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Background Medulloblastomas with chromothripsis developing in children with Li-Fraumeni Syndrome (germline TP53 mutations) are highly aggressive brain tumors with dismal prognosis. Conventional photon radiotherapy and DNA-damaging chemotherapy are not successful for these patients and raise the risk of secondary malignancies. We hypothesized that the pronounced homologous recombination deficiency in these tumors might offer vulnerabilities that can be therapeutically utilized in combination with high linear energy transfer carbon ion radiotherapy. Methods We tested high-precision particle therapy with carbon ions and protons as well as topotecan with or without PARP inhibitor in orthotopic primary and matched relapsed patient-derived xenograft models. Tumor and normal tissue underwent longitudinal morphological MRI, cellular (markers of neurogenesis and DNA damage-repair), and molecular characterization (whole-genome sequencing). Results In the primary medulloblastoma model, carbon ions led to complete response in 79% of animals irrespective of PARP inhibitor within a follow-up period of 300 days postirradiation, as detected by MRI and histology. No sign of neurologic symptoms, impairment of neurogenesis or in-field carcinogenesis was detected in repair-deficient host mice. PARP inhibitors further enhanced the effect of proton irradiation. In the postradiotherapy relapsed tumor model, median survival was significantly increased after carbon ions (96 days) versus control (43 days, P < .0001). No major change in the clonal composition was detected in the relapsed model. Conclusion The high efficacy and favorable toxicity profile of carbon ions warrants further investigation in primary medulloblastomas with chromothripsis. Postradiotherapy relapsed medulloblastomas exhibit relative resistance compared to treatment-naïve tumors, calling for exploration of multimodal strategies.

Advancements in state-of-the-art molecular profiling techniques have resulted in better understanding of pediatric cancers and driver events. It has become apparent that pediatric cancers are significantly more heterogeneous than previously thought as evidenced by the number of novel entities and subtypes that have been identified with distinct molecular and clinical characteristics. For most of these newly recognized entities there are extremely limited treatment options available. The ITCC-P4 consortium is an international collaboration between several European academic centers and pharmaceutical companies, with the overall aim to establish a sustainable platform of >400 molecularly well-characterized PDX models of high-risk pediatric cancers, their tumors and matching controls and to use the PDX models for in vivo testing of novel mechanism-of-action based treatments. Currently, 251 models are fully characterized, including 182 brain and 69 non-brain PDX models, representing 112 primary models, 92 relapse, 42 metastasis and 4 progressions under treatment models. Using low coverage whole-genome and whole exome sequencing, somatic mutation calling, DNA copy number and methylation analysis we aim to define genetic features in our PDX models and estimate the molecular fidelity of PDX models compared to their patient tumor. Based on DNA methylation profiling we identified 43 different tumor subgroups within 18 cancer entities. Mutational landscape analysis identified key somatic and germline oncogenic drivers. Ependymoma PDX models displayed the C11orf95-RELA fusion event, YAP1, C11orf95 and RELA structural variants. Medulloblastoma models were driven by MYCN, TP53, GLI2, SUFU and PTEN. High-grade glioma samples showed TP53, ATRX, MYCN and PIK3CA somatic SNVs, along with focal deletions in CDKN2A in chromosome 9. Neuroblastoma models were enriched for ALK SNVs and/or MYCN focal amplification, ATRX SNVs and CDKN2A/B deletions. Tumor mutational burden across entities and copy number analysis was performed to identify allele-specific copy number detection in tumor-normal pairs. Large chromosomal aberrations (deletions, duplications) detected in the PDX models were concurrent with molecular alterations frequently observed in each tumor type -isochromosome 17 was detected in 5 medulloblastoma models, while deletion of chromosome arm 1p or gain of parts of 17q in neuroblastomas which correlate with tumor progression. We observe clonal evolution of somatic variants not only in certain PDX-tumor pairs but also between disease states. The multi-omics approach in this study provides insight into the mutational landscape and patterns of the PDX models thus providing an overview of molecular mechanisms facilitating the identification and prioritization of oncogenic drivers and potential biomarkers for optimal treatment therapies. Citation Format: Apurva Gopisetty, Aniello Federico, Didier Surdez, Yasmine Iddir, Sakina Zaidi, Alexandra Saint-Charles, Joshua Waterfall, Elnaz Saberi-Ansari, Justyna Wierzbinska, Andreas Schlicker, Norman Mack, Benjamin Schwalm, Christopher Previti, Lena Weiser, Ivo Buchhalter, Anna-Lisa Böttcher, Martin Sill, Robert Autry, Frank Estermann, David Jones, Richard Volckmann, Danny Zwijnenburg, Angelika Eggert, Olaf Heidenreich, Fatima Iradier, Irmela Jeremias, Heinrich Kovar, Jan-Henning Klusmann, Klaus-Michael Debatin, Simon Bomken, Petra Hamerlik, Maureen Hattersley, Olaf Witt, Louis Chesler, Alan Mackay, Johannes Gojo, Stefano Cairo, Julia Schueler, Johannes Schulte, Birgit Geoerger, Jan J. Molenaar, David J. Shields, Hubert N. Caron, Gilles Vassal, Louis F. Stancato, Stefan M. Pfister, Natalie Jaeger, Jan Koster, Marcel Kool, Gudrun Schleiermacher. ITCC-P4: Genomic profiling and analyses of pediatric patient tumor and patient-derived xenograft (PDX) models for high throughput in vivo testing [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 234.

