Patent Application
20160299149
The polyamines (putrescine 1, spermidine 2a and spermine 3,
In contrast to inorganic cations, polyamines can be biosynthesized and catabolized. Intracellular levels of polyamines are maintained by a subtle equilibrium between a complex enzymatic machinery (controlling biosynthesis and catabolism), and systems that allow import and export1. Although the molecular biology of polyamine metabolism in mammalian cells is extensively known, polyamine transport remains an observable, yet elusive phenomenon. While polyamine transporters have been characterized in the bacteria and unicellular eukaryotes, the identities of the genes involved in the polyamine transport system (PTS) in any multicellular organism were, until recently, unknown(2-13).
Even with recent advances, in most respects the mammalian PTS is still a black box. Mammalian cells import polyamines by carrier-mediated, energy- and Natdependent mechanisms(15-17). Observations in many cell-types suggest that transport may be more complex than a single transporter. Transporters with different affinities for putrescine, spermidine and spermine have been biochemically characterized(18-25), and differences in Na+-dependence for import have been reported(20-23).
The PTS is fully integrated into the cellular regulatory system for the control of intracellular polyamine levels(19, 24-25).
Many cancer cell types have high polyamine biosynthesis and high polyamine transport activity, presumably to sustain their rapid growth rates. While polyamine biosynthesis inhibitors like difluoromethylornithine (DFMO) are known and have a mixed clinical history in the treatment of cancers26, there is no commercially-available polyamine transport inhibitor (PTI)(27-29).
These transport inhibitors are needed because cancers can circumvent DFMO therapy by importing exogenous polyamines24. Indeed, there is an opportunity to uncover new biomarkers which can help clinicians identify tumors which will best respond to DFMO therapy or combination therapies involving DFMO and a PTI. Unfortunately, both PTI-design and biomarker development are hampered without molecular knowledge of the PTS itself.
There are at least two models that describe polyamine entry into cells, either of which may be operative depending on cell type and/or the molecular structure of the polyamine itself (
The membrane-bound polyamine is then imported into cells via caveolin dependent endocytosis(35-36).
A similar model invoking receptor-mediated endocytosis as a route for polyamine entry is also supported by the initial work of Poulin, Soulet, et al., who used polyamines conjugated to fluorescent probes to explore polyamine transport and polyamine intracellular trafficking.
Belting observed dramatic reduction of spermine uptake in proteoglycan-deficient CHO cell mutants, which suggested that proteoglycans are necessary for polyamine transport. In support of Model B, Belting demonstrated a high-affinity binding site for spermine on HS and showed that cells with an inability to process HS (via nitric oxide) had significantly reduced spermine uptake. However, there is contradictory evidence which showed that polyamine transport was not significantly altered (20% reduction) in CHO cells defective in HS biosynthesis(31-32). Intriguingly, these HS-deficient cells had up-regulated chondroitin sulfate (CS) suggesting that a different carbohydrate could also mediate uptake(31-32). These observations suggest that cells can alter their cell-surface glycan composition to bind and import polyamines from outside the cell. Due to the fact that neither spermidine nor putrescine were evaluated in the prior art, further studies would be required to delineate the nature of the transport receptor and if the other polyamines share the same uptake mechanism.
The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
The term “therapeutically effective amount” as used herein means an amount that achieves the intended therapeutic effect of terminating targeted cells treated with a drug, or reducing the number of the cells targeted by the drug in a subject. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations per day for successive days. A series of doses administered over the course of a period of time is referred to herein as a “regimen.”
The term “preventing” a disease as used herein refers to reducing or delaying the onset of the disease in a subject who is at high risk of developing the disease. In a non-limiting embodiment, the disease may include colorectal cancer, and in a further non-limiting embodiment, the disease may include inflammatory bowel diseases (IBD) like Crohn's disease and ulcerative colitis.
The term “treating” refers to a disorder or disease described herein including but not limited to cancer or other proliferative disorders. In a non-limiting embodiment, the disease may include pancreatic cancer, and in a further non-limiting embodiment, the disease may include a primary pancreatic cancer and/or a metastatic pancreatic cancer.
The term “sample” as used herein refers to a cell-containing sample of a patient, including a tissue, or a tumor, blood, tears, urine or feces sample, in non-limiting embodiments. In some embodiments described herein, the term sample is referred to as a blood sample, but any other type of sample described herein or known to those skilled in the art may be used as a substitute sample, or in addition to a blood sample. The term “cell sample” as used herein may include whole cells, lysed cells, cell homogenate, a cell tissue layer, or any other type of cells ample described herein or known to those skilled in the art.
The terms “animal” “patient” or “subject” as used interchangeably herein refer to any animal for example mammals, including, but not limited to humans, primates, dogs, cattle, cows, horses, kangaroos, pigs, sheep, goats, cats, rabbits, rodents, and transgenic non-human animals, and the like, which are to be the recipient of a particular treatment. Typically, the terms “animal” “subject” and “patient” are used interchangeably herein in reference to a human subject or a rodent. The preferred animal, patient, or subject is a human.
The term “solid support” or “substrate” refers to any material that can be modified to contain discrete individual sites appropriate for the attachment or association of a capture binding ligand. Suitable substrates include metal surfaces such as gold, electrodes, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes, Teflon, derivatives thereof, etc.), polysaccharides, nylon or nitrocellulose, resins, mica, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, fiberglass, ceramics, GETEK (a blend of polypropylene oxide and fiberglass) and a variety of other polymers and their functionalized derivatives.
The term “measuring” means methods which include detecting the presence or absence of marker(s) in the sample, quantifying the amount of marker(s) in the sample, and/or qualifying the type of biomarker. Measuring can be accomplished by methods known in the art and those further described herein, including but not limited to microarray analysis (with Significance Analysis of Microarrays (SAM) software), SELDI, quantitative polymerase chain reaction (PCR), quantitative reverse transcription polymerase chain reaction-(RT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunoassays to measure either RNA and/or protein levels in biological samples. Any suitable methods can be used to detect and measure one or more of the markers described herein. These methods include, without limitation, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry, Western blot analysis, ELISA, immunoassays, RT-PCR or PCR and atomic force microscopy.
The term “label” may include a radioactive, a fluorescent, a chemiluminescent, a dye, an enzyme, or a histidine label, or any other suitable label known in the art.
The term “binding molecule(s)” may include antibodies or other binding molecules specific to and configured to bind other antibodies, in a non-limiting embodiment. In other non-limiting embodiments, the binding molecules may bind multiple antibodies. The binding molecules may be provided as attached to or associated with a substrate to which an antibody-bound molecule can bind.
“Detect” refers to identifying the presence, absence or amount of the object to be detected.
The phrase “differentially present” refers to differences in the quantity and/or the frequency of a marker present in a sample taken from subjects having symptoms of cancer as compared to a control subject or a reference subject or sample. For example, some markers described herein are present at an elevated level in samples of subjects compared to samples from control subjects, e.g., c-myc, Cav-2 and ATP13A3. In contrast, other markers described herein are present at a decreased level in samples of cancer subjects compared to samples from control subjects, e.g., Cav-1. Furthermore, a marker can be a polypeptide or a nucleic acid, which is detected at a higher frequency or at a lower frequency in samples of human cancer subjects compared to samples of control subjects.
Furthermore, a marker can be a polypeptide or nucleic acid, which is detected at a higher frequency or at a lower frequency in samples of unaffected tissue from subjects with cancer compared to samples of affected tissue from subjects with cancer (e.g., melanoma).
Furthermore, a marker can be a polypeptide or nucleic acid, which is detected at a higher frequency or at a lower frequency in samples of human unaffected tissue from cancer subjects (e.g., melanoma) compared to samples of control subjects (e.g., without cancer).
Furthermore, a marker can be a polypeptide or nucleic acid, which is detected at a higher frequency or at a lower frequency in samples of human affected tissue from cancer subjects (e.g., melanoma) compared to samples of control subjects (without cancer).
A marker can be differentially present in terms of quantity, frequency or both.
“Affected tissue,” as used herein refers to tissue from a cancer subject that is grossly diseased tissue (e.g., skin (epidermis and/or dermis) lymph nodes, metastatic sites or tumor tissue, e.g., pancreas, brain, lung, bone, liver, skin, or melanocytes).
“Unaffected tissue,” as used herein refers to a tissue from a cancer subject that is from a portion of tissue that does not have gross disease present, for example tissue that is about 1, 2, 5, 10, 20 or more cm from grossly-diseased tissue.
“Normal cells” as used herein refers to cells of unaffected tissue.
