Patent Application
20130345164
There is a need for drugs and reagents for treating various disorders. For example, effective chemotherapy for treating cancer is a continuing goal in the oncology field. Many potential anticancer treatments have been developed. Yet, it has become increasingly clear that specific tumor types respond differently to the many potential treatments.
This invention is based, at least in part, on an unexpected discovery of a new methodology for determining whether a test treatment is effective for treating a disorder.
Accordingly, one aspect of this invention features a method for constructing a model for determining whether a test treatment is effective for treating a disorder. In particular, the method is suitable for determining whether a test treatment is effective for treating a disorder that has a specific feature. The method includes steps of:
Score=the Responsive Fraction×the Non-Responsive Fraction,
In the method, the test treatment can be any treatments, including one selected from the group consisting of a test compound, a microorganism (e.g., a virus), a radiation, a force, a field, a thermal energy, and a lack of a material. Examples of the test compound include, but are not limited to, a small molecule compound, a peptide, a polypeptide, a protein (e.g., an antibody), a nucleic acid, a carbohydrate, or a lipid.
In one embodiment, the test treatment is a test Compoundj and, in that case, the responsiveness of a case cell line or a control cell line in the panel to test Compoundj is obtained by a process having steps of: obtaining an IC50 value of said test Compoundj against each of the cell lines in the panel of cell lines; and calculating a Relative Sensitivity (RS) of said test Compoundj against a Cell Linei over all of the cell lines in the panel according to Formula II-1:
RSij=log10IC50ij−Average(log10IC50)j,
In Formula II-1, RSij represents the RS of Compoundj against Cell Linei; IC50ij represents the log10IC50 of test Compoundj against Cell Linei; Average(log10IC50)j represents the average IC50 of test Compoundj against all of the cell lines in the panel, i=1, 2, . . . , n; and j=1, 2, . . . m. Cell Linei is determined to be (a) responsive to test Compoundj if RSij is less than 2σ, or (b) non-responsive to test Compoundj if RSij is no less than 2σ. The IC50 value is the concentration of a compound that is necessary to inhibit by 50% the growth of treated cells relative to untreated cells; σ represents one standard deviation; 2σ represents two standard deviations.
In another embodiment, the test treatment is ionizing radiation and the responsiveness of a case cell line or a control cell line in the panel to ionizing radiation is obtained by a process having steps of obtaining an ID50 value of the ionizing radiation (i.e., the dose of the radiation that is necessary to inhibit by 50% the growth of treated cells relative to untreated cells) against each of the cell lines in the panel of cell lines; and calculating a Relative Sensitivity (RS) of ionizing radiation against a Cell Linei in the panel over all of the cell lines in the panel according to Formula II-2:
RSi=ID50i−Average(ID50),
In Formula II-2, RSi represents the RS of the ionizing radiation against Cell Linei; ID50i represents the ID50 of the ionizing radiation against Cell Linei; Average(ID50) represents the average ID50 of the ionizing radiation against all of the cell lines in the panel, and i=1, 2, . . . , n. The Cell Linei is determined to be (a) responsive to the ionizing radiation if RSi is less than 2σ, or (b) non-responsive to the ionizing radiation if RSi is no less than 2σ. In certain embodiments, n is at least 2, 3, 5, 10, 15, 20, 30, 50, 60, or 100.
In a second aspect, the invention features a method of determining whether a test treatment is effective for treating a disorder. The method includes steps of obtaining a Score or P-value of the test treatment using the model constructed by the method described above. A Score value for a treatment, if no less than or greater than 0.25 (e.g., 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, or 0.60) indicates that the treatment should be effective in treating the disorder. The P-value, if no larger than or less than 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, 0.005, or, 0.001), also indicates that the test treatment is effective for treating the disorder. For example, the P-value, if no larger than 0.05, or/and the Score, if no less than 0.5, indicates that the test treatment is effective for treating the disorder.
In each of the above-described methods, the record can have one or more of the following values:
In one embodiment, the disorder is a cellular proliferative disorder. A cellular proliferative disorder refers to a disorder characterized by uncontrolled, autonomous cell growth, including malignant and non-malignant growth. Examples include various cancers. The feature of the disorder can be a mutation in a gene, a gene copy alteration, overexpression or loss of a cellular gene, an alteration in a signal transduction pathway, or a resistance to a drug. In one example, the gene is an oncogene or a tumor suppressor gene, including, but not limited to, the BRAF gene, the p53 gene, the PTEN gene, or the RAS gene.
In a third aspect, the invention features a machine-readable medium for carrying out the methods described above. The machine-readable medium has machine-readable instructions encoded thereon which, when executed by a processor, cause a machine having or linked to the processor to execute each of the methods. This invention also features a computer system having the machine-readable medium and a user interface capable of receiving the above-mentioned data and displaying the above-mentioned record.
In a fourth aspect, the invention features a machine-readable medium on which is stored a database capable of configuring a computer to respond to queries based on a plurality of records or values belonging to the database. Each of the records has one or more of the following values:
In any of the above-mentioned machine-readable media, the test treatment is selected from the group consisting of a test compound, a microorganism, a radiation, a force, a field, a thermal energy, and a lack of a material. In any of the above-mentioned computer systems, the user interface is capable of displaying the record in the format of a relative responding histogram (RRH) or of a response graph (RG). In this invention, the above-mentioned n or m can be at least 2, 3, 5, 10, 15, 20, 30, 50, 60, or 100 in certain embodiments.
In a fifth aspect, the invention features a method for treating a cellular proliferative disorder in a subject. The method includes administering to a subject in need thereof an effective amount of a compound selected from the group consisting of those compounds in the four sub-groups listed below or a pharmaceutically acceptable salt of the compound.
1. Compounds specific for disorders characterized by a mutation in the p53 gene: NSC319726, NSC319725, NSC328784, NSC612941, NSC155694, NSC694266, and NSC93739.
2. Compounds specific for disorders characterized by a V600E mutation in the BRAF gene: NSC656238, NSC682449, NSC690432, NSC741078, NSC706829, NSC669995, NSC361127, NSC263637, and NSC354462.
3. Compounds specific for disorders characterized by a mutation in the KRAS gene: NSC613327, NSC146268, NSC740, NSC696558, NSC666787, NSC682306, NSC117356, NSC739, NSC680417, NSC363981, and NSC266046.
