US20130331381A1 - Treatment of Cancer WIth Dopamine Receptor Antagonists - Google Patents

Treatment of Cancer WIth Dopamine Receptor Antagonists Download PDF

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US20130331381A1
US20130331381A1 US14/001,899 US201214001899A US2013331381A1 US 20130331381 A1 US20130331381 A1 US 20130331381A1 US 201214001899 A US201214001899 A US 201214001899A US 2013331381 A1 US2013331381 A1 US 2013331381A1
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Mickie Bhatia
Eleftherios Sachlos
Ruth Munoz Risueno
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McMaster University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/9413—Dopamine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the disclosure relates to methods for the prognosis or treatment of cancer and particularly to methods for the prognosis or treatment of acute myeloid leukemia (AML) that target dopamine receptors.
  • AML acute myeloid leukemia
  • cancer stem cells (Dick, 2009; Jordan, 2009; Reya et al., 2001) that are uniquely able to initiate and sustain disease.
  • conventional chemotherapeutics characterized by their ability to inhibit cell proliferation of cancer cell lines (Shoemaker, 2006) or reduce tumor burden in murine models (Frese and Tuveson, 2007), are ineffective against human CSCs (Guan et al., 2003; Li et al., 2008).
  • Normal and neoplastic SCs are functionally defined by a tightly controlled equilibrium between self-renewal vs. differentiation potential. In the case of CSCs, this equilibrium shifts towards enhanced self-renewal and survival leading to limited differentiation capacity that eventually allows for tumor growth. In contrast to direct toxic effects that equally affect normal SCs, an alternative approach to eradicate CSCs is by modification of this equilibrium in favor of differentiation in an effort to exhaust the CSC population. The identification of molecules that selectively target somatic CSCs while sparing healthy SC capacity would therefore be useful for the development of novel diagnostics and therapeutic treatments to selectively target human CSCs.
  • Hematological malignancies are types of cancer that affect blood, bone marrow and lymph nodes. Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. Examples of myeloid malignancies include acute myeloid leukemia and chronic myeloid leukemia.
  • myeloid malignancies are all generally considered to arise from precursors of the myeloid lineage in the bone marrow, they are highly divergent in presentation, pathology and treatment.
  • Myeloproliferative Neoplasms See Tefferi et al. Cancer , September 1 st , pp. 3842-3847 (2009); also Vannucchi et al. Advances in Understanding and Management of Myeloproliferative Neoplasms CA Cancer J. Clin.
  • CML chronic myelogenous leukemia
  • Preferred treatments for leukemias would target leukemic cells without unduly affecting hematopoietic stem cell populations.
  • Thioridazine is a dopamine receptor antagonist that belongs to the phenothiazine drug group and is used as an anti-psychotic. It has been in clinical use since 1959, however because of concerns about cardiotoxicity and retinopathy at high doses this drug is not commonly prescribed, and is reserved for patients who have failed to respond to, or have contraindications for more widely used antipsychotics. Schizophrenic patients receiving dopamine receptor antagonist medication at doses deemed effective for schizophrenia have been reported to have a reduced incidence of rectum, colon, and prostate cancer compared to the general population.
  • dopamine receptor antagonists such as thioridazine or chlorpromazine are cytotoxic to cancer cells and in particular acute myeloid leukemia (AML). Furthermore, dopamine receptors antagonists at concentrations toxic to cancer cells have been found to have a relatively limited effect on normal stem cells such as hematopoietic stem cells. It has also been determined that dopamine receptors are expressed in AML cell lines and in primary AML cells, but show relatively less expression in cell lines enriched for normal hematopoietic stem cells. In addition, the expression of dopamine receptors in AML cells is shown to correlate with that of the monoblastic marker CD14. Dopamine receptor antagonists such as thioridazine are cytotoxic to AML cells that express CD14.
  • a method of treating a cancer or precancerous disorder in a subject comprising administering to the subject in need thereof a dopamine receptor (DR) antagonist.
  • a dopamine receptor (DR) antagonist for the treatment of cancer or a precancerous disorder.
  • the cancer or precancerous disorder is a myeloproliferative disease or leukemia.
  • the cancer is acute myeloid leukemia (AML).
  • the DR antagonist preferentially induces the differentiation of cancer stem cells relative to hematopoietic or normal stem cells.
  • the dopamine receptor antagonist is a phenothiazine derivative such as thioridazine or chlorpromazine.
  • the dopamine receptor antagonist is a multi-receptor antagonist that antagonizes more than one dopamine receptor.
  • the dopamine receptor antagonist is a D 2 family dopamine receptor antagonist.
  • the DR antagonist is a compound selected from those listed in Table 1.
  • a method for reducing the proliferation of a cancer cell comprising contacting the cancer cell with a dopamine receptor antagonist.
  • a dopamine receptor antagonist for reducing the proliferation of a cancer cell.
  • contacting the cell with a dopamine receptor antagonist induces cell death or differentiation of a cancer cell or precancerous cell.
  • the cancer cell is a cancer stem cell and contacting the cancer stem cell with a dopamine receptor antagonist induces differentiation of the cancer stem cell.
  • the cell may be in vivo or in vitro.
  • the precancerous cell is a myeloproliferative cell.
  • the cancer cell is a leukemic cell, such as an acute myeloid leukemia (AML) cell or a monocytic leukemic cell.
  • the cell is CD14 positive.
  • the dopamine receptor antagonist is a phenothiazine derivative such as thioridazine.
  • the dopamine antagonist is a compound selected from those listed in Table 1.
  • the method includes determining the expression of one or more dopamine receptors in a sample of cancer cells from the subject.
  • subjects with cancer cells that express one or more dopamine receptors are identified as suitable for treatment with dopamine receptor antagonists.
  • the cancer is leukemia and the cancer cells are leukemic cells.
  • the leukemia is acute myeloid leukemia or monocytic leukemia.
  • the cancer is breast cancer.
  • the method of identifying a subject with cancer comprises testing the sample for the expression of CD14.
  • a method for determining a prognosis for a subject with cancer comprising determining the expression level of one or more dopamine receptor biomarkers in a sample from the subject and comparing the level of expression of the one or more biomarkers to a control.
  • the method provided herein include providing or obtaining a sample of cancer cells from the subject.
  • increased expression of one or more biomarkers compared to the control indicates a more severe form of cancer.
  • the dopamine receptor biomarkers are DR3 and/or DR5.
  • the cancer is leukemia or breast cancer and the sample comprises leukemic cells or breast cancer cells.
  • the leukemia is acute myeloid leukemia or monocytic leukemia.
  • the methods include determining the expression level of one or more dopamine receptors in a sample from the subject and comparing the level of expression of the one or more dopamine receptors to a control.
  • sample comprises white blood cells and/or the method further comprises providing a sample comprising white blood cells from the subject.
  • increased expression levels of one or more dopamine receptors compared to a control is indicative of a subject with leukemia, such as acute myeloid leukemia, or monocytic leukemia.
  • the method further comprises testing for CD14.
  • kits for screening compounds for anti-cancer activity comprising identifying compounds that are dopamine receptor antagonists.
  • the anti-cancer activity is reduced proliferation of AML cells or monocytic cells.
  • the methods include identifying compounds that preferentially induce the differentiation of cancer stem cells relative to hematopoietic or normal stem cells.
