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Characterization of lung cancer radiosensitizers from genome-wide RNAi screens

David Kozono

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National Institutes of Health (NIH)
My career goal as a radiation oncologist is to improve lung cancer outcomes through basic and translational research initiated in the laboratory and validated in pre-clinical models to provide rationale for Phase I/II studies. I am currently dedicating 75% effort to research, and the remaining 25% in the clinic performing consultations, treatment planning and follow-ups for thoracic oncology patients. My Ph.D. thesis project with Nobel laureate Dr. Peter Agre was on aquaporin structure-function relationships, and I developed techniques for the purification, reconstitution and biophysical characterization of these water channel proteins in a wide range of species. Toward the end of my studies, my father became diagnosed with lung cancer and died 16 months later. I ultimately sought training in radiation oncology to join the fight against the disease. As a result, my research efforts took different direction compared to my Ph.D., and I sought mentorship under Dr. Alan D'Andrea to study DNA repair biology. I spent 18 months during residency on the Holman Research Pathway in his laboratory, and after graduating I joined the faculty at the Dana-Farber Cancer Institute to continue the research I started. I received one of two 2011 American Society for Radiation Oncology (ASTRO) Junior Faculty Research Training Awards. It is the expectation of this award that I apply for and obtain K08 or equivalent funding to further my training in cancer biology research. My aim is to improve tumor control via development of lung cancer radiosensitizers identified by whole genome RNAi screens. ENVIRONMENT: Considering the relatively short time that I have dedicated to cancer biology research, I will benefit greatly fromcontinued mentorship under Dr. Alan D'Andrea. His expertise in both DNA repair biology and deubiquitinating enzymes will be invaluable for the undertaking of all three Specific Aims of this proposal. Co-mentorship under Dr. Kwok-Kin Wong, who developed the genetically engineered mouse models (GEMMs) of lung cancer that will be studied in Specific Aims 1 and 2, will also be invaluable, as I have relatively less experience in animal studies and none to date in GEMMs. The project also makes use of key departmental resources, including our recently commissioned small animal radiation research platform (SARRP) for CT-guided targeted mouse radiotherapy. Key elements of the career development plan also include courses at the Harvard School of Public Health on genomic data manipulation and statistical methods, seminars at the Countway Library of Medicine, and resources offered by Harvard Catalyst on grant writing and translational research. RESEARCH: Radiation therapy (RT) is a critical modality in the treatment of lung cancer. The majority of patients with potentially curable disease, however, present with locally advanced tumors. The dose of RT that can be safely administered is limited due to infiltration of, or proximity to, radiosensitive structures including the lungs, spinal cord, esophagus and heart, resulting in locoregional failure rates of around 30% despite optimal treatment. To identify radiosensitizers that may improve the therapeutic index, I performed whole genome pooled shRNA screens in non-small cell lung cancer (NSCLC) cell lines treated with or without daily irradiation over a period of 2-3 weeks. Based on top hits, I identified two potential strategies for NSCLC radiosensitization. In Specific Aim 1, I hypothesize that proteasome inhibition may serve by inhibiting DNA double strand break (DSB) repair. I plan to explore the mechanism by which proteasome inhibition impacts homologous recombination and non-homologous end joining using established reporter constructs and assays. In Specific Aim 2, I hypothesize that USP9X deubiquitinating enzyme inhibition potentiates apoptosis. I plan to assess this in NSCLC using several established apoptosis assays, and to observe effects of USP9X inhibition on expression of Bcl-2 family proteins. In both Aims 1 and 2, I will attempt to demonstrate radiosensitization in genetically engineered mouse models with our SARRP. In Specific Aim 3, I propose to identify potential biomarkers for patient selection for treatment withconcurrent chemoradiotherapy and proteasome and/or USP9X inhibitors, by profiling tumors obtained from multiple GEMMs of lung cancer treated with radiation (?) proteasome and/or USP9X inhibitors. I will also analyze publically available, clinically annotated patient datasets t identify associations of gene expression and other alterations and patient outcomes. In so doing, I hope to provide rationale for testing proteasome and/or USP9X inhibition in Phase I/II trials, and to identify biomarkers for patient selection.

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