In situ assay for topoisomerase 2-mediated DNA damage

In situ assay for topoisomerase 2-mediated DNA damage AbstractTopoisomerase 2 (Top2) is essential for DNA replication and transcription, but this enzyme generates double-strand DNA breaks during its normal catalytic cycle. This activity of Top2 is a key contributor to cancer initiation and progression in several tumor types. Paradoxically, amplification of Top2 DNA damage became the basis of the most successful strategy for cancer treatment. It produced some of the best therapeutic agents used to treat human malignancies. Virtually every form of cancer can be treated with regimens targeting Top2. The development of novel non-toxic and selective Top2 inhibitors became one of the prioritized targets in precision anticancer medicine.Therefore, a technology which detects and quantifies Top2-produced DNA breaks will be broadly important in both molecular and clinical research fields. The most advantageous method should detect its target directly in sections of normal and cancer tissues. Such an in situ assay would enable efficient deciphering of cancer initiation mechanisms and would enhance discovery of new Top2 anticancer drugs. However, currently there are no in situ assays specific for Top2-mediated DNA breakage.The goal of this project is to introduce the first in situ assay for Top2 DNA damage. The new technology will quantitatively visualize Top2-generated DNA breaks directly in tissue sections and in individual cells. This new ability is essential for molecular pathology studies and for the assessment of anticancer therapies. The assay will have high commercial potential because it will offer advantages of the new detection format, speed, specificity and cost over the current biochemical approaches. It will use a novel molecular labeling method specific for signature Top2 breaks, which carry homodimeric adducts linked to extruding 4-nucleotide overhangs on the 5-end of DNA.The Specific Aims of this project are: 1. To develop and validate in the context of commercial use the first tissue section technology specifically labeling Top2-produced DNA breaks with 5’Top2 adducts and 4-base overhangs. The technology will use the new and original in situ 5’ tyrosine enabled labeling method. To employ model systems with controlled production of Top2 DNA breaks to validate labeling specificity of the new approach, and to ensure its adequate sensitivity and reliability of detection. 2. To apply the new imaging technology to fixed tissue sections. To validate the assay in the context of commercial use in models related to cancer. To assess and verify the specific, robust, and sensitive detection of Top2 DNA cleavage by using tissue sections with Top2 DNA breaks. To test and optimize sensitivity, specificity and commercial applicability of the new assay.Narrative Statement The purpose of this project is to introduce an enabling method to analyze many varieties of cancer. This will produce a new and useful histological assay with broad commercial applications in the development of more efficient therapies and for molecular research in cancer and other pathologies where tissues sections are routinely used.