  • the method comprises determining whether a cell expresses one or more biomarkers selected from dopamine receptor (DR) 1, DR2, DR3, DR4 and DR5. In one embodiment, the expression of dopamine receptor (DR) 1, DR2, DR3, DR4 and DR5 is indicative that the cell is a cancer stem cell.
  • DR dopamine receptor
  • the population of cells comprises cells isolated from a mammal or cells in culture such as cell culture. In one embodiment, the population of cells comprises pluripotent stem cells. In one embodiment, the population of cells comprises cancer cells such as hematological cancer cells or pre-cancerous cells.
  • the method includes testing the cell for the expression of polynucleotides or polypeptides that code for DR1, DR2, DR3, DR4 or DR5. In one embodiment, a cell that expresses DR1, DR2, DR3, DR4 and DR5 is identified as a cancer stem cell. In some embodiments, the methods described herein also include isolating cancer stem cells from a population of cells.
  • cancer stem cells can be isolated from a population of cells or other material using methods known in the art such as flow cytometry, fluorescence activated cell sorting, panning, affinity column separation, or magnetic selection.
  • cancer stem cells are isolated using antibodies to one or more of DR1, DR2, DR3, DR4 and DR5.
  • FIG. 1 shows thioridazine at 10 ⁇ M is cytotoxic to leukemic cell lines HL-60, MV4-11 and OCI3.
  • FIG. 2 shows that thioridazine 10 ⁇ M has limited affects on the colony forming potential of normal HSCs (2A) while significantly reducing AML blast forming potential.
  • FIG. 3 shows cell pellets of CFU colonies generated from normal HSC and AML treated with Thioridazine.
  • FIG. 4 shows that both 10 ⁇ M chlorpromazine and 10 ⁇ M thioridazine is cytotoxic to leukemic cell lines HL-60, MV4-11 and OCI3.
  • FIG. 5 shows the expression of dopamine receptors DR1, DR2, DR3, DR4 and DR5.
  • DR expression was observed in AML cell lines, some primary AML and mononuclear cells (MNC) but not in HSC enriched cells (CB lin( ⁇ )).
  • FIG. 6 shows that multiple DR antagonists are cytotoxic to AML cell lines.
  • FIG. 7 shows FACS data showing that dopamine receptors are expressed in the population of CD14+ cells in primary AML.
  • FIG. 8 shows that thioridazine selectively targets and reduces the normalized frequency of CD14+ cells in primary AML.
  • FIG. 9 shows the identification of mefloquine and thioridazine using chemical screening for compounds that differentiate neoplastic hPSC.
  • A Schematic of screening strategy.
  • C Summary of responses seen with 590 compounds.
  • D Chemical structure of candidate compounds; thioridazine, azathioprine and mefloquine.
  • E Representative GFP, Hoechst and merged microscopic images of v1H9-Oct4-GFP cells treated with candidate compounds at 10 ⁇ M.
  • F Histogram of GFP intensity of these images.
  • FIG. 10 shows the effect of salinomycin, mefloquine and thioridazine on normal and neoplastic populations.
  • A-B Flow cytometry analysis of frequency of Oct4+ cells in (A) H9 and (B) v1H9-Oct4-GFP cells treated with salinomycin (SAL), mefloquine (MQ) and thioridazine (THIO) at 10 ⁇ 7 -10 ⁇ 6 M.
  • SAL salinomycin
  • MQ mefloquine
  • THIO thioridazine
  • G-K Hematopoietic multilineage and clonogenic potential in response to compound treatment detected using methycellulose assays. Representative colony forming unit (CFU) pellets of (G) hematopoietic stem and progenitor cells (HSPC) versus (H) AML blast CFUs pellets following compound treatment.
  • CFU colony forming unit
  • (L) Frequency of normalized CD11b granulocytic cells in cultured patient AML cells treated with thioridazine 10 ⁇ M (THIO 10 ⁇ M) or DMSO vehicle (CTRL) for up to 96 hours. Each bar n 3, mean+/ ⁇ SD. (*) p ⁇ 0.05, (**) p ⁇ 0.01, (***) p ⁇ 0.001, (****) p ⁇ 0.0001.
  • FIG. 11 shows the effect of salinomycin, mefloquine and thioridazine on fibroblast-derived iPSC and HSPC.
  • A Flow cytometry analysis of frequency of Oct4+ cells in fibroblast-derived iPSC (Fib-iPS) treated with salinomycin (SAL), mefloquine (MQ) and thioridazine (THIO) at 10 ⁇ 7 -10 ⁇ 6 M.
  • SAL salinomycin
  • MQ mefloquine
  • THIO thioridazine
  • C Hematopoietic lineage potential of CBlin ⁇ treated with thioridazine. Colony forming units (CFUs) of erythoblast (CFU-E), macrophage (CFU-M) and granulocyte (CFU-G) colonies generated in methylcellulose assays.
  • D Composition of CFU generated from CBlin ⁇ treated with salinomycin, mefloquine and thioridazine.
  • FIG. 12 shows thioridazine's effect on HSC and LSC engraftment.
  • A Frequency of human CD45+ cells in the bone marrow following HSPC treatment with thioridazine 10 ⁇ M (THIO 10 ⁇ M) or mefloquine 10 ⁇ M (MQ 10 ⁇ M). Values normalized to DMSO-treated HSPC control (CTRL) samples. Total of two HSPC samples evaluated. Mean+/ ⁇ SEM.
  • B Representative flow cytometry plots of side scatter (SSC) versus myeloid (CD33) or lymphoid (CD19) markers within the hCD45+ population.
  • C Frequency of CD45+ CD33+ AML blast cells in the bone marrow (BM) following treatment of AML with thioridazine 10 ⁇ M (THIO 10 ⁇ M) or mefloquine 10 ⁇ M (MQ 10 ⁇ M). Values normalized to DMSO-treated AML control (CTRL) samples. Total of two AML patient samples evaluated.
  • C Representative flow plots of CD33 vs CD45 in DMSO-treated control (CTRL) populations versus thioridazine treated (THIO 10 ⁇ M).
  • E Ratio of normalized percent hCD45 HSPC engraftment per normalized percent CD45 CD33 AML blast engraftment.
  • * p ⁇ 0.05
  • FIG. 13 shows in vivo response to drug treatment.
  • A The normalized frequency of human CD45+ cells in the bone marrow following HSPC treatment with salinomycin 1 ⁇ M (SAL 1 ⁇ M) relative to DMSO-treated (CTRL) samples. Total of two HSPC samples evaluated. Mean+/ ⁇ SEM. (****) p ⁇ 0.0001
  • B Thioridazine's effect on HSC and LSC splenic engraftment.
  • B, top Frequency of human CD45+ cells in the spleen following HSPC treatment with thioridazine 10 ⁇ M (THIO 10 ⁇ M). Values normalized to DMSO-treated HSPC control (CTRL) samples.
  • Red blood cells are defined by glycophorin A positivity and platelets by CD41a.
  • D Confirmation of myeloid leukemic engraftment of xenotransplants with AML. Flow cytometry of side scatter versus CD19, a marker of lymphoid cells. Inset number represents mean+/ ⁇ SEM.
  • E-F Thioridazine's effect on HSC and LSC in vivo self-renewal.
  • THIO 10 ⁇ M thioridazine
  • CRL DMSO control
  • FIG. 14 shows dopamine receptors expressed on neoplastic stem cells.
  • A-B Flow cytometry of (A) normal H9 and (B) neoplastic v1H9-Oct4-GFP cells stained with SSEA3 and all five dopamine receptor (DR) subtypes. DR expression in the SSEA3+ fraction is shown.
