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Background Pediatric low-grade gliomas (pLGG) are the most common pediatric central nervous system tumors, with driving alterations typically occurring in the MAPK pathway. The ERK1/2 inhibitor ulixertinib (BVD-523) has shown promising responses in adult patients with mitogen-activated protein kinase (MAPK)-driven solid tumors. Methods We investigated the antitumoral activity of ulixertinib monotherapy as well as in combination with MEK inhibitors (MEKi), BH3-mimetics, or chemotherapy in pLGG. Patient-derived pLGG models reflecting the two most common alterations in the disease, KIAA1549:BRAF-fusion and BRAFV600E mutation (DKFZ-BT66 and BT40, respectively) were used for in vitro and in vivo (zebrafish embryos and mice) efficacy testing. Results Ulixertinib inhibited MAPK pathway activity in both models, and reduced cell viability in BT40 with clinically achievable concentrations in the low nanomolar range. Combination treatment of ulixertinib with MEKi or BH3-mimetics showed strong evidence of antiproliferative synergy in vitro. Ulixertinib showed on-target activity in all tested combinations. In vivo, sufficient penetrance of the drug into brain tumor tissue in concentrations above the in vitro IC50 and reduction of MAPK pathway activity was achieved. In a preclinical mouse trial, ulixertinib mono- and combined therapies slowed tumor growth and increased survival. Conclusions These data indicate a high clinical potential of ulixertinib for the treatment of pLGG and strongly support its first clinical evaluation in pLGG as single agent and in combination therapy in a currently planned international phase I/II umbrella trial.

Recent advances in molecular profiling methods led to the identification of multiple new molecularly defined tumor types and subtypes, distinguished by distinct molecular markers and characteristic DNA methylation signatures. While the analysis of human methylome using microarrays has become an affordable and a routine in many labs, this technology until recently was not available for murine samples. In the past years, we have successfully generated a variety of mouse models for childhood tumors (e.g brain tumors and sarcomas) using different techniques, most of which faithfully reflect the human tumor counterparts at the histological level. With the recently released Infinium Mouse Methylation BeadChip, we now set out to use our models to generate the first DNA methylation database for murine pediatric tumors. We profiled more than 70 mouse models of pediatric tumors including gliomas, medulloblastomas, ependymomas and sarcomas, as well as 40 normal brain and muscle control tissues. We are currently performing a cross-species comparative analysis of established mouse models and the human counterparts. This will assess, how faithfully each models reflect the human situation and examine the effects of multiple passages of allografting. We will also analyze purified immune cell populations and use the derived methylation signatures to assess the model-specific immune microenvironment. Furthermore, we will investigate the methylomes of multiple putative cells-of-origin, which is hardly possible in the human context due to the lack of purified material. We will correlate these to murine tumor samples and thereby provide novel insights into tumor origins. In summary, our study will generate a validated biobank of murine models for pediatric cancers and provide a valuable resource for future developmental studies and preclinical trials.

Pediatric high-grade glioma is a heterogeneous group of highly malignant tumors of the central nervous system, with a median overall survival of less than two years after diagnosis, demanding novel treatment options. One innovative approach is gene therapy, which has so far been hampered for cancer treatment owing to the lack of a system targeting tumor cells specifically. To overcome this limitation, we established a novel strategy for gene therapy, combining tumor cell-specific adeno-associated virus (AAV) variants with oncogene-specific CRISPR-Cas nucleases. We screened 177 different Cas9/gRNA combinations targeting the genes encoding H3K27M or BRAFV600E, and identified highly specific nucleases that edited the oncogenic allele but left the respective WT loci intact, which we validated by PCR amplicon sequencing. Next, we intravenously injected an AAV library engineered to encode its own capsid DNA into mice harboring patient-derived xenograft tumors driven by H3K27M or BRAFV600E. After 21 days, we resected neoplasms and separated mCherry-labeled tumor cells from normal surrounding cells by fluorescence-activated cell sorting. Using the DNA from tumor cells as template, we generated a second AAV library, which was utilized in another round of in vivo selection. At the end of each screen, DNA from tumor cells, surrounding cells, and control tissues (liver and spleen) was analyzed by amplicon sequencing. Strikingly, we identified multiple AAV variants that were highly and recurrently enriched in the analyzed tumor tissues. We are currently validating these variants by intravenously injecting selected, GFP-encoding AAVs to tumor-bearing mice and by subsequently analyzing their distribution throughout the aforementioned tissues. We will combine oncogene-specific nucleases with these validated AAV variants and analyze their anti-tumoral efficacy in a preclinical setting. Furthermore, we plan to adapt this approach to allografted mice, evaluating its feasibility and efficacy in syngeneic models.

