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Pediatric central nervous system (CNS) cancers are the leading disease-related cause of death in children and there is urgent need for curative therapeutic strategies for these tumors. To address the urgency, Children’s Brain Tumor Network (CBTN) has advanced an open science model to accelerate the research discovery for pediatric brain tumors. In first phase of Open Pediatric Brain Tumor Atlas (OpenPBTA) effort CBTN together with Pacific Pediatric Neuro-Oncology Consortium (PNOC) with support of Gabriella Miller Kids First Data Resource Center (KFDRC) created and comprehensively characterized over 1000 clinically annotated pediatric brain tumors. In the second phase of the OpenPBTA effort, through resource awards and collaboration across KFDRC, the NCI Childhood Cancer Data Initiative (CCDI), NCI’s Clinical Proteomic Tumor Analysis Consortium (CPTAC), NCI Center for Cancer Research and additional partnered institutions and foundations, CBTN has expanded OpenPBTA to support high throughput molecular characterization for an additional 1900 pediatric brain tumor patients and their families. This includes the processing and characterization of over 8000 specimens across >50 brain tumor diagnoses. The cohort expansion builds on >1000 previously characterized samples with a portfolio of multimodal data including whole genome sequencing, RNA sequencing, miRNA sequencing, methylation sequencing, proteomics, lipidomics and/or metabolomics. Molecular data is linked to patient longitudinal clinical data, imaging data (MRIs and radiology reports), histology slide images, and pathology reports. To inform novel discovery and clinical implementation of genomic approaches for diagnostic/therapeutic purposes, the data is deposited the cloud-based research environment of the NCI’s CCDI and the KFDRC to provide near real-time integration, dissemination, processing, and sharing of associated petabyte-scale harmonized data. The approach leverages the DRC platform’s cloud-based computational environment in CAVATICA. Processed annotations are facilitated via CAVATICA-enabled shareable pipelines and can be explored through PedcBioPortal, a data visualization/analysis application further integrating additional public and deposited datasets. This expansion phase of OpenPBTA is released with no embargo period and provides one of the largest deeply characterized cohorts of pediatric brain tumor samples and associated clinical data for >3000 pediatric brain tumor patients. CBTN’s open-science, rapid-release model aims to advance novel biomarkers and therapeutic exploratory research, supporting new clinical trial development and accelerated discovery on behalf of changing the outcome for kids with brain tumors. Citation Format: Mateusz P. Koptyra, Komal Rahti, Yuankun Zhu, Bailey Farrow, Daniel Miller, Adam Kraya, Yiran Guo, Peter Madsen, Nicholas Van Kuren, Xiaoyan Huang, Miguel A. Brown, Jennifer L. Mason, Meen Chul Kim, Allison P. Heath, Brian M. Ennis, Bo Zhang, Jena V. Lilly, Jo Lynne Rokita, Christopher Friedman, Ximena P. Cuellar, Catherine A. Sullivan, Noel Coleman, Trang Duros, Thinh Q. Nguyen, Emmett C. Drake, Zeinab Helili, Beth A. Frenkel, Gerri R. Trooskin, Ariana Familiar, Karthik Viswanathan, Christopher M. Beck, Madison L. Hollawell, Valerie P. Baubet, Cassie Kline, Mariarita Santi, Tatiana S. Patton, Stephanie Stefankiewicz, Arya Kamnaa, Ryan A. Velasco, Dani Cardona, Phillip J. Storm, Adam C. Resnick, o/b/o Children's Brain Tumor Network. Expansion of the Pediatric Brain Tumor Atlas: Children's Brain Tumor Network, Kids First Data Resource and Childhood Cancer Data Initiative Open Science effort. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3566.

One of the greatest barriers to curative treatment of neuroblastoma is its frequent metastatic outgrowth prior to diagnosis, especially in cases driven by amplification of the MYCN oncogene. However, only a limited number of regulatory proteins that contribute to this complex MYCN-mediated process have been elucidated. Here we show that the growth arrest-specific 7 (GAS7) gene, located at chromosome band 17p13.1, is preferentially deleted in high-risk MYCN-driven neuroblastoma. GAS7 expression was also suppressed in MYCN-amplified neuroblastoma lacking 17p deletion. GAS7 deficiency led to accelerated metastasis in both zebrafish and mammalian models of neuroblastoma with overexpression or amplification of MYCN. Analysis of expression profiles and the ultrastructure of zebrafish neuroblastoma tumors with MYCN overexpression identified that GAS7 deficiency led to (i) downregulation of genes involved in cell–cell interaction, (ii) loss of contact among tumor cells as critical determinants of accelerated metastasis, and (iii) increased levels of MYCN protein. These results provide the first genetic evidence that GAS7 depletion is a critical early step in the cascade of events culminating in neuroblastoma metastasis in the context of MYCN overexpression. Significance: Heterozygous deletion or MYCN-mediated repression of GAS7 in neuroblastoma releases an important brake on tumor cell dispersion and migration to distant sites, providing a novel mechanism underlying tumor metastasis in MYCN-driven neuroblastoma. See related commentary by Menard, p. 2815