“Basal level” as used herein in relation to a level of a protein relates to a level of the protein in a normal cell.
“Relative to normal cells” as used herein to describe a protein level in a cell of affected tissue pertains to relative comparison of the protein level in a cell of unaffected tissue. Typically, the cell of the unaffected tissue is of a similar source to that of the affected tissue. For example, a protein level of cells of affected tissue in the breast is compared to the protein level of cells in unaffected tissue in the breast.
“At least 50-100% higher than basal level” as used herein means that a determined value is at least 50% or at least 100% higher than a basal level, or at least a percentage value higher than basal level of each integer percent within the range of 50-100%. For example, at least 50-100% higher than basal level includes at least 51%, 52%, 60%, 70%, 85% etc. higher than basal level.
As used herein, “array” or “microarray” refers to an array of distinct polynucleotides, oligonucleotides, polypeptides, or oligopeptides synthesized on a substrate, such as paper, nylon, or other type of membrane, filter, chip, glass slide, or any other suitable solid support. Arrays also include a plurality of antibodies immobilized on a support for detecting specific protein products. There are several microarrays that are commercially available. A microarray may include one or more biomarkers disclosed herein. A panel of about 20 biomarkers as nucleic acid fragments can be included in an array. The nucleic acid fragments may include oligonucleotides or amplified partial or complete nucleotide sequences of the biomarkers. The term “consisting essentially of” generally refers to a collection of markers that substantially affects the determination of the disorder and may include other components such as controls.
In an embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al.; PCT application WO95/11995, Chee et al.; Lockhart et al., 1996. Nat Biotech., 14:1675-80; and Schena et al., 1996. Proc. Natl. Acad. Sci. 93:10614-619, all of which are herein incorporated by reference to the extent they relate to methods of making a microarray. Arrays can also be produced by the methods described in Brown et al., U.S. Pat. No. 5,807,522. Arrays and microarrays may be referred to as “DNA chips” or “protein chips.”
A variety of clustering methods are available for microarray-based gene expression analysis. See for example, Shamir & Sharan (2002) Algorithmic approaches to clustering gene expression data. In Current Topics In Computational Molecular Biology (Edited by: Jiang T, Xu Y, Smith T). 2002, 269-300; Tamames et al., (2002): Bioinformatics methods for the analysis of expression arrays: data clustering and information extraction, J Biotechnol, 98:269-283.
The dose administered to an animal, particularly a human, in accordance with the present invention should be sufficient to effect the desired response in the animal over a reasonable time frame. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the strength of the particular compositions employed, the age, species, condition, and body weight of the animal. The size of the dose also will be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular composition and the desired physiological effect. It will be appreciated by one of ordinary skill in the art that various conditions or desired results, may require prolonged treatment involving multiple administrations.
Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
The amount of the compound of the invention administered per dose or the total amount administered per day may be predetermined or it may be determined on an individual patient basis by taking into consideration numerous factors, including the nature and severity of the patient's condition, the condition being treated, the age, weight, and general health of the patient, the tolerance of the patient to the compound, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetics and toxicology profiles of the compound and any secondary agents being administered, and the like. Patients undergoing such treatment will typically be monitored on a routine basis to determine the effectiveness of therapy. Continuous monitoring by the physician will insure that the optimal amount of the compound of the invention will be administered at any given time, as well as facilitating the determination of the duration of treatment. This is of particular value when secondary agents are also being administered, as their selection, dosage, and duration of therapy may also require adjustment. In this way, the treatment regimen and dosing schedule can be adjusted over the course of therapy so that the lowest amount of compound that exhibits the desired effectiveness is administered and, further, that administration is continued only so long as is necessary to successfully achieve the optimum effect.
Disclosed herein are new biomarkers associated with polyamine transport and cell lines expressing high levels of specific proteins provided herein are shown to have high polyamine transport activity and to be especially sensitive to therapies which target polyamine transport and polyamine metabolism. This property is shown in several assays where cell lines containing active polyamine transport were specifically targeted by compounds which utilize the polyamine transport system for cell entry. Therefore, the inventors have discovered herein, new specific biomarkers wherein their relative expression can be used to identify tumors (and patients) who will best respond to therapies which target cell growth processes which rely on polyamines, i.e., polyamine metabolism. The discovery of a new polyamine transport gene has provided opportunities to develop diagnostic tests to allow more directed and specific treatment of cancers and other proliferative disorders with drugs that target polyamine metabolism. Various assays and test kits can be used as described herein to rapidly identify and assess the relative expression of these biomarkers in biopsies of patient tissue, tumors, or other patient derived samples such as blood, urine, feces, tears, etc. These tests can be used in clinical labs, for example, or may be used in a healthcare provider office setting, or for at-home use in other, non-limiting embodiments.
Assays, therefore, which allow one to conveniently determine the relative expression of these biomarkers will assist clinicians performing clinical trials on polyamine based drugs or drugs that target polyamine metabolism. This technology forms the basis of a new personalized medicine where one can analyze a patient sample for these specific markers and know that that patient will respond to the polyamine targeted therapy. This will reduce the number of patients subjected to medications which will not help their specific cancer or disease state. Assays that may be used to determine presence of a particular marker in a biological sample include, but are not limited to, immunoassay, spectrometry, mass spectrometry, Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry, microscopy, northern blot, western blot, southern blot, isoelectric focusing, SDS-PAGE, PCR, RT-PCR, gel electrophoresis, protein microarray, DNA microarray, antibody microarray, ELISA, and combinations thereof.
Despite more than two decades of study, the bulk of the macromolecules involved in polyamine transport and the details of polyamine trafficking events within the cell remain elusive until the discovery of a particular biomarker described herein. In addition to improving our understanding of a fundamental biological process, knowledge of how polycationic materials are imported and sequestered in eukaryotic cells can lead to new drug-delivery and gene-delivery technologies that are selective against malignant cells and infectious protozoan parasites (Gardner, R. A.; Delcros, J-G.; Konate, F.; Breitbeil III, F.; Martin, B.; Sigman, M.; Huang, M.; Phanstiel IV, O. N1-Substituent Effects in the Selective Delivery of Polyamine-Conjugates into Cells Containing Active Polyamine Transporters. J. Med. Chem. 2004, 47, 6055-6069; Gardner, R. A.; Belting, M.; Svensson, K.; Phanstiel IV, O. Synthesis and transfection efficiencies of new lipophilic polyamines. J. Med. Chem. 2007, 50, 308-318; Blagbrough I. S.; Geall A. J. Homologation of polyamines in the synthesis of lipo-spermine conjugates and related lipoplexes. Tetrahedron Lett. 1998, 39, 443-446; Azzam T.; Eliyahu H.; Shapira L.; Linial M.; Barenholz Y.; Domb A. J. Polysaccharide-oligoamine based conjugates for gene delivery. J. Med. Chem. 2002, 45, 1817-1824).
C-Myc has been shown to be overexpressed in pancreatic cancers (both primary and metastatic) and was thought in 2002 to be “involved in early neoplastic development and progression rather than in locoregional spread of invasive cancer.” (Ref: C. Schleger, Ph.D., C. Verbeke, M. D., R. Hildenbrand, M. D., H. Zentgraf, Ph.D., U. Bleyl, M. D. c-MYC Activation in Primary and Metastatic Ductal Adenocarcinoma of the Pancreas: Incidence, Mechanisms, and Clinical Significance. Modern Pathology 2002, 15, 462-469) However, a 2011 review paper on the role of c-Myc in pancreatic cancer (Ref: Anouchka Skoudy, Inmaculada Hernandez-Muñoz, Pilar Navarro. Pancreatic Ductal Adenocarcinoma and Transcription Factors: Role of c-Myc. J Gastrointest Canc 2011, 42, 76-84.) stated that c-Myc function is highly dose and cell dependent and that c-Myc has the ability to reprogram somatic cells towards a pluripotent stem like state. This means that therapies which can reduce c-Myc and early neoplastic growth could provide effective therapies against pancreatic and other cancers. In this regard c-myc is a known oncogene which increases the transcription of polyamine biosynthetic genes like ODC (Casero, Jr., R. and Marton, L. Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases. Nature Reviews, 2007, 6, 373-390.)
The discovery of CATP-5 as an ATPase involved in polyamine transport in C. elegans (i.e., worms) provided clues as to the Drosophila and human genes via sequence similarities. (Heinick A, Urban K, Roth S, Spies D, Nunes, F Phanstiel O 4th, Liebau E, Luersen K. Caenorhabditis elegans P5B-type ATPase CATP-5 operates in polyamine transport and is important for norspermidine-mediated suppression of RNA interference. FASEB J. 2010, 24:206-217.) This area of polyamine transport was recently reviewed by Poulin R, Casero R A, Soulet D, where the possibility of ATPases playing a role in human polyamine transport were suggested. See Poulin et al., Recent advances in the molecular biology of metazoan polyamine transport. Amino Acids. 2012, 42, 711-723.
