Patent Application
20080318803
The present invention relates to biomarkers useful for monitoring the effects of inhibition of IMPDH in a patient.
The synthesis of nucleotides in organisms is required for the cells in those organisms to divide and replicate. Nucleotide synthesis in mammals may be achieved through one of two pathways: the de novo synthesis pathway or the salvage pathway. Different cell types use these pathways to a different extent.
Inosine-5′-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is an enzyme involved in the de novo synthesis of guanine nucleotides. IMPDH catalyzes the NAD-dependent oxidation of inosine-5′-monophosphate (IMP) to xanthosine-5′-monophosphate (XMP) [Jackson R. C. et. al., Nature, 256, pp. 331-333, (1975)].
IMPDH is ubiquitous in eukaryotes, bacteria and protozoa [Y. Natsumeda & S. F. Carr, Ann. N.Y. Acad., 696, pp. 88-93 (1993)]. The prokaryotic forms share 30-40% sequence identity with the human enzyme. Two isoforms of human IMPDH, designated type I and type II, have been identified and sequenced [F. R. Collart and E. Huberman, J. Biol. Chem., 263, pp. 15769-15772, (1988); Y. Natsumeda et. al., J. Biol. Chem., 265, pp. 5292-5295, (1990)]. Each is 514 amino acids, and they share 84% sequence identity. Both IMPDH type I and type II form active tetramers in solution, with subunit molecular weights of 56 kDa [Y. Yamada et. al., Biochemistry, 27, pp. 2737-2745 (1988)].
The de novo synthesis of guanosine nucleotides, and thus the activity of IMPDH, is particularly important in B and T-lymphocytes. These cells depend on the de novo, rather than salvage pathway to generate sufficient levels of nucleotides necessary to initiate a proliferative response to mitogen or antigen [A. C. Allison et. al., Lancet II, 1179, (1975) and A. C. Allison et. al., Ciba Found. Symp., 48, 207, (1977)]. Thus, IMPDH is an attractive target for selectively inhibiting the immune system without also inhibiting the proliferation of other cells. Although resting lymphocytes can utilize the alternate salvage nucleotide synthesis pathway (Fairbanks et al, 1995), rapidly proliferating lymphocytes rely predominantly on the de novo pathway for satisfying their requirement for nucleotides. This observation makes enzymes of the de novo pathway attractive targets for pharmacological intervention aimed at inhibiting lymphocyte proliferation (Allison and Eugui, 2000). Blocking IMPDH enzyme activity results in a decrease of cellular guanine nucleotide levels, thereby inhibiting DNA and RNA synthesis, resulting ultimately in antiproliferative, immunosuppressive and antiviral effects (reviewed in Franchetti and Grifantini, 1999).
It is also known that IMPDH plays a role in other metabolic events. Increased IMPDH activity has been observed in rapidly proliferating human leukemia cell lines and other tumor cell lines, indicating IMPDH as a target for anti-cancer as well as immunosuppressive chemotherapy [M. Nagai et. al., Cancer Res., 51, pp. 3886-3890, (1991)].
Inhibitors of IMPDH have been extensively investigated to treat various diseases, e.g., tumors and cancers, immunosuppression, and viral diseases, See, e.g., WO 00/56331. Mycophenolic acid (“MPA”) and VX-944 are two known IMPDH inhibitors. VX-944 is currently being investigated for potential use as an anti-cancer agent. Several IMPDH inhibitors have been used clinically and more are being developed for the treatment of immune cell-mediated, chronic inflammatory, antineoplatic and antiviral indications (Jain et al, 2001, Jain et al, 2002, Dhar et al, 2002). In addition, several new, non-nucleoside IMPDH inhibitors have been designed using a rational drug design approach (Sintchak et al, 1996, reviewed in Saunders and Raybuck, 2000). Of these, VX-497, is currently being investigated in a Phase II trial in combination with pegylated IFN and ribavirin for the treatment of Hepatitis C (Markland et al, 2000, Jain et al, 2001). VX-944 inhibits both IMPDH isozymes with Ki values of 7-10 nM (Jain et al, 2003, ASH poster). VX-944 is also very potent in inhibiting the proliferation of human peripheral lymphocytes stimulated with either T or B-cell mitogens with IC50 values ranging from 20-100 nM, and is more potent than VX-497 or MPA (Eugui et al, 1991a, Jain et al, 2001, Jain et al, 2003 ASH poster, Jain et al, 2004 ASH poster). The inhibition of lymphocyte proliferation by VX-944 is alleviated by the addition of 50 μM guanosine, demonstrating its specificity towards the IMPDH enzyme.
There is a need to develop a reliable biomarker that can be readily used for tracking and monitoring the efficacy of such an IMPDH inhibitor. Such a biomarker should be sensitive to inhibition of IMPDH and be readily detectable by methods that are not unduly burdensome.
The present invention provides biomarkers that are useful in monitoring the effect of inhibition of IMPDH in a patient. Applicants have identified a group of genes that are up- or downregulated as a result of IMPDH inhibition. Exemplary expression data showing genes that are up- or down-regulated in response to IMPDH inhibition after 24, 48 and 72 hours of exposure to exemplary IMPDH inhibitors are provided in Table IX. Determining the expression of these genes in a biological sample obtained from a subject treated with an IMPDH inhibitor will therefore allow an assessment of whether the individual will be responsive to IMPDH inhibitor therapy.
The invention describes a nucleic acid array consisting essentially of at least 4 polynucleotides selected from the polynucleotides listed in any one or more of Tables I through VIII, wherein said polynucleotides are immobilized on a solid surface, and wherein said array further contains one or more calibration points and one or more housekeeping genes. In some embodiments the nucleic acid array consists of at least 4 polynucleotides selected from the polynucleotides listed in any one or more of Tables I through VIII. The array may have between 1 and 10 calibration spots and also or alternatively may have between 1 to 10 housekeeping genes to serve as controls for the array. The polynucleotides may be cDNAs or oligonucleotides. The arrays may consist of at least 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300 or more polynucleotides. The polynucleotides may be preferred polynucleotides presented herein in Tables I through Table VIII or alternatively may be fragments or variants of those polynucleotides. In one preferred aspect the microarray is one which comprises all of the polynucleotides set forth in Table VI. Another preferred microarray contains all of the polynucleotides set forth in Table V. Another preferred microarray contains all of the polynucleotides set forth in Table IV. In preferred embodiments, the array contains more than one polynucleotide hybridizing to the same gene.
Also encompassed by the invention is a nucleic acid array consisting essentially of at least 4 distinct nucleic acid sequences selected from the group consisting of the polynucleotides listed in Table I, immobilized on the surface at discrete and known positions, wherein said nucleic acids hybridize to nucleic acids in a sample of a subject that are either up-regulated or down-regulated in response to inhibition of IMPDH. In other embodiments, there is provided a nucleic acid array consisting of at least 4 distinct nucleic acid sequences selected from the group consisting of the polynucleotides listed in Table I. Another embodiment is directed to a nucleic acid array consisting essentially of at least 4 distinct nucleic acid sequences selected from the group consisting of the polynucleotides listed in Table IV, immobilized on the surface at discrete and known positions, wherein said nucleic acids hybridize to nucleic acids in a sample of a subject that are either up-regulated or down-regulated in response to inhibition of IMPDH. Another embodiment is directed to a nucleic acid array consisting of at least 4 distinct nucleic acid sequences selected from the group consisting of the polynucleotides listed in Table IV. Still more preferably, the nucleic acids on said microarray are selected from the group consisting, or consisting essentially, of the genes from Table V. Alternatively, the nucleic acids on said microarray are selected from the group consisting, or consisting essentially, of the genes from Table VI.
Other exemplary nucleic acid arrays are those that consist, or consist essentially, of at least 4 distinct nucleic acid sequences selected from the group consisting of the polynucleotides listed in Table VII, immobilized on the surface at discrete and known positions, wherein said nucleic acids hybridize to nucleic acids in a sample of a subject that are either up-regulated or down-regulated in response to inhibition of IMPDH. In the arrays the surface is typically selected from the group consisting of a metal, silicon, a polymer plastic, paper, ceramic, quartz, gallium arsenide, metal, metalloid, cellulose, celluose acetate, nitrocellulose, and a glass. More specifically, the plastic is selected from the group consisting of nylon, polycarbonate, polyethylene, polystyrene, teflon, polypropylene, poly(4-methylbutene), polystyrene/latex, polymethacrylate, poly(ethylene terephthalate), rayon, polyvinylbutyrate, and polyvinylidene difluoride. Preferably, the arrays have at least one control spot consisting of one or more nucleic acids that are known not to be modulated with IMPDH inhibition.
In specific embodiments the array surface comprises a plurality of microarrays separated from each other with a hydrophobic polymer strip. In preferred aspects the hydrophobic polymer strip is selected from the group of polyethylene, silicone, paraffin, and Teflon®.
Also contemplated by the invention is a set of polynucleotides for use in the detection of IMPDH inhibition, wherein said polynucleotides hybridize to 4 to 314 genes selected from the group consisting of the genes set forth in any one or more of Table I, Table II, Table III, Table IV, Table V, Table VI, Table VII and Table VIII wherein the expression of each said nucleic acid is either up- or down-regulated in response to inhibition of IMPDH.
Another aspect of the invention is related to a set of polynucleotides for use in the prediction of efficacy of an IMPDH inhibitor in non-proliferating cells, wherein the set of polynucleotides hybridize to 4 to 38 genes selected from the group consisting of the genes set forth in Table I. Still another aspect is directed to a set of polynucleotides for use in the prediction of efficacy of an IMPDH inhibitor in a proliferating cell, wherein the set of polynucleotides hybridize to 4 to 300 genes selected from the group consisting of the genes set forth in Table II, Table III, and Table IV. Preferably, the proliferating cell is from a hematological cancer and said genes are selected from the group of genes set forth in Table V. Alternatively, the genes are selected from the group of genes set forth in Table VI.
Also part of this invention is a set of nucleic acids for use in the prediction of anti-viral efficacy of an IMPDH inhibitor, wherein the set of polynucleotides hybridize to 4 to 9 genes selected from the group consisting of the genes set forth in Table VII. These are not the only genes identified that may predict antiviral activity of VX-944. The genes listed in Table VII are from the intersection of the genes of Table I and IFN-responsive genes. The skilled person may identify additional anti-viral genes by obtaining the intersection between Table IV and IFN-responsive genes.
Another aspect of the invention is a set of nucleic acids for use in the prediction of efficacy of an IMPDH inhibitor as an anti-cancer agent, wherein the set of polynucleotides hybridize to the 4 genes set forth in Table VIII.
The invention specifically contemplates a method for predicting whether a candidate IMPDH inhibitory agent will produce a therapeutic effect in a subject comprising: contacting a biological sample with said inhibitory agent and determining the expression level of four or more prognostic genes selected from the group consisting of the genes set forth in one or more of Gene Table I, Gene Table II, Gene Table III, Gene Table IV, Gene Table V, Gene Table VI, Gene Table VII and Gene Table VIII in said biological sample, wherein a modulation of the expression level of four or more of genes similar to the modulation resulting from a known IMPDH inhibitor is indicative that said agent is a therapeutically effective IMPDH inhibitory agent.
A candidate IMPDH inhibitor produces an expression pattern that is “similar” to a known IMPDH inhibitor, e.g., VX-944 or MPA, if the qualitative, but not necessarily quantitative, effect on gene expression of the candidate and known inhibitors reliably resemble or mimic each other from the perspective of one of skill in the art. The qualitative effect on expression (i.e., the number of genes exhibiting modulated expression and whether that modulation reflects up- or down-regulation) is distinct from the quantitative effect on expression (i.e., the magnitude of a given change in expression). Preferably, a candidate IMPDH inhibitor is identified as producing an expression pattern similar to a known IMPDH inhibitor if the two modulators exert the same qualitative effect (i.e., up-regulated or down-regulated) on a percentage of the modulated genes that is at least 50%, 60%, 70%, 75%, 80%, 90%, 95% or 99%.
Preferably, the effect is a clinical response. More particularly, the subject is a mammal, more specifically a human patient. The response also may be monitored in vitro in tissues or cells lines from a cancer patient. In the methods described herein the biological sample is preferably a sample from a cancer patient, and wherein said cancer is selected from the group consisting of myeloproliferative diseases, leukemia, breast cancer, ovarian cancer, gastric cancer, colorectal cancer, prostate cancer; pancreatic cancer, lung cancer, etc. The methods are such that the alteration of expression in response to said IMPDH inhibitory agent is preferably similar to the alteration in expression seen in response to administration of VX-944.
Preferably, the subject is a human patient. Or human cell lines or tissues or cells derived from cancers. More preferably, the subject is one who is suffering form a proliferative disorder. Where the proliferative disorder is cancer, the biological sample is a sample from a cancer patient, wherein said cancer is selected from the group consisting of myeloproliferative diseases, leukemia, breast cancer, ovarian cancer, gastric cancer, colorectal cancer, prostate cancer; pancreatic cancer, lung cancer, etc. The method may involve comparing the alteration of expression in response to said IMPDH agent to the level of alteration in expression seen in response to administration of VX-944.
Preferably, the sample is a tissue sample comprising cancer cells. The tissue or cells may be fixed, paraffin-embedded, fresh, or frozen. Preferably, the tissue is obtained through a biopsy, such as for example, a fine needle aspiration, bronchial lavage, or transbronchial biopsy. In specific embodiments, the expression level of said prognostic RNA transcript or transcripts is determined by PCR, immunohistochemistry, in situ hybridization, and the like. The assay for the measurement of said prognostic RNA transcripts or their expression products is provided in the form of a kit or kits.
Also contemplated is a method of preparing a prognostic profile for a subject's response to an IMPDH inhibitor comprising the steps of exposing ex vivo cells of said subject to an IMPDH inhibitor, subjecting RNA extracted from the cells of step (a) to gene expression profiling; determining the expression level of at least four genes selected from the group consisting of the genes set forth in Table I, Table II, Table III, Table IV, Table V, Table VI, Table VII and Table VIII in said cells; and comparing the expression levels obtained in step (c) to expression levels obtained in the absence of said IMPDH inhibitor, wherein a modulation of the expression level of said four or more of genes in response to said IMPDH inhibitor indicates that said subject is likely to be responsive to said inhibitor.
Also contemplated is a method of preparing a prognostic profile for a subject's response to an IMPDH inhibitor comprising the steps of administering to said subject to an IMPDH inhibitor; subjecting RNA extracted from the cells of said subject to gene expression profiling; determining the expression level of at least four genes selected from the group consisting of the genes set forth in Table I, Table II, Table III, Table IV, Table V, Table VI, Table VII and Table VIII in said cells of said subject; and comparing the expression levels obtained in step (c) to expression levels obtained in the absence of said administration of said IMPDH inhibitor, wherein a modulation of the expression level of said four or more of genes in response to said IMPDH inhibitor administration indicates that said IMPDH inhibitor will likely predict the subject's response to said IMPDH inhibitor administration. These methods can be used to monitor a response such as a therapeutic response, toxicity, dose range, and patient stratification.
In these methods, the cells may be cancer cells, e.g., cells selected from the group consisting of breast cancer, ovarian cancer, hematological cancer, gastric cancer, colorectal cancer, pancreatic cancer, and lung cancer. The cells can be fixed or may be from a fresh biopsy or may be from a cancer cell culture. In preferred embodiments, the cancer cells are cultured ex vivo. In the above methods the expression profile may be compiled into a report that includes recommendation for a treatment for said subject with an IMPDH inhibitor. In further embodiments, if altered expression of one or more of the genes selected from the genes set forth in Tables I, II, III, IV, V, VI, VII, and VIII, or the corresponding expression product is determined, said report includes a prediction that said subject is a suitable candidate for IMPDH inhibition-based therapy. In certain embodiments, the method may further comprise treating said patient with an IMPDH inhibitory agent. The biological sample used may be a blood sample, a tissue biopsy, or a tumor cell isolated from a tumor biopsy. The subset of genes could also be useful in conducting animal models or defining biomarkers in animal models used to determine efficacy or PK-PD correlation for IMPDH inhibitors.
The treatment method is one in which subject has been treated with any IMPDH inhibitor. In this regard, several IMPDH inhibitors have been used clinically and more are being developed for the treatment of immune cell-mediated, chronic inflammatory, antineoplatic and antiviral indications (Jain et al, 2001, Jain et al, 2002, Dhar et al, 2002). Mycophenolic acid (MPA), has been demonstrated to inhibit the proliferation of lymphocytes in vitro and in vivo (Eugui et al, 1991a, Allison and Eugui, 2000). An ester pro-drug of MPA, mycophenolate mofetil (CellCept®) is prescribed for the prevention of acute rejection in kidney, heart and liver transplantation (reviewed in Mele and Halloran, 2000), in combination with steroids and cyclosporine A (CsA). Mizoribine (Bredinin®) and ribavirin (Virazole®, Rebetol®) are nucleoside analogs which following intracellular phosphorylation are competitive IMPDH inhibitors (Franchetti and Grifantini, 1999, Saunders and Raybuck, 2000). Mizoribine is approved in Japan for multiple indications including prevention of rejection after renal transplantation, idiopathic glomerulonephritis, lupus nephritis, and rheumatoid arthritis (Ishikawa 1999). Ribavirin is used extensively as an inhaled antiviral agent for treatment of respiratory syncytial virus (RSV) and, orally in combination with interferon-a, for the treatment of chronic hepatitis C viral (HCV) infection (Davis et al 1998, McHutchison et al, 1998, Poynard et al 1998). Antineoplastic and antiviral activities of other nucleoside and NAD analogs, such as tiazofurin for the treatment of CML (reviewed in Jayaram et al, 1999), have been described but these are not yet clinically approved (reviewed in Saunders and Raybuck, 2000). The diverse therapeutic indications for these and other IMPDH inhibitors that are being developed illustrate the unmet clinical need for potent, reversible and lymphocyte-selective inhibitors that are safe, well-tolerated, and suitable for long-term dosing. The methods of the invention will facilitate such evaluations.
