mutations in acute myeloid leukemia promote interchromosomal homologous recombination and the potential for copy neutral loss of heterozygosity (CN-LOH)

Acquired copy neutral loss-of-heterozygosity (CN-LOH) is a frequent occurrence in myeloid malignancies and is often associated with resistance to standard therapeutic modalities and poor survival. Here we show that constitutive signaling driven by mutated FLT3 and JAK2 confers inter-chromosomal homologous recombination (iHR), a precedent for CN-LOH. Using a targeted recombination assay, we determined significant iHR activity in internal tandem duplication FLT3 (FLT3-ITD) and JAK2V617F mutated cells. Sister chromatid exchanges, a surrogate measure of iHR, was significantly elevated in primary FLT3-ITD normal karyotype acute myeloid leukemia (NK-AML) compared to wild type FLT3 NK-AML. Homologous recombination (HR) was harmonized to S phase of the cell cycle to repair broken chromatids and prevent iHR. Increased HR activity in G 0 arrested primary FLT3-ITD NK-AML in contrast to wild-type FLT3 NK-AML. Cells expressing mutated FLT3-ITD demonstrated a relative increase in mutation frequency as detected by thymidine kinase (TK) gene mutation assay. Moreover, resistance was associated with CN-LOH at the TK locus. Treatment of FLT3-ITD and JAK2V617F mutant cells with the antioxidant N-acetylcysteine diminished reactive oxygen species (ROS), restoring iHR and HR levels. Our findings show that mutated FLT3-ITD and JAK2 augment ROS production and HR, shifting the cellular milieu towards illegitimate recombination events such as iHR and CN-LOH. Therapeutic reduction of ROS may thus prevent leukemic progression and relapse in myeloid malignancies. DNA FLT3 activation a subsequent in and repair The We show that the FLT3-ITD and JAK2V617F mutation confer increased inter (between chromosomes) homologous recombination (iHR) activity compared to WT kinase. Moreover, iHR activity was reliant on the FLT3-ITD and JAK2V617F mutation dependent elevation of ROS. NK-AML patients with FLT3-ITD mutations also possessed significantly increased sister chromatid exchanges (SCE) (a surrogate for iHR activity), whilst a thymidine kinase (TK) reporter assay demonstrated FLT3-ITD activity preferentially increases resistance to TK through the creation of CN-LOH. Our results pinpoint a common pathway whereby constitutive FLT3 and JAK2 signalling confers iHR and the subsequent propagation of CN-LOH in normal karyotype myeloid malignancy. in FLT3-ITD and JAK2V617F cells. STAT5 dependent activation of NADPH oxidase in association with RAC1 results in increased ROS, whilst AKT activates FOXO3 to sister by HR. Prior to DNA replication in G 1 , NHEJ repairs DSB, however, illegitimate HR expression in G 1 has the capacity to repair breaks between homologous chromosomes resulting in iHR events and CN-LOH. Further, we show that mutated FLT3 also confers increased expression of the cyclin dependent kinase inhibitor; p21 in primary NK-AML; mediating G 1 arrest and slower progression to S phase increasing the likelihood for iHR. The proposal that CN-LOH is not merely a stochastic event brought about by selection pressure of a leukemic clone, but a direct result of constitutional FLT3 and JAK2 activity has considerable relevance to the biology and therapy of myeloid malignancies. The disparate and independent activities of a single oncogene having the capacity to combine to create CN-LOH may be a common mechanism in these diseases. The identification of this mechanism of CN-LOH propagation will also open up further avenues of investigation. The TK assay revealed a bias in FLT3-ITD transfected cells to specific sites and sizes of CN-LOH. The uniformity of the CN-LOH suggests that common breakpoints at the TK locus were responsible for the CN-LOH. Similarly, CN-LOH at 13q may be a reflection of hotspots of recombination at the locus rather than simply a selection of a mutant FLT3 homozygous clone. Targeting integration of DNA repair substrates at CN-LOH rich loci could be utilised to determine preferential sites of recombination and their correlation to fragile sites. ROS induced DNA damage has been postulated to induce mutations to accelerate leukemic progression and relapse (9). The present study makes a valid case for the use of anti-oxidants in haematological malignancy given the observed ROS accumulation in CML, AML, MPN and CLL (30). Such treatments working alone or in combination with conventional therapies could prevent the accumulation of mutations and the acquisition of CN-LOH in NK-AML slowing the rate of disease progression. Furthermore, the side-effects of using high dose DNA damaging chemotherapy could be avoided if low doses can be combined with anti-oxidants. Our findings suggest that novel therapies targeting the generation of ROS would prove to be of great importance clinically for treatment of mutant FLT3 and JAK2 myeloid malignancies.