Histone H3 point mutations have been identified in incurable pediatric brain cancers, but the mechanisms through which these mutations drive tumorigenesis are incompletely understood. Here, we provide evidence that RACK7 (ZMYND8) recognizes the histone H3.3 patient mutation (H3.3G34R) in vitro and in vivo. We show that RACK7 binding to H3.3G34R suppresses transcription of CIITA, which is the master regulator of MHC (major histocompatibility complex) class II molecules and genes involved in vesicular transport of MHC class II molecules to the cell surface, resulting in suppression of MHC class II molecule expression and transport. CRISPR-based knock-in correction of the H3.3G34R mutation in human pediatric glioblastoma (pGBM) cells significantly reduces overall RACK7 chromatin binding and derepresses the same set of genes as does knocking out RACK7 in the H3.3G34R pGBM cells. By demonstrating that H3.3G34R and RACK7 work together, our findings suggest a potential molecular mechanism by which H3.3G34R promotes cancer.

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Pediatric brain tumors comprise a distinct spectrum of diseases compared to adult brain tumors and are distinguished by their unique clinical and histopathological features, developmental context, mutation burden, and genomic, epigenomic, and transcriptomic alterations. Access to in vivo models that recapitulate pediatric brain tumors has been limited and inadequate to represent these heterogeneous diseases. Here we introduce the Pediatric Brain Tumor Portal (PBTP, pbtp.stjude.cloud), an open resource to access molecular characterization, including whole-genome sequencing, whole-exome sequencing, RNA-seq, and DNA methylome profiling, of patient-derived orthotopic xenograft (PDOX) models of pediatric brain tumors. The portal will host more than 70 models, which currently include pediatric High-Grade Glioma (pHGG), Medulloblastoma, Atypical Teratoid/Rhabdoid Tumors (AT/RT), Ependymoma, and Embryonal Tumors with Multilayered Rosettes (ETMR), and reflects close to ten years of effort to generate and extensively characterize in vivo models that recapitulate primary pediatric brain tumors. PBTP is a database-driven and user-friendly platform that enables multi-omics views of PDOX and matched patient tumor and germline samples at multiple levels to help identify appropriate lines for studies of tumorigenesis or preclinical testing. To evaluate the representation of different molecular features within a disease subgroup, we implemented various visualization tools for searching and comparing somatic mutations, gene expression profiles, and methylation groups of our models and matched patient tumors. Details for each tumor including de-identified clinical data, histology, growth characteristics, DNA methylation classification, mutation status, and copy number variations are also readily available for side-by-side comparison. Furthermore, in vitro chemical sensitivity profiling is presented for selected models with matched cell lines. Users can investigate genes of interest for single nucleotide variants, small insertions and deletions, copy number variations, gene fusions, and mRNA expression in PDOX and matched patient tumors. This portal has been integrated into the St. Jude Cloud platform, through which users can explore patient and PDOX sequencing data in the context of larger cohort data sets and download raw genomics data files. PBTP provides a platform to share PDOX models with in-depth genome/epigenome-wide characterization to support advances in basic and translational research in pediatric brain tumors. *co-first, #co-corresponding Citation Format: Paige S. Dunphy, Ke Xu, Darrell T. Gentry, Chen He, Kimberly Mercer, Xiaoyan Zhu, Kyle Smith, Brian Gudenas, Sarah Robinson, Junyuan Zhang, Lawryn H. Kasper, Chang-Hyuk Kwon, Laura D. Hover, Jon D. Larson, Nathaniel Twarog, Aksana Vasilyeva, Nedra Robison, Daniel Alford, Cynthia Williams, Anthony Woodard, Xin Zhou, Edgar Sioson, J. Robert Michael, Austyn Trull, Irina McGuire, Brandon McMahan, Swapnali Mohite, Ashok Boddu, Kirby Birch, Clay McLeod, Michael A. Dyer, Paul Klimo, Frederick A. Boop, Amar Gajjar, Christopher L. Tinkle, Giles Robinson, Brent A. Orr, Jason Chiang, Paul A. Northcott, Jinghui Zhang, Keith Perry, Gang Wu, Anang A. Shelat, Ed Suh, Martine F. Roussel, Suzanne J. Baker. St. Jude Pediatric Brain Tumor Portal: Cloud-based resource for patient-derived orthotopic xenograft (PDOX) models of pediatric high-grade glioma, ependymoma, and CNS embryonal tumors [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 6144.

Oncogenic fusion events involving c-MET have been observed in up to 12% of pediatric high-grade glioma (pHGG). MET inhibitors have displayed potent initial responses in MET rearranged tumors but acquired resistance to single agent modalities invariably occurs. To identify new treatment options against these tumors, we established two novel orthotopic mouse models including an immunocompetent, murine allograft and an intracranial patient-derived xenograft (PDX), both harboring distinct MET fusions. We analyzed the pharmacokinetic and pharmacodynamic profiles of two MET inhibitors, crizotinib and capmatinib, and examined their efficacy against tumor cell cultures derived from the aforementioned models. Capmatinib outperformed crizotinib in terms of specificity, potency and brain availability, resulting in a highly differential cellular response compared to crizotinib treatment. We evaluated the efficacy of both compounds in combination with radiotherapy (RT) and found that radiation further potentiated the inhibitory effect of capmatinib on tumor cell growth. We then utilized both models to assess the combinatorial effect of capmatinib and radiation on intracranial tumors in vivo and found that the combination therapy significantly increased overall survival in both cohorts. In the PDX model, the combination, relative to either intervention alone, induced a remarkable decrease of tumor burden, which persisted throughout the observation period in all treated animals. RNA-sequencing of capmatinib-treated tumors and tumor cell cultures revealed impaired expression of DNA repair genes. Further, we showed that capmatinib enhanced radiation-induced DNA damage, as demonstrated by increased γ-H2AX foci in treated cells, providing mechanistic insight for the cooperative effects of the combined treatment. Our results validate capmatinib as an effective inhibitor of MET in pHGG and demonstrate the outstanding efficacy of capmatinib and radiation against MET-driven pHGG in two complementary preclinical models, informing future clinical trials.