Methods for assessing and treating leukemia

Abstract

Methods for treating leukemia patients include analyzing gene expression profiles of a patient to determine whether the patient is likely to respond to treatment with farnesyl transferase inhibitor (FTI) and, optionally, other therapeutics. The methods are also useful for monitoring patient therapy and for selecting a course of therapy. Genes modulated in response to FTI treatment are provided and are used in formulating the profiles.

Description

BACKGROUND

[0002] Some molecules, such as Ras, that are implicated in cancers must be farnesylated by the farnesyl transferase enzyme in order to interact with the inner leaflet of the plasma membrane of the cell and become involved in various signaling pathways. Ras is not the only protein implicated in cancer that has a CAAX box that is prenylated. Farnesyl transferase inhibitors (FTIs) are therapeutic agents that inhibit the covalent attachment of the carbon farnesyl moieties to the C-terminal CAAX motif of various proteins. They have utility in the treatment of cancers and proliferative disorders such as leukemia. Acute myclogenous leukemia (AML) is among the diseases that can most beneficially be addressed with FTIs.

[0003] As is true in the case of many treatment regimens, some patients respond to treatment with FTIs and others do not. Prescribing the treatment to a patient who is unlikely to respond to it is not desirable. Thus, it would be useful to know how a patient could be expected to respond to such treatment before a drug is administered so that non-responders would not be unnecessarily treated and so that those with the best chance of benefiting from the drug are properly treated and monitored. Further, of those who respond to treatment, there may be varying degrees of response. Treatment with therapeutics other than FTIs or treatment with therapeutics in addition to FTIs may be beneficial for those patients who would not respond to FTIs or in whom response to FTIs alone is less than desired.

SUMMARY OF THE INVENTION

[0004] The invention is a method of treating a patient with leukemia with an FTI. In one such method, the patient's gene expression profile is analyzed to determine whether the patient is likely to respond to the FTI and treating a patient with the FTI if they are likely to respond.

[0005] In another aspect of the invention, a patient with leukemia is monitored for treatment with an FTI in which the patient's gene expression profile is analyzed to determine whether the patient is responding to the FTI and treating a patient with the FTI if they are likely to respond in a desirable fashion.

[0006] In yet another aspect of the invention, a patient is treated if the gene expression profile shows up regulation of one or more particular genes indicative of FTI responders.

[0007] In yet another aspect of the invention, gene expression profiles indicative of FTI responders are those which show at least a 1.5, 1.7, or 2 fold difference relative to FTI non-responders.

[0008] In yet another aspect of the invention, a patient is treated if the gene expression profile shows down regulation of one or more particular genes indicative of FTI responders In yet another aspect of the invention, a patient is treated if the gene expression profile shows modulation of a gene selected from the group of genes identified in Tables 1-3 infra.

[0009] In yet another aspect of the invention, the FTI is a quinilone or quinoline derivative.

[0010] In yet another aspect of the invention, the FTI is (B)-6-[amino(420 chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone).

[0011] Articles used in practicing the methods are also an aspect of the invention. Such articles include gene expression profiles or representations of them that are fixed in computer readable media. Other articles according to the invention include nucleic acid arrays used to determine the gene expression profiles of the invention.

[0012] In another aspect of the invention, a method of treating a patient with leukemia comprises administering an FTI and a therapeutic composition that modulates the MAPK/ERK signaling pathways, TGFβ, WNT or apoptotic pathways.

[0013] In another aspect of the invention, the patient is treated with an FTI and a therapeutic composition selected from the group consisting of tyrosine kinase inhibitors, MEK kinase inhibitors, PI3K kinase inhibitors, MAP kinase inhibitors, apoptosis modulators and combinations thereof.

[0014] In yet another aspect of this invention, the gene expression profile of a patient with leukemia is analyzed to determine whether the patient is likely to respond to an FTI or if the patient would likely benefit from the combination of an FTI and another drug. The patient is then treated with such combination or, if the patient is unlikely to respond to an FTI, the patient is treated with drug selected from the group consisting of tyrosine kinase inhibitors, MEK kinase inhibitors, PI3K kinase inhibitors, MAP kinase inhibitors, apoptosis modulators and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is an example of a graphical display of gene expression patterns used to analyze the gene expression profiles of this invention.

[0016] FIG. 2 is a schematic diagram of the MAPK/ERK pathway.

[0017] FIG. 3 is a schematic diagram of the TGFβ and Wnt pathway.

[0018] FIG. 4 is a schematic diagram of the apoptotic pathway.

DETAILED DESCRIPTION

[0019] The therapeutic agents referred to in this specification are FTIs. They take on a multitude of forms but share the essential inhibitory function of interfering with or lessening the farnesylation of proteins implicated in cancer and proliferative diseases. Preferably, the FTIs are those indicated for the treatment of leukemias such as AML. A patient who responds to an FTI is one in whom a reduction of more than 50% of blast cells is seen in bone marrow following treatment with the FTI.

[0020] Numerous FTIs are within the scope of the invention and include those described in U.S. Patents: U.S. Pat. No. 5,976,851 to Brown et al; U.S. Pat. No. 5,972,984 to Anthony et al.; U.S. Pat. No. 5,972,966 to deSolms; U.S. Pat. No. 5,968,965 to Dinsmore et al.; U.S. Pat. No. 5,968,952 to Venet et al.; U.S. Pat. No. 6,187,786 to Venet et al.; U.S. Pat. No. 6,169,096 to Venet et al.; U.S. Pat. No. 6,037,350 to Venet et. al.; U.S. Pat. No. 6,177,432 to Angibaud et al.; U.S. Pat. No. 5,965,578 to Graham et al.; U.S. Pat. No. 5,965,539 to Sebti et al.; U.S. Pat. No. 5,958,939 to Afonso et al.; U.S. Pat. No. 5,939,557 to Anthony et al.; U.S. Pat. No. 5,936,097 to Commercon et al.; U.S. Pat. No. 5,891,889 to Anthony et al.; U.S. Pat. No. 5,889,053 to Baudin et al.; U.S. Pat. No. 5,880,140 to Anthony; U.S. Pat. No. 5,872,135 to deSolms; U.S. Pat. No. 5,869,682 to deSolms; U.S. Pat. No. 5,861,529 to Baudoin; U.S. Pat. No. 5,859,015 to Graham et al.; U.S. Pat. No. 5,856,439 to Clerc; U.S. Pat. No. 5,856,326 to Anthony et al.; U.S. Pat. No. 5,852,010 to Graham et al.; U.S. Pat. No. 5,843,941 to Marsters et al.; U.S. Pat. No. 5,807,852 to Doll; U.S. Pat. No. 5,780,492 to Dinsmore et al.; U.S. Pat. No. 5,773,455 to Dong et al.; U.S. Pat. No. 5,767,274 to Kim et al.; U.S. Pat. No. 5,756,528 to Anthony et al.; U.S. Pat. No. 5,750,567 to Baudoin et al.; U.S. Pat. No. 5,721,236 to Bishop et al,; U.S. Pat. No. 5,700,806 to Doll et al.; U.S. Pat. No. 5,661,161 to Anthony et al.; U.S. Pat. No. 5,602,098 to Sebti et al.; U.S. Pat. No. 5,585,359 to Breslin et al.; U.S. Pat. No. 5,578,629 to Ciccarone et al.; U.S. Pat. No. 5,534,537 to Ciccarone et al.; U.S. Pat. No. 5,532,359 to Marsters et al.; U.S. Pat. No. 5,523,430 to Patel et al.; U.S. Pat. No. 5,504,212 to de Solms et al.; U.S. Pat. No. 5,491,164 to deSolms et al.; U.S. Pat. No. 5,420,245 to Brown et al.; and U.S. Pat. No. 5,238,922 to Graham et al. each of which is incorporated herein by reference. Non-peptidal, so-called “small molecule” therapeutics are preferred. More preferred FTIs are quinolines or quinoline derivatives such as:

[0021] 7-(3-chlorophenyl)-9-[(4-chlorophenyl)-1H-imidazol-1-ylmethyl]-2,3-dihydro-1H,5H-benzo[ij]quinolizin-5-one,

[0022] 7-(3-chlorophenyl)-9-[(4-chlorophenyl)-1H-imidazol-1-ylmethyl]-1,2-dihydro-4H-pyrrolo[3,2,1-ij]quinoline-4-one,

[0023] 8-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-6-(3-chlorophen yl)-1,2-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one, and

[0024] 8-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-6-(3-chloropheny l)-2,3-dihydro-1H,5H-benzo[ij]quinolizin-5-one.

