PROJECT SUMMARY The long-term goal of this study is to develop new strategies to mitigate and protect radiation-induced hematopoietic injury by delineating the molecular mechanisms responsible for enhancing hematopoietic stem cells (HSC) self-renewal and survival. Radiation therapy commonly results in not only acute hematopoietic suppression but also long-term bone marrow (BM) injury. To date, no effective treatment has been developed to prevent or treat these pathological consequences. Enhancing HSC survival and maintaining their genomic integrity upon radiation are crucial for preservation of HSC self-renewal function and for protection against radiation-induced BM injury. However, the molecular mechanisms for regulating HSC survival and self-renewal are not well defined. HSC number and function demonstrate natural variations, and underlying genetic diversity is an important, yet largely unknown, mechanism involved in the regulation of HSC function. Latexin (Lxn) was previously identified as the first stem cell regulatory gene whose natural variation negatively contributes to the HSC population size in different mouse strains. In preliminary studies, constitutive gene ablation approaches were developed to define the role of Lxn in HSCs and hematopoiesis in normal and radiation stress conditions. Results showed that Lxn inactivation protects HSCs and hematopoietic progenitor cells (HPCs) from radiation- induced cell death, thus mitigating acute hematopoietic suppression and conferring a survival advantage. Moreover, Lxn knockout mice do not develop hematological malignancies following single or fractionated dosages of radiation. Mechanistically, Lxn inactivation in vivo increases HSC self-renewal and survival. At the molecular level, our published and preliminary work showed that ribosomal protein subunit 3 (Rps3) is a novel Lxn-binding protein, and Lxn overexpression inhibits Rps3 activity, thus enhancing radiation sensitivity and toxicity. In addition, the number of human HSCs/HPCs negatively correlates with Lxn expression level, indicating the potential role of Lxn in regulating human hematopoiesis. These findings lead to the hypothesis that Lxn maintains homeostatic HSC and hematopoiesis and inhibition of Lxn function protects against radiation-induced hematopoietic injury via Rps3-dependent prosurvival pathways. Specific aims are to: 1) determine the cellular mechanisms by which Lxn inactivation enhances HSC survival and self-renewal and confers radioprotection, 2) identify the molecular mechanisms by which Lxn inactivation enhances Lxn-Rps3- NF-kB prosurvival pathway, and 3) determine the role of Lxn in mitigating human hematopoietic cell toxicity from radiation injury. This research will advance understanding of a critical but understudied mechanism involved in the natural regulator of HSC function and self-renewal. By using mouse and human model systems and state-of-the-art molecular and genomic techniques, findings from the proposed study may facilitate to the development of novel mechanism-based therapeutic strategy to protect against radiation-induced BM injury, which could be beneficial for the patients receiving cancer therapy and BM transplantation.