4. Compounds specific for disorders characterized by a mutation in the PTEN gene: NSC706744, NSC735493, NSC734294, NSC681640, NSC681645, NSC681634, NSC681638, NSC606499, NSC606498, NSC606497, NSC364830, NSC639174, NSC620256, NSC363979, NSC363980, NSC363981, NSC378734, NSC378735, NSC378727, NSC355447, NSC368891, and NSC48006.
In a preferred embodiment, the cellular proliferative disorder is characterized by a mutation in a gene, such as the p53 gene, the RAS gene, the BRAF gene, or the PTEN gene. In one example, the cellular proliferative disorder is characterized by a mutation in the p53 gene and the compound is selected from the group consisting of those in Subgroup 1. In another example, the cellular proliferative disorder is characterized by a mutation in the BRAF gene and the compound is selected from the group consisting of those in Subgroup 2. In a third example, the cellular proliferative disorder is characterized by a mutation in the KRAS gene and the compound is selected from the group consisting of those in Subgroup 3. In a further example, the cellular proliferative disorder is characterized by a mutation in the PTEN gene and the compound is selected from the group consisting of those in Subgroup 4.
The invention also features use of a compound selected from the group consisting of NSC669995, NSC682449, NSC656238, NSC612941, NSC155694, NSC319726, NSC319725, NSC694266, and NSC93739, or a pharmaceutically acceptable salt of the compound, for the treatment of a cellular proliferative disorder. The invention further features use of a compound selected from the group consisting of NSC669995, NSC682449, NSC656238, NSC612941, NSC155694, NSC319726, NSC319725, NSC694266, and NSC93739, or a pharmaceutically acceptable salt of the compound, in the manufacture of a medicament for the treatment of a cellular proliferative disorder.
The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.
a and 4b are a set of diagrams showing structures of six compounds that were identified as candidates for treating cancer and GI50s of the six compounds against various tumor cell lines.
a-b are a set of diagrams showing (a) chemical identities and structure scheme of two thiosemicarbazone family compounds, NSC319725 and NSC319726, identified by a method disclosed in the application, (b) clustering of cell lines with sensitivity to the two compounds, based on computational analysis of the NCI60 database, and (c) validation of the two compounds in mouse embryonic fibroblast (MEF) cells with individual p53 mutations, using 96-well MTS assay.
a-c are a set of diagrams showing allele specificity of p53 R175 mutations to NSC319726, which is demonstrated with (a) sensitivity to NSC319726 of MEF cells with endogenous p53R172H mutation (equivalent to human p53R175H mutation), (b) sensitivity to of NSC319726 of human cells with wild type p53 (H460, HCT116, MCF7 and WI38) and in p53-null cell line (SKOV3), and (c) sensitivity to NSC319726 of human tumor cells with various p53 mutations.
a-c are a set of diagrams showing induction of p53R175 mutation-dependent apoptosis by NSC319726 in MEF cells with exogenous p53 mutations (b) and in three human ovarian carcinoma cell lines with p53 mutations (TOV112D (p53R175H), OVCAR3 (p53R248W) and SKOV3 (p53-null)) (b), and p53-dependent growth inhibition in TOV112D cells where siRNA to p53 abrogated the growth inhibition by NSC319726 (c).
a-c are a set of photographs and diagrams showing conformational change induced by NSC319726, but not NSC319725, in p53R175H mutant protein.
a-e are a set of photographs and diagrams showing functional recovery of the p53 protein upon NSC319726 treatment: (a) Induction of p21 protein in TOV112D cells but not in SKOV3 upon NSC319726 treatment, shown in Western blot; (b) Induction of the p53 mutant protein binding to the 20-bp, p53-recognition element (p53RE) in the p21 promoter region, shown in luciferase activity assay; (c) Induction of the p53-target genes (p21, PUMA and MDM2) as shown in quantitative real-time PCR; (d) Microarray analysis of the p53 target genes in the TOV112D cells with treatment of NSC319725 and NSC319726. Each treatment had three independent repeats (labeled as 1, 2, and 3); (e) Comparison of the p53 expression signatures of 319726 treated cells with that of p53 wild type, p53 null and p53R175H cell lines in response to gamma-irradiation.
a-c are a set of diagrams, a set of photographs, and a table showing in-vivo evidence of p53 mutant synthetic lethality in response to 319726 treatment: a) Toxicity assays performed in p53 wild type, p53 null, p53+/R172H and p53R172H/R172H mice indicating dependence of toxicity on TP53R175H expression; b) Cleaved caspase-3 immunostaining in spleen and thymus tissues of NSC319726 treated p53 wild type and p53R172H/R172H mice demonstrating increased apoptosis in the p53R172H/R172H mice, and c) Comparative expression of p53 targets by quantitative PCR across various tissues of NSC319726 treated p53 wild type and p53R172H/R172H mice indicating significant upregulation in several tissues of the p53R172H/R172H mice.
This invention relates to methods and systems for identifying candidate treatments for treating various disorders, e.g., treatments for cancer with increased activity in specific tumor types. The invention also relates to compositions and methods for treating cellular proliferative disorders.
In one aspect, this application provides a highly efficient analysis methodology for identifying candidate treatments for treating various disorders. The methodology is based, at least in part, on a rigorous statistical definition of good response and the use of a Score function to rank test treatments (e.g., compounds) according to their selective activity in a specific group of disease cell lines (e.g., tumor-derived cell lines). The methodology involves processing multi-dimensional input raw data sets across a large number of studies and experiments from diverse technologies as well as different biological and chemical assays, data types, and organisms. For example, the above-mentioned data of responsiveness includes one or more values identifying, among others, a disorder, a test treatment, a panel of cell lines, each of the cell lines in the panel, responsiveness of each of the cell lines in the panel to the test treatment, a feature characterized of the disorder, the plurality of case cell lines/Case Group, and the plurality of control cell lines/Control Group. The methodology allows one to combine orthogonal types of data and available public knowledge to identify treatments for various disorders, such as cellular proliferative disorders.
In one example, application of this methodology proceeds by, first, identifying a group of cell lines representing the specified tumor type and an appropriate control, and, second, ranking the treatments in the screen according to their specific anticancer activity in one of the groups but not in the other. In one embodiment, the input multi-dimensional raw data sets include responsiveness of a panel of tumor derived cell lines and control/normal cell lines to a panel of potential anticancer treatments. The responsiveness can be measured using different protocols, including but not limited to, growth inhibition assays.
