Use of a patient-derived cell-line (A1ATD) in research on cancer genes
Research type
Research Study
Full title
Use of a patient-derived cell-line (A1ATD) in research on genes involved in cancer
IRAS ID
183328
Contact name
David Adams
Contact email
Duration of Study in the UK
5 years, 0 months, 1 days
Research summary
Research Summary
iPSCs (induced Pluripotent Stem Cells) mimic stem cells as they are able to divide and maintain themselves indefinitely, and also have the potential to differentiate (develop) into other specialised cell types. This is called 'pluripotency'.
The A1ATD iPSC line was derived from a patient suffering from hepatic disease as a result of a DNA change (genetic mutation) in the patient's genome. The A1ATD (corrected) cell-line has had this mutation 'corrected' to normal in the laboratory.
In brief, this study broadly seeks to characterise (both physically and genetically) and compare the A1ATD and A1ATD (corrected) lines, alter the genetic make-up of these cell-lines and create new cell-lines, look for genes involved in cancer progression, and use the original and newly created cell-lines to screen for potential therapies.
The study will use a variety of assays, including altering genes through targeted mutations (changes in the DNA) in specific genes or deletion of whole genes; differentiation (changing from one cell type to another) of A1ATD, A1ATD (corrected) and any new mutated A1ATD lines in order to investigate how mutations affect their function; and for modelling the processes by which mutated A1ATD lines differentiate into different cell types.
Furthermore, A1ATD, A1ATD (corrected) and the new cell-lines we create will be used to examine the accumulation of mutations (changes in the DNA) in the absence of critical DNA repair proteins.
Summary of Results
The development of targeted therapies has had a significant impact on cancer survival rates. However, targeting cancers that are driven by loss of tumour suppressor genes remains a major challenge. One promising approach to treat these cancers is the exploitation of synthetic lethal interactions. Synthetic lethality describes an interaction between two genes, where loss of one gene alone does not affect viability but loss of both genes induces cell death. Inhibiting the synthetic lethal partner of a tumour suppressor gene should specifically kill tumour cells, and so these represent potential therapeutic targets. However, very few synthetic lethal interactions have been well-established.
The aim of this project was to systematically screen for synthetic lethal partners of known tumour suppressor genes. To do so, isogenic human induced pluripotent stem cell lines were generated, each carrying a loss-of-function mutation in a single tumour suppressor gene. These cells have a normal genetic background, thus making it simpler to accurately identify interactions. CRISPR/Cas9 technology was applied as it allows for large-scale, unbiased screening of genetic interactions. A genome-wide guide RNA library was prepared and implemented for knockout screening in the isogenic cell line panel. Analysis was performed to identify genes that were specifically essential for cell fitness/survival in the mutant lines. Particular focus was placed on four tumour suppressor genes that encode subunits of the PBAF/BAF complexes. Approximately 20% of human cancers harbour mutations in subunits of these complexes, so identifying dependencies associated with these could have broad therapeutic potential. Candidate synthetic lethal interactions with these genes were investigated using low-throughput assays in the stem cells and in a cancer cell line. The data obtained suggests that screening in stem cells produces highly variable results. Although potential vulnerabilities associated with all of the tumour suppressor genes were identified, further work is required to validate these and to assess the quality of the results.REC name
West Midlands - Coventry & Warwickshire Research Ethics Committee
REC reference
15/WM/0276
Date of REC Opinion
3 Aug 2015
REC opinion
Further Information Favourable Opinion