PROJECT SUMMARY Melanoma is the fifth most common cancer in the US and while the prognosis for early- stage melanoma is favorable, late-stage disease was long considered incurable. Immune checkpoint inhibitors (ICIs) have revolutionized metastatic melanoma treatment; nevertheless, ~40% of patients still do not respond to therapy and ~80% of ICI-treated patients experience immune-related adverse events (irAEs). Identifying reliable pre-treatment biomarkers stratifying patients for maximal efficacy while minimizing toxicity is an urgent clinical task, as existing tumor or tumor/immune surrogates are limited. Recent research suggests that baseline host immunity is controlled by specific regulatory networks impacting T-cell activation and differentiation, including CD8+ T cells (the primary targets of ICI). Accumulated data show that the regulatory genome (the “regulome”), composed of transcription factors, enhancers, and other elements (e.g. lncRNAs), controls the transcriptional reprogramming of cytotoxic T-cells, playing a critical role in host anti-tumor immune response. We and others have reported that the regulome is enriched by underlying genetic variation associated with autoimmune risk and that this genetic susceptibility associates with ICI outcomes. Based on these intriguing observations, I hypothesize that baseline “regulome” states of peripheral immune cells, determined by the interplay of inherited genomic and transcriptomic factors, represent novel biomarkers of ICI resistance and susceptibility to irAEs. I postulate that these transcriptional regulatory networks (TRNs) govern T cell activity, potentially revealing biological pathways to reduce toxicity and enhance ICI efficacy. For the F99 phase, I will develop a genomic approach capturing TRNs of pre-treatment peripheral CD8+ T-cell (pre-pCD8+) activity as surrogates of ICI efficacy in metastatic melanoma using samples from ICI-treated metastatic melanoma patients from a large clinical trial led by Bristol-Myers Squibb. I will identify transcriptional signatures associated with ICI outcomes in pre-pCD8+ cells; single-cell RNA-seq will offer insight into immune cell subtypes in the ICI context (Aim 1.1). Combining Assay for Transposase-Accessible Chromatin (ATAC)-seq, RNA seq, and whole-genome sequencing, I will determine CD8+-specific regulome TRNs as modulators of ICI outcomes in advanced melanoma patients (Aim 1.2). For the K00 phase, I will expand this hypothesis to explore pre- and post-treatment changes at the single-cell level, correlating alterations in specific immune cell types with ICI response and irAEs using melanoma patient PBMCs (Aim 2). I will also develop models linking ICI-associated single-cell PBMC cell types and transcriptomic signatures with specific tumor neo-epitopes for therapeutic targeting. Identifying patients most likely to respond to ICI using these biomarkers may also point to novel biological pathways for more efficacious and less toxic treatments, furt...