JP2015515261A - Screening assay for identification and validation of drugs targeting cancer stem cells - Google Patents

Abstract

Methods are described for the identification and validation of drugs that selectively target cancer stem cells (CSCs) but have no effect on normal stem cells (NSCs). An agent is identified by the ability of the agent to inhibit cancer stem cell surrogate (sCSC) cell proliferation or induce cell differentiation or death, but does not have a similar effect on normal stem cells. Cancer stem cell surrogate (sCSC) is a human embryonic stem cell (t-hPSC) or transformation-induced pluripotent stem cell (t-iPSC) that acquires a naturally transformed property but retains pluripotency . Dopamine receptors (DRs) are identified as biomarkers for cancer stem cells. Dopamine receptor blockers induce the differentiation of cancer stem cells (CSCs) and eradicate the ability of cancer stem cells (CSCs) to seed cancer. [Selection] Figure 27

Description

  The present invention relates to a method for prognosis or treatment of cancer and a screening method, and more particularly to a method for prognosis or treatment of cancer targeting dopamine receptors, and a screening method for identification and verification of drugs targeting cancer stem cells. It is about.

  This application has priority over US patent application 13 / 605,609 filed September 6, 2012 (Patent Document 1) and PCT international application PCT / CA2012 / 000175 filed February 28, 2012 (Patent Document 2). All of which are hereby incorporated by reference.

  Much evidence is attributed to the rare cell population whose cancer / tumor expression is called cancer stem cells (CSC) (Dick, 2009; Jordan, 2009; Reya et al., 2001) that can independently initiate and maintain disease I suggested that. Further, experimental results show that conventional chemotherapeutic agents characterized by the ability to suppress the growth of cancer cell lines (Shoemaker, 2006) or reduce tumor burden in mouse models (Frese and Tubeson, 2007) are human cancer stem cells (CSC) was shown to be ineffective (Guan et al., 2003; Li et al., 2008). This resistance to chemotherapeutic agents is often combined with indiscriminate cytotoxicity that affects healthy stem and progenitor cells, leading to dose limitations and requiring protective treatment (Smith et al., 2006). A recent example along this direction is the selective induction of cell death (Gupta et al., 2009; Raj et al., 2011), which still remains tested in normal stem cells and human systems. Therefore, the identification of drugs that target only cancer stem cells is an urgent issue in order to provide truly selective anticancer drugs for preclinical testing.

  Normal and neoplastic stem cells are functionally defined by a tightly controlled equilibrium between self-renewal and differentiation potential. In the case of cancer stem cells, this equilibrium state leads to increased self-renewal and survival, resulting in limited differentiation capacity, which ultimately allows tumor growth. In contrast to the direct toxic effects that equally affect normal stem cells, another means of eradicating cancer stem cells is to change this equilibrium to differentiation in an effort to exhaust the cancer stem cell population. by. Therefore, identifying molecules that selectively target somatic cancer stem cells while preserving healthy stem cell ability is to selectively target human cancer stem cells for the development of innovative diagnostics and treatments. Useful for.

  Methods have been described for discovering anti-cancer compounds that are selectively effective on cancer cells compared to the effects of certain compounds on normal cells, thereby finding anti-cancer stem cells (US Pat. No. 6,180,357). ). However, progress in technology was required to identify cancer stem cells. Several advances are needed to discover selective anticancer stem cell compounds. Among them, both cancer stem cells and normal stem cells are grown in a sufficiently large number, these cells are maintained in an undifferentiated state, have stem cells that are strong enough for high-throughput screening including robotic manipulation, Development, development of relevant stem cell endpoints that are compatible with high throughput screening. The number of cells that can be used for high throughput is a major technical limitation. Since stem cells are rare in vivo, primary isolation is challenging and therefore culturing of stem cells allows for an increase in the number of stem cells. Stem cells in culture tend to differentiate and do not retain their stem cell properties in culture. One example of the difference between stem cell culture and general cell culture is that stem cell culture requires conditions without antibiotics to prevent differentiation. Limitation of cell number is evident in the paper by Kondo et al. (WO 2006/051405 A2), because selection of side population (as a surrogate for cancer stem cells) by Hoechst 3334 makes this dye toxic to stem cells This is because there is a limit (Machalinski et al., 1998). In progenitor culture, side populations form two cell populations, side population cells and non-side population cells, as disclosed in Kondo's paper. The culture does not maintain a pure group of side population cells and requires re-isolation of the side population with Hoechst 3334, thereby reducing the cell number. This problem also hinders the determination of mixed cell endpoints in culture because pure side population is not directly monitored. Furthermore, subculture and subpopulation analysis is not possible because cell culture forms a mixed population. Kondo et al. Disclaims a method for discovering selective cancer stem cell compounds, but in a high-throughput manner, maintains a pure side population, shows the effect of the same compound on normal cells, And the inability to compare them with the side populations disclosed as cancer stem cell models is a limitation of existing techniques for discovering selective anti-cancer stem cell drugs.

  Similarly, Tyer's patent (US Pat. No. 8,058,243) shows other limitations of the prior art. The literature describes clonal native neurospheres that identify potent and / or selective regulators of neural precursor cells, neural progenitor cell proliferation, differentiation regeneration, and / or self-renewal and / or pluripotent neural stem cells An assay method is disclosed. The screen is directed to compounds that are active in the stem cell assay and does not necessarily target cancer stem cells. The counter screen was an astrocyte cell line rather than a normal stem cell line so as to detect selective cancer stem cell versus normal stem cell activity rather than stem cell vs differentiated cell activity. The document discloses testing 12 subsets of medulloblastoma precursor cells among active compounds that are rich in cancer stem cells but are not a pure population of cancer stem cells. The discovery of anticancer stem cell compounds relies on the discovery of active compounds against neurosphere cells and further testing. Since neurospheres contain neural stem and progenitor cells, facilities for discovering selective anticancer stem cell compounds are excluded because the first step of the screen is directed at normal stem cells . Thus, Tyers's invention is self-evident in trying to discover anti-cancer stem cell compounds at high throughput because the invention first identified active stem cell compounds by high-throughput screening and then enriched with subsets of cancer stem cells. This is because he teaches testing on various cells.

  Hematological tumors are a type of cancer that affects the blood, bone marrow, and lymph nodes. Hematological tumors originate from one of two major blood cell differentiation lineages: myeloid or lymphoid cell lines. Examples of hematological tumors include acute myeloid leukemia and chronic myeloid leukemia.

  All hematological tumors are generally thought to arise from precursors of the myeloid lineage within the bone marrow, but they are highly diverse in symptoms, pathology and treatment. For example, the 2008 WHO Myeloproliferative Tumor Classification (Tefferi et al., Cancer September 1 issue, pages 3842-3847 (2009); and Vannucci et al. Advances in understanding and managing myeloproliferative tumors CA Cancer J. Clin. 2009; 59: 171-191, both referenced and incorporated herein) presents five different classification schemes for myeloid tumors, and acute myeloid leukemia (AML) as chronic myelogenous leukemia (CML) and others It is classified into a different category from myeloproliferative tumors. In addition, chronic myeloid leukemia (CML) is often characterized as containing a BCR-Abl translocation, which is not present in acute myeloid leukemia (AML). A suitable treatment for leukemias such as bone marrow tumors will target leukemia cells without unduly affecting the hematopoietic stem cell population.

Thioridazine is a dopamine receptor blocker belonging to the phenothiazine drug group and is used as an antipsychotic. It has been used in hospitals since 1959, but due to concerns about cardiotoxicity and retinopathy at high doses, this drug is not generally prescribed and is a more universally used antipsychotic drug, Set aside for patients who fail to respond or are contraindicated. Patients with schizophrenia who are treated with dopamine receptor blockers at doses that are considered effective for schizophrenia have a lower incidence of rectal, colon, and prostate cancer than the general population It has been reported.
There is a need for new methods for the treatment and prognosis of cancer, particularly for the treatment and prognosis of acute myeloid leukemia. There is also a need for new methods for identifying and validating drugs that target cancer stem cells.

US patent application 13 / 605,609 PCT International Application PCT / CA2012 / 000175

  The discovery of drugs that target cancer stem cells can be approached through several methodologies. The molecular mechanism that distinguishes cancer stem cells from normal stem cells has not been fully elucidated. Although there are markers associated with cancer stem cells, such markers are deficient and they may not necessarily be targets for drug discovery. In addition, selection of compounds that can target either stem cells and potentially deplete the pool of stem cells through differentiation induction or reduce the number of stem cells through various mechanisms is normal stem cells Should be selective in order to have a similar effect and to avoid giving excessive therapeutic toxicity as a result. Therefore, there is a need for a methodology for discovering drugs that have different effects on cancer stem cells compared to other cells.

  The present specification describes methods that can be used to identify compounds that exhibit different levels of biological activity or that are selective for cancer stem cells compared to other types of cells, such as normal stem cells. I will provide a. This method functions as a surrogate and has characteristics related to tumor progression, thereby enabling high-throughput testing for the anti-cancer stem cell activity of thousands of compounds and is referred to herein as transformed tumor stem cells. Use reproducible cell lines. A suitable cell line for this test is provided in PCT / CA2010 / 001340. The methods provided herein provide a multifactorial approach to the process of comparing the effect of one compound on multiple cancer stem cells with one or more preset values or results. Such results can be determined by measuring, among other things, cell death, loss of pluripotency, morphology, cell number, and cell stress response. Furthermore, the method provides a calculation of the selective activity potency ratio (SAPR), defined as the ratio of the EC50 vs surrogate cell line EC50 to normal cells, which allows those skilled in the art to more accurately target compounds than before. Allows you to select in. The use of a series of dose range comparisons is preferred because at any single dose or small range of doses, the difference in the effect of the compound on each class of cells may not be apparent. In certain embodiments, the method further comprises comparing the effect of the compound on one or more normal stem cells with the effect of the compound on one or more cancer stem cells, such as mutant neoplastic stem cells. In certain embodiments, the effect of a compound on one or more normal stem cells is tested by contacting one or more stem cells with the compound and detecting the effect of the compound on one or more stem cells. Where the effect is indicative of the biological activity of the compound.

The inventors have determined that the use of a two-step screening method is particularly useful for identifying and validating selective anti-cancer stem cell agents. In the first stage, to select a test agent, the agent causes changes in both pluripotency and / or cell number of the mutated neoplastic stem cell, and in some cases pluripotency and cell Screened for causing changes in both number changes. In the second stage, the test agent selected in the first stage as causing pluripotency and / or a decrease in the number of cells in the mutated neoplastic stem cell compared to the normal stem cell in the number of mutated neoplastic stem cells. To test for differential effects. The use of the two-step method offers a number of advantages over conventional screening methods, and the identification of selective anti-cancer stem cell drugs that may have been classified as non-selective or unlikely in other conventional screening methods. to enable. Furthermore, normal human stem cells are generally difficult to grow in culture compared to mutant neoplastic stem cells. Before looking for differential activity with normal human stem cells, screening for first-stage drugs using neoplastic stem cells is the time and resources required to identify and validate selective anti-cancer stem cell drugs To save money. The identification of selective anti-cancer stem cell agents increases the chance that these agents will progress through the drug development stage because they are unlikely to affect normal stem cells, thereby making them non-selective This is because it reduces the possibility of side effects compared to drugs.

The inventors have determined that thioridazine is cytotoxic to cancer stem cells and specifically cytotoxic to cancer stem cells that cause acute myeloid leukemia (AML). Furthermore, thioridazine has been found to have a relatively limited effect on normal stem cells such as hematopoietic stem cells at concentrations that are toxic to cancer stem cells. The present disclosure seeks a multifactorial process for quantifying the ability of thioridazine-like compounds that are characterized as having a structure similar to thioridazine and exhibit a Tanimoto coefficient> 0.6 to have cancer stem cell effects.

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Surprisingly, it has been found that only 5% of the 167 known or currently used therapeutic agents show an anti-cancer stem cell effect. Accordingly, the present invention provides a compound that has not been used clinically to treat cancer patients even when clinical cancer compounds have such a low possibility of having an anticancer stem cell effect. It fulfills a long and urgent need in the art to provide a method for elucidating as having.

Accordingly, in one aspect of the invention,
A method of identifying and verifying a test agent as a selective anticancer stem cell agent comprising:
i) contacting one or more mutant neoplastic stem cells with the test agent and contacting one or more normal stem cells with the test agent;
ii) detecting a change in the number of cells of the mutated neoplastic stem cell in response to the test agent, and detecting a change in the number of cells of the normal stem cell in response to the test agent;
iii) Contact with the test agent induces a decrease in the number of cells of the mutated neoplastic stem cell, while inducing a decrease in the number of cells of the normal stem cell comparable to the decrease in the number of cells of the mutated neoplastic stem cell If not, identifying the test agent as a selective anti-cancer stem cell agent;
There is provided a method characterized by comprising:

  In certain embodiments, the method includes detecting changes in cell number in response to different test concentrations of test agent. Thus, in certain embodiments, the method contacts a mutated neoplastic stem cell and a normal stem cell with a test agent at a plurality of test concentrations, and a plurality of cell number changes relative to one or more mutated neoplastic stem cells and normal stem cells. Detecting at a test concentration of.

  In a related aspect of the invention, detecting a change in pluripotency and cell number of a mutated neoplastic stem cell in response to contact with the drug, the drug detects loss of pluripotency of the mutated neoplastic stem cell and / or Alternatively, there is provided a step of selecting an agent as a test agent when inducing a decrease in cell number. In certain embodiments, the method comprises contacting one or more mutant neoplastic stem cells with the test agent and contacting one or more normal stem cells with the test agent and responding to the test agent. Detecting changes in the number of neoplastic stem cells, detecting changes in the number of normal stem cells in response to the test drug, and the test drug is equivalent to a decrease in the number of mutant neoplastic stem cells. Identifying the test agent as a selective anti-cancer stem cell agent if it induces a decrease in the number of mutant neoplastic stem cells without inducing a decrease in cell number.

  Anti-cancer stem cell drugs exhibit complex drug response curves, and drug selectivity and / or activity may not be easily determined by extrapolation of test results using a single concentration or a small number of concentrations of the drug or test agent It was judged. The methods described herein optionally include contacting the mutated neoplastic stem cells and / or normal stem cells with a test agent at multiple test concentrations.

  In some embodiments, the plurality of test concentrations varies by at least about 3, 4 or 5 orders of magnitude or more. In certain embodiments, the plurality of test concentrations vary from at least about 0.01 μM to 2 μM, or from at least 10 nM to about 20 μM. In some cases, multiple test concentrations have a dilution series such as 8 or 10 points.

  In certain embodiments, the methods described herein are dose-response curves for changes in cell numbers of mutant neoplastic stem cells in response to contacting a test agent at multiple test concentrations, and tested at multiple test concentrations. Comparing a dose-response curve for changes in the number of normal stem cells in response to contact with the agent. One skilled in the art will appreciate that many different methods can be used to compare dose response curves. For example, in certain embodiments, a half maximal effective concentration (EC50) for a reduction in the number of cells of the mutated neoplastic stem cell is measured for a test agent and a half maximal effective for a reduction in the number of cells of the normal stem cell The concentration (EC50) is measured.

  In certain embodiments, EC50 values or similar indicators can be compared to provide a selective activity ratio for a test agent. For example, in one embodiment, the method calculates the ratio of the half maximal effective concentration (EC50) for a reduction in the number of normal stem cells to the half maximal effective concentration (EC50) for a reduction in the number of cells of the mutant neoplastic stem cell. Including the step of measuring. Optionally, the methods described herein include identifying a test agent as a selective anti-cancer stem cell agent if the half maximal effective concentration (EC50) ratio is greater than 3.

  In one aspect of the invention, the mutated neoplastic stem cells and / or normal stem cells are seeded in a container prior to contacting the cells with a drug or test drug. In certain embodiments, the mutated neoplastic stem cells are seeded at about 3000 to 7000 cells / receptacle in a first receptacle, and normal stem cells are seeded at about 8000 to 12000 cells / receptacle in a second receptacle. In certain embodiments, the mutated neoplastic stem cells are seeded at about 4000 to 6000 cells / receptacle, and in some cases at about 5000 cells / receptacle. In certain embodiments, normal stem cells are seeded at about 9000 to 11000 cells / receptacle, and in some cases at about 10,000 cells / receptacle.

    In some cases, the first receptacle and the second receptacle are wells on the same microtiter plate or are wells on different microtiter plates. In certain embodiments, the microtiter plate is a 96 well microtiter plate, and optionally a 96 well coated microtiter plate.

  In certain embodiments, the change in cell number of the mutated neoplastic stem cell is detected between about 48 hours and 96 hours, optionally about 72 hours after contacting the cells with the test agent, and normal stem cells This change in cell number is detected between about 4 and 6 days, and in some cases, about 5 days after contacting the cells with the test agent.

    In certain embodiments of the methods described herein, the mutated neoplastic stem cells are transformed human pluripotent stem cells (t-hPSCs), optionally V1H9-Oct4-GFP cells, or transformation induced pluripotency Sex stem cells (t-iPSCs). In certain embodiments, the mutated neoplastic stem cell is a human mutated neoplastic stem cell.

  In one aspect of the invention, the change in cell number is deoxyribonucleic acid (DNA) content analysis, nuclear markers such as histones, or nuclear laminin, detection of nuclear enrichment, and / or Hoechst, 4 ′, 6-diamidino-2 -Measured by detection of nuclei such as by staining with DNA-binding fluorescent dyes such as phenylindole (DAPI), acridine orange, DRAQ5 or safarin. Those skilled in the art will understand that detecting changes in cell number includes identifying and / or counting individual cells. In some cases, changes in cell number are detected using high content analysis, and possibly nuclear image analysis.

  In certain embodiments, the methods described herein include screening test agents identified as selective anti-cancer stem cell agents for activity against cancer cell lines from cancer subjects. In certain embodiments, the cancer cell line is a leukemia cell line such as an acute myeloid leukemia (AML) cell line. In certain embodiments, the activity is cytotoxicity, induction of apoptosis, loss of pluripotency, induction of differentiation, or a combination thereof. In certain embodiments, the activity is the presence / absence or level of one or more markers.

  In certain embodiments, the methods described herein identify a test agent identified as a selective anticancer stem cell agent with one or more stress response genes, optionally one or more annexin V, p53. And / or screening for induction of expression of apoptotic biomarkers such as p21.

  In another aspect of the invention, prior to step i), to identify one or more test agents that induce loss of pluripotency of the mutated neoplastic stem cells and a decrease in cell number, Or further screening for more drugs. These test agents are then used to select selective anti-cancers using the methods described herein, for example, by comparing changes in cell numbers in response to test agents between mutated neoplastic and normal stem cells. Identified and verified as a stem cell drug.

  In certain embodiments, the method detects contacting a mutated neoplastic stem cell with a drug prior to step i) and detecting a pluripotency change and cell number change of the mutated neoplastic stem cell in response to the drug. And selecting an agent as a test agent if the agent induces loss of pluripotency of the mutated neoplastic stem cells and a decrease in cell number. In certain embodiments, the mutated neoplastic stem cell is contacted with the agent at about 10 μM. In certain embodiments, the plurality of agents are based on a standard deviation threshold from an average on a microtiter plate, in some cases from an average, and in some cases from a whole plate average, a Z score threshold of at least 3 standard deviations. Are screened for pluripotency changes and cell number changes and identified as test agents. In certain embodiments, mutant neoplastic stem cells treated with bone morphogenetic protein-4 (BMP4) to induce loss of potency are used as a positive control, and in some cases, the cells are at least 100 ng / mL. Treated with BMP4.

  A person skilled in the art uses many different methods of existing technology to detect changes in the pluripotency of mutant tumor stem cells by detecting changes in the expression level of one or more pluripotency markers. Understand what is possible. In certain embodiments, detecting a change in the expression level of one or more pluripotency markers comprises using an antibody selective for the one or more pluripotency markers. In certain embodiments, the expression of one or more pluripotency markers in one or more mutated neoplastic stem cells is operably linked to a reporter gene such as GFP. In certain embodiments, the pluripotency marker is selected from Oct4 and Sox2. In certain embodiments, the pluripotency marker is Oct4, Sox2, Nanog, SSEA3, SSEA4, TRA-1-60, TRA-1-81, IGF1 receptor, connexin 43, E-cadherin, alkaline phosphatase, REX1, CRIPTO , CD24, CD90, CD29, CD9 and CD49f, and mixtures thereof.

In one aspect, the invention provides:
A method of identifying and verifying a test agent as a selective anticancer stem cell agent comprising:
i) contacting one or more mutant neoplastic stem cells with the test agent and contacting one or more normal stem cells with the test agent;
ii) detecting a change in the activity of one or more of the mutated neoplastic stem cells in response to the test agent and detecting a change in the activity of one or more normal stem cells in response to the test agent. When,
iii) if the contact with the test agent induces one or more activities of the mutated neoplastic stem cell, while not inducing the activity of normal stem cells comparable to that of the mutated neoplastic stem cell; Identifying a test agent as a selective anticancer stem cell agent;
A method characterized by comprising:

  In certain embodiments, the activity is apoptosis, necrosis, proliferation, cell division, differentiation, migration or movement, the presence or absence of one or more biomarkers, the level of one or more biomarkers, or them Is the induction of

In other aspects;
A two-step method for identifying and validating a drug as a selective anti-cancer stem cell drug, comprising:
i) detecting a change in the activity of one or more of the mutated neoplastic stem cells in response to contact with the agent, wherein the agent induces one or more activities of the mutated neoplastic stem cell Selecting the agent as a test agent; and ii) contacting one or more mutated neoplastic stem cells with the test agent and contacting one or more normal stem cells with the test agent; Detecting a change in the activity of one or more of the mutated neoplastic stem cells in response to the test agent, detecting a change in the activity of one or more normal stem cells in response to the test agent; and If the test agent induces one or more activities of the mutated neoplastic stem cell without inducing the activity of a normal stem cell comparable to that of the mutated neoplastic stem cell, the test Identifying the agent as a selective anti-cancer stem cell agent;
There is provided a method characterized by comprising:

  In certain embodiments, the activity is apoptosis, necrosis, proliferation, cell division, differentiation, migration or movement, the presence or absence of one or more biomarkers, the level of one or more biomarkers, or them Is the induction of

    In another aspect of the invention, it has surprisingly been determined that compounds structurally similar to thioridazine are effective in inducing cancer stem cell differentiation or killing cancer stem cells. Also provided is a method of treating cancer in a subject comprising administering to the subject a compound that is structurally similar to thioridazine.

  In certain embodiments, the compound has a Tanimoto modulus of at least 0.6, similar to thioridazine. In certain embodiments, the compound has a Tanimoto modulus of at least 0.7, 0.8, 0.9, or 0.95 that is similar to thioridazine. Optionally, the compound is selected from the group consisting of thioridazine, prochlorperazine, trifluoperazine, fluphenazine, perphenazine, and triflupromazine. In certain embodiments, the compound is a phenothiazine derivative. In certain embodiments, the compound preferentially induces differentiation of cancer stem cells relative to normal stem cells. For example, in certain embodiments, the compound preferentially induces differentiation of mutant neoplastic stem cells relative to normal stem cells such as H9 cells.