[0025] The most preferred FTI is (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone).

[0026] In the aspect of the invention comprising treating leukemia with FTIs and other therapeutic agents, The therapeutic agents referred to in this specification are those that have an effect on the biological pathway explicated through the gene expression analysis of leukemic cells subjected to treatment with quinilone-based FTIs.

[0027] The mere presence of nucleic acid sequences having the potential to express proteins or peptides (“genes”) within the genome is not determinative of whether a protein or peptide is expressed in a given cell. Whether or not a given gene capable of expressing proteins or peptides does so and to what extent such expression occurs, if at all, is determined by a variety of complex factors. Irrespective of difficulties in understanding and assessing these factors, assaying gene expression can provide useful information about the cellular response to a given stimulus such as the introduction of a drug or other therapeutic agent. Relative indications of the degree to which genes are active or inactive can be found in gene expression profiles. The gene expression profiles of this invention are used to identify and treat patients who will likely benefit from a given therapy or exclude patients from a given therapy where the patient likely would experience little or no beneficial response to the drug or therapy.

[0028] Preferred methods for establishing gene expression profiles (including those used to arrive at the explication of the relevant biological pathways) include determining the amount of RNA that is produced by a gene that can code for a protein or peptide. This is accomplished by reverse transcription PCR (RT-PCR), competitive RT-PCR, real time RT-PCR, differential display RT-PCR, Northern Blot analysis and other related tests. While it is possible to conduct these techniques using individual PCR reactions, it is best to amplify copy DNA (cDNA) or copy RNA (cRNA) produced from mRNA and analyze it via microarray. A number of different array configurations and methods for their production are known to those of skill in the art and are described in U.S. Patents such as: U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637; the disclosures of which are herein incorporated by reference.

[0029] Microarray technology allows for the measurement of the steady-state mRNA level of thousands of genes simultaneously thereby presenting a powerful tool for identifying the effect of FTIs on cell biology and the likely effect of treatment based on analysis of such effects. Two microarray technologies are currently in wide use. The first are cDNA arrays and the second are oligonucleotide arrays. Although differences exist in the construction of these chips, essentially all downstream data analysis and output are the same. The product of these analyses are typically measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to a nucleic acid sequence at a known location on the microarray. Typically, the intensity of the signal is proportional to the quantity of cDNA, and thus mRNA, expressed in the sample cells. A large number of such techniques are available and useful. Preferred methods for determining gene expression can be found in U.S. Pat. No. 6,271,002 to Linsley, et al.; U.S. Pat. No. 6,218,122 to Friend, et al.; U.S. Pat. No. 6,218,114 to Peck, et al.; and U.S. Pat. No. 6,004,755 to Wang, et al., the disclosure of each of which is incorporated herein by reference.

[0030] Analysis of the expression levels is conducted by comparing such intensities. This is best done by generating a ratio matrix of the expression intensities of genes in a test sample versus those in a control sample. For instance, the gene expression intensities from a tissue that has been treated with a drug can be compared with the expression intensities generated from the same tissue that has not been treated with the drug. A ratio of these expression intensities indicates the fold-change in gene expression between the test and control samples.

[0031] Gene expression profiles can also be displayed in a number of ways. The most common method is to arrange a ratio matrix into a graphical dendogram where columns indicate test samples and rows indicate genes. The data is arranged so genes that have similar expression profiles are proximal to each other (e.g., FIG. 1). The expression ratio for each gene is visualized as a color. For example, a ratio less than one (indicating down-regulation) may appear in the blue portion of the spectrum while a ratio greater than one (indicating up-regulation) may appear as a color in the red portion of the specrtum. Commercially available computer software programs are available to display such data including “OMNIVIZ PRO” software from Batelle and “TREE VIEW” software from Stanford The genes that are differentially expressed are either up regulated or down regulated in diseased cells following treatment with an FTI. Up regulation and down regulation are relative terms meaning that a detectable difference (beyond the contribution of noise in the system used to measure it) is found in the amount of expression of the genes relative to some baseline. In this case, the baseline is the measured gene expression of the untreated diseased cell. The genes of interest in the treated diseased cells are then either up regulated or down regulated relative to the baseline level using the same measurement method. Preferably, levels of up and down regulation are distinguished based on fold changes of the intensity measurements of hybridized microarray probes. A 1.5 fold difference is preferred for making such distinctions. That is, before a gene is said to be differentially expressed in treated versus untreated diseased cells, the treated cell is found to yield at least 1.5 times more, or 1.5 times less intensity than the untreated cells. A 1.7 fold difference is more preferred and a 2 or more fold difference in gene expression measurement is most preferred. Table 3 lists genes that were commonly modulated across all cell lines and in responder samples.

[0032] A portfolio of genes is a set of genes grouped so that information obtained about them provides the basis for making a clinically relevant judgment such as a diagnosis, prognosis, or treatment choice. In this case, the judgments supported by the portfolios involve the treatment of leukemias with FTIs. Portfolios of gene expression profiles can be comprised of combinations of genes shown in Tables 1-3.

[0033] One method of the invention involves comparing gene expression profiles for various genes to determine whether a person is likely to respond to the use of a therapeutic agent. Having established the gene expression profiles that distinguish responder from nonresponder, the gene expression profiles of each are fixed in a medium such as a computer readable medium as described below. A patient sample is obtained that contains diseased cells (such as hematopoietic blast cells in the case of AML) is then obtained. Sample RNA is then obtained and amplified from the diseased patient cell and a gene expression profile is obtained, preferably via micro-array, for genes in the appropriate portfolios. The expression profiles of the samples are then compared to those previously determined as responder and non-responder. If the sample expression patterns are consistent with an FTI responder expression pattern then treatment with an FTI could be indicated (in the absence of countervailing medical considerations). If the sample expression patterns are consistent with an FTI non-responder expression pattern then treatment with an FTI would not be indicated. Preferably, consistency of expression patterns is determined based on intensity measurements of micro-array reading as described above.

[0034] In similar fashion, gene expression profile analysis can be conducted to monitor treatment response. In one aspect of this method, gene expression analysis as described above is conducted on a patient treated with an FTI at various periods throughout the course of treatment. If the gene expression patterns are consistent with a responder then the patient's therapy is continued. If it is not, then the patient's therapy is altered as with additional therapeutics such as tyrosine kinase inhibitor, changes to the dosage, or elimination of FTI treatment. Such analysis permits intervention and therapy adjustment prior to detectable clinical indicia or in the face of otherwise ambiguous clinical indicia.

[0035] It is possible to attain ambiguous results in which some gene expression profiles are recorded that are in some respects indicative of a responder and in other respects indicative of a non-responder. For example, the profiles may show that three genes are up-regulated consistent with a responder but that another gene is not up-regulated as would ordinarily be the case for a responder. In such a case, statistical algorithms can be applied to determine the probability that the patient will respond or not respond to the drug. Statistical algorithms suitable for this purpose are well known and are available.

[0036] Articles of this invention are representations of the gene expression profiles useful for treating, diagnosing, prognosticating, staging, and otherwise assessing diseases that are reduced to a medium that can be automatically read such as computer readable media (magnetic, optical, and the like). The articles can also include instructions for assessing the gene expression profiles in such media. For example, the articles may comprise a CD ROM having computer instructions for comparing gene expression profiles of the portfolios of genes described above. The articles may also have gene expression profiles digitally recorded therein so that they may be compared with gene expression data from patient samples. Alternatively, the profiles can be recorded in different representational format. A graphical recordation is one such format. FIG. 1 shows an example of the graphical display of such a recordation. Clustering algorithms such as those incorporated in “OMNIVIZ” and “TREE VIEW” computer programs mentioned above can best assist in the visualization of such data.

