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PDF file, 48KB, Induction of apoptosis in HOP62 and HepG2 cells after ACLY silencing.

ATP citrate lyase (ACLY) is a cytosolic enzyme that catalyzes generation of acetyl-CoA, which is a vital building block for fatty acid, cholesterol, and isoprenoid biosynthesis. ACLY is upregulated in several types of cancer, and its inhibition induces proliferation arrest in certain cancer cells. As ACLY is involved in several pathways, its downregulation may affect multiple processes. Here, we have shown that short hairpin RNA-mediated ACLY silencing in cell lines derived from different types of cancers induces proliferation, cell-cycle arrest, and apoptosis. However, this antiproliferative effect of ACLY knockdown was observed only when cells were cultivated under lipid-reduced growth conditions. Proliferation arrest induced by ACLY silencing was partially rescued by supplementing the media with fatty acids and/or cholesterol. This indicates that the ACLY knockdown-mediated growth arrest might be the result of either fatty acid or cholesterol starvation or both. In the absence of ACLY, the cancer cells displayed elevated expression of sterol regulatory element binding protein–regulated downstream genes involved in de novo fatty acid and cholesterol biosynthesis. Furthermore, ACLY suppression resulted in elevated expression of acyl-CoA synthetase short-chain family member 2 (ACSS2), an enzyme that also produces acetyl-CoA using acetate as a substrate. Acetate supplementation partially rescued the cancer cells from ACLY suppression–induced proliferation arrest. We also observed that the absence of ACLY enhanced ACSS2-dependent lipid synthesis. These findings provide new insights into the role of ACLY in cancer cell growth and give critical information about the effects of ACLY silencing on different pathways. This information is crucial in understanding the possible application of ACLY inhibition in cancer therapeutics. Mol Cancer Ther; 11(9); 1925–35. ©2012 AACR.

PDF file, 56KB, Reduction in protein expression of ACLY by IPTG-inducible shRNA targeting ACLY in HOP62 and HepG2 cells.

PDF file, 137KB, Growth curves for PC3M cells with/without simvastatin and/or oleic acid supplementation.

PDF file, 27KB, Reduction in mRNA expression of ACLY by IPTG-inducible shRNA targeting ACLY in HOP62 and HepG2 cells.

PDF file, 59KB, Growth curves for ACLY-knockdown population of PC3M cells with/without Geranylgeranyl pyrophosphate (GGPP) supplementation.

PDF file, 85KB.

Supplementary Figure 5 from De novo Lipogenesis Protects Cancer Cells from Free Radicals and Chemotherapeutics by Promoting Membrane Lipid Saturation

Supplementary Methods from De novo Lipogenesis Protects Cancer Cells from Free Radicals and Chemotherapeutics by Promoting Membrane Lipid Saturation

Supplementary Figure 6 from De novo Lipogenesis Protects Cancer Cells from Free Radicals and Chemotherapeutics by Promoting Membrane Lipid Saturation

Supplementary Figure 1 from De novo Lipogenesis Protects Cancer Cells from Free Radicals and Chemotherapeutics by Promoting Membrane Lipid Saturation

Activation of de novo lipogenesis in cancer cells is increasingly recognized as a hallmark of aggressive cancers and has been implicated in the production of membranes for rapid cell proliferation. In the current report, we provide evidence that this activation has a more profound role. Using a mass spectrometry–based phospholipid analysis approach, we show that clinical tumor tissues that display the lipogenic phenotype show an increase in the degree of lipid saturation compared with nonlipogenic tumors. Reversal of the lipogenic switch in cancer cells by treatment with the lipogenesis inhibitor soraphen A or by targeting lipogenic enzymes with small interfering RNA leads to a marked decrease in saturated and mono-unsaturated phospholipid species and increases the relative degree of polyunsaturation. Because polyunsaturated acyl chains are more susceptible to peroxidation, inhibition of lipogenesis increases the levels of peroxidation end products and renders cells more susceptible to oxidative stress–induced cell death. As saturated lipids pack more densely, modulation of lipogenesis also alters lateral and transversal membrane dynamics as revealed by diffusion of membrane-targeted green fluorescent protein and by the uptake and response to doxorubicin. These data show that shifting lipid acquisition from lipid uptake toward de novo lipogenesis dramatically changes membrane properties and protects cells from both endogenous and exogenous insults. These findings provide important new insights into the role of de novo lipogenesis in cancer cells, and they provide a rationale for the use of lipogenesis inhibitors as antineoplastic agents and as chemotherapeutic sensitizers. Cancer Res; 70(20); 8117–26. ©2010 AACR.

