Abstract BACKGROUND A major challenge in the treatment of brain tumors is the limited penetration of many drugs through the blood-brain barrier (BBB). BBB characteristics include endothelial cells connected by tight junction proteins (e.g. Claudin5, ZO1, occludin) and the expression of efflux transporters (e.g. P-gp) with the physiological role to limit the accumulation of potentially toxic substances in brain tissue. Our study aims to enhance our understanding of the BBB composition across different molecular groups of ependymoma (EPN) with the goal of leveraging this knowledge to improve therapeutic strategies against these tumors. METHODS We applied a multi-omics approach integrating single-nuclear RNA sequencing and ultra-high content imaging to unravel BBB composition at both transcriptomic and proteomic levels. Patient-derived xenograft (PDX) models were utilized to explore differences in BBB penetration between tumor and healthy brain tissue following drug treatments. RESULTS Expression of tight junction and transporter proteins revealed strong dependencies specific to molecular groups, but were independent of the corresponding brain regions. Human tissue of ST-EPN-ZFTA exhibited highest expression of claudin5, while both ST-EPN-ZFTA and PF-EPN-A represented upregulation of occludin and ZO1 in comparison to healthy tissue. These findings on claudin5 and ZO1 were further confirmed in PDX models. Single-cell data analysis from patients localized the expression of relevant BBB factors to tumor-associated endothelial cells. Treatment of PDX models with drugs revealed disparities in drug penetration between EPN tumors and healthy brain regions. Notably, for most drugs BBB penetration was lower in ST-EPN-ZFTA tumors than healthy brain region. CONCLUSIONS Molecular BBB specifics may contribute to drug resistance of aggressive EPN particularly in ST-EPN-ZFTA tumors, which presented high tight junction expressions and lower drug penetration. This resource aims to improve BBB penetration prediction in EPN, to identify combination therapies targeting BBB components and to select innovative drug delivery approaches.

Abstract Increasing evidence highlights the diverse functions of extracellular vesicles (EVs) as intercellular messengers in all facets of cancer progression, pointing towards a potential new avenue in cancer treatment. Limited understanding of tumor-specific deregulations and lack of suitable drug compounds have hampered clinical translation of targeted interference with EV signaling. The most aggressive types of intracranial ependymoma (EPN) in children and adults are resistant to chemotherapy. Despite enormous diagnostic advancements in classification and stratification, EPN lack recurrent effective molecular targets precluding access of affected patients to precision-based oncology therapeutics. Here, we characterized the proteome of ninety-five EPN and five matching healthy control brain tissue samples. Employing a mulit-omic data integration approach, we identified EVs as an excessively augmented pathway on the functional level. Isolation and molecular characterization of EPN EVs combined with single cell sequencing analysis, and ultra-high content imaging unraveled perivascular mesenchymal cell populations as predominant EV originators. Targeting EV pathways through chemical inhibition of sphingomyelinases (SMPD1, SMPD2, SMPD3) including re-purposed FDA approved compounds revealed growth inhibition, reduced invasion capacity in vitro and significant survival probability in vivo. Transcriptomic analysis and scanning electron microscopy delineated transition of mesenchymal-to neuroepithelial subpopulations in cells and patient-derived xenograft (PDX) models of EPN upon reduction of EV release. This study showcases the inhibition of EV signaling as a promising therapeutic strategy against mesenchymal tumor persister cells, which are the suspected origins of repeated EPN relapses. Our study provides a framework to leverage cell state-specific vulnerabilities and drug repurposing to attack the most aggressive cell populations in cancers with unfavorable prognosis.

Suppl. Table S1 - A summary of the human cohort used for DNA methylation-profiling_DRG3003

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.

