EP1235568A2

EP1235568A2 - Depletion of cellular coenzyme-a levels as a means to selectively kill cancer cells

Info

Publication number: EP1235568A2
Application number: EP00977179A
Authority: EP
Inventors: Ellen S. Pizer; Craig A. Townsend; Francis P. Kuhajda
Original assignee: Johns Hopkins University; School of Medicine of Johns Hopkins University
Current assignee: Johns Hopkins University; School of Medicine of Johns Hopkins University
Priority date: 1999-11-12
Filing date: 2000-11-13
Publication date: 2002-09-04
Also published as: CA2390501A1; JP2003516938A; KR20020060737A; WO2001034202A2; WO2001034202A3; AU1485401A

Abstract

The invention describes a method to inhibit growth or kill cancer cells by acute depletion of free cellular Coenzyme A (CoA). This invention encompasses: any method to selectively decrease CoA in cancer cells by increasing the utilization of CoA and/or reducing its synthesis. As demonstrated herein, depletion of free CoA may be accomplished by inhibiting fatty acid synthase (FAS). This invention also includes depletion of free cellular Coenzyme A by interventions in addition to or alternative to FAS inhibition.

Description

DEPLETION OF CELLULAR COENZYME-A LEVELS AS A MEANS TO SELECTIVELY KILL CANCER CELLS

Review of Related Art

A number of studies have demonstrated surprisingly high levels of fatty acid synthase expression (FAS, E.C. 2.3.1.85) in virulent human breast cancer (Alo, P. L., Visca, P., Marci, A., Mangoni, A., Botti, C, and Di Tondo, U. Expression of fatty acid synthase (FASO as a predictor of recurrence in stage I breast carcinoma patients., Cancer. 77: 474-482, 1996; Jensen, V., Ladekarl, M., Holm- Nielsen, P., Melsen, F., and Soerensen, F. B. The prognostic value of oncogenic antigen 519 (OA-519) expression and prohferative activity detected by antibody MIB-1 in node-negative breast cancer., Journal of Pathology. 776: 343-352, 1995), as well as other cancers (Rashid, A., Pizer, E. S., Moga, M., Milgraum, L. Z.. Zahurak, M., Pasternack, G. R., Kuhajda, F. P., and Hamilton, S. R. Elevated expression of fatty acid synthase and fatty acid synthetic activity in colorectal neoplasia., American Journal of Pathology. 150: 201-208, 1997; Pizer, E., Lax, S., Kuhajda, F., Pasternack, G., and Kurman, R. Fatty acid synthase expression in endometrial carcinoma: correlation with cell proliferation and hormone receptors., Cancer. 83: 528-537, 1998). FAS expression has also been identified in intraductal and lobular /; situ breast carcinoma; lesions associated with increased risk for the development of infiltrating breast cancer (Milgraum, L. Z., Witters, L. A., Pasternack, G. R.. and Kuhajda, F. P. Enzymes of the fatty acid synthesis pathway are highly expressed in in situ breast carcinoma.. Clinical Cancer Research. 3: 21 15- 2120, 1997). FAS is the principal synthetic enzyme of fatty acid synthesis (FA synthesis) which catalyzes the NADPH dependent condensation of malonyl-CoA and acetyl-CoA to produce predominantly the 16-carbon saturated free fatty acid, palmitate (Wakil, S. Fatty acid synthase, a proficient multifunctional enzyme., Biochemistry. 28: 4523-4530, 1989). Ex vivo measurements in tumor tissue have revealed high levels of both FAS and FA synthesis indicating that the entire genetic program is highly active consisting of some 25 enzymes from hexokinase to FAS (Rashid, et al, 1997). Cultured human cancer cells treated with inhibitors of FAS, including the fungal product, cerulenin, and the novel compound, C75, demonstrated a rapid decline in FA synthesis, with subsequent reduction of DNA synthesis and cell cycle arrest, culminating in apoptosis (Pizer, E. S.. Jackisch, C, Wood, F. D., Pasternack, G. R., Davidson, N. E., and Kuhajda, F. Inhibition of fatty acid synthesis induces programmed cell death in human breast cancer cells., Cancer Research. 56: 2745- 2747, 1996, Pizer, E. S., Chrest, F. J., DiGiuseppe, J. A., and Han, W. F. Pharmacological inhibitors of mammalian fatty acid synthase suppress DNA replication and induce apoptosis in tumor cell lines.. Cancer Research. 58: 461 1 - 4615, 1998). Pharmacological inhibition of mammalian fatty acid synthase activity lead to inhibition of DNA replication within about 90 minutes of drug application. These findings suggested a vital biochemical link between FA synthesis and cancer cell growth. While generating a great deal of interest, the question of how inhibition of fatty acid synthase triggered this phenomenon remained unknown. Importantly, these effects occurred despite the presence of exogenous fatty acids in the culture medium derived from fetal bovine serum. While it has been possible to rescue the cytotoxic effect of cerulenin on certain cells in fatty acid-free culture conditions by the addition of exogenous palmitate, most cancer cells were not rescued from FA synthesis inhibition by the pathway endproduct (data not shown) (Pizer, E. S., Wood, F. D.. Pasternack, G. R., and Kuhajda, F. P. Fatty acid synthase (FAS): A target for cytotoxic antimetabolities in HL60 promyelocytic leukemia cells., Cancer Research. 1996: 745-751, 1996). Thus, it has been unresolved whether the cytotoxic effect of FA synthesis inhibition on most cancer cells resulted from end product starvation, or from some other biochemical mechanism.

Summary of the Invention

This invention describes a method to inhibit growth or kill cancer cells by acute depletion of free cellular Coenzyme A (CoA). This invention encompasses: any method to selectively decrease CoA in cancer cells by increasing the utilization of CoA and/or reducing its synthesis. This therapeutic strategy will lead to novel chemotherapeutic agents for a wide variety of human cancers. In addition, as this is a novel pathway leading to apoptosis not shared by other cancer drugs, it may be anticipated that this therapeutic strategy may potentiate other commonly utilized cancer therapeutic agents.

In one embodiment, this invention provides a method for inhibiting growth of tumor cells in an organism comprising administering to the organism a composition which causes acute depletion of intracellular free Coenzyme A in cancer cells in said organism. In particular, upon administration of the composition, intracellular malonyl CoA in cells of the organism rises abruptly, preferably within 3 hours of the administration. It is expected that intracellular malonyl CoA rises prior to growth inhibition of the cells, and preferably, the rise in intracellular malonyl CoA is correlated with reduced consumption of malonyl CoA. More preferably, the rise in intracellular malonyl CoA occurs prior to any increase in rate of consumption of malonyl CoA. In one mode, the rise in intracellular malonyl CoA is correlated with reduced intracellular activity of malonyl CoA decarboxylase (MCD) or reduced intracellular activity of fatty acid synthase, and the composition may comprises an inhibitor of MCD. In another mode, the rise in intracellular malonyl CoA is correlated with increased synthesis of malonyl CoA.

In another embodiment, this invention provides a method for inhibiting growth of tumor cells in an organism comprising administering to the organism a composition which causes acute depletion of intracellular free Coenzyme A in cancer cells in said organism and the rise in intracellular malonyl CoA is correlated with increased intracellular activity of acetyl-CoA carboxylase (ACC). Alternatively, the composition may comprises an activator of ACC, an activator of citrate synthase, an inhibitor of 5'-AMP-actιvated protein kinase (AMPK), and/or an inhibitor of acyl CoA synthase. In a preferred mode, a second chemotherapeutic agent is also administered to the organism.

In the method of this invention, intracellular malonyl CoA level prior to administration of said composition is preferably at least 2-fold above normal malonyl CoA level in non-malignant cells. Generally, intracellular level of malonyl CoA is elevated and intracellular level of acetyl CoA and free CoA are reduced relative to pre-treatment levels. Preferably, fatty acid synthesis rate in some cells of the organism is at least 2-fold above normal prior to administration of the composition, and administration of the composition is cytotoxic to the cells. The fall in intracellular free Coenzyme A level may be expected to be correlated with appoptosis of cells having decreased Coenzyme A. In a preferred embodiment of the method of this invention, the composition comprises an inhibitor of Pantothenate kinase, an inhibitor of Phosphopantothenoylcysteine synthetase, an inhibitor of

Phosphopantothencylcysteine decarboxylase, and/or an inhibitor of Phosphopantotheine adenylyltransferase. In another preferred mode, the composition comprises a substrate capable of esterifi cation to CoA. Typically, the organism treated according to this invention comprises tumor cells having elevated fatty acid synthesis rates and cell number of said tumor cells is reduced subsequent to administration of said composition.

In another embodiment, this invention provides a screening method to assist in detecting compositions which are selectively cytotoxic to tumor cells comprising administering a target composition to a target cell, monitoring intracellular levels of free and/or derivatized Coenzyme A in said cell subsequent to said administration, wherein an abrupt decrease in intracellular free Coenzyme A is indicative of selective cytotoxicity. In yet another embodiment, this invention provides a screening method to assist in classifying compositions which are selectively cytotoxic to tumor cells comprising administering a target composition to a target cell, in the absence, and in parallel, in the presence of sufficient ACC inhibitor to limit the production of malonyl-CoA, wherein a difference in cytotoxicity is indicative of a cytotoxic activity derived from an effect on intracellular levels of free and/or derivatized Coenzyme A.

Brief Description of the Figures

Figure 1 shows the fatty acid synthesis pathway, and the effect of various fatty acid synthase inhibitors on fatty acid synthesis and tumor cell growth.

Figure 2 shows malonyl CoA levels under various conditions. Figure 3 show the results of clonogenic assays and apoptosis assays on breast cancer cells treated with various inhibitors.

Figure 4 shows various parameters in tumor cells and liver cells.

Figure 5 shows malonyl CoA levels in tumor cells and liver cells.

Detailed Description of the Embodiments

If fatty acid starvation mediated the cytotoxic effects of cerulenin and C75, then any other FA synthesis inhibitor of similar potency should produce similar effects. To test this idea, we compared the effects on cancer cells of inhibition of acetyl-CoA carboxylase (ACC, E.C. 6.4.1.2), the rate limiting enzyme of fatty acid synthesis, with the effects of FAS inhibitors. The inventors have now demonstrated that inhibition of FAS leads to high levels of malonyl-CoA which occurs within 30 minutes of C75 treatment. These superphysiological levels of malonyl-CoA, not low levels of endogenously synthesized fatty acids, are responsible for breast cancer cell apoptosis. In addition, this is a novel pathway which leads to selective apoptosis of cancer cells.

Figure 1A outlines the portion of the FA synthesis pathway containing the target enzymes of the inhibitors used in this study. TOFA (5- (tetradecyloxy)-2-furoic acid) is an allosteric inhibitor of acetyl-CoA carboxylase (ACC, E.C. 6.4.1.2), blocking the carboxylation of acetyl-CoA to malonyl-CoA. Once esterified to coenzyme-A, TOFA-CoA allosterically inhibits ACC with a mechanism similar to long chain acyl-CoA's, the physiological end-product inhibitors of ACC (Halvorson, D. L. and McCune, S. A. Inhibition of fatty acid synthesis in isolated adipocytes by 5-(tetradecyloxy)-2-furoic acid., Lipids. 19: 851 - 856, 1984). Both cerulenin (Funabashi, H., Kawaguchi, A., Tomoda, H., Omura, S., Okuda, S., and Iwasaki, S. Binding site of cerulenin in fatty acid synthetase., J. Biochem. 105: 751-755, 1989) and C75 (Pizer, et al., 1998) are inhibitors of FAS, preventing the condensation of malonyl-CoA and acetyl-CoA into fatty acids. Cerulenin is a suicide inhibitor, forming a covalent adduct with FAS (Moche, M., Schneider, G., Edwards, P., Dehesh, K., and Lindqvist, Y. Structure of the complex between the antibiotic cerulenin and its target, beta-ketoacyl carrier protein synthase., J Biol Chem. 274: 6031-6034, 1999), while C75 is likely a slow-binding inhibitor (Kuhajda FP, Pizer ES, Mani NS, Pinn ML, Han WF, Chrest FJ. and CA, T. Synthesis and anti-tumor activity of a novel inhibitor of fatty acid synthase., Proceeding of the American Association for Cancer Research. 40: 121 , 1999). Using TOFA, the inventors have achieved FA synthesis inhibition in human breast cancer cell lines comparable to inhibition by cerulenin or C75. Surprisingly, however, TOFA was essentially non-toxic to human breast cancer cells. These data indicate that fatty acid starvation is not a major source of cytotoxicity to cancer cells in serum supplemented culture. An alternative effect of FAS inhibition, production of high levels of the substrate, malonyl-CoA, resulting specifically from inhibition of FAS, appears to mediate cytotoxicity of cerulenin and C75.

Malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase (ACC, E.C. 6.4.1.2), is a key regulatory molecule in cellular metabolism. In addition to its role as a substrate in fatty acid synthesis, malonyl-CoA regulates β- oxidation of fatty acids through its interaction with camitine palmitoyltransferase-1 (CPT-1 ) at the outer membrane of the mitochondria. CPT-1 regulates β-oxidation of fatty acids in the mitochondrion by controlling the passage of long-chain acyl-CoA derivatives such as palmitoyl-CoA through the outer mitochondrial membrane. Physiologically, cytoplasmic malonyl-CoA levels are higher during fatty acid synthesis. The higher steady state level of malonyl-CoA blocks entry of long-chain acyl-CoA's into the mitochondrion thus preventing the futile cycle of oxidizing endogenously synthesized fatty acids.

Coenzyme-A is a vital cofactor for cellular processes involved in energy generation, lipid biosynthesis, and energy regulation. For example, acetyl- CoA and malonyl-CoA are substrates for fatty acid and cholesterol synthesis. All fatty acids must be esterified to CoA before they can be incorporated into cellular structures, or oxidized in the mitochondria for energy. Succinyl-CoA is an intermediate of the TCA cycle. Thus, maintenance of an adequate supply of CoA is vital for cell survival. Many types of cancer cells have high levels of fatty acid synthesis. As expected, cells with high levels of fatty acid synthesis have high steady state levels of malonyl-CoA, at least six times the levels in normal cells (see Example 6). To deplete the available supply of free CoA, intracellular malonyl CoA levels can be selectively and abruptly raised to superphysiological levels in tumor cells by treating them with inhibitors of FAS. This maneuver raises malonyl-CoA levels by both blocking utilization of malonyl-CoA as a substrate in fatty acid synthesis and concomitantly stimulating malonyl-CoA synthesis by relieving fatty acyl-CoA inhibition of ACC (Figure 1A). Since FAS is preferentially expressed in cancer cells, the malonyl-CoA elevation is largely restricted to tumors cells. This leads to cancer cell apoptosis and sparing of normal tissues as occurs in human cancer xenografts treated with FAS inhibitors (See Example 5). By abruptly increasing malonyl-CoA levels, adequate free CoA is not available for other cellular processes, leading to cell death. Free CoA levels may be manipulated using a variety of methods and target enzymes. The Examples demonstrate reduction of free CoA in conjunction with elevation of malonyl-CoA levels through reduced utilization and simultaneous enhanced production of malonyl CoA. Evidence utilizing metabolic labeling with [U-¹⁴C] acetate documents the high levels of fatty acid synthesis in human cancer cells (Kuhajda, F. P., Jenner, K., Wood, F. D.. Hcnnigar, R. Λ., Jacobs, L. B., Dick, J. D., and Pasternack, G. R. Fatty acid synthesis: a potential selective target for antineoplastic therapy., Proceedings of National Academy of Science. 91: 6379- 6383, 1994; Rashid, A., Pizer, E. S., Moga, M., Milgraum, L. Z., Zahurak, M., Pasternack. G. R., Kuhajda, F. P., and Hamilton, S. R. Elevated expression of fatty acid synthase and fatty acid synthetic activity in colorectal neoplasia., American Journal of Pathology. 150: 201-208, 1997). This high level of fatty acid synthesis in human cancer cells allows for the selective manipulation of malonyl-CoA levels to induce apoptosis. Acute increase in malonyl-CoA levels leads to the selective destruction of cancer cells via apoptosis, leaving normal cells unaffected. This therapeutic strategy identifies potential new targets and strategies for cancer chemotherapy based upon alteration of malonyl-CoA levels. Preferably, manipulation of free Coenzyme A levels according to this invention is accomplished by administeπng a composition (or multiple compositions) to an organism m need thereof. The composition administered to the organism may contain an agent having a biological effect of reducing the available supply of free CoA. Agents which interfere with biosynthesis of CoA, or agents that are incorporated into CoA-esters, reducing the pool of free CoA, may be used alone or together with other agents of this invention Preferred agents have the effect, at least part, of raising intracellular malonyl-CoA levels Typically, the organism will be a mammal, such as a mouse, rat. rabbit, guinea pig, cat dog, horse, cow, sheep, goat, pig. or a pπmate, such as a chimpanzee, baboon, or preferably a human Usually, the organism will contain neoplastic (malignant) cells The method of this invention is directed to selectively affecting malignant cells, and having less effect (or more preferably no effect) on normal (non-malignant) cells

The agent in the composition administered to the organism will preferably raise intracellular malonyl-CoA levels in at least a portion of the malignant cells in the organism Preferably the malonyl CoA level will be raised at least 2-fold, more preferably at least 5-fold Preferably, the agent will raise the intracellular malonyl-CoA concentration in the malignant cells to a level higher than the level in surrounding normal cells Suitable agents may raise the malonyl CoA level by any of a number of methods (see alternative mechanisms listed below) In some embodiments, two or more agents aie administered, and some or all of these agents may affect malonyl CoA level by a different mechanism Agents acting b> any of the modes of the following list may be used m compositions of this invention Assays for the following activities are available in the literature, and determination of whether a particular agent exhibits one of these activities is within the skill in the art.

Methods to acutely decrease free CoA for cancer treatment. Acute (i.e., abrupt or preciptous) decrease m free CoA levels leads to the selective destruction of cancer cells via apoptosis This therapeutic strategy identifies potential new targets and strategies for cancer chemotherapy based upon alteration of malonyl-CoA levels that occur selectively in cancer cells, with coordinate changes in free CoA levels.

Agents for increasing malonyl-CoA production:

Acetyl-CoA carboxylase (ACC)effectors: Agents which increase ACC activity, reduce ACC inhibition, or increase the mass of active ACC enzyme will lead to increased levels of malonyl-CoA.

5' c-AMP protein kinase effectors: 5' c-AMP protein kinase inhibits ACC by phosphorylation leading to acute reduction of malonyl-CoA. Inhibitors of this kinase would lead to acutely increased levels of malonyl-CoA by releasing inhibition of ACC.

Citrate synthase effectors: Increasing mitochondrial citrate would provide substrate for fatty acid synthesis and citrate also acts as a "feed-forward" activator of ACC causing increase malonyl-CoA synthesis.

Acyl-CoA synthase effectors: Inhibition of acyl-CoA synthase would reduce cellular fatty acyl-CoA concentration releasing inhibition of ACC. This would result in increased ACC activity and malonyl-CoA levels. Agents to decrease malonyl-CoA utilization:

Malonyl-CoA decarboxylase (MCD) effectors: This enzyme catalyzes an ATP dependent decarboxylation of malonyl-CoA back to acetyl-CoA. Inhibition of MCD would acutely raise malonyl-CoA levels.

Simultaneously decreased malonyl-CoA utilization and increased production:

Fatty acid synthase (FAS) effectors: Inhibition of FAS leads to decreased utilization of malonyl-CoA by blocking its incorporation into fatty acids. FAS inhibition also leads to reduced fatty acyl-CoA levels which will activate ACC. Exemplary FAS inhibitors may be obtained as described in U.S. Patent Nos.

5,759.837 and 5,981,575, incorporated herein by reference.

These strategies for acutely increasing malonyl-CoA levels may be used together or in concert with other drugs to enhance apoptosis of cancer cells. Preferably, at least one agent in the compositions of this invention raises the level of malonyl-CoA by a mechanism other than inhibiting FAS. Decreasing CoA synthesis:

Pantothenate kinase (PanK) effectors: this enzyme catalyses an ATP dependent phosphorylation of pantothenic acid, the first step in Coenzyme A synthesis, and reduction in its activity may be expected to reduce the total amount of CoA with the consequent lowering of available free CoA.

Phosphopantothenoylcysteine synthetase effectors: this enzyme catalyses the ATP dependent addition of cysteine to 4-phosphopantothenic acid to form 4- phosphopantothenoyl-L-cysteine, the second step in Coenzyme A synthesis, and reduction in its activity may be expected to reduce the total amount of CoA with the consequent lowering of available free CoA.

Phosphopantothenoylcysteine decarboxylase effectors: this enzyme catalyses the removal of the alpha-carboxyl group of cysteine from 4-phosphopantothenoyl-L- cysteine to form 4-phosphopantotheine, the third step in Coenzyme A synthesis, and reduction in its activity may be expected to reduce the total amount of CoA with the consequent lowering of available free CoA.

Phosphopantotheine adenylyltransferase (also called dephospho-CoA pyrophosphorylase) effectors: this enzyme catalyses the addition of adenine to 4- phosphopantotheine, consuming ATP and producing dephospho-CoA and pyrophosphate, the fourth step in Coenzyme A synthesis. The final step in CoA synthesis, ATP dependent phosphorylation of dephospho-CoA to CoA, is performed by dephospho-CoA kinase, which is probably an additional catalytic activity of the phosphopantotheine adenylyltransferase enzyme. Inhibition of PanK, or of phosphopantothenoylcysteine synthetase, or of phosphopantothenoylcysteine decarboxylase, or of phosphopantotheine adenylyltransferase would acutely inhibit CoA synthesis, and would decrease free CoA.

Sequestration of cellular CoA in the form of stable CoA-esters:

Certain synthetic agents are taken up by cells and esterified with CoA by various cellular enzymes to form stable CoA-esters. A direct effect of such agents is to decrease free CoA by the amount of CoA that is incorporated into stable CoA- esters. These CoA-esters may or may not have additional biological activities within the cell. Two examples of such synthetic agents are TOFA and etomoxir. Administration of a sufficiently large dose of such an agent to a tumor cell would sequester enough CoA in the form of its stable CoA-ester to decrease free CoA by a functionally significant amount.

ADMINISTRATION OF THE COMPONENTS Therapeutic agents according to this invention are preferably formulated in pharmaceutical compositions containing the agent and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain other components so long as the other components do not reduce the effectiveness of the agent according to this invention so much that the therapy is negated. Pharmaceutically acceptable carriers are well known, and one skilled in the pharmaceutical art can easily select carriers suitable for particular routes of administration (Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 1985).

The pharmaceutical compositions containing any of the agents of this invention may be administered by parenteral (subcutaneously, intramuscularly, intravenously, intraperitoneally, intrapleurally, intravesicularly or intrathecally), topical, oral, rectal, or nasal route, as necessitated by choice of drug. The concentrations of the active agent in pharmaceutically acceptable carriers may range from 0.01 mM to 1 M or higher, so long as the concentration does not exceed an acceptable level of toxicity at the point of administration.

Dose and duration of therapy will depend on a variety of factors, including the therapeutic index of the drugs, disease type, patient age, patient weight, and tolerance of toxicity. Dose will generally be chosen to achieve serum concentrations from about 0.1 μg/ml to about 100 μg/ml. Preferably, initial dose levels will be selected based on their ability to achieve ambient concentrations shown to be effective in in-vitro models, such as those described herein, and in-vivo models and in clinical trials, up to maximum tolerated levels. Standard clinical procedure prefers that chemotherapy be tailored to the individual patient and the systemic concentration of the chemotherapeutic agent be monitored regularly. The dose of a particular drug and duration of therapy for a particular patient can be determined by the skilled clinician using standard pharmacological approaches in view of the above factors The response to treatment may be monitored by analysis of blood or body fluid levels of the agent according to this invention, measurement of activity of the agent or its levels in relevant tissues or monitoring disease state in the patient The skilled clinician will adjust the dose and duration of therapy based on the response to treatment revealed by these measurements

EXAMPLES

In order to facilitate a more complete understanding of the invention, a number of Examples are provided below However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only

Example 1. Inhibition of FAS in cells /^'/; vitro

TOFA. Cerulenin. and C75 all inhibited fatty acid synthesis in human breast cancer cells The human breast cancer cell lines, SKBR3 and MCF7 were maintained in RPMI with 10% fetal bovine serum Cells were screened periodically for Mycoplasma contamination (Gen-probe) All inhibitors were added as stock 5 mg/ml solutions in DMSO For fatty acid synthesis activity determinations, 5xl0⁴ cells/well in 24 well plates were pulse labeled with [U- C]-acetate after exposure to drug, and hpids were extracted and quantified as described previously (Pizer, et al , 1988) For MCF7 cells, pathw ay activ ity w as determined aftei 2 hours of lnhibitoi exposure SKBR3 cells demonstrated slower response to FAS inhibitors, possibl) because of their extremelv high FAS content so pathw av actmtv was determined after 6 hours of inhibitor exposure

In standard pulse labeling expeπments in which breast cancer cell lines, SKBR3 and MCF7 were labeled for 2 hours after exposure to FA synthesis inhibitors, TOFA. C75, and cerulenin all inhibited [U ⁴C-acetate] incorporation into hpids to a similar extent (Figure IB and D) In numerous similar expeπments (not shown), TOFA maximally inhibited FA synthesis in the 1 to 5μg/ml dose range in all cell lines tested, and cerulenin and C75 maximally inhibited FA synthesis in the range of 1 Oμg/ml. Example 2. Effect of the same inhibitors on cell growth

TOFA, Cerulenin, and C75 all inhibited fatty acid synthesis in human breast cancer cells, but showed differential cytotoxicity. Cells and inhibitors were as described for Example 1. For clonogenic assays, 4x 10⁵ cells were plated in 25 cm² flasks with inhibitors added for 6 hours in concentrations listed. Equal numbers of treated cells and controls were plated in 60 mm dishes. Clones were stained and counted after 7 to 10 days.

Although all inhibitors reduced FA synthesis to a similar degree, TOFA was non-toxic or stimulatory to the cancer cell growth in the dose range for ACC inhibition, as measured by clonogenic assays, while cerulenin and C75 were significantly cytotoxic in the dose range for FAS inhibition (Figure 1 C and E). The profound difference between the cytotoxic effects of ACC and FAS inhibition demonstrate that the acute reduction of fatty acid production per se is not the major source of cell injury after FAS inhibition.

Example 3. Measurement of malonyl-CoA.

The most obvious difference in the expected results of inhibiting these two enzymes was that malonyl-CoA levels should fall after ACC inhibition, but should increase after FAS inhibition. Although not previously investigated in eukaryotcs, recent data in E. coli have demonstrated elevated levels of malonyl-CoA resulting from exposure to cerulenin (Chohnan, et al., 1997, "Changes in the size and composition of intracellular pools of non-csterified coenzyme A and coenzyme A thioesters in aerobic and facultatively anaerobi bacteria," Applied and Environmental Microbiology, 63:555-560). Malonyl-CoA levels were measured in cells subjected to FAS inhibition and to inhibition by TOFA under conditions described in Example 2.

Malonyl-CoA levels were measured in MCF-7 cells using the HPLC method of Corkey, et al ("Analysis of acyl-coenzyme A esters in biological samples, "Methods in Enzymology, 166:55-70). Briefly, 2.5 x 10⁵ cells/well in 24 well plates were subjected to 1.2 ml of 10% TCA at 4° C after various drug treatments. The pellet mass was recorded and the supernatant was washed 6 times with 1.2 ml of ether and reduced to dryness using vacuum centrifugation at 25° C. Coenzyme-A esters were separated and quantitated using reversed phase HPLC on a 5 μ Supelco C18 column with a Waters HPLC system running Millenium" software mo tonng 254 nm as the maximum absorbance for coenzyme-A The following gradients and buffers were utilized: Buffer A: 0.1 M potassium phosphate, pH 5.0, Buffer B' 0.1 M potassium phosphate, pH 5 0, with 40% acetonitπle. Following a 20 mm. isocratic run with 92% A, 8% B at 0.4 ml/min, flow was increased to 0 8 ml/min over one minute whereupon a linear gradient to 10% B was run until 24 min then held at 10% B until 50 min. where a linear gradient was run to 100% B at 55 min., completing at 60 min The following coenzyme-A esters (Sigma) were run as standards- malonyl-CoA, acetyl-CoA, glutathione-CoA, succinyl-CoA, HMG-CoA, and free CoA Samples and standards were dissolved in 50 μl of buffer A Coenzyme-A esters eluted sequentially as follows malonyl-CoA, glutathione-CoA, free CoA, succmyl-CoA, HMG-CoA, and acetyl-CoA Quantitation of coenzyme-A esters was performed by the Millenium " software. Direct measurement of coenzyme-A deπvatives in MCF-7 cells by reversed phase HPLC of acid soluble extracts from drug treated cells confinncd that both cerulenin and C75 caused a rapid increase in malonyl-CoA levels while TOFA reduced malonyl-CoA levels Figure 2A is a representative chromatograph demonstrating the separation and identification of coenzyme-A derivatives important in cellular metabolism Malonyl-CoA is the first of these to elute, with a column retention time of 19-22 minutes The overlay of chromatographs in Figure 2B shows that cerulenin treatment lead to a marked increase in malonyl-CoA over the control while TOFA caused a significant reduction. The chemical identity of the malonyl-CoA was independently confirmed by spiking samples with standards (not shown)

Malonyl-CoA levels were markedly increased with FAS inhibition and reduced by TOFA. Analysis of multiple expeπments in Figure 2C demonstrated that following a 1 hour exposure to cerulenin or C75 at 10 μg/ml, malonyl-CoA levels increased by 930% and 370% respectively, over controls, while TOFA treatment (20 μg/ml) led to a 60% reduction of malonyl-CoA levels The concentration of TOFA required for maximal reduction of malonyl-CoA levels was 4 fold higher than the dose for pathway inhibition m Figure IB and D However, optimal cultures for extraction of CoA deπvatives had 5 fold higher cell density than the cultures used in the other biochemical and viability assays presented

The remarkable increase malonyl -CoA after FAS inhibition can be attributed in part to the release of long-chain fatty acyl-CoA inhibition of ACC leading to an increase in ACC activity (Figure 1A) Moreover, the cerulenin- mduced increase in malonyl-CoA levels occurred within 30 minutes of treatment (930 +/-15% increase over control, not shown), withm the time frame of FA synthesis inhibition, and well before the onset of DNA synthesis inhibition or early apoptotic events (Pizer, et al , 1998) Thus, high levels of malonyl-CoA were a characteristic effect of FAS inhibitors and temporally preceded the other cellular responses, including apoptosis

The levels of cerulenin or C75 which induce high levels of malonyl- CoA are cytotoxic to human breast cancer cells as measured by clonogenic assays and flow-cytometric analysis of apoptosis using merocyanfn 450 staining (Pizer, et al., 1998) FAS inhibition causes high malonyl-CoA levels by inhibiting its consumption through FAS inhibition, with concomitant stimulation of synthesis by relieving the inhibitory effect of long-chain acyl-CoA's upon ACC activity (Figure 2)

Example 4. TOFA rescue of FAS inhibition

TOFA rescue of FAS inhibition demonstrates that high levels of malonyl-CoA are responsible for cancer cell cytotoxicity. If the elevated levels of malonyl-CoA resulting from FAS inhibition were responsible for cytotoxicity, then it should be possible to rescue cells from FAS inhibition by reducing malonyl-CoA accumulation with TOFA Co-adm istration of TOFA and cerulenin to SKBR3 cells (Figure 3A) abrogated the cytotoxic effect of cerulenin alone in clonogenic assays performed as descπbed in Example 2 In MCF7 cells (Figure 3C), TOFA produced a rescue of both cerulenin and C75 under similar expeπmental conditions

Representative flow cytometπc analyses of SKBR3 cells (Figure 3B) and MCF7 (Figure 3D) substantiated these findings, since TOFA rescued cells from cerulenin induced apoptosis Apoptosis was measured by multiparameter flow cytometry using a FACStar^plus flow cytometer equipped with argon and krypton lasers (Becton Dickinson). Apoptosis was quantified using merocyanine 540 staining (Sigma), which detects altered plasma membrane phospholipid packing that occurs early in apoptosis, added directly to cells from culture (Pizer, et al., 1998; Mower, et al., 1994, "Decreased membrane pospholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis, J. Immunol, 152:4832-4842). In some experiments, chromatin conformational changes of apoptosis were simultaneously measured as decreased staining with LDS-751 (Exciton) (Frey, et al, 1995, "Nucleic acid dyes for detection of apoptosis in live cells," Cylomeliγ, 21 :265-274). Merocyanine 540 [ lOμg/ml] was added as a 1 mg/ml stock in water. Cells were stained with LDS-751 at a final concentration of l OOnM from a ImM stock in DMSO. The merocyanine 540-positive cells were marked by an increase in red fluorescence, collected at 575 +/- 20 nm, 0.5 to 2 logs over merocyanine 540-negative cells. Similarly, the LDS-751 dim cells demonstrated a reduction in fluorescence of 0.5 to 1.5 logs relative to noπnal cells, collected at 660 nm with a DF20 band pass filter. Data were collected and analyzed using CellQuest software (Becton Dickinson).

In these experiments, all LDS-751 dim cells were merocyanine 540 bright, however a population of merocyanine 540 bright cells were detected that were not yet LDS-751 dim. All merocyanine 540 bright cells were classified as apoptotic. These experiments also confiniicd the differential cytotoxicity between TOFA (<5% increase in apoptosis; no reduction in clonogcnicity) compared to cerulenin (>85% apoptosis; 70% reduction in clonogenicity). Taken together, these studies show that high malonyl-CoA levels play a role in the cytotoxic effect of FAS inhibitors on cancer cells.

Example 5. Effect of FAS inhibitors on tumor cell growth in vivo

To determine if the effects of FAS inhibition seen in vitro would translate to an in vivo setting requiring systemic activity, C75 was tested against subcutaneous MCF-7 xenografts in athymic nude mice, to quantitate effects on FA synthesis and the growth of established solid tumor. Previous studies have demonstrated local efficacy of cerulenin against a human cancer xenograft (Pizer, et al., 1996, "Inhibition of fatty acid synthesis delays disease progression in a xenograft model of ovarian cancer," Cancer Res., 56: 1189-1193), but were limited by the failure of cerulenin to act systemically. The similar responses of breast cancer cells to cerulenin and C75 in vitro suggested that C75 might be effective in vivo against xenografted breast cancer cells.

Subcutaneous flank xenografts of the human breast cancer cell line, MCF-7 in nu/nu female mice (Harlan) were used to study the anti-tumor effects of C75 in vivo. All animal expeπments complied with institutional animal care guidelines. All mice received a 90-day slow-release subcutaneous estrogen pellet (Innovative Research) in the anteπor flank 7 days before tumor inoculation. 10 MCF-7 cells were xenografted from culture in DMEM supplemented with 10% FBS and insulin 10 μg/ml.

Treatment began when measurable tumors developed about 10 days after inoculation. Eleven mice (divided among two separate expeπments of 5 and 6 mice each) were treated intraperitoneally with weekly doses of C75 at 30 mg/kg in 0.1 ml RPMI. Dosing was based on a single dose LDio determination of 40 mg kg in BALB/c mice; 30 mg/kg has been well tolerated in outbred nude mice. Eleven control mice (divided in the same way as the treatment groups) received RPMI alone Tumor volume was measured with calipers in three dimensions Experiment was terminated when controls reached the surrogate endpoint.

In a parallel experiment to determine fatty acid synthesis activity in treated and control tumors, a group of MCF-7 xenografted mice were treated with C75 or vehicle at above doses and sacrificed after 3 hours. Tumor and hver tissue were ex vivo labeled with [U^,4*C], lipids were extracted and counted as described (Rashid, et al, 1997).

In an additional parallel expeπment to histologically examine treated and control tumors, 6 C75 treated and 6 vehicle control mice were sacπficed 6 hours after treatment. Tumor and normal tissues were fixed in neutral-buffered formalin, processed for routine histology, and immunohistochemistry for FAS was performed. Immunohistochemistry for FAS was performed on the MCF-7 xenografts using a mouse monoclonal anti-FAS antibody (Alo, et al., 1996) at 1 :2000 on the Dako Immunostainer using the LS AB2 detection kit.

Fatty acid synthesis pathway activity in tissues of xenografted mice was determined by ex vivo pulse labeling with [U ⁴C]-acetate. The tumor xenografts had 10-fold higher FA synthesis activity than liver, highlighting the difference in pathway activity between benign and malignant tissues (Figure 4A). FAS expression in the MCF-7 xenograft paralleled the high level of FA synthesis activity (Figure 4B). Intraperitoneal injections of C75 at 30 mg/kg reduced fatty acid synthesis in ex vivo labeled liver by 76% and in the MCF-7 xenografts by 70% within 3 hours (Figure 4A). These changes in FA synthesis preceded histological evidence of cytotoxicity in the xenograft, which became evident 6 hours after treatment (Figures 4 C and 4D). The C75 treated xenografts showed numerous apoptotic bodies throughout the tumor tissue, which were not seen in vehicle treated tumors. Histological analysis of liver and other host tissues following C75 treatment showed no evidence of any short or long term toxicity (not shown).

C75 treatment of the xenografts leads to cytotoxicity and reduction in tumor growth without injury to normal tissues. Tumor histology 6 hours following a 30 mg/kg dose of C75 demonstrates significant cytotoxicity compared to control tumor (Figures 4 C and 4D, attached preprint). Note the evidence of apoptotic bodies in the C75 treated xenograft while examination of liver and other organs show no evidence of tissue injury (data not shown). Weekly intraperitoneal C75 treatment retarded the growth of established subcutaneous MCF-7 tumors compared to vehicle controls, demonstrating a systemic anti-tumor effect (Figure 4E). After 32 days of weekly treatments, there was a greater than eight-fold difference in tumor growth in the treatment group compared to vehicle controls. Similar to cerulenin, transient reversible weight loss was the only toxicity noted (Pizer, et al., 1996).

The systemic pharmacologic activity of C75 provided the first analysis of the outcome of systemic FAS inhibitor treatment. The significant anti- tumor effect of C75 on a human breast cancer xenograft in the setting of physiological levels of ambient fatty acids was similar to the in vitro result in serum supplemented culture, and was consistent with a cytotoxic mechanism independent of fatty acid starvation.

Example 6. Human cancer cells have high steady state levels of malonyl-CoA in vivo. The result in Example 5 suggested that malonyl-CoA accumulation may not be a significant problem in normal tissues, possibly because FA synthesis pathway activity is normally low, even in lipogenic organs such as the liver. It is of further interest that, while malonyl-CoA was the predominant low molecular weight

CoA conjugate detected in breast cancer cells in these experiments, other studies have reported predominantly succinyl-CoA and acetyl-CoA in cultured hcpatocytes (Corkey, 1988). The high level of malonyl-CoA in the tumor tissues reflects the high level of fatty acid synthesis in the tumor cells compared to liver(Rashid. et al., 1997).

Using the MCF7 human breast cancer xenograft model of Example 5, malonyl-CoA levels were measured in the tumor xenograft and liver from the same animal using high-performance liquid chromato graph y. Figure 3 below shows high levels of malonyl-CoA in the tumor tissue compared to the liver. In addition, the distribution of other CoA derivatives are markedly altered. For example, while liver has about 10 fold less malonyl-CoA compared to the xenograft, it has about 10 fold higher levels of acetyl-CoA, and higher levels of other CoA derivatives, particularly succinyl-CoA. Differences in CoA derivative profiles may be indicative of larger differences in energy metabolism between cancer cells and hepatocytes.

For purposes of clarity of understanding, the foregoing invention has been described in some detail by way of illustration and example in conjunction with specific embodiments, although other aspects, advantages and modifications will be apparent to those skilled in the art to which the invention pertains. The foregoing description and examples are intended to illustrate, but not limit the scope of the invention. Modifications of the above-described modes for carrying out the invention that are apparent to persons of skill in medicine, immunology, hybridoma technology, pharmacology, and/or related fields are intended to be within the scope of the invention, which is limited only by the appended claims. All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMS:

1. A method for inhibiting growth of tumor cells in an organism comprising administering to the organism a composition which causes acute depletion of intracellular free Coenzyme A in cancer cells in said organism.

2. The method of claim 1 , wherein intracellular malonyl CoA in cells of said organism rises abruptly.

3. The method of claim 1 , wherein intracellular malonyl CoA in cells of said organism rises within 3 hours of said administration.

4. The method of claim 1 , wherein intracellular malonyl CoA rises prior to growth inhibition of the cells.

5. The method of claim 1 , wherein said rise in intracellular malonyl CoA is correlated with reduced consumption of malonyl CoA.

6. The method of claim 1 , wherein said rise in intracellular malonyl CoA occurs prior to any increase in rate of consumption of malonyl CoA.

7. The method of claim 1, wherein said rise in intracellular malonyl CoA is correlated with reduced intracellular activity of malonyl CoA decarboxylase (MCD) or reduced intracellular activity of fatty acid synthase.

8. The method of claim 1 , wherein said composition comprises an inhibitor of MCD.

9. The method of claim 1 , wherein said rise in intracellular malonyl CoA is correlated with increased synthesis of malonyl CoA.

10. The method of claim 1 , wherein said rise in intracellular malonyl CoA is correlated with increased intracellular activity of acetyl-CoA carboxylase (ACC).

11. The method of claim 1 , wherein said composition comprises an activator of ACC, an activator of citrate synthase, an inhibitor of 5'-AMP- activated protein kinase (AMPK), and/or an inhibitor of acyl CoA synthase.

12. The method of claim 1, wherein a second chemotherapeutic agent is administered to the organism.

13. The method of claim 1 , wherein intracellular malonyl CoA level prior to administration of said composition is at least 2-fold above normal malonyl CoA level in non-malignant cells.

14. The method of claim 1, wherein intracellular level of malonyl CoA is elevated and intracellular level of acetyl CoA and free CoA are reduced relative to pre-treatment levels.

15. The method of claim 1 , wherein fatty acid synthesis rate in some cells of said organism is at least 2-fold above normal prior to administration of said composition, and administration of said composition is cytotoxic to said cells.

16. The method of claim 1, wherein fall in intracellular free Coenzyme A level is correlated with appoptosis of cells having decreased Coenzyme A.

17. The method of claim 1 , wherein said composition comprises an inhibitor of Pantothenate kinase, an inhibitor of Phosphopantothenoylcysteine synthetase, an inhibitor of Phosphopantothenoylcysteine decarboxylase, and/or an inhibitor of Phosphopantotheine adenylyltransferase.

18. The method of claim 1 , wherein said composition comprises a substrate capable of esterification to CoA.

19. The method of claim 1, wherein said organism comprises tumor cells having elevated fatty acid synthesis rates and cell number of said tumor cells is reduced subsequent to administration of said composition.

20. A screening method to assist in detecting compositions which are selectively cytotoxic to tumor cells comprising administering a target composition to a target cell, monitoring intracellular levels of free and/or derivatized Coenzyme A in said cell subsequent to said administration, wherein an abrupt decrease in intracellular free Coenzyme A is indicative of selective cytotoxicity.

21. A screening method to assist in classifying compositions which are selectively cytotoxic to tumor cells comprising administering a target composition to a target cell, in the absence, and in parallel, in the presence of sufficient ACC inhibitor to limit the production of malonyl-CoA, wherein a difference in cytotoxicity is indicative of a cytotoxic activity derived from an effect on intracellular levels of free and/or derivatized Coenzyme A.