  Cancer stem cells are in vivo, in vitro, or ex vivo. In certain embodiments, cancer stem cells are found in a subject's in vivo. In certain embodiments, the cancer is leukemia, such as acute myeloid leukemia. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is lung cancer, colon cancer, brain cancer, or prostate cancer.

The methods described herein are also useful in determining treatment, monitoring, or treatment strategies for subjects suffering from or suspected of having cancer being treated with an anti-cancer stem cell agent. In certain embodiments, the anti-cancer stem cell agent is a selective anti-cancer stem cell agent.
For example, in certain embodiments;
Providing a sample from a subject undergoing treatment with an anti-cancer stem cell drug, suffering from cancer or suspected of having cancer;
Measuring the level of cancer stem cells in the sample;
If the sample does not contain cancer stem cells, the subject determines that no further treatment with an anti-cancer stem cell drug is needed, and if the sample contains cancer stem cells, the subject treats with an anti-cancer stem cell drug Identifying as a subject in need of further,
There is provided a method characterized by comprising:

In some embodiments, also;
Treating a subject suffering from or suspected of having cancer with an anti-cancer stem cell agent;
Measuring the level of cancer stem cells in a sample from a subject;
If the sample does not contain cancer stem cells, the subject determines that no further treatment with the anti-cancer stem cell drug is required, and if the sample contains cancer stem cells, the subject further requires treatment with the anti-cancer stem cell drug. Identifying as a subject to perform,
There is provided a method characterized by comprising:

In certain embodiments;
Providing a sample from a subject undergoing treatment with an anti-cancer stem cell drug, suffering from cancer or suspected of having cancer;
Measuring the level of cancer stem cells in the sample;
Comparing the level of cancer stem cells in the sample with the level of cancer stem cells in the control sample is also provided.

  Optionally, the method includes determining whether to continue treatment with the anti-cancer stem cell agent on the subject based on an increase or decrease in the level of cancer stem cells in the sample compared to the control sample. In certain embodiments, the control sample is a temporally previous sample from the subject.

  In certain embodiments, the anti-cancer stem cell agent is thioridazine, a structurally similar compound to thioridazine, or a phenothiazine derivative.

  In certain embodiments, measuring the level of cancer stem cells in the sample comprises measuring the level of cancer stem cells in the sample compared to other cell types in the sample, such as differentiated cells. In some cases, the methods described herein include measuring a time change in the ratio of cancer stem cells to differentiated cells in a sample.

  In certain embodiments, measuring the level of cancer stem cells in the sample comprises detecting biomarkers associated with cancer stem cells and / or biomarkers associated with differentiated cells. In some cases, the level of cancer stem cells in a sample can be measured using xenografts known in the art.

  In certain embodiments, the methods described herein include determining the level of cancer stem cells in a sample by testing the sample for expression of biomarkers associated with cancer stem cells and / or differentiated cells. In certain embodiments, the biomarker associated with cancer stem cells is selected from dopamine receptor (DR) 1+, DR2 +, DR3 +, DR4 +, CD14 +, CD34 + and CD44 +. In certain embodiments, the biomarker associated with differentiated cells is selected from CD11b +, CD24 +, CD114 + and CD16 +.

  In certain embodiments, the sample consists of leukemia cells or suspected leukemia cells, and measuring the level of cancer stem cells comprises testing for one or more dopamine receptors and CD14, wherein Cells expressing one or more dopamine receptors and CD14 (DR +; CD14 +) are identified as leukemia stem cells. In certain embodiments, the sample consists of leukemia cells or cells suspected of being leukemic cells, and the step of determining the presence or absence of cancer stem cells is tested against one or more dopamine receptors and CD14 +, optionally CD34 +. Includes steps.

In certain embodiments, the sample consists of breast cancer cells or cells suspected of being breast cancer cells, and measuring the level of breast cancer stem cells comprises CD44 expression and absence of CD24 expression (CD44 + / CD24− or CD44 + / CD24 _low ) Testing the sample against. In certain embodiments, determining the level of breast cancer stem cells comprises the expression of one or more dopamine receptors selected from DR1, DR2, DR3, DR4 and DR5, and optionally CD44 and CD24 expression (DR + / CD44 + / CD24− or CD24 _low ). In certain embodiments, measuring the level of breast cancer stem cells comprises testing the sample for DR5 and optionally CD44 and CD24 expression. The methods described herein optionally include testing the sample for biomarkers associated with other cancer stem cells or differentiated cells known in the art.

  In another aspect of the invention, there is provided a method of treating cancer in a subject comprising administering to the subject a compound having a Tanimoto coefficient of at least 0.6 similar to thioridazine. Also provided is a method of using a compound having a Tanimoto coefficient of at least 0.6 similar to thioridazine for the treatment of cancer. In certain embodiments, the compound has a Tanimoto modulus of at least 0.7, 0.8, 0.9, or 0.95 that is similar to thioridazine. In some cases, compounds include, but are not limited to, thioridazine, prochlorperazine, trifluoperazine, fluphenazine, perphenazine, and triflupromazine. In certain embodiments, the compound is a phenothiazine derivative. In certain embodiments, the compound preferentially differentiates cancer stem cells relative to normal stem cells. In certain embodiments, the cancer is acute myeloid leukemia.

  In one aspect of the invention, it has surprisingly been identified that dopamine receptor blockers such as thioridazine or chlorpromazine are cytotoxic to cancer cells, particularly those of acute myeloid leukemia (AML). Further, it has been found that dopamine receptor blockers at concentrations that are toxic to cancer cells have a relatively limited effect on normal stem cells such as hematopoietic stem cells. Still further, dopamine receptors were expressed in AML cell lines and primary AML cells, but relatively low expression was seen in cell lines rich in normal hematopoietic stem cells. Furthermore, the expression of dopamine receptor in AML cells was shown to correlate with the expression of the monoblast marker CD14. Dopamine receptor blockers such as thioridazine are cytotoxic to AML cells that express CD14.

  Accordingly, in one aspect, a method for treating a cancer or precancerous disease in a subject is provided, comprising administering to a subject in need of a dopamine receptor blocker. In a similar aspect, the present disclosure describes methods of using dopamine receptor (DR) blockers for the treatment of cancer or precancerous diseases. In certain embodiments, the cancer or precancerous disease is a myeloproliferative disease or leukemia. In certain embodiments, the cancer is acute myeloid leukemia (AML). In certain embodiments, the dopamine receptor (DR) blocker preferentially induces cancer stem cell differentiation compared to hematopoietic or normal stem cells.

In certain embodiments, the dopamine receptor blocker is a phenothiazine derivative such as thioridazine or chlorpromazine. As an option, the dopamine receptor blocker is a multi-receptor blocker that blocks one or more dopamine receptors. In some embodiments, the dopamine receptor blocker is D ₂ family of over dopamine receptor blockers. In certain embodiments, the dopamine receptor blocker is one compound selected from the compounds listed in Table 1.

  In another aspect, a method for reducing cancer cell growth is provided, comprising contacting the cancer cell with a dopamine receptor blocker. In a similar aspect, a method of using a dopamine receptor blocker to reduce cancer cell growth is provided. In certain embodiments, contacting a cell with a dopamine receptor blocker induces cell death or differentiation of a cancer cell or precancerous cell. In certain embodiments, the cancer cell is a cancer stem cell, and contacting the cancer stem cell with a dopamine receptor blocker induces differentiation of the cancer stem cell. The cells may be in vivo or in vitro. In certain embodiments, the precancerous cell is a myeloproliferative cell. As an option, the cancer cells are leukemia cells such as acute myeloid leukemia (AML) cells or monocytic leukemia cells. In certain embodiments, the cell is CD14 positive. In certain embodiments, the dopamine receptor blocker is a phenothiazine derivative such as thioridazine. In certain embodiments, the dopamine receptor blocker is one compound selected from the compounds listed in Table 1.

  In another aspect, a method for identifying a subject having a cancer suitable for treatment with a dopamine receptor blocker is provided. In certain embodiments, the method includes measuring the expression of one or more dopamine receptors in a cancer cell sample from the subject. In certain embodiments, a subject having cancer cells that express one or more dopamine receptors is identified as being suitable for treatment with a dopamine receptor blocker. In certain embodiments, the cancer is leukemia and the cancer cell is a leukemia cell. In certain embodiments, the leukemia is acute myeloid leukemia or monocytic leukemia. In certain embodiments, the cancer is breast cancer. In certain embodiments, a method of identifying a subject having cancer comprises testing a sample for CD14 expression.

  In one aspect, a method for determining the prognosis of a cancer subject is provided. It comprises measuring the expression level of one or more dopamine receptor biomarkers in a sample from the subject and comparing the expression level of one or more biomarkers to a control. As an option, the methods provided herein comprise providing or obtaining a sample of cancer cells from a subject. In certain embodiments, an increased expression level of one or more biomarkers relative to a control indicates that the cancer is a more severe form compared to a control. In certain embodiments, the dopamine receptor biomarker is DR3 and / or DR5. In certain embodiments, the cancer is leukemia or breast cancer and the sample consists of leukemia cells or breast cancer cells. In certain embodiments, the leukemia is acute myeloid leukemia or monocytic leukemia.

  Also provided is a method for identifying leukemia subjects. In certain embodiments, the method comprises measuring the expression level of one or more dopamine receptors in a sample from the subject and comparing the expression level of one or more dopamine receptors to a control. Optionally, the sample consists of white blood cells and / or the method further comprises providing a sample consisting of white blood cells from the subject. In certain embodiments, an increased expression level of one or more dopamine receptors relative to a control indicates that the subject has leukemia, such as acute myeloid leukemia or monocytic leukemia. In certain embodiments, the method further comprises testing for CD14.

  Further provided are methods for screening for compounds with anticancer activity. It includes identifying a compound that is a dopamine receptor blocker. In certain embodiments, the anti-cancer activity is a decrease in proliferation of acute myeloid leukemia (AML) cells or monocytes. As an option, the method comprises identifying compounds that preferentially induce differentiation of cancer stem cells relative to hematopoietic or normal stem cells.

  In one aspect, a method for distinguishing cancer stem cells from a cell population is provided. In certain embodiments, the method comprises determining whether the cell expresses one or more biomarkers selected from dopamine receptor (DR) 1, DR2, DR3, DR4 and DR5. In certain embodiments, the expression of DR1, DR2, DR3, DR4 and / or DR5 indicates that the cell is a cancer stem cell.

  In certain embodiments, the cell population is cells isolated from a mammal or cells in culture, such as a cell culture medium. In certain embodiments, the population of cells is pluripotent stem cells. In certain embodiments, the population of cells comprises cancer cells such as hematological cancer cells or precancerous cells. As an option, the method comprises testing the cell for expression of a polynucleotide or polypeptide encoding DR1, DR2, DR3, DR4 or DR5. In certain embodiments, cells that express DR1, DR2, DR3, DR4 and DR5 are identified as cancer stem cells. In some embodiments, the methods described herein also include isolating cancer stem cells from the cell population. For example, cells identified as cancer stem cells can be isolated from cell populations or other materials using existing techniques such as flow cytometry, fluorescence labeled cell sorting, panning, affinity column separation, or magnetic sorting. is there. In certain embodiments, cancer stem cells are isolated using antibodies to one or more of DR1, DR2, DR3, DR4 and DR5.

  Other features and advantages of the present disclosure will become apparent from the following detailed description. However, the detailed description and specific examples, while indicating the preferred embodiment of the present disclosure, are provided for purposes of illustration only and many variations and modifications will occur to those skilled in the art having read this description within the spirit and scope of the disclosure. It is self-explanatory.

One or more embodiments of the invention are described with reference to the figures;
FIG. 10 shows that 10 μM thioridazine is cytotoxic to HL-60, MV4-11 and OCI3 leukemia cell lines. 10 μM thioridazine has a limited effect on the colony forming potential of normal hematopoietic stem cells (HSC) (FIG. 2A), but significantly reduces the blast forming potential of acute myeloid leukemia (AML). It is. (FIG. 3) shows a cell pellet of CFU colonies formed from normal hematopoietic stem cells (HSC) and acute myeloid leukemia (AML) treated with thioridazine. (FIG. 4) shows that 10 μM chlorpromazine and 10 μM thioridazine are both cytotoxic to HL-60, MV4-11 and OCI3 leukemia cell lines. It is a figure which shows expression of dopamine receptor DR1, DR2, DR3, DR4 and DR5. Dopamine receptor (DR) expression is found in acute myeloid leukemia (AML) cell lines, some primary AML and mononuclear cells (MNC), but cells rich in hematopoietic stem cells (HSC) ( It was not found in umbilical cord blood CBlin (-)) to strain depletion. FIG. 2 shows that multiple dopamine receptor (DR) blockers are cytotoxic to acute myeloid leukemia (AML) cell lines. SKF = (R)-(+)-SKF-38393 hydrochloride; 7OH = R (+)-7-hydroxy-DPAT hydrobromide; GR = GR 103691; SCH = R (+)-SCH-23390 hydrochloric acid CLOZ = clozapine; CHL = chlorpromazine hydrochloride; THIO = thioridazine. FIG. 4 is a diagram of fluorescence labeled cell sorting (FACS) data showing that dopamine receptors are expressed in CD14 + cell populations in primary AML. FIG. 5 shows that thioridazine selectively targets CD14 + cells and reduces their normalized frequency of occurrence in primary AML. FIG. 5 shows the discrimination between mefloquine and thioridazine using chemical screening for compounds that differentiate neoplastic human pluripotent stem cells (hPSCs). (FIG. 9A) shows an outline of the screening procedure. (FIG. 9B) is an XY scatter plot of the green fluorescent protein (GFP) and Hoechst signal% residual activity (% RA) for 590 compound screens. The enclosed area shows a loss of pluripotency (LOP) defined by reduced GFP and reduced Hoechst. Each point is n = 3, average +/− standard deviation. (FIG. 9C) shows a summary of the reaction of 590 compounds. (FIG. 9D) shows the chemical structural formulas of candidate compounds; thioridazine, azathioprine and mefloquine. (FIG. 9E) is a representative microscopic image of GFP, Hoechst and their combined v1H9-Oct4-GFP cells treated with 10 μM candidate compound. (FIG. 9F) is a histogram of the GFP intensity of these images. (FIG. 9G) is the calculated value of the dose response curve and 50% effective concentration (EC ₅₀ ) of v1H9-Oct4-GFP. Each point is n = 3, average +/− standard deviation. FIG. 5 shows the effects of salinomycin, mefloquine and thioridazine on normal and neoplastic populations. (FIGS. 10A-B) Within H 9 (FIG. 10A) and v1H9-Oct4-GFP (FIG. 10B) treated with 10 ⁻⁷ -10 ⁻⁶ M salinomycin (SAL), mefloquine (MQ) and thioridazine (THIO). It is a flow cytometry analysis of the appearance frequency of Oct4 + cell. Each bar is n = 3, average +/− standard deviation. Values are normalized to control samples treated with dimethyl sulfoxide (DMSO); (−) DMSO average, (−−) average minus 1 standard deviation, (−)% Oct4 + level in BMP4 treated samples. (FIG. 10C) Ratio of normalized frequency of Oct4 + cells in H9 to% normalized frequency of Oct4 + cells in v1H9-Oct4-GFP at the same concentration of the same compound. Neoplastic human staining positive for p53 (FIG. 10D) and p21 (FIG. 10E) after treatment with 10 μM etoposide, thioridazine (THIO), BMP4 for 24 hours, and in dimethyl sulfoxide (DMSO) treatment (control, CTRL) Percentage of pluripotent stem cells (hPSC) is shown. Each bar is n = 3, average +/− standard deviation. Representative images after etoposide, thioridazine treatment are included. Arrows indicate p53 + and p21 + in etoposide and thioridazine treated cells. (FIG. 10F) is a diagram showing genes associated with differentiation increased more than 2-fold after thioridazine treatment of neoplastic human pluripotent stem cells (hPSC). The genes are divided into rows: endoderm (ENDO), mesoderm (MESO), germ cells (GERM), neurons (NEURO) and trophoblasts (TROPH). Each bar is the average of two separate experiments. (FIGS. 10G-K) show hematopoietic multiple lineage and clonogenic potential in response to compound treatment detected using the methylcellulose assay. (FIG. 10G) shows representative colony forming unit (CFU) pellets from hematopoietic stem and progenitor cells (HSPC) after compound treatment. (FIG. 10H) shows a representative blast CFU pellet from AML after compound treatment. (FIGS. 10I-J) show the quantification results of CFU and blast CFU respectively generated from HSPC (FIG. 10I) and AML blasts (FIG. 10J) after compound treatment. Numbers are normalized to control samples treated with dimethyl sulfoxide (DMSO); (−) DMSO average, (−−) average minus 1 standard deviation. Each HSPC bar is n = 7 individual samples, mean +/− standard deviation. Each AML bar is at least n = 5 individual patient samples, mean +/− standard deviation. (FIG. 10K) Ratio of HSFU CFU to normalized AML blast CFU at the same concentration of the same compound. (FIG. 10L) Normalized frequency of CD11b granulocyte cells in cultured patient AML cells after treatment with thioridazine 10 μM (THIO 10 μM) or DMSO vehicle (CTRL) for up to 96 hours. Each bar is n = 3, average +/− standard deviation. (*) P <0.05 (**) p <0.01 (***) p <0.001 (***) p <0.0001. It is a figure which shows the effect with respect to a fibroblast origin induced pluripotent stem cell (iPSC) and a hematopoietic stem cell, and a progenitor cell (HSPC) of salinomycin, mefloquine, and thioridazine. (FIG. 11A) Flow cytometry of the frequency of occurrence of Oct4 + cells in fibroblast-derived iPSCs (Fib-iPS) treated with 10 ⁻⁷ -10 ⁻⁶ M salinomycin (SAL), mefloquine (MQ) and thioridazine (THIO). It is analysis. Each bar is n = 3; mean +/− standard deviation. Numbers are normalized to control samples treated with dimethyl sulfoxide (DMSO); (−) DMSO average, (−−) average minus 1 standard deviation, (−)% Oct4 + level of BMP4 treated samples. (FIG. 11B) Expanded dose response of compounds to neoplastic human pluripotent stem cells (hPSC). Each point is the mean +/- standard deviation. (FIG. 11C) shows the hematopoietic lineage potential of umbilical cord blood (CBlin−) to line depletion treated with thioridazine. The colony forming units (CFU) of erythroblasts (CFU-E), macrophages (CFU-M) and granulocytes (CFU-G) colonies generated in the methylcellulose assay are shown. (FIG. 11D) shows the organization of CFU generated from cord blood (CBlin-) to line depletion treated with salinomycin, mefloquine and thioridazine. The% composition of CFU produced with 0.1 μM, 1 μM, 10 μM salinomycin (SAL), mefloquine (MQ) and thioridazine (THIO) treatment is shown. (*) P <0.05 (**) p <0.01. It is a figure which shows the influence of the thioridazine with respect to hematopoietic stem cell (HSC) and leukemia stem cell (LSC) engraftment. Frequency of human CD45 + cells in bone marrow after treatment of hematopoietic stem and progenitor cells (HSPC) with thioridazine 10 μM (THIO 10 μM) or mefloquine 10 μM (MQ 10 μM). Numbers are normalized to a control sample (CTRL) treated with dimethyl sulfoxide (DMSO). A total of two HSPC samples were verified. Mean +/- standard deviation. Representative flow cytometry plot of myeloid marker (CD33) or lymphoid marker (CD19) versus side scatter (SSC) within the hCD45 + population. It is a figure which shows the expression frequency of CD45 + CD33 + AML blast cells in the bone marrow (BM) after acute myeloid leukemia (AML) treatment with thioridazine 10 μM (THIO 10 μM) or mefloquine 10 μM (MQ 10 μM). Numbers are normalized to a control sample (CTRL) treated with dimethyl sulfoxide (DMSO). A total of two AML samples were verified. (FIG. 12D) Representative flow plot of CD45 against CD33 in DMSO treated control (CTRL) population and thioridazine treated (THIO 10 μM). (FIG. 12E) Ratio of normalized% hCD45 CD33AML blast engraftment to% normalized hCD45 hematopoietic stem and progenitor cell (HSPC) engraftment. (*) P <0.05. FIG. 4 shows in vivo response to drug treatment. The normalized frequency of occurrence of human CD45 + cells in the bone marrow after treatment of hematopoietic stem and progenitor cells (HSPC) with 1 μM salinomycin (SAL 1 μM) is shown in comparison to a DMSO-treated control (CTRL). A total of two HSPC samples were verified. Mean +/- standard deviation. (***) p <0.0001. It is a figure which shows the effect of the thioridazine with respect to spleen engraftment of a hematopoietic stem cell (HSC) and a leukemia stem cell (LSC). (FIG. 13B top) shows the frequency of appearance of human CD45 + cells in the spleen after treatment of hematopoietic stem cells and progenitor cells (HSPC) with thioridazine 10 μM (THIO 10 μM). Numbers are normalized to the DMPC-treated HSPC control sample (CTRL). A total of two HSPC samples were verified. Mean +/- standard deviation. (FIG. 13B bottom) shows the frequency of appearance of CD45 + CD33 + blasts in the spleen after AML treatment with 10 μM thioridazine (THIO 10 μM). Numbers are normalized to AML control samples (CTRL) treated with DMSO. A total of two AML patient samples were verified. FIG. 13C is a diagram showing the effect of thioridazine on the regeneration of erythrocytes and megakaryocytes. Human blood cell composition detected in xenograft bone marrow injected with thioridazine 10 μM (THIO 10 μM) or DMSO treated (CTRL) HSPC. Red blood cells (RBC) are defined by glycophorin A positivity and platelets are defined by CD41a. (FIG. 13D) shows confirmation of myeloid leukemia engraftment of xenografts with AML. Flow cytometry of CD19, a marker of side scatter versus lymphoid cells. The numbers in the box are mean +/- standard deviation. It is a figure which shows the effect of the thioridazine regarding the in vivo self-renewal of a hematopoietic stem cell (HSC) and a leukemia stem cell (LSC). Live of hCD45 + cells in bone marrow of secondary xenografts receiving the same number of hCD45 cells explanted from cord blood (CBlin−) to primary line depletion treated with thioridazine 10 μM (THIO 10 μM) or DMSO (CTRL) Indicates the wear level. Each bar is n = 3 mice, mean +/− standard deviation. FIG. 6 shows engraftment levels of hCD45 + CD33 + in bone marrow of secondary xenografts receiving the same number of hCD45 cells explanted from thioridazine 10 μM (THIO 10 μM) or DMSO treated (CTRL) primary AML engraftment. It is a figure which shows the dopamine receptor expressed by the neoplastic stem cell. Flow cytometry of normal H9 (FIG. 14A) and neoplastic v1H9-Oct4-GFP cells (FIG. 14B) stained with SSEA3 and all five dopamine receptor (DR) subtypes. Dopamine receptor (DR) expression within the SSEA3 + fragment is shown. Flow cytometry of umbilical cord blood (hematopoietic stem and progenitor cells (HSPC)) depleted of differentiation lineage stained with CD34, CD38 and all five dopamine receptor (DR) subtypes. Dopamine receptor (DR) expression is shown in the gated population. Flow cytometry of 13 AML patient samples with FAB classification stained with all 5 dopamine receptor (DR) subtypes. (FIG. 14E) demonstrates DRD5 co-localization in triple negative (ER-, PR-, and HER2-) primary human breast cancers stained with CD44 and CD24. (FIG. 14F) Frequency of expression of triple negative breast cancer stem cells (CSC) (CD44 + CD24− / ^lo ) within the DRD3 and DRD5 populations. Each bar consists of 3 primary triple negative breast cancers with mean +/- standard deviation. Frequency of occurrence of AML blasts (CD33 + CD45 +) from patient samples that are positive for DRD3 (FIG. 14G) and DRD5 (FIG. 14H). A total of 8 patient samples were validated for leukemic potential in xenograft recipients. Leukemia onset was defined as 0.1% human engraftment> CD33 + hCD45 + in mouse bone marrow. Of the 22 mice, 4 leukemic onset AML samples were assayed, while 4 out of 17 mice were assayed for non-leukemic AML samples. Total n = 8 AML samples, mean +/− standard deviation. Flow cytometry of SSEA3 + fragments in fibroblast-derived human induced pluripotent stem cells (hiPSC) (FIG. 15A) and umbilical cord blood-derived hiPSC (FIG. 15B) stained with all five types of dopamine receptors. Figure 2 shows dopamine receptor expression in human blood populations. Flow cytometry of umbilical cord blood mononuclear cells, which are stained with red blood cells (glycophorin A) and megakaryocytes (CD41a). Flow cytometry of umbilical cord blood mononuclear cells, stained with T-cells (CD3) and B-cells (CD19). Flow cytometry of umbilical cord blood mononuclear cells, stained with monocytes (CD14) and granulocytes (CD15). Staining by all five dopamine receptors in the gated population is shown as a histogram. (FIG. 15F) Summary of localization of dopamine receptors within the blood population. (FIG. 15G) is a flow cytometry of AML patients, showing dopamine receptors in the gated population. It is a figure which shows dopamine receptor expression in triple negative human breast cancer. Breast cancer stem cells are defined as CD44 + CD24− / ^lo (Al-Hajj et al., 2003). Co-localization of each dopamine receptor (DR) within the CD44 and CD24 population is shown for three triple negative (ER-, PR- and HER2-) breast cancers. It is a figure which shows that thioridazine suppresses dopamine receptor signal transduction in AML. Figure 2 shows dopamine receptor expression in AML-OCI2 and AML-OCI3 cell lines. (FIG. 16B) is the number of AML-OCI2 and AML-OCI3 cells treated with three dopamine receptor blockers. Numbers are normalized to control samples treated with DMSO. Each bar is n = 3; mean +/− standard deviation. (FIG. 16C) Viable cell count of the same cell line treated with the dopamine receptor D2 family stimulant 70H-DPAT under serum-free conditions (7AAD−, Hoechst +). Numbers are normalized to control samples treated with DMSO. Each bar is n = 3; mean +/− standard deviation. Number of viable cells in the same cell line treated with SKF38393, a dopamine receptor D1 family stimulant under serum-free conditions (7AAD−, Hoechst +). FIG. 6 is a scatter plot of cell number and pluripotency changes in v104 cells treated with compounds from a chemical library. In an illustrative embodiment, compounds within the dashed box are identified based on their ability to lose pluripotency and a moderate reduction in the number of mutant neoplastic stem cells. Above this box are compounds that do not significantly reduce cell number and increase or decrease pluripotency. Compounds located just below the box were considered potentially toxic due to the magnitude of the effect on cell number, but some of these compounds were similar to many of the current chemotherapeutic drugs It may be useful as an anti-cancer stem cell drug because of its toxic effect. Thioridazine is indicated by a black star surrounded by a dotted circle, while the thioridazine analog is indicated by a black star in the box. Chlorpromazine is indicated by a black star in a solid circle. Thioridazine analogs (compounds similar in structure to thioridazine) have been shown disproportionately in causing a relative decrease in cell number and loss of pluripotency in mutant neoplastic stem cells. FIG. 18 is a plot showing the induction of apoptosis after treatment of the AML cancer cell lines AML-OCI2 and AML-OCI3 with the compounds identified from FIG. 17 (grey dots) (based on a decrease in the percentage of normal nuclei present). Signs of apoptosis increase towards the origin. Black dots indicate thioridazine and thioridazine analogs. Thioridazine induces cell death in AML cancer cell lines, whereas thioridazine analogs are inactive. Thioridazine analogs having activity against mutant neoplastic stem cells (v1O4 cells) do not induce cell death in AML cancer cell lines. Mutant neoplastic stem cells (v1O4 cells) treated with thioridazine show signs of cellular stress / death compared to cells treated with thiostrepton as a control known to induce apoptosis It is a figure which shows that there are few. Microscopic image of v1O4 cells treated with 10 μM compound stained with Hoechst nuclear staining after 3 days of treatment. Thiostrepton is known to induce apoptosis and cytarabine is a known chemotherapeutic agent for the treatment of AML. Nuclear swelling and condensation is a sign of cell stress or cell death induction. FIG. 6 shows that screening with mutant neoplastic stem cells (v1O4 cells) identifies a small subset of anticancer compounds that are also active against cancer stem cells (CSC). All known / current anticancer therapeutic compounds were identified from our combinatorial chemical library (NIH, PWK, TOCRIS, CCC: 167 in total) and plotted against cell number and loss of pluripotency (LOP) It was. Only 5% of known anti-cancer compounds have been identified as anti-cancer stem cell compounds that cause a loss of pluripotency and a modest reduction in cell number of mutant neoplastic stem cells. Therefore, anticancer compounds that are also anticancer stem cell agents are surprisingly rare. FIG. 6 shows a two-step process workflow for identifying and validating compounds that selectively target cancer stem cells and not normal stem cells. EC50 values from mutant neoplastic stem cells (v1O4 cells) and dose response curves of normal stem cells (H9 cells) are used to calculate the selective activity potency ratio of each compound. FIGS. 22A-C show the determination of optimal duration and initial seeding density for normal stem cells (H9 cells) and v1O4 cells for use in high content analysis to determine pluripotency and cell number loss. is there. Figure Ai: Day 6 after seeding was determined as the optimal time point for H9. Figure Aii: The optimal seeding density for H9 was determined to be 10K. Based on the range of v1O4 cell seeding density for Hoechst staining (FIG. Bi) and GFP signal (FIG. Bii), the fourth day after seeding was determined as the optimal time point. 5K was determined as the optimal seeding density of v1O4 cells for Hoechst staining (FIG. Ci) and GFP signal (FIG. Cii). H9 and v1O4 cells were seeded on 96 well plates and treated with +/− BMP4 (0.1% DMSO) one day after seeding. The loss of pluripotency (LOP) response of v1O4 was quantified 4 days after seeding using plate fluorescence quantification, and the number of H9 cells was quantified by high content analysis. It is a bar graph which identifies the compound which has the highest selective activity ratio with respect to a cancer stem cell (v1O4 cell). FIG. 2 shows dose response curves of selective active compounds that are selective for mutant tumor stem cells at 10 μM. FIG. 5 shows dose response curves of selectively active compounds that do not necessarily show selectivity at 10 μM but nevertheless show selectivity at other concentrations. Cell numbers are normalized to untreated controls. The dashed line is the 10 μM concentration. Accordingly, screening compounds at multiple test concentrations is useful for identifying compounds that are selective for anticancer agents. FIG. 24 shows a plot of the percentage of v1O4 or H9 cells that stain positive for p53 after treatment with the highly selective active compound identified in FIG. 23 (gray). High levels of p53 indicate activation of p53-dependent stress responses. The black dots represent the levels of p53 in v1O4 and H9 cells treated with thioridazine and compounds similar to the structure of thioridazine. Highly selective compounds exhibit varying degrees of activation in the p53 stress response. The thioridazine-analogs shown in FIG. 25 that have little or no activation of the p53 stress response include triflupromazine, prochlorperazine, trifloperazine, fluphenazine and perphenazine. FIG. 3 shows that thioridazine selectively targets cancer stem cells expressing dopamine receptors. FIG. 26A: Breast cancer cell line MDA-MB-231 is very rich in cancer stem cells (CSC). Flow cytometry showed that 99% of MDA-MB-231 cells expressed the CD44 + CD24 _{− / low} breast cancer stem cell antigen phenotype. FIG. 26B: Histograms revealing that MDA-MB-231 cells express various dopamine receptors. FIG. 26C shows that thioridazine treatment of MDA-MB-231 cells reduces the cell population expressing the D1 dopamine receptor DRD1 (also known as DR1). Cells were treated with 10 μM thioridazine for 3 days. FIG. 26D shows that thioridazine treatment of MDA-MB-231 cells increases the overall expression of CD24. Thioridazine treatment also resulted in a decrease in total cell number compared to untreated cells (data not shown). FIG. 3 is a scatter plot of selective anti-cancer stem cell agents identified and validated using an exemplary embodiment of the two-stage screening method described herein. In the first stage, mutant neoplastic stem cells (V104 cells) were treated with compounds from the chemical library. Compounds that elicited loss of pluripotency and decreased cell number and passed a predetermined threshold (shown in dashed lines in FIG. 27) were classified as test agents. In the second stage, a series of dilutions of the test agent was tested on mutant neoplastic stem cells and normal human stem cells to generate a dose-response curve. EC50 values from dose-response curves from each cell line are then calculated, and compounds that showed EC50 values more than 3-fold different from normal stem cells versus mutated neoplastic stem cells are selective anti-cancer stem cell drugs. Identified (indicated by black stars in FIG. 27).