Flamigni has suggested the existence of an oncogene-polyamine metabolic loop, where the products of oncogenes like Kras and Raf result in downstream proteins c-Myc and AP-1, which regulate ODC transcription and therefore polyamine biosynthesis. Note: AP-1 is either a homodimer of Jun or a heterodimer of Jun and Fos, which bind to a common DNA binding site located in introns 3, 5, and 11 of the ODC gene. The Myc protein, on the other hand, is a transcription factor that regulates the expression of genes by binding to the specific DNA sequence CACGTG. Among the target genes for Myc regulation is ODC. Both Myc and AP-1 are substrates of phosphorylation by MAPK. (Flamigni F, Facchini A, Capanni C, Stefanelli C, Tantini B, and Caldarera C M. p44/42 Mitogen-activated protein kinase is involved in the expression of ornithine decarboxylase in leukemia L1210 cells. Biochem J. 1999, 341, 363-369. And see Uriel Bachrach, Yong-Chun Wang, and Amalia Tabib. Polyamines: New Cues in Cellular Signal Transduction. News Physiol. Sci. 2001, 16, 106-109.
Therefore, strategies which result in decreased intracellular polyamine content (reduced levels of Spermidine and putrescine are known to be caused by DFMO+PTI therapies, see Muth A., Madan M., Archer J. J., Ocampo N., Rodriguez L., Phanstiel O. 4th. Polyamine transport inhibitors: design, synthesis, and combination therapies with difluoromethylornithine. J Med Chem. 2014, 57, 348-363) as well as reduced c-Myc expression (as seen with DFMO and DFMO+GW5074 (
It has been discovered that certain key proteins are associated with increased polyamine import in mammalian cells. These proteins include: c-myc, Cav-1 and ATP13A3 (also referred to herein as PTS markers or biomarkers). Specifically, in pancreatic cancer cells, for example, the inventors have observed that cells with high expression of c-myc and/or ATP13A3 and low Cav-1 expression have high polyamine transport activity. Moreover, ATP13A3 and its role in polyamine transport was unknown until its discovery by the inventors as provided herein.
It has been identified herein that human pancreatic cancer cell lines which have high c-myc expression have high basal polyamine import rates as measured by the uptake of radiolabeled spermidine (high Vmax values). In this regard, high c-myc expression (as measured by Western blot and c-myc protein/beta-actin protein ratios) is a good indicator of a cell's high polyamine import rates. Thus, tumors with high c-myc expression would be expected to be sensitive to compounds which enter and kill cells via the polyamine transport system.
It has been observed that the expression of ATP13A3 increases when cells are treated with a polyamine biosynthesis inhibitor, difluoromethylornithine (DFMO) and that spermidine can modulate the levels of this ATP13A3 protein. These observations are consistent with ATP13A3 playing a role in polyamine transport. Indeed, the related protein, CATP-5, in the C. elegans model system was shown to play a role in polyamine transport. In addition, cells with high basal ATP13A3 expression have been shown to readily escape DFMO therapy via their increased import of exogenous spermidine. In this regard, ATP13A3 is an indicator as to the potential of the cell type or tumor to escape DFMO only therapy. This finding is important as ATP13A3 indicates the ability of cancer cells to escape DFMO via polyamine import. In short, cells or tumors with high ATP13A3 expression are predicted to be good at escaping DFMO-only therapy via their enhanced ability to import exogenous spermidine. These cells are still sensitive to a combination therapy (DFMO+PTI) involving DFMO and a polyamine transport inhibitor (PTI), where this escape pathway has been blocked via the PTI. In short, higher doses of the PTI have been shown to be able to overcome this increased ATP13A3 expression by blocking polyamine import in vitro. Thus, ATP13A3 represents a good biomarker for polyamine uptake and ultimately DFMO resistance.
The discoveries described herein are critical for the development of new cancer therapies predicated upon targeting polyamine transport and inducing sustained intracellular polyamine depletion. In a non-limiting embodiment, these cancer therapies may be accomplished using a combination therapy involving DFMO and a PTI (Burns, M. R.; Graminski, G. F.; Weeks, R. S.; Chen, Y.; O'Brien, T. G. Lipophilic Lysine-Spermine Conjugates Are Potent Polyamine Transport Inhibitors for Use in Combination with a Polyamine Biosynthesis Inhibitor, J Med Chem 2009, 52, 1983-1993; Aaron Muth, Jennifer Archer, Nicolette Ocampo, Meenu Madan, Luis Rodriguez, and Otto Phanstiel IV, Polyamine transport inhibitors: Design, Synthesis and Combination therapies with Difluoromethylornithine, J. Med. Chem. 2014, 57, 348-363).
Caveolin-1 (Cav-1) levels also provide a predictive tool for how tumors may respond to DFMO therapy. For example, DFMO is expected to lower intracellular Cav-1 levels as well as Cav-1 mRNA (Altaf Mohammed, Naveena B. Janakiram, Venkateshwar Madka, Rebekah L. Ritchie, Misty Brewer, Laura Biddick, Jagan Mohan R. Patlolla, Michael Sadeghi, Stan Lightfoot, Vernon E. Steele, and Chinthalapally V. Rao* Eflornithine (DFMO) Prevents Progression of Pancreatic Cancer by Modulating Ornithine Decarboxylase Signaling, Cancer Research Prevention 2014, in press, doi: 10.1158/1940-6207. CAPR-14-0176). This outcome however may be cell line dependent as shown in
Recently, DFMO has also been shown to affect thymidine metabolism and affect thymidine levels in cancer cells. (Witherspoon, M., et al Cancer Discov; 2013, 3(9); 1072-1081). In this regard, DFMO therapy may affect other metabolic cycles linked to polyamine metabolism, some of which may adversely affect cell growth. One endpoint that can be considered in DFMO+PTI therapy is intracellular thymidine and SAM levels, which are expected to decrease as cells futilely attempt to generate decarboxylated SAM for polyamine biosynthesis. These endpoints could be used to provide a direct measure of therapy efficacy.
It is disclosed that measuring relative c-myc expression levels as well as Cav-1 and ATP13A3 levels provides a critical step in understanding to what degree a tumor is committed to polyamine import as a means to supply its polyamine pools needed for growth, and therefore, the potential for effectiveness of polyamine targeting drugs on the tumor. Measuring relative Cav-2 levels has also provided important information in determining to what degree the tumor is committed to polyamine import for growth, and the effectiveness of polyamine targeting drugs on these cells. As shown in
Providing a convenient assay to specifically look at these markers will have tremendous clinical utility in helping physicians understand the tumors they are dealing with and to prescribe effective therapies which take advantage of this phenotype of high polyamine transport activity by ferrying in toxic polyamine based compounds (like compound A), or blocking the uptake of polyamine growth factors with DFMO+PTI, etc. In short, this discovery can help inform clinicians as to the nature of the tumors they seek to treat and provide additional confidence that the polyamine-based intervention will be successful.
Examples of biological samples typically obtained for use in certain method embodiments include but are not limited to a fluid, cell, stool sample, tissue, or tissue lysate obtained from the subject. Examples of fluids include but are not limited blood, serum, semen, urine, saliva, tears, perspiration, breath condensate, or vaginal fluid, cerebral spinal fluid, epithelial mucus or synovial fluid. Biological samples can be obtained from a subject, which may be a human or non-human mammal.
In non-limiting embodiments provided herein, a high or higher level of a biomarker includes a level at least 50% higher or at least two times the 50% higher level relative to basal levels in normal cells. In non-limiting embodiments provided herein basal level refers to the level in normal cells. In other non-limiting embodiments provided herein, a lower level of a biomarker includes a level within 20% of the basal level.
Embodiments of the invention include diagnostic tests, testing methods and assays to determine the presence of certain PTS markers disclosed herein. Various types of assays and tests may be used, including, but not limited to immunoassays, spectrometry, mass spectrometry, Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry, microscopy, northern blot, western blot, southern blot, isoelectric focusing, SDS-PAGE, PCR, RT-PCR, gel electrophoresis, protein microarray, DNA microarray, and antibody microarray, or combinations thereof. Preferably, the level of the one or more biomarkers is determined using an immunoassay. An immunoassay uses an antibody or antibodies to a specific antigen to determine the levels of the antigen.
