Investigating a novel role for Top2a in R-loop resolution and in YM155 mechanism of action

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R-loops are DNA:RNA hybrids formed when newly transcribed RNA binds to its complementary DNA template, preventing reannealing of the two DNA strands. R-loops occur naturally and play roles in normal physiological processes including immunoglobulin class switch, transcription regulation, and mitochondrial replication initiation. Pathologic R-loop accumulation has also been demonstrated in disease states and has been linked to DNA damage and genome instability. Various factors are involved in resolving R-loops as part of normal cellular physiology including mRNA binding proteins, helicases, and RNaseH as well as topoisomerases 1 and 3b. Based on preliminary data investigating YM155, a small molecule, in the treatment of anaplastic thyroid cancer, this project will investigate two hypotheses - Topoisomerase 2α (Top2α) plays a novel role in the resolution of R-loops, and Top2α inhibition and R-loop accumulation are important for YM155 mechanism of action which will be tested in two aims: 1) to assess the role of Top2α in R-loop resolution and 2) to investigate the role of Top2α and R-loop accumulation in YM155 mechanism of action. R-loop formation (immunofluorescence), DNA damage (COMET assay and p-H2AX foci), and replication fork stalling (DNA fiber assay) will be measured after Top2α knockdown. The interaction between Top2α and YM155 will be characterized via site-directed mutagenesis. RNaseH is specific to RNA in DNA:RNA hybrids and is often used to confirm R-loop accumulation. We will see if RNaseH1 overexpression rescues cells from Top2α knockdown and YM155 treatment. Currently, there is no known role for Top2α in R-loop resolution. While R-loops have been implicated in cancer, little is known about whether increased R-loop accumulation can be turned into a therapeutic strategy. This project will further understanding of R-loop biology as well as cancer pathophysiology. This proposal also describes an integrated research and clinical training plan for an MD-PhD student. Activities under this proposal include development of lab methods, first author publication, and presentation skills including institutional seminars and national scientific meetings, and teaching students in graduate healthcare programs. The student will also gain valuable experience in research design, statistics, and data interpretation and evaluation by participating in literature review clubs and graduate coursework. A clinical training sponsor is identified in the student’s specialty of interest with opportunities for clinical instruction and observation, for clinical research, and for mentored guidance to facilitate transition into a research-oriented residency program. Research and clinical training will be performed at Louisiana State University Health Sciences Center – Shreveport (LSUHSC-S). LSUHSC-S has a medical school and graduate school with faculty performing research in basic science and clinical disciplines. LSUHSC-S has various resources for investigators including dedicated lab space and research core facilities to assist with the latest technologies as well as seminars, focus groups, and professional development workshops in grant writing, conduct of research, biostatistics and data analysis.