Patent Application
20110030074
The present invention relates generally to the use of a genome unstable animal cancer model for cancer gene discovery.
Cancer is a genetic disease driven by the stochastic acquisition of mutations and shaped by natural selection. Genomic instability, a hallmark of many human cancers, propagates these mutations, allowing cells to overcome critical barriers to unregulated growth, and may therefore herald a defining event in malignant transformation. Genomic instability is manifested by chromosomal aberrations, such as translocations and amplifications. How and when during the course of tumor progression significant genomic instability arises, and whether a cancer can be cured or even contained after that point, represent pivotal and largely unanswered questions.
Animal models for human carcinomas are valuable tools for the investigation and development of cancer therapies. Murine models having oncogenes incorporated into its genome, or tumor suppressor genes suppressed have been widely used for human cancer research. However, an impediment towards maximal utilization of murine models for guiding human cancer gene discovery efforts is the relatively benign cytogenetic profiles of most standard genetically engineered mouse models of cancer (see, e.g., N. Bardeesy, et al., Proc Natl Acad Sci USA 103 (15), 5947 (2006); M. Kim, et al., Cell 125 (7), 1269 (2006); L. Zender, et al., Cell 125 (7), 1253 (2006); A. Sweet-Cordero, et al., Genes Chromosomes Cancer 45 (4), 338 (2006)). These models do not reflect the global chromosomal aberrations associated with many types of human cancers.
Several cancer-prone murine models have recently been developed that more closely simulate the rampant chromosomal instability of human cancers. For example, Artandi et al. describe the development of epithelial cancers in a telomerase-definition p53-mutant mouse model (Nature 406 (6796), 641 (2000)); Zhu et. al describe oncogene translocation and amplification in a mouse model that is deficient in both p53 and nonhomologous end-joining (NHEJ) (Cell 109 (7), 811 (2002)); Olive et. al describe a Li-Fraumeni Syndrome mouse model having dominant p53 mutant alleles (Cell 119 (6), 847 (2004)); Lang et. al describe a Li-Fraumeni Syndrome mouse model having p53 missense mutations (Cell 119 (6), 861 (2004)); and Hingorani et. al describe a mouse model of pancreatic ductal adenocarcinoma, expressing mutant forms of TP53 and KRAS2 (Cancer Cell 7 (5), 469 (2005)). However, the frequency of chromosomal aberrations in these mouse models are relatively low, and the transgenic mice do not necessarily develop malignant cancer. To facilitate oncogenomic anlayses, there is a need to create new mammal models that are genetically modified to develop cancer, having chromosomal aberrations at a frequency that is comparable to human cancers.
Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the cancer process. Here, we engineered lymphoma-prone mice with chromosomal instability to assess the utility of animal models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number alterations. Integrating with targeted re-sequencing, our comparative oncogenomic studies identified FBXW7 and PTEN as commonly deleted or mutated tumor suppressors in human T-cell acute lymphoblastic leukemia/lymphoma (T-ALL). More generally, the murine cancers acquire widespread recurrent clonal amplifications and deletions targeting loci syntenic to alterations present in not only human T-ALL but also diverse tumors of hematopoietic, mesenchymal and epithelial types. These results thus support the view that murine and human tumors experience common biological processes driven by orthologous genetic events as they evolve towards a malignant phenotype. The highly concordant nature of genomic events encourages the use of genome unstable animal cancer models in the discovery of biologically relevant driver events in human cancer.
In one aspect, the invention provides a non-human transgenic mammal that is genetically modified to develop cancer, such that the genome of a cancer cell from the mammal comprises chromosomal structural aberrations at a frequency that is at least 5-fold higher than the frequency of chromosomal structural aberrations in such mammal without the genetic modification. In certain embodiments, the mammal is a rodent. In certain embodiments, the mammal is a mouse.
In certain embodiments, the mammal comprises engineered inactivation of: at least one allele of one or more genes encoding a protein involved in DNA repair function (such as a protein involved in non-homologous end joining (NHEJ), a protein involved in homologous recombination, or a DNA repair helicase), and at least one allele of one or more genes encoding a component that synthesizes and maintains telomere length. Alternatively, the mammal may comprise engineered inactivation of: at least one allele of one or more genes encoding a protein involved in DNA repair function and at least one allele of one or more genes encoding a DNA damage checkpoint protein. Alternatively, the mammal may comprise engineered inactivation of: at least one allele of one or more genes encoding a DNA damage checkpoint protein and at least one allele of one or more genes encoding a component that synthesizes and maintains telomere length.
In certain embodiments, the genome of the mammal further comprises at least one additional cancer-promoting modification, such as an activated oncogene, an inactivated tumor suppressor gene, or both.
In another aspect, the invention provides a method of identifying a chromosomal region of interest for the identification of a gene or genetic element that is potentially related to human cancer, comprising the step of: identifying a DNA copy number alteration in a population of cancer cells from a non-human mammal that is engineered to produce chromosomal instability. The chromosomal region of the DNA copy number alteration is a chromosomal region of interest for identifying a gene or genetic element that is potentially related to human cancer.
In certain embodiments, the DNA copy number alteration is recurrent in two or more cancer cells from the non-human mammal. The DNA copy number alteration can be a DNA gain or a DNA loss.
In another aspect, the invention provides a method of identifying a chromosomal region of interest for the identification of a gene or genetic element that is potentially related to human cancer, comprising the step of: identifying a chromosomal structural aberration in a population of cancer cells from a non-human mammal that is engineered to produce genome instability. A chromosomal region containing the chromosomal structural aberration is a chromosomal region of interest for identifying a gene or genetic element that is potentially related to human cancer.
In certain embodiments, the method further comprises the steps of: (1) identifying a DNA copy number alteration in the population of cancer cells from the non-human mammal, and (2) identifying a chromosomal region in the genome of the cancer cell of the non-human mammal that contains a chromosomal structural aberration and a DNA copy number alteration. The chromosomal region containing a chromosomal structural aberration and a DNA copy number alteration is a chromosomal region of interest for identifying a gene and genetic element that is potentially related to human cancer. In certain embodiments, the method further comprises the step of determining the uniform copy number segment boundary of the DNA copy number alteration.
In another aspect, the invention provides a method for identifying a potential human cancer-related gene, comprising the steps of: (a) identifying a chromosomal region of interest (e.g., comprising a gene or genetic element that is potentially related to human cancer); (b) identifying a gene or genetic element within the chromosomal region of interest in the non-human mammal, and (c) identifying a human gene or genetic element that corresponds to the gene or genetic element identified in step (b). The human gene or genetic element is a potential human cancer-related gene or genetic element. In certain embodiments, the human gene is orthologous, paralogous, or homologous to the gene or genetic element identified in step (b). In certain embodiments, the method further comprises the step of detecting a mutation in the non-human mammalian gene or genetic element identified in step (b), the human gene or genetic element identified in step (c), or both.
In another aspect, the invention provides a method of identifying a potential human cancer-related gene or genetic element, comprising the steps of: (a) detecting a DNA copy number alteration in a population of cancer cells from a non-human mammal that is engineered to produce genome instability, (b) identifying a gene or genetic element located within the boundaries of the DNA copy number alteration detected in step (a), and (c) identifying a human gene or genetic element that corresponds to the gene or genetic element identified in step (b) and that is located within the boundaries of a DNA copy number alteration or of a chromosomal structural aberration in a human cancer cell. The human gene or genetic element identified in step (c) is a gene or genetic element potentially related to human cancer.
In another aspect, the invention provides a method of identifying a potential human cancer-related gene or genetic element, comprising the steps of (a) detecting a chromosomal structural aberration in a population of cancer cells from a non-human mammal that is engineered to produce genome instability, (b) identifying a gene or genetic element located at the site of the chromosomal structural aberration detected in step (a), and (c) identifying a human gene or genetic element that corresponds to the gene or genetic element identified in step (b) and that is located within the boundaries of a DNA copy number alteration or at the site of a chromosomal structural aberration in a human cancer cell. The human gene or genetic element identified in step (c) is a gene or genetic element potentially related to human cancer. In certain embodiments, the method further comprises the step of detecting a mutation in the non-human mammalian gene or genetic element identified in step (b), the human gene or genetic element identified in step (c), or both.
In certain embodiments, the method further comprises the step of defining the minimum common region (MCR) of a recurrent gene copy number alteration. In certain embodiments, the MCR is defined by boundaries of overlap between two or more samples. In certain embodiments, the MCR is defined by the boundaries of a single tumor against a background of larger alteration in at least one other tumor.
In another aspect, the invention provides a method for identifying subjects with T-cell acute lymphoblastic leukemia (T-ALL) who may have a decreased response to γ-secretase inhibitor therapy, comprising detecting the expression or activity of FBXW7 in a tumor cell from the subject. A decreased expression or activity of FBXW7, as compared to a control, is indicative that the subject may have a decreased response to γ-secretase inhibitor therapy.
In certain embodiments, the method further comprises detecting the expression or activity of NOTCH1 in a tumor cell from the subject. An increased expression or activity of NOTCH1, as compared to a control, is indicative that the subject may have a decreased response to γ-secretase inhibitor therapy.
In another aspect, the invention provides a method for identifying subjects with T-ALL that may benefit from treatment with a PI3K pathway inhibitor, comprising detecting the expression or activity of PTEN in a tumor cell from the subject. A decreased expression or activity of PTEN, as compared to a control, is indicative that the subject may benefit from a treatment with a PI3K inhibitor. In certain embodiments, the method further comprises treating the subject with a PI3K inhibitor.
In another aspect, the invention provides a method of assessing whether a subject is afflicted with cancer or at risk for developing cancer, comprising: determining the expression or activity level of at least one cancer gene or candidate cancer gene located in an amplified MCR in Table 1 in a biological sample from the subject. An increase in the expression or activity the gene, as compared to a control, indicates that the subject is afflicted with cancer or at risk for developing cancer. Alternatively, if there is a decrease in the expression or activity of a cancer gene or candidate cancer gene located in a deleted MCR in Table 1, as compared to a control, the decreased expression or activity level also indicates that the subject is afflicted with cancer or at risk for developing cancer.
In another aspect, the invention provides a method of assessing whether a subject is afflicted with cancer or at risk for developing cancer, the method comprising: determining the copy number of at least one amplified minimal common region (MCR) listed in Table 1 in a biological sample from the subject. An increased copy number of the MCR in the sample, as compared to the normal copy number of the MCR, indicates that the subject is afflicted with cancer or at risk for developing cancer. Alternatively, a decreased copy number of a deleted MCR (also listed in Table 1) in the sample, as compared to the normal copy number of the MCR, also indicates that the subject is afflicted with cancer or at risk for developing cancer. The normal copy number of an MCR is typically one per chromosome.
