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• W2414985384 abstract "•Fasnall is a thiophenopyrimidine targeting fatty acid synthase•Fasnall has anti-proliferative activity and induces apoptosis in breast cancer cells•Fasnall promotes fatty acid uptake, ceramide and diacylglycerol accumulation•Fasnall is well tolerated and shows efficacy against HER2+ breast tumors in vivo Many tumors are dependent on de novo fatty acid synthesis to maintain cell growth. Fatty acid synthase (FASN) catalyzes the final synthetic step of this pathway, and its upregulation is correlated with tumor aggressiveness. The consequences and adaptive responses of acute or chronic inhibition of essential enzymes such as FASN are not fully understood. Herein we identify Fasnall, a thiophenopyrimidine selectively targeting FASN through its co-factor binding sites. Global lipidomics studies with Fasnall showed profound changes in cellular lipid profiles, sharply increasing ceramides, diacylglycerols, and unsaturated fatty acids as well as increasing exogenous palmitate uptake that is deviated more into neutral lipid formation rather than phospholipids. We also showed that the increase in ceramide levels contributes to some extent in the mediation of apoptosis. Consistent with this mechanism of action, Fasnall showed potent anti-tumor activity in the MMTV-Neu model of HER2+ breast cancer, particularly when combined with carboplatin. Many tumors are dependent on de novo fatty acid synthesis to maintain cell growth. Fatty acid synthase (FASN) catalyzes the final synthetic step of this pathway, and its upregulation is correlated with tumor aggressiveness. The consequences and adaptive responses of acute or chronic inhibition of essential enzymes such as FASN are not fully understood. Herein we identify Fasnall, a thiophenopyrimidine selectively targeting FASN through its co-factor binding sites. Global lipidomics studies with Fasnall showed profound changes in cellular lipid profiles, sharply increasing ceramides, diacylglycerols, and unsaturated fatty acids as well as increasing exogenous palmitate uptake that is deviated more into neutral lipid formation rather than phospholipids. We also showed that the increase in ceramide levels contributes to some extent in the mediation of apoptosis. Consistent with this mechanism of action, Fasnall showed potent anti-tumor activity in the MMTV-Neu model of HER2+ breast cancer, particularly when combined with carboplatin. In humans, de novo fatty acid synthesis is active in a limited number of tissues such as liver, adipose, cycling endometrium, and lactating mammary gland. This contrasts with the other bodily tissues, which largely meet their fatty acid requirements from dietary sources (Brusselmans and Swinnen, 2009Brusselmans K. Swinnen J. The lipogenic switch in cancer.in: Costello L. Singh K. Mitochondria and Cancer. Springer, New York2009: 39-59Crossref Scopus (9) Google Scholar) (Iwanaga et al., 2009Iwanaga T. Tsutsumi R. Noritake J. Fukata Y. Fukata M. Dynamic protein palmitoylation in cellular signaling.Prog. Lipid Res. 2009; 48: 117-127Crossref PubMed Scopus (89) Google Scholar) (Swinnen et al., 2006Swinnen J.V. Brusselmans K. Verhoeven G. Increased lipogenesis in cancer cells: new players, novel targets.Curr. Opin. Clin. Nutr. Metab. Care. 2006; 9: 358-365Crossref PubMed Scopus (472) Google Scholar). However, some pathological conditions promote cells to become dependent on de novo fatty acid synthesis including solid tumors, leukemic cells, and host cells of certain viruses (Ameer et al., 2014Ameer F. Scandiuzzi L. Hasnain S. Kalbacher H. Zaidi N. De novo lipogenesis in health and disease.Metab. Clin. Exp. 2014; 63: 895-902Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Fatty acid synthase I (FASN) catalyzes the final steps leading to the synthesis of long-chain fatty acids in vivo. The active form of FASN is composed of a homodimer where each monomer has seven different catalytic