Christin Schmidt

STAT3 Blockade Inhibits Radiation-Induced Malignant Progression in Glioma

High grade gliomas (HGG) are classified into four subgroups based on transcriptional signatures and phenotypic characteristics. In particular, the proneural-to-mesenchymal transition (PMT) is associated with increased malignancy, poor prognosis, and disease recurrence, but the underlying causes of PMT are still unclear. In this study, we investigated whether radiotherapy promotes PMT using a genetically engineered mouse model of proneural HGG. We found that cranial ionizing radiation induced robust and durable PMT in tumors. Additionally, we isolated primary proneural HGG cells from mouse and human tumors and demonstrate that radiation induced a sustained cell-intrinsic mesenchymal transition associated with increased invasiveness and resistance to the alkylating agent temozolomide. Expectedly, irradiation-induced PMT was also associated with activation of the STAT3 transcription factor, and the combination of STAT3 blockade using JAK2 inhibitors with radiation abrogated the mesenchymal transition and extended survival of mice. Taken together, our data suggest that clinical JAK2 inhibitors should be tested in conjunction with radiation in patients with proneural HGG as a new strategy for blocking the emergence of therapy-resistant mesenchymal tumors at relapse.

Matching mice to malignancy: molecular subgroups and models of medulloblastoma

IntroductionMedulloblastoma, the largest group of embryonal brain tumors, has historically been classified into five variants based on histopathology. More recently, epigenetic and transcriptional analyses of primary tumors have subclassified medulloblastoma into four to six subgroups, most of which are incongruous with histopathological classification.DiscussionImproved stratification is required for prognosis and development of targeted treatment strategies, to maximize cure and minimize adverse effects. Several mouse models of medulloblastoma have contributed both to an improved understanding of progression and to developmental therapeutics. In this review, we summarize the classification of human medulloblastoma subtypes based on histopathology and molecular features. We describe existing genetically engineered mouse models, compare these to human disease, and discuss the utility of mouse models for developmental therapeutics. Just as accurate knowledge of the correct molecular subtype of medulloblastoma is critical to the development of targeted therapy in patients, we propose that accurate modeling of each subtype of medulloblastoma in mice will be necessary for preclinical evaluation and optimization of those targeted therapies.

Loss of Smarc Proteins Impairs Cerebellar Development

SMARCA4 (BRG1) and SMARCB1 (INI1) are tumor suppressor genes that are crucially involved in the formation of malignant rhabdoid tumors, such as atypical teratoid/rhabdoid tumor (AT/RT). AT/RTs typically affect infants and occur at various sites of the CNS with a particular frequency in the cerebellum. Here, granule neurons and their progenitors represent the most abundant cell type and are known to give rise to a subset of medulloblastoma, a histologically similar embryonal brain tumor. To test how Smarc proteins influence the development of granule neurons and whether this population may serve as cellular origin for AT/RTs, we specifically deleted Smarca4 and Smarcb1 in cerebellar granule cell precursors. Respective mutant mice displayed severe ataxia and motor coordination deficits, but did not develop any tumors. In fact, they suffered from a severely hypoplastic cerebellum due to a significant inhibition of granule neuron precursor proliferation. Molecularly, this was accompanied by an enhanced activity of Wnt/β-catenin signaling that, by itself, is known to cause a nearly identical phenotype. We further used an hGFAP-cre allele, which deleted Smarcb1 much earlier and in a wider neural precursor population, but we still did not detect any tumor formation in the CNS. In summary, our results emphasize cell-type-dependent roles of Smarc proteins and argue against cerebellar granule cells and other progeny of hGFAP-positive neural precursors as the cellular origin for AT/RTs.

Preclinical drug screen reveals topotecan, actinomycin D, and volasertib as potential new therapeutic candidates for ETMR brain tumor patients

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.

Antisecretory Factor-mediated Inhibition of Cell Volume Dynamics Produces Anti-tumor Activity in Glioblastoma

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AFs ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 777–90. ©2018 AACR.

Abstract 3093: Unravelling the biology of aggressive and therapy-resistant embryonal tumors with multilayered rosettes (ETMR)

Embryonal tumor with multilayered rosettes (ETMR) is a highly aggressive embryonal CNS tumor, which predominantly affects children under the age of three to four years and is associated with a highly aggressive disease course with reported overall survival times ranging from 5-30 months. As these tumors have often been misdiagnosed as medulloblastoma or CNS-PNETs it was thought that ETMR is a very rare tumor. However, now molecular tools are available to detect ETMR and distinguish them from other brain tumors it has become clear that it is one of the most common brain tumors among infants. Amplification of a miRNA cluster at 19q13.42 and high expression of LIN28A have been identified as molecular hallmarks of ETMR, affecting 95-100% of samples tested and are considered unifying molecular diagnostic markers to detect them and distinguish from other brain tumors. Three histological variants of ETMR are known. These include embryonal tumor with abundant neuropil and true rosettes (ETANTR), ependymoblastoma (EBL), and medulloepithelioma (MEPL). A comprehensive clinical, pathological, and molecular analysis of 97 cases of these fatal brain neoplasms identified uniform molecular signatures in all tumors irrespective of histological patterns, indicating that ETANTR, EBL, and MEPL comprise a single biological entity. In particular, DNA methylation (Illumina 450k arrays) and gene expression data (Affymetrix 133plus2.0 arrays) showed that the three histological variants of ETMR are biologically indistinguishable but together highly distinct from other pediatric brain tumors. In order to better understand the biology of these highly aggressive pediatric CNS malignancies, we performed whole genome DNA sequencing of 15 tumor-normal pairs including 3 recurrences, complemented by (mi)RNA sequencing of tumor RNA. Mutations detected included mutations in TP53, CTNNB1, and mutations affecting the miRNA processing pathway. Chromothripsis was detected in several cases and in all cases affecting chromosome 19q. Finally, as DNA sequencing identified only very few somatic mutations per tumor, we next studied the epigenome of these tumors by performing whole genome bisulfite sequencing. Integrating these high throughput genomic analyses may now lead to alternative treatment strategies for these highly aggressive and therapy-resistant tumors. Citation Format: Marcel Kool, Natalie Jager, Dominik Sturm, David T.W. Jones, Volker Hoverstadt, Ivo Buchhalter, Pascal Johann, Christin Schmidt, Marina Ryzhova, Paul A. Northcott, Pablo Landgraf, Marc Remke, Michael D. Taylor, Martin Hasselblatt, Ulrich Schuller, Annie Huang, Marie-Laure Yaspo, Andreas von Deimling, Roland Eils, Peter Lichter, Andrey Korshunov, Stefan M. Pfister. Unravelling the biology of aggressive and therapy-resistant embryonal tumors with multilayered rosettes (ETMR). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2014-3093