bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract B02: 5-Hydroxymethylcytosine profiles in circulating cell-free DNA are biomarkers of disease burden in children with neuroblastoma
Authors: Applebaum Mark A. , Barr Erin K. , Karpus Jason , Oliva Meritxell , Chlenski Alexandre , Salwen Helen R. , Wilkinson Emma , Dobratic Marija , Sokol Elizabeth A. , Stranger Barbara E. , He Chuan , Cohn Susan L. 
Published: August 2020

Abstract
Background: 5-Hydroxymethylcytosine (5-hmC) is an epigenetic marker of open chromatin and active gene expression. The level of 5-hmC expression serves as a biomarker in adults with cancer, and we recently demonstrated the prognostic significance of tumor whole-genome 5-hmC profiling in children with neuroblastoma. Here, we used Nano-hmC-Seal technology to profile 5-hmC in circulating cell-free DNA (cfDNA) in blood samples from patients with neuroblastoma and age-matched healthy children.
Methods: Plasma was collected from 32 children with neuroblastoma at various time points during therapy and 34 well children. 5-hmC sequencing was performed using 100ng of cfDNA extracted from plasma. Metastatic disease burden was classified as high, moderate, low, or none based on Curie MIBG scores, the percentage of tumor cells in bone marrow samples, and/or measurable disseminated soft tissue disease.
Results: One hundred cfDNA 5-hmC profiles were generated from 27 high-risk, 3 intermediate-risk, and 2 low-risk patients and 34 well controls. Hierarchical clustering based on 5-hmC levels of 347 genes separated the samples into three main clusters corresponding to high, moderate, and low/no disease burden in the bone, bone marrow, and/or soft tissue. Using functional gene pathways, two subsets of patients within the low/no cluster were revealed that correlated with low tumor burden or no clinically detectable metastatic disease (sensitivity 70%, specificity 76.9%). 5-hmC profiles tracked closely with changes in disease status in 15 patients with serial samples. Samples from patients with a detectable tumor burden had genes with elevated 5-hmC in pathways of neuronal stem cell maintenance and the epigenetic regulatory complexes PRC2 and CTCF/cohesion.
Conclusions: cfDNA 5-hmC profiles correlate with disease burden in children with neuroblastoma and may serve as sensitive biomarkers of treatment response. Analysis of transcriptional networks regulated by these epigenomic modifications may lead to a deeper understanding of the pathways that drive resistance to treatment.
Citation Format: Mark A. Applebaum, Erin K. Barr, Jason Karpus, Meritxell Oliva, Alexandre Chlenski, Helen R. Salwen, Emma Wilkinson, Marija Dobratic, Elizabeth A. Sokol, Barbara E. Stranger, Chuan He, Susan L. Cohn. 5-Hydroxymethylcytosine profiles in circulating cell-free DNA are biomarkers of disease burden in children with neuroblastoma [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 B02.
Abstract B06: Candidate differentiation stall in epithelial mesenchymal transition in H3K27M diffuse midline glioma
Authors: Cheney Allison R. , Sanders Lauren M. , Seninge Lucas , Beale Holly C. , Kephart Ellen Towle , Pfeil Jacob , Learned Katrina , Lyle A. Geoffrey , Bjork Isabel , Haussler David , Salama Sofie R. , Vaske Olena M. 
Published: August 2020

Abstract
The purpose of our study was to elucidate the molecular mechanisms by which the histone H3 K27M mutation drives tumorigenesis of pediatric gliomas. Though it has potential implications on the treatment of diffuse midline glioma as a disease driver, the timing, cell type of origin, and effect of the H3K27M mutation on early embryonic brain development are not fully characterized. Here, we performed differential expression analysis on a cohort of H3K27M and H3WT pediatric gliomas, revealing that genes in the epithelial-mesenchymal transition (EMT) pathway were significantly differentially expressed. SNAI1, the EMT master regulator, was significantly overexpressed in the H3K27M tumor cohort. Overall, pre-EMT genes were overexpressed in H3K27M tumors while post-EMT genes were underexpressed. We hypothesized that H3K27M may lead to gliomagenesis by stalling an EMT in early brain development and employed single-cell and bulk RNA sequencing data from cerebral organoids at multiple developmental timepoints to test this hypothesis. We observed that a long noncoding RNA (lncRNA) signature identified as transiently expressed in early brain development was preferentially expressed in H3K27M tumors. Cell type-specific lncRNA signatures had higher expression in H3K27M tumors for pre-EMT cell types, and higher expression in H3WT tumors for post-EMT cell types. Finally, t-SNE clustering of single-cell glioma RNA sequencing data with single-cell organoid data revealed transcriptional similarities between H3K27M and pre-EMT neural stem cells. In conclusion, we observed aberrant activity of the EMT in H3K27M gliomas. Our data suggest that the H3K27M mutation is associated with a pre-EMT cell phenotype, and that this mutation may cause EMT arrest or de-differentiation.