[0037] Additional articles according to the invention are nucleic acid arrays (e.g. cDNA or oligonucleotide arrays), as described above, configured to discern the gene expression profiles of the invention.

[0038] Using clustering analysis (including the algorithms mentioned above) one can compare the expression levels of patient samples to establish regulatory relationships among genes with a certain statistical confidence. A dynamic map was constructed based upon such expression data. Such a genetic network map is useful for drug discovery. For example, once basic genes of interest were identified, a list of potential up-stream regulatory genes was found using such a genetic network map. The genes so identified or their expression products were then analyzed for their use as drug targets. In some embodiments, the regulatory function of the particular genes identified was used to identify therapeutics for use in treating leukemia.

[0039] The regulation of transcription, RNA processing and RNA editing are all accomplished by proteins which are coded by their own genes. In addition, DNA sequences can exert long range control over the expression of other genes by positional effects. Therefore, the expression of genes is often regulated by the expression of other genes. Those regulatory genes are called upstream genes, relative to the regulated or down-stream genes. In a simple regulatory pathway:

A++>B−−>C++>D

[0040] where: A, B, C, D are genes

[0041] ++up-regulates

[0042] −−down-regulates

[0043] Gene A is an up-stream gene of gene B and B is an up-stream gene of C. One of skill in the art would appreciate that the network is frequently looped and inter-connected. In some instances, the expression of a gene is regulated by its own product as either a positive or negative feedback.

[0044] Cluster analysis methods were used to group genes whose expression level is correlated. Methods for cluster analysis are described in detail in Harfigan (1975) Clustering Algorithms, NY, John Wile and Sons, Inc, and Everritt, (1980) Cluster Analysis 2nd. Ed. London Heineman Educational books, Ltd., incorporated herein for all purposed by reference. Path analysis was used to decompose relations among variables and for testing causal models for the genetic networks. Multiple primary targets of a drug in leukemic cells were identified as were drugs/drug classes useful in treating such cells. According to the current invention, drugs are any compounds of any degree of complexity that perturb a biological system.

[0045] The biological effect of a drug may be a consequence of drug-mediated changes in the rate of transcription or degradation of one or more species of RNA, the rate or extent of translation or post-translational processing of one or more polypeptides, the rate or extent of the degradation of one or more proteins, the inhibition or stimulation of the action or activity of one or more proteins, and so forth. In addition to the FTIs that are preferred, the preferred drugs of this invention are those that modulate the MAPK/ERK signaling pathways, TGFβ, WNT or apoptotic pathways. These include, without limitation, tyrosine kinase inhibitors, MEK kinase inhibitors, P13K kinase inhibitors, MAP kinase inhibitors, apoptosis modulators and combinations thereof. Exemplary drugs that are most preferred among these are the “GLEEVEC” tyrosine kinase inhibitor of Novartis, U-0126 MAP kinase inhibitor, PD-098059 MAP kinase inhibitor, SB-203580 MAP kinase inhibitor, and antisense, ribozyme, and DNAzyme Bcl-XL anti-apoptotics. Examples of other useful drugs include, without limitation, the calanolides of U.S. Pat. No. 6,306,897; the substituted bicyclics of U.S. Pat. No. 6,284,764; the indolines of U.S. Pat. No. 6,133,305; and the antisense oligonucleotides of U.S. Pat. No. 6,271,210.

[0046] As noted, the drugs of the instant invention can be therapeutics directed to gene therapy or antisense therapy. Oligonucleotides with sequences complementary to a mRNA sequence can be introduced into cells to block the translation of the mRNA, thus blocking the function of the gene encoding the mRNA. The use of oligonucleotides to block gene expression is described, for example, in, Strachan and Read, Human Molecular Genetics, 1996, incorporated herein by reference.

[0047] These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2′-O-alkylRNA, or other antisense oligonucleotide mimetics. Antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence.

[0048] In the case of gene therapy, the gene of interest can be ligated into viral vectors that mediate transfer of the therapeutic DNA by infection of recipient host cells. Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like. Alternatively, therapeutic DNA can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy. Protocols for molecular methodology of gene therapy suitable for use with the gene is described in Gene Therapy Protocols, edited by Paul D. Robbins, Human press, Totawa N.J., 1996.

[0049] Pharmaceutically useful compositions comprising the drugs of this invention may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the drug. The effective amount of the drug may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.

[0050] The drugs of this invention include chemical derivatives of the base molecules of the drug. That is, they may contain additional chemical moieties that are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.

[0051] Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition or activity while minimizing any potential toxicity. In addition, coadministration or sequential administration of other agents may be desirable.

[0052] The drugs of this invention can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the drugs can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as a modulating agent.

[0053] The daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per patient, per day. For oral administration, the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day. The dosages are adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.

[0054] Advantageously, compounds or modulators used in the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds or modulators for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen., For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.

[0055] The dosage regimen utilizing the compounds or modulators in the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular drug employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

[0056] The drugs of this invention can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as “carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0057] For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

[0058] For liquid forms the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents that may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations, which generally contain suitable preservatives, are employed when intravenous administration is desired.

[0059] The drugs in the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

[0060] Drugs in the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The drugs in the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the drugs in the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

[0061] For oral administration, the drugs may be administered in capsule, tablet, or bolus form or alternatively they can be mixed with feed. The capsules, tablets, and boluses are comprised of the active ingredient in combination with an appropriate carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate. These unit dosage forms are prepared by intimately mixing the active ingredient with suitable finely-powdered inert ingredients including diluents, fillers, disintegrating agents, and/or binders such that a uniform mixture is obtained. An inert ingredient is one that will not react with the drugs and which is non-toxic to the animal being treated. Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils, and the like. These formulations may contain a widely variable amount of the active and inactive ingredients depending on numerous factors such as the size and type of the animal species to be treated and the type and severity of the infection. The active ingredient may also be administered by simply mixing the compound with the feedstuff or by applying the compound to the surface of the foodstuff.

[0062] The compounds or modulators may alternatively be administered parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Injection may be either intramuscular, intraruminal, intratracheal, or subcutaneous. The injectable formulation consists of the active ingredient mixed with an appropriate inert liquid carrier. Acceptable liquid carriers include the vegetable oils such as peanut oil, cotton seed oil, sesame oil and the like as well as organic solvents such as solketal, glycerol formal and the like. As an alternative, aqueous parenteral formulations may also be used. The vegetable oils are the preferred liquid carriers. The formulations are prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from 0.005 to 10% by weight of the active ingredient.

[0063] The invention is further illustrated by the following nonlimiting examples.

EXAMPLE 1

Cell Culture

[0064] The AML-like cell lines HL-60 (promyelocytic) and U-937 (promonocytic) were obtained from the ATCC. AML-193 (monocytic) and THP-1 (monocytic) cells were obtained from the RW Johnson Pharmaceutical Research Center, San Diego. Cells were grown in Roswell Park Memorial Institute medium (RPMI) with 20% Fetal Bovine Serum (FBS). AML-193 was also supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/ml), insulin (0.005 mg/ml), and transferrin (0.005 mg/ml).

EXAMPLE 2

Toxic Dose Assay

[0065] The cells of Example 1 were inoculated into 6-well plates at an initial concentration of 1×105 cells/ml. (B)-6-[amino(4-chlorophenyl)(1-methyl-i 1H-imidazol-5-yl)methyl]-4(3-chlorophenyl)-1-methyl-2(1H)-quinolinone) was added at concentrations ranging form 0.5 to 500 nM in 3 μl of DMSO directly to the culture medium. Control cells from Example 1 were grown in medium alone or in medium supplemented with vehicle (0.1% DMSO). Cell numbers were counted at days four and seven in a hemocytometer and cell viability was determined by trypan blue exclusion assay. The IC50 was defined as the dose at which the number of viable cells in the treated sample was 50% of that in the control at day seven. Calculations were made based on duplicate runs of the experiment. The IC50 of the four cell lines was calculated after seven days of treatment with the FTI. AML-193 had an IC50 of 134 nM, HL-60 had an IC50 of 24 nM, THP-1 had an IC50 of 19 nM, and U-937 had an IC50 of 44 nM. This indicated that the four AML-like cell lines were sensitive to FTI treatement.