Neuroblastoma is a childhood cancer with heterogeneous clinical outcomes. To comprehensively assess the impact of telomere maintenance mechanism (TMM) on clinical outcomes in high-risk neuroblastoma, we integrated the C-circle assay [a marker for alternative lengthening of telomeres (ALT)], TERT mRNA expression by RNA-sequencing, whole-genome/exome sequencing, and clinical covariates in 134 neuroblastoma patient samples at diagnosis. In addition, we assessed TMM in neuroblastoma cell lines (n = 104) and patient-derived xenografts (n = 28). ALT was identified in 23.4% of high-risk neuroblastoma tumors and genomic alterations in ATRX were detected in 60% of ALT tumors; 40% of ALT tumors lacked genomic alterations in known ALT-associated genes. Patients with high-risk neuroblastoma were classified into three subgroups (TERT-high, ALT+, and TERT-low/non-ALT) based on presence of C-circles and TERT mRNA expression (above or below median TERT expression). Event-free survival was similar among TERT-high, ALT+, or TERT-low/non-ALT patients. However, overall survival (OS) for TERT-low/non-ALT patients was significantly higher relative to TERT-high or ALT patients (log-rank test; P < 0.01) independent of current clinical and molecular prognostic markers. Consistent with the observed higher OS in patients with TERT-low/non-ALT tumors, continuous shortening of telomeres and decreasing viability occurred in low TERT–expressing, non-ALT patient-derived high-risk neuroblastoma cell lines. These findings demonstrate that assaying TMM with TERT mRNA expression and C-circles provides precise stratification of high-risk neuroblastoma into three subgroups with substantially different OS: a previously undescribed TERT-low/non-ALT cohort with superior OS (even after relapse) and two cohorts of patients with poor survival that have distinct molecular therapeutic targets. Significance: These findings assess telomere maintenance mechanisms with TERT mRNA and the ALT DNA biomarker C-circles to stratify neuroblastoma into three groups, with distinct overall survival independent of currently used clinical risk classifiers.

Relapsed neuroblastomas are enriched with activating mutations of the RAS–MAPK signaling pathway. The MEK1/2 inhibitor trametinib delays tumor growth but does not sustain regression in neuroblastoma preclinical models. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP1 as an additional driver of relapsed neuroblastomas, as well as a mediator of trametinib resistance in other cancers. Here, we used a highly annotated set of high-risk neuroblastoma cellular models to modulate YAP1 expression and RAS pathway activation to test whether increased YAP1 transcriptional activity is a mechanism of MEK1/2 inhibition resistance in RAS-driven neuroblastomas. In NLF (biallelic NF1 inactivation) and SK-N-AS (NRAS Q61K) cell lines, trametinib caused a near-complete translocation of YAP1 protein into the nucleus. YAP1 depletion sensitized neuroblastoma cells to trametinib, while overexpression of constitutively active YAP1 protein induced trametinib resistance. Mechanistically, significant enhancement of G1–S cell-cycle arrest, mediated by depletion of MYC/MYCN and E2F transcriptional output, sensitized RAS-driven neuroblastomas to trametinib following YAP1 deletion. These findings underscore the importance of YAP activity in response to trametinib in RAS-driven neuroblastomas, as well as the potential for targeting YAP in a trametinib combination. Significance: High-risk neuroblastomas with hyperactivated RAS signaling escape the selective pressure of MEK inhibition via YAP1-mediated transcriptional reprogramming and may be sensitive to combination therapies targeting both YAP1 and MEK.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and is currently divided into WNT, SHH, Group 3 and Group 4 subtypes. Even with multimodal chemotherapy, radiotherapy and surgery, many children with Group 3 MBs do not survive. While the molecular aberrations underlying WNT- and SHH-driven MBs are relatively well understood, the oncogenic drivers that lead to Group 3/4 MBs are poorly defined, limiting therapeutic progress. In addition to genetic mutations and alterations, cancers display dysregulated transcription and translation. RNA-binding proteins (RBPs) play key roles in both transcription and translation, and a subset of RBPs are differentially expressed in many different cancers. Indeed, we have previously demonstrated an oncogenic role for the RBP LIN28B in neuroblastoma and it is known to be upregulated in Wilms tumor, hepatoblastoma, germ cell tumors, leukemia among others. LIN28B is a key regulator of let-7 family miRNAs, which in turn inhibit LIN28B and other oncogenes. We hypothesize that LIN28B plays an important role in Group 3 MB and that a better understanding of LIN28B and LIN28B-driven networks will reveal novel therapeutic vulnerabilities. In support of our hypothesis we find that among the four subtypes, LIN28B levels are highest in Group 3 MB, and that overexpression is associated with significantly worse survival. Down-regulation of LIN28B results in significant reduction in cell proliferation by CellTiter-Glo and increased apoptosis by Caspase-Glo (as well as induction of cleaved PARP on immunoblots). In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation. In addition we find that PDZ-binding kinase (PBK) a downstream target of LIN28B is downregulated when LIN28B is depleted. PBK knock down also leads to decreased proliferation of Group 3 MB cells. Finally, in order to robustly define the signaling networks downstream from LIN28B that are involved in Group 3 MB metastasis, we have performed who transcriptome RNA-seq profiling of two group 3 cell lines following LIN28B depletion and plan to interrogate a subset of these based on expression change and functional relevance to LIN28B-mediated Group 3 MB metastasis. This work will help define the role for LIN28B in Group 3 MB aggressiveness and pave the way for similar studies in other cancers. Citation Format: Shubin W. Shahab, Jo Lynne Rokita, Kyle Juraschka, Sachin Kumar, Michael Taylor, Robert W. Schnepp, Tobey J. MacDonald, Anna M. Kenney. Targeting the RNA binding protein LIN28B in Group 3 medulloblastoma decreases proliferation and promotes apoptosis [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 3024.