Supplementary Figure 2 from De novo Lipogenesis Protects Cancer Cells from Free Radicals and Chemotherapeutics by Promoting Membrane Lipid Saturation

Supplementary Figure Legends 1-6 from De novo Lipogenesis Protects Cancer Cells from Free Radicals and Chemotherapeutics by Promoting Membrane Lipid Saturation

Supplementary Figure 3 from De novo Lipogenesis Protects Cancer Cells from Free Radicals and Chemotherapeutics by Promoting Membrane Lipid Saturation

We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC50 = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement.

The guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the αC-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII.

The aberrant blood lipoprotein levels in cancer patients are reported to be associated with cancer risk and mortality incidents however, there are several discrepancies in the previous reports. Hence the clinical usefulness of plasma/serum levels in risk stratification of a variety of cancers remains elusive. The present review highlights and compiles findings from different research groups regarding association of plasma lipoprotein levels with the risk of developing various types of cancer. We will discuss some prospective underlying mechanisms for this reported association. In addition to that the potential roles of plasma lipids in promoting carcinogenesis will be conferred.

ATP-citrate lyase (ACLY) is a cytosolic enzyme that catalyzes generation of acetyl-CoA from citrate. Acetyl-CoA is a vital building block for the endogenous biosynthesis of fatty acids and cholesterol and is involved in isoprenoid-based protein modifications. Acetyl-CoA is also required for acetylation reactions that modify proteins such as histone acetylation. In the present review some of the known features of ACLY such as tissue distribution, subcellular localization, enzymatic properties, gene regulation and associated physiological conditions are highlighted.

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel green fluorescent protein ZsGreen into the first exon of the c-Kit gene, creating a null allele called WZsGreen. In the gastrointestinal tract of heterozygous WZsGreen/+ mice, tiny ZsGreen fluorescent dots were observed in all KIT-expressing ICC populations, with exception of ICC at the deep muscular plexus in small intestine. During development of the gastrointestinal tract, ZsGreen expression followed KIT expression in a spatiotemporal way. Stellate and basket KIT-expressing cells in the molecular layer of the cerebellum also exhibited ZsGreen dots, whereas no ZsGreen was detected in skin, testis, and bone marrow. ZsGreen dot-containing intestinal cells could be isolated from jejunum and maintained alive in culture for at least 3 days. ZsGreen is a suitable alternative to EGFP in transgenic animals. The novel WZsGreen/+ model reported here appears to be a promising tool for live studies of KIT-expressing cells in the gastrointestinal tract and cerebellum and for the further analysis of pacemaker mechanisms.

Background Cancer cells consume large amount of glutamine for growth and proliferation. In normal mitochondria, glutamine is converted to a-ketoglutarate, which is further oxidized through the TCA cycle [1]. Recent studies find that under certain extreme conditions, such as mitochondria dysfunction and hypoxia, cells use glutamine through reductive carboxylation (RC) to generate acetyl-CoA for lipogenesis. During tumor development, the acquisition of anchorage independence enables cancer cells to survive without their natural extracellular matrix, but this comes at the price of increased oxidative stress [2]. Here we examined reprogramming of glutamine metabolism during adaptation to growth of anchorage independent tumor spheroids, including reprogramming of pathways that counteract production of reactive oxygen species (ROS).

Increased lipogenesis is a hallmark of a wide variety of cancers and is under intense investigation as potential antineoplastic target. Although brisk lipogenesis is observed in the presence of exogenous lipids, evidence is mounting that these lipids may adversely affect the efficacy of inhibitors of lipogenic pathways. Therefore, to fully exploit the therapeutic potential of lipid synthesis inhibitors, a better understanding of the interrelationship between de novo lipid synthesis and exogenous lipids and their respective role in cancer cell proliferation and therapeutic response to lipogenesis inhibitors is of critical importance. Here, we show that the proliferation of various cancer cell lines (PC3M, HepG2, HOP62 and T24) is attenuated when cultured in lipid-reduced conditions in a cell line-dependent manner, with PC3M being the least affected. Interestingly, all cell lines--lipogenic (PC3M, HepG2, HOP62) as well as non-lipogenic (T24)--raised their lipogenic activity in these conditions, albeit to a different degree. Cells that attained the highest lipogenic activity under these conditions were best able to cope with lipid reduction in term of proliferative capacity. Supplementation of the medium with very low density lipoproteins, free fatty acids and cholesterol reversed this activation, indicating that the mere lack of lipids is sufficient to activate de novo lipogenesis in cancer cells. Consequently, cancer cells grown in lipid-reduced conditions became more dependent on de novo lipid synthesis pathways and were more sensitive to inhibitors of lipogenic pathways, like Soraphen A and Simvastatin. Collectively, these data indicate that limitation of access to exogenous lipids, as may occur in intact tumors, activates de novo lipogenesis is cancer cells, helps them to thrive under these conditions and makes them more vulnerable to lipogenesis inhibitors. These observations have important implications for the design of new antineoplastic strategies targeting the cancer cell's lipid metabolism. ispartof: PLoS One vol:9 issue:9 ispartof: location:United States status: published

