Oncogenomic Screening Strategies to Identify Tumour Suppressor Genes on Chromosome 12 in Acute Myeloid Leukaemia

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 impart a poor prognosis (Harrison et al J Clin Oncol 2010). Despite multiple deletion mapping studies, a single gene has not been identified from this region that is responsible for driving leukaemic progression, thus it is clear that a functional approach is required. This study aimed to functionally implicate a significant gene through the use of a competitive selection assay.

Publicly available array data from 8 studies were used to determine copy number from AML patient samples to define a minimal region of 12p deletion (MRD). Data were obtained from 866 samples, which had been analysed on either the Affymetrix SNP Array 6.0 or the Affymetrix GeneChip 500K Array platforms. Data files from matched remission samples were also analysed in 576 cases. In total 58 samples (6.7%) were found to have deletions of 12p and were used to determine the MRD.

Multiplex ligation dependent probe amplification, a polymerase chain reaction based technique to determine copy number was utilised to further define the MRD. A set of probes targeted to genes from within the MRD was designed and 75 karyotypically normal AML patients were screened. 5 samples (6.6%) were found to have deletions of the MRD region, and these were confirmed by fluorescence in situ hybridisation.

AML cell lines with deletions of 12p (NKM-1 and GDM-1) were selected to investigate the functional relevance of the genes within the MRD. Both 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, genes within the MRD were expressed from integrated lentiviral vectors in these cell lines. 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. The effects on growth of 11 candidate genes were assessed. 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 assay.

Expression levels of CDKN1B were measured in a range of AML cell lines and patient samples with and without 12p deletion, and the downstream effects of high and low CDKN1B protein levels were investigated. Overexpression of CDKN1B in AML cell lines carrying a 12p deletion was shown to inhibit growth through G1 phase cell cycle arrest. The sensitivity of AML cell lines to treatment with the cyclin dependent kinase inhibitor Flavopiridol was assessed and low expression levels of CDKN1B were found to moderately correlate with increased sensitivity to Flavopiridol (n = 8, Spearman’s rho = 0.4048).

Data obtained from this project provide valuable insight into the mechanisms giving rise to this complex disease and represent the initial step towards developing novel targeted therapies specifically tailored to individual patients with high risk AML. These assays may also be used to study other regions of interest as well as other cancers.