Also part of the invention are kits which comprise the nucleic acid arrays described herein in combination with appropriate containers, buffers, reagents and instructions for use in diagnostic and/or therapeutic methods. Such kits may further comprise nucleic acids of a reference subject as calibration standards. Further, the kits also may comprise detectable labels for the compositions of the invention.
Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
Traditional therapeutic monitoring of drugs involves the measurement of drug concentrations in patient blood. Several biochemical approaches to measure the biological effect of MPA or MMF (Langman et al 1995, Langman et al 1996, Griesmacher et al, 1997, Gummert et al, 1999, Albrecht et al, 2000, Budde et al, 2000, Qiu et al, 2000, Daxecker et al, 2001), mizoribine (Hirohata and Yamagida, 1995), and ribavirin (Fairbanks et al, 1995, Montero et al, 1995) have been described. In the present invention, the inventors have evaluated gene expression changes in response to VX-944 as an example of a potent and specific IMPDH inhibitor. MPA, a structurally unrelated but mechanistically similar IMPDH inhibitor, was included in some studies to compare its effect with VX-944. Given the similar mechanism of action of the two compounds, genes affected by both VX-944 and MPA are more likely to represent biomarkers for IMPDH inhibition. These results will be valuable in optimizing the dosing regimen and therapeutic benefit of IMPDH-based therapies in a broad range of therapeutic indication. Monitoring these patterns of gene expression changes are also likely to be predictive of a beneficial outcome with other unknown IMPDH pathway inhibitors. The gene expression changes observed with VX-944 and MPA may also allow selection of drugs that may enhance the effect of IMPDH inhibitors in combination treatments. Examples of gene expression changes have been published for Gleevec/STI-571. Based on the two compounds targeting two complementary pathways, we predict that a combination of the two agents is likely to be synergisitic in treating cancer.
The present invention is directed to methods and compositions for using biomarkers that are responsive to changes in IMPDH activity levels. More particularly, in certain preferred embodiments, the invention is directed to biomarkers that are responsive to IMPDH inhibition. In preferred aspects of the present invention there are provided nucleic acid arrays or sets of nucleic acids that are specifically up-regulated or down-regulated in response to IMPDH inhibition. Having identified these specific nucleic acids, it is now possible to prepare microarrays and specific collections of nucleic acids that may be used in order to identify additional IMPDH inhibitors, to verify that a known IMPDH inhibitor is exerting a desired effect in vitro or in vivo, and to provide a genetic profile of a subject under investigation in order to determine whether the individual is responsive to IMPDH inhibition. In one embodiment, the inventors obtained 18 gene chips that contained a total of 8793 genes on the arrays. Out of these 8793 genes there were 3421 genes that were common to all 18 gene chips. Of these, the inventors identified 1355 genes that were responsive to IMPDH inhibition. Gene sets 2 and 3 identified in the examples provided below contain the preferred subset of genes that are responsive to IMPDH inhibition. The following table summarizes the response of those genes to such inhibition and from these analyses the genes that were identified as particularly useful exemplary biomarkers for IMPDH inhibition are listed immediately beneath the table.
Of the above genes, particularly useful exemplary biomarkers include, but are not limited to: ARF4; CCNE2; CGR19; CPR8; EDF1; GTF2F1; LGALS1; PDGFC; PSMC5; PTK9; RABGGTA; SCAP2; SERPINB2; TBPL1.
Monitoring gene expression patterns offers an alternative approach for optimizing the therapeutic window as compared with the traditional method of measuring drug concentrations in blood and correlating either the AUC or Cmax levels with a clinical response. Towards that end, the inventors have comprehensively evaluated several genes affected by treatment with two distinct IMPDH inhibitors in vitro, as potential readouts for assessing their biological activity. The genes that are listed in Tables I through VIII are particularly useful in the present invention. These genes were identified using the specific methods as follows: Most cell culture reagents, unless specified otherwise, were obtained from Gibco-BRL or JRH Biologicals. Complete RPMI was prepared by adding up to a final of 10% heat-inactivated fetal bovine serum (FBS), 55 μM β mercaptoethanol, 50 units/mL penicillin with 50 μg/mL streptomycin, 300 μg/ml L-glutamine and 10 mM HEPES pH 7.5 to RPMI1640. Stock solutions of Phyto-hemagglutinin (PHA-P, Difco) was prepared in FBS-free RPMI and stored at −20° C. VX-944, and MPA (Sigma-Aldrich or Calbiochem), were dissolved in DMSO at a concentration of 20 mM and stored at −20° C.
Peripheral blood mononuclear cells (PBMCs) and T cells were isolated as follows: Human venous blood was drawn from healthy volunteers using heparin as an anti-coagulant. PBMCs were isolated from blood by centrifugation over Ficoll-paque gradient or CPT tubes (Becton-Dickinson, Calif.), using standard conditions. PBMCs were harvested, washed and resuspended in complete RPMI, counted and diluted to 1×106 cells/ml.
In order to monitor PBMC proliferation assay, human PBMC derived T cells (5×104 cells/well), were added to 96-well plates. PHA was added up to a final concentration of 10-20 μg/mL per well for stimulating T cells. The mitogen SPAS was used at a final concentration of 2 mg/mL. DMSO was maintained at a final concentration of 0.1%.
The methods described here are standard techniques that can be implemented at many clinical laboratories. The readouts can be adapted further for pharmacodynamic monitoring of IMPDH inhibitors in a variety of therapeutic indications, ultimately helping to optimize their therapeutic window in patients.
The methods and compositions of the present invention will typically employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, 3rd edition (Sambrook et al., 2000); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology”, 4.sup.th edition (D. M. Weir & C. C. Blackwell, eds., Blackwell Science Inc., 1987); “Gene Transfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymnerase Chain Reaction”, (Mullis et al., eds., 1994); ); Current Protocols in Immunology.
Exemplary aspects of the present invention will involve partial and/or complete gene expression profiling of a patient in order to assess whether the patient has or will be responsive to IMPDH inhibition. Such gene expression profiling methods include methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, and proteomics-based methods. Common methods for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)); and PCR-based methods, such as reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)). Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).
PCR-based methods of expression profiling include, for example, RT-PCR, differential display (Liang and Pardee, Science 257:967-971 (1992)); amplified fragment length polymorphism (iAFLP) (Kawamoto et al., Genome Res. 12:1305-1312 (1999)); BeadArray™ technology (Illumina, San Diego, Calif.; Oliphant et al., Discovery of Markers for Disease (Supplement to Biotechniques), June 2002; Ferguson et al., Analytical Chemistry 72:5618 (2000)); BeadsArray for Detection of Gene Expression (BADGE), using the commercially available Luminex100 LabMAP system and multiple color-coded microspheres (Luminex Corp., Austin, Tex.) in a rapid assay for gene expression (Yang et al., Genome Res. 11:1888-1898 (2001)); high coverage expression profiling (HiCEP) analysis (Fukumura et al., Nucl. Acids. Res. 31(16) e94 (2003)), and MassARRAY-based gene expression profiling method, which employs a mass spectrometry detection system.
A TaqMan assay is used to detect and optionally measure specific sequences in Polymerase Chain Reaction (PCR) products by employing the 5′Δ3′ exonuclease activity of Taq DNA polymerase. The TaqMan probe disabled from extension at the 3′ end, consists of a site-specific sequence labeled with a fluorescent reporter dye and a fluorescent quencher dye. During PCR the TaqMan probe set are derived from the genes of Tables I through VIII hybridizes to its complementary single stranded DNA sequence within the PCR target. When amplification occurs the TaqMan probe is degraded due to the 5′→3′ exonuclease activity of Taq DNA polymerase, thereby separating the quencher from the reporter during extension. Due to the release of the quenching effect on the reporter, the fluorescence intensity of the reporter dye increases. During the entire amplification process this light emission increases exponentially, the final level being measured by spectrophotometry after termination of the PCR. Because increase of the fluorescence intensity of the reporter dye is only achieved when probe hybridization and amplification of the target sequence has occurred, the TaqMan assay offers a sensitive method to determine the presence or absence of specific sequences. Therefore, this technique is particularly useful in diagnostic applications, such as the screening of samples for the presence or incorporation of favorable traits and the detection of pathogens and diseases. The TaqMan assay allows high sample throughput because no gel-electrophoresis is required for detection. Each TaqMan® Gene Expression Assay consists minimally of two unlabeled primers and a labeled probe. A more detailed description of this technique is contained in e.g., Bustin, S A. (2000). Journal of Molecular Endocrinology, 25: 169-193.
Sample preparation for use in analysis. In order to conduct sample analysis in the above gene expression profiling methods, a sample containing target polynucleotides is provided. The samples derived from can be any source containing target polynucleotides and obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. In preferred embodiments the target nucleic acid is obtained from a biological sample obtained from an individual that has undergone therapy based on IMPDH inhibition. In some embodiments, the expression profile of such a patient is obtained before and after IMPDH inhibition in order to assess the change in gene expression as a result of IMPDH therapy. Such expression profiling may be ongoing throughout the IMPDH therapy in order to assess whether the inhibition is having a sustained effect throughout the therapy.
DNA or RNA can be isolated from the biological sample according to any of a number of methods well known to those of skill in the art. In certain embodiments, the nucleic acid is isolated from a variety of primary tumors, including breast, lung, colorectal, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc., tumor, or tumor cell lines, with pooled DNA from healthy donors. More generally, the nucleic acid is isolated from hyperproliferative cells such as those found in immune system disorders such as transplant rejection and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, inflammatory bowel disease, as well as in the treatment of cancer and tumors, such as lymphomas and leukemia, vascular diseases, such as restenosis, and viral replication diseases, such as retroviral diseases, hepatitis C, and herpes. If the source of mRNA is a primary tumor or primary tissue from a hyperproliferative disorder, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples. Other methods of purification of nucleic acids are described in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Elsevier, New York N.Y. For example, total RNA is can be isolated using the TRIZOL reagent (Life Technologies, Gaithersburg Md.), and mRNA is isolated using oligo d(T) column chromatography or glass beads. Alternatively, when target polynucleotides are derived from an mRNA, the target polynucleotides can be a cDNA reverse transcribed from an mRNA to generate more stable cDNAs, an RNA transcribed from that cDNA, a DNA amplified from that cDNA, an RNA transcribed from the amplified DNA, and the like. When the target polynucleotide is DNA, it can be derived from a DNA source or from an RNA source by reverse transcription. In yet another embodiment, the targets are target polynucleotides prepared by more than one method.
General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andrs et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen™, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MasterPure™ Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), and Paraffin Block RNA Isolation Kit, (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumor can be isolated, for example, by cesium chloride density gradient centrifugation. Note that as RNA cannot serve as a template for PCR, the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction.
When target polynucleotides are amplified it is desirable to amplify the nucleic acid sample and maintain the relative abundances of the original sample, including low abundance transcripts. Total mRNA can be amplified by reverse transcription using a reverse transcriptase and a primer consisting of oligo d(T) and a sequence encoding the phage T7 promoter to provide a single-stranded DNA template. The second DNA strand is polymerized using a DNA polymerase and a RNAse (e.g., RNAse H) which assists in breaking up the DNA/RNA hybrid. After synthesis of the double-stranded DNA, T7 RNA polymerase can be added, and RNA transcribed from the second DNA strand template (Van Gelder et al. U.S. Pat. No. 5,545,522). RNA can be amplified in vitro, in situ or in vivo (See Eberwine U.S. Pat. No. 5,514,545).
Controls to quantitate the product may be included within the sample to assure that amplification and labeling procedures do not change the relative representation of target polynucleotides in a sample. For this purpose, a sample is spiked with a known amount of a control target polynucleotide and the composition of polynucleotide probes includes reference polynucleotide probes which specifically hybridize with the control target polynucleotides. After hybridization and processing, the hybridization signals obtained should reflect accurately the amounts of control target polynucleotide added to the sample.
Prior to hybridization, it may be desirable to fragment the nucleic acid target polynucleotides. Fragmentation improves hybridization by minimizing secondary structure and cross-hybridization to other nucleic acid target polynucleotides in the sample or noncomplementary polynucleotide probes. Fragmentation can be performed by mechanical or chemical means.
The target polynucleotides may be labeled with one or more labeling moieties to allow for detection of hybridized probe/target polynucleotide complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. The labeling moieties include radioisotopes, such as 32P, 33P or 35S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent. markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like.
Exemplary dyes include quinoline dyes, triarylmethane dyes, phthaleins, azo dyes, cyanine dyes and the like. Preferably, fluorescent markers absorb light above about 300 nm, preferably above 400 nm, and usually emit light at wavelengths at least greater-than 10 nm above the wavelength of the light absorbed. Preferred fluorescent markers include fluorescein, phycoerythrin, rhodamine, lissamine, and C3 and C5 available from Amersham Pharmacia Biotech (Piscataway N.J.).
Labeling can be carried out during an amplification reaction, such as polymerase chain and in vitro transcription reactions, or by nick translation or 5′ or 3′-end-labeling reactions. When the label is incorporated after or without an amplification step, the label is incorporated by using terminal transferase or by kinasing the 5′ end of the target polynucleotide and then incubating overnight with a labeled oligonucleotide in the presence of T4 RNA ligase.
Alternatively, the labeling moiety can be incorporated after hybridization once a probe/target complex has formed.
Nucleic Acid Microarrays: The samples prepared above are hybridized to nucleic acid microarrays of the invention. An array of the invention typically will be a nucleic acid array consisting essentially of at least 4 polynucleotides selected from the polynucleotides listed in any one or more of Gene Tables I through Gene Table VIII, wherein said polynucleotides are immobilized on a solid surface, and wherein the array further contains one or more calibration points and one or more housekeeping genes.
The housekeeping genes are provided as controls for the chip so as to provide signals corresponding to nucleic acids that are constitutively expressed at a known level. These genes are presumed to produce the minimally essential transcripts necessary for normal cellular physiology, and are expressed at a similar level in almost all cells. These controls are useful in normalizing the data, for example to quantify expression levels. As a control gene, any known reference gene can be used, including, for example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, U-snRNP-associated cyclophilin (USA-CYP), ribosomal protein LPO; 18S ribosomal RNA; Glucoronidase beta; and Ribosomal protein L32. Alternatively, normalization can be achieved by correcting for differences between the total of all signals of the tested gene sets (global normalization strategy). The report may include a prognosis for the outcome of the treatment of the patient. The method may additionally comprise the step of treating the subject, e.g. a human patient, if a good prognosis is indicated.
The calibration spot is a signal that is contained on the microarray that will be used to ensure that the microarray is approprotately aligned and being read by a detection device. Typically, such a calibration spot comprises a mixture of the plurality of nucleic acids isolated from the sample that are known not to be modulated with IMPDH inhibition.
In the present invention, 5 or more nucleic acids selected from the distinct sequences of listed in Gene Tables I-VIII may be presented in a DNA microarray for the analysis and expression of these genes in various cell types. Microarray chips are well known to those of skill in the art (e.g., see U.S. Pat. No. 6,308,170; U.S. Pat. No. 6,183,698; U.S. Pat. No. 6,306,643; U.S. Pat. No. 6,297,018; U.S. Pat. No. 6,287,850; U.S. Pat. No. 6,291,183, each incorporated herein by reference. These are merely exemplary patents that disclose nucleic acid microarrays and those of skill in the art will be aware of numerous other methods and compositions for producing microarrays). Given the identification of the above nucleic acids that are specifically responsive to IMPDH inhibition it is possible to use commercially available microarray chips that contain probes that will hybridize to such nucleic acids regardless of any other nucleic acid probes contained on such chips. In this regard a preferred chip is the Affymetrix HG-FOCUS arrays (Affymetrix, CA catalog #900377).
The present invention provides for a composition comprising a plurality of polynucleotide probes for use in detecting changes in expression of a large number of genes from cells in response to IMPDH inhibition. As used in this section, the term “polynucleotide probe” refers to a nucleic acid that hybridize to any one of the nucleic acids listed in Tables I-VIII or hybridizes to any fragment thereof or any nucleic acid that hybridizes specifically to one such nucleic acid. Particularly preferred exemplary markers of IMPDH inhibition are provided in gene sets I through VIII. Other exemplary markers include ARF4; CCNE2; CGR19;CPR8; EDF1; GTF2F1; LGALS1; PDGFC; PSMC5; PTK9; RABGGTA; SCAP2; SERPINB2; TBPL1. Preferably the fragment is longer than 20 base pairs. Such a composition can be employed for the diagnosis and for monitoring the treatment of any disease in which abnormal IMPDH is found, and preferably diseases in which overexpression or increased activity of IMPDH is implicated. IMPDH-mediated disease refers to any disease state in which the IMPDH enzyme plays a regulatory role in the metabolic pathway of that disease. Examples of IMPDH-mediated diseases include transplant rejection and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, and inflammatory bowel disease, as well as inflammatory diseases, cancer, viral replication diseases and vascular diseases. In particular embodiments, the compositions of the invention may be used to monitor/track the treatment of transplant rejection (e.g., kidney, liver, heart, lung, pancreas (islet cells), bone marrow, cornea, small bowel and skin allografts and heart valve xenografts), rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, inflammatory bowel disease (Crohn's disease, ulcerative colitis), lupus, diabetes, mellitus myasthenia gravis, psoriasis, dermatitis, eczema, seborrhea, pulmonary inflammation, eye uveitis, hepatitis, Grave's disease, Hashimoto's thyroiditis, Behcet's or Sjorgen's syndrome (dry eyes/mouth), pernicious or immunohaemolytic anaemia, idiopathic adrenal insufficiency, polyglandular autoimmune syndrome, and glomerulonephritis, scleroderma, lichen planus, viteligo (depigmentation of the skin), autoimmune thyroiditis, and alveolitis, inflammatory diseases such as osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma and adult respiratory distress syndrome, as well as in the treatment of cancer and tumors, such as solid tumors, lymphomas and leukemia, vascular diseases, such as restenosis, stenosis and atherosclerosis, and DNA and RNA viral replication diseases, such as retroviral diseases, and herpes.
