THE PCNA-PARI PATHWAY OF GENOME STABILITY IN CANCER

Personnel: Dr. George-Lucian Moldovan, the applicant, has a longstanding interest in mechanisms of carcinogenesis, focusing on DNA repair and genomic instability. Dr. Moldovan made essential contributions to the topic areas of genetic cancer research and cancers related to radiation exposure, in particular through his discovery of PCNA post-translational modifications that regulate DNA repair. Dr. Wafik El-Deiry, the designated mentor, is a cancer physician and scientist who is renowned for the discovery of p21(WAF1) 20 years ago. His research on colorectal cancer, cancer susceptibility factors, and the genetic basis of cancer has been published in over 300 papers and funded by numerous federal programs.Career Development: Successful completion of this research proposal would uncover a previously unanticipated connection between DNA repair regulation and leukemia differentiation. This will allow the Principal Investigator to expand his research program in the topic area of blood cancers and to frame an overarching model for carcinogenesis spanning both solid and blood cancers.Research: Background: DNA damage is a major cause of cancer. Genomic instability caused by exposure to genotoxic agents or inactivation of DNA repair mechanisms allows cancer cells to accumulate mutations and acquire malignant phenotypes. In particular, hyper-replicating cancer cells are under increased pressure to synthesize DNA in a short amount of time and thus are faced with increased replication stress. This results in inability of the replication machinery to bypass difficult-to-replicate DNA regions (such as DNA lesions or repetitive sequences), generating DNA strand breaks. To deal with replication stress, cancer cells highjack normal DNA repair processes. We previously identified such a mechanism that controls DNA repair in cancer cells, centered on the protein PARI. We showed that PARI is a major determinant of genomic instability, carcinogenesis, and tumor growth. Recently, we found that PARI is expressed at high levels in leukemia cells, where it is required to suppress spontaneous DNA damage and to protect leukemia cells against differentiation. Moreover, we found that PARI downregulation in leukemia cells induces a stress response characterized by activation of DNA damage checkpoint and NFkappaB pathways.Objectives/Hypothesis: We propose that PARI is at the center of a novel mechanism that controls the leukemic differentiation barrier. We hypothesize that PARI promotes leukemia proliferation by alleviating replication stress, which would otherwise result in DNA damage, activating a leukemic differentiation program mediated by the NFkappaB pathway.Specific Aims: We will investigate this hypothesis using an innovative approach that integrates DNA replication and repair assays, leukemia specific assays, and transcriptomic analyses. The first specific aim is to study the role of PARI in differentiation of leukemia cells. The second specific aim is to study how PARI modulates the NFkappaB pathway.Study Design: In the first aim, we will investigate the mechanism of differentiation that is activated upon PARI knockdown. We will test the hypothesis that PARI promotes leukemia by blocking DNA damage-induced differentiation, as well as the alternative hypothesis that PARI directly functions to regulate one of the signaling pathways known to be involved in initiating myeloid differentiation. We will employ DNA combing assay, differentiation assays, and mouse xenograft studies to investigate the impact of replication stress in PARI-depleted cells on leukemia tumorigenicity. In the second aim, we propose to study how PARI affects NFkappaB signaling and the implication of this interaction in leukemic differentiation. We will test the hypothesis that PARI inhibits NFkappaB activation by silencing the DNA damage checkpoint and that NFkappaB activation in PARI-depleted cells promotes leukemic differentiation. We will employ cellular, molecular, pharmacological, and transcriptomic approaches to investigate the impact of NFkappaB pathway activation by PARI depletion or exogenous DNA damage on the induction of leukemic differentiation.Military Relevance: Radiation exposure is a well-known, military-relevant risk factor. Radiation creates DNA damage, which can lead to mutations, genomic instability, and carcinogenesis. In particular, radiation exposure results in increased incidence of leukemia. Our research investigates a new pathway that repairs radiation-induced DNA damage and explores its impact on leukemia development and treatment.