Biology subjects

Roberts, M. A.

Publications and source records attributed to Roberts, M. A..

4 recordsLinked to original sources

bioRxiv2022

Identification of structurally diverse FSP1 inhibitors that sensitize cancer cells to ferroptosis

Ferroptosis is a regulated form of cell death associated with the iron-dependent accumulation of lipid peroxides. Inducing ferroptosis is a promising approach to treat therapy resistant cancer. Ferroptosis suppressor protein 1 (FSP1) promotes ferroptosis resistance in cancer by generating the antioxidant form of coenzyme Q10 (CoQ). Despite the important role of FSP1, few molecular tools exist that target the CoQ-FSP1 pathway. Exploiting a series of chemical screens, we identify several structurally diverse FSP1 inhibitors. The most potent of these compounds, ferroptosis sensitizer 1 (FSEN1), is an uncompetitive inhibitor that acts selectively through on target inhibition of FSP1 to sensitize cancer cells to ferroptosis. Furthermore, a synthetic lethality screen reveals that FSEN1 synergizes with endoperoxide-containing ferroptosis inducers, including dihydroartemisinin, to trigger ferroptosis. These results provide new tools that catalyze the exploration of FSP1 as a therapeutic target and highlight the value of combinatorial therapeutic regimes targeting FSP1 and additional ferroptosis inducers.

cancer biology↗

bioRxiv2022

Parallel CRISPR-Cas9 screens reveal mechanisms of PLIN2 and lipid droplet regulation

Lipid droplets (LDs) are lipid storage organelles that consist of a central core of neutral lipids surrounded by a phospholipid monolayer decorated with a unique set of integral and peripheral proteins. Invariably, at least one member of the perilipin family of proteins (PLIN1-5) associates with LDs in all cell types. Despite key roles of PLIN2 in governing hepatic lipid metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we develop a set of genome-edited PLIN2 reporter cell lines that facilitate the analysis of genes that regulate PLIN2 and LD abundance. Leveraging these reporter cells in a series of CRISPR-Cas9 loss-of-function screens, we generate a comprehensive inventory of genes that influence PLIN2 levels under different metabolic conditions. Moreover, we uncouple their effects on PLIN2 expression and post-translational stability. Identified genetic modifiers include canonical genes that control LD metabolism (e.g., ACSL3, DGAT2, PNPLA2, ABHD5) as well as genes with less characterized roles in PLIN2 and LD regulation such as ubiquitination machinery (e.g., MARCH6, UBE2J2), transcription regulators (e.g., HNF4A, HDAC3), mitochondrial pathways (e.g., electron transport chain and mitochondrial fatty acid synthesis), and others. These CRISPR screens, and several published screens that focus on different aspects of lipid metabolism, provide the foundation for CRISPRlipid (http://crisprlipid.org), a versatile, online data commons for lipid-related functional genomics data. Together, our study uncovers new mechanisms of PLIN2 regulation and provides an extensive, phenotype-rich resource for the exploration of LD biology and lipid metabolism.

cell biology↗

bioRxiv2022

VPS13A and VPS13C influence lipid droplet abundance

Lipid transfer proteins mediate the exchange of lipids between closely apposed membranes at organelle contact sites and play key roles in lipid metabolism, membrane homeostasis, and cellular signaling. A recently discovered novel family of lipid transfer proteins, which includes the VPS13 proteins (VPS13A-D), adopt a rod-like bridge conformation with an extended hydrophobic groove that enables the bulk transfer of membrane lipids for membrane growth. Loss of function mutations in VPS13A and VPS13C cause chorea acanthocytosis and Parkinsons disease, respectively. VPS13A and VPS13C localize to multiple organelle contact sites, including endoplasmic reticulum (ER) - lipid droplet (LD) contact sites, but the functional roles of these proteins in LD regulation remains mostly unexplored. Here, we employ CRISPR-Cas9 genome editing to generate VPS13A and VPS13C knockout cell lines in U-2 OS cells via deletion of exon 2 and introduction of an early frameshift. Analysis of LD content in these cell lines revealed that loss of either VPS13A or VPS13C results in reduced LD abundance under oleate-stimulated conditions. These data implicate VPS13A and VPS13C in LD regulation and raise the intriguing possibility that VPS13A and VPS13C-mediated lipid transfer facilitates LD biogenesis.

cell biology↗

bioRxiv2022

Ribosome stalling during selenoprotein translation exposes a ferroptosis vulnerability in cancer

Ferroptosis is a regulated, iron-dependent form of necrosis that is triggered by the accumulation of oxidatively damaged phospholipids1-3. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting phospholipid hydroperoxides into non-toxic lipid alcohols4, 5. Ferroptosis has been implicated in the pathology of several degenerative conditions and inhibiting GPX4 activity has emerged as a therapeutic strategy to induce cancer cell death1, 2. However, many cancer cell lines are resistant to GPX4 inhibition6, and the mechanisms that regulate GPX4 activity and ferroptosis resistance remain incompletely understood. Here, employing a synthetic lethal CRISPR-Cas9 screen in a triple negative breast cancer (TNBC) cell line, we identify LRP8 (also known as ApoER2) as a ferroptosis resistance factor. LRP8 is upregulated in cancer, and we find that it promotes ferroptosis resistance in cancer cells in both 2-dimensional (2-D) cell culture and 3-dimensional (3-D) spheroid models. Mechanistically, loss of LRP8 decreases cellular selenium levels, resulting in the reduced expression of a subset of selenoproteins, including GPX4. Remarkably, the reduction in GPX4 is not due to the classic hierarchical selenoprotein regulatory program7, 8. Instead, our findings demonstrate that the translation of GPX4 is severely impaired in the selenium-deficient LRP8 knockout (KO) cells due to extensive ribosome stalling at the inefficiently decoded GPX4 selenocysteine (SEC) UGA codon, which results in ribosome collisions and early translation termination. Thus, our findings reveal ribosome stalling and collisions during GPX4 translation as targetable ferroptosis vulnerabilities in cancer cells.

cancer biology↗