Medulloblastoma (WHO grade 4) is the most common malignant brain tumor of childhood. Despite the high importance of radiotherapy for disease control, the mechanisms underlying response and resistance to radiotherapy are incompletely understood. Therefore, we assessed the radiosensitivity and DNA repair capacity of medulloblastoma cell lines in-vitro and of patient-derived xenograft (PDX) models ex-vivo. Cell survival after irradiation of seven medulloblastoma cell lines displaying different subgroups was assessed via colony formation assay (DAOY, UW228, UW473, SJMM4, ONS-76, HDMB-03, D283). The ONS-76 and the mouse SJMM4 cell line were the most radioresistant strains (surviving fraction after 6 Gy (SF6): 0.33 and 0.31, respectively), followed by UW473, UW 228 and DAOY cells (SF6 0.16-0.21). The non-WNT/non-SHH-activated cell lines HDMB-03 and D283 cells demonstrated profoundly higher cellular radiosensitivity (SF6

INTRODUCTION: Disorders with Abnormal DNA Damage Response/Repair (DADDRs) are inherited conditions caused by constitutional mutations of DNA damage response and repair genes and are characterized by an increased cancer risk. Furthermore, affected individuals also show an elevated risk of secondary neoplasms as well as excessive toxicity, poor therapy response and increased mortality when treated with standard radiation and chemotherapy regimens. The main aim of this project is to screen for potential novel chemotherapeutic approaches for these cancer entities, and to employ faithful PDX models for in vivo validation. METHODS: In vitro drug screening was performed using a custom library composed of 345 compounds targeting 61 different proteins. For two specific DADDRs, Li-Fraumeni syndrome (LFS) and Constitutional Mismatch Repair Deficiency (CMMRD), two cancerous (glioblastoma and medulloblastoma) and one non-cancerous cell lines were selected to model each of these conditions. Performance of each drug was assessed based on its efficacy (sensitivity score) and genotoxicity (micronucleus assay). For DADDR PDX model establishment tumor material from DADDR patients is currently being injected orthotopically (brain tumors) or subcutaneously (non-brain tumors) into NSG mice. Following engraftment and expansion, the PDX models will be characterized molecularly and compared with original patient material. RESULTS AND OUTLOOK: In vitro screening revealed n=26 drugs that fulfilled the following criteria: a) favorable toxicity in cancerous cell lines compared to non-cancerous cell lines, b) little to no genotoxic effect in non-cancerous cell lines. These characteristics qualify them as potentially suitable candidates for novel therapeutic approaches specifically for DADDR patients. The hits included inhibitors of ATM/ATR, CHK1/CHK2, DHFR, mTOR and PI3K, as well as microtubule-associated compounds. Combination testing and further validation of these hits using disease-specific in vitro and in vivo PDX models is ongoing.

YAP1 fusion-positive supratentorial ependymomas predominantly occur in infants, but the molecular mechanisms of oncogenesis are unknown. Here we show YAP1-MAMLD1 fusions are sufficient to drive malignant transformation in mice, and the resulting tumors share histo-molecular characteristics of human ependymomas. Nuclear localization of YAP1-MAMLD1 protein is mediated by MAMLD1 and independent of YAP1-Ser127 phosphorylation. Chromatin immunoprecipitation-sequencing analyses of human YAP1-MAMLD1-positive ependymoma reveal enrichment of NFI and TEAD transcription factor binding site motifs in YAP1-bound regulatory elements, suggesting a role for these transcription factors in YAP1-MAMLD1-driven tumorigenesis. Mutation of the TEAD binding site in the YAP1 fusion or repression of NFI targets prevents tumor induction in mice. Together, these results demonstrate that the YAP1-MAMLD1 fusion functions as an oncogenic driver of ependymoma through recruitment of TEADs and NFIs, indicating a rationale for preclinical studies to block the interaction between YAP1 fusions and NFI and TEAD transcription factors., The molecular mechanisms driving proliferation in the pediatric brain cancer epdendymoma are poorly understood. Here the authors show that a YAP1- MAMLD1 fusion drives tumor formation in mice and show that the fusion protein can collaborate with the TEAD and NFI transcription factors.

Brain and spinal cord tumors are the second most common group of cancers in children. For children who experience relapses of their tumors, usually after very intensive first-line therapy, curative treatment options are scarce. Thus, the need for predictive preclinical platforms explicitly for pediatric brain tumors is an urgent need. In the framework of the ITCC-P4 public-private partnership, we thus far fully established 23 pediatric brain tumor-derived PDX models. A selection of four high grade glioma, one medulloblastoma and one ependymoma models were used to validate a protocol for fluorescence-based optical imaging. Tumor growth characteristics, latency and histopathology were evaluated for the un-transduced and iRFP713-transduced models side by side. In case of iRFP713-transduced PDX models, tumor load was determined twice a week with the Pearl trilogy system (LiCor, Germany). All animals were examined for neurological symptoms daily and body weight was examined twice a week. The latency of the tumor models ranged from 29 days to 180 days. The take rate was 100% across all the models with n=12 NSG mice per setting. The transduction did not influence the take rate, but 30 - 40% more donor material was needed due to viability loss during the overnight transduction. It was not possible to determine the transduction efficiency for iRFP713 in the overnight culture as the signal is getting upregulated only 48h - 72h post transduction, e.g., when the cells are already implanted. The signal was stable for up to 180 days in the slowest tumor model ependymoma HN0579. The fastest model, high grade glioma HG0068, reached termination criteria within 24 days. Histopathological examination was strictly correlated with the tumor load determined by optical imaging in situ and ex vivo (organ imaging). The histopathological investigation of the mouse brains displayed no differences in tumor localization, size, and invasiveness between the transduced and the un-transduced lines. The proliferation rate determined by Ki-67 staining was not influenced by the modification of the cells. Further molecular and phenotypic characterization of the transduced vs the un-transduced PDX will increase the utility of this platform for the development of new drugs and the identification of innovative drug targets. Citation Format: Eva Oswald, Kanstantsin Lashuk, Johannes Gojo, Dorothee Lenhard, Norman Mack, Sonja Krausert, Daniela Lötsch, David Jones, Marcel Kool, Till Milde, Walter Berger, Stefan M. Pfister, Julia Schüler. Establishment and characterization of pediatric brain tumor models in an orthotopic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1673.

