Abstract The ITCH ubiquitin E3 ligase has been well characterized as a key molecule in immune cells. The roles of ITCH in tumorigenesis are rather controversial due to its versatile functions in both tumor and immune cells. Therefore, an in-depth characterization of ITCH in tumorigenesis considering both tumor and immune cells is warranted to fill the gap in our current knowledge of ITCH’s function in cancer. We recently reported an oncogenic function of ITCH through promoting BRAF activation in BRAF wild-type melanoma cells. Our newly obtained preliminary results unveil a multifaceted role of ITCH in melanoma cells by modulating CRAF and other oncogenic signals. Our results also suggest an immunosuppressive function of ITCH in the melanoma tumor immune microenvironment. Based on these data, we hypothesize that in BRAF wild-type melanoma cells, ITCH is a therapeutic vulnerability in both melanoma cells and the tumor immune microenvironment. In melanoma cells, ITCH sustains oncogenic pathways including RAF-MEK-ERK and others to maintain their fitness; activation of ITCH in immune cells, on the other hand, fosters an immunosuppressive tumor microenvironment. Hence targeting ITCH could be a novel strategy to alter the cancer-immune set point. Three aims are proposed to test our hypothesis. In Aim 1, we will define how K27-linked ubiquitination of RAF proteins by ITCH modulates MEK-ERK signaling. We will characterize if ITCH facilitates the atypical ubiquitination and subsequent activation of CRAF in a similar way to BRAF, especially in the cellular contexts where CRAF plays a central role in activating MEK-ERK signals. Moreover, we will determine if the B55-PP2A phosphatase functions as the reader protein for the poly-ubiquitin chains assembled on RAF proteins to remove the inhibitory N-terminal phosphorylations. In Aim 2, we will characterize RAF-independent oncogenic functions of ITCH. We will examine if ITCH facilitates the activation of other oncogenic pathways that fulfill its pro-survival and pro-metastatic roles. In Aim 3, we will focus on investigating both melanoma cell-autonomous and non-autonomous functions of ITCH in vivo. We will dissect these roles by using several mouse melanoma models. To define if ITCH creates an immunosuppressive microenvironment, mouse syngeneic melanoma models will be generated using Itch+/+ and Itch-/- mice, and immuno-profiling will be carried out to search for the underlying mechanism(s). Further, Itch+/+ and Itch-/- mice will be crossed with well-characterized murine melanoma models to assess its roles in both the tumor cell and immune microenvironment.