  • C Flow cytometry of lineage-depleted cord blood (HSPC) stained with CD34, CD38 and all five DR subtypes. DR expression is presented in the gated populations.
  • D Flow cytometry of 13 AML patient samples stained for all five DRs along with associated FAB classification.
  • E Co-localization of DRD5 in triple-negative (ER ⁇ , PR ⁇ and HER2 ⁇ ) primary human breast tumor stained with CD44 and CD24.
  • F The frequency of triple-negative breast CSC (CD44+CD24 ⁇ / lo ) within the DRD3 and DRD5 population. Each bar composed of 3 primary triple-negative breast tumors, mean+/ ⁇ SEM.
  • G-H Frequency of AML blast cells (CD33+CD45+) from patient samples which are also positive for (G) DRD3 and (H) DRD5. A total of 8 AML patient samples were assessed for leukemic-initiation potential in xenotransplantation recipients.
  • Leukemic-initiating was defined as human engraftment >0.1% of CD33+ hCD45+ in mouse bone marrow.
  • Four leukemic-initiating AML samples were assayed in 22 mice while 4 non-initiating AML samples were assayed in 17 mice.
  • Total n 8 AML samples, mean+/ ⁇ SEM.
  • FIG. 15 (A-B) Flow cytometry SSEA3+ fraction in (A) fibroblast-derived hiPSC and (B) umbilical cord blood-derived hiPSC stained for all five dopamine receptors.
  • C Dopamine receptors expression of human blood populations. Flow cytometry of cord blood mononuclear cells stained for (C) erythroid (glycophorin A), (C) megakaryocytes (CD41a); (D) T-cells (CD3), (D) B-cells (CD19); (E) monocytes (CD14) and (E) granulocytes (CD15). Staining for all five DRs in the gated populations are shown as histograms.
  • FIG. 1 Summary of DR localization in the blood populations.
  • G Flow cytometry of AML patient showing DR in gated populations.
  • H Dopamine receptor expression in triple-negative human breast tumors.
  • Breast CSC are defined as CD44+ CD24 ⁇ / lo (Al-Hajj et al., 2003). Co-localization of each DR within the CD44 and CD24 population is shown for three triple-negative (ER ⁇ , PR ⁇ and HER2 ⁇ ) breast tumors.
  • FIG. 16 shows that thioridazine inhibits dopamine receptor signalling in AML.
  • A DR expression of AML-OCI2 and AML-OCI3 cell lines.
  • cancer refers to one of a group of diseases caused by the uncontrolled, abnormal growth of cells that can spread to adjoining tissues or other parts of the body. Cancer cells can form a solid tumor, in which the cancer cells are massed together, or exist as dispersed cells, as in leukemia.
  • cancer cell refers a cell characterized by uncontrolled, abnormal growth and the ability to invade another tissue or a cell derived from such a cell.
  • Cancer cell includes, for example, a primary cancer cell obtained from a patient with cancer or cell line derived from such a cell.
  • a “hematological cancer cell” refers to a cancer cell deriving from a blood cell or bone marrow cell. Examples of cancer cells include, but are not limited to, cancer stem cells, breast cancer cells, rectum cancer cells, colon cancer cells, prostate cancer cells and hematological cancer cells such as myelomas, leukemic cells or lymphoma cells.
  • cancer stem cell refers to a cell that is capable of self-renewal and differentiating into the lineages of cancer cells that comprise a tumor or hematological malignancy. Cancer stem cells are uniquely able to initiate and sustain the disease.
  • precancerous disorder refers to one of a group of hyperproliferative disorders that can develop into cancer, including for example precancerous blood disorders, such as myeloproliferative disease or myelodysplastic syndrome which is a premalignant condition that is related to and/or can develop into acute myeloid leukemia (AML).
  • precancerous blood disorders such as myeloproliferative disease or myelodysplastic syndrome which is a premalignant condition that is related to and/or can develop into acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • precancerous cell refers to a cell characterized by uncontrolled, abnormal growth or a cell derived from such a cell.
  • precancerous cell includes, for example, a primary precancerous cell obtained from a patient with precancerous disorder or cell line derived from such a cell or a cancer stem cell.
  • a “hematological precancerous cell” refers to a precancerous cell deriving from a blood cell or bone marrow cell. In one embodiment, the hematological precancerous cell is a myeloproliferative cell.
  • leukemia refers to any disease involving the progressive proliferation of abnormal leukocytes found in hemopoietic tissues, other organs and usually in the blood in increased numbers.
  • Leukemic cells refers to leukocytes characterized by an increased abnormal proliferation of cells. Leukemic cells may be obtained from a subject diagnosed with leukemia.
  • AML acute myeloid leukemia
  • AML acute myelogenous leukemia
  • the term “monocytic leukemia” refers to a subtype of leukemia characterized by the expression of CD14, and includes Acute Monocytic Leukemia, which is a subtype of acute myeloid leukemia.
  • a subject is identified as having acute monocytic leukemia if they have greater than 20% blasts in the bone marrow, and of these, greater than 80% are of the monocytic lineage.
  • dopamine receptor antagonist refers to a compound that produces any detectable or measurable reduction in the function or activity of one or more dopamine receptors.
  • the dopamine receptors are selected from DR1, DR2, DR3, DR4 and DR5.
  • Dopamine receptor antagonists may be selective for one or multiple dopamine receptors, i.e. a “multi-receptor antagonist”. Examples of multi-receptor dopamine antagonists include thioridazine and chlorpromazine.
  • Dopamine receptors are commonly grouped in D 1 -family dopamine receptors (DR1 and DR5) and D 2 -family dopamine receptors (DR2, DR3 and DR4).
  • the dopamine receptor antagonist is a compound selected from those listed in Table 1.
  • Dopamine antagonists suitable for use in the methods described herein.
  • Dopamine Receptor Antagonist Mechanism of Action Acetopromazine maleate salt
  • Dopaminergic antagonist Amisulpride D2 and D3 receptor antagonist
  • Amoxapine Dopamine-reuptake inhibitor
  • Azaperone Dopaminergic receptor antagonist
  • Benperidol Dopamine antagonist Benzo[a]phenanthridine-10,11-diol, D1 ligand Bromopride Dopamine antagonist Bromperidol
  • Dopamine antagonist Chlorpromazine hydrochloride D2 antagonist, selective D1, D3, D4 & D5 Chlorprothixene hydrochloride D2 dopamine receptor antagonist Clomipramine hydrochloride chlorpromazine derivative Disulfiram Dopamine beta-hydroxylase inhibitor DO 897/99 D3 antagonist Domperidone Dopamine Antagonists DROPERIDOL D2 (dopamine receptor) antagonist Ethopropazine hydrochloride Thiorida
  • phenothiazine or “phenothiazine derivative” refers to a compound that is derived from or contains a phenothiazine moiety or backbone.
  • Phenothiazine has the formula S(C 6 H 4 ) 2 NH and phenothiazine derivatives comprise one or more substitutions or additions to phenothiazine.
  • some phenothiazine derivatives have a three-ring structure in which two benzene rings are linked by a nitrogen and a sulfur.
  • phenothiazine derivatives include thioridazine, chlorpromazine, levomepromazine, mesoridazine, fluphenazine, perphenazine, prochlorperazine, and trifluoperazine. Additional examples of phenothiazine derivatives for use in the methods of the present disclosure are set out in Table 1.
