bluuebunny/crossref_metadata_2025 huggingface dataset:
System Identification of Neural Signal Transmission Based on Backpropagation Neural Network
Authors: Li Xiangyu , Yuan Chunhua , Shan Bonan
Published: August 2020

The identification method of backpropagation (BP) neural network is adopted to approximate the mapping relation between input and output of neurons based on neural firing trajectory in this paper. In advance, the input and output data of neural model is used for BP neural network learning, so that the identified BP neural network can present the transfer characteristics of the model, which makes the network precisely predict the firing trajectory of the neural model. In addition, the method is applied to identify electrophysiological experimental data of real neurons, so that the output of the identified BP neural network can not only accurately fit the neural firing trajectories of neurons participating in the network training but also predict the firing trajectories and spike moments of neurons which are not involved in the training process with high accuracy.
The Influence of High‐Temperature Nitrogen Plasma‐Based Ion Implantation on Niobium Creep Behavior
Authors: Oliveira Aline Capella de , Reis Joares Lidovino dos , Oliveira Rogerio Moraes , Reis Danieli Aparecida Pereira
Published: August 2020

Niobium has been considered for applications in the aerospace sector, but its use at high temperatures is restricted, due to the great affinity of refractory metals with oxygen, which results in the formation of oxide layers and a decrease in their mechanical resistances. In the present work, Nb samples were submitted to High‐Temperature Nitrogen Plasma‐Based Ion Implantation (HT‐NPBII). The process runs at a working pressure between 3 and 4 mbar and negative high voltage pulses of 7 kV/30 μs/300 Hz were applied to samples heated to 1000°C, at treatment times of 1 h, 4 h, and 8 h, respectively. Microstructural and mechanical characterizations of the treated samples revealed the formation of a layer of Nb2N, with 3.0 μm thickness and increase in the surface hardness from 225 HV for the untreated material up to about 2498 HV, for samples treated during 8 h. Creep tests were performed at 500°C and with loads varying from 25 to 40 MPa. Results indicated a decrease in the secondary creep rate for treated specimens when compared to the untreated ones. This behavior can be attributed to the formation of a nitride layer on the surface of Nb that acts as barrier to avoid the oxygen diffusion into the material under high temperature conditions.
Detection for Multisatellite Downlink Signal Based on Generative Adversarial Neural Network
Authors: Guan Qing-yang , Shuang Wu
Published: August 2020

A method for satellite downlink signal detection based on a generative adversarial network is proposed. The generator adversarial network and adversarial network are established, respectively. The generator network realizes the local generator of satellite signals, and the adversarial network is used for high-precision signal detection. The error network is generated by the error signal to form the satellite link downlink. The network reconstructs the optimal weights by generating errors, forms an error matrix for different satellite downlink, and then forms an adaptive matrix weight adjustment. Through the reconstruction of the optimal detection matrix, detection for the downlink signals of multiple satellites is completed. The proposed generative adversarial network can realize the high-precision detection for the downlink signal.
Expression of the Androgen Receptor Governs Radiation Resistance in a Subset of Glioblastomas Vulnerable to Antiandrogen Therapy
Authors: Werner Christian K. , Nna Uchechi J. , Sun Hanshi , Wilder-Romans Kari , Dresser Joseph , Kothari Ayesha U. , Zhou Weihua , Yao Yangyang , Rao Arvind , Stallard Stefanie , Koschmann Carl , Bor Tarik , Debinski Waldemar , Hegedus Alexander M. , Morgan Meredith A. , Venneti Sriram , Baskin-Bey Edwina , Spratt Daniel E. , Colman Howard , Sarkaria Jann N. , Chinnaiyan Arul M. , Eisner Joel R. , Speers Corey , Lawrence Theodore S. , Strowd Roy E. , Wahl Daniel R.
Published: August 2020

New approaches are needed to overcome intrinsic therapy resistance in glioblastoma (GBM). Because GBMs exhibit sexual dimorphism and are reported to express steroid hormone receptors, we reasoned that signaling through the androgen receptor (AR) could mediate therapy resistance in GBM, much as it does in AR-positive prostate and breast cancers. We found that nearly half of GBM cell lines, patient-derived xenografts (PDX), and human tumors expressed AR at the transcript and protein level—with expression levels overlapping those of primary prostate cancer. Analysis of gene expression datasets also revealed that AR expression is higher in GBM patient samples than normal brain tissue. Multiple clinical-grade antiandrogens slowed the growth of and radiosensitized AR-positive GBM cell lines and PDXs in vitro and in vivo. Antiandrogens blocked the ability of AR-positive GBM PDXs to engage adaptive transcriptional programs following radiation and slowed the repair of radiation-induced DNA damage. These results suggest that combining blood–brain barrier permeable antiandrogens with radiation may have promise for patients with AR-positive GBMs.
Abstract A15: Improving immune recognition of shared tumor-associated antigens in pediatric tumors using a multimodal oncolytic virus
Authors: Ghonime Mohammed G. , Roth Justin C. , Barker Naomi J. , Miller Katherine E. , Mardis Elaine R. , Walker Christopher M. , Cassady Kevin A.
Published: August 2020

