding transcriptome has the potential to provide novel biomarkers for early detection, diagnostics, prognostics and targets for therapy.

The purpose of this study was to develop a method of classifying cancers to specific diagnostic categories based on their gene expression signatures using artificial neural networks (ANNs). We trained the ANNs using the small, round blue-cell tumors (SRBCTs) as a model. These cancers belong to four distinct diagnostic categories and often present diagnostic dilemmas in clinical practice. The ANNs correctly classified all samples and identified the genes most relevant to the classification. Expression of several of these genes has been reported in SRBCTs, but most have not been associated with these cancers. To test the ability of the trained ANN models to recognize SRBCTs, we analyzed additional blinded samples that were not previously used for the training procedure, and correctly classified them in all cases. This study demonstrates the potential applications of these methods for tumor diagnosis and the identification of candidate targets for therapy.

Microarray analysis of complementary DNA (cDNA) allows large-scale, comparative, gene expression profiling of two different cell populations. This approach has the potential for elucidating the primary transcription events and genetic cascades responsible for increased glioma cell motility in vitro and invasion in vivo. These genetic determinants could become therapeutic targets. We compared cDNA populations of a glioma cell line (G112) exposed or not to a motility-inducing substrate of cell-derived extracellular matrix (ECM) proteins using two sets of cDNA microarrays of 5,700 and 7,000 gene sequences. The data were analyzed considering the level and consistency of differential expression (outliers) and whether genes involved in pathways of motility, apoptosis, and proliferation were differentially expressed when the motility behavior was engaged. Validation of differential expression of selected genes was performed on additional cell lines and human glioblastoma tissue using quantitative RT-PCR. Some genes involved in cell motility, like tenascin C, neuropilin 2, GAP43, PARG1 (an inhibitor of Rho), PLCy, and CD44, were over expressed; other genes, like adducin 3y and integrins, were down regulated in migrating cells. Many key cell cycle components, like cyclin A and B, and proliferation markers, like PCNA, were strongly down regulated on ECM. Interestingly, genes involved in apoptotic cascades, like Bcl-2 and effector caspases, were differentially expressed, suggesting the global down regulation of proapoptotic components in cells exposed to cell-derived ECM. Overall, our findings indicate a reduced proliferative and apoptotic activity of migrating cells. cDNA microarray analysis has the potential for uncovering genes linking the phenotypic aspects of motility, proliferation, and apoptosis.

Neuroblastoma is one of the most genomically heterogeneous childhood malignances studied to date, and the molecular events that occur during the course of the disease are not fully understood. Genomic studies in neuroblastoma have showed only a few recurrent mutations and a low somatic mutation burden. However, none of these studies has examined the mutations arising during the course of disease, nor have they systemically examined the expression of mutant genes. Here we performed genomic analyses on tumors taken during a 3.5 years disease course from a neuroblastoma patient (bone marrow biopsy at diagnosis, adrenal primary tumor taken at surgical resection, and a liver metastasis at autopsy). Whole genome sequencing of the index liver metastasis identified 44 non-synonymous somatic mutations in 42 genes (0.85 mutation/MB) and a large hemizygous deletion in the ATRX gene which has been recently reported in neuroblastoma. Of these 45 somatic alterations, 15 were also detected in the primary tumor and bone marrow biopsy, while the other 30 were unique to the index tumor, indicating accumulation of de novo mutations during therapy. Furthermore, transcriptome sequencing on the 3 tumors demonstrated only 3 out of the 15 commonly mutated genes (LPAR1, GATA2, and NUFIP1) had high level of expression of the mutant alleles, suggesting potential oncogenic driver roles of these mutated genes. Among them, the druggable G-protein coupled receptor LPAR1 was highly expressed in all tumors. Cells expressing the LPAR1 R163W mutant demonstrated a significantly increased motility through elevated Rho signaling, but had no effect on growth. Therefore, this study highlights the need for multiple biopsies and sequencing during progression of a cancer and combinatorial DNA and RNA sequencing approach for systematic identification of expressed driver mutations.

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.