In another aspect, the invention provides a method for monitoring the progression of cancer in a subject, the method comprising: a) determining in a biological sample from the subject at a first point in time, the expression or activity level of a cancer gene or a candidate cancer gene listed in Table 1; b) repeating step a) at a subsequent point in time; and c) comparing the expression or activity of the gene in steps a) and b), and therefrom monitoring the progression of cancer in the subject.
In another aspect, the invention provides a method of assessing the efficacy of a test agent for treating a cancer in a subject, comprising: a) determining the expression or activity level of at least one cancer gene or a candidate cancer gene located in an amplified MCR in Table 1 in a biological sample from the subject in the presence of the test agent; and b) determining the expression or activity level of the gene in a biological sample from the subject in the absence of the test agent. A decreased expression or activity of the gene in step (a), as compared to that of (b), is indicative of the test agent's potential efficacy for treating the cancer in the subject. Alternatively, if the test agent increases the expression or activity of at least one cancer gene or a candidate cancer gene located in a deleted MCR in Table 1, the test agent is also potentially effective for treating the cancer in a subject.
In another aspect, the invention provides a method of assessing the efficacy of a therapy for treating cancer in a subject, the method comprising: a) determining the expression or activity level of at least one cancer gene or a candidate cancer gene located in an amplified MCR in Table 1 in a biological sample from the subject prior to providing at least a portion of the therapy to the subject; and b) determining the expression or activity level of the gene in a biological sample from the subject following provision of the portion of the therapy. A decreased expression or activity of the gene in step (a), as compared to that of (b), is indicative of the therapy's efficacy for treating the cancer in the subject. Alternatively, if the therapy increases the expression or activity of at least one cancer gene or a candidate cancer gene located in a deleted MCR in Table 1, the therapy is also potentially effective for treating the cancer in a subject.
In another aspect, the invention provides a method of treating a subject afflicted with cancer comprising administering to the subject an agent that decreases the expression or activity level of at least one cancer gene or candidate cancer gene located in am amplified MCR in Table 1. Alternatively, the invention provides a method of treating a subject afflicted with cancer comprising administering to the subject an agent that increases the expression or activity level of at least one cancer gene or candidate cancer gene located in a deleted MCR in Table 1.
In certain embodiments, the agent is an antibody, or its antigen-binding fragment thereof, that specifically binds to a cancer gene or candidate cancer gene listed in Table 1.
In another aspect, the invention provides a method of assessing whether a subject is afflicted with cancer or at risk for developing cancer, the method comprising: determining the copy number of at least one minimal common region (MCR) listed in Table 5 in a biological sample from the subject. A change of copy number of the MCR in the sample, as compared to the normal copy number of the MCR, indicates that the subject is afflicted with cancer or at risk for developing cancer. The normal copy number of an MCR is typically one per chromosome.
In certain embodiments, the cancer is lymphoma. In certain embodiments, the lymphoma is T-ALL.
In another aspect, the invention provides a method of assessing whether a subject is afflicted with cancer or at risk for developing cancer, by comparing the copy number of an MCR, identified using a genome-unstable non-human mammal model (including a genome-unstable mouse model of the invention), with the normal copy number of the MCR. The normal copy number of an MCR is typically one per chromosome.
In vivo cancer models used for the discovery of cancer-related genes and therapeutic cancer targets typically produce cancer cells with benign chromosomal profiles, i.e., nearly normal chromosomal stability. In contrast, in naturally occurring human cancer, cancer cell genomes display widespread instability as evidenced by chromosomal structural aberrations. Accordingly, the present invention provides an in vivo cancer model with a destabilized genome (“genome unstable”).
The genomes of cancer cells from the genome unstable model of the invention simulate the chromosomal instability displayed by human cancer cell genomes The genome unstable cancer model of the invention, thus, provides significant advantages for the discovery of genes and genetic elements involved in human cancer initiation, maintenance and progression. The chromosomal aberrations in cancer cells from the model, particularly recurrent aberrations, permit investigation of chromosomal events in cancer that is not possible in cancer models with “benign” chromosomal profiles. Such chromosomal aberrations also focus attention on particular regions of the genome more likely to harbor cancer-related elements. The validation herein of a genome unstable mouse cancer model that generates chromosomal and genetic events that mirror those in multiple types of human cancers provides an important new tool for the discovery of cancer-related genes and therapeutic targets of relevance to human cancer. Although useful by itself to discover genes and genetic elements relevant to human cancer, the genome unstable model of the invention also can be used as a background for establishing other cancer models, including known cancer models. Layering genetic modifications in known oncogenes and/or tumor suppressors onto the genome unstable model of the invention provides improved models that more closely replicate naturally occurring cancer. Even more importantly, the genome unstable model of the invention permits cross-species comparison with human cancer genomes to identify shared chromosomal and genetic events. Such shared events provide a powerful guide for the discovery of cancer-related genes and therapeutic targets.
Throughout this specification and embodiments, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, cell and cancer biology, virology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.
Most standard genetically engineered mouse models of cancer have relatively benign cytogenetic profiles. These genomically stable models do not reflect the widespread chromosomal instability that is typical of human genomes in cancer. It has been reported that in most “genome-stable” murine tumor models, about 20 to 40 chromosomal aberrations were detected per genome, or, less than 0.1 chromosomal rearrangements per chromosome.
Accordingly, in one aspect, the invention provides a non-human animal that is genetically modified to develop cancer, wherein the genomes of cancer cells from the animal display enhanced chromosomal instability as evidenced by a frequency of chromosomal structural aberration that approaches or matches that seen in human cancer cells. In various embodiments, the frequency of chromosomal structural aberrations in a population of cancer cells from the non-human animal model is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or 10-fold higher than the frequency of chromosomal structural aberrations in such mammal without the genetic modification, whether defined on a per-genome or per-chromosome basis.
The frequency of chromosomal abnormalities can be based on the average number of such abnormalities per genome or per chromosome, or the average number of a particular type of chromosomal abnormality per genome, or the average number of aberrations in a particular chromosome. Methods of measuring chromosomal alterations are known in the art (see, e.g., R. C. O'Hagan, et al., Cancer Res 63 (17), 5352 (2003); N. Bardeesy, et al., Proc Natl Acad Sci USA 103 (15), 5947 (2006); M. Kim, et al., Cell 125 (7), 1269 (2006); L. Zender, et al., Cell 125 (7), 1253 (2006)), and are further disclosed below. Cancer cells from the genome unstable non-human animal model of the invention will have an enhanced frequency of chromosomal aberrations compared to cells derived from comparable non-human animal models lacking the genome destabilizing mechanisms described above, by at least one of the aforementioned parameters.
A chromosomal structural aberration may be any chromosomal abnormality resulting from DNA gains or losses, DNA amplification, DNA deletion, and DNA translocation. Exemplary chromosomal structural aberrations include, for example, sister chromatid exchanges, multi-centric chromosomes, inversions, gains, losses, reciprocal and non-reciprocal translocations (NRTs), p-p robertsonian-like translocations of homologous and/or non-homologous chromosomes, p-q chromosome arm fusions, and q-q chromosome arm fusions.
The genetic modifications in the genome unstable animal model of the invention can be in any gene or genetic element that renders the animal cancer-prone and affects genome structure or genome stability, so that the modifications destabilize the genome, as evidenced by an increased frequency of chromosomal structural aberrations in the genomes and/or chromosomes of cancer that develops in the animal compared to genomes and/or chromosomes in comparable animal models lacking such genome destabilizing mechanisms. Genetic elements include [DNA that is not translated to produce a protein product such as micro RNA, expression control sequences including DNA transcription factor binding sites, RNA transcription initiation sites, promoters, enhancers, response elements and the like. In some embodiments the genetic modifications inactivate a gene or genetic element involved in chromosomal structural stability or integrity. Inactivation may be by directly inactivating the gene or genetic element, by suppressing the expression, or by inactivating or inhibiting the activity of a gene product, which can be a nucleic acid product including RNA or a protein gene product
In some embodiments, the genetic modifications comprise inactivation of at least one allele of one or more genes or genetic elements involved in DNA repair and inactivation of at least one allele of one or more genes or genetic elements involved in a DNA damage checkpoint. In some embodiments, the genetic modifications further comprise inactivation of at least one allele of a gene or genetic element involved in telomere maintenance. In any of the foregoing embodiments, both alleles of the DNA repair related, DNA damage checkpoint related and/or telomere maintenance related genes or genetic elements may be inactivated.
Any gene or genetic element involved in DNA repair or in a DNA damage checkpoint can be inactivated in the genome unstable model of the invention. Many such genes and genetic elements in humans an other mammals will be known to those of skill in the art. See, for example, R. D. Wood et al., Human DNA Repair Genes, Science, 291: 1284-1289 (February 2001); R A Bulman, S D Bouffler, R Cox and T A Dragani, Locations of DNA Damage Response and Repair Genes in the Mouse and Correlation with Cancer Risk Modifiers, National Radiological Protection Board Report, October 2004 (ISBN 0-85951-544-3). The mouse DNA repair gene database is available at the UK Health Protection Agency website.
They include, for example, genes encoding base excision repair (BER) proteins such as ung, smug1, mbd4, tdg, off1, myh, nth1, mpg, ape1, ape2, lig3, xrcc1, adprt, adprtl2 and adprtl3 or species homologs thereof; mismatch excision repair proteins such as msh2, msh3, msh4, msh5, msh6, pms1, pms3, mlh1, mlh3, pms2l3 and pms2l4 or species homologs thereof; nucleotide excision repair (NER) proteins, non-homologous end joining (NHEJ) proteins, homologous recombination proteins, DNA polymerases, editing and processing nucleases and DNA repair helicases, among others. Wood et al., supra.
Exemplary NHEJ proteins include Ligase4, XRCC4, H2AX, DNAPKcs, Ku70, Ku80, Artemis, Cernunnos/XLF, MRE11, NBS1, and RAD50. Exemplary homologous recombination proteins include RAD51, RAD52, RAD54, XRCC3, RAD51C, BRCA1, BRCA2 (FANCD1), FANCA, FANCB, FANCC, FANCD2; FANCE, FANCF, FANCG, FANCJ (BRIP1/BACH1), FANCL, and FANCM. Exemplary DNA repair helicases include BLM and WRN.
Any gene or genetic element involved in a DNA damage checkpoint can be used in the genome unstable model of the invention. Information about many such genes and genetic elements is readily available and will be well-known those of skill in the art. Exemplary DNA checkpoint proteins include sensor proteins such as RAD1, RAD9, RAD17, HUS1, MRE11, Rad50, and NBS1; mediators such as ATRIP; phosphoinositide 3-kinase related kinase (PIKK) family proteins such as ATM, ATR, SMG-1 and DNA-PK; checkpoint kinases such as Chk1 and Chk2; and effector proteins such as p53, p63, p73, CDC25A, B and C, p21 and 14-3-3β,γ,ξ,σ,ε,η,τ APC; BRCA1, MDM2, MDM4, NBS1, RAD24, RAD 25, RAD50, MDC1, SMC1, and claspin.
