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Development of high throughput in vivo oncogenomic screening strategies in acute leukaemia

Final Report Summary - ONCOGENOMICS (Development of high throughput in vivo oncogenomic screening strategies in acute leukaemia)

The main aim of this project was to develop new ways to identify genes that contribute to childhood acute lymphocytic leukaemia (ALL) through loss (tumour suppressor genes) or gain (oncogenes). To this end we devised assays where effects on growth or survival of leukaemia cells of increasing expression, of candidate tumour suppressor genes, or of reducing expression of candidate oncogenes, was investigated. Initially we investigated genes on the long arm of chromosome 6 (6q) because although deletions of this region are seen in about 10% of childhood ALL the tumour suppressor genes had not been identified. By increasing expression of pools of candidate genes, in an ALL cell line with a deletion of 6q, we identified two that produced very clear negative effects on cell growth. The effects were seen not only in tissue culture but also when the cells were grown in mice. We therefore had strong evidence that these two candidates are tumour suppressor genes that contribute to childhood ALL and have further investigated them to understand the mechanism by which their loss contributes to disease. This data is currently being summarised in a manuscript for publication.
A PhD student on the project used similar assays to investigate candidate tumour suppressor genes on the short arm of chromosome 12 (12p) and candidate oncogenes on 11q, regions affected respectively by deletion or amplification in patients with acute myeloid leukaemia (AML). After defining a common region of deletion on 12q in patient material, the student tested a number of genes for their potential role as tumour suppressors, identifying one strong candidate. The role of this gene in AML was further investigated in patient samples and in other functional studies and has been summarised for publication. To identify oncogenes on 11q the approach was to use reagents (shRNAs) that reduced expression of candidate genes. However this study produced only inconclusive results, possibly because the shRNA were not sufficiently active. The student completed his PhD graduating in 2015.
A major interest of our group is intrachromosomal amplification of chromosome 21(iAMP21), a cytogenetic abnormality seen in about 2% of childhood ALL patients, that have a poor response to standard therapy. These abnormalities have regions of chromosome 21 that are amplified or deleted in complex patterns that are unique to each patient. To identify candidate chromosome 21 oncogenes for analysis, in collaboration with the Sanger institute, we used a combination of state of the art techniques to characterise iAMP21 abnormalities in ALL patients. This preliminary analysis was published in the prestigious journal Nature. To create assays for the analysis of the candidate oncogenes, because no cell lines are available, we developed models, known as xenografts, by transplanting iAMP21-ALL patient cells into mice. The xenografts have been used to produce substantial stocks of cells which have been extensively characterised and shown to carry iAMP21 abnormalities that closely resemble those in the patients from which they originated. Leukaemia in the xenografts has also been characterised by live imaging and histological analysis and we have summarised this data in a paper which is currently being submitted for publication. We have also performed assays in mice, using the xenograft cells and shRNAs that target the candidate chromosome 21 oncogenes. This approach did not identify any of the candidates, as contributing to iAMP21 ALL, so we have adopted an alternative technique that makes use of CRISPR-cas9 ‘molecular scissors’ to knock out these genes. Although there was insufficient time to perform these assays within the course of the grant we have been awarded further funding to continue this work.
In summary we have achieved our principle objective of developing a new type of assay for the identification of genes contributing to leukaemia. Thus far the assay has been more successful for the identification tumour suppressor genes than for oncogenes, but with further funding we will continue to refine these techniques and expect to overcome this problem.