Despite the success of multimodal therapies, the mortality and morbidity remains substantial for patients with high-risk neuroblastoma (NBL). Sequencing of paired tumor/normal DNA of NBL has revealed a low somatic mutation burden and few recurrent somatically-mutated genes. Here we hypothesize that the integrated analysis of DNA sequencing with whole transcriptome sequencing (WTS) in patients with high-risk NBL tumor will yield valuable insights into the biology of this disease. We performed WTS of 139 NBLs (118 high-risk stage 4 and 21 stage 4S tumors) which had whole genome sequencing or whole exome sequencing of case-matched tumor/normal pairs through the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. We identified expressed mutations, fusion genes, and correlations between gene expression and clinical parameters of patients such as survival to provide understandings of high-risk NBL biology. Out of 1500 protein-coding changing somatic nucleotide variants detected by DNA sequencing, 614 variants (41%) were also detected in the transcriptome. Twenty-four genes known to be recurrently mutated in NBL showed the exact mutations in their transcriptome as seen in the DNA, including ALK (9.4%), ATRX (2.2%) and MYCN (1.4%). Fusion gene analysis identified in-frame fusions involving ALK (n = 2) and FOXR1 genes (n = 4). All tumors positive for ALK- and FOXR1-fusions expressed transcripts containing ALK or FOXR1 sequences at much higher levels (>10 folds) than those without fusion in these respective genes. Consensus clustering using tumor gene expression profiles revealed 4 subgroups with distinct survival probability. Among them, several molecular signatures including MYC activation and tumor microenvironment were observed. Intriguingly, 58% tumors without MYCN-amplification showed a MYC activation signature significantly associated with worse overall survival (p = 0.0017). Further examination of these tumors with the MYC activation signature revealed different somatic alterations including MYCN P44L mutations, high expression of other MYC family members (MYC and MYCL), mutations in the RAS pathway, and FOXR1 fusions. Interestingly, a gene expression signature representing tumor-associated macrophages (TAM) and regulatory T-cells significantly correlated with a worse outcome in NBLs with normal MYCN copy number, similar to that seen in tumors with the MYC activation signature. In contrast, NBL patients with tumors showing a signature of cytotoxic T-cells and B-cells have better outcomes. Furthermore, tumors of the latter subgroup express significantly more somatic SNVs comparing to the other two subgroups of worse outcome with MYC activation or TAM signatures, suggesting that expressed neo-antigens may elevate cytotoxic T-cell response in these tumors. Our study suggests that patients with high-risk neuroblastoma may benefit from immune-based therapies including check point inhibitors in the future trials. Citation Format: Jun S. Wei, Shile Zhang, Young K. Song, Shahab Asgharzadeh, Sivasish Sindiri, Xinyu Wen, Rajesh Patidar, Jaime M. Guidry Auvil, Daniela S. Gerhard, Robert Seeger, John M. Maris, Javed Khan. The transcriptome landscape of high-risk neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 126.

PAX-fusion negative rhabdomyosarcoma (RMS) arises from skeletal muscle precursors that have failed to differentiate normally despite the expression of the myogenic master transcription factor, MYOD1. The cure rate for relapsed or refractory fusion negative RMS is poor despite aggressive multi-modality treatment. Novel treatment approaches such as the use of targeted therapies including those that induce skeletal muscle differentiation might improve overall survival for patients with fusion negative RMS. Genetic studies have shown that the most common single nucleotide variant in fusion negative RMS is an oncogenic change in one of the RAS isoforms, namely NRAS, HRAS or KRAS. In this study, we hypothesized that targeting aberrant RAS activity releases the differentiation block in fusion negative RMS and sought to unravel the underlying epigenetic mechanisms through which RAS signaling drives oncogenic transcription in RMS. To achieve this goal, we combined high-throughput drug screening with biochemical, RNAseq and ChIPseq assays across a panel of RMS cell lines driven by oncogenic RAS mutations. Critically, we demonstrated that expression of oncogenic RAS was necessary for survival of these RAS-mutated RMS cells. In addition, overexpression of mutant RAS isoforms in C2C12 myoblasts inhibited myogenic differentiation induced by low-serum conditions. This differentiation block was mediated primarily by engagement of the RAF-MEK-ERK MAP kinase pathway. In corroboration with these observations, an unbiased screen of the ability of small molecules to impact cell viability demonstrated that inhibitors of the MAP kinase pathway were the most potently selective class of molecules for RAS-mutated RMS. In particular, trametinib, an allosteric, non-ATP competitive inhibitor of MEK1/2, was the most consistently potent MEK inhibitor in RAS-mutated RMS cell lines. Trametinib treatment induced G1 arrest and skeletal muscle differentiation in RAS-mutated RMS cell lines. Trametinib also slowed tumor growth and prolonged survival in xenograft models of RAS-mutated RMS. To determine the mechanism by which MEK inhibition induced skeletal muscle differentiation in RAS-mutated RMS, we analyzed changes in gene expression, transcription factor deposition and histone modification in RMS cells treated with trametinib. Trametinib treatment increased expression of myogenic transcription factors, such as MYOG and MEF2C, and decreased expression of transcription factors important for proliferation, such as MYC and ID3, in RAS-mutated RMS cells. ChIPseq experiments demonstrated that this transcriptional reprogramming was driven in part by changes in the active enhancer landscape, since H3K27ac deposition at MYH3, TTNT2 and other muscle-specific loci increased with trametinib treatment. Both MYC and MYOD1 bound the active enhancers induced by trametinib treatment in RAS-mutated RMS, despite an overall decrease in MYC expression. Finally, we found significant ERK2 deposition on the MYOG promoter in the untreated cells. ERK2 is known to recruit the Polycomb repressive machinery at developmental loci in embryonic stem cells and therefore aberrant ERK2 activity may facilitate repression of MYOG expression in RAS-mutated RMS. In summary, our data support a model of RAS-driven RMS in which aberrant ERK activity drives tumor cell proliferation, in part through increased expression and stability of MYC, and prevents myogenic differentiation, in this case through alterations in the enhancer landscape and interactions with the Polycomb repressive machinery. Future work is aimed at identifying rational combinations of trametinib and direct epigenetic modulators that synergistically drive RAS-mutated RMS differentiation with the goal of providing measurable clinical benefit in relapsed or refractory RAS-mutated RMS. Citation Format: Marielle E. Yohe, Berkley E. Gryder, Jack F. Shern, Young K. Song, Hongling Liao, Hsein-Chao Chou, Sivasish Sindiri, Arnulfo Mendoza, Xiaohu Zhang, Rajarashi Guha, Diana C. Haines, James P. Madigan, Jun S. Wei, Marc Ferrer, Craig J. Thomas, Javed Khan. Reprogramming RAS-driven rhabdomyosarcoma via MEK inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A25.

