Patent Application
20220193270
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created about Oct. 4, 2021, is named 56699_729_301SL.txt and is 129 kb in size.
The present application relates to methods for enhancing engraftment of tumor in experimental animals. The present application also relates to a method of making cancer stem cells.
Mice, rodents and other animals traditionally, used in drug discovery, do not accurately mimic humans in cancer drug studies using mouse models or xenograft experiments. First, the response that mice have to drug candidates often does not reflect how those drug candidates do in human studies, as is evidenced by the number of lawsuits against pharmaceutical companies for ‘bad’ drugs. The FDA and drug companies have raised the bar significantly in terms of the level of pre-clinical testing required for permission to test in humans, as well as increasing the numbers of patients tested in clinical trials. Still, compounds that showed no toxicities in mice, and other test animals, continue to cause significant adverse events, including death, when administered to humans. It appears that there are critical differences between mouse and human biology that confound drug safety testing in mice as well as in other test animals, especially, non-primate test animals. A great improvement to the current practice would be to develop methods that make animals, especially rodents, respond more like humans to cells that are implanted or for the study of basic science, drug efficacy, toxicity or dosing.
Secondly, it is hard to get some cancer cells or cancer cell lines to engraft in mice. Many cancer cell lines that are routinely used in in vitro testing simply have engraftment rates that are too low for reliable animal studies. For example, T47D breast cancer cell line is typically used for in vitro studies because of its overexpression of the oncogene MUC 1. However, T47D cells are infrequently used in mouse xenograft studies because of their poor engraftment rate. Animal models have been selected or are genetically altered such that they mimic certain human diseases. Some animal models spontaneously get certain types of cancer. However, these models are generally only useful for studying disease that arises from a specific mutation or do not exactly mimic human disease. As in traditional mouse studies, the effects of a drug candidate in mice are often not predictive of the effects the drug will have in humans. Thus, a significant improvement over the state of the art would be to develop methods that increase the engraftment rate of human cancer cells into a test animal, especially rodent test animals.
In a related matter, there is currently no practical method for evaluating the efficacy, toxicity or dosing of compounds, biologicals or drugs on tumor initiating cells or cancer stem cells. Oncomed, for example has a protracted method for doing so in which a surviving tumor from a first animal is then transplanted into a second animal, then surviving tumor cells from the second animal, which may have survived treatment with an anti-cancer agent, are then implanted into a third animal which is then tested with an agent to test its ability to inhibit the growth of the surviving cancer cells, proposed to be cancer stem cells or tumor initiating cells. These methods were based on research showing that only a small percentage of cancer cells have the ability to metastasize and the identification of markers of these ‘cancer stem cells’, such as high expression of CD44 accompanied by low expression of CD24 (Clarke M F et al 2006, Chen K et al 2013). The receptor CXCR4 has also been identified as a metastatic receptor whose expression is elevated in cancer stem cells (Darash-Yahana M, Pikarsky E, Abramovitch R, Zeira E et al 2004). However, to date, there has been no evidence that it is possible to expose cancer cells to an agent or agents which causes them to be transformed to cancer stem cells. Thus, a significant improvement over the state of the art would be to develop or identify agents or methods that when added to cancer cells transform some of the cells to cancer stem cells. Drugs and drug candidates for the treatment or progression of metastatic cancers could then be tested on these cancer stem cells. Additionally, the agents and methods would provide a practical method for enriching a population of cancer cells for cancer stem cells which could also be used for the identification of additional yet still unknown markers of a metastatic cancer cells, which would then be new targets for anti-cancer drugs.
It is also important to develop in vitro methods to accelerate the transformation of local cancer cells into the highly aggressive and metastatic cancer stem cells so that metastatic drug targets can be identified and so that drugs can be developed that specifically are able to kill those cancer stem cells that can evade chemotherapy. However, it is also important Animals xenografted with NME-7 induced cancer stem cell characteristics not only generated tumors from very low numbers of implanted cancer cells, but also developed metastatic cancer with multiple tumors developing, including ones at locations remote from the implantation site.
Essentially, there are but a handful of cancer cell lines that are repeatedly used to study the basic science of cancer biology and to test for drug efficacy as well as drug toxicities. This is because human cells, unlike mouse cells, can only divide in culture a very limited number of times before they senesce. The cancer cell lines that researchers use today are either naturally immortalized cancer cells isolated from pleural effusions of a single metastatic cancer patient or induced to become immortalized by fusing to an immortalized cell line, often of mouse origin, or more recently by transfecting the cancer cells with an immortalizing gene. The main point is that the methods used to get these cells to continue to self-replicate significantly alter the molecular characteristics of the original or primary cancer cell. The reality is that these cell lines are cancer cells from patients that lived and died years ago and may in no way resemble a particular cancer that a particular patient is stricken with. Thus a significant improvement over the state of the art would be to develop methods to study cancer cells from a patient in a way that does not alter the molecular characteristics of the patient's cancer cells or if the molecular characteristics are altered, those alterations would ideally mimic the progression of that cancer in the patient's body. For example, an acceptable alteration would be if the method for increasing the number of divisions that a patient's cancer cell could undergo transformed the cancer cell or cells into a more aggressive form of that cancer or a metastatic form of that cancer. Then, the efficacy, toxicity or dosing of a compound, biological or drug could be evaluated on the patient's own cancer cells.
Similarly, the efficacy, safety testing and dosing schedule of candidate drugs is established to a first order approximation in rodents using just a handful of immortalized cancer cell lines, which as stated above, bear little or no resemblance to the particular cancer of a particular patient. Thus, a significant improvement over the state of the art would be to develop methods that enable engraftment of a patient's cancer cells into an animal, preferably a rodent, and optionally evaluating the efficacy, toxicity or dosing of a compound, biological or drug on the patient's cancer in the test animal.
In addition to the problems stated above that essentially prevent the engraftment of patient cancer cells into a test animal using current methods, approximately 4 to 6 million cancer cells must be implanted into a test animal in order to establish a tumor in a host animal. The reason for this is that scientists recently discovered that not every cell within a tumor has the ability to give rise to a tumor; the vast majority cannot. The small population of cancer cells that can give rise to a tumor if implanted are called ‘tumor initiating cells’ or ‘cancer stem cells’. It is estimated that as few as 1 in 100,000 cancer cells can give rise to a tumor and established protocols usually call for implantation of millions of cancer cells in order to generate a tumor in a test animal. The problem of studying patient cancer cells in test animals then becomes a problem numbers in addition to the previously described problem of immortalization. A typical tumor biopsy simply would not yield enough cells to implant hundreds of mice with 4-6 million cancer cells each in order to do a proper evaluation of the efficacy, toxicity or dosing of a proposed candidate drug. Recall also that primary patient cells would not be immortalized so would not proliferate as in a patient for the duration of the evaluation experiment. Thus, a significant improvement over the state of the art would be to develop methods that enable engraftment of a very few patient cancer cells into an animal.
In one aspect, the present invention is directed to a method of testing for efficacy of a potential drug agent against cancerous cells in a non-human mammal, comprising: (i) generating the cancer cells in the non-human mammal; (ii) contacting the cancer cells with a potential drug agent by administering the potential drug agent to the mammal; and (iii) measuring effect of the potential drug agent on the cancer cells, wherein reduction of number of cancer cells in the mammal may be indicative of efficaciousness of the potential drug agent against cancerous cells, wherein the method comprises contacting the cancer cells with an agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve state before carrying out step (i), after carrying out step (i), or both before and after carrying out step (i).
In the method above, the agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve state may be an NME protein, 2i, 5i, chemical, or nucleic acid. The NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME6 dimer, or bacterial NME.
The mammal may be a rodent, such as a mouse or rat. The cancer may be spontaneously generated or implanted from a human being.
In the method above, the non-human mammal may be transgenic, wherein the mammal expresses human MUC1 or MUC1* or NME protein in the germ cells or somatic cells, wherein the germ cells and somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said mammal. The gene expressing the human MUC1 or MUC1* or NME protein may be under control of an inducible promoter. The promoter may be inducibly responsive to a naturally occurring protein in the non-human mammal. The amount of cells implanted into the mammal may be at least about 30, about 30 to about 1,000,000, about 50 to about 500,000, about 50 to 100,000, or from about 1,000 to about 1,000,000. The NME protein may be present in serum-free media as the single growth factor.
In another aspect, the invention may be directed to a method for engrafting human tumor in a non-human mammal, comprising injecting or implanting human tumor cells into the mammal, the method comprising contacting the tumor cells with an agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state before carrying out the injecting or implanting step, after carrying out the injecting or implanting step, or both before and after carrying out the injecting or implanting step.
In the method above, the agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state may be an NME protein, 2i, 5i, chemical, or nucleic acid. The NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME6 dimer, or bacterial NME. The mammal may be a rodent, such as a mouse or rat.
In the method above, the non-human mammal may be transgenic, wherein the mammal expresses human MUC1 or MUC1* or NME protein in the germ cells or somatic cells, wherein the germ cells and somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said mammal. The gene expressing the human MUC1 or MUC1* or NME protein may be under control of an inducible promoter. The promoter may be inducibly responsive to a naturally occurring protein in the non-human mammal. The amount of cells implanted into the mammal may be at least about 30, about 30 to about 1,000,000, about 50 to about 500,000, about 50 to 100,000, or from about 1,000 to about 1,000,000. The NME protein may be present in serum-free media as the single growth factor.
In still another aspect, the present invention is directed to a method of generating cancer stem cells, comprising contacting cancer cells with an agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve state.
In the method above, the agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state may be an NME protein, 2i, 5i, chemical, or nucleic acid. The NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME6 dimer, or bacterial NME.
In this method, the agent may suppress expression of MBD3, CHD4, BRD4 or JMJD6. The agent may be siRNA made against MBD3, CHD4, BRD4 or JMJD6, or siRNA made against any gene that encodes a protein that upregulates expression of MBD3, CHD4, BRD4 or JMJD6. The cancer stem cell may be characterized by increased expression of CXCR4 or E-cadherin (CDH1) compared with cancer cells or normal cells.
In yet another aspect, the invention is directed to a method of generating metastatic tumors in a non-human mammal, comprising transferring cancer cells into a mammal, wherein the method comprises contacting the cancer cells with an agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve state, before carrying out the transferring step, after carrying out the transferring step, or both before and after carrying out the transferring step.
In the method above, the agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve state may be an NME protein, 2i, 5i, chemical, or nucleic acid. The NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME6 dimer, or bacterial NME.
