Project Summary/Abstract The ultimate goal of this proposal is to address the fundamental gap in knowledge on the mechanisms driving resistance to immune checkpoint blockade (ICB), particularly in melanoma patients who exhibit downregulation or deletion of p16 (~40% of cases). The research plan focuses on elucidating the mechanistic role of p16 loss in regulating zinc (Zn) homeostasis through the Zn transporter SLC39A9. The goal is to explore whether manipu- lating Zn levels enhance the efficacy of current ICB in p16low melanomas. Drawing on a compelling in vivo CRISPR knockout screen, our data reveal that the knockdown of SLC39A9 sensitizes p16low melanomas to anti- PD1 ICB. Additionally, increased SLC39A9 expression correlates with unfavorable outcomes in melanoma pa- tients undergoing ICB. Notably, loss of p16 expression leads to an increase in plasma membrane SLC39A9, resulting in the sequestration of Zn from the microenvironment and creating a Zn-depleted tumor microenviron- ment. We propose two major consequences of this reprogramed Zn homeostasis in p16low tumors that contribute to impaired ICB response. Firstly, increased intracellular Zn in p16low cancer cells decreases cGAS-STING sig- naling and downregulates CXCL10, a T cell chemokine. Secondly, limited access to microenvironmental Zn compromises CD8 T cell effector function, given its crucial role in early TCR activation. This research plan is structured around two overarching scientific aims: 1) Interrogate the mechanism underlying Zn-dependent atten- uation of cGAS-STING signaling as a consequence of SLC39A9 and 2) Determine the contribution of low TME Zn on CD8 T cell recruitment and differentiation and ICB response. Successful completion of these aims will not only yield new insights into the mechanistic role of p16 in Zn metabolism but will also position the modulation of Zn homeostasis as a promising strategy to enhance therapeutic outcomes for melanoma patients with low p16 expression. Given the p