  • thioridazine has the IUPAC name 10- ⁇ 2-[(RS)-1-Methylpiperidin-2-yl]ethyl ⁇ -2-methylsulfanylphenothiazine.
  • one or more racemic forms of a phenothiazine derivative such as thioridazine are used in the methods described herein.
  • reducing the proliferation of a cancer cell refers to a reduction in the number of cells that arise from a cancer cell as a result of cell growth or cell division and includes cell death or differentiation of a cancer stem cell.
  • cell death as used herein includes all forms of cell death including necrosis and apoptosis.
  • differentiation of a cancer stem cell refers to the process by which a cancer stem cell loses the capacity to self-renew and cause the lineages of cancer cells that comprise a tumor or hematological malignancy.
  • determining a prognosis refers to a prediction of the likely progress and/or outcome of an illness, which optionally includes defined outcomes (such as recovery, symptoms, characteristics, duration, recurrence, complications, deaths, and/or survival rates).
  • control refers to a comparative sample or a pre-determined value. In one embodiment, “control” refers to a level of expression of a biomarker as described herein. In one embodiment, the control is representative of normal, disease-free cell, tissue, or blood. In one embodiment, the control is representative of subjects with cancer for whom the clinical outcome or severity of the disease is known. For example, in one embodiment the “control” is representative of subjects who have survived for at least 5 years after a diagnosis with AML. In one embodiment, the “control” is representative of subjects with cancer who have a particular stage of grade of the disease. In one embodiment, the “control” is representative of stem cells that are not cancer stem cells.
  • an effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • an effective amount is an amount that for example induces remission, reduces tumor burden, and/or prevents tumor spread or growth of leukemic cells compared to the response obtained without administration of the compound. Effective amounts may vary according to factors such as the disease state, age, sex and weight of the animal.
  • the amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • pharmaceutically acceptable means compatible with the treatment of animals, in particular, humans.
  • subject as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.
  • subject includes mammals that have been diagnosed with cancer or are in remission.
  • treating means an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease (e.g. maintaining a patient in remission), preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.
  • Treating” and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Treating” and “treatment” as used herein also include prophylactic treatment.
  • treatment methods comprise administering to a subject a therapeutically effective amount of a dopamine receptor antagonist as described herein and optionally consists of a single administration, or alternatively comprises a series of administrations.
  • DR antagonists are cytotoxic to AML lines and primary AMLs while being much less toxic to normal hematopoietic stem cells.
  • the DR antagonist thioridazine significantly reduced leukemic stem cell (LSC) function while preserving normal hematopoietic stem cell capacity.
  • a method of treating a cancer or precancerous disorder in a subject comprising administering to the subject in need thereof a dopamine receptor antagonist.
  • a dopamine receptor antagonist for the treatment of cancer or a precancerous disorder.
  • the methods or uses described herein are useful to treat a precancerous disorder, such as a myeloproliferative disease.
  • the cancer is leukemia such as acute myeloid leukemia (AML), or monocytic leukemia.
  • AML acute myeloid leukemia
  • monocytic leukemia monocytic leukemia.
  • the methods and uses described herein are particularly useful for the treatment of cancer cells that express dopamine receptors.
  • the methods and uses described herein are useful for the treatment of cancer cells that express the monocytic marker CD14.
  • the dopamine receptor antagonist preferentially induces the differentiation of cancer stem cells in the subject relative to hematopoietic or normal stem cells.
  • the cancer stem cells are leukemic cancer stem cells.
  • the subject has AML and the cancer stem cells are AML cancer stem cells.
  • the dopamine receptor antagonists are antagonists for one or more of dopamine receptors (DR) such as DR1, DR2, DR3, DR4, and DR5.
  • DR dopamine receptors
  • the DR antagonist is a multi-receptor antagonist, or is specific for a single dopamine receptor subtype.
  • the DR antagonist is a phenothiazine derivative such as thioridazine, or chlorpromazine.
  • the DR antagonist is selected from the compounds listed in Table 1. A person of skill in the art would readily be able to identify additional dopamine receptor antagonists that are useful for the treatment of cancer as described herein.
  • the methods or uses described herein involve a phenothiazine derivative such as thioridazine or chlorpromazine.
  • a phenothiazine derivative such as thioridazine or chlorpromazine.
  • a person skilled in the art would readily be able to identify additional phenothiazine derivatives that are dopamine receptor antagonists and useful for the treatment of cancer as described herein.
  • the phenothiazine derivatives have a differential toxicity for cancer cells, such as leukemic cells, compared to normal stem cells or hematopoietic stem cells.
  • the dopamine receptor antagonists and/or phenothiazine derivatives are prepared for administration to a subject in need thereof as known in the art.
  • Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003—20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • the cancer cell is a cancer stem cell.
  • the DR antagonist induces differentiation or cell death of a cancer stem cell.
  • the DR antagonist induces cell death of a cancer cell.
  • the cancer cell may be in vivo or in vitro.
  • the cancer cell may be a precancerous cell such as a myelodyplastic or myeloproliferative cell.
  • the cancer cell is a hematological cancer cell.
  • the cancer cell is a leukemic cell, such as a cell from a subject with AML.
  • the DR receptor antagonist is a phenothiazine derivative such as thioridazine or chlorpromazine.
  • the DR antagonist is selected from the compounds listed in Table 1.
  • the Applicants have surprisingly shown that some AML cell lines and primary AML cells exhibit a relative increase in the expression of dopamine receptors compared to normal hematopoietic stem cells. Screening subjects with cancer for the expression of dopamine receptors in cancer cells may therefore serve to identify subjects who would benefit from treatment with dopamine receptor antagonists. Accordingly, in one aspect of the disclosure there is provided a method for identifying a subject with cancer suitable for treatment with dopamine receptor antagonists. In one embodiment, the method comprises determining the expression of one or more dopamine receptors in a sample of cancer cells from a subject. Subjects with cancer cells that express one or more dopamine receptors are thereby identified as suitable for treatment with dopamine receptor antagonists.
  • the expression of one or more dopamine receptors in a sample of cancer cells can be determined by testing the cancer cells for polypeptides or polynucleotides that encode for dopamine receptors as described herein.
  • the method includes obtaining or providing a sample of cancer cells from the subject and/or testing the sample for the expression of one or more dopamine receptors.
  • the cancer is leukemia and the cancer cells are leukemic cells.
  • the cancer is AML.
  • the method includes determining additional markers known to be associated with cancer, hematological malignancies, leukemia or AML or markers associated with specific treatment regimes.
  • cancer cells are also tested for the monocytic marker CD14.
  • the expression of dopamine receptors has been observed in samples of breast cancer and AML and can serve as a biomarker for the severity of disease. As shown in Example 11 and FIGS. 14 g - h , high levels of DR expression correlate with poor prognosis while low levels demonstrate a better prognosis. Accordingly, in one aspect of the disclosure there is provided a method of determining a prognosis for a subject with cancer. In one embodiment, the method comprises determining the expression of one or more biomarkers selected from dopamine receptor (DR) 1, DR2, DR3, DR4, DR5 and CD14 in a sample of cancer cells from the subject and comparing the level of expression of the one or more biomarkers to a control.
  • DR dopamine receptor
  • an increase in the level of expression of the one or more biomarkers relative to the control indicates that the subject has a more severe form of cancer.
  • the methods described herein include providing or obtaining a sample of cancer cells from the subject such as a blood sample containing leukemic cells or a tumour sample.