EXAMPLE 3

Time Course Assay

[0066] Duplicate cultures of the cells of Example 1 were inoculated into 6-well plates at an initial concentration of 1×105 cells/ml. (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone) was supplemented at a concentration of 100 nM in 3 μl of DMSO directly to the culture medium. The concentration of 100 nM was chosen for the subsequent time course experiments to normalize the treatment protocol based, in part, on the results of Example 2. Duplicate control cultures were grown in medium containing 0.1% DMSO. Duplicate cultures were harvested daily for a total of six days. Cells were counted, assayed for viability, and total RNA isolated according to the manufacturer's protocol (Qiagen RNeasy). The analysis showed that cells from different cell lines were effected at different times. RNA was treated with DNase1 (Qiagen DNase1 kit) to remove any residual genomic DNA. Linear amplification of RNA was conducted according to the procedure described in U.S. Pat. No. 5,545,522 to Van Gelder et. al. Aliquots of 5 μg of aRNA were then prepared for hybridization to cDNA arrays.

EXAMPLE 4

Bone Marrow Processing

[0067] Bone marrow aspirates were obtained from two patients diagnosed with AML who had been treated with FTI. These AML patients were administered 600 mg (B)-6[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1 methyl-2(1H)-quinolinone) twice daily over a 21 day period. Bone marrow aspirates were taken at baseline and once a week for the three weeks of treatment. One of these patients did not respond (RH) while the other responded (BS) to the FTI. Response was determined as a reduction of more than 50% of blast cells in bone marrow aspirates. The aspirates were diluted to 15 ml with PBS and Ficoll-density centrifuged. White blood cells were washed twice with PBS, resuspended in FBS with 10% DMSO and immediately frozen at −80° C. Cells were cryogenically preserved to maintain cell viability. Samples were thawed at 37° C. and 10× volume of RPMI with 20% FBS was added drop-wise over a period of 5 min. Cells were centrifuged at 1600 rpm for 10 min and resuspended in 10 ml PBS with 2 mM EDTA and 0.5% BSA. Samples were then passed through a 70 μM filter to remove any cell clumps. Cell viability was determined by Trypan Blue assay. If sample viability was less than 50% a Miltenyi Dead Cell Removal Kit was employed to enrich for the live cell fraction. 2×105 viable cells were then double labeled with CD33-FITC and CD34-PE antibodies (Pharminigen) and FACS analysis was performed. Post (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone)-treated bone marrow samples were enriched for leukemic cells by magnetic bead cell separation using either CD33 or CD34 antibodies (Miltenyi). The extracted cells had RNA extracted as described in Example 3.

EXAMPLE 5

Probe Preparation

[0068] RNA samples obtained in Examples 3 and 4 were prepared for hybridization to cDNA microarrays according to the following procedure. One to two rounds of linear amplification was performed on total RNA depending on the amount of starting material. Initially, 1-10 μg total RNA was reverse transcribed using the Superscript cDNA transcription kit (Gibco BRL). Ten μl total RNA was first mixed with 1 μl of 0.5 mg/ml T7-oligodT primer, incubated at 70° C. for 10 min, and then chilled on ice. Next, 8 μl of 5×first-strand reaction buffer, 0.1M DTT, 10 mM dNTPs, and 1 μl Rnase Block were added, and the solution incubated at 42° C. for 5 min. One μl Superscript II was then added and the reaction was incubated at 42° C. for 2 hr. The reaction was heat deactivted at 70° C. for 10 min and 1 μl was removed for PCR. Next, 92 μl Rnase-free water, 30 μl 5×second-strand reaction buffer, 3 μl 10 mM dNTP, 4 μl DNA polymerase 1, 1 μl E. Coli Rnase H, 1 μl E. Coli DNA ligase were added and the mixture incubated at 16° C. for 2 hr. cDNA was linear amplified using the Ampliscribe T7-transcription kit (Epicenter). If required, a second round of RNA amplification was performed by the random hexamer approach. Fluorescently labeled cDNA probes were synthesized by priming aRNA with random hexamers and including Cy3-dCTP in the nucleotide mix. Reactions were purified using a QIAquick PCR purification kit (Qiagen), the volumes of probe normalized using relative fluoresence (Cytofluor), and resuspended in 50 μl of Version 2 hybridization buffer (Amersham Pharmacia Biotech, Pistcataway, N.J.) with 50% formamide and human Cot1 DNA (Life Technologies).

EXAMPLE 6

Array Hybridization and Analysis

[0069] The arrays contained 7452 cDNAs from the IMAGE consortium (Integrated Molecular Analysis of Genome and their Expression: Research Genetics, Huntsville, Ala.) and Incyte libraries. Micro-arrays were generated as follows and probes hybridized as described in Example 5. cDNAs were printed on amino silane-coated slides (Corning) with a Generation III Micro-array Spotter (Molecular Dynamics). The cDNAs were PCR amplified, purified (Qiagen PCR purification kit), and mixed 1:1 with 10 M NaSCN printing buffer. Prior to hybridization micro-arrays were incubated in isopropanol at room temperature for 10 min. The probes were incubated at 95° C. for 2 min, at room temperature for 5 min, and then applied to three replicate slides. Cover slips were sealed onto the slides with DPX (Fluka) and incubated at 42° C. overnight. Slides were then washed at 55° C. for 5 min in 1×SSC/0.2% SDS and 0.1×SSC/0.2% SDS, dipped in 0.1×SSC and dried before being scanned by a GenIII Array Scanner (Molecular Dynamics). The fluorescence intensity for each spot was analyzed with AUTOGENE software (Biodiscovery, Los Angeles).

[0070] The intensity level of each micro-array was normalized so that the 75th percentile of the expression levels was equal across micro-arrays. Clones displaying a coefficient of variance (CV) greater than 50% of the mean were excluded from the analysis. Since background intensity was a maximum of 32 units for all experiments a threshold of 32 was assigned to all clones exhibiting an expression level lower than this. A ratio matrix was then generated based on pair-wise analysis of treated and control samples and Hierarchical clustering was performed using an euclidean metric and average linkage (Omniviz Pro™).

[0071] Each sample was hybridized to three identical arrays and the mean signal intensity was compared by scatter-plot analysis. High correlation coefficients were also observed when control samples were compared to treated samples from the same day. This indicated there were no gross changes in gene expression due to treatment with (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone). In addition, the variation between control samples from different days was examined. Cells were mock-treated and RNA was isolated after 1, 2, 3, 4, 5, and 6 days. Following labeling and hybridization the mean intensity of duplicate samples and the coefficient of variance (CV) of each clone (3 spots per clone) were calculated. Data points which displayed a CV of more than 50% were discarded from further analysis.

EXAMPLE 7

Differential Gene Expression in Treated Cell Line Samples

[0072] Hierarchical clustering was performed on the time-course data sets using the OmniViz Pro™ software (Battelle). Initially, fold-changes of 1.5, 1.7, and 2.0 were used as filters for the treated versus control intensity ratios for each day of the time-course. The gene expression profiles of genes modulated beyond these thresholds were analyzed to examine those genes that were commonly modulated between the three data sets and identify gene clusters that shared similar expression profiles. Results are shown in Tables 1-3 below.

[0073] Genes analyzed according to this invention are identified in the tables below by reference to Gene ID Numbers (internally generated) and accession numbers in the Genbank database where such genes have been entered in the Genbank database. The attached sequence listing, incorporated herein by reference, shows sequences corresponding to the Gene ID Number and are named with those Gene ID Numbers. In some cases, the listed sequences are to full length nucleic acid sequences that code for the production of a protein or peptide. One skilled in the art will recognize that identification of full-length sequences is not necessary from an analytical point of view. That is, portions of the sequences or ESTs can be selected according to well-known principles for which probes can be designed to assess gene expression for the corresponding gene. Further, it should be noted that some of the sequences in the listing contain the letter “N” in place of a nucleotide designation. One skilled in the art will recognize that the “N” indicates placement of any nucleotide in that portion of the sequence.

