Thesis
Functional translation of malignant peripheral nerve sheath tumour driver gene variants using CRISPR screens
- Abstract:
-
Malignant peripheral nerve sheath tumours (MPNSTs) are aggressive neural-crest cell derived sarcoma with limited treatment options, and only margin-free surgery as a potentially curative treatment option. The dire prognosis of these patients warrants the need for novel therapeutics. The advancements in genome-engineering now allow for rapid functional interrogation of cancer genomes using genome-wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout screens. This study aimed to optimise and utilise an enAsCas12a-based dual-targeting CRISPR library screen workflow to identify dependencies in MPNSTs in various context, including potential synthetic lethality relationships. During the optimisation of the CRISPR screen workflow, variability in initial guide RNA (gRNA) representation emerged as a significant confounding factor. This variability contributed significantly to discrepancies between replicate experiments and across different CRISPR libraries, leading to substantial alterations in observed gene effects and increasing the risk of false-positive results. Notably, this confounding factor was found to be prevalent and unaddressed in other external CRISPR screen datasets, including the entire DepMap dataset. Although further refinement in correcting for gRNA representation bias is possible, adjustments were made in this study to mitigate its impact while identifying robust genetic dependencies in MPNSTs across eight cell line models and 15 independent CRISPR screen experiments. These identified dependencies were either predicted to be druggable or involved in synthetic lethality relationships.
Key candidate genes included well-established cancer dependencies, such as MYC, aurora kinases, cyclin-dependent kinases, and MTOR, as well as several less well-characterized genes. Additionally, a more focused CRISPR screen was conducted to identify synthetic lethality partners associated with the loss of polycomb repressive complex 2 (PRC2) function, a common feature in MPNST. This was achieved by degrading PRC2 components using a targeted Proteolysis-targeting Chimera (PROTAC) approach in immortalized human Schwann cells. The candidate target genes identified in this study hold promise, but further functional and mechanistic validation is necessary to confirm their roles and therapeutic potential.
Actions
Authors
Contributors
- Institution:
- University of Oxford
- Division:
- MSD
- Department:
- Pathology Dunn School
- Role:
- Supervisor
- Institution:
- University of Oxford
- Division:
- MSD
- Department:
- Pathology Dunn School
- Role:
- Supervisor
- Funder identifier:
- https://ror.org/054225q67
- Grant:
- BST00200
- Programme:
- DPhil in Cancer Science
- DOI:
- Type of award:
- DPhil
- Level of award:
- Doctoral
- Awarding institution:
- University of Oxford
- Language:
-
English
- Keywords:
- Subjects:
- Deposit date:
-
2025-03-05
Terms of use
- Copyright holder:
- Paul Metz
- Copyright date:
- 2024
- Rights statement:
- This thesis is made available for non-commercial academic use only. No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the author or as otherwise permitted by law. This thesis may be made available online for open access through the University of Oxford's institutional repository, and users are permitted to view and download this document solely for personal, academic, or research purposes.
If you are the owner of this record, you can report an update to it here: Report update to this record