Oncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.

Redox lipid reprogramming commands susceptibility of macrophages and microglia to ferroptotic death.

Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NO•-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO• donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO• donors and/or suppression of NO• production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.

Pseudomonas aeruginosa utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium.

Ferroptosis is a death program executed via selective oxidation of arachidonic acid-phosphatidylethanolamines (AA-PE) by 15-lipoxygenases. In mammalian cells and tissues, ferroptosis has been pathogenically associated with brain, kidney, and liver injury/diseases. We discovered that a prokaryotic bacterium, Pseudomonas aeruginosa, that does not contain AA-PE can express lipoxygenase (pLoxA), oxidize host AA-PE to 15-hydroperoxy-AA-PE (15-HOO-AA-PE), and trigger ferroptosis in human bronchial epithelial cells. Induction of ferroptosis by clinical P. aeruginosa isolates from patients with persistent lower respiratory tract infections was dependent on the level and enzymatic activity of pLoxA. Redox phospholipidomics revealed elevated levels of oxidized AA-PE in airway tissues from patients with cystic fibrosis (CF) but not with emphysema or CF without P. aeruginosa. We believe that the evolutionarily conserved mechanism of pLoxA-driven ferroptosis may represent a potential therapeutic target against P. aeruginosa-associated diseases such as CF and persistent lower respiratory tract infections.

Omega-3 and omega-6 fatty acid differentially impact cardiolipin remodeling in activated macrophage.

BACKGROUND: The macrophage plays an important role in innate immunity to induce immune responses. Lipid replacement therapy has been shown to change the lipid compositions of mitochondria and potentially becomes an alternative to reduce the inflammatory response. METHODS: We examined the effects of omega-6 arachidonic acid (AA), omega-3 eicosapentaenoic acid (EPA), and omega-3 docosahexaenoic acid (DHA) supplementation on the activated the macrophage cell line RAW264.7 via KdO2-lipid A (KLA). The mitochondrial cardiolipin (CL) and monolysocardiolipin (MLCL) were analyzed by LC-MS. RESULTS: After macrophage activation by KLA, CL shifted to saturated species, but did not affect the quantity of CL. Inhibition of delta 6 desaturase also resulted in the same trend of CL species shift. We further examined the changes in CL and MLCL species induced by polyunsaturated fatty acid supplementation during inflammation. After supplementation of AA, EPA and DHA, the MLCL/CL ratio increased significantly in all treatments. The percentages of the long-chain species highly elevated and those of short-chain species reduced in both CL and MLCL. CONCLUSIONS: Comparisons of AA, EPA and DHA supplementation revealed that the 20-carbon EPA (20:5) and AA (20:4) triggered higher incorporation and CL remodeling efficiency than 22-carbon DHA (22:6). EPA supplementation not only efficiently extended the chain length of CL but also increased the unsaturation of CL.

Guanine nucleotide induced conformational change of Cdc42 revealed by hydrogen/deuterium exchange mass spectrometry.

Cdc42 regulates pathways related to cell division. Dysregulation of Cdc42 can lead to cancer, cardiovascular diseases and neurodegenerative diseases. GTP induced activation mechanism plays an important role in the activity and biological functions of Cdc42. P-loop, Switch I and Switch II are critical regions modulating the enzymatic activity of Cdc42. We applied amide hydrogen/deuterium exchange coupled with liquid chromatography mass spectrometry (HDXMS) to investigate the dynamic changes of apo-Cdc42 after GDP, GTP and GMP-PCP binding. The natural substrate GTP induced significant decreases of deuteration in P-loop and Switch II, moderate changes of deuteration in Switch I and significant changes of deuteration in the α7 helix, a region far away from the active site. GTP binding induced similar effects on H/D exchange to its non-hydrolysable analog, GMP-PCP. HDXMS results indicate that GTP binding blocked the solvent accessibility in the active site leading to the decrease of H/D exchange rate surrounding the active site, and further triggered a conformational change resulting in the drastic decrease of H/D exchange rate at the remote α7 helix. Comparing the deuteration levels in three activation states of apo-Cdc42, Cdc42-GDP and Cdc42-GMP-PCP, the apo-Cdc42 has the most flexible structure, which can be stabilized by guanine nucleotide binding. The rates of H/D exchange of Cdc42-GDP are between the GMP-PCP-bound and the apo form, but more closely to the GMP-PCP-bound form. Our results show that the activation of Cdc42 is a process of conformational changes involved with P-loop, Switch II and α7 helix for structural stabilization.

Cell cycle arrest and cell survival induce reverse trends of cardiolipin remodeling.

Cell survival from the arrested state can be a cause of the cancer recurrence. Transition from the arrest state to the growth state is highly regulated by mitochondrial activity, which is related to the lipid compositions of the mitochondrial membrane. Cardiolipin is a critical phospholipid for the mitochondrial integrity and functions. We examined the changes of cardiolipin species by LC-MS in the transition between cell cycle arrest and cell reviving in HT1080 fibrosarcoma cells. We have identified 41 cardiolipin species by MS/MS and semi-quantitated them to analyze the detailed changes of cardiolipin species. The mass spectra of cardiolipin with the same carbon number form an envelope, and the C64, C66, C68, C70 C72 and C74 envelopes in HT1080 cells show a normal distribution in the full scan mass spectrum. The cardiolipin quantity in a cell decreases while entering the cell cycle arrest, but maintains at a similar level through cell survival. While cells awakening from the arrested state and preparing itself for replication, the groups with short acyl chains, such as C64, C66 and C68 show a decrease of cardiolipin percentage, but the groups with long acyl chains, such as C70 and C72 display an increase of cardiolipin percentage. Interestingly, the trends of the cardiolipin species changes during the arresting state are completely opposite to cell growing state. Our results indicate that the cardiolipin species shift from the short chain to long chain cardiolipin during the transition from cell cycle arrest to cell progression.