Brain tumors are the most frequent category of solid tumors in children and have the highest mortality rate among all pediatric cancers. Although diagnosis and treatment have improved prognosis over the past decades for some childhood brain tumors, others remain lethal and current treatments are highly toxic to the developing brain, resulting in severe sequelae and considerably affecting the patient’s quality of life. Thus, new therapeutic options with reduced secondary effects are urgently needed. From this perspective, immunotherapies have gained a lot of attention due to their effectiveness in targeting tumor cells specifically. Chimeric-antigen receptor (CAR) T-cells recognize the target antigen on the surface of. CD276 is an immune checkpoint molecule that is expressed in a variety of solid tumor entities, including pediatric brain tumors. We analyzed the CD276 expression in our Patient-Derived-Xenograft (PDX) biobank of brain tumors and found that CD276 is ubiquitously expressed (ATRT, MB, EPN, GBM, ETMR, etc). Flow cytometry of MB PDX (n=4) confirmed CD276 expression of 97-99% of tumor cells, indicating that CD276 might be a good antigen target for CAR-T cell therapy of MBG3 and MBSHH. We found that second generation CAR-T cells targeting CD276 antigen significantly decreased tumor burden of the most aggressive MB subgroups (G3 and SHH-TP53mut PDX models) in NSG mice. We further treated NSG mice carrying a high tumor burden of the aggressive SHH-TP53mut PDX BT084 with second (CD28) and third generation (CD28-41BB) CD276-CAR-T cells. While both 2nd and 3rd generation improved the survival rates compared with CD19-CAR-T control cells, we found no difference in survival between the CD276 CAR-T generations, with no severe secondary effect during treatment. In conclusion, CD276 is a good antigen target for medulloblastoma, and warrants further evaluation for the treatment of medulloblastoma patients at relapse or as a maintenance therapy after standard treatment.

Previously, we have found that Embryonal Tumors with Multilayered Rosettes (ETMR) tumor cells harboring high levels of R-loops, a potential marker for replication stress and genomic instability, are vulnerable to a combination of topoisomerase and PARP inhibitors. To follow up on this, we investigated whether other pediatric brain tumor types with high levels of R-loops, such as MYC-amplified Group 3 medulloblastoma (MB) and ZFTA-fusion positive ependymoma, are also sensitive to these inhibitors. First, we performed in vitro drug screens using HD-MB03, a Group 3 MB cell line, and the ETMR cell line BT183, and in both screens PARP inhibitors were identified as the most synergistic combination partners for the topoisomerase inhibitor Irinotecan, respectively the active metabolite SN-38. Normal Astrocytes were not sensitive to these combinations. Secondly, we performed in vivo studies using patient-derived xenograft (PDX) models injected subcutaneously or intracranially into NSG mice, and treated with the PARP inhibitor Pamiparib, Irinotecan or a combination of both. For a MYC-amplified Group 3 MB and a ZFTA-fusion positive Ependymoma model, both injected intracranially, treatment with Irinotecan or the combination led to a significant survival benefit and inhibition of tumor growth including transient tumor shrinkage, but addition of Pamiparib did not add any further benefit in vivo, even though intratumoral PARP was inhibited by at least 80%. In contrast, in the subcutaneously injected ETMR model, the combination treatment with Irinotecan and Pamiparib led to a synergistic effect and complete regression of the tumors. Further refinements of the treatment strategy as dose adaptations and the use of a pegylated version of SN-38 (PLX038A) did also not induce a synergistic effect of the drugs for the intracranial tumors. Additional in vivo studies to evaluate the differences in efficacy and whether these are tumor specific or due to incomplete brain penetrance of the drugs are ongoing.

Thanks to state-of-the-art molecular profiling techniques we by now have a much better understanding of pediatric cancers and what is driving them. On the other hand, we have also realized that pediatric cancers are much more heterogeneous than previously thought. Many new types and subtypes of pediatric cancers have been identified with distinct molecular and clinical characteristics. However, for many if not most of these new types and subtypes there is no specific treatment available, yet. In order to develop specific treatment protocols and to increase survival rates for pediatric cancer patients further, both at diagnosis and relapse/metastasis, we need a large collection of well-characterized preclinical models representing all the different types and subtypes. These models can be used for preclinical drug testing to prioritize the pediatric development of anticancer drugs that would be best targeting pediatric tumor biology. The ITCC-P4 consortium, which is a collaboration between many academic centers across Europe, several companies involved in in vivo preclinical testing, and ten pharmaceutical companies, started in 2017 with the overall aim to establish a sustainable platform of >400 molecularly well-characterized PDX models of high-risk pediatric cancers and to use them for in vivo testing of novel mechanism-of-action based treatments. Currently, 340 models have been fully established, including 87 brain tumor models and 253 non-brain tumor models, together representing many different tumor types both from primary and relapsed/metastatic disease. Out of these 340 models, 252 have been fully molecularly characterized, most of them together with their matching original tumors, and almost of all these models are currently being subjected to in vivo testing using three standard of care drugs and six novel mechanism-of-action based drugs. In this presentation, an update on the current status of the ITCC-P4 platform and the data we collectively have generated thus far will be presented.