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed a siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of NB cell lines, those with MYCN amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of its known target histone H3, and upregulation of p53 pathway in MYCN-amplified NB cells with wild-type TP53. Both wild-type and p53-mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials. Citation Format: Dominik Bogen, Jun S. Wei, David O. Azorsa, Pinar Ormanoglu, Eugen Buehler, Rajarshi Guha, Jonathan M. Keller, Lesley A. Mathews Griner, Marc Ferrer, Young K. Song, Hongling Liao, Arnulfo Mendoza, Berkley E. Gryder, Sivasish Sindri, Jianbin He, Xinyu Wen, Xinyu Wen, Shile Zhang, John F. Shern, Marielle E. Yohe, Sabine Taschner-Mandl, Jason Shohet, Craig J. Thomas, Scott E. Martin, Peter F. Ambros, Javed Khan. Combined siRNA and small molecule screening identifies Aurora B kinase as an effective target in MYCN-driven neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B31.

Technological advances now allow us to rapidly produce CARs and other antibody-derived therapeutics targeting cell surface receptors. To maximize the potential of these new technologies, relevant extracellular targets must be identified. The Pediatric Oncology Branch of the NCI curates a freely accessible database of gene expression data for both pediatric cancers and normal tissues, through which we have defined discrete sets of over-expressed transcripts in twelve pediatric cancer subtypes as compared to normal tissues. We coupled gene expression profiles to current annotation databases (i.e., Affymetrix, Gene Ontology, Entrez Gene), in order to categorize transcripts by their subcellular location. In this manner we generated a list of potential immune targets expressed on the cell surface, ranked by their difference from normal tissue. Global differences from normal between each of the pediatric tumor types studied varied, indicating that some malignancies expressed transcript sets that were more highly diverged from normal tissues than others. The validity of our approach is seen by our findings for pre-B cell ALL, where targets currently in clinical trials were top-ranked hits (CD19, CD22). For some cancers, reagents already in development could potentially be applied to a new disease class, as exemplified by CD30 expression on sarcomas. Moreover, several potential new targets shared among several pediatric solid tumors are herein identified, such as MCAM (MUC18), MTDH (metadherin), and GPC2 (glypican-2). These targets have been identified at the mRNA level and are yet to be validated at the protein level. The safety of targeting these antigens has yet to be demonstrated and therefore the identified transcripts should be considered preliminary candidates for new CAR and therapeutic antibody targets. Prospective candidate targets will be evaluated by proteomic analysis including Westerns and immunohistochemistry of normal and tumor tissues.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with an undifferentiated status and generally poor prognosis, but the basis for these characteristics remains unknown. In this study, we show that upregulation of the Polycomb protein histone methyltransferase EZH2, which limits differentiation in many tissues, is critical to maintain the undifferentiated state and poor prognostic status of NB by epigenetic repression of multiple tumor suppressor genes. We identified this role for EZH2 by examining the regulation of CASZ1, a recently identified NB tumor suppressor gene whose ectopic restoration inhibits NB cell growth and induces differentiation. Reducing EZH2 expression by RNA interference–mediated knockdown or pharmacologic inhibiton with 3-deazaneplanocin A increased CASZ1 expression, inhibited NB cell growth, and induced neurite extension. Similarly, EZH2−/− mouse embryonic fibroblasts (MEF) displayed 3-fold higher levels of CASZ1 mRNA compared with EZH2+/+ MEFs. In cells with increased expression of CASZ1, treatment with histone deacetylase (HDAC) inhibitor decreased expression of EZH2 and the Polycomb Repressor complex component SUZ12. Under steady-state conditions, H3K27me3 and PRC2 components bound to the CASZ1 gene were enriched, but this enrichment was decreased after HDAC inhibitor treatment. We determined that the tumor suppressors CLU, NGFR, and RUNX3 were also directly repressed by EZH2 like CASZ1 in NB cells. Together, our findings establish that aberrant upregulation of EZH2 in NB cells silences several tumor suppressors, which contribute to the genesis and maintenance of the undifferentiated phenotype of NB tumors. Cancer Res; 72(1); 315–24. ©2011 AACR.

