Molecular mechanisms of copy-neutral loss of heterozygosity

Cancer is one of the deadliest human diseases; a continuously increasing threat to human health. Understanding the mechanisms that lead to malignancy is central to the development of therapeutic strategies. The alternation of one of the two copies of a gene that suppresses tumor formation is often followed with by additional events leading to the loss and replacement of the remaining healthy copy by a replicate of the mutated one. This is a common occurrence in human malignancies and leads to the complete inactivation of the affected gene. Such events are called copy-neutral loss of heterozygosity or in short cnLOH. Despite the frequency and relevance of cnLOH events, no mechanism has been discovered to-date. To understand more about these events we will use a groundbreaking 3D system, developed in our lab, which models human-specific aspects of brain development, called cerebral organoids. We will track these events in different organoid model which recreate brain tumor formation caused by alterations in different genes where the emerging tumors are known to undergo cnLOH events. The simplicity of this system will allow us to detect such events in very high throughput and help determine where and when they occur during tumorigenesis in cerebral organoids. We will perform genetic screens to uncover the mechanism(s) that controls cnLOH events and test their relevance during tumorigenesis in cerebral organoids. By comparing data from these screens we will assess whether the same or distinct mechanisms lead to cnLOH. Applying a wide panel of approaches has the potential to finally uncover the mechanisms governing cnLOH events during tumorigenesis and could give rise to unique new therapeutic strategies in the treatment of relevant pathologies and cancer in general.

This project aims at understanding a biological phenomenon called copy-neutral loss of heterozygosity (cnLOH). In this process, a cell that originally had two different versions (or "copies") of a gene ends up with two identical versions, both of which may be faulty. This change can lead to cancerous growth, but the exact mechanism by which cnLOH happens-especially in the human brain-remains poorly understood. Our goal is to use human stem cells to model the process in cell culture, avoiding animal experiments and obtaining results that are directly relevant to human biology and medicine. We used cerebral organoids, miniature brain-like structures grown from human stem cells in the lab. These 3D models allow us to study human brain development and disease in a way that closely mimics what happens in real life. A key focus of the project was Tuberous Sclerosis Complex (TSC), a severe brain disorder caused by mutations in the TSC2 gene. TSC pathology involves the formation of tumours in the brain. In our earlier work, we found that brain organoids with one faulty copy of TSC2 could form tumour-like structures, and that cnLOH occurred as part of this process. During the course of the project, we expanded our efforts to also include neurospheres-small balls of brain-like cells grown from specific cell types known to be involved in early tumours. This model is simpler than organoids but still mimics certain aspects of tumour development. These experiments led us to discover a type of cell that might be particularly vulnerable for this disease. We could identify characteristic alterations in the types of neurons generated by this cell type in TSC patients, a defect that might contribute to TSC. Experiments to investigate cnLOH and to analyse its consequences specifically in this cell type are underway. The results of the project are currently being prepared for publication.