Four main medulloblastoma (MB) molecular subgroups are known, including the sonic hedgehog (SHH) subgroup, which represents ~25% of MB cases. The 5-year overall survival of SHH-MB is ~80%. However, survival between patients is highly diverse and dependent on the driver mutation(s) of the tumor. Patients with TP53 mutated tumors (often accompanied with MYCN and/or GLI2 amplifications) don’t respond well to current therapies and have a 10-year overall survival below 20%. Therefore, there is a need for new and more tailored therapies for these patients. In this study we aim to screen patient-derived organoid models of TP53-mutated SHH MB with a library of ~200 different compounds. We have optimized the cultures of two PDX-derived and one patient-derived organoid line in vitro. The lines will be screened in a high-throughput manner and the best hits and combinations will be validated in corresponding in vivo PDX models. To further assess the role of specific mutations in therapy outcome of TP53-mutated SHH MB, cerebellar organoids generated from human iPSCs were genetically modified with overexpression of dominant-negative P53 (DNP53) alone or in combination with MYCN and/or GLI2. Introduction of DNP53 and MYCN overexpression in cerebellar organoids at day 28/35 leads to the outgrowth of a Ki67-positive proliferating mass after three weeks, indicating tumor growth. Further analyses are ongoing to see how they match SHH-MB patient tumors. These genetically engineered organoid models may elucidate the role of specific mutations in therapy response and/or resistance. In addition, as tumors in these genetically engineered cerebellar organoids arise in a microenvironment of normal cerebellar cell types, initial safety of drugs on cerebellar cells can be assessed. In conclusion, different organoid models of TP53-mutated SHH MB will enable us to find more effective treatments and to better understand how to treat patients with different mutation combinations.

Medulloblastoma is a malignant childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH) subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs), and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional corepressor BCOR are recurrent and enriched in SHH medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a genetically engineered mouse model to delete exons 9/10 of Bcor (BcorΔE9–10) in GNPs during development. This mutation leads to reduced expression of C-terminally truncated BCOR (BCORΔE9–10). While BcorΔE9–10 alone did not promote tumorigenesis or affect GNP differentiation, BcorΔE9–10 combined with loss of the SHH receptor gene Ptch1 resulted in fully penetrant medulloblastomas. In Ptch1+/−;BcorΔE9–10 tumors, the growth factor gene Igf2 was aberrantly up-regulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/− GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/−;BcorΔE9–10 tumors. Overall, our data suggests that BCOR–PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.

Brain tumors are the leading cause of cancer-related death in children. Genomic studies have provided insights into molecular subgroups and oncogenic drivers of pediatric brain tumors that may lead to novel therapeutic strategies. To evaluate new treatments, better preclinical models adequately reflecting the biological heterogeneity are needed. Through the Children’s Oncology Group ACNS02B3 study, we have generated and comprehensively characterized 30 patient-derived orthotopic xenograft models and seven cell lines representing 14 molecular subgroups of pediatric brain tumors. Patient-derived orthotopic xenograft models were found to be representative of the human tumors they were derived from in terms of histology, immunohistochemistry, gene expression, DNA methylation, copy number, and mutational profiles. In vivo drug sensitivity of targeted therapeutics was associated with distinct molecular tumor subgroups and specific genetic alterations. These models and their molecular characterization provide an unprecedented resource for the cancer community to study key oncogenic drivers and to evaluate novel treatment strategies.

Background Embryonal tumor with multilayered rosettes (ETMR) is a rare and aggressive embryonal brain tumor that solely occurs in infants and young children and has only recently been recognized as a separate brain tumor entity in the World Health Organization classification for CNS tumors. Patients have a very dismal prognosis with a median survival of 12 months upon diagnosis despite aggressive treatment. The aim of this study was to develop novel treatment regimens in a preclinical drug screen in order to inform potentially more active clinical trial protocols. Methods We have carried out an in vitro and in vivo drug screen using the ETMR cell line BT183 and its xenograft model. Furthermore, we have generated the first patient-derived xenograft (PDX) model for ETMR and evaluated our top drug candidates in an in vitro drug screen using this model. Results BT183 cells are very sensitive to the topoisomerase inhibitors topotecan and doxorubicin, to the epigenetic agents decitabine and panobinostat, to actinomycin D, and to targeted drugs such as the polo-like kinase 1 (PLK1) inhibitor volasertib, the aurora kinase A inhibitor alisertib, and the mammalian target of rapamycin (mTOR) inhibitor MLN0128. In xenograft mice, monotherapy with topotecan, volasertib, and actinomycin D led to a temporary response in tumor growth and a significant increase in survival. Finally, using multi-agent treatment regimens of topotecan or doxorubicin combined with methotrexate and vincristine, the response in tumor growth and survival was further increased compared with mice receiving single treatments. Conclusions We have identified several promising candidates for combination therapies in future clinical trials for ETMR patients.