Citation Format: Allison R. Cheney, Lauren M. Sanders, Lucas Seninge, Holly C. Beale, Ellen Towle Kephart, Jacob Pfeil, Katrina Learned, A. Geoffrey Lyle, Isabel Bjork, David Haussler, Sofie R. Salama, Olena M. Vaske. Candidate differentiation stall in epithelial mesenchymal transition in H3K27M diffuse midline glioma [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 B06.
Abstract B12: PAX3 translocations co-opt super enhancers and intrinsically disordered fusion partners in rhabdomyosarcoma
Authors: Gryder Berkley E. , Wachtel Marco , Ewert Winston , Chang Kenneth , El Demerdash Osama , Song Young , Schäfer Beat W , Vakoc Christopher R. , Khan Javed 
Published: August 2020

Abstract
Chromosomal translocations drive many types of childhood cancer. Fusion-positive rhabdomyosarcoma (FP-RMS) tumors are found most frequently with a fusion between PAX3 and FOXO1, but less frequent translocation partners have been discovered, including INO80D and NCOA1. We hypothesized that all FP-RMS translocations are selecting simultaneously for (1) enhancers active in a myoblast-like epigenome and (2) protein partners with high-levels of intrinsic disorder to give PAX3 enhanced transcriptional strength. Analysis of primary tumors revealed these diverse PAX3 fusions recapitulate a near-identical transcriptome, suggesting uniform underlying molecular mechanisms. ChIP-seq evidence from cell lines and primary tumors suggested that in all FP-RMS tumors, large super-enhancer (SE) elements were present near each chosen translocation partner (distal to FOXO1, INO80D, or NCOA1). Using tools to determine the 3-D folding of chromatin (3C, 4C-seq, and HiChIP), we discovered an extensive network of hijacked FOXO1 enhancers and a SE that physically interact together and with the PAX3 promoter, only in PAX3-FOXO1 positive cells. Furthermore, pooled CRISPR tiling of cis-regulatory elements revealed special dependence on the FOXO1 SE, or certain CTCF boundary elements that facilitate enhancer interactions. ChIP-seq paired to short-term CRISPR experiments shows PAX3-FOXO1 transcription depends on an extended network of related enhancers distal to FOXO1. We find these enhancers are unique to early myoblast stages of differentiation and are bound by myogenic TFs in RMS, suggesting miswiring of normal myogenic enhancer logic. While many SE-driven genes exist in FP-RMS, most are never selected for translocation partners. We found that FOXO1, INO80D, and NCOA1 are all highly disordered proteins, a theme they hold in common despite having no amino acid sequence homology. Initial evidence suggests that PAX3-FOXO1 enables the formation of phase condensates in the nucleus that recruit high-levels of transcriptional machinery such as BRD4. Ongoing work is exploring the mechanistic chemical determinants of these interactions, and how phase condensates are manipulated by drugs that block PAX3-FOXO1’s transcriptional output. Together our studies are illuminating new paradigms for understanding how fusion transcription factors drive cancer.
Citation Format: Berkley E. Gryder, Marco Wachtel, Winston Ewert, Kenneth Chang, Osama El Demerdash, Young Song, Beat W Schäfer, Christopher R. Vakoc, Javed Khan. PAX3 translocations co-opt super enhancers and intrinsically disordered fusion partners in rhabdomyosarcoma [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 B12.
Abstract B24: Dysregulated Transcription Drives Splicing Defect in Ewing Sarcoma
Authors: Lawrence L. , Gorthi A. , Miller H. , Bishop A. J. R. 
