The relationship between ionizing radiation (IR) and human health is paradoxical; IR is both a potent carcinogen and a highly effective therapeutic agent for the treatment of cancers. This proposal exploits this relationship?utilizing genome sequencing of rare, genetically radiosensitive individuals to identify novel genes that act in the cellular response to IR and then targeting the products of these genes for functional studies and preliminary drug development. Since the mutations identified in these individuals confer radiation sensitivity without significantly compromising survival, the genes identified may constitute particularly favorable targets for the design of drugs intended to radiosensitize tumors while limiting cytotoxicity to normal tissue. For more than 25 years we have worked to elucidate the genetics of human radiation sensitivity, focusing on individuals displaying extremes of IR hypersensitivity as a means of identifying genes with the most significant effects on cellular DNA damage responses. Over the course of these studies, a steady stream of patients has been referred to us for diagnostic testing for Ataxia-telangiectasia, Nijmegen Breakage Syndrome or Ligase IV Syndrome. Cell lines established from some of these patients displayed radiation hypersensitivity in clonogenic survival assays but did not harbor causative mutations in ATM, NBN or LIG4. In preliminary studies we have utilized whole exome sequencing which, in some subjects, revealed deleterious alleles at genes known to be involved in DNA damage responses, such as ERCC6 and MCPH1. More intriguing, however, were 5 genes where causative mutations were found and confirmed to be radiosensitizing, but whose roles in established DNA damage response pathways were both unknown and unanticipated. In this application we propose to build upon these preliminary findings using whole genome sequencing (WGS) to capture a greater fraction of the causative variants in the remaining unsequenced radiosensitive cell lines, improved bioinformatic tools to filter those variants, and high throughput functional screening assays to efficiently identify the genes they impact. Functional studies will be applied to two of the novel genes we have identified because they show promise as potential chemoradiosensitization targets. The first of these, MTPAP, is a non-canonical poly-A polymerase and terminal uridyltransferase, which, when mutated or knocked down, radiosensitizes by a novel mechanism involving disruption of histone homeostasis. The second gene, ATIC, encodes a bifunctional enzyme that catalyzes the final two steps of de novo purine synthesis. Knockdown or chemical inhibition of ATIC depletes cellular ATP, alters the cell cycle distribution of cells and results in increased DNA double-strand breaks and impaired survival after irradiation. For each of these two molecules we will define their mechanisms of action in the cellular response to ionizing radiation exposure and evaluate the efficacy of small molecule inhibitors which we have already obtained from in silico screening to reproduce these radiosensitive phenotypes in cells in culture.
Studies of individuals with rare genetic disorders that result in cellular hypersensitivity to the lethal effects of IR have been instrumental in elucidating key components of human cellular DNA damage response pathways. Current sequencing technologies now allow for the identification of the causative genes in disorders where only one or a few affected individuals exist. Application of this approach to individuals with unexplained IR hypersensitivity can provide insights into novel mechanisms and pathways used by human cells to respond to ionizing radiation exposure. An understanding of these pathways, and their potential interactions, may facilitate the rational design of new radiosensitizing or radioprotective agents.
|Liu, Xiangfei; Paila, Uma Devi; Teraoka, Sharon N et al. (2018) Identification of ATIC as a Novel Target for Chemoradiosensitization. Int J Radiat Oncol Biol Phys 100:162-173|
|Bernstein, Jonine L; WECARE Study Collaborative Group; Concannon, Patrick (2017) ATM, radiation, and the risk of second primary breast cancer. Int J Radiat Biol 93:1121-1127|