Data from individual tumors/cell lines or treatments may be correlated with other orthogonal data and public information, e.g., that available from National Cancer Institute (NCI), such as the NCI60 screen data, which reported the IC50s of 47,624 compounds against 60 tumor derived cell lines (NCI60 screen, October 2009 release, which is available at dtp.nci.nih.gov/docs/cancer/cancer_data.html).
As more of such data becomes publicly available, the data can be correlated with previous findings on relevant cell lines, gene mutations, and treatments. For example, the multi-dimensional data sets can include data of tumor cell lines, related gene mutations, related gene methylations, and related chromosomal aberrations. The multi-dimensional data sets can include data from studies of various new treatments on the cells or related tumors. The methodology disclosed in this application allows the data sets to be combined and used to elucidate tumors' sensitivity or resistance to the treatments.
Shown in
First, input IC50 data are received (102). Each data point represents the IC50 value of a test compound against each of the cell lines in the panel of cell lines. The IC50 values can be obtained via assays known in the art. Alternatively, one can obtain such data from publicly available databases, such as that maintained as NCI, in the manner shown in the working example below.
Then, responsiveness data of the cell lines to the compounds are then calculated (104). For example, a Relative Sensitivity (RS) of a test Treatmentj (e.g., Compoundj) against a Cell Linei over all of the cell lines in the panel can be calculated according to Formula II-1 mentioned above:
RSij=log10IC50ij−Average(log10IC50)j.
Generally, the data is given by a matrix reporting the response sensitivity of a panel of tumor-derived cell lines to a panel of test treatments. This matrix is transformed into Relative Sensitivities (RSes), by reporting the sensitivity measurement for each treatment relative to its average value across the entire panel of cell lines. For each pair of treatment and cell line, one obtains a value quantifying the relative response sensitivity of that cell line to that treatment.
In Formula II-1, RSij represents the RS of Treatmentj (e.g., Compoundj) against Cell Linei: IC50ij represents the IC50 of Treatmentj (e.g., Compoundj) against Cell Linei; and Average(log10IC50)j represents the average IC50 of Treatmentj (e.g., Compoundj) against all of the cell lines in the panel. The variables “i” and “j” are used to designate the cell lines and the treatments (e.g., compounds), respectively, where i=1, 2, . . . , n; and j=1, 2, . . . , m. Cell Linei is determined (a) to be responsive (or have a good response) to Treatmentj if RSij is less than two standard deviations (i.e., −2σ), or (b) to be non-responsive (or have a bad response) if RSij is no less than two standard deviations.
Once RSes are calculated, one can evaluate the relative sensitivities for all pairs of treatment-cell line by constructing a Relative Responding Histogram (RRH), a histogram of relative response sensitivities across all treatments and cell lines (106). RRH is informative about whether the right variable is being used to quantify the response sensitivity. Ideally the RRH should follow an approximately symmetric distribution, centered at zero and with fast decaying left and right tails. In such an ideal scenario, good responses can be clearly distinguished from bad responses, as both are located at the opposite tails with the typical or expected behavior in between. If this is not the case, some transformation should be applied to the sensitivity measurement such that the resulting RRH satisfy those desired properties. For example, for small molecule compounds, a typical response sensitivity measurement is the IC50, the concentration necessary to inhibit the growth of treated cells 50% relative to untreated controls. The histogram of the IC50s is, however, often squeezed towards the zero concentration and has a right tail only. This is because some cell lines are highly insensitive to treatments of some small molecules, with IC50s above the micro molar range. Thus, when plotting all measurements together, responses between nano- and micro-molar IC50s are practically indistinguishable. A way around this problem is to work with the log10IC50 and define the relative sensitivity as RS=log10IC50-average(log10IC50) as shown above. With the RSes defined in this way, a symmetric RRH is obtained, centered at zero, with a left tail for the good responses (low IC50) and a right tail for the bad responses (high IC50s) (Vazquez, A., BMC Syst Biol, 2009. 3: p. 81). In the following it is assumed that the good and bad responses are located at the left and the right tails, respectively.
Shown in
In addition to constructing RRH, one can also construct a Response Graph (RG) to evaluate and visualize the relative sensitivities for all pairs of treatments and cell lines (108). Using the definition of good response provided above, the treatment-cell line response graph (RG) is constructed with a class of nodes representing treatments, another class of nodes representing cell lines, and a line connecting a treatment to a cell lines whenever the latter has a good response to the former (Vazquez, A., BMC Syst Biol, 2009. 3: p. 81). The RG represents a discrete version of the response sensitivity matrix. It has the advantage of distinguishing good responses from the rest of the data.
Shown in
To use the methodology described herein for determining whether a test treatment (e.g., a test compound) is effective for treating a disorder, one can obtain data of responsiveness of a panel of cell lines for that disorder to a test treatment. Specifically, one can divide the panel into two groups based on whether a feature characterized of the disorder is shared by various cell lines: (1) a Case Group consisting of a plurality of case cell lines each of which has a feature characterized of the disorder and (2) a Control Group consisting of a plurality of control cell lines each of which lacks the feature (110).
One can then calculate Scores and P-values for the compounds as shown in
Score=the Responsive Fraction×the Non-Responsive Fraction.
In other words, the Score is the product of the fraction of good responders in the Case Group and the fraction of non-good responders in the Control Group. This Score function ranks higher those treatments with an enrichment of good responders in the Case Group, while simultaneously having a depletion of good responders (thus an enrichment of not good responders) in the Control Group. The statistical significance, P-value, for enrichment of responsive cell lines in the Case Group relative to the Control Group can be obtained using Fischer's exact test.
The value of the Score of a test treatment indicates effectiveness of the test treatment for treating the disorder and the P-value quantifies statistical significance. For example, a Score value for a treatment, if no less than or greater than 0.25 (e.g., 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, or 0.60) indicates that the treatment should be effective in treating the disorder. The P-value, if no larger than 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, 0.005, or, 0.001), also indicates that the test treatment is effective for treating the disorder. Scores for all the treatments can be obtained in the same way and ranked according to their values. In a preferred embodiment, the Scores of all treatments are ranked, starting at 1, in a decreasing order (114). If necessary, a validation process (116) can be further conducted.
