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BackgroundRecent genomic studies have shed light on the biology and inter-tumoral heterogeneity underlying pineal parenchymal tumors, in particular pineoblastomas (PBs) and pineal parenchymal tumors of intermediate differentiation (PPTIDs). Previous reports, however, had modest sample sizes and lacked power to integrate molecular and clinical findings. The different proposed subgroup structures also highlighted a need to reach consensus on a robust and relevant classification system.MethodsWe performed a meta-analysis on 221 patients with molecularly characterized PBs and PPTIDs. DNA methylation profiles were analyzed through complementary bioinformatic approaches and molecular subgrouping was harmonized. Demographic, clinical and genomic features of patients and samples from these pineal tumor subgroups were annotated.FindingsFour clinically and biologically relevant consensus PB subgroups were defined: PB-miRNA1 (n=96), PB-miRNA2 (n=23), PB-MYC/FOXR2 (n=34) and PB-RB1 (n=25); with PPTID (n=43) remaining as a molecularly distinct entity. Genomic and transcriptomic profiling allowed the characterization of oncogenic drivers for individual subgroups, specifically, alterations in the microRNA processing pathway in PB-miRNA1/2, MYC amplification and FOXR2 overexpression in PB-MYC/FOXR2, RB1 alteration in PB-RB1, and KBTBD4 insertion in PPTID. Age at diagnosis, sex predilection and metastatic status varied significantly among tumor subgroups. While patients with PB-miRNA2 and PPTID had superior outcome, survival was intermediate for patients with PB-miRNA1, and dismal for those with PB-MYC/FOXR2 and PB-RB1.InterpretationWe systematically interrogated the clinical and molecular heterogeneity within pineal parenchymal tumors and proposed a consensus nomenclature for disease subgroups, laying the groundwork for future studies as well as routine use in tumor classification.

Pineoblastoma (PB) is a rare and aggressive childhood brain tumor with highly variable age and treatment-associated outcomes. Our recent bulk tumor analyses of DNA methylation and mutational landscapes uncovered four discrete PB molecular subgroups (PB-miRNA1, PB-miRNA2, PB-MYC/FOXR2, and PB-RB), providing a major advance in our understanding of biological and clinical heterogeneity. However, developmental origins of PB subgroup heterogeneity and mechanisms governing how specific genetic alterations promote malignancy remain unknown. To resolve the cellular origins of PB, we assembled a large single-nucleus RNA-sequencing cohort (n=32) of primary PB tumors, including representatives from each subgroup. Transcriptomic analysis identified subgroup-specific gene expression programs driving intra-tumoral heterogeneity. In addition, we discovered substantial differences in the expression of miRNA biogenesis genes between the PB-miRNA1 and PB-miRNA2 subgroups, providing mechanistic support for their distinct subgroup identities despite overlapping driver events. The MYC/FOXR2 subgroup was characterized by over-expression of the FOXR2 proto-oncogene in bulk RNA-seq, which we validated in single-nuclei and identified co-expressed downstream target genes. To map PB subgroups to their putative developmental beginnings, we created a single-cell transcriptional atlas of the murine pineal gland across 11 developmental stages (E11-P21). Trajectory inference within the developing pineal gland revealed a differentiation continuum of early, mid, and mature alpha-/beta pinealocytes. Cross-species correlation and deconvolution identified significant associations between multiple PB subgroups and specific differentiation states of the pinealocyte lineage, suggestive of developmental origins. Characterization of pinealocyte development informed generation of biologically faithful disease models, including a novel genetically engineered mouse model of the PB-RB subgroup. PB-Rb1 mouse tumors were histologically and molecularly validated for their fidelity to human tumor counterparts, exhibiting up-regulation of key pinealocyte lineage markers that are diagnostic in patients. Finally, high-throughput drug screening identified several promising pharmacological candidates that may attenuate consequences of Rb1 deficiency in affected children.

BackgroundMalignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas. Somatic inactivation of NF1 and cooperating tumor suppressors, including CDKN2A/B, PRC2, and p53, is found in most MPNST. Inactivation of the LATS1/2 kinases of the Hippo pathway was recently shown to cause tumors resembling MPNST histologically, although Hippo pathway mutations are rarely found in MPNST. Because existing genetically engineered mouse (GEM) models of MPNST do not recapitulate some of the key genetic features of human MPNST, we aimed to establish a mouse MPNST model that recapitulated the human disease genetically, histologically, and molecularly.MethodsWe combined two genetically modified alleles, an Nf1;Trp53 cis-conditional allele and an inducible Plp-CreER allele (NP-Plp), to model the somatic, possibly postnatal, mutational events in human MPNST. We also generated conditional Lats1;Lats2 knockout mice. We performed histopathologic analysis of mouse MPNST models and transcriptomic comparison of mouse models and human nerve sheath tumors.ResultsPostnatal Nf1;Trp53 cis-deletion resulted in GEM-MPNST that was histologically more similar to human MPNST than the widely used germline Nf1;Trp53 cis-heterozygous (NPcis) model and showed partial loss of H3K27me3. At the transcriptome level, Nf1;p53-driven GEM-MPNST were distinct from Lats-driven GEM-MPNST and resembled human MPNST more closely than do Lats-driven tumors.ConclusionsThe NP-Plp model recapitulates human MPNST genetically, histologically, and molecularly.Key PointsPostnatal Nf1;p53 cis-deletion in NP-Plp mice results in tumors similar to MPNST.The transcriptomes of Nf1;p53-driven and Lats-driven MPNST models are distinct.NP-Plp model resembles human MPNST genetically, histologically, and molecularly.Importance of the StudyMalignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with a poor prognosis and limited treatment options. Existing genetically engineered mouse (GEM) models of MPNST do not recapitulate some of the key genetic features of human MPNST. To model the somatic, possibly postnatal, mutational events seen in MPNST patients, we generated a GEM-MPNST model by combining two genetically modified alleles, an Nf1;Trp53 cis-conditional allele and a Plp-CreER allele. Our histologic and transcriptomic analyses showed that this NP-Plp model resembles human MPNST genetically, histologically, and molecularly—more so than the widely used NPcis model and the recently published Lats-driven model. The NP-Plp model is genetically simple, making it easy to maintain and an ideal platform for preclinical studies. Given its tamoxifen-inducible nature, this model can be used to study the time/stage dependency of the tumorigenic potential of Schwann cells.

Germline loss-of-function (LOF) mutations in Elongator complex protein 1 (ELP1) are found in 15-20% of childhood SHH medulloblastoma (MB) and are exceedingly rare in non-SHH-MB or other cancers. ELP1 germline carriers that develop SHH-MB harbor frequent somatic PTCH1 mutations and universally sustain loss-of-heterozygosity of the remaining ELP1 allele through chromosome 9q deletion. ELP1 functions as a scaffolding subunit of the Elongator complex that is required for posttranscriptional modification of tRNAs and maintenance of efficient translational elongation and protein homeostasis. However, the molecular, biochemical, and cellular mechanisms by which ELP1/Elongator LOF contribute to SHH-MB tumorigenesis remain largely unknown. Herein, we report that mice harboring germline Elp1 monoallelic loss (i.e., Elp1+/-) exhibit hallmark features of malignant predisposition in developing cerebellar granule neuron progenitors (GNPs), the lineage-of-origin for SHH-MB. Elp1+/- GNPs are characterized by increased replication stress-induced DNA damage, upregulation of the homologous recombination repair pathway, aberrant cell cycle, and attenuation of p53-dependent apoptosis. CRISPR/Cas9-mediated Elp1 and Ptch1 gene targeting in mouse GNPs reproduces highly penetrant SHH-MB tumors recapitulating the molecular and phenotypic features of patient tumors. Reactivation of the p53 pathway through MDM2 and PAK4 inhibitors promotes selective cell death in patient-derived xenograft tumors (PDX) harboring deleterious ELP1 mutations. Together, our findings reveal that germline Elp1 deficiency heightens genomic instability and survival in GNPs, providing a mechanistic model for the subgroup-restricted pattern of predisposition and malignancy associated with pathogenic ELP1 germline carriers. These results provide rationale for further preclinical studies evaluating drugs that overcome p53 pathway inhibition in ELP1-associated SHH-MB and a renewed outlook for improving treatment options for affected children and their families.*, # Contributed equally