In one embodiment of the genome unstable model of the invention, the non-human transgenic animal further comprises engineered inaction of at least one allele of one or more genes or genetic elements involved in synthesizing or maintaining telomere length. In some embodiments, the non-human transgenic mammal is engineered for decreased telomerase activity, for example by inactivation of telomerase reverse transcriptase, Tert, or telomerase RNA (Terc). In some embodiments the genetic modification decreases the activity of a protein affecting telomere structure such as capping function. Exemplary proteins that affect telomere structure include TRF1, TRF2, POT1a, POT1b, RAP1, TIN2, and TPP1.
The non-human genome unstable model of the invention may be any animal, including, fish, birds, mammals, reptiles, amphibians. Preferably, the animal is a mammal, including rodents, primates, cats, dogs, goats, horses, sheep, pigs, cows. In preferred embodiments, the mammal is a mouse.
The genome unstable animal models of the invention include animals in which all or only some portion of cells comprise the genetic modifications that create genome instability. In some embodiments, the germ cells of the animal comprise the genetic modifications.
In some embodiments, the genome unstable model comprises inactivation of one or both alleles of atm, terc or p53 or any combination of those genes. In a particular embodiment, one or both alleles of all three genes are inactivated. In some embodiments both alleles of atm are inactivated. In a particular embodiment, both alleles of all three genes are inactivated.
Also within the invention are tissues and cells from the genome unstable model of the invention, including somatic cells, germ cells, stem cells including embryonic stem cells, differentiated cells and undifferentiated cells. The cells may be cancer cells, non-cancer cells, or pre-cancer cells.
Inactivation of a gene or a genetic element in the genome unstable animal model of the invention can be achieved by any means, many of which are well-known to those of skill in the art. Such means include deletion of all or part of the gene or genetic element or introducing an inactivating mutation (lesion) in the gene or genetic element. Deletion of all or a portion of a gene or genetic element may be by knock-out such as by homologous recombination or techniques using Cre recombinase (e.g., a Cre-Lox system). Deletions including knock-outs can be conditional knock-outs, where alteration of a nucleic acid sequences can occur upon, for example, exposure of the animal to a substance that promotes gene alteration, introduction of an enzyme that promotes recombination at the gene site (e.g., Cre in the Cre-lox system), or other method for directing the gene alteration. Conditional or constitutive knock-outs can be tissue-specific, temporally-specific (e.g., occurring during a particular developmental stage) or both.
Inactivating mutations may be introduced using any means, many of which are well known. Such methods include site directed mutagenesis for example using homologous recombination or PCR. Such mutations may be introduced in the 5′ untranslated region (UTR) of a gene, including in an expression control region, in a coding region (intron or exon) or in the 3′ UTR.
The expression or activity of a gene or genetic element also may be accomplished by any means including but not limited to RNA interference, antisense including triple helix formation and ribozymes including RNaseP, leadzymes, hairpin ribozymes and hammerhead ribozymes.
In some embodiments, the genome unstable animal model of the invention further comprises one or more additional cancer-promoting genetic modifications including but not limited to the introduction of one or more activated oncogenes, modifications to increase the expression of one or more oncogenes, targeted inactivation of one or more tumor-suppressors, or combinations of the foregoing. Such additional cancer-promoting modifications may be inducible, tissue specific, temporally specific or any combination of the three. For example, an oncogene can be introduced into the genome using an expression cassette that includes in the 5′-3′ direction of transcription, a transcriptional and translational initiation region that is associated with gene expression in a specific tissue type, an oncogene, and a transcriptional and translational termination region functional in the host animal. One or more introns may also be present. In addition to the oncogene of interest, a detectable marker, such as GFP (and its variants), luciferase, and lacZ may be optionally operably linked to the oncogene and co-expressed. Similarly, a tumor-suppressor-gene may be inactivated using, for example, gene targeting technology.
Introducing additional cancer-promoting modifications into a genome-unstable animal model described herein creates a powerful tool for cancer gene discovery. For example, Kras activation and p53 mutation in pancreas are known to cause pancreas cancer in human. A genome-unstable model having pancreas-specific Kras activation, p53 inactivation (and optionally, a decreased telomere function) would greatly facilitate the discovery of pancreas cancer gene in human.
The cancer in the genome unstable model any type of cancer, including carcinoma, sarcoma, myeloma, leukemia, lymphoma or mixed cancer types. The cancer can arise from any tissue type including epithelial tissue, mesenchymal tissue, nervous tissue and hematopoietic tissue and be located in any organ or tissue of the body. The frequency of chromosomal aberrations can be determined in cells from any of the aforementioned cancers and can be from a primary tumor, a secondary tumor, a metastatic tumor, a tumor recurrence perhaps normal cells derived from said genomically unstable model that were genetically manipulated in vitro, through additional oncogene activation and tumor suppressor gene inactivation introduced by those knowledgeable in the art, to become cancerous
The genome unstable mouse model of the invention may develop any cancer including but not limited to acral lentiginous melanoma, actinic keratoses, adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, anaplastic glioma, astrocytic tumors, astrocytomas, bartholin gland carcinoma, basal cell carcinoma, biliary tract cancer, bone cancer, bile duct cancer, bladder cancer, brain stem glioma, brain tumors, breast cancer, bronchial gland carcinomas, capillary carcinoma, carcinoids, carcinoma, carcinosarcoma, cavernous, central nervous system lymphoma, cerebral astrocytoma, cervical cancer, connective tissue cancer, cholangiocarcinoma, chondosarcoma, choroid plexus papilloma/carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal, ependymoma, epitheloid, esophageal cancer, Ewing's sarcoma, extragonadal germ cell tumor, eye cancer, fibrolamellar, focal nodular hyperplasia, gallbladder cancer, gangliogliomas, gastric cancer, gastrinoma, germ cell tumors, gestational trophoblastic tumor, glioblastoma multiforme, glioma, glucagonoma, head and neck cancer, hemangiblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, childhood, insulinoma, intaepithelial neoplasia, interepithelial squamous cell neoplasia, intraocular melanoma, intra-epithelial neoplasm, invasive squamous cell carcinoma, large cell carcinoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lentigo maligna melanomas, leukemia-related disorders, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, malignant mesothelial tumors, malignant thymoma, medulloblastoma, medulloepithelioma, melanoma, meningeal, merkel cell carcinoma, mesothelial, metastatic carcinoma, mucoepidermoid carcinoma, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neurofibromatosis, neuroepithelial adenocarcinoma nodular melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oat cell carcinoma, oligodendroglial, oligoastrocytomas, oral cancer, oropharyngeal cancer, osteosarcoma, pancreatic polypeptide, ovarian cancer, ovarian germ cell tumor, pancreatic cancer, papillary serous adenocarcinoma, pineal cell, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, parathyroid cancer, penile cancer, pheochromocytoma, pineal and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell carcinoma, cancer of the respiratory system, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, skin cancer, small cell carcinoma, small intestine cancer, soft tissue carcinomas, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, stomach cancer, stromal tumors, submesothelial, superficial spreading melanoma, supratentorial primitive neuroectodermal tumors, testicular cancer, thyroid cancer, undifferentiatied carcinoma, urethral cancer, uterine sarcoma, uveal melanoma, verrucous carcinoma, vaginal cancer, vipoma, vulvar cancer, Waldenstrom's macroglobulinemia, well differentiated carcinoma, and Wilm's tumor.
The animal models described herein are typically obtained using transgenic technologies. Transgenic technologies are well known in the art. For example, transgenic mouse can be prepared in a number of ways. A exemplary method for making the subject transgenic animals is by zygote injection. This method is described, for example in U.S. Pat. No. 4,736,866. The method involves injecting DNA into a fertilized egg, or zygote, and then allowing the egg to develop in a pseudo-pregnant mother. The zygote can be obtained using male and female animals of the same strain or from male and female animals of different strains. The transgenic animal that is born is called a founder, and it is bred to produce more animals with the same DNA insertion. In this method of making transgenic animals, the exogenous DNA typically randomly integrates into the genome by a non-homologous recombination event. One to many thousands of copies of the DNA may integrate at one site in the genome.
In another aspect, the invention provides methods for identifying genes and genetic elements involved in cancer initiation, maintenance and/or progression in humans utilizing the genome unstable model of the invention. The gene discovery and identification methods are based on the surprising discovery described herein that chromosomal structural aberrations, copy number alterations and mutations in cancer cells in a genome unstable mouse model have syntenic counterparts (i.e., occurring in evolutionarily related chromosomal regions) in human cancer cells.
Accordingly, in one embodiment, the invention provides a method of identifying a chromosomal region of interest for the identification of a gene that is potentially related to human cancer, comprising the step of identifying a DNA copy number alteration in a population of cancer cells from a non-human, genome-unstable mammal described above. The chromosomal region where the DNA copy number alteration occurred is a chromosomal region of interest for the identification of a gene or genetic element (such as microRNAs) that is potentially related to human cancer.
A DNA copy number alteration may be a DNA gain (such as amplification of a genomic region) or a DNA loss (such as deletion of a genomic region). Methods of evaluating the copy number of a particular genomic region are well known in the art, and include, hybridization and amplification based assays. According to the methods of the invention, DNA copy number alterations may be identified using copy number profiling, such as comparative genomic hybridization (CGH) (including both dual channel hybridization profiling and single channel hybridization profiling (e.g. SNP-CGH)). Other suitable methods including fluorescent in situ hybridization (FISH), PCR, nucleic acid sequencing, and loss of heterozygosity (LOH) analysis may be used in accordance with the invention.
In one embodiment of the invention, the DNA copy number alterations in a genome are determined by copy number profiling.
In some embodiments of the invention, the DNA copy number alterations are identified using CGH. In comparative genomic hybridization methods, a “test” collection of nucleic acids (e.g. from a tumor or cancerous cells) is labeled with a first label, while a second collection (e.g. from a normal cell or tissue) is labeled with a second label. The ratio of hybridization of the nucleic acids is determined by the ratio of the first and second labels binding to each fiber in an array. Differences in the ratio of the signals from the two labels, for example, due to gene amplification in the test collection, is detected and the ratio provides a measure of the gene copy number, corresponding to the specific probe used. A cytogenetic representation of DNA copy-number variation can be generated by CGH, which provides fluorescence ratios along the length of chromosomes from differentially labeled test and reference genomic DNAs.