  • the cancer cells are leukemic cells or breast cancer cells and increased expression of one or more biomarkers compared to the control indicates a more severe form of leukemia or breast cancer.
  • additional biomarkers known to be associated with cancer or severity of disease are also tested and compared to control samples.
  • control that is representative of a particular prognosis in subjects with cancer such that observing a difference or similarity in the level of the one or more of the biomarkers described herein between the test sample with the control provides a corresponding prognosis for the test subject.
  • the control represents subjects diagnosed with AML known to have a particular outcome or prognosis and observing an increase in the level of expression of one or more dopamine receptors relative to the control indicates a worse prognosis for the subject relative to the control.
  • a method for identifying a subject with leukemia comprising providing a sample from a subject and testing the sample for the expression of one or more biomarkers selected from DR1, DR2, DR3, DR4 and DR5.
  • the sample comprises cancer cells such as leukemic cells and/or white blood cells.
  • the method comprises comparing the level of expression of one or more biomarkers to a control. In one embodiment, increased expression of DRs in the sample compared to the control is indicative of cancer.
  • an increased expression of DRs in the subject compared to the control is indicative of leukemia. In one embodiment, increased expression of DRs in the subject compared to the control is indicative AML or monocytic leukemia.
  • the methods described herein may be used in combination with other diagnostic methods for the identification of cancers or leukemia as known to a person of skill in the art.
  • dopamine receptor expression demarcates human cancer stem cells from other cells such as normal hPSCs that express the pluripotent marker SSEA3. Accordingly, in one embodiment, there is provided a method of identifying a cancer stem cell from a population of cells comprising determining whether a cell expresses one or more biomarkers selected from dopamine receptor (DR) 1, DR2, DR3, DR4 and DR5. In one embodiment, expression of DR1, DR2, DR3, DR4 or DR5 is indicative that the cell is a cancer stem cell. Optionally, expression of 2 or more, 3 or more, 4 or more or all 5 DRs is indicative that the cell is a cancer stem cell. In one embodiment, a cell that expresses DR1, DR2, DR3, DR4 and DR5 is identified as a cancer stem cell.
  • DR dopamine receptor
  • the cancer stem cell is identified from a population of cells.
  • the population of cells contains more than one cell type, such as somatic cells, pluripotent stem cells, cancer cells and/or cancer stem cells.
  • the population of cells is a plurality of cells in cell culture, such as tissue culture.
  • the population of cells is from a mammal, such as a primary tissue sample or blood sample.
  • the population of cells is from a mammal with cancer or suspected of having cancer.
  • the population of cells includes stem cells, somatic stem cells and/or pluripotent stem cells as well as one or more cancer stem cells.
  • the population of cells includes cancer cells or pre-cancerous cells such as hematological cancer cells.
  • the population of cells includes monocytic cells.
  • the population of cells includes breast cancer cells.
  • the population of cells is from a tissue sample, such as a tumor sample, that has been dissociated into single cells.
  • the step of determining whether the cell expresses one or biomarkers comprises testing the cell for the expression of polynucleotides or polypeptides that code for DR1, DR2, DR3, DR4 or DR5.
  • methods known in the art such a RT-PCR or reporter genes that detect the expression of polynucleotides, or immunohistochemical methods that detect expression of polypeptides, can be used for determining the expression of a biomarker such as DR1, DR2, DR3, DR4 or DR5.
  • the biomarkers are cell surface biomarkers and the method involves detecting DR1, DR2, DR3, DR4 or DR5 expressed on the surface of the cell.
  • the methods for identifying a cancer stem cell described herein include determining the level of expression of one or more biomarkers selected from dopamine receptor (DR) 1, DR2, DR3, DR4 and DR5 and then comparing the level of expression to a control level.
  • the control represents cells that are not cancer stem cells, such as somatic stem cells, hematopoietic stem cells or cells that express the pluripotency marker SSEA3, and cells that have an increased level of expression of the biomarkers DR1, DR2, DR3, DR4 and/or DR5 compared to the control are identified as cancer stem cells.
  • cells that have an increased amount of expression compared to the control are identified as cancer stem cells (e.g. at least 2 ⁇ , 5 ⁇ , or 10 ⁇ etc.).
  • the method can also comprise: (a) providing a population of cells (b) contacting the population with an agent that specifically binds to one or more biomarkers selected from DR1, DR2, DR3, DR4 and DR5; and (c) selecting cells that specifically bind to the agent of (b) thereby identifying and/or isolated cancer stem cells from a population of cells.
  • the agent is an antibody that selectively binds to a biomarker.
  • the methods described herein can optionally include two or more selection or isolation steps. The methods described herein can also include a negative step selection, e.g., excluding cells that express one or more markers expressed in cells that are not cancer stem cells, or excluding cells that show reduced levels of expression of a particular marker.
  • the present disclosure includes isolating cancer stem cells from a population of cells.
  • cells that are identified as cancer stem cells are isolated from cells that are not cancer stem cells or from other materials in a sample by selecting for or isolating cells that express one or more biomarkers selected from DR1, DR2, DR3, DR4 and DR5.
  • the cancer stem cells are isolated or selected using methods known in the art for sorting cells based on the expression of one or more biomarkers.
  • the step of isolating the cancer stem cells form the population of cells comprises flow cytometry, fluorescence activated cell sorting, panning, affinity column separation, or magnetic selection.
  • cells that express one or more dopamine receptors are isolated using a binding agent that selectively bind to dopamine receptors that is conjugated to a support such the matric in a separation column or magnetic beads.
  • the methods described herein include determining the level of one or more biomarkers in a sample from a subject, such as the level of one or more dopamine receptors.
  • the sample comprises cancer cells or is suspected of comprising cancer cells or pre-cancerous cells.
  • the sample can comprise a blood sample, for example a peripheral blood sample, a fractionated blood sample, a bone marrow sample, a biopsy, a frozen tissue sample, a fresh tissue specimen, a cell sample, and/or a paraffin embedded section.
  • the subject has or is suspected of having AML and the sample comprises mononuclear cells.
  • the sample is processed prior to detecting the biomarker level.
  • a sample may be fractionated (e.g. by centrifugation or using a column for size exclusion or by FACS using a biomarker for monocytes), concentrated or processed, depending on the method of determining the level of biomarker employed.
  • the level of expression of the biomarkers described herein can be determined by methods commonly known to one of skill in the art.
  • 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.
  • expression of the one or more biomarkers is determined by detecting the cell surface expression of DR1, DR2, DR3, DR4 and/or DR5.
  • the methods described herein include detecting a biomarker using immunohistochemistry, such as by using an antibodies specific for the biomarker or another biomarker-specific detection agent. Examples of dopamine receptor antibodies suitable for use in the methods described herein are also listed in Example 7 of the present disclosure.
  • the level of an mRNA encoding for a biomarker is determined by quantitative PCR such as RT-PCR, serial analysis of gene expression (SAGE), use of a microarray, digital molecular barcoding technology or Northern blot.
  • quantitative PCR such as RT-PCR, serial analysis of gene expression (SAGE), use of a microarray, digital molecular barcoding technology or Northern blot.
  • mass spectrometry approaches such as multiple reaction monitoring (MRM) and product-ion monitoring (PIM)
  • PIM product-ion monitoring
  • antibody based methods such as immunoassays such as Western blots and enzyme-linked immunosorbant assay (ELISA).
  • the step of determining the expression of a biomarker such as one or more dopamine receptors as described herein comprises using immunohistochemistry and/or an immunoassay.