Based upon the immunohistochemical demonstration of neuropeptides in the skeleton, including vasoactive intestinal peptide (VIP), we have addressed the question of whether neuropeptides may exert regulatory roles on bone tissue metabolism or not. In the present communication, we have investigated if VIP can affect anabolic processes in osteoblasts. Osteoblasts were isolated from neonatal mouse calvariae by time sequential enzyme-digestion and subsequently cultured for 2-28 days in the presence of VIP and other modulators of cyclic AMP formation. VIP (10(-6) M) stimulated ALP activity and calcium content. The cyclic AMP phosphodiesterase inhibitors ZK 62 711 (10(-4) M) and isobutyl-methylxanthine (10(-4) M) stimulated ALP activity and synergistically potentiated the effect of VIP. Neither VIP, nor isobutyl-methylxanthine or ZK 62 711, in the absence or presence of VIP, affected cell number. The stimulatory effect of VIP on ALP activity, in the presence of ZK 62 711, was dependent on time and concentration of VIP. The stimulatory effects of VIP and ZK 62 711 on ALP activity was seen also in cells stained for ALP. VIP (10(-6) M), in the presence of ZK 62 711 (10(-6) M), significantly enhanced mRNA for tissue non-specific ALP. VIP (10(-6) M), in the presence of ZK 62 711, stimulated cyclic AMP production. Forskolin and choleratoxin stimulated ALP activity and cyclic AMP formation in a concentration-dependent manner, without affecting cell number. VIP (10(-6) M) and ZK 62 711 (10(-5) M) stimulated, and their combination synergistically enhanced, calcium content in bone noduli. These data show that VIP, without affecting cell proliferation, can stimulate osteoblastic ALP biosynthesis and bone noduli formation by a mechanism mediated by cyclic AMP. Our observations suggest a possibility that anabolic processes in bone are under neurohormonal control.

One of the most important metabolic hallmarks of cancer cells is enhanced lipogenesis. Depending on the tumor type, tumor cells synthesize up to 95% of saturated and mono-unsaturated fatty acids (FA) de novo in spite of sufficient dietary lipid supply. This lipogenic conversion starts early when cells become cancerous and further expands as the tumor cells become more malignant. It is suggested that activation of FA synthesis is required for carcinogenesis and for tumor cell survival. These observations suggest that the enzymes involved in FA synthesis would be rational therapeutic targets for cancer treatment. However, several recent reports have shown that the anti-tumor effects, following inhibition of endogenous FA synthesis in cancer cell lines may be obviated by adding exogenous FAs. Additionally, high intake of dietary fat is reported to be a potential risk factor for development and poor prognosis for certain cancers. Recently it was reported that breast and liposarcoma tumors are equipped for both de novo fatty acid synthesis pathway as well as LPL-mediated extracellular lipolysis. These observations indicate that lipolytically acquired FAs may provide an additional source of FAs for cancer. This review focuses on our current understanding of lipogenic and lipolytic pathways in cancer cell progression.

ATP-citrate lyase (ACLY) is a cytosolic enzyme that catalyzes the generation of acetyl CoA from citrate. Acetyl CoA is a vital building block for the endogenous biosynthesis of fatty acids and cholesterol and is involved in isoprenoid-based protein modifications. Acetyl CoA is also required for acetylation reactions that modify proteins, such as histone acetylation. ACLY is upregulated or activated in several types of cancers, and its inhibition is known to induce proliferation arrest in cancer cells both in vitro and in vivo. The present review highlights current knowledge about the role of ACLY in cancer cells, with special reference to the different pathways that are linked by ACLY. Cancer Res; 72(15); 3709–14. ©2012 AACR.