In some embodiments of the present invention, the DNA copy number alterations are analyzed by microarray-based CGH (array-CGH). Microarray technology offers high resolution. For example, the traditional CGH generally has a 20 Mb limited mapping resolution; whereas in microarray-based CGH, the fluorescence ratios of the differentially labeled test and reference genomic DNAs provide a locus-by-locus measure of DNA copy-number variation, thereby achieving increased mapping resolution. Details of various microarray methods can be found in the literature. See, for example, U.S. Pat. No. 6,232,068; Pollack et al., Nat. Genet., 23(1):41-6, (1999), Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) Nature Biotechnology 14:1685; Chee (1995) Science 274: 610; WO 96/17958, Pinkel et al. (1998) Nature Genetics 20: 207-211 and others.
The DNA used to prepare the CGH arrays is not critical. For example, the arrays can include genomic DNA, e.g. overlapping clones that provide a high resolution scan of a portion of the genome containing the desired gene or of the gene itself. Genomic nucleic acids can be obtained from, e.g., HACs, MACs, YACs, BACs, PACs, PIs, cosmids, plasmids, inter-Alu PCR products of genomic clones, restriction digests of genomic clones, cDNA clones, amplification (e.g., PCR) products, and the like. Arrays can also be obtained using oligonucleotide synthesis technology. For example, see, e.g., light-directed combinatorial synthesis of high density oligonucleotide arrays U.S. Pat. No. 5,143,854 and PCT Patent Publication Nos. WO 90/15070 and WO 92/10092.
The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other suitable methods include are the nucleic acid sequence based amplification (NASBAO, Cangene, Mississauga, Ontario) and Q Beta Replicase systems.
In one embodiment of the invention, the DNA copy number alterations in a genome are determined by single channel profiling, such as single nucleotide polymorphism (SNP)-CGH. Traditional CGH data consists of two channel intensity data corresponding to the two alleles. The comparison of normalized intensities between a reference and subject sample is the foundation of traditional array-CGH. Single channel profiling (such as SNP-CGH) is different in that a combination of two genotyping parameters are analyzed: normalized intensity measurement and allelic ratio. Collectively, these parameters provide a more sensitive and precise profile of chromosomal aberrations. SNP-CGH also provides genetic information (haplotypes) of the locus undergoing aberration. Importantly, SNP-CGH has the capability of identifying copy-neutral LOH events, such as gene conversion, which cannot be detected with array-CGH.
In another embodiment, FISH is used to determine the DNA copy number alterations in a genome. Fluorescence in situ hybridization (FISH) is known to those of skill in the art (see Angerer, 1987 Meth. Enzymol., 152: 649). Generally, in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization, and (5) detection of the hybridized nucleic acid fragments.
In a typical in situ hybridization assay, cells or tissue sections are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The targets (e.g., cells) are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained.
The probes used in such applications are typically labeled, for example, with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to enable specific hybridization with the target nucleic acid(s) under stringent conditions.
In some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, human genomic DNA, or Cot-1 DNA is used to block non-specific hybridization.
In another embodiment, Southern blotting is used to determine the DNA copy number alterations in a genome. Methods for doing Southern blotting are known to those of skill in the art (see Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York, 1995, or Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed. vol. 1-3, Cold Spring Harbor Press, NY, 1989). In such an assay, the genomic DNA (typically fragmented and separated on an electrophoretic gel) is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal genomic DNA (e.g., genomic DNA from the same or related cell, tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid.
In one embodiment, amplification-based assays, such as PCR, are used to determine the DNA copy number alterations in a genome. In such amplification-based assays, the genomic region where a copy number alteration occurred serves as a template in an amplification reaction. In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the copy number of the genomic region.
Methods of “quantitative” amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided, for example, in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.
Real time PCR can be used in the methods of the invention to determine DNA copy number alterations. (See, e.g., Gibson et al., Genome Research 6:995-1001, 1996; Heid et al., Genome Research 6:986-994, 1996). Real-time PCR evaluates the level of PCR product accumulation during amplification. To measure DNA copy number, total genomic DNA is isolated from a sample. Real-time PCR can be performed, for example, using a Perkin Elmer/Applied Biosystems (Foster City, Calif.) 7700 Prism instrument. Matching primers and fluorescent probes can be designed for genes of interest using, for example, the primer express program provided by Perkin Elmer/Applied Biosystems (Foster City, Calif.). Optimal concentrations of primers and probes can be initially determined by those of ordinary skill in the art, and control (for example, beta-actin) primers and probes may be obtained commercially from, for example, Perkin Elmer/Applied Biosystems (Foster City, Calif.). To quantitate the amount of the specific nucleic acid of interest in a sample, a standard curve is generated using a control. Standard curves may be generated using the Ct values determined in the real-time PCR, which are related to the initial concentration of the nucleic acid of interest used in the assay. Standard dilutions ranging from 10-106 copies of the gene of interest are generally sufficient. In addition, a standard curve is generated for the control sequence. This permits standardization of initial content of the nucleic acid of interest in a tissue sample to the amount of control for comparison purposes.
Methods of real-time quantitative PCR using TaqMan probes are well known in the art. Detailed protocols for real-time quantitative PCR are provided, for example, for RNA in: Gibson et al., 1996, A novel method for real time quantitative RT-PCR. Genome Res., 10:995-1001; and for DNA in: Heid et al., 1996, Real time quantitative PCR. Genome Res., 10:986-994.
A TaqMan-based assay also can be used to quantify a particular genomic region for DNA copy number alterations. TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5′ fluorescent dye and a 3′ quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3′ end. When the PCR product is amplified in subsequent cycles, the 5′ nuclease activity of the polymerase, for example, AmpliTaq, results in the cleavage of the TaqMan probe. This cleavage separates the 5′ fluorescent dye and the 3′ quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, for example, http://www2.perkin-elmer.com).
Other suitable amplification methods include, but are not limited to ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4:560, Landegren et al. (1988) Science 241:1077, and Barringer et al. (1990) Gene 89:117), transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173), self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87:1874), dot PCR, and linker adapter PCR, etc.
In one embodiment, DNA sequencing is used to determine the DNA copy number alterations in a genome. Methods for DNA sequencing are known to those of skill in the art.
In one embodiment, karyotyping (such as spectral karyotyping, SKY) is used to determine the chromosomal structural aberrations in a genome. Methods for karyotyping are known to those of skill in the art. For example, for SKY, a collection of DNA probes, each complementary to a unique region of one chromosome, may be prepared and labeled with a fluorescent color that is designated for a specific chromosome. DNA amplification, deletion, translocations or other structural abnormalities may be determined based on fluorescence emission of the probes.
In certain embodiments, tumor samples from two or more genome-unstable animal models of the invention are analyzed for DNA copy number alterations, and the common genomic regions where the copy number alterations occurred in at least two of the samples are identified. Such recurrent DNA copy number alterations are of particular interest.
A minimum common region (MCR) of the recurrent DNA copy number alteration may be defined when copy number alterations of two or more samples are compared. In one embodiment, the MCR is defined by the boundaries of overlap between two samples, or by boundaries of a single tumor against a background of larger alterations in at least one other tumor.
Methods for determining MCRs is known in the art (see, e.g., D. R. Carrasco, et al., Cancer Cell 9 (4), 313 (2006); A. J. Aguirre, et al., Proc Natl Acad Sci USA 101 (24), 9067 (2004)). Briefly, a “segmented” dataset was generated by determining uniform copy number segment boundaries and then replacing raw log 2 ratio for each probe by the mean log 2 ratio of the segment containing the probe. A threshold representing minimal copy number alterations (CNAs) is then chosen to filter out noise. For example, the median log 2 ratio of a two-fold change for the platform may be chosen as a threshold. In an exemplary embodiment, the thresholds representing CNAs are +/−0.6 (Agilent 22K a-CGH platform) and +/−0.8 (Agilent 44K/244K a-CGH platform), and the width of MCR is less than 10 Mb.
The boundaries of MCRs can be mapped by any method that is known in the art, such as southern blotting, or PCR.
Genes and genetic elements located within an MCR are potentially related to human cancer and such genes and genetic elements can be subject to additional analyses to further characterize them. For example, a gene that is initially identified by array-CGH may be quantitatively amplified. Quantitative amplification of either the identified genomic DNA or the corresponding RNA can confirm DNA gain or loss. Alternatively, if the sequence encodes a protein, the mRNA level, protein level, or activity level of the encoded protein may be measured. An increase in RNA/protein/activity level, as compared to a control, confirms DNA amplification; a decrease in RNA/protein/activity level, as compared to a control, confirms DNA deletion.
The gene or genetic element identified through initial screening may also be re-sequenced to confirm amplification or deletion. Further, DNA sequencing and protein expression profiling may also be used to identify genetic mutations that may be associated with tumorigenesis.
In another aspect, the invention provides a method of identifying a chromosomal region of interest for the identification of a gene or genetic element that is potentially related to human cancer, comprising the step of identifying a chromosomal structural aberration in a population of cancer cells from a genome-unstable animal models of the invention. A chromosomal region containing the chromosomal structural aberration is a chromosomal region of interest for the identification of a gene or genetic element that is potentially related to human cancer.
In some embodiments, the chromosomal structural aberration is detected using karyotyping, such as SKY. In some embodiments, the method further comprises determining the DNA copy number alteration, as described above. A chromosomal region containing the both chromosomal structural aberration and a DNA copy number alteration is a chromosomal region of interest for the identification of a gene or genetic element that is potentially related to human cancer.
In another aspect, the invention provides a method of identifying a potential human cancer-related gene or genetic element, comprising the steps of (a) identifying a chromosomal region of interest as described herein; (b) identifying a gene or a genetic element within the chromosomal region of interest in the non-human animal, and (c) identifying a human gene or genetic element that corresponds to the gene or genetic element identified in step (b).
Additionally, many public and private databases provide cancer gene information (for example, Sanger's Cancer Gene Census, at http://www.sanger.ac.uk/genetics/CGP/Census), and the information may be used to map known cancer genes to a particular chromosomal region.
If a gene or a genetic element is found to be potentially relevant to human cancer, the corresponding human gene may be identified by homolog mapping, ortholog mapping, paralog mapping, among other methods. As used herein, a homolog is a gene related to a second gene by descent from a common ancestral DNA sequence, an ortholog is a gene in a different species that evolved from a common ancestral gene by speciation, and a paralogs is a gene related by duplication within a genome.
In one embodiment, human homologs are identified by using, for example, the NCBI homologene website, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=homologene.
In some embodiments, the method further comprises detecting a mutation in the identified non-human gene or genetic element. In another embodiment, a mutation in the corresponding human gene or genetic element is identified. In another embodiment, mutations in the both the non-human gene or genetic element and the human gene or genetic element are identified, and the mutations are compared.
