Breast cancer (BC), the most common cancer among military servicewomen than in the general population. This makes BC a critical concern for female Veterans and VA health care. VA women has an increased risk of BC due to service-related exposures. Women diagnosed with triple negative breast cancer (TNBC) have a more aggressive clinical course, a higher propensity for metastases, and a worse outcome. Therefore, development of novel therapeutic strategies represents a clear unmet need. Genome instability is a hallmark of cancer and DNA replication represents one of the most vulnerable cellular processes that dysfunction therein leads to genome instability in cancer cells. Topoisomerase inhibitors (TIs) are shown to enhance replication stress (RS), and several TIs are approved by FDA for TNBC therapy. However, clinical utility is limited by toxicity and the development of resistance. Therefore, understanding the molecular basis of RS and DNA repair will provide the requisite foundation to develop new therapeutic targets and combination therapies. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) plays a critical role in TNBC progression. PELP1 status is a prognostic indicator of poor BC survival. There is a major knowledge gap regarding the mechanisms by which PELP1 contribute to cancer progression. We have developed a first-in-class inhibitor of PELP1 (SMIP34) that has activity on TNBCs and preliminary studies suggested SMIP34 enhance the utility of TIs in treating TNBC. The Objective of this proposal is to define the molecular mechanism of action of PELP1 in RS, DNA repair and to pave the path for a novel combination therapy. Our overarching hypothesis is that PELP1 signaling in DNA repair represents an attractive therapeutic target for SMIP34 in combination with TI in TNBC. In Aim 1, we will define the mechanisms by which PELP1 promotes DNA repair, determine the role of PELP1 in RS mediated DNA damage and identify PELP1 interactome under conditions of DNA damage. In Aim 2, we will characterize the role of PELP1 interactions with topoisomerases, establish the biological activity of SMIP34+TI combination therapy using multiple TNBC models and determine the mechanisms by which SMIP34 enhances efficacy of topoisomerase inhibitor. In Aim3, we will test the utility of SMIP34 + TI combination therapy in treating TNBC using orthotopic xenograft models, determine its utility in reducing TNBC metastases and establish the translatability of SMIP34 + TI therapy in treating TNBC using organoids (PDOs) and patient derived xenografts (PDX). This proposal tests the novel concept that SMIP34 blocks oncogenic DNA repair pathways which plays a critical role in TNBC progression leading to therapy resistance; and that disruption of PELP1 signaling with SMIP34 will enhance therapeutic effect of currently FDA approved chemotherapy using TI. Successful completion of the proposed studies will lead to the development of novel SMIP34+TI combination therapy that a...