ATP citrate lyase (ACLY) is a cytosolic enzyme that catalyzes generation of acetyl-CoA, which is a vital building block for fatty acid, cholesterol, and isoprenoid biosynthesis. ACLY is upregulated in several types of cancer, and its inhibition induces proliferation arrest in certain cancer cells. As ACLY is involved in several pathways, its downregulation may affect multiple processes. Here, we have shown that short hairpin RNA-mediated ACLY silencing in cell lines derived from different types of cancers induces proliferation, cell-cycle arrest, and apoptosis. However, this antiproliferative effect of ACLY knockdown was observed only when cells were cultivated under lipid-reduced growth conditions. Proliferation arrest induced by ACLY silencing was partially rescued by supplementing the media with fatty acids and/or cholesterol. This indicates that the ACLY knockdown-mediated growth arrest might be the result of either fatty acid or cholesterol starvation or both. In the absence of ACLY, the cancer cells displayed elevated expression of sterol regulatory element binding protein–regulated downstream genes involved in de novo fatty acid and cholesterol biosynthesis. Furthermore, ACLY suppression resulted in elevated expression of acyl-CoA synthetase short-chain family member 2 (ACSS2), an enzyme that also produces acetyl-CoA using acetate as a substrate. Acetate supplementation partially rescued the cancer cells from ACLY suppression–induced proliferation arrest. We also observed that the absence of ACLY enhanced ACSS2-dependent lipid synthesis. These findings provide new insights into the role of ACLY in cancer cell growth and give critical information about the effects of ACLY silencing on different pathways. This information is crucial in understanding the possible application of ACLY inhibition in cancer therapeutics. Mol Cancer Ther; 11(9); 1925–35. ©2012 AACR.

Activation of de novo lipogenesis in cancer cells is increasingly recognized as a hallmark of aggressive cancers and has been implicated in the production of membranes for rapid cell proliferation. In the current report, we provide evidence that this activation has a more profound role. Using a mass spectrometry-based phospholipid analysis approach, we show that clinical tumor tissues that display the lipogenic phenotype show an increase in the degree of lipid saturation compared with nonlipogenic tumors. Reversal of the lipogenic switch in cancer cells by treatment with the lipogenesis inhibitor soraphen A or by targeting lipogenic enzymes with small interfering RNA leads to a marked decrease in saturated and mono-unsaturated phospholipid species and increases the relative degree of polyunsaturation. Because polyunsaturated acyl chains are more susceptible to peroxidation, inhibition of lipogenesis increases the levels of peroxidation end products and renders cells more susceptible to oxidative stress-induced cell death. As saturated lipids pack more densely, modulation of lipogenesis also alters lateral and transversal membrane dynamics as revealed by diffusion of membrane-targeted green fluorescent protein and by the uptake and response to doxorubicin. These data show that shifting lipid acquisition from lipid uptake toward de novo lipogenesis dramatically changes membrane properties and protects cells from both endogenous and exogenous insults. These findings provide important new insights into the role of de novo lipogenesis in cancer cells, and they provide a rationale for the use of lipogenesis inhibitors as antineoplastic agents and as chemotherapeutic sensitizers. ispartof: CANCER RESEARCH vol:70 issue:20 pages:8117-8126 ispartof: location:United States status: published

Interstitial cells of Cajal (ICC) are the so-called pacemaker cells of the gut. W-LacZ/Wv and Sl/Sld mice lack ICC surrounding the myenteric plexus (MP) in the jejunum. We compared the gene expression profile of wild type (WT) and W-LacZ/Wv and Sl/Sld mice using suppression subtractive hybridization (SSH), generating a cDNA library of 1303 clones from which 48 unique sequences were differentially expressed with Southern blot. Among them, we identified heme oxygenase2, TROY, and phospholamban in ICC using immunohistochemistry. Using RT-qPCR, c-Kit and two new transcripts Dithp and prenylcysteine oxidase1 were significantly lower expressed in Sl/Sld and W-LacZ/Wv versus WT. Prenylcysteine oxidase1 appeared cytotoxic for COS-7 cells and was highly expressed in liver while Dithp was mainly expressed in small intestine. The combination of SSH, Southern blot, RT-qPCR, and immunohistochemistry turned out to be a useful approach to identify rarely expressed genes and genes with small differences in expression. (c) 2006 Elsevier Inc. All rights reserved. ispartof: Biochemical and Biophysical Research Communications vol:346 issue:2 pages:491-500 ispartof: location:United States status: published