In another aspect, the invention provides a method of identifying a potential human cancer-related gene or genetic element, comprising the steps of (a) detecting a DNA copy number alteration in a population of cancer cells from a non-human mammal, wherein the genome of the non-human mammal is engineered to produce genome instability, (b) identifying a gene or genetic element located within the boundaries of the copy number alteration detected in step (a), (c) identifying a human gene or genetic element that corresponds to the gene or genetic element identified in step (b) and that is located within the boundaries of a copy number alteration or of a chromosomal structural aberration in a human cancer cell. The human gene or genetic element identified in step (c) is a gene potentially related to human cancer.
Methods for detecting a copy number alteration or a chromosomal structural aberration have been described above in detail. Methods for identifying a gene or genetic element located within the boundaries of the copy number alteration are also described above in detail.
In one embodiment, a copy number alteration or a chromosomal structure aberration in the non-human animal model of the invention is compared with a copy number alteration or a chromosomal structural aberration in human cancer cell. A potentially relevant human cancer related gene or genetic element is identified based on synteny. Synteny describes the preserved order and orientation of genes between related species. Comparisons of non-human animal model and human cancer syntenic chromosomal regions may reveal the conserved nature of certain genetic modification in tumorgenesis.
The cross-species comparison based on synteny has several advantages. First is the ability to narrow the chromosomal regions of interest—certain genomic modification is more focal in one species than the other, and a cross-species comparison may eliminate such species-specific event. Second, a minimal common region (MCR) typically contains a number of genes; a cross-species comparison of syntenic regions allows an efficient way to reduce the gene numbers because the syntenic regions of the genome between non-human mammals (in particular, mice) and humans may be in relatively small portions. Genes located within syntenic MCRs may be highly relevant to human cancers.
In another aspect, the invention provides a method of identifying a potential human cancer-related gene or genetic element, comprising the steps of (a) detecting a chromosomal structural aberration in a population of cancer cells from a non-human mammal, wherein the genome of the non-human mammal is engineered to produce genome instability, (b) identifying a gene or genetic element located within the boundaries of the copy number alteration detected in step (a), (c) identifying a human gene or genetic element that corresponds to the gene or genetic element identified in step (b) and that is located within the boundaries of a copy number alteration or of a chromosomal structural aberration in a human cancer cell. The human gene or genetic element identified in step (c) is a gene potentially related to human cancer.
In one aspect, the present invention provides a method for identifying subjects with T-cell acute lymphoblastic leukemia (T-ALL) who may have a decreased or increased response to γ-secretase inhibitor therapy, based on the discovery that inactivation of FBXW7 is associated with human T-cell malignancy.
In one embodiment, the method for identifying subjects with T-ALL who may have a decreased response to a γ-secretase inhibitor therapy comprises: detecting in a cancer cell from the subject the expression level or activity level of FBXW7; a decreased expression/activity of FBXW7, as compared to a control, indicates that the subject may have a decreased response to a γ-secretase inhibitor therapy. The expression or activity level of NOTCH1 in the cancer cell may also be determined simultaneously; an increased expression/activity of NOTCH1, as compared to a control, further indicates that the subject may have a decreased response to a γ-secretase inhibitor therapy. Conversely, an increased expression/activity of FBXW7 (together with a decreased expression/activity of NOTCH1, optionally), as compared to a control, indicates that the subject may be sensitive to a γ-secretase inhibitor therapy.
γ-Secretase is a complex composed of at least four proteins, namely presenilins (presenilin 1 or -2), nicastrin, PEN-2, and APH-1. Several proteins have been identified as substrates for γ-secretase cleavage, include Notch and the Notch ligands Delta1 and Jagged2, ErbB4, CD44, and E-cadherin (Wong, G. T. et. al, J. Biol. Chem., Vol. 279, Issue 13, 12876-12882, Mar. 26, 2004). The cleavage of Notch by γ-secretase has been studied most extensively. Notch plays an evolutionarily conserved role in regulating cell growth and lineage specification particularly during embryonic development. Notch is activated by several ligands (Delta, Jagged, and Serrate) and is then proteolytically processed by a series of ligand-dependent and -independent cleavages. γ-Secretase catalyzes the terminal cleavage event (S3 cleavage), which releases a fragment known as the Notch intracellular domain (NICD). The NICD fragment then translocates to the nucleus where it acts as a nuclear transcription factor. As expected from its role in Notch S3 cleavage, γ-secretase inhibitors have been shown to block NICD production in vitro. In vivo, Notch function appears to be critical for the proper differentiation of T and B lymphocytes, and γ-secretase inhibitors reduce the thymocyte number and block thymocyte differentiation at an early stage in fetal thymic organ cultures.
The FBXW7 gene (also called hCDC4) encodes a key component of the E3 ubiquitin ligase that is implicated in the control of chromosome stability (Mao J. et. al, Nature 432, 775-779 (2004)). FBXW7 is responsible for binding the PEST domain of intracellular NOTCH1, leading to ubiquitination and degradation by the proteasome. Because there exists a statistically significant anti-correlation between PEST domain mutations in NOTCH1 and FBXW7 mutation in human T-ALL, T-ALL cells having a reduced expression/activity of FBXW7 will less likely to respond to γ-secretase inhibitors.
One of the recurring problems of cancer therapy is that a patient in remission (after the initial treatment by surgery, chemotherapy, radiotherapy, or combination thereof) may experience relapse. The recurring cancer in those patients is frequently resistant to the apparently successful initial treatment. In fact, certain cancers in patients initially diagnosed with the disease may be already resistant to conventional cancer therapy even without first being exposed to such treatment. γ-secretase inhibitor therapy can be physically exhausting for the patient. Side effects of secretase inhibitors include weight loss, changes in gastrointestinal tract architecture, accumulation of necrotic cell debris, dilation of crypts and infiltration of inflammatory cells, nausea, vomiting, weakness, diarrhea elevation in white blood cell count, and esophageal failure (Siemers E. et al, 2005 May-June; 28(3):126-32; Wong, G T. et al, J Biol Chem. 2004 Mar. 26; 279 (13):12876-82). Thus there is a need to determine whether a cancer patient may benefit from a chemotherapeutic treatment prior to the commencement of the treatment.
In one embodiment, a cancer patient is screened based on the expression level of FBXW7 and optionally, NOTCH1, in a cancer cell sample.
The expression level of FBXW7 or NOTCH1 may be measured by DNA level, mRNA level, protein level, activity level, or other quantity reflected in or derivable from the gene or protein expression data. For example, a genetic alteration may result in a decreased expression of FBXW7. Common genetic alterations include deletion of at lease one FBXW7 gene from the genome, or a mutation in at least one allele of an FBXW7 gene. The mutation may be a mis-sense mutation; a non-sense mutation; an insertion, deletion, or substitution of one or more nucleotides; a truncation from the 5′ terminal (either untranslated region or coding region), 3′ terminal (either untranslated region or coding region), or both; a substitution of one or more nucleotides in the 5′ untranslated region, 3′ untranslated region, coding region (which results in an amino acid change), or combinations of the three. Exemplary genetic alterations include a mutation in the third WD40 domain or the fourth WD40 domain of the FBXW7, G423V, R465C, R465H, R479L. R479Q, R505C and D527G mutations. A genetic alteration may also result in an increased expression of NOTCH1, such as translocation or copy number amplification of NOTCH1 gene.
The mRNA level of FBXW7 or NOTCH 1 may be measured using any art-known method, such as PCR, northern blotting, RNase Protection Assay, or microarray hybridization. For example, Real-time polymerase chain reaction, also called quantitative real time PCR (QRT-PCR) or kinetic polymerase chain reaction, is widely used in the art to measure mRNA level of a target gene. The QRT-PCR procedure follows the general pattern of polymerase chain reaction, but the DNA is quantified after each round of amplification. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA. QRT-PCR can be combined with reverse transcription polymerase chain reaction to quantify low abundance messenger RNA (mRNA), enabling one to quantify relative gene expression at a particular time, or in a particular cell or tissue type.
The expression level of FBXW7 or NOTCH1 may also be measured by protein level using any art-known method. Traditional methodologies for protein quantification include 2-D gel electrophoresis, mass spectrometry and antibody binding. Frequently used methods for assaying target protein levels in a biological sample include antibody-based techniques, such as immunoblotting (western blotting), immunohistological assay, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or protein chips. Gel electrophoresis, immunoprecipitation and mass spectrometry may be carried out using standard techniques. Additionally, NOTCH1 expression may be measured by detection of cleaved, intranuclear (ICN) form of NOTCH1 protein in cells.
The expression level of FBXW7 or NOTCH1 may also be measured by the activity level of the gene product using any art-known method, such as transcriptional activity of NOTCH1 or ligase activity of FBXW7. For example, NOTCH1 activity may be measured by a increased binding of ICN of NOTCH1. Alternatively, the expression level of a transcriptional downstream target of NOTCH1 may be measured as an indicator of NOTCH1 activity, such as c-Myc, PTCRA, Hes1, etc.
In certain embodiments, it is useful to compare the expression/activity level of FBXW7 or NOTCH1 to a control. The control may be a measure of the expression level of FBXW7 or NOTCH1 in a quantitative form (e.g., a number, ratio, percentage, graph, etc.) or a qualitative form (e.g., band intensity on a gel or blot, etc.). A variety of controls may be used. Levels of FBXW7 or NOTCH1 expression from a non-cancer cell of the same cell type from the subject may be used as a control. Levels of FBXW7 or NOTCH1 expression from the same cell type from a healthy individual may also be used as a control. Alternatively, the control may be expression levels of FBXW7 or NOTCH1 from the individual being treated at a time prior to treatment or at a time period earlier during the course of treatment. Still other controls may include expression levels present in a database (e.g., a table, electronic database, spreadsheet, etc.) or a pre-determined threshold.
The present invention further discloses methods of treating a T-ALL subject who will likely be sensitive a treatment with γ-secretase inhibitors (identified using the methods described above), comprising administering to the patients a γ-secretase inhibitor. γ-secretase inhibitors are known in the art, exemplary γ-secretase inhibitors include LY450139 Dihydrate and LY411575.
The present invention further discloses methods of treating a T-ALL subject who will has a decreased expression/activity of FBXW7 (identified using the methods described above) with an agent that increases the expression/activity of FBXW7. The agent may be a recombinant FBXW7 protein or a functionally active fragment or derivative thereof, a nuclei acid that encodes FBXW7 protein or a functionally active fragment or derivative thereof, or an agent that activates FBXW7. A “functionally active” PBXW7 fragment or derivative exhibits one or more functional activities associated with a full-length, wild-type FBXW7 protein, such as antigenic or immunogenic activity, ability to bind natural cellular substrates, etc. The functional activity of FBXW7 proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science, Coligan et al., eds., John Wiley & Sons, Inc., Somerset, N.J. (1998)).