The immunoassay may be an enzyme linked immunoassay (ELISA), a sandwich assay, a radioimmunoassay, a Western Blot, an immunoassay using a biosensor, an immunoprecipitation assay, an agglutination assay, a turbidity assay or a nephelometric assay.
The one or more antibodies may be synthetic, monoclonal, polyclonal, bispecific, chimeric or humanised. A chimeric antibody includes portions derived from different animals. Humanised antibodies are antibodies from non-human species having one or more complementarity determining regions from the non-human species and a framework region from a human immunoglobulin molecule. Chimeric and humanised antibodies can be produced by recombinant techniques well known in the art.
The one or more antibodies may comprise a tag or a label selected from the group comprising a radioactive, a fluorescent, a chemiluminescent, a dye, an enzyme, or a histidine tag or label, or any other suitable label or tag known in the art.
The use of subscript A, B, (e.g. AntibodyA, AntibodyB, etc.) unless otherwise indicated, is an arbitrary designation to indicate that they are different antibodies, or antibodies that are each specific to a different protein.
The presence, and possibly even the level, of particular proteins in a sample may also be determined by using mass spectrometry techniques. Mass spectrometry techniques may be used to detect gas phase ions which correlate to specific proteins or parts of proteins, such as, trypsin peptides. Examples of mass spectrometers include time of flight, magnetic sector, quadruple filter, ion trap, ion cyclotron resonance, electrostatic sector analyser and hybrids of these. The mass spectrometer may use laser desorption/ionisation.
MALDI (Matrix-assisted laser desorption/ionization) peptide mass fingerprinting may be used to identify the presence of particular biomarker proteins from 2D gel analysis and RPE/SDS PAGE analysis.
MALDI-MS and ANN analysis may be used to profile identified proteins and tryptic biomarker signatures to determine that certain cancer cells are susceptible to PTS targeting.
In a particular embodiment, disclosed is the use of immobilized antibodies which target and identify c-myc, ATP13A3, Cav-1 or Cav-2 proteins, or a combination thereof. These could be in the form of an ELISA test, in a non-limiting embodiment, which may quantify the relative expression levels of ATP13A3, c-myc Cav-1 and/or Cav-2. High expression of ATP13A3, Cav-2 and c-myc would indicate high polyamine transport activity by the tumor (or sample), in one embodiment. Low Cav-1 expression would indicate increased polyamine transport activity, in an embodiment. In a further embodiment, low Cav-1 expression where c-myc, Cav-2, and/or ATP13A3 activity is high could indicate increased polyamine transport activity. In a further embodiment, low Cav-1 expression in samples where ATP13A3 expression is high could indicate increased polyamine transport activity in the presence of DFMO and indicate a higher level of PTI dosing to ensure efficacy. Further, low Cav-1 expression in samples where Cav-2 expression is high could indicate sensitivity to polyamine transport inhibitors like trimer44NMe or other polyamine transport targeting compounds, such as, in non-limiting examples, Compounds A and/or B as described in more detail herein. Endpoints could be measured in terms of tumor size, weight and histological examination of tumor biopsies looking for these markers by immunohistochemistry (IHC) or by Western blot analysis using a standard protein like beta actin or GADPH for normalization and quantification.
The ability to easily quantify these markers would provide guidance for treatment using effective and specific treatment therapies, including many polyamine targeting therapies like DFMO alone or in combinations with NSAIDs, for example, or smart drugs which selectively target cells with active polyamine transport (Ant44 and compounds A and B) (Development of Polyamine Transport Ligands with Improved Metabolic Stability and Selectivity against Specific Human Cancers. Aaron Muth, Joseph Kamel, Navneet Kaur, Allyson C. Shicora, Iraimoudi S. Ayene, Susan K. Gilmour, and Otto Phanstiel IV J. Med. Chem. 2013, 56, 5819-5828.), in non-limiting embodiments. In other non-limiting embodiments, combination therapies which use a polyamine biosynthesis inhibitor (e.g., DFMO) in concert with a polyamine transport inhibitor (PTI) may be used to target these cells. (Polyamine transport inhibitors: Design, Synthesis and Combination therapies with Difluoromethylornithine, Aaron Muth, Jennifer Archer, Nicolette Ocampo, Meenu Madan, Luis Rodriguez, and Otto Phanstiel IV. J. Med. Chem. 2014, 57, 348-363.) All of these medical interventions will be more effective against tumors with high expression of the biomarkers ATP13A3, c-myc, and/or Cav-2 (and in some instances concomitant lower expression of Cav-1). A multitude of therapies can be enhanced by the pre-selection of patients (and/or tumors) who will best respond to the therapies identified with the tests and methods described herein.
In Table A below, therapeutic suggestions based on biomarker levels are provided in non-limiting examples. For example, high ATP13A3 and low Cav-1 levels are indicative of high polyamine transport, and consequently, treatment with DFMO-only would be less effective than a combination treatment with DFMO and PTI. Low Cav-1 and high ATP13A3 indicate high potential of that cell to escape DFMO therapy via polyamine import. Consequently effective treatment strategies would include increased PTI to overcome the increased transport activity, for example (see Table A).
While protein levels can be used for this detection purpose, other biomolecules like RNA and DNA which encode for these specific proteins can also be used. This broadens the application to include detection methods for these nucleic acid sequences which correspond to the listed protein biomarkers (e.g., ATP13A3, Cav-2, etc).
Quantitative polymerase chain reaction (QPCR) tests may be used, in another embodiment, to measure the relative gene expression of the PTS markers described herein. Both DNA (QPCR) and RNA (via reverse transcriptase and RT-PCR) can be measured by this method to provide relative mRNA and DNA levels of each of these genes in cells/tumors. This method is very sensitive and has real cost saving value as there exist commercial instruments to perform these routine experiments.
DNA microarray may also be used to quantify the relative gene expression of these PTS markers. A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot may contain picomoles (10−12 moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.
Other examples for identifying these PTS markers may include any process of assessing protein expression including flow cytometry, immunohistochemistry, immunocytochemistry, ELISA, Western blot, and immunoaffinity chromatography, HPLC, mass spectrometry, protein microarray analysis, PAGE analysis, isoelectric focusing, 2-D gel electrophoresis, or any enzymatic assay.
Other methods used to assess expression include the use of natural or artificial ligands capable of specifically binding a marker. Such ligands include antibodies, antibody complexes, conjugates, natural ligands, small molecules, nanoparticles, or any other molecular entity capable of specific binding to a marker. Antibodies may be monoclonal, polyclonal, or any antibody fragment including an Fab, F(ab)2, Fv, scFv, phage display antibody, peptibody, multispecific ligand, or any other reagent with specific binding to a marker. Ligands may be associated with a label such as a radioactive isotope or chelate thereof, dye (fluorescent or nonfluorescent,) stain, enzyme, metal, or any other substance capable of aiding a machine or a human eye from differentiating a cell expressing a marker from a cell not expressing a marker. Additionally, expression may be assessed by monomeric or multimeric ligands associated with substances capable of killing the cell. Such substances include protein or small molecule toxins, cytokines, pro-apoptotic substances, pore forming substances, radioactive isotopes, or any other substance capable of killing a cell.
Differential expression encompasses any detectable difference between the expression of a marker in one sample relative to the expression of the marker in another sample. Differential expression may be assessed by a detector, an instrument containing a detector, or by aided or unaided human eye. Examples include but are not limited to differential staining of cells in an IHC assay configured to detect a marker, differential detection of bound RNA on a microarray to which a sequence capable of binding to the marker is bound, differential results in measuring RTPCR measured in ΔCt or alternatively in the number of PCR cycles necessary to reach a particular optical density at a wavelength at which a double stranded DNA binding dye (e.g. SYBR Green) incorporates, differential results in measuring label from a reporter probe used in a real-time RTPCR reaction, differential detection of fluorescence on cells using a flow cytometer, differential intensities of bands in a Northern blot, differential intensities of bands in an RNAse protection assay, differential cell death measured by apoptotic markers, differential cell death measured by shrinkage of a tumor, or any method that allows a detection of a difference in signal between one sample or set of samples and another sample or set of samples.
The expression of the marker in a sample may be compared to a level of expression predetermined to predict the presence or absence of a particular physiological characteristic, or compared to levels of the biomarkers in normal cells. The level of expression may be derived from a single control or a set of controls. A control may be any sample with a previously determined level of expression. A control may comprise material within the sample or material from sources other than the sample. Alternatively, the expression of a marker in a sample may be compared to a control that has a level of expression predetermined to signal or not signal a cellular or physiological characteristic. This level of expression may be derived from a single source of material including the sample itself or from a set of sources. Comparison of the expression of the marker in the sample to a particular level of expression results in a prediction that the sample exhibits or does not exhibit the cellular or physiological characteristic.