A particularly useful composition of the present invention is a collection of hybridizable array elements in a microarray for monitoring the expression of a plurality of target polynucleofides. The microarray typically comprises a solid substrate and hybridizable array elements positioned on said substrate. The microarray can be used, for example, in the assessment of the efficacy of IMPDH inhibition on the treatment of a cancer, in immune disorders, in neuropathologies, and the like.
Tables I through VIII herein list the exemplary sequences disclosed herein as being responsive to IMPDH inhibition. In set I there are listed specific genes that are responsive to VX-944 in resting PBMC cells, i.e., genes from normal non-proliferating lymphocytes that were found to be specifically responsive (i.e., their expression levels were either increased or decreased) to VX-944. Gene set I can be described as a set of genes whose expression changes in changes in blood obtained from patients suffering from cancer, autoimmune, inflammatory diseases upon treatment with IMPDH inhibitors. These genes were: thrombospondin 1; interleukin 18 receptor 1; aldehyde dehydrogenase 1 family, member A1; complement component 1, q subcomponent, beta polypeptide; chitinase 3-like 1 (cartilage glycoprotein-39); BCR downstream signaling 1; XIAP associated factor-1; protein kinase N2; interferon-induced protein with tetratricopeptide repeats 5; CD38 antigen (p45); peroxisomal biogenesis factor 19; defensin, alpha 1, myeloid-related sequence; cleavage and polyadenylation specific factor 1, 160 kDa; hemochromatosis; chromosome 1 open reading frame 29; apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B; interferon, alpha-inducible protein 27; ubiquitin specific protease 18; interferon-induced protein with tetratricopeptide repeats 1; tissue inhibitor of metalloproteinase 2; myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse); chemokine (C-C motif) ligand 8; interferon regulatory factor 7; 2′-5′-oligoadenylate synthetase 3, 100 kDa; sialoadhesin; interferon-induced protein 44; myxovirus (influenza virus) resistance 2 (mouse); phospholipid scramblase 1; hect domain and RLD 5; interferon, alpha-inducible protein (clone IFI-6-16); interferon, alpha-inducible protein (clone IFI-15K); suppressor of cytokine signaling 1; chromosome 6 open reading frame 11; chemokine (C-C motif) ligand 13; chemokine (C-C motif) ligand 2; interleukin 1 receptor antagonist; chemokine (C-X-C motif) ligand 10; chemokine (C-X-C motif) ligand 9. The sequences of these genes are provided below in Table 1.
In set II there are listed specific genes that are responsive to VX-944 in resting or non-proliferating and proliferating cells. As such, these genes are those whose gene expression changes in normal human lymphocytes upon treatment with VX-944 or a mitogen or both mitogen and VX-944. This set was determined as follows: the genes altered by VX-944 administration alone in resting lymphocytes were identified by comparing genes altered in VX-944 treated cells as compared to untreated cells. These genes from resting cells were compared with gene expression profile seen in proliferating cells by comparing genes altered in HL60 cells treated with VX-944 compared to untreated HL60 cells. Subsequently, gene expression pattern changes attenuated in proliferating human lymphocytes in the presence of with VX-944 treatment were determined by comparing genes altered in PHA-treated cells compared to untreated cells as compared with genes altered by PHA+VX-944 treated cells. These genes were: chitinase 3-like 1 (cartilage glycoprotein-39) and interleukin 1 receptor antagonist. In gene set III, an assessment was made of the genes that are responsive to VX-944 in resting PBMC cells as well as in HL-60 cells and also genes that were responsive to MPA, another IMPDH inhibitor. These studies again showed that both chitinase 3-like 1 (cartilage glycoprotein-39) and interleukin 1 receptor antagonist are particularly responsive. As such, these two genes will be particularly predictive biomarkers of responsiveness of a given cell to IMPDH inhibition. Gene set II can be monitored in lymphocytes or blood obtained from cancer patients that have been treated with an IMPDH inhibitor such as VX-944.
Set IV shows the genes that could be monitored in proliferating cells from patients treated with VX-944. This set is typical of the gene expression changes that can be monitored in lymphocytes or blood obtained from cancer, autoimmune, transplant-patients, virally infected or otherwise diseased subjects that have been treated with VX-944. These genes represent a subset of genes that are up- or down-regulated in lymphocytes upon stimulation with a mitogen such as PHA (T1), that are modulated in the presence of VX-944.
These genes were identified by first inducing PBMC cells to proliferate, e.g., by stimulating the cells with PHA and determining which genes have an altered expression upon proliferation alone. The PBMC's also were analyzed to obtain the differential expression seen in response to both stimulation with PHA and treatment with VX-944. The overlap between the genes that were expressed in both sets of experiments is listed in Set IV as genes that should preferably be monitored in proliferating lymphocytes or cells of patients treated with VX-944. These genes are: RAD54 homolog B (homolog of S. cerevisiae); chemokine (C-C motif) ligand 2; kinesin family member 4A; spleen focus forming virus (SFFV) proviral integration oncogene spi1; chromosome condensation 1; Fc fragment of IgG, high affinity Ia, receptor for (CD64); asialoglycoprotein receptor 2; aurora kinase B; a disintegrin and metalloproteinase domain 28; growth arrest-specific 7; mucolipin 1; activator of S phase kinase; hexokinase 3 (white cell); nudix (nucleoside diphosphate linked moiety X)-type motif 1; Fc fragment of IgE, low affinity II, receptor for (CD23A); hemopoietic cell kinase; kinesin family member 11; SNARE protein Ykt6; 3-hydroxyisobutyryl-Coenzyme A hydrolase; cytochrome P450, family 1, subfamily B, polypeptide 1; chemokine (C motif) ligand 1; cyclin F; primase, polypeptide 2A, 58 kDa; Charcot-Leyden crystal protein; alpha-2-macroglobulin; exonuclease 1; CDC6 cell division cycle 6 homolog (S. cerevisiae); matrix metalloproteinase 1 (interstitial collagenase); protein regulator of cytokinesis 1; leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 1; KIAA0146 protein; CDC28 protein kinase regulatory subunit 1B; baculoviral IAP repeat-containing 5 (survivin); Rac GTPase activating protein 1; zinc finger homeobox 1b; glycoprotein (transmembrane) nmb; IGF-II mRNA-binding protein 2; solute carrier family 27 (fatty acid transporter), member 2; cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity phosphatase); chemokine (C-X3-C motif) receptor 1; sperm associated antigen 5; formyl peptide receptor 1; cadherin 3, type 1, P-cadherin (placental); origin recognition complex, subunit 1-like (homolog of yeast); chromosome condensation 1-like; protein tyrosine phosphatase, non-receptor type 12; myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (homolog of mouse); solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1; solute carrier family 7 (cationic amino acid transporter, y+ system), member 7; serine hydroxymethyltransferase 2 (mitochondrial); nuclear receptor subfamily 4, group A, member 2; CDC28 protein kinase regulatory subunit 2; aldehyde dehydrogenase 2 family (mitochondrial); matrix metalloproteinase 19; development and differentiation enhancing factor 2; dipeptidylpeptidase 3; aryl hydrocarbon receptor; chemokine (C-C motif) ligand 8; chromosome 1 open reading frame 29; tensin-like SH2 domain containing 1; killer cell lectin-like receptor subfamily F, member 1; tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor); cat eye syndrome chromosome region, candidate 1; splicing factor 1 [BLAST]; v-fos FBJ murine osteosarcoma viral oncogene homolog; pleckstrin homology-like domain, family A, member 1; geminin, DNA replication inhibitor; hyaluronan-mediated motility receptor (RHAMM); chromosome 1 open reading frame 33; regulator of G-protein signalling 2, 24 kDa; transforming growth factor, beta-induced, 68 kDa; serine/threonine kinase 6; Fc fragment of IgG, low affinity IIa, receptor for (CD32); ribonuclease T2; arachidonate 5-lipoxygenase; cytokine receptor-like factor 2; purinergic receptor P2Y, G-protein coupled, 5; chromobox homolog 5 (HP1 alpha homolog of Drosophila); inositol(myo)-1 (or 4)-monophosphatase 2; 2′-5′-oligoadenylate synthetase 3, 100 kDa; transcription factor EC; chondroitin sulfate proteoglycan 2 (versican); histone 1, H2ac; metallothionein 1X; aquaporin 3; apolipoprotein A-II; chemokine (C motif) ligand 1; RAD51 homolog (RecA homolog of E. coli) (homolog of S. cerevisiae); C-type (calcium dependent, carbohydrate-recognition domain) lectin, superfamily member 5; TYRO protein tyrosine kinase binding protein; polymerase (DNA directed), epsilon 2 (p59 subunit); FBJ murine osteosarcoma viral oncogene homolog B; fucosyltransferase 8 (alpha (1,6) fucosyltransferase); CD14 antigen; GLI pathogenesis-related 1 (glioma); CD209 antigen; thymidine kinase 1, soluble; interleukin 9; dihydropyrimidine dehydrogenase; lectin, galactoside-binding, soluble, 2 (galectin 2); Kruppel-like factor 4 (gut); ribonucleotide reductase M2 polypeptide; proliferating cell nuclear antigen; cyclin B2; killer cell lectin-like receptor subfamily B, member 1; chemokine (C-C motif) receptor 1; syntaxin 11; mannose receptor, C type 1; lung type-I cell membrane-associated glycoprotein; guanidinoacetate N-methyltransferase; alanyl (membrane) aminopeptidase (aminopeptidase N, aminopeptidase M, microsomal aminopeptidase, CD13, p150); zinc finger protein 36, C3H type-like 1; 24-dehydrocholesterol reductase; hematological and neurological expressed 1; M-phase phosphoprotein 1; activation-induced cytidine deaminase; interleukin 1 receptor, type II; transforming growth factor, alpha; collagen, type VI, alpha 3; deoxythymidylate kinase (thymidylate kinase); pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 1; TTK protein kinase; ataxia telangiectasia mutated (includes complementation groups A, C and D); histidine decarboxylase; acyloxyacyl hydrolase (neutrophil); G protein-coupled receptor 77; serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 2; solute carrier family 7, (cationic amino acid transporter, y+ system) member 11; splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing factor); glutaminyl-peptide cyclotransferase (glutaminyl cyclase); solute carrier family 16 (monocarboxylic acid transporters), member 6; CCAAT/enhancer binding protein (C/EBP), alpha; SKB1 homolog (S. pombe); v-myb myeloblastosis viral oncogene homolog (avian)-like 2; CDC42 effector protein (Rho GTPase binding) 1; integrin, alpha M (complement component receptor 3, alpha; also known as CD11b (p170), macrophage antigen alpha polypeptide); centromere protein F, 350/400 ka (mitosin); cystatin C (amyloid angiopathy and cerebral hemorrhage); sialic acid binding Ig-like lectin 7; C-type (calcium dependent, carbohydrate-recognition domain) lectin, superfamily member 14 (macrophage-derived); serum/glucocorticoid regulated kinase; uridine monophosphate kinase; Kruppel-like factor 2 (lung); fructose-1,6-bisphosphatase 1; neutrophil cytosolic factor 2 (65 kDa, chronic granulomatous disease, autosomal 2); frizzled homolog 2 (Drosophila); integrin, beta 7; interleukin 12 receptor, beta 2; colony stimulating factor 1 receptor, formerly McDonough feline sarcoma viral (v-fms) oncogene homolog; kinesin family member C3; N-acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase); tensin; complement component 1, q subcomponent, receptor 1; leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 4; kinesin-like 7; chemokine (C-C motif) ligand 7; myeloid cell nuclear differentiation antigen; BCL2/adenovirus E1B 19 kDa interacting protein 3; pituitary tumor-transforming 1; interferon-induced protein 44; chemokine (C-X-C motif) ligand 2; SLAM family member 8; transferrin receptor 2; integrin, alpha X (antigen CD11C (p150), alpha polypeptide); serine/threonine kinase 6; ficolin (collagen/fibrinogen domain containing) 1; chromodomain helicase DNA binding protein 1-like; Fc fragment of IgG, low affinity IIb, receptor for (CD32); secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1); CD86 antigen (CD28 antigen ligand 2, B7-2 antigen); achalasia, adrenocortical insufficiency, alacrimia (Allgrove, triple-A); polo-like kinase 1 (hormolog of Drosophila); ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin); nuclear receptor interacting protein 1; thymidylate synthetase; DNA replication complex GINS protein PSF2; CD9 antigen (p24); T-LAK cell-originated protein kinase; RAB31, member RAS oncogene family; centromere protein E, 312 kDa; ZW10 interactor; fibrinogen-like 2; bombesin-like receptor 3; matrix metalloproteinase 12 (macrophage elastase); dual specificity phosphatase 6; chemokine (C-X-C motif) ligand 3; MAD2 mitotic arrest deficient-like 1 (homolog of yeast); leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 2; CD72 antigen; cellular repressor of E1A-stimulated genes; CHK1 checkpoint homolog (homolog of S. pombe); interferon, gamma-inducible protein 30; ubiquitin-conjugating enzyme E2S; ribokinase; serine/threonine kinase 3 (STE20 homolog of yeast); v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (homolog of avian); exonuclease NEF-sp; vascular endothelial growth factor; stathmin 1/oncoprotein 18; MCM2 minichromosome maintenance deficient 2, mitotin (homolog of S. cerevisiae); ribonuclease, RNase A family, k6; G-protein signalling modulator 2 (AGS3-like, homolog of C. elegans); amylase, alpha 2B; pancreatic; tribbles homolog 1 (homolog of Drosophila); C-type (calcium dependent, carbohydrate-recognition domain) lectin, superfamily member 6; Ig superfamily protein; baculoviral IAP repeat-containing 1; transgelin; kinesin family member 20A; interleukin 3 (colony-stimulating factor, multiple); oxidised low density lipoprotein (lectin-like) receptor 1; Similar to ribosomal protein L13a; 60S ribosomal protein L13a; 23 kD highly basic protein (LOC399810), mRNA [BLAST]; endoplasmic reticulum chaperone SIL1, homolog of yeast; telomerase-associated protein 1; Fc fragment of IgG, receptor, transporter, alpha; nerve growth factor receptor (TNFRSF 16) associated protein 1; BUB1 budding uninhibited by benzimidazoles 1 homolog (homolog of yeast); cyclin A2; BUB1 budding uninhibited by benzimidazoles 1 homolog beta (homolog of yeast); gamma-glutamyltransferase-like activity 1; immunoglobulin superfamily, member 6; hypothetical protein CG003; fer-1-like 3, myoferlin (homolog of C. elegans); cell division cycle 2, G1 to S and G2 to M; erythrocyte membrane protein band 4.1-like 3; cell division cycle 25C; thyroid hormone receptor interactor 13; thrombospondin 1; TPX2, microtubule-associated protein homolog (homolog of Xenopus laevis); CDC20 cell division cycle 20 homolog (homolog of S. cerevisiae); lymphotoxin beta receptor (TNFR superfamily, member 3); membrane-spanning 4-domains, subfamily A, member 4; polo-like kinase 4 (homolog of Drosophila); cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4); toll-like receptor 8; histamine N-methyltransferase; interleukin 3 receptor, alpha (low affinity); cathepsin L; transforming, acidic coiled-coil containing protein 3; sialyltransferase 4A (beta-galactoside alpha-2,3-sialyltransferase); exosome component 9; BRCA1 associated RING domain 1; reticulon 1; leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member. 1; Transcribed sequences; H2A histone family, member Z; RAD54-like (homolog of S. cerevisiae); chromosome condensation protein G; glutathione transferase zeta 1 (maleylacetoacetate isomerase); kinesin family member 2C; ubiquitin-conjugating enzyme E2C; spinocerebellar ataxia 1 (olivopontocerebellar ataxia 1, autosomal dominant, ataxin 1); v-myb myeloblastosis viral oncogene homolog (homolog of avian); thrombomodulin; centromere protein A, 17 kDa; cytochrome b-245, beta polypeptide (chronic granulomatous disease); endoglin (Osler-Rendu-Weber syndrome 1); formyl peptide receptor-like 2; nucleolar and spindle associated protein 1; tripartite motif-containing 26; BRCA2 and CDKN1A interacting protein; ubiquitin specific protease 18; acid phosphatase 5, tartrate resistant; metallothionein 1H; NIMA (never in mitosis gene a)-related kinase 2; dual specificity phosphatase 1; breast cancer 1, early onset; fatty acid desaturase 2; topoisomerase (DNA) II alpha 170 kDa; neuropilin 1; LIM domain only 2 (rhombotin-like 1); tissue inhibitor of metalloproteinase 2; oncostatin M; S100 calcium binding protein A9 (calgranulin B); chemokine (C-C motif) ligand 24; BTB and CNC homology 1, basic leucine zipper transcription factor 1; leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 3; ribonuclease, RNase A family, 1 (pancreatic); pro-platelet basic protein (chemokine (C-X-C motif) ligand 7); phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma). These genes are listed in Table IV. These genes will be useful in assessing treatment of cancer, autoimmune diseases, inflammatory diseases and the like by VX-944 and are likely to represent attenuation of proliferation by VX-944 in diseases where cell proliferation is implicated. A combination of gene set I and gene set IV reveals markers that can be monitored in hematological malignancies. By hematological malignancies the present application refers to any hematological cancer or hematological disorder that manifests in the proliferation or hyperproliferation of blood cells.
