Zinc Finger Nuclease (ZFN) Targeted Homologous Recombination Assay Detects Elevated Inter Homologous Recombination Activity and Copy Neutral Loss of Heterozygosity (CN-LOH) in Internal Tandem Duplication Mutated FLT3 (FLT3-ITD) Acute Myeloid Leukaemia (AML)

Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in normal karyotype acute myeloid leukaemia (NK-AML). The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH at the FLT3 locus, 13q associated with a FLT3-ITD mutation in NK-AML that results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined.

To elucidate the mechanisms that produce CN-LOH in NK-AML we developed a Zinc Finger Nuclease (ZFN) inter homologous recombination (iHR) targeting assay. The assay, based on the DR-GFP reporter developed by Pierce et al (1999), (Genes Dev. 13(20):2633-8) relies on the sequential targeting of two overlapping, but disrupted GFP repeat sequences to the adeno associated virus integration site 1 (AAVS1) on separate 19q alleles. Productive recombination between heterologous chromosomes in the region of 19q would result in restoration of GFP expression. The AML cell lines NB4 and THP-1 were transfected with a ZFN specific for AAVS1 integration (CompZr, Sigma) and the PZDonor AAVS1 puromycin derived construct DR1-Puro (Figure 1A,C) that possesses a 5' GFP sequence interrupted by an 18bp recognition sequence for the rare restriction enzyme, I-SCEI. Puromycin selected cells were then transfected with pZDonor derived DR2-Blast (Figure 1B,D) possessing the 3' GFP sequence and a blastocidin resistance gene. Puromycin and blastocidin selected cells were verified for correct integration of the plasmids to separate AAVS1 loci using location specific PCR and FISH, Figure 1F-H. Clones with correct integration were then expanded and transfected with FLT3-ITD, WT FLT3 expressing or empty vectors. Transfected cells were treated with an I-SCEI expression viral supernatant to induce a double strand break in DR1-Puro. Cells were cultured for a further 5 days before FACS analysis. FLT3-ITD transfected cells demonstrated GFP positive cells (inter HR events, 0.15% compared to WT-FLT3 and empty vector transfected cells (0.15% vs 0%, p<0.05, n=3) (Figure 1I). Furthermore, we were able to abolish inter HR events with co-culture with the FLT3 inhibitor, ACC220. To verify that GFP positive cells in this assay were the result of HR and not false positives, hot start PCR using primers designed at the DR1 locus was carried out on FACS sorted GFP positive cells. PCR products from GFP positive cells could only be digested by Bcg1 and not I-SCEI in primary AML confirming the presence of iHR dependent GFP positive cells.

Evaluation of HR events in primary AML was also investigated using ZFN targeted integration.  Using the PZDonor AAVS1 puromycin derived construct HR substrate, PZD-DR1/DR2-Puro (Figure 1C), primary FLT3-ITD demonstrated significantly increased single locus targeted HR activity compared to primary WT FLT3, (0.62 vs 0.17%, p<0.01, n=22). Spontaneous HR activity (without I-SCEI cleavage) was observed in more than half the primary FLT3-ITD and was not seen in primary WT FLT3. Moreover, HR activity was significantly inhibited in primary AML with ACC220 (0.65% vs 0.1%, p<0.01, n=3) correlating with the reduction in RAD51 expression.

The findings that FLT3-ITD mutation directly confers iHR activity provide a strong proof of principle that constitutive FLT3-ITD kinase activity augments HR activity, shifting the cellular milieu in favour of illegitimate recombination events such as CN-LOH that are selected for leukaemic progression. As both JAK2 and p53 mutations demonstrate enhanced HR repair and acquire CN-LOH at 9p and 17p respectively, we have identified a common mechanism of mutagenesis that furthers significantly our understanding of leukaemic progression and relapse in NK-AML.