  • the immunoassay is an ELISA.
  • the ELISA is a sandwich type ELISA.
  • the term “level” as used herein refers to an amount (e.g. relative amount or concentration) of biomarker that is detectable or measurable in a sample.
  • the level can be a concentration such as ⁇ g/L or a relative amount such as 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 or greater a control level, standard or reference level.
  • a control is a level such as the average or median level in a control sample.
  • the level of biomarker can be, for example, the level of protein, or of an mRNA encoding for the biomarker such as a dopamine receptor.
  • the levels of the two or more biomarkers can be used to generate an expression profile for the subject.
  • the methods described herein include determining a level for two or more biomarkers in the sample, generating an expression profile based on the level of the two or more biomarkers and comparing the expression profile to a control expression profile. A difference or similarity in the test sample expression profile and the control expression profile is then used to provide a prognosis for the test subject, identify the subject as having cancer, or indicate whether the subject is suitable for treatment with a dopamine receptor antagonist.
  • a further aspect of the disclosure includes the use of a dopamine receptor antagonist for the treatment of cancer or a precancerous disorder.
  • a dopamine receptor antagonist for use in the treatment of cancer or a precancerous disorder in one embodiment the cancer is leukemia.
  • the leukemia is acute myeloid leukemia or monocytic leukemia.
  • the dopamine receptor antagonist is a phenothiazine derivative such thioridazine or chlorpromazine.
  • the DR antagonist is selected from the compounds listed in Table 1.
  • Also disclosed herein is the use of a dopamine receptor antagonist for the manufacture of a medicament for the treatment of a cancer and/or a precancerous disorder.
  • a further aspect of the disclosure includes methods of screening compounds for anti-cancer activity comprising identifying compounds that antagonize one or more dopamine receptors.
  • compounds that antagonize dopamine receptors are identified as having anti-cancer activity.
  • the methods include screening compounds to identify those that reduce the proliferation of cancer stem cells relative to normal stem cells such as hematopoietic stem cells as set out in Examples 7 and 8 of the present description.
  • Thioridazine is Cytotoxic to Leukemic Cell Lines
  • HL-60 leukemic cells lines
  • MV4-11 leukemic cell lines
  • OCI-AML3 leukemic cell lines
  • HL-60 was derived from promyelocytic AML
  • MV 4-11 and OCI-AML3 are representative of AML.
  • Each compound was incubated with the cells for 72 h.
  • the control was DMSO (ie the vehicle used for the compound) for 72 h. Each condition had three replicates.
  • Normal HSCs and progenitors were sourced from either mobilized peripheral blood or umbilical cord blood of health patients.
  • Primary AML cells were taken from patients diagnosed with AML. Both normal HSCs and primary AML cells were cultured under standard in vitro methocellulose assay conditions (see http://www.stemcell.com/en/Products/All-Products/MethoCult-H4434-Classic.aspx as well as Clinton Campbell et al.
  • the human stem cell hierarchy is defined by a functional dependence on Mcl-1 for self-renewal capacity. Blood 116 (9) 1433-1442 (Jun. 4, 2010), hereby incorporated by reference) for at least 14 days before the number of colonies were recorded. As shown in FIG.
  • 10 ⁇ M thioridazine has a differential effect on normal HSCs versus AML cells. 10 ⁇ M thioridazine reduced the colony forming potential of normal HSCs from about 100 (CTRL treated with DMSO) to about 66 total colonies ( FIG. 2A ), but had a much more significant effect on AML cells reducing the number of CFU colonies to about 22 blast colonies ( FIG. 2B ) to 1.6 blast colonies.
  • FIG. 3 shows cell pellets of CFU colonies generated from normal HSC and AML treated with thioridazine. At a dose of 10 ⁇ M, pelleted cells are still visible for HSCs, but not for AML cells. Thioridazine therefore selectively targets Blast-CFU Potential of AML cells.
  • Chlorpromazine is Toxic to AML Cell Lines
  • the dopamine receptor antagonist and phenothiazine-related compound chlorpromazine was also investigated for effects on the AML cell lines HL-60, MV4-11 and OCI-AML3. Testing was performed as set out in Example 1. As shown in FIG. 4 , 10 ⁇ M Chlorpromazine is toxic to AML cell lines.
  • the expression of the dopamine receptors DR1, DR2, DR3, DR4 and DR5 were analyzed in AML cell lines HL-60, MV4-11, AML-OCI2 and AML-OCI3), Primary AML cells (AML22101, AML29428, AML22174, AML29560) isolated from AML patients, normal blood mononuclear cells (MNC) (MPB21471 and MPB28137; healthy patient blood) as well as umbilical cord blood primary cells enriched for normal Human Stem Cells or progenitors (CB107, CB108 and CB109) using StemSep® Human Hematopoieitc Progenitor Cell enrichment kit (http://www.stemcell.com/en/Products/All-Products/StemSep-Human-Hematopoietic-Progenitor-Cell-Enrichment-Kit.aspx) and enrichment levels of HSCs/Human Progenitor cells confirmed
  • dopamine receptors are expressed on primary AML, AML cell lines and normal mononuclear blood cells (MNC) but not in blood enriched for normal HSCs (CB(lin ⁇ ).
  • MNC normal mononuclear blood cells
  • CB(lin ⁇ ) normal mononuclear blood cells
  • subjects may be pre-screened for the expression of dopamine receptors in order to identify subjects suitable for AML treatment with DR antagonists.
  • pre-screening of subjects may encompass all five DR subtypes, or specific subtypes or combination of subtypes.
  • a series of dopamine receptor agonists, D 3- antagonists, DR 1 & 5- antagonists and multi-receptor antagonists were tested for cytotoxicity against three AML cell lines HL-60, OCI-AML2 and OCI-AML3. Testing was performed as set out in Example 1.
  • CLOZ at higher concentrations as well as CHL and THIO have a significant effect on cytotoxicity of AML cell lines. Without being limited by theory, the cytotoxic effect may require inhibition of multiple dopamine receptors.
  • THIO, CHL and CLOZ being multireceptor antagonists work to eradicate the AML cell lines while the D 3 and DR 1 & 5 -specific antagonists only reduce cell count to 60%.
  • dopamine receptor subtypes The expression of dopamine receptor subtypes was analyzed in primary AML cells.
  • the expression of the CD14 monocytic marker is correlated with the expression of each DR subtype.
  • thioridazine were also examined on a subpopulation of CD14+ cells in primary AML.
  • Primary AML cells were cultured under control (DMSO vehicle) or 10 uM thioridazine for 72 h and then stained for with antibodies specific to CD14.
  • the number of CD14+ cells in both control and thioridazine treated samples was determined using flow cytometry and the frequency of CD14+ cells was found to be lower in the thioridazine treated sample, suggesting that this compound selectively targets the CD14+ subpopulation in AML cells.
  • 10 ⁇ M thioridazine also reduced the normalized frequency of CD14+ cells in primary AML cells, showing that thioridazine selectively targets CD14+ cells.
  • the AML control group contained a fraction of CD14+ cells. This fraction is reduced with thioridazine treatment and is represented as a reduction in the normalized frequency of the control (100%) versus treated (20%).
  • CSCs cancer stem cells
  • SCs normal stem cells
  • Neoplastic hPSC EOS-GFP Lines Generation of Neoplastic hPSC EOS-GFP Lines.