(I. Definition)
As used herein, "cancer" refers to one of a group of diseases caused by uncontrolled abnormal cell growth that spreads to adjacent tissues or other parts of the body. Cancer cells can form solid tumors in which cancer cells lump, or exist as dispersed cells like leukemia.

  “Cancer cells” refer to cells characterized by uncontrolled abnormal growth and the ability to invade other tissues, or cells derived from such cells. Cancer cells include, for example, primary cancer cells obtained from cancer patients, or cell lines derived from such cells. Similarly, “blood system cancer cells” refers to cancer cells derived from blood cells or bone marrow cells. Examples of cancer cells include, but are not limited to, cancer stem cells, breast cancer cells, rectal cancer cells, colon cancer cells, prostate cancer cells, and hematological cancer cells such as myeloma, leukemia cells or lymphoma cells.

  “Cancer stem cell” refers to a cell that has the ability to self-renew and to differentiate into a differentiated lineage of cancer cells consisting of tumors or hematological tumors. Cancer stem cells can independently initiate and maintain the disease. Mutant neoplastic stem cells are pluripotent cells that exhibit the characteristics of cancer stem cells and are useful in the screening methods described herein for identifying and / or verifying anti-cancer stem cell agents.

As used herein, a “biomarker associated with a cancer stem cell” refers to a biomarker such as a nucleic acid, polypeptide or fragment thereof, wherein its expression or lack of expression by a cell indicates that the cell is a cancer stem cell It is suggested. For example, in certain embodiments, the expression of one or more dopamine receptors (DR +) and CD14 (CD14 +) suggests that the cell is a leukemia cancer stem cell. In certain embodiments, cells that do not express CD24 (CD24-) or express CD24 at a low level (CD24 _low ), and express CD44 (CD44 +), are indicative of breast cancer stem cells.

As used herein, a “differentiated cell related biomarker” refers to a biomarker, such as a nucleic acid, polypeptide or fragment thereof, whose expression or lack of expression by a cell Suggesting that it is not a stem cell such as a normal stem cell or a cancer stem cell. For example, in certain embodiments, expression of CD11b (CD11b +) suggests that the cell is in a differentiated state and not a cancer stem cell.

As used herein, the term “mutant neoplastic stem cell” refers to a cell that can differentiate into multiple cell types and does not require Oct4 for self-renewal or survival. Mutant neoplastic stem cells are easily distinguished from normal stem cells. For example, some mutant neoplastic stem cells co-express FGFR1 and IGFR1. In contrast, normal human embryonic stem cells (hESS) express IGF1R, but FGFR1 is also expressed exclusively in fibroblast-like cells that are also found in human embryonic stem cell cultures. Some mutant neoplastic stem cells do not require basic fibroblast growth factor (bFGF) in culture to maintain an undifferentiated state. As used herein, an “undifferentiated state” refers to a cell that is pluripotent or can differentiate into one or more cell types. Some mutant neoplastic stem cells maintain SSEA3 expression in the absence of bFGF and / or require Nanog for self-renewal and cell survival.

In one aspect of the invention, the mutated neoplastic stem cells described herein can be passaged as single cells. As used herein, “passaging as a single cell” means that a single cell can be isolated individually, transferred to a culture vessel, and then the cells can form multiple cells. It means that there is. Optionally, the mutated neoplastic stem cell comprises one or more vectors or reporter constructs such as vectors, wherein expression of one or more pluripotency genes is operably linked to the reporter gene. In certain embodiments, the mutated neoplastic stem cell has neoplastic characteristics and exhibits poor differentiation potential in vitro. In certain embodiments, the mutated neoplastic stem cells are niche independent and have increased tumor initiation activity. In particular, in certain embodiments, the mutated neoplastic stem cells exhibit significantly reduced neuronal differentiation and lack hematopoietic capacity. As used herein, the term “mutant neoplastic stem cell” refers to transformed human pluripotent stem cells (t-hPSCs), or transformation-induced pluripotent stem cells, as described in PCT Publication No. WO2011026222. (T-iPSCs), which is incorporated herein by reference in its entirety. Mutant neoplastic stem cells show similarities to somatic cancer stem cells in functional properties and are therefore useful as surrogate for somatic cancer stem cells. Mutant neoplastic stem cells are also amenable to high content and high throughput screening in vitro. In certain embodiments, the mutated neoplastic stem cell is an induced mutated neoplastic stem cell. In certain embodiments, the mutant neoplastic stem cell is a human mutant neoplastic stem cell. In some cases, the mutated neoplastic stem cells are derived from a somatic or embryonic source.

  A “precancerous disorder” is one of a group of hyperproliferative disorders that can develop into cancer, including precancerous blood disorders such as myeloproliferative diseases or myelodysplastic syndromes, which include acute bone marrow It is a premalignant symptom that is associated with and / or can develop into sexual leukemia (AML).

  “Pre-cancerous cell” refers to a cell characterized by uncontrolled abnormal growth or a cell derived from such a cell. “Pre-cancerous cells” include primary pre-cancerous cells obtained from patients with pre-cancerous disorders, or cell lines derived from such cells or cancer stem cells. Similarly, “blood system precancerous cells” refers to precancerous cells derived from blood cells or bone marrow cells. In certain embodiments, the hematological precancerous cell is a myeloproliferative cell.

  “Leukemia” refers to any disease involving the progressive proliferation of abnormal white blood cells found in increased numbers in hematopoietic tissues, other organs and normal blood. “Leukemia cells” refer to white blood cells that are characterized by increasing cell abnormal proliferation. Leukemia cells may be obtained from a subject diagnosed with leukemia.

  “Acute myeloid leukemia (AML)” refers to a myeloid cancer of blood cells and is characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. Pro-leukemia symptoms such as myelodysplastic or myeloproliferative syndromes can also develop into AML.

  “Monocytic leukemia” refers to a subtype of leukemia characterized by expressing CD14 and includes acute monocytic leukemia, a subtype of acute myeloid leukemia. In certain embodiments, a subject is identified as having acute monocytic leukemia if he has more than 20% blasts in the bone marrow and more than 80% are monocytic.

  A “dopamine receptor blocker” refers to a compound that produces any detectable or measurable decrease in the function or activity of one or more dopamine receptors. In certain embodiments, the dopamine receptor (DR) is selected from DR1, DR2, DR3, DR4 and DR5. A dopamine receptor blocker may be selective for one dopamine receptor or selective for multiple dopamine receptors (ie, a “multiple receptor blocker”). Examples of multiple receptor blockers include thioridazine and chlorpromazine. Dopamine receptors are generally grouped into D1 family dopamine receptors (DR1 and DR5) and D2 family dopamine receptors (DR2, DR3 and DR4). In certain embodiments, the dopamine receptor blocker is one compound selected from the compounds listed in Table 1.

“Phenothiazine” or “phenothiazine derivative” refers to a compound derived from or containing a phenothiazine molecule or backbone. The general formula of phenothiazine is S (C ₆ N ₄ ) ₂ NH, and phenothiazine derivatives have one or more substitutions or additions to phenothiazine. For example, some phenothiazine derivatives have a tricyclic structure in which two benzene rings are connected by nitrogen and sulfur. Examples of phenothiazine derivatives include thioridazine, chlorpromazine, levomepromazine, mesoridazine, fluphenazine, perphenazine, prochlorperazine, and trifluoperazine. Additional examples of phenothiazine derivatives used in the method of the present invention are shown in Table 1. In certain embodiments, the thioridazine has the IUPAC name 10- {2-[(RS) -1-methylpiperidin-2-yl] ethyl} -2-methylsulfanylphenothiazine.
As an option, racemates of one or more phenothiazine derivatives such as thioridazine are used in the methods described herein.

  “Reducing the proliferation of cancer cells” means a decrease in the number of cells generated from cancer cells as a result of cell growth or cell division, and includes cell death or differentiation of cancer stem cells. “Cell death” includes all forms of cell death including necrosis and apoptosis. “Cancer stem cell differentiation” refers to the process by which cancer stem cells lose the ability to self-renew and cause lineage differentiation of cancer cells, including tumors or hematological tumors. “Killing cancer stem cells” includes all forms of cell death including necrosis and apoptosis.

  “Determining prognosis” means predicting the likely progression and / or outcome of the disease and, optionally, a defined outcome (recovery, signs, characteristics, duration, recurrence, complications, death and / or Including survival rate).

  “Control” means a comparative sample or preset value. In certain embodiments, “control” refers to the expression level of a biomarker, as described herein. In certain embodiments, a control refers to normal disease free cells, tissues or blood. In certain embodiments, a control refers to a cancer subject with a known clinical outcome or disease severity. For example, in one embodiment, a control refers to a subject who has survived at least 5 years after diagnosis of AML. In certain embodiments, a control refers to a cancer subject at a particular grade stage of the disease. In certain embodiments, a control refers to a stem cell that is not a cancer stem cell. In certain embodiments, the term “control” refers to a sample previously obtained from a subject.

  “Effective quantity” or “therapeutically effective quantity” refers to an effective quantity in administration and in the time period necessary to obtain the desired result. For example, a quantity that is effective in the context or treatment of cancer, such as AML, may e.g. induce remission, reduce tumor burden, and / or tumor spread or relative to the response obtained without administration of the compound. The quantity that prevents the growth of leukemia cells. The effective quantity varies with factors such as stage, animal age, sex, and weight. The quantity of a given compound corresponding to such a number will vary depending on many factors, such as a given drug or compound, pharmaceutical formulation, route of administration, type of disease or disorder, the identity of the subject or host being treated, Nevertheless, it can be determined routinely by those skilled in the art.

  “Pharmaceutically acceptable” means compatible with the treatment of animals, particularly humans.

  “Subject” includes all members of the animal kingdom, including mammals, and preferably refers to humans. As an option, the “subject” includes a mammal diagnosed with cancer or in remission.

  “Treat” or “treatment” means an approach for obtaining beneficial or desired results, including clinical outcome, as is well known to those skilled in the art. Beneficial or desired results include, but are not limited to, alleviation or amelioration of one or more signs or symptoms, diminished severity of disease, stable (ie, not exacerbated) medical condition (whether detectable or impossible) May include, for example, keeping the patient in remission), preventing the spread of the disease, slowing or slowing the progression of the disease, recovering or alleviating the condition, attenuating the recurrence of the disease, and remission (partial or complete remission). . “Treat” or “treatment” may mean prolonging survival relative to expected survival if not receiving treatment. “Treat” or “treatment” also includes prophylactic treatment. In certain embodiments, the treatment regimen comprises administering to a subject a therapeutically effective amount of a dopamine receptor blocker as described herein, and optionally, a single dose or a continuous dose. Including steps.

(II. Method and use)
Surprisingly, it has been discovered that dopamine receptor (DR) blockers are cytotoxic to the AML system and primary AML, while less cytotoxic to normal hematopoietic stem cells. As shown in cases 2 and 10, the dopamine receptor (DR) blocker thioridazine significantly reduced leukemia stem cell (LSC) function while maintaining normal hematopoietic stem cell capacity.

Accordingly, in certain embodiments, a method of treating a cancer or precancerous disorder in a subject is provided, comprising administering a dopamine receptor (DR) blocker to the subject in need thereof.
Also provided are methods of using dopamine receptor (DR) blockers to treat cancer or precancerous disorders. In certain embodiments, the methods or methods described herein are useful for treating precancerous disorders such as myeloproliferative diseases. In certain embodiments, the cancer is AML or leukemia such as monocytic leukemia. The methods or uses described herein are particularly useful for cancer cells that express dopamine receptors. In certain embodiments, the methods or uses described herein are useful for the treatment of cancer cells that express the monocyte marker CD14. In certain embodiments, the dopamine receptor (DR) blocker preferentially induces differentiation of a subject's cancer stem cells relative to hematopoietic or normal stem cells. In certain embodiments, the cancer stem cell is a leukemia cancer stem cell. In certain embodiments, the subject suffers from AML and the cancer stem cell is an AML cancer stem cell.

  In certain embodiments, the dopamine receptor (DR) blocker is a blocker for one or more dopamine receptors (DR), such as DR1, DR2, DR3, DR4 and DR5. As an option, dopamine receptor (DR) blockers are multiple receptor blockers or are specific for a single dopamine receptor (DR) subtype. In certain embodiments, the dopamine receptor (DR) blocker is a phenothiazine derivative such as thioridazine or chlorpromazine. In certain embodiments, the dopamine receptor (DR) blocker is selected from the compounds listed in Table 1. One skilled in the art will readily be able to identify additional dopamine receptor (DR) blockers useful for the treatment of cancer as described herein.

  In certain embodiments, the methods or uses described herein include phenothiazine derivatives such as thioridazine or chlorpromazine. One skilled in the art will readily be able to identify additional phenothiazine derivatives that are dopamine receptor (DR) blockers useful for the treatment of cancer as described herein. In certain embodiments, the phenothiazine derivative is differentially toxic to cancer cells such as leukemia cells compared to normal or hematopoietic stem cells.

  In certain embodiments, dopamine receptor (DR) blockers and / or phenothiazine derivatives are prepared as known in the art for administration to a subject in need thereof. Prior art procedures and raw materials for selection and preparation of compatible formulations are described, for example, in Remington's Pharmaceutical Sciences (2003, 20th edition) and The United States Pharmacopeia: The National Formula 24 (1999) published in 1999. It is described in.

  In certain embodiments, there is also provided a method of reducing cancer cell growth, comprising contacting the cell with a dopamine receptor (DR) blocker. In a similar embodiment, the use of a dopamine receptor (DR) blocker to reduce cancer cell growth is provided. In certain embodiments, the dopamine receptor (DR) blocker induces cancer stem cell differentiation or cell death. In certain embodiments, the dopamine receptor (DR) blocker induces cell death of cancer cells. As an option, the cancer cells are in vivo or in vitro. The cancer cell may be a precancerous cell such as a myelodysplastic cell or a myeloproliferative cell. In certain embodiments, the cancer cell is a leukemia cell, such as a cell from an AML patient. In certain embodiments, the dopamine receptor (DR) blocker is a phenothiazine derivative such as thioridazine or chlorpromazine. In certain embodiments, the dopamine receptor (DR) blocker is selected from the compounds listed in Table 1.