In this method, the agent may suppress expression of MBD3, CHD4, BRD4 or JMJD6. The agent may be siRNA made against MBD3, CHD4, BRD4 or JMJD6, or siRNA made against any gene that encodes a protein that upregulates expression of MBD3, CHD4, BRD4 or JMJD6. The cancer stem cell may be characterized by increased expression of CXCR4 or E-cadherin (CDH1) compared with cancer cells or normal cells.
In the method above, the mammal may be a rodent, such as a mouse or rat. The non-human mammal may be transgenic, wherein the mammal expresses human MUC1 or MUC1* or NME protein in the germ cells or somatic cells, wherein the germ cells and somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said mammal. The gene expressing the human MUC1 or MUC1* or NME protein may be under control of an inducible promoter. The promoter may be inducibly responsive to a naturally occurring protein in the non-human mammal. The amount of cells implanted into the mammal may be at least about 30, about 30 to about 1,000,000, about 50 to about 500,000, about 50 to 100,000, or from about 1,000 to about 1,000,000. The NME protein may be present in serum-free media as the single growth factor.
In another aspect, the present invention is directed to a method of testing for efficacy of a potential drug agent against a patient's cancerous cells in a non-human mammal, comprising: (i) transferring the patient's cancer cells into the non-human mammal; (ii) contacting the cancer cells with a potential drug agent by administering the potential drug agent to the mammal; and (iii) measuring effect of the potential drug agent on the cancer cells, wherein reduction of number of the patient's cancer cells in the mammal may be indicative of efficaciousness of the potential drug agent against cancerous cells, wherein the method comprises contacting the patient's cancer cells with an agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve state before carrying out step (i), after carrying out step (i), or both before and after carrying out step (i).
In the method above, the agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve state may be an NME protein, 2i, 5i, chemical, or nucleic acid. The NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME6 dimer, or bacterial NME.
In this method, the agent may suppress expression of MBD3, CHD4, BRD4 or JMJD6. The agent may be siRNA made against MBD3, CHD4, BRD4 or JMJD6, or siRNA made against any gene that encodes a protein that upregulates expression of MBD3, CHD4, BRD4 or JMJD6. The cancer stem cell may be characterized by increased expression of CXCR4 or E-cadherin (CDH1) compared with cancer cells or normal cells.
In the method above, the mammal may be a rodent, such as a mouse or rat. The non-human mammal may be transgenic, wherein the mammal expresses human MUC1 or MUC1* or NME protein in the germ cells or somatic cells, wherein the germ cells and somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said mammal. The gene expressing the human MUC1 or MUC1* or NME protein may be under control of an inducible promoter. The promoter may be inducibly responsive to a naturally occurring protein in the non-human mammal. The amount of cells implanted into the mammal may be at least about 30, about 30 to about 1,000,000, about 50 to about 500,000, about 50 to 100,000, or from about 1,000 to about 1,000,000. The NME protein may be present in serum-free media as the single growth factor.
In another aspect, the invention is directed to a method for generating tissue from xenograft in a non-human mammal, comprising: (i) generating a transgenic non-human mammal, wherein the mammal expresses human MUC1 or MUC1* or NME protein in the germ cells and somatic cells, wherein the germ cells and somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said mammal, wherein the expression of the gene sequence may be under control of an inducible and repressible regulatory sequence; (ii) transferring stem cells or progenitor cells that are xenogeneic in origin to the non-human mammal such that the gene may be induced to be expressed so as to multiply the number of stem or progenitor cells; and (iii) repressing the gene expression so as to generate tissue from the xenografted stem cells.
In this method, in step (iii), the gene expression repression may be carried out by contacting the stem cells with a tissue differentiation factor, or in step (iii) the gene expression repression may be carried out naturally in the mammal in response to naturally produced host tissue differentiation factor. The transferred cells may be human. The tissue may be an organ. The NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME6 dimer, or bacterial NME. The mammal may be a rodent, such as a mouse or rat.
In another aspect, the invention is directed to a method of generating cancer preventative peptide comprising: (i) generating a non-human transgenic host mammal, wherein the mammal expresses human MUC1 or MUC1* or NME protein in the germ cells or somatic cells, wherein the germ cells and somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said mammal; (ii) immunizing the mammal with a fragment of NME protein; (iii) implanting human tumor cells into the mammal; and (iv) comparing tumor engraftment, tumor growth rate or tumor initiating potential of cells in the mammal with a control transgenic non-human mammal such that peptide causing significantly reduced tumor engraftment, tumor growth rate, or tumor initiating potential are selected as an immunizing peptide.
In this method, the fragment of NME protein may be a peptide selected from peptide number 1 to 53 in
The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;
As used herein, the “MUC1*” extra cellular domain is defined primarily by the PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNLTIS DVS VSD VPFPFS AQS GA (SEQ ID NO:6)). Because the exact site of MUC1 cleavage depends on the enzyme that clips it, and that the cleavage enzyme varies depending on cell type, tissue type or the time in the evolution of the cell, the exact sequence of the MUC1* extra cellular domain may vary at the N-terminus.
As used herein, the term “PSMGFR” is an acronym for Primary Sequence of MUC 1 Growth Factor Receptor as set forth as GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFP FSAQSGA (SEQ ID NO:6). In this regard, the “N-number” as in “N-10 PSMGFR”, “N-15 PSMGFR”, or “N-20 PSMGFR” refers to the number of amino acid residues that have been deleted at the N-terminal end of PSMGFR. Likewise “C-number” as in “C-10 PSMGFR”, “C-15 PSMGFR”, or “C-20 PSMGFR” refers to the number of amino acid residues that have been deleted at the C-terminal end of PSMGFR.
As used herein, the “extracellular domain of MUC1*” refers to the extracellular portion of a MUC1 protein that is devoid of the tandem repeat domain. In most cases, MUC1* is a cleavage product wherein the MUC1* portion consists of a short extracellular domain devoid of tandem repeats, a transmembrane domain and a cytoplasmic tail. The precise location of cleavage of MUC1 is not known perhaps because it appears that it can be cleaved by more than one enzyme. The extracellular domain of MUC1* will include most of the PSMGFR sequence but may have an additional 10-20 N-terminal amino acids.
As used herein, “NME family proteins” or “NME family member proteins”, numbered 1-10, are proteins grouped together because they all have at least one NDPK (nucleotide diphosphate kinase) domain. In some cases, the NDPK domain is not functional in terms of being able to catalyze the conversion of ATP to ADP. NME proteins were formally known as NM23 proteins, numbered H1, H2 and so on. Herein, the terms NM23 and NME are interchangeable. Herein, terms NME1, NME2, NME6 and NME7 are used to refer to the native protein as well as NME variants. In some cases these variants are more soluble, express better in E. coli or are more soluble than the native sequence protein. For example, NME7 as used in the specification can mean the native protein or a variant, such as NME7-AB that has superior commercial applicability because variations allow high yield expression of the soluble, properly folded protein in E. coli. “NME1” as referred to herein is interchangeable with “NM23-H1”. It is also intended that the invention not be limited by the exact sequence of the NME proteins. The mutant NME1-5120G, also called NM23-5120G, are used interchangeably throughout the application. The 5120G mutants and the P96S mutant are preferred because of their preference for dimer formation, but may be referred to herein as NM23 dimers or NME1 dimers.
NME7 as referred to herein is intended to mean native NME7 having a molecular weight of about 42 kDa, a cleaved form having a molecular weight between 25 and 33 kDa, a variant devoid of the DM10 leader sequence, NME7-AB or a recombinant NME7 protein, or variants thereof whose sequence may be altered to allow for efficient expression or that increase yield, solubility or other characteristics that make the NME7 more effective or commercially more viable.
As used herein, an “an agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve state” refers to a protein, small molecule or nucleic acid that alone or in combination maintains stem cells in the naïve state, resembling cells of the inner cell mass of an embryo. Examples include but are not limited to NME1 dimers, human or bacterial, NME7, NME7-AB, 2i, 5i, nucleic acids such as siRNA that suppress expression of MBD3, CHD4, BRD4, or JMJD6.
As used herein, in reference to an agent being referred to as a “small molecule”, it may be a synthetic chemical or chemically based molecule having a molecular weight between 50 Da and 2000 Da, more preferably between 150 Da and 1000 Da, still more preferably between 200 Da and 750 Da.
As used herein, in reference to an agent being referred to as a “natural product”, it may be chemical molecule or a biological molecule, so long as the molecule exists in nature.
As used herein, “2i inhibitor” refers to small molecule inhibitors of GSK3-beta and MEK of the MAP kinase signaling pathway. The name 2i was coined in a research article (Silva J et al 2008), however herein “2i” refers to any inhibitor of either GSK3-beta or MEK, as there are many small molecules or biological agents that if they inhibit these targets, have the same effect on pluripotency or tumorigenesis.
As used herein, FGF, FGF-2 or bFGF refer to fibroblast growth factor.
As used herein, “Rho associated kinase inhibitors” may be small molecules, peptides or proteins (Rath N, et al, 2012). Rho kinase inhibitors are abbreviated here and elsewhere as ROCi or ROCKi, or Ri. The use of specific rho kinase inhibitors are meant to be exemplary and can be substituted for any other rho kinase inhibitor.
As used herein, the term “cancer stem cells” or “tumor initiating cells” refers to cancer cells that express levels of genes that have been linked to a more metastatic state or more aggressive cancers. The terms “cancer stem cells” or “tumor initiating cells” can also refer to cancer cells for which far fewer cells are required to give rise to a tumor when transplanted into an animal. Cancer stem cells and tumor initiating cells are often resistant to chemotherapy drugs.
As used herein, the terms “stem/cancer”, “cancer-like”, “stem-like” refers to a state in which cells acquire characteristics of stem cells or cancer cells, share important elements of the gene expression profile of stem cells, cancer cells or cancer stem cells. Stem-like cells may be somatic cells undergoing induction to a less mature state, such as increasing expression of pluripotency genes. Stem-like cells also refers to cells that have undergone some de-differentiation or are in a meta-stable state from which they can alter their terminal differentiation. Cancer like cells may be cancer cells that have not yet been fully characterized but display morphology and characteristics of cancer cells, such as being able to grow anchorage-independently or being able to give rise to a tumor in an animal.
Sequence Listing Free Text
As regards the use of nucleotide symbols other than a, g, c, t, they follow the convention set forth in WIPO Standard ST.25, Appendix 2, Table 1, wherein k represents t or g; n represents a, c, t or g; m represents a or c; r represents a or g; s represents c or g; w represents a or t and y represents c or t.