EXAMPLE 8

Identification of Gene Networks

[0074] Genes that were regulated in two or more cell lines by at least 1.5-fold in drug treated cell lines (Table 1) were identified as described above. The list of these genes was employed to identify major gene pathways that were being modulated by the most preferred FTI, (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone). If clones did not perfectly match a known gene annotations from the best BLAST result of the clone sequence were used. Since the level of regulation of these genes varied over the course of treatment of the cell lines, the gene expression profiles from the primary AML tissue that responded to this FTI were determined.

[0075] It was found that many genes in the MAPK/ERK (FIG. 2) signaling pathways were being down-regulated and that genes in the TGFβ (FIG. 3) and WNT (FIG. 3) signaling pathways were generally up-regulated, while apoptotic pathways were also activated (FIG. 4). This allowed the identification of other gene targets sensitive to treatment with known or novel drug compounds. For example, beneficial treatment can result from FTIs used in conjunction with tyrosine kinase, MEK kinase, PI3K and/or MAP kinase inhibitors to obtain a more potent effect. In addition, given the finding that apoptotic pathways are activated in FTI treated cells, drugs that modulate apoptosis could be expected to have beneficial effect when employed in conjunction with an FTI. Examples of these types of compounds include tyrosine kinase inhibitors (eg Gleevec, Novartis), MAP kinase inhibitors (eg U-0126, PD-098059, SB-203580), and inhibitors of anti-apoptotic genes such as Bcl-XL (eg antisense, ribozymes, DNAzymes).

TABLE 1
Genes (by Genbank Accession Number) modulated at least 1.5 fold
in 2 or more of the cell lines over the 6 day time course.
Gene ID   Accession No.
3434105F7 AB026898
3881595H1 AC000134
AI939481  AC005155
AA961061  AC005670
3918104H1 AC006023
AI025519  AC008427
5543360F8 AC009220
AA744682  AC009289
AA932129  AC009756
AI148008  AC011473
Y00052    AC013722
R52476    AC021078
AA864819  AC022087
AI815593  AC022150
AI141943  AC026448
AI300541  AC073585
2515486H1 AF161372
1731618H1 AJ003147
BE222911  AJ400879
R13802    AK022901
AI656222  AL021155
AI553823  AL022313
AA010251  AL034397
AA237071  AL035420
2445101F6 AL049824
AI638342  AL122004
AA779424  AL136980
H81171    AL137073
R77754    AL137790
AI209040  AL139082
6421806H1 AL139396
H61066    AL161787
R59209    AL355136
AA486141  AL355352
AI339252  AP001630
AW023438  BC009732
914979H1  BC013834
AA455969  D00015
T64335    D00017
X02308    D00596
X67098    D00596
X01023    D10493
D10493    D10493
Y00396    D10493
X69292    D10667
AA736561  D11094
S68252    D11139
M25315    D12592
AI186110  D13118
H84153    D13639
AA598561  D14043
D14695    D14695
2206642T6 D16889
AI878943  D17004
U38846    D17080
J03801    D21235
AI762926  D23660
D25215    D25215
U41078    D26512
AA485961  D30648
AA456408  D38441
AI311090  D38616
1869911H1 D42084
D43950    D43950
AW006368  D44467
U29092    D45050
D45887    D45887
W68193    D49489
3633286H1 D63874
N76967    D66904
AA985407  D82326
AI962797  D83260
M59829    D85730
3107995H1 D86322
AA279906  D86550
AI016874  D86586
556963H1  D86955
AI810687  D86997
AF045581  D87462
U41070    D89078
D90209    D90209
AA812265  D90767
2135769H1 J02763
1876511H1 J03072
J04088    J04088
AI590075  J04973
H30357    J05451
K00558    K03460
L10413    L00634
L01087    L01087
AI027898  L02426
L06139    L06139
U28936    L07914
M86400    L07955
AA428915  L08634
AA464627  L09604
M94859    L10284
R78541    L10717
L15189    L11066
L11284    L11284
T87908    L12387
T80827    L13802
L08044    L15203
AI278029  L16862
M60278    L17032
M60278    L17032
M65128    L18980
W49672    L20861
L22005    L22005
AI380522  L23822
AA988469  L25591
L26336    L26336
AI074564  L32866
M63175    L35233
2470939H1 L35848
AA190648  L36055
AI243166  L38716
L39833    L39833
M97347    L41415
2005142H1 L42324
M11723    L43615
U04045    L47574
AA284067  L76938
M13142    M13142
W68291    M15395
5560880H1 M19483
3171275H1 M20199
M22489    M22489
AA070627  M22810
H39560    M24736
M25897    M25897
M27492    M27492
AA570304  M29366
205581R6  M29696
M84739    M32294
M84739    M32294
M35857    M32315
3801801H1 M33680
M63904    M63904
AI091579  M63971
M69215    M69215
M74782    M74782
M80254    M80254
M80647    M80647
M84526    M84526
6805226H1 M84526
S93414    M86553
M91556    M91556
M95678    M95678
2017923F6 M96326
H73054    NM_000551
AA488324  NM_001211
N73242    NM_001274
AF013611  NM_001335
AW163686  NM_001524
AI921879  NM_002287
AI652785  NM_002333
AI423526  NM_003332
3028719F6 NM_003600
AB010882  NM_003601
AF030424  NM_003642
AW194791  NM_003668
AF075599  NM_003969
AF031141  NM_004223
2676931T6 NM_004412
AF053304  NM_004725
AF119815  NM_004885
AI816398  NM_004888
2185556H1 NM_004917
3357511H1 NM_004917
AA047585  NM_005109
AA448972  NM_005592
AW629084  NM_005817
AJ001015  NM_005854
AJ001016  NM_005856
U85055    NM_006480
3618886F6 NM_006536
AA906714  NM_006573
AF060153  NM_007037
1467864F6 NM_012089
AI097079  NM_012100
AF204944  NM_012105
W44673    NM_012428
AI522316  NM_013386
AI700673  NM_013439
AA057781  NM_014172
AI299795  NM_014251
1931159F6 NM_014397
AW630208  NM_014413
2821685T6 NM_014967
1539060H1 NM_015343
AI762738  NM_015449
AA401397  NM_015596
AW243944  NM_015596
AI436551  NM_016141
AI651159  NM_016440
AA527334  NM_016625
g922698   NM_017555
1693028H1 NM_017636
002783H1  NM_017860
AI368583  NM_017874
AW170305  NM_017903
H62827    NM_018321
M78706    NM_019020
BE048230  NM_020216
AI214466  NM_020334
AA452802  NM_021196
AI808824  NM_022082
W77977    NM_022336
AA535015  NM_022570
AA861140  NM_022829
AW078834  NM_023080
5122087H1 NM_024056
AF017182  NM_024101
AA449040  NM_024116
2792728F6 NM_024902
BE218593  NM_025230
AA669885  NM_030763
AI339565  NM_030908
AF038564  NM_031483
AI126706  NM_032038
1961084H1 NM_032188
4609810F6 NM_032554
AA745592  NM_032844
AW612141  NM_033050
AI740538  NM_033280
U58522    S51016
M57703    S63697
AA873257  S73591
AA521213  S77359
N77754    S79873
AA984230  S80071
R79935    S81439
T71391    T71391
AA598776  U05340
U11053    U11053
T55353    U12597
AA456616  U14970
U18300    U18300
AF017306  U20657
AA459663  U25182
U27699    U27699
AI884916  U29171
U29171    U29171
1671033F6 U33429
AA017042  U40989
AI126520  U48405
U48807    U48807
U49395    U49395
AA186542  U50078
U51586    U51586
AW150605  U54558
AA455800  U55206
AF029777  U57316
I19355    U58913
319095H1  U58913
AF027964  U59911
U60519    U60519
AI371158  U65378
U69883    U69883
H30148    U73641
R80718    U75283
U77180    U77180
U78180    U78180
U83115    U83115
AA938905  U86218
AI401546  U88844
2526581H1 U90904
AA773114  U95740
AA176596  U96781
X13274    V00543
I16618    V00595
R91899    X00226
AW519155  X00318
X87344    X00369
X02910    X01394
M15840    X02532
AA401046  X02592
X02812    X02812
X87344    X03066
X03084    X03084
M10901    X03225
X03225    X03225
R81823    X03742
X04011    X04011
K02400    X04076
X07036    X04408
X07036    X04408
Y00816    X05309
N20475    X05344
M11233    X05344
X02544    X05784
X52192    X06292
R33755    X06547
X06989    X06989
X07979    X07979
X14723    X08004
M20566    X12830
M20566    X12830
X13197    X13197
M21304    X13709
X00351    X13839
X60236    X14008
H57180    X14034
M33011    X14758
X14768    X14768
M31625    X14768
M31626    X14768
M30816    X14768
X52882    X14983
AA598758  X15187
X15606    X15606
K03515    X16539
AA868186  X17093
X51416    X51416
M23699    X51439
AA455222  X51675
X51757    X51757
X52195    X52195
U06434    X53682
J03198    X54048
M32304    X54533
AA487812  X56134
X56134    X56134
M16985    X56257
N31660    X56257
H27379    X57198
X57522    X57522
X57830    X57830
M57765    X58377
X58528    X58528
M81182    X58528
S60489    X60111
AI739095  X61157
R76314    X61587
M83665    X62534
X65921    X65921
M37722    X66945
AA453816  X69516
AA187162  X69654
X69819    X69711
X70070    X70070
X70697    X70697
S40706    X71427
T53775    X71874
X71877    X71877
X73458    X73458
X74801    X74801
AA454585  X75755
R43734    X76939
AI189206  X77303
2496221H1 X77303
H17504    X80692
R26434    X80910
X83688    X83688
U24231    X84709
3576337H1 X85030
X87212    X87212
T56477    X87212
AA464034  X89401
X89576    X89576
AA187458  X92396
M15887    X94565
X94991    X94991
X96427    X96427
X97058    X97058
AA425120  X98262
3283686H1 XM_005825
AW027188  XM_005958
1525902F6 XM_006646
R48796    XM_008099
AK000599  XM_027140
AA044653  XM_031608
AW665954  XM_035574
H86407    XM_037453
R00285    XM_038150
X57447    XM_039395
778372H1  XM_040459
R82530    XM_041024
AI580830  XM_042041
3097063H1 XM_044784
3038910H1 XM_046691
H53340    XM_048213
1654210F6 XM_048530
L42856    XM_054964
2707270F6 XM_056259
M23468    Y00062
Y00649    Y00649
Y00757    Y00757
M17017    Y00787
M28130    Y00787
L02932    Y07619
Y10256    Y10256
AA454813  Y12395
AA149850  Y12670
704183H1  Y13710
059476H1  Y13829
Y13834    Y13834
L11016    Y14768
3141315H1 Y17803
AI341167  Y18391
AI707852  Z12962
U51278    Z23115
AI686653  Z26876
AA043102  Z35102
AA136533  Z35481
U49083    Z49148
R70234    Z56852
257274R6  Z58168
U62027    Z73157
391237F1  Z73157
510997F1  Z73157
AI808621  Z82214
AA460801  Z98749
2673259F6 Z98752
R22977    Z98946
L03380    Z99995
AA425422
AA460392
AA508510
AA552028
AA576785
AA663307
AI015248
AI024468
AI086865
AI190605
AI203269
AI264420
AI333013
AI435052
AI671268
AI796718
AI990816
AW027164
AW167520
H24679
H66015
H91370
W07570
1274737F6
195337H1
2398102H1
2531082H1
264639H1
2794246F6
3290073H1
335737H1
4539942F8
6300669H1
938765H1