As a proof of principles, compounds with specific activity against tumor cell lines carrying a mutation in the tumor suppressor gene p53 or the oncogene BRAF were evaluated using the methodology disclosed herein and their actual specificity demonstrated. As shown in the examples below, the methodology successfully identified effective compounds. Accordingly, the methodology can be used for identifying a cancer treatment having a better response for a specific cell type which comprises identifying both a group of cell lines representing the specified cell type and a group of cell lines which constitute an appropriate control group and ranking an anticancer compound according to its selective activity in the specific group of derived cell lines and its inactivity in the control group. The methodology can accelerate the process of discovery of treatments for personalized anticancer treatment.
The methodology disclosed in this application has a wide range of applications. In addition to search of treatments with specific activity in cells carrying a mutation in B-Raf and p53, this methodology can be applied to identify candidate compounds with an increased activity in tumors carrying other somatic mutations in cancer related genes. In particular, this is useful for somatic mutations with high prevalence in tumors. Examples include mutations in the PTEN and KRAS genes. Within the above-mentioned NCI60 screen, there are 12 cell lines with a PTEN mutation or deletion and 11 cell lines with a KRAS mutation. Thus, this methodology can be applied to find compounds with specific activity in tumors carrying a PTEN or KRAS alteration, using as input the publicly available NCI60 screen data.
The methodology can be also applied to search for treatments with increased activity in tumors with defined alterations, provided there are enough samples representing the case and control groups. Examples of these alterations include, but are not limited to, somatic mutations, gene copy alterations, overexpression or under-expression of gene, or even more complex molecular phenotypes such as pathway alterations, and any combination thereof. This set of alterations can be used to define the case (carrying the alterations) and control (not carrying the alterations) groups.
The methodology can be used to identify treatments (e.g., compounds) for adjuvant therapy. For all available anticancer treatments, a significant number of patients manifest resistance to the treatments. A strategy to overcome this problem is to use an adjuvant therapy that targets those tumors insensitive to the original treatment. A search for adjuvant therapies can be optimized using the methodology disclosed herein. To this end, given a specified treatment, the case group will contain cell lines that are resistant to that treatment (as defined above) while the control group will contain the cell lines that respond well to treatment (as defined above). Taking as input these case and control groups, the methodology disclosed in this application can be used to identify compounds with increased activity in tumors that are resistant to the given treatment, which could be used as adjuvant therapy.
Furthermore, the methodology disclosed herein can be applied to search for treatments with increased activity in any specified tumor group, provided a definition of the case and control groups and enough samples representing them. The input data can be obtained from any anticancer treatment screen, including but not limited to the NCI60 screen. A treatment as used herein refers to any stimulus. Examples include exposure to a compound, such as small molecules (as in the NCI60 screen), peptides, and antibodies, or to others, such as ionizing radiation, or any other entity with anticancer potential. This methodology applies to all such treatments, provided a meaningful definition of relative sensitivity can be obtained.
In the above-described methodology, certain raw data for a disorder and related cell lines are needed. In one embodiment, the data include genetic features, such as gene point mutations, SNP patterns (e.g., haplotype blocks), portions of genes (e.g., exons/introns or regulatory motifs), regions of a genome or chromosome spanning more than one gene. Other examples include phenotypic features such as the morphology of cells and cellular organelles (e.g., cytoskeleton) or their behaviors (e.g., drug-resistance, proliferation, differentiation, cell death, and metastasis). Examples of technology for producing such raw data include, but are not limited to, microarray platforms including RNA and miRNA expression, SNP genotyping, protein expression, protein-DNA interaction and methylation data and amplification/deletion of chromosomal regions platforms, quantitative polymerase chain reaction gene expression platforms, identified novel genetic variants, copy-number variation detection platforms, detecting chromosomal aberrations (amplifications/deletions) and whole genome sequencing. While the description here concerns genetic mutation data, the methods described may be extrapolated to other types of data, e.g., protein sequences and phenotypic features.
The methodology described in this application involves biological experiments in which a stimulus (i.e., a test treatment) acts on a biological sample such as a tissue or cell culture. Often the biological experiment will have associated clinical parameters such as tumor stage, patient history, etc. The sample may be exposed to one or more stimuli or treatments to produce test data. Control data may also be produced in the same way. The stimulus is chosen as appropriate for the particular study undertaken. Examples of stimuli that may be employed can be an exposure to particular materials or compositions, radiation (including all manner of electromagnetic and particle radiation), forces (including mechanical (e.g., gravitational), electrical, magnetic, and nuclear), fields, thermal energy, and the like. General examples of materials that may be used as stimuli include organic and inorganic chemical compounds, biological materials such as nucleic acids, carbohydrates, peptides, polypeptides, proteins (e.g., antibodies), lipids, microorganisms (e.g., viruses, including those useful in gene therapy), various infectious agents, mixtures of the foregoing, and the like. Other examples of stimuli include the lack of a particular material e.g., a nutrient or a growth factor.
An “antibody” includes intact molecules as well as fragments thereof, such as Fab, F(ab′)2, Fv, scFv (single chain antibody), and dAb (domain antibody). A derivative of an antibody refers to a protein or a protein complex having a polypeptide variant of this invention. An antibody or derivative in this invention can be made by co-expressing corresponding light and heavy chain CDRs-containing polypeptides in a suitable host cell by methods known in the art. See, e.g., Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
A nucleic acid refers to a DNA molecule (e.g., a cDNA or genomic DNA), an RNA molecule (e.g., an mRNA), or a DNA or RNA analog. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. Examples include an antisense nucleic acid and an RNAi agent.
In a preferred embodiment, the stimulus is an exposure to therapeutic agents (including agents suspected of being therapeutic but not yet proven to have this property). Often the therapeutic agent is a chemical compound such as a drug or drug candidate or a compound present in the environment. The biological impact of chemical compounds is manifest as a change in a feature such as a level gene expression or a phenotypic characteristic, including cell growth, cell proliferation, cell differentiation, and cell death.
The methodology of this invention can be incorporated into a multiplicity of suitable computer products, systems, and/or information instruments. User interface methods known in the information processing art can be used in the systems of this invention.