Immunotherapy is an attractive treatment approach for children because of its precision and reduced toxicity. Unfortunately, pediatric solid tumors often evade immune recognition. Like adult cancers, the immunosuppressive tumor microenvironment can restrict immune activity. Further complicating this, pediatric cancers have low mutation rates, creating fewer antigenic targets for the immune response. We therefore developed a multimodal oncolytic virus that harnesses the antiviral immune response and redirects it against shared tumor-associated antigen (TAA) in the tumor. Engineering a TAA, Ephrin A2 (EphA2), into the oncoviral (OV) genome circumvents transcriptional/translational arrest and allows TAA overexpression during infection. Our results show that virus-based EphA2 expression (C57BL/6 sequence) induces an immune-mediated antitumor response in syngeneic C57BL/6-based brain and peripheral tumor models, improving survival (e.g., CT2A brain tumor median survival: 43d-TAA virus vs. 30d-parent virus, 29d-Saline; overall survival: 44.4%-TAA virus vs. 12%-parent virus vs 0%-Saline, *p=0.0233). TAA-virus treatment increases CD8 memory effector-like cells in tumor infiltrates (CD62L+, CD44+) and rechallenge studies show abscopal effect in TAA-virus treated survivors. Peptide pulsing studies using splenocytes from survivors show that viral TAA expression induces a circulating EphA2-specific CD8 effector-like (CD8+, CD25+, Granzyme B+) population (14.1%-TAA virus vs 2.6%-parent virus, 0.6% Saline, **p≤0.0098). This multimodal viral platform harnesses the oncolytic and immunostimulatory properties of a next-generation OV to enhance immune activity against tumors expressing shared tumor antigens. Our results suggest that this flexible viral-based platform provides an effective in situ antitumor vaccination approach and could be engineered against multiple antigens for low mutational load tumors.

Citation Format: Mohammed G. Ghonime, Justin C. Roth, Naomi J. Barker, Katherine E. Miller, Elaine R. Mardis, Christopher M. Walker, Kevin A. Cassady. Improving immune recognition of shared tumor-associated antigens in pediatric tumors using a multimodal oncolytic virus [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 A15.
Abstract A20: Single-cell immune TCR repertoire profiling in the context of immunotherapy by using three 10x Genomics libraries
Authors: Qi Yuan , Im Jin Seon , Su Xiaoping
Published: August 2020

10x Genomics single-cell sequencing provides a comprehensive and scalable solution for cell characterization and gene expression profiling of hundreds to tens of thousands of cells. For performance comparison of single-cell immune TCR repertoire profiling, we tested three libraries: (1) 5-prime gene expression, (2) direct enrichment for TCR, and (3) post-cDNA-amplification enrichment for TCR, on two invariant natural killer T (iNKT) cells, on B240 and B241, with iNKT purity at 90% and 45%, respectively. We also performed correlation analysis between gene expression data and clonotype data by library 3. About 1,100 cells of B240 and 580 cells of B241 were sequenced. The numbers of productive clonotypes of B240 were identified as 165, 284, and 332 for the three libraries, respectively, while 47 clonotypes overlapped among the libraries. The numbers of productive clonotypes of B241 were 88, 152, and 148, respectively, while 35 clonotypes overlapped. Each sample is a mixture of iNK T cells and non-invariant NK T cells. We were able to identify the clusters of iNKT cells using the 5’ gene expression data by library 1 in tSNE plots for both B240 and B241. More importantly, the clusters correlated strongly with the TCR clonotypes identifying iNKT cells by library 3. Based upon our data, we noticed that the number of clonotypes identified is proportional to the number of cells sequenced regardless of libraries. Library 1 has low sensitivity in terms of clonotype detection, due to un-enrichment of TCR genes, and mainly detected single chain in each cell (beta chain most of the time). The other two libraries are able to identify high and similar number of clonotypes. However, library 3 is able to associate gene expression pattern with TCR clonotype usage, which is a big advantage over the other two libraries for 10x Genomics single-cell immune TCR repertoire profiling.