Determining the level of expression that signifies a physiological or cellular characteristic may be assessed by any of a number of methods. The skilled artisan will understand that numerous methods may be used to select a level of expression for a particular marker or a plurality of markers that signifies a particular physiological or cellular characteristic. In diagnosing the presence of a disease, a threshold value may be obtained by performing the assay method on samples obtained from a population of patients having a certain type of disease (cancer for example) and from a second population of subjects that do not have the disease. In assessing disease outcome or the effect of treatment, a population of patients, all of which have, a disease such as cancer, may be followed for a period of time. After the period of time expires, the population may be divided into two or more groups. For example, the population may be divided into a first group of patients whose disease progresses to a particular endpoint and a second group of patients whose disease does not progress to the particular endpoint. Examples of endpoints include disease recurrence, death, metastasis or other states to which disease may progress. If expression of the marker in a sample is more similar to the predetermined expression of the marker in one group relative to the other group, the sample may be assigned a risk of having the same outcome as the patient group to which it is more similar.
Cancer cells include any cells derived from a tumor, neoplasm, cancer, pre-cancer, cell line, malignancy, or any other source of cells that have the potential to expand and grow to an unlimited degree. Cancer cells may be derived from naturally occurring sources or may be artificially created. Cancer cells may also be capable of invasion into other tissues and metastasis when placed into an animal host. Cancer cells further encompass any malignant cells that have invaded other tissues and/or metastasized. One or more cancer cells in the context of an organism may also be called a cancer, tumor, neoplasm, growth, malignancy, or any other term used in the art to describe cells in a cancerous state.
According to other embodiments, provided are kits designed for determining PTS markers in a biological sample. The kit may further include one or more of a buffer solution, a conjugating solution for developing the signal of interest, or a detection reagent for detecting the signal of interest, each in separate packaging, such as a container. In another example, the kit includes a plurality of size-associated marker of target nucleic acid sequences for hybridization with a detection array. The kit can also include instructions in a tangible form, such as written instructions or in a computer-readable format.
Kits comprising a primer or probe that is complementary to and specifically hybridizes to or binds to a target genes/mRNA in a sample and enzymes suitable for amplifying target genes/mRNA are provided in certain embodiments of the invention. The primer or probe may be labeled with a radioisotope, a fluorophore, a chromophore, a dye, an enzyme, or TOF carrier. In these kits, binding may be detected by in situ hybridization, PCR RT-PCR, fluorescence resonance energy transfer, chemiluminescence enzymatic signal amplification, electron dense particles magnetic particles and capacitance coupling. The probe is selected to allow the target genes/mRNA to be sequenced if wanted, or for quantitation of the respective different target genes/mRNA as compared to the wild-type sequence. These reagents in certain embodiments may comprise one or more nucleic acid probes, may be in the form of a microarray, are suitable for primer extension and can comprise controls indicative of a healthy individual.
Nucleotides may be amplified to obtain amplification products. Suitable nucleic acid amplification techniques are well known to a person of ordinary skill in the art, and include polymerase chain reaction (PCR) as for example described in Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, Inc. 1994-1998) (and incorporated herein by reference). The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR).
PCR is well known in this field and comprehensive description of this type of reaction is provided in E. van Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Springer, 2008. PCR is a powerful technique that amplifies a target DNA sequence against a background of complex DNA. If RNA is to be amplified (by PCR), it must be first transcribed into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Thereafter, the resulting cDNA is amplified by PCR. PCR is an exponential process that proceeds as long as the conditions for sustaining the reaction are acceptable. The components of the reaction are: i. pair of primers—short single strands of DNA with around 10-30 nucleotides complementary to the regions flanking the target sequence (typically the reporter nucleotide of the marker complex); ii. DNA polymerase—a thermostable enzyme that synthesizes DNA; iii. deoxyribonucleoside triphosphates (dNTPs)—to provide the nucleotides that are incorporated into the newly synthesized DNA strand; and iv. buffer—to provide the optimal chemical environment for DNA synthesis.
PCR typically involves placing these reactants in a small tube (˜10-50 μL) containing the extracted nucleic acids. The tube is placed in a thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. The standard protocol for each thermal cycle involves a denaturation phase, an annealing phase, and an extension phase. The extension phase is sometimes referred to as the primer extension phase. In addition to such three-step protocols, two-step thermal protocols can be employed, in which the annealing and extension phases are combined. The denaturation phase typically involves raising the temperature of the reaction to 90-95° C. to denature the DNA strands; in the annealing phase, the temperature is lowered to 50-60° C. for the primers to anneal; and then in the extension phase the temperature is raised to the optimal DNA polymerase activity temperature of 60-72° C. for primer extension. This process is typically repeated cyclically around 20-40 times, in one example.
There are a number of variants to the standard PCR protocol such as multiplex PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCR, and reverse-transcriptase PCR, amongst others, which have been developed for molecular diagnostics.
Multiplex PCR uses multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several experiments. Optimization of multiplex PCR is more difficult though and requires selecting primers with similar annealing temperatures, and amplicons with similar lengths and base composition to ensure the amplification efficiency of each amplicon is equivalent.
Linker-primed PCR, also known as ligation adaptor PCR, is a method used to enable nucleic acid amplification of essentially all DNA sequences in a complex DNA mixture without the need for target-specific primers. The method firstly involves digesting the target DNA population with a suitable restriction endonuclease (enzyme). Double-stranded oligonucleotide linkers (also called adaptors) with a suitable overhanging end are then ligated to the ends of target DNA fragments using a ligase enzyme. Nucleic acid amplification is subsequently performed using oligonucleotide primers which are specific for the linker sequences. In this way, all fragments of the DNA source, which are flanked by linker oligonucleotides, can be amplified.
Direct PCR describes a system whereby PCR is performed directly on a sample without any, or with minimal, nucleic acid extraction. It has long been accepted that PCR reactions are inhibited by the presence of many components of unpurified biological samples, such as the heme component in blood. Traditionally, PCR has required extensive purification of the target nucleic acid prior to preparation of the reaction mixture. With appropriate changes to the chemistry and sample concentration, however, it is possible to perform PCR with minimal DNA purification, or direct PCR. Adjustments to the PCR chemistry for direct PCR include increased buffer strength, the use of polymerases which have high activity and processivity, and additives.
Tandem PCR utilizes two distinct rounds of nucleic acid amplification to increase the probability that the correct amplicon is amplified. One form of tandem PCR is nested PCR in which two pairs of PCR primers are used to amplify a single locus in separate rounds of nucleic acid amplification. The first pair of primers hybridize to the nucleic acid sequence at regions external to the target nucleic acid sequence. The second pair of primers (nested primers) used in the second round of amplification bind within the first PCR product and produce a second PCR product containing the target nucleic acid, that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake during the first round of nucleic acid amplification, the probability is very low that it would also be amplified a second time by a second pair of primers and thus ensures specificity.
Real-time PCR, or quantitative PCR, is used to measure the quantity of a PCR product in real time. By using a fluorophore-containing probe or fluorescent dyes along with a set of standards in the reaction, it is possible to quantitate the starting amount of nucleic acid in the sample. This is particularly useful in molecular diagnostics where treatment options may differ depending on the pathogen load in the sample.
If the reporter nucleotide is an RNA sequence, it can be amplified or converted into complementary DNA (cDNA), such as by using RT-PCR. Reverse-transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. Reverse transcriptase is an enzyme that reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. It is also used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus. cDNA is DNA synthesized from a messenger RNA (mRNA) template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase.
Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which comprises a capture binding ligand. An “array location,” “addressable location,” “pad” or “site” herein means a location on the substrate that comprises a covalently attached capture binding ligand. An “array” herein means a plurality of capture binding ligands in a regular, ordered format, such as a matrix. The size of the array will depend on the composition and end use of the array. Arrays containing from about two or more different capture binding ligands to many thousands can be made. Generally, the array will comprise 3, 4, 5, 6, 7 or more types of capture binding ligands depending on the end use of the array. In the present invention, the array can include controls, replicates of the markers and the like. Exemplary ranges are from about 3 to about 50. In some embodiments, the compositions of the invention may not be in array format; that is, for some embodiments, compositions comprising a single capture ligand may be made as well. In addition, in some arrays, multiple substrates may be used, either of different or identical compositions. Thus for example, large arrays may comprise a plurality of smaller substrates.
