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Precision Medicine DTP – Development of clinically relevant models of undifferentiated pleomorphic sarcoma using a genetic screening approach


Project Description

Background

Sarcomas are relatively uncommon cancers thought to arise from pluripotent mesenchymal stem cells. Undifferentiated pleomorphic soft tissue sarcoma (UPS), though one of the commonest sarcoma subtypes encountered, exemplify the limitations of historical progress in sarcoma therapy. Management of the metastatic case relies currently on standard chemotherapy agents with a very long track record but limited efficacy, such as doxorubicin and/or ifosfamide. Benefits are short lived, treatment palliative in nature, and median survival remains in the range of one to two years.1 Recent efforts to develop new agents and regimes have provided only limited benefits. One confounding factor is the lack of preclinical models that accurately recapitulate the human disease which is coupled with a lack of understanding of the underlying biology of the disease. A recognised feature of UPS is its pronounced chromosomal instability2 which has been proposed to be a manifestation of extra chromosomal DNA (ecDNA) which segregates stochastically into daughter cells, resulting in extreme heterogeneity of copy-number between tumour cells.3

Aims

To develop and annotate a mouse model of undifferentiated pleomorphic sarcoma to gain insight into the underlying biology of the disease and to provide a platform for testing new therapies based on the mutational landscape of individual tumours.

The project aims to generate immune competent mouse models of autochthonous tumour development using an adeno‐associated virus (AAV)‐mediated in vivo CRISPR screening approach.4 Informed by genomic data generated from whole exome sequencing efforts of UPS in Edinburgh and in publicly available databases, focused sgRNA libraries of mouse tumour suppressor genes corresponding to the top‐ranked mutated genes in UPS identified using the IntOgen pipeline, will be made. In particular, the computational analysis of existing tumour data will aim to identify impactful somatic genetic changes that precede the acquisition of chromosomal instability, with the intention of recapitulating that molecular phenotype in the in vivo model system. AAV8‐based vectors containing Cas9 from Staphylococcus aureus (SaCas9) and a sgRNA library (5sgRNAs per gene plus nontargeting controls) will be constructed and injected in combination with gain-of-function mutant Trp53, which is highly prevalent in human UPS.2 Local delivery of the virus increases transduction efficiency in the cell of interest within the anatomical location found in the human disease. This provides an unbiased approach for identification of multiple tumour drivers, co‐operation between drivers and will provide a greater understanding of the mutational landscape that drives disease progression in the native microenvironment. DNA isolated from tumours that develop will be sequenced to identify barcodes and corresponding overrepresented gRNAs, which indicate genetic alterations that provide a growth advantage. Detailed histopathological analysis of the tumours will be undertaken to establish that the models recapitulate human disease. Mechanistic studies will be carried out to provide insight into the biological function of the genetic relationships identified. PCR, microscopy and genome sequencing approaches will be applied to quantify chromosomal instability in tumours and heterogeneity between tumour cells. If we are able to establish the induced generation of tumours with ecDNA in an in vivo model this would provide a uniquely powerful system in which to explore the consequences of extreme copy-number heterogeneity on cancer development and adaptation. Based on ongoing dug screening efforts in UPS (Brunton, Salter), tumour derived organoids will be established and used for preclinical evaluation of potential new therapeutic targets, with lead hits being followed up in vivo.

Training Outcomes

This studentship will provide interdisciplinary training in genomics, computational biology, molecular biology, cancer cell biology and mouse modeling.

This MRC programme is joint between the Universities of Edinburgh and Glasgow. You will be registered at the host institution of the primary supervisor detailed in your project selection.

All applications should be made via the University of Edinburgh, irrespective of project location. For those applying to a University of Glasgow project, your application along with any supporting documents will be shared with University of Glasgow.

http://www.ed.ac.uk/studying/postgraduate/degrees/index.php?r=site/view&id=919

Please note, you must apply to one of the projects and you must contact the primary supervisor prior to making your application. Additional information on the application process is available from the link above.

For more information about Precision Medicine visit:
http://www.ed.ac.uk/usher/precision-medicine

Funding Notes

Start: September 2020

Qualifications criteria: Applicants applying for a MRC DTP in Precision Medicine studentship must have obtained, or will soon obtain, a first or upper-second class UK honours degree or equivalent non-UK qualification, in an appropriate science/technology area.
Residence criteria: The MRC DTP in Precision Medicine grant provides tuition fees and stipend of at least £15,009 (RCUK rate 2019/20) for UK and EU nationals that meet all required eligibility criteria.

Full eligibility details are available: View Website

Enquiries regarding programme:

References

1. Carvalho et al., Pleomorphic sarcomas: the state of the art. Surg Pathol Clin (2019) 12:63-105

2. Steele et al., Undifferentiated Sarcomas Develop through Distinct Evolutionary Pathways. Cancer Cell (2019) 35:441-456

3. Turner et al., Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity. Nature 543:122-125

4. Chow et al., AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma. Nature Neurosci (2017) 20:1329-1341

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