domains. These domains include the acyl carrier protein, which is responsible for substrate channeling from one domain to another; the ketoacyl synthetase domain, which catalyzes the condensation step; ketoacyl reductase and enoyl reductase, which both are responsible for saturating the acyl chain; the dehydratase domain, responsible for removing a water molecule from the acyl chain between the two reduction steps; malonylacetyl transferase, which catalyzes the transfer of both malonyl-coenzyme A (CoA) and acetyl-CoA; and the thioesterase domain, which clips the palmitate off the enzyme after reaching the desired acyl-chain length (Maier et al., 2008Maier T. Leibundgut M. Ban N. The crystal structure of a mammalian fatty acid synthase.Science. 2008; 321: 1315-1322Crossref PubMed Scopus (337) Google Scholar). In breast cancer, the level of FASN expression is correlated with tumor progression, where high FASN expression leads to more tumor aggressiveness and poor prognostic outcome (Alo et al., 1996Alo P.L. Visca P. Marci A. Mangoni A. Botti C. Di Tondo U. Expression of fatty acid synthase (FAS) as a predictor of recurrence in stage I breast carcinoma patients.Cancer. 1996; 77: 474-482Crossref PubMed Scopus (276) Google Scholar). Inhibiting FASN activity in vitro by pharmacological means or its message levels by small interfering RNA (siRNA) has been shown to stop cancer cell growth and induce apoptosis. As a consequence, many research groups have tried to exploit FASN as a target for cancer by developing inhibitors including C75, C93, EGCG (epigallocatechin gallate), G28UCM, orlistat, GSK2194069, and GSK837149A (Hardwicke et al., 2014Hardwicke M.A. Rendina A.R. Williams S.P. Moore M.L. Wang L. Krueger J.A. Plant R.N. Totoritis R.D. Zhang G. Briand J. et al.A human fatty acid synthase inhibitor binds beta-ketoacyl reductase in the keto-substrate site.Nat. Chem. Biol. 2014; 10: 774-779Crossref Scopus (64) Google Scholar, Kuhajda et al., 2000Kuhajda F.P. Pizer E.S. Li J.N. Mani N.S. Frehywot G.L. Townsend C.A. Synthesis and antitumor activity of an inhibitor of fatty acid synthase.Proc. Natl. Acad. Sci. USA. 2000; 97: 3450-3454Crossref PubMed Scopus (508) Google Scholar, Landis-Piwowar et al., 2007Landis-Piwowar K.R. Huo C. Chen D. Milacic V. Shi G. Chan T.H. Dou Q.P. A novel prodrug of the green tea polyphenol (-)-epigallocatechin-3-gallate as a potential anticancer agent.Cancer Res. 2007; 67: 4303-4310Crossref PubMed Scopus (201) Google Scholar, McFadden et al., 2005McFadden J.M. Medghalchi S.M. Thupari J.N. Pinn M.L. Vadlamudi A. Miller K.I. Kuhajda F.P. Townsend C.A. Application of a flexible synthesis of (5R)-thiolactomycin to develop new inhibitors of type I fatty acid synthase.J. Med. Chem. 2005; 48: 946-961Crossref PubMed Scopus (70) Google Scholar, Oliveras et al., 2010Oliveras G. Blancafort A. Urruticoechea A. Campuzano O. Gomez-Cabello D. Brugada R. Lopez-Rodriguez M.L. Colomer R. Puig T. Novel anti-fatty acid synthase compounds with anti-cancer activity in HER2+ breast cancer.Ann. N. Y. Acad. Sci. 2010; 1210: 86-92Crossref PubMed Scopus (28) Google Scholar, Orita et al., 2007Orita H. Coulter J. Lemmon C. Tully E. Vadlamudi A. Medghalchi S.M. Kuhajda F.P. Gabrielson E. Selective inhibition of fatty acid synthase for lung cancer treatment.Clin. Cancer Res. 2007; 13: 7139-7145Crossref PubMed Scopus (91) Google Scholar, Puig et al., 2009Puig T. Turrado C. Benhamu B. Aguilar H. Relat J. Ortega-Gutierrez S. Casals G. Marrero P.F. Urruticoechea A. Haro D. et al.Novel inhibitors of fatty acid synthase with anticancer activity.Clin. Cancer Res. 2009; 15: 7608-7615Crossref PubMed Scopus (84) Google Scholar, Puig et al., 2011Puig T. Aguilar H. Cufi S. Oliveras G. Turrado C. Ortega-Gutierrez S. Benhamu B. Lopez-Rodriguez M.L. Urruticoechea A. Colomer R. A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines.Breast Cancer Res. 2011; 13: R131Crossref PubMed Scopus (74) Google Scholar, Thupari et al., 2002Thupari