James Arthur Olzmann - US grants

Abstract: We propose cooperative partnerships between California State University, Northridge (CSUN) and five outstanding Ph.D.-granting institutions. The partnerships described in this proposal are a model for establishing a program between a comprehensive Hispanic Serving Institution (HSI), CSUN, and several pre-eminent Research Extensive universities (UC Berkeley, UC Davis, UC Riverside, UC Santa Barbara, and Stanford University). Students participating in this Bridges to the PhD program will have the opportunity to choose a MA/MS concentration at CSUN among biology, psychology, chemistry/biochemistry, physics, and dietetics and nutrition. Linkages between the Ph.D.-granting institutions and CSUN will be established or enhanced through these partnerships. Activities and interactions that will foster and strengthen these ties include seminars at CSUN by faculty from the Ph.D.-granting institutions, visits of CSUN students to the Ph.D.-granting institutions, and the opportunity for CSUN students to collaborate on research projects at the partnership schools. CSUN students will participate in a special workshop that focuses on scientific rigor and reproducibility of results as well as responsible conduct of research. The proposed activities will further enhance the preparation of the participating Bridges Trainees for entry into Ph.D. programs, and ultimately to their earning a Ph.D. in a bio-medically relevant program at the partner institutions or a comparable one. We anticipate that over the five-year timeframe, 42 trainees will have participated in the program. Of these, 35 students will have completed the program (95% to have graduated with a master?s degree), while seven will still be in the program after the first cycle of funding. We expect 100% of those who earn a master?s degree to apply to Ph.D. programs in a basic bio-medically relevant field. Based on past success at CSUN, we expect 90% of the trainees to enter a Ph.D. program in a bio-medically relevant field and to have a retention/completion rate of 95%.

Ferroptosis is a regulated form of lipotoxic cell death that involves iron-dependent generation of reactive oxygen species (ROS) and the accumulation of oxidatively damaged lipids (e.g. lipid peroxides). Ferroptosis has been implicated in the etiology of degenerative diseases, such as neurodegeneration associated with iron accumulation. Cells contain a protective pathway in which the glutathione-dependent peroxidase GPX4 repairs lipid peroxides and blocks cell death. Targeted induction of ferroptosis by inhibiting GPX4 has proven to be an efficacious treatment in in vitro and in vivo models of cancer, including drug-resistant forms of cancer. Despite the excitement from these recent findings, our understanding of the mechanisms underlying ferroptosis remains limited. Furthermore, many cancer cells are resistant to ferroptosis and the mechanisms of ferroptosis resistance in cancer remains mostly unknown. To overcome this critical gap in knowledge, we performed a synthetic lethal, whole-genome CRISPR screen to identify factors that protect cancer cells from ferroptosis. Our findings identify the lipid droplet oxidoreductase AIFM2 as a key factor that promotes ferroptosis resistance in cancer. Deletion of AIFM2 dramatically sensitizes cells to ferroptosis and AIFM2 levels correlate with cancer resistance across hundreds of cancer lines, indicating that AIFM2 is a biomarker of ferroptosis resistance and suggesting that it is broadly involved in ferroptosis resistance across many types of cancer. Our proposed research builds on our discovery and employs a combination of functional genomic, cell biology, and biochemistry strategies to achieve the following goals: 1) elucidate the mechanism by which AIFM2 prevents lipid damage and ferroptosis, 2) define the relationship between lipid droplets, fatty acid metabolism, and ferroptosis, and 3) identify new factors involved in protecting cancer cells from ferroptosis. These goals are potentially transformative because they focus on new mechanisms of ferroptosis resistance in cancer cells that act in parallel to the canonical glutathione-based protective system.

PROJECT SUMMARY / ABSTRACT Lipid droplets (LDs) are neutral lipid storage organelles that act as cellular hubs of lipid homeostasis. Dysregulation in LD function has been implicated in prevalent metabolic diseases such as obesity, diabetes, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD). Indeed, the pathological hallmark of NAFLD is the accumulation of large hepatic LDs. In addition to metabolic diseases, LDs have also been implicated in cancer proliferation and survival, host-pathogen interactions, and neurodegeneration. Thus, understanding how LDs are regulated has the potential to broadly impact our understanding of human health and disease. LDs are ER-derived organelles that have a unique ultrastructure, consisting of a core of neutral lipid surrounded by a phospholipid monolayer decorated with integral and peripheral proteins. While recent findings have advanced our understanding of LD biogenesis, how LDs are regulated under different metabolic conditions and how the composition of the LD proteome remain poorly understood. To overcome these critical gaps in knowledge and define the mechanisms that regulate neutral lipid storage, we performed a series of CRISPR-Cas9 screen in human cells using a fluorescence-based neutral lipid reporter under different metabolic conditions. We also employed genetic screens to examine the mechanisms that regulate PLIN2, a near ubiquitous Class II LD protein that plays important roles in regulating LD stability. Our findings establish a compendium of neutral lipid storage regulators, revealing interesting novel regulators that are condition specific. Furthermore, we identify several ubiquitination factors that influence neutral lipid storage and the stability of PLIN2. The current proposal aims to build on the foundation provided by our extensive preliminary data to characterize new mechanisms of LD regulation. In aim 1, we will complete our validation experiments to establish an extensive, phenotypic-rich resource for the community that is hypothesis generating. We will also examine the concept that metabolic state-dependent regulation of LDs is a significant contributor to cellular lipid homeostasis. Finally, we will characterize high priority candidates in iPSC-derived hepatocytes and examine the hypothesis that a subset of regulators governs LD stability as part of a host response to pathogens. In Aim 2, we will define the role of new ubiquitination pathways in regulating lipid homeostasis, examining the hypothesis that the identified factors regulate LD stability by controlling the degradation of PLIN2 during lipolysis. These findings will provide new global and mechanistic insights in to LD proteome remodeling and regulation under different metabolic conditions.