BCR/ABL and Other Oncogenic Fusion Tyrosine Kinases (FTKs) Stimulate Single Strand Annealing (SSA), the Rare and Extremely Unfaithful DNA Double-Strand Break Repair Pathway To Facilitate Genomic Instability in Hematologic Malignancies.

Chromosomal translocations are responsible for the appearance of oncogenes encoding BCR/ABL-related fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFR and NPM/ALK, which induce hematological malignancies such as chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelomonocytic leukemia (CMML), and anaplastic large cell lymphoma (ALCL). We showed that cells transformed by BCR/ABL and other FTKs generate higher levels of reactive oxygen species (ROS) than normal counterparts. Enhanced ROS production in malignant cells causes extensive oxidative DNA damage resulting in elevated numbers of DNA double strand breaks (DSBs) measured by immunofluorescence detecting histone H2AX phosphorylated on serine 139 (γ-H2AX) and localized near DSB sites. Since DSBs may be lethal for cells, they are usually repaired by homologous recombination and non-homologous end-joining. Occasionally, a third mechanism called single-strand annealing (SSA) may be involved, too. During SSA an exonuclease degrades one strand at the DNA ends until homology is revealed (for example between two Alu sequences) allowing the broken ends to anneal and the intervening sequence to be clipped off. Therefore, SSA is extremely unfaithful, resulting in a deletion of sequences between the repeats. Because ∼50% of the mammalian genome consists of repeat sequences, SSA is potentially an important pathway of mutagenesis. For example, intrachromatid SSA events can give rise to interstitial deletions leading to a loss of a tumor suppressor gene. To determine the activity of SSA, a well-defined genetic model designed to exclusively measure SSA was applied. The model consists of a reporter cassette containing two fragments of the gene encoding green fluorescent protein (GFP) with a unique I-SceI restriction site introduced into one fragment. Expression of I-SceI restrictase will cause a DSB, which if repaired by SSA will result in recovery of a functional GFP gene generating a GFP+ cell. A single copy of the SSA reporter cassette was integrated in the genomic DNA of parentals and FTK-transformed cells. Cells were transfected with an expression vector encoding I-SceI restrictase and red fluorescent protein with mitochondrial localization (RFP-Mito) to control transfection efficiency. The number of double-positive (GFP+/RFP-Mito+) cells was scored in the population of RFP-Mito+ cells. FTKs induced an approximately 3–30 fold increase of the frequency of SSA events. Therefore, elevated levels of ROS-induced DSBs combined with increased frequency of a relatively rare but extremely unfaithful SSA repair may contribute to genetic instability in hematologic malignancies induced by FTKs.