J.N. Landree L.E. Ronnett G.V. Kuhajda F.P. C75 increases peripheral energy utilization and fatty acid oxidation in diet-induced obesity.Proc. Natl. Acad. Sci. USA. 2002; 99: 9498-9502Crossref PubMed Scopus (215) Google Scholar, Tian, 2006Tian W.X. Inhibition of fatty acid synthase by polyphenols.Curr. Med. Chem. 2006; 13: 967-977Crossref PubMed Scopus (99) Google Scholar, Turrado et al., 2012Turrado C. Puig T. Garcia-Carceles J. Artola M. Benhamu B. Ortega-Gutierrez S. Relat J. Oliveras G. Blancafort A. Haro D. et al.New synthetic inhibitors of fatty acid synthase with anticancer activity.J. Med. Chem. 2012; 55: 5013-5023Crossref PubMed Scopus (52) Google Scholar, Ueda et al., 2009Ueda S.M. Mao T.L. Kuhajda F.P. Vasoontara C. Giuntoli R.L. Bristow R.E. Kurman R.J. Shih Ie M. Trophoblastic neoplasms express fatty acid synthase, which may be a therapeutic target via its inhibitor C93.Am. J. Pathol. 2009; 175: 2618-2624Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, Vazquez et al., 2008Vazquez M.J. Leavens W. Liu R. Rodriguez B. Read M. Richards S. Winegar D. Dominguez J.M. Discovery of GSK837149A, an inhibitor of human fatty acid synthase targeting the beta-ketoacyl reductase reaction.FEBS J. 2008; 275: 1556-1567Crossref PubMed Scopus (41) Google Scholar, Wang and Tian, 2001Wang X. Tian W. Green tea epigallocatechin gallate: a natural inhibitor of fatty-acid synthase.Biochem. Biophys. Res. Commun. 2001; 288: 1200-1206Crossref PubMed Scopus (165) Google Scholar, Zhou et al., 2007Zhou W. Han W.F. Landree L.E. Thupari J.N. Pinn M.L. Bililign T. Kim E.K. Vadlamudi A. Medghalchi S.M. El Meskini R. et al.Fatty acid synthase inhibition activates AMP-activated protein kinase in SKOV3 human ovarian cancer cells.Cancer Res. 2007; 67: 2964-2971Crossref PubMed Scopus (129) Google Scholar). Despite these efforts, however, the majority of FASN inhibitors have failed to even advance in translational studies largely due selectivity issues in vivo resulting in unexpected toxicities. The only FASN inhibitor advanced to clinical trial for the treatment of advanced solid tumors to date is the FASN inhibitor TVB-2640. This molecule is based on a potent imidazopyridine scaffold and also has anti-hepatitis C virus activity (Oslob et al., 2013Oslob J.D. Johnson R.J. Cai H. Feng S.Q. Hu L. Kosaka Y. Lai J. Sivaraja M. Tep S. Yang H. et al.Imidazopyridine-based fatty acid synthase inhibitors that show anti-HCV Activity and in vivo target modulation.ACS Med. Chem. Lett. 2013; 4: 113-117Crossref PubMed Scopus (29) Google Scholar). One of the common themes among current FASN inhibitors is a mechanism of action favoring competition with substrate intermediates over co-factor binding. Even in the case of GSK2194069, despite acting on the β-ketoacyl reductase step, the triazolone is only competitive with trans-1-decalone binding and is uncompetitive with NADPH (Hardwicke et al., 2014Hardwicke M.A. Rendina A.R. Williams S.P. Moore M.L. Wang L. Krueger J.A. Plant R.N. Totoritis R.D. Zhang G. Briand J. et al.A human fatty acid synthase inhibitor binds beta-ketoacyl reductase in the keto-substrate site.Nat. Chem. Biol. 2014; 10: 774-779Crossref Scopus (64) Google Scholar). Inhibitors targeting the FASN co-factor domain therefore remain largely unexplored. Targeting of the substrate domains may in part explain the toxicities and lack of efficacy in vivo of the majority of FASN inhibitors, since in order to act competitively the molecules are lipid like in nature. A second concern relates to the broader physiological consequences of selectively inhibiting FASN in vivo, either acutely or chronically. The de novo fatty acid synthesis pathway is highly regulated at several steps and is therefore highly prone to compensatory adaptive responses that would potentially mitigate the efficacy of any selective FASN inhibitor in vivo. Likely compensations could include increased overexpression of FASN itself, increased uptake of exogenous dietary