Accordingly, in one aspect, the invention provides a composition comprising a solid support comprising a capture binding ligand for each biomarker of a biomarker panel. In various embodiments, the capture ligand is a nucleic acid. In various embodiments, the capture binding ligand is an antibody. In various embodiments, the composition further comprises a soluble binding ligand for each biomarker of a biomarker panel.
A number of different biochip array platforms as known in the art may be used. For example, the compositions and methods of the present invention can be implemented with array platforms such as GeneChip® (Affymetrix), CodeLink™ Bioarray (Amersham), Expression Array System (Applied Biosystems), SurePrint microarrays (Agilent), Sentrix® LD BeadChip or Sentrix® Array Matrix (Illumina) and Verigene (Nanosphere).
In various exemplary embodiments, detection and measurement of biomarkers utilizes colorimetric methods and systems in order to provide an indication of binding of a target analyte or target species. In colorimetric methods, the presence of a bound target species such as abiomarker will result in a change in the absorbance or transmission of light by a sample or substrate at one or more wavelengths. Detection of the absorbance or transmission of light at such wavelengths thus provides an indication of the presence of the target species.
A detection system for colorimetric methods includes any device that can be used to measure colorimetric properties as discussed above. Generally, the device is a spectrophotometer, a colorimeter or any device that measures absorbance or transmission of light at one or more wavelengths. In various embodiments, the detection system comprises a light source; a wavelength filter or monochromator; a sample container such as a cuvette or a reaction vial; a detector, such as a photoresistor, that registers transmitted light; and a display or imaging element.
In various exemplary embodiments, a ClonDiag chip platform is used for the colorimetric detection of biomarkers. In various embodiments, a ClonDiag ArrayTube (AT) is used. One unique feature of the ArrayTube is the combination of a micro probe array (the biochip) and micro reaction vial. In various embodiments, where a target sequence is a nucleic acid, detection of the target sequence is done by amplifying and biotinylating the target sequence contained in a sample and optionally digesting the amplification products. The amplification product is then allowed to hybridize with probes contained on the ClonDiag chip. A solution of a streptavidin-enzyme conjugate, such as Poly horseradish peroxidase (HRP) conjugate solution, is contacted with the ClonDiag chip. After washing, a dye solution such as o-dianisidine substrate solution is contacted with the chip. Oxidation of the dye results in precipitation that can be detected colorimetrically. Further description of the ClonDiag platform is found in Monecke S, Slickers P, Hotzel H et al., Clin Microbiol Infect 2006, 12: 718-728; Monecke S, Berger-Bächi B, Coombs C et al., Clin Microbiol Infect 2007, 13: 236-249; Monecke S, Leube I and Ehricht R, Genome Lett 2003, 2: 106-118; Monecke S and Ehricht R, Clin Microbiol Infect 2005, 11: 825-833; German Patent DE 10201463; US Publication US/2005/0064469 and ClonDiag, ArrayTube (AT) Experiment Guideline for DNA-Based Applications, version 1.2, 2007, all incorporated by reference in their entirety. One of skill in the art will appreciate that numerous other dyes that react with a peroxidase can be utilized to produce a colorimetric change, such as 3,3′,5,5′-tetramethylbenzidine (TMB). For information on specific assay protocols, see www.clondiag.com/technologies/publications.php.
In various embodiments, where a target species is a protein, the ArrayTube biochip comprises capture binding ligands such as antibodies. A sample is contacted with the biochip, and any target species present in the sample is allowed to bind to the capture binding ligand antibodies. A soluble capture binding ligand or a detection compound such as a horseradish peroxidase conjugated antibody is allowed to bind to the target species. A dye, such as TMB, is then added and allowed to react with the horseradish peroxidase, causing precipitation and a color change that is detected by a suitable detection device. Further description of protein detection using ArrayTube is found in, for example, Huelseweh B, Ehricht R and Marschall H-J, Proteomics, 2006, 6, 2972-2981; and ClonDiag, ArrayTube (AT) Experiment Guideline for Protein-Based Applications, version 1.2, 2007, all incorporated by reference in their entirety.
Transmission detection and analysis is performed with a ClonDiag AT reader instrument. Suitable reader instruments and detection devices include the ArrayTube Workstation ATS and the ATR 03.
In addition to ArrayTube, the ClonDiag ArrayStrip (AS) can be used. The ArrayStrip provides a 96-well format for high volume testing. Each ArrayStrip consists of a standard 8-well strip with a microarray integrated into the bottom of each well. Up to 12 ArrayStrips can be inserted into one microplate frame enabling the parallel multiparameter testing of up to 96 samples. The ArrayStrip can be processed using the ArrayStrip Processor ASP, which performs all liquid handling, incubation, and detection steps required in array based analysis. In various embodiments, where a protein is detected, a method of using the ArrayStrip to detect the protein comprises conditioning the AS array with buffer or blocking solution; loading of up to 96 sample solutions in the AS wells to allow for binding of the protein; 3× washing; conjugating with a secondary antibody linked to HRP; 3× washing; precipitation staining with TMB; and AS array imaging and optional data storage.
Those skilled in the art will be familiar with numerous additional immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein. See generally E. Maggio, Enzyme-Immunoassay, (CRC Press, Inc., Boca Raton, Fla., 1980); see also U.S. Pat. Nos. 4,727,022; 4,659,678; 4,376,110; 4,275,149; 4,233,402; and 4,230,767.
Antibodies
Antibodies for ATP13A3, Cav-1 and c-myc to use in the various detection assays described herein are commercially available. c-Myc Antibody (e10: (sc-40 Santa Cruz Biotechnology), ATP13A3 antibody (E-18) (sc102335, Santa Cruz Biotechnology). Caveolin-1 Antibody (7C8) (sc-53564, Santa Cruz Biotechnology). HPA-029471 is a Sigma polyclonal antibody that binds only to the intact ATP13A3 protein target. The ATP13A3 polyclonal antibody, HPA-029471, provides a band at approximately 120 kDa. In embodiments as described herein, HPA-029471 is used as the antibody to bind ATP13A3.
Sequences
Other known polyamine transport genes may also respond in this way and could be of value for tracking sample response to therapy including Oct2 and DAX.
Sequences of ATP13A3, Cav-1 c-myc and Cav-2 are provided below:
In view of the above known polypeptide sequences, probes and primers can be generated for use in the detection assays described above. Furthermore, known, commercially available primers and probe assays for the noted sequences are provided by Bio-Rad. Additionally improved antibodies can be developed using information gained from these initial products to improve target specificity.
Experiments were designed to identify the levels of expression of ATP13A3 and/or c-myc correlate with polyamine transport. Cells with high levels c-myc have high basal levels of polyamine import and are sensitive to polyamine targeting therapies. In addition, cells with high basal levels of ATP13A3 readily escape DFMO only therapy by importing exogenous polyamines and require DFMO+PTI therapy in order to be effective in reducing tumor progression. Moreover, DFMO escape via exogenous polyamine import is especially facile in cells with high basal levels of ATP13A3 and low Cav-1 expression. These cell types (samples) which can escape DFMO-only therapy require DFMO+PTI therapy to effectively reduce tumor progression. Endpoints for this DFMO+PTI combination therapy (or for DFMO-only therapy if transport is not appreciably invoked by the DFMO treated cells) can include significantly altered intracellular polyamine levels (especially reduced putrescine and/or spermidine), S-adenosylmethionine (SAM), thymidine levels and CD-47. (See, David R. Soto-Pantoja, Erica V. Stein, Chengyu Liu, Abdel G. Elkahloun, Michael L. Pendrak, Alina Nicolae, Satya P. Singh, Zuqin Nie, David Levens, Jeffrey S. Isenberg & David D. Roberts. Thrombospondin-1 Signaling through CD47 Inhibits Self-renewal by Regulating c-Myc and Other Stem Cell Transcription Factors. Scientific Reports 2013, 3, 1673; doi:10.1038/srep01673). Cells with high levels of Cav-1 and low levels of Cav-2 are found in some of the most resistant cancer cells (i.e., melanomas), whereas cells with low Cav-1 and high Cav-2 levels are particularly sensitive to compounds A and B which are described in greater detail below. Compound A is a cytotoxic polyamine transport ligand that outcompetes native polyamines for cell entry (see
The inventors used Polyamine Transport Assays to show, for example, that cells like CHO, L3.6pl and PanO2 cells which have high polyamine transport activity also have high c-myc expression and are susceptible to therapies which target polyamine transport or metabolism. Additionally, cells which had high ATP13A3 expression were shown to readily import spermidine to escape DFMO treatment. Caveolin-1 by itself did not track well with basal polyamine uptake but low Cav-1 and high ATP13A3 were hallmarks of cells which readily imported spermidine to escape DFMO treatment. In some examples described below and shown in the FIGS, cells treated with compound A and/or compound B reduced in viability, whereas cells treated with compound A and varying quantities of exogenous Spermidine (Spd) had varying levels of viability indicating that compound A, competes with Spd for cell entry, for example. This confirms compound A is a polyamine transport targeting compound (see
Polyamine transport inhibitor (PTI) Assay 1 was performed to block the uptake of a cytotoxic polyamine probe (Ant44) by a polyamine transport inhibitor (PTI) as shown in the diagram of
A successful PTI will block the entry of the cytotoxic Ant44 probe into cells and result in significantly enhanced cell viability versus the Ant44 treated control. The inventors have found that a non toxic PTI rescues cells from the toxic Ant44 probe. Cells that do not have active transport will be much less sensitive to the Ant44 probe, i.e., CHO vs CHO-MG cells. See “N1-Substituent Effects in the Selective Delivery of Polyamine-Conjugates into Cells Containing Active Polyamine Transporters” Gardner, R. A.; Delcros, J-G.; Konate, F.*; Breitbeil III, F.; Martin, B.; Sigman, M.; Huang, M.; Phanstiel IV, O. J. Med. Chem. 2004, 47, 6055-6069. Using this assay 1 seen in
The results of the assay 1 can be seen in the table and graph of
Assay 2 was used to screen the GW5074 PTI in L3.6pl cells which has high expression of c-myc and ATP13A3. Using L3.6 pl cells, 500 cells/well, incubated for 48 h at 37° C. along with 250 μM aminoguanidine (AG, to inhibit the amine oxidases in the fetal bovine serum which could degrade the spermidine added), the inventors showed that DFMO alone and in combination with the GW5074 polyamine transport inhibitor is effective against L3.6pl cells containing high expression of the two biomarkers.