  • Neoplastic v1H9 or v2H9 hPSC cells were transduced with lentivirus bearing the EOS-C3+ or EOS-S4+ vectors provided by Dr James Ellis (Hotta et al., 2009). After lentiviral transduction cells were selected using Puromycin, and subsequently sorted as single cells into a 96-well plate based on GFP expression using a FASCAria II (Becton-Dickinson).
  • H9 hESC, v1H9, v1H9-Oct4-GFP, v2H9-Oct4-GFP, v1H9-Sox2-GFP and fibroblast-derived iPSCs were cultured as previously described (Chadwick et al., 2003; Werbowetski-Ogilvie et al., 2009).
  • peripheral blood and/or bone marrow was collected at the time of clinical presentation.
  • Healthy hematopoietic cells were obtained from umbilical cord blood samples. All samples were obtained following informed consent according to Research Ethics Board approved protocols at McMaster University and the London Health Sciences Centre. Human breast tumor samples were obtained from reduction mammoplasty surgeries following informed consent according to Research Ethics Board approved protocols at McMaster University.
  • Confluent H9 & fibroblast-derived iPSC were seeded at 10,000 cells per well in MEFCM supplemented with 8 ng/ml bFGF. 24 hours later the cells were treated with compounds at 10 ⁇ M and 0.1% DMSO, 0.1% DMSO ( ⁇ BMP4) or 100 ng/ml of BMP4 and 0.1% DMSO (+BMP4). Fresh MEFCM supplemented with compounds was exchanged daily for 5 days. On day 5, hPSC's were fixed and prepared for automated imaging and plate reader analysis. See supplementary experimental procedures for further details.
  • NOD.Cg-Prkdc scid II2rg tm1Wjl /SzJ adult mice were sub-lethally irradiated with 315 rads 24 hours prior transplantation.
  • 0.8 ⁇ 1.0 ⁇ 10 7 AML MNCs or 1.5 ⁇ 1.8 ⁇ 10 5 CB lin ⁇ hematopoietic cells treated with compound or DMSO-vehicle for 24 h were injected via tail vein (IV).
  • IV tail vein
  • the H9 hESC, v1H9, v1H9-Oct4-GFP, v2H9-Oct4-GFP, v1H9-Sox2-GFP and fibroblast-derived iPSCs were cultured on MatrigelTM-coated (BD Biosciences 353234) plates with mouse embryonic fibroblast-conditioned (MEFCM) media supplemented with 8 ng/ml bFGF (GIBCO 13256-029).
  • MEFCM is composed of KO-DMEM (GIBCO 10829-018), 20% KO-Serum Replacement (GIBCO 10828-028), 1% Non-Essential Amino Acids (GIBCO 11140-050), 1 mM L-Glutamine, 0.1 mM ⁇ -mercaptoethanol (Sigma Aldrich M7522).
  • Cell lines were passaged every 7 days using 100 Units/mL of Collagenase IV (GIBCO 17104-019) for 2-3 minutes. Cell seeding density, assay duration and DMSO vehicle concentration in 96 wells were optimized for v1H9-Oct4-GFP cells and normal H9 hPSC.
  • v1H9-Oct4-GFP For v1H9-Oct4-GFP, an optimum initial seeding density of 5,000 cells per well for 72 h of treatment was selected based on maximal levels of GFP and z′ discrimination between ⁇ BMP4 controls. For normal hPSC, an optimal seeding density of 10,000 cells per well was selected based on maximal z′-prime discrimination between ⁇ BMP4 controls.
  • Mononuclear cells were prepared using Ficoll-Paque Premium (GE Healthcare). For hematopoietic cells, lineage depletion was performed using EasySep (StemCell Technologies) following manufacturer's recommendations.
  • AML cell lines namely, OCI-AML2 (M4), OCI-AML3 (M4), HL-60 (M2) and MV-4-11(M5) were cultured in RPMI (Gibco) supplemented with 5% heated-inactivated FBS (HyClone).
  • RPMI Gibco
  • FBS heated-inactivated FBS
  • serum-free conditions were employed instead due to the prevalence of dopamine in FBS (Little et al., 2002).
  • AML patient blasts were cultured in IMDM supplemented with 5% heated inactivated FBS (HyClone), 5 ng/mL IL3 (R&D systems), 5 ⁇ 10 ⁇ 5 M ⁇ -mercaptoethanol (Sigma) and BIT (StemCell Technologies).
  • HSC media contained IMDM supplemented with 1% BSA (Sigma), 100 ng/mL SCF (R&D systems), 100 ng/mL Flt-3L (R&D systems) and 20 ng/mL TPO (R&D systems).
  • Patient HSPC and AML samples were treated with compound or DMSO-vehicle (0.1%) for 24 h prior to CFU plating or xenotransplantation studies.
  • Antibodies used for immunocytochemistry were the following: Oct3/4 (BD Trunsduction Laboratories, cat#611203), Sox2 (R&D, cat#AF2018).
  • Oct3/4 BD Trunsduction Laboratories, cat#611203
  • Sox2 R&D, cat#AF2018
  • FITC anti-CD33, PE anti-CD13, FITC anti-CD41a, FITC anti-HLA DR, and PE anti-CD19 antibodies were obtained from BD Pharmingen.
  • PE anti-CD14, PE anti-CD15 and PE anti-GlyA were acquired from Immunotech Beckman Coulter.
  • PE anti-SSEA3 (BD Biosciences) and PE- or AlexaFluor488 anti-Oct4 (BD Biosciences).
  • Rabbit anti-human dopamine receptor antibodies; DRD1 (Cat#324390), DRD2 (Cat#324393), DRD3 (Cat#324402), DRD4 (Cat#324405) and DRD5 (Cat#324408) were sourced 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 IgG sourced 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 sourced from BD Pharmingen.
  • Image analysis was performed using custom scripts in Acapella software (Perkin Elmer). Nuclear objects were segmented from the Hoechst signal. For neoplastic cell lines, object intensity analysis was performed on GFP positive cells only. For normal cell lines, the fraction of Alexa-Fluor-647-positive cells was quantified. Images and well-level data were stored and analysed in a Columbus Database (Perkin Elmer) and further data analysis, compounds registration and hit identification in ActivityBase (IDBS).
  • IDBS ActivityBase
  • Each reaction sample was dispensed into loading wells on the array card and centrifuged twice at 336 ⁇ g for 1 min each time, sealed, and placed in the thermal cycler. The following cycling conditions were used for all array card applications: 45° C. for 10 min, 94° C. for 10 min, and 40 cycles of 94° C. for 30 s followed by 60° C. for min.
  • Array data were normalized to 18S RNA and GAPDH and comparisons were performed using data analysis 2.0 software (Applied Biosystems).
  • AML patient or CB lin ⁇ cells were cultured 24 hours in the presence of compound or DMSO-vehicle (0.1%) control.
  • AML cells were plated at 50 000 cells/mL in Methocult GF H4434 (Stem Cell Technologies).
  • CB lin ⁇ cells were plated at 1000 cells/mL in Methocult GF H4434 (Stem Cell Technologies). Colonies were scored after 14 days of culture using standard morphological criteria.
  • the trypsin-collagenase solution consisted of RPMI 1640 (Gibco #11875093), 2% penicillin/streptomycin (Invitrogen #15140163), 1% Fungizone Antimycotic (Invitrogen #15290018), 2% FBS, 3 mg/mL Collagenase A (Roche Diagnostics #11088793001), and 0.1% of 2.5% trypsin (Gibco #15090).
