DNA damage is the principle mechanism of activity of most chemotherapeutic and radiation treatments given to patients with hematologic malignancies. Understanding the mechanisms by which malignant cells repair DNA and become resistant is of paramount clinical significance. There is data to indicate a role of Pso4 in DNA repair from yeast to humans. Saccharomyces cerevisiae Mutant pso4.1 displays pleitropic phenotype with major defects in DNA repair particularly in response to Interstrand Cross- Linking (ICL) agents. Human Pso4 was isolated as an interacting partner of multiple DNA repair proteins including the Non Homologous End Joining (NHEJ) repair protein Metnase, the Werner Syndrome Protein (WRN), and the antibody diversity protein Terminal Deoxynucleotide Transferase (TDT). hPso4 complex binds checkpoint protein Ataxia-Telangiectasia and Rad3-related (ATR). Depletion of hPso4 by siRNA resulted in a defective S-phase cell-cycle checkpoint and cellular sensitivity in response to replication-fork blocking agents. This depletion also leads to enhanced apoptosis in response to multiple DNA damaging agents. hPso4 is present in a complex with the Transcription-Coupled Repair (TCR) protein XAB2 which suggests a role in this important DNA repair pathway. Apart from its contribution to ICL repair in humans, it is unknown if it participates in other repair pathways. hPso4 contains WD40 domains which are present in other proteins that bind Histones. In this application, we will attempt to answer the following question: 1- What is the molecular mechanism of hPso4 activity in DNA repair? The molecular interactions of hPso4 with Histones and other proteins known to be involved in DNA repair will be investigated. Since hPso4 contains U-box that displays ubiquitin E3 ligase activity, we will investigate the role of this ubiquitination in DNA repair and Histone modifications. 2- What is (are) the repair pathway (s) through which hPso4 exerts its DNA repair activity? We will investigate the contribution of hPso4 to other DNA repair pathways aside from ICL repair. 3- Does hPso4 predict resistance to chemotherapy in hematologic malignancies? We will investigate a potential role of hPso4 in mediating chemotherapy resistance in a lymphoma cell line. We will collect samples prospectively from lymphoma patients to correlate hPso4 expression with response to chemotherapy and risk of relapse. We will also evaluate whether hPso4 depletion sensitize cell lines to Poly (ADP-Ribose) Polymerase (PARP) inhibitors. The postulated E2 conjugating enzyme for hPso4 is Ubc3. We will deplete this E2 and evaluate if this will achieve the same chemosensitivity as depleting hPso4. This will open the opportunity to identify Ubc3 small molecule inhibitors as cancer therapy.
Most drugs used to treat hematological malignancies exert activity by damaging DNA and triggering apoptosis. Malignant cells appear to be more susceptible to these drugs than normal cells, but they develop resistance quickly by different mechanisms. Our project aims to characterize the precise role of the DNA repair protein human Pso4 in this process and to investigate its mechanism of action and its potential role in chemotherapy resistance.
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