MTPGTQSPFF LLLLLTVLTV VTGSGHASST PGGEKETSAT QRSSVPSSTE KNAVSMTSSV LSSHSPGSGS STTQGQDVTL APATEPASGS AATWGQDVTS VPVTRPALGS TTPPAHDVTS APDNKPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDNRPALGS TAPPVHNVTS ASGSASGSAS TLVHNGTSAR ATTTPASKST PFSIPSHHSD TPTTLASHST KTDASSTHHS SVPPLTSSNH STSPQLSTGV SFFFLSFHIS NLQFNS SLED PSTDYYQELQ RDISEMFLQI YKQGGFLGLS NIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPAR DTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASANL (SEQ ID NO:1) describes full-length MUC1 Receptor (Mucin 1 precursor, Genbank Accession number: P15941).
SEQ ID NOS:2, 3 and 4 describe N-terminal MUC-1 signaling sequence for directing MUC1 receptor and truncated isoforms to cell membrane surface. Up to 3 amino acid residues may be absent at C-terminal end as indicated by variants in SEQ ID NOS:2, 3 and 4.
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALL VLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTD RSPYEKVSAGNGGSSLSYTNPAVAAASANL (SEQ ID NO:5) describes a truncated MUC1 receptor isoform having nat-PSMGFR at its N-terminus and including the transmembrane and cytoplasmic sequences of a full-length MUC1 receptor.
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:6) describes the extracellular domain of Native Primary Sequence of the MUC1 Growth Factor Receptor (nat-PSMGFR—an example of “PSMGFR”):
TINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:7) describes the extracellular domain of Native Primary Sequence of the MUC1 Growth Factor Receptor (nat-PSMGFR—An example of “PSMGFR”), having a single amino acid deletion at the N-terminus of SEQ ID NO:6).
GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:8) describes the extracellular domain of “SPY” functional variant of the native Primary Sequence of the MUC1 Growth Factor Receptor having enhanced stability (var-PSMGFR—An example of “PSMGFR”).
TINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:9) describes the extracellular domain of “SPY” functional variant of the native Primary Sequence of the MUC1 Growth Factor Receptor having enhanced stability (var-PSMGFR—An example of “PSMGFR”), having a single amino acid deletion at the C-terminus of SEQ ID NO:8).
tgtcagtgccgccgaaagaactacgggcagctggacatctttccagcccgggatacctaccatcctatgagcgagtaccccacc taccacacccatgggcgctatgtgccccctagcagtaccgatcgtagcccctatgagaaggtttctgcaggtaacggtggcagcagcctctcttac acaaacccagcagtggcagccgcttctgccaacttg (SEQ ID NO:10) describes MUC1 cytoplasmic domain nucleotide sequence.
CQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNG GSSLSYTNPAVAAASANL (SEQ ID NO:11) describes MUC1 cytoplasmic domain amino acid sequence.
gagatcctgagacaatgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgag cttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctnttaaagcggaccaaatatgataacctgcacttggaa gatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaa gaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactc aaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtc ctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttct gaaagcattagagccctattggaacagatggcataagaaatgcagcgcatggccctgattcttngcttctgcggccagagaaatggagttgttttt tccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggtatgttgaat acactatattcagtacattttgttaataggagagcaatgtttattttcttgatgtactttatgtatagaaaataa (SEQ ID NO:12) describes NME7 nucleotide sequence (NME7: GENBANK ACCESSION AB209049).
DPETMNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRT KYDNLHLEDLFIGNKVNVF SRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIIN KAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLG PANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNC TCCIVKPHAVSEGMLNTLYSVHFVNRRAMFIFLMYFMYRK (SEQ ID NO:13) describes NME7 amino acid sequence (NME7: GENBANK ACCESSION AB209049).
atggtgctactgtctactttagggatcgtctttcaaggcgaggggcctcctatctcaagctgtgatacaggaaccatggccaactgt gagcgtaccttcattgcgatcaaaccagatggggtccagcggggtcttgtgggagagattatcaagcgttttgagcagaaaggattccgccttgtt ggtctgaaattcatgcaagatccgaagatcttctcaaggaacactacgttgacctgaaggaccgtccattctttgccggcctggtgaaatacatgc actcagggccggtagttgccatggtctgggaggggctgaatgtggtgaagacgggccgagtcatgctcggggagaccaaccctgcagactcc aagcctgggaccatccgtggagacttctgcatacaagttggcaggaacattatacatggcagtgattctgtggagagtgcagagaaggagatcg gcttgtggtttcaccctgaggaactggtagattacacgagctgtgctcagaactggatctatgaatga (SEQ ID NO:14) describes NM23-H1 nucleotide sequence (NM23-H1: GENBANK ACCESSION AF487339).
MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKRFEQK GFRLVGLKFMQASEDLLKEHYVDLKDRPFF AGLVKYMHSGPVVAMVWEGLNVVKTGRV MLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVESAEKEIGLWFHPEELVDYTSCAQNWIYE (SEQ ID NO:15) NM23-H1 describes amino acid sequence (NM23-H1: GENBANK ACCESSION AF487339).
atggtgctactgtctactttagggatcgtctttcaaggcgaggggcctcctatctcaagctgtgatacaggaaccatggccaactgt gagcgtaccttcattgcgatcaaaccagatggggtccagcggggtcttgtgggagagattatcaagcgttttgagcagaaaggattccgccttgtt ggtctgaaattcatgcaagcttccgaagatcttctcaaggaacactacgttgacctgaaggaccgtccattctttgccggcctggtgaaatacatgc actcagggccggtagttgccatggtctgggaggggctgaatgtggtgaagacgggccgagtcatgctcggggagaccaaccctgcagactcc aagcctgggaccatccgtggagacttctgcatacaagttggcaggaacattatacatggcggtgattctgtggagagtgcagagaaggagatcg gcttgtggtttcaccctgaggaactggtagattacacgagctgtgctcagaactggatctatgaatga (SEQ ID NO:16) describes NM23-H1 5120G mutant nucleotide sequence (NM23-H1: GENBANK ACCESSION AF487339).
MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKRFEQK GFRLVGLKFMQASEDLLKEHYVDLKDRPFF AGLVKYMHSGPVVAMVWEGLNVVKTGRV MLGETNPADSKPGTIRGDFCIQVGRNIIHGGDSVESAEKEIGLWFHPEELVDYTSCAQNWIY E (SEQ ID NO:17) describes NM23-H1 5120G mutant amino acid sequence (NM23-H1: GENBANK ACCESSION AF487339).
atggccaacctggagcgcaccttcatcgccatcaagccggacggcgtgcagcgcggcctggtgggcgagatcatcaagcgct tcgagcagaagggattccgcctcgtggccatgaagttcctccgggcctctgaagaacacctgaagcagcactacattgacctgaaagaccgacc attcttccctgggctggtgaagtacatgaactcagggccggttgtggccatggtctgggaggggctgaacgtggtgaagacaggccgagtgatg cttggggagaccaatccagcagattcaaagccaggcaccattcgtggggacttctgcattcaggttggcaggaacatcattcatggcagtgattca gtaaaaagtgctgaaaaagaaatcagcctatggtttaagcctgaagaactggttgactacaagtcttgtgctcatgactgggtctatgaataa (SEQ ID NO:18) describes NM23-H2 nucleotide sequence (NM23-H2: GENBANK ACCESSION AK313448).
MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDL KDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRN IIHGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE (SEQ ID NO:19) describes NM23-H2 amino acid sequence (NM23-H2: GENBANK ACCESSION AK313448).
Human NM23-H7-2 sequence optimized for E. coli expression:
Making Mouse Respond More Like a Human to Drug Testing Against Cancer Cells by Allowing the Human Engrafted Cancer Cells in Mouse to Contact NME7 or NME7-AB
NME family member proteins can function to promote cancer. Mice, rodents and other animals traditionally, used in drug discovery, do not accurately mimic humans in their response to drugs being tested for efficacy or toxicity. Several ‘bad drugs’ have been the subject of lawsuits for causing severe adverse reactions in humans, including death, whereas the same drugs showed no toxicities in mice. One reason for the discrepancy between the effect in mice and the effect in humans is that the molecular mechanisms of disease as well as healthy functions is different in mice compared to humans.
We have discovered that human stem cells and human cancer cells grow by the same mechanism. Both cancer cells and pluripotent stem cells overexpress MUC1* which is a potent growth factor receptor (Mahanta S et al 2008). The ligand of MUC1*, NM23-H1, also called NME1, in dimeric form is alone sufficient to make human stem cells grow in the pluripotent state, without the need for feeder cells, conditioned media, or any other growth factors or cytokines (Hikita S et al 2008). We previously showed that NM23-H1 dimers are ligands of the MUC1* growth factor receptor, wherein MUC1* is the remaining transmembrane portion after most of the extra cellular domain has been cleaved and shed from the cell surface. The remaining portion of the extra cellular domain is comprised essentially of the PSMGFR sequence. NM23 dimers bind to and dimerize the MUC1* receptor. Competitive inhibition of the NME-MUC1* interaction, using a synthetic PSMGFR peptide, induced differentiation of pluripotent stem cells, which shows that pluripotent stem cell growth is mediated by the interaction between NM23 dimers and MUC1* growth factor receptor. Similarly, a large percentage of cancers grow by virtually the same mechanism. MUC1 is cleaved to the MUC1* form on over 75% of all human cancers. Like human stem cells, human cancer cells secrete NM23 where, after dimerization, it binds to the MUC1* receptor and stimulates growth of human cancer cells. Competitive inhibition of the interaction by either addition of the PSMGFR peptide or by adding the Fab of an anti-MUC1* (anti-PSMGFR) antibody caused a great reduction in the growth of human tumor cells in vitro and in vivo.
We discovered a new growth factor of the NME family, NME7, that makes human stem cells grow and inhibits their differentiation, in the absence of FGF or any other growth factor. NME7 is only reportedly expressed in the embryo but not in any adult tissue except in testes. Surprisingly, we discovered that many human cancers, especially aggressive cancers, express NME7 and secrete it in an activated form that has undergone post-translational cleavage to produce an NME7 species that runs with an apparent molecular weight of −33 kDa. This is in contrast to full-length NME7 that has a calculated molecular weight of −42 kDa and which appears to be restricted to the cytoplasm. Secreted NME7 functions as a growth factor for human stem cells and human cancers. Like NM23 dimers, NME7 binds to and dimerizes the MUC1* growth factor receptor; however, NME7 does so as a monomer as it has two binding sites for the extracellular domain of MUC1*. Thus, human stem cells and human cancers grow via the same growth factors: NM23, also called NME1, in dimer form or NME7.
