Abstract LB323: Thioredoxin interacting protein (TXNIP), a regulator of oxidative stress, mediates the RAPGEF3/4 (EPAC1/2) signaling required for melanoma progression

Abstract Exchange proteins directly activated by cyclic AMP (EPACs) are RAP guanine nucleotide exchange factors (RAPGEFs). We showed that EPAC signaling is required for the survival and growth of human primary, but not metastatic, melanoma cell lines, and this dependency of primary melanoma on EPACs is mediated by activation of mTOR signaling. Here, we show that low expression of EPAC1/2 mRNA (TCGA dataset) and activated RAP1 (immunohistochemistry for RAP1-GTP in melanoma TMA) in primary tumors correlate with better disease-free survival. We investigated the role of EPACs in melanoma in vivo by administration of EPAC1/2 inhibitor (ESI-09; i. p. 25mg/Kg/day for 3 weeks) to Braf/Pten/Tyr-CreERT2 (BPT) mice 3 weeks after initiating melanomagenesis by topical application of tamoxifen on shaved flanks. Treatment with ESI-09 significantly inhibited tumor growth and improved the survival of mice. We also generated BRaf CA , PtenloxP, EPAC1/2loxP, Tyr::CreERT2 mice and compared tumor incidence, latency, tumor growth and survival between Epac WT (BPT) control and Epac1/2-null mice. Loss of Epac1/2 delayed tumor initiation, inhibited tumor growth and improved survival of the mice. These data confirmed the important role EPAC signaling plays in melanoma in vivo. To identify the mechanisms underlying the dependency of primary melanoma cells on EPAC signaling, we performed bulk RNA-seq analysis of a set of patient-matched primary and metastatic melanoma cell lines treated with ESI-09. Our analysis showed that 12h of treatment with EPAC inhibitor significantly altered the expression of more genes in primary melanoma cells than in metastatic cells (288 vs 2 gene downregulated, and 332 vs 0 genes upregulated in primary vs metastatic). Differential gene expression analysis showed TXNIP (thioredoxin interacting protein), a major regulator of cellular redox signaling, as the most significantly downregulated gene (>15-fold decrease) in primary melanoma. qRT-PCR and western blotting showed a progressive loss of the effect of EPAC inhibitor on TXNIP from primary to lymph node metastases and to distant metastases. Gene enrichment analysis showed that EPAC signaling regulates genes several genes within the cancer Hallmark pathways (E2F targets, G2-M checkpoint, Glycolysis) suggesting a critical role for TXNIP melanoma development and progression. Our data suggest that EPAC/TXNIP signaling pathway is a potential target for prevention and early interception of cutaneous malignant melanoma. Citation Format: Sireesh Kumar Teertam, Mithalesh Singh, Sarah Altameemi, Sonia Gude, Vijayasaradhi Setaluri. Thioredoxin interacting protein (TXNIP), a regulator of oxidative stress, mediates the RAPGEF3/4 (EPAC1/2) signaling required for melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB323.