lipids, alteration in expression of enzymes regulating malonyl-CoA levels, such as acetyl-CoA carboxylase or malonyl-CoA decarboxylase, or even switching of the cell to a glycolytic phenotype. In this paper, we have set out to define new scaffolds specifically targeting the largely unexplored sites of purine interaction within FASN. Three of the FASN enzymatic activities (ketoacyl reductase, enoyl reductase, and malonyl/acetyltransferase) use purine-containing co-factors in the form of NADPH, acetyl-CoA, and malonyl-CoA. Importantly, inhibitors targeting purine-utilizing enzymes are generally not lipophilic and have formed the basis of many drugs in clinical use from reverse transcriptase inhibitors to the newer cutting-edge inhibitors targeting protein kinases or heat-shock proteins (Felder et al., 2012Felder E.R. Badari A. Disingrini T. Mantegani S. Orrenius C. Avanzi N. Isacchi A. Salom B. The generation of purinome-targeted libraries as a means to diversify ATP-mimetic chemical classes for lead finding.Mol. Divers. 2012; 16: 27-51Crossref PubMed Scopus (16) Google Scholar, Haystead, 2006Haystead T.A. The purinome, a complex mix of drug and toxicity targets.Curr. Top. Med. Chem. 2006; 6: 1117-1127Crossref PubMed Scopus (49) Google Scholar, Knapp et al., 2006Knapp M. Bellamacina C. Murray J.M. Bussiere D.E. Targeting cancer: the challenges and successes of structure-based drug design against the human purinome.Curr. Top. Med. Chem. 2006; 6: 1129-1159Crossref Scopus (16) Google Scholar, Murray and Bussiere, 2009Murray J.M. Bussiere D.E. Targeting the purinome.Methods Mol. Biol. 2009; 575: 47-92Crossref Scopus (18) Google Scholar). We now report the identification of a thiophenopyrimidine-based FASN inhibitor (Fasnall) that was discovered with the native enzyme using the chemoproteomic platform FLECS (fluorescence-linked enzyme chemoproteomic strategy; Carlson et al., 2013Carlson D.A. Franke A.S. Weitzel D.H. Speer B.L. Hughes P.F. Hagerty L. Fortner C.N. Veal J.M. Barta T.E. Zieba B.J. et al.Fluorescence linked enzyme chemoproteomic strategy for discovery of a potent and selective DAPK1 and ZIPK inhibitor.ACS Chem. Biol. 2013; 8: 2715-2723Crossref PubMed Scopus (35) Google Scholar). Fasnall has potent broad anti-tumor activity against various breast cancer cell lines and in detailed lipidomic analysis produced a profound change in the global cellular lipid profiles remarkably consistent with selective inhibition of FASN. Based on these studies we evaluated Fasnall for safety, its pharmacokinetic properties in normal mice, and efficacy in the mouse mammary tumor virus (MMTV)-Neu mouse model of human epidermal growth factor receptor 2-positive (HER2+) breast cancer. We show that Fasnall is well tolerated, exhibits bioavailability in vivo, and acts a potent inhibitor of HER2+ breast tumor growth. To specifically identify inhibitors of FASN targeting its nucleotide binding pockets, we utilized Cibacron blue Sepharose. This medium has been used previously to purify NAD and NADP binding proteins from crude tissue extracts (Miyaguchi et al., 2011Miyaguchi Y. Sakamoto T. Sasaki S. Nakade K. Tanabe M. Ichinoseki S. Numata M. Kosai K. Simple method for isolation of glyceraldehyde 3-phosphate dehydrogenase and the improvement of myofibril gel properties.Anim. Sci. J. 2011; 82: 136-143Crossref Scopus (4) Google Scholar, Muratsubaki et al., 1994Muratsubaki H. Enomoto K. Ichijoh Y. Tezuka T. Katsume T. Rapid purification of yeast cytoplasmic fumarate reductase by affinity chromatography on blue Sepharose CL-6B.Prep. Biochem. 