NME proteins promote growth and pluripotency of embryonic and iPS cells as well as inducing cells to revert to a stem-like state. Because much of the genetic signature of a stem-like state and a cancerous state is now shared (Kumar S M et al 2012, Liu K et al 2013, Yeo J C et al 2014, Oshima N et al 2014, Wang M L et al 2013), we conclude that NME family member proteins are also able to induce a cancerous state. In a preferred embodiment the NME family member protein is NME1 or an NME protein having greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity to NME1, wherein said protein is a dimer. In a more preferred embodiment, the NME family member protein is NME7 or an NME protein having greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity to at least one of the NME7 domains A or B and able to dimerize the MUC1* growth factor receptor.
Here, we report that NME1 in dimer form, a bacterial NME1 in dimer form, NME7 or NME7-AB were able to: a) fully support human ES or iPS growth and pluripotency, while inhibiting differentiation; b) revert somatic cells to a more stem-like or cancer-like state; and c) transform cancer cells to the highly metastatic cancer stem cell state, also referred to as tumor initiating cells.
We made recombinant human NM23, also called NME1, dimers that bear the 5120G mutation that stabilizes dimers. We previously reported that human NME1 dimers bind to the PSMGFR portion of the extracellular domain of the MUC1* receptor (Smagghe et al. 2013). We also made recombinant human NME7-AB that is secreted as a monomer.
Additional experiments were performed that showed that bacterial NMEs with greater than 30%, or more preferably 40%, identity to human NME1 or NME7 function like the human NMEs that promote cancer and stem cell growth and survival. Many of the bacterial NMEs that had this high sequence identity to the human NMEs were reported to be implicated in human cancers. Here we report that bacterial NME proteins that have high sequence identity to the human NMEs are able to function like human NMEs to promote the growth of cancers and to promote the transformation of cancer cells to cancer stem cells or more metastatic cancer cells. The bacterial NME was tested in functional assays against human NME1 and NME7.
However, it is known that mouse stem cells grow by a different mechanism than human stem cells. Mouse LIF, leukemia initiating factor, can fully support mouse stem cell growth, whereas LIF has no effect on the growth of human stem cells. The primary growth factor used in the culture of human stem cells is bFGF (Xu C et al 2005), or basic fibroblast growth factor. bFGF cannot support mouse stem cell growth. This shows that the basic biology of mouse stem cell growth is very different from that of human stem cell growth. This fact becomes very important when considering the testing of cancer drugs in mice.
The current practice for testing cancer drugs in mice and other animals is to inject human cancer cells into the animal and either immediately or after several days or weeks of engraftment, inject the animal with the test drug. However, this approach is fundamentally flawed because the host does not naturally produce the growth factors that the human cancer cells need to grow or engraft. Additionally, because the host does not produce the growth factor or the same levels of the growth factor or the human form of the growth factor, drugs being tested in the animals will not have the same effect as they would in humans. Mouse NME7 is only 84% homologous to human NME7 and is not expressed in the adult. Therefore, current xenograft methods for anti-cancer drug testing often fall short in predicting human response to those drugs. This problem could be solved by introducing NM23 dimers or NME7 into the mice so that the human tumor cells have their cognate growth factor to feed the tumor. NM23 dimers or NME7 can be introduced into an animal by a variety of methods. It can be mixed in with the tumor cells prior to implantation, or it can be injected into the animal bearing the tumor.
In a preferred embodiment, a transgenic animal is generated that expresses human NME7 or a fragment thereof. The NME7 may be carried on an inducible promoter so that the animal can develop naturally, but expression of the NME7 or the NME7 fragment can be turned on during implantation of human tumors or for the evaluation of drug efficacies or toxicities. In a preferred embodiment, the NME7 species that is introduced to the test animal is NME7-AB.
Alternatively, a transgenic animal can be made wherein the animal expresses human MUC1, MUC1*, NME7 and/or NM23-H1 or -H2, a variant of H1 or H2 that prefers dimer formation, single chain constructs or other variants that form dimers. Because NME proteins and MUC1 are parts of a feedback loop in humans, wherein expression of one can cause upregulation of the other, it could be advantageous to generate transgenic animals that express human NME protein(s) and MUC1 or the cleaved form MUC1*. A natural or an engineered NME species can be introduced into animals, such as mice, by any of a variety of methods, including generating a transgenic animal, injecting the animal with natural or recombinant NME protein or a variant of an NME protein, wherein NME1 (NM23-H1), NME6 and NME7 proteins or variants are preferred and NME NME1 (NM23-H1), NME6 and NME7 proteins or variants that are able to dimerize MUC1*, specifically the PSMGFR peptide, are especially preferred. In a preferred embodiment, the NME species is a truncated form of NME7 having an approximate molecular weight of 33 kDa. In a more preferred embodiment, the NME7 species is devoid of the DM10 domain of its N-terminus. In a still more preferred embodiment, the NME7 species is human.
NME family proteins, especially NME1, NME6 and NME7 are expressed in human cancers where they function as growth factors that promote the growth and metastasis of human cancers. Therefore, human NME protein or active forms of NME protein should be present for proper growth, evolution and evaluation of human cancers and for determining their response to compounds, biologicals or drugs.
The Presence of NME7 Increases the Engraftment Rate of Human Cancer Cells in Non-Human Animals
Another problem with testing of human cancers and drugs to treat them in rodents and other animals is that many human cancers do not easily engraft in the host animal. Many cancer cell lines that are routinely used in in vitro testing simply have engraftment rates that are too low for reliable animal studies. For example, T47D breast cancer cell line is typically used for in vitro studies because of its overexpression of the oncogene MUC 1. However, T47D cells are infrequently used in mouse xenograft studies because of their poor engraftment rate.
Like many human cancers, T47D cells express and secrete NME1 and NME7. A problem that arises when verifying in vitro results in animals bearing human tumors is that the rate of T47D engraftment into mice is usually between 25-35%. That means that one needs to start with at least four-times as many mice as the experiment requires and wait several weeks in order to identify those mice whose tumors engraft. More importantly, it is problematic that the engraftment rate is so low and raises the concern that the host is not producing an environment that mimics the human and therefore, the drug testing results may be of limited use.
Engraftment of human cancers into rodents and other test animals is greatly enhanced by the presence of an NME protein, wherein the NME protein can be NME1, NME6 or NME7. There are many methods known to those skilled in the art for introducing the NME protein into a test animal. The NME protein can be injected into the test animal or the transgenic animals that express the NME protein can be generated. In some instances, it is desirable to be able to control the timing of the expression of the NME protein. In these cases, the protein expression may be linked to an inducible genetic element such as a regulatable promoter. In a preferred embodiment, the NME protein that is introduced into an animal to increase engraftment of human stem cells or cancer cells is human NME7. In a yet more preferred embodiment, the NME7 protein is a fragment that is −33 kDa. In a still more preferred embodiment, the NME protein is human NME7-AB.
In mouse studies, animals injected with human NME7-AB displayed an increase in the engraftment rate of human tumor cells. Engraftment of MUC1*-positive tumor cells (T47D) was greatly enhanced by mixing the cancer cells with NME7 prior to implantation into the animals and further enhanced by daily injections of NME7 into the tumor-bearing mice. In one experiment, the average growth increase from Day 7 until day 21 was 144% when tumors were mixed with NME7 prior to implantation and 57% without it, see
Cancer Stem Cells
In a related matter, there is currently no practical method for evaluating the efficacy, toxicity or dosing of compounds, biologicals or drugs on ‘tumor initiating cells’, which are also called ‘cancer stem cells’. Cancer stem cells are the minority of cells in a tumor that have the ability to initiate tumor formation. These cells are thought to be the ones responsible for metastasis since a single cell that breaks free from a tumor could in theory give rise to a distant metastasis. Researchers have also developed evidence that cancer stem cells can escape the killing effects of chemotherapy (Fessler S et al 2009, Lissa Nurrul Abdullah and Edward Kai-Hua Chow 2013). Cancer stem cells were first identified by the presence of specific molecular markers such as CD44-high and CD24-low. More recently, the receptor CXCR4 has been identified as a metastatic receptor whose expression is elevated in cancer stem cells and metastatic cancer cells. There are now a panel of genes thought to be markers of cancer stem cells (Miki J et al 2007, Jeter C R et al 2011, Hong X et al 2012, Faber A et al 2013, Mukherjee D et al 2013, Herreros-Villanueva M et al, 2013, Sefah K et al, 2013; Su H-T et al 2013).
The experiment considered to be definitive proof of cancer stem cells is if very small numbers of the cells are able to generate a tumor in an animal. Whereas the typical number of cancer cells required for tumor engraftment into the flank of a nu/nu mouse is 4,000,000 to 6,000,000, cancer stem cells are able to initiate tumor formation from as few as 100 or so cells, albeit in most published reports these cancer stem cells were implanted into the mammary fat pad and required months to develop. Although there are methods for identifying cancer stem cells, to date, there has been no evidence that it is possible to expose cancer cells to an agent or agents which causes them to be transformed to cancer stem cells. Therefore, there is currently no practical way of testing compounds, biologicals or drugs for their ability to inhibit these metastatic cancer stem cells. A vast improvement over the current state of the art would be to develop a way to influence regular cancer cells to rapidly become cancer stem cells so that in addition to the original cancer cells, the metastatic cancer stem cells could also be screened to identify treatments that would inhibit them. In this way a patient could be treated with drugs to inhibit the primary cancer as well with drugs that would inhibit the sub-population of cancer stem cells that could kill the patient years later when the cancer stem cells metastasize.
Agents that are able to revert primed stem cells to the naïve state (Nichols J, Smith A 2009, Hanna J et al 2010, Smagghe B et al 2013) or able to maintain naïve stem cells in the naïve state are also able to transform cancer cells into a more metastatic state also sometimes referred to as cancer stem cells or tumor initiating cells. For example, NME proteins are able to maintain stem cells in the naïve state and are able to revert primed state stem cells to the naïve state. In particular, NME1 and NME6 in the dimeric form or NME7 as a monomer are able to maintain stem cells in the naïve state and are also able to transform cancer cells to a more aggressive or metastatic state. NME7 transforms cancer cells into cancer stem cells.
NME1 and NME7 have the ability to transform cancer cells to the more metastatic cancer stem cell state, also called tumor initiating cells. A panel of cancer cells were cultured in a serum-free minimal base media with human NME7-AB or human NME1 dimers (‘NM23’ in figures) as the only growth factor or cytokine. After several days in this media, cells began to float off the surface and continued to grow in solution. The ‘floaters’ were collected and separately analyzed by PCR. Cells in other wells were treated with a rho kinase inhibitor (‘Ri in figures’). Quantitative PCR measurements show an increase of the cancer stem cell markers, some of which used to be thought of as stem cell markers only.