Pediatric brain tumors frequently harbor MYC- or MYCN-amplification or show high overexpression and are associated with poor outcome. Examples for which current treatments are unsuccessful are for instance the medulloblastoma (MB) subgroups SHH (MYCN-amplified), Group 3 (MYC amplified) and embryonal tumors with multilayered rosettes (ETMR). Since MYC(N) is normally expressed in rapidly dividing cells but not in quiescent tissue stem cells, amplification or high expression in tumors leads to increased transcription, which can be in conflict with DNA replication and subsequently can cause replication stress and DNA damage. We hypothesized that high expression or amplification of MYC(N) in tumors makes them vulnerable to DNA damage response inhibitors (DDRi) and even more vulnerable to combinations of DDRi and chemotherapeutics. To test this hypothesis we performed in vitro drug experiments using different Group 3 MB cell lines and the BT183 ETMR cell line. We evaluated the IC50-values of the topoisomerase inhibitor Topotecan and the DDRi BGB-290, a PARP-inhibitor, in monotherapy. All cell lines were sensitive for Topotecan and showed IC50-values in the low nM-range but PARP-inhibitors were ineffective (for MB) or showed only slight effects (for ETMR cells). However, a significant decrease in IC50 can be observed when Topotecan and BGB-290 are used in combination. For in vivo treatments, we injected NSG mice with luciferase labelled ETMR cells, monitored tumor growth via IVIS and started the non-toxic single and combination treatments when a predefined threshold of tumor growth was reached. When effective in this ETMR in vivo model, we will repeat the experiments with additional ETMR and MB models. In vivo treatments are ongoing and results will be presented at the conference.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive childhood tumor of the brainstem with currently no curative treatment available. The vast majority of DIPGs carry a histone H3 mutation leading to a lysine 27-to-methionine exchange (H3K27M). We engineered human induced pluripotent stem cells (iPSCs) to carry an inducible H3.3-K27M allele in the endogenous locus and studied the effects of the mutation in different disease-relevant neural cell types. H3.3-K27M upregulated bivalent promoter-associated developmental genes, producing diverse outcomes in different cell types. While being fatal for iPSCs, H3.3-K27M increased proliferation in neural stem cells (NSCs) and to a lesser extent in oligodendrocyte progenitor cells (OPCs). Only NSCs gave rise to tumors upon induction of H3.3-K27M and TP53 inactivation in an orthotopic xenograft model recapitulating human DIPGs. In NSCs, H3.3-K27M leads to maintained expression of stemness and proliferative genes and a premature activation of OPC programs that together may cause tumor initiation.

Pediatric brain tumors harboring amplifications or high overexpression of MYC-/MYCN are often associated with poor outcome. High MYC(N) expression in these tumors leads to increased transcription, which can be in conflict with DNA replication and subsequently can cause replication stress, R-loops and DNA damage. We hypothesize that high MYC(N) expression makes them vulnerable to DNA damage response inhibitors (DDRi) and even more vulnerable to combinations of DDRi and chemotherapeutics. To test this hypothesis we performed in vitro drug experiments using Group 3 medulloblastoma (MB) and ETMR cell lines. IC50-values were evaluated of topoisomerase inhibitor Irinotecan (SN-38) and Pamiparib (BGB-290), a brain-penetrant PARP-inhibitor, in monotherapy. All cell lines were sensitive for SN-38 and showed IC50-values in the low nM-range but PARP-inhibitors were ineffective. However, a significant decrease in IC50 can be observed when SN-38 and Pamiparib are used in combination. For in vivo treatments, we injected NSG mice with luciferase labelled patient-derived xenograft- (PDX-) cells of various models (MB Group 3, MB SHH, ETMR, RELA EPN), monitored tumor growth via IVIS and randomized the mice into four groups (vehicle, BGB-290, Irinotecan and Irinotecan+Pamiparib) when a predefined threshold of tumor growth was reached. Mice were treated with Irinotecan (or vehicle) once per day i.p. and Pamiparib (or vehicle) twice per day per oral gavage. Treatment with Pamiparib did not show any survival benefit, but mice treated with Irinotecan or the combination showed a clear survival benefit. Treatments are ongoing and more results will be presented at the conference.

Introduction: The majority of pediatric supratentorial (ST) ependymomas (EPN) is driven by distinct gene fusions between C11orf95 and RELA. The resultant molecular group of ST-EPN-RELA tumors is characterized by constitutive activation of NF-κB signaling and deregulation of the p53 pathway. In contrast to surgery and radiotherapy, chemotherapy has failed to demonstrate significant benefit in the management of affected children. Alternative strategies including enhanced drug delivery, combination treatments, or application of new selective compounds are needed to tackle this disease. Material and Methods: RNAi and drug screening methods were applied to identify potential therapeutic approaches using ST-EPN-RELA cell lines. In order to identify optimal dosing strategies of selected drugs and to assess effects of combinatorial treatment approaches on blood-brain barrier (BBB) penetration, cerebral microdialysis combined with ultraperformance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) was applied. This approach allowed for exact, continuous, and time-dependent drug quantification in tumors or healthy tissue in freely moving experimental mice. Patient-derived xenograft models of ST-EPN-RELA were treated to investigate toxicity and outcome parameters. Results: Regulation of p53 signaling and nuclear protein shuttling were identified as promising therapeutic approaches. While low-dose dactinomycin could successfully reestablish p53 function in ST-EPN-RELA cells in vitro, penetration of the drug across the BBB was found to be very poor and did not result in a survival benefit of tumor-bearing mice. Preliminary results of alternative strategies such as combination with efflux pump inhibitors, liposomal packaging, and inhibition of XPO1 being the sole nuclear exporter of p53 hold promise to overcome these constraints. Conclusion: Oncogenic dependencies of ST-EPN-RELA are currently difficult to target. Preclinical evaluation of effective drug disposition combined with long-term treatment studies may help to better select promising compounds and thereby increase success rates of early clinical trials in patients with ST-EPN-RELA in the future. Citation Format: Julia Benzel, Max Sauter, Norman Mack, Abigail Davis, Johanna Weiss, Philipp Uhl, Jürgen Burhenne, Kendra K. Maass, Jens-Martin Hübner, Hendrik Witt, Anang Shelat, Amar Gajjar, Santhosh A. Upadhyaya, Aylin Camgoz, Frank Buchholz, Sina Oppermann, Marcel Kool, Daisuke Kawauchi, Olaf Witt, Walter E. Haefeli, Stefan M. Pfister, Clinton Stewart, Kristian W. Pajtler. Evaluation of Drug Disposition in Supratentorial Ependymoma [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 A25.