As shown in Case 4 and FIG. 5, applicants have surprisingly found that some AML cell lines and primary AML cells show a relative increase in dopamine receptor (DR) expression compared to normal hematopoietic stem cells. It was. Thus, screening cancer subjects by dopamine receptor (DR) expression in cancer cells would help identify subjects that would benefit from treatment with dopamine receptor (DR) blockers.
Accordingly, in one aspect of the invention, a method for identifying a subject suitable for treatment with a dopamine receptor (DR) blocker is provided. In certain embodiments, the method comprises measuring the expression of one or more dopamine receptors (DR) in a cancer cell sample from the subject. A subject having cancer cells that express one or more dopamine receptors (DR) is thereby identified as a subject suitable for treatment with a dopamine receptor (DR) blocker. For example, the expression of one or more dopamine receptors (DR) in a cancer cell sample can be measured by testing cancer cells against a polypeptide or polynucleotide encoding for dopamine receptors (DR). . In certain embodiments, the method comprises obtaining or providing a sample of cancer cells from a subject and / or testing the sample for expression of one or more dopamine receptors (DR). In certain embodiments, the cancer is leukemia and the cancer cell is a leukemia cell. In certain embodiments, the cancer is AML. As an option, the method comprises measuring additional markers known to be associated with cancer, hematological tumors, leukemia, or AML, or markers associated with a particular treatment regime. In certain embodiments, cancer cells are also tested against the monocyte marker CD14.

  Dopamine receptor (DR) expression has been observed in breast and AML samples, which serves as a biomarker of disease severity. As shown in Case 11 and FIGS. 14G, H, high levels of dopamine receptor (DR) expression are associated with poor prognosis, while low levels of dopamine receptor (DR) expression mean better prognosis. Accordingly, in one aspect of the invention, a method for determining the prognosis of a cancer subject is provided. In one embodiment, the method measures the expression of one or more biomarkers selected from dopamine receptor (DR) DR1, DR2, DR3, DR4, DR5 and CD14 in a cancer cell sample from a subject; And comparing the expression level of one or more biomarkers with a control. In certain embodiments, an increase in the expression level of one or more biomarkers compared to a control indicates that the subject has a more severe form of cancer than the control. As an option, the methods described herein comprise providing or obtaining a cancer cell sample from a subject, such as a blood sample or tumor sample containing leukemia cells. In certain embodiments, the cancer cell is a leukemia cell or breast cancer cell, and an increase in the expression level of one or more biomarkers compared to a control indicates that the leukemia or breast cancer is a more severe form than the control. As an option, additional biomarkers known to be associated with cancer or disease severity are also tested and compared to a control sample. One skilled in the art can select a control that is representative of a particular prognosis of a cancer subject and observe the difference or similarity between the test sample and the subject at the level of one or more biomarkers to determine the subject's corresponding prognosis Will understand that you provide. For example, in certain embodiments, the control is representative of a subject diagnosed with AML known to have a particular outcome or prognosis, and the expression level of one or more dopamine receptors (DR) is increased relative to the control. Observing the presence suggests a worse prognosis than the subject's control.

  The methods described herein are also useful for identifying cancer subjects. In certain embodiments, the methods described herein are useful for identifying leukemia subjects such as AML or monocytic leukemia. Thus, in one embodiment, leukemia comprising providing a sample from a subject and testing the sample for expression of one or more biomarkers selected from DR1, DR2, DR3, DR4 and DR5. A method for identifying a subject is provided. In certain embodiments, the sample consists of cancer cells such as leukemia cells and / or white blood cells. In certain embodiments, the method comprises comparing the expression level of one or more biomarkers to a control. In certain embodiments, increased dopamine receptor (DR) expression in a sample compared to a control is indicative of cancer. In certain embodiments, increased expression of dopamine receptor (DR) in a subject compared to a control is indicative of leukemia. In certain embodiments, increased expression of dopamine receptor (DR) in a subject compared to a control is indicative of AML or monocytic leukemia. As an option, the methods described herein may be used in combination with other diagnostic methods for the identification of cancer or leukemia known to those skilled in the art.

  As shown in Case 11, dopamine receptor (DR) expression separates human cancer stem cells from other cells such as normal hPSCs that express pluripotent marker stage-specific fetal antigen 3 (SSEA3). Thus, in certain embodiments, cancer stem cells are cell populations comprising identifying whether the cells express one or more biomarkers selected from dopamine receptors (DR) DR1, DR2, DR3, DR4 and DR5. A method of identifying from is provided. In certain embodiments, the expression of DR1, DR2, DR3, DR4, or DR5 indicates that the cell is a cancer stem cell. As an option, the expression of two or more, three or more, four or more, or all five dopamine receptors (DR) suggests that the cell is a cancer stem cell. In certain embodiments, cells expressing DR1, DR2, DR3, DR4 and DR5 are identified as cancer stem cells.

  In certain embodiments, cancer stem cells are identified from a population of cells. In certain embodiments, the cell population has one or more cell types, such as somatic cell lines, pluripotent stem cells, cancer cells and / or cancer stem cells. In certain embodiments, the cell population is a plurality of cells in a cell culture, such as a tissue culture. In certain embodiments, the cell population is a population of cells from a mammal, such as a primary tissue sample or a blood sample. In certain embodiments, the cell population is a population of cancer cells or suspected cancer cells from a mammal. In certain embodiments, the cell population includes not only one or more types of cancer stem cells, but also stem cells, somatic stem cells and / or pluripotent stem cells. In certain embodiments, the cell population comprises cancer cells such as hematological cancer cells or precancerous cells. In certain embodiments, the cell population comprises monocytic cells. In certain embodiments, the cell population comprises breast cancer cells. As an option, the cell population is a population of cells from a tissue sample, such as a tumor sample, dissociated into single cells.

  In one aspect of the method, determining whether the cell expresses one or more biomarkers comprises testing the cell for expression of a polynucleotide or polypeptide encoding DR1, DR2, DR3, DR4 or DR5. There is a step to do. For example, in an existing method, reverse transcription polymerase chain reaction (RT-PCR) or a reporter gene that detects expression of a polynucleotide, or an immunohistochemical method that detects expression of a polypeptide is DR1, DR2, DR3. , DR4 or DR5 can be used to measure the expression of biomarkers. In certain embodiments, the biomarker is a cell surface biomarker, and the method includes detecting DR1, DR2, DR3, DR4 or DR5 expressed on the surface of the cell.

  In certain embodiments, the method of identifying cancer stem cells or determining the level of cancer stem cells described herein comprises one or more biologics selected from dopamine receptors (DR) DR1, DR2, DR3, DR4 and DR5. Measuring the expression level of the marker and then comparing the expression level to a control level. For example, in certain embodiments, a control refers to a cell that is not a cancer stem cell, such as a somatic stem cell, a blood stem cell or a cell that expresses the pluripotency marker SSEA3, and DR1, DR2, DR3, DR4 and / or Alternatively, cells in which the DR5 biomarker has an increased expression level compared to controls are distinguished from cancer stem cells. As an option, cells with increased expression levels (eg, at least 2-fold, 5-fold, or 10-fold, etc.) relative to controls are distinguished from cancer stem cells.

  In certain embodiments, the method also includes (a) providing a cell population; (b) an agent that specifically binds to one or more biomarkers selected from DR1, DR2, DR3, DR4 and DR5. Contacting the cell population; and (c) selecting cells that specifically bind to the agent of (b), thereby identifying and / or isolating cancer stem cells from the cell population. In certain embodiments, the agent is an antibody that selectively binds to a biomarker. In certain embodiments, the methods described herein may optionally have more than one selection or isolation step. The methods described herein can also be eliminated by selection, eg, excluding cells that express one or more biomarkers expressed in cells that are not cancer stem cells, or exclude cells that have low expression levels of a particular marker. Steps may be included.

  In certain embodiments, the invention includes isolating cancer stem cells from the cell population. For example, in certain embodiments, a cell identified as a cancer stem cell is not a cancer stem cell by selecting or isolating cells that express one or more biomarkers selected from DR1, DR2, DR3, DR4 and DR5 Isolated from cells or from other materials in the sample. As an option, cancer stem cells are isolated or selected by a method of classification based on the expression of one or more markers known in the art. For example, in certain embodiments, isolating cancer stem cells from a cell population comprises flow cytometry, fluorescence activated cell sorting, panning, affinity column separation, or magnetic selection. In certain embodiments, cells expressing one or more dopamine receptors (DR) are isolated using a binding agent that selectively binds to the dopamine receptor (DR), wherein the binding agent is a separation column or Bond to a matrix-like support in a magnetic bead. In certain embodiments, the method detects cancer in a sample by detecting one or more biomarkers related to cancer stem cells as described herein or one or more biomarkers related to differentiated cells. Determining the level of stem cells.

  In one aspect of the invention, the method described herein comprises measuring the level of one or more biomarkers, such as the level of one or more dopamine receptors (DR) in a sample from a subject. Including. In certain embodiments, the sample has cancer cells or is suspected of having cancer cells or precancerous cells. For example, the sample may comprise a blood sample, such as a peripheral blood sample, a fractionated blood sample, a bone marrow sample, a biopsy, a frozen tissue sample, a fresh tissue sample, a cell sample, and / or a paraffin-embedded section. In certain embodiments, the subject has or is suspected of having AML and the sample consists of mononuclear cells. In certain embodiments, the sample is processed prior to detecting biomarker levels. For example, the sample may be fractionated (eg, by centrifugation or using a column for size exclusion or FACS using monocyte biomarkers, depending on the biomarker level measurement method used. ), Concentrated or processed.

  The expression level of the biomarkers described herein is measured by methods well known to those skilled in the art. For example, in certain embodiments, the level of one or more biomarkers is determined by measuring or detecting the level of a nucleic acid, such as mRNA, or the level of a protein or polypeptide. In certain embodiments, the expression of one or more biomarkers is measured by detecting cell surface expression of DR1, DR2, DR3, DR4 and / or DR5. In certain embodiments, the methods described herein detect biomarkers using immunohistochemistry, eg, using an antibody specific to the biomarker or other detection reagent specific to the biomarker. Including the steps of: Examples of dopamine receptor (DR) antibodies suitable for use in the methods described herein are listed in Case 7 of the present invention.

  In certain embodiments, the level of mRNA encoding for a biomarker is determined by quantitative polymerase chain reaction (PCR), such as reverse transcription (RT) -PCR, SAGE, use of microarrays, digital molecular barcode technology, or Measured using Northern blot. One skilled in the art will appreciate that many methods can be used to measure biomarker levels. They include mass spectrometry approaches such as multiple reaction monitoring (MRM) and product ion monitoring (PIM), and antibody-based methods such as immunoassays such as Western blots and enzyme-linked immunosorbent assays (ELISA). In certain embodiments, measuring the expression of a biomarker such as one or more dopamine receptors (DR) described herein includes using immunohistochemistry and / or immunoassays. In certain embodiments, the immunoassay is an ELISA. In a further embodiment, the ELISA is a sandwich type ELISA.

  “Level” refers to a quantity (relative quantity or concentration) of a biomarker or cell type that is measurable or detectable in a sample. For example, the level may be a concentration or relative value such as μg / L, eg, control level, standard or reference level 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 .8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0 , 4.2, 4.4, 4.6, 4.8, 5.0, 10, 15, 20, 25, 30, 40, 60, 80, and / or 100 times larger. As an option, the control is one level, such as the mean or median level in the control sample. The biomarker level is, for example, the level of a protein or mRNA that encodes for a biomarker such as a dopamine receptor (DR).

  In certain embodiments, once two or more biomarker levels are measured, the two or more biomarker levels can be used to generate an expression profile for the subject. For example, in certain embodiments, the methods described herein include measuring levels of two or more biomarkers in a sample, and generating an expression profile based on the levels of two or more biomarkers. And comparing the expression profile with a control expression profile. The difference or similarity between the test sample expression profile and the control expression profile then determines the level of cancer stem cells in the sample, provides a prognosis for the test subject, identifies it as a cancer subject, or the subject is a dopamine receptor Used to indicate whether it is compatible with treatment with a blocking agent.

  Further aspects of the invention include the use of dopamine receptor blockers for the treatment of cancer or precancerous disorders. In a similar aspect, dopamine receptor blockers for use in the treatment of cancer or precancerous disorders are provided. In certain embodiments, the cancer is leukemia. In certain embodiments, the leukemia is acute myeloid leukemia or monocytic leukemia. In certain embodiments, the dopamine receptor blocker is a phenothiazine derivative such as thioridazine or chlorpromazine. In certain embodiments, the dopamine receptor blocker is selected from the compounds listed in Table 1.

  Further disclosed herein is the use of a dopamine receptor blocker for the manufacture of a medicament for the treatment of cancer and / or precancerous disorders.

  A further aspect of the invention includes a method of screening for compounds for anti-cancer activity comprising identifying compounds that block one or more dopamine receptors. In certain embodiments, compounds that block dopamine receptors are identified as having anti-cancer activity. In certain embodiments, the method screens compounds to identify compounds that reduce cancer stem cell proliferation as compared to normal stem cells such as hematopoietic stem cells, as described in cases 7 and 8 , Including steps.

The present invention also provides:
A method of identifying and verifying a test agent as a selective anticancer stem cell agent comprising:
i) contacting one or more mutant neoplastic stem cells with the test agent and contacting one or more normal stem cells with the test agent;
ii) detecting a change in the number of mutant neoplastic stem cells in response to the test agent and detecting a change in the number of normal stem cells in response to the test agent;
iii) If contact with the test agent does not induce a decrease in the number of mutant neoplastic stem cells, whereas it does not induce a decrease in the number of normal stem cells comparable to the decrease in the number of mutant neoplastic stem cells Identifying the agent as a selective anti-cancer stem cell agent;
A method characterized by comprising:

In some cases, the methods described herein may include the loss of pluripotency of a mutated neoplastic stem cell and the test agent prior to screening the test agent for differential activity on the number of normal and mutated neoplastic stem cells. Screening for an agent to select a test agent that induces a reduction in cell number. In certain embodiments, the method screens one or more agents to identify one or more test agents that induce loss of pluripotency and decreased cell number of the mutated neoplastic stem cells. including. Accordingly, the embodiments described herein are also:
A two-step method for identifying and validating a drug as a selective anti-cancer stem cell drug, comprising:
i) Detecting pluripotency change and cell number change of mutated neoplastic stem cells in response to contact with the drug, the drug losing pluripotency and / or reducing the number of mutated neoplastic stem cells Selecting the agent as a test agent when provoking; and ii) contacting one or more mutant neoplastic stem cells with the test agent and contacting one or more normal stem cells with the test agent; Detecting a change in the number of mutant tumor stem cells in response to the test agent, detecting a change in the number of normal stem cells in response to the test agent, and Identifying a test agent as a selective anti-cancer stem cell agent if it induces a decrease in the number of mutant tumor stem cells without inducing a decrease in the number of normal stem cells comparable to the decrease in number;
A method characterized by comprising:

Select a test agent that induces loss of pluripotency and a decrease in cell number of the mutated neoplastic stem cell before screening the test agent for differential activity on the effect of the test agent on normal and mutated neoplastic stem cells There are many advantages to screening for drugs. Screening test agents for differential activity using normal stem cells is resource intensive and time consuming. The use of a two-step screening method allows a more targeted approach to validate drugs as selective anticancer stem cell agents. In particular, culturing normal stem cells is relatively difficult compared to culturing neoplastic stem cells. Testing these agents for differential activity using normal stem cells to induce loss of pluripotency and / or a decrease in the number of cells in the mutated neoplastic stem cell would otherwise be an anti-cancer stem cell agent Minimize the estimated resources and time needed to verify

More particularly, 1) induces a loss of pluripotency and a decrease in the number of cells in the mutated neoplastic stem cells, and 2) a cell number of the mutated neoplastic stem cells without inducing a relative decrease in normal stem cells. It has been found that identifying agents that induce a decrease in the activity is particularly effective for the identification and validation of anti-cancer stem cell agents that selectively target the self-renewal ability of cancer stem cells.

  In one aspect of the invention, screening a test agent against a mutated neoplastic stem cell and a normal human stem cell at a plurality of test concentrations for a differential change in cell number is a selective anti-cancer stem cell of the test agent. It was judged useful for identification and verification as a drug. On the other hand, many high-throughput screening methods test for activity at a single or a few test concentrations, but some selective anticancer stem cell agents have differential cell number activity at a single or a few test concentrations. It is not easily identified by this test. As shown in FIG. 24 and Example 18, 8-azaguanine and parbendazole showed similar levels at about 10 nM, or about 10 μM test concentration for reduction of normalized cell number, but at about 1 μM test concentration. Showed different activities. Floxuridine showed much more significant differential activity at 10 nM compared to high concentrations such as 1 μM or 10 μM.

  Accordingly, in some embodiments, the methods of the invention involve contacting a test agent at multiple test concentrations with a mutant neoplastic stem cell and a normal stem cell, and one or more mutant neoplastic stem cells and normal stem cells. Detecting a change in the number of cells at a plurality of test concentrations. In some embodiments, the plurality of test concentrations differ by 3 orders of magnitude, and in some cases 4-5 orders of magnitude. In certain embodiments, the plurality of test concentrations varies from at least about 0.01 μM to 2 μM, or from at least about 10 nM to about 20 μM. In certain embodiments, the plurality of test concentrations varies from at least about 1 nM to about 10 μM. In certain embodiments, the plurality of test concentrations varies by at least 3, 4 or 5 orders of magnitude and comprises at least one test concentration between 10 nM and 10 μM, 10 nM and 1 μM, or 0.01 μM and 2 μM. In certain embodiments, the test concentration has a dilution series, such as a 5-point, 8-point, 10-point dilution series, where in some cases the plurality of test concentrations varies by at least 3, 4 or 5 orders of magnitude and 10 nM And at least one test concentration between 10 μM, 10 nM and 1 μM, or between 0.01 μM and 2 μM.

  In one aspect of the invention, the test agent is contacted with normal stem cells at one or more test concentrations, and the test agent is contacted with mutant neoplastic stem cells at one or more test concentrations, and normal stem cells. A test agent is identified as a selective anti-cancer stem cell agent if it induces a decrease in the number of mutant neoplastic stem cells without inducing an equivalent decrease in. Identification of test agents that show differential activity against reducing the number of mutated neoplastic stem cells compared to the number of normal stem cells is selective for cancer stem cells and low toxicity to normal stem cells Useful for identifying drugs that exhibit

One skilled in the art can use a variety of methods to determine whether a test agent induces a reduction in the number of mutant neoplastic stem cells without inducing an equivalent reduction in normal stem cells. For example, in certain embodiments, a dose response curve for changes in cell number of mutant neoplastic stem cells in response to contact with a test agent at multiple test concentrations is compared to a dose response curve for changes in cell number of normal stem cells. Is done. In certain embodiments, the normalized number of mutant neoplastic stem cells at a single test concentration is at least 10%, 20%, 30%, 40% or 50 compared to the normalized number of normal stem cells. If% low, the test agent is considered to induce a decrease in the number of mutant neoplastic stem cells without inducing an equivalent decrease in normal stem cells. In certain embodiments, a test agent induces an equivalent decrease in normal stem cells if there is a statistically significant difference between the mutated neoplastic stem cells in response to the test agent and the normalized number of normal stem cells Without inducing a decrease in the number of mutant neoplastic stem cells. In certain embodiments, the difference between the normalized number of mutant neoplastic stem cells and normal stem cells in response to a test agent, such as by calculating and / or comparing the area under the dose response curve, is simply Calculated and / or compared over one concentration or multiple test concentrations.

In certain embodiments, the methods described herein include determining a quantifiable method of cell number change in mutant neoplastic and normal stem cells in response to a test agent. For example, in one embodiment, a semi-effective concentration (EC50) value for a reduction in the number of mutant neoplastic stem cells and an EC50 value for a reduction in the number of normal stem cells are measured. In certain embodiments, the ratio of the EC50 value of normal stem cells to that of neoplastic stem cells for a given test agent is measured, and if the ratio value of EC50 values is greater than a predetermined value, the test agent is selected Identified as an anti-cancer stem cell drug. For example, in certain embodiments, a test agent is identified as a selective anti-cancer stem cell agent if the ratio of the EC50 value to a decrease in the number of normal stem cells and its ratio to neoplastic stem cells is greater than 2, 3 or 4. In certain preferred embodiments, a test agent is identified as a selective anti-cancer stem cell agent if the ratio of EC50 values is greater than 3.

In another aspect of the invention, the effect of seeding density of mutated neoplastic stem cells and normal stem cells on methods for identifying and validating an agent or test agent as a selective anticancer stem cell agent was investigated. As shown in FIG. 22 and Example 16, seeding mutant tumor stem cells and normal stem cells at a specific seeding density allows for rapid and effective detection of the selective anti-cancer stem cell agents described herein. To do. Using specific densities for seeding mutant neoplastic and normal stem cells as described above avoids the problems of reproducibility, vagal cell proliferation, and poor signal strength in screening methods using these cells To help.

In certain embodiments, the mutated neoplastic stem cells are seeded in the first receptacle at about 3,000 to 7,000 cells per receptacle and the normal stem cells are seeded in the second receptacle at about 8000 to 12,000 cells per receptacle. Is done. In certain embodiments, the mutated neoplastic stem cells are seeded at about 4,000 to 6,000 cells per receptacle, preferably about 5,000 cells per receptacle. In certain embodiments, normal stem cells are seeded at about 9,000 to 11,000 cells per receptacle, preferably about 10,000 cells per receptacle.

As a selective anti-cancer stem cell agent, the methods described herein for identifying and validating agents or test agents may optionally use microtiter plates or similar receptacles that allow physical separation of samples. It can be carried out. For example, in certain embodiments, mutant neoplastic stem cells are seeded in a first receptacle, normal stem cells are seeded in a second receptacle, and the first and second receptacles are wells on the same microtiter plate. Or wells on separate microtiter plates. The microtiter plate is a 96 well microtiter plate and may optionally be a coated 96 well microtiter plate or a similar container having multiple receptacles useful for automating screening assays.

In certain embodiments, the methods of the invention comprise detecting a change in the number of mutant neoplastic or normal stem cells in response to a test agent. In certain embodiments, a change in cell number of the mutated neoplastic stem cell is detected between about 48 hours and 96 hours, and optionally 72 hours after contacting the cell with a test agent. In certain embodiments, a change in the number of normal stem cells is detected between about 4 and 6 days and optionally about 5 days after contacting the test agent with the cells. In certain embodiments, optionally after seeding the mutated neoplastic stem cells at about 3,000 to 7,000 cells per receptacle and normal stem cells at about 8,000 to 12,000 cells per receptacle. After about 12 to 36 hours, preferably about 24 hours, the mutated neoplastic stem cells and normal stem cells are contacted with the agent or test agent.