[0076]

TABLE 2
Genes (by GenBank Accession Number) modulated
at least 1.7 fold in primary AML Sample.
Gene ID   Accession No.
T94331    AB026898
3881595H1 AC000134
1329021F6 AC002073
2858615H1 AC002325
AI791539  AC002428
AI821217  AC004258
AA774798  AC004671
H29666    AC004845
T95173    AC005071
AA814523  AC005160
5905620T9 AC005212
5986963H1 AC005280
5825251H1 AC005306
N36113    AC005670
1700438H1 AC005682
R48756    AC005757
5538589F6 AC005839
3918104H1 AC006023
AA443719  AC007240
AI867297  AC007883
R63067    AC008073
AW022174  AC008382
5537789F6 AC008525
H00249    AC008733
1436240H1 AC008860
2668191F6 AC008949
5543360F8 AC009220
AA737674  AC009892
5104579H1 AC009892
3335217F6 AC010311
BE326380  AC010521
3746214H1 AC011088
1671315F6 AC011500
H60969    AC012351
AA926944  AC012377
3100089H1 AC012454
Y00052    AC013722
R52476    AC021078
N45149    AC021106
AI742120  AC022137
4177228F6 AC022224
2676312H1 AC022415
H73476    AC022740
AA652121  AC046170
AI308320  AC046170
1956982H1 AC046170
1428534F6 AC051619
2914934H1 AC055707
AA621370  AC064807
5514511R6 AC073333
AI698737  AC074331
3406131H1 AC079118
N20072    AC096579
5911413H1 AC096667
AI458182  AF042782
2291436H1 AF074333
W32067    AF136745
6755801J1 AF157623
2397317F6 AF235100
R53190    AF384819
1731618H1 AJ003147
2959801H1 AJ003147
3123232H1 AJ003147
2760110H1 AJ006345
X64073    AJ239325
3986782F7 AJ249275
AI366098  AJ276674
AI695385  AJ289236
BE222911  AJ400879
AI400473  AK017738
AI299633  AK021499
R13802    AK022901
1489075H1 AK025775
AI656222  AL021155
W96144    AL021155
2459540H1 AL031282
3461693F6 AL031588
4333034H1 AL031726
3332309H1 AL031728
R61661    AL032821
U71321    AL033519
AA935151  AL034374
AA010251  AL034397
U43431    AL035367
AA237071  AL035420
AA609779  AL049610
AA167461  AL049612
4228729H2 AL049742
6712339H1 AL049766
AI051176  AL049872
1747028H1 AL078600
5164454H1 AL109840
7007735H1 AL117382
AA526337  AL121601
AI638342  AL122004
4835576H1 AL122035
W01596    AL133243
U64205    AL133367
4820983H1 AL135786
5594552H1 AL136381
H12102    AL136979
H81171    AL137073
AA151374  AL137790
AA578089  AL138787
AI209040  AL139082
6421806H1 AL139396
H60498    AL157776
3721604H1 AL160271
H61066    AL161787
2798009H1 AL162252
2225447F6 AL162430
AI885557  AL162729
2918417F6 AL163279
AA489975  AL355151
U77456    AL355794
5375277T9 AL356266
AI051860  AL356489
4019605F6 AL356489
U29607    AL356801
AA861429  AL359512
AA767859  AL359915
1362587H1 AL391122
R09122    AL391194
R93094    AP000173
AA954331  AP000432
R10535    AP000555
5327443H1 AP000936
1569726H1 AP001347
R92422    AP001672
3422674H1 AP002800
AI310451  AP002812
3568042H1 AP003900
AA455969  D00015
AF030575  D00015
T64335    D00017
D12614    D00102
X67098    D00596
R27585    D00759
AA465593  D00762
M80436    D10202
M80436    D10202
M80436    D10202
M80436    D10202
AA464600  D10493
AI147046  D10653
S68252    D11139
M25315    D12592
AI186110  D13118
S57708    D13515
D13626    D13626
AA682625  D13641
AA598561  D14043
D14695    D14695
D14825    D14825
855326R1  D16234
L20046    D16305
V00496    D17206
AA629808  D17554
M57285    D21214
J03801    D21235
AI700360  D21878
D25216    D25216
U41078    D26512
AF245447  D28468
AF245447  D28468
AA070997  D29012
2134847H1 D30756
AI147295  D30756
AA455067  D31839
AI311090  D38616
AW629690  D42084
1869911H1 D42084
5122374H1 D43701
D43950    D43950
D45887    D45887
W68193    D49489
X72498    D50326
L11667    D63861
D63874    D63874
3633286H1 D63874
X61598    D83174
AA279906  D86550
AA729988  D86550
D86956    D86956
L36719    D87116
D89078    D89078
U41070    D89078
AI821897  D89675
D90209    D90209
2135769H1 J02763
J03040    J03040
1876511H1 J03072
J03258    J03258
J03571    J03571
J04111    J04111
AI125073  J04132
1634342H1 J04794
H30357    J05451
K02054    K02054
X02415    K02569
K03000    K03000
H58873    K03195
AI791949  K03474
L10413    L00634
H22919    L03558
L04288    L04288
AA405769  L05144
H62473    L07594
L08177    L08177
L08177    L08177
AA234897  L08895
AA464627  L09604
M94859    L10284
R78541    L10717
L15189    L11066
M15400    L11910
L12168    L12168
L12350    L12350
L12350    L12350
T87908    L12387
L09600    L13974
M14221    L16510
M60278    L17032
M60278    L17032
M60278    L17032
M60278    L17032
2745317H1 L17411
M65128    L18980
W49672    L20861
AI380522  L23822
AA988469  L25591
NM_001168 L26245
R20939    L31848
2470939H1 L35848
AA442810  L36034
L36148    L36148
M11723    L43615
M14745    M14745
W68291    M15395
M16038    M16038
339598H1  M16038
M17783    M17783
3171275H1 M20199
5189380H1 M21121
4130807F7 M22440
M22612    M22612
AA070627  M22810
1445982H1 M23254
M28638    M24906
R45525    M28215
AI051962  M28983
736837R6  M29696
M29870    M29870
AI264247  M30309
1512407F6 M30310
M30471    M30471
M30704    M30703
AW467649  M31158
M84739    M32294
M84739    M32294
U52165    M32315
M35857    M32315
5077322H1 M32315
N72918    M34175
M63193    M58602
M59465    M59465
2294719H1 M60858
2992331H1 M63005
AA069596  M63582
M63904    M63904
AI091579  M63971
M74782    M74782
AA410680  M77016
M80647    M80647
M84526    M84526
S93414    M86553
AI310138  M91463
M95678    M95678
2017923F6 M96326
R60624    NM_000702
AA488324  NM_001211
AA488341  NM_001336
AF006823  NM_002246
1322305T6 NM_002250
AI921879  NM_002287
AW129770  NM_002349
AJ004977  NM_002873
AI423526  NM_003332
4516963H1 NM_003576
3028719F6 NM_003600
AB010882  NM_003601
AF030424  NM_003642
AF029899  NM_003814
AF055993  NM_003864
AI220935  NM_004142
AW665782  NM_004142
AI191941  NM_004226
1392516T6 NM_004621
AA449579  NM_004769
1810447H1 NM_004917
AA047585  NM_005109
4181072F6 NM_005468
AA448972  NM_005592
AA742351  NM_005739
3406436F6 NM_005845
AJ001015  NM_005854
3118530H1 NM_005880
AA906714  NM_006573
AI016020  NM_006672
AW770551  NM_006770
AW009940  NM_006871
864164H1  NM_007194
1467864F6 NM_012089
AF204944  NM_012105
W23427    NM_012115
3363678H2 NM_012226
AI652076  NM_012243
346874T6  NM_013308
AI522316  NM_013386
AI338030  NM_013439
AI700673  NM_013439
4540025H1 NM_014322
W00842    NM_014331
AW511388  NM_014358
AW630208  NM_014413
H63640    NM_014834
AI743175  NM_014959
2821685T6 NM_014967
W38474    NM_015542
AW243944  NM_015596
W07181    NM_015701