1. Computer Software
For example, the above-disclosed methodology or components thereof can be embodied in a fixed or non-transitory medium (e.g., a computer accessible/computer readable medium program component containing logic instructions or data, or both), that when loaded into an appropriately configured computing device can cause that device to perform operations to the invention. In various embodiments a fixed medium component containing logic instructions can be delivered to a viewer on a fixed medium for physically loading into a viewer's computer or a fixed medium containing logic instructions can reside on a remote server that a viewer can access through a communication medium in order to download a program component.
Examples of a tangible computer-readable medium suitable for use computer program products and computational apparatus of this invention include, but are not limited to, magnetic media such as hard disks, floppy disks, and magnetic tape; optical media such as CD-ROM disks; magneto-optical media; semiconductor memory devices (e.g., flash memory), and hardware devices that are specially configured to store and perform program instructions, such as read-only memory devices (ROM) and random access memory (RAM) and sometimes application-specific integrated circuits (ASICs), programmable logic devices (PLDs) and signal transmission media for delivering computer-readable instructions, such as local area networks, wide area networks, and the Internet. The data and program instructions provided herein may also be embodied on a carrier wave or other transport medium (including electronic or optically conductive pathways). The data and program instructions of this invention may also be embodied on a carrier wave or other transport medium (e.g., optical lines, electrical lines, and/or airwaves).
2. Database
The above-described raw data sets and information generated using the methodology of this invention can be used to establish a database, i.e., a collection of data, which can be used to analyze and respond to queries. In one embodiment, the database includes one or more records for organizing the raw data sets and information sets in a particular hierarchy or directory (e.g., a hierarchy of studies and projects). In addition, the database may include information correlating the records to one another, a list of globally unique terms or identifiers for cancers, tumors, cell lines, genes, treatments (e.g., compounds), or other features. Such a database also contains a taxonomy that contains a list of all tags (keywords) for different tissues, disease states, treatment types, phenotypes, cells, as well as their relationships.
In one embodiment, the database contains data from a number of sources, including data from external sources, such as public databases, including those maintained at the National Cancer Institute and the National Center for Biotechnology Information (NCBI). In addition, the database can include proprietary data obtained and processed by the database developer or user. A database may be updated by a developer or user as new public or private information from biological or chemical experiments becomes available. Once imported, all data are correlated with other information in the database so as to enable users to interrogate tumors, cell lines, biological features, compounds, and responsiveness thereto across the entire information space.
3. Computer Hardware
In another aspect, the invention provides an apparatus for performing the above-mentioned operations. This apparatus may be specially designed and/or constructed for the required purposes, or it may be a general-purpose computer selectively configured by one or more computer programs and/or data structures stored in or otherwise made available to the computer. The processes presented herein are not inherently related to any particular computer or other apparatus. In particular, various general-purpose machines may be used with programs written in accordance with the teachings herein, or it may be more convenient to construct a more specialized apparatus to perform the required method steps.
CPU 602 is also coupled to an interface 610 that connects to one or more input/output devices such as video monitors, track balls, mice, keyboards, microphones, touch-sensitive displays, transducer card readers, magnetic or paper tape readers, tablets, styluses, voice or handwriting recognition peripherals, USB ports, or other well-known input devices such as other computers. Finally, CPU 602 optionally may be coupled to an external device such as a database or a computer or telecommunications network using an external connection as shown generally at network 614. With such a connection, it is contemplated that the CPU might receive information from the network, or might output information to the network in the course of performing the method steps described herein.
In one embodiment, a system such as computer system 600 is used as a special purpose data import, data correlation, and querying system capable of performing some or all of the tasks described herein. System 600 may also serve as various other tools associated with database described above and querying such as a data capture tool. Information and programs, including data files can be provided via a network connection 614 for access or downloading from server system 616. Alternatively, such information, programs and files can be provided to the researcher on a storage device. In a specific embodiment, the computer system 600 is directly coupled to a data acquisition system such as a high-throughput screening system that captures data from samples. Data from such systems are provided via interface 610 for analysis by system 600. Alternatively, the data processed by system 600 are provided from a data storage source such as a database or other repository of relevant data. Once in apparatus 600, a memory device such as primary storage 606 or mass storage 608 buffers or stores, at least temporarily, relevant data. The memory may also store various routines and/or programs for importing, analyzing and presenting the data, including importing the above-described data sets, correlating data sets with one another and with feature groups, generating and running queries, etc.
In certain embodiments, the system of this invention may include one or more user terminals (618). User terminals can include any type of computer (e.g., desktop, laptop, tablet, etc.), media computing platforms (e.g., cable, satellite set top boxes, digital video recorders, etc.), handheld computing devices (e.g., PDAs, e-mail clients, etc.), cell phones or any other type of computing or communication platforms. A server (616) in communication with a user terminal may include a server device or decentralized server devices, and may include mainframe computers, mini computers, super computers, personal computers, or combinations thereof. A plurality of server systems may also be used without departing from the scope of the present invention. User terminals and a server system may communicate with each other through the network 614. The network may comprise, e.g., wired networks such as LANs (local area networks), WANs (wide area networks), MANs (metropolitan area networks), ISDNs (Intergrated Service Digital Networks), etc. as well as wireless networks such as wireless LANs, CDMA, Bluetooth, and satellite communication networks, etc. without limiting the scope of the present invention.
Within the scope of this invention is a composition that contains a suitable carrier and one or more of the compounds described above, such as NSC669995, NSC682449, NSC656238, NSC612941, NSC155694, NSC319726, NSC319725, NSC694266, and NSC93739, or a pharmaceutically acceptable salt of the compound.
The composition can be a pharmaceutical composition that contains a pharmaceutically acceptable carrier, a dietary composition that contains a dietarily acceptable suitable carrier, or a cosmetic composition that contains a cosmetically acceptable carrier.
The term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A “pharmaceutically acceptable carrier,” after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient and, preferably, capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active compound. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.
The above-described composition, in any of the forms described above, can be used for treating cellular proliferative disorders. An effective amount refers to the amount of an active compound that is required to confer a therapeutic effect on a treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on the types of diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.
A pharmaceutical composition of this invention can be administered parenterally, orally, nasally, rectally, topically, or buccally. The term “parenteral” as used herein refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.
A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acid, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long chain alcohol diluent or dispersant, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purpose of formulation.
A composition for oral administration can be any orally acceptable dosage form including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions. In the case of tablets, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.
