Breast cancer remains a major killer of women due to the ineffectiveness of current drugs against metastatic and drug-resistant cancers. Additionally, African American (AA) women suffer disproportionately from breast cancer mortality in part because they develop the aggressive Triple- Negative Breast Cancer (TNBC) subtype more frequently than other ethnic groups. Thus, agents effective against drug-resistant and metastatic cancers and TNBCs may improve the survival of breast cancer patients. These aggressive cancers evade cell death through a variety of mechanisms including overactivation of the pro-survival HER-family of Receptor Tyrosine Kinases (RTKs), including EGFR/HER1, HER2, and HER3 (HER1-3), and inactivation of pro-apoptotic signaling. Tumors expressing HER1-3 are difficult to treat due to the partial redundancy among these receptors, their oncogenic signaling as heterodimers, and their ability to aberrantly heterodimerize with non-HER RTKs such as MET and IGF1R. Thus, resistance to current HER-targeted agents is a significant clinical problem. Defective cancer cell apoptosis can result from inactivation of the TNF Receptor Apoptosis Inducing Ligand (TRAIL)/Death Receptor 4/5 (DR4/5) pathway, which selectively kills cancer cells, while not affecting normal cells. Tumor resistance to TRAIL and other DR4/5 agonists results primarily from poor pharmacological properties of the agonists and the ability of cancer cells to downregulate DR4/5. Consequently, agents that could inactivate the EGFR/HER2/HER3 signaling axis and upregulate and activate DR4/5 independently of the TRAIL ligand may be efficacious against breast cancers unresponsive to current medicines. Disulfide bond Disrupting Agents (DDAs) are a new class of anti-cancer agents that induce regression of primary tumors and metastatic lesions of drug-resistant patient-derived tumors in animal models. In addition to the structural uniqueness of DDAs, recent studies indicate that DDAs are the first identified active site inhibitors of the Protein Disulfide Isomerases (PDIs) ERp44 and AGR2. Further, DDA inhibition of the PDIs ERp44, AGR2, and PDIA1 alters the disulfide bonding of HER1-3 and DR4/5, resulting in HER1-3 downregulation, DR5 upregulation, and disulfide bond-mediated oligomerization and activation of DR4/5. The objective of the current project is to move DDAs toward clinical trials. The two Specific Aims proposed to achieve this objective are to 1) optimize DDA pharmacological properties and dosing for future IND-enabling studies, and 2) validate biomarkers to predict tumor sensitivity to DDAs and to monitor target engagement, and thoroughly evaluate any adverse effects of DDAs on normal tissues or animal health. Based on their unique mechanisms of action and preclinical efficacy, we expect DDAs to benefit breast cancer patients with treatment-refractory breast cancers.