2997457H1 NM_015938
AA631149  NM_016205
AA527334  NM_016625
5543749F6 NM_017414
AW170305  NM_017903
AA160974  NM_018155
AA625433  NM_018404
AA074666  NM_018834
767295H1  NM_018983
M78706    NM_019020
AF245447  NM_020126
AF245447  NM_020126
4294821H1 NM_020344
2490994H1 NM_021624
3556218H1 NM_021634
2435705R6 NM_022048
3092423H1 NM_022054
W77977    NM_022336
AA429219  NM_023930
1001514R6 NM_024022
AI031531  NM_024083
AA449040  NM_024116
2803571H1 NM_024586
1390130H1 NM_024671
3241088H1 NM_024850
H96170    NM_030779
1540906H1 NM_030779
AI824146  NM_030811
W90438    NM_032127
AA430653  NM_032177
3495438F6 NM_032294
AW612141  NM_033050
AA417237  NM_033225
AI740538  NM_033280
M57703    S63697
780099H1  S63912
AA714835  S67156
AA777347  S76736
AA521213  S77359
U39231    S79852
N77754    S79873
AA984230  S80071
U00672    U00672
U02478    U02478
AA019459  U02680
3401107H1 U03019
AI580044  U04816
3041874H1 U07563
2457652H1 U12465
U39318    U13175
U13666    U13666
U13695    U13695
AA056652  U14176
AA456616  U14970
U18242    U18242
U18300    U18300
AA465444  U18422
U20537    U20536
U25128    U25128
U35237    U26174
AI884916  U29171
AA481076  U31278
NM_002411 U33147
1671033F6 U33429
AA664389  U35048
6313632H1 U43030
R09288    U43522
AA488645  U47007
U47077    U47077
5801413H1 U48449
2405358R6 U48729
AA186542  U50078
U51586    U51586
1355140F1 U51586
AA455800  U55206
U56390    U56390
U83410    U58088
AA121261  U58675
AF027964  U59911
U60519    U60519
2836805T6 U62293
U62433    U62433
3188135H1 U66673
3188135H1 U66673
3188135H1 U66673
3188135H1 U66673
1360938T6 U66679
809631T6  U66684
AA454652  U67058
AI214335  U68755
U69883    U69883
R98589    U81375
5695322H1 U82671
AA745989  U82979
AA188256  U83661
2526581H1 U90904
AA434064  U95000
AA773114  U95740
AA514978  U96776
Y07503    V00510
X96754    V00557
N67917    V01512
V01514    V01514
X87344    X00369
N53169    X00567
X02910    X01394
X01451    X01451
X01451    X01451
X01451    X01451
X01451    X01451
AA401046  X02592
5537736F6 X02592
X87344    X03066
M10901    X03225
M54894    X04403
M54894    X04403
M54894    X04403
M54894    X04403
X07036    X04408
X07036    X04408
N75719    X04744
M19507    X04876
Y00816    X05309
M11233    X05344
AA479102  X05972
N24824    X06182
R33755    X06547
N41062    X06820
M86511    X06882
X07549    X07549
1686702H1 X07730
X07979    X07979
X14723    X08004
J03561    X12510
J03561    X12510
J03561    X12510
J03561    X12510
M20566    X12830
M20566    X12830
M20566    X12830
M20566    X12830
U76549    X12882
M21304    X13709
X00351    X13839
X14830    X14830
X52882    X14983
AA598758  X15187
H27564    X15729
W15277    X15940
AA393214  X15949
M23502    X16166
K03515    X16539
M28880    X166P9
2403512H1 X16674
AA868186  X17093
J03236    X51345
X51416    X51416
AA411440  X51521
AA058828  X51602
AA455222  X51675
X51804    X51804
T72877    X52015
X52195    X52195
X52947    X52947
U06434    X53682
3081284F6 X53702
M36821    X53799
AA490256  X54048
J03198    X54048
M60761    X54228
M11025    X55283
M33294    X55313
M33294    X55313
M31627    X55543
X55544    X55544
AA487812  X56134
X56134    X56134
X56777    X56777
H27379    X57198
M83652    X57748
X58528    X58528
M81182    X58528
S60489    X60111
X60592    X60592
R76314    X61587
M83665    X62534
R11490    X62947
AI436567  X63422
X63465    X63465
AA083577  X63527
X63547    X63546
2159360H1 X63692
X64074    X63926
X63926    X63926
X64083    X63926
2535659H1 X69168
AA187162  X69654
X69819    X69711
AI310990  X71491
T53775    X71874
3285272H1 X73568
U11087    X75299
X75299    X75299
AA454585  X75755
X75821    X75821
X75918    X75918
X76029    X76029
R43734    X76939
AI189206  X77303
H17504    X80692
R26434    X80910
AI521155  X81892
AA088861  X83228
U10440    X84849
407169H1  X84909
3576337H1 X85030
T55802    X85117
4407508H1 X85337
AA025432  X85373
T56477    X87212
AA464034  X89401
X89576    X89576
X89576    X89576
R83270    X89750
917064H1  X91249
X91809    X91809
X92106    X92106
AA187458  X92396
AJ000519  X92962
X94991    X94991
X96427    X96427
R85213    X98022
X98296    X98296
X99585    X99585
R48796    XM_008099
R50354    XM_009915
W15172    XM_016514
AK000599  XM_027140
7157414H1 XM_031246
AA044653  XM_031608
L16953    XM_032556
1266202T6 XM_033674
AA805691  XM_033788
AA861582  XM_036492
H86407    XM_037453
778372H1  XM_040459
AA016239  XM_041087
AI580830  XM_042041
AI732875  XM_042637
AA463411  XM_045320
AA648280  XM_046411
3038910H1 XM_046691
H63831    XM_047328
1654210F6 XM_048530
AA460131  XM_049228
5539620F6 XM_049755
AA682896  XM_050250
L42856    XM_054964
1483347H1 XM_056259
AI307255  XM_058135
H74265    Y00062
Y00064    Y00064
M17017    Y00787
M28130    Y00787
L02932    Y07619
AA504415  Y09781
AI809036  Y12336
AA516206  Y12851
000527H1  Y13829
059476H1  Y13829
Y13834    Y13834
L11016    Y14768
3141315H1 Y17803
551234R6  Y17803
AA426103  Y18000
H97778    Z13009
AA402431  Z15005
L07555    Z22576
U51278    Z23115
M58525    Z26491
AW772610  Z26652
Z29090    Z29090
H19371    Z32684
AA136533  Z35481
Z48810    Z48810
U49083    Z49148
R70234    Z56852
4902714H1 Z69918
150224T6  Z80147
M29871    Z82188
AI808621  Z82214
AA699919  Z83821
5538394H1 Z83843
5020377F9 Z97832
AA460801  Z98749
AI625585  Z98750
2673259F6 Z98752
R22977    Z98946
AA007595
AA188574
AA280754
AA283874
AA460392
AA508510
AA515469
AA526772
AA576785
AA634241
AA663307
AA663482
AA713864
AA714520
AA828809
AA868502
AI061445
AI086865
AI264420
AI378131
AI440504
AI567491
AI693066
AI709066
AI766478
AI821337
AI949694
AW439329
AW630054
H24679
H29257
H51856
H66015
H72339
N57580
N54592
W07570
T75463
R88730
R91509
T56441
T77711
W92423
1274737F6
1338107F6
1508571F6
1548205H1
1594182F6
1594701F6
1879290H1
1902928H1
194370H1
195337H1
198381H1
2021568H1
205203T6
2194064H1
224922R6
2398102H1
2531082H1
2630745F6
264639H1
2704982H1
2716787H1
2798810F6
2832401H1
2894096F6
2919406F6
2937644F6
2950021H1
3010621F6
3123948H1
3253054R6
3290073H1
3330472H1
335737H1
3674358H1
3749346F6
3820429H1
3978404F6
4031124H1
4056384H1
4097060H1
4288779H1
4301823H1
4558488F6
4570377H1
5058893F9
5541621H1
5546249F6
5546336H1
5771839H1
5804485H1
5849807H1
6530555H1
656258H1
6591535H1
859993H1
930273R6
938765H1