Pharmaceutical compositions for topical administration according to the present invention can be formulated as solutions, ointments, creams, suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils. Alternatively, topical formulations can be in the form of patches or dressings impregnated with active ingredient(s), which can optionally comprise one or more excipients or diluents. In some preferred embodiments, the topical formulations include a material that would enhance absorption or penetration of the active agent(s) through the skin or other affected areas. The topical composition is useful for treating cellular proliferative disorders in the skin, such as melanoma.
A topical composition contains a safe and effective amount of a dermatologically acceptable carrier suitable for application to the skin. A “cosmetically acceptable” or “dermatologically-acceptable” composition or component refers a composition or component that is suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic response, and the like. The carrier enables an active agent and optional component to be delivered to the skin at an appropriate concentration(s). The carrier can thus act as a diluent, dispersant, solvent, or the like to ensure that the active materials are applied to and distributed evenly over the selected target at an appropriate concentration. The carrier can be solid, semi-solid, or liquid. Preferably, it is in the form of a lotion, a cream, or a gel, in particular one that has a sufficient thickness or yield point to prevent the active materials from sedimenting. The carrier can be inert or possess dermatological benefits. It should also be physically and chemically compatible with the active components described herein, and should not unduly impair stability, efficacy, or other use benefits associated with the composition.
The invention also features methods for treating in a subject a cellular proliferative disorder (e.g., cancer). A cellular proliferative disorder refers to a disorder characterized by uncontrolled, autonomous cell growth, including malignant and non-malignant growth. Examples of this disorder include colon cancer or colorectal cancer, breast cancer, prostate cancer, hepatocellular carcinoma, melanoma, lung cancer, glioblastoma, brain or CNS tumor, hematopoeitic malignancies, leukemia, retinoblastoma, renal cell carcinoma, head and neck cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and squama cell carcinoma.
In one embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the p53 gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (Ia) or (Ib):
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the p53 gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (Ia) or (Ib), wherein:
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the p53 gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (Ia) or (Ib), wherein said compound is selected from the group consisting of NSC319726, NSC319725, and NSC328784.
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the p53 gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (II):
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the p53 gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (II), wherein:
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the p53 gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (II), wherein said compound is selected from the group consisting of NSC612941, NSC155694, and NSC620256.
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the BRAF gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (IIIa) or (IIIb):
36. The method of claim 35, wherein:
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the KRAS gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (IV):
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the KRAS gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (IV), wherein said compound is selected from the group consisting of NSC613327, NSC146268, and NSC48006.
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the KRAS gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (V):
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the KRAS gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (V), wherein:
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the KRAS gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (V), wherein said compound is selected from the group consisting of NSC740, NSC696558, NSC666787, NSC682306, NSC117356, and NSC739.
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the PTEN gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (VI):
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the PTEN gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (VI), wherein:
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the PTEN gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (VI), wherein said compound is selected from the group consisting of NSC706744, NSC735493, and NSC734294.
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the PTEN gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (VII):
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the PTEN gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (VII), wherein:
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the PTEN gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (VII), wherein said compound is selected from the group consisting of NSC681640, NSC681645, NSC681634, NSC681638, NSC606499, NSC606498, NSC606497, NSC364830, and NSC639174.
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the PTEN gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (VIII):
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the PTEN gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (VIII), wherein said compound is one of cephalostatins.
In another embodiment, the present invention provides a method for treating a cellular proliferative disorder in a subject, characterized by a mutation in the PTEN gene, comprising administering to the subject in need thereof an effective amount of a compound of Formula (VIII), wherein said compound is selected from the group consisting of cephalostatin 1 (NSC363979), cephalostatin 2 (NSC363980), cephalostatin 3 (NSC363981), cephalostatin 4 (NSC378727), cephalostatin 8 (NSC378734), and cephalostatin 9 (NSC378735).
It is understood that when a chiral center exists in any of the above structures, the chiral center can take either an R- or an S-configuration. Thus, the structures would encompass all possible stereoisomers, including but not limited to enantiomers and diastereomers.
A “subject” refers to a human and a non-human animal. Examples of a non-human animal include all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), dog, rodent (e.g., mouse or rat), guinea pig, cat, and non-mammals, such as birds, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model. A subject to be treated for a cellular proliferative disorder can be identified by standard diagnosing techniques for the disorder.
Optionally, the subject can then be examined for mutation (e.g., one or more of the mutations discussed herein), expression level, or activity level of an oncogene or a tumor suppressor gene (e.g., the BRAF gene, the p53 gene, the PTEN gene, or the RAS gene) or polypeptide by methods known in the art or described above. If the subject has a particular mutation in the gene, or if the gene expression or activity level is, for example, lower in a sample from the subject than that in a sample from a normal person, the subject is a candidate for treatment.
“Treating” or “treatment” refers to administration of a compound or agent to a subject, who has a disorder (such as a cellular proliferative disorder), with the purpose to cure, alleviate, relieve, remedy, delay the onset of, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder.
An “effective amount” or “therapeutically effective amount” refers to an amount of the compound that is capable of producing a medically desirable result, e.g., as described above, in a treated subject. The treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
The agent can be administered in vivo or ex vivo, alone or co-administered in conjunction with other drugs or therapy. As used herein, the term “co-administration” or “co-administered” refers to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents/therapies used may vary.
In an in vivo approach, a compound is administered to a subject. Generally, the compound is suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100 mg/kg. Variations in the needed dosage are to be expected in view of the variety of compounds available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
In the following examples, the applicability of the methodology disclosed herein was tested and corroborated. The input data was obtained from the National Cancer Institute anticancer drug screen, NCI60 screen (October 2009 release, which is available at dtp.nci.nih.gov/docs/cancer/cancer_data.html).
Oncogenenic mutations in the BRAF gene correlate with increased severity and decreased response to chemotherapy in a wide variety of tumors. The BRAF gene encodes a protein kinase (B-Raf) and mutations in this gene can result in a constitutive activation of its kinase, promoting cell proliferation. Among these oncogenic mutations, BRAFV600E is one of the most prevalent mutations in tumors and the most frequent oncogenic protein kinase mutations known. A potent inhibitor of the B-Raf activity (AZD6244) has been recently reported (Yeh, T. C., et al., Clin Cancer Res, 2007. 13(5): p. 1576-83). AZD6244 manifests selective growth inhibition of tumor derived cell lines with mutations in B-Raf and members of the Ras receptor kinases. This compound is in Phase II clinical trials in cancers with BRAF mutations (NCT00888134) and was tested within the NCI60 screen, offering a unique opportunity to test the applicability of the methodology described above.