Oncogenic fusions containing RELA or YAP1 have recently been identified as genetic hallmarks of distinct molecular subgroups of supratentorial ependymomas (ST-EPN), designated as ST-EPN-RELA and ST-EPN-YAP1, respectively. The lack of adequate models for ST-EPN-YAP1 has so far hindered the development of effective targeted therapies for these tumors. In an attempt to model this subgroup, YAP1-MAMLD1 was constructed and cloned upstream of IRES-Luciferase into the pT2K transposable vector. The resulting plasmid was injected together with the Tol2 transposase into the lateral ventricle of E13.5 wildtype mouse embryos followed by transfection using an electroporation-based in vivo gene transfer approach. After birth, YAP1-MAMLD1-expressing tumors, monitored using luciferase-based in vivo bioluminescence imaging, developed rapidly with 100% penetrance, indicating that the fusion alone is enough to initiate tumors. Resulting tumors resembled molecular characteristics of their human counterparts and were distinct from mouse tumors driven by the C11orf95-RELA fusion. As in human tumors the YAP1-MAMLD1 protein was predominantly compartmentalized to the nuclei even when phosphorylated at serine residue 127, which normally retains YAP1 in the cytoplasm. The MAMLD1 domain was found to be crucial for nuclear translocation of the fusion protein. Intriguingly, amino acid replacement of serine 94 with alanine, which prevents interaction of YAP1 with Tead transcription factors, inhibited tumor formation. Conserved tumorigenic potential of transformed cells was confirmed by subsequent orthotopic transplantation of these tumor cells into immunocompromised recipient mice. We thus provide a novel model for ST-EPN-YAP1 ependymomas, which is currently entering first preclinical settings, especially by targeting YAP1-Tead interaction with preexisting drugs.

Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive pediatric brain tumors that are characterized by a recurrent mutation (K27M) within the histone H3 encoding genes H3F3A or HIST1H3A/B/C. These mutations have been shown to induce a global reduction in the repressive histone modification H3K27me3. Together with global changes in DNA methylation patterns this results in a broad diversity of downstream effects which hampers the identification of single therapeutic targets based on a molecular rationale. Therefore, our aim is to identify critical nodes of tumor development and maintenance using a DECIPHER pooled shRNA screening approach in combination with next-generation sequencing. The shRNA library targets more than 5,000 genes, with multiple shRNAs per target as well as internal controls. Currently, four patient-derived H3.3K27M mutated DIPG vs. two H3.3WT control high-grade glioma cell lines are being screened for shRNAs inducing cell death. Preliminary hits unexpectedly included the H3.3-specific histone chaperone complex components ATRX (alpha thalassemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein) - targets which are sometimes mutated in DIPG. This suggests a more complex role of H3.3-dependent heterochromatin formation in DIPGs than is currently appreciated. These and other promising hits will be validated in an expanded cell line panel using a custom CRISPR/Cas9 library, with top candidates further taken forward to in vivo pre-clinical testing for identification of drug targets.

Genomic studies have shown that multiple molecular subtypes of pediatric brain tumors exist that are biologically and clinically highly distinct. These findings ask for novel subtype specific treatments. To develop these we need more and better preclinical models that correctly reflect the proper tumor (sub)type. Orthotopic patient-derived xenograft (PDX) models generated by intracranial injection of primary patient material into the brain of NSG mice offer the unique possibility to test novel substances in primary patient tissue in an in vivo environment. Prior to drug selection and testing, extensive molecular characterizations of PDX and matching primary tumor/blood (DNA methylation, DNA sequencing, and gene expression) are needed to see how the PDX represents the original disease and to learn about targetable oncogenic drivers in each model. In collaboration with several groups around the world we have generated and fully characterized thus far 75 PDX models reflecting 15 distinct subtypes of pediatric brain cancer. PDX models always retain their molecular subtype and in the vast majority of cases also mutations and copy number alterations compared to matching primary tumors. Most aggressive tumors, harboring MYC(N) amplifications, are overrepresented in the cohort, but also subtypes which have not been available for preclinical testing before due to lack of genetically engineered mouse models or suitable cell lines, such as Group 4 medulloblastoma, are included. All models and corresponding molecular data will become available for the community for preclinical research. Examples of such preclinical experiments will be presented. PDX models of pediatric brain tumors are still quite rare. Our repertoire of PDX models and corresponding molecular characterizations allow researchers all over the world to find the right models for their specific scientific questions. It will provide an unprecedented resource to study tumor biology and pave the way for improving treatment strategies for children with malignant brain tumors.

