Simultaneous inhibition of Sirtuin 3 and cholesterol homeostasis targets acute myeloid leukemia stem cells by perturbing fatty acid ß-oxidation and inducing lipotoxicity.

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapeutic regimens to fully eradicate disease-initiating leukemia stem cells (LSC). Previous studies have demonstrated that oxidative phosphorylation (OXPHOS) is an essential process that is targetable in LSC. Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation, has been shown to regulate OXPHOS in cancer models; however, it has not yet been studied in the context of LSC. Thus, we sought to identify if SIRT3 is important for LSC function. Using RNAi and a SIRT3 inhibitor (YC8-02), we demonstrate that SIRT3 is a critical target for the survival of primary human LSC but is not essential for normal human hematopoietic stem and progenitor cell function. In order to elucidate the molecular mechanisms by which SIRT3 is essential in LSC we combined transcriptomic, proteomic, and lipidomic approaches, showing that SIRT3 is important for LSC function through the regulation of fatty acid oxidation (FAO) which is required to support OXPHOS and ATP production in human LSC. Further, we discovered two approaches to further sensitize LSC to SIRT3 inhibition. First, we found that LSC tolerate the toxic effects of fatty acid accumulation induced by SIRT3 inhibition by upregulating cholesterol esterification. Disruption of cholesterol homeostasis sensitizes LSC to YC8-02 and potentiates LSC death. Second, SIRT3 inhibition sensitizes LSC to the BCL-2 inhibitor venetoclax. Together, these findings establish SIRT3 as a regulator of lipid metabolism and potential therapeutic target in primitive AML cells.

S-acylation of ATGL is required for lipid droplet homoeostasis in hepatocytes.

Lipid droplets (LDs) are organelles specialized in the storage of neutral lipids, cholesterol esters and triglycerides, thereby protecting cells from the toxicity of excess lipids while allowing for the mobilization of lipids in times of nutrient deprivation. Defects in LD function are associated with many diseases. S-acylation mediated by zDHHC acyltransferases modifies thousands of proteins, yet the physiological impact of this post-translational modification on individual proteins is poorly understood. Here, we show that zDHHC11 regulates LD catabolism by modifying adipose triacylglyceride lipase (ATGL), the rate-limiting enzyme of lipolysis, both in hepatocyte cultures and in mice. zDHHC11 S-acylates ATGL at cysteine 15. Preventing the S-acylation of ATGL renders it catalytically inactive despite proper localization. Overexpression of zDHHC11 reduces LD size, whereas its elimination enlarges LDs. Mutating ATGL cysteine 15 phenocopies zDHHC11 loss, causing LD accumulation, defective lipolysis and lipophagy. Our results reveal S-acylation as a mode of regulation of ATGL function and LD homoeostasis. Modulating this pathway may offer therapeutic potential for treating diseases linked to defective lipolysis, such as fatty liver disease.

Legionella pneumophila Infection of Human Macrophages Retains Golgi Structure but Reduces O-Glycans.

Legionella pneumophila is an accidental pathogen that replicates intracellularly within the Legionella-containing vacuole (LCV) in macrophages. Within an hour of infection, L. pneumophila secretes effectors to manipulate Rab1 and intercept ER-derived vesicles to the LCV. The downstream consequences of interrupted ER trafficking on the Golgi of macrophages are not clear. We examined the Golgi structure and function in L. pneumophila-infected human U937 macrophages. Intriguingly, the size of the Golgi in infected macrophages remained similar to uninfected macrophages. Furthermore, TEM analysis also did not reveal any significant changes in the ultrastructure of the Golgi in L. pneumophila-infected cells. Drug-induced Golgi disruption impacted bacterial replication in human macrophages, suggesting that an intact organelle is important for bacteria growth. To probe for Golgi functionality after L. pneumophila infection, we assayed glycosylation levels using fluorescent lectins. Golgi O-glycosylation levels, visualized by the fluorescent cis-Golgi lectin, Helix pomatia agglutinin (HPA), significantly decreased over time as infection progressed, compared to control cells. N-glycosylation levels in the Golgi, as measured by L-PHA lectin staining, were not impacted by L. pneumophila infection. To understand the mechanism of reduced O-glycans in the Golgi we monitored UDP-GalNAc transporter levels in infected macrophages. The solute carrier family 35 membrane A2 (SLC35A2) protein levels were significantly reduced in L. pneumophila-infected U937 and HeLa cells and L. pneumophila growth in human macrophages benefitted from GalNAc supplementation. The pronounced reduction in Golgi HPA levels was dependent on the translocation apparatus DotA expression in bacteria and occurred in a ubiquitin-independent manner. Thus, L. pneumophila infection of human macrophages maintains and requires an intact host Golgi ultrastructure despite known interference of ER-Golgi trafficking. Finally, L. pneumophila infection blocks the formation of O-linked glycans and reduces SLC35A2 protein levels in infected human macrophages.