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

Neuroblastoma (NB) is a heterogeneous pediatric tumor. To better understand the biological pathways involved in the development of high-risk neuroblastoma, we performed parallel global protein and mRNA expression profiling on NB tumors of stage 4 MYCN-amplified (4+) and stage 1 MYCN-not-amplified (1-) using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. A total of 1461 proteins represented by 2 or more peptides were identified from the quantitative ICAT analysis, of which 433 and 130 proteins are up- or down-regulated, respectively, in 4+ tumor compared to the 1- tumor. Pathway analysis of the differentially expressed proteins showed the enrichment of glycolysis, DNA replication and cell cycle processes in the up-regulated proteins and cell adhesion, nervous system development and cell differentiation processes in the down-regulated proteins in 4+ tumor; suggesting a less mature neural and a more invasive phenotype of 4+ tumor. Myc targets and ribosomal proteins are overrepresented in the 4+ tumors as expected; functional gene sets reported to be enriched in neural and embryonic stem cells are significantly enriched in the 4+ tumor, indicating the existence of a stemness signature in MYCN-amplified stage 4 tumor. In addition, protein and mRNA expression are moderately correlated (r = 0.51, p < 0.0001), as approximately half of the up-regulated proteins in 4+ tumor have elevated mRNA level (n = 208), and one-third of down-regulated proteins have lower mRNA expression (n = 47). Further biological network analysis revealed that the differentially expressed proteins closely interact with other proteins of known networks; the important role of MYCN is confirmed and other transcription factors identified in the network may have potential roles in the biology of NB tumor. We used global genomic and proteomic analysis to identify biologically relevant proteins and pathways important to NB progression and development that may provide new insights into the biology of advanced neuroblastoma.

Purpose: microRNAs have been shown to be involved in different human cancers. We therefore have performed expression profiles on a panel of pediatric tumors to identify cancer-specific microRNAs. We also investigated if microRNAs are coregulated with their host gene. Experimental Design: We performed parallel microRNAs and mRNA expression profiling on 57 tumor xenografts and cell lines representing 10 different pediatric solid tumors using microarrays. For those microRNAs that map to their host mRNA, we calculated correlations between them. Results: We found that the majority of cancer types clustered together based on their global microRNA expression profiles by unsupervised hierarchical clustering. Fourteen microRNAs were significantly differentially expressed between rhabdomyosarcoma and neuroblastoma, and 8 of them were validated in independent patient tumor samples. Exploration of the expression of microRNAs in relationship with their host genes showed that the expression for 43 of 68 (63%) microRNAs located inside known coding genes was significantly correlated with that of their host genes. Among these 43 microRNAs, 5 of 7 microRNAs in the OncomiR-1 cluster correlated significantly with their host gene MIRHG1 (P < 0.01). In addition, high expression of MIRHG1 was significantly associated with high stage and MYCN amplification in neuroblastoma tumors, and the expression level of MIRHG1 could predict the outcome of neuroblastoma patients independently from the current neuroblastoma risk-stratification in two independent patient cohorts. Conclusion: Pediatric cancers express cancer-specific microRNAs. The high expression of the OncomiR-1 host gene MIRHG1 correlates with poor outcome for patients with neuroblastoma, indicating important oncogenic functions of this microRNA cluster in neuroblastoma biology. (Clin Cancer Res 2009;15(17):5560–8)

Gain of chromosome 17 is the most prevalent genetic abnormality identified in neuroblastoma (NB) and distal 17q gain has prognostic significance in NB. In this report, we have combined array-based comparative genomic hybridization (A-CGH) and gene expression analysis to investigate gene copy number changes and its impact on the gene expression level as well as their association with prognosis genes located on chromosome 17 in NB tumors. We observed differential gains of chromosome 17 between Stages 4- and 4S tumors. We found that WSB1, mapping to 17q11.1, which was frequently gained in 4S- tumors and not changed in 4- tumors, showed strong correlations between expression level and copy number. Furthermore, the increase of WSB1 gene expression is associated with good outcome in patients with NB of all stages. WSB1 also enhances the prognostic prediction when combined with other current prognostic factors in NB. Our results demonstrate that WSB1 copy number correlates with its expression level and that its high expression associates with good prognosis suggesting a possible role of this gene in the biology of favorable outcome NB. This article contains Supplementary Material available at http://www. interscience.wiley.com/jpages/1045-2257/suppmat. Published 2006 Wiley-Liss, Inc. {

In this study, gene expression profiling was performed on 103 neuroblastoma (NB) tumors, stages 1–4 with and without MYCN amplification, using cDNA microarrays containing 42 578 elements. Using principal component analysis (PCA) to analyse the relationships among these samples, we confirm that the global patterns of gene expression reflect the phenotype of the tumors. To explore the biological processes that may contribute to increasing aggressive phenotype of the tumors, we utilized a statistical approach based on PCA. We identified a specific subset of the cell cycle and/or chromosome segregation genes that distinguish stage 4 NB tumors from all lower stage tumors, including stage 3. Furthermore, the control of the kinetochore assembly emerges from the Gene Ontology analysis as one of the key biological processes associated with an aggressive NB phenotype. Finally, we establish that these genes are further upregulated in the most aggressive MYCN-amplified tumors.