Within the cell lines in the NCI60 screen, 11 lines carry a BRAFV600E mutation (Ikediobi, O. N., et al., Mol Cancer Ther, 2006. 5(11): p. 2606-12). Furthermore, 10 of those also carry a mutation in TP53, CDKN2A or both. The TP53 gene encodes the transcription factor p53 that plays a central role in tumor suppression. In turn, ARF, one of the gene products of the CDKN2A gene, is a positive regulator of p53. Inactivating alterations of these two genes promote tumor formation and correlate with decreased response to chemotherapy. This correlation, between carrying a BRAFV600E mutation and a mutation in TP53 or CDKN2A, should be taken into account when constructing the control group of cell lines with wild-type BRAF. Thus, as a control group, the 10 NCI60 cell lines with wild-type BRAF but carrying a mutation in either TP53 or CDKN2A were selected. The resulting groups are the following:
Using the protocols described above, the NCI60 treatment-cell line RG was constructed, taking RS=log10IC50-average(log10IC50) as a relative response sensitivity measurement and computing the average(log10IC50) over all the 60 cell lines. The Case and Control groups listed above were chosen, and the score using Formula I was computed for the 47,624 compounds in the NCI60 screen, including AZD6244 under the NCI designation NSC741078.
Six compounds with highest score were identified and shown in
Mutations in the TP53 gene correlate with increased severity and decreased response to chemotherapy in a wide variety of tumors. The TP53 gene encodes a transcription factor that plays a central role in tumor suppression and mutations in this gene can result in lost of the p53 tumor suppressor function and gain of function as well. TP53 mutations are the most frequent mutations in tumors. Most occurring mutations are observed within the p53 DNA binding domain, with hot spots at amino acids 273 and 248 in the p53 protein, right at the point of contact with DNA, and at amino acid 175, in the zinc pocket region.
Among the NCI60 cell lines, 16 have wild-type p53 and 44 carry a p53 alteration (Ikediobi, O. N., et al., Mol Cancer Ther, 2006. 5(11): p. 2606-12). Mutations at amino acids 273 and 248 are the most frequent, with 5 and 4 cell lines, respectively. Given the abundance of p53 mutations at positions 273 and 248 within the NCI60 cell lines, and their location in the p53-DNA contact, cells carrying these mutations were chosen to form a case group to identify compounds with increased activity in tumors with a mutant p53. On the other hand, cells with a wild-type p53 were chosen to form a control group. The resulting groups are the following:
Using the protocols described above, the NCI60 treatment-cell line RG was constructed, taking RS=log10IC50-average(log10IC50) as a relative response sensitivity measurement and computing the average(log10IC50) over all the 60 cell lines. The Case and Control groups listed above were chosen, and the score in Formula I was computed for the 47,624 compounds in the NCI60 screen. The six compounds ranking highest were identified as NSC612941 (0.55), NSC155694 (0.55), NSC319726 (0.52), NSC319725 (0.50), NSC694266 (0.50) and NSC93739 (0.50), their scores being indicated within the parenthesis.
None of these compounds have been previously reported for their selective activity in cells with a mutant p53. To provide further evidence for their specificity, growth inhibition assays were performed on isogenic cell lines. Previously developed murine fibroblast cell line (10)3 (p53 null) and 10(3) with transfection of cytomegalovirus-human mutant p53 constructs (273, 248 and 175) (Dittmer, D., et al., Nature Genetics, 1993. 4(1): p. 42-6) were used as cases, while the murine fibroblast cell line 3T3 was used as a p53 wild type control. The six compounds ranking highest were requested from the NCI and three of them (NSC155694, NSC319725 and NSC319726) were obtained.
It was found that Compounds NSC319725 and NSC319726 exhibited a selective activity as predicted, with a higher sensitivity in cells with a p53 alteration (
In this example, NSC319725 and NSC319726 were found to be synthetic lethal for cell lines expressing mutant p53. These two compounds belong to the same family—the thiosemicarbazones. The chemical structures for these compounds are shown in
The compounds were validated using several different cell line systems. The first system used was a mouse embryonic fibroblast (MEF) system in which a p53 null MEF line, 10(3), was used as a parental line from which several stable CMV-mutant p53 transfectants were derived. The p53 mutations investigated were the “hot spot” mutants 175, 248, and 273. NIH3T3 fibroblasts were used as a p53 wild type control. It was found that both of the compounds exhibited markedly higher sensitivity in cell lines expressing mutant p53 as compared to the wild type control,
Assays were carried out to further validate NSC319726 in other p53 mutant cell line systems. Using a system of mouse embryonic fibroblast (MEF) cell lines derived from the same strain, the sensitivities of MEF-p53 wild type, MEF-p53 null, and MEF-p53R172H cells to NSC319726 were compared. The sensitivity was measured in 12-well cultures for viability assay using the Guava PCA instrument following the manufacture protocol. It was found that again NSC319726 exhibited a much higher sensitivity for the MEF-p53R172H as compared to the p53 wild type and p53 null controls,
Next, assays were carried out to examine the sensitivity to NSC319726 of a human tumor cell line system that was made up of 4 cell lines expressing wild type p53, one cell line expressing p53 null cell in (SKOV3), and one ovarian carcinoma cell line containing a p53R175H (TOV112D). It was noted that the TOV112D cell line had a marked sensitivity and the p53 wild type cell lines showed a relative lack of sensitivity at a concentration rage from 0.1 μM to 10 μM of 319726,
In this example, assays were performed to demonstrate that the mechanism of NSC319726's mutant synthetic lethality is by induction of apoptosis that is p53R175H dependent.
Briefly, apoptosis was examined by Annexin V staining using standard methods. The Annexin V staining of NSC319726 treated MEF cells indicated that the mechanism of growth inhibition was by induction of apoptosis. As shown in
In addition, treatment of three ovarian carcinoma cell lines (TOV112D-p53R175H, OVCAR3-p53 R248W and SKOV3-p53 null) revealed an induction of apoptosis in all three that was maximally seen in the TOV112D cell line. As shown in
Because NSC319726 displayed such a marked sensitivity for cell lines expressing the R175H p53 mutant allele, it was hypothesized that the mechanism involved the p53R175H mutant protein. In fact, when expression of the p53R175H mutant protein was silenced in the TOV112D cells by siRNA, it was observed that the sensitivity of TOV112D was reduced by four-fold,
As apoptosis induced by NSC319726 was dependent on the p53R175H mutant protein, assays were carried out to determine if this compound induced a conformational change in the mutant protein structure.