Others have reported that inhibitors called ‘2i’ (Silva J et al 2008) and ‘5i’ (Theunissen T W et al 2014) are able to maintain stem cells in the naïve state. We reasoned that they would also be able to transform cancer cells to a cancer stem cell or metastatic state. Our experiments demonstrate that this is so. Treatment of cancer cells with human recombinant NME7-AB for 7-10 days dramatically increased the expression of markers of cancer stem cells. Similarly, culture in NME1 dimers, bacterial NME1 dimers or treatment with the ‘2i’ inhibitors or ‘5i’ inhibitors caused cancer cells to be transformed into cancer stem cells. 2i refers to inhibitors of the MAP kinase pathway and GSK3 inhibitors such as PD0325901 and CHIR99021. Expression of the metastasis receptor CXCR4 was greatly increased as were other markers of cancer stem cells, such as E-cadherin, Oct4 and Nanog, in some cases. Cancer cells cultured in human NME7-AB increased expression of CXCR4 by nearly 200-fold. In addition, the morphology of the cancer cells changed after exposure to NME1 dimers, NME7, bacterial NME1 dimers or inhibitors that make human primed stem cells revert to the naïve state. The NME treated cells, which are normally adherent floated off the surface and grew in suspension. Analysis showed that the cells that remained adherent had less of an increase in the expression of markers of cancer stem cells.
In one experiment, MUC 1-positive T47D breast cancer cells were cultured in either normal RPMI growth media, or a serum free minimal media to which was added recombinant human NME7-AB as the single growth factor. A portion of those cells became non-adherent and floated off the surface and were collected while ‘±Ri’ refers to Rho kinase inhibitor that was added to the media so that all the cells would remain attached to the surface, thus giving an average reading of the adherent and the non-adherent cells.
Similarly, bacterium HSP593 expresses an NME1 that forms dimers and can fully support stem cell growth while inhibiting differentiation. Further, it can maintain naive stem cells in the naive state. Here we show that bacterial NME1 also transforms cancer cells to a cancer stem cell state, see
Others previously reported that kinase inhibitors were able to revert stem cells from a primed state to a naive state and maintain them in the naive state. 2i inhibitors, which are small molecule inhibitors of GSK3-beta and MEK of the MAP kinase signaling pathway, have been reported (Silva J et al, 2008) to revert mouse primed stem cells to the naive state. We wondered whether these inhibitors could also revert human cancer cells to the cancer stem cell state. Here we report that the 2i inhibitors induce cancer cells to become transformed to cancer stem cells or a more metastatic state. The invention envisions any combination of genes, proteins, nucleic acids or chemical agents that make human stem cells in the primed state revert to the less mature naive state can also be used to transform cancer cells into a more metastatic state often called cancer stem cells or tumor initiating cells.
Here we show that these ‘2i’ inhibitors also transform cancer cells to a cancer stem cell state displaying markers of a metastatic state.
Many types of cancer cells undergo this transition to a more metastatic state when treated with agents such as NME1, NME6 dimers, NME7, 2i or 5i, which are able to revert stem cells from the primed state to the less mature naive state. In one example, DU145 prostate cancer cells that were cultured in regular cancer growth media or serum-free media to which was added one of these agents, also displayed an increase in the expression of stem cell, cancer stem cell and metastatic markers, including CDH1, also called E-cadherin is often elevated in metastatic prostate cancers. Prostate cancer cells were also cultured as described above with either human NME1 dimers, bacterial NME1 dimers, NME7-AB, or 2i inhibitors. Unlike the T47D cells, the prostate cancer cells did not become non-adherent. Thus, the RT-PCR measurements of the cancer stem cell markers are lower than that of the T47D cells, which could be explained by it being the measure of transformed cells and non-transformed cells. In the case of breast cancer cells, we were able to isolate the fully transformed cancer stem cells by collecting the floating cells, but were not able to do so here. DU145 MUC1-positive prostate cancer cells were cultured in RPMI media as a control and in minimal media to which was added recombinant human NME1/NM23 dimers, bacterial HSP593 dimers, or human NME7-AB.
PC3 MUC1-negative prostate cancer cells were also tested for their ability to transform into cancer stem cells when cultured in agents that are able to maintain stem cells in the naive state. PC3 cells were cultured as described above with either human NME1 dimers, bacterial NME1 dimers or NME7-AB. Like DU145 prostate cancer cells, PC3 cells did not become non-adherent. Thus, the RT-PCR measurements of the cancer stem cell markers may be lower because measurements will be the average of transformed cells and non-transformed cells.
Other agents have been reported to maintain stem cells in the naive state or revert primed stem cells to the naive state. Chromatin re-arrangement factors MBD3 and CHD4 were recently reported to block the induction of pluripotency (Rais Y et al, 2013). For example, siRNA suppression of the chromatin re-arrangement factors MBD3 and CHD4 were shown to be key components of a method for reverting human primed stem cells to the naive state. Transcription factors BRD4 and co-factor JMJD6 reportedly suppress NME7 and up-regulate NME1 (Lui W et al, 2013). We found that these factors were expressed at lower levels in naive stem cells than they were in the later stage primed stem cells,
We observed that these four (4) genes, MBD3, CHD4, BRD4 and JMJD6, are naturally suppressed in cancer cells that were cultured in 2i, NME1 dimers or NME7.
Agents that are able to maintain stem cells in the naive state are also able to transform somatic cells to a cancer or stem-like state. Human NME1 dimer or human NME7 are able to make somatic cells revert to a less mature state, expressing stem and cancer cell markers. Bacterial NME from HSP 593 was tested alongside the human homologs to determine if it could mimic their function by being able to revert somatic cells to a cancer-like state. Human fibroblasts were cultured in a serum-free minimal base media with either HSP 593, human NME1 dimers or human NME7-AB as the only growth factor or cytokine. RT-PCR measurement showed that like the human NMEs, bacterial NME1 HSP 593 reverted somatic cells to an OCT4-positive stage by Day 19. Recalling that stem cells and metastatic cancer cells can grow anchorage-independently, we repeated the experiments but this time a rho kinase inhibitor was added to one set of cells to make the cells adhere to the surface. When the floating cells were forced to adhere to the surface, RT-PCR showed that there had actually been a 7-fold increase in stem/cancer marker OCT4 and as high as a 12-fold increase in the stem/cancer markers Nanog. Photos of the experiment show the dramatic change in morphology as the fibroblasts revert when cultured in human or bacterial NME,
Agents that transform cancer cells to a more metastatic cancer stem cell state decrease expression of pluripotency-blocking genes BRD4, JMJD6, MBD3 and CHD4. These agents also decreased expression of BRD4, JMJD6, MBD3 and CHD4 in somatic cells. Thus, NME1 dimers, NME7 and bacterial NME1 dimers cause somatic cells to revert to a less mature cancer/stem-like state.
Factors that are able to maintain stem cells in the naive state are also able to transform cells to a cancer-like or stem-like state and are also able to transform cancer cells to a cancer stem cell state or a more metastatic state. Herein, we have shown several examples of bacterial NME1 being able to function like human NME1 dimers, which are also able to function like NME7 or NME7-AB. However, the primary example that these factors function in a very similar way is their ability to promote the growth of stem cells, inhibit differentiation and maintain them in the naive state. We have demonstrated that human NME1 dimers, also called NM23-H1, bacterial NME1 and NME7-AB promote the growth of embryonic stem cells and induced pluripotent stem cells, inhibit their differentiation and maintain them in a naive state as evidenced by global genetic analysis, having both X chromosomes in the active state if stem cell donor is human and by having the ability to form teratomas in a host animal. Human HES-3 embryonic stem cells were cultured in a serum-free minimal base media with either HSP 593, human NME1 or NME7-dimers or human NME7-AB as the only growth factor or cytokine. Just as human NME1 and NME7 fully supported human stem cell growth, so did bacterial NME from HSP 593.
A method for the identification or testing of agents to treat cancer, and metastatic cancer in particular, is comprised of the steps of: 1) contacting cancer cells with an entity that is able to maintain stem cells in the naive state; 2) contacting the cancer cells with an agent; 3) assessing the ability of the agent to inhibit the growth or metastatic potential of the cancer cells; 4) concluding that agents that inhibit the growth or metastatic potential of the cancer cells are suitable for treating a patient with cancer; and 5) administering the agent to the patient. An inhibition of the metastatic potential of cancer cells is indicated by a decrease in the expression of CXCR4, E-cadherin, MUC1, NME1, NME7 or stem cell markers OCT4, SOX2, NANOG, KLF2 or KLF4, or an increase in the expression of MBD3, CHD4, BRD4 or JMJD6.
Alternatively, cells that have been cultured in the presence of an agent that is able to maintain stem cells in a naive state are then transplanted into a test animal. Since cancer cells cultured in the presence of an agent that maintains stem cells in the naive state become cancer stem cells and more metastatic, they can be implanted at very low numbers into test animals. Drugs and drug candidates can then be tested for efficacy, toxicity or to establish dosing regimens in animals implanted with cancer stem cells generated in this way.
In one experiment, T47D breast cancer cells were cultured with human recombinant NME7-AB for 7 days. The floating cells were collected and analyzed by RT-PCR which showed an increase in the expression of genes associated with cancer stem cells. In particular the expression of the metastasis receptor CXCR4, which is a key indicator of metastatic potential, was 130-times higher than that of the parent cells. These cells were implanted into immune-deficient, nu/nu female mice. The number of cells implanted was 50, 100, 1,000 or 10,000. Recall that 4,000,000 to 6,000,000 cells are normally required to get tumor engraftment, whereas as few as 100 cancer stem cells have been known to give rise to a tumor in an immune-compromised mouse, albeit with very slow growth rates, such as several months for tumor development.
Additionally, half of the mice were injected daily with recombinant human NME7-AB. Tumor engraftment was achieved even for the group implanted with only 50 cells. Surprisingly, the groups that were injected daily with NME7 had increased rate of engraftment and some of the animals in that group also formed multiple tumors. That is to say, the cancers of the group injected with NME7 daily metastasized after about 50 days and gave rise to multiple tumors at sites remote to the initial implantation site. In this particular experiment, 67% of the 24 mice implanted with the NME7-induced cancer stem cells developed tumors. However, a closer look at the data shows that only 50% of the mice that did not having circulating NME7 formed tumors, while 83% of the mice receiving daily injections of NME7 formed tumors. Of that 83% that formed tumors, 80% developed cancer metastasis as they had multiple tumors by approximately Day 50 of the experiment.
In general, the animal need not be injected per se with the agent that makes stem cells revert to the naive state such as NME1 dimers, NME7-AB, 2i, 5i and the like. In an alternative approach, the agent can be administered to the animal by injection, adsorption, or ingestion.