Set V provides a more select marker set of genes from set IV whose expression also is altered in response to VX-944 in AML or PML cancers or other diseases in which the proliferation of lymphocytes is implicated. Thus, gene expression changes that can be monitored in lymphocytes (PBMCs, blood) obtained from hematological cancers, or other diseases in which proliferation of lymphocytes is implicated in the presence of VX-944 to provide an assessment of the efficacy of VX-944 or IMPDH inhibition in general. This subset of genes includes: kinesin family member 4A; Fc fragment of IgG, high affinity Ia, receptor for (CD64); aurora kinase B; growth arrest-specific 7; solute carrier family 27 (fatty acid transporter), member 2; cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity phosphatase); sperm associated antigen 5; formyl peptide receptor 1; chromosome condensation 1-like; tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor); pleckstrin homology-like domain, family A, member 1; hyaluronan-mediated motility receptor (RHAMM); regulator of G-protein signalling 2, 24 kDa; serine/threonine kinase 6; arachidonate 5-lipoxygenase; inositol(myo)-1(or 4)-monophosphatase 2; metallothionein IX; C-type (calcium dependent, carbohydrate-recognition domain) lectin, superfamily member 5; ribonucleotide reductase M2 polypeptide; cyclin B2; chemokine (C-C motif) receptor 1; mannose receptor, C type 1; collagen, type VI, alpha 3; histidine decarboxylase; G protein-coupled receptor 77; serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 2; CCAAT/enhancer binding protein (C/EBP), alpha; integrin, alpha M (complement component receptor 3, alpha; also known as CD11b (p170), macrophage antigen alpha polypeptide); serum/glucocorticoid regulated kinase; neutrophil cytosolic factor 2 (65 kDa, chronic granulomatous disease, autosomal 2); N-acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase); pituitary tumor-transforming 1; integrin, alpha X (antigen CD11C (p150), alpha polypeptide); serine/threonine kinase 6; polo-like kinase 1 (homolog of Drosophila); RAB31, member RAS oncogene family; CD72 antigen; interferon, gamma-inducible protein 30; v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (homolog of avian); tribbles homolog 1 (homolog of Drosophila); oxidised low density lipoprotein (lectin-like) receptor 1; BUB1 budding uninhibited by benzimidazoles 1 homolog beta (homolog of yeast); hypothetical protein CG003; CDC20 cell division cycle 20 homolog (homolog of S. cerevisiae); toll-like receptor 8; kinesin family member 2C; ubiquitin-conjugating enzyme E2C; v-myb myeloblastosis viral oncogene homolog (homolog of avian); centromere protein A, 17 kDa; cytochrome b-245, beta polypeptide (chronic granulomatous disease); nucleolar and spindle associated protein 1; metallothionein 1H; S100 calcium binding protein A9 (calgranulin B); and are listed herein below in Table V.
The genes in Table IV were further analysed and narrowed to those genes that are responsive to VX-944 and to the unrelated IMPDH inhibitor MPA to provide a subset of IV, that is further narrower than subset V by using genes commonly affected by both VX-944 and MPA in HL60 cells. This subset of genes included the following genes: kinesin family member 4A; Fc fragment of IgG, high affinity Ia, receptor for (CD64); growth arrest-specific 7; solute carrier family 27 (fatty acid transporter), member 2; cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity phosphatase); sperm associated antigen 5; formyl peptide receptor 1; chromosome condensation 1-like; tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor); pleckstrin homology-like domain, family A, member 1; regulator of G-protein signalling 2, 24 kDa; arachidonate 5-lipoxygenase; inositol(myo)-1(or 4)-monophosphatase 2; metallothionein 1X; C-type (calcium dependent, carbohydrate-recognition domain) lectin, superfamily member 5; ribonucleotide reductase M2 polypeptide; chemokine (C-C motif) receptor 1; collagen, type VI, alpha 3; histidine decarboxylase; serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 2; integrin, alpha M (complement component receptor 3, alpha; also known as CD11b (p170), macrophage antigen alpha polypeptide); serum/glucocorticoid regulated kinase; neutrophil cytosolic factor 2 (65 kDa, chronic granulomatous disease, autosomal 2); N-acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase); integrin, alpha X (antigen CD11C (p150), alpha polypeptide); RAB31, member RAS oncogene family; CD72 antigen; interferon, gamma-inducible protein 30; v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (homolog of avian); tribbles homolog 1 (homolog of Drosophila); v-myb myeloblastosis viral oncogene homolog (homolog of avian); cytochrome b-245, beta polypeptide (chronic granulomatous disease); nucleolar and spindle associated protein 1; metallothionein 1H; S100 calcium binding protein A9 (calgranulin B); and the sequences of these genes are listed in Table VI.
VX-944 also has been used as an antiviral agent. Genes that are predictive of the antiviral efficacy of VX-944 are found in Set VII (see Table VII), which were identified as the subset of genes from set I which are also modified in response to interferon. These genes will be useful for monitoring gene expression changes that in lymphocytes (PBMCs, blood) obtained from patients suffering from viral diseases such as HCV, or other diseases in which interferon treatment is useful. These genes were: interferon, alpha-inducible protein (clone IFI-6-16); interferon, alpha-inducible protein (clone IFI-15K); interferon-induced protein with tetratricopeptide repeats 5; interferon-induced protein with tetratricopeptide repeats 1; interferon-induced protein 44; myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse); chemokine (C-C motif) ligand 2; chemokine (C-X-C motif) ligand 10; chemokine (C-X-C motif) ligand 9 and the sequences of these genes is provided in Table VII.
In set VIII there are listed the apoptosis genes that are responsive to VX-944. This is a narrower subset of genes from subset IV whose gene expression changes can predict beneficial response to VX-944 and be monitored in lymphocytes (PBMCs, blood) obtained from cancer patients. Out of the 67 genes implicated in cancers, 4 are down-regulated by VX-944 in proliferating lymphocytes. These genes will be predictive of the anti-cancer cancer efficacy of VX-944 in leukemias and/or solid tumors. The sequences of these genes are listed below in Table VIII.
Alternatively, the above subsets of genes may be segregated according to function and it may be desirable to prepare microarrays that will be useful in providing an indication of the effects of VX-944 or any other IMPDH inhibitor on a specific functional pathway. In this regard, genes involved in apoptosis and whose expression is altered in response to IMPDH inhibition are: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105); TNF receptor-associated factor 2; TRK-fused gene; lymphotoxin alpha (TNF superfamily, member 1); inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon; nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; tumor necrosis factor receptor superfamily, member 5; tumor necrosis factor (ligand) superfamily, member 6; ADP-ribosyltransferase (NAD+; poly (ADP-ribose) polymerase); Fas (TNFRSF6)-associated via death domain; nerve growth factor, beta polypeptide; TNFRSF1A-associated via death domain; BH3 interacting domain death agonist; receptor (TNFRSF)-interacting serine-threonine kinase 1; tumor protein p53 (Li-Fraumeni syndrome); Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (homolog of mouse); caspase 7, apoptosis-related cysteine protease; tumor necrosis factor (TNF superfamily, member 2); myeloid cell leukemia sequence 1 (BCL2-related); caspase 10, apoptosis-related cysteine protease; BCL2-related ovarian killer; tumor necrosis factor receptor superfamily, member 1A; baculoviral IAP repeat-containing 3; B-cell CLL/lymphoma 2; BCL2-associated X protein; TRK-fused gene; tumor necrosis factor receptor superfamily, member 1B; caspase 6, apoptosis-related cysteine protease; tumor necrosis factor (ligand) superfamily, member 10; caspase 9, apoptosis-related cysteine protease; protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)-associated phosphatase); TNF receptor-associated factor 6; cytochrome c, somatic; mitogen-activated protein kinase kinase 4; v-myc myelocytomatosis viral oncogene homolog (homolog of avian); BCL2-like 2; v-jun sarcoma virus 17 oncogene homolog (homolog of avian); BCL2-antagonist/killer 1; mitogen-activated protein kinase kinase kinase 14;splicing factor, arginine/serine-rich 2, interacting protein; caspase 2, apoptosis-related cysteine protease (neural precursor cell expressed, developmentally down-regulated 2); apoptotic protease activating factor; perforin 1 (pore forming protein); BCL2-antagonist of cell death; BCL2-related protein A1; harakiri, BCL2 interacting protein (contains only BH3 domain); tumor necrosis factor receptor superfamily, member 6; tumor necrosis factor (ligand) superfamily, member 5 (hyper-IgM syndrome); mitogen-activated protein kinase 10; TNF receptor-associated factor 3; TNF receptor-associated factor 1; caspase 8, apoptosis-related cysteine protease; granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1); baculoviral IAP repeat-containing 2; caspase 4, apoptosis-related cysteine protease; CASP2 and RIPK1 domain containing adaptor with death domain; caspase 1, apoptosis-related cysteine protease (interleukin 1, beta, convertase); DNA fragmentation factor, 45 kDa, alpha polypeptide; DNA fragmentation factor, 40 kDa, beta polypeptide (caspase-activated DNase); baculoviral IAP repeat-containing 4; inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma; caspase 3, apoptosis-related cysteine protease; nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor); v-rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gene enhancer in B-cells 3, p65 (homolog of avian); nerve growth factor receptor (TNFR superfamily, member 16). Thus a microarray may be prepared to determine the efficacy of IMPDH inhibition by monitoring genes involved in apoptosis wherein the microarray comprises at least two or more of the above VX-944-responsive apoptosis-associated genes.
Genes involved in cellular metabolism that are responsive to VX-944 and IMPDH inhibition include: dehydrogenase/reductase (SDR family) member 4; dehydrogenase/reductase (SDR family) member 10; prion protein (p27-30) (Creutzfeld-Jakob disease, Gerstmann-Strausler-Scheinker syndrome, fatal familial insomnia); phosphomannomutase 1; arylsulfatase B; UDP glycosyltransferase 1 family, polypeptide A10; branched chain keto acid dehydrogenase E1, alpha polypeptide (maple syrup urine disease); 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1; chitinase 1 (chitotriosidase); ATPase, Ca++ transporting, plasma membrane 3; tyrosinase-related protein 1; 1-acylglycerol-3-phosphate O-acyltransferase 1 (lysophosphatidic acid acyltransferase, alpha); aldehyde dehydrogenase 1 family, member B1; dehydrogenase/reductase (SDR family) member 6; 3-hydroxyisobutyryl-Coenzyme A hydrolase; isocitrate dehydrogenase 3 (NAD+) alpha; pyruvate dehydrogenase complex, component X; 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2; galactokinase 2; pyruvate dehydrogenase (lipoamide) alpha 1; hydroxyprostaglandin dehydrogenase 15-(NAD); flightless I homolog (homolog of Drosophila); glutathione S-transferase M3 (brain); ATPase, H+/K+ exchanging, alpha polypeptide; ATPase, Ca++ transporting, type 2C, member 1; N-sulfoglucosamine sulfohydrolase(sulfamidase); aldehyde dehydrogenase 1 family, member A3; 5,10-methenyltetrahydrofolate synthetase(5-formyltetrahydrofolate cyclo-ligase); succinate-CoA ligase, ADP-forming, beta subunit; carboxypeptidase E; alkaline phosphatase, intestinal; chromodomain protein, Y-like; aldehyde dehydrogenase 5 family, member A1 (succinate-semialdehyde dehydrogenase); aldehyde dehydrogenase 4 family, member A1; branched chain aminotransferase 2, mitochondrial; isocitrate dehydrogenase 3 (NAD+) beta; acyl-CoA synthetase long-chain family member 3; hydroxysteroid (17-beta) dehydrogenase 4; ankyrin 2, neuronal; tryptophan hydroxylase 1 (tryptophan 5-monooxygenase); fibroblast growth factor 16; N-acetyltransferase 2 (arylamine N-acetyltransferase); solute carrier family 27 (fatty acid transporter), member 2; peroxisomal trans-2-enoyl-CoA reductase; phosphoserine aminotransferase 1; alanine-glyoxylate aminotransferase (oxalosis I; hyperoxaluria I; glycolicaciduria; serine-pyruvate aminotransferase); tafazzin (cardiomyopathy, dilated 3A (X-linked); endocardial fibroelastosis 2; Barth syndrome); liver-specific bHLH-Zip transcription factor; UDP glycosyltransferase 8 (UDP-galactose ceramide galactosyltransferase); pyruvate dehydrogenase (lipoamide) alpha 2; arylsulfatase A; alanine-glyoxylate aminotransferase (oxalosis I; hyperoxaluria I; glycolicaciduria; serine-pyruvate aminotransferase); ATPase, Na+/K+ transporting, alpha 1 polypeptide; arylsulfatase F; hydroxysteroid (17-beta) dehydrogenase 12; G protein-coupled receptor 56; phosphoribosyl pyrophosphate amidotransferase; eyes absent homolog 4 (homolog of Drosophila); hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit; lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase); family with sequence similarity 16, member A, X-linked; alkaline phosphatase, placental (Regan isozyme); acyl-CoA synthetase long-chain family member 1; alkaline phosphatase, liver/bone/kidney; eosinophil chemotactic cytokine; hydroxysteroid (11-beta) dehydrogenase 1; isocitrate dehydrogenase 2 (NADP+), mitochondrial; pyruvate carboxylase; galactosamine (N-acetyl)-6-sulfate sulfatase (Morquio syndrome, mucopolysaccharidosis type IVA); phenylalanine hydroxylase; matrix metalloproteinase 24 (membrane-inserted); nucleolar protein 5A (56 kDa with KKE/D repeat); phosphoglycerate mutase 2 (muscle); AU RNA binding protein/enoyl-Coenzyme A hydratase; retinol dehydrogenase 8 (all-trans); glutathione S-transferase A1; glutamine-fructose-6-phosphate transaminase 1; phosphatidylglycerophosphate synthase; WW domain containing oxidoreductase; serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 4; glutamine-fructose-6-phosphate transaminase 2; phosphoserine phosphatase; aspartoacylase (aminoacylase 2, Canavan disease); oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide); eyes absent homolog 3 (homolog of Drosophila); glyoxylate reductase/hydroxypyruvate reductase; aldehyde dehydrogenase 3 family, member B1; glutathione S-transferase M4; hydroxysteroid (17-beta) dehydrogenase 1; ATP citrate lyase; iduronate 2-sulfatase (Hunter syndrome); WW domain containing oxidoreductase; chitinase 3-like 1 (cartilage glycoprotein-39); propionyl Coenzyme A carboxylase, alpha polypeptide; dopachrome tautomerase (dopachrome delta-isomerase, tyrosine-related protein 2); inorganic pyrophosphatase 2; methylmalonyl Coenzyme A mutase; aldehyde dehydrogenase 1 family, member A2; hydroxysteroid (17-beta) dehydrogenase 3; chitobiase, di-N-acetyl-; ATPase, Ca++ transporting, cardiac muscle, slow twitch 2; UDP glycosyltransferase 2 family, polypeptide B17; selenocysteine lyase; ATPase, Ca++ transporting, ubiquitous; quinoid dihydropteridine reductase; peroxisomal D3,D2-enoyl-CoA isomerase; SA hypertension-associated homolog (homolog of rat); chromosome 1 open reading frame 10; insulin induced gene 1; NEL-like 1 (homolog of chicken); rTS beta protein; sepiapterin reductase (7,8-dihydrobiopterin:NADP+ oxidoreductase); phosphoserine phosphatase; pyrophosphatase (inorganic); branched chain aminotransferase 1, cytosolic; UDP glycosyltransferase 1 family, polypeptide A10; oviductal glycoprotein 1, 120 kDa (mucin 9, oviductin); chromosome 5 open reading frame 4; retinol dehydrogenase 5 (11-cis and 9-cis); chromosome 5 open reading frame 4; ATPase, Na+/K+ transporting, alpha 3 polypeptide; arylsulfatase D; dehydrogenase/reductase (SDR family) member 2; UDP glycosyltransferase 2 family, polypeptide A1; solute carrier family 27 (fatty acid transporter), member 5; glyceronephosphate O-acyltransferase; steroid sulfatase (microsomal), arylsulfatase C, isozyme S; acyl-CoA synthetase long-chain family member 1; glutathione S-transferase pi; aldehyde dehydrogenase 8 family, member A1; 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4; ATPase, Ca++ transporting, cardiac muscle, fast twitch 1; aconitase 1, soluble; hydroxysteroid (17-beta) dehydrogenase 7; 1-acylglycerol-3-phosphate O-acyltransferase 3; UDP-glucose pyrophosphorylase 2; chromosome 12 open reading frame 5; glutathione S-transferase M5; phospholipase D1, phophatidylcholine-specific; dehydrogenase/reductase (SDR family) member 8; phospholipase D2; fumarylacetoacetate hydrolase (fumarylacetoacetase); glutathione S-transferase omega 1; 3-hydroxysteroid epimerase; ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide; 2,4-dienoyl CoA reductase 1, mitochondrial; arylsulfatase E (chondrodysplasia punctata 1); 2,4-dienoyl CoA reductase 2, peroxisomal; E-1 enzyme; chitinase 3-like 2; solute carrier family 27 (fatty acid transporter), member 6; phosphoglycerate mutase 1 (brain); hydroxysteroid (17-beta) dehydrogenase 2; 1-acylglycerol-3-phosphate O-acyltransferase 4 (lysophosphatidic acid acyltransferase, delta); aldehyde dehydrogenase 3 family, member A1; iduronate 2-sulfatase (Hunter syndrome); UDP glycosyltransferase 1 family, polypeptide A10; dehydrogenase/reductase (SDR family) member 3; ATPase, Cu++ transporting, alpha polypeptide (Menkes syndrome); matrix metalloproteinase 24 (membrane-inserted); galactokinase 1; ATPase, Ca++ transporting, plasma membrane 1; isocitrate dehydrogenase 1 (NADP+), soluble; 1-acylglycerol-3-phosphate O-acyltransferase 2 (lysophosphatidic acid acyltransferase, beta); succinate-CoA ligase, GDP-forming, alpha subunit; glutathione S-transferase M1; glutathione S-transferase A4; UDP-N-acteylglucosamine pyrophosphorylase 1; glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID); ATPase, Ca++ transporting, plasma membrane 4; glutathione S-transferase A3; fatty acid 2-hydroxylase; 1-acylglycerol-3-phosphate O-acyltransferase 1 (lysophosphatidic acid acyltransferase, alpha); dehydrogenase/reductase (SDR family) member 9; glutathione S-transferase M4; 3-oxoacid CoA transferase 1; sterol-C4-methyl oxidase-like; carboxylesterase 1 (monocyte/macrophage serine esterase 1); acetyl-Coenzyme A carboxylase alpha; acyl-CoA synthetase long-chain family member 5; methylcrotonoyl-Coenzyme A carboxylase 1 (alpha); UDP glycosyltransferase 1 family, polypeptide A10; hydroxysteroid (11-beta) dehydrogenase 2; COP9 constitutive photomorphogenic homolog subunit 8 (Arabidopsis); ATPase, H+/K+ transporting, nongastric, alpha polypeptide; integrin beta 4 binding protein; ATPase, Cu++ transporting, beta polypeptide (Wilson disease); eyes absent homolog 1 (homolog of Drosophila); 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; asparagine synthetase; formiminotransferase cyclodeaminase; eyes absent homolog 2 (homolog of Drosophila); 2,3-bisphosphoglycerate mutase; amiloride binding protein 1 (amine oxidase (copper-containing)); alpha-methylacyl-CoA racemase; ubiquitin associated and SH3 domain containing, A; acetyl-Coenzyme A carboxylase beta; proprotein convertase subtilisin/kexin type 1; tyrosinase (oculocutaneous albinism IA); ATPase, Ca++ transporting, plasma membrane 2; dicarbonyl/L-xylulose reductase; phosphomannomutase 2; carbonyl reductase 3; arylacetamide deacetylase (esterase); carbonyl reductase 1; NFS1 nitrogen fixation 1 (homolog of S. cerevisiae). As such another contemplated microarray would be one which comprises two or more of the above genes to specifically monitor the efficacy of IMPDH inhibition on metabolic pathways in general.