A variety of methods known in the art can be used to detect changes in the number of mutant neoplastic or normal stem cells in response to an agent or test agent. For example, in some embodiments, changes in cell number may include deoxyribonucleic acid (DNA) content analysis, detection of nuclear markers such as histone or nuclear laminin, nuclear condensation and / or Hoechst, 4 ′, 6-diamidino-2 -Measured by detection of nuclei, such as by staining with a DNA-binding fluorescent dye such as phenylindole (DAPI), acridine orange, DRAQ5 or safarin. Other changes suitable for use in high-throughput screening assays to detect changes in cell number using high content analysis, in some cases nuclear imaging or detecting changes in cell number in response to drug or test agent It is possible to detect using this technique.

The methods of the invention involve the use of mutant neoplastic stem cells and / or normal stem cells. For example, in certain embodiments, the mutated neoplastic stem cells are transformed such as transformed human pluripotent stem cells (t-hPSCs), optionally v1H9-Oct4-GFP cells, or those described in PCT Publication No. WO2011026222. Induced pluripotent stem cells (t-iPSCs). PCT Publication No. WO2011026222 is hereby incorporated by reference in its entirety. In certain embodiments, the normal stem cells are pluripotent stem cells or induced pluripotent stem cells, optionally H9 stem cells or fibroblast derived derived pluripotent stem cells.

In some embodiments of the methods described herein, detecting a pluripotency change and a change in cell number of a mutated neoplastic stem cell in response to contact with the drug, wherein the drug is a mutated tumor Selecting an agent as a test agent when induced to lose pluripotency of sex stem cells and / or reduce cell number. In a preferred embodiment, an agent is selected as a test agent when the agent induces a loss of pluripotency and a decrease in cell number of the mutated neoplastic stem cell. In a preferred embodiment, the mutant neoplastic stem cells are contacted with the agent at a single test concentration to select the test agent. The use of a single test concentration in the first step to select test agents minimizes the resources and time required to select and identify anti-cancer stem cell agents in the methods described herein. . For example, in one embodiment, the mutated neoplastic stem cell is contacted with the agent at 10 μM. In some cases, if the test agent induces a decrease in the number of mutant neoplastic stem cells without inducing an equivalent decrease in the number of normal stem cells, the test is identified to identify the test agent as a selective anticancer stem cell agent. The agent is then contacted with mutant neoplastic stem cells and normal stem cells at multiple test concentrations.

In certain embodiments, an agent is selected as a test agent if the agent induces loss of pluripotency and / or decreased cell number of the mutated neoplastic stem cell. In some embodiments, an agent is selected as a test agent if the agent statistically significantly induces mutated tumor stem cell pluripotency and / or decreases cell number. For example, in one embodiment, multiple drugs are screened on a microtiter plate and selected as test drugs based on a threshold of standard deviation from the mean value. In certain embodiments, an agent is selected as a test agent for pluripotency changes and cell number changes based on a Z-score threshold of at least 3 standard deviations from an average, optionally from an average value across the plate. .

In some embodiments, the methods described herein include the use of positive and / or negative controls. For example, in one embodiment, a mutant neoplastic stem cell treated with a substance such as BMP4 that induces loss of pluripotency of the mutant neoplastic stem cell is used as a positive control. In certain embodiments, cells treated with at least 100 ng / ml BMP4 are used as a positive control. In other embodiments, differentiation agents such as activin A, retinoic acid, ascorbic acid, nicotinamide, insulin or transferrin may be employed.

A variety of methods known in the art can be used to detect loss of pluripotency of mutant neoplastic stem cells in response to drugs. For example, in certain embodiments, a change in pluripotency of a mutated neoplastic stem cell is detected by detecting a change in the expression level of one or more pluripotency markers. Changes in the expression level of pluripotency markers can use the use of immunohistochemical detection such as antibodies selective for pluripotency markers, or other methods for analyzing gene expression such as quantitative RT-PCR Can be detected. The expression of one or more pluripotency markers can be functionally linked to a reporter gene of the mutated neoplastic stem cell and the product of the reporter gene can be detected. In preferred embodiments, the reporter gene encodes a green fluorescent protein (GFP) gene or a gene that produces an expression product that is readily detected using high-throughput screening methods such as image analysis. In certain embodiments, the pluripotency marker is selected from Oct4 and Sox2. In certain embodiments, the pluripotency marker is Oct4, Sox2, Nanog, SSEA3, SSEA4, TRA-1-60, TRA-1-81, IGF1 receptor, connexin 43, E-cadherin, alkaline phosphatase, REX1, CRIPTO, Selected from CD24, CD90, CD29, CD9 and CD49f, and mixtures thereof.

In some embodiments, the method of identifying and validating an agent or test agent as a selective anti-cancer stem cell agent screens the test agent identified as a selective anti-cancer stem cell agent for activity against cancer cell lines from cancer patients. The method further includes the step of: For example, in certain embodiments, the cancer cell line may be a leukemia cell line, optionally an acute myeloid leukemia (AML) cell line. In certain embodiments, the activity is cancer cell line cytotoxicity, induction of apoptosis, loss of pluripotency, induction of differentiation or a combination thereof.

In some embodiments, a method of identifying and verifying an agent or test agent described herein as a selective anti-cancer stem cell agent, wherein the test agent identified as a selective anti-cancer stem cell is one or more stress response genes. Optionally further screening for the expression of an apoptotic biomarker such as Annexin V, p53 and / or p21.

In another aspect of the invention, it has surprisingly been found that compounds structurally similar to thioridazine are effective in inducing cancer stem cell differentiation or killing cancer stem cells. Specifically, compounds structurally similar to thioridazine are useful for selectively inducing cancer stem cell differentiation or reducing cancer stem cell proliferation. As shown in FIG. 26 and Example 20, treatment of cancer stem cell (CSC) -rich cell lines with thioridazine selectively targets CSC populations that express dopamine receptors and, consequently, cells that express CD24. Increase.

Thus, in certain embodiments, a method of selectively inducing cancer stem cell differentiation or reducing cancer stem cell proliferation, wherein the cancer stem cell is contacted with a compound having a Tanimoto coefficient of at least 0.6 similar to thioridazine There is provided a method characterized by comprising the steps of: In certain embodiments, the compound is selected from the group consisting of thioridazine, prochlorperazine, trifluoperazine, fluphenazine, perphenazine, and triflupromazine. In certain embodiments, the compound having a Tanimoto coefficient of at least 0.6 similar to thioridazine is a phenothiazine derivative. In certain embodiments, the compound preferentially induces differentiation of cancer stem cells relative to normal stem cells. Cancer stem cells are optionally in vivo, in vitro, or ex vivo. In some embodiments, the cancer stem cells are in a subject's in vivo.

Aspects of the present invention provide a method for determining treatment of a subject suffering from cancer or suspected of cancer, as well as treatment and / or monitoring of a subject suffering from or suspected of cancer, or determining the course of treatment. including. As described herein, anti-cancer stem cell agents such as dopamine receptor blockers selectively target cancer stem cells, and are particularly useful for inducing differentiation or reducing proliferation of cancer stem cells It is. Other cancer treatment approaches that target cancer cells and not cancer stem cells result in failure to eradicate the source of the cancer, thereby leading to possible recurrence of the disease or progression to a more severe form of disease . In general, cancer patients are treated until either the disease is perceived to have been eradicated, symptoms are controlled in case of remission, or side effects limit further treatment . If clinical disease cannot be perceived and treatment stops, recurrence of the disease may be due to the presence of cancer stem cells that were not eliminated by conventional therapy. Embodiments described herein for selectively identifying cancer stem cells and targeting cancer stem cells are useful for monitoring a subject and / or treating a subject with an anti-cancer stem cell agent. is there. For example, a subject's treatment, monitoring, or therapeutic decision based on detection of cancer stem cells in the subject does not simply continue treatment until there is a reduction or eradication of cancer cells or cancer biomarkers, Ensure that treatment continues until removed from the subject. This aspect of the invention is significantly different from other clinical paradigms because continuing cancer treatment in patients who do not develop neoplastic disease has generally not been shown necessary and deviates from most conventional practices. Because. For example, detection of cancer stem cells from cells harvested from bone marrow or peripheral blood by use of the markers described herein can be used to determine the duration of treatment of an anti-cancer stem cell agent described herein. . Similarly, for solid tumors such as breast cancer, samples from lymph node biopsies can be obtained to test for the presence of cancer stem cells.

Thus in certain embodiments:
Providing a sample from a subject undergoing treatment with an anti-cancer stem cell drug, suffering from cancer or suspected of having cancer;
Measuring the level of cancer stem cells in the sample;
If the sample does not contain cancer stem cells, the subject determines that no further treatment with the anti-cancer stem cell drug is required, and if the sample contains cancer stem cells, the subject further requires treatment with the anti-cancer stem cell drug. Identifying as a subject to perform,
There is provided a method characterized by comprising:

In other embodiments;
Treating a subject suffering from or suspected of having cancer with an anti-cancer stem cell agent;
Measuring the level of cancer stem cells in a sample from the subject;
If the sample does not contain cancer stem cells, the subject determines that no further treatment with an anti-cancer stem cell drug is needed, and if the sample contains cancer stem cells, the subject treats with an anti-cancer stem cell drug Identifying as a subject in need of further,
There is provided a method characterized by comprising:

In another embodiment;
Providing a sample from a subject undergoing treatment with an anti-cancer stem cell drug, suffering from cancer or suspected of having cancer;
Measuring the level of cancer stem cells in the sample;
Comparing the level of cancer stem cells in the sample with the level of cancer stem cells in the control sample;
A method is also provided.

In certain embodiments, the method further comprises determining whether to continue treatment with the anti-cancer stem cell agent on the subject based on an increase or decrease in the level of cancer stem cells in the sample compared to the control sample. In some cases, the control sample is a preset value or threshold.
In certain embodiments, the control sample is a temporally previous sample from the subject.

  In some embodiments, the anti-cancer stem cell agent comprises a dopamine receptor blocker or an agent that selectively targets cancer stem cells described herein, including but not limited to the agent described in FIG. In certain embodiments, the dopamine receptor blocker has a titanimoto factor of at least 0.6, similar to oridazine. Optionally the dopamine receptor blocker is selected from thioridazine, prochlorperazine, trifluoperazine, fluphenazine, perphenazine, and triflupromazine.

  In certain aspects, the methods described herein may be directed to treatment by reducing cancer stem cells in response to anti-cancer stem cell drugs and / or loss of cancer stem cell differentiation or pluripotency, or by anti-cancer stem cell drugs. Useful for monitoring subjects undergoing treatment. Thus, in certain embodiments, measuring the level of cancer stem cells in the sample comprises measuring the ratio of cancer stem cells to differentiated cells in the sample. In certain embodiments, detection of a change in the ratio of cancer stem cells to differentiated cells over time in a sample from a subject, or a change compared to a control sample, determines a treatment strategy, or a subject suffering from or suspected of having cancer. Useful for monitoring.

  In certain embodiments, the level of cancer stem cells in the sample is the expression or signature of one or more biomarkers associated with cancer stem cells, and / or the expression or expression of one or more biomarkers associated with differentiated cells or It can be measured by detecting the biomarker signature. For example, a cancer stem cell is obtained by testing a sample for expression of one or more dopamine receptors (DR) selected from DR1, DR2, DR3, DR4, DR5, and one or more CD14, CD34 and CD44. It can be detected in the sample. As shown in FIG. 14, the expression of DR suggests the presence of cancer stem cells. In certain embodiments, the sample comprises leukemia cells or cells suspected of being leukemia cells and the biomarker associated with differentiated cells is selected from CD11b +, CD114 + and CD16 +. In certain embodiments, the sample comprises breast cancer cells or cells suspected of being breast cancer cells, and the biomarker associated with differentiated cells is CD24-.

In certain embodiments, a change in the level of cancer stem cells in a sample or the level of cancer stem cells in a sample is the expression of biomarkers associated with cancer stem cells relative to expression of biomarkers, It can measure by comparing and detecting. For example, in certain embodiments, an increase in the proportion of cells that express CD11b relative to cells that express biomarkers associated with leukemia cancer stem cells (such as DR +, CD14 +) is an effect of differentiating cancer stem cells of an anti-cancer stem cell agent. And does not simply suggest the death of cancer cells. In certain embodiments, an increase in the percentage of cells expressing CD24 suggests the effect of anticancer stem cell agents to differentiate breast cancer stem cells and does not simply suggest death of cancer cells. In certain embodiments, an increase in the proportion of cells expressing CD24 relative to cells expressing CD44, and optionally an increase in one or more dopamine receptors, suggests the effect of an anti-cancer stem cell agent to differentiate breast cancer stem cells. And does not simply suggest the death of cancer cells.

  In certain embodiments, the level of cancer stem cells in a sample can be measured by detecting the expression of biomarkers known in the art to be associated with cancer stem cells. For example, the literature by Eppert et al. (2011) and Al-Haj et al. (2003), which is incorporated herein by reference in its entirety, describes biomarkers and gene expression associated with specific cancer stem cells such as leukemia cancer stem cells. The signature is described.

The sample may be an immunohistochemical method for detecting protein levels, a fluorescence-based method such as FACS, or a RT-PCR or quantitative RT-PCR based method for detecting the level of a biomarker transcript, Or using the aforementioned or other methods known in the art can be tested for expression of biomarkers associated with cancer stem cells or differentiated cells.

In certain embodiments, a method of measuring the level of cancer stem cells in a sample comprises detecting the presence or absence of cancer stem cells in the sample. Other methods for determining the presence or absence of cancer stem cells include the use of xenograft studies in animal models such as mice.

  In certain embodiments, the sample comprises cancer cells or cells suspected of being cancer cells. In certain embodiments, the sample is from a subject suffering from or suspected of having cancer. In some cases, the sample may be a peripheral blood sample or a bone marrow sample. In some embodiments, the sample may be a tissue sample, such as a tissue biopsy taken from cancer or a subject suspected of having cancer, or another biological sample. For example, in some embodiments, the sample is from a subject suffering from leukemia and contains leukemia cells or cells suspected of being leukemia cells, or from a subject suffering from breast cancer, Includes cells suspected of being breast cancer cells. In some cases, the sample is processed prior to determining the level of cancer stem cells, such as to remove other types of cells or cell debris, or to select cells that are cancer cells that contain cancer stem cells. . For example, in certain embodiments, the sample is a peripheral blood sample containing leukemia cells, and the sample is enriched for a cell population that is CD34 + or CD34 + CD38− prior to determining the level of cancer stem cells. In certain embodiments, the sample is a breast cancer sample, and the sample is enriched for the CD44 + CD24− cell population prior to measuring the level of cancer stem cells.

  In certain embodiments, the sample is leukemia cells or cells suspected of being leukemia cells and the step of determining the presence or absence of cancer stem cells is associated with cancer stem cells such as CD14 and DR1, DR2, DR3, DR4 and DR5. Testing the sample for one or more biomarkers. In certain embodiments, expression of CD14 and one or more dopamine receptors suggests the presence of cancer stem cells in the sample. In certain embodiments, expression of CD14, CD34, and one or more dopamine receptors suggests the presence of leukemia cancer stem cells in the sample.

In certain embodiments, the sample is a breast cancer cell or a cell suspected of being a breast cancer cell, and determining the presence or absence of cancer stem cells comprises testing the sample for expression of CD44 and CD24. In certain embodiments, detection of cells that express CD44 but not CD24 suggests the presence of cancer stem cells.

The following non-limiting examples illustrate the invention:

(Example)
(Case 1: Thioridazine is cytotoxic to leukemia cell lines)
The effect of thioridazine on normalized cell numbers was verified with three leukemia cell lines: HL-60, MV4-11 and OCI-AML3. All three cell lines are leukemic cell lines. HL-60 is derived from promyelocytic AML, while MV4-11 and OCI-AML3 are representative of AML. Each compound is incubated with the cells for 72 hours. Control is 72 hours in dimethyl sulfoxide (DMSO) (ie the solvent used for the compound). Each condition was repeated three times.

  As shown in FIG. 1, administration of 0.1 μM and 1 μM thioridazine had little effect on the normalized cell number, whereas at 10 μM the normalized cell number decreased to almost zero.

(Case 2: Different activity of thioridazine on blast formation potential of AML and colony formation potential of normal stem cells)
The effect of thioridazine on blast formation in AML cell lines was compared with that of thioridazine on colony formation in normal human stem cells.

  Normal human stem cells (HSCs) and progenitor cells were derived from either peripheral blood or umbilical cord blood with increased progenitor cells in healthy patients. Primary AML cells were collected from patients diagnosed with AML. Both normal HSCs and primary AML cells were tested in standard in vitro methocellulose assay conditions (http://www.stemcell.com/en/Products/All-Products/MethoCult-H4434-Classic.aspx and Clinton Campb et al.). (See “Human stem cell order is defined by functional dependence on Mcl-1h for self-renewal potential”, Blood 116 (9) 1433-1442 (June 4, 2010), which is incorporated herein by reference) For at least 14 days, after which the number of colonies was recorded. As shown in FIG. 2, 10 μM thioridazine showed different effects in normal HSC and AML cells. 10 μM thioridazine reduced the colony formation potential of normal HSCs from approximately 100 (control, DMSO treatment) to a total colony count of 66. (FIG. 2A). However, for AML cells, the number of CFU colonies was greatly reduced from about 22 blast colonies to 1.6 blast colonies (FIG. 2B).

FIG. 3 shows a cell pellet of CFU colonies formed from normal human stem cells (HSC) treated with thioridazine and AML. At a dose of 10 μM, pelleted cells are visible on HSC but not on AML cells. Thus, thioridazine selectively targets the blast-CFU potential of AML cells.

(Case 3: Chlorpromazine is toxic to AML cell lines)
Chlorpromazine, a dopamine receptor blocker and phenothiazine-related compound, was also investigated for effects on the AML cell lines HL-60, MV4-11 and OCI-AML3. The test was performed as shown in Case 1. As shown in FIG. 4, 10 μM chlorpromazine is toxic to the AML cell line.

(Case 4: Dopamine receptor expression in normal blood vs. leukemia)
Expression of the dopamine receptors DR1, DR2, DR3, DR4 and DR5 is detected in AML cell lines (HL-60, MV4-11, AML-OCI2 and AML-OCI3), primary AML cells isolated from AML patients (AML22101). AML29428, AML22174, AML29560), normal blood mononuclear cells (MNC) (MPB21471 and MPB28137; healthy patient blood) and StemSep® human hematopoietic progenitor cell enrichment kit (http://www.stemcell.com). /En/Products/All-Products/StemSep-Human-Hematopoietic-Progenitor-Cell-Enrichment-Kit.aspx) and HSC Human progenitor cells enrichment level was confirmed by flow cytometry and analyzed with respect to it rich in the normal human stem cells or precursor umbilical cord blood primary cells (CB107, CB108 and CB109). Isotype expression was measured as background. The peak on the right side of the isotype peak means positive expression of the dopamine receptor (DR) marker.

  As shown in FIG. 5, dopamine receptors are expressed in primary AML, AML cell lines and normal mononuclear blood cells (MNC) but normal human stem cells (HSC) (line cord-depleted cord blood (CBlin−)). It is not expressed in blood rich in blood. The data show that if the sample is positive for dopamine receptor (DR) expression, all five DR subtypes are usually present.

  Not all primary AML expressed dopamine receptors. Accordingly, subjects may be pre-screened for dopamine receptor expression to identify subjects suitable for AML treatment with dopamine receptor blockers. As an option, subject pre-screening may span all five DR subtypes, or specific subtypes, or combinations of subtypes.

(Case 5: Multiple DR blockers are cytotoxic to AML cell lines)
A series of dopamine receptor stimulants, D3 _- blockers, DR _{1 & 5-} blockers and multiple receptor blockers were tested for cytotoxicity against three AML cell lines HL-60, OCI-AML2, and OCI-AML3. The test was carried out according to the settings of case 1.

As shown in FIG. 6, high concentrations of clozapine (CLOZ), and chlorpromazine hydrochloride (CHL), thioridazine (THIO) have significant cytotoxicity against AML cell lines. Without being limited by theory, cytotoxic effects may require inhibition of multiple dopamine receptors. Multiple receptor blockers CLOZ, CHL, THIO act to eradicate AML cell lines, while specific blockers D _3- , DR _{1 & 5-} only reduce the number of cells to 60%. is there.

(Case 6: Dopamine receptor is expressed in CD14 + cell population of primary AML)
Expression of dopamine receptor subtypes was analyzed in primary AML cells. Primary AML cells obtained from AML patients contained DR subtype and CD14 specific antibodies prior to analysis using flow cytometry. The majority of DR + cells were found to be CD14 positive.

  As shown in FIG. 7, the expression of the CD14 monocyte marker correlates with the expression of each DR subtype.

  The effect of thioridazine was also tested in the CD14 + cell subpopulation within primary AML. Primary AML cells were cultured for 72 hours under control (DMSO solvent) or 10 μM thioridazine and stained with antibodies specific for CD14. The number of CD14 + cells in both the control and thioridazine treated samples was measured using flow cytometry, and the appearance frequency of CD14 + cells was lower in thioridazine treated samples, indicating that this compound is selectively expressed in AML cells. Suggested targeting the middle CD14 + subpopulation.

  As shown in FIG. 8, 10 μM thioridazine also decreased the normalization frequency of CD14 + cells in primary AML cells, indicating that thioridazine selectively targets CD14 + cells. The AML control group contained a portion of CD14 + cells. This fraction is reduced by thioridazine treatment and is shown as a decrease in normalized frequency of appearance of control (100%) vs. treated (20%).

(Case 7: Identification and characterization of drugs that induce human pluripotent stem cell (hPSC) differentiation)
Identification of drugs that target cancer stem cells (CSC) without affecting normal stem cells (SC) is ideal for future cancer treatments, but human cancer stem cells that can accept a vast biological screen Limited due to the lack of both (CSC) and normal stem cell assays. As described in the examples below, the neoplastic vs normal human pluripotent stem cell (hPSC) differentiation platform is used to induce differentiation to overcome tumor self-renewal of cancer stem cells (CSC) The compound is identified. Of several candidates for identified anti-cancer stem cell (CSC) drugs, the approved antipsychotic drug thioridazine is a human somatic cancer stem cell capable of initiating leukemia in vivo ( CSC) could be selectively targeted while not affecting the ability of normal blood stem cells (SC). Blocking dopamine receptor (DR) signaling by thioridazine underlies the selective targeting of cancer stem cells (CSC), and dopamine receptors (DR) are biomarkers of hematopoietic tumors and breast cancer-derived cancer stem cells (CSC) It was revealed that.

(Experimental procedure)
(Generation of neoplastic human pluripotent stem cells (hPSC) EOS-GFP system)
Neoplastic v1H9 or v2H9 human pluripotent stem cells (hPSC) (Werbowetski-Ogilvie et al., 2009) were transduced with a lentivirus carrying the EOS-C3 + EOS-S4 + vector provided by Dr James Ellis (Hota et al., 2009) . After lentiviral transduction, cells were selected using puromycin and then sorted into 96-well plates as single cells based on green fluorescent protein (GFP) expression using FASCaria II (Becton-Dickinson). It was. Colonies formed from single cell clones are used to establish v1H9-Oct4-GFP (EOS-C3 +), v2H9-Oct4-GFP (EOS-C3 +) and v1H9-Sox2-GFP (EOS-S4 +) systems It was done.

(Cell culture medium)
H9 human embryonic stem cells (hESC), v1H9, v1H9-Oct4-GFP, v2H9-Oct4-GFP, v1H9-Sox2-GFP and fibroblast-derived induced pluripotent stem cells (iPSC) were cultured as described above ( Chadwick et al. 2003; Werbowetski-Ogilvie et al., 2009).

(Primary human sample)
For AML specimens, peripheral blood and / or bone marrow was collected at the clinical presentation. Healthy hematopoietic cells were obtained from umbilical cord blood samples. All samples were collected at McMaster University and the London Health Science Center, following informed consent following a Research Ethics Board approved protocol. Human breast cancer samples were collected from breast reduction surgery at MacMaster University according to informed consent following a research ethics committee approved protocol.

(In vitro culture platform for normal and neoplastic human pluripotent stem cells (hPSC))
The chemical screen is v1H9-Oct4- seeded in mouse embryonic fibroblast conditioned medium (MEFCM) supplemented with 8 ng / ml basic fibroblast growth factor (bFGF) at 5,000 cells per well. GFP cells were included. After 24 hours, the medium was replaced with MEFCM containing 10 μM compound and 0.1% DMSO, 0.1% DMSO (−BMP4) or BMP4 100 ng / ml and 0.1% DMSO (+ BMP4) for 48 hours, after which Replaced with fresh medium containing compound over 24 hours (total compound treatment 72 hours). It was then fixed and prepared for automated imaging and plate reader analysis. Confluent H9 and fibroblast derived induced pluripotent stem cells (iPSC) were seeded in MEFCM supplemented with 8 ng / ml basic fibroblast growth factor (bFGF) at 10,000 cells / well. After 24 hours the cells were treated with 10 μM compound and 0.1% DMSO, 0.1% DMSO (−BMP4) or BMP4 100 ng / ml and 0.1% DMSO (+ BMP4). Fresh MEFCM with compound was exchanged daily for 5 days. On day 5, human pluripotent stem cells (hPSC) were fixed and prepared for automated imaging and plate reader analysis. See supplemental experimental procedures for further details.

(Teratoma assay)
400,000 H9 human pluripotent stem cells (hPSC) or v1H9-Oct4-GFP were injected into the testes of male non-obese diabetic (NOD) / severe combined immunodeficiency (SCID) mice and teratomas were described above for Oct4. (Werbowetski-Ogilvie et al., 2009).

(Xenograft assay)
NOD. Cg-Prkdc ^Scid Il2rg ^tm1wjl / SzJ adult mice (NSG mice) were sublethally irradiated with 315 rads 24 hours before transplantation. 0.8-1.0 × 10 ⁷ AML monocytic cells (MNC) or 1.5-1.8 × 10 ⁵ line-depleted cord blood (CB lin-) hematopoietic cells treated with compound or DMSO solvent for 24 hours in the tail vein Injected via (intravenous injection (IV)). After 6-10 weeks, the animals were killed and the bone marrow and spleen were analyzed by flow cytometry for the presence of human cells (LSRII, BD) and the data were analyzed using FlowJo software (Tree Star Inc). For secondary human pluripotent stem cell (hPSC) transplantation, the same number of transplanted human cells from umbilical cord blood (CB lin-) engraftment to the lineage depletion was intravenously transferred to adult irradiated NSG mice as described above. Infused by internal injection (IV).

(Statistical analysis)
Data are expressed as mean ± standard error of mean or mean ± standard deviation. Significant differences between groups were determined using independent two-way or one-way t tests.

(Pluripotent stem cell culture)
H9 human embryonic stem cells (hESC), v1H9, v1H9-Oct4-GFP, v2H9-Oct4-GFP, v1H9-Sox2-GFP and fibroblast-derived induced pluripotent stem cells (iPSC) were 8 ng / ml bFGF (GIBCO 13256) -02) was cultured on Matrigel®-coated (BD Bioscience 353234) plates with mouse embryonic fibroblast conditioned medium (MEFCM) added. MEFCM is KO-DMEM (GIBCO10829-018), 20% KO-serum substitute (GIBCO10828-028), 1% non-essential amino acid (GIBCO11140-050), 1 mM L-glutamine, 0.11 mM β-mercaptoethanol (Sigma Aldrich M7522) Consists of The cell line was passaged every 7 days using 100 units / mL Collagenase IV (GIBCO 17104-019) for 2-3 minutes. Cell density, assay duration, and DMSO solvent concentration in 96 wells were optimized for v1H9-Oct4-GFP cells and normal H9 human embryonic stem cells (hESC). For v1H9-Oct4-GFP, the optimal initial cell density was selected for 5,000 cells / well for 72 hours treatment based on z ′ discrimination between GFP maximum levels and ± BMP4 controls. For normal human embryonic stem cells (hESC), an optimal cell seeding density of 10,000 cells / well was selected based on maximum z'-prime discrimination.

(Primary human sample)
Mononuclear cells were prepared using Ficoll-Paque Premium (GE Healthcare). For hematopoietic cells, lineage depletion was performed according to the manufacturer's recommendations using EasySep (StemCell Technologies).

(AML / HPSC cell culture)
AML cell lines, namely OCI-AML2 (M4), OCI-AML3 (M4), HL-60 (M2) and MV-4-11 (M5) are 5% overheated-inactive fetal bovine serum (FBS) (HyClone). ) In Roswell Park Memorial Laboratory Medium (RPMI) (Gibco). For dopamine receptor (DR) stimulant studies with R (+)-7-hydroxy-DPAT hydrobromide (Sigma), due to the prevalence of dopamine in fetal bovine serum (FBS) (Little et al., 2002) Serum-free conditions were applied. AML patient blast cells with 5% overheat inactivated fetal bovine serum (FBS) (HyClone), 5 ng / mL IL3 (R & D systems), 5 × 10 ⁻⁵ M β-mercaptoethanol (Sigma) and BIT (StemCell Technologies) Cultured in modified Dulbecco's medium (IMDM). Human stem cell (HSC) medium is Iskov modified Dulbecco medium (IMDM) supplemented with 1% bovine serum albumin (BSA) (Sigma), 100 ng / mL Flt-3L (R & D systems) 20 ng / mL thrombopoietin (TPO) (R & D systems) Included. The patient's human pluripotent stem cell (hPSC) and acute myeloid leukemia (AML) samples are treated with compound or DMSO vehicle (0.1%) for 24 hours before colony forming unit (CFU) plating or xenograft studies. It was implemented.

(antibody)
The antibodies used for immunocytochemistry were as follows: Oct3 / 4 (BD Truncations Laboratories, cat # 611203), Sox2 (R & D, cat # AF2018). In order to detect human hematopoietic cells, anti-human CD45 with Pacific Blue-, phycoerythrin (PE)-, allophycocyanin (APC)-or fluorescein isothiocyanate (FITC) label was used (BD Bioscience). FITC anti-CD33, PE anti-CD13, FITC anti-CD41a, FITC anti-human leukocyte antigen (HLA) dopamine receptor (DR), and PE anti-CD19 antibody were obtained from BD Pharmingen. PE anti-CD14, PE anti-CD15 and PE anti-GlyA were obtained from Immunotech Beckman Coulter. PE anti-SSEA2 (BD Bioscience) and PE- or AlexaFluor 488 anti-Oct4 (BD Bioscience) were used to measure pluripotency. Rabbit anti-human dopamine receptor antibodies DRD1 (Cat # 324390), DRD2 (Cat # 324393), DRD3 (Cat # 324402), DRD4 (Cat # 324405), DRD5 (Cat # 324408) were obtained from EMD Chemical. Anti-rabbit Alexa-Fluor-488 (Molecular Probes) was used as the secondary antibody. Primary anti-p53 (Cat # 2527) and anti-p21 (Cat # 2947) rabbit immunoglobulin G (IgG) obtained from Cell Signaling Technology were used to stain fixed and permeabilized cells. Anti-rabbit Alexa-Fluor-546 (Molecular Probes) was used as the secondary antibody. APC anti-CD44 and PE-CD24 were obtained from BD Pharmingen for breast cancer staining.

(Automated imaging and analysis)
(Imaging of tumorous human pluripotent stem cells (hPSC))
Cells were fixed with 2% paraformaldehyde and stained with Combi Multidrop Dispenser (Thermo) with 10 μg / mL Hoechst 33342 (Invitrogen). For experiments related to Oct4 immunocytology, a monoclonal antibody for Oct4 (BD) was used with Alexs-Fluor-6472 secondary antibody (Invitrogen). Immunocytological staining used Janus automatic liquid handler (Perkin Elmer). The image is 10 × N. Acquired by means of epifluorescence illumination and standard filter sets using an Arrayscan HCS VTI reader (Cellomics) at A.

(Imaging of normal human pluripotent stem cells (hPSC))
Cells were fixed with 2% paraformaldehyde and stained with 10 μg / mL Hoechst 33342 (Invitrogen). Standard fluorescent immunocytochemistry techniques were used to stain cells with Oct4 monoclonal antibody (BD) and Alexs-Fluor-647 secondary antibody (Invitrogen). All steps were performed using a Janus automatic liquid handler (Perkin Elmer). Images were acquired by means of epifluorescence illumination and a standard filter set using an Arrayscan HCS VTI reader (Cellomics) at 5x.

(Image analysis)
Image analysis was performed using a custom script of Acapella software (Perkin Elmer). Nuclear material was distinguished from Hoechst signals. For neoplastic cell lines, object luminance analysis was performed only on green fluorescent protein (GFP) positive cells. For normal cell lines, Alexs-Fluor-647 positive cells were quantified. Image and well level data were stored and analyzed in the Columbias database (Perkin Elmer), and further data analysis, compound registration, and hit identification were performed in ActivityBase (IDBS).

(Gene expression analysis)
Cells under specific conditions are collected and RNA is extracted using the RNeasy kit (Qiagen) and complementary DNA (cDNA) is generated using the SuperScript III® cDNA synthesis kit (Invitrogen) Amplification and TaqMan® array reactions (Applied Biosystems) were performed as per manufacturer's instructions. The gene expression profile of each treated cell population was analyzed on a ViiA 7 real-time PCR system (Applied Biosystems) using a TaqMan® stem cell pluripotency array card. Each reaction sample was loaded into a well on the array card and centrifuged twice at 336 × g for 1 minute each, sealed and placed in a thermal cycler. The following cycle conditions were applied to all array card uses: 40 cycles of 45 ° C. for 10 minutes, 84 ° C. for 10 minutes, and (94 ° C. for 30 seconds then 60 ° C. for 1 minute). Array data was normalized to 18S RNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and compared using Data Analysis 2.0 software (Applied Biosystems).

(Methylcellulose colony formation assay)
AML patients and lineage depleted cord blood (CBlin-) cells were cultured for 24 hours in the presence of compound or in DMSO vehicle (0.1%) control. AML cells were plated in Methodoc GF H4434 (Stem Cell Technologies) at 50,000 cells / mL. Lineage depleted umbilical cord blood (CBlin-) cells were plated at 1,000 cells / mL in Methocult GF H4434 (Stem Cell Technologies). Colonies were scored using standard morphological criteria after 14 days of culture.

(Volumetric cell count)
The number of AML-OCI2 and AML-OCI3 cells present after 72 hours treatment with dopamine receptor blockers (FIG. 16B) and dopamine receptor stimulants (FIGS. 16C, D) is defined by forward scatter and side scatter clustering. 7AAD− and Hoechst +, counted by measuring the number of events in a fixed volume according to the lattice strategy.

(Human breast cancer sample processing)
Human breast cancer samples were collected from breast reduction surgery at MacMaster University according to informed consent following a research ethics committee approved protocol. Breast cancer meat pieces were cut into small pieces (1 mm or less) with scissors and a scalpel. Thereafter, 3 mL of the base solution (1 mL of 0.5 M ethylenediaminetetraacetic acid (EDTA) dissolved in 1 L of 1 × phosphate buffered saline (PBS)) and 7 mL of trypsin-collagen degrading enzyme solution were added to 1 g of tumor tissue. And incubated at 37 ° C. for 30 minutes. Trypsin-collagenase solution is RPMI 1640 medium (gibco # 11875093), 2% penicillin / streptomycin (Invitrogen # 15140163), 1% fungizone antifungal (Invitrogen # 15290018), 2% fetal bovine serum (FBS), 3 mg / Consists of mL Collagenase A (Roche Diagnostics # 110887933001) and 0.1% 2.5% trypsin (Gibco # 15090). The same volume of RPMI 1640 with 2% fetal bovine serum (FBS) was then added to the tissue suspension. Tissue suspension was filtered with a 40 micron nylon strainer (Falcon # 352340). The supernatant was discarded and the cell pellet was resuspended in 10 mL F-12 + GlutaMAX Nutrient Mix Ham 1X (Gibco # 31765) with 2% penicillin / streptomycin and 1% fungizone antifungal. Viable cells were counted using a hemocytometer and Trypan blue solution and prepared for flow cytometry. Antibody staining included rabbit anti-human dopamine receptor antibodies; DRD1 (Cat # 324390), DRD2 (Cat # 324393), DRD3 (Cat # 324402), DRD4 (Cat # 324405) and DRD5 (Cat # 324408) were EMD Obtained from Chemical. Anti-rabbit Alexa-Fluor-488 (Molecular Probes) was then used as a secondary antibody with allophycocyanin (APC) anti-CD44 and phycoerythrin (PE) -CD24 obtained from BD Pharmingen.

(Case 8: High-productivity screening identification of compounds that induce differentiation of neoplastic human pluripotent stem cells (hPSCs))
The inventors have previously demonstrated a variant human pluripotent that exhibits neoplastic characteristics including increased self-renewal and survival with vagal blockade of terminal differentiation ability in vitro and in vivo. The sex stem cell (hPSC) line has been described (Werbowetski-Ogilvie et al., 2009). Based on the similarity of functional features with somatic cancer stem cells (CSC), the body allows the screening of high content and high productivity of in vitro human pluripotent stem cells (hPSC) It was tested as a surrogate for cell line cancer stem cells (CSC). The screening platform has been developed to selectively identify small molecules that target neoplastic human pluripotent stem cells (hPSCs) while having little effect on normal hPSCs. This differentiation screening platform can identify powerful candidate drugs that selectively target somatic cancer stem cells (CSC), while leaving the ability of normal stem cells (SC).

  Oct4 and Sox2 provide reliable indicators for loss of self-renewal pluripotent state and induction of differentiation of normal and neoplastic human pluripotent stem cells (hPSC). In order to provide a more direct way to detect the loss of Oct4 and Sox2 during the induced differentiation of neoplastic human pluripotent stem cells (hPSCs), the GFP reporter system is an EOS-GFP reporter (v1H9-respectively Oct4-GFP and v1H9-Sox2-GFP) were formed by transducing neoplastic human pluripotent stem cells (hPSCs) (Hota et al., 2009). GFP brightness was observed to correlate with Oct4 and Sox2 expression in treatments that favor the stability of self-renewal and conditions that induce differentiation by the addition of BMP4. This response is not limited to the Sox2 reporter system (v1H9-Sox2-GFP), but an additional neoplastic human pluripotent stem cell (hPSC) system transduced with the same EOS lentiviral GFP reporter (v2H9-Oct4-GFP), Seen consistently using v2H9 (Werbowetski-Ogilvie et al., 2009).

  Uniform response to differentiation and maintenance of pluripotency in all human pluripotent stem cell (hPSC) lines generated also reveals that virus integration or clone selection by EOS reporter-forming insertion is independent of responsiveness I made it. This result suggested that compounds that induce differentiation are distinguishable based on the decrease in GFP brightness in the neoplastic human pluripotent stem cell (hPSC) line and are available for chemical screening. Therefore, conditions for automated high content microscopy and fluorometric based high productivity screening were used to detect a decrease in pluripotency marker expression in human pluripotent stem cells (hPSC). Microscopic analysis of normal hPSCs showed that clear Oct4 + cells disappeared after BMP4 treatment. Similarly, the reduction of both Oct4 and GFP by BMP4 treatment of neoplastic Oct4-GFP hPSC was quantified by high content microscopy and plate reader-based fluorimetry. In order to identify ideal candidates targeting cancer stem cells (CSCs), both normal and neoplastic hPSC differentiation in response to compound treatment were verified in parallel.

  Given the effectiveness of the screening platform, a chemical library consisting of 590 established annotated compounds from the NIH Clinical Collection and the Canadian Compound Collection was screened. These collections have previously been scrutinized in many other mammalian cell lines (Diallo et al., 2010; Shoemaker, 2006). Fluorometric quantitative high productivity screening (HTC) and high content screening (HCS) platforms were used to reduce the pluripotency of 51 defined compounds (GFP RFU and average GFP intensity per cell, respectively) and cell count (respectively Following the demonstration of giving equivalent measurements to (Hoechst RFU and cell count), high productivity screening (HTC) was chosen as a suitable platform for screening compound libraries faster (FIG. 9A). Of the 590 compounds screened (at 10 μM based on previous studies (Inglese et al., 2007)), 11 compounds are shown by a decrease in both GFP% residual activity (% RA) and Hoechst% RA As identified as inducing differentiation (FIGS. 9B-C). A total of four of these compounds, indatraline, thioridazine, azathioprine, and mefloquine, were identified as candidate compounds based on clustering and Hoechst% RA> 30% (FIG. 9B).

A secondary high content analysis revealed that indatraline was a questionable candidate and was excluded. However, both high content analysis and high productivity screening (HTC) confirmed that thioridazine, azathioprine, and mefloquine are candidate compounds (FIG. 9D), and these compounds were selected for further testing (FIG. 9E). -G). Compared to control-treated human pluripotent stem cells (hPSC), each compound induced distinct morphological changes in neoplastic human pluripotent stem cells (hPSC) (FIG. 9E). The decrease in GFP brightness was confirmed using image analysis (FIG. 9F) and further verified over a wide range of doses to calculate the maximum half dose effective concentration (EC50) of each compound (FIG. 9G). ). Only thioridazine and mefloquine have EC50 values below the target threshold of 10 μM (FIG. 9G), so that the compounds are able to produce neoplastic human pluripotent stem cells (hPSC) and somatic cancer stem cells (CSC) from patients. Defined as a candidate for further detailed verification to use.

(Case 9: Thioridazine selectively induces differentiation of neoplastic human pluripotent stem cells (hPSCs) and reduces human AML blasts without affecting normal hematopoietic stem / progenitor cells)
In order to detect Oct4 loss and to reveal the extent of differentiation, responses to thioridazine and mefloquine were detected in three normal (Fig. 10A) and neoplastic (Fig. 10B) human pluripotent stem cells (hPSC). The concentration was verified using quantitative flow cytometry. Salinomycin, a previously reported selective inhibitor of breast cancer stem cells (Gupta et al., 2009) was included for comparison. At 10 μM, all compounds reduced cell numbers. However, Oct4 levels in residual normal human pluripotent stem cells (hPSC) did not fall below the levels observed with BMP4 treatment (FIG. 10A). This same response was reproduced in a fibroblast-derived human induced pluripotent stem cell (iPSC) that represents an additional normal human pluripotent stem cell (hPSC) line derived from a defined adult (FIG. 11A). Suggested that the effect was not specific to embryonic origin. When the same compound was used to treat neoplastic human pluripotent stem cells (hPSCs), thioridazine and mefloquine treatment resulted in a decrease in the number of cells and Oct14 levels in neoplastic human pluripotent stem cells (hPSCs). Only thioridazine was able to reduce Oct4 levels below the BMP4 differentiation control (FIG. 10B), suggesting that thioridazine has the ability to overcome the inhibition of neoplastic human pluripotent stem cell (hPSC) differentiation. To confirm this response, an exhaustive dose response of all compounds to neoplastic human pluripotent stem cells (hPSC) was performed (FIG. 11B).

  To quantitatively identify compounds that selectively differentiate neoplastic human pluripotent stem cells (hPSCs), the ratio between normal and neoplastic hPSCs in normalized% of Oct4 + cells in response to these compounds is Measured. For example, a ratio of 1 means that differentiation is equivalent, while a ratio> 1 means that neoplastic hPSCs are relatively more differentiated than normal hPSCs. Only thioridazine had a significant impact on the differentiation of neoplastic hPSCs versus normal hPSCs at both 1 and 10 μM (FIG. 10C). The rapid accumulation of the cellular stress marker p53 (FIG. 10D) and its transcription target p21 (FIG. 10E) was used to further distinguish differentiation induction from cytotoxicity. Treatment of neoplastic hPSC with the toxic chemotherapeutic agent etoposide resulted in high levels of p53 and p21 after 24 hours. However, treatment with 10 μM thioridazine or BMP4 did not result in p53 or p21 accumulation, unlike agents that induce toxicity alone. This was consistent with induced differentiation rather than a stress response program. Furthermore, thioridazine treatment resulted in the expression of differentiation genes within neoplastic hPSCs, which were quantified by TaqMan Low-Density Array-qPCR. In treated neoplastic human pluripotent stem cells (hPSC), an increase was observed in 21 out of 50 genes associated with differentiation (FIG. 10F), which was consistent with the differentiation-inducing effect of thioridazine.

  To validate the potential similarity of neoplastic human pluripotent stem cells (hPSC) to somatic cancer stem cells (CSC) in chemical response, normal and neoplastic populations of the human hematopoietic system were validated. Experimentally, the self-renewal and differentiation of both human hematopoietic stem-progenitor cells (HSPC) and leukemia stem cells (LSC) is a powerful and well-established in vitro that can be used uniquely for hematopoietic systems. And can be investigated by in vivo assays. It makes it an ideal tissue to validate the potential surrogate of using normal and neoplastic hPSCs as primary screening tools for anti-cancer stem cell (CSC) compounds. Umbilical cord blood (CBlin-) is a highly abundant hematopoietic stem-progenitor cell (HSPC), and normal somatic stem cells (SC) capable of self-renewal and multilineage differentiation into all blood lineages Is a highly reliable source. Acute myeloid leukemia (AML) is a hematological tumor characterized by inhibition of differentiation of mature bone marrow maintained by self-renewing leukemia stem cells (LSC) (Bonnet and Dick, 1997; Lapidot et al., 1994).

  Thus, precursor assays in methylcellulose were performed on hematopoietic stem-progenitor cells (HSPC) and 5 acute myeloid leukemia (AML) patient samples; each sample was in vitro for each compound Were treated with thioridazine, mefloquine or salinomycin to verify their clonogenic and multilineage hematopoietic differentiation impact. Representative cell pellets generated from the total colony forming unit (CFU) HSPC (FIG. 10G) and AML (FIG. 10H) and treated with the respective compounds are shown. Thioridazine treatment reduced the proliferative / clonogenic capacity of AML while maintaining multiple lineage differentiation of HSPC (FIG. 11C). Changes in multiple lineage differentiation were quantified based on enumeration of colony forming units (CFU) formed after treatment of HSPC samples (FIG. 10I) and AML patient samples (FIG. 10J) with these compounds. Salinomycin decreased the AML-blast CFU potential at both 1 μM and 10 μM (FIG. 10J), but at the same time decreased the CFU potential of HSPC at all test dose concentrations (FIG. 10I). This suggested non-specific toxicity in the sex system. In contrast, thioridazine and mefloquine reduced AML-blast CFU formation (FIG. 10J), while having little effect on HSPC CFU potential (FIG. 10I) and multilineage organization (FIG. 11D). Suggested that thioridazine and mefloquine did not alter normal hematopoiesis.