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. Our aim was to identify ICC-specific genes and their function in the mouse jejunum. Suppression subtractive hybridization using two independent ICC-deficient mouse models identified 56 genes putatively downregulated in the muscularis propria compared with wild-type littermates. Differential expression was confirmed by real-time quantitative PCR for the tyrosine kinase receptor KIT, the established marker for ICC, and for the Na+-K+-2Cl−cotransporter (NKCC1). Immunoreactivity for NKCC1 was detected in myenteric ICC but not in the ICC population located at the deep muscular plexus. NKCC1 was also expressed in enteric neurons and mucosal crypts. Bumetanide, an NKCC1 inhibitor, reversibly affected the shape, amplitude, and frequency of the slow waves. Similar alterations were observed in NKCC1 knockout mice. These data support the hypothesis that NKCC1 expressed in myenteric ICC is involved in the mechanism of slow waves in the murine jejunum.

The failure of many drugs in the clinic - drugs proven to be active in pre-clinical assays - has raised question marks on the predictive power of these models, often based on cell line data. The divergence of these cell lines from the original tumors, and their rapid growth as simple monocultures on 2D plastic were highlighted as potentially causing these issues. Implantation of the cell lines into mice adds complexity to these models, but the crosstalk with mouse stroma also leads to confounding results (eg, HGF, IL-6). PREDECT, a European consortium funded by the Innovative Medicines Initiative (IMI), has the goal to build and characterise more predictive Oncology model platforms for three pathologies (lung, breast and prostate). Signalling pathways and heterogeneity of these model platforms are compared to each other as well as to primary human tissue using a central TMA-based platform. For prostate cancer, a 3D tumor growth assay system was established recently using RFP/FLuc-labelled LNCaP, PC346C, or VCaP human prostate cancer cells grown in a matrix of Matrigel, collagen, or a mix of the two, in the presence or absence of GFP/RLuc labelled human stromal cells (WPMY, or an immortalised cancer-associated fibroblast cell line, CAF). Cell densities, ratios, and matrix concentrations/volumes were optimised for cells cultured for 3 weeks. In an approach to improve tumor-stroma crosstalk in vivo, LNCaP cells pre-grown in 3D cultures with or without human stromal cells were implanted orthotopically into SCID mice. Growth of the tumor and stromal cells was monitored by in vivo bioluminescence, and the effect of the co-implantation on the primary tumor and metastasis was analysed via ex-vivo luciferase assays and IHC. Whereas LNCaP and PC346C cells readily formed spheroids in 3D culture, VCaPs remained as single cells. Growth of VCaPs was facilitated by pre-forming spheroids in vitro, followed by matrix embedding. Addition of stromal cells stimulated growth of not only the tumor, but also the stromal cells in some conditions, as monitored using RFP and GFP live cell imaging. 3D, as well as stromal cell-mediated treatment resistance was observed. Using the fluorescent dyes Hoechst, EdU, and NucView, the proliferative status of individual cells within spheroids could be analysed in situ. Finally, paraffin-embedding of the 3D cultures allowed for TMA analysis and comparison to other models and patient samples.Results will also be shown on the orthotopic growth of LNCaP tumors and the effect of stromal cell co-implantation. Introducing complexity to cell culture may help to generate more realistic, and thus also more predictive models - in a first step using even standard cell lines. Citation Format: Suzana Vidic, Norbert Esser, Ronald de Hoogt, Ingrid Verberne, Ira Kogan-Sakin, Yan Stein, Varda Rotter, Michael Barbier, Yolanda Chong, Sabine De Breucker, Karine Smans, Malin Akerfelt, Matthias Nees, Peter King, Ian Hickson, Wytske van Weerden, Ralph Graeser. Complex in vitro and in vivo prostate cancer models for the PREDECT consortium. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2023. doi:10.1158/1538-7445.AM2014-2023

