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Supervisors: Dr David Michod, Dr Paula Alexandre
Background:
Diffuse midline Glioma (DMG) is a tumour located in the pons of the brain stem which affects mostly young children. DMG has a zero percent survival rate with a median overall survival of children diagnosed with DMG of approximately 9 months, which makes it one of the most devastating paediatric malignancies. Clinical studies conducted over the past 20 years, most of them using drugs approved for adults, have failed to demonstrate any significant improvement in survival for these patients. DMG is characterized by a specific mutation resulting in the expression of a mutant histone 3 protein where lysine 27 is replaced by a methionine (H3K27M). Expression of this mutation has a dominant effect leading to a global reduction of methylation of histone H3 lysine 27 (H3K27), an important mark for the modulation of gene expression. In addition, because of the global H3K27 reduction in methylation, the DMG epigenome shows increased H3K27 acetylation.
Aims/Objectives:
Drug discovery for DMG is a very active research topic at GOSICH, with several groups active in this research area (Michod, de Boer, Hargrave and Martinez-Barbera labs). However, translation to the clinic of our research is affected by the absence of in vivo model to test the potential of our candidate drugs. We therefore propose to use zebrafish to generate two experimental systems that model DMG. A transgenic model, which will recapitulate the molecular and cellular evolution of DMG and an orthotopic model which will recapitulate the biology and pathology of the human malignancy.
Methods:
Aim1: To create a transgenic DMG fish model
To generate a zebrafish transgene that induces DMG, we will first produce constructs that are necessary for the expression of the histone mutant H3K27M under the control of a promoter that will induce its expression in the zebrafish central nervous system during development. As p53 expression is lost in DMG, we will inject our constructs in a p53 zebrafish mutant. The DNA constructs will be co-injected in p53 knockout zebrafish embryos at 1-cell stage and screened for fluorescence 24 hours post injection. The mosaic labelled zebrafish will be monitored for tumour formation in the central nervous system using a wide-field fluorescence and confocal microscope. Tumour cells expansion, movements and association with the brain vasculature will be monitored in a wide-field fluorescence and confocal microscope. Tumour will also be dissected for analyses by RNA-seq and ChiP-seq techniques.
Aim2: To establish an orthotopic DMG zebrafish model
To directly test candidate drugs in human DMG primary cancer cells, we will develop an orthotopic zebrafish model using an approach that has been successfully used in the past to study the brain tumour glioblastoma multiform. Human primary DMG cells expressing red fluorescent proteins will be transplanted into zebrafish at blastula-stage. After micro-injection, embryos will be screened for successful transplantation. Tumour cells expansion, movements and association with the brain vasculature will be monitored in a wide-field fluorescence and confocal microscope to assess for similarities with progression in human. Tumour will also be dissected for analyses by RNA-seq and ChiP-seq techniques. The orthotopic DMG zebrafish model will also be used to test candidate drugs develop in the laboratory.
Aim3: To test the effect of drugs using the 2 models.
Both zebrafish models will then be used to test candidate drugs developed in our laboratory and will also be used in collaboration with JP Martinez-Barbera lab, who is also testing senolytic drugs against DMG. This aim will extensively use novel microscopy technology available at GOSICH.
The project involves the use of a broad range of molecular and cellular techniques that will be necessary to produce the different zebrafish models. It will also involve the analysis of high throughput data produced by RNA-seq and ChIP-seq experiments. The candidate will benefit from the expertise of Paula Alexdandre’s Lab in manipulating zebrafish and brain development and David Michod’s lab in paediatric brain tumour and epigenetics.
References:
1. D. Srikanthan et al., Diffuse intrinsic pontine glioma: current insights and future directions. Chin Neurosurg J 7, 6 (2021)
2. C. J. Veinotte, G. Dellaire, J. N. Berman, Hooking the big one: the potential of zebrafish xenotransplantation to reform cancer drug screening in the genomic era. Dis Model Mech 7, 745-754 (2014)
3. C. J. Eden et al., Orthotopic models of pediatric brain tumors in zebrafish. Oncogene 34, 1736-1742 (2015).