  The most desirable outcome for clinical use of the identified compounds is to preferentially eliminate AML-blast CFU formation while maintaining the progenitor capacity of normal hematopoietic stem-progenitor cells (HSPC). To reveal the highest selectivity for targeting AML, the ratio of AML-blasts to total CFU generated from HSPC was calculated (FIG. 10K). A ratio of 1 means that the potentials of normal and neoplastic precursors are equal. A ratio> 1 defines compounds that selectively reduce the AML-blast CFU potential. Salinomycin (1 μM), mefloquine (10 μM) and thioridazine (10 μM) administration showed the highest ratio values for each compound (FIG. 10K) and were selected for in vivo validation. Thioridazine 10 μM, in particular, showed the highest ratio among all compounds, but most importantly is the only compound that showed significantly lower AML-blast CFU potential compared to that of normal HSPC. Points (FIG. 10K).

To discuss whether the specificity of thioridazine that reduces the clonogenic potential of AML-blast CFU is due to induction of differentiation, the emergence of granulocytic maturation marker CD11b in patient AML cells in response to thioridazine treatment The frequency was tested (Figure 10L). A significant increase in the frequency of granulocytic AML-blasts was observed during treatment (FIG. 10L), suggesting that thioridazine represents specific targeting of AML cells via induction of differentiation. Yes. This finding is similar to the induction of differentiation seen in neoplastic human pluripotent stem cells (hPSCs), and this screening platform is a powerful readout for identifying agents that can differentiate neoplastic cells Confirm. This result also suggests that thioridazine is the best candidate for specific targeting of AML cancer stem cells (CSCs) and needs to be tested using in vivo human-mouse xenograft assays.

(Case 10: Thioridazine reduces leukemia stem cell (LSC) function while leaving normal hematopoietic stem-progenitor cells (HSPC))
Functionally leukemia stem cells (LSCs) and hematopoietic stem cells (HSCs) to depict whether the suppression of AML-blasts detected in vitro is due to compounds that affect the neoplastic stem cell compartment A xenograft study (Dick, 2008) was defined (Figure 12). Treatment of hematopoietic stem-progenitor cells (HSPC) with salinomycin (1 μM) reduces hematopoietic engraftment to near undetectable levels (FIG. 13A), which interferes with normal hematopoiesis from HSPC. And this compound was excluded from further validation because it is unlikely to provide selective targeting of the desired anti-cancer stem cells (CSC). In contrast, mefloquine (10 μM) treatment showed a slight but not significant decrease in hematopoietic stem cell (HSC) capacity compared to the control (FIG. 12A). However, mefloquine was found to be ineffective in reducing AML leukemia stem cell (LSC) capacity and therefore lacked a selective effect, and further validation was discontinued (FIG. 12C).

  In contrast to both salinomycin and mefloquine, treatment of HSPC with thioridazine (10 μM) showed the same levels of bone marrow engraftment (FIG. 12A) and spleen engraftment (FIG. 13B) as control solvent-treated cells. Multilineage reconstitution capacity is the same in control and thioridazine-treated human hematopoietic stem cells (HSCs), myeloid (Figure 12B), lymphoid (Figure 12B), erythroid (Figure 13D), and megakaryocyte expression (Figure 13D) was not affected at all. Thioridazine treatment did not affect hematopoietic stem cell (HSC) self-renewal compared to control treated samples, as measured in a series of secondary engraftment (FIG. 13F). However, in stark contrast to salinomycin and mefloquine, thioridazine treatment significantly reduced leukemic disease initiating AML leukemia stem cells (LSC) (FIGS. 12C-D; FIGS. 13C; 13E).

Calculation of the ratio of HSPC normal hematopoietic regeneration (% hCD45 +) to AML leukemia induction (% CD33 + hCD45 + blast) shows that thioridazine significantly reduced leukemia stem cell (LSC) function while retaining normal hematopoietic stem cell (HSC) ability It was clarified that (FIG. 12E). In the absence of thioridazine, it was observed that there was no difference in leukemia engraftment levels in secondary transplant recipients. This suggested that continuous exposure to this compound was necessary for secondary recipients to suppress leukemogenesis. These data indicated that thioridazine selectively targets somatic cancer stem cells (CSCs) in vivo while not affecting normal stem cell function. Since thioridazine has been identified through in vitro use of a novel differentiation screening platform that uses normal and neoplastic human pluripotent stem cells (hPSCs), the functional effect of thioridazine has been demonstrated in somatic cancers. Provides an example of the predictive value of using human pluripotent stem cells (hPSC) to understand stem cells (CSC).

(Case 11: Dopamine receptor clearly separates human cancer stem cells (CSC))
Thioridazine is known to act via the dopamine receptor (DR1-5) (Beaulieu and Gainetdinov, 2011; Seeman and Lee, 1975). In order to verify whether the mechanism of thioridazine action that selectively interferes with human cancer stem cells (CSCs) compared to normal stem cells is mediated by dopamine receptor blocking, cell surface expression of dopamine receptors (DR) Was analyzed. To date, five dopamine receptors (DR) have been identified and divided into D1 family receptors (D1 and D5) and D2 family receptors (D2, D3, and D4) (Sibreley and Monsma, 1992). Normal human pluripotent stem cells (hPSC) expressing the pluripotency marker SSEA3 lack dopamine receptor (DR) expression (FIGS. 14A and 15A-B). In contrast, neoplastic human pluripotent stem cells (hPSC) expressed all five dopamine receptors (DR) (FIG. 14B). The observed differential expression of dopamine receptor (DR) and selective inhibition of thioridazine on tumor human pluripotent stem cells (hPSCs) is related to suppression of dopamine receptor (DR) signaling and normal stem cells (SC). This suggests a possible role for selective targeting to human cancer stem cells (CSC) compared.

  In order to expand the potential role of dopamine receptor (DR) in cancer stem cells (CSC) based on the functional role of thioridazine treatment, we have identified that dopamine receptor (DR) blockade is a leukemic stem cell (LSC) after thioridazine treatment. We examined whether we could explain the loss of function. DR1-5 expression in human hematopoietic mononuclear cells (FIGS. 15C-F) and AML patient samples (FIGS. 14D and 15G) from hematopoietic stem-progenitor cells (HSPC) and normal umbilical cord blood (CB) was analyzed. (FIG. 14C). Dopamine receptors (DR) were not observed in cord blood primitive hematopoietic stem cells (HSC) or precursor populations (identified as CD34 + 38− or CD34 + 38 +, respectively (Bhatia et al., 1997)) (FIG. 14C). This suggested that hematopoietic stem cells (HSC) and progenitors do not express thioridazine targets.

  Similarly, dopamine receptor (DR) was undetectable in erythroid (Figure 15C), megakaryocyte (Figure 15C) and lymphoid cells (Figure 15D). Only half of the monocytes defined as CD14 + and the granulocyte population defined as CD15 + expressed dopamine receptors (DR) (FIGS. 15E-F). All 13 AML patient samples analyzed contained a population of DR + blasts with different levels of all five receptors, and they were detected predominantly in CD34 + / CD14 + cells (FIG. 15G). However, unlike normal hematopoietic stem cells (HSC), CD34 + cells do not correlate with leukemic stem cell (LSC) ability in human AML (Taussig et al., 2008) and have recently been identified in many subfractions lacking CD34 or CD38. (Eppert et al., 2011). Differential expression of dopamine receptors (DR) in normal and AML human hematopoietic samples is heterogeneous in human AML leukemia stem cells (LSC) and drug targeting should be based on molecular pathways rather than alternative phenotypic descriptions I strongly suggested that there was.

  Apart from hematopoietic tissue, somatic cancer stem cells (CSCs) have recently been identified and verified in human breast cancer and have a CD44 + CD24− / lo phenotype (Al-Haj et al., 2003). Use human primary breast cancer with worst prognosis, negative for estrogen receptor (ER-), negative for progesterone receptor (PR-) and negative for human epidermal growth factor receptor 2 gene (HER2-) (Dent et al., 2007), we show that dopamine receptors (DR) co-localize with CD44 + CD24− / lo breast cancer stem cells (n = 3 patients) (FIGS. 14E-F and FIG. 15H). This finding is consistent with the low levels of dopamine receptors (DR) found in normal mammary tissue, while benign breast tumors show intermediate levels of these receptors and breast cancer shows high levels of these receptors (Carlo 1986).

It was investigated whether dopamine receptor (DR) expression in AML blasts correlates with the incidence of leukemic stem cells (LSC) in AML patients. AML samples with large fragments of DRD3 + blasts and DRD5 + blasts differ from AML patient samples with significantly lower levels of dopamine receptor (DR) that do not have leukemia stem cells (LSCs) and are different from xenograft recipients Had leukemia stem cells (LSC) because leukemia could be initiated in AML patient samples containing leukemia stem cells (LSC) correlated with poor prognosis, while samples without LSC showed good prognosis (Eppert et al., 2011). High level dopamine receptor (DR) appearance correlated with poor prognosis, while low level DR appearance showed good prognosis (FIGS. 14G-H). This suggests that dopamine receptor (DR) validation has use as a prognostic biomarker and is simpler than the molecular signature or leukemia stem cell (LSC) count for each AML patient. Based on the initial identification of neoplastic human pluripotent stem cells (hPSCs), these overall results show a potential for dopamine receptor (DR) appearance in human somatic cancer stem cells (CSC) compared to what is expected. Suggests the existence of a more generalized role and certifies the dopamine receptor (DR) as a candidate biomarker for other human cancer stem cells (CSC).

(Case 12: Dopamine receptor (DR) blockade of thioridazine suppresses human AML)
To better understand the functional role of dopamine receptors (DR) in human AML, two patient-derived AML cell lines; AML-OCI2 and AML-OCI3 were used (Koistinen et al., 2001).

  Like the primary sample, these two cell lines showed much higher levels of expression than the patient sample for each DR1-5 (FIG. 16A). Due to the bioavailability of dopamine in fetal bovine serum (FBS) (Little et al., 2002), serum-free conditions were employed to verify the role of dopamine receptors (DR) in AML. Both AML systems were treated with thioridazine and compared to other known dopamine receptor (DR) blockers clozapine and chlorpromazine (Seeman and Lee, 1975). All three DR blockers reduced AML cell numbers after treatment (FIG. 16B). To verify the specificity of dopamine receptor (DR) to target human AML cells, patient AML samples were divided into DR + and DR− subfractions by fluorescence-activated cell sorting, followed by DMSO solvent or thioridazine for 24 hours. Treated and subsequently assayed for blast colony forming unit (CFU) content.

  Decreased blast colony forming unit (CFU) formation was only seen in the thioridazine treated DR + subfraction (FIG. 17A), while not in the thioridazine treated DR-subfraction (FIG. 17B). Conversely, addition of the dopamine receptor (DR) D2 family stimulant 7OH-DPAT increased AML cells (FIG. 16C). The dopamine receptor (DR) D2 and D1 families add the opposite effect to intercellular signaling, resulting in different biological effects (Self et al., 1996). Treatment with the dopamine receptor (DR) D1 family stimulant SKF38393 significantly reduced AML cells, confirming that D2 family signaling is required for AML cell survival (FIG. 16D). Combining these results suggests that the mechanism of action of thioridazine is due to blockade of the D2 family dopamine receptor (DR), not due to untargeting, and via dopamine receptor (DR) signaling This suggests that we have identified a novel path for cancer stem cell (CSC) targeting.

(Case 13: Thioridazine analog as an anti-cancer stem cell drug)
FIG. 17 shows the identification of active compounds, i.e. induced a loss of pluripotency (LOP, measurement based on detection of Oct4 level reporter, in this case GFP signal output) and decreased cell number (per acquired image). FIG. 2 shows a scatter plot of mutant neoplastic stem cells (v1O4, v1H9-Oct4-GFP cells) treated with different chemical libraries to identify compounds that induce 750 cells or less). Compounds with reduced cell numbers below 100 were classified as potentially toxic, but many current chemotherapeutic agents show similar toxic effects, so these compounds are still useful as anticancer stem cell agents. It is possible.

Briefly, v1O4 cells were seeded in Matrigel-coated 96 well plates (5000 cells / well) containing mouse embryonic fibroblast conditioned medium (MEFCM) supplemented with 8 ng / ml bFGF, and dimethyl sulfoxide (DMSO) ) For 72 hours. The final concentration of each compound used for treatment was either 10 μM or 1 μM (n = 3). Control wells were treated with 0.1% DMSO (low control) or 100 ng / ml BMP4 (high control to induce LOP). At the end of 72 hours, the cells were fixed, stained with Hoechst and imaged with an automated microscope. GFP intensity and Hoechst signal are quantified as indicators of LOP and cell number, respectively, and compounds with a Z score greater than 3 standard deviations from the mean for reduced cell number and LOP are selected for further study. It was.

Thioridazine (FIG. 17, dotted circle star) and six compounds with similar chemical structure (thioridazine analog) (FIG. 17, star in dotted box) were selected for further study. Thioridazine analogs identified for further study included prochlorperazine, trifluoperazine, fluphenazine, perphenazine, and triflupromazine. The structural similarity of the analogs was determined based on these compounds with a Tanimoto similarity factor> 0.6 (calculated by IDBS software SARView) when compared to thioridazine. Surprisingly, thioridazine analogs caused a relative loss in the number of mutated neoplastic stem cells and loss of pluripotency in the total frequency of occurrence of thioridazine analogs against the entire chemical library (22/1594). = 1.4%), it appeared more disproportionately (6/64 = 9%), which was larger than the difference of 6 times. This suggests that structural features present in thioridazine and related compounds cause a decrease in the number of neoplastic cancer stem cells and LOP.

The phenothiazine-related compound chlorpromazine is cytotoxic to several leukemia and other types of cancer cell lines and has potency comparable to thioridazine. However, chlorpromazine had no equivalent effect on v1O4 cells (FIG. 17, circled asterisk). Thus, the effect of chlorpromazine depends on the cells to which it is exposed, and having some anticancer properties does not necessarily mean that it has broad activity on all cancer cells or cancer stem cells .

(Example 14: Thioridazine analogs active on mutant neoplastic stem cells do not induce cell death in AML cancer cell lines.)
Cancer cell lines have been used in high-throughput screening assays to identify potential anticancer therapeutic agents. Cell viability is an output that has been commonly used to assay a compound's potential anticancer activity. However, cancer therapeutics that rely solely on induction of cell death may not represent the most effective way to eradicate cancer stem cells. Identification of compounds that specifically target cancer stem cells and induce differentiation may represent another type of targeted therapy. The difficulty in identifying these types of compounds is due, in part, to the lack of a suitable surrogate for cancer stem cells. Based on the characteristic features and characteristics of mutated neoplastic stem cells, mutated neoplastic stem cells are useful for identifying anti-cancer compounds that do not necessarily induce cell death.

Two observations demonstrate that mutant neoplastic stem cells are useful in identifying compounds that were or were not identified as anti-cancer compounds in other systems. Both observations are related to the response of AML cancer cell lines AML-OC12 and AML-OC13 to thioridazine and thioridazine analogs.

Prior to this specification, thioridazine has been shown to reduce cell numbers in both AML-OC12 and AML-OC13 (see, eg, PCT / CA2010 / 000175). Cells were treated with thioridazine in AML-OCI2 and AML-OCI3 cells and analyzed for signs of apoptosis-induced cell death. As shown in FIG. 18, thioridazine exerts its effect on these cell lines via cell death with equal efficiency to AML-OC12 and AML-OC13. This is in contrast to the differentiation-inducing effect of thioridazine on mutant tumor stem cells. Further demonstration of the utility of the methods disclosed herein is shown by the response of cancer cell lines to thioridazine analogs. FIGS. 17 and 18 showed that these compounds exhibited thioridazine-like activity, including the ability to reduce cell number and pluripotency, and generate similar differentiation responses in v1 cells. However, thioridazine analogs did not significantly induce apoptosis in any AML cancer cell line; these thioridazine analogs probably have not been identified on the viability screen using these cells.

Thus, the method of use of the screening assays disclosed herein that use mutant neoplastic stem cells is significantly different from other screening processes that use cancer cell lines.

(Example 15: Compounds identified as anticancer compounds are rarely anticancer stem cell compounds.)
The chemical library used to screen for neoplastic pluripotent stem cells (v1O4 cells) contains compounds that are described as known or current anticancer therapeutics. Many of these anticancer therapeutics have probably been toxic to cancer cell lines.

Meta-drug search was performed on low molecular weight drugs with available structures used to treat human cancers (tumors). This search found 167 such anti-cancer compounds from a combination of NIH, PWK, TOCRIS and CCC libraries. These anti-cancer compounds were plotted as shown in FIG. 20, and only a small subset (5%) of them was identified as having activity against mutant neoplastic stem cells (v1O4 cells). This suggests that the screening platform herein as described in Example 16 is highly rigorous or identifies anti-cancer compounds in a unique manner. Furthermore, compounds identified as anticancer compounds are unlikely to be anticancer stem cell agents.

Example 16: Improved workflow for high-throughput identification of compounds that selectively target cancer stem cells but not normal stem cells
Previously, we treated mutant tumor stem cells and normal using quantitative flow cytometry to measure Oct4 levels in both cell types after treatment with three different concentrations of thioridazine. The effect of thioridazine on H9 stem cells was described (PCT / CA2010 / 000175). Only at the highest concentration tested (10 mM) was there a significant difference in loss of pluripotency (based on Oct4 expression) between neoplastic v1 cells and normal H9 cells. However, quantitative flow cytometry cannot be easily changed to high throughput analysis.

Here, the inventors disclose an improved screening method for identifying and verifying compounds that selectively target cancer stem cells as compared to normal stem cells, as shown in FIG.
The inventors have sought to seed normal stem cells and mutant neoplastic stem cells at a specific seeding density as shown in FIG. 22 to improve screening assays and to provide a rapid and efficient selection of selective anti-cancer stem cell drugs. It was decided that it would be possible to detect. Exemplary candidate compounds identified using this secondary screening procedure have selective activity or are sometimes referred to as selective actives, but are shown in FIG.

The first step of the screening process, the primary screening, is shown in FIG. 21 Step 1 and described in FIG. 17 and Example 13. In this initial screening step, compounds that reduce the number of mutated neoplastic stem cells and cause LOP are identified as active compounds. These compounds are then subjected to a further set of analyzes described in FIG. This stage represents an improvement over previous quantitative flow cytometry methods for determining compound potency and detecting response differences between mutant neoplastic and normal stem cells. To generate a dose response curve, 8 or 10 point dilutions for each compound are tested against mutant neoplastic stem cells (V1O4) and normal stem cells (H9 cells). For each compound, an effective concentration value (EC50) for a 50% reduction in cell number is extrapolated from the dose-response curves of treated v1O4 and H9 cells.

Dose response data was fitted to a four parameter Hill equation to derive EC50, slope, minimum and maximum values using IDBS ActivityBase software. The EC50 value is then used to calculate the selective activity potency ratio (EC50 for H9 / EC50 for V104). The ratio value above 1 indicates that the compound is more potent against v1O4 cells than against H9 cells. The value of this ratio is then used as a basis for the identification of highly selective active compounds that potentially induce cancer stem cell differentiation and not normal stem cell differentiation. Testing compounds at many different concentrations against mutant neoplastic and normal stem cells allows for the generation of dose response curves and is not identified by screening at a single concentration or a limited concentration range Enabling the identification of compounds that express selective activity. The effectiveness of each compound can be quantified by its analysis if the minimum fits the four parameter Hill equation. An alternative metric for selective activity is effectiveness comparison.
Selective efficacy = minimum fit for H9-minimum fit for v1O4.

Example 17: Anti-cancer stem cell compound identified using high selectivity ratio
The selective activity ratio [EC50 (v1O4) / EC50 (H9)] was calculated for a number of screened compounds using the method described above as in Example 15. A ratio value of “3” was selected as a threshold for identifying selectively highly active compounds. These compounds selectively induce differentiation / toxicity in cancer stem cells, but are expected to have minimal effects on normal stem cells. The compound with the highest selective activity ratio identified using the screening assay is shown in FIG. Surprisingly, the selective activity potency ratio for thioridazine and thioridazine analogs is too low to be included in FIG. 23, suggesting that other compounds disclosed herein have a large therapeutic dose range. To do.

(Example 18: Analysis of dose-response curves allows identification of anti-cancer stem cell drugs)
Most major high-throughput screening methods use a single concentration point to examine the assay response resulting from treatment with a compound. The present invention describes assays and conditions for manipulating mutant neoplastic and normal stem cells to develop stem cell-based assays for identifying and validating compounds selective for cancer stem cells To do. In particular, the method allows the generation of dose-response curves as shown in FIGS. Data from these curves was used to distinguish differences in the response of mutated neoplastic and normal stem cells to different medicinal compounds.

FIG. 24A shows seven selectively active compounds that may have been identified by testing cells at a single concentration point of 10 μM. FIG. 24B shows dose response curves for the other 12 selectively active compounds. Based on a single concentration point of 10 μM, many of these compounds, such as 8-azaguanine, parbendazole or 31-8220, would not be considered selective for cancer stem cells. As shown in Example 17, these 19 selectively active compounds appear to have greater selectivity for cancer stem cells than thioridazine.

(Example 19: Some highly selective active compounds have low activity for p53 stress response activation.)
Acute myeloid leukemia (AML) is characterized by neoplastic hematopoietic cells that have been inhibited from their ability to differentiate into mature cells. Similarly, mutated neoplastic stem cells are resistant to normal differentiation cues (see Werbowetski-Ogilive et al., 2009). Agents that can induce neoplastic progenitor / stem cell differentiation represent a promising strategy for the treatment of certain cancers. Treatment of acute promyelocytic leukemia (APL) using all-trans retinoic acid (ATRA) and arsenic trioxide is an exemplary use of this strategy. These compounds are believed to eradicate cancer stem cells that maintain cancer by inducing differentiation.

In order to identify compounds that were demonstrated to induce differentiation efficiently and have high selectivity (FIG. 23), treated mutant neoplastic stem cells were analyzed for changes in p53-dependent cytotoxic stress response. . Mutant neoplastic stem cells (v1O4 cells) and normal H9 stem cells were fixed and stained for p53 expression after treatment with selective active compounds. The percentage of v1O4 and H9 stained cells that stained positive for p53 was plotted for each compound as shown in FIG. A high level of p53 activity suggested high cytotoxicity. Although selectively active compounds caused different levels of p53 activation in both v1O4 and H9 cells, v1O4 cells appeared to be more sensitive overall compared to normal H9 cells.

Highly selective active compounds clustered in the lower left corner did not significantly increase the p53-dependent stress response in v1O4 and H9 cells. This group may contain potential candidates for compounds that selectively differentiate v1O4 cells. P53 levels of mutant neoplastic stem cells treated with thioridazine and thioridazine analogs were measured and are shown as black dots in FIG. As shown in FIG. 25, thioridazine analogs appear in the same lower left corner, consistent with previous observations that they act on mutant neoplastic stem cells by inducing differentiation.