Therefore, a method for transforming cancer cells to cancer stem cells is to culture cancer cells in NME1 dimers, NME7, bacterial NME1 dimers or inhibitors that make primed stem cells revert to the naive state. In a preferred embodiment, the agents that make primed stem cells revert to the naive state are the 2i inhibitors or the 5i inhibitors. In a preferred embodiment, the NME7 is human NME7. In a more preferred embodiment the NME7 is NME7-AB. Drugs and drug candidates for the treatment of cancers or to inhibit the progression of cancers, especially metastatic cancers are then tested on these cancer stem cells. Additionally, cancer stem cells generated by these methods can be analyzed in order to identify still unknown markers of metastatic cancer cells, which would then be new targets for anti-cancer drugs.
Therefore, a method for evaluating compounds, biologicals or drugs for the treatment of cancers or for the prevention of metastasis or for the inhibition of metastasis comprises: 1) transforming cancer cells into more metastatic cancer cells by culturing the cells in a media that contains NME1 dimers that may be human or bacterial, NME7 or a fragment thereof or NME7-AB, 2i inhibitors or 5i inhibitors; 2) contacting the cells with a test agent in vitro or implanting the cells in an animal then treating the animal with the test agent; 3) evaluating the effect of the test agent on tumor growth or metastasis; 4) concluding that a test agent that inhibits tumor growth or metastasis to remote sites is an agent suitable for treatment of patients diagnosed with cancer, patients with cancer recurrence or metastatic cancers or patients at risk for developing cancers. In a preferred embodiment, the test animal is injected with an NME protein, 2i or 5i, or is a transgenic animal that expresses human NME or human NME7 or NME7-AB. Alternatively, the animal is a transgenic animal in which the kinases inhibited by 2i or 5i are suppressed or agents to suppress the kinases are administered to the test animal.
Metastasis in Mouse
As discussed above, low numbers of cancer cells were able to initiate tumors in animals or cancer cells metastasized in animals that were injected with NME7-AB. However, the same effect is readily accomplished by generating transgenic animals that express human NME7 or NME7-AB. In one aspect of the invention, the animal is a rodent. A transgenic mouse expressing human NME7, human NME1 or mutants that prefer dimerization or bacterial NME, human MUC1 or MUC1* would be of great use in drug discovery, for growing cancer cells in vivo and for testing the effects of immunizing NME-derived peptides as elements of an anti-cancer vaccine and for generating animals that could support the growth of human stem cells in their developing organs to yield an animal, such as a mouse, with some human heart tissue, and the like. Murine NME proteins differ significantly from human NME proteins, which are required for human cancer growth and for human stem cell growth. Therefore a normal mouse does not produce the requisite proteins to support the growth of human cancer cells or human stem cells. A mouse or other mammal that would spontaneously form tumors, or respond more like a human to drugs being tested or that would better allow human tumor engraftment, would be advantageous. Transgenic animals that better support the growth of human cancer cells or human stem cells are therefore generated by making the animal express human NME genes. In a preferred embodiment the human NME gene is human NME7. In a more preferred embodiment, the human NME gene is human NME7-AB. Transgenic animals are generated using a number of methods. In general, a foreign gene is transferred into the germ cells of a host animal. The transgene can be integrated into the host animal's genome. Some of the methods for transgene integration enable site specific integration. One of the advantages of some methods of site-specific integration is that they allow the expression of the transgene to be controlled by the expression of a naturally occurring gene in the host animal. Methods for generating transgenic animals are known to those skilled in the art. Such methods include knock-in, knock-out, CRISPR, TALENS and the like. The invention envisions using any method for making the mammal express human NME1, bacterial NME1, NME7 or NME7-AB.
In a preferred embodiment, a transgenic animal that expresses human NME7 or NME7-AB is generated. Because NME1, human or bacterial, and NME7 inhibit differentiation of stem cells, it may be advantageous to use technology in which the timing of expression of the NME protein, preferably NME7 or NME7-antibody, in the transgenic animal can be controlled. It would be advantageous to have the human NME7 on an inducible promoter, for example to avoid potential problems of NME7 expression during development of the animal. Methods for making the expression of foreign genes inducible in the host animal are known to those skilled in the art. Expression of NME7 or NME7-AB can be inducible using any one of many methods for controlling expression of transgenes that are known in the art.
Alternatively, the expression or timing of expression, of NME7 may be controlled by the expression of another gene which may be naturally expressed by the mammal. For example, it may be desirable for the NME7 or NME7 variant to be expressed in a certain tissue, such as the heart. The gene for NME7 is then operably linked to the expression of a protein expressed in the heart such as MHC. In this instance, the expression of NME7 is turned off when and where the MHC gene product is expressed. Similarly, one may want to have the expression of human NME1, NME6 or NME7 turn on or off in the prostate such that the location and timing of its expression is controlled by the expression of for example, a prostate specific protein. Similarly, the expression of human NME6 or NME7 in a nonhuman mammal can be controlled by genes expressed in mammary tissues. For example, in a transgenic mouse, human NME6 or human NME7 is expressed from the prolactin promoter, or a similar gene. In this way, it would be possible to induce or repress expression of the human NME protein in a site specific manner.
Animals xenografted with human tumors and also injected with human NME7 developed metastatic cancers. Therefore, an animal model for the development of cancer metastasis is generated by making a transgenic animal that expresses human NME7 or more preferably NME7-AB. Optimally the NME7 is on an inducible promoter to allow the animal to correctly develop. Alternatively, a metastatic animal model, preferably rodent, is made by making a transgenic animal that expresses human NME or human NME7 or NME7-AB. Alternatively, the animal is a transgenic animal in which the kinases inhibited by 2i or 5i are suppressed via inducible promoters or agents to suppress the kinases are administered to the test animal. Metastatic animal models are then used to study the basic science of the development or progression of cancers as well as to test the effects of compounds, biologicals, drugs and the like on the development of cancers.
Thus a method for the identification and selection of agents to treat cancer and metastatic cancer in particular is comprised of the steps of: 1) generate a transgenic animal that expresses a form of NME or NME7; 2) implant the animal with human cancer cells; 3) treat the animal with a candidate drug for the treatment of cancer or metastatic cancer; 4) evaluate the effect of the candidate drug on the size or spread of the cancer; and 5) conclude that candidate drugs that inhibited the growth or spread of the cancer in the test animal are suitable for the treatment of cancers or metastatic cancers in humans.
Personalized Drug Discovery by Extending the Period of Time that Patient Cancer Cells Proliferate to Test for Response to Candidate Drugs
Nearly all pre-clinical drug testing and drug selection is done using a very few established cancer cell lines. Those cell lines were derived from the tumor of a patient that lived and died decades ago and have been propagated through millions of generations, so that the resultant test cells bear little or no resemblance to the donor's original cancer and even less resemblance to a new patient's cancer. It would therefore be of great benefit to be able to screen drugs and even to establish dosing using the cells of the patient to be treated as the test cells. Unfortunately, it is difficult if not impossible to grow cancer cells from a patient in a dish and even more difficult to propagate patient cells in an animal. This is because human cells, unlike mouse cells, can only divide in culture a very limited number of times before they senesce. The cancer cell lines that researchers use today are either naturally immortalized cancer cells isolated from pleural effusions of a single metastatic cancer patient or induced to become immortalized by fusing to an immortalized cell line or by transfecting the cancer cells with an immortalizing gene. The latter methods significantly alter the molecular characteristics of the original or primary cancer cell.
A method that is useful for propagating cancer cells from a particular patient is to culture the cells using methods that transform cancer cells into cancer stem cells. Another method is to culture the patient cells using reagents and methods that are used to revert primed stem cells to the naïve state. The resultant cancer stem cells are heartier, live longer and propagate for longer periods of time. As described in the section above, patient cancer cells can be propagated by culturing them in NME1 dimers that may be human or bacterial, NME7 or a fragment thereof or NME7-AB, 2i inhibitors or 5i inhibitors. The resultant cancer cells can be divided into two fractions: those that adhere to the surface and those that float off the surface. The adherent cells will closely resemble the original tumor cells while those that float will be cancer stem cells and will look like the metastatic cancer cells that the patient may develop in the future or in response to treatment with chemotherapy drugs. These patient cells are then used to identify and select drugs and candidate drugs that will effectively inhibit that particular patient's cancer as well as identify drugs that will inhibit the progression of their cancer to a metastatic state caused by the survival of cancer stem cells. In another aspect of the invention, a patient's cancer cells propagated in this way are used to determine optimal combinations of drugs that would effectively inhibit that particular patient's cancer or used to establish dosing of a drug or drugs.
Personalized Drug Discovery by Transplanting Patient Cancer Cells into Multiple Animals for Drug Testing Due to Small Number of Required Cells for Engraftment
Recall that cancer cells that have been cultured in NME1 dimers that may be human or bacterial, NME7 or a fragment thereof or NME7-AB, 2i inhibitors or 5i inhibitors are able to initiate tumors in test animals with very low numbers of cells implanted. Patient cancer cells cultured using these methods are then able to form tumors in multiple test animals because as few as 50 or 100 cells are required per mouse rather than millions. The reduced numbers of cells required to initiate a tumor then makes it feasible to implant a patient's cancer cells into test animals, some of which can be injected with NME1 dimers that may be human or bacterial, NME7 or a fragment thereof or NME7-AB, 2i inhibitors or 5i inhibitors or may be transgenic animals as described above, for example such that the animal expresses human NME7 or NME7-AB.
In one embodiment, the host animal is injected with candidate drugs or compounds and efficacy is assessed in order to predict the patient's response to treatment with the candidate drug or compound. In another instance, the first line treatments or drugs that are being administered to the patient or are being considered for treatment of the patient, are administered to the animal bearing the patient's cancer cells which are being reverted to a less mature state. The first line treatments likely influence which mutations the cancer cells adopt in order to escape the first line treatments. The resultant cancer cells can then be removed from the host animal and analyzed or characterized to identify mutations that are likely to occur in response to certain treatments. Alternatively, the cancer cells can remain in the host animal and the host animal is then treated with other therapeutic agents to determine which agents inhibit or kill the resistant cells or cancer stem cells.
Thus a method of the invention for identifying suitable treatments to inhibit the growth or spread of a patient's cancer comprises the steps of: 1) obtaining cancer cells from a patient; 2) culturing the patient's cancer cells in the presence of NME1 dimers that may be human or bacterial, NME7 or a fragment thereof or NME7-AB, 2i inhibitors or 5i inhibitors; 3) contacting said cells with a test agent for the inhibition of cancer or metastasis; 4) evaluating the efficacy, toxicity or dosing of a compound, biological or drug on the patient's cancer cells or evaluating the effect of the test agent on the levels of cancer stem cell markers expressed by the cells; and 5) determining that test agents that reduce viability of the cancer cells or reduce expression of genes known as cancer stem cell markers are suitable treatments for the patient.