Note: This abstract was not presented at the conference.

Citation Format: Yuan Qi, Jin Seon Im, Xiaoping Su. Single-cell immune TCR repertoire profiling in the context of immunotherapy by using three 10x Genomics libraries [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 A20.
Abstract A22: YAP1 knockout in vitro models of medulloblastoma showed improved response to sonidegib
Authors: Cruzeiro Gustavo Alencastro Veiga , de Almeida Magalhães Taciani , de Sousa Ribeiro Graziella , Ferreira das Chagas Pablo , Silva Ricardo Bonfim , Chatterje Sampurna , Scrideli Carlos Alberto , Tone Luiz Gonzaga
Published: August 2020

Background: Medulloblastoma (MB) is the most common malignant brain tumor in childhood. Despite improvement in current treatment, children still suffer from long-term sequelae resulting from standard care therapy. World Health Organization considers MB as 4 distinct entities: SHH, WNT, Group 3, and Group 4. The most aggressive variant of SHH MB is the bearer of TP53 mutation and it is resistant to standard chemo/radiation therapy. Therefore, novel treatments modalities are urgently needed. In this study we aimed to evaluate the biologic relevance of YAP1 in SHH MB human cell lines bearers of TP53 mutation and its potential as a new therapeutic target.

Methods: We generated YAP1 CRISPR/CAS9 knockout single-cell clones of human cell lines DAOY and UW228 assigned as “MB SHH-like” and bearers of TP53 mutation. We picked sonidegib as a standard-care chemotherapy for in vitro SHH MB model and assessed the apoptosis/necrosis using Annexin V/Propidium-Iodide staining to identify positive cells through flow cytometry. Proliferation and colony formation were assessed by cell counting Kit 8 proliferation assay and GIEMSA staining, respectively. Additionally, we used verteporfin, a YAP1 inhibitor, and evaluated its effect on cell proliferation.

Findings: Using single-cell cloning, we successfully generated DAOY and UW228 cell lines with depleted YAP1 protein. YAP1 knockout cell lines DAOY and UW228 showed improved response to sonidegib regarding i) decrease in proliferation (p<0.05), ii) apoptosis induction (p<0.01), and iii) colony formation abrogation (p<0.001). In order to step towards translation, we tested verteporfin (YAP1 inhibitor) and found that this compound successfully reduced proliferation in both cell lines (p<0.05).

Conclusion: Although YAP1 depletion showed association with better response to sonidegib, a smoothened inhibitor, more experiments are needed to evaluate if the in vitro findings might shed new light on a potential synergistic combination between sonidegib and verteporfin and provide a good rationale for targeted therapy.

Citation Format: Gustavo Alencastro Veiga Cruzeiro, Taciani de Almeida Magalhães, Graziella de Sousa Ribeiro, Pablo Ferreira das Chagas, Ricardo Bonfim Silva, Sampurna Chatterje, Carlos Alberto Scrideli, Luiz Gonzaga Tone. YAP1 knockout in vitro models of medulloblastoma showed improved response to sonidegib [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 A22.
Abstract A33: HIF2 in pediatric high-grade glioma and its targeting
Authors: Fuchs Quentin , Blandin Anne Florence , Rebel Isabelle Lelong , Pierrevelcin Marina , Dontenwill Monique , Entz-Werlé Natacha
Published: August 2020

Pediatric high-grade gliomas (pHGGs) represent a very dismal disease that is needing new innovative compound for treatment. Despite the past discovery of histone H3 driver mutations, we are not able for instance to stop this induced process on epigenetic remodulation. Therefore, there is since a decade proactive works aimed to discover new targetable proteins in these tumors. In our recent previous works in pHGGs, we highlighted HIF2alpha as a biomarker of worse prognosis and outcome and a key in treatment resistance. Therefore, this new project was designed to determine in several patient-derived cell lines (5 PDCLs) the presence of HIF2alpha, its role and its induction in normoxic and hypoxic microenvironment concomitantly by immunofluorescence and Western blot assessments and RNA sequencing analyses. Complementary ChipSeq was also performed using HIF2 antibodies to determine its role as a transmission factor. Specific promoters were involved. After the confirmation of its frequent presence in multiple PDCLs initiated from thalamic pHGGs and DIPG, we used allosteric inhibitors to target HIF2alpha. Surprisingly, this protein was expressed constantly in hypoxic and normoxic conditions and specifically in PDCLs bearing stem cell features. Specific expression associations were established between stemness markers and HIF2alpha. To go further, we tested specific HIF2A inhibitors, which were having an impact on cell proliferation and on the decrease of the target itself. In conclusion, HIF2 seem to be a major biomarker in pHGGs that might be targeted, and it is a useful new opportunity for pHGGs treatments.