Suppl. Table S7 - Antibody list

Suppl. Table S4 - Histopathological evaluation of 25 cases

Novel drug treatments for pediatric patients with cancer are urgently needed. Success of drug development in pediatric oncology has been promising, but many drugs still fail in translation from preclinical to clinical phases. To increase the translational potential, several improvements have been implemented, including the use of clinically achievable concentrations in the drug testing phase. Although pharmacokinetic (PK) parameters of numerous investigated drugs are published, a comprehensive PK overview of the most common drugs in pediatric oncology could guide preclinical trial design and improve the translatability into clinical trials. A review of the literature was conducted for PK parameters of 74 anticancer drugs, from the drug sensitivity profiling library of the INdividualized Therapy FOr Relapsed Malignancies in Childhood (INFORM) registry. PK data in the pediatric population were reported and complemented by adult parameters when no pediatric data were available. In addition, blood–brain barrier (BBB)‐penetration assessment of drugs was provided by using the BBB score. Maximum plasma concentration was available for 73 (97%), area under the plasma concentration‐time curve for 69 (92%), plasma protein binding for 66 (88%), plasma half‐life for 57 (76%), time to maximum concentration for 54 (72%), clearance for 52 (69%), volume of distribution for 37 (49%), lowest plasma concentration reached by the drug before the next dose administration for 21 (28%), and steady‐state concentration for 4 (5%) of drugs. Pediatric PK data were available for 48 (65%) drugs. We provide a comprehensive review of PK data for 74 drugs studied in pediatric oncology. This data set can serve as a reference to design experiments more closely mimicking drug PK conditions in patients, and may thereby increase the probability of successful clinical translation.

Suppl. Table S5 - GO analysis for C11orf95-target models specific DEGs

Suppl. Table S2 - Copy number alterations for all 613 tumors

Suppl. Table S6 - Primer list

Suppl. Table S3 - Differentially expressed genes of ST-EPN-RELA and cluster 2 - 4

BACKGROUND: A major challenge in the treatment of brain tumors is the limited penetration of many drugs through the blood-brain barrier (BBB). BBB characteristics include endothelial cells connected by tight junction proteins (e.g. Claudin5) and expression of efflux transporters (e.g.P-gp) with the physiological role to limit the accumulation of potentially toxic substances in brain tissue. While BBB permeability shows anatomical region-specific characteristics and changes with age, brain tumors can also impact its integrity. To achieve therapeutically relevant drug concentrations in the tumor, characterization of the pathophysiological BBB is fundamental when developing (pre)clinical trials. Our study seeks to increase our understanding of the BBB composition in various molecular groups of ependymoma. RESULTS: Transcriptional BBB characteristics were assessed for primary ependymoma (n=440), cell lines (n=3), mouse models (n=22), and healthy brain controls (n=200). T-distributed stochastic neighbor embedding (tSNE)-based clustering analyses based on most relevant tight junction and efflux transporter gene sets revealed distinct molecular ependymoma group-specific expression patterns. While patient-derived xenografts models (PDX, n=20) showed high similarity with patient tumor samples, in utero electroporation-based (IUE, n=2) mouse models did not fully recapitulate these BBB characteristics. Supratentorial ependymoma with ZFTA fusions revealed a higher transcriptional expression level of important tight junctions (e.g.Claudin5) compared to other groups and normal brain which was confirmed at protein level in respective PDX models by using both western blot and ultra-high content imaging. CONCLUSION: Analyses of important BBB markers revealed significant differences between molecular groups of ependymoma, which may partly explain drug resistance of ependymoma with ZFTA fusion caused by a low BBB permeability. BBB characteristics of corresponding models suggest that IUE and PDX models representing ZFTA fusion-driven tumors cannot equally be used within preclinical drug trials. Findings will be further validated in preclinical studies while molecular BBB characterization will be expanded to other brain tumors.