Published: August 2020

Abstract
Ewing sarcoma (EwS) is a pediatric and young adult cancer that is driven by the EWSR1-FLI1 translocation. Despite decades of work, this cancer is still an enigma, with poorly understood biology and no targeted treatments. Our recent work published in Nature demonstrated a previously overlooked consequence of EWSR1-FLI1, that this fusion causes hyperphosphorylated RNA polymerase II (pRNAPII) due to loss of EWSR1 inhibition of CDK7 and CDK9. We observed high levels of transcription, with high levels of R-loops present in locations that R-loops normally (physiologically) occur. Based upon these findings, we began to reconsider cellular phenotypes of EwS to identify the molecular basis of these phenotypes and ask whether these changes provide a fundamental defect in all EwS. One phenotype that was previously identified in EwS is that these cells display altered splicing profiles. In our prior work we reported that the normal dephosphorylation of pRNAP in response to damage does not occur, and this results in trapping of BRCA1 protein with the active transcription complex. It was recently shown that the splicing machinery is bound to pRNAPII. This observation raises the interesting possibility that in EwS, like BRCA1 protein, the splicing machinery is also sequestered with pRNAPII. Interestingly, in recent years, there have been several reports linking R-loops to splicing, with splicing defects causing R-loop accumulation and R-loops being associated with sites of alternative splicing. We therefore asked if there is an association between the R-loops and alternative splicing phenotype in EwS cells and whether this results in a defect that can be leveraged to specifically target EwS cells and tumors.
Citation Format: L. Lawrence, A. Gorthi, H. Miller, A. J. R. Bishop. Dysregulated Transcription Drives Splicing Defect in Ewing Sarcoma [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 B24.
Abstract B25: HNRNPH1-dependent splicing of the fusion oncogene EWS-FLI1 reveals a targetable RNA G-quadruplex interaction
Authors: Neckles Carla , Boer Robert E. , Aboreden Nicholas , Cross Allison M. , Walker Robert L. , Kim Bong-Hyun , Kim Suntae , Schneekloth John S. , Caplen Natasha J. 
Published: August 2020

Abstract
The primary oncogenic event in ~85% of Ewing sarcomas is a chromosomal translocation that generates a fusion gene encoding an aberrant transcription factor EWS-FLI1. In a third of EWS-FLI1 driven tumors, translocations that retain exon 8 of EWSR1 must exclude this exon to synthesize a protein-encoding mRNA. We have previously demonstrated that the heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) binds to G-rich sequences within EWSR1 exon 8 and that this binding event is required to express an in-frame EWS-FLI1 mature mRNA. G-rich nucleic acids may fold into stable tertiary structures, such as RNA guanine quadruplexes (rG4s). Our current studies are focused on the tertiary RNA structure of the G-rich regions within EWSR1 exon 8 and its role in HNRNPH1-dependent RNA processing. Here, we show that the processing of distinct EWS-FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of rare transcript variants of wild-type EWSR1. We demonstrate that guanine-rich sequences within EWSR1 exon 8 can fold into RNA G-quadruplex structures and that HNRNPH1 preferentially binds these RNAs when in a folded state. Critically, we also demonstrate that transfection of a single-stranded RNA oligomer corresponding to one of these G-rich sequences (rG1) reduces the expression of EWS-FLI1 mRNA and EWS-FLI1 protein and decreases its transcriptional activity. Small molecules that can displace HNRNPH1 binding may provide a therapeutic vulnerability in a subset of Ewing sarcoma. To evaluate if this protein-RNA interaction is amenable to small molecules, we performed displacement assays with HNRNPH1 protein and an EWSR1 exon 8 RNA oligomer (rG1) at varying concentrations of a G4-binding molecule, pyridostatin (PDS). We found that PDS disrupts the formation of the HNRNPH1-rG1 complex (IC50 of 7 micromolar). Furthermore, treatment with PDS selectivity inhibits the growth of Ewing sarcoma cell lines harboring EWSR1 exon 8 fusions, decreases EWS-FLI1 activity in cell-based reporter assays, and restores mRNA expression of EWS-FLI1 deregulated transcriptional targets. Our findings illustrate that modulation of the alternative splicing of EWS-FLI1 pre-mRNA is a promising strategy for future therapeutics against this fusion oncogene expressed in a third of Ewing sarcoma.