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.

Ulixertinib (BVD-523) is a well-tolerated, orally delivered, catalytic ERK1/2 inhibitor, which has shown promising responses in adult patients with mitogen-activated protein kinase (MAPK)-driven solid tumors. Pediatric low grade gliomas (pLGGs) are the most common pediatric brain tumors, with the most frequent driving alterations (KIAA:BRAF fusion, BRAF V600E mutation) occurring in the MAPK pathway. To investigate the anti-tumoral activity of ulixertinib in pLGG, cell lines recapitulating both main MAPK alterations were used: DKFZ-BT66 (pilocytic astrocytoma; KIAA:BRAF fusion) and BT40 (pleomorphic xanthoastrocytoma; BRAF V600E mutation and CDKN2A/B deletion). The potential synergism of combinations with MEK inhibitors, senolytics, and chemotherapy was investigated in vitro using metabolic activity and MAPK activity assays. The most promising combinations were validated in vitro by analysis of viable, dead, and apoptotic cells through high-content microscopy. The most clinically relevant combinations were further validated in vivo: 1) in two zebrafish embryo models (respectively, BT40 and DKFZ-BT66 yolk sac injection) and 2) in NSG mice (BT40 orthotopic PDX) including in vivo pharmacokinetic and -dynamic analyses. Our data demonstrate ulixertinib’s ability to inhibit MAPK pathway activity in all used models. Ulixertinib treatment reduced cell viability in the BRAF V600E mutated cell line at a remarkably low concentration of 62.7 nM (compared to other cell lines’ IC50 from the Genomics of Drug Sensitivity in Cancer database). In vivo pharmacokinetic and -dynamic analyses showed good penetrance of the drug into mouse brain tissue, with concentrations above the in vitro IC50 and reduction of MAPK activity as assessed by Western blot. Furthermore, ulixertinib treatment slowed tumor growth and significantly increased survival in NSG mice with orthotopic BT40 xenografts. Ulixertinib showed indications for anti-proliferative synergy in vitro, according to the Loewe and Bliss independence models, in combination with MEK inhibitors (trametinib, binimetinib) or senolytics (navitoclax, A1331852). Combinations with chemotherapy (carboplatin, vinblastine) were at most additive. Indications for synergy with binimetinib and navitoclax were confirmed in the zebrafish embryo xenograft models for both MAPK-altered backgrounds. The combination of ulixertinib with navitoclax was further investigated in the BT40 PDX mouse model, where tumor growth and survival were comparable to ulixertinib monotherapy. In conclusion, our data indicate a strong potential for ulixertinib as a clinically relevant therapeutic option for the treatment of pLGG to be further investigated in upcoming clinical trials. Potential synergism with MEK inhibitors and senolytics was noted and warrants further investigation. Citation Format: Romain Sigaud, Lisa Rösch, Charlotte Gatzweiler, Julia Benzel, Laura von Soosten, Heike Peterziel, Sara Najafi, Simay Ayhan, Nina Hofmann, Kathrin I. Förster, Jürgen Burhenne, Rémi Longuespée, Cornelis M. van Tilburg, David T. Jones, Stefan M. Pfister, Deborah Knoerzer, Brent Kreider, Max Sauter, Kristian W. Pajtler, Marc Zuckermann, Ina Oehme, Olaf Witt, Till Milde. The first-in-class ERK inhibitor ulixertinib (BVD-523) shows activity in MAPK-driven pediatric low-grade glioma models as single agent and in combination with MEK inhibitors or senolytics [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 5221.

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.