[0077]

TABLE 3
Genes (By Genbank Accession Number) modulated at least 1.5 fold in
all cell lines and at least 1.7 fold in patient responder sample.
Gene ID   Accession No.
5543360F8 AC009220
AA237071  AL035420
AA455969  D00015
M25315    D12592
U41078    D26512
L10413    L00634
AA464627  L09604
2470939H1 L35848
M84526    M84526
AI921879  NM_002287
AF204944  NM_012105
W77977    NM_022336
AA449040  NM_024116
AA521213  S77359
AA984230  S80071
AA456616  U14970
AI884916  U29171
U60519    U60519
X00351    X13839
AA868186  X17093
H27379    X57198
AA454585  X75755
X89576    X89576
AI580830  XM_042041
U49083    Z49148
2398102H1
2531082H1

[0078]

Claims

1. A method of determining whether a patient will respond to treatment with an FTI by analyzing the expression of a gene that is differentially modulated in the presence of an FTI.

2. The method of claim 1 wherein the differential modulation is at least 1.5 fold.

3. The method of claim 1 wherein the differential modulation is at least 1.7 fold.

4. The method of claim 1 wherein the analysis is of the expression of more than one gene.

5. The method of claim 1 wherein the gene correlates with one or more nucleic acid sequences identified in Tables 1-3

6. The method of claim 1 used to monitor the therapy of a patient.

7. The method of claim 5 wherein the FTI is (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone).

8. The method of claim 1 wherein the analysis is of the expression of a group of genes correlating with nucleic acid sequences identified in Tables 1-3 and wherein the FTI is (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone).

9. A method of treating a patient comprising: a) analyzing the gene expression profile of said patient to determine whether the patient will respond to treatment with an FTI, and b) treating the patient with the FTI if the analysis indicates that the patient will respond.

10. The method of claim 9 wherein the analysis is of the expression of more than one gene.

11. The method of claim 9 wherein the FTI is selected from the group consisting of quinolines or quinoline derivatives.

12. The method of claim 11 wherein the FTI is selected from the group consisting of 7-(3-chlorophenyl)-9-[(4-chlorophenyl)-1H-imidazol-1-ylmethyl]-2,3-dihydro-1H,5H-benzo[ij]quinolizin-5-one, 7-(3-chlorophenyl)-9-[(4-chlorophenyl)-1H-imidazol-1-ylmethyl]-1,2-dihydro-4H-pyrrolo[3,2,1-ij]quinoline-4-one, 8-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-6-(3-chlorophen yl)-1,2-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one, 8-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-6-(3-chloropheny l)-2,3-dihydro-1H,5H-benzo[ij]quinolizin-5-one, and (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone).

13. The method of claim 12 wherein the FTI is (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone)

14. The method of claim 10 wherein the genes correlate with one or more nucleic acid sequences identified in Tables 1-3.

15. The method of claim 9 wherein the treatment comprises the administration of an FTI and another therapeutic composition.

16. The method of claim 15 wherein said another therapeutic composition modulates MAPK/ERK signaling pathways, TGFβ, WNT or apoptotic pathways.

17. The method of claim 16 wherein said another composition is selected from the group consisting of tyrosine kinase inhibitors, MEK kinase inhibitors, PI3 kinase inhibitors, MAP kinase inhibitors, apoptosis modulators, and combinations thereof.

18. Articles for assessing the efficacy of treatment of a patient with an FTI comprising a medium with which patient gene expression profiles indicative of FTI response are determined.

19. The articles of claim 18 wherein the gene expression profiles are obtained from a group of genes correlating to more than one nucleic acid sequences identified in Tables 1-3.

20. The articles of claim 19 wherein the nucleic acid sequences are found in Table 3.

21. The articles of claim 18 comprising representations of gene expression profiles fixed to a medium.

22. The articles of claim 18 wherein the medium is computer readable.

23. Kits comprising articles for obtaining gene expression profiles for determining response to FTI treatment.

24. The kits of claim 23 further comprising instructions.