Summary Sterol Regulatory Element-Binding Proteins (SREBPs) activate genes involved in the synthesis and trafficking of cholesterol and other lipids, and therefore are critical for maintaining lipid homeostasis. Aberrant SREBP activity, however, can result in excess stored fat and contribute to obesity, fatty liver disease and insulin resistance, hallmarks of metabolic syndrome. Our studies identify a conserved regulatory circuit in which SREBP-1 controls production of the methyl donor S-adenosylmethionine (SAMe). Methylation is critical for synthesis of phosphatidylcholine (PC), a major membrane component, and we find that blocking SAMe or PC synthesis in C. elegans, mouse liver and human cells causes elevated SREBP-1-dependent transcription and lipid droplet accumulation. Distinct from negative regulation of SREBP-2 by cholesterol, our data suggest a mechanism where maturation of nuclear, transcriptionally active SREBP-1 is controlled by levels of PC. Thus, nutritional or genetic conditions limiting SAMe or PC production may activate SREBP-1, contributing to human metabolic disorders.

The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD + -dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in dietinduced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBPdependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis. Lipids and sterols play key roles in diverse biological processes in eukaryotes, such as membrane biosynthesis, intra-and extracellular signaling, and energy storage. In humans, aberrant lipid and cholesterol homeostasis has been linked to a number of diseases prevalent in the developed world, including metabolic syndrome-a constellation of conditions and diseases that includes obesity, insulin resistance, liver steatosis, and hypertension, as well as type 2 diabetes, cardiovascular disease, and cancers (Cornier et al. 2008). An improved understanding of the molecular mechanisms governing lipid/cholesterol homeostasis might lead to novel therapeutic strategies to ameliorate such diseases.Fasting (short-term food deprivation) produces a rapid metabolic shift from lipid/cholesterol synthesis and fat storage to mobilization of fat, and recent studies have suggested that fasting may improve conditions associated with metabolic syndrome (Varady and Hellerstein 2008;Fontana et al. 2010). There is thus keen interest in determining the mechanism of fasting-dependent regulation of lipid/cholesterol metabolism to facilitate the development

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Background Developmental processes, including neuronal differentiation, require precise regulation of transcription. The RE‐1 silencing transcription factor (Rest), is often called a “master neuronal regulator” due to its large number of neural‐specific targets. Rest recruits CoRest (Rcor) and Sin3 corepressor complexes to gene regulatory sequences. CoRest not only associates with Rest, but with other transcription regulators. In this study, we generated zebrafish rcor1 mutants using transcription activator‐like effector nucleases (TALENS), to study its requisite role in repression of Rest target genes as well as Rest‐independent Rcor1 developmental functions. Results While rcor1 mutants have a slight decrease in fitness, most survived and produced viable offspring. We examined expression levels of RE1‐containing genes in maternal zygotic rcor1 (MZrcor1) mutants and found that Rcor1 is generally not required for the repression of Rest target genes at early stages. However, MZrcor1 mutants undergo more rapid neurogenesis compared to controls. We found that at gastrula stages, Rcor1 acts as a repressor of her gene family, but at later stages, her6 decreased in the MZrcor1 mutant. Conclusions Based on these findings, the central role of CoRest1 in neurogenesis is likely due to a Rest‐independent role rather than as a Rest corepressor.

Background Despite some robust initial responses, anti-CD19 Chimeric Antigen Receptor (CAR) T-cell therapy can be associated with significant short-term (cytokine release syndrome) and long-term (B-cell deficiency) toxicities. CAR-engineered natural killer (NK) cells potentially provide a safer alternative while maintaining efficacy. Activated Natural Killer (aNKTM) cells are a clinical grade cell line derived from the NK-92R cell line that has demonstrated potent cytotoxicity towards a broad spectrum of malignant cell lines as well as safety and efficacy in phase I trials. Variants of the aNKcell line are currently in Phase I/II clinical trials: a CAR-expressing aNK cell line and the haNKTM cell line, which have been engineered to carry a high-affinity version of the CD16/FcγRIII receptor to allow for combination therapy with monoclonal antibodies. haNK cells have also been genetically modified to express an endoplasmic reticulum-retained version of IL-2 (ERIL-2), which provides IL-2 independence and limits IL-2 secretion to sub-physiological, safe levels. A key factor for the efficacy of cellular immunotherapies against a given target is biodistribution, which affects the local effector to target ratio. Inability to reach the tumor cells, either by lack of homing or by the accumulation of extracellular matrix (ECM) surrounding a tumor, can be responsible for the clinical failure of even the most effective CAR. The chemokines CCL19 and CCL21 drive recruitment of CCR7-expressing immune cells to secondary lymphoid organs. Engineering aNK cells to express the CCR7 receptor is likely to improve their efficacy by increasing their targeted migration to lymphoma tumor sites. Methods and Results Clinical grade aNK cells were electroporated with a non-viral vector containing the CCR7 receptor, an anti-CD19 CAR, and a high affinity CD16 receptor. To assay the migration of these engineered cell lines, a modified Boyden Chamber assay was performed using Matrigel coated Transwells. K562 cells or modified K562 cells engineered to express CCL19 (K-19) were placed in the destination chamber and CFSE-stained effector cells were placed in the top well. After 24 hours, cells in the bottom well were analyzed by flow cytometry to measure the number of effectors which had migrated through the Matrigel (Fig 1a). The excellent activity of the CAR in stably transfected cells was confirmed against SUP-B15 cells (aNK-resistant), while the ADCC activity was tested against a SUP-B15 variant expressing CD20, but engineered to lack the CD19 antigen (Sup-B15 CD19-, CD20+). Migration towards human lymph node chemokine CCL19 was also tested in vivo in NSG mice with bilateral subcutaneous tumors - with parental K562 in one flank and K-19 tumors on the contralateral flank. CFSE-stained effector cells were delivered via tail vein injection once average tumor size reached 100mm3 and following randomization. Tumors were then harvested at multiple time points, dissociated, and the number of infiltrating effectors in each tumor compared by flow cytometric analysis. (Fig 1b). In testing, monoclonal cell lines expressing all components of the polycistronic system displayed preferential migration towards CCR7 chemokines both in vitro and in vivo, as well as robust cytotoxicity vs. K562 (92.4% +/- 2.4% at 5:1 E:T), Sup-B15(97% +/- 0.6% at 5:1 E:T), and Sup-B15(CD19-, CD20+) when pre-incubated with Rituximab(83.2% +/- 2.8% at 5:1 E:T) but not with control antibody Trastuzumab (22.3% +/- 1.1% at 5:1 E:T) in standard cytotoxicity and ADCC assays. Conclusion We show here that the incorporation of a CCR7 receptor into an off the shelf CAR engineered NK cell line improves their homing towards lymph node chemokines both in vitro and in vivo. This improved homing should result in a greater ratio of effector to target in lymphoid tissue, and maximize the immunogenic cell death. Disclosures Schomer: NantKwest, Inc.: Employment, Equity Ownership. Boissel:NantKwest, Inc.: Employment, Equity Ownership. Jiang:NantKwest, Inc.: Employment, Equity Ownership. Klingemann:NantKwest, INc.: Employment, Equity Ownership, Patents & Royalties. Lee:NantKwest, Inc.: Employment, Equity Ownership. Soon-Shiong:NantKwest, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.