Genes involved in the citric acid cycle that are responsive to VX-944 and IMPDH inhibition include: malate dehydrogenase 2, NAD (mitochondrial); isocitrate dehydrogenase 3 (NAD+) alpha; succinate dehydrogenase complex, subunit A, flavoprotein (Fp); isocitrate dehydrogenase 3 (NAD+) beta; dihydrolipoamide dehydrogenase (E3 component of pyruvate dehydrogenase complex, 2-oxo-glutarate complex, branched chain keto acid dehydrogenase complex); aconitase 2, mitochondrial; isocitrate dehydrogenase 3 (NAD+) gamma; succinate dehydrogenase complex, subunit B, iron sulfur (Ip); isocitrate dehydrogenase 2 (NADP+), mitochondrial; pyruvate carboxylase; fumarate hydratase; phosphoenolpyruvate carboxykinase 1 (soluble); oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide); aconitase 1, soluble; isocitrate dehydrogenase 1 (NADP+), soluble; malate dehydrogenase 1, NAD (soluble); succinate-CoA ligase, GDP-forming, alpha subunit. As such another contemplated microarray would be one which comprises two or more of the above genes to specifically monitor the efficacy of IMPDH inhibition on glycolysis and the citric acid cycle in general.
An array designed to monitor the IMPDH inhibition on genes involved in purine metabolism will advantageously determine the expression levels of two or more genes selected from the group consisting of adenosine monophosphate deaminase 2 (isoform L); hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan syndrome); methylenetetrahydrofolate dehydrogenase (NADP+ dependent), methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase; adenosine kinase; adenylosuccinate synthase; IMP (inosine monophosphate) dehydrogenase 1; ATP-binding cassette, sub-family G (WHITE), member 1; phosphoribosyl pyrophosphate amidotransferase; cat eye syndrome chromosome region, candidate 1; thiopurine S-methyltransferase; solute carrier family 28 (sodium-coupled nucleoside transporter), member 2; phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase; adenosine monophosphate deaminase (isoform E); IMP (inosine monophosphate) dehydrogenase 2; adenosine monophosphate deaminase 1 (isoform M); phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase; adenylosuccinate lyase; nucleoside phosphorylase; 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase; 8-oxoguanine DNA glycosylase; guanine monphosphate synthetase; adenosine deaminase; adenine phosphoribosyltransferase. Specific microarrays comprising these genes are contemplated as part of the invention.
Another advantage array may be one which can be used to determine the expression levels of lysosomal enzymes that are altered in response to IMPDH inhibition. Such specific arrays may comprise two or more of the following genes: arylsulfatase B; prolylcarboxypeptidase (angiotensinase C); scavenger receptor class B, member 2; acid phosphatase 2, lysosomal; cystinosis, nephropathic; coronin, actin binding protein, 1A; lysosomal-associated membrane protein 2; CD63 antigen (melanoma 1 antigen); lysosomal-associated membrane protein 3.
An array for monitoring chemokines that are responsive to IMPDH inhibition will advantageously comprise a plurality of genes selected from the group consisting of: chemokine (C-X-C motif) ligand 14;chemokine (C-X-C motif) ligand 11; chemokine (C motif) ligand 1; chemokine-like factor; chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); platelet factor 4 (chemokine (C-X-C motif) ligand 4); chemokine (C-C motif) receptor-like 1; chemokine (C-X3-C motif) ligand 1; Burkitt lymphoma receptor 1, GTP binding protein (chemokine (C-X-C motif) receptor 5); chemokine (C-X3-C motif) receptor 1; chemokine-like receptor 1; chemokine (C-X-C motif) receptor 6; chemokine (C-C motif) ligand 8; chemokine (C-C motif) ligand 25; chemokine (C motif) ligand 1; tumor necrosis factor receptor superfamily, member 1B; chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated); chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant); G protein-coupled receptor 17; chemokine binding protein 2; chemokine (C-C motif) ligand 3; chemokine (C-X-C motif) ligand 5; chemokine (C-C motif) ligand 1; chemokine (C-C motif) ligand 17; chemokine (C-C motif) receptor-like 2; chemokine(C-X-C motif) ligand 2; chemokine (C-C motif) ligand 20; cardiotrophin-like cytokine; chemokine (C-C motif) ligand 16; G protein-coupled receptor 2; chemokine (C-X-C motif) ligand 3; colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage); chemokine (C-C motif) ligand 27; chemokine (C-C motif) ligand 22; chemokine (C-C motif) ligand 15 [BLAST]; chemokine (C-C motif) ligand 21; chemokine (C-X-C motif) ligand 9; chemokine (C-C motif) ligand 19; chemokine (C-C motif) ligand 13; platelet factor 4 variant 1; chemokine (C-C motif) ligand 24; and interleukin 18 (interferon-gamma-inducing factor).
A cell-adhesion microarray that contains genes responsive to IMPDH inhibition also is part of the invention and will comprise a plurality of genes selected from the group consisting of: chemokine (C-C motif) ligand 2; protocadherin gamma subfamily C, 3; brain-specific angiogenesis inhibitor 1; cell adhesion molecule with homology to L1CAM (close homolog of L1); catenin (cadherin-associated protein), alpha 1, 102 kDa; sushi-repeat-containing protein, X-linked; EGF-like repeats and discoidin I-like domains 3; cadherin 1, type 1, E-cadherin (epithelial); Clone IMAGE:5301388, mRNA; Rho GTPase activating protein 5; retinal outer segment membrane protein 1; bystin-like; myelin associated glycoprotein; protein disulfide isomerase, pancreatic; limbic system-associated membrane protein; cadherin 17, LI cadherin (liver-intestine); poliovirus receptor-related 1 (herpesvirus entry mediator C; nectin); cadherin 9, type 2 (T1-cadherin); CD226 antigen; parvin, alpha; trophinin associated protein (tastin); protocadherin gamma subfamily C, 3; selectin P (granule membrane protein 140 kDa, antigen CD62); ephrin-B1; plakophilin 2; sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4D; desmocollin 2; selectin E (endothelial adhesion molecule 1); cadherin, EGF LAG seven-pass G-type receptor 2 (flamingo homolog of Drosophila); calsyntenin 2; myosin binding protein C, cardiac; ARS component B; sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A; neural precursor cell expressed, developmentally down-regulated 9; intercellular adhesion molecule 2; alpha-1-microglobulinlbikunin precursor; sialic acid binding Ig-like lectin 5; plakophilin 3; laminin, gamma 1 (formerly LAMB2); flotillin 2; integrin, alpha E (antigen CD103, human mucosal lymphocyte antigen 1; alpha polypeptide); myosin binding protein C, slow type; retinoschisis (X-linked, juvenile) 1; desmocollin 1; CD44 antigen (homing function and Indian blood group system); thrombospondin 2; fasciculation and elongation protein zeta 1 (zygin I); BH-protocadherin (brain-heart); protocadherin beta 12; leupaxin; insulin-like growth factor binding protein, acid labile subunit; pinin, desmosome associated protein; parvin, beta; chemokine (C-X3-C motif) ligand 1; scavenger receptor class B, member 2; amyloid beta (A4) precursor-like protein 1; Rho GDP dissociation inhibitor (GDI) alpha; parvin, beta; collagen, type V, alpha 3; sialophorin (gpL115, leukosialin, CD43); laminin, gamma 3; hyaluronan and proteoglycan link protein 2; collagen, type XIX, alpha 1; cadherin 15, M-cadherin (myotubule); chemokine (C-X3-C motif) receptor 1; laminin, alpha 2 (merosin, congenital muscular dystrophy); cadherin 3, type 1, P-cadherin (placental); bone gamma-carboxyglutamate (gla) protein (osteocalcin); lectin, galactoside-binding, soluble, 7 (galectin 7); protocadherin 11 X-linked; protocadherin alpha 2; chondroitin sulfate proteoglycan 4 (melanoma-associated); tumor necrosis factor (TNF superfamily, member 2); myosin binding protein H; TYRO3 protein tyrosine kinase; plakophilin 1 (ectodermal dysplasia/skin fragility syndrome); neuropilin 2; vascular cell adhesion molecule 1; acetylcholinesterase (YT blood group); erbb2 interacting protein; a disintegrin and metalloproteinase domain 2 (fertilin beta); polycystic kidney disease 1 (autosomal dominant); thrombospondin 4; multimerin 1; laminin, beta 3; cadherin 11, type 2, OB-cadherin (osteoblast); contactin 1; Lutheran blood group (Auberger b antigen included); protein tyrosine phosphatase, receptor type, U; catenin (cadherin-associated protein), alpha 2; desmoglein 3 (pemphigus vulgaris antigen); collagen, type XVI, alpha 1; chemokine (C-X-C motif) receptor 3; desmoglein 2; tenascin R (restrictin, janusin); growth arrest-specific 6; comeodesmosin; transforming growth factor, beta-induced, 68 kDa; collagen, type VIII, alpha 1; FXYD domain containing ion transport regulator 5; transforming growth factor beta 1 induced transcript 1; spondin 2, extracellular matrix protein; dystonin; intercellular adhesion molecule 3; DiGeorge syndrome critical region gene 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); CD99 antigen; L1 cell adhesion molecule (hydrocephalus, stenosis of aqueduct of Sylvius 1, MASA (mental retardation, aphasia, shuffling gait and adducted thumbs) syndrome, spastic paraplegia 1); laminin, beta 1; calcium and integrin binding 1 (calmyrin); collagen, type V, alpha 1; WNT1 inducible signaling pathway protein 1; cadherin 7, type 2; tissue specific transplantation antigen P35B; collagen, type XVIII, alpha 1; vinexin beta (SH3-containing adaptor molecule-1); desmocollin 3; brevican; protocadherin beta 6; collagen, type XV, alpha 1; cadherin, EGF LAG seven-pass G-type receptor 1 (flamingo homolog of Drosophila); amine oxidase, copper containing 3 (vascular adhesion protein 1); glycoprotein Ib (platelet), alpha polypeptide; CD209 antigen; integrin, alpha 7; trophoblast glycoprotein; pancreatitis-associated protein; crumbs homolog 1 (homolog of Drosophila); oligodendrocyte myelin glycoprotein; sialic acid binding Ig-like lectin 7; integrin, alpha 2b (platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41B); discs, large homolog 5 (homolog of Drosophila); slit homolog 2 (homolog of Drosophila); NEL-like 2 (homolog of chicken); protocadherin beta 11; protocadherin alpha 2; nephronophthisis 1 (juvenile); neuroligin 3; intercellular adhesion molecule 5, telencephalin; protocadherin beta 5; collagen, type VI, alpha 3; phosphoglucomutase 5; contactin 2 (axonal); deleted in liver cancer 1; cadherin 5, type 2, VE-cadherin (vascular epithelium); slit homolog 3 (homolog of Drosophila); AE binding protein 1; ATPase, Ca++ transporting, cardiac muscle, slow twitch 2; chemokine (C-C motif) receptor 3; spastic paraplegia 7, paraplegin (pure and complicated autosomal recessive); myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog of Drosophila); translocated to, 4; CD209 antigen-like; heparan sulfate proteoglycan 2 (perlecan); activated leukocyte cell adhesion molecule; cadherin 12, type 2 (N-cadherin 2); PTK2B protein tyrosine kinase 2 beta; nidogen (enactin); chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1); glycoprotein V (platelet); discoidin domain receptor family, member 1; epithelial V-like antigen 1; chromosome 1 open reading frame 10; lectin, galactoside-binding, soluble, 3 binding protein; NEL-like 1 (homolog of chicken); neuronal cell adhesion molecule; amyloid beta (A4) precursor protein-binding, family A, member 1 (X11); melanoma cell adhesion molecule; sialic acid binding Ig-like lectin 7; protocadherin alpha 2; CD84 antigen (leukocyte antigen); colony stimulating factor 3 receptor (granulocyte); protocadherin 8; sialoadhesin; protein tyrosine phosphatase, receptor type, S; neural cell adhesion molecule 2; ectonucleoside triphosphate diphosphohydrolase 1; vitronectin (serum spreading factor, somatomedin B, complement S-protein); mesothelin; adhesion regulating molecule 1; coagulation factor V (proaccelerin, labile factor); intercellular adhesion molecule 1 (CD54), human rhinovirus receptor; protocadherin alpha 2; junction plakoglobin; chemokine (C-C motif) receptor 8; CD22 antigen; complement component 1, q subcomponent, receptor 1; neural cell adhesion molecule 1; protocadherin beta 1; aggrecan 1 (chondroitin sulfate proteoglycan 1, large aggregating proteoglycan, antigen identified by monoclonal antibody A0122); protocadherin alpha 2; integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II); CD33 antigen (gp67); stromal interaction molecule 1; armadillo repeat gene deletes in velocardiofacial syndrome; laminin, beta 2 (laminin S); signal-induced proliferation-associated gene 1; tetraspan 2; von Willebrand factor; elastin microfibril interfacer 1; reelin; endothelial differentiation, sphingolipid G-protein-coupled receptor, 1; protein tyrosine phosphatase, receptor type, F; protocadherin 9; cadherin 16, KSP-cadherin; alpha-2-glycoprotein 1, zinc; CD36 antigen (collagen type I receptor, thrombospondin receptor); symplekin; contactin 6; sialic acid binding Ig-like lectin 8; chondroitin sulfate proteoglycan 3 (neurocan); cadherin, EGF LAG seven-pass G-type receptor 2 (flamingo homolog of Drosophila); CD9 antigen (p24); nephrosis 1, congenital, Finnish type (nephrin); selectin L (lymphocyte adhesion molecule 1); natural killer cell transcript 4; FAT tumor suppressor homolog 1 (homolog of Drosophila); cadherin 8, type 2; coagulation factor VIII, procoagulant component (hemophilia A); integrin, beta 2 (antigen CD18 (p95), lymphocyte function-associated antigen 1; macrophage antigen 1 (mac-1) beta subunit); Kallmann syndrome 1 sequence; proline-serine-threonine phosphatase interacting protein 1; mucosal vascular addressin cell adhesion molecule 1; periostin, osteoblast specific factor; sarcospan (Kras oncogene-associated gene); hyaluronan binding protein 2; Rho family GTPase 1; catenin (cadherin-associated protein), delta 1; glycoprotein Ib (platelet), beta polypeptide; amyloid beta (A4) precursor protein (protease nexin-II, Alzheimer disease); protocadherin beta 8; junctional adhesion molecule 2; a disintegrin and metalloproteinase domain 22; CD72 antigen; tenascin C (hexabrachion); collectin sub-family member 10 (C-type lectin); vinculin; catenin (cadherin-associated protein), beta 1, 88 kDa; trophinin; catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein); vascular endothelial growth factor; protein tyrosine phosphatase, non-receptor type substrate 1; CD44 antigen (homing function and Indian blood group system); CD2 antigen (p50), sheep red blood cell receptor; cysteine-rich secretory protein 3; C-type (calcium dependent, carbohydrate-recognition domain) lectin, superfamily member 6; cadherin 6, type 2, K-cadherin (fetal kidney); Usher syndrome 2A (autosomal recessive, mild); myosin binding protein C, fast type; cell adhesion molecule-related/down-regulated by oncogenes; fibronectin leucine rich transmembrane protein 3; scavenger receptor class B, member 1; carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4; hyaluronan synthase 1; retinal degeneration, slow; roundabout, axon guidance receptor, homolog 1 (homolog of Drosophila); WNT1 inducible signaling pathway protein 2; chromosome 1 open reading frame 38; egf-like module containing, mucin-like, hormone receptor-like 1; immunoglobulin superfamily, member 1; tumor necrosis factor (ligand) superfamily, member 5 (hyper-IgM syndrome); desmoglein 1; intercellular adhesion molecule 4, Landsteiner-Wiener blood group; selectin P ligand; spastic paraplegia 7, paraplegin (pure and complicated autosomal recessive); claudin 1; leucine rich repeat neuronal 5; likely ortholog of mouse gene rich cluster, C10 gene; protocadherin beta 3; contactin associated protein 1; glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase; CD6 antigen; milk fat globule-EGF factor 8 protein; KIT ligand; calcium/calmodulin-dependent serine protein kinase (MAGUK family); zyxin; discoidin domain receptor family, member 2; adenomatosis polyposis coli; tetraspan 1; glycoprotein IX (platelet); thrombospondin 1; sialic acid binding Ig-like lectin 6; embryonal Fyn-associated substrate; protocadherin gamma subfamily C, 3; claudin 10; tumor necrosis factor, alpha-induced protein 6; lectin, galactoside-binding, soluble, 4 (galectin 4); FAT tumor suppressor homolog 2 (homolog of Drosophila); formyl peptide receptor-like 1; osteomodulin; carbohydrate sulfotransferase 10; Rho GDP dissociation inhibitor (GDI) beta; collagen, type VIII, alpha 1; neogenin homolog 1 (homolog of chicken); a disintegrin and metalloproteinase domain 10; protocadherin gamma subfamily C, 3; CD97 antigen; kininogen 1; protocadherin gamma subfamily C, 3; lymphocyte antigen 6 complex, locus D; cadherin 4, type 1, R-cadherin (retinal); lysyl oxidase-like 2; GDP-mannose 4,6-dehydratase; collagen, type XI, alpha 1; chemokine (C-C motif) ligand 11; RAB13, member RAS oncogene family; integrin beta 3 binding-protein (beta3-endonexin); protocadherin gamma subfamily C, 3; cartilage oligomeric matrix protein; DiGeorge syndrome critical region gene 6; spleen tyrosine kinase; connective tissue growth factor; cadherin, EGF LAG seven-pass G-type receptor 3 (flamingo homolog of Drosophila); scavenger receptor class F, member 1; hyaluronan and proteoglycan link protein 1; CD151 antigen; cadherin 2, type 1, N-cadherin (neuronal); neurotrimin; a disintegrin and metalloproteinase domain 12 (meltrin alpha); plexin C1; endoglin (Osler-Rendu-Weber syndrome 1); ret proto-oncogene (multiple endocrine neoplasia and medullary thyroid carcinoma 1, Hirschsprung disease); cadherin 19, type 2; immunoglobulin superfamily containing leucine-rich repeat; protocadherin gamma subfamily C, 3; protocadherin 12; dermatopontin; CD58 antigen, (lymphocyte function-associated antigen 3); ADAM-like, decysin 1; CD164 antigen, sialomucin; neurexin 3; tumor necrosis factor receptor superfamily, member 12A; PTK7 protein tyrosine kinase 7; cysteine-rich, angiogenic inducer, 61; CD34 antigen; protocadherin alpha 2; neuropilin 1; laminin, alpha 4; serum amyloid A2; nidogen 2 (osteonidogen); plakophilin 4; tuberous sclerosis 1; cadherin 18, type 2; transmembrane protein 8 (five membrane-spanning domains); macrophage erythroblast attacher; opioid binding protein/cell adhesion molecule-like; interleukin 18 (interferon-gamma-inducing factor); and ninjurin 2.