Conformational change to wild type in TOV112D (p53R175H) was studied by immuno-fluorescent staining. Briefly, TOV112D cells were grown on coverslips, treated with NSC319726 and stained with conformation-specific antibodies PAB1620, which only recognizes wild type p53 conformation, PAB240, which only recognizes mutant p53 conformation, and PAB1801, which can recognize both. As shown in
This conformational change was confirmed by immunoprecipitation of lysates from NSC319726-treated TOV112D cells using the mutant specific antibody PAB240. To that end, cell lysates were precipitated with PAB240 and detected with p53 (DO-1) antibody which recognizes all types of p53 protein conformations. It was found that NSC319726 treatment, but not NSC319725, greatly reduced the amount of p53 R175H that can be immunoprecipitated by the mutant specific antibody,
To determine if the conformation change caused by NSC319726 was functional, Western Blot assays were performed to examine p21 protein levels in TOV112D (p53R175H) and SKOV3 (p53 null) cells treated with NSC319725 and NSC319726.
It was found that NSC319726 treatment induced p21 in the TOV112D line but not in the SKOV3 line,
To provide further evidence that NSC319726 restored site-specific p53 transactivational function. To that end, luciferase activity assays were performed. Briefly, a 20-bp p53 response element (p53RE) was subcloned in the pGL3 vector to generate a luciferase reporter plasmid, which contained 20 base pairs of the p53 response element in the p21 promoter. The sequences of the 5′ site and 3′ site at −2.27 kb and −1.38 kb (SEQ ID NOs: 1 and 3) and their reverse complements (SEQ ID NOs: 2 and 4) are listed below:
The reporter plasmid was transfected to the TOV112D cells or MEF cells with exogenous 248 or 273 mutations. Cells thus-transfected were treated with NSC319726 and subjected to luciferase assays. It was found that, upon NSC319726 treatment, a 2.5 fold increase in luciferase activity in the TOV112D cells was observed. This effect was not seen in MEF cell lines expressing the 248 and 273 alleles,
Assays were carried out to compare the mRNA levels of several p53 targets (p21, PUMA, and MDM2) in the TOV112D (p53R175H), OVCAR3 (p53R248W) and SKOV3 (p53 null) upon treatment with the compounds. The relative gene expression level was normalized with the actin and the ratio of the treated level vs. untreated level was shown for each cell line. It was found that NSC319726 induced all three targets in the TOV112D cells, particularly the apoptotic gene PUMA, while no such effect was observed upon NSC319725 treatment,
To examine the transcriptional activity of the NSC319725 and NSC19726-treated cells on a larger scale, microarrays were used to analyze control- and compound-treated TOV112D cells. Shown in
Next, assays were performed to compare the signature of the NSC319726-treated cells to a signature that would be indicative of wild type p53 upon an activating stimulus such radiation. As shown in
Toxicity assays were performed in p53 wild type, p53 null, and p53R172H mice to determine if in vivo p53 mutant synthetic lethality could be detected. It was hypothesized that p53R172H mice would experience greater toxicity for a given dose of NSC319726 as compared to the p53 wild type or null mice.
First, three groups of mice: p53+/R172H wild type, p53+/R172H/R172H mice, were compared. NSC319726 was injected intraperitoneally at 10 mg/kg daily for seven days. It was found that by day 3 all seven of the p53R172H/R172H mice had died while only 1 in 9 p53 wild type mice had died. By day 4, the survival of the p53 wild type mice fell to 70% while the p53+/R172H had fallen to 30%, suggesting a clear effect that is dependent on the TP53 status. By day seven approximately 40% of the wild type mice were alive, suggesting that there is some toxicity of the drug at this dose that is not p53 dependent.
Next, the dose was lowered to 5 mg/kg and three groups of mice (p53 wild type, p53 null, and p53R172H/R172H) were compared. At this dose, it was found that, by day seven, the p53 wild type and p53 null mice exhibited a 100% survival compared to only 30% in the p53R172H/R172H mice.
Tissues of p53 wild type and p53R172H/R172H mutant mice after treatment for 24 hours with NSC319726 were examined for evidence of apoptosis by cleaved Caspase-3 immuno-staining as well as gene expression of a panel of p53 targets by quantitative-PCR. As shown in
Xenograft tumors were created by subcutaneous flank injections of 8×106 human tumor cells and allowed to grow to an initial size ranging from 50-200 mm3 prior to initiation of NSC319726 (1 μg/g) (versus control, “ctl”) administered intravenously once daily. As shown in
The methodology described in this application was used to further screen compounds in the NCI60 screen in the manner described in Examples 1 and 2 above for compounds having increased activity against cells having mutations in four genes: p53, BRAF, KRAS, and PTEN. Listed below are 46 identified compounds:
1. 7 compounds identified as being specific for disorders characterized by a mutation in the p53 gene:
2. 9 compounds identified as being specific for disorders characterized by a V600E mutation in the BRAF gene:
3. 11 compounds identified as being specific for disorders characterized by a mutation in the KRAS gene:
4.22 compounds identified as being specific for disorders characterized by a mutation in the PTEN gene:
NSC706744, NSC735493, NSC734294, NSC681640, NSC681645, NSC681634, NSC681638, NSC606499, NSC606498, NSC606497, NSC364830, NSC639174, NSC620256, NSC363979, NSC363980, NSC363981, NSC378734, NSC378735, NSC378727, NSC355447, NSC368891, and NSC48006.
The chemical identities and structure schemes of some of the 46 compounds are shown in
The foregoing example and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims.
This application claims priority of U.S. Provisional Application No. 61/326,490, filed on Apr. 21, 2010, and U.S. Provisional Application No. 61/438,678, filed on Feb. 2, 2011. The contents of the applications are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/33386 | 4/21/2011 | WO | 00 | 5/9/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61326490 | Apr 2010 | US | |
| 61438678 | Feb 2011 | US |