Citation Format: Carla Neckles, Robert E. Boer, Nicholas Aboreden, Allison M. Cross, Robert L. Walker, Bong-Hyun Kim, Suntae Kim, John S. Schneekloth, Natasha J. Caplen. HNRNPH1-dependent splicing of the fusion oncogene EWS-FLI1 reveals a targetable RNA G-quadruplex interaction [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 B25.
Abstract B30: ARID1A is a haploinsufficient tumor suppressor for N-Myc transformation of neural crest cells
Authors: Wallace Kirby , Garcia-Lopez Jesus , Otero Joel , Olsen Rachelle , DeVaux Chelsea , King Ashton , Davidoff Andrew , Freeman Kevin 
Published: August 2020

Abstract
Large segmental chromosomal alterations are common to cancer and a feature of high-risk neuroblastoma (NBL). Though they are early or initiating events in cancer, including often being found in precancerous lesions, their contribution to tumorigenesis is poorly understood. An unproven model is that these changes promote cancer through the cumulative effect of multiple dosage-sensitive genes. Loss of heterozygosity (LOH) at 1p36 is a frequent structural rearrangement in a broad range of human cancers including NBL. Approximately 70% of MYCN amplified NBL have 1p36 LOH with both mutations independently contributing to tumor aggressiveness in NBL. Two tumor suppressor regions have been proposed to exist in 1p36, a distal and proximal region, which have MYCN-independent and dependent roles. Through CRISPR/Cas9 genome editing of primary mouse neural crest cells (NCCs), a source for NBL, we found that loss of the chromatin remodeling factor Chd5 conferred most of the tumor-suppressor effects of 1p36 LOH in in vitro cell transformation assays in cells with endogenous levels of N-Myc. In contrast, when N-Myc was overexpressed, tumor evolution of NCCs genome edited to have randomly sized 1p36 deletions showed a reduction in tumor latency that significantly correlated with deletion of Arid1a. The Arid1a deletions that were selected for ranged from small indels up to large 1p36 deletions, indicating that large deletions are tolerated to achieve loss of a single critical tumor suppressor. Further, using lentiviral Cre-induced deletion of floxed Arid1a in isolated NCCs, we found that Arid1a is a haploinsufficient tumor suppressor in MYCN-driven transformation of NCCs. As Arid1a is a subunit of the chromatin remodeling complex SWI/SNF, which is mutated in 20% of cancers, Arid1a synthetic lethal therapies are being developed for adult malignancies. We are currently verifying those proposed therapies in NBL and are using small-molecule screening of cells lines derived from Arid1a wild-type, het, or null tumors to identify additional synthetic lethalities. Our findings indicate that context, such as the status of MYCN as an oncogene, dictates which 1p36 gene is the critical tumor suppressor, establishing 1p36 LOH as multifaceted not cumulative, as was previously believed.
Citation Format: Kirby Wallace, Jesus Garcia-Lopez, Joel Otero, Rachelle Olsen, Chelsea DeVaux, Ashton King, Andrew Davidoff, Kevin Freeman. ARID1A is a haploinsufficient tumor suppressor for N-Myc transformation of neural crest cells [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 B30.
Abstract B42: Mutant RAS represses CASZ1, a novel regulator of MYOD and MYOG, to inhibit embryonal rhabdomyosarcoma differentiation
Authors: Liu Zhihui , Zhang Xiyuan , Lei Haiyan , Lam Norris , Yockey Oliver , Xu Max , Mendoza Arnulfo , Wei Jun S , Khan Javed , Yohe Marielle E. , Shern Jack F. , Thiele Carol J. 
