PROJECT SUMMARY Melanoma is a devastating skin cancer that kills nearly 8,000 patients in the US annually. Patients with thicker melanomas, invading deeper and closer to lymphatics and blood vessels, are more likely to have metastasis to regional lymph nodes (LNs). For patients without clinically evident LN metastases, the presence of occult metastases in the sentinel LN (SLN) or first tumor-draining LN (TDLN) is the most important prognostic factor. However, why melanoma metastasizes to LNs in some patients and not in others remain poorly understood; therefore, it is critical to determine the factors regulating metastasis to inform the development of novel therapeutic approaches to prevent and target metastasis. We demonstrated that not only is the gut microbiome a critical modulator of response to immune checkpoint blockade (ICB) but also that a favorable gut microbiome can augment anti-tumor immunity at distant sites, including the skin. We have also identified differences in the gut microbiome of melanoma patients with early-stage (0-II) compared to late-stage (III-IV) disease. The gap in knowledge is the effect of the gut microbiome on the natural evolution of melanoma at the primary tumor and on metastasis to regional LNs. Our hypothesis is that microbes in the gut of melanoma patients influence primary tumor development and risk for LN metastasis, such that targeted modulation of the gut microbiome can prevent melanoma LN metastases. To test this hypothesis, we will: 1) Identify gut microbial and immunologic features associated with tumor progression and LN metastasis in melanoma patients. We will compare gut microbiome profiles and paired tissue immune profiles using GeoMx digital spatial profiling to characterize the immune microenvironment (IME) of the primary melanoma and SLN, and 16S gene amplicon sequencing and/or whole metagenomic sequencing to analyze fecal samples of melanoma patients with: 1.1) Low-risk vs High-risk primary tumors; 1.2) Negative vs Positive SLNs; and 1.3) Clinical Stage III melanoma receiving neoadjuvant ICB. Statistical correlation analyses will be applied to identify gut microbial features associated with immune modulation, melanoma progression and metastasis. 2) Delineate strategies to modulate gut microbes to prevent melanoma progression and LN metastasis. We will investigate the causal relationship between the gut microbiome, tumor progression, and LN metastasis using an inducible model of murine melanoma (cKit:ERT2;BrafV600E;Ptenfl/fl) by assessing the: 2.1) Impact of FMT from responder and non- responder patients on primary tumor progression and LN metastasis; 2.2) IME in both the primary tumor and SLN to identify the microbiome-dependent immunological mechanisms of tumor progression; 2.3) Effect of perturbation of the gut microbiota on the development of regional LN metastasis. The impact of this work will be recognition of the gut microbiome as a critical determinant of melanoma progression at the primary and metastasis to regional LNs, aiding the development of next generation approaches to prevent LN metastasis in melanoma and other cancers.

PROJECT NARRATIVE Melanoma is a deadly skin cancer that kills thousands of people in the US each year, despite revolutionary treatments such as immunotherapy; more forms of treatment are needed to improve outcomes for patients with advanced melanoma and to keep melanoma from spreading in patients with early-stage disease. Our group previously demonstrated that bacteria in the gut influence response to immunotherapy and showed that modifying these bacteria can improve treatment responses in patients with advanced melanoma. We will now study the impact of gut microbes in early-stage melanoma, with the goal of developing next-generation therapeutic strategies to better treat, intercept, and ultimately prevent melanoma.