Dysfunctional Double-Strand DNA Break Repair In Primary t-AML/t-MDS Myeloblasts.

Abstract 3366

The gross chromosomal aberrations in treatment-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/t-MDS) cells suggest that disease initiation and progression may result from the inability of cells to appropriately respond to double-strand DNA breaks (DSBs) induced by prior exposure to radiation, alkylator, or topoisomerase II inhibitor therapy. We hypothesize that dysregulation of DSB repair by homology-directed repair (HDR) or nonhomologous end joining (NHEJ) contributes to the development of t-AML/t-MDS. Dysregulation of DSB repair in t-AML/t-MDS may result from inherited variants or acquired mutations in HDR/NHEJ pathway genes. To directly test this possibility, we used next-generation sequencing technology and an array CGH platform to identify inherited and somatic genetic variants, including small indels and copy number alterations, in 21 canonical HDR and 9 NHEJ DNA repair genes, as well as a subset of 7 DNA damage response genes using tumor DNA and paired normal DNA obtained from 30 t-AML/t-MDS patients. All the data has been acquired and the analysis is ongoing. Because dysregulation of DNA repair pathways can result from epigenetic changes or post-translational modifications in DNA repair genes and would not be detected using sequencing and array CGH, we are also performing functional studies to interrogate DSB repair using primary bone marrow cells from 16 of these t-AML/t-MDS patients and CD34+ cells from 5 normal healthy controls. We performed a flow-based assay for DSB formation and repair by measuring the phosphorylated form of the variant histone H2AX (pH2AX), which is rapidly phosphorylated upon DSB formation, in myeloblasts (CD45 dim, low side scatter) and lymphocytes (a surrogate for normal cells) from leukemic bone marrow. Baseline measurements of unmanipulated primary bone marrow cells and a time course to measure the kinetics of DSB repair after gamma irradiation are used to assess a cell's basal DSB burden and the response to acute damage. We have validated this assay in a defined genetic system using isogenic cells deficient or not in BRCA2, a gene central to the HDR pathway, and were able to demonstrate that cells lacking BRCA2 have elevated pH2AX levels at 4–24 hours post DSB induction compared to controls (p=0.01). In addition, we have evaluated the ability of the cells to form nuclear foci of pH2AX and RAD51 (a protein central to the repair of DSB by HDR) by quantifying the relative numbers of immunofluorescent nuclear foci upon irradiation. We have performed these functional assays for 8 t-AML/t-MDS samples and 3 normal donors. We identified one t-AML/t-MDS sample whose myeloblasts show 2–3 fold greater pH2AX at baseline, compared to control CD34+ cells (p=0.006). Interestingly, the clearance of pH2AX is significantly faster in this sample compared to controls (p=0.001) despite showing elevated baseline pH2AX levels. This patient had a translocation involving chromosome 11q23. One additional patient in our cohort with an 11q23 translocation also showed similar increased baseline pH2AX levels. In addition, we identified abnormalities in a third t-AML/t-MDS sample that trends towards impaired maximal pH2AX induction (p=0.052). Furthermore, myeloblasts from a fourth t-AML/t-MDS sample, a known heterozygous BRCA2 mutation carrier, show a trend towards delayed pH2AX clearance (p=0.07). As predicted, bone marrow cells from this BRCA2 carrier also have 3.5 fold less RAD51 nuclear foci formation compared to control cells (8.6% vs 30%). Collectively, these results show that primary t-AML bone marrow cells can be used to assess the functional integrity of DSB response, and suggest that a large proportion of samples (4/8) may contain genetic alterations in DNA DSB response and/or repair genes. Functional studies of an additional 8 samples are ongoing. Next generation sequencing and array CGH data for the 37 HDR/NHEJ genes has been completed and will be correlated with the functional studies for each sample. Identification of abnormal DSB repair in leukemic patients may not only elucidate the molecular pathogenesis of the disease, but may provide rationale for testing agents to achieve selective killing of leukemic cells, as defects in one DNA repair pathway may increase reliance on another, making these cancer cells particularly susceptible to killing by inhibitors targeting the extant pathway(s).