Fatty Acid Synthase (FASN) is a multi-domain protein that carries out de novo fatty acid (palmitate) synthesis from acetate and malonate in mammalian cells. FASN is up-regulated in cancer cells, providing fatty acid building blocks for rapid cell growth and cell division. Increased FASN expression is correlated with disease progression and poor prognosis in many cancers including prostate, breast, ovary, colon, and lung. FASN has been demonstrated to play an important role in carcinogenesis by protecting cells from apoptosis. Herein we report a new series of potent, selective and orally bioavailable FASN inhibitors. Recent publications disclose several FASN inhibitor chemotypes that share a common pharmacophore, wherein an aromatic group and an acylated cyclic amine are attached to a central scaffold. We postulated that a spirocyclic imidazolinone core would be an acceptable and drug-like scaffold, inspired by the precedent of irbesartan, an approved antihypertensive drug in which a spirocyclopentyl-imidazolinone core replaces the substituted imidazole ring of losartan, an older approved agent from the same drug class. This hypothesis led to a new spirocyclic imidazolinone based FASN inhibitors. Extensive SAR efforts resulted in FASN inhibitors with potent enzyme and cell activity, selectivity, and oral bioavailability exemplified by JNJ-54302833. JNJ-54302833 is a potent inhibitor of human FASN (IC50 = 28 nM) and also potently inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced medium. This cellular activity can be rescued by addition of palmitate, demonstrating on-target effects. JNJ-54302833 is also potent in many other cells, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposures. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with JNJ-54302833 resulted in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate. This novel series potently inhibits the FASN KR domain (IC50 = 54 nM for JNJ-54302833); specific binding to KR was confirmed by crystal structures.In summary, we have designed and discovered a new series of FASN inhibitors that are potent both in enzyme and in cell proliferation assays, are highly bioavailable, and bind to KR domain. Additionally, palmitate rescue of lipid-reduced cellular activity suggests selectivity and pharmacodynamics studies confirm target engagement. Citation Format: Tianbao Lu, Richard Alexander, Gilles Bignan, James Bischoff, Peter Connolly, Max Cummings, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Bruce Grasberger, Boudewijn Janssens, Donald Ludovici, Lieven Meerpoel, Christophe Meyer, Michael Parker, Danielle Peeters, Carsten Schubert, Karine Smans, Luc Van Nuffel, Peter Vermeulen. Design and synthesis of a series highly potent and bioavailable FASN KR domain inhibitors for cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4747. doi:10.1158/1538-7445.AM2014-4747

Fatty Acid Synthase (FASN) catalyzes the final step in palmitate (PA) synthesis, using acetyl-CoA, malonyl-CoA and NADPH. Most normal tissues express low levels of FASN and rely on uptake of fatty acids (FA) from the diet. It has been proposed that FASN overexpressing tumors including prostate and breast tumors depend on de novo FA synthesis, which is advantageous to tumors by providing lipids for membrane synthesis and increased growth factor receptor expression/signaling in lipid rafts. Overexpression of FASN leads to a higher amount of saturated lipids in membranes which can lead to resistance to cytotoxic chemotherapy. Lastly, NADPH consumption during PA synthesis keeps the redox balance in check. All of the above imply that FASN represents a potential therapeutic target for the treatment of multiple cancer types. At this AACR we report two novel chemical series (posters Connolly et al., Lu et al.). JNJ-53793220 and JNJ-54302833 potently and selectively (< 100nM) inhibit the FASN enzyme and proliferative activity in cells in lipid reduced medium (LRM). Using these, we investigated the underlying hypothesis that tumor cells do not utilize circulating FA and are dependent on de novo synthesis of FA. In a lipid reduced environment many cell lines, particularly of prostate, breast, ovarian or heme origin, proved to be sensitive to JNJ-53793220. However co-administration of PA dose-dependently reversed the anti-proliferative effects. Also androgen driven proliferation of LNCaP cells was potently blocked by JNJ-53793220 (EC50 30 nM), and decreased PSA levels. Both effects were partially rescued by the addition of PA. While the rescue of tumor cells by PA confirmed the on-target activity of the compounds, it also suggested that cancer cells are capable of using external FA. To extend these findings, we screened more than 400 cell lines in lipid containing medium (LCM) for their sensitivity to JNJ-53793220. In LCM sensitivity to FASN inhibition was lower than in LRM conditions. In most, but not all, cases the addition of PA reverted the antiproliferative effects of JNJ-53793220, although target engagement was not reduced in LCM conditions. The EC50 of 14C-acetate incorporation in lipids of ∼30 nM corresponded well with enzymatic and anti-proliferative effects in LRM (27 and 13 nM respectively). Furthermore, growth of pre-established LNCaP xenografts in vivo was not blocked significantly by JNJ-53793220, even though malonyl-CoA levels were increased as expected upon FASN inhibition in the tumor. While circulating lipids in vivo are likely culprits for the lack of efficacy, other factors may play a role as well. In a 3D culture model (poster Vidic et al.) the growth of LNCaP and PC346c spheroids was blocked by JNJ-54302833 (1µM), but growth of PC346c spheroids co-cultured with cancer associated fibroblasts was not inhibited. Taken together our data suggest that the outcome of FASN inhibition is influenced by the tumor environment. Citation Format: Karine A. Smans, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Ralph Graeser, Boudewijn Janssen, Lieven Meerpoel, Danielle Peeters, Geert Van Hecke, Luc Van Nuffel, Yolanda Chong, Peter Vermeulen, Gilles Bignan, James Bischoff, Peter Connolly, Bruce Grasberger, Tianbao Lu, Donald Ludovici, Carsten Schubert, Michael Parker, Christophe Meyer, Suzana Vidic. Sensitivity of cell lines to Fatty Acid Synthase inhibitors depends on the lipid content in the cellular environment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 801. doi:10.1158/1538-7445.AM2014-801