(Example 20: Thioridazine selectively targets breast cancer stem cells expressing dopamine receptors.)
As shown in Example 12, dopamine receptors (DR) co-localize in the cancer stem cell (CSC) fraction of primary triple negative breast cancer tissue. MDA-MB-231 is a triple-negative breast cancer-derived cell line that is highly enriched for cells with the identified and approved breast cancer CSC phenotype, CD44 + CD24− / low (FIG. 26A) (Al-Hajj et al. 2003; Mani et al., 2008). FIG. 26B shows that breast cancer CSCs co-express different subtypes of DR. Treatment of cells with thioridazine resulted in a lower frequency of cells expressing D1-type DR (DRD1) (FIG. 26C). This was accompanied by increased expression of CD24 as shown in FIG. 26D, which was a negative determinant of breast cancer CSC and was associated with decreased regrowth ability (see Sleeman et al. 2006). These results indicate that thioridazine selectively targets a population of breast cancer CSCs that express DR via induced differentiation and cell death, and these results indicate that mutant neoplastic human stem cells and AML blasts Consistent with the data in FIG. 10 and Example 9 showing thioridazine-induced differentiation.

Example 21: Identification of selective anti-cancer stem cell drugs using a two-step screening method.
FIG. 27 is a scatter diagram showing the results of screening a chemical library using a two-step screening method for the detection of selective anti-cancer stem cell drugs. The primary screening step identified compounds (test agents) with anti-cancer stem cell activity based on their ability to induce loss of pluripotency (LOP) and cell number reduction of mutant tumor stem cells (dashed line in FIG. 27). All compounds below). This first step helps to identify compounds that target the stem cell (or self-renewal) properties of cancer stem cells. The scatter plot shows various properties of the compound library tested, for example, some compounds induced LOP with a gradual decrease in cell number, while other compounds induced LOP with high cytotoxicity. In general, more cytotoxic compounds appeared to induce more LOP. Targeting drugs that induce both loss of pluripotency and a decrease in cell number has resulted in a high rate of identifying drugs as selective anticancer stem cell drugs in the second stage of the screening procedure. The next step in the two-step method is to target the mutated neoplastic stem cell and reduce its cell number by testing the test agent at multiple test concentrations and determining the EC50 if necessary, but normal stem cells Identifying an anti-cancer stem cell agent that does not reduce the number of cells. Compounds that exceed a predetermined selectivity value are listed in FIG. 23 as selective active compounds and are indicated by black stars in FIG.

  Although the present invention has been described with reference to examples presently considered to be preferred, it should be understood that the invention is not limited to the disclosed examples. On the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the claims.

  All publications, patents and patent applications are hereby incorporated by reference in their entirety as if each publication, patent and patent application were specifically and individually incorporated by reference in their entirety.

(reference)

Claims (93)

A method of identifying and verifying a test agent as a selective anticancer stem cell agent comprising:
i) contacting one or more mutant neoplastic stem cells with the test agent and contacting one or more normal stem cells with the test agent;
ii) detecting a change in the number of cells of the mutated neoplastic stem cell in response to the test agent and detecting a change in the number of cells of the normal stem cell in response to the test agent;
iii) Contact with the test agent induces a decrease in the number of cells of the mutated neoplastic stem cell, while inducing a decrease in the number of cells of the normal stem cell comparable to the decrease in the number of cells of the mutated neoplastic stem cell If not, identifying the test agent as a selective anti-cancer stem cell agent;
A method characterized by comprising:   The mutant neoplastic stem cells and the normal stem cells are contacted with the test agent at a plurality of test concentrations, and the number of the one or more mutant neoplastic stem cells and the normal stem cells at the plurality of test concentrations The method of claim 1, further comprising the step of detecting a change in.   3. The method of claim 2, wherein the plurality of test concentrations varies by at least about 3, 4 or 5 orders of magnitude.   3. The method of claim 2, wherein the plurality of test concentrations vary from at least about 0.01 [mu] M to 2 [mu] M, or from at least about 10 nM to about 20 [mu] M.   The method of claim 2, wherein the plurality of test concentrations have a dilution series of at least 8 points.   The step iii) comprises a dose-response curve relating to a change in the number of cells of the mutated neoplastic stem cell in response to contacting the mutated neoplastic stem cell with the test agent at a plurality of test concentrations, and the normal The method of claim 2, comprising comparing a dose-response curve for changes in the number of normal stem cells in response to contacting stem cells with the test agent at a plurality of test concentrations. the method of.   Measuring a half-maximal effective concentration (EC50) for reducing the number of cells of the mutated neoplastic stem cell and a half-maximal effective concentration (EC50) for reducing the number of cells of the normal stem cell for the test agent; The method of claim 6 further comprising:   Measuring the ratio of the half maximal effective concentration (EC50) for reduction of the number of normal stem cells to the half maximal effective concentration (EC50) for reduction of the number of cells of the mutant neoplastic stem cells, The method according to claim 7.   9. The step of iii) comprises identifying the test agent as a selective anticancer stem cell agent if the ratio of the half maximal effective concentration (EC50) is greater than 3. The method described.   The mutant neoplastic stem cells are seeded in a first receptacle at about 3000 to 7000 cells / receptacle, and the normal stem cells are seeded in a second receptacle at about 8000 to 12000 cells / receptacle. The method of claim 1.   11. The method of claim 10, wherein the mutated neoplastic stem cells are seeded at about 4000 to 6000 cells / receptacle, and in some cases at about 5000 cells / receptacle.   11. The method of claim 10, wherein the normal stem cells are seeded at about 9000 to 11000 cells / receptacle, and in some cases at about 10,000 cells / receptacle.   11. The method of claim 10, wherein the first receptacle and the second receptacle are wells on the same microtiter plate or are wells on different microtiter plates.   14. The method of claim 13, wherein the microtiter plate is a 96 well microtiter plate and optionally a 96 well coated microtiter plate.   The change in cell number of the mutated neoplastic stem cell is detected between about 48 hours and 96 hours after contacting the cell with the test agent, and in some cases after about 72 hours, and in the normal stem cell The change in cell number is detected between about 4 and 6 days, and in some cases after about 5 days after contacting said cells with said test agent. Method.   The mutant neoplastic stem cells are transformed human pluripotent stem cells (t-hPSCs), and in some cases are V1H9-Oct4-GFP cells, or transformation-induced pluripotent stem cells (t-iPSCs); The method of claim 1, wherein:   2. The method of claim 1, wherein the normal stem cell is a pluripotent stem cell or an induced pluripotent stem cell, optionally an H9 stem cell or fibroblast derived induced pluripotent stem cell.   Changes in cell number may include deoxyribonucleic acid (DNA) content analysis, nuclear markers such as histones, or nuclear laminin, detection of nuclear enrichment, and / or Hoechst, 4 ′, 6-diamidino-2-phenylindole (DAPI), The method according to claim 1, characterized in that it is measured by nuclear detection, such as by staining with a DNA-binding fluorescent dye such as acridine orange, DRAQ5 or safarin.   The method of claim 1, wherein the change in cell number is detected using high content analysis, and possibly nuclear image analysis.   2. The method of claim 1, further comprising screening a test agent identified as a selective anti-cancer stem cell agent for activity against a cancer cell line derived from a cancer subject.   21. The method of claim 20, wherein the cancer cell line is a leukemia cell line, and optionally an acute myeloid leukemia (AML) cell line.   21. The method of claim 20, wherein the activity is cytotoxicity, induction of apoptosis, loss of pluripotency, induction of differentiation, or a combination thereof.   A test agent identified as a selective anti-cancer stem cell agent is expressed by one or more stress response genes, optionally one or more apoptotic biomarkers such as annexin V, p53 and / or p21 The method of claim 1, further comprising screening for induction of.   Screen one or more agents prior to step i) to identify one or more test agents that induce loss of pluripotency and decreased cell number of the mutated neoplastic stem cells The method of claim 1, further comprising the step of:   Before the step of i), the step of contacting the mutant neoplastic stem cell with the drug, and the step of detecting the pluripotency change and the cell number change of the mutant neoplastic stem cell in response to the drug And selecting the drug as the test drug when the drug induces a loss of pluripotency and a decrease in cell number of the mutated neoplastic stem cells. the method of.   26. The method of claim 25, wherein the mutant neoplastic stem cell is contacted with the agent at about 10 [mu] M.   Multiple drugs are screened on microtiter plates and, in some cases, from the mean, and optionally from the average of the entire plate, based on a standard deviation threshold from the mean for changes in pluripotency and changes in cell number 26. The method of claim 25, wherein the method is identified as a test agent based on a Z-score threshold of at least 3 standard deviations.   Mutant neoplastic stem cells treated with bone morphogenetic protein-4 (BMP4) to induce loss of pluripotency are used as a positive control, and in some cases, the cells are treated with at least 100 ng / mL BMP4. 25. The method of claim 24, wherein:   26. The method of claim 25, wherein the pluripotency change of the mutated neoplastic stem cell is detected by detecting a change in the expression level of one or more pluripotency markers. .   Detecting a change in the expression level of one or more pluripotency markers comprises using an antibody selective for said one or more pluripotency markers. 30. The method of claim 29.   30. The method of claim 29, wherein the expression of one or more pluripotency markers in one or more mutated neoplastic stem cells is operably linked to a reporter gene.   30. The method of claim 29, wherein the pluripotency marker is selected from Oct4 and Sox2.   The pluripotency markers are Oct4, Sox2, Nanog, SSEA3, SSEA4, TRA-1-60, TRA-1-81, IGF1 receptor, connexin 43, E-cadherin, alkaline phosphatase, REX1, CRIPTO, CD24, CD90 30. The method of claim 29, wherein the method is selected from: CD29, CD9 and CD49f, and mixtures thereof. A two-step method for identifying and validating a drug as a selective anticancer stem cell drug, comprising:
i) detecting a change in pluripotency and cell number of a mutated neoplastic stem cell in response to contact with the drug, and the drug detects a loss of pluripotency of the mutated neoplastic stem cell and / or cell number Selecting said agent as a test agent if it induces a decrease in the amount of; and ii) contacting one or more mutated neoplastic stem cells with said test agent and one or more normal stem cells Contacting with the test agent, detecting a change in the number of cells of the mutated neoplastic stem cell in response to the test agent, detecting a change in the number of cells of the normal stem cell in response to the test agent, and the test When the drug induces a decrease in the number of cells of the mutant neoplastic stem cell without inducing a decrease in the number of cells comparable to the decrease in the number of cells in the mutant neoplastic stem cell, Identifying a test agent as a selective anticancer stem cell agent;
A method characterized by comprising:   35. The step ii) comprises detecting a change in the number of cells of the mutated neoplastic stem cell and a change in the number of cells of the normal stem cell at a plurality of test concentrations. The method described in 1.   36. The method of claim 35, wherein the plurality of test concentrations varies by at least about 3, 4 or 5 orders of magnitude.   36. The method of claim 35, wherein the plurality of test concentrations vary from at least about 0.01 [mu] M to 1 [mu] M, or from at least 10 nM to about 10 [mu] M.   36. The method of claim 35, wherein the plurality of test concentrations have a dilution series of at least 8 points. The step iii) comprises a dose-response curve relating to a change in the number of cells of the mutated neoplastic stem cell in response to contacting the mutated neoplastic stem cell with the test agent at a plurality of test concentrations, and the normal 36. Comparing a dose-response curve for changes in the number of normal stem cells in response to contacting stem cells with the test agent at a plurality of test concentrations. the method of.   Measuring a half-maximal effective concentration (EC50) for reducing the number of cells of the mutated neoplastic stem cell and a half-maximal effective concentration (EC50) for reducing the number of cells of the normal stem cell for the test agent; 36. The method of claim 35, further comprising:   Measuring the ratio of the half maximal effective concentration (EC50) for reduction of the number of normal stem cells to the half maximal effective concentration (EC50) for reduction of the number of cells of the mutant neoplastic stem cells, 41. The method of claim 40, wherein:   42. The method of claim 41, further comprising identifying the test agent as a selective anti-cancer stem cell agent if the ratio of the half maximal effective concentration (EC50) is greater than 3.   In step ii), the mutated neoplastic stem cells are seeded in a first receptacle at about 3000 to 7000 cells / receptacle, and the normal stem cells are seeded in a second receptacle at about 8000 to 12000 cells / receptacle. 36. The method of claim 35, wherein:   44. The method of claim 43, wherein the mutant neoplastic stem cells are seeded at about 4000 to 6000 cells / receptacle, and in some cases at about 5000 cells / receptacle.   44. The method of claim 43, wherein the normal stem cells are seeded at about 9000 to 11000 cells / receptacle, and in some cases at about 10,000 cells / receptacle.   44. The method of claim 43, wherein the first receptacle and the second receptacle are wells on the same microtiter plate or are wells on different microtiter plates.   47. The method of claim 46, wherein the microtiter plate is a 96 well microtiter plate, and optionally a 96 well coated microtiter plate.   The change in cell number of the mutated neoplastic stem cell is detected between about 48 hours and 96 hours after contacting the cell with the test agent, and in some cases after about 72 hours, and in the normal stem cell A change in cell number is detected between about 4 and 6 days after contacting the cells with the test agent, and in some cases after about 5 days, and the pluripotency of the mutated neoplastic stem cells. The change is between about 48 hours and 96 hours after contacting the mutant neoplastic stem cells with the test agent, and optionally about 72 hours after contacting the mutant neoplastic stem cells with the test agent. 35. The method of claim 34, wherein the method is detected later.   The mutant neoplastic stem cells are transformed human pluripotent stem cells (t-hPSCs), and in some cases are V1H9-Oct4-GFP cells, or transformation-induced pluripotent stem cells (t-iPSCs); 35. The method of claim 34, wherein:   35. The method of claim 34, wherein the normal stem cells are pluripotent stem cells or induced pluripotent stem cells, optionally H9 stem cells or fibroblast derived induced pluripotent stem cells.   Changes in cell number may include deoxyribonucleic acid (DNA) content analysis, nuclear markers such as histones, or nuclear laminin, detection of nuclear enrichment, and / or Hoechst, 4 ′, 6-diamidino-2-phenylindole (DAPI), 35. The method of claim 34, wherein the method is measured by nuclear detection, such as by staining with a DNA-binding fluorescent dye such as acridine orange, DRAQ5, or safarin.   35. The method of claim 34, wherein the change in cell number is detected using high content analysis, and possibly nuclear image analysis.   35. The method of claim 34, further comprising screening test agents identified as selective anti-cancer stem cell agents for activity against cancer cell lines from cancer subjects.   54. The method of claim 53, wherein the cancer cell line is a leukemia cell line, and optionally an acute myeloid leukemia (AML) cell line.   54. The method of claim 53, wherein the activity is cytotoxicity, induction of apoptosis, loss of pluripotency, induction of differentiation, or a combination thereof.   A test agent identified as a selective anti-cancer stem cell agent is expressed by one or more stress response genes, optionally one or more apoptotic biomarkers such as annexin V, p53 and / or p21 35. The method of claim 34, further comprising screening for induction of.   35. The method of claim 34, wherein step i) comprises contacting the mutant neoplastic stem cells with the agent at about 10 [mu] M.   Mutant neoplastic stem cells treated with bone morphogenetic protein-4 (BMP4) to induce loss of pluripotency are used as a positive control, and in some cases, the cells are treated with at least 100 ng / mL BMP4. 35. The method of claim 34, wherein:   35. The method of claim 34, wherein the pluripotency change of the mutated neoplastic stem cell is detected by detecting a change in the expression level of one or more pluripotency markers. .   Detecting a change in the expression level of one or more pluripotency markers comprises using an immunohistochemical detection such as an antibody selective for said one or more pluripotency markers. 60. The method of claim 59, comprising:   60. The method of claim 59, wherein the expression of one or more pluripotency markers in one or more mutated neoplastic stem cells is operably linked to a reporter gene.   60. The method of claim 59, wherein the pluripotency marker is selected from Oct4 and Sox2.   The pluripotency markers are Oct4, Sox2, Nanog, SSEA3, SSEA4, TRA-1-60, TRA-1-81, IGF1 receptor, connexin 43, E-cadherin, alkaline phosphatase, REX1, CRIPTO, CD24, CD90 60. The method of claim 59, wherein the method is selected from: CD29, CD9 and CD49f, and mixtures thereof. A method of selectively inducing cancer stem cell differentiation or reducing cancer stem cell proliferation comprising:
Contacting said cancer stem cells with a compound having a Tanimoto coefficient of at least 0.6 similar to thioridazine. 65. The method of claim 64, wherein the compound is selected from the group consisting of thioridazine, prochlorperazine, trifluoperazine, fluphenazine, perphenazine, and triflupromazine.   65. The method of claim 64, wherein the compound is a phenothiazine derivative.   65. The method of claim 64, wherein the compound preferentially induces cancer stem cell differentiation compared to normal stem cells.   65. The method of claim 64, wherein the cancer stem cells are in vivo, in vitro, or ex vivo.   65. The method of claim 64, wherein the cancer stem cells are in vivo in a subject.   70. The method of claim 69, wherein the subject has cancer or is suspected of having cancer.   71. The method of claim 70, wherein the cancer is acute myeloid leukemia. A method of identifying and verifying a test agent as a selective anticancer stem cell agent comprising:
i) contacting one or more mutant neoplastic stem cells with the test agent and contacting one or more normal stem cells with the test agent;
ii) detecting a change in the activity of one or more of the mutated neoplastic stem cells in response to the test agent and a change in the activity of one or more of the normal stem cells in response to the test agent Detecting steps,
iii) Contact with the test agent induces one or more activities of the mutated neoplastic stem cells, while the normal stem cells exhibit an activity comparable to the activity elicited in the mutated neoplastic stem cells. If not induced in step, identifying the test agent as a selective anti-cancer stem cell agent;
A method characterized by comprising:   Said activity is apoptosis, necrosis, proliferation, cell division, differentiation, migration or movement, presence or absence of one or more biomarkers, level of one or more biomarkers, or induction thereof 73. The method of claim 72, wherein: A two-step method for identifying and validating a drug as a selective anticancer stem cell drug, comprising:
i) detecting a change in the activity of one or more of the mutated neoplastic stem cells in response to contact with the agent, wherein the agent induces one or more activities of the mutated neoplastic stem cell Selecting said agent as a test agent; and ii) contacting one or more mutated neoplastic stem cells with said test agent and contacting one or more normal stem cells with said test agent. Detecting one or more activity changes in the mutant neoplastic stem cells in response to the test agent, detecting one or more activity changes in the normal stem cells in response to the test agent; Then, the test agent does not induce an activity comparable to that induced in the mutant neoplastic stem cell in the normal stem cell, but the mutant neoplastic stem cell. Identifying said test agent as a selective anti-cancer stem cell agent when one or more activities are induced in a cell;
A method characterized by comprising:   Said activity is apoptosis, necrosis, proliferation, cell division, differentiation, migration or movement, presence or absence of one or more biomarkers, level of one or more biomarkers, or induction thereof 75. The method of claim 74, wherein: Providing a sample from a subject undergoing treatment with an anti-cancer stem cell drug, suffering from cancer or suspected of having cancer;
Measuring the level of cancer stem cells in the sample;
If the sample does not contain cancer stem cells, the subject determines that no further treatment with an anti-cancer stem cell drug is required, and if the sample contains cancer stem cells, treats the subject with an anti-cancer stem cell drug Identifying as a subject in need of further,
A method characterized by comprising: Treating a subject suffering from or suspected of having cancer with an anti-cancer stem cell agent;
Measuring the level of cancer stem cells in a sample from the subject;
If the sample does not contain cancer stem cells, the subject determines that no further treatment with an anti-cancer stem cell drug is required, and if the sample contains cancer stem cells, treats the subject with an anti-cancer stem cell drug Identifying as a subject in need of further,
A method characterized by comprising: Providing a sample from a subject undergoing treatment with an anti-cancer stem cell drug, suffering from cancer or suspected of having cancer;
Measuring the level of cancer stem cells in the sample;
Comparing the level of cancer stem cells in the sample with the level of cancer stem cells in a control sample;
Determining whether to continue treatment with an anti-cancer stem cell agent for the subject based on an increase or decrease in the level of cancer stem cells in the sample compared to a control sample;
A method characterized by comprising:   79. The method of claim 78, wherein the control sample is a temporally prior sample from the subject.   80. The method of any one of claims 76-79, wherein the anti-cancer stem cell agent is a dopamine receptor blocker.   81. The method of claim 80, wherein the dopamine receptor blocker has a Tanimoto coefficient of at least 0.6 similar to thioridazine.   81. The method of claim 80, wherein the dopamine receptor blocker is a phenothiazine derivative. 81. The method of claim 80, wherein the dopamine receptor blocker is selected from thioridazine, prochlorperazine, trifluoperazine, fluphenazine, perphenazine, and triflupromazine.   84. Measuring the level of cancer stem cells in the sample comprises measuring the ratio of cancer stem cells to differentiated cells in the sample, according to any one of claims 76-83. Method.   Comparing the level of cancer stem cells in the sample with the level of cancer stem cells in the control sample comprises the ratio of cancer stem cells to differentiated cells in the sample and the ratio of cancer stem cells to differentiated cells in the control sample. 79. The method of claim 78, comprising the step of comparing.   Measuring the level of cancer stem cells in the sample comprises detecting one or more biomarkers associated with cancer stem cells and / or one or more biomarkers associated with differentiated cells. 86. A method according to any one of claims 76-85.   87. The method of claim 86, wherein the one or more biomarkers associated with cancer stem cells are selected from dopamine receptor (DR) 1, DR2, DR3, DR4 and DR5, and CD14. . 87. The sample of claim 86, wherein the sample comprises leukemia cells or cells suspected of being leukemic cells, and the one or more biomarkers associated with differentiated cells are selected from CD11B, CD114, and CD16. The method described. 87. The method of claim 86, wherein the sample comprises breast cancer cells or cells suspected of being breast cancer cells, and the biomarker associated with differentiated cells is CD24.   Measuring the level of cancer stem cells in the sample comprises detecting the expression of one or more dopamine receptors selected from DR1, DR2, DR3, DR4 and DR5. 87. The method of any one of claims 76-86, wherein the expression of the dopamine receptor is indicative of the presence of cancer stem cells.   The sample comprises leukemia cells or cells suspected of being leukemic cells, and the step of measuring the level of cancer stem cells comprises one or more of the samples selected from CD14 and DR1, DR2, DR3, DR4 and DR5 87. Any of claims 76-86, comprising testing for dopamine receptors, wherein expression of said CD14 and one or more dopamine receptors is indicative of the presence of cancer stem cells. The method according to one.   The sample includes breast cancer cells or cells suspected of being breast cancer cells, and the step of measuring the level of cancer stem cells comprises testing the sample against CD44 and CD24, wherein the CD44 and CD24 87. The method of any one of claims 76-86, wherein expression is indicative of the presence of cancer stem cells.   The sample comprises breast cancer cells or cells suspected of being breast cancer cells, and the step of measuring the level of cancer stem cells is one selected from CD44, CD24 and DR1, DR2, DR3, DR4 and DR5. Testing for or more dopamine receptors, wherein the one or more dopamine receptors and CD44 expression, and the absence of CD24 expression indicates the presence of cancer stem cells, 87. A method according to any one of claims 76-86.

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