To determine the role of microRNA regulation in brain tumor development, we incorporated a conditional allele of the microRNA processing enzyme Dicer to a previously characterized glioma mouse model based on inactivation of the tumor suppressors Nf1, Trp53, and Pten using the Nestin-creERT2 transgene. Loss of Dicer and tumor suppressors at adult ages led to glioma development; however, mutant mice tamoxifen induced at early postnatal ages developed medulloblastoma instead of glioma. The switch in tumor spectrum occurred with 100% penetrance and tumors were histologically indistinguishable from human medulloblastoma (MB). The minimum genetic mutations required for MB formation were Dicer and Trp53. Nf1 was dispensable, while additional loss of Pten produced more invasive tumors and leptomeningeal metastases. The time window for initiation of tumorigenesis was until the 2nd postnatal week, coinciding with the disappearance of the external granule layer (EGL), where cerebellar granule neuron precursors (CGNPs) undergo proliferation. Analysis of pre-symptomatic mutant mice showed proliferative defects and retained cells in the EGL, suggesting that the tumors may arise from CGNPs. However, targeting a subset of CGNPs using Math1-creERT2 did not lead to MB development, suggesting that an earlier EGL precursor may be required for tumorigenesis. Analysis of tumor transcriptome and MB subtype-specific genes and markers show that Dicer tumors most resemble extremely high risk p53-mutated SHH MB. Small RNA and mRNA sequencing analyses showed downregulation of microRNAs and dysregulation of its targets such as N-Myc. These studies demonstrate a role for microRNAs in MB development and show a fully penetrant genetic mouse model of highly metastatic MB.

Understanding the interplay between normal development and tumorigenesis, including the identification and characterization of lineage-specific origins of MB, is a fundamental challenge in the field. Recent studies have highlighted novel associations between biologically distinct MB subgroups and diverse murine cerebellar lineages via cross-species single-cell transcriptomics. Specifically, Group 4-MB correlated with the unipolar brush cell lineage and Group 3-MB resembled Nestin+ stem cells of the early cerebellum. However, these analyses were hampered by low resolution due to the sparsity of pertinent cerebellar cell types and the cross-species nature of the approach. Herein, we profoundly expand the depth of these rare developmental populations in the murine cerebellum using a combination of lineage tracing and integrative multi-omics. Isolation and enrichment of spatially and temporally unique developmental trajectories of key rhombic lip-derived glutamatergic lineages provided an enhanced reference for mapping MB subgroups based on molecular overlap, especially for poorly defined Group 3- and Group 4-MB. Further comparisons to a novel single-cell atlas of the human fetal cerebellum, companioned with laser-capture microdissected transcriptional and epigenetic datasets, reinforced developmental insights extracted from the mouse. Characterization of compartment-specific transcriptional programs and co-expression networks identified in the human upper rhombic lip implicated convergent cellular correlates of Group 3- and Group 4-MB, suggestive of a common developmental link. Together, our results strongly implicate developmental lineages of the upper rhombic lip as the probable origins of poorly defined Group 3- and Group 4-MB. These important findings will shape future efforts to accurately model the biological heterogeneity underlying these subgroups and provide unprecedented opportunities to explore their cellular and mechanistic basis.

Medulloblastoma (MB), a common malignant pediatric brain tumor, comprises at least four distinct molecular entities: WNT, SHH, Group 3, and Group 4. SHH-MB is driven by aberrant activation of the Sonic hedgehog (SHH) pathway in granule neuron progenitors (GNPs) and is associated with hereditary cancer predisposition syndromes including Li Fraumeni and Gorlin. We recently identified germline loss of function (LoF) mutations affecting IKBKAP/ELP1, the primary scaffolding subunit of the Elongator complex in a subset of SHH-MB patients. Germline ELP1 mutations account for ~15% of all pediatric SHH-MBs and position ELP1 as the most prevalent hereditary predisposition gene in MB. We genetically engineered Elp1 LoF in mouse GNPs to determine Elp1 function in cerebellar development and SHH-MB. Results of both mechanistic and phenotypic experiments demonstrate that GNPs harboring Elp1 loss exhibit ribosome pausing and protein aggregation, reinforcing the critical role of Elp1 in translational elongation and protein homeostasis. Further, we generated new transgenic mouse models mimicking germline ELP1 LoF mutations observed in SHH-MB patients. Elp1+/- transgenic mice exhibit purkinje cell degeneration and an increased DNA damage response. These mice are currently being evaluated for their capacity to recapitulate ELP1-associated SHH-MB. Additional analyses carried out on SHH-MB patient-derived xenografts showed that ELP1-mutant tumor cells specifically exhibit defects in tRNA biogenesis. Therefore, the function of ELP1 as a translational regulator is severely impaired in ELP1-mutant SHH-MBs. Our ongoing molecular and functional studies will provide important insights into the most common MB predisposition gene and will lay the foundation for future preclinical studies.