Citation Format: Quentin Fuchs, Anne Florence Blandin, Isabelle Lelong Rebel, Marina Pierrevelcin, Monique Dontenwill, Natacha Entz-Werlé. HIF2 in pediatric high-grade glioma and its targeting [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 A33.
Abstract A38: Validation of potential therapies for treatment of fatal pediatric brain tumors DIPG and AT/RT using a novel rapid intracranial model in zebrafish
Authors: Kaur Harpreet , Guo Huizi , White David , Green Peter , Hector Alara M. , Akhtarkhavari Sepehr , Bhargava Anukriti , Shah Smit , Eberhart Charles G. , Mumm Jeffrey , Raabe Eric H.
Published: August 2020

Diffuse intrinsic pontine glioma (DIPG) and atypical teratoid/rhabdoid tumors (AT/RT) are deadly pediatric brain tumors. Developing new targets and therapeutics are urgently needed. We have previously shown that multiple primary brain tumors and cell lines express increased amounts of the epigenetic modifier and DNA binding oncoprotein high-mobility group AT-hook 2 (HMGA2). Targeting HMGA2 using short hairpins decreased AT/RT and glioma growth in xenografted mice. We hypothesized that pharmacologic inhibition of HMGA proteins using DNA minor-groove binding drugs such as quinacrine would decrease tumor growth due to displacement of HMGA proteins from DNA. Quinacrine is a safe and widely used treatment for pediatric malaria and parasitic infections. We used quinacrine in six patient-derived cell lines: three AT/RT (BT37, CHLA-05, CHLA-266) and three DIPG (JHHDIPG1, SUDIPGXIII, SF7761). Using quinacrine fluorescence as a surrogate, we can achieve micromolar concentration of quinacrine in the mouse and zebrafish brain after oral administration without overt toxicity. In both tumor cell lines, quinacrine causes a dose-dependent reduction in proliferation (BrdU) and increase in apoptosis (cleaved caspase-3 and cleaved PARP) compared to vehicle-treated cells (P<0.01). Quinacrine had no effect on growth of normal hindbrain neural stem and progenitor cells. Treatment of mice bearing ATRT flank tumors with quinacrine resulted in decreased tumor volume compared to vehicle-treated mice (P<0.05). Current mouse intracranial DIPG models can take 6-9 months due to long latency. To validate quinacrine orthotopically in DIPG, we developed a novel model in zebrafish wherein we injected fluorescent DIPG cells into the developing zebrafish blastula. Five days post injection, DIPG cells had homed to the zebrafish brain. Treatment of xenografted zebrafish with quinacrine for 48 hours decreased DIPG growth by 40% as measured by fluorescence, suggesting that minor groove binding drugs such as quinacrine are a viable treatment strategy for these tumors. Future studies are aimed at investigating the mechanism of quinacrine in these tumors.

Citation Format: Harpreet Kaur, Huizi Guo, David White, Peter Green, Alara M. Hector, Sepehr Akhtarkhavari, Anukriti Bhargava, Smit Shah, Charles G. Eberhart, Jeffrey Mumm, Eric H. Raabe. Validation of potential therapies for treatment of fatal pediatric brain tumors DIPG and AT/RT using a novel rapid intracranial model in zebrafish [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 A38.
Abstract A43: Effects of transcriptional dysregulation on the DNA damage response in Ewing’s sarcoma
Authors: Rollins Matthew G. , Schwartz Jacob C.
Published: August 2020

Ewing’s sarcoma is a soft-tissue bone malignancy characterized by a translocation event wherein the N-terminal low complexity domain of EWSR1 is fused with the DNA binding domain of the ETS transcription factor Fli1. The resulting EWS-Fli1 fusion protein drives expression of oncogenes and knockdown of tumor-suppressor proteins. More specifically, EWS-Fli1 acts as transcriptional regulator by helping recruit RNA Pol II to promoter regions, which is subsequently phosphorylated by CDK9 to escape initiation into elongation. Interestingly, Ewing’s sarcoma is particularly susceptible to chemotherapy compared to other cancers; however, the underlying reason for this sensitivity to DNA damage has not been elucidated. Given EWS-Fli1’s role in disrupting transcription, it was hypothesized that EWS-Fli1 is concurrently disrupting the DNA damage response. Here, a combination of CDK9 inhibition with DNA damage was found to drastically diminish cell viability in Ewing’s cell lines but not in non-Ewing’s osteosarcoma or HEK293 cells. Ordinarily, after DNA damage γH2A.X phosphorylation occurs on histones near sites of damaged DNA early during the DNA damage response to signal DNA damage repair initiation. Further work has shown Ewing’s cells undergoing the combination treatment above have a marked decrease in the DNA damage marker γH2A.X as compared to non-Ewing’s cells. Together, these results suggest Ewing’s sarcoma cells are sensitized to DNA damage due in part to transcriptional dysregulation.

