AD_________________ Award Number: DAMD17-02-1-0431 TITLE: Fatty Acid Synthase Inhibitor Cytotoxicity: Depletion of the Coenzyme-A Pool PRINCIPAL INVESTIGATOR: Francis P. Kuhajda, M.D. CONTRACTING ORGANIZATION: Johns Hopkins University School of Medicine Baltimore, MD 21205 REPORT DATE: April 2005 TYPE OF REPORT: Final, Revised PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.

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Fatty Acid Synthase Inhibitor Cytotoxicity: Depletion of the Coenzyme-A Pool 5b. GRANT NUMBER DAMD17-02-1-0431

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6. AUTHOR(S) Francis P. Kuhajda, M.D.

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E-Mail: fkuhajda@jhmi.edu 5f. WORK UNIT NUMBER

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Johns Hopkins University School of Medicine Baltimore, MD 21205

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14. ABSTRACT: Inhibition of fatty acid synthase (FAS) with C75 in human cancer cells leads to cytotoxicity without evidence of DNA damage. Based on this and other biochemical observations, attention was focused on the cytoplasm as the site for the origin of C75 cytotoxicity to human cancer cells. We have shown that eukaryotic initiation factor 2 alpha (EIF2α), a key regulator of protein synthesis and the ER stress response, is involved in the cytotoxic mechanism of C75 against human breast cancer cells. Based on these observations, we synthesized a series of novel thiazolidinedione (TZD) compounds. TZD’s likely induce cancer cell death through inhibition of translation initiation mediated by phosphorylation of eukaryotic initiation factor 2α (EIF2α) rendering it inactive. In summary, our novel TZD are cytotoxic to human colon cancer cells in vitro and xenografts in athymic mice. These compounds also phosphorylate EIF2α similar to C75. Based on these observations, novel class of potent TZD derivatives which may be useful for the treatment of a wide variety of human cancers.

15. SUBJECT TERMS fatty acid synthase, thiazolidinediones, eukaryotic initiation factor 2α, HCT116 human colon cancer cells, endoplasmic reticulum stress response 16. SECURITY CLASSIFICATION OF:

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Table of Contents

Introduction…………………………………………………………………………………………………………4

Body………………………………………………………………………………………………………………………………4

Key Research Accomplishments………………………………………………………………7

Reportable Outcomes………………………………………………………………………………………7

Conclusions……………………………………………………………………………………………………………7

References………………………………………………………………………………………………………………7

Appendices………………………………………………………………………………………………………………8

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INTRODUCTION Initially, this proposal, written by Dr. Ellen Pizer at Johns Hopkins, sought to investigate coenzyme-A levels following inhibition of fatty acid synthase (FAS). Since we could not document consistent changes in coenzyme-A synthesis following fatty acid synthase inhibition, I suggested a change in the Statement of Work which was approved. FAS inhibition with C75 significantly inhibited protein synthesis in MCF7 cells within 4 hours. This reduction in protein synthesis is seen during apoptosis and is likely mediated by the ER stress response. Because of these findings, the role of eukaryotic initiation factor 2 alpha (EIF2α) was probed in regard to its effect of the regulation of protein translation. Interestingly, these studies have dovetailed with our overall interest in energy metabolism in cancer. BODY Inhibition of fatty acid synthase (FAS) in human cancer cells leads to cytotoxicity without evidence of DNA damage. C75, the index FAS inhibitor that has been the focus of our mechanistic studies to determine how FAS inhibition triggers apoptosis, has been recently shown to also stimulate fatty acid oxidation (5, 6). It was conceivable that increased fatty acid oxidation could be the cause of cytotoxicity rather than FAS inhibition. In our recent paper, we have shown that FAS inhibition, not fatty acid oxidation stimulation is the trigger for apoptosis in human cancer cells (8). Given the dual effects of C75 on fatty acid synthesis and oxidation, we needed an additional compound with a different chemical scaffold to determine the relationship between the pharmacological manipulation of EIF2α phosphorylation fatty acid synthesis inhibition, and cancer cell death before we could publish our results. As part of our ongoing synthetic chemical effort, a series of novel thiazolidinedione (TZD) class of compounds were synthesized. During screening against human tumor cells, one particular compound, M133, inhibited fatty acid synthesis and was cytotoxic to MCF7 cells. The TZD class of compounds are well known ligands of peroxisome proliferators-activated receptor (PPAR) γ known to induce adipocyte differentiation and increase insulin sensitivity (4). This class of compounds have shown in vitro activity against lung (7), breast (1), colon (2), and prostate cancers (3), and against animal models of breast cancer (1). It was initially thought that TZD’s were inducing apoptosis in human cancer cells by acting as PPARγ ligands. Recently, however, it was demonstrated that TZD’s likely induce cancer cell death through inhibition of translation initiation mediated by phosphorylation of eukaryotic initiation factor 2α (EIF2α) rendering it inactive (4). We have now shown that similar to C75, M133 also rapidly inhibits fatty acid synthesis, phosphorylates EIF2, and has antitumor activity in vitro and in vivo. Using these data from disparate chemical scaffolds, we will now be able to demonstrate convincingly that inhibition of fatty acid synthesis leads to an ER stress response mediated through EIF2α phosphorylation. Recently, we have synthesized and tested a second novel TZD compound, M164 to determine if the anti-tumor effect of M133 was restricted to a single compound, potentially indicating an idiosyncratic effect, or common to this class of compounds. M164 also has significant anti-tumor activity both in vitro and in vivo demonstrating that these compound have potential as anti-tumor agents. Through these series of experiments, we will outline the relationship FAS inhibition and the role of eukaryotic initiation factor 2 alpha (EIF2α) in the cytotoxic response using two novel TZD derivatives, M133 and M164. A. M133 inhibits fatty acid synthesis and is cytotoxic to human cancer cells. Using our well established method of [14C]acetate labeling to measure fatty acid synthesis, we have demonstrated that M133 inhibits fatty acid synthesis in MCF7 human breast cancer cells with an IC50 of 15.4 µg/ml within 2 h of drug application. As demonstrated by the in vitro XTT cytotoxicity assay, M133 has an IC50 of 9.1 and 11.3 µg/ml after 72h of drug

