Oncogenomic screening strategies to identify driver genes in acute myeloid leukaemia /

Abstract

Deletions of the short arm of chromosome 12 (12p) are found in around 6% of acute myeloid leukaemia (AML). Particularly in paediatric AML they often occur as the sole cytogenetic change and are associated with a poor prognosis. Amplifications of the long arm of chromosome 11 (11q) are rarer, found in around 1% of AML, but are particularly prevalent in the poor prognosis complex karyotype group. Despite multiple deletion mapping studies, single genes have not been identified from these regions that are responsible for driving leukaemic progression, thus it is clear that a functional approach is required. This study aimed to functionally implicate significant genes through the use of competitive selection assays. Publicly available array data from eight studies were used to determine copy number from AML patient samples to define minimal regions of copy number alteration. Data were obtained from 866 samples which had been analysed on Affymetrix SNP array platforms. In total 58 samples (6.7%) were found to have deletions of 12p and were used to determine a minimally deleted region (MDR), whilst five samples (0.6%) were identified with amplifications of 11q and used to define a minimally amplified region (MAR). AML cell lines with deletions of 12p (NKM-1 and GDM-1) and amplification of 11q (UoC-M1) were selected to investigate the functional relevance of the genes within the regions of interest. Cell lines were used in vitro and in immunocompromised mouse models, where leukaemia was established by intrafemoral injection and monitored by luminescent imaging of luciferase expression constructs. Taking a pooled approach, 11 genes within the 12p MDR were overexpressed and 30 genes from the 11q MAR were knocked down using integrated lentiviral vectors. To evaluate effects of expression on leukaemia growth or survival, changes in construct copy number after cell line expansion in vitro and in vivo were determined by targeted high throughput sequencing on the Illumina MiSeq platform. Demonstrating a strong anti leukaemic effect for expression of this gene, a construct for cyclin dependent kinase inhibitor 1B (CDKN1B) was rapidly selected against in the 12p assay, whilst knockdown of MPZL3 and UBE4A from 11q were selected against in the 11q assay. Functional follow up work mainly focussed on CDKN1B, where its expression was measured in a range of a cell lines and patient samples, its effects on the cell cycle assessed and correlation of CDKN1B expression and treatment with a cyclin dependent kinase inhibitor (Flavopiridol) was established.