ATP13A3 is the human analogue of CATP-5, a gene shown to be involved in polyamine transport in C. elegans (see Caenorhabditis elegans P5B-type ATPase CATP-5 operates in polyamine transport and is crucial for norspermidine-mediated suppression of RNA interference. Heinick, A. Urban, K.; Roth, S.; Spies, D.; Nunes, F.; Phanstiel IV, O.; Liebau, E. Lüersen, K. FASEB Journal 2010, 24, 206-217.)
The results provided herein supports the finding that ATP13A3 plays an important role in human polyamine transport because only those cell lines which had significant expression of this marker were shown to be rescued by exogenous spermidine indicating this marker as a biomarker of DFMO escape via polyamine import. This marker was also shown to be significantly upregulated in L3.6pl cells in the presence of DFMO (72 h incubation) and modulated (lowered) by exogenous spermidine levels in the presence of DFMO (
The results identified in the in vitro studies provide that high c-myc and ATP13A3 expression levels were good indications of cell lines that had high polyamine import activity (high Vmax) and were rescue-able by exogenous spermidine (in the presence of DFMO). These cells were still sensitive to blockade of polyamine import but required higher doses of PTI to be effective. PTIs 6a and 6b (
These experiments demonstrate that cell lines like L3.6pl and PanO2 with high polyamine transport and high expression of c-myc and ATP13A3 are sensitive to polyamine targeting therapies in vitro. The extension of these findings is that cells which have high c-myc and ATP13A3 expression will be sensitive to the therapy described herein. These therapies were evaluated in vivo. The PTI, GW5074 compound (MW=520 g/mol), was used at 1 mg/kg and compared with the polyamine based PTI 6b at an equimolar dose (1.8 mg/kg; MW=962.19 g/mol) in three animal studies.
The first set of experiments was in nude mice (immune compromised) using human L3.6pl pancreatic cancer cells which have high ATP13A3 and high c-myc expression and high Vmax. The An experiment was conducted to look at combination therapies of the GW5074 compound with DFMO (an inhibitor of ODC),
An additional animal study used immune competent C57Bl/6 mice and the PanO2 mouse pancreatic cancer cell line, (
Table 1 compares unmethylated Compound A (containing primary amine termini), and N-methylated Compound A (containing N-methyl termini). Methylated Compound A (having terminal N-methyl groups) was found to be metabolically stable to amine oxidases. Due to its metabolic stability, methylated Compound A was found to outlast unmethylated Compound A, particularly in the presence of amine oxidases, in a non-limiting embodiment. As shown in Table 1 below, without the protective aminoguanidine (AG) amine oxidase inhibitor, unmethylated Compound A molecules were less selective and less metabolically stable when compared to the sample containing AG (used to inhibit the degradative action of amine oxidases on the primary amine present in the unmethylated compound A). However, N-methylated Compound A was highly selective in targeting CHO cells with active PTS (ratio >2200). This is due to the presence of the terminal N-methyl groups in A.
Table 2 below provides a comparison of methylated vs. unmethylated Compound A in selective targeting of melanoma cells.
aA high CHO-MG*/CHO IC50 ratio denotes high selectivity in killing cells with an active polyamine transport system, PTS. Aminoguanidine (AG) is added to cell culture to inhibit the action of amine oxidases found in calf serum. When this protective AG agent is removed, compound A retains its high selectivity in targeting CHO cells with active PTS (ratio >2200). This is due to the presence of the terminal N-methyl groups in A. In contrast when these N—Me groups are not present (top entry) and AG is not added, there is a dramatic loss in selectivity (IC50 ratio changes from >4545 to 9.5). Thus, we have designed around this issue via N—Me groups.
ba high IC50 ratio of MALME-3/MALME-3M denotes selective targeting of melanoma cells. Both compound demonstrate selective killing of melanomas in the presence of aminoguanidine (1 mM), which was added to allow the comparison as the top entry is unstable without AG present (See Table 1). Compound A (which has N-methyl groups) provides specific targeting ability and enhanced stability which obviates the need to add an amine oxidase inhibitor.
Table 3 shows a comparison of the different protein markers and the Vmax and degree of Spermidine rescue in a side by side comparison.
cEntries represent protein expression (normalized by beta-actin expression) and expressed in relative %, where the expression level of each protein found in L3.6pl cells was set to 100%. To illustrate how the relative protein expression patterns relate to the polyamine transport properties of the human cell lines, the Vmax value for 3H-Spd import (nmoles Spd/mg protein/min) and the Spd rescue index observed with DFMO-treated cells are re-listed for comparisons. Cell lines with high ATP13A3 and high c-myc had high Vmax and high Spd rescue outcomes, which indicates that these cell types will circumvent DFMO via increased polyamine import and would be good candidates to be treated with DFMO + PTI therapy.
In embodiments provided herein, a diagnostic and therapeutic treatment method for treating cancer cells in a patient are provided including, determining a level of Cav-1 and a level of Cav-2 in a cell sample from the patient, comparing the levels of Cav-1 and Cav-2 in the cell sample to the levels of Cav-1 and Cav-2 a control sample or to standard values of Cav-1 and Cav-2, wherein a lower level of Cav-1 and a higher level of Cav-2 in the cell sample relative to the control sample or to the standard values is indicative of cancer cells that are susceptible to treatment with a polyamine transport inhibitor (PTI), and treating the cancer cells with the PTI, DFMO, or a combination thereof.
In one non-limiting embodiment, the method is provided wherein the cancer cells are melanoma cells. In a further non-limiting embodiment, the melanoma cells are MALME-3M cells. In other non-limiting embodiments, the melanoma cells include: M14, MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, or UACC-62 cells.
In one particular non-limiting embodiment, the method may be provided wherein the PTI includes Compound A, Compound B, or a combination thereof.
In another embodiment, a method of detecting cancer cells susceptible to treatment with a polyamine transport inhibitor (PTI) in a patient is provided. The method includes determining a level of ATP13A3 and a level of Cav-1 in a cell sample from the patient and comparing the levels of ATP13A3 and Cav-1 in the cell sample to the levels of ATP13A3 and Cav-1 in a control sample or standard values of ATP13A3 and Cav-1, wherein higher level of ATP13A3 and a lower level of Cav-1 in the sample relative to the control sample or standard values is indicative of cancer cells that are susceptible to treatment with a PTI. The method may further include determining a level of Cav-2 in the cell sample and comparing the level of Cav-2 in the cell sample to the level of Cav-2 in a control sample or to a standard value of Cav-1, wherein a higher level of Cav-2 in the cell sample relative to the control sample or the standard value for Cav-1 is indicative of cancer cells in the patient that are susceptible to treatment with a PTI. The method may further include treating the cancer cells with a polyamine biosynthesis inhibitor. In one non-limiting embodiment, the polyamine biosynthesis inhibitor comprises DFMO (difluoromethylornithine).