Background Recurrent non-random genomic alterations are the hallmarks of cancer and the characterization of these imbalances is critical to our understanding of tumorigenesis and cancer progression. Results We performed array-comparative genomic hybridization (A-CGH) on cDNA microarrays containing 42,000 elements in neuroblastoma (NB). We found that only two chromosomes (2p and 12q) had gene amplifications and all were in the MYCN amplified samples. There were 6 independent non-contiguous amplicons (10.4–69.4 Mb) on chromosome 2, and the largest contiguous region was 1.7 Mb bounded by NAG and an EST (clone: 757451); the smallest region was 27 Kb including an EST (clone: 241343), NCYM, and MYCN. Using a probabilistic approach to identify single copy number changes, we systemically investigated the genomic alterations occurring in Stage 1 and Stage 4 NBs with and without MYCN amplification (stage 1-, 4-, and 4+). We have not found genomic alterations universally present in all (100%) three subgroups of NBs. However we identified both common and unique patterns of genomic imbalance in NB including gain of 7q32, 17q21, 17q23-24 and loss of 3p21 were common to all three categories. Finally we confirm that the most frequent specific changes in Stage 4+ tumors were the loss of 1p36 with gain of 2p24-25 and they had fewer genomic alterations compared to either stage 1 or 4-, indicating that for this subgroup of poor risk NB requires a smaller number of genomic changes are required to develop the malignant phenotype. Conclusions cDNA A-CGH analysis is an efficient method for the detection and characterization of amplicons. Furthermore we were able to detect single copy number changes using our probabilistic approach and identified genomic alterations specific to stage and MYCN amplification.

Treatment of Wilms tumor has a high success rate, with some 85% of patients achieving long-term survival. However, late effects of treatment and management of relapse remain significant clinical problems. If accurate prognostic methods were available, effective risk-adapted therapies could be tailored to individual patients at diagnosis. Few molecular prognostic markers for Wilms tumor are currently defined, though previous studies have linked allele loss on 1p or 16q, genomic gain of 1q, and overexpression from 1q with an increased risk of relapse. To identify specific patterns of gene expression that are predictive of relapse, we used high-density (30 k) cDNA microarrays to analyze RNA samples from 27 favorable histology Wilms tumors taken from primary nephrectomies at the time of initial diagnosis. Thirteen of these tumors relapsed within 2 years. Genes differentially expressed between the relapsing and nonrelapsing tumor classes were identified by statistical scoring (t test). These genes encode proteins with diverse molecular functions, including transcription factors, developmental regulators, apoptotic factors, and signaling molecules. Use of a support vector machine classifier, feature selection, and test evaluation using cross-validation led to identification of a generalizable expression signature, a small subset of genes whose expression potentially can be used to predict tumor outcome in new samples. Similar methods were used to identify genes that are differentially expressed between tumors with and without genomic 1q gain. This set of discriminators was highly enriched in genes on 1q, indicating close agreement between data obtained from expression profiling with data from genomic copy number analyses. © 2004 Wiley-Liss, Inc.

We recently reported that the first detectable expression of SMC-specific proteins during coronary smooth muscle cell (CoSMC) differentiation from isolated proepicardial cells was restricted to cells undergoing epithelial-to-mesenchymal transformation (EMT). The objectives of this study were to examine more closely the relation between actin cytoskeletal rearrangements and serum response factor (SRF)-dependent transcription, and to specifically test whether rhoA‐GTPase signaling is required for CoSMC differentiation. We report here that PDGF-BB stimulates EMT and promotes SRF-dependent expression of SMC marker genes calponin, SM22a, and SMgactin (SMgA) in proepicardial cells. C3 exoenzyme or rhoGDI, inhibitors of rhoA signaling, blocked PDGF-BB-induced EMT, prevented actin reorganization into stress fibers, and inhibited CoSMC differentiation. Incubation with the selective p160 rho-kinase (p160RhoK) inhibitor Y27632 (RKI) blocked EMT, prevented the appearance of calponin and SMgA-positive cells, and abolished expression and nuclear localization of SRF. To test the role of RhoK signaling for CoSMC differentiation in vivo, quail proepicardial organs (PEOs) were pretreated with RKI or vehicle and then grafted into age-matched host chick embryos to produce a chimeric epicardium. The ability of grafted cells to participate in coronary vessel formation was monitored by staining with antibodies for quail cell nuclear antigen and SMC marker proteins. Proepicardial cells pretreated with RKI failed to form CoSMCs in vivo. Time course studies traced this deficiency to a failure of epicardial-derived mesenchymal cells to migrate into or survive within the myocardium. In summary, these data point to important roles for rhoA‐RhoK signaling in molecular pathways controlling cytoskeletal reorganization, SRF-dependent transcription, and cell survival that are required to produce CoSMCs from proepicardial cells. © 2001 Elsevier Science