Desorption/ionization (DI)-mass spectrometric (MS) methods offer considerable advantages of rapidity and low-sample input for the analysis of solid biological matrices such as tissue sections. The concept of desorption electrospray ionization (DESI) offers the possibility to ionize compounds from solid surfaces at atmospheric pressure, without the addition of organic compounds to initiate desorption. However, severe drawbacks from former DESI hardware stability made the development of assays for drug quantification difficult. In the present study, the potential of new prototype source setups (High Performance DESI Sprayer and Heated Transfer Line) for the development of drug quantification assays in tissue sections was evaluated. It was demonstrated that following dedicated optimization, new DESI XS enhancements present promising options regarding targeted quantitative analyses. As a model compound for these developments, ulixertinib, an inhibitor of extracellular signal-regulated kinase (ERK) 1 and 2 was used.

Desorption/ionization mass spectrometry (DI-MS) approaches allow for the rapid quantification of drugs in biological matrices using assays that can be validated according to regulatory guidelines. However, specific adaptations must be applied to create reliable quantification methods, depending on the approach and instrumentation used. In the present article, we demonstrate the importance of the molecular weight, the fragmentation pattern, and the purity of the internal standard for the development of matrix-assisted laser desorption/ionization (MALDI)-ion mobility (IM)-tandem MS and MS/MS methods. We present preliminary results of method development for the quantification of selinexor in microdialysis fluids with a stable isotopically labeled internal standard. In addition, we discuss the selection of internal standards for MALDI-MS assays using different instrumentations.

Abstract INTRODUCTION: The majority of pediatric ependymoma (EPN) comprise either supratentorial EPN characterized by ZFTA-fusions (ST-EPN-ZFTA) or posterior fossa group A EPN (PF-EPN-A), for both of which only limited therapeutic options are available. Because pediatric EPNs have a relatively low mutational burden, identification and characterization of tumor-associated pathways and molecular processes are of critical importance to reveal potential therapeutic targets. Data from previous transcriptional studies and a cross-species in vivo screen implied aberrant vesicular pathways in ST-EPN-ZFTA, prompting further investigation of their putative role in EPN pathogenesis. METHODS: We investigated EPN group-specific differences in extracellular vesicle (EV) biogenesis pathways in human EPN transcriptome and proteome datasets. In addition, we characterized isolated EPN EVs by mass spectrometry. EPN-specific EV cargo was further investigated by immunofluorescence staining and western blotting. This enhanced understanding of EPN vesicular signaling allowed for a pre-selection of inhibitors targeting specific EV biogenesis pathways. In vitro proliferation and invasion assays as well as in vivo treatment studies were performed on EPN model systems. RESULTS: Integration of multi-omic data from both EPN tissues and EPN-EV-associated proteome led to the identification of ST-EPN-ZFTA-specific EV populations. We could spatially map specific EV markers to the perivascular niche that primarily harbors undifferentiated ST-EPN-ZFTA cell populations. Targeting EV biogenesis pathways by inhibiting factors of the lipid metabolism reduced the abundance of released EVs resulting in altered growth behavior and decreased invasion of tumor cells in vitro. In vivo validation of EV release inhibitors in an orthotopic ST-EPN-ZFTA PDX model significantly reduced tumor growth and increased survival. OUTLOOK: In summary, we have leveraged ST-EPN-ZFTA-specific EV pathways as a potential therapeutic vulnerability. Further mechanistic investigations on EPN EV biogenesis, release, or uptake are expected to improve our understanding of the cross-talk between tumor cells and cells of the microenvironment and may lead to potential new therapeutic avenues.

Pituitary adenomas (PAs) are mostly benign endocrine tumors that can be treated by resection or medication. However, up to 10% of PAs show an aggressive behavior with invasion of adjacent tissue, rapid proliferation, or recurrence. Here, we provide an overview of target structures in aggressive PAs and summarize current clinical trials including, but not limited to, PAs. Mainly, drug targets in PAs are based on general features of tumor cells such as immune checkpoints, so that programmed cell death 1 (ligand 1) (PD-1/PD-L1) targeting may bear potential to cure aggressive PAs. In addition, epidermal growth factor receptor (EGFR), mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and their downstream pathways are triggered in PAs, thereby modulating tumor cell proliferation, migration and/or tumor angiogenesis. Temozolomide (TMZ) can be an effective treatment of aggressive PAs. Combination of TMZ with 5-Fluorouracil (5-FU) or with radiotherapy could strengthen the therapeutic effects as compared to TMZ alone. Dopamine agonists (DAs) are the first line treatment for prolactinomas. Dopamine receptors are also expressed in other subtypes of PAs which renders Das potentially suitable to treat other subtypes of PAs. Furthermore, targeting the invasive behavior of PAs could improve therapy. In this regard, human matrix metalloproteinase (MMP) family members and estrogens receptors (ERs) are highly expressed in aggressive PAs, and numerous studies demonstrated the role of these proteins to modulate invasiveness of PAs. This leaves a number of treatment options for aggressive PAs as reviewed here.

