Project Summary-Abstract Tumor microenvironment (TME) consists of many cell types that co-exist to promote tumor progression. Most cancer therapeutics are designed to target one molecule in one defined cell type. For example, vemurafenib (BRAF inhibitor) kills melanoma cells through targeting mutated BRAF; whereas pembrolizumab (anti-PD-1 antibody) blocks PD-1 on T cells, re-activating anti-tumor immunity. Our overarching goal is to identify targetable molecules/pathways that are critical for multiple cell types within the TME. Using the published single cell RNAseq (scRNAseq) datasets, we searched for these molecules/pathways meeting the following criteria: 1) they should have important functions in cancer cells and immune suppressive cell types such as regulatory T cells (Tregs), exhausted T cells (Texh), and myeloid-derived suppressor cells (MDSCs) etc.; 2) they are not important for effector function of major immune cells such as effect T cells (CD4+ or CD8+ Teff) or nature killer cells (NK); 3) they should be targetable with known inhibitors. NR4A1 fits all 3 criteria and represents a valid target for cancer immunotherapy. In the current proposal, we intend to use proteolysis-targeting chimera (PROTAC) technology to develop a first-of-its-kind NR4A1 degrader for melanoma therapy. Aim 1. Rational design of novel celastrol-based NR4A1-Ps by modifying celastrol and linkers. Aim 2. Determine cellular and molecular mechanisms by which NR4A1-Ps work to inhibit melanoma. Aim 3. Explore the therapeutic potential of NR4A1- Ps as a single agent or in combination. The outcome is to define the rationale for the future clinical translation of NR4A1-Ps to enhance ICI therapy responses in melanoma.