Background: Our ongoing genome-wide association study (GWAS) in neuroblastoma has identified multiple common and rare polymorphisms highly associated with disease susceptibility and phenotype. SNPs at the LMO1 locus showed one of the most robust associations and were associated with LMO1 overexpression and oncogenicity in preclinical models (Nature, 2011). Here we seek to define the DNA polymorphism(s) responsible for the GWAS association and determine the mechanism by which common variation determines tumor aggressiveness. Methods: Using 1000 Genomes data, we imputed all known variants within a 500-kilobase window around the LMO1 gene in a discovery set of 2,101 cases and 4,202 controls of European ancestry using the IMPUTE2 algorithm and performed association testing with SNPTEST. We further prioritized significant variants by evolutionary conservation based on comparative genome analysis of 46 mammalian species. Identification of non-coding variant regions with regulatory potential was performed through analysis of neuroblastoma-specific DNase I hypersensitivity mapping and chromatin immunoprecipitation (ChIP) sequencing data generated through the ENCODE consortium. Identification of canonical transcription factor binding motifs was performed using the MATCH-TM algorithm and JASPAR motif database. Independent replication of the top candidate causal SNP was performed by PCR-based genotyping in case series from Italy (420 cases, 751 controls) and the United Kingdom (369 cases, 1,108 controls). For statistical testing, homozygous and heterozygous odds ratios (OR) were computed from a comparison of (TT vs. GG) and (GT vs. GG) genotypes, respectively, and a single chi-squared p-value was computed from the 2x3 contingency table of phenotype vs. genotype. Results: The most significantly associated SNP at the LMO1 locus (rs2168101, homozygous OR=0.55, heterozygous OR=0.69, p-value=6.18x10-21) resides within an active enhancer region inferred by DNase I sequencing and histone modification ChIP-seq profiling in the SKNSH neuroblastoma cell line. Furthermore, rs2168101 lies within a GATA motif with perfect evolutionary conservation (5′-AGATAA-3′, phastCons score=100% across 46 mammalian species) that robustly binds to GATA2 and GATA3 transcription factors by ChIP-seq profiling. Consistent with an enhancer model, the risk “G” allele preserves the GATA motif and is associated with higher levels of LMO1 expression across a panel of 24 neuroblastoma cell lines (t-test p=0.047). Additionally, primary tumors with heterozygous G/T genotypes exhibit a greater degree of allele-specific expression imbalance relative to homozygous tumors by RNA-sequencing (t-test p=4.42x10-4), consistent with an effect in cis. In follow-up to imputation-based analysis, robust replication of the rs2168101 neuroblastoma association was observed by direct genotyping in an independent Italian cohort (homozygous OR=0.40, heterozygous OR=0.57, chi-squared p=9.94x10-6) and UK cohort (homozygous OR=0.31, heterozygous OR=0.51, chi-squared p=2.44x10-9). Furthermore, rs2168101 genotype was also associated with high-risk neuroblastoma and outcome (homozygous OR=0.67, heterozygous OR=0.74, p-value=2.16x10-4). Conclusions: Integrative genomic analysis provides mechanistic insight into how germline variation within non-coding DNA can impact neuroblastoma susceptibility and prognosis, supporting the hypothesis that the risk “G” allele at rs2168101 promotes oncogenic LMO1 overexpression through increased GATA transcription factor binding within an active enhancer region. Functional validation experiments are currently underway, including ChIP-PCR of the enhancer locus and targeted genome editing of the GATA binding site. Citation Format: Derek A. Oldridge, Andrew Wood, Cindy Winter, Maura Diamond, Ian Crimmins, Shile Zhang, Jun Wei, Javed Khan, Mario Capasso, Nazneen Rahman, Sharon J. Diskin, John M. Maris. A noncoding polymorphism in a GATA-containing enhancer element drives the association of LMO1 with neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A27.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. With the development of multimodal chemotherapy regimens, relapse-free survival rates have improved to 70-80% in patients with localized disease albeit with significant toxicity. Despite these gains, survival for those patients with metastatic or recurrent disease remains dismal. Further characterization of the genetic events underlying this tumor type is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. In a collaborative effort between the National Cancer Institute, the Children's Oncology Group, and the Broad Institute, we used a combination of whole-genome, whole-exome and transcriptome sequencing along with high resolution SNP arrays to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two distinct genotypes are evident in RMS tumors; those characterized by the PAX3 or PAX7 fusion proteins that includes novel fusions with cryptic partners (35% of cases) and those that lack a PAX3/7 fusion but harbor mutations in key signaling pathways (65% of cases). Consistent with other pediatric cancer types, the overall burden of somatic mutations in RMS is relatively low (0.31 somatic protein coding mutations per megabase) especially in tumors that harbor a PAX3/7 gene fusion (0.1 somatic mutations per megabase). In addition to genes previously reported as altered in RMS including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1 we discovered novel recurrent mutations in FBXW7, and the chromatin remodeling gene BCOR, which provide new potential avenues for therapeutic intervention. Transcriptome analysis showed that 58% of the verified genomic mutations were expressed with a marked enrichment in cell cycle (P=2e-6), protein phoshorylation (P=6.9e-5) and muscle cell differentiation (P=3.3e-4) and many of the tumors appear to accumulate multiple genetic hits within these same pathways. Finally, we identify alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of RMS cases which provides a framework for genomics-directed therapies that might improve outcomes for RMS patients. Citation Format: Jack F. Shern, Li Chen, Juliann Chmielecki, Jun Wei, Rajesh Patidar, Young Song, Hongling Liao, Andy Brohl, Daniel Catchpoole, Thomas Badgett, Gad Getz, Jaume Graupera, James Anderson, Stephen X. Skapek, Frederic G. Barr, Douglas S. Hawkins, Javed Khan. Integrative genome and transcriptome sequencing defines the landscape of genetic alterations underlying pediatric rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A21.