Citation Format: Matthew G. Rollins, Jacob C. Schwartz. Effects of transcriptional dysregulation on the DNA damage response in Ewing’s 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 A43.
Abstract A57: Feasibility of physical activity in children with cancer: A multidisciplinary program in the context of the VIE study
Authors: Caru Maxime , Duhamel Gabrielle , Marcil Valérie , Sultan Serge , Meloche Caroline , Bouchard Isabelle , Drouin Simon , Bertout Laurence , Laverdière Caroline , Sinnett Daniel , Curnier Daniel
Published: August 2020

Purpose: Cancer is one of the leading causes of death in the world. The physiologic and psychological benefits of physical activity have been shown in children with cancer. However, almost one in two cancer patients do not follow the physical activity guidelines. The aim of this study will be to assess the feasibility of a physical activity program intervention in pediatric oncology and to assess the barriers and facilitators in the success or failure of this physical activity program.

Methods: The VIE (Valorization, Implication and Education) intervention is a multidisciplinary program including physical activity, nutritional, and psychological interventions in pediatric oncology. This study involves one intervention group that will be followed over two years (evaluations and physical activity interventions) and one control group that will participate in only one evaluation. The feasibility of this program will be measured through a comparison between sessions performed and sessions scheduled, while the security will be measured according to the number of reported incidents.

Preliminary Results: An adhesion rate of about 40% despite restrictions and organizational conflicts specific to the hospital context was reported. A minor incident in 1 of 278 sessions performed with 35 children with cancer was observed.

Conclusion: This study is the first to examine the effects of exercise in pediatric oncology, from diagnosis to the expected end of treatment. At this time, our findings show the feasibility of this physical activity program in a safe context. This is an important study in the exercise and oncology field to help patients and their family during and after cancer treatments.

Citation Format: Maxime Caru, Gabrielle Duhamel, Valérie Marcil, Serge Sultan, Caroline Meloche, Isabelle Bouchard, Simon Drouin, Laurence Bertout, Caroline Laverdière, Daniel Sinnett, Daniel Curnier. Feasibility of physical activity in children with cancer: A multidisciplinary program in the context of the VIE study [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 A57.
Abstract A70: Aqueous humor is superior to blood as a liquid biopsy for retinoblastoma
Authors: Xu Liya , Berry Jesse L , Polski Ashley , Jubran Rima , Kuhn Peter , Kim Jonathan W. , Hicks James
Published: August 2020

Purpose: To evaluate whether blood demonstrates similar potential as aqueous humor (AH) to be used as a liquid biopsy for retinoblastoma (RB) and whether tumor-derived cell-free DNA (cfDNA) can be isolated as effectively from the blood as AH.

Methods: AH was extracted via clear corneal limbal paracentesis from RB eyes at diagnosis or during intravitreal injection of chemotherapy. Matched peripheral venous blood samples were drawn. Shallow whole-genome sequencing was performed to assess for cell-free tumor DNA fractions and highly recurrent somatic copy number alterations (SCNAs) in the blood and AH samples.

Results: Seven samples of AH taken at diagnosis and 13 samples at the time of intravitreal injection were compared to matched blood samples. The presence of any detectable SCNA in the AH was 11/20 and 0/20 in the blood (p=<0.001). The median size distribution of cfDNA molecules in the AH was 157.5 bp versus 181.5 bp in the blood (p=<0.001).

Conclusions: The AH appears to be superior to the blood as a source of cell-free tumor DNA for retinoblastoma, thus a better target for development as a liquid biopsy for this cancer.

Citation Format: Liya Xu, Jesse L Berry, Ashley Polski, Rima Jubran, Peter Kuhn, Jonathan W. Kim, James Hicks. Aqueous humor is superior to blood as a liquid biopsy for retinoblastoma [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 A70.
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

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

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

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

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

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

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

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<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<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

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.