1994; 24: 289-296PubMed Google Scholar). FASN-enriched extract from lactating pig mammary gland was bound to the resin and labeled with cysteine reactive fluorescein. Having established that labeled FASN could be competitively released from the resin with adenine-containing nucleotides (Figure S2), we subsequently screened the bound enzyme against a single concentration of an in-house small-molecule library comprising compounds with structural similarity to any purine or known purine analog scaffold (Carlson et al., 2013Carlson D.A. Franke A.S. Weitzel D.H. Speer B.L. Hughes P.F. Hagerty L. Fortner C.N. Veal J.M. Barta T.E. Zieba B.J. et al.Fluorescence linked enzyme chemoproteomic strategy for discovery of a potent and selective DAPK1 and ZIPK inhibitor.ACS Chem. Biol. 2013; 8: 2715-2723Crossref PubMed Scopus (35) Google Scholar). Of the 3,379 compounds screened, 247 were found to yield a fluorescent signal at 488ex/522em nm (Figure 1). One hundred fifty-five of the molecules selectively eluted FASN from the resin, and 20 potential lead compounds progressed according to their FASN selectivity (assessed with SDS-PAGE, silver staining, and mass spectrometry [MS]). These 20 compounds were reduced to 13 based on the absence of any obvious chemical liabilities. Next, the molecules were tested for their ability to inhibit FASN activity in a HepG2 cell-based assay that measured the incorporation of [3H]glucose into lipids (Figures 1B and S1). Of the 13 molecules tested, Fasnall was the most potent inhibitor. In more detailed cell-based assays, Fasnall potently blocked both acetate and glucose incorporation into total lipids, with IC50 values of 147 and 213 nM, respectively, in HepG2 cells (Figure 2A ). Subsequently, we confirmed direct inhibition of FASN using the purified human enzyme isolated from the BT474 cell line (IC50 = 3.71 μM, Figure 2B). We also synthesized the enantiomers of Fasnall (HS-79 and HS-80) and a truncated version of Fasnall (HS-102) as a negative control (Figure 2C). The molecules were tested in the acetate incorporation assay in BT474 cells. Both of the Fasnall enantiomers were able to inhibit the incorporation of tritiated acetate (IC50 5.84 μM, 1.57 μM, and 7.13 μM for Fasnall, HS-79, and HS-80, respectively) into lipids. The truncated molecule HS-102 had no effect on acetate incorporation. The molecules were also tested in the blue Sepharose elution assay (Figure S2) and there was no significant difference between them except for HS-102, which had no activity. Finally, as an additional testament to the selectivity of Fasnall, we surveyed data derived from prior screens against our in-house library (Carlson et al., 2013Carlson D.A. Franke A.S. Weitzel D.H. Speer B.L. Hughes P.F. Hagerty L. Fortner C.N. Veal J.M. Barta T.E. Zieba B.J. et al.Fluorescence linked enzyme chemoproteomic strategy for discovery of a potent and selective DAPK1 and ZIPK inhibitor.ACS Chem. Biol. 2013; 8: 2715-2723Crossref PubMed Scopus (35) Google Scholar) (Figure 3). None of the previously screened proteins (ACC [acetyl-CoA carboxylase], Hsp90, Hsp70, TRAP-1, DAP kinase 3, IRAK 2, AMPK α and γ subunits, NEK9, dengue nonstructural protein 5, malarial kinase PfPK9, and HSF-1) were targeted by Fasnall. In addition, we tested the ability of Fasnall to eluted proteins of ATP Sepharose loaded with BT474 lysate and found that Fasnall does not elute any proteins over that observed with the vehicle DMSO (Figure S3D), further confirming selectivity.Figure 3Selectivity of FasnallShow full captionIndividual compounds were assayed for their ability to elute proteins from Cibacron blue resin. Blue-red color spectrum indicates protein concentration as measured by fluorescence (see FLECS Methods). SDS-PAGE and MS analysis showed that Fasnall selectively elutes FASN compared with strong (HS-206160) and weak (HS-202889) hits. Bottom (red graph): compound library was screened for inhibitory activity against the following enzymes: ACC, ZipK, AMPKα, AMPKγ, TRAP1, HSP70, NS5, and IRAK2; Fasnall was a potent inhibitor of FASN (only).View Large Image Figure ViewerDownload (PPT) Individual compounds were assayed for their ability to elute proteins from Cibacron blue resin. Blue-red color spectrum indicates protein concentration as measured by fluorescence (see FLECS Methods). SDS-PAGE and MS analysis showed that Fasnall selectively elutes FASN