Published: August 2020

Abstract
Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Zinc finger transcription factor CASZ1 is a tumor suppressor and orchestrates cell-fate specification, commitment, and differentiation in many cell types during development. CASZ1 is expressed during skeletal myogenesis; however, whether CASZ1 plays a role in skeletal muscle cell differentiation and RMS pathogenesis is unknown. Here we uncover that CASZ1 crosstalks with myogenic regulatory factors Myf5, MyoD, and Myog to regulate myogenesis in C2C12 cells. Public transcriptome data show that CASZ1 mRNA levels are lower in ERMS compared to normal muscle (p&lt;0.001). CASZ1 levels decrease upon oncogenic activation of RAS-MEK pathway and increase when ERMS undergoes terminal differentiation after MEK inhibitor trametinib (MEKi) treatment. Analysis of super-enhancer landscape in MEKi-induced differentiated ERMS cells reveals a core transcriptional regulatory circuitry (CRC) including CASZ1, MYOD, and MYOG. Loss of CASZ1 attenuates MEKi-induced myogenic programming in ERMS. Restoration of CASZ1 in ERMS cells induces muscle differentiation. ChIP-seq, ATAC-seq, and RNA-seq results show that CASZ1 directly upregulates expression of skeletal muscle differentiation genes and represses non-muscle genes through affecting chromatin accessibility, enhancer maintenance, and super-enhancer activity. Next-generation sequencing of primary RMS tumors identified a nonsynonymous single-nucleotide variant causing an arginine-to-cysteine switch (R25C). When evaluated in 293T and ERMS cells, R25C impairs CASZ1 nuclear localization and fails to activate target genes’ transcription. Tumor xenograft studies show that CASZ1 suppresses RMS tumor growth (p&lt;0.005), while the CASZ1-R25C mutant has no effect. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis.
Citation Format: Zhihui Liu, Xiyuan Zhang, Haiyan Lei, Norris Lam, Oliver Yockey, Max Xu, Arnulfo Mendoza, Jun S Wei, Javed Khan, Marielle E. Yohe, Jack F. Shern, Carol J. Thiele. Mutant RAS represses CASZ1, a novel regulator of MYOD and MYOG, to inhibit embryonal rhabdomyosarcoma differentiation [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 B42.
Abstract B43: The c-Myc/AKT1/TBX3 axis is important to target in the treatment of embryonal rhabdomyosarcoma
Authors: Sims Danica Anne , Maranyane Hapiloe Mabaruti , Peres Jade , Prince Sharon 
Published: August 2020

Abstract
Rhabdomyosarcoma (RMS), a pediatric cancer of the skeletal muscle, is the most common malignant soft-tissue sarcoma in children and adolescents. There are two main subtypes: embryonal RMS (ERMS), which is more prevalent in children and accounts for approximately 75% of RMS cases, and alveolar RMS (ARMS), which presents in adolescents and young adults and accounts for the remaining 25% of cases. Little is known about the molecular mechanisms underpinning ERMS, and there is a need for new and effective therapeutic approaches. Here we investigate the status and role of the T-Box transcription factor 3 (TBX3), which has been well characterized for its function in development and, in particular,r the congenital disorder ulnar mammary syndrome that results from its haploinsufficiency. Conversely, TBX3 has been reported to be overexpressed in numerous cancers including a range of sarcoma subtypes and shown to contribute to oncogenic processes such as cell proliferation, migration, and invasion, in vitro and in vivo tumor-forming ability. To determine the status of TBX3 in ERMS, immunohistochemistry was performed using RMS patient tissue and Western blotting on a panel of RMS cell lines compared to normal myoblast cells. Cell culture models in which TBX3 was either stably knocked down or overexpressed were established, and in vitro and in vivo assays were performed to determine the impact of TBX3 on key hallmarks of cancer including cell proliferation (growth curves), migration (scratch motility assay), as well as tumor formation and invasion in nude mice. Our data showed high TBX3 levels in RMS patient tumor tissue sections as well as in ERMS cell lines tested. Preliminary data reveal that the mechanisms involved in upregulating TBX3 in ERMS involve c-Myc at a transcriptional level and AKT1 at a post-translational level. Importantly, TBX3 was found to promote ERMS cell proliferation and migration, anchorage-independent growth, tumor formation, and metastasis. This study describes for the first time that TBX3 is an oncogene in ERMS and identifies c-Myc/AKT1/TBX3 as an important axis that could be targeted as novel therapeutic approaches for the treatment of this cancer.
Citation Format: Danica Anne Sims, Hapiloe Mabaruti Maranyane, Jade Peres, Sharon Prince. The c-Myc/AKT1/TBX3 axis is important to target in the treatment of embryonal rhabdomyosarcoma [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 B43.