De novo synthesis of fatty acids in mammalian cells is catalyzed by Fatty Acid Synthase (FASN), a complex dimeric protein with seven catalytic domains that processes acetyl-CoA and malonyl-CoA into palmitic acid. Palmitate and other fatty acids are essential for normal physiological processes like energy storage and production and are key intermediates in the biosynthesis of hormones and other essential biomolecules. The role of FASN in cancer has been well documented over the past decade. While FASN is expressed at low levels in most normal tissue (except liver and adipose tissue), it is highly expressed in many tumors including prostate, breast, and colon. FASN overexpression and activity is correlated with poor prognosis and disease progression. Numerous publications describing anti-cancer effects of small molecule FASN inhibitors have appeared in the scientific and patent literature. Recently, several groups reported anticancer and antiviral activities of N-benzoyl arylpiperidine FASN inhibitors. In this work we describe the discovery of novel N-benzoyl arylpiperidine and arylazetidine compounds that potently inhibit FASN enzymatic activity in vitro and antiproliferative activity in FASN-sensitive cell lines. We conducted focused structure-activity (SAR) studies exploring the benzoyl group and the aryl substituent on the piperidine or azetidine heterocycle to optimize potency, improve properties and oral bioavailability, and achieve in vivo FASN-driven pharmacodynamic (PD) activity. X-ray crystal structures of these molecules confirm a unique binding mode within the KR subdomain of FASN. Using structural information and molecular modeling, we were able to rationalize SAR trends and design FASN inhibitors with excellent in vitro potency and cellular activity. These efforts resulted in the discovery of compounds exemplified by JNJ-54380482, a potent FASN inhibitor (IC50 = 26 nM) that inhibits proliferation of A2780 ovarian cells in lipid-reduced medium (LRM, IC50 = 8.9 nM). Engagement of FASN in cellular assays is confirmed by rescue of proliferation upon addition of palmitate. JNJ-54380482 is orally bioavailable in mice (F% = 51) and exhibits excellent plasma exposure when dosed at 10 mg/kg (Cmax = 2.6 µM, T1/2 = 5 h). In a PD study using H460 lung xenograft-bearing mice, oral treatment with 30 mg/kg of JNJ-54380482 results in 36-fold higher tumor levels of malonyl-CoA vs vehicle. In summary, we synthesized several hundred compounds having the N-benzoyl arylpiperidine and arylazetidine chemotypes. Utilizing structural information from X-ray co-crystals to understand SAR and guide the design of more potent molecules, we identified >100 analogs with highly potent FASN enzymatic (IC50 ≤ 50 nM) and antiproliferative activities in A2780 cells in LRM (IC50 ≤ 100 nM). Key examples from these series have good in vitro properties, are orally bioavailable, and demonstrate FASN target engagement in a mouse PD model. Citation Format: Peter J. Connolly, Gilles Bignan, James Bischoff, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Bruce Grasberger, Boudewijn Janssens, Tianbao Lu, Donald Ludovici, Lieven Meerpoel, Christophe Meyer, Michael Parker, Danielle Peeters, Carsten Schubert, Karine Smans, Luc Van Nuffel, Peter Vermeulen. Structure-activity relationships of novel N-benzoyl arylpiperidine and arylazetidine FASN inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2535. doi:10.1158/1538-7445.AM2014-2535