compared with strong (HS-206160) and weak (HS-202889) hits. Bottom (red graph): compound library was screened for inhibitory activity against the following enzymes: ACC, ZipK, AMPKα, AMPKγ, TRAP1, HSP70, NS5, and IRAK2; Fasnall was a potent inhibitor of FASN (only). To evaluate the potential of Fasnall in breast cancer, we first tested its effects on proliferation across a panel of non-tumorigenic (MCF10A) and aggressive tumor-forming breast cancer cell lines including estrogen receptor positive (ER+) (MCF7), triple negative (MDA-MB-468), and HER2+ (BT474 and SKBR3). Fasnall inhibited the proliferation of aggressive cell lines with potency similar to that of C75, but showed lower activity in the non-tumorigenic cell line MCF10A (Figures 4A–4E ). The weaker effects of Fasnall in MCF10A cells correlated with low expression of FASN in this cell line relative to the more aggressive lines, suggesting that the former cells are less dependent on FASN for growth (Figure 4G). Fasnall treatment of BT474 cells did not induce cell-cycle arrest except for an increase in the Sub 2N cell population (Figure 4F). To determine the effects of Fasnall on the whole-cell lipid profile, we carried out lipidomic analysis by liquid chromatography-tandem MS (LC-MS/MS) following 2 hr of exposure to 10 μM Fasnall in BT474 cells (Figure 5A). Using electrospray ionization (ESI+ and ESI−) profiling, more than 3,000 lipid features can be simultaneously quantified, and our analysis showed that Fasnall induced more than 2-fold change in abundance of 167 specific molecules (p < 0.01 relative to vehicle). Many fatty acids were found to increase over the control, most significantly the polyunsaturated lipids (Figure 5B), suggesting a compensatory effect as a result of their uptake from the media. This was confirmed by a [14C]palmitate uptake assay whereby Fasnall treatment increased 14C labeling of free fatty acids (Figure 5C). Other lipids of particular note that increased many fold with Fasnall are ceramides, which are considered as pro-apoptotic lipids. The increase of ceramides would be expected due to malonyl-CoA (the direct substrate of FASN) accumulation and its effects on CPT-1 inhibition (Bandyopadhyay et al., 2006Bandyopadhyay S. Zhan R. Wang Y. Pai S.K. Hirota S. Hosobe S. Takano Y. Saito K. Furuta E. Iiizumi M. et al.Mechanism of apoptosis induced by the inhibition of fatty acid synthase in breast cancer cells.Cancer Res. 2006; 66: 5934-5940Crossref PubMed Scopus (132) Google Scholar). As a consequence, any free fatty acids (derived primarily from the extracellular media) are likely to be condensed to 3-keto dihydrosphingosine and on through a series of reduction and acylation steps to various ceramides such as dihydroceramide and ceramide. Diacylglycerols were also found to increase significantly, which can indicate an overall increase in the lipolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) or an increase in de novo synthesis of diacylglycerols. Increase in diacylglycerol accumulation would be expected as a consequence of FASN inhibition, since this would be predicted to promote accumulation of glycerol, a precursor of triglyceride and diacylglycerols. This is because flux of carbons normally supplied by glycolysis for de novo fatty acid is now blocked at the level of FASN itself, causing accumulation of all upstream intermediates (Haystead et al., 1989Haystead T.A. Sim A.T. Carling D. Honnor R.C. Tsukitani Y. Cohen P. Hardie D.G. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism.Nature. 