Recent genomic studies have revealed multiple molecular subtypes of pediatric brain cancers that are not only biologically but also clinically distinct. In order to develop novel treatment strategies for these often fatal diseases we need more preclinical models like orthotopic patient-derived xenograft (PDX) models that correctly reflect the many different tumor types. Prior to drug selection and testing, extensive molecular characterizations are needed to precisely assign a distinct molecular subgroup to each PDX model and to learn about its targetable oncogenic drivers. In an international effort we aim to characterize a large repertoire of PDX models reflecting the many different molecular subtypes of pediatric brain cancer. Thus far, we have collected and characterized 64 established PDX models from 6 atypical teratoid rhabdoid tumors (AT/RT), 7 ependymomas (EPN), 16 high-grade gliomas (HGG), 32 medulloblastomas (MB), and 3 primitive neuroectodermal tumors (PNET). All PDX models and their matching primary tumors (if available) are analyzed by whole-exome and low-coverage whole-genome sequencing, as well as DNA methylation and gene expression profiling. The DNA methylation and gene expression data showed that PDX models always cluster together with their respective brain tumor reference samples and in most cases very close to their matching primary tumor. Tumor subtype-specific oncogenic lesions could be detected by both sequencing technologies. By comparing PDX models to their primary tumor, we showed that they retain the molecular subtype, mutations and copy number alterations. Only in rare cases we observed additional aberrations in PDX models such as chromothriptic events in one MB (Group 3) or chromosome 1q gain in one EPN (posterior fossa subtype A). However, these additional aberrations are typical for this tumor subtype and were therefore most likely already present in the primary lesion at a subclonal level. Analysis of our entire cohort identified a overrepresentation of the most aggressive tumor subtypes, but also subtypes which have not been available for preclinical testing before due to lack of genetically engineered mouse models or suitable cell lines, such as Group 4 MBs. Our molecular characterizations of PDX models provide an unprecedented resource to study tumor biology and pave the way for improving treatment strategies of malignant pediatric brain tumors. Citation Format: Sebastian Brabetz, Huriye Seker-Cin, Susanne N. Gröbner, Norman L. Mack, Volker Hovestadt, David T. W. Jones, Till Milde, Madison T. Wise, Jessica M. Rusert, Kyle Pedro, Karina Bloom, Xiao-Nan Li, Robert J. Wechsler-Reya, James M. Olson, Stefan M. Pfister, Marcel Kool. Molecular characterization of patient-derived xenograft models of pediatric brain tumors. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A07.

Solid tumors of the nervous system are the most common childhood cancers after leukemias. Although brain tumors are the leading cause of cancer-related mortality in children, there are not enough adequate model systems to study their biology. We therefore started a pediatric preclinical testing program in Heidelberg to generate orthotopic patient-derived xenograft (PDX) models for a large variety of pediatric brain tumors. Freshly dissected primary material from multiple centers is being sent to us immediately after surgical resections. One part of the tumor is being reserved for pathological and molecular analysis and the other part is being dissociated into a single cell suspension and injected into the brain of immunodeficient mice. After successful engraftment and passaging, extensive molecular characterization of the PDX tumor and the matching primary tumor are being performed. Thus far, we have injected 95 tumors: 36 low-grade gliomas (LGG), 23 medulloblastomas (MB), 13 ependymomas (EPN), 7 high-grade gliomas (HGG), 6 atypical teratoid rhaboid tumors (AT/RT), 3 meningeal tumors (MT), 3 embryonal tumors with multilayered rosettes (ETMR), 2 gliomatosis cerebri (GC), 1 dysembryoplastic neuroepithelial tumor (DNT) and 1 primitive neuroectodermal tumor (PNET). No engraftment was observed for any of the low-grade tumors (LGG, MT, DNT). For high-grade tumors we established initial engraftments of MB (5/23, 22%), EPN (5/13, 38%), HGG (2/7, 29%), AT/RT (2/6, 33%), ETMR (1/3, 33%) and PNET (1/1, 100%). 11 out of 16 (69%) established PDX models were already passaged at least twice in mice and can be used for preclinical experiments. We conclude that it is possible to generate preclinical models for most malignant pediatric brain tumor entities, but not for low-grade tumors using our current protocol. However, even for the malignant entities there seems to be a selection for only the most aggressive subtypes that successfully engraft. Therefore, due to the low engraftment rate of some tumor types, the rarity of pediatric brain tumors and the multitude of different subtypes, international collaborations are absolutely necessary in this field in order to generate and characterize a broad repertoire of PDX models for all pediatric brain tumor subtypes for preclinical testing. Citation Format: Norman L. Mack, Sebastian Brabetz, Florian Selt, Xanthopolous Christina, David Sumerauer, Heidi Bächli, Arnulf Pekrun, Martin U. Schuhmann, Stefan M. Pfister, Olaf Witt, Till Milde, Marcel Kool. Establishment of orthotopic patient-derived xenograft models of pediatric brain tumors – the Heidelberg experience. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A25.

