Analysis of Oncogenic Drivers in Supratentorial Brain Tumors

Pediatric brain tumors are a leading cause of cancer mortality among children and adolescents (age 0-19) because of the paucity of effective treatment regimens. Especially for ependymoma, surgical intervention combined with focal radiotherapy is the current standard of care in routine clinical practice while this regimen very often induces irreversible damage on the developing brain and patients frequently still suffer from tumor recurrence. Thus, generating de novo representative tumor models to decipher the underlying molecular mechanisms of tumorigenesis is imminent and crucial to provide more precise and mechanism-of-action based treatment plans. In my thesis, I applied various techniques to create in vivo models for several brain tumor types and identified potential therapeutic vulnerabilities. Chapter 2 focuses on dissecting the role of oncogenic fusion genes in C11orf95 fusion- positive supratentorial ependymoma (ST-EPN), a type of pediatric brain tumor with poor prognosis. C11orf95 is a zinc finger protein that binds to DNA but has not yet been well characterized. I performed in-utero electroporation in mouse embryos and found all tested C11orf95 fusion genes were able to drive malignant transformation in the cerebral cortex. The tumors faithfully recapitulated molecular characteristics of their human counterparts. The zinc finger domain and the fusion partners were essential for tumor formation. Cross-species genomic analyses demonstrated that C11orf95-related fusions can increase the expression of a sonic hedgehog mediator gene, GLI2. Targeting GLI2 with arsenic trioxide prolonged survival in mouse models, providing a basis for further preclinical studies for C11orf95 fusion-positive tumors. Based on these findings, C11orf95 is now officially designated as zinc finger translocation associated (ZFTA) by the HUGO Gene Nomenclature Committee. In the latest edition of the WHO classification of central nervous tumors, the group of ST-EPN with ZFTA fusion genes is now named as Supratentorial ependymoma, ZFTA fusion-positive (ST- EPN-ZFTA). In Chapter 3, I investigated on a novel group of neuroepithelial tumors harboring PLAGL1 fusion (NET_PLAGL1) that has been identified in 2021 only. Mouse model generation via in-utero electroporation unfortunately failed. However, after I had performed substantial methodological optimization, overexpression of PLAGL1 fusion gene through a doxycycline-mediated system in human induced pluripotent stem cell-derived neural stem cells, followed by in vivo orthotopic transplantation successfully led to brain tumor formation in mice. This inducible in vivo system offers a reliable model to study NET_PLAGL1 tumors as well as a versatile tool to answer various biological questions behind brain tumorigenesis.Array-based DNA methylation analysis to accurately classify tumors has been developed as a routine diagnostic tool for brain tumors and sarcomas. Since mouse models are the most widely used in vivo systems in pediatric cancer research, it is important to assess the molecular similarity across species based on the methylome. In Chapter 4, I describe the approach of generating a mouse model biobank for pediatric cancers. I collected and profiled 86 murine tumor models and 40 normal tissue controls. DNA methylation-based clustering showed that samples from the same model clustered together and the copy number alteration pattern of ependymoma and glioma (e.g TFG-MET fusion-driven) mouse models recapitulate their human counterparts. This validated biobank will serve as a beneficial resource for future developmental studies such as identifying cellular origin of the tumor and decoding the composition of tumor immune microenvironment

Primary cilia sensitize endothelial cells to BMP and prevent excessive vascular regression

Blood flow shapes vascular networks by orchestrating endothelial cell behavior and function. How endothelial cells read and interpret flow-derived signals is poorly understood. Here, we show that endothelial cells in the developing mouse retina form and use luminal primary cilia to stabilize vessel connections selectively in parts of the remodeling vascular plexus experiencing low and intermediate shear stress. Inducible genetic deletion of the essential cilia component intraflagellar transport protein 88 (IFT88) in endothelial cells caused premature and random vessel regression without affecting proliferation, cell cycle progression, or apoptosis. IFT88 mutant cells lacking primary cilia displayed reduced polarization against blood flow, selectively at low and intermediate flow levels, and have a stronger migratory behavior. Molecularly, we identify that primary cilia endow endothelial cells with strongly enhanced sensitivity to bone morphogenic protein 9 (BMP9), selectively under low flow. We propose that BMP9 signaling cooperates with the primary cilia at low flow to keep immature vessels open before high shear stress-mediated remodeling

Automatically Extracting Rubber Tree Stem Shape from Point Cloud Data Acquisition Using a B-Spline Fitting Program

Natural rubber is an important and strategic raw material, used in tires, gloves, and insulating products, that is mainly obtained by cutting the bark of rubber trees. However, the complex contour curve of the rubber tree trunk is hard to fit using a tapping machine. Thus, a trunk contour curve collection would be useful for the development of tapping machines. In this study, an acquisition system based on laser-ranging technology was proposed to collect the point cloud data of rubber tree trunks, and a B-spline fitting program was compiled in Matrix Laboratory (MATLAB) to extract the trunks’ contour curves. The acquisition system is composed of power, a controller, a driver, a laser range finder, and data transmission modules. An automatic extraction experiment on the contour curves of rubber tree trunks was carried out to verify the feasibility and accuracy of using the acquisition system. The results showed that the degree of rubber tree trunk characteristic recognition reached 94.67%, which means that the successful extraction of the rubber tree trunk contour curves and the B-spline fitting program are suitable for the extraction of irregular curves of rubber tree trunks. The coefficient of variation of repeated collection was 0.04%, which indicates that changes in relative positions and acquisition directions have little influence on the extraction and the accuracy of the acquisition system, which are high and stable. Therefore, it was unnecessary to adjust the position of the acquisition device before the collecting process, which helped to improve the efficiency of acquisition considerably. The acquisition system proposed in this study is meaningful to the practical production and application of agroforestry and can not only improve the precision of the rubber tapping process by combining with an automatic rubber tapping machine but can also provide technical support for the prediction of rubber wood volume and the development of ring-cutting equipment for other fruit trees

Cross-Species Genomics Reveals Oncogenic Dependencies in ZFTA/C11orf95 Fusion-Positive Supratentorial Ependymomas

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

Cross-species genomics reveals oncogenic dependencies in ZFTA/C11orf95 fusion-positive supratentorial ependymomas.

International audienceMolecular 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 histological 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

Recurrent fusions in PLAGL1 define a distinct subset of pediatric-type supratentorial neuroepithelial tumors

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2- and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients