WO2006024026A2 - Molecular profile of statin responsive cancers and uses thereof - Google Patents

Abstract

Inhibitors of the mevalonate pathway are shown to affect the phosphorylation and activation of oncogenic signaling molecules, demonstrating a mechanism for the anti-proliferative activity of such agents. Methods are provided for predicting whether a tumor will be responsive to therapy. The ability to predict responsiveness to therapy allows patient selection, optimization of treatment, determination of whether on whether to proceed with a specific therapy, and how to optimize dose, and choice of treatment. An oncogenic profile is obtained from the subject cells, where the phosphorylation status of specific oncogenic signaling molecules associated by a responsive phenotype is determined. Tumors having an activated myc signaling pathway, as evidenced by the phosphorylation status of proteins in the pathway, are shown to be responsive to treatment with mevalonate pathway inhibitors, including statins.

Description

MOLECULAR PROFILE OF STATIN RESPONSIVE CANCERS AND USES THEREOF

[01] Cancer is caused by multiple genetic events that result in the activation of proto- oncogenes and/or the inactivation of tumor suppressor genes. In some areas of the world, cancer has become or shortly will become the leading disease-related cause of death of the human population. For example, in the United States, cancer is the second leading cause of death behind cardiovascular disease, and it is projected that cancer will become the leading cause of death within a few years. The medical treatment of cancer still has many unmet needs. Surgery and radiation are generally only successful if the cancer is found at an early, localized stage. Once the disease has progressed to locally advanced cancer or metastatic disease, these therapies are less successful. Existing chemotherapeutic treatments are largely palliative in these advanced tumors, particularly in the case of the common epithelial tumors such as lung, colorectal, breast, prostate, and pancreatic cancers. Although a few chemotherapeutic regimens have yielded lasting remissions or cures (for example, in testicular cancer and childhood leukemias), it is clear that new therapeutic options are necessary.

[02] Mechanism-based approaches have been explored for cancer therapy.

Antimetabolite drugs (for example, methotrexate and mercaptopurine) were developed on the basis of a scientific understanding of key enzyme steps in nucleotide biosynthesis and the sensitivity of tumor cells to alterations in these pathways. Overall, though, the identification of successful agents with clinical utility has been a mostly empirical process up to now, not least because of the focus on the antitumor efficacy of potential new agents in cell-culture cytotoxicity assays that do not effectively reflect the complexities of human cancer. This often makes it difficult to assess why a particular drug can be successful or why others may fail. In the absence of a specific mechanistic understanding, particularly with regard to a molecular target, it is difficult to learn from the successes and failures and understand why different tumor types have different susceptibilities.

[03] A genetic defect in a tumor may identify a potential target, but it can only serve as a successful target for drug discovery if it is pharmaceutically tractable. For example, there is strong evidence that if one were to disrupt protein-protein interactions such as Myc/Max dimerization, specific SH2 domain interactions, or Ras/Raf binding, one could inhibit the function of pathways essential to certain tumor cells. Progress to develop inhibitors of these protein-protein interactions has, however, proven problematic. Model peptides have been found, but they are often difficult to convert into molecules with appropriate pharmaceutical properties. This process often entails the development of low molecular weight (<600 daltons) organic molecules with sufficient potency, pharmacokinetic, and safety profiles to be considered a drug suitable for testing in humans. [04] Knowledge of cellular signaling pathways can also be helpful for exploiting rational targets that prove difficult to inhibit. For example, many of the early approaches to inhibit Ras function failed but knowledge of the pathways afforded new targets in Raf and MEK. This strategy used to identify "secondary" targets is known as synthetic lethality.

[05] 3-hydroxy-3methylglutaryl coenzyme A (HMGcoA) reductase is a key enzyme of the cholesterol biosynthesis pathway and also functions in the synthesis of isoprenoids, such as dolichols, ubiquinones and protein prenylation. Mevalonate, the product of HMGcoA reductase, is important for cholesterol synthesis, which is essential in membrane fluidity, and is also required for biosynthesis of isoprenoid intermediates which serve as lipid attachments for the posttranslational modification of signal transducing proteins. Although perturbations in the level of cholesterol in cell membranes have also been associated with some cancers the role of HMGcoA reductase in tumorigenesis has not been established.

[06] Statins specifically inhibit the activity of HMGcoA reductase, resulting in the disruption of the synthesis of mevalonate. The pleiotropic effects of inhibiting the mevalonate pathway have gained interest in regulating the pathophysiologies of coronary heart disease, angiogenesis, and reduction of inflammation. Recently statins have been demonstrated to treat autoimmune disease in several multiple sclerosis murine models by promoting differentiation of ThO cells into Th2 cells.

[07] The transformation and malignant growth of tumor cells is a complex process, which can be variable even within a particular tissue type. Analytical methods that can define the phenotype of tumor cells are useful in determining appropriate therapy, and are therefore of clinical interest. Additionally, knowledge of the mechanism by which a chemotherapeutic agent acts is useful determining optimal formulation and dosage of such agents; in screening for agents effective in treating cancer; and in following patients through a course of treatment.

Relevant Literature [08] The review articles Dimitroulakos et al. (2001) Clinical Cancer Research, 7: 158-

167; Kelvin et al. (2003) Clinical Cancer Research, 9: 10-19; Wong et al. (2002) Leukemia,

16: 508-519; and Chan et al. (2003) Clin. Cancer Res. 9:10-19 provide a useful overview of the field. [09] The effect of statins on leukemias was reported by Clutterbuck et al. (1998) Br. J.

Haematology, 102: 522-527; Li et al. (2003) Blood, 101 : 3628-3634; van de Donk et al.

(2002) Leukemia, 16: 1362-1371; and by Wong et al. (2001) Clin. Cancer Res. 7:2067-

2075. [10] The effect of statins on brain tumors is discussed by Bouterfa et al. (2000)

Anticancer Research, 20: 2761-2772; Kikuchi et al. (1997) J. Neurooncology, 34: 233-239. [11] The effect of statins on breast cancer is discussed by Denoyelle et al. (2001)

Carcinogenesis, 22: 1139-1148; Seeger et al. (2003) Exp. Clin. Endocrinol. Diabetes, 111 :

47-48; and Shibata et al. (2003) Carcinogenesis, 24: 453-459. [12] The effect of statins on colon tumorigenesis is discussed by Narisawa et al. (1994)

Carcinogenesis, 15: 2045-2048; and on human lung carcinoma in Polo and de Bravo (2001)

Arch. Physiological Biochemistry, 109: 435-440. [13] Other references regarding the effects of statins include Katano et al. (2004) Proc

Natl Acad Sci U S A 101, 4960-5; Otsuki et al., (2004) Oncol Rep 11, 1053-8; Chan et al.

(2003) Clin Cancer Res 9, 10-9; Furst et al. (2002) Cell Physiol Biochem 12, 19-30;

Denoyelle et al. (2001) Carcinogenesis 22, 1139-48; Feleszko et a/. (2000) Clin Cancer Res

6, 2044-52. [14] Induction of apoptosis by statins is discussed by Wang et al. (2003) Clin. Endocrinol.

Metabolism, 88: 3021-3026; and Wong et al. (2001) Clinical Cancer Research, 7: 2067-

2075.

SUMMARY OF THE INVENTION

[15] Methods are provided for predicting whether a tumor will be responsive to therapy that is targeted to inhibit the mevalonate pathway, which therapy may include administration of statins. Responsive tumors have a specific profile of oncogenic signaling molecule activation. The signaling molecules are activated/inactivated by inhibition of the mevalonate synthesis pathway. The ability to predict responsiveness to therapy allows patient selection, optimization of treatment, determination of whether on whether to proceed with a specific therapy, and how to optimize dose, choice of treatment, and the like. In another embodiment, methods are provided for high throughput screening of candidate mevalonate pathway inhibitors for efficacy as anti-cancer agents. Methods of preventing tumor development through administration of mevalonate pathway inhibitors are also provided.

[16] In practicing the methods, an oncogenic profile is obtained from the subject cells, where the phosphorylation status of specific oncogenic signaling molecules associated by a responsive phenotype is determined. In one embodiment of the invention, the phosphorylated signaling molecules comprise one or more members of the myc oncogenic signaling profile, e.g. raf, erk, myc, etc. Tumors having an activated myc signaling profile, as evidenced by the phosphorylation status of proteins in the pathway, are shown to be responsive to treatment/prevention with mevalonate inhibitors, including statins.

[17] In some embodiments of the invention, the dephosphorylation of proteins in oncogenic signaling pathways is monitored in response to the presence of a mevalonate inhibitor in order to asses the efficacy of therapy; as a method of compound screening; for optimizing drug formulations and drug doses for cancer treatment; for assessment of combination therapies, and the like. Of particular interest are methods of optimizing the dose of mevalonate inhibitors, and combinations of agents. It is found that combinations of statins can provide for a greater effectiveness than single agents.

[18] Reagents and kits thereof that find use in practicing the subject methods are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[19] Figure 1A-D. Statins reversed the neoplastic properties of mouse and human lymphomas induced by different oncogenes. A) The HMG-CoA reductase enzyme mediates the production of many metabolites required for the post-transcriptional regulation and activation of oncogenes known to be required to sustain tumorigenesis. B) The influence of Statins on cell proliferation was analyzed for murine lymphomas derived from a conditional transgenic model of MYC-induced lymphomagenesis utilizing the Tet system. Tumor derived cell lines were treated with 10μM Atorvastatin (AT), 10μM Simvastatin (SM), 10μM Lovastatin (LOV) and 10μM Pravastatin (PRA) analyzed after 24 and 48 hours. Representative data is shown from results from one of six tumor-derived cell lines from our conditional MYC transgenic model. C) Influence of Atovastatin (10 and 50 μM) on the cellular proliferation in murine trasnsgenic lymphomas induced through the expression of MYC, RAS or MYC and BCL2 compared with inactivation of these respective oncogenes. Tumor derived cell lines were generated from transgenic mice that conditionally expressed oncogenes under the Tet system. To suppress oncogene expression, cell lines were treated with Doxycycline (Dox) (20 ng/ml). D) Influence of Atorvastatin on human lymphomas (Jurkat, EL-4) or leukemia (CML) was measured. For both murine and human tumors, the effects of Atorvastatin were also analyzed in the presence of the down-stream product of HMG-CoA reductase, mevalonate (100 μM) (Mev). Proliferation was measured by [³H]thymidine incorporation. Results are presented as stimulation index (Sl) measured as the incorporation of [³H]thymidine in the presence versus the absence of treatment with Statins, Doxycycline and/or mevalonate. Experiments were performed in triplicate. ** PO.0001 * P<0.001.

[20] Figure 2A-G. Atorvastatin reversed tumorigenesis in vivo. A) Survival of mice transplanted with a MYC-induced lymphoma cell line. Mice that were moribund with tumor were either non-treated (square) or treated with Doxycycline in their drinking water (100 μg/ml) to inactivate MYC (circle) or treated with Atorvastatin at doses of 1mg/kg (triangle), or 10mg/kg (star). Each cohort consisted of 8 mice. Representative picture of mouse B) prior to treatment and C) after treatment with Atorvastatin (1 mg/kg) for 8 days. D) Histology of a tumor prior to treatment and E) after treatment with Atorvastatin, stained with haematoxylin and eosin. F) TUNEL assay was performed prior to treatment and G) after treatment with Atorvastatin. Representative data is shown from 1 of 8 mice. Similar results were seen from the injection of three different tumor derived lymphoma cell lines.

[21] Figure 3. Atorvastatin prevented the onset of MYC-induced lymphomagenesis.

Kaplan-Meyer survival curves of transgenic mice conditionally overexpressing the MYC transgene in murine T-cell lymphocytes using the Tet system that were either treated with Doxycycline to suppress MYC expression, not treated with Doxycycline and thus overexpressing MYC in their lymphocytes, or not treated with doxycycline, but treated with Atorvostatin at 1 , 10, or 100 mg/kg as indicated. Each cohort consisted of 11-15 mice.

[22] Figure 4A-C. Atorvastatin disrupted prenylation and phosphorylatoin of oncogenes in lymphomas. A) Measurement of the famesylation of Ras (Ras), Rho A, Rho B, Rap and Rac and geranylgeranylation of DNA-J2 were performed in a MYC-induced lymphoma cell line in which MYC was expressed (ON) or not expressed (OFF) after Doxycycline treatment (20 ng/ml) in the absence or presence of Atorvastatin (10 μM) and/or mevalonate (100 μM), as measured by Western blot analysis. B) Kinetic analysis of changes in phosphoprotein expression analyzed by FACS analysis. MYC-induced lymphoma cell lines were treated with Atorvastatin (10 μM) (AT) or with Atorvastatin (10 μM) and mevalonate (100 μM) (AT + Mev) for 24, 36 and 48 hours, as indicated. The boxes identify clusters of proteins that exhibited increased (uppper box) or decreased (lower box) phosphorylation associated with treatment with Atorvastatin (AT). C) Analysis of levels of protein expression and phosphorylation in which MYC was expressed (ON) or not expressed (OFF) after Dox treatment (20 ng/ml) in the absence or presence of Atorvastatin (10 μM) or mevalonate (100 μM). Levels of ERK1/2, phosphorylated-ERK1/2 (P-ERK1/2), Akt and phosphorylated-Akt (P-s473) (P-Akt), HMG-CoA reductase, MYC, and phosphorylated MYC at Thr58 and Ser62 (P-MYC) measured by Western analysis.

[23] Figure 5A-B. Atorvastatin induced apoptosis is associated with direct mitochondrial depolarization. A) Tumor cells overexpressing MYC (MYC ON), not expressing MYC (MYC OFF) or overexpressing MYC and treated with 100 μM mevalonate (MYC ON, Mev), 10 μM Atorvastatin (MYC ON, AT) or 10 μM Atorvastatin together with 100 μM mevalonate (MYC ON, AT + Mev) were analyzed for apoptosis by FACS for cleavage of Caspase 3. B) Integrity of mitochondria was analyzed by JC-1 staining of tumor cells overexpressing MYC (Control) or cells treated with 10 μM Atorvastatin (AT) or Atorvastatin (10 μM) together with mevalonate (100 μM) (AT + Mev).

[24] Figure 6A-C. Atorvastatin disrupted ERK1/2 and MYC phosphorylation prior to inducing apoptosis. A) MYC-induced lymphoma cell lines were treated with Atorvastatin and analyzed at indicated times for phosphorylation of ERK1/2 and B) for phosphorylation of MYC by FACS. ^"C) ^'Αtorvastat^'iή treated cells were analyzed for cleavage of Caspase 3 by FACS.

[25] Figure 7A-C. Inactivation of HMG-CoA A reductase by RNAi was sufficient to induce loss of neoplastic phenotype. A) HMG-CoA reductase and MYC protein expression in MYC- induced lymphoma cell line transfected with siRNA directed against HMG-CoA reductase versus mock transfected cells, by Western analysis. B) Levels of expression of HMG-CoA reductase, phosphorylated ERK1/2, and phosphorylated MYC after transfection of siRNA directed against HMG-CoA reductase, as measured by FACS analysis. Three color clustering of cellular populations was performed in FlowJo and unique populations are color coded non transfected (blue/purple) and transfected cells (orange). Cells were analyzed 36 hours after transfection.

[26] Figure 8. Atorvastatin induced dose dependent inhibition of proliferation. Conditional transgenic lymphoma cell lines induced using the Tet system by different oncogenes were treated with different doses of Atorvastatin and growth was measured by [³H]thymidine incorporation. Results are presented as stimulation index (Sl), as described in Figure 1. Cells were treated with Atorvastatin at doses as indicated and analyzed after 24 and 48 hours (h) of treatment. The results are presented as mean of three different experiments performed in triplicate.

[27] Figure 9. Atorvastatin induced proliferative arrest and apoptosis in tumor cells but not in normal cells. MYC-induced lymphoma cell lines and normal murine thymic T cells were examined for cell cycle transit by BRDU incorporation and apoptosis by AnnexinV- PE/7AAD staining. Cells were non-treated (Control) or treated with Dox (20 ng/ml) to inactivate MYC transgene expression, mevalonate (100 μM) (Mev), Atorvastatin (10 μM) (AT) or Atorvastatin (10μM) and mevalonate (100 μM) (AT + MEV). S-phase (upper box), G1 phase (lower left box) and G2 phase (lower right box) are indicated.

[28] Figure 10A-B. Atorvastatin induced apoptosis of tumor cells is independent of changes in expression of Bax, Bak, Bad or Bcl2. A) Cleavage of Caspase 3 and protein expression of Bak and Bax were measured by Western analysis B) Expression of Bad and Bcl2 were measured by FACS.

[29] Figure 11. Effect of combinations of statins on tumor growth in vivo.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[30] Mevalonate pathway inhibitors, including statins, are shown to inhibit tumor cell growth through deactivation of specific oncogenic signaling pathways. The responsiveness of a specific tumor to statin therapy is predicted by obtaining an oncogenic profile from the tumor cells of interest, in which profiles are obtained of the oncogenic signaling molecules that are activated in the tumor cell. In some embodiments of the invention, the activation of oncogenic signaling molecules is determined through the phosphorylation status of the molecules. The method may include comparing the obtained profile to a reference profile, e.g. a tumor cell known to be responsive, a normal cell of the same tissue type as the tumor, and the like. Optionally the tumor cells of interest are exposed to the mevalonate pathway inhibitor, and the effect of the inhibitor on the phosphorylation state of the oncogenic signaling molecules then determined.

[31] The oncogenic profile of a clinical sample is used in patient selection for therapy, to monitor treatment, modify therapeutic regimens, and to further optimize the selection of therapeutic agents; which may be administered as one or a combination of agents. With this approach, therapeutic regimens can be individualized and tailored according to the data obtained prior to, and at different times over the course of treatment, thereby providing a regimen that is individually appropriate. Oncogenic profiles may also be used in research settings to screen candidate mevalonate pathway inhibitors for anti-proliferative activity; determine the efficacy of combination therapies, and the like. The combination and dose of statins is found to be an important aspect of the formulation for anti-cancer therapies.

[32] In one embodiment of the invention, the presence of activated signaling molecules in the myc signaling pathway is detected, e.g. akt, erk1/2, myc, etc. Tumors having an activated oncogenic signaling profile, as evidenced by the phosphorylation status of proteins in the pathway, are shown to be responsive to treatment with mevalonate inhibitors, including statins.

[33] The subject invention provides a method of determining whether a patient is responsive to anti-proliferative therapy, where the method includes (a) obtaining a response profile for a sample from said subject in the absence or presence of said therapy; and (b) comparing said obtained profile to a reference response profile to determine whether said subject is susceptible to said therapy. In one embodiment, the response profile comprises determination of the phosphorylation status of a molecule in the myc signaling pathway.

[34] The subject invention also provides a kit for use in determining the susceptibility phenotype of a tumor sample, said kit comprising one or more specific binding reagents for activated oncogenic signaling molecules, and may additionally comprise one or more mevalonate pathway inhibitors. The kit may further comprise a software package for data analysis of oncogenic profiles, which may include reference profiles for comparison with the test profile.

[35] Oncogenic profile. As used herein, the term "oncogenic profile" refers to a determination of the specific signaling proteins that are activated / inactivated in a tumor cell of interest, as compared to normal levels of activation in a normal cell. The presence of certain activated / inactivated signaling molecules is predictive of whether the tumor will be responsive to mevalonate pathway inhibitors, i.e. that contacting the tumor cell with the mevalonate pathway inhibitor will inhibit growth of the tumor cell. Such oncogenic signaling molecules may be referred to as "responsive" oncogenic signaling molecules.

[36] In one embodiment of the invention, the activation of the oncogenic signaling molecule is determined by the level of post-translational phosphorylation of the protein, typically phosphorylation of serine or threonine on proteins whose activity is regulated by this modification. Typically a binding member specific for the phosphoprotein is brought into contact with the cell, or a lysate of the cell, and the level of binding determined, relative to appropriate controls.

[37] In some embodiments of the invention, the responsive oncogenic signaling molecule is myc, or a member of the myc signaling pathway. Such pathway members include myc, erk1/2, akt, etc. Myc is a member of a mitogen-activated protein kinase (MAPK) cascade. MAP kinases lie within protein kinase cascades. Each cascade consists of no fewer than three enzymes that are activated in series: a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK) and a MAP kinase (MAPK). At least 14 MAPKKKs, 7 MAPKKs, and 12 MAPKs have been identified in mammalian cells.

[38] The stimulation of tyrosine kinase receptors (RTKs) provokes the activation of

MAPKs in a multistep process. For example, the essential linkers from cell surface receptors to MAP kinase may include adaptor protein Grb2, a guanine nucleotide exchange protein, such as Sos, a small GTP binding protein, p21^ras, a cascade of protein kinase defined sequentially as MAPKKK (represented by c-Raf-1), and MAPKK such as MEK1 and MEK2. MEKs ultimately phosphorylate p44 MAPK and p42 MAPK, also known as ERK1 and ERK2 respectively, thereby increasing their enzymatic activity. Then the activated ERKs translocate to the nucleus and transactivate transcription factors, changing gene expression to promote growth, differentiation or mitosis. G protein-coupled receptors (GPCRs) can also lead to activation of MAPKs mediated by stimulation of a large number of complex cascades.

[39] Several cytokine receptors activate the ERK pathway through the activation of JAK

(JAK1 , 2, 3 and Tyk2). JAK can phosphorylate She leading to activation of the ERK1/2 pathway. Several cytoplasmic proteins have been shown to be substrates for ERK1/2 including RSK (90KDa ribosomal S6 kinase, p90rsk, also known as MAPKAP-K1), cytosolic phospholipase A2 and several microtubule-associated proteins (MAP), including MAP-1 , MAP-2, MAP-4 and Tau.

[40] ERK1/2 can activate the C-terminal kinase of RSK, leading to activation of the N- terminal kinase. The substrates of RSK include transcription factors like CREB, ERa, IKBOC/NFKB, C-FOS and glycogen synthase kinase 3 (GSK 3). RSK is implicated in cell cycle regulation by inactivation of the Myt1 protein kinase leading to activation of the cyclin- dependent kinase p34^eαcZ RSK can also phosphorylates the Ras GTP/GDP-exchange factor, Sos leading to feedback inhibition of the Ras-ERK pathway.

[41] ERK can translocate to the nucleus and phosphorylate different transcription factors, including the ternary complex factor (TCF) Elk-1 , serum response factor accessory protein Sap-1a, Ets1 , c-Myc, TaI etc. One of Ras-induced cellular responses is transcriptional activation of multiple genes, such as the immediate early gene c-fos. So the ERK pathway can link G₀ZG₁ mitogenic signals to the immediate early response.

[42] The c-Myc protein is a DNA binding protein that is involved in transcriptional control of gene expression and has been shown to be essential for cell proliferation. Coexpression of Ras with Myc allows the generation of cyclin E-dependent kinase activity and the induction of S phase. The c-Myc protein drives the p2l^kιp1 protein out of Cdk2/CyclinE complexes, which then facilitates the phosphorylation of p27 and thereby marks the protein for ubiquitination and degradation.

[43] In other embodiments, the responsive phosphorylated oncogenic signaling molecule is one or more of STAT5(Y694); STAT1 (Y701); STAT3(Y705); STAT3(S727); p44/42 (T202/Y204); p38(T180/Y182); Plcγ1 (Y783); Syk(Y352); Lck(Y505); Zap70(Y319); Ikkα (S32/36); Gsk3β (Y216); Lck(S158); β1(Y785); PKCΘ(S695); PKCΘ(T538); Slp76(Y145); PKCΘ(S676); Src(Y418); β3(Y785); β3(Y773); lkkα(S176/180); cRaf(S259); cRaf(S338); cRaf(S43); cRaf(S621); Gsk3α(S9); JNK(YI 83/T185); AKT(S473); AKT(T308); Pyk2(Y402); FAK(Y397); Lat(Y132); Lck(Y192); PKCδ(T505); cJun(S73); Bad(S112); Bad(S136); Mek(T394); Mek(S298); NFκB(S259); Vav1(Y160); cJun(S63); Syk(Y525/526); Mek1/2(S217/221); PKC-PAN; PKCδ(S643); MYC(T58/S62); p-erk1/2; p-akt(S473); and the like, where the phosphorylated residue is noted in parentheses.

[44] In a typical assay, a sample is assayed for the presence of activated / inactivated oncogenic signaling molecules by combining the sample with a specific binding member, and detecting directly or indirectly the presence of the complex formed between the two members. The term "specific binding member" as used herein refers to a member of a specific binding pair, i.e. two molecules where one of the molecules through chemical or physical means specifically binds to the other molecule.

[45] In addition to antigen and antibody specific binding pairs, peptide-MHC antigen and

T cell receptor pairs; complementary nucleotide sequences (including nucleic acid sequences used as probes and capture agents in DNA hybridization assays); peptide ligands and receptor, where at least one ligand of is the activated oncogenic signaling molecule, and the like. The specific binding pairs may include analogs, derivatives and fragments of the original specific binding member. For example, an antibody directed to a protein antigen may also recognize peptide fragments, chemically synthesized peptidomimetics, labeled protein, derivatized protein, etc. so long as an epitope is present.

[46] Monoclonal antibodies specific for oncogenic signaling molecules set forth above are commercially available. Alternatively, monoclonal or polyclonal antibodies are raised to the proteins. The antibodies may be produced in accordance with conventional ways, immunization of a mammalian host, e.g. mouse, rat, guinea pig, cat, dog, etc., fusion of resulting splenocytes with a fusion partner for immortalization and screening for antibodies having the desired affinity to provide monoclonal antibodies having a particular specificity. These antibodies can be used for affinity chromatography, ELISA, RIA, and the like. The antibodies may be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other label which will allow for detection of complex formation between the labeled antibody and its complementary epitope.

[47] Mevalonate Pathway Inhibitors. The methods according to the present invention may comprise the use of agents that are inhibitors of mevalonate synthesis or effector pathways. Mevalonate metabolites are involved in modification of G-proteins, such as Ras. Inhibitors of the mevalonate pathway have been used to inhibit isoprenylation of Ras proteins and the Raf/MAP kinase cascade (Kikuchi et a!., J. Biol. Chem., 269:20054-20059 (1994)). HMG-CoA reductase and mevalonate pyrophosphate decarboxylase are two useful targets for inhibition in the mevalonate pathway.

[48] Inhibitors of this pathway include inhibitors of mevalonate-PP kinase, e.g. Q- fluromevalonate; inhibitors of famesyl protein transferases (FTP I₁ II, III, IV) such as gliotoxin; L-744,832; α-Hydroxyfamesylphosphoric acid; Manumycin A; FTI 276, FTI 277; etc.; methyltransferase inhibitor FTS (S-trans, trans Famesylthiosalic acid) Ras inhibitor; geranylgeranyl transferase inhibitors (GGTI), e.g. GGTI-287, GGTI-297.GGTI 298,GGTI 2133, GGTI-2147; GGTI -286, which inhibits the geranylation of Rap1; squalene synthease inhibitors, e.g. squalestatin (zaragonic acid A); squalene epoxidase inhibitors, e.g. NB-598; and the like.

[49] In a preferred embodiment, the mevalonate pathway inhibitor is a statin. Statins refer to a known class of of HMG-CoA reductase inhibitors. These agents are described in detail, for example, mevastatin and related compounds as disclosed in U.S. Pat. No. 3,983,140, lovastatin (mevinolin) and related compounds as disclosed in U.S. Pat. No. 4,231 ,938, pravastatin and related compounds such as disclosed in U.S. Pat. No. 4,346,227, simvastatin and related compounds as disclosed in U.S. Pat. Nos. 4,448,784 and 4,450,171 ; fluvastatin and related compounds as disclosed in U.S. Pat. No. 5,354,772; atorvastatin and related compounds as disclosed in U.S. Pat. Nos. 4,681,893, 5,273,995 and 5,969,156; and cerivastatin and related compounds as disclosed in U.S. Pat. Nos. 5,006,530 and 5,177,080. Additional compounds are disclosed in U.S. Pat. Nos. 5,208,258, 5,130,306, 5,116,870, 5,049,696, RE 36,481, and RE 36,520. Recently the "super statin" rosuvastatin has been commercialized. The lipophilicity of certain statins make them particularly suitable for subcutaneous delivery.

[50] The term HMG-CoA inhibitor further includes all HMG-CoA reductase inhibitors described in Winokur, PCT Appl. US98/21901 , filed Oct. 16, 1998, published as WO99/20110 entitled Combination Therapy for Reducing the Risks Associated with Cardio and Cerebrovascular Disease," and the compounds and substances which are HMG-CoA inhibitors in Nichtberger, U.S. Pat. 6,136,804, Oct. 24, 2000, entitled "Combination therapy for treating, preventing, or reducing the risks associated with acute coronary ischemic syndrome and related conditions." Compactin is also described as a fungi derived competitive inhibitor of HMG-CoA reductase. An HMG-CoA reductase inhibitor with the natural structure of lovastatin can also be isolated from red rice yeast and is an HMG-CoA reductase inhibitor.

[51] The term HMG-CoA inhibitor encompasses the pharmaceutically acceptable salts of

HMG-CoA inhibitor selected. The invention includes pharmaceutically active salts of an HMG-CoA inhibitor, which may include non-toxic salts of the compounds employed in this invention which are generally prepared by reacting the free acid with a suitable organic or inorganic base. Examples of salt forms of HMG-CoA reductase inhibitors may include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, camsylate, carbonate, chloride, citrate, dihydrochloride, edentate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laureate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mutate, napsylate, mitrate, oleate, oxalate, pamaote, palpitate, panthothenate, phosphate/diphosphate, polygalacturonate, potassium sodium, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate.

[52] Ester derivatives of the above described compounds included HMG-CoA inhibitors may act as prodrugs which, when absorbed into the bloodstream of a warm-blooded animal, may cleave in such a manner as to release the drug form and permit the drug to afford improved therapeutic efficacy.

[53] The formulation and administration of statins is well known, and will generally follow conventional usage. The dosage required to treat cancer may vary from the levels used for management of cholesterol, and in some instances will be higher doses, around about 5 fold increase over conventional dosage (where conventional dosage is intended to refer to approved dosage for management of cholesterol); around about 10 fold increase over conventional dosage, and may be as much as 20 fold increase, or more. [54] In some aspects of the invention, combinations of statins are administered as anti¬ cancer therapies. Combinations comprise two or more chemically distinct statins. The statins may be complementary in terms of pharmacologic properties. Variability is noted in a myriad of contexts including their efficacy in lowering cholesterol, ability to affect phosphorylation of IkappaB, and differences in metabolism and half-life. Combining statins of different properties con provide synergistic effects. Further, variable processing, variable binding and variable half-lives, and the like, allow for particular combinations of drug to be more effective in particular cases of disease. The methods of the invention provide a new analysis of specific molecular distinctions between different types of cancers and the effect of statins on such cancers, offering opportunities for improved formulations and patient- specific therapies.

[55] Some properties of statins and provided in the following table:

Based on a 40-mg oral dose, with the exception of fluvastatin XL (80 mg).Adapted from data in Corsini A, et al. Pharmacol Ther. 1999;84:413-428, and White CM. J Clin Pharmacol.2002;42:963- 970. [56] Tumors. Tumor cells are characterized by uncontrolled growth, invasion to surrounding tissues, and metastatic spread to distant sites. Growth and expansion may utilize an ability not only to proliferate, but also to down-modulate cell death (apoptosis) and activate angiogenesis to produce a tumor neovasculature.

[57] Tumors of interest for treatment include carcinomas, e.g., colon, duodenal, prostate, breast, melanoma, ductal, hepatic, pancreatic, renal, endometrial, stomach, dysplastic oral mucosa, polyposis, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell urinary carcinoma etc.; neurological malignancies; e.g., neuroblastoma, gliomas, etc.; hematological malignancies, e.g., childhood acute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus, etc.; and the like.

[58] In one embodiment of the invention, the tumor of interest is is a type that has been associated with aberrant activation of the myc signaling pathway (see Facchini and Penn (1998) FASEB J. 12 (9):633 for a review). C-myc expression has been shown to be altered in at least a portion of human breast, colon, and cervical carcinomas, small cell lung carcinomas, osteosarcomas, glioblastomas, and hematopoietic tumors such as Burkitt lymphoma, large B-cell lymphoma, lymphoblastic lymphoma, and multiple myeloma. Translocations involving c-myc and an Ig locus have been reported as a rare occurrence in human multiple myeloma (MM).

[59] Amplification of n-myc is associated with a poor prognosis in patients with neuroblastoma. N-MYC amplification is found in approximately 3% of stage I and II, 50% of stage III and IV and 10% of stage IVs patients. N-myc amplification can also be found in small-cell lung cancer; alveolar rhabdomyosarcoma; and retinoblastoma.

[60] Chemotherapeutic agents. In some cancer patients it will be desirable to combine mevalonate pathway inhibitors with other chemotherapeutic agents, as well as non- chemotherapeutic agents such as radiation, surgery, etc.

[61] Chemotherapeutic agents may induce DNA damage, for example the topoisomerase inhibitors anthracyclines, including the compounds daunorubicin, adriamycin (doxorubicin), epirubicin, idarubicin, anamycin, MEN 10755, and the like. Other topoisomerase inhibitors include the podophyllotoxin analogues etoposide and teniposide, and the anthracenediones, mitoxantrone and amsacrine. Other DNA-damaging agents include such as nucleotide analogs, alkylating agents, efc Alkylating agents include nitrogen mustards, e.g. mechlorethamine, cyclophosphamide, melphalan (L-sarcolysin), etc.; and nitrosoureas, e.g. carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, efc. Nucleotide analogs include pyrimidines, e.g. cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FUdR), efc; purines, e.g. thioguanine (6-thioguanine), mercaptopurine (6-MP), pentostatin, fluorouracil (5-FU) efc; and folic acid analogs, e.g. methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8- dideazatetrahydrofolic acid (DDATHF), leucovorin, efc

[62] Other chemotherapeutic agents of interest include metal complexes, e.g. cisplatin

(cis-DDP), carboplatin, oxaliplatin, efc; ureas, e.g. hydroxyurea; and hydrazines, e.g. N- methylhydrazine. [63] Other chemotherapeutic agents interfere with microtubule assembly, e.g. the family of vinca alkaloids. Examples of vinca alkaloids include vinblastine, vincristine; vinorelbine (NAVELBINE); vindesine; vindoline; vincamine; etc.

[64] Samples. Methods and compositions are provided for the classification of clinical samples, e.g. tumor biopsy samples; blood samples for leukemias and lymphomas and metastatic cancers; patient tissue samples, cells, fluids, extracts of tissues, etc., according to the physiological status of cells present in the sample. The information thus derived is useful in prognosis and diagnosis, including responsiveness to therapy. The state of the cells provided in the clinical sample may be classified according to the activation of oncogenic signaling pathways of interest. The cells can also be classified as to their ability to respond to therapeutic agents and treatments.

[65] Clinical samples for use in the methods of the invention may be obtained from a variety of sources, including blood, lymph, cerebrospinal fluid, synovial fluid, tissue biopsies, skin, saliva, lavage, and the like. Such samples can comprise complex populations of cells, which can be assayed as a population, or separated into sub-populations. Such cellular and acellular sample can be separated by centrifugation, elutriation, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with Hypaque, etc. By using antibodies specific for markers identified with particular cell types, a relatively homogeneous population of cells may be obtained. Alternatively, a heterogeneous cell population can be used. Once a sample is obtained, it can be used directly, frozen, or maintained in appropriate culture medium for short periods of time. Usually a sample will comprise at least about 10² cells, more usually at least about 10³ cells, and preferable 10⁴ or more cells.

[66] The methods of the invention find use in a wide variety of animal species, including mammalian species. Animal models, particularly small mammals, e.g. murine, lagomorpha, etc. are of interest for experimental investigations. Humans are of particular interest for both diagnostic and prognostic applications of the method.

[67] Methods of particular interest include Western blots and flow cytometry. Where the sample is to be analyzed by flow cytometry, the cells will be maintained in an intact form, but are usually permeabilized in order to contact the cytoplasmic oncogenic signaling proteins. Brefeldin A is commonly utilized to prevent secretion of intracellular substances. Flow cytometry methods are known in the art, and described in the following: Flow Cytometry and Cell Storing (Springer Lab Manual), Radbruch, Ed., Springer Verlag, 2000; Ormerod, Flow Cytometry, Springer Verlag, 1999; Flow Cytometry Protocols (Methods in Molecular Biology , No 91), Jaroszeski and Heller, Eds., Humana Press, 1998; Current Protocols in Cytometry, Robinson et al., eds, John Wiley & Sons, New York, NY, 2000. Plug-flow flow cytometry that has the potential to automate the delivery of small samples from unpressurized sources at rates compatible with many screening and assay applications, may allow higher throughput, compatible with high throughput screening, Edwards et al. (1999) Cytometry 37:156-9. Both single cell multiparameter and multicell multiparameter multiplex assays, where input cell types are identified and parameters are read by quantitative imaging and fluorescence and confocal microscopy are used in the art, see Confocal Microscopy Methods and Protocols (Methods in Molecular Biology Vol. 122.) Paddock, Ed., Humana Press, 1998. These methods are described in U.S. Patent no. 5,989,833 issued Nov. 23, 1999.

[68] Where the assay relies on blotting techniques, the sample is lysed. Methods of lysis are known in the art, including sonication, non-ionic surfactants, etc. Non-ionic surfactants include the Triton™ family of detergents, e.g. Triton™ X-15; Triton™ X-35; Triton™ X-45; Triton™ X-100; Triton™ X-102; Triton™ X-114; Triton™ X-165, etc. Brij™ detergents are also similar in structure to Triton™ X detergents in that they have varying lengths of polyoxyethylene chains attached to a hydrophobic chain. The Tween™ detergents are nondenaturing, nonionic detergents, which are polyoxyethylene sorbitan esters of fatty acids. Tween™ 80 is derived from oleic acid with a Ci₈ chain while Tween™ 20 is derived from lauric acid with a C₁₂ chain. The zwitterionic detergent, CHAPS, is a sulfobetaine derivative of cholic acid. This zwitterionic detergent is useful for membrane protein solubilization when protein activity is important. The surfactant is contacted with the cells for a period of time sufficient to lyse the cells.

METHODS OF OBTAINING AND USING ONCOGENIC PROFILES

[69] The responsiveness of an individual tumor to therapeutic use of a mevalonate pathway inhibitor is predicted by obtaining an oncogenic profile from the tumor cells of interest, which may be referred to as a test profile. The oncogenic profile determines whether the tumor cells have increased levels, relative to a normal control (or reference profile), of activated oncogenic signaling molecules. Mevalonate pathway inhibitors are shown herein to have anti-proliferative activity by deactivating certain of such oncogenic signaling molecules. The presence of increased levels of a targeted oncogenic signaling molecule is indicative that the tumor will be responsive to therapy, and thus is useful in patient selection.

[70] In a related embodiment of the invention, the patient thus selected is treated with a therapeutically effective dose of the mevalonate pathway inhibitor. The patient may be followed during the course of therapy to determine the effectiveness of the regimen by profiling additional samples over time, and determining of there has been a change in the oncogenic profile in response to therapy. In another related embodiment, oncogenic profiles may be obtained after the sample has been exposed to one or more mevalonate pathway inhibitors, in order to further assess the responsiveness of the tumor cells.

[71] In one embodiment of the invention, the oncogenic profile determines the presence of increased levels of phosphorylated oncogenic signaling molecules, relative to normal control cells. Of particular interest is the presence of increased levels of activated myc pathway proteins, e.g. n-myc, c-myc, akt, erk1/2, etc. The presence of such proteins is indicative that a tumor will be responsive to treatment with statins. For example, it is shown that atorvastatin treatment decreases phosphorylation of akt at serine 473 and threonine 308; erk V₂ and myc.

[72] The relationship between mevalonate pathway inhibitors and oncogenic signaling molecules is readily determined by one of skill. For example, a panel of cells may be assessed for increased levels of oncogenic signaling molecules, particularly those that are activated by phosphorylation. The cells are then exposed to a mevalonate pathway inhibitor, and the changes in phosphorylation of oncogenic signaling molecules is detected.

[73] Cells are classified by any method that quantitates the presence of the specific phosphorylated oncogenic signaling molecule of interest. Such methods may include radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA), homogeneous enzyme immunoassays, and related non-enzymatic techniques, Western blots, whole cell staining and flow cytometry, etc. These techniques utilize specific antibodies as reporter molecules, which are particularly useful due to their high degree of specificity for attaching to a single molecular target. U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for modified protein parameters. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Flow cytometry methods are useful for measuring intracellular parameters.

[74] Where the sample is analyzed by flow cytometry, the cells are dispersed into a single cell suspension, e.g. by enzymatic digestion with a suitable protease, e.g. collagenase, dispase, etc; and the like. An appropriate solution is used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hanks balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES₁ phosphate buffers, lactate buffers, etc. The cells may be fixed, e.g. with 3% paraformaldehyde, and are usually permeabilized, e.g. with ice cold methanol; HEPES-buffered PBS containing 0.1% saponin, 3% BSA; covering for 2 min in acetone at -20⁰C; and the like as known in the art. [75] The specific binding agents, e.g. antibodies, are added to a suspension of cells, and incubated for a period of time sufficient to bind the available antigens. The incubation will usually be at least about 5 minutes and usually less than about 30 minutes. It is desirable to have a sufficient concentration of antibodies in the reaction mixture, such that the efficiency is not limited by lack of antibody. The appropriate concentration is determined by titration. The medium will be any medium that maintains the integrity of the cells. A preferred medium is phosphate buffered saline containing from 0.1 to 0.5% BSA. Various media are commercially available and may be used. Methods of analysis by flow cytometry are well-known in the art. Readouts from such assays may be the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these.

[76] The oncogenic profile readout is measured, desirably normalized, and the resulting profile may then be evaluated by comparison to reference profiles. The reference profiles may include tumor samples having known levels of phosphorylated oncogenic signaling molecules; normal (non-transformed) cells, preferably of the same tissue type as the tumor sample, e.g. a leukemia or lymphoma sample might be compared to a sample of peripheral blood mononuclear cells; etc.

[77] The comparison of a profile obtained from test cells, and a reference profile(s) is accomplished by the use of suitable deduction protocols, Al systems, statistical comparisons, etc. Similarity to reference profiles from normal cells, cells from similarly diseased tissue, from cells with responses induced by the presence of mevalonate pathway inhibitors, and the like, may be performed. A database of reference profiles can be compiled. These databases may include reference profiles from a plurality of tumor and non-tumor tissue samples.

[78] It may be convenient in a clinical setting to perform an immunoassay in a self- contained apparatus. A number of such methods are known in the art. The apparatus will generally employ a continuous flow-path of a suitable filter or membrane, having at least three regions, a fluid transport region, a sample region, and a measuring region. The sample region is prevented from fluid transfer contact with the other portions of the flow path prior to receiving the sample. After the sample region receives the sample, it is brought into fluid transfer relationship with the other regions, and the fluid transfer region contacted with fluid to permit a reagent solution to pass through the sample region and into the measuring region. The measuring region may have bound to it a conjugate of an enzyme with a specific antibody. Alternatively, kits may be provided for flow cytometry analysis of patient samples. Such kits may comprise specific binding reagents; buffers for permeabilizing and suspending cells; and/or control cells for use as a reference. [79] In some embodiments of the invention, oncogenic profiles are obtained for patient samples and other transformed and non-transformed cells in the presence of a mevalonate pathway inhibitor or candidate mevalonate pathway inhibitor. Such profiling find use in optimizing formulations for the use of mevalonate pathway inhibitors in the treatment of cancer. For example, the dose, combination of statins, excipient, combination with other anti-proliferative agents, etc. may be determined. Such profiling also finds use in optimizing selection of an agent for a patient or tumor type. Testing of candidate mevalonate pathway inhibitors for their therapeutic effectiveness against tumor cells may also be performed, e.g. in a high-throughput screening method.

[80] Cells of interest for such profiles include patient samples, as described above. Cells may further include including primary cells, normal and transformed cell lines, transduced cells and cultured cells; e.g. carcinomas of different cell origins, lymphomas of different cell types, etc. The American Type Culture Collection (Manassas, VA) has collected and makes available over 4,000 cell lines from over 150 different species, over 950 cancer cell lines including 700 human cancer cell lines. The National Cancer Institute has compiled clinical, biochemical and molecular data from a large panel of human tumor cell lines, these are available from ATCC or the NCI (Phelps et al. (1996) Journal of Cellular Biochemistry Supplement 2.:32-91). Included are different cell lines derived spontaneously, or selected for desired growth or response characteristics from an individual cell line; and may include multiple cell lines derived from a similar tumor type but from distinct patients or sites.

[81] The mevalonate pathway inhibitor or candidate mevalonate pathway inhibitor is conveniently added to the cells in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.

[82] Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself. [83] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.

[84] Based on changes in the oncogenic profile in response to the agent(s), information is derived that is useful in determining the effectiveness of an agent on a particular cell. Changes in response to therapeutic agents provides information that is informative of a patient's ability to respond to a drug. In a method of selecting an agent for treatment of a patient, an oncogenic profile of a patient sample is performed in the absence and presence of a candidate mevalonate pathway inhibitor. The ability of such an agent to dephosphorylate and inactivate relevant oncogenic signaling molecules is indicative that the agent is effective in treatment of the patient. In one embodiment of the invention, such a selecting step is following by administration of a therapeutically effective dose of the agent.

[85] The change in oncogenic profile following administration of a candidate mevalonate pathway inhibitor at different doses, in combination with, for example, other agents such as statins, conventional chemotherapeutic drugs; excipients; and the like may also be determined. The ability of a test formulation to dephosphorylate and inactivate relevant oncogenic signaling molecules is indicative of the efficacy of the formulation. In one embodiment of the invention, the formulation is tested at varying doses, and the dose that optimizes dephosphorylation and inactivation of the relevant oncogenic signaling molecules is selected for therapeutic use, or for further testing in an in vivo setting.

[86] Changes in oncogenic profile are also useful in determining the efficacy of a candidate mevalonate pathway inhibitor in the treatment of cancer. Such candidate agents may include analogs and derivatives of statins, and related compounds. In addition to the testing the efficacy of a candidate agent on an oncogenic profile, the agent may also be tested for inhibition of the mevalonate pathway, e.g. by determining if addition of mevalonate reverses the effect, monitoring anti-cholesterol properties; activity on HMG-CoA reductase, and the like.

[87] The term "agent" as used herein describes any mevalonate pathway inhibitor molecule, usually a pharmaceutical compound. Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

[88] Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Test agents can be obtained from libraries, such as natural product libraries or combinatorial libraries, for example.

[89] Libraries of candidate compounds can also be prepared by rational design. {See generally Cho et al., Pac. Symp. Biocompat. 305-16, 1998); Sun et al., J. Comput Aided MoI. Des. 12:597-604, 1998); each incorporated herein by reference in their entirety). For example, libraries of phosphatase inhibitors can be prepared by syntheses of combinatorial chemical libraries (see generally DeWitt et al., Proc. Nat. Acad. ScL USA 90:6909-13, 1993; International Patent Publication WO 94/08051 ; Baum, Chem. & Eng. News, 72:20-25, 1994; Burbaum et al., Proc. Nat. Acad. Sci. USA 92:6027-31, 1995; Baldwin et al., J. Am. Chem. Soc. 117:5588-89, 1995; Nestler et al., J. Org. Chem. 59:4723-24, 1994; Borehardt et al., J. Am. Chem. Soc. 116:373-74, 1994; Ohlmeyer et al., Proc. Nat. Acad. Sci. USA 90:10922- 26, all of which are incorporated by reference herein in their entirety.)

[90] A "combinatorial library" is a collection of compounds in which the compounds comprising the collection are composed of one or more types of subunits. Methods of making combinatorial libraries are known in the art, and include the following: U.S. Patent Nos. 5,958,792; 5,807,683; 6,004,617; 6,077,954; which are incorporated by reference herein. The subunits can be selected from natural or unnatural moieties. The compounds of the combinatorial library differ in one or more ways with respect to the number, order, type or types of modifications made to one or more of the subunits comprising the compounds. Alternatively, a combinatorial library may refer to a collection of "core molecules" which vary as to the number, type or position of R groups they contain and/or the identity of molecules composing the core molecule. The collection of compounds is generated in a systematic way. Any method of systematically generating a collection of compounds differing from each other in one or more of the ways set forth above is a combinatorial library.

[91] A combinatorial library can be synthesized on a solid support from one or more solid phase-bound resin starting materials. The library can contain five (5) or more, preferably ten (10) or more, organic molecules that are different from each other. Each of the different molecules is present in a detectable amount. The actual amounts of each different molecule needed so that its presence can be determined can vary due to the actual procedures used and can change as the technologies for isolation, detection and analysis advance. When the molecules are present in substantially equal molar amounts, an amount of 100 picomoles or more can be detected. Preferred libraries comprise substantially equal molar amounts of each desired reaction product and do not include relatively large or small amounts of any given molecules so that the presence of such molecules dominates or is completely suppressed in any assay.

[92] Combinatorial libraries are generally prepared by derivatizing a starting compound onto a solid-phase support (such as a bead). In general, the solid support has a commercially available resin attached, such as a Rink or Merrifield Resin. After attachment of the starting compound, substituents are attached to the starting compound. Substituents are added to the starting compound, and can be varied by providing a mixture of reactants comprising the substituents. Examples of suitable substituents include, but are not limited to, hydrocarbon substituents, e.g. aliphatic, alicyclic substituents, aromatic, aliphatic and alicyclic-substituted aromatic nuclei, and the like, as well as cyclic substituents; substituted hydrocarbon substituents, that is, those substituents containing nonhydrocarbon radicals which do not alter the predominantly hydrocarbon substituent (e.g., halo (especially chloro and fluoro), alkoxy, mercapto, alkylmercapto, nitro, nitroso, sulfoxy, and the like); and hetero substituents, that is, substituents which, while having predominantly hydrocarbyl character, contain other than carbon atoms. Suitable heteroatoms include, for example, sulfur, oxygen, nitrogen, and such substituents as pyridyl, furanyl, thiophenyl, imidazolyl, and the like. Heteroatoms, and typically no more than one, can be present for each carbon atom in the hydrocarbon-based substituents. Alternatively, there can be no such radicals or heteroatoms in the hydrocarbon-based substituent and, therefore, the substituent can be purely hydrocarbon. [93] Compounds that are initially identified by any screening methods can be further tested to validate the apparent activity. The basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a model for humans and then determining the effects on cancer. The animal models utilized in validation studies generally are mammals. Specific examples of suitable animals include, but are not limited to, primates, mice, and rats.

[94] The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment of cancer. The agents may be administered in a variety of ways, orally, topically, parenterally e.g. subcutaneously, intraperitoneal^, by viral infection, intravascularly, intratumor, etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt. %.

[95] The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.

[96] The agents of the present invention can be used in native form or can be modified to form a chemical derivative. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of present invention can be administered concurrently with, prior to, or following the administration of the other agent.

[97] The agents of the present invention are administered to the mammal in a pharmaceutically acceptable form and in a therapeutically effective concentration.- A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient. [98] The agents of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton Pa. (1980)). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of one or more of the agents of the present invention, together with a suitable amount of carrier vehicle.

[99] Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb one or more of the agents of the present invention. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate agents of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization.for example, hydroxymethylcelluloseor gelatine microcapsules; and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

[100] Therapeutic regimens may comprise co-formulation or separate formulation of additional chemotherapeutic drugs, as previously described. Preferably the combination therapy provides for improved effectiveness, as compared to the individual agents.

[101] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar to those described herein can be used in the practice or testing of the present invention, only exemplary methods and materials are described.

[102] The terms "a," "an," and "the" as used herein are not limiting and include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a complex" includes a plurality of such complexes and reference to "the formulation" includes reference to one or more formulations and equivalents thereof known to those skilled in the art, and so forth.

[103] As used herein, the term "treating" is used to refer to both prevention of disease and treatment of pre-existing conditions.

[104] The terms "effective amount" and "effective dose" as used herein are synonymous.

An "effective dose" in context of administration of an agent refers to an amount of a molecule that is sufficient to inhibit growth or survival of cancer cells in a subject so as to inhibit the occurrence or ameliorate one or more symptoms of the target disease. Thus, an effective dose of an agent is the dose that, when administered for a suitable period of time, will evidence a reduction in the severity of the disease. A suitable period of time for administration that will evidence a reduction in the severity of the disease is usually at least about one week, and may be about two weeks, or more, up to a period of about 4 weeks. It will be understood by those of skill in the art that an initial dose may be administered for such periods of time, followed by maintenance doses, which, in some cases, will be at a reduced dosage.

[105] Further, an effective amount of an agent is administered according to the methods of the present invention in an "effective regime." The term "effective regime" refers to a combination of amount of the agent and dosage frequency adequate to accomplish treatment or prevention of the disease.

EXPERIMENTAL

[106] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

[107] Cancer is largely caused by genomic catastrophes that result in the activation of oncogenes that usurp physiological programs mandating relentless cellular proliferation and growth. The targeted inactivation of oncogenes may be a specific and effective therapy for treating cancer. Experimental results in animal models validate that even the brief inactivation of a single oncogene can be sufficient to reverse tumorigenesis. Drugs that target specific oncogenes have been identified and shown to be effective in the treatment of certain cancers. Hence, cancers are susceptible to therapies that target their molecular underpinnings. [108] However, cancers can escape dependence upon oncogenes, as has been observed upon targeted inactivation of oncogenes in transgenic mouse models as well as in human patients. The most effective therapeutic approaches likely will incorporate the targeted inactivation of multiple oncogenes or alternatively, essential genes that regulate multiple oncogene-dependent pathways. The best proteins to target to treat cancer may not be those expressed from the usual suspected oncogenes.

[109] The 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase is the rate limiting enzyme in the cholesterol biosynthesis pathway. HMG-CoA reductase serves an important role in the synthesis of metabolites that regulate many other physiological processes (Figure 1A) and is required for the synthesis of isoprenoids, such as farnesyl and geranyl moieties, as well as dolichols and ubiquinones, lsoprenoid intermediates serve as attachments in the lipid membranes and are important for the posttranslational modification of signal transduction proteins that mediate cellular signaling. Isoprenoids thus participate in the post-translational modification (famesylation and geranylgeranylation) of proteins, which plays a fundamental role in the regulation of cell signaling, for example by the regulation of G-proteins, such as Ras. Thus, in addition to playing a key role in the production of cholesterol, HMG-CoA reductase coordinates the synthesis of many biological intermediates required for the regulation of gene function.

[110] Statins are a class of drugs that target the inactivation of HMG-CoA reductase to reduce cholesterol levels. Multiple reports document that statins are efficacious in the treatment of many other pathological conditions. Statins have proven to be highly effective clinically in the treatment of high cholesterol and in the prevention of atherosclerosis. Statins have been shown to be effective in the inhibition of angiogenesis, inflammation, prevention and reversal of experimental autoimmunity and of inflammatory arthritis. Statins also may be useful in the treatment of cancer. Statins can induce proliferative arrest and apoptosis in tumor cells. Epidemiological studies suggest that statins may reduce the frequency of some cancers. However, the mechanisms by which statins induce tumor- specific cell cycle arrest and apoptosis are not known.

[111] The mevalonate biosynthetic pathway plays an integral role in the regulation of cellular physiology leading us to speculate that statins may mediate their effects by inhibiting the function of critical proteins required to sustain aberrant states of cellular proliferation and growth. To address this possibility, we examined the affects of statins on the neoplastic properties of tumors derived from a series of novel defined oncogene- induced transgenic models employing the Tet system, permitting the conditional regulation of oncogene expression. Previously, we and others have used this strategy to demonstrate that oncogene-induced tumorigenesis is reversible. Now, we describe that the inhibition of HMG-CoA reductase using Atorvastatin reverses established tumorigenesis both in vitro and in vivo, and prevents oncogene activation from inducing tumorigenesis as measured by changes in the phosphorylation of key proteins. Atorvastatin exhibited comparable efficacy to the direct inactivation of an oncogene. Moreover, the inhibition of HMG-CoA reductase through RNAi similarly resulted in the loss of the neoplastic state, confirming that HMG-CoA reductase is required to sustain a neoplastic state. Results

[112] Statins can induce proliferative arrest and apoptosis in oncogene-induced tumors.

Several statins were found to induce proliferative arrest and apoptosis in a wide range of tumor cell lines derived from hematopoietic, epithelial or mesenychmal cells including: B and T-cell lymphomas; breast, colon and ovarian cancer; and osteogenic sarcoma (Table 1). The effect of statin on tumor cells was dose dependent and similar results were observed for Atorvastatin, Simvastatin and Lovastatin, but not for Pravastatin. The down¬ stream product of HMG-CoA reducaste, mevolanate, prevented statins from inducing the proliferative arrest and apoptosis of tumor cells (Figure 1 B, C, D, Figure 8 and 9). Pravastatin is known to bind less effectively to HMG-CoA reductase, perhaps accounting for it's observed attenuated effects on tumor cells. Hence, a broad range of human and mouse tumor cell lines exhibited cooperative and dose-dependent sensitivity to a variety of statins. Further studies were focused on Atorvastatin since this drug exhibited the most potent anti¬ neoplastic properties in our studies.

Tabic 1. Atorvastatin prevents the survival of human and mouse tumors.

⁰ =P<0.0001, ^b=P<0.001, °=P<0.01

*Rektive number of cells treated with Atorvastatin (1 OμM) vs untreated cells after 24 hours. Data are the mean of three different experiments done in triplicates.

Previously, we have employed the Tet system to generate conditional transgenic models of oncogene-induced tumorigenesis. Using this approach, oncogene-induced tumorigenesis has been shown to be reversible. A panel of hematopoietic tumors was generated utilizing transgenic lines that were induced through the conditional expression of MYC, RAS, and/or the BCL2 oncogenes in lymphocytes. Regardless of the oncogene that initiated tumorigenesis, tumors treated in vitro with Atorvastatin were found responsive with a dose dependent proliferative arrest and apoptosis (Table 1 , Figures 8, 9). Clinically it is relevant that Atorvastatin induced the reversion of a neoplastic phenotype at doses that are not toxic to normal lymphocytes (Figures 8, 9). The efficacy of Atorvastatin was comparable to the efficacy seen with inactivation of the conditional MYC or RAS oncogenes with doxycycline (Figure 1). However, Atorvastatin induced apoptosis with more rapid kinetics than MYC inactivation, as measured by FACS analysis of AnnexinV/7AAD stained cells. The majority of the cells treated with Atorvastatin (62%) stained for 7AAD compared to cells where MYC was inactivated (33%). In contrast, Atorvastatin did not induce proliferative arrest or apoptosis in mitogen-stimulated normal T-cell lymphocytes (Figure 9). Thus, Atorvastatin induced the proliferative arrest and apoptosis in tumor cells regardless of the oncogene that initiated tumor formation and with similar or better efficacy to the inactivation of that same oncogene.

[114] Atorvastatin reverses and prevents tumorigenesis. Next, we examined the ability of

Atorvastatin treatment to induce tumor regression. Mice sick with tumor burden were either not treated or treated with doxycycline to inactivate the MYC oncogene, or treated with Atorvostatin, at doses in mice equivalent to the maximum dose used to treat hypercholesteremia in humans. Untreated mice rapidly succumbed to tumor burden (Figure 2 A, B), whereas, treatment with Atorvastatin, similar to inactivation of MYC, induced the rapid regression of tumors within one week of treatment (Figure 2A, C). Treatment with Atorvastatin resulted in the apoptosis of tumor cells, as observed by examination of histological specimens and measured in vivo by TUNEL assay (Figure 2D-G). Thus, Atorvastatin can reverse oncogene-induced tumorigenesis in established tumors.

[115] Epidemiological studies suggest that statins may be capable of preventing the onset of tumorigenesis in certain cancers. To directly address if statins can prevent oncogene- initiated tumorigenesis, we examined the affects of Atorvastatin treatment on the development of lymphoma in our transgenic model of MYC-induced lymphomagenesis. Cohorts of mice were either not treated or treated with doxycycline to suppress expression of MYC or treated with Atorvastatin (Figure 3). Untreated mice developed tumors with a frequency of 100% and a latency of tumor onset within 5-24 weeks. As we have previously shown, inactivation of MYC with doxycycline, suppressed tumorigenesis. Similarly, mice treated with Atorvastatin (100 mg/kg) failed to develop lymphoma and when treated at a lower dose (1 or10 mg/kg) 30% of mice remained disease free for 6 months after treatment. Therefore, Atorvastatin can prevent the onset MYC-induced tumorigenesis in transgenic mice.

[116] Atorvastatin inactivates multiple oncogene pathways. To account for the dramatic effects of statins on the reversal and prevention of onocogene-induced tumorigenesis we reasoned that they may be inhibiting the function of oncogenes. Previous reports from many laboratories have shown that the inhibition of even a single oncogene can be sufficient to reverse tumorigenesis. The inhibition of HMG-CoA reductase by statins results in the depletion of mevalonate, which could inactivate oncogene signaling pathways, inhibit cellular proliferation and induce apoptosis (see above Figure 1A). Mevalonate is required for prenylation and activation of many gene products, including the Ras family of oncogenes that regulate signaling and apoptosis, as well as other gene products such as DNA-J2 (heat shock protein 40). As predicted, Atorvastatin treatment of lymphomas abolished protein prenylation of Ras, RhoA/B, Rap, Rac and DNA-J2, as seen by Western analysis (Figure 4A. Hence, Atorvastatin broadly inhibits prenylation, which could result in the disruption multiple signaling pathways.

[117] To examine the effects of statins on diverse signal transduction pathways, a multi¬ parameter phospho-proteomic flow cytometric approach (Phos-Flow) was applied to assess the phosphorylation of multiple proteins in tumor cells after treatment with Atorvastatin. Single cell analysis of phosphorylation changes was accomplished by intracellular staining of phosphorylation epitopes using directly conjugated multi-color phospho-specific antibodies. Cancer cells were treated with Atorvastatin, mevalonate, or Atorvastatin plus mevalonate for 24, 36, or 48 hrs. Cells were then stained with multicolor phospho-specific antibody cocktails and processed by multidimensional flow cytometry. The fluorescent intensities values of the detecting phospho-specific antibodies were computed, made relative to non-treated media control, and analyzed by unsupervised hierarchical clustering. Each square in the grid represents data acquired for at least 30,000 cell events. The response to Atorvastatin, mevalonate and Atorvastatin with mevalonate were determined and compared with the basal state, shown in the profile as normalized to black.

[118] Atorvastatin treatment induced the phosphorylation of several molecules, namely residues of cRaf, Stati , Plcγi , Bad, and cJun at one or more time points (upper box in Figure 4B). Alternatively, there were proteins whose phosphorylation was reduced with Atorvastatin, but not with treatment with Atorvastatin and mevalonate including: lκBα, cRAF(S269), ERK1/2 (p44/p42), Vav, and Lck (lower box in Figure 4B). These changes of phosphorylation were not observed in the combination of Atorvastatin plus mevalonate. Interestingly, mevalonate alone also induced some changes in phosphorylation. At 24 hrs, mevalonate alone displayed increases in phosphorylation of c-Raf, Stat3, lκκ-α, NF-κB, and p38. Between 36-48 hrs, mevalonate treated cells displayed enhanced ERK1/2 (p44/p42), Vav, Lck, Jnk, and Stati (Figure 4B). These results suggest that the inhibition of mevalonate biosynthesis via Atorvastatin inactivation of HMG-CoA reductase results in the dephosphorylation of many integral signaling components of the ERK1/2 MAPK pathway, cell survival pathways, as well as T cell signaling molecules. The results also demonstrate that artificially supplementing the cells with excess mevalonate induces changes in patterns of phosphorylation that can overcome some of the effects of Atorvastatin but not lead to death of the cancer cells when applied alone. Therefore, the regulatory balance of mevalonate production is integrated to several signaling networks. Hence, Atorvastatin induced specific temporal changes in phosphorylation of many proteins that are known to regulate cellular proliferation and apoptosis.

[119] Atorvastatin was confirmed to inhibit the phosphorylation of ERK1/2 by Western analysis (Figure 4C). ERK1/2 and Ras are known to be required for phosphorylation of MYC. Indeed, Atorvastatin treatment resulted in the dephosphorylation of MYC, as measured by Western analysis (Figure 4C). ERK1/2 is also known to be required for phosphorylation of Akt at serine 473 and threonine 308, two sites required for cell survival signaling. Thus, treatment with Atorvastatin resulted in the inactivation of multiple oncoproteins known to be required to maintain a neoplastic phentoype: Ras, ERK1/2, Akt and MYC.

[120] Atorvastatin induces apoptosis through mitochondrial depolarization. Atorvastatin treatment did not result in changes in the levels of protein expression of apoptosis regulatory gene products Bad, Bax, Bak, Bcl-xl and Bcl2 as measured by FACS or Western blot analysis (Figure 10). However, Atorvastatin rapidly induced activation of apoptosis measured by examining cleavage of Caspase-3, by FACS (Figure 5A) and confirmed by Western analysis (Figure 10). Since mitochondrial dysfunction precedes Caspase 3 cleavage, Atorvastatin could be targeting the mitochondrial membrane rather than modulating the levels of expression of proteins involved in apoptosis. Therefore, we assessed the integrity of the mitochondrial potential in the presence of Atorvastatin. Tumor cells treated with Atorvastatin were stained with JC-1 , a dye that exhibits a potential- dependent accumulation in the mitochondria that results in the detection of a shift in the ratio of red/green fluorescence. Atorvastatin treated cells exhibited a depolarized membrane potential compared to control cells indicating disruption of mitochondrial integrity (Figure 5B).

[121] To further assess the temporal effect of Atorvastatin treatment of tumor cells we analyzed the sequence of events that occur prior to cell death by simultaneous staining of phospho-ERK1/2, phospho-MYC and cleaved Caspase-3 as a function of time. Within 6 hours of Atorvastatin treatment dephosphorylation of ERK1/2 occurred (Figure 6A) followed by the decrease in phosphorylation in MYC after 12 hours (Figure 6B) and the cleavage of Caspase 3 after 24 hours of treatment (Figure 6C). Thus, Atorvastatin treatment results in dephosphorylation of ERK1/2 and MYC followed by depolarization of the mitochondrial membrane and apoptosis.

[122] Inactivation of HMG-CoA reductase is sufficient to reverse tumorigenesis.

Atorvastatin both inhibited and decreased levels of HMG-CoA reductase in our tumor models (Figure 4C). The addition of the down-stream product of the HMG-CoA reductase enzyme reaction, mevalonate, was sufficient to reverse cell cycle arrest and apoptosis induced by Atorvastatin (see above, Figure 1C, D and 5A, B). To further validate that Atorvastatin reversed tumorigenesis by the inactivation of HMG-CoA reductase, we targeted HMG-CoA reductase with specific siRNA oligomers. Transfection of these oligomers decreased HMG-CoA reductase protein expression by 75%, as measured by Western analysis (Figure 6A). By monitoring individual tumor cells and simultaneously assessing phospho-ERK1/2, phospho-MYC, and HMG-CoA reductase expression, targeting of HMG- CoA reductase was shown to be sufficient to inhibit phosphorylation of ERK1/2 and MYC (Figure 6B). Inhibition of HMG-CoA reductase (orange), exhibited decreased levels of phosphorylated ERK1/2 and MYC than cells that had unaltered levels of HMG-CoA reductase and phosphorylated ERK1/2 and MYC (blue/purple). Thus, the direct targeting of HMG-CoA reductase is sufficient to inhibit oncogene signaling in a manner that could be sufficient to reverse and prevent tumorigenesis.

[123] Effect of statin on tumor burden in vivo. Mice were injected S. Q. with 50 x 10⁶, transgenic MYC conditional T-cell lymphoma cells. When sick with tumor burden mice were treated orally with doxcycline to inactivate MYC expression or with 10 & 100 mg/kg statins including Atorvastatin, Simvastatin or a mixture of Atorvastatin & Simvastatin. Mice sick with tumor burden were also injected directly into the tumor site with 10 & 100 mg/kg Atorvastatin. Specific statins, combination of statins or specific doses inhibit tumor growth at different rates. 10 mg/kg mix of Atorvastatin and Simvastatin suppresses tumor growth. Data is shown in Figure 11.

[124] Statins are some of the most commonly prescribed medications in humans, in use now for almost two decades for the treatment of hypercholesteremia and atherosclerosis. However, their mechanisms of action are more complicated than inhibiting the production of cholesterol. The immediate product of HMG-CoA reductase is mevalonate which is required for protein prenylation, that regulates the function of many signaling proteins. The inhibition of HMG-CoA reductase by statins has been shown to be effective in the treatment of several diseases. Statins have been suggested to have anti-neoplastic properties, but HMG-CoA reductase has not been previously suggested to play an important role in tumor maintenance. Here we demonstrate that the inhibition of HMG-CoA reductase by Atorvastatin results in the disruption of multiple signaling pathways that are required to initiate or sustain tumor growth. The inhibition of HMG-CoA reductase reversed established tumors and prevented oncogene-induced tumorigenesis. Therefore, HMG-CoA reductase is an example of a protein not otherwise thought to be involved in the pathogenesis of cancer, but nevertheless essential for the initiation and maintenance of a neoplastic phenotype.

[125] Atorvastatin was found to be effective in both the treatment of established oncogene-induced tumors as well as the prevention of oncogene induced tumorigenesis. Atorvastatin induced in vitro the rapid and dose-dependent proliferative arrest followed by apoptosis of a broad spectrum of human and murine tumor cell lines in vitro. Our results expand upon previous reports that statins induce cell death in vitro in tumor cells. Atorvastatin induced the rapid regression of established tumors at doses (1-10 mg/kg) comparable to those used to treat hypercholestermia in humans. Moreover, Atorvastatin induced the regression of tumors both in vitro and in vivo comparable in efficacy to the inactivation of the MYC or the RAS oncogenes, which we were able to demonstrate through conditional transgenic model systems that employed the Tet system.

[126] Atorvastatin treatment prevented tumor development of MYC-induced tumorigenesis in transgenic mouse models, also at doses comparable to those used to treat humans for hypercholesteremia. Thus, we provide direct proof that Atorvastatin can prevent tumorigenesis. Interestingly, the optimum dose of Atorvastatin required to induce tumor regression of established tumors was lower than the optimum dose required to prevent tumorigenesis, suggesting that the mechanism of inhibition of tumorigenesis may be different. Therefore, statins provide an effective, easily tolerated, oral administered medication for the treatment of some human cancers, as well as for the prevention of cancer in patients with a high susceptibility to tumorigenesis. Our results obtained in vitro in cell lines and in vivo in transgenic models will require validation in humans.

[127] The statins tested here mediate, their broad anti-neoplastic effects by globally altering cell signaling pathways and potentiating mitochondrial mediated apoptosis. In particular, we have shown that statins inhibit Ras and Myc mediated oncogenic pathways. Thus, there are oncogene pathways that are required for the initiation and maintenance of tumorigenesis, which are affected by statins. Experimental animal models have demonstrated that the inhibition of Myc, Ras, BCR-AbI and many other oncogenes can reverse tumorigenesis.

[128] The present results demonstrate that Atorvastatin mediates its effect by inhibiting multiple oncogenic pathways simultaneously, through the disruption of essential regulatory modifications of proteins essential in cancer cells for the cellular proliferation and the prevention of apoptosis. Atorvastatin generally inhibits prenylation, inhibiting the Ras family of oncogenes and preventing the phosphorylation and activation of ERK1/2, Akt and the Myc oncogene. The inactivation of these oncoproteins by Atorvastatin can be responsible for its ability to reverse and prevent tumorigenesis. In addition, statins were found to potentiate intrinsic pathways resulting in- the destabilization of mitochondria. However, Atorvastatin did not adversely effect the proliferation or induce apoptosis in normal lymphocytes at doses that were sufficient to reverse the neoplastic properties of tumor cells. Hence, statins through the combined inactivation of proteins that support oncogenic signaling pathways and the potentiation of apoptotic pathways are generically effective in reversing established tumors and preventing the onset of tumorigenesis, without apparent effects on normal cells. [129] Atorvastatin also promotes apoptosis through a mechanism influencing mitochondrial depolarization. We were unable to observe any changes in levels of proteins that regulate apoptosis; including Bad, Bim, Bax, Bak and Bcl2. Instead, we found that statins induced the depolarization of mitochondria leading to the subsequent cleavage of Caspase 3. Thus, statins may induce apoptosis in tumor cells both by inhibiting oncogenic signaling and by activating mitochondrial-mediated apoptosis.

[130] One of the virtues of statins for treating cancer is the diversity of signaling pathways where they act. Here we have demonstrated that statins have widespread effects on oncogenic signaling pathways. The present results demonstrate that the inhibition of HMG- CoA reductase by statins can impact multiple critical oncogenic signaling pathways, which may also be required in many pathologic disorders mediated through the disruption of proliferation, inflammation and/or apoptosis.

[131] One of the major limitations of targeting individual proteins for the treatment of cancer is that tumors even can become independent of oncogenes that initiated tumorigenesis and thereby acquire resistance to the drugs that target these genes. The present results validate that HMG-CoA reductase, as a key regulator of many oncogenic pathways is a valuable potential therapeutic target for the treatment of cancer. Several observations confirmed that the inhibition of HMG-CoA reductase mediated by statins was responsible for the loss of neoplastic features. Several statins exhibited clinical efficacy in reversing the neoplastic properties of tumors, demonstrating effects that are not unique to any particular drug in this class. The metabolite product of HMG-CoA reductase, mevalonate, reversed the ability of statins to induce proliferative arrest and apoptosis. RNAi directed at HMG-CoA reductase was sufficient to induce the loss of the neoplastic features of tumor cells. We conclude that HMG-CoA reductase is required for the maintenance of a neoplastic phenotype. HMG-CoA reductase is an example of a gene product that otherwise does not play a direct role in tumorigenesis, but whose inactivation is effective in the prevention and treatment of cancer.

[132] Given the proclivity of statins to block multiple pathological pathways, and their well known risk-benefit ratio when administered in humans for hypercholesterolemia, statins offer significant benefits as an adjunct to other therapeutic approaches. A major hurdle for the treatment of cancer is to identify agents that are not only effective, but well-tolerated and easily administered, so that they can be used to not only treat cancer, but also prevent cancer. Statins are a class of drugs that are promising in all these regards. Materials and Methods

[133] Transgenic mice: Tet system was used to generate transgenic mice that conditionally express the human MYC cDNA in T-cell lymphocytes, as previously described by Felsher et al. (1989) MoI Cell 4, 199-207; and Jain et al. (2002) Science 297, 102-4. [134] Histology. Tissues were fixed in 10% buffered formalin, paraffin embedded and 5 μm paraffin sections were stained with hematoxylin and eosin.

[135] TUNEL Assay. Apoptotic cells were detected by the TUNEL assay in situ death detection kit (Roche Diagnostic, IN) as described by supplier. Cells were counterstained with DAPI (Vector Lab, CA).

[136] Proliferation Assay. Cell were grown in their respective media requirements and cultures were pulsed for 18 h with 1 μCi per well of [3H]thymidine. Cells were harvested and [³H] thymidine incorporation was measured. Results are shown as mean of triplicates with standard error mean (s.e.m) given in percentage.

[137] Flow Cytometry. Intracellular probes for active kinases were made by conjugating phospho-specific antibodies to the Alexa Fluor dye series as described and used in phospho-protein optimized conditions. Cells were fixed in tissue culture plates at 1x10⁶ cells/mL (2% PFA final concentration) for 15 min at 37⁰C, and permeabilized with methanol and stored at -20⁰C until stained. Cells were seeded into 96 well plates, washed in PBS, and stained with antibody cocktails in 4% FCS, for 30 min, washed 3 times and then processed for flow cytometry. All centrifugation, staining, and flow cytometric processes were performed on ice with ice-cold buffers using pre-titred antibodies at optimal antibody concentrations and fluorophore FTP ratios. Flow cytometry data are representative of 3 independent experiments. Four-color flow cytometry was collected on a FACSCalibur machine. 6 or 9-color flow cytometry was collected on a FACSCAN or FACSARIA. Data was analyzed using Flowjo software (Treestar). Phosphorylated kinase clustering was performed n Hierarchical Cluster Explorer.

[138] Antibodies: Phospho-specific antibodies to STAT5(Y694), STAT1(Y701),

STAT3(Y705), STAT3(S727), p44/42 (T202/Y204), p38(T180/Y182), Plcγ1 (Y783), Lck(Y505) conjugated to alexa488, PE, or alexa647 were from BD-Biosciences. Phospho- specific antibodies raised against lκB-α (S32/36), lkkα(S176/180), cRaf(S259), cRaf(S338), cRaf(S43), cRaf(S621), JNK(YI 83/T185), AKT(S473), AKT(T308), cJun(S73), Bad(S112), Bad(S136), Mek(T394), Mek(S298), NFκB(S259) and Vav1(Y160) were from Biosource International. Antibodies against HMG-CoA reductase and MYC were from Upstate Technologies. Cleaved Caspase-3-PE, cleaved PARP-FITC, Bad-FITC, Bcl2-Fitc, Annexin- Cy5 were from BD-Biosciences. Alexa fluor dye series 405, 430, 488, 546, 568, 647, 700 were from Molecular Probes.

[139] Antibodies used in western blots: ERK1/2, p-ERK1/2, Akt, p-Akt(S473), were from

BD-Biosciences. Antibodies against tubilin and Bax from Upstate cell signaling solutions, Ras from BD Biosciences, DNA J from Neo markers, Caspase 3 and cleaved Caspase 3 for Western Cell Signaling Technologies, Rho A, B Rap and Rac are from Santa Cruz Biotechnology.

[140] lmmunoblotting and Kinase Assays. Cell extracts were prepared by washing 2x10⁶ cells (treated as indicated) in ice cold PBS and harvesting in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCI 1 mM EDTA 1 mM EGTA, 1% Triton X-100, 2.5 mM Na₂PO₄, 1 mM β - glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml Leupeptin, 1 mM PMSF, protease inhibitor cocktail tablet (Boehringer Mannheim). Extracts were centrifuged 14,000 RPM (5 min, 4⁰C) and 10-20 μg (BCA protein assay (Pierce)) were immunoblotted using standard procedures. Blots were incubated with the indicated antibodies and developed using ECL (Amersham). lmmunoblots stripped by incubating with stripping buffer (62.5 mM Tris, pH 6.8, 10% SDS, 1% β-mercaptoethanol) (30 min, 55⁰C) and reprobed.

[141] Atorvastatin treatment. Atorvastatin (Lipitor® Pfizer Inc.) (prescription formulation) was brought into suspension in PBS. Atorvastatin was administered orally in 0.5 ml (for example, 0.04 mg ml^"1 for 1 mg kg^"1 dose or 0.4 mg ml^"1 for 10 mg kg^"1 dose) twice weekly using 20-mm feeding needles (Popper and Sons Inc.). PBS was administered as control. Purified Atorvastatin (Carbomer Inc), was used for in vitro studies.

Claims

WHAT IS CLAIMED IS:

1. A method of phenotyping a cancer, the method comprising: contacting a sample of said cancer with a binding member specific for an activated form of at least one responsive oncogenic signaling molecule to quantitate the presence of said activated form; wherein the presence of increased levels of said activated form of a responsive oncogenic signaling molecule is indicative that said cancer is responsive to a mevalonate pathway inhibitor.

2. The method according to Claim 1 , wherein said activated form of a responsive oncogenic signaling molecule is phosphorylated.

3. The method according to Claim 2, wherein said oncogenic signaling molecule is in a member of myc signaling pathway.

4. The method according to Claim 3, wherein said oncogenic signaling molecule is one or more of c-myc, n-myc, erk ^ΛA and akt.

5. The method according to Claim 1, wherein said mevalonate pathway inhibitor is a statin, statin analog or statin derivative.

6. The method according to Claim 5, wherein administration of said statin to said cancer causes dephosphorylation of said oncogenic signaling molecule.

7. The method according to Claim 1 , wherein said sample is a patient sample.

8. The method according to Claim 7, further comprising administering a therapeutic regimen to said patient of said mevalonate pathway inhibitor.

9. The method according to Claim 8, further comprising obtaining a sample from said patient following said therapeutic regimen; contacting said sample with a binding member specific for an activated form of at least one responsive oncogenic signaling molecule to quantitate the presence of said activated form; wherein the presence of decreased levels of said activated form of a responsive oncogenic signaling molecule is indicative that said cancer has responded to said mevalonate pathway inhibitor.

10. A method of screening a candidate therapeutic agent for cancer treatment, the method comprising: contacting a cancer cell with a candidate therapeutic agent, wherein said agent is a mevalonate pathway inhibitor, then contacting said cancer cell with a binding member specific for an activated form of at least one responsive oncogenic signaling molecule to quantitate the presence of said activated form; wherein the presence of decreased levels of said activated form of a responsive oncogenic signaling molecule is indicative that said cancer cell has responded to said mevalonate pathway inhibitor.

11. The method according to Claim 10, wherein said mevalonate pathway inhibitor is a statin, statin analog or statin derivative.

12. The method according to Claim 10, wherein said activated form of a responsive oncogenic signaling molecule is phosphorylated.

13. The method according to Claim 12, wherein said oncogenic signaling molecule is in a member of myc signaling pathway.

14. The method according to Claim 13, wherein said oncogenic signaling molecule is one or more of c-myc, n-myc, erk 14 and akt.

15. A method of optimizing a formulation of a mevalonate pathway inhibitor for cancer treatment, the method comprising: contacting a cancer cell with a two or more candidate formulations comprising a mevalonate pathway inhibitor, then contacting said cancer cell with a binding member specific for an activated form of at least one responsive oncogenic signaling molecule to quantitate the presence of said activated form; wherein the presence of decreased levels of said activated form of a responsive oncogenic signaling molecule is indicative that said candidate formulation has enhanced activity against said cancer cell.

16. The method according to Claim 15, wherein said mevalonate pathway inhibitor is a statin, statin analog or statin derivative.

17. The method according to Claim 16, wherein said activated form of a responsive oncogenic signaling molecule is phosphorylated.

18. The method according to Claim 17, wherein said oncogenic signaling molecule is in a member of myc signaling pathway.

19. The method according to Claim 18, wherein said oncogenic signaling molecule is one or more of c-myc, n-myc, erk ^ΛA and akt.

20. The method according to Claim 15, wherein said two or more candidate formulations vary in at least one of dose, excipient, and additional therapeutic agent.

21. The method according to Claim 15, wherein said two or more candidate formulations vary combinations of chemically distinct mevalonate pathway inhibitors.