1989; 337: 78-81Crossref PubMed Scopus (705) Google Scholar). We were able to confirm the accumulation of neutral lipids by performing an oil red O stain for lipid droplets under different serum conditions, showing an increase in lipid droplet formation when BT474 are exposed to Fasnall in full serum (Figure 5D). Inhibition of FASN in rapidly proliferating tumorigenic cells would be predicted to have two major effects; first, limiting the oxidative capacity of the mitochondria through increasing malonyl-CoA levels; second, triggering program cell death pathways mostly via accumulation of ceramide. To investigate the latter mechanism, we examined caspase-3 and -7 activation in response to Fasnall and C75 (Figure 6A). Consistent with their tumorigenic capacities, SKBR3 and BT474 cells had 2- to 10-fold, respectively, higher caspase activity than MCF10A cells in response to Fasnall or C75 treatment. The ability of Fasnall to induce apoptosis was also confirmed by detecting the presence of phosphatidylserine and phosphatidylcholine on the outer leaflet of the plasma membrane using fluorescently labeled annexin V and flow cytometry (Figure S5A). To validate the on-target effect of Fasnall, we performed an siRNA knockdown of FASN (Figure S4A) and assessed its effect on Fasnall induction of apoptosis and reduction of viability and proliferation. FASN siRNA pretreatment was able to reduce Fasnall-induced toxicity in BT474 cells, suggesting that Fasnall-induced toxicity is due to FASN inhibition (Figures S4C–S4E). To further confirm that Fasnall induction of apoptosis is directly related to the inhibition of FASN, we tried to rescue the cells by pretreating them with different combinations of palmitate (the end product of FASN) and the ACC inhibitor 5-tetradecyloxy-2-furoic acid (TOFA) to prevent malonyl-CoA accumulation (Figure 6C). However, in our hands TOFA treatment was able to completely reverse the effect of Fasnall only in BT474 cells, which was not due to a general anti-apoptotic activity of TOFA (Figure S5B), while palmitate, or the combination of both palmitate and TOFA, did not fully reverse the effect of the inhibitor. In SKBR3 cells, TOFA, palmitate, and the combination of both was able to partially reverse the effect of Fasnall. We tried further verify whether ceramide accumulation is the main cause of apoptosis by treating the cells with different ceramide synthesis inhibitors targeting various enzymes in both the de novo and salvage ceramide synthesis pathway (Figure S5C). Only myriocin, a serine palmitoyl transferase inhibitor, was able to partially reverse the Fasnall-induced apoptosis (Figure S5D), indicating that ceramide accumulation is not the only cause of apoptosis. In an acute toxicity study, FVB/J mice received 5, 20, or 80 mg/kg Fasnall via intraperitoneal injection on days 1 and 3, and blood was collected on day 4. Fasnall was toxic at 80 mg/kg, but at 5–20 mg/kg was well tolerated with no adverse effects on white blood cell counts, hemoglobin levels, kidney, or liver functions (Table S2). To test for the long-term effects of Fasnall, we administered biweekly intraperitoneal injections of 5, 10, or 15 mg/kg Fasnall to mice for 8 weeks. None of these doses induced any signs of toxicity, stress, or significant change in mouse weight (Figure 7A). Next, we carried out pharmacokinetic studies to determine the uptake and biodistribution of Fasnall in MMTV-Neu mice by LC-MS (Figure S6). These studies showed that Fasnall appears rapidly in the plasma within 5 min of the injection and is cleared rapidly (T1/2 = 9.81 ± 0.02 min, n = 3). Similar uptake and clearance was also observed in liver and kidney (liver T1/2 = 9.84 ± 0.09 min, n = 3; kidney T1/2 = 9.90 ± 0.01 min, n = 3). Although our MS analysis focused primarily on the parent compound (amu 339Da), preliminary examination of the entire LC profile following drug extraction of the tissues did not reveal any obvious Fasnall metabolites (data not shown). These findings suggest that Fasnall is rapidly cleared through the kidney and liver in its parent ion state. Having determined that Fasnall was well tolerated in mice, we next tested its efficacy on tumor progression in the MMTV-Neu model of HER2+ breast cancer (Muller et al., 1988Muller W.J. Sinn E. Pattengale P.K. Wallace R. Leder P. Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene.Cell. 1988; 54: 105-115Abstract Full Text PDF PubMed Scopus (937) Google Scholar) (Jackson Laboratory strain 002376). Cohorts of MMTV-Neu mice were treated with a biweekly intraperitoneal injection of 15 mg/kg Fasnall (Figures 7B and 7C). When given alone, Fasnall reduced tumor volume compared with vehicle-treated animals (day 21, Fasnall treatment volume 436 ± 218 [SD of mean] mm, n = 7, control volume 628 ± 381 mm, n = 9; p = 0.85). Significantly, Fasnall also increased the median survival of the MMTV-Neu mice to 63 days (p = 0.049) compared with animals treated with vehicle alone (Figure 7D). Importantly, MS analysis of tumor tissue verified Fasnall uptake and also showed a significantly longer elimination time (T1/2 = 65.71 ± 0.32 min, n = 3) than all other tissues tested. The long duration of treatment in our studies suggest that the dosing frequency of Fasnall can be greatly increased to achieve greater effects on survival and tumor volume. These findings are consistent with effects of Fasnall as an anti-proliferative agent in tumors. More dramatic acute tumor responses were observed when Fasnall was combined with 50 mg/kg of the platinum-based chemotherapeutic agent carboplatin administered weekly. Here, 88% of tumors achieved an objective response rate of stable disease or better compared with carboplatin only at 25% (Fisher's exact test, p = 0.01). This response was not durable, however, as there was no long-term benefit of the combination therapy at this dosing regimen (Figure 7D). As often seen in the clinic, tumors that are responsive initially will develop resistance, which is likely the case here. These findings are consistent with the actions of two compounds acting independently of one another whereby one anti-neoplastic develops resistance while the other maybe unaffected. Importantly, carboplatin is a front-line chemotherapeutic agent for the treatment of" @default.
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• W2414985384 title "Fasnall, a Selective FASN Inhibitor, Shows Potent Anti-tumor Activity in the MMTV-Neu Model of HER2 + Breast Cancer" @default.
• W2414985384 cites W135473883 @default.
• W2414985384 cites W1540335219 @default.
• W2414985384 cites W1577348758 @default.
• W2414985384 cites W1589402586 @default.
• W2414985384 cites W1602678530 @default.
• W2414985384 cites W1757385007 @default.
• W2414985384 cites W1759422567 @default.
• W2414985384 cites W1818464951 @default.
• W2414985384 cites W1878036365 @default.
• W2414985384 cites W1966541730 @default.
• W2414985384 cites W1978442696 @default.
• W2414985384 cites W1986261554 @default.
• W2414985384 cites W1987231965 @default.
• W2414985384 cites W1996586460 @default.
• W2414985384 cites W2003552185 @default.
• W2414985384 cites W2005794853 @default.
• W2414985384 cites W2015312498 @default.
• W2414985384 cites W2028642327 @default.
• W2414985384 cites W2032344256 @default.
• W2414985384 cites W2042819247 @default.
• W2414985384 cites W2045530900 @default.
• W2414985384 cites W2048548143 @default.
• W2414985384 cites W2054474037 @default.
• W2414985384 cites W2056122422 @default.
• W2414985384 cites W2059010575 @default.
• W2414985384 cites W2059609101 @default.
• W2414985384 cites W2074446289 @default.
• W2414985384 cites W2079816467 @default.
• W2414985384 cites W2090694532 @default.
• W2414985384 cites W2100799417 @default.
• W2414985384 cites W2113163520 @default.
• W2414985384 cites W2113892466 @default.
• W2414985384 cites W2117597975 @default.
• W2414985384 cites W2118486580 @default.
• W2414985384 cites W2134027848 @default.
• W2414985384 cites W2135715116 @default.
• W2414985384 cites W2136192861 @default.
• W2414985384 cites W2136816004 @default.
• W2414985384 cites W2137642531 @default.
• W2414985384 cites W2139126860 @default.
• W2414985384 cites W2141833284 @default.
• W2414985384 cites W2146033919 @default.
• W2414985384 cites W2163181319 @default.
• W2414985384 cites W2229416282 @default.
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