Project Summary Advanced NSCLC still remains an incurable disease, at least in part due to resilient populations of stem-like, cancer initiating cells (CSCs) that survive conventional therapies and reignite post-therapy relapse and metastatic dissemination. This proposal will test the hypothesis that the mitochondrial citrate carrier, Slc25a1, acts as a key metabolic hub through which NSCLC acquire resistance to different therapeutic agents. Given that the genetic spectrum of mutations continues to evolve during the course of therapy, newly emerging mutations may not always be targetable with currently available drugs. Thus, the development of therapies that act regardless of the mutational profile of tumors is an attractive concept. We have recently shown that Slc25a1 promotes CSC expansion and self-renewal, enhancing the energetic output of this population by promoting the mitochondrial entry of citrate with consequent induction of mitochondrial metabolism and oxidative phosphorylation. The scope of the current project is to identify the metabolic hallmarks of drug resistance in patient-derived tumors. Our preliminary data show that resistance to inhibitors of the Epidermal Growth Factor Receptor, EGFR or to platinum therapy involves a switch- and a dependency- towards Slc25a1-driven mitochondrial metabolism accompanied by the induction of a stemness phenotype. Hence, CTPI-2 is synthetic lethal with cisplatin or with EGFR inhibitor co-treatment and restores sensitivity to these agents in vitro and in vivo. Further, we provide evidence that Slc25a1 induces an Interferon type I (IFN-I) anti-viral innate immune response, likely driven by oxidative stress and by accumulation of mitochondrial DNA (mtDNA) in the cytoplasm. We link this signature to the therapy resistance phenotype induced by Slc25a1. With this in mind, the scopes of the current project are to test the hypothesis that Slc25a1 allows drug-resistant cells to endure and survive therapeutic attacks in an energetically favorable state and that tumors resistant to different types of drugs rely upon common metabolic traits driven by Slc25a1. Second, we will clarify whether IFN-I is involved in the drug resistant phenotype driven by Slc25a1. In Aim 1 we will use a newly developed organoid system that allows for the expansion of primary tumors derived from patients to determine whether Slc25a1 drives different types of drug resistance independently of the primary driver mutations. In Aim 2 we will determine whether components of the newly identified Slc25a1-mtDNA-IFN-I loop are responsible for induction of the stemness phenotype and the insensitivity to drugs that act predominantly on highly proliferating cells. Together, these studies will provide a major advance in enlightening novel mechanisms underlying NSCLC pathogenesis, will fill a gap in knowledge elucidating unexpected mechanistic links between the mitochondria, stemness and drug-resistance, which we will ultimately hope will open new...