Ulixertinib (BVD-523) is a catalytic ERK1/2 inhibitor that showed promising responses in adult patients with mitogen-activated protein kinase (MAPK)-driven solid tumors. Pediatric low-grade gliomas (pLGG) are the most common pediatric brain tumors, with the most frequent driving alterations in the MAPK pathway. The anti-tumor activity of ulixertinib in pLGG and its potential synergism in combination with MEK inhibitors, senolytics, and chemotherapy were investigated in vitro using metabolic activity, MAPK reporter assay and high-content microscopy in pLGG-derived cell lines (DKFZ-BT66 - KIAA:BRAF fusion; BT40 - BRAF V600E mutation and CDKN2A/B deletion). The most clinically relevant combinations were further validated in vivo: 1) in zebrafish embryo models (BT40 and DKFZ-BT66 yolk sac injection) and 2) in NSG mice (BT40 orthotopic PDX) including in vivo pharmacokinetic and -dynamic analyses. Ulixertinib inhibited MAPK pathway activity in all models and reduced cell viability in the BRAF V600E mutated cell line at concentrations in the nanomolar range. In vivo pharmacokinetic and -dynamic analyses showed penetrance of the drug into mouse brain tissue and on-target activity, with concentrations above the in vitro IC50 and reduction of MAPK activity. Ulixertinib treatment slowed tumor growth and significantly increased survival in NSG mice with BT40 xenografts. Ulixertinib showed indications for anti-proliferative synergy in vitro in combination with MEK inhibitors (trametinib, binimetinib) or BH3 mimetics (navitoclax, A-1331852). Combinations with chemotherapy (carboplatin, vinblastine) were at most additive. Indications for synergy with binimetinib and navitoclax were confirmed in the zebrafish embryo models. In the NSG mouse model, the combination of ulixertinib with senolytics induced effects on tumor growth and survival comparable to ulixertinib monotherapy. Ulixertinib shows promising potential as a clinically relevant therapeutic option for the treatment of pLGG to be further investigated in upcoming clinical trials. Potential synergism with MEK inhibitors and BH3 mimetics was noted and warrants further investigation.

Medulloblastoma (MB), the most common embryonal tumour of the Central Nervous System, occurs in the cerebellum. Treatment regimens involve surgery, craniospinal radiotherapy, and chemotherapy. The greatest mortality is associated with disseminated disease, almost exclusively found in the leptomeningeal space. Unfortunately, knowledge about the aetiology of MB spread is limited and the need for kinder and efficacious therapy remains an unmet goal. Of the four molecular classified MB groups, Group3 (Gr3) MB presents with a high frequency of metastasis at diagnosis, with the worst overall survival. Gr3 MB tumours are dominated by primitive progenitor-like cells and cMYC deregulation; often, p53 deficiency is observed at relapse. To dissect the biology of primary and metastatic Gr3 MB, we have developed a new germline genetically engineered mouse model (GEMM), harbouring cMYC amplification in a Tamoxifen-inducible p53 functional background (Trp53ERTAM strain). A novel LSL-cMYC-CopGFP-Luciferase transgene was integrated into the Rosa-26 locus of the mouse genome. Transgenic mice were crossed with a strain expressing Cre recombinase under the Blbp promoter targeting embryonic neural progenitors, and subsequently bred to Trp53ERTAM mice. As result, the cMYC overexpression was sufficient to generate tumours. Tumour penetrance was observed in all the expected tumour bearing genotypes, with increased aggressiveness in a non-functional p53 background. Bioluminescence imaging demonstrated tumour onset in the brain and dissemination along the spinal cord. CopGFP positive tumour cells were isolated from primary and metastatic tumours. Pathological interrogation confirmed that tumours present large cell/anaplastic (LCA) histology. Analysis of preliminary transcriptional profiling data proved that tumours cluster with human Gr3 MB. Ongoing methylation profiling and multi-omics approaches will inform on the tumour cells of origin and clonal divergence of primary tumour versus metastasis. In conclusion, we have successfully developed a novel immunocompetent mouse model of metastatic Gr3 MB with which we can investigate therapeutic vulnerabilities of MB.