In other embodiments, a method for detecting cancer cells susceptible to treatment with a polyamine transport inhibitor (PTI) is provided. The method includes receiving a cell sample from a patient, presenting a first reagent and a second reagent to the cell sample to detect differential levels of Cav-1 and Cav-2, or nucleic acid sequences encoding the same, in the cell sample, wherein the first reagent is specific to Cav-1, the second reagent is specific to Cav-2, under conditions to detect binding of the first and second reagents to Cav-1 and Cav-2, respectively, or hybridization of the first and second reagents to the nucleic acid sequences, and determining that cancer cells in the patient are treatable with a polyamine transport inhibitor if a higher level of Cav-2 and a lower level of Cav-1, or nucleic acid sequences encoding same, than in normal cells was determined to be present in the cell sample.
Another embodiment includes a method of treating cancer cells susceptible to treatment with a polyamine transport inhibitor (PTI). The method includes receiving a cell sample from a patient, presenting a first reagent and a second reagent to the cell sample to detect differential levels of Cav-1 and Cav-2, or nucleic acid sequences encoding the same, in the cell sample, wherein the first reagent is specific to Cav-1, the second reagent is specific to Cav-2, under conditions to detect binding of the first and second reagents to Cav-1 and Cav-2, respectively, or hybridization of the first and second reagents to the nucleic acid sequences, determining that cancer cells in the patient are treatable with a polyamine transport inhibitor if a higher level of Cav-2 and a lower level of Cav-1, or nucleic acid sequences encoding same, than in normal cells was determined to be present in the cell sample, exposing the cancer cells to the polyamine transport inhibitor to cause a reduction in viability of the cancer cells.
In still another embodiment, an ELISA microarray testing kit for detecting cancer cells susceptible to treatment with a polyamine transport inhibitor (PTI) is provided. The testing kit may include a component comprising a substrate to which ATP13A3 and Cav-1 in a cell sample can bind, wherein the substrate comprises an array of individual antibody loci comprising antibodies specific to ATP13A3 and/or Cav-1, one or more reagents configured to detect ATP13A3 and Cav-1 bound to the substrate, wherein the detection of a high level of ATP13A3 and a low level of Cav-1 in the cell sample when compared to a control is indicative of cancer cells susceptible to treatment with a polyamine transport inhibitor. The ELISA microarray testing kit may further include a substrate to which c-myc in the cell sample can bind, wherein the substrate comprises individual antibody loci comprising antibodies specific to c-myc, the microarray testing kit further including one or more reagents configured to detect c-myc bound to the substrate, wherein the detection of a high level of c-myc in the cell sample when compared to a control is indicative of cancer cells susceptible to treatment with a polyamine transport inhibitor.
The ELISA microarray testing kit may further include a substrate to which Cav-2 in the cell sample can bind, wherein the substrate comprises individual antibody loci comprising antibodies specific to Cav-2, the microarray testing kit further including one or more reagents configured to detect c-myc bound to the substrate, wherein the detection of a high level of Cav-2 in the cell sample when compared to a control is indicative of cancer cells susceptible to treatment with a polyamine transport inhibitor, in a non-limiting example.
Primary antibodies covalently attached to respective fluorophores can be used to directly measure the relative expression levels of these biomarkers in histological samples either separately or together to create a mosaic readout of differential protein expression of the key biomarkers. Accordingly, in another embodiment, specific antibodies for ATP13A3, c-myc, cav-1 and Cav-2 are either covalently linked to different fluorophores (different colors) or detected by differently colored fluorescent secondary antibodies could be added to a histological sample and show a colored panel readout of the relative expression of each protein by immunocytochemistry, for example. These methods are further described in Controls for Immunocytochemistry: An Update, Burry, Richard W., Journal of Histochemistry & Cytochemistry 59(1) 6-12, 2011, which is incorporated by reference herein. This would provide a snapshot of the relative protein expression patterns of each of the biomarkers in the tissue sample. Another related embodiment pertains to a method that involves subjecting a tissue sample to a primary antibodyA that detects ATP13A3, a primary antibodyB that detects c-myc, a primary antibodyC that detects Cav-1, and a primary antibodyD that detects Cav-2 in a tissue sample, wherein upon subjecting the tissue sample to the solution, the primary antibodiesA-D bind specifically to proteins ATP13A3, c-myc, Cav-1, and Cav-2, respectively, in the sample, wherein the primary antibodiesA-D are each individually linked to different colored fluorophores Upon subjecting the tissue sample to the antibodies, the relative levels of the noted proteins can be visualized to provide a color mosaic that will indicate whether the tissue sample includes cells having an active PTA (polyamine transport activity).
Various polyamine biosynthesis inhibitors and PTI's are described herein, including, but not limited to, DFMO, N1-(9-anthrylmethyl)-homospermidine (Ant44), MeN44Nap44NMe or GW5074. DFMO is a polyamine biosynthesis inhibitor. Polyamine transport inhibitors (PTI's) include, for example, compounds which inhibit the transport of polyamines into the cell, and compounds having cytotoxic properties which inhibit transport of polyamines into the cells, such as Compound A as described herein, in a non-limiting example. Polyamine transport inhibitors therefore include Compound A, Compound B in addition to other PTI's which may be used to inhibit polyamine transport in cells identified by methods described herein as being susceptible to polyamine transport inhibition. Additional PTI's which may be used can be found in the following: Polyamine transport inhibitors: Design, Synthesis and Combination therapies with Difluoromethylornithine. Aaron Muth, Jennifer Archer, Nicolette Ocampo, Meenu Madan, Luis Rodriguez, and Otto Phanstiel IV. J. Med. Chem. 2014, 57, 348-363; “Fluorescent Cytotoxic Compounds Specific for the Cellular Polyamine Transport System.” U.S. Pat. No. 8,410,311 (Apr. 2, 2013); “Polyamine Transporter Selective Compounds as Anti-Cancer Agents.” Otto Phanstiel, U.S. Pat. No. 8,497,398 (Jul. 30, 2013); “Polyamine Transport Selective Therapeutic Agents with Enhanced Stability.” Otto Phanstiel IV and Aaron Muth, World Patent, WO 2013148230 A1, Oct. 3, 2013; “Fluorescent Cytotoxic Compounds Specific for the Cellular Polyamine Transport System”, Otto Phanstiel IV, U.S. Patent 2013-0337494-A1 (Dec. 19, 2013); “Polyamine Transporter Selective Compounds as Anticancer agents”, Otto Phanstiel, U.S. Patent US-2014-0057989-A1 (Feb. 27, 2014); “Polyamine Transport Inhibitors as Novel Therapeutics”, US-2012-0172449-A1, Pub Date: Jul. 5, 2012; see also, U.S. patent application Ser. No. 14/388,856, U.S. Patent Application No. 62/097,896, U.S. patent application Ser. No. 12/113,540, U.S. patent application Ser. No. 13/953,667, U.S. patent application Ser. No. 14/862,907, U.S. patent application Ser. No. 13/379,191, U.S. patent application Ser. No. 12/113,540, U.S. patent application Ser. No. 12/754,962, U.S. patent application Ser. No. 13/835,708, and US Patent Publication No. 2012/01725499A1, all of which are herein incorporated by reference to the extent they relate to polyamine transport inhibitors and polyamine biosynthesis inhibitors.
It should be borne in mind that all patents, patent applications, patent publications, technical publications, scientific publications, and other references referenced herein are hereby incorporated by reference in this application in order to more fully describe the state of the art to which the present invention pertains. The teachings of all patents and other references cited herein are incorporated herein by reference in their entirety to the extent they are not inconsistent with the teachings herein.
Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed.
It is important to an understanding of the present invention to note that all technical and scientific terms used herein, unless defined herein, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. The techniques employed herein are also those that are known to one of ordinary skill in the art, unless stated otherwise.
While a number of embodiments of the present invention have been shown and described herein in the present context, such embodiments are provided by way of example only, and not of limitation. Numerous variations, changes and substitutions will occur to those of skill in the art without materially departing from the invention herein. For example, the present invention need not be limited to best mode disclosed herein, since other applications can equally benefit from the teachings of the present invention. Also, in the claims, means-plus-function and step-plus-function clauses are intended to cover the structures and acts, respectively, described herein as performing the recited function and not only structural equivalents or act equivalents, but also equivalent structures or equivalent acts, respectively. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims, in accordance with relevant law as to their interpretation.
| Number | Date | Country | |
|---|---|---|---|
| 62134895 | Mar 2015 | US |