In another aspect, the present invention provides a method for identifying subject with T-ALL who may benefit from treatment with a phosphatidylinositol 3-kinase (PI3K) pathway inhibitor, based on the discovery that PTEN inactivation is associated with human T-cell malignancy.
PTEN has been characterized as a tumor suppressor gene that regulates cell cycle. PTEN functions as a phosphodiesterase and an inhibitor of the PI3K/AKT pathway, by removing the 3′ phosphate group of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). When PTEN is inactivated, increased production of PIP3 activates AKT (protein kinase B). The AKT pathway promotes tumor progression by enhancing cell proliferation, growth, survival, and motility, and by suppressing apoptosis. AKT is activated by two phosphorylation events catalyzed by the phosphoinositide dependent kinase PDK1, an enzyme that is activated by PI3K.
In one embodiment, the method for identifying subject with T-ALL who may benefit from treatment with a PI3K pathway inhibitor comprises: detecting in a tumor cell from the subject the expression level or activity level of PTEN. A decreased expression/activity of FBXW7, as compared to a control, indicates that the subject may benefit from a PI3K inhibitor therapy.
The phospho-AKT level in the cancer cell from the subject may also be determined simultaneously; an increased phospho-AKT level, as compared to a control, further indicates that the subject may benefit from a PI3K inhibitor therapy.
The expression level of PTEN may be measured by DNA level, mRNA level, protein level, activity level, or other quantity reflected in or derivable from the gene or protein expression data. For example, a genetic alteration may result in a decreased expression of PTEN. Common genetic alterations include deletion of at least one PTEN gene from the genome, or a mutation in at least one allele of a PTEN gene. The mutation may be a mis-sense mutation; a non-sense mutation; an insertion, deletion, or substitution of one or more nucleotides; a truncation from the 5′ terminal (either untranslated region or coding region), 3′ terminal (either untranslated region or coding region), or both; a substitution of one or more nucleotides in the 5′ untranslated region, 3′ untranslated region, coding region (which results in an amino acid change), or combinations of the three.
The expression level of PTEN may also be measured by mRNA level using any method known in the art, such as PCR, Northern blotting, RNase Protection Assay, and microarray hybridization.
The expression level of PTEN may also be measured by protein level using any method known in the art, such as 2-D gel electrophoresis, mass spectrometry and antibody binding
The expression level of PTEN may also be measured by the activity level of PTEN using any art-known method, such as measuring the phosphatase activity. Additionally, the expression or activity of other proteins involved in the PI3K/AKT pathway may also be measured as a proxy for PTEN activity. For example, the phospho-AKT level in a cell generally reflects the PTEN activity, therefore may be measured as a marker for PTEN activity.
In certain embodiments, a control may be used to compare the expression/activity level of PTEN. As described in detail above, a control may be derived from a non-cancer cell of the same type from the subject, same cell type from a healthy individual, a predetermined value, etc.
The present invention further discloses methods of treating a T-ALL subject who may benefit from a treatment with PI3K inhibitors (identified using the methods described above), comprising administering to the patients a PI3K inhibitor. PI3K inhibitors are well know in the art (e.g., Pinna, L A and Cohen, P T W (eds.) Inhibitors of Protein Kinases and Protein Phosphates, Springer (2004) and Abelson, J N, Simon, M I, Hunter, T, Sefton, B M (eds.) Methods in Enzymology, Volume 201: Protein Phosphorylation, Part B: Analysis of Protein Phosphorylation, Protein Kinase Inhibitors, and Protein Academic Press (2007)).
The present invention further discloses methods of treating a T-ALL subject who will has a decreased expression/activity of PTEN (identified using the methods described above) with an agent that increases the expression/activity of PTEN. The agent may be a recombinant PTEN protein or a functionally active fragment or derivative thereof, a nuclei acid that encodes PTEN protein or a functionally active fragment or derivative thereof, or an agent that activates PTEN.
In another aspect, the invention provides a method of assessing whether a subject is afflicted with cancer or at risk for developing cancer, comprising: determining the expression or activity level of at least one cancer gene or candidate cancer gene located in an amplified MCR in Table 1 in a biological sample from the subject. An increase in the expression or activity the gene, as compared to a control, indicates that the subject is afflicted with cancer or at risk for developing cancer. Alternatively, if there is a decrease in the expression or activity of a cancer gene or candidate cancer gene located in a deleted MCR in Table 1, as compared to a control, the decreased expression or activity level also indicates that the subject is afflicted with cancer or at risk for developing cancer.
In another aspect, the invention provides a method of assessing whether a subject is afflicted with cancer or at risk for developing cancer, the method comprising: determining the copy number of at least one amplified minimal common region (MCR) listed in Table 1 in a biological sample from the subject. An increased copy number of the MCR in the sample, as compared to the normal copy number of the MCR, indicates that the subject is afflicted with cancer or at risk for developing cancer. Alternatively, a decreased copy number of a deleted MCR (also listed in Table 1) in the sample, as compared to the normal copy number of the MCR, also indicates that the subject is afflicted with cancer or at risk for developing cancer. The normal copy number of an MCR is typically one per chromosome.
In another aspect, the invention provides a method for monitoring the progression of cancer in a subject, the method comprising: a) determining in a biological sample from the subject at a first point in time, the expression or activity level of a cancer gene or a candidate cancer gene listed in Table 1; b) repeating step a) at a subsequent point in time; and c) comparing the expression or activity of the gene in steps a) and b), and therefrom monitoring the progression of cancer in the subject.
In another aspect, the invention provides a method of assessing the efficacy of a test agent for treating a cancer in a subject, comprising: a) determining the expression or activity level of at least one cancer gene or a candidate cancer gene located in an amplified MCR in Table 1 in a biological sample from the subject in the presence of the test agent; and b) determining the expression or activity level of the gene in a biological sample from the subject in the absence of the test agent. A decreased expression or activity of the gene in step (a), as compared to that of (b), is indicative of the test agent's potential efficacy for treating the cancer in the subject. Alternatively, if the test agent increases the expression or activity of at least one cancer gene or a candidate cancer gene located in a deleted MCR in Table 1, the test agent is also potentially effective for treating the cancer in a subject.
In another aspect, the invention provides a method of assessing the efficacy of a therapy for treating cancer in a subject, the method comprising: a) determining the expression or activity level of at least one cancer gene or a candidate cancer gene located in an amplified MCR in Table 1 in a biological sample from the subject prior to providing at least a portion of the therapy to the subject; and b) determining the expression or activity level of the gene in a biological sample from the subject following provision of the portion of the therapy. A decreased expression or activity of the gene in step (a), as compared to that of (b), is indicative of the therapy's efficacy for treating the cancer in the subject. Alternatively, if the therapy increases the expression or activity of at least one cancer gene or a candidate cancer gene located in a deleted MCR in Table 1, the therapy is also potentially effective for treating the cancer in a subject.
In another aspect, the invention provides a method of treating a subject afflicted with cancer comprising administering to the subject an agent that decreases the expression or activity level of at least one cancer gene or candidate cancer gene located in am amplified MCR in Table 1. Alternatively, the invention provides a method of treating a subject afflicted with cancer comprising administering to the subject an agent that increases the expression or activity level of at least one cancer gene or candidate cancer gene located in a deleted MCR in Table 1.
In certain embodiments, the agent is an antibody, or its antigen-binding fragment thereof, that specifically binds to a cancer gene or candidate cancer gene listed in Table 1. Optionally, the antibody may be conjugated to a toxin, or a chemotherapeutic agent.
Alternatively, the agent may be an RNA interfering molecule (such as an shRNA or siRNA molecule) that inhibits expression of a cancer gene or candidate cancer gene in an amplified MCR in Table 1, or an antisense RNA molecule complementary to a cancer gene or candidate cancer gene in an amplified MCR in Table 1.
Alternatively, the agent may be a peptide or peptidomimetic, a small organic molecule, or an aptamer.
Preferrably, the agent is administered in a pharmaceutically acceptable formulation.
In another aspect, the invention provides a method of assessing whether a subject is afflicted with cancer or at risk for developing cancer, the method comprising: determining the copy number of at least one minimal common region (MCR) listed in Table 5 in a biological sample from the subject. A change of copy number of the MCR in the sample, as compared to the normal copy number of the MCR, indicates that the subject is afflicted with cancer or at risk for developing cancer. The normal copy number of an MCR is typically one per chromosome.
In certain embodiments, the cancer is lymphoma. In certain embodiments, the lymphoma is T-ALL.
In another aspect, the invention provides a method of assessing whether a subject is afflicted with cancer or at risk for developing cancer, by comparing the copy number of an MCR, identified using a genome-unstable non-human mammal model (including a genome-unstable mouse model of the invention), with the normal copy number of the MCR. The normal copy number of an MCR is typically one per chromosome.
In this example, we created a murine lymphoma model system that combines the genome-destabilizing impact of Atm deficiency and telomere dysfunction to effect T lymphomagenesis in a p53-dependent manner.
We interbred mTerc Atm p. 53 heterozygous mice and maintained them in pathogen-free conditions. We intercrossed the null alleles of mTerc, Atm and p53 to generate various genotypic combinations from this “triple”-mutant colony (for simplicity, hereafter designated as “TKO” for all genotypes from this colony).
We monitored animals for signs of ill-health every other day. Moribund animals were euthanized and subjected to complete autopsy; mice found dead were subject to necropsy specifically for signs of lymphoma. We performed all animal uses and manipulations according to approved IACUC protocol. Tumors were harvested from TKO mice and partitioned in the following manner. One section was snap-frozen for DNA and RNA extraction, a second portion was processed for histology, and the remaining portion was disaggregated for in vitro culture. Suspensions of tumor cells were maintained in RPMI supplemented with 50 μM beta-mercaptoethanol, 10% Cosmic Calf serum (HyClone), 0.5 ng/ml recombinant IL-2, and 4 ng/ml recombinant IL-7 (both from Peprotech). Tumor cells were immunostained with antibodies against CD4, CD8, CD3, and B220/CD45R (eBioscience) and subjected to FACS analysis.
We prepared DNA frozen tumors with the PureGene kit according to manufacturer's instructions (Gentra Systems). We prepared RNA by an initial extraction with Trizol (Invitrogen) according to the manufacturer's instructions. Pelleted total RNA was then digested with RQ1 DNase (Promega) and subsequently purified through RNA purification columns (Gentra). Proteins were obtained either from cell lines or tumor pieces by dis-aggregation in lysis buffer (according to Cell Signaling Technology) followed by sonication in a bath sonicator for 30 s. Lysates were clarified by centrifugation prior to quantification according to manufacturer's instructions (BioRad Protein Assay) and separation on 4-12% NuPage gels (Invitrogen).
