PROJECT SUMMARY Glutathione (GSH) is a small thiol antioxidant that is critical in maintaining redox homeostasis, and increases in glutathione occur in KRAS-mutant lung adenocarcinoma. Glutathione can also lead to S-glutathionylation (PSSG), the conjugation of GSH to reactive cysteines in proteins, which can be reversed by the de- glutathionylase, glutaredoxin (GLRX). While the glutaredoxin/PSSG system has emerged as a key regulator of redox signaling, little is known about its disruption in lung adenocarcinoma (LUAD). We have made the exciting discovery that GLRX expression is decreased in human LUAD and that KrasG12D-induced lung tumorigenesis is enhanced in mice lacking Glrx. In a redox proteomics screen we revealed increases in S-glutathionylation of ovarian tumor deubiquitinase, OTUB1, which was recently identified as a regulator of System XC-. System XC- is a cystine/glutamate antiporter that exports glutamate and imports cystine, which is in turn reduced intracellularly to cysteine, the rate limiting amino acid in glutathione synthesis. System XC- is composed of SLC7A11 and SLC3A2. OTUB1 binds to SLC7A11 to prevent its ubiquitination and proteasomal degradation. We have made the exciting discovery that S-glutathionylation of OTUB1 is important in the stabilization of SLC7A11, leading to increases in GSH. Compared to adjacent control tissue, expression of OTUB1 and SLC7A11 are increased in LUAD. These exciting findings led us to hypothesize that decreases in glutaredoxin expression in mutant KRAS-driven lung adenocarcinoma cause increases in system XC- activity through the S- glutathionylation of ovarian tumor deubiquitinase 1 (OTUB1), augmenting glutathione and promoting survival of lung cancer cells. Furthermore, we hypothesize that avenues to augment glutaredoxin will diminish GSH levels leading to increased potency of cisplatin-induced killing of lung cancer cells. In Specific Aim 1 we will determine whether GLRX status controls KrasG12D-induced tumorigenesis. Specific Aim 2 proposes to address the importance of OTUB1 S-glutathionylation and System XC- in the augmentation of GSH in KrasG12D- induced tumors while in Specific Aim 3 we aim to address the impact of AAV-mediated transduction of GLRX on cisplatin-induced tumor killing in primary LUAD tumor organoids and KrasG12D-driven tumors in mice. Completion of this proposal will provide new insights into the role of the GLRX-protein S-glutathionylation redox system in the pathogenesis of LUAD and offers a rationale for augmenting GLRX in lung tumors in order to promote killing. The pharmaceutical development of GLRX has the potential to offer new therapeutic strategies to combat LUAD, in combination with standard of care therapy.

NARRATIVE The environment of lung tumors is hostile requiring tumors to adapt to be able to survive and grow. Part of this adaptation is a change in metabolism and increases the antioxidant, glutathione to combat the augmented oxidative stress. In this proposal we will address the mechanism whereby this increase in glutathione occurs and offer new strategies to prevent the increases in glutathione from happening. We will test whether this strategy can be used to augment the therapeutic efficacy of the chemotherapeutic drug, cisplatin.