Increased Mutation Frequency Induced by Oxidative Stress in the NUP98-HOXD13 MDS Mouse Model

Abstract 1705

The myelodysplastic syndromes (MDS) comprise a group of hematologic disorders characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML) in a subset of cases. The causative agents for de novo or therapy-related MDS (t-MDS) include exposure to known genotoxins, such as anti-cancer chemotherapy, or ionizing radiation. It is known that MDS is associated with chromosomal aberrations including deletions, amplifications, inversions, and translocations. However, the molecular mechanisms that lead to progression of MDS to AML have not been completely elucidated. Recently, several studies have reported that oxidative stress contributes to disease progression of MDS. Reactive oxygen species (ROS) play a role in regulating several biologic phenomena including activation of signaling pathways in response to cytokines, and the gene expression pattern induced by this signaling. ROS is also known to induce oxidative DNA damage, which can lead to activation of proto-oncogenes or inactivation of tumor suppressor genes. A mouse model for MDS, generated by expression of a NUP98-HOXD13 (NHD13) fusion gene, was exploited to investigate ROS levels. Lineage negative (Lin^neg) bone marrow mononuclear cell (BMNC) from NHD13 mice with MDS had a 6.0 fold increased level of ROS compared with wild-type (WT) Lin^neg BMNC. In order to address potential mechanism(s) leading to this increased ROS, a Real-Time Quantitative PCR array, which profiles the expression of 84 genes related to oxidative stress, was utilized and the data demonstrated down regulation of Myeloperoxidase (Mpo; −9.9 fold), Eosinophil peroxidase (Epx; −8.8 fold) and Lactoperoxidase (Lpo; −5.2 fold) in the Lin^neg BMNC from NHD13 mice. The increased ROS level in NHD13 Lin^neg BMNC was associated with a 2 fold increase in G2/M phase cells along with increased expression of p16^INK4A (43 fold) and p21 (10 fold), possibly to allow for DNA repair at cell cycle checkpoints. In order to investigate genomic DNA damage by ROS, immunocytochemistry for γH2AX foci was performed and demonstrated a 2 fold increase in γH2AX foci in the NHD13 BMNC compared to WT BMNC. To determine if mis-repair of the ROS-induced DNA damage led to mutations of genomic DNA in vivo, mutation frequencies (MF) were determined using the Big Blue^®cII mutation detection assay. Big Blue mice are transgenic mice that have a λLIZ shuttle vector, which is derived from the coliphage lambda. Selection of mutation is based on the ability of the λ phage to multiply through either the lytic or lysogenic cycle in E. coli host cells. Big Blue mice were crossed to NHD13 mice, and NHD13/Big Blue^® mice were compared with littermate controls that were positive for the BigBlue transgene but negative for the NHD13 transgene. BMNC from the NHD13/BigBlue mice showed a 1.8-fold increased MF compared to control BMNC (p value = 0.0371). Mutations were comfirmed by sequencing the cII target gene, and revealed that the NHD13/BigBlue mice had a 3-fold higher frequency of frameshift mutations compared to the control animals. These results suggest that the oxidative stress induced by the NHD13 fusion may contribute to disease progression of MDS to AML through DNA damage and mutation.