Glycosylation-associated genes that were found to be responsive to IMPDH inhibition and that could be used to prepare a microarray include: core 2 beta-1,6-N-acetylglucosaminyltransferase 3; sialyltransferase 8E (alpha-2,8-polysialyltransferase); mannosyl(alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase; low density lipoprotein-related protein 2; glucosaminyl (N-acetyl) transferase 3, mucin type; mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase; UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1); stress-associated endoplasmic reticulum protein 1; transmembrane 4 superfamily member 5; lipase, hepatic; asparagine-linked glycosylation 3 homolog (yeast, alpha-1,3-mannosyltransferase); mannosyl(alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase; UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3; mannosidase, alpha, class 1A, member 1; UDP-glucose ceramide glucosyltransferase-like 2; mannosyl (beta-1,4-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; sialyltransferase 9 (CMP-NeuAc:lactosylceramide alpha-2,3-sialyltransferase; GM3 synthase); fucosyltransferase 1 (galactoside 2-alpha-L-fucosyltransferase); fucosyltransferase 9 (alpha (1,3) fucosyltransferase); sialyltransferase 8D (alpha-2,8-polysialyltransferase); fucosyltransferase 4 (alpha (1,3) fucosyltransferase, myeloid-specific); asparagine-linked glycosylation 6 homolog (homolog of yeast, alpha-1,3-glucosyltransferase); ribophorin I; Kell blood group; low density lipoprotein receptor (familial hypercholesterolemia); UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 3; beta-1,3-glucuronyltransferase 1 (glucuronosyltransferase P); transmembrane 4 superfamily member 4; UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 4; tissue specific transplantation antigen P35B; UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GaINAc-T2); phosphatidylinositol glycan, class B; protein-O-mannosyltransferase 1; sialyltransferase 8C (alpha2,3Galbeta1,4GlcNAcalpha 2,8-sialyltransferase); sialyltransferase 1 (beta-galactoside alpha-2,6-sialyltransferase); ribophorin II; interleukin 17 (cytotoxic T-lymphocyte-associated serine esterase 8); fucosyltransferase 7 (alpha (1,3) fucosyltransferase); UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 2; dolichyl-phosphate mannosyltransferase polypeptide 1, catalytic subunit; component of oligomeric golgi complex 2; fucosyltransferase 5 (alpha (1,3) fucosyltransferase); stromal cell-derived factor 2; integral membrane protein 1; sialyltransferase 8A (alpha-N-acetylneuraminate: alpha-2,8-sialyltransferase, GD3 synthase); galactose-3-O-sulfotransferase 1; glucosaminyl (N-acetyl) transferase 2, I-branching enzyme; protein-O-mannosyltransferase 2; O-linked N-acetylglucosamine (GIcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase); mannosidase, alpha, class 1C, member 1; UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 4; transmembrane 4 superfamily member 3; asparagine-linked glycosylation 5 homolog (homolog of yeast, dolichyl-phosphate beta-glucosyltransferase); UDP-glucose ceramide glucosyltransferase-like 1; transmembrane 4 superfamily member 2; mannosidase, alpha, class 1B, member 1; sialyltransferase 4C (beta-galactoside alpha-2,3-sialyltransferase); alpha-1,4-N-acetylglucosaminyltransferase; sialyltransferase 7 ((alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl galactosaminide alpha-2,6-sialyltransferase) B; CD37 antigen; fucosyltransferase 6 (alpha (1,3) fucosyltransferase); sialyltransferase 10 (alpha-2,3-sialyltransferase VI); UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1; CDNA clone IMAGE:5093665, partial cds; sialyltransferase 4A (beta-galactoside alpha-2,3-sialyltransferase); sialyltransferase 7 ((alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl galactosaminide alpha-2,6-sialyltransferase) E; glycophorin C (Gerbich blood group); glucosaminyl (N-acetyl) transferase 1, core 2 (beta-1,6-N-acetylglucosaminyltransferase); UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 1; mannosidase, alpha, class 2B, member 1; fucosyltransferase 3 (galactoside 3(4)-L-fucosyltransferase, Lewis blood group included); sialyltransferase 7D ((alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl galactosaminide alpha-2,6-sialyltransferase); lipase A, lysosomal acid, cholesterol esterase (Wolman disease); UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 5; sialyltransferase 8B (alpha-2,8-sialyltransferase); dolichyl-diphosphooligosaccharide-protein glycosyltransferase; dolichyl-phosphate mannosyltransferase polypeptide 2, regulatory subunit; phosphomannomutase 2; and glucosidase I.
Microarrays that comprise chemotaxis-associated genes that are responsive to IMPDH inhibition would advantageously include a plurality of genes selected from the group consisting of: chemokine (C-C motif) receptor 7; chemokine (C-C motif) ligand 2; chemokine (C-X-C motif) ligand 14;chemokine (C-X-C motif) ligand 11; interleukin 8; chemokine (C-C motif) receptor 9; chemokine (C motif) ligand 1; chemokine-like factor; interleukin 4; chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); platelet factor 4 (chemokine (C-X-C motif) ligand 4); leukocyte cell-derived chemotaxin 2; chemokine (C-C motif) receptor-like 1; fibroblast growth factor 2 (basic); chemokine (C-X3-C motif) ligand 1; sialophorin (gpL115, leukosialin, CD43); plasminogen activator, urokinase receptor; chemokine (C-X3-C motif) receptor 1; chemokine-like receptor 1; chemokine (C-C motif) ligand 8; complement component 5; interleukin 8 receptor, beta; chemokine (C-C motif) ligand 25; azurocidin 1 (cationic antimicrobial protein 37); complement component 3a receptor 1; chemokine (C motif) receptor 1; chemokine (C-X-C motif) receptor 3; G protein-coupled receptor 77; phosphoinositide-3-kinase, catalytic, beta polypeptide; ribonuclease, RNase A family, 2 (liver, eosinophil-derived neurotoxin); chemokine (C motif) ligand 1; chemokine (C-C motif) receptor 5; chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated); slit homolog 2 (homolog of Drosophila); chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant); v-ral simian leukemia viral oncogene homolog A (ras related); chemokine (C-C motif) receptor 3; G protein-coupled receptor 77; chemokine binding protein 2; chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1); mitogen-activated protein kinase 14;chemokine (C-X-C motif) ligand 2; chemokine (C-X-C motif) ligand 5; chemokine (C-C motif) ligand 1; chemokine (C-C motif) ligand 17; chemokine (C-X-C motif) ligand 10; serine (or cysteine) proteinase inhibitor, clade D (heparin cofactor), member 1; chemokine (C-C motif) receptor 8; endothelial cell growth factor 1 (platelet-derived); chemokine (C-X-C motif) ligand 2; FOS-like antigen 1; secretogranin III; chemokine (C-C motif) ligand 23; interleukin 10; secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1); ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin); platelet-activating factor receptor; chemokine (C-C motif) ligand 20; chemokine (C-C motif) receptor 6; phospholipase D1, phophatidylcholine-specific; chemokine (C-C motif) ligand 16; Kallmann syndrome 1 sequence; chemokine (C-C motif) ligand 15; chemokine (C-X-C motif) ligand 3; roundabout, axon guidance receptor, homolog 3 (homolog of Drosophila); mitogen-activated protein kinase 1; ralA binding protein 1; plasminogen activator, urokinase; defensin, beta 4; roundabout, axon guidance receptor, homolog 1 (homolog of Drosophila); interleukin 16 (lymphocyte chemoattractant factor); chemokine (C-C motif) ligand 27; chemokine (C-C motif) ligand 22; formyl peptide receptor-like 1; small inducible cytokine subfamily E, member 1 (endothelial monocyte-activating); v-Ha-ras Harvey rat sarcoma viral oncogene homolog; Transcribed sequences; chemokine (C-C motif) ligand 11; spleen tyrosine kinase; chemokine (C-C motif) receptor 4; chemokine (C-C motif) ligand 21; chemokine (C-X-C motif) ligand 9; formyl peptide receptor-like 2; chemokine (C-C motif) ligand 19; mitogen-activated protein kinase kinase 1; chemokine (C-C motif) ligand 13; cysteine-rich, angiogenic inducer, 61; serum amyloid A2; chemokine (C-C motif) ligand 24; and pro-platelet basic protein (chemokine (C-X-C motif) ligand 7).
Microarrays that are specific for cell-cell signaling pathway response to IMPDH would include a plurality of genes selected from the group consisting of G protein-coupled receptor 50; glutamyl aminopeptidase (aminopeptidase A); chemokine (C-X-C motif) ligand 14;chemokine (C-X-C motif) ligand 11; vasoactive intestinal peptide; interferon, alpha 8; ephrin-A5; gap junction protein, beta 1, 32 kDa (connexin 32, Charcot-Marie-Tooth neuropathy, X-linked); interferon, alpha 10; a disintegrin and metalloproteinase domain 17 (tumor necrosis factor, alpha, converting enzyme); S100 calcium binding protein A6 (calcyclin); poliovirus receptor-related 1 (herpesvirus entry mediator C; nectin); netrin 1; interleukin 8; carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross reacting antigen); neuregulin 2; ephrin-B1; pan-hematopoietic expression; plakophilin 2; adrenergic, alpha-1A-, receptor; lymphotoxin alpha (TNF superfamily, member 1); interferon, gamma; fatty acid binding protein 1, liver; somatostatin receptor 3; wingless-type MMTV integration site family, member 4; chemokine (C motif) ligand 1; granulin; sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A; leptin (obesity homolog of mouse); TGFB inducible early growth response; intercellular adhesion molecule 2; thioredoxin; tachykinin, precursor 1 (substance K, substance P, neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha, neuropeptide K, neuropeptide gamma); sonic hedgehog homolog (homolog of, Drosophila); chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); tumor necrosis factor (ligand) superfamily, member 6; ephrin-A4; fibroblast growth factor 17; CD44 antigen (homing function and Indian blood group system); nerve growth factor, beta polypeptide; fibroblast growth factor 4 (heparin secretory transforming protein 1, Kaposi sarcoma oncogene); adrenergic, alpha-2C-, receptor; fibroblast growth factor 2 (basic); CD80 antigen (CD28 antigen ligand 1, B7-1 antigen); fibroblast growth factor 8 (androgen-induced); fibroblast growth factor 3 (murine mammary tumor virus integration site (v-int-2) oncogene homolog); myelin protein zero-like 1; fibroblast growth factor 16; gap junction protein, alpha 3, 46 kDa (connexin 46); adenosine A1 receptor; bone morphogenetic protein 10; tumor suppressor candidate 2; fibroblast growth factor 1 (acidic); thyrotropin-releasing hormone degrading ectoenzyme; tumor necrosis factor (TNF superfamily, member 2); bone morphogenetic protein 3 (osteogenic); gap junction protein, alpha 1, 43 kDa (connexin 43); colony stimulating factor 3 (granulocyte); vasoactive intestinal peptide receptor 2; proprotein convertase subtilisin/kexin type 5; vascular cell adhesion molecule 1; transforming growth factor, beta 3; tumor necrosis factor (ligand) superfamily, member 8; chemokine (C-C motif) ligand 8; ephrin-A1; somatostatin receptor 2; chorionic somatomammotropin hormone 1 (placental lactogen); IK cytokine, down-regulator of HLA II; tumor necrosis factor (ligand) superfamily, member 9; sprouty homolog 2 (homolog of Drosophila); fibroblast growth factor 6; motilin; TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous and mucosal); cardiotrophin 1; FXYD domain containing ion transport regulator 5; WNT inhibitory factor 1; intercellular adhesion molecule 3; GRB2-related adaptor protein 2; gonadotropin-releasing hormone 1 (leutinizing-releasing hormone); estrogen receptor 2 (ER beta); bone morphogenetic protein 2; WNT1 inducible signaling pathway protein 1; steroid-5-alpha-reductase, alpha polypeptide 2 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 2); calcitonin/calcitonin-related polypeptide, alpha; desmoplakin, tumor necrosis factor (ligand) superfamily, member 7; fibroblast growth factor 14;nitric oxide synthase 1 (neuronal); microsomal glutathione S-transferase 2; chemokine (C motif) ligand 1; thyrotropin-releasing hormone; protocadherin beta 6; interferon, alpha 4; epiregulin; proprotein convertase subtilisin/kexin type 2; CD209 antigen; chemokine (C-C motif) receptor 5; interleukin 9; nuclear receptor subfamily 5, group A, member 1; growth factor receptor-bound protein 10; crumbs homolog 1 (homolog of Drosophila); GRB2-related adaptor protein; pre-B-cell colony enhancing factor 1; chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated); discs, large homolog 5 (homolog of Drosophila); angiotensinogen (serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 8); fibroblast growth factor 20; protocadherin beta 11; interleukin 17 (cytotoxic T-lymphocyte-associated serine esterase 8); nephronophthisis 1 (juvenile); ephrin-A2; somatostatin receptor 1; proprotein convertase subtilisin/kexin type 6; intercellular adhesion molecule 5, telencephalin; protocadherin beta 5; transforming growth factor, alpha; tumor necrosis factor (ligand) superfamily, member 10; chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant); phosphoglucomutase 5; adrenergic, alpha-1D-, receptor; proopiomelanocortin (adrenocorticotropin/beta-lipotropin/alpha-melanocyte stimulating hormone/beta-melanocyte stimulating hormone/beta-endorphin); thyroid stimulating hormone, beta; agouti signaling protein, nonagouti homolog (homolog of mouse); interferon, alpha 10; phosphate regulating endopeptidase homolog, X-linked (hypophosphatemia, vitamin D resistant rickets); placental growth factor, vascular endothelial growth factor-related protein; prostaglandin I2 (prostacyclin) receptor (IP); inhibin, alpha; c-mer proto-oncogene tyrosine kinase; lymphotoxin beta (TNF superfamily, member 3); myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog of Drosophila); translocated to, 4; CD209 antigen-like; endothelin 3; tumor necrosis factor receptor superfamily, member 11a, activator of NFKB; adrenergic, alpha-2B-, receptor; chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1); wingless-type MMTV integration site family, member 8B; tumor necrosis factor (ligand) superfamily, member 18; chromosome 1 open reading frame 10; SH2 domain containing phosphatase anchor protein 1; tumor necrosis factor (ligand) superfamily, member 4 (tax-transcriptionally activated glycoprotein 1, 34 kDa); interleukin 11; interleukin 6 (interferon, beta 2); neuronal cell adhesion molecule; endometrial bleeding associated factor (left-right determination, factor A; transforming growth factor beta superfamily); chemokine (C-X-C motif) ligand 5; protocadherin 8; sialoadhesin; adrenomedullin; chemokine (C-C motif) ligand 1; ectonucleoside triphosphate diphosphohydrolase 1; wingless-type MMTV integration site family, member 5A; chemokine (C-C motif) ligand 17; chemokine (C-X-C motif) ligand 10; amphiregulin (schwannoma-derived growth factor); intercellular adhesion molecule 1 (CD54), human rhinovirus receptor; complement component 1, q subcomponent, receptor 1; furin (paired basic amino acid cleaving enzyme); erythropoietin; endothelial cell growth factor 1 (platelet-derived); neuromedin B; interleukin 22; neural cell adhesion molecule 1; thyroid stimulating hormone receptor; CD33 antigen (gp67); stromal interaction molecule 1; sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4F; ephrin-B2; growth differentiation factor 5 (cartilage-derived morphogenetic protein-1); insulin; chemokine (C-C motif) ligand 23; fibroblast growth factor binding protein 1; interleukin 10; secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1); proenkephalin; CD86 antigen (CD28 antigen ligand 2, B7-2 antigen); fibroblast growth factor 13; lactalbumin, alpha-; stanniocalcin 2; steroid-5-alpha-reductase, alpha polypeptide 1 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1); wingless-type MMTV integration site family, member 3; stanniocalcin 1; chemokine (C-C motif) ligand 20; adenylate cyclase activating polypeptide 1 (pituitary); ephrin-B3; insulin-like 4 (placenta); FAT tumor suppressor homolog 1 (homolog of Drosophila); chemokine (C-C motif) ligand 16; nudix (nucleoside diphosphate linked moiety X)-type motif 3; interferon, alpha 21; integrin, beta 2 (antigen CD18 (p95), lymphocyte function-associated antigen 1; macrophage antigen 1 (mac-1) beta subunit); arginine vasopressin (neurophysin II, antidiuretic hormone, diabetes insipidus, neurohypophyseal); interferon, alpha 2; transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma); interleukin 1 receptor-like 1 ligand; peptide YY; chemokine (C-C motif) ligand 15; WNT1 inducible signaling pathway protein 3; catenin (cadherin-associated protein), delta 1; junctional adhesion molecule 2; prostaglandin D2 synthase, hematopoietic; fibroblast growth factor 21; endothelin 1; CD44 antigen (homing function and Indian blood group system); CD2 antigen (p50), sheep red blood cell receptor; cysteine-rich secretory protein 3; discs, large (Drosophila) homolog-associated protein 1; endothelin 2; wingless-type MMTV integration site family, member 7A; carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4; platelet-derived growth factor alpha polypeptide; membrane metallo-endopeptidase (neutral endopeptidase, enkephalinase, CALLA, CD10); interleukin 3 (colony-stimulating factor, multiple); islet amyloid polypeptide; WNT1 inducible signaling pathway protein 2; interleukin 17B; major intrinsic protein of lens fiber; growth hormone releasing hormone; parathyroid hormone-like hormone; intercellular adhesion molecule 4, Landsteiner-Wiener blood group; ephrin-A3; protocadherin beta 3; sema domain, immunoglobulin domain,(Ig), short basic domain, secreted, (semaphorin) 3B; pro-melanin-concentrating hormone; neurotrophin 3; zyxin; interleukin 13; sialic acid binding Ig-like lectin 6; chemokine (C-C motif) ligand 27; interleukin 15; chorionic gonadotropin, beta polypeptide; Norrie disease (pseudoglioma); glycoprotein hormones, alpha polypeptide; tumor necrosis factor, alpha-induced protein 6; androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease); chemokine (C-C motif) ligand 22; bone marrow stromal cell antigen 2; midkine (neurite growth-promoting factor 2); fibroblast growth factor 9 (glia-activating factor); wingless-type MMTV integration site family, member 11; SH2 domain protein 1A, Duncan's disease (lymphoproliferative syndrome); discs, large (homolog of Drosophila) homolog-associated protein 2; neogenin homolog 1 (homolog of chicken); a disintegrin and metalloproteinase domain 10; fibroblast growth factor 1 (acidic); CD97 antigen; small inducible cytokine subfamily E, member 1 (endothelial monocyte-activating); SH2 domain protein 1A, Duncan's disease (lymphoproliferative syndrome); inhibin, beta A (activin A, activin AB alpha polypeptide); parathyroid hormone; jagged 2; collagen, type XI, alpha 1; chemokine (C-C motif) ligand 11; interleukin 1 family, member 9; complement component 1, q subcomponent, alpha polypeptide; leukemia inhibitory factor (cholinergic differentiation factor); chemokine (C-C motif) ligand 21; hydroxysteroid (11-beta) dehydrogenase 2; chemokine (C-X-C motif) ligand 9; adrenergic, alpha-1B-, receptor; cocaine- and amphetamine-regulated transcript; interferon, alpha-inducible protein (clone IFI-15K); fibroblast growth factor 5; CD58 antigen, (lymphocyte function-associated antigen 3); interleukin 7; anti-Mullerian hormone; chemokine (C-C motif) ligand 13; proprotein convertase subtilisin/kexin type 1; interleukin 1, beta; interleukin 26; neuropilin 1; oncostatin M; sorbin and SH3 domain containing 1; S100 calcium binding protein A9 (calgranulin B); chemokine (C-C motif) ligand 24; interleukin 18 (interferon-gamma-inducing factor); and insulin-degrading enzyme.