exposure against MCF7 and OVCAR3 human ovarian cancer cells. Both inhibition of fatty acid synthesis and cytotoxicity occur at concentrations ~30 µM. B. M133 induces EIF2α phosphorylation. MCF7 cells were treatedbelow 0.1% and is includphosphorylation within 4at 24 h (upper right panelower panels show the ex C. M133 causes signific In this pilot study, we utthe flanks of athymic mibegan at 60 mg/kg, i.p., DMSO vehicle. There w By day nine, there was astudy was terminated on

M133 µg/ml

as indicated with M133 dissolved in DMSO. The DMSO concentration was kept ed in the control wells. At concentrations well below the IC50, M133 induces EIF2α h as shown by immunoblot (upper left panel) and persists at the 2 µg/ml concentration l). The higher drug concentrations likely caused significant cell injury by 24 h. The pression of non-phosphorylated EIF2α as a control.

ant growth inhibition of the HCT116 human colon cancer xenograft.

ilicefoer

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zed that fast growing, FAS expressing HCT116 human colon cancer cells grafted into . After 7 days of engraftment, with an average size of 52 mm3, treatment with M133 r the first two days, and 120 mg/kg on days 2,3, and 6. Control animals received 50 µl e five animals in each group and tumors were measured with calipers in 3 dimensions.

statistically significant difference between the controls and M133 treated animals. The ay 10 due to compound limitations. Importantly, M133 does not cause the weight loss

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seen with the C75 class of compounds because M133 does not induce fatty acid oxidation. Thus, we have achieved the separation of weight loss from cytotoxicity of fatty acid synthesis inhibitors. D. M121 has a significant anti-tumor effect both in vitro and in vivo. M121 is a second novel TZD developed by our group. The rationale for developing and testing a second compound was to determine if the significant anti-tumor effects of M133 were idiosyncratic, or common to this class of compounds. M164 was test against 3 human cancer cell lines in vitro with the following IC50’s: MCF7 (human breast cancer) 5.5 µg/ml, OVCAR-3 (human ovarian cancer) 3.7 µg/ml, and HCT116 (human colon cancer) 5.6 µg/ml. Fatty acid synthesis inhibition was measured by 14Cactetate inhibition into total lipids in MCF7 cells with an IC50 of ~ 30 µg/ml. Using a similar treatment protocol to M133 described above, M164 has significant anti-tumor activity against the HCT116 xenograft in athymic mice as shown below.

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Interestingly, compared to troglitazone against MCF7 xenografts, we achieved a superior result with 5 treatments over 9 days with both M133 and M164, than daily treatment at 1000 mg/kg for 9 weeks with troglitazone delivered orally (3). While this is an early study, we now have demonstrated that we have a TZD derivative that is likely significantly more potent in vivo than troglitazone. Since TZD’s have been used in humans to treat diabetes, this compound may be similarly non-toxic, and if so, M133 or another derivative could be moved rapidly to the clinic. E. FAS inhibition activates AMP-activated protein kinase (AMPK). We have recently demonstrated that FAS inhibition activates AMPK (9) (Zhou et al, 2007, Appendix I). Activated AMPK inhibits protein translation through inactivation if EIF as well. These data further indicate that inhibition of protein synthesis through a stress response or AMPK activation both demonstrate significant anti-tumor effects.

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KEY RESEARCH ACCOMPLISHMENTS

A. Similar to C75, M133 inhibits fatty acid synthesis and is cytotoxic to human breast, colon, and ovarian cancer cells that express high levels of fatty acid synthesis.

