Thursday, November 21, 2024
11/21/2024
PROJECT ABSTRACT Abnormalities in genome maintenance pathways offer an opportunity to develop therapeutic approaches that selectively target cancer cells. Furthermore, because of their proliferative nature, cancer cells are susceptible to replication-associated DNA damage and more dependent upon the pathways that deal with this type of damage. In a recent collaborative study, we found that the levels of DNA ligase I (LigI), the major enzyme joining Okazaki fragments at the replication fork, correlates with outcome and therapy resistance in ovarian cancer. Further, we found that ovarian cancer cells, which have a defect in homology-dependent repair (HDR), are sensitive to LigI inhibition. In preliminary studies we compared the effects of LIG1 deletion with chemical inhibition. In LIG1 null cells, there were elevated levels of XRCC1, the partner protein of LigIII, and PARP2 associated with newly synthesized DNA, consistent with the PARP- and LigIII-dependent back-up pathway that joins Okazaki fragments and enables cells to replicated in the absence of LigI. In contrast, incubation with the LigI inhibitor did not result in increased levels of XRCC1 or PARP2. Instead, there were elevated levels of the Mre11Rad50Nbs1 complex, that initiates the resection step of homology-dependent repair (HDR). In aim 1, we will use a combination of genetic, cell biological and biochemical approaches to determine the mechanism of the HDR pathway induced by LigI inhibition. Since HDR deficiency occurs in both inherited and sporadic forms of ovarian cancer and we have identified LIG1 as a biomarker of outcome and therapy resistance in ovarian cancer, we will examine the activity of the LigI inhibitor alone and in combination with DNA repair inhibitors and/or genotoxic chemotherapeutics in genetically-defined ovarian cancer cell lines. Building upon our existing LigI inhibitors in aim 2, we will use a combination of molecular modeling and medicinal chemistry to design and synthesize putative LigI inhibitors that are predicted to have enhanced potency and selectivity as well as improved pharmacological properties. The activity of the putative LigI inhibitors will be evaluated in parallel biochemical assays with purified human DNA ligases and cell-based assays with genetically-defined ovarian cancer cell lines. In aim 3, we will first examine the activity of existing and new LigI inhibitors for activity in genetically-characterized patient-derived organoids alone and in combination with DNA repair inhibitors and chemotherapeutics used to treat ovarian cancer. Subsequently, combinations with the highest activity will evaluated in mouse xenograft models of ovarian, including patient-derived xenografts. Together, the proposed studies will provide mechanistic insights into replication-associated PARP-dependent and HDR pathways that are critical in LigI deficient cells, develop improved LigI inhibitors and identify specific DNA alterations that confer sensitivity to the LigI inhibitor alone or in combination with DNA damaging agents and/or DNA repair inhibitors in preclinical ovarian cancer models, providing the rationale for the further development of LigI inhibitors as a novel therapeutic for ovarian cancer.
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