We found that TKO mice which are p53+/− or p53−/− succumbed to lethal lymphoma with shorter latency and higher penetrance relative to TKO animals wildtype for p53 (
To quantify chromosomal rearrangements, we used Spectral Karyotype (SKY) analyses according to the following protocol. Metaphase preparations were typically obtained within 48 hours of establishment, although in a few instances establishment of the cell line was required to obtain good quality metaphases. Harvested cells were incubated in 105 mM KCl hypotonic buffer for 15 min prior to fixation in 3:1 methanol-acetic acid. Spectral karyotyping was done using the SkyPaint Kit and SkyView analytical software (Applied Spectral Imaging, Carlsbad, Calif.) according to manufacturer's protocols. Chromosome aberrations were defined using the rules from the Committee on Standard Genetic Nomenclature for Mice. T-test comparison between G0 and G1-G4 cytogenetics is based on 90 SKY profiles each set (ten metaphase spreads for each of TKO lymphomas).
To assess the degree of syntenic overlap in the murine lymphoma-prone TKO instability model and in human T-ALL and other cancers, we applied and integrated multiple genome analysis technologies to survey cancer-associated alterations for comparison with T-ALL and a diverse set of major human cancers.
Synteny describes the preserved order and orientation of genes between species. Disruption of synteny, caused by chromosome rearrangement, is an indication of divergent evolution. Comparisons of TKO mouse model and human T-ALL syntenic chromosomal regions may reveal the conserved nature of certain genetic modification in tumorigenesis.
Because TKO lymphomas harbored a large number of complex nonreciprocal translocations (NRTs), we sought to determine whether these genome-unstable tumors possess increased numbers of recurrent amplifications and deletions. To this end, we compiled high-resolution genome-wide array-CGH profiles for 35 TKO tumors (Table 3) and 26 human T-ALL cell lines and tumors (Tables 4A and 4B) for comparison.
T-ALL cell lines used in this example, and in Examples 3-7 are listed in Table 4A. A subset was subjected to both array-CGH (described in detail below) and re-sequencing, as indicated.
We used two cohorts of clinical human T-ALL samples in this example. A cohort of 8 samples (Table 4B) comprised of cryopreserved lymphoblasts or lymphoblast cell lysates, obtained with informed consent and IRB approval at the time of diagnosis from pediatric patients with T-ALL treated on Dana-Farber Cancer Institute study 00-001. We subjected these samples to genome-wide array-CGH profiling.
For genome-wide array-CGH profiling, we used the following protocol. Genomic DNA processing, labeling and hybridization to Agilent CGH arrays were performed as per manufacturer's protocol (http://www.home.agilent.com/agilent/home.jspx). Murine tumors were profiled against individual matched normal DNA (e.g., non-tumor cell of the same cell type from the same individual) or, when not available, pooled DNA of matching strain background. Labeled DNAs were hybridized onto 44K or 244K microarrays for mouse, and 22K or 44K microarrays for human. The Mouse 44K array contained 42,404 60-mer elements for which unique map positions were defined (National Center for Biotechnology Information, Mouse Build 34). The median interval between mapped elements was 21.8 kb, 97.1% of intervals of <0.3 megabases (Mb), and 99.3% are <1 Mb. The 244K array contained 224,641 elements for which unique map positions were defined based on the same mouse genome build. The Human 22K array contained 22,500 elements designed for expression profiling for which 16,097 unique map positions were defined with a median interval between mapped elements of 54.8 kb. The Human 44K microarray contained 42,494 60-mer oligonucleotide probes for which unique map positions were defined (National Center for Biotechnology Information, Human Build 35). The 244K array contained 226,932 60-mer oligonucleotide probes for which unique map positions were defined based on the same human genome build.
Profiles generated on 244K density arrays were extracted for the same 42K probes on the 44K microarrays to allow combination of profiles generated on the two different platforms. Fluorescence ratios of scanned images were normalized and calculated as the average of two paired (dye swap), and copy number profile was generated based on Circular Binary Segmentation, an algorithm that uses permutation to determine the significance of change points in the raw data (A. B. Olshen, et al., Biostatistics 5 (4), 557 (2004)).
TKO profiles revealed marked genome complexity with all chromosomes exhibiting recurrent CNAs—both regional and focal in nature (
The pathogenetic relevance of these recurrent genomic events, and of this instability model, is supported by integrated array-CGH and SKY analyses of a high amplitude genomic event on chromosome 2 in several independent TKO tumors. These CNAs shared a common boundary defined by array-CGH and contained a recurrent NRT involving the A3 band of chromosome 2 with different partner chromosomes by SKY (
For further comparison of genomic events in the TKO model and in human T-All, we used a separate series of 38 human clinical specimens (Table 4C) for re-sequencing of NOTCH 1, FBXW7 and PTEN (see Examples 5-6). These T-ALL samples were collected from 8 children and adolescents diagnosed at the Royal Free Hospital, London, and 30 adult patients enrolled in the MRC UKALL-XII trial. Appropriate informed consent was obtained from the patients (if over 18 years of age) or their guardians (if under 18 years), and the study had Ethics Committee approval.
1. HPLC and Sequencing. Gene mutation status was established by denaturing high-performance liquid chromatography (see, e.g., M. R. Mansour, et al., Leukemia 20 (3), 537 (2006)), and by bidirectional sequencing. Briefly, genomic DNA was extracted using the Qiagen (Hilden, Germany) genomic purification kit. PCR primers were designed to amplify exons and flanking intronic sequences. PCR amplification and direct sequencing were done according to art-known methods (for details, see H. Davies, et al., Cancer Res 65 (17), 7591 (2005)). Sequence traces were analysed using a combination of manual analysis and software-based analyses, where deviation from normal is indicated by the presence of two overlapping sequencing traces (indicating the presence of one normal allelic and one mutant allelic DNA sequence), or the presence of a single sequence trace that deviates from normal (indicating the presence of only a mutant DNA allele). All variants were confirmed by bidirectional sequencing of a second independently amplified PCR product.
2. Expression profiling. Biotinylated target cRNA was generated from total sample RNA from a TKO model and hybridized to mouse oligonucleotide probe arrays against normal control murine thymus RNA (Mouse Development Oligo Microarray, Agilent, Palo Alto, Calif.) according to manufacturer's protocols. Expression values for each gene were mapped to genomic positions based on National Center for Biotechnology Information Build 34 of the mouse genome.
3. Real-Time PCR. To confirm genetic loci, Real-time PCR was performed with a Quantitect SYBR green kit (Qiagen USA, Valencia, Calif.) using 2 ng DNA from each tumor run in triplicate, on Applied Biosystems or Stratagene MX3000 realtime thermocyclers. Each triplicate run was performed twice; quantification was performed using the standard curve method and the average fold change for the combined run was calculated. Primer sequences are listed in Table 8.
4. Western Blotting. Western blots were performed on clarified tumor lysates on PVDF membranes using the following antibodies: PTEN (9552), Akt (9272), phospho-Akt (9271), Notch1, activated Notch1 Val1744 (2421) (Cell Signaling Technology, Ipswich, Mass.), and tubulin (Sigma Chemical, St. Louis, Mo.), according to the manufacturer's instructions and developed with HRP-labeled secondary antibodies (Pierce; Rockford, Ill.) and enhanced chemiluminescent substrate.
5. Common Boundary Analysis of NOTCH1. Detailed structural analysis of the common boundary of CNAs revealed Notch1 locus alterations with rearrangement close to the 3′ region of the Notch1 gene in four TKO tumors, and focal amplifications encompassing Notch1 in two additional tumors (
In this Example, We further assessed the CNAs in the TKO mouse model by defining and characterization the minimal common regions of CNAs.
Synteny describes the preserved order and orientation of genes between species. Disruption of synteny, caused by chromosome rearrangement, is an indication of divergent evolution. Comparisons of TKO mouse model and human T-ALL syntenic chromosomal regions may reveal the conserved nature of certain genetic modification in tumorigenesis.
The observation of physiological deletion of TCR loci and human-like pattern of Notch1 genomic and mutational events prompted us to assess the extent to which the highly unstable genome of the TKO model engendered CNAs targeting loci syntenic to CNAs in human T-ALL using ortholog mapping of genes resident within the minimal common regions (MCRs) of copy number alterations.
1. Definition of MCRs. To facilitate this comparison, we first defined the MCRs in TKO genome by an established algorithm (see, e.g., D. R. Carrasco, et al., Cancer Cell 9 (4), 313 (2006); A. J. Aguirre, et al., Proc Natl Acad Sci USA 101 (24), 9067 (2004)) with criteria of CNA width<=10 Mb and amplitude>0.75 (log 2 scale). Briefly, a “segmented” dataset was generated by determining uniform copy number segment boundaries according to the method of Olshen (A. B. Olshen, et al., Biostatistics 5 (4), 557 (2004) and then replacing raw log 2 ratio for each probe by the mean log 2 ratio of the segment containing the probe. For 22K and 44K profiles, thresholds representing minimal CNA were chosen at ±0.15 and ±0.3, respectively.
Thresholds representing CNAs were chosen at ±0.4 and ±0.6, respectively. Higher thresholds were used for 44K profiles comparing to 22K profiles to adjust for signal-to-noise detection difference in platform performance. For examples 3-6, w selected minimal common region (MCR) by requiring at least one sample to show an extreme CNA event, defined by a log 2 ratio of ±0.60 and ±0.75 for 22K and 44K profiles, respectively, and the width of MCR is less than 10 Mb.
2. Homolog Mapping. We identified human homologs of genes identifies in regions of chromosomal structural alteration of CNAs within mouse TKO MCRs using NCBI HOMOLOGENE database. In parallel, we identified CNAs in seven human tumor datasets (pancreatic, glioblastoma, melanoma, lung, colorectal and multiple myeloma). The human homolog gene list was then used to merge with genes within CNAs of each of the seven human tumor datasets.
3. Cancer Gene Mapping. For cancer gene mapping, the mouse homologs were obtained based on Sanger's Cancer Gene Census55 (http://www.sanger.ac.uk/genetics/CGP/Census). The mouse cancer genes were then mapped to TKO's MCRs.
We obtained a list of 160 MCRs with average sizes of 2.12 Mb (0.15-9.82 Mb) and 2.33 Mb (0.77-9.6 Mb) for amplifications and deletions, respectively (Table 5). This frequency of genomic alterations is comparable to that of most human cancer genomes (e.g.