Another method of the invention for identifying suitable treatments to inhibit the growth or spread of a patient's cancer comprises the steps of: 1) obtaining cancer cells from a patient; 2) culturing the patient's cancer cells in the presence of NME1 dimers that may be human or bacterial, NME7 or a fragment thereof or NME7-AB, 2i inhibitors or 5i inhibitors; 3) implanting the cells in a test animal that may also be injected with NME1 dimers that may be human or bacterial, NME7 or a fragment thereof or NME7-AB, 2i inhibitors or 5i inhibitors or is a transgenic animal that expresses NME proteins, NME7, NME7-AB or suppresses expression of the targets of 2i or 5i; 4) administering to the test animal a test agent for the inhibition of cancer or metastasis; 5) evaluating the efficacy, toxicity or dosing of a compound, biological or drug on the ability of the patient's cells to engraft in the animal, tumor size, or development of metastasis; and 6) determining that test agents that reduce engraftment rate, tumor growth rate or development of metastasis are suitable treatments for the patient.
Alternatively, the methods described above that enable the proliferation or implantation of patient cancer cells may be used for the benefit of patients other than the donor. Patient cancer cells proliferated or implanted according to the methods of the invention will more accurately resemble a naturally occurring cancer, so could replace standard cancer cell lines in use today which have been artificially immortalized by transfecting with immortalizing genes. In particular, cancer cells cultured in NME1 dimers, NME7, NME7-AB, 2i inhibitors or 5i inhibitors that express high levels of stem cell markers, cancer stem cell markers or CXCR4 can be used for the discovery, testing and selection of drugs to treat metastatic cancers in vitro, ex vivo or in vivo.
Generation of Animals that Express Human Tissue
Other applications are envisioned wherein an animal transgenic for human NME1, bacterial NME1 or human NME7, preferable NME7-AB, is implanted or engrafted with human cells which may be stem cells or progenitor cells. For example in some cases it is desirable to generate an animal, such as a mouse, that will grow human tissue in its heart, liver, skin or other organ. One method for doing so is to generate a kind of chimeric animal by implanting human stem cells into an animal that has been made to express human NME7 or human NME7-AB. The human stem cells or progenitor cells can be implanted at various stages of the animal's development, including in vitro and in vivo, at the blastocyst, embryo or fetus stage of development. Because NME7 inhibits differentiation, the NME7 or NME7-AB transgene would be linked to a method by which the timing of its expression is controllable. Methods are known to those skilled in the art which could be used such that expression of the human NME7 or NME7-AB is turned off or decreased at times or locations where it is desirable to have differentiation or maturation occur. One method for making the transgene, preferably NME7, inducible or repressible is to link its expression or repression to the expression of a gene that is only expressed later in development. In such cases, one would make a transgenic animal in which expression of NME7 or NME7-AB is linked to the expression of a later gene expressed in heart or in heart progenitor cells. Thus, the expression or timing of expression, of NME7 is controlled by the expression of another gene which may be naturally expressed by the mammal. For example, it may be desirable for the NME7 or NME7 variant to be expressed in a certain tissue, such as the heart. The gene for the NME7 is then operably linked to the expression of a protein expressed in the heart such as MHC. In this instance, the expression of NME7 is decreased or turned off when and where the MHC gene product is expressed. Similarly, one may want to have the expression of human NME1, NME6 or NME7 turn on in the prostate such that the location and timing of its expression is controlled by the expression of for example, a prostate specific protein. Similarly, the expression of human NME1 or NME7 in a non-human mammal can be controlled by genes expressed in mammary tissues. For example, in a transgenic mouse, human NME1 or human NME7 can be expressed or repressed by the prolactin promoter, or a similar gene.
In this way, an animal transgenic for human NME7 or NME7-AB can be allowed to grow to a point, then implanted with human stem or progenitor cells, where they proliferate because of contact with human NME protein. The expression of the human NME is then turned off such that a specific organ or part of an organ in the animal would develop as a human tissue.
The invention contemplates many applications of animals transgenic for human NME1, bacterial NME1 or human NME7, or NME7-AB. In one aspect of the invention, human stem or progenitor cells are implanted in the NME transgenic animal or germ cells of what will be a transgenic animal. Expression of the NME may be inducible or repressible. Depending on the site and timing of the implantation of the stem or progenitor cells, the resulted animal can be made to express human heart, liver, neuronal cells or skin.
Thus human tissues can be generated in a transgenic non-human mammal, wherein the mammal expresses human MUC1 or MUC1* or NME protein in the germ cells or somatic cells, wherein the germ cells or somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said mammal, wherein the expression of the gene sequence can be induced or repressed either by introduction of an external composition or by linking its expression or repression to the expression or repression of a naturally occurring gene of the host animal. Stem cells or progenitor cells that are xenogeneic in origin to the non-human mammal are transferred to the transgenic animal such that the gene is induced to be expressed so as to multiply the stem or progenitor cells and then repressing the gene expression so as to generate tissue from the xenografted stem cells. One method by which repression of the transgene is carried out is by contacting the stem cell or progenitor cells with a tissue differentiation factor. Transgene repression is also carried out naturally in the mammal in response to naturally produced host tissue differentiation factors. These animals can be used for drug discovery. They can also be used for toxicity testing, to use an animal to determine the effects of a compound, biological or drug on human tissue or on the development of human tissue. Alternatively, the transgenic animal implanted with human stem or progenitor cells is used to grow human tissue for transplant into a human patient. In some cases, the stem or progenitor cells that are implanted are from a patient who will be the recipient of the human tissue harvested from the transgenic animal.
In one aspect, the MUC1, MUC1* or NME protein expression may be induced until the amount of transferred stem or progenitor cells are sufficiently large. The MUC1, MUC1* or NME protein expression may then be shut down by injecting the host mammal with a substance that represses the expression of MUC1, MUC1* or NME protein. The population of stem or progenitor cells may be induced to differentiate by either natural methods such as by the expression in the mouse of a differentiation inducing factor for a particular tissue or organ type, or chemical or protein substances may be injected into the host at the site of stem or progenitor cell transference to cause differentiation to desired tissue type.
Induction, differentiation/transformation agents for endoderm cell tissue may include without limitation the following agents: hepatocyte growth factor, oncostatin-M, epidermal growth factor, fibroblast growth factor-4, basic-fibroblast growth factor, insulin, transferrin, selenius acid, BSA, linoleic acid, ascorbate 2-phosphate, VEGF, and dexamethasone, for the following cell types: liver, lung, pancreas, thyroid, and intestine cells. Induction, differentiation/transformation agents for mesoderm tissue include without limitation the following agents: insulin, transferrin, selenous acid, BSA, linoleic acid, TGF-[31, TGF-[33, ascorbate 2-phosphate, dexamethasone, P-glycerophosphate, ascorbate 2-phosphate, BMP, and indomethacine, for the following cell types: cartilage, bone, adipose, muscle, and blood cells.
Induction, differentiation/transformation agents for ectoderm tissue include without limitation the following agents: dibutyryl cyclin AMP, isobutyl methylxanthine, human epidermal growth factor, basic fibroblast growth factor, fibroblast growth factor-8, brain-derived neurotrophic factor, and/or other neurotrophic growth factor, for the following cell types: neural, skin, brain, and eye cells.
Animals Transgenic for NME Protein for Discovery and Testing of Vaccines
A transgenic animal expressing human NME, especially NME7-AB, would also be useful for assessing which immunizing peptides could safely be used for the generation of antibodies against NME proteins, including NME1, bacterial NME and NME7. For example, mice transgenic for human NME1, NME7, or NME7-AB could be immunized with one or more of the immunizing peptides set forth as in
Regulators of NME Protein or Downstream Effectors of NME Protein can Substitute for the NME Protein
These studies have shown that one way in which NME proteins function to promote stem-like or cancer-like growth is by binding to a clipped form of the MUC1 transmembrane protein, herein referred to as MUC1*, which consists primarily of the PSMGFR sequence. Dimerization of the MUC1* extracellular domain stimulates growth and de-differentiation of somatic cells, stem cells and cancer cells, making them more metastatic. Another way that NME proteins exert their effects is by being transported to the nucleus where they function directly or indirectly to stimulate or suppress other genes. It has been previously reported (Boyer et al, 2005) that OCT4 and SOX2 bind to the promoter sites of MUC1 and its cleavage enzyme MMP16. The same study reported that SOX2 and NANOG bind to the promoter site of NME7. We conclude, on the basis of our experiments that these ‘Yamanaka’ pluripotency factors (Takahashi and Yamanaka, 2006) up-regulate MUC1, its cleavage enzyme MMP16 and its activating ligand NME7. It has also been previously reported that BRD4 suppresses NME7, while its co-factor JMJD6 up-regulates NME1 (Thompson et al), which we have demonstrated is a self-regulating stem cell growth factor that is expressed later than NME7 in embryogenesis. Still others recently reported that siRNA suppression of Mbd3 or Chd4 greatly reduced resistance to iPS generation (Rais Y et al 2013 et al.) and was able to maintain stem cells in the naive state. Evidence presented here shows that there is a reciprocal feedback loop wherein NME7 suppresses BRD4 and JMJD6, while also suppressing inhibitors of pluripotency Mbd3 and CHD4. We note that in naive human stem cells, these four factors BRD4, JMJD6, Mbd3 and CHD4 are suppressed compared to their expression in later stage ‘primed’ stem cells. We also note that the 2i inhibitors (inhibitors of Gsk3(2, and MEK) that revert mouse primed stem cells to the naive state, also down regulated the same four factors BRD4, JMJD6, Mbd3 and CHD4.
We have also discovered that NME7 up-regulates SOX2 (>150×), NANOG (−10×), OCT4 (−50×), KLF4 (4×) and MUC1 (10×). Importantly, we have shown that NME7 up-regulates cancer stem cell markers including CXCR4 (200×) and E-cadherin (CDH1). Taken together these multiple lines of evidence point to the conclusion that NME7 is the most primitive stem cell growth and pluripotency mediator and that it is a powerful factor in the transformation of somatic cells to a cancerous state as well as transforming cancer cells to the more metastatic cancer stem cells.
Therefore, the present invention contemplates substituting genes and gene products that increase expression of NME7 for NME7. Similarly, the invention contemplates substituting downstream effectors of NME7 for NME7. For example, alone or in combination, agents that suppress MBD3, CHD4, BRD4 or JMJD6 can be substituted in any of the methods described herein, for NME7, which we have shown suppresses MBD3, CHD4, BRD4 or JMJD6.
