PROJECT SUMMARY/ABSTRACT Our application focus on DNA-dependent protein kinase (DNA-PK), a DNA repair factor with newly identified role in RNA metabolism and a target of cancer therapy, and will use genetic, cell biology and single molecule approaches to dissect the role of DNA-PK during lymphoma and leukemia-genesis and therapy. Genomic instability is the hallmark of human cancer. The Non-Homologous End-Joining (NHEJ) is a major DNA double-strand breaks (DSBs) repair pathway and is required for physiological gene-rearrangements and oncogenic chromosomal translocations in developing lymphocytes. DNA-PK, composed of KU70-KU80 heterodimer (KU) and the large catalytic subunit (DNA-PKcs), is a NHEJ factor critical for both end-processing (e.g., hairpin opening) and end-ligation during NHEJ. DNA-PKcs inhibitors is in phase I/IIa clinical trials for cancer therapy. During NHEJ, KU binds to DNA ends, recruits and activates DNA-PKcs. Loss of DNA-PKcs abrogate Artemis endonuclease mediated end-processing without abolishing end-ligation. We showed that expression of kinase-dead (KD) (DNA-PKcsKD/KD) abrogates end-ligation without affecting end-processing, uncovering an end- protection role of DNA-PKcs that is regulated by its own kinase activity. End-processing in DNA-PKcsKD/KD mice is blocked by ATM inhibition, indicating end-processing requires DNA-PKcs protein, and the kinase activity from either DNA-PKcs or the related ATM kinase in vivo. DNA-PKcs is the best characterized substrate of DNA-PK and can also be phosphorylated by ATM. Mice carrying phosphorylation-deficient (DNA-PKcs5A/5A) DNA-PKcs display mild end-ligation defects and are sensitive to ATM inhibition. Thus, we propose that once assembled on KU-bound DNA, DNA-PK phosphorylation regulates end-processing and eventually the release of DNA-PKcs to licence end-ligation. Moreover, we found that Ku can also direct the assembly of DNA-PKcs on structured RNA (e.g., rRNA and snoRNA), where phosphorylation defective (DNA-PK5A) or KD DNA-PKcs (DNA-PKcsKD/KD) blocks rRNA processing, protein translation, and erythropoiesis, leading to Trp53-dependent bone marrow failure. These findings uncovered a NHEJ-independent role of DNA-PK in mammals. And two-third of DNA- PKcsKD/KDTrp53-/- mice succumbed to ribosomal stress induced myeloid leukemia and one-third died of lymphomas with IgH-Myc translocations, highlighting the critical role of DNA-PK in tumor suppression. Based on these and other findings, we hypothesize that DNA-PKcs kinase activity and auto-phosphorylation regulates KU-dependent assembly of DNA-PK on DNA and RNA to suppress lymphoma and leukemia genesis. To test this, we will 1) characterize and compare KU and DNA-PK dynamics on RNA vs DNA; 2) elucidate how KU- depletion impact RNA processing in human cells; 3) determine the physiological function of KU80 C-terminal domain and tail in lymphoma and leukemia genesis and the recruitment and stabilization of DNA-PKcs. The results will reveal the regul...