Thales Papagiannakopoulos, Ph.D. - US grants

? DESCRIPTION (provided by applicant): Lung cancer is the leading cause of cancer-related deaths in the United States and worldwide (Herbst et al. 2008). Non-small-cell lung cancer (NSCLC), accounts for ~85% of all lung cancer cases. 20-30% of human NSCLC tumors acquire mutations in anti-oxidant transcription factor Nrf2 (gain-of-function (GOF)) or its negativ regulator Keap1 (LOF) suggesting an important role for oxidative stress homeostasis to maintain cancer cell survival during lung carcinogenesis. Despite the high frequency of mutations observed in this pathway, little is know about its role in lung tumor initiation and progression. To study NSCLC our laboratory utilizes a genetically engineered mouse model (GEMM) of NSCLC that faithfully recapitulates the histologic, molecular, progression features of human NSCLC. Tumors develop in the autochthonous (native) tissue context, in the presence of an intact tumor microenvironment and in the absence of confounding tobacco mutagens. I have recently developed a rapid and precise in vivo method that bypasses the need for time- consuming manipulation of the murine germline in order to engineer novel alleles of interest. I have combined the power of sophisticated Cre/loxP-based GEMMs with the highly precise genome editing CRISPR/Cas9 system. Using this powerful new approach I will functionally investigate the tumor cell- autonomous role of Nrf2 and Keap1 in the lung adenocarcinoma initiation and progression using the NSCLC GEMM. The research proposed within this application for a K22 NIH career transition award focuses on elucidating the role of the Nrf2 anti-oxidant pathway in initiation and progression by rigorous in vivo experimental approaches combining both genetic and biochemical approaches. The goals and timeline of these well-defined and achievable experiments outlined within are to: * Dissect the functional importance of Nrf2 in NSCLC tumorigenesis * Identify the metabolic changes mediated by Nrf2 in lung cancer * Identify the Nrf2 targets that are important for mediating its anti-oxidant functions in vivo * Provide a platform for experimental cross talk between observations in NSCLC GEMMs and human cell lines, which will prove valuable for future research The project described within this application has been shaped by my long-standing dedication to the field of cancer biology and more importantly my ongoing efforts in Dr. Tyler Jacks' laboratory to uncover the fundamental mechanisms involved in initiation and progression in this autochthonous mouse model of NSCLC. I have benefited from the research environment in the Jacks Laboratory, MIT, and the surrounding area that offers unmatched opportunities for scientific discussion, collaboration, and training. The scientific community at MIT, the Broad Institute, and Harvard Medical School offers countless seminars and workshops that continue to foster my scientific development. Additionally, I supervise have supervised three undergraduate MIT student, three summer students and two technical assistant that work directly with me on experiments pertaining to the proposed research project. This is an incredible experience that will endow me with many of the necessary skills to manage a laboratory during the independent phase of this award and beyond. This proposal represents my dedicated scientific investment towards identifying the fundamental drivers of NSCLC tumorigenesis, by uniting collaborators' analyses and the use of a sophisticated genetically engineered mouse. It is my intention to start an independent research program that will capitalize on the insight I obtain from the in vivo animal model with the goal to translate my findings in human systems. For the long-term, I am confident that these experiments will provide a solid foundation on which my research program can be built upon. I look forward to educating and recruiting students and postdocs that share my passion for cancer research.

SUMMARY Treating KRAS mutant lung adenocarcinoma (LUAD) remains a major challenge for clinical oncology. Approximately 20% of KRAS mutant LUAD tumors carry loss-of-function mutations in KEAP1, a negative regulator of NRF2, which is the master transcriptional regulator of the endogenous antioxidant response. Using CRISPR/Cas9-based somatic editing in a genetically engineered mouse model of KRAS-driven LUAD we demonstrated that loss of Keap1 hyper- activates Nrf2 and dramatically accelerates KRAS-driven LUAD. Combining CRISPR/Cas9- based genetic screening and metabolic analyses, we showed that Keap1 mutant cells are dependent on increased glutamine metabolism, and this property can be therapeutically exploited through the pharmacological inhibition. In this application we focus on characterizing the molecular mechanisms and therapeutic potential of targeting glutamine metabolism in KRAS- driven KEAP1 mutant LUAD, and other cancers with hyperactivation of the NRF2 antioxidant pathway. This application aims to: 1) Assess the therapeutic potential of inhibiting glutamine utilization in both human and murine KRAS-driven LUAD models with KEAP1 mutations, 2) Characterize the metabolic mechanisms underlying glutamine dependency in KEAP1 mutant LUAD, and 3) Determine the therapeutic potential of inhibiting glutaminolysis in cancers with hyperactivation of the NRF2 pathway. Our studies will provide a rationale for sub-stratification of patients with hyperactivation of the NRF2 pathway as treatment responders to glutaminase inhibitors, which is pertinent to the goals of precision medicine. !

SUMMARY Treating KRAS mutant lung adenocarcinoma (LUAD) remains a major challenge for clinical oncology. Approximately 20% of KRAS mutant LUAD tumors carry loss-of-function mutations in KEAP1, a negative regulator of NRF2, which is the master transcriptional regulator of the endogenous antioxidant response. Using CRISPR/Cas9-based somatic editing in a genetically engineered mouse model of KRAS-driven LUAD we demonstrated that loss of KEAP1 hyper-activates NRF2 and dramatically accelerates KRAS-driven LUAD. Our data are in line with mounting evidence demonstrating that, contrary to popular belief, antioxidants can promote cancer progression. Combining CRISPR/Cas9-based genetic screening and metabolic analyses, we have identified novel synthetic lethal interactions in KEAP1 mutant cells. We observe that the ability of KEAP1 mutant tumors to divert their metabolism towards antioxidant production comes with a cost, creating metabolic vulnerabilities that may be targeted by novel therapeutic strategies. In preliminary studies, we observed a dependency of KEAP1 mutant tumors on the amino acid serine. In this application we focus on elucidating this newly appreciated metabolic vulnerability of KRAS-driven KEAP1 mutant tumors to serine and explore the therapeutic potential of targeting serine metabolism in highly relevant pre-clinical mouse and human models. This application aims to: 1) Determine the therapeutic potential of inhibiting the serine transporter SLC1A5 and serine uptake in KRAS-driven LUAD models with KEAP1 mutations, 2) Define the metabolic mechanisms underlying serine dependency in LUAD and other KEAP1 mutant cancers, 3) Determine whether dietary serine restriction can selectively affect the growth of KEAP1 mutant tumors, 4) Dissect the metabolic crosstalk of glutamine and serine dependency in cancers with hyperactivation of the NRF2 pathway. Our studies will provide a rationale for sub-stratification of patients with hyperactivation of the NRF2 pathway as treatment responders to therapies targeting serine metabolism, which is pertinent to the goals of precision medicine.