Example 1: Immune-compromised nu/nu mice between 40-60 days old were implanted with 90-day estrogen release pellets to foster the engraftment and growth of breast tumor cells. Human T47D breast cancer cells were mixed 1:2 with Matrigel then injected into the flank of the mouse (4 million each mouse), six (6) mice per group. In a second group, cancer cells were mixed with Matrigel at the same 1:2 ratio (100 uL cells: 200 ul Matrigel) except, human recombinant NME7-AB was added into the cell/Matrigel mix to a final NME7 concentration of 16 nM (6 mice). Of those six (6) mice, three (3) were randomly chosen to receive NME7 injections daily (100 uL at 32 nM), after day 14.
Example 2: Several groups of immune-compromised mice between 40-60 days old were implanted with 90-day estrogen release pellets to foster the engraftment and growth of breast tumor cells and implanted into the flank of each mouse. Human T47D breast cancer cells were mixed 1:2 with Matrigel (100 uL cells: 200 ul Matrigel) then injected into one flank of each mouse (4 million each mouse). Animals were untreated and tumor growth was measured to track the rate of tumor growth and assess the percentage tumor engraftment of this cell line.
Example 3: Agents that are able to maintain stem cells in the naïve state transform cancer cells into cancer stem cells wherein only a small number of cells are required for engraftment. Cancer cells were cultured according to standard practice. In the controls, the cancer cells were cultured in their normal media: RPMI for T47D breast cancer cells, DU145 and PC3 prostate cancer cells. However, the test agents, recombinant proteins human NME1 dimers, bacterial HSP593 NME1 dimers, human NME7-AB and 2i, were added to a serum-free minimal media to eliminate potential interference from the many growth factors and cytokines in serum. As a further control, cancer cells were cultured in minimal media that did not contain NME1, NME7 or 2i, but did not proliferate nor did the resultant cells up-regulate markers of cancer stem cells,
Serum-free Minimal Media 500 mL includes the following components: 394 mL DMEM/F12, GlutaMAX; 100 mL Knockout™ Serum Replacement; 5.0 mL 100×MEM Non-essential Amino Acid Solution; 0.9 mL P-mercaptoethanol, 55 mM stock.
To this serum-free minimal media, rhNME1 (aka NM23) was added to a final concentration of 8 nM, or bacterial HSP593 recombinant NME1 was added to a final concentration of 8 nM, or rhNME7-AB was added to a final concentration of 4 nM, or 2i inhibitors, PD0325901 and CHIR99021, which inhibit the MAP kinase pathway and GSK3, respectively, were added to a final concentration of 3 μM and 1 μM.
When a rho kinase inhibitor, “Ri” or “ROCi”, was added it was Y27632 from Stemgent (Cambridge, Mass.), added immediately before use to a final concentration of 10 uM.
Example 3a: Breast cancer T47D cells were cultured either in the traditional RPMI growth media or in a minimal stem cell media to which was added either human NME1 dimers, also known as NM23-H1, bacterial HSP593 NME1 dimers, human NME7-AB, or kinase inhibitors known as ‘2i’. What these agents have in common is that they are each able to promote the growth of stem cells in the naive state or are able to revert primed state stem cells to the less mature naive state. We observed that some of the T47D cancer cells cultured in any of these agents became non-adherent. These ‘floaters’ were also morphologically different. The addition of a rho kinase inhibitor, called ‘Ri’ or ‘ROCP here caused most of the cells to remain attached to the surface. RT-PCR measurements shown in the graphs of
In
The result of culturing T47D breast cancer cells in the presence of the ‘2i’ kinase inhibitors is shown in
Example 3b: Prostate cancer cells were also cultured as described above with either human NME1 dimers, bacterial NME1 dimers, NME7-AB, or 2i inhibitors. Unlike the T47D cells, the prostate cancer cells did not become non-adherent. Thus, the RT-PCR measurements of the cancer stem cell markers are lower than that of the T47D cells, which could be explained by it being the measure of transformed cells and non-transformed cells. In the case of breast cancer cells, we were able to isolate the fully transformed cancer stem cells by collecting the floating cells, but were not able to do so here. DU145 MUC1-positive prostate cancer cells were cultured in RPMI media as a control and in minimal media to which was added recombinant human NME1/NM23 dimers, bacterial HSP593 dimers, or human NME7-AB.
PC3 MUCl-negative prostate cancer cells were also tested for their ability to transform into cancer stem cells when cultured in agents that are able to maintain stem cells in the naïve state. PC3 cells were cultured as described above with either human NME1 dimers, bacterial NME1 dimers or NME7-AB. Like DU145 prostate cancer cells, PC3 cells did not become non-adherent. Thus, the RT-PCR measurements of the cancer stem cell markers may be lower because measurements will be the average of transformed cells and non-transformed cells.
Example 3c: In this example, the effect of ‘2i’ inhibitors on cancer cells was tested. Recall that 2i is the name given to 2 small molecules, CHIR99021 and PD0325901 that inhibit the MAP kinase pathway and GSK3 respectively. In stem cell experiments, it was demonstrated that 2i is able to revert stem cells in the primed state back to the less mature naïve state. In this experiment, we cultured MUCl-positive T47D breast cancer cells in minimal media to which was added either 2i or NME7-AB. RT-PCR measurements were taken that showed that 2i as well as NME7-AB suppressed expression of chromatin rearrangement factors MBD3 and CHD4, see
Example 3d: In this example, fibroblast cells were cultured in the presence of recombinant human NME1/NM23 dimers, bacterial HSP593 NME1 dimers or NME7-AB. The fibroblasts were cultured in their normal media as a control, which is for 500 mL, 445 mL DMEM high glucose base media, 5 mL GlutaMAX and 50 mL of fetal bovine serum (FBS). After 15-20 days in culture with either NME1/NM23 dimers, bacterial HSP593 NME1 dimers or NME7-AB, the morphology of the cells completely changed so that they no longer were recognizable as fibroblasts. RT-PCR showed that the resultant cells greatly increased expression of stem cell marker genes OCT4 and NANOG, see
Example 4: Cancer stem cells generated by culture in NME7-AB initiate tumors in mice with implantation of as few as 50 cells.
T47D breast cancer cells were cultured as described in Example 3a above. After 7-10 days culture in minimal media to which was added rhNME7-AB to a final concentration of 4 nM, floating cells were collected. RT-PCR measurements showed that expression of CXCR4 was increased 130-times over the control cells that were cultured in RPMI media. The floating cells were counted and re-suspended in PBS. The NME7-induced cancer stem cells were mixed 1:2 with reduced growth factor Matrigel; that is 2 parts Matrigel to 1 part cells. A range of ratios of cancer cells to NME7 or the injection schedule of NME7 is expected to vary from one mouse strain to another and from one tumor type to another.
The cancer stem cells were implanted in the flank of female nu/nu mice which had previously been implanted with a 90-day release estrogen pellet in the shoulder area. Four (4) groups of six (6) mice each were implanted with either 50, 100, 1,000 or 10,000 T47D cancer stem cells. In each case the total volume injected into each mouse was 100 uL. Half of the mice in each group were injected daily with 200 uL of recombinant human NME7-AB in the flank near but not at the cancer stem cell implantation site.
Animals were monitored for food intake and body weight, which were normal and stable. Two (2) independent and blinded tumor measurements were taken once a week and recorded.
Tumor engraftment was achieved even for the group implanted with only 50 cells. Surprisingly, the groups that were injected daily with NME7 had increased rate of engraftment and some of the animals in that group also formed multiple tumors. That is to say, the cancers of the group injected with NME7 daily metastasized after about 50 days and gave rise to multiple tumors at sites remote to the initial implantation site. In this particular experiment, 67% of the 24 mice implanted with the NME7-induced cancer stem cells developed tumors. However, a closer look at the data shows that only 50% of the mice that did not having circulating NME7 formed tumors, while 83% of the mice receiving daily injections of NME7 formed tumors. Of that 83% that formed tumors, 80% developed cancer metastasis as they had multiple tumors by approximately Day 50 of the experiment.
All of the references cited herein are incorporated by reference in their entirety.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.
This application is a continuation of U.S. Ser. No. 14/468,106, filed Aug. 25, 2014. U.S. Ser. No. 14/468,106 is a continuation in part of PCT/US14/50773, filed Aug. 12, 2014, and is a continuation in part of PCT/US14/17515, filed Feb. 20, 2014. PCT/US14/50773 claims priority to U.S. 61/865,092, filed Aug. 12, 2013. PCT/US14/17515 claims priority to U.S. 61/767,206, filed Feb. 20, 2013; U.S. 61/768,992, filed Feb. 25, 2013; U.S. 61/774,558, filed Mar. 7, 2013; U.S. 61/837,560, filed Jun. 20, 2013; U.S. 61/865,092, filed Aug. 12, 2013; U.S. 61/894,365, filed Oct. 22, 2013; U.S. 61/901,343, filed Nov. 7, 2013; U.S. 61/925,190, filed Jan. 8, 2014; U.S. 61/925,601, filed Jan. 9, 2014; U.S. 61/938,051, filed Feb. 10, 2014. PCT/US14/17515 is a Continuation in-part of PCT/US2013/050563, filed Jul. 15, 2013; is a Continuation in-part of PCT/US2013/051899, filed Jul. 24, 2013; and is a Continuation in-part of PCT/US2013/055015, filed Aug. 14, 2013. The entire contents of the aforementioned patent applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61865092 | Aug 2013 | US | |
| 61767206 | Feb 2013 | US | |
| 61768992 | Feb 2013 | US | |
| 61774558 | Mar 2013 | US | |
| 61865092 | Aug 2013 | US | |
| 61894365 | Oct 2013 | US | |
| 61901343 | Nov 2013 | US | |
| 61925190 | Jan 2014 | US | |
| 61925601 | Jan 2014 | US | |
| 61938051 | Feb 2014 | US | |
| 61837560 | Jun 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14468106 | Aug 2014 | US |
| Child | 17449932 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US14/50773 | Aug 2014 | US |
| Child | 14468106 | US | |
| Parent | PCT/US14/17515 | Feb 2014 | US |
| Child | 14468106 | US | |
| Parent | PCT/US2013/050563 | Jul 2013 | US |
| Child | PCT/US14/17515 | US | |
| Parent | PCT/US2013/051899 | Jul 2013 | US |
| Child | PCT/US2013/050563 | US | |
| Parent | PCT/US2013/055015 | Aug 2013 | US |
| Child | PCT/US2013/051899 | US |
