Project Summary In this revised R01 application we propose to characterize a novel mechanism underlying the control of apoptosis by the mitochondrial enzyme GCDH, and to extend the development of small molecule GCDH inhibitors to treat melanoma. In our preliminary results we discovered that addiction to GCDH activity is critical for cell survival in melanoma, but not liver, breast or prostate tumor cells, a discovery that was confirmed in patient specimens where inverse correlation between GCDH expression and survival is seen in melanoma. Our studies reveal induction of apoptosis in melanoma cell lines following GCDH inhibition, a phenotype dependent on the upstream DHTKD1 enzyme. Key in mediating GCDH activities in melanoma is NRF2, which is subjected to glutarylation upon inhibition of GCDH. NRF2 glutarylation promotes its stability and transactivation of an apoptotic UPR signaling consisting of ATF4, ATF3, CHOP and CHAC1, the latter being components of the Unfolded Protein Response (UPR). Inhibition of GCDH effectively induces apoptotic UPR signaling in melanoma but not in liver or breast cancer cells, substantiating the selectivity of the pathway as mapped in cell lines and patient tumor samples. These observations provide the foundation for our hypothesis that GCDH-mediated regulation of NRF2-UPR signaling constitutes a novel pathway controlling melanoma cell survival. Our studies will (i) map the NRF2 glutarylation pathway in melanoma, generate antibodies specific to glutarylated NRF2, monitor NRF2 glutarylation in specimens from melanoma patients both responsive and non-responsive to therapy (ii) assess how GCDH impacts tumor development, progression and response to targeted and immuno therapy in genetic mouse models. YUMM1.7, MaRas, B16F10 lines will be modified to express inducible KD of GCDH before their inoculation into syngeneic WT or GCDH KO mouse models which will be monitored prior and following therapy (iii) advance development GCDH inhibitors as novel therapeutic modalities. SBI-0690564 was confirmed in vitro and in cultured melanoma cells, where it phenocopies genetic inactivation of GCDH and inhibits melanoma tumors in mice. We will characterize and further develop a novel class of inhibitors to target GCDH and determine their effectiveness in culture and in vivo using mouse models. Our studies will establish novel paradigm for GCDH signaling as we define mechanisms underlying GCDH control melanoma tumor fate. Understanding the addiction to GCDH in melanoma provides the foundation for the development and evaluation of novel GCDH inhibitors to selectively target these and possibly other select cancers.