Abstract 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.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has rarely been used in the field of therapeutic drug monitoring, partly because of the complexity of the ionization processes between the compounds to be quantified and the many MALDI matrices available. The development of a viable MALDI-MS method that meets regulatory guidelines for bioanalytical method validation requires prior knowledge of the suitability of (i) the MALDI matrix with the analyte class and properties for ionization, (ii) the crystallization properties of the MALDI matrix with automation features, and (iii) the MS instrumentation used to achieve sensitive and specific measurements in order to determine low pharmacological drug concentrations in biological matrices. In the present hybrid article/white paper, we review the developments required for the establishment of MALDI-MS assays for the quantification of drugs in tissues and plasma, illustrated with concrete results for the different steps. We summarize the necessary parameters that need to be controlled for the successful development of fully validated MALDI-MS methods according to regulatory authorities, as well as currently unsolved problems and promising ways to address them. Finally, we propose an expert opinion on future perspectives and needs in order to establish MALDI-MS as a universal method for therapeutic drug monitoring.

Actinomycin D is a potent cytotoxic drug against pediatric (and other) tumors that is thought to barely cross the blood–brain barrier. To evaluate its potential applicability for the treatment of patients with central nervous system (CNS) tumors, we established a cerebral microdialysis model in freely moving mice and investigated its CNS disposition by quantifying actinomycin D in cerebral microdialysate, brain tissue homogenate, and plasma. For this purpose, we developed and validated an ultraperformance liquid chromatography–tandem mass spectrometry assay suitable for ultra-sensitive quantification of actinomycin D in the pertinent biological matrices in micro-samples of only 20 µL, with a lower limit of quantification of 0.05 ng/mL. In parallel, we confirmed actinomycin D as a substrate of P-glycoprotein (P-gp) in in vitro experiments. Two hours after intravenous administration of 0.5 mg/kg, actinomycin D reached total brain tissue concentrations of 4.1 ± 0.7 ng/g corresponding to a brain-to-plasma ratio of 0.18 ± 0.03, while it was not detectable in intracerebral microdialysate. This tissue concentration exceeds the concentrations of actinomycin D that have been shown to be effective in in vitro experiments. Elimination of the drug from brain tissue was substantially slower than from plasma, as shown in a brain-to-plasma ratio of approximately 0.53 after 22 h. Because actinomycin D reached potentially effective concentrations in brain tissue in our experiments, the drug should be further investigated as a therapeutic agent in potentially susceptible CNS malignancies, such as ependymoma.

Abstract Molecular groups of supratentorial ependymomas comprise tumors with ZFTA–RELA or YAP1-involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylation–based classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histologic features containing alternative translocations that shared ZFTA as a partner gene. Somatic overexpression of ZFTA-associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation in vivo, and cross-species comparative analyses identified GLI2 as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion–positive tumors. Significance: ZFTA–RELA fusions are a hallmark feature of supratentorial ependymoma. We find that ZFTA acts as a partner for alternative transcriptional activators in oncogenic fusions of supratentorial tumors with various histologic characteristics. Establishing representative mouse models, we identify potential therapeutic targets shared by ZFTA fusion–positive tumors, such as GLI2. This article is highlighted in the In This Issue feature, p. 2113