Despite improvement of survival using multimodal chemo- and immunotherapy, high mortality and morbidity is still substantial for pediatric patients with metastatic cancers. Recent large-scale sequencing studies of pediatric tumors including rhabdomyosarcoma (RMS) and neuroblastoma (NB) have been focusing on somatic mutations, and revealed a low somatic mutation rate and surprisingly few recurrently somatic mutated genes in these childhood tumors. Currently, only a small portion of pediatric cancer cases can be explained by somatic driver events; whereas the cause for the majority of these diseases remains unknown. Because both these two types of tumors are uncommon, here we hypothesize that infrequent germline mutations (frequency To test this hypothesis, we utilized sequencing data from two cancer patient cohorts consisting of RMS (n=133) and NB (n=222) patients, of which latter is a part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative for pediatric cancers. First, high-quality protein-coding changing single nucleotide variants (SNVs) were called in both paired germline and tumor genomic DNAs. Then we excluded common variants with frequency >5% in a normal human population using the 1000 Genomes data. Due to our interest in the enriched variants, we further required the frequencies of variants in our rhabdomyosarcoma and neuroblastoma patient cohorts are higher than those in the ESP dataset, a non-cancer control population comprising 6503 individuals. There are 63247 SNVs fulfilled these selection criteria. Among them, 1589 have been reported in these pediatric cancers or in other malignancies in the Cancer Genome Atlas (TCGA) project; and 1178 variants are present in the Human Gene Mutation Database (HGMD). Of these HGMD variants, 49 have been reported in human diseases and 34 of them are known disease-causing mutations for human cancers and genetic disorders including TP53, ALK, CHEK2, and PINK1. Interestingly, the most frequent germline mutations in these pediatric tumors were rarely found in the TCGA project which mostly consists of adult cancers. This observation suggests a very different genetic background of pediatric cancer patients from that of the adult cancers, and warrants a careful examination of germline mutations in these cancers. Furthermore, previous studies have highlighted the importance of expression of variant genes (including tumor suppressor genes) for identification of driver mutations in cancers. Therefore we will use 178 transcriptome sequencing experiments available for these tumors (RMS=84; NB=93) to identify expressed variants in tumor. Statistical and pathway analyses are currently underway to determine potential pathological or casual germline mutations associated with neuroblastoma and rhabdomyosarcoma. Citation Format: Jun S. Wei, Rajesh Patidar, John Shern, Shile Zhang, Trevor Pugh, Sharon J. Diskin, Sivasish Sindiri, Young K. Song, Hongling Liao, Xinyu Wen, Jianjun Wang, Stephen X. Skapek, James R. Anderson, Frederic G. Barr, Robert C. Seeger, John M. Maris, Douglas S. Hawkins, Javed Khan. Systematic identification of germline mutations in rhabdomyosarcoma and neuroblastoma using massively paralleled sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5081. doi:10.1158/1538-7445.AM2014-5081

Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes.

Neuroblastoma is the most common extra-cranial solid tumor of childhood, and is one of the most genomically heterogeneous malignances studied to date. Despite advance of multimodal therapies, the current overall survival rate for patient with metastatic neuroblastoma is Citation Format: Jun S. Wei, Peter Johansson, Li Chen, Young K. Song, Catherine Tolman, Samuel Li, Laura Hurd, Rajesh Patidar, Xinyu Wen, Thomas C. Badgett, Adam Cheuk, Jean-Claude Marshall, Patricia Steeg, José Pedro Vaqué Díez, J Silvio Gutkind, Javed Khan. Whole genome and transcriptome sequencing identifies an activating mutation of LPAR1 in a patient with metastatic neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2619. doi:10.1158/1538-7445.AM2013-2619