Microarrays affecting translation in response to IMPDH inhibitors would include a plurality of genes selected from the group consisting of methionine-tRNA synthetase; alanyl-tRNA synthetase; solute carrier family 22 (organic cation transporter), member 17; tyrosyl-tRNA synthetase; histidyl-tRNA synthetase; seryl-tRNA synthetase; phenylalanine-tRNA synthetase 1 (mitochondrial); N-acylaminoacyl-peptide hydrolase; valyl-tRNA synthetase 2; aminoacylase 1; aspartoacylase (aminoacylase 2, Canavan disease); isoleucine-tRNA synthetase; arginyl-tRNA synthetase; transcription factor binding to IGHM enhancer 3; lysyl-tRNA synthetase; phenylalanine-tRNA synthetase-like, alpha subunit; threonyl-tRNA synthetase; cysteinyl-tRNA synthetase; tryptophanyl tRNA synthetase 2 (mitochondrial); glycyl-tRNA synthetase; asparaginyl-tRNA synthetase; glutaminyl-tRNA synthetase; solute carrier family 22 (organic cation transporter), member 17; aspartyl-tRNA synthetase; small inducible cytokine subfamily E, member 1 (endothelial monocyte-activating); glutamyl-prolyl-tRNA synthetase; lysyl-tRNA synthetase; and tryptophanyl-tRNA synthetase.
Microarrays that are specific for genes required for mitochondrial function that are responsive to IMPDH would include a plurality of geres selected from the group consisting of succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa; mitochondrial ribosomal protein L3; malate dehydrogenase 2, NAD (mitochondrial); hexokinase 2; uncoupling protein 3 (mitochondrial, proton carrier); death associated protein 3; 4-aminobutyrate aminotransferase; ubiquinol-cytochrome c reductase binding protein; mitochondrial ribosomal protein L12; mitochondrial intermediate peptidase; ubiquinol-cytochrome c reductase complex (7.2 kD); translocase of inner mitochondrial membrane 17 homolog A (yeast); ATP-binding cassette, sub-family F (GCN20), member 2; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6; solute carrier family 25 (mitochondrial carrier, brain), member 14; thiosulfate sulfurtransferase (rhodanese); cytochrome c oxidase subunit VIa polypeptide 2; BH3 interacting domain death agonist; aldehyde dehydrogenase 4 family, member A1; translocase of outer mitochondrial membrane 40 homolog (yeast); heat shock 60 kDa protein 1 (chaperonin); mercaptopyruvate sulfurtransferase; NADH dehydrogenase (ubiquinone) Fe—S protein 6, 13 kDa (NADH-coenzyme Q reductase); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2, 8 kDa; mitochondrial ribosomal protein S12; glycerol kinase; NADH dehydrogenase (ubiquinone) Fe—S protein 4, 18 kDa (NADH-coenzyme Q reductase); myeloid cell leukemia sequence 1 (BCL2-related); NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2, 14.5 kDa; translocase of inner mitochondrial membrane 22 homolog (homolog of yeast); acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain; solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 3; cytochrome c oxidase subunit Vb; popeye domain containing 2; peptidase (mitochondrial processing) beta; mitochondrial ribosomal protein L23; NADH dehydrogenase (ubiquinone) Fe—S protein 1, 75 kDa (NADH-coenzyme Q reductase); dynamin 1-like; translocase of inner mitochondrial membrane 13 homolog (yeast); NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6, 14 kDa; cytochrome c oxidase subunit VIa polypeptide 1; sarcosine dehydrogenase; B-cell CLL/lymphoma 2; voltage-dependent anion channel 1; 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase (hydroxymethylglutaricaciduria); BCL2-associated X protein; NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1, 6 kDa; omithine aminotransferase (gyrate atrophy); camitine palmitoyltransferase II; solute carrier family 25 (mitochondrial oxodicarboxylate carrier), member 21; monoamine oxidase B; NADH dehydrogenase (ubiquinone) Fe—S protein 2, 49 kDa (NADH-coenzyme Q reductase); ornithine carbamoyltransferase; NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16 kDa; ubiquinol-cytochrome c reductase core protein I; solute carrier family 25 (mitochondrial carrier; omithine transporter) member 15; surfeit 1; voltage-dependent anion channel 3; hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), beta subunit; translocase of inner mitochondrial membrane 8 homolog B (homolog of yeast); ubiquinol-cytochrome c reductase (6.4 kD) subunit; electron-transferring-flavoprotein dehydrogenase; NADH dehydrogenase (ubiquinone) Fe—S protein 8, 23 kDa (NADH-coenzyme Q reductase); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3, 12 kDa; phosphoprotein enriched in astrocytes 15 [BLAST]; carbonic anhydrase VA, mitochondrial; NADH dehydrogenase (ubiquinone) Fe—S protein 5, 15 kDa (NADH-coenzyme Q reductase); inner membrane protein, mitochondrial (mitofilin); translocase of inner mitochondrial membrane 9 homolog (yeast); oxidase (cytochrome c) assembly 1-like; transcription factor A, mitochondrial; translocase of outer mitochondrial membrane 20 homolog (homolog of yeast); DnaJ (Hsp40) homolog, subfamily A, member 3; NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6, 17 kDa; endothelial cell growth factor 1 (platelet-derived); hypothetical protein LOC114971; NADH dehydrogenase (ubiquinone) flavoprotein 1, 51 kDa; electron-transfer-flavoprotein, beta polypeptide; transcription factor A, mitochondrial; mitochondrial folate transporter/carrier; NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa; v-raf-1 murine leukemia viral oncogene homolog 1; ubiquinol-cytochrome c reductase core protein II; solute carrier family 25 (mitochondrial carrier; peroxisomal membrane protein, 34 kDa), member 17; NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19 kDa; uncoupling protein 1 (mitochondrial, proton carrier); START domain containing 3; BCL2-antagonist/killer 1; nicotinamide nucleotide transhydrogenase; translocase of outer mitochondrial membrane 34; holocytochrome c synthase (cytochrome c heme-lyase); mitochondrial carrier family protein; ATP-binding cassette, sub-family B (MDR/TAP), member 8; BCL2-antagonist of cell death; cytochrome c oxidase subunit IV isoform 2; succinate dehydrogenase complex, subunit D, integral membrane protein; solute carrier family 25 (camitine/acylcamitine translocase), member 20; solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), member 11; translocase of inner mitochondrial membrane 17 homolog B (yeast); solute carrier family 25 (mitochondrial carrier, Aralar), member 12; uncoupling protein 2 (mitochondrial, proton carrier); NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5, 13 kDa; cytochrome c-1; methylcrotonoyl-Coenzyme A carboxylase 1 (alpha); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 4, 15 kDa; isovaleryl Coenzyme A dehydrogenase; mitochondrial ribosomal protein S15; benzodiazapine receptor (peripheral); fracture callus 1 homolog (homolog of rat); protein (peptidyl-prolyl cis/trans isomerase) NIMA-interacting, 4 (parvulin); cytochrome c oxidase subunit VIIb; metaxin 2; mitochondrial ribosomal protein L40; NADH dehydrogenase (ubiquinone) flavoprotein 2, 24 kDa; translocase of inner mitochondrial membrane 44 homolog (yeast); translocase of inner mitochondrial membrane 23 homolog (homolog of yeast); electron-transfer-flavoprotein, alpha polypeptide (glutaric aciduria II); solute carrier family 25, and member 13 (citrin).
Microarrays that are specific for genes from cGMP biosynthesis pathway that are responsive to IMPDH inhibitors would include a plurality of genes selected from the group consisting of guanylate cyclase 1, soluble, beta 3; natriuretic peptide receptor B/guanylate cyclase B (atrionatriuretic peptide receptor B); protein kinase, cGMP-dependent, type II; phosphodiesterase 6B, cGMP-specific, rod, beta (congenital stationary night blindness 3, autosomal dominant); phosphodiesterase 6C, cGMP-specific, cone, alpha prime; phosphodiesterase 6A, cGMP-specific, rod, alpha; guanylate cyclase 2F, retinal; natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A); guanylate cyclase 1, soluble, alpha 2; phosphodiesterase 3B, cGMP-inhibited; phosphodiesterase 9A; phosphodiesterase 3B, cGMP-inhibited; guanylate cyclase 2D, membrane (retina-specific); phosphodiesterase 2A, cGMP-stimulated; cyclic nucleotide gated channel alpha 3; phosphodiesterase 6G, cGMP-specific, rod, gamma; phosphodiesterase 6H, cGMP-specific, cone, gamma; cyclic nucleotide gated channel alpha 1; and phosphodiesterase 3A, cGMP-inhibited.
Microarrays that are specific for genes from cAMP biosynthesis pathway that are responsive to IMPDH inhibitors would include a plurality of genes selected from the group consisting of phosphodiesterase 4D, cAMP-specific (phosphodiesterase E3 dunce homolog of Drosophila); cholinergic receptor, muscarinic 2; adenylate cyclase 7; protein kinase, cGMP-dependent, type I; adenylate cyclase 6; Rap guanine nucleotide exchange factor (GEF) 3; glucagon-like peptide 2 receptor; protein kinase, X-linked; melanocortin 4 receptor; adrenergic, beta-3-, receptor; calcitonin/calcitonin-related polypeptide, alpha; phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 dunce homolog, Drosophila); cAMP responsive element modulator; adenylate cyclase 3; calcium-binding tyrosine-(Y)-phosphorylation regulated (fibrousheathin 2); adrenergic, alpha-1D-, receptor; protein kinase, AMP-activated, gamma 1 non-catalytic subunit; cyclic nucleotide gated channel beta 1; chemokine (C-C motif) receptor 3; testicular soluble adenylyl cyclase; adenylate cyclase 8 (brain); phosphodiesterase 7B; protein kinase, cAMP-dependent, catalytic, alpha; adrenomedullin; Rap guanine nucleotide exchange factor (GEF) 2; A kinase (PRKA) anchor protein 4; adenylate cyclase 9; Rap guanine nucleotide exchange factor (GEF) 4; phosphodiesterase 4C, cAMP-specific (phosphodiesterase E1 dunce homolog of Drosophila); prostaglandin E receptor 4 (subtype EP4); G protein-coupled receptor kinase 5; protein kinase (cAMP-dependent, catalytic) inhibitor alpha; galanin receptor 2; protein kinase, cAMP-dependent, regulatory, type I, alpha (tissue specific extinguisher 1); arginine vasopressin receptor 2 (nephrogenic diabetes insipidus); protein kinase, cAMP-dependent, catalytic, beta; corticotropin releasing hormone receptor 2; growth hormone releasing hormone; parathyroid hormone-like hormone; adenosine A2a receptor; protein kinase, cAMP-dependent, regulatory, type II, alpha; corticotropin releasing hormone receptor 1; parathyroid hormone; hyperpolarization activated cyclic nucleotide-gated potassium channel 2; adrenergic, alpha-1B-, receptor; ret proto-oncogene (multiple endocrine neoplasia and medullary thyroid carcinoma 1, Hirschsprung disease); phosphodiesterase 4A, cAMP-specific (phosphodiesterase E2 dunce homolog of Drosophila); protein kinase, cAMP-dependent, regulatory, type II, beta; and protein kinase (cAMP-dependent, catalytic) inhibitor gamma.
The term “microarray” refers to an ordered arrangement of hybridizable array elements (i.e., the above-described polynucleotide probes). The array elements are arranged so that there are preferably at least one or more different array elements, more preferably at least 100 array elements, and most preferably at least 1,000 array elements, on a 1 cm2 substrate surface. The hybridization signal from each of the array elements is individually distinguishable. In a preferred embodiment, the array elements comprise polynucleotide probes.
A “polynucleotide” refers to a chain of nucleotides, preferably a single-stranded chain. Preferably, the chain has from about 75 to 10,000 nucleotides, more preferably from about 100 to 3,500 nucleotides. The term “probe” refers to a polynucleotide sequence capable of hybridizing with a target sequence to form a polynucleotide probe/target complex. A “target polynucleotide” refers to a chain of nucleotides to which a polynucleotide probe can hybridize by base pairing. In some instances, the sequences will be complementary (no mismatches) when aligned. In other instances, there may be up to a 10%. mismatch.
A “plurality” is given its ordinary meaning of 2 or more. In the present context, a plurality of polynucleotide probes (elements) refers preferably to a group of at least least 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300 or more polynucleotides or more elements, more preferably to a group of at least about 100, and even more preferably to a group of at least about 1,000, elements. The maximum number of members is unlimited, but is at least about 100,000 members.
The term “gene” or “genes” refers to polynucleotide sequence of a gene which may be the partial or complete gene and may comprise regulatory, untranslated, or coding regions.
The microarray can be used for large-scale genetic or gene expression analysis of a number of target polynucleotides. The microarray can also be used in the diagnosis of diseases and in the monitoring of treatments. Further, the microarray can be employed to investigate an individual's predisposition to a disease. Furthermore, the microarray can be employed to investigate cellular responses to carcinogens, mitogens, drug treatment, and the like.