Pineoblastoma (PB) is an aggressive embryonal brain tumor comprising 1% of pediatric CNS tumors. The clinico-molecular heterogeneity and developmental origins underlying PB are poorly understood; therefore, we have assembled a molecular cohort of histologically defined PBs (n=43) with corresponding outcome data. Methylation profiling revealed four molecularly and clinically distinct PB subgroups, including two novel entities. Mutational and transcriptional analysis identified characteristic molecular features of each subgroup, such as mutations in the miRNA processing pathway or FOXR2 proto-oncogene overexpression. Furthermore, subgroups exhibited differences in propensity for metastasis, cytogenetics, and clinical outcomes. To dissect PB developmental origins and resolve PB subgroup biology, we have employed a combination of single-cell genomics and genetically engineered mouse modeling. We created a single-cell transcriptional atlas of the developing murine pineal gland across 11 timepoints and are currently integrating these data with single nuclei RNA-seq data of human PB (n=25). Single-cell analysis of the developing pineal gland revealed three distinct populations of pinealocytes, referred to as early, mid and late pinealocytes, which segregate by developmental stage yet lie along a single developmental trajectory. Preliminary results implicate significant associations between PBs and the early pinealocyte population as well as subgroup-specific differences in intratumoral heterogeneity. Furthermore, this knowledge has informed the downstream generation of biologically faithful disease models, including a transgenic mouse model of the PB-RB subgroup. Remarkably, this model shows up-regulation of key markers of PB such as Crx, Asmt and Otx2 and substantiates early pinealocytes as the probable cell-of-origin for this PB subgroup.

BACKGROUND Our previous analysis of established cancer predisposition genes in medulloblastoma (MB) identified pathogenic germline variants in ~5% of all patients. Here, we extended our analysis to include all protein-coding genes. METHODS Case-control analysis performed on 795 MB patients against >118,000 cancer-free children and adults was performed to identify an association between rare germline variants and MB. RESULTS Germline loss-of-function variants of Elongator Complex Protein 1 (ELP1; 9q31.3) were strongly associated with SHH subgroup (MBSHH). ELP1-associated-MBs accounted for ~15% (29/202) of pediatric MBSHH cases and were restricted to the SHHα subtype. ELP1-associated-MBs demonstrated biallelic inactivation of ELP1 due to somatic chromosome 9q loss and most tumors exhibited co-occurring somatic PTCH1 (9q22.32) alterations. Inheritance was verified by parent-offspring sequencing (n=3) and pedigree analysis identified two families with a history of pediatric MB. ELP1-associated-MBSHH were characterized by desmoplastic/nodular histology (76%; 13/17) and demonstrated a favorable clinical outcome when compared to TP53-associated-MBSHH (5-yr OS 92% vs 20%; p-value=1.3e-6) despite both belonging to the SHHα subtype. ELP1 is a subunit of the Elongator complex, that promotes efficient translational elongation through tRNA modifications at the wobble (U34) position. Biochemical, transcriptional, and proteomic analyses revealed ELP1-associated-MBs exhibit destabilization of the core Elongator complex, loss of tRNA wobble modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response. CONCLUSIONS We identified ELP1 as the most common MB predisposition gene, increasing the total genetic predisposition for pediatric MBSHH to 40%. These results mark MBSHH as an overwhelmingly genetically-predisposed disease and implicate disruption of protein homeostasis in MBSHH development.

Pineoblastomas (PB) are rare, aggressive pineal gland tumours with poor global OS of 50–70% and only 15–49% OS for patients

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.