PCM-16. MOLECULAR CHARACTERIZATION OF ORTHOTOPIC PATIENT-DERIVED XENOGRAFT MODELS OF PEDIATRIC BRAIN TUMORS Sebastian Brabetz1, Susanne N. Groebner1, Huriye Seker-Cin1, Norman L. Mack1, Volker Hovestadt1, David T. W. Jones1, Florian Selt1,2, Till Milde1,2, Madison T. Wise3, Jessica M.Rusert4, Kyle Pedro3, Karina Bloom3, Olaf Witt1,2, Sarah E. Leary3, Xiao-Nan Li5, Robert J. Wechsler-Reya4, James M. Olson3, Stefan M. Pfister1,2, and Marcel Kool1; German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany; Center for Individualized Pediatric Oncology (ZIPO) and Pediatric Brain Tumors, Department of Pediatric Oncology, University Hospital and National Center for Tumor Diseases (NCT), Heidelberg, Germany; Fred Hutchinson Cancer Research Center and Seattle Children’s Hospital, Seattle, WA, USA; Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA; Baylor College of Medicine, Houston, TX, USA Orthotopic patient-derived xenograft (PDX) models are an excellent platform for biomarker and preclinical drug development. Prior to drug selection and testing, extensive molecular characterization is needed to precisely determine the distinct molecular subgroup and constellation of genetic alterations for each PDX model, and thus identify its targetable oncogenic drivers. In an international effort we have characterized a large repertoire of PDX models reflecting many different molecular subtypes of pediatric brain tumors and assessed inter-tumoral heterogeneity within these subtypes. Thus far, we have collected and characterized 70 established PDX models from 6 ATRTs, 8 ependymomas, 16 high-grade gliomas, 38 medulloblastomas, and 2 CNS-PNETs. All PDX models and matching primary tumors (if available) have been analyzed by whole-exome and low-coverage wholegenome sequencing, as well as DNA methylation and gene expression profiling.PDXmodels always retain theirmolecular subtypeand in thevastmajority of cases also the mutations and copy number alterations when compared to their primary tumors. Only in rare cases do we observe additional aberrations, which most likely represent outgrowths of subclones from the primary tumor. Analysis of our entire cohort identified an overrepresentation of the most aggressive tumor subtypes, but also subtypes which have not been available for preclinical testing before due to lack of genetically engineered mouse models or suitable cell lines, such as Group 4 medulloblastoma. Our molecular characterization of PDX models will provide an unprecedented resource to study tumorbiologyandpave theway for improving treatment strategies forchildren with malignant brain tumors. Neuro-Oncology 18:iii139–iii144, 2016. doi:10.1093/neuonc/now080.16 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

EPN-30. YAP1-MAMLD1 FUSIONS ALONE ARE SUFFICIENT TO FORM SUPRATENTORIAL EPENDYMOMA-LIKE TUMORS IN MICE Kristian W. Pajtler1,2, Sebastian Brabetz1, Monika Mauermann1, Norman Mack1, Laura Sieber1, David T. W. Jones1, Hendrik Witt1,2, Huiqin Korkel-Qu3, Marc Zuckermann4, Jan Gronych4, Andrey Korshunov5,6, David Capper5,6, Hai-Kun Liu3, Stefan M. Pfister1,2, Marcel Kool1, and Daisuke Kawauchi1; German Cancer Research Center, Division of Pediatric Neurooncology, Heidelberg, Germany; University Hospital Heidelberg, Department of Pediatric Oncology, Hematology and Immunology, Heidelberg, Germany; German Cancer Research Center, Molecular Neurogenetics, Heidelberg, Germany; German Cancer Research Center, Molecular Genetics, Heidelberg, Germany; GermanCancer Research Center, Clinical Cooperation Unit Neuropathology, Heidelberg, Germany; University Hospital Heidelberg, Department of Neuropathology, Heidelberg, Germany Oncogenic fusions containing RELA orYAP1have recentlybeen identified as genetic hallmarks of distinct molecular subgroups of supratentorial ependymomas (ST-EPN), designated ST-EPN-RELA and ST-EPN-YAP1, respectively. ST-EPN-YAP1 tumors, exclusively found in pediatric patients, are molecularly andclinicallydifferentfromST-EPN-RELAtumors, suggesting that theyhave to be treated differently. YAP1 acts as a transcriptional regulator in the HIPPO tumor suppressor pathway. The lack of adequate models for ST-EPN-YAP1 has so far hindered efforts to develop effective targeted therapies for these tumors. In an attempt to model this subgroup, the most frequent fusion type, YAP1-MAMLD1, was constructed and cloned upstream of IRES-Luciferase into the pT2K transposable vector. The resulting vector was injected together with the Tol2 transposase into the lateral ventricle of E13.5 wildtype mouse embryos followed by transfection using an electroporation-based in vivo gene transferapproach.Afterbirth,YAP1-MAMLD1-expressing tumors,monitored using luciferase-based in vivo bioluminescence imaging, developed rapidly with 100% penetrance. The animals had to be sacrificed due to severe neurological symptoms on average at P20. Resulting tumors resembled molecular characteristics of their human counterparts. The YAP1-MAMLD1 protein was predominantly compartmentalized to the nuclei of tumor cells even when phosphorylated at serine residue 127, which normally retains YAP1 in the cytoplasm, implying constitutive activation of the YAP1 fusion protein. Conserved tumorigenic potential of transformed cells was confirmed by subsequent orthotopic transplantation of these tumor cells into immunocompromised recipient mice. We thus provide a novel model for ST-EPN-YAP1, which is currently entering first preclinical drug testings. Neuro-Oncology 18:iii30–iii39, 2016. doi:10.1093/neuonc/now070.29 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.