In Table 1, each murine TKO MCR with syntenic overlap with an MCR in the human T-ALL dataset is listed, separated by amplification and deletion, along with its chromosomal location (Cytoband/Chr) and base number (Start and End, in Mb). The minimal size of each MCR is indicated in bp. Peak ratio refers to the maximal log 2 array-CGH ratio for each MCR. Rec refers to the number of tumors in which the MCR was defined. Cancer genes and candidate cancer genes located in the amplified MCRs and deleted MCRs are also listed. The NCBI accession numbers and identification numbers for these cancer genes and candidate cancer genes are listed in Table 9.
To calculate the statistic significance of MCR overlap between mouse TKO and each of the human cancers of different histological types, we implemented a permutation test to determine the expected frequency of achieving the same degree of overlap between two genomes by chance alone. Specifically, we randomly generated simulated mouse genome containing the same number and sizes of amplification MCRs in the corresponding chromosomes as the actual TKO genome a similar set was created for each of the human cancer genomes. The number of overlapping amplifications between mouse and each human genome was calculated and stored. This simulation process was repeated 10,000 times. The p value for significance of amplification overlap was then calculated by dividing the frequency of randomly achieving the same or greater degree of overlap as actually observed during the 10,000 permutations by 10,000. p values for deletion overlap were calculated in a similar fashion.
We concluded that this degree of overlap was not by chance. First, statistic significance (p=0.001 and 0.004 for deletions and amplifications, respectively) supports this conclusion, as demonstrated by the rigorous permutation testing to validate the significance of the cross-species overlap. Second, we identified several genes already known or implicated in T-ALL biology, such as Crebbp, Ikaros, and Abl, present within these identified syntenic MCRs. Together, these data support the relevance of this engineered murine model to a related uman cancer and its usefulness.
In this example, We identified Fbxw7 gene as a target of frequent inactivation or deletion in the TKO mouse model.
We observed that a few TKO tumors with minimal Notch1 expression exhibited elevated Notch4 or Jagged1 (Notch ligand) mRNA levels (data not shown). To investigate this observation, we conducted a more detailed examination of the genomic and expression status of known components in the Notch pathway The four core elements of the Notch signaling system include the Notch receptor, DSL (Delta, Serrate, Lag-2) ligands, CSL (CBF1, Suppressor of hairless, Lag-1) transcriptional cofactors, and target genes. Upon binding ligand the Notch signaling converts CSL from a transcriptional repressor to a transcriptional activator. TKO sample A577 was one of the two tumors harboring a syntenic MCR encompassing the Fbxw7 gene (MCR #18, Table 1). In human T-ALL, focal FBXW7 deletions including one case with a single-probe event were detected (
FBXW7 is a key component of the E3 ubiquitin ligase responsible for binding the PEST domain of intracellular NOTCH1, leading to ubiquitination and degradation by the proteasome (N. Gupta-Rossi, et al., J Biol Chem 276 (37), 34371 (2001); C. Oberg, et al., J Biol Chem 276 (38), 35847 (2001); G. Wu, et al., Mol Cell Biol 21 (21), 7403 (2001)). PEST domain mutations in human T-ALL are thought to prolong the half-life of intracellular NOTCH1, raising the possibility that loss of FBXW7 function may cause similar effects on this pathway. To address this, we additionally characterized the human cell lines and clinical samples for NOTCH1 mutations (Table 2; Tables 4A, 4C, 6). Interestingly, there was no association between known functional mutations of NOTCH1 (HD-N, HD-C and PEST domains) and FBXW7 mutations (p=0.16). However, among samples with NOTCH1 mutations, FBXW7 mutations were found less frequently in samples with a mutated PEST domain (4/19; 21%) than samples with mutations of only the HD-N or HD-C domain (13/20; 65%; p=0.009 by Fisher exact test). One explanation of this observation is that mutations of FBXW7 and the PEST domain of NOTCH1 target the same degradation pathway, and little selective advantage accrues to the majority of leukaemias from mutating both components. At the same time, the lack of NOTCH1 and FBXW7 mutual exclusivity may suggest non-overlapping activities by FBXW7 on pathways other than NOTCH signaling.
In this example, We identified Pten gene as a target of frequent inactivation or deletion in the TKO mouse model.
Focal deletion on chromosome 19, centering on the Pten gene, was among the most common genomic event in TKO lymphomas (Table 1,
In addition to these genomic and genetic alterations, Northern and Western blot analyses and transcriptome profiling of the TKO and human T-ALL samples revealed a broader collection of tumors with low to undetectable PTEN expression (
In examples 3-6, Applicant identified and characterized Fbxw7 and Pten using the TKO mouse model. Both Fbxw7 and Pten have been previously identified as tumor suppressor genes. Thus their identification as mutated in human T-ALL provided proof of principle for the Applicants' approach and demonstrated that the mouse model described herein provides a powerful tool to cancer gene discovery. In this example, Applicants extended the cross-species genomic analyses to other human cancers.
While above cross-species comparison showed numerous concordant lesions in cancers of T cell origin, the fact that this instability model is driven by mechanisms of fundamental relevance (e.g., telomere dysfunction and p53 mutation) to many cancer types, including non-hematopoietic malignancies, suggested potentially broader relevance to other human cancers. A case in point is the Pten example above, in that PTEN is a bona fide tumor suppressor for multiple cancer types49,50. To assess this, we extended the cross-species comparative genomic analyses to 6 other human cancer types (n=421) of hematopoietic, mesenchymal and epithelial origins, including multiple myeloma (n=67)53, glioblastoma (n=38) (unpublished) and melanoma (n=123) (unpublished), as well as adenocarcinomas of the pancreas (n=30) (unpublished), lung (n=63)54 and colon (n=74) (unpublished).
Compared against similarly defined MCR lists (i.e. MCR width<=10 Mb; see Example 4 and
Next, Applicants determined whether these syntenic MCRs targeted known cancer genes to provide an additional level of validation for these TKO genomic events. Among the 363 genes listed on the Cancer Gene Census55, 237 genes have a mouse homolog based on NCBI homologene (see Example 4). Of these, 24 known cancer genes were found to be resident within one of the 104 syntenic MCRs (Table 7). These included 17 oncogenes in amplifications and 7 tumor suppressor genes in deletions. The majority of these syntenic MCRs do not contain known cancer genes, raising the strong possibility that re-sequencing focused on resident genes of syntenic MCRs may provide a high-yield strategy to identify somatic mutations in human cancers, a thesis supported by the FBXW7 and PTEN examples.
The practice of the various aspects of the present invention may employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Current Protocols in Molecular Biology, by Ausubel et al., Greene Publishing Associates (1992, and Supplements to 2003); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y. (1997); Bast et al., Cancer Medicine, 5th ed., Frei, Emil, editors, BC Decker Inc., Hamilton, Canada (2000); Lodish et al., Molecular Cell Biology, 4th ed., W. H. Freeman & Co., New York (2000); Griffiths et al., Introduction to Genetic Analysis, 7th ed., W. H. Freeman & Co., New York (1999); Gilbert et al., Developmental Biology, 6th ed., Sinauer Associates, Inc., Sunderland, Mass. (2000); and Cooper, The Cell—A Molecular Approach, 2nd ed., Sinauer Associates, Inc., Sunderland, Mass. (2000). All patents, patent applications and references cited herein are incorporated in their entirety by reference.
Mm Dvl1 cDNA (Homo sapiens)
Mm DVL1 protein (Homo sapiens)
Ccnl2 cDNA (Homo sapiens)
CCNL2 protein (Homo sapiens)
Aurkaip1 cDNA (Homo sapiens)
AURKAIP1 Protein (Homo sapiens)
Myb cDNA (Homo sapiens)
MYB Protein (Homo sapiens)
Ahi1 cDNA (Homo sapiens)
AHl1 Protein (Homo sapiens)
Runx1 cDNA (Homo sapiens)
RUNX1 Protein (Homo sapiens)
Ets2 cDNA (Homo sapiens)
ETS2 Protein (Homo sapiens)
Tmprss2 cDNA (Homo sapiens)
TMPRSS2 Protein (Homo sapiens)
Ripk4 cDNA (Homo sapiens)
RIPK4 Protein (Homo sapiens)
Erg cDNA (Homo sapiens)
ERG Protein (Homo sapiens)
Gnb2 cDNA (Homo sapiens)
GNB2 Protein (Homo sapiens)
Perq1 cDNA (Homo sapiens)
PERQ1 Protein (Homo sapiens)
Tox cDNA (Homo sapiens)
TOX Protein (Homo sapiens)
Set cDNA (Homo sapiens)
SET Protein (Homo sapiens)
Fnbp1 cDNA (Homo sapiens)
FNBP1 Protein (Homo sapiens)
Abl1 cDNA (Homo sapiens)
ABL1 Protein (Homo sapiens)
Nup214 cDNA (Homo sapiens)
NUP214 Protein (Homo sapiens)
Trp53 cDNA (Homo sapiens)
TRP53 Protein (Homo sapiens)
Bcl6 cDNA (Homo sapiens)
BCL6 Protein (Homo sapiens)
Negr1 cDNA (Homo sapiens)
NEGR1 Protein (Homo sapiens)
Baalc cDNA (Homo sapiens)
BAALC Protein (Homo sapiens)
Fzd6 cDNA (Homo sapiens)
FZD6 Protein (Homo sapiens)
Crebbp cDNA (Homo sapiens)
CREBBP Protein (Homo sapiens)
C2ta cDNA (Homo sapiens)
C2TA Protein (Homo sapiens)
Mxi1 cDNA (Homo sapiens)
MXI1 Protein (Homo sapiens)
Hes3 cDNA (Homo sapiens)
HES3 Protein (Homo sapiens)
Rpl22 cDNA (Homo sapiens)
RPL22 Protein (Homo sapiens)
Chd5 cDNA (Homo sapiens)
CHD5 Protein (Homo sapiens)
Ikaros cDNA (Homo sapiens)
IKAROS Protein (Homo sapiens)
Ptprn2 cDNA (Homo sapiens)
PTPRN2 Protein (Homo sapiens)
Tcrb cDNA (Partial Sequence) (Homo sapiens)
TCRB Protein (Homo sapiens)
Gnaq cDNA (Homo sapiens)
GNAQ Protein (Homo sapiens)
Pten cDNA (Homo sapiens)
PTEN Protein (Homo sapiens)
Fbxw7 cDNA (Homo sapiens)
FBXW7 Protein (Homo sapiens)
This application claims the benefit of and priority to U.S. Provisional Application No. 60/931,294, filed on May 21, 2007, the contents of which is hereby incorporated by reference in its entirety.
The work described herein was funded, in whole or in part, by Grant Number CA84628 (RO1) and CA84313 (UO1). The United States government may have certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US08/06583 | 5/21/2008 | WO | 00 | 6/11/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60931294 | May 2007 | US |