Neuroblastoma (NBL) is a cancer arising from the peripheral nervous system and is the most common solid tumor outside the brain in infants and children. Despite improved treatment over the past 20 years, only 45% of those with high-risk disease survive emphasizing the need for new molecular targets for therapy. To identify genetic mutations that encode such targets, we present an analysis of 162 whole exome sequences from 81 tumor/normal pairs from high-risk NBL patients (N=27 with MYCN amplification). From each sample, genomic DNA fragments encoding 185,961 exons from 18,380 genes were isolated using in-solution hybrid capture and sequenced using the Illumina sequencing platform to a median of 195X coverage. A wide diversity of somatic variation was observed with non-silent mutation counts ranging from 5 to 149 per tumor (median=24). Novel hyper-mutation phenotypes (> 125 candidate coding somatic mutations) were observed in four tumors including one case with both a deletion and a truncating mutation in MLH1. Several genes were mutated across the set at a statistically significant level including ALK (6 tumors, q=0.006) and PTPN11 (3 tumors, q=0.012). There are 27 other genes with candidate mutation frequencies in the 4-10% range, but no single gene identified to date with a mutation frequency >10%. Importantly, multiple paired blood samples (acquired at diagnosis) have sufficient numbers of circulating tumor DNA to potentially interfere with our SNV detection algorithm; refinement of the analytic pipeline using SNP-derived “contamination” metrics is ongoing. We anticipate validating candidate somatic candidates using an orthogonal genotyping method and further defining their true frequency in an expanded sample set that includes non-high-risk cases as well. Finally, we plan a concerted effort to catalogue potentially detrimental germline variations in order to further extend our understanding of the heritable component of NBL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4756. doi:10.1158/1538-7445.AM2011-4756

Casz1 is a C2H2 Zn-finger transcriptional factor that localizes to a region of LOH on Chr1p36.22 in tumors of Neuroblastoma patients who have a poor prognosis. Moreover, high CASZ1 expression is found in tumors of patients with a good prognosis while tumors from patients with a poor prognosis express low levels of CASZ1 (58 patients, p= 2.07×10-6). Studies indicate that CASZ1 functions as a tumor suppressor gene. Our finding that treatment of NB cells with HDAC inhibitors results in a 10-fold increase in CASZ1 expression suggests that epigenetic silencing contributes to the functional loss of CASZ1 expression from the remaining Chr1p36.22 allele. In studying the mechanism of CASZ1 epigenetic silencing, we found a bivalent chromatin modification region around the CASZ1 Transcription Start Site (TSS) that contains both a H3K27 methylation mark as well as an H3K4 methylation mark. The H3K27 methylation is thought to be a gene repressive marker, which is catalyzed by Polycomb repressor complex 2 (PRC2) proteins. PRC2 contains three core components, EZH2, EED and SUZ12, in which EZH2 (enhancer of zeste 2) is a methyltransferase catalyzing H3K27 methylation. Using Chromatin Immunoprecipitation (ChIP) we identified a 5-fold decrease in binding of EZH2, EED and SUZ12 to the bivalent region after HDAC inhibitor treatment. Consistent with decreased PRC2 binding is the finding of decreased tri-methylation of H3K27 and an increase in H3K4me3, a histone mark associated with transcriptionally active genes. The changes in histone modifications are associated with the activation of the CASZ1 gene. RNA silencing of EZH2 also leads to increases in CASZ1 expression. Consistent with this, CASZ1 expression is also up-regulated (3- fold) in EZH2 knockout MEFs compared to EZH2 wild type MEFs. This indicates that CASZ1 expression on the remaining allele is silenced by epigenetic modifications mediated by EZH2. High expression of EZH2 and EED are also found in tumor samples of neuroblastoma patients with a poor prognosis (58 patients, p=2.6×10-3 for EED and p=5.87×10-4 for EZH2). This is consistent with a model in which aberrant epigenetic gene silencing of the tumor suppressor gene CASZ1 contributes to neuroblastoma tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4880.

Introduction: High-risk neuroblastoma (H-NB) is an aggressive tumor of the sympathetic nervous system for which there are few predictors of therapeutic response. We sought to determine the association of global alternative splicing (AS) gene expression patterns with NB aggressiveness and MYCN gene status. Methods: As part of the NCI-sponsored Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, we profiled 190 primary human NBs using Affymetrix Human Exon arrays. Tumors were obtained from children who participated in Children's Oncology Group trials with informed consent. The high-risk cohort included tumors with (H-NBA n=48) and without MYCN amplification (H-NBN n=111), a key molecular determinant of clinical aggressiveness. Twenty-four samples from low-risk NB lacking MYCN amplification (L-NB) were also profiled as a comparison group. Robust multichip analysis (RMA) was used for summarization of probe selection regions (PSR) and transcript regions. Finding isoforms using RMA (FIRMA) was used to generate PSR isoform scores. After an unsupervised pre-filtering step based on coefficient of variation, ANOVA analyses were conducted comparing H-NBA or H-NBN against the L-NB group for differential transcript and isoform expression. Last, gene set enrichment analyses (GSEA) comparing high-risk to low-risk NBs were conducted using unfiltered transcript data and the Molecular Signature Database's (MsigDB) canonical gene sets. Results: 1914 out of the 22,011 core transcripts passed our strict prefiltering criteria that required a high coefficient of variation. There was a larger number of genes with significant differential expression between H-NBA vs. L-NB than H-NBN vs. L-NB (23.5% versus 4.6%, P Conclusion: Significant differences in gene expression and pathway expression are identified among H-NBA compared to H-NBN tumors. In addition, we show for the first time a high degree of alternative splicing events in the H-NBA tumors. These findings could aid in understanding the regulatory networks in the two subgroups of high-risk neuroblastomas and suggest possible therapeutic targets for further validation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5249.