B. M133 does not cause significant weight loss similar to C75 because it does not stimulate fatty acid oxidation. C. M133 is potent against the HCT116 human colon cancer xenografts. D. M133 is a novel thiazolidinedione (TZD) derivative, a class of compounds used widely in the treatment of diabetes. As such, M133 could prove to be a relatively non-toxic compound with significant anti-tumor efficacy against a wide variety of human cancer. E. M164 is a second novel TZD derivative with significant in vitro and in vivo anti-tumor activity.

REPORTABLE OUTCOMES 1. Publications: We are planning an abstract for the AACR meeting in 2008. 2. Degrees obtained: Minerva Hughes who synthesized these compound obtained her Ph. D. in Chemistry from the Johns Hopkins University. 3. Patents: We are in discussions over a patent for these compounds (composition of matter patent) for M133 and M164. The structures are patentable since they have not been previously synthesized. 4. Based on these data, we plan to apply for NIH funding during the following year in collaboration with the Townsend chemistry laboratory at JHU. CONCLUSIONS Fatty acid synthase (FAS) is upregulated in most common human carcinomas. Inhibition of FAS has led to apoptosis in a variety of human cancer cell lines, and C75 has had significant anti-tumor activity in breast, prostate, colon, lung, and mesothelioma xenografts. Understanding the mechanism of cytotoxicity of FAS inhibitors is key to both the basic understanding of the biology and development of FAS as a drug target. Through this exploration of the mechanism of action of C75, we now have a novel class of potent TZD derivatives which may be useful for the treatment of a wide variety of human cancers. REFERENCES 1. Elstner E, Muller C, Koshizuka K, Williamson EA, Park D, Asou H, Shintaku P, Said JW, Heber D, and Koeffler HP. Ligands for peroxisome proliferator-activated receptorgamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc Natl Acad Sci U S A 95: 8806-8811, 1998. 2. Kitamura S, Miyazaki Y, Shinomura Y, Kondo S, Kanayama S, and Matsuzawa Y. Peroxisome proliferator-activated receptor gamma induces growth arrest and differentiation markers of human colon cancer cells. Jpn J Cancer Res 90: 75-80, 1999. 3. Kubota T, Koshizuka K, Williamson EA, Asou H, Said JW, Holden S, Miyoshi I, and Koeffler HP. Ligand for peroxisome proliferator-activated receptor gamma (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo. Cancer Res 58: 3344-3352, 1998.

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4. Palakurthi SS, Aktas H, Grubissich LM, Mortensen RM, and Halperin JA. Anticancer effects of thiazolidinediones are independent of peroxisome proliferator-activated receptor gamma and mediated by inhibition of translation initiation. Cancer Res 61: 6213-6218, 2001. 5. Thupari JN, Kim EK, Moran TH, Ronnett GV, and Kuhajda FP. Chronic C75 Treatment of Diet-Induced Obese Mice Increases Fat Oxidation and Reduces Food Intake to Reduce Adipose Mass. Am J Physiol Endocrinol Metab, 2004. 6. Thupari JN, Landree LE, Ronnett GV, and Kuhajda FP. C75 increases peripheral energy utilization and fatty acid oxidation in diet-induced obesity. Proc Natl Acad Sci U S A 99: 9498-9502, 2002. 7. Tsubouchi Y, Sano H, Kawahito Y, Mukai S, Yamada R, Kohno M, Inoue K, Hla T, and Kondo M. Inhibition of human lung cancer cell growth by the peroxisome proliferator-activated receptor-gamma agonists through induction of apoptosis. Biochem Biophys Res Commun 270: 400-405, 2000. 8. Zhou W, Simpson PJ, McFadden JM, Townsend CA, Medghalchi SM, Vadlamudi A, Pinn ML, Ronnett GV, and Kuhajda FP. Fatty acid synthase inhibition triggers apoptosis during S phase in human cancer cells. Cancer Res 63: 7330-7337, 2003. 9. Zhou W, Han WF, Landree LE, Thupari JN, Pinn ML, Bililign T, Kim EK, Vadlamudi A, Medghalchi SM, El Meskini R, Ronnett GV, Townsend CA, Kuhajda FP. Fatty acid synthase inhibition activates AMP-activated protein kinase in SKOV3 human ovarian cancer cells. Cancer Res 67: 2964-2971, 2007. APPENDICES . Zhou W, Han WF, Landree LE, Thupari JN, Pinn ML, Bililign T, Kim EK, Vadlamudi A, Medghalchi SM, El Meskini R, Ronnett GV, Townsend CA, Kuhajda FP. Fatty acid synthase inhibition activates AMP-activated protein kinase in SKOV3 human ovarian cancer cells. Cancer Res 67: 2964-2971, 2007.

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