The hybridizable array elements in a microarray of the invention may be organized in an ordered fashion so that each element is present at a specified location on the substrate. As such, each of the nucleic acids on the array will have its own “address” such that hybridization to that nucleic acid will allow specific identification of the complementary nucleic acid in a biological sample. Because the array elements are at specified locations on the substrate, the hybridization patterns and intensities can be interpreted in terms of expression levels of particular genes. Collectively, the expression profile obtained with the microarrays of the invention are tightly correlated to a particular disease or condition or treatment. Hence, the invention provides dramatically enhanced reliability in profiling and obtaining prognostic indicators of response to IMPDH inhibition.
The composition comprising a plurality of polynucleotide probes can also be used to purify a subpopulation of mRNAs, cDNAs, genomic fragments and the like, in a sample. This may be particularly useful in identifying subsets of the above-identified nucleic acids that are more highly indicative of modulated/abnormal IMPDH activity.
The nucleic acids identified herein as being responsive to IMPDH inhibition will be used in microarray production and can be genomic DNA or cDNA or mRNA, or any RNA-like or DNA-like material, such as peptide nucleic acids, branched DNAs and the like. The polynucleotide probes can be sense or antisense polynucleotide probes. Where target polynucleotides are double-stranded, the probes may be either sense or antisense strands. Where the target polynucleotides are single-stranded, the nucleotide probes are complementary single strands.
In one embodiment, the polynucleotide probes are cDNAs. The size of the DNA sequence of interest may vary and is preferably from 20 to 10,000 nucleotides, more preferably from 20 to 200 nucleotides. The polynucleotide probes can be prepared by a variety of synthetic or enzymatic schemes which are well known in the art. The probes can be synthesized, in whole or in part, using chemical methods well known in the art Caruthers et al. (1980) Nucleic Acids Res. Symp. Ser. 215-233). Alternatively, the probes can be generated, in whole or in part, enzymatically.
Nucleotide analogues can be incorporated into the polynucleotide probes by methods well known in the art. The only requirement is that the incorporated nucleotide analogues must serve to base pair with target polynucleotide sequences. For example, certain guanine nucleotides can be substituted with hypoxanthine which base pairs with cytosine residues. However, these base pairs are less stable than those between guanine and cytosine. Alternatively, adenine nucleotides can be substituted with 2,6-diaminopurine which can form stronger base pairs than those between adenine and thymidine.
Additionally, the polynucleotide probes can include nucleotides that have been derivatized chemically or enzymatically. Typical chemical modifications include derivatization with acyl, alkyl, aryl or amino groups.
The polynucleotide probes can be immobilized on a substrate. Preferred substrates are any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the polynucleotide probes are bound. Preferably, the substrates are optically transparent.
Complementary DNA (cDNA) can be arranged and then immobilized on a substrate. The probes can be immobilized by covalent means such as by chemical bonding procedures or UV. In one such method, a cDNA is bound to a glass surface which has been modified to contain epoxide or aldehyde groups. In another case, a cDNA probe is placed on a polylysine coated surface and then UV cross-linked (Shalon et al. PCT publication WO95/35505, herein incorporated by reference). In yet another method, a DNA is actively transported from a solution to a given position on a substrate by electrical means (Heller et al. U.S. Pat. No. 5,605,662). Alternatively, individual DNA clones can be gridded on a filter.
Furthermore, the probes do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups are typically about 6 to 50 atoms long to provide exposure to the attached polynucleotide probe. Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with one of the terminal portions of the linker to bind the linker to the substrate. The other terminal portion of the linker is then functionalized for binding the polynucleotide probe.
The polynucleotide probes can be attached to a substrate by dispensing reagents for probe synthesis on the substrate surface or by dispensing preformed DNA fragments or clones on the substrate surface. Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions simultaneously.
Hybridization of Nitcleic Acids from Biological Sample to Microarray. In order to detect the presence of a given nucleic acid in a biological sample it will be desirable to hybridize the nucleic acid isolated from that sample to the microarray. Hybridization causes a denatured polynucleotide probe and a denatured complementary target to form a stable duplex through base pairing. Hybridization methods are well known to those skilled in the art (See, e.g., Ausubel (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., units 2.8-2.11, 3.18-3.19 and 4-6-4.9). Conditions can be selected for hybridization where exactly complementary target and polynucleotide probe can hybridize, i.e., each base pair must interact with its complementary base pair. Alternatively, conditions can be selected where target and polynucleotide probes have mismatches but are still able to hybridize. Suitable conditions can be selected, for example, by varying the concentrations of salt in the prehybridization, hybridization and wash solutions, or by varying the hybridization and wash temperatures. With some membranes, the temperature can be decreased by adding formamide to the prehybridization and hybridization solutions.
Hybridization can be performed at low stringency with buffers, such as 6 x SSPE with 0.005% Triton X-100 at 37° C., which permits hybridization between target and polynucleotide probes that contain some mismatches to form target polynucleotide/probe complexes. Subsequent washes are performed at higher stringency with buffers, such as 0.5×SSPE with 0.005% Triton X-100 at 50° C., to retain hybridization of only those target/probe complexes that contain exactly complementary sequences. Alternatively, hybridization can be performed with buffers, such as 5×SSC/0.2% SDS at 60° C. and washes are performed in 2×SSC/0.2% SDS and then in 0.1×SSC. Background signals can be reduced by the use of detergent, such as sodium dodecyl sulfate, Sarcosyl or Triton X-100, or a blocking agent, such as salmon sperm DNA.
After hybridization, the microarray is washed to remove nonhybridized nucleic acids, and complex formation between the hybridizable array elements and the target polynucleotides is detected. Methods for detecting complex formation are well known to those skilled in the art. In a preferred embodiment, the target polynucleotides are labeled with a fluorescent label, and measurement of levels and patterns of fluorescence indicative of complex formation is accomplished by fluorescence microscopy, preferably confocal fluorescence microscopy. An argon ion laser excites the fluorescent label, emissions are directed to a photomultiplier, and the amount of emitted light is detected and quantitated. The detected signal should be proportional to the amount of probe/target polynucleotide complex at each position of the microarray. The fluorescence microscope can be associated with a computer-driven scanner device to generate a quantitative two-dimensional image of hybridization intensity. The scanned image is examined to determine the abundance/expression level of each hybridized target polynucleotide.
Typically, microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions. In a preferred embodiment, individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.
Microarray Expression Profiles. As discussed above an important aspect of the present invention is a method of obtaining an expression profile, using the microarray compositions of the invention, of a subject that has or is about to undergo therapy based on IMPDH inhibition. The expression profile can be used to detect changes in the expression of genes in response to such inhibition and to provide a prognosis of a patient's response to an IMPDH inhibitor comprising the steps of: (a) subjecting RNA extracted from the cells obtained from the patient to gene expression analysis on one of the microarrays of the invention in the presence and absence of said IMPDH inhibitor. In doing so, the expression level of at least one gene selected from the genes in Tables I through VIII is determined and compared to the amount of expression found in a corresponding reference tissue set that has not been treated with an IMPDH inhibitor. Subsequently, a report summarizing the data obtained by such gene expression analysis can be prepared and used to determine whether the patient will likely be responsive to IMPDH inhibition.
The expression profile comprises determining the level of expression the nucleic acids that have been identified herein as being responsive to IMPDH inhibition and may further involve categorizing said nucleic acids into functional categories (e.g., the gene has a cell-cycle function, a cell proliferation function, is involved in lipid metabolism some other metabolic pathway, and the like). It is contemplated that at least one of the nucleic acids identified herein, and preferably a plurality thereof, is hybridized to a complementary target polynucleotide forming at least one, and preferably a plurality, of complexes. A complex is preferably detected by incorporating at least one labeling moiety in the complex as described above. The expression profiles provide “snapshots” that can show unique expression patterns that are characteristic of that individual's response to IMPDH inhibition.
After performing hybridization experiments and interpreting detected signals from a microarray, particular polynucleotide probes can be identified and selected based on their expression patterns (e.g., those that are consistently and dramatically up- or down-regulated upon IMPDH inhibition). Such polynucleotide probe sequences can be used to clone a full length sequence of the gene for further analysis, provide an alternative diagnostic tool, or to produce the encoded polypeptide.
In one situation, the microarray is used to monitor the progression of disease and the response of that disease to IMPDH inhibition. Researchers can assess and catalog the differences in gene expression between healthy and diseased tissues or cells. By analyzing changes in patterns of gene expression, disease can be diagnosed at earlier stages before the patient is symptomatic. The invention can also be used to monitor the efficacy of treatment. For some treatments with known side effects, the microarray is employed to “fine tune” the treatment regimen. A dosage of IMPDH inhibitor is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment.
Alternatively, animal models which mimic a disease, rather than patients having the disease, can be used to characterize expression profiles associated with a particular inhibitor. This gene expression data may be useful in diagnosing and monitoring the course of disease in a patient, in determining gene targets for intervention, and in testing treatment regimens.
Also, researchers can use the microarray to rapidly screen large numbers of candidate IMPDH inhibitory drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs e.g., VX-944, MPA, Nucleoside analogs such as tiazofurin, ribavirin and mizoribine, and other agents listed in e.g., U.S. Pat. Nos. 5,807,876, 5,932,600, 6,054,472, 6,344,465, 6,395,763, 6,399,773, 6,420,403, 6,867,299, 6,826,488, 6,825,224, 6,653,309, 6,624,184, 6,617,323, 6,541,496, 6,518,291, and 6,498,178 (each specifically incorporated herein by reference in its entirety for its teaches of IMPDH inhibitor compositions and methods of administering the same for the treatment of IMPDH related disorders), with the expectation that molecules with the same expression profile will have similar therapeutic effects. Thus, the invention provides the means to determine the molecular mode of action of an IPDH inhibitor or IMPDH pathway inhibitor, as well as to facilitate identification of new such drugs.
The following examples are included to demonstrate certain embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus are considered to constitute certain aspects for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Inosine monophosphate dehydrogenase (IMPDH) catalyzes a rate-limiting step in guanine nucleotide biosynthesis, the NAD-dependent reduction of IMP to XMP. Inhibition of IMPDH activity results in the cessation of DNA synthesis and is an important target for immunosuppressive, anticancer and antiviral therapy.
Mycophenolate mofetil, an approved immunosuppressive agent (CellCept® from Roche) used for the prevention of kidney, heart or liver transplant rejections is the 2-morpholinoethyl ester of mycophenolic acid (MPA). VX-944 is currently being developed for the treatment of hematological malignancies. Both MPA and VX-944 are selective inhibitors of IMPDH with VX-944 being 3-4 fold more potent in cell assays (see
In order to arrive at the conclusion that the expression of certain nucleic acids is modulated in response to IMPDH inhibition, the following inhibitors were employed:
The above agents were tested in HL-60 cells at a concentration of 500 nM for VX-944 and 1000 nM for MPA or with 0.1% DMSO as the vehicle control. The cells were harvested at 14 or 30 hours post addition of compound or vehicle. Briefly, the culture medium was removed at the appropriate time-points and the cells washed with PBS. The cells were then harvested, and pelleted by-gentle centrifugation. The supernatant was discarded and the pellets snap-frozen on an ethanol/dry-ice slurry. RNA was isolated from cells by using a RNEasy kit from Qiagen. RNAs were quality controlled using the Agilent 2100 Bioanalyzer to determine the presence of RNAs.
The results from the Affymetrix array analyses are summarized in the Table below. Data from the arrays corresponding to VX-944 and MPA at the two time-points—14 hours (Early) and 30 hours (Late) were analyzed using Spotfire™, microarray data analysis software. Some of the data analysis parameters used are outlined below:
a) All the arrays were normalized by using trimmed means, with a 5% exclusion limit
b) Only genes present in at least 5 of the 6 chips used for the analyses were included in the genesets
c) Only statistically significant genes (ANOVA, p<0.05) were included in the final analyses to generate gene lists.
A global view of datasets identified by comparing VX-944 and MPA at the early (14 h) time-point showed that there were 77 genes in common between VX-944 and MPA. At the late (30 h) time-point, 335 genes were identified as overlapping between the VX-944 and MPA datasets. A global view of datasets identified by comparing VX-944 and MPA at the two time-points, 14 h (EARLY) and 30 h (LATE) showed that with VX-944 102 genes modulated at the early time-point were also modulated at the late time-point. With MPA 66 genes are in common between the two time-points. As to a comparison between the two data sets, of the 102 genes modulated by VX-944 and the 66 genes modulated by MPA, 43 genes modulated at 14 hours were also modulated at 30 hours. To add additional value to the gene lists and provide additional information to the investigator, the genes underlying each of these expression patterns was queried against publicly available databases. These databases included Locus Link, Gene Cards, KEGG, GO annotations, OMIM, PubMed. The information gathered from such an analysis is organized below in data tables.
VX-944 (Early) vs. MPA (Early): 77 genes were found to be common between these two comparisons. These genes were further assigned to the following functional categories:
Interestingly, for this dataset a large number of genes were classified into the Nucleotide metabolism functional category. Some of the other major categories include protein metabolism, response to biotic stimulus, biosynthesis, signal transduction, cell proliferation, lipid metabolism and response to pest/pathogen/parasite. However, 30/77 genes could not be readily classified using the GO functional categorization. Even with these genes, every effort has been made to provide as much information as possible.
The genes in some of these categories are shown below:
VX-944 (Late) vs. MPA (Late): 335 genes were found to be common between these two comparisons. These genes were further assigned to the following functional categories:
VX-944 (Early) vs. VX-944 (Late): 102 genes found common between these two comparisons were binned into the following functional categories.
MPA (Early) vs. MPA (Late): 66 genes were binned into this category. These genes were further binned into the following functional categories.
VX-944 (Early n Late) vs. MPA (Early ∩ Late): 43 genes were found to be in common for this stringent comparison. These genes were binned into the following functional
polyphemus)
For each of the comparative analyses set up for this project, the processed data in the form of gene lists with hyperlinks to various databases has been prepared and is represented in Appendix III.
A snapshot of one of these gene lists is shown below. Each of the columns referring to an external database (GENE CARDS at the Weizmann Institute, Locus Link and OMIM at the NIH) has a hyperlink, which when accessed will pull in data corresponding to that gene.
indicates data missing or illegible when filed
In addition, a subset of the data was mapped onto relevant pathways, using the KEGG database. The following gene sets 2 and 3 also may be used in the arrays of the invention and are useful predictors of IMPDH inhibition. Particularly preferred exemplary markers of IMPDH inhibition are ARF4 (SEQ ID NO:______); CCNE2 (SEQ ID NO:______); CGR19 (SEQ ID NO:______); CPR8 (SEQ ID NO:______); EDF1 (SEQ ID NO:______); GTF2F1 (______); LGALS1 (SEQ ID NO:______); PDGFC (SEQ ID NO:______); PSMC5 (SEQ ID NO:______); PTK9 (SEQ ID NO:______); RABGGTA (SEQ ID NO:______); SCAP2 (SEQ ID NO:______); SERPINB2 (SEQ ID NO:______); TBPL1 (SEQ ID NO:______).
Homo sapiens cDNA FLJ36224 fis, clone
Homo sapiens cDNA FLJ43274 fis, clone
Homo sapiens mRNA; cDNA DKFZp434B142 (from
polyphemus)
polyphemus)
Homo sapiens G21VN02 mRNA, mRNA sequence
Homo sapiens cDNA FLJ36224 fis, clone
Homo sapiens mRNA; cDNA DKFZp434B142 (from
polyphemus)
Homo sapiens cDNA FLJ43274 fis, clone
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The references cited herein throughout, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are all specifically incorporated herein by reference. Each of the tables, pages and figures of priority application No. 60/575,076 filed on May 27, 2004 are specifically incorporated by reference in their entirety. The following references are exemplary of the state of the art of the development and use of IMPDH inhibitors and are incorporated herein by reference in their entirety:
sapiens IL1.0 receptor antagonist IL1.0Ra
S. cerevisiae exonuclease 1
Drosophila)
sapiens cDNA, 3 end /clone = IMAGE-
ataxia
telangiectasia
ataxia telangiectasia mutated (includes
S. pombe) homolog
sapiens cDNA, 3 end /clone = IMAGE-
laevis)
spinocerebellar
ataxia 1
spinocerebellar ataxia 1
spinocerebellar ataxia 1
sapiens kinesin family member 4A
sapiens mRNA for Aik2, complete
sapiens fatty-acid-Coenzyme A
sapiens isolate BX-01 cyclin-
sapiens mitotic spindle coiled-coil
sapiens formyl peptide receptor 1
sapiens chromosome
sapiens tissue inhibitor of
sapiens regulator of G-protein
sapiens serinethreonine kinase 15
sapiens arachidonate 5-
sapiens inositol(myo)-1(or 4)-
sapiens metallothionein 1X
sapiens C-type (calcium
sapiens, ribonucleotide reductase
sapiens cyclin B2 (CCNB2),
sapiens mannose receptor, C type
sapiens collagen, type VI, alpha 3
sapiens histidine decarboxylase
sapiens complement component 5
sapiens serine (or cysteine)
sapiens CCAATenhancer binding
sapiens integrin, alpha M
sapiens serumglucocorticoid
sapiens, Similar to neutrophil
sapiens hypothetical protein
sapiens pituitary tumor-
sapiens serinethreonine kinase 6
sapiens polo (Drosophia)-like
sapiens clone TCBAP0702 mRNA
sapiens interferon, gamma-
sapiens Kreisler (mouse) maf-
sapiens phosphoprotein regulated
sapiens oxidized low-density
sapiens budding uninhibited by
sapiens putative gene product
sapiens CDC20 (cell division cycle
sapiens Toll-like receptor 8
sapiens testis specific mitotic
sapiens ubiquitin carrier protein E2
sapiens v-myb avian
sapiens centromere protein A
sapiens cytochrome b-245, beta
sapiens clone HQ0310
sapiens metallothionein 1H
sapiens S100 calcium-binding
indicates data missing or illegible when filed
The present application claims the benefit of priority of 60/575,076 filed on May 27, 2004. The entire text of this priority application and its appendices is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/18551 | 5/27/2005 | WO | 00 | 9/28/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60575076 | May 2004 | US |