Despite substantial advances in the molecular classification of pediatric brain tumors over the last decade, many tumors are still incurable, have a poor prognosis or therapy-related detrimental long term effects. To develop innovative and modern therapy approaches, sophisticated mouse models, faithfully reflecting the human disease are urgently needed. However, the development of transgenic mouse lines is a time consuming process that is confronted with an increasing number of candidate genes resulting from next generation sequencing of tumor specimens. To cope with this discrepancy, more adaptable animal models are needed. Using somatic gene transfer to manipulate cells in situ holds great advantages in terms of speed and flexibility. We therefore established a system in which we are using the CRISPR/Cas9 system for somatic disruption of candidate tumor suppressor genes (TSGs) in combination with transposon-mediated overexpression of candidate oncogenes to induce murine brain tumors. By deleting Ptch1 in murine cerebella, we induced Sonic Hedgehog (SHH) subgroup medulloblastoma with high penetrance and short latency. We characterized the induced mutations in depth and did not detect recurrent off-targets using unsupervised whole genome sequencing. We further demonstrated that this method is suited to induce other brain tumor entities by targeting multiple TSGs at the same time or by combining TSG targeting with overexpression of certain oncogenes. To date we established five novel mouse models using this system. Two of these resulted from simultaneous deletion of Nf1, Pten and Trp53 or from combining overexpression of a MET fusion with a loss of Trp53, respectively. Both of these combinations led to efficient glioblastoma induction in the murine forebrain. Our results demonstrate a fast and flexible system for validating novel candidate genes and for generating faithful animal models of human cancer. Citation Format: Marc Zuckermann, Britta Ismer, Volker Hovestadt, Christiane B. Knobbe-Thomsen, Marc Zapatka, Paul A. Northcott, Martine F. Roussel, Guido Reifenberger, Peter Lichter, Stefan M. Pfister, Daisuke Kawauchi, Jan Gronych, David T. Jones. Somatic CRISPR/Cas9-mediated gene editing enables versatile brain tumor modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5109.

Pilocytic astrocytoma, the most common childhood brain tumor, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression and often becoming a chronic disease with substantial morbidities. Here we describe whole-genome sequencing of 96 pilocytic astrocytomas, with matched RNA sequencing (n = 73), conducted by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. We identified recurrent activating mutations in FGFR1 and PTPN11 and new NTRK2 fusion genes in non-cerebellar tumors. New BRAF-activating changes were also observed. MAPK pathway alterations affected all tumors analyzed, with no other significant mutations identified, indicating that pilocytic astrocytoma is predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in the NF1 gene. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.

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.

Modeling cancer in mice through engineering of candidate genes in the germline has long been the gold standard for the validation of putative oncogenes or tumor suppressor genes (TSGs). For TSGs, whereby loss-of-function (LOF) mutations act as a driver for malignant transformation, this has traditionally been accomplished using constitutive or cell type-specific knockout strategies mediated by homologous recombination in embryonic stem cells. While this allows evaluation of cell type-specific susceptibility to malignant transformation, generation of genetically engineered mouse models (GEMMs) is a time consuming process. For in vivo investigation of a large number of molecular alterations, such as the many new candidates currently emerging from large-scale tumor genome sequencing efforts, a faster and more flexible method is required. We therefore sought to adapt the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-guided endonuclease technique for the somatic disruption of candidate TSGs and thereby complement aforementioned already existing models like GEMM. To provide a flexible and effective method for investigating somatic LOF alterations and their influence on tumorigenesis in vivo, we established in vivo somatic gene transfer of CRISPR/Cas9-encoding vectors using polyethylenimine or in utero electroporation, respectively, allowing for in vivo targeting of TSGs in the developing murine brain. We demonstrate the utility of this approach by deleting the tumor suppressor Ptch1, which resulted in the development of cerebellar tumors resembling sonic hedgehog (SHH) subgroup medulloblastoma both at the histopathological as well as the molecular level. Furthermore, we show that multiple genes can be disrupted with this approach, using in utero electroporation of guide RNAs (gRNAs) targeting Trp53, Pten and Nf1 into the forebrain of mice. This resulted in induction of glioblastoma with 100% penetrance. Using whole genome sequencing (WGS) we characterized the MB-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumors. Taken together, these approaches provide a fast and convenient way for validating the emerging wealth of novel candidate TSGs and the generation of faithful animal models of human cancer. Citation Format: Marc Zuckermann, Volker Hovestadt, Christiane B. Knobbe-Thomsen, Marc Zapatka, Paul A. Northcott, Kathrin Schramm, Jelena Belic, David T.W. Jones, Barbara Tschida, Branden Moriarity, David Largaespada, Martine F. Roussel, Andrey Korshunov, Guido Reifenberger, Stefan M. Pfister, Peter Lichter, Daisuke Kawauchi, Jan Gronych. Somatic CRISPR/Cas9-mediated tumor suppressor disruption enables versatile brain tumor modeling. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr PR02.