Oxidation and reduction of thiol proteins are considered the major mechanisms by which reactive oxidants control cellular signaling pathways. Protein disulfide isomerase (PDI) is a member of the thioredoxin superfamily. PDI is a multifunctional protein primarily involved in the thiol disulfide exchanges in native ER proteins. Emerging evidence from our group indicates a functional association between PDI and NADPH oxidase family members (Nox). We have shown that PDI can regulate Nox activation and expression in multiple cell types. Therefore, PDI/Nox may represent a new redox interaction that regulates cellular processes such as protein folding and signal transduction. Changes in the cellular redox state is one mechanism by which the cell integrates information and coordinates complex signaling pathways involved in tumor progression. Melanoma is the most aggressive form of skin cancer. Half of the patient's tumors have the BRAF V600E mutation and therefore, develop resistance to BRAF inhibitor vemurafenib (iBRAF). Targeting redox‐regulated signaling pathways activated in melanoma may represent a new therapeutic opportunity for this disease. Herein, we sought to investigate the effect of PDI and Nox on the mechanism that enables resistance of melanoma cells to iBRAF. Using the melanoma SKMEL‐28 cell line, exposed to low concentrations of iBRAF for 4–6 weeks, we generated vemurafenib resistant cells (SKMEL‐28R). The combination of a cell surface PDI inhibitor (rutin, 100 μM, 48 h) and iBRAF (3 μM, 48 h) resulted in SKMEL‐28R survival of 30% compared to rutin (100% survival) or iBRAF (70%) monotherapy. In order to investigate the role of Noxes in the iBRAF resistance, we treated melanoma cells with increasing concentrations of the flavoenzyme inhibitor DPI (1,25–10 μM). The combination of DPI and iBRAF completely impaired cell survival in both parental and resistant cells when compared to DPI or iBRAF only. These findings were confirmed by analysis of the subG1 population, which revealed a 50% increase in apoptosis upon combined treatment. SKMEL‐28R cells had greater migratory potential than SKMEL‐28 cells, which was dose‐dependently reversed by rutin (50–300 μM). Additionally, rutin (10 and 200 μM) long term treatment in combination with iBRAF, decreased the colony forming ability of SKMEL‐28R. Next, we evaluated Nox4 and P4HB (PDI) gene expression data from 54 and 27 melanoma or non‐malignant nevus patients' samples, respectively, that were obtained from GEO profile database (GSE98394) and from primary (n=103) and metastatic (n=369) melanoma samples from TGCA Provisional Study (cBioPortal). In melanoma patients, Nox4 and P4HB mRNA levels were increased compared to non‐malignant nevus samples (p<0.0001). High Nox4 and low P4HB expression were also found in metastatic compared to primary site samples (p<0.01). These data indicate that both, Nox4 and PDI, are involved in melanoma development, but with distinct roles during tumor progression. Therefore, we propose that PDI could represent a novel therapeutic target to overcome resistance to BRAF inhibitors in melanoma.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.