  • An equal volume of RPMI 1640 with 2% FBS was then added to the tissue suspension. The tissue suspension was filtered through a 40 ⁇ m nylon strainer (Falcon #352340).
  • 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 sourced from EMD Chemical and Anti-rabbit Alexa-Fluor-488 (Molecular Probes) was used as the secondary antibody along with APC anti-CD44 and PE-CD24, both sourced from BD Pharmingen.
  • hPSC human pluripotent stem cell
  • Oct4 and Sox2 provide a reliable indicator of loss of self-renewing pluripotent state and differentiation induction of normal and neoplastic hPSCs.
  • GFP-reporter lines were generated by transduction of neoplastic hPSCs with the EOS-GFP reporter (v1H9-Oct4-GFP and v1H9-Sox2-GFP, respectively) (Hotta et al., 2009). GFP intensity was observed to be correlated with Oct4 and Sox2 expression in treatments that favored self-renewal stability and conditions that induce differentiation with the addition of BMP4.
  • HTS fluorometric highthroughput screening
  • HCS high content screening
  • Thioridazine Selectively Induces Neoplastic hPSC Differentiation and Reduces Human AML Blasts without Affecting Normal Hematopoietic Stem/Progenitor Cells
  • the ratio of normalized percentage of Oct4+ cells between normal and neoplastic hPSCs in response to these compounds was determined. For example, a ratio of 1 suggests equivalent differentiation whereas a ratio>1 defines relatively more differentiation in neoplastic hPSCs vs. normal hPSCs. Only thioridazine, at both 1 ⁇ M and 10 ⁇ M, had a significant impact on inducing differentiation of neoplastic hPSCs over normal hPSCs ( FIG. 10 c ). Rapid accumulation of the cell stress marker p53 ( FIG. 10 d ) and its transcriptional target p21 ( FIG.
  • HSPCs human hematopoietic stem-progenitor cells
  • LSCs Leukemic Stem Cells
  • Lineage-depleted umbilical cord blood (CB lin ⁇ ) is highly enriched for HSPCs and is a reliable source of normal somatic SCs capable of self-renewal and multilineage differentiation to all blood lineages.
  • Acute myeloid leukemia (AML) is a hematological neoplasia characterized by a block in mature myeloid differentiation that is sustained by a self-renewing LSC (Bonnet and Dick, 1997; Lapidot et al., 1994).
  • progenitor assays in methylcellulose were conducted with HSPCs and 5 AML patient samples; each treated with thioridazine, mefloquine, or salinomycin in order to assess each compound's impact on in vitro clonogenic and multilineage hematopoietic differentiation.
  • Representative cell pellets of the total colony-forming units (CFUs) generated from HSPCs ( FIG. 10 g ) and AML ( FIG. 10 h ) treated with each compound are shown.
  • Thioridazine treatment resulted in a reduction in AML proliferation/clonogenic capacity while retaining HSPC multilineage differentiation ( FIG. 11 c ).
  • the most desired outcome of compounds identified toward clinical use would entail preferential elimination of AML-blast CFU generation while preserving normal HSPC progenitor capacity.
  • the ratio between total CFUs generated from HSPC vs. AML-blasts to reveal the highest selectivity for targeting AML was calculated ( FIG. 10 k ).
  • a ratio of 1 suggests equivalent normal to neoplastic progenitor potential whereas a ratio>1 defines a compound that selectively reduces AML-blast CFU potential.
  • Salinomycin (1 ⁇ M), mefloquine (10 ⁇ M), and thioridazine (10 ⁇ M) doses yielded the highest ratio values for each compound ( FIG. 10 k ) and were thus selected for in vivo evaluation.
  • Thioridazine 10 ⁇ M demonstrated the highest ratio of all compounds, but most importantly was the only compound to show a significantly lower AML-blast CFU potential relative to normal HSPC CFU potential ( FIG. 10 k ).
  • the frequency of CD11b a marker of granulocytic maturation, in patient AML cells was assayed in response to thioridazine treatment ( FIG. 10I ).
  • a marked increase in the frequency of granulocytic AML-blast cells was observed with treatment duration ( FIG.
  • mefloquine (10 ⁇ M) treatment displayed a slight, yet insignificant, reduction in HSC capacity relative to controls ( FIG. 12 a ).
  • mefloquine proved ineffective in reducing AML LSC capacity and was thus discontinued from further evaluation due to absence of selective effects ( FIG. 12 c ).
  • thioridazine treatment did not affect HSC self-renewal as compared to control-treated samples ( FIG. 13 f ). However, in sharp contrast to salinomycin and mefloquine, thioridazine treatment was able to significantly reduce leukemic disease-initiating AML LSCs ( FIGS. 12 c - d ; FIG. 13 c ; FIG. 13 e ).
  • Calculating the ratio of HSPC normal hemaotopoietic regeneration (% hCD45+) to AML leukemogenesis (% CD33+hCD45+ blasts) revealed that thioridazine significantly reduced LSC function while preserving normal HSC capacity ( FIG. 12 e ).
  • thioridazine selectively targets somatic CSCs whilst having no effect on normal SC properties in vivo.
  • thioridazine was identified through the use of a novel differential screening platform using normal and neoplastic hPSCs in vitro, the functional effects of thioridazine provide an example of the predictive value of using human PSCs to understand somatic CSCs.
  • Thioridazine is known to act through the dopamine receptors (DR 1-5) (Beaulieu and Gainetdinov, 2011; Seeman and Lee, 1975). To assess whether the mechanism of thioridazine action to selectively interfere with human CSCs vs. normal SCs is via DR antagonism, DR cell surface expression was analyzed. To date, five DRs have been identified and divided into D 1 -family (D1 and D5) and D 2 -family (D2, D3, and D4) receptors (Sibley and Monsma, 1992). Normal hPSCs expressing the pluripotent marker SSEA3 were devoid of DR expression ( FIG. 14 a and FIG. 15 a - b ).
  • neoplastic hPSCs expressed all five DRs ( FIG. 14 b ).
  • the observed differential expression of DRs and the selective inhibition of thioridazine for neoplastic hPSCs suggest that inhibition of DR signaling may play a role in selective targeting of human CSCs vs. normal SCs.
  • DR antagonism could account for the loss of LSC function following thioridazine treatment.
  • Expression of DR1-5 was analyzed in HSPCs ( FIG. 14 c ) and human hematopoietic mononuclear cells from normal CB ( FIGS. 15 c - f ) and AML patient samples ( FIG. 14 d and FIG. 15 g ).
  • DRs were not observed in the primitive HSCs or progenitor populations of CB (identified as the CD34+38 ⁇ or CD34+38+ fractions, respectively (Bhatia et al., 1997)) ( FIG.
  • CD34+ cells do not correlate with LSC capacity in human AML (Taussig et al., 2008) and have recently been identified in numerous subfractions devoid of CD34 or CD38 (Eppert et al., 2011). Observations of differential DR expression in normal and AML human hematopoietic samples strongly suggest the human AML LSCs are heterogeneous and drug targeting should be based on molecular pathways instead of surrogate phenotype predications.
  • somatic CSCs have recently been identified and validated in human breast tumors and have a CD44+CD24 ⁇ /lo phenotype (Al-Hajj et al., 2003).
  • ER ⁇ estrogen receptor
  • PR ⁇ progesterone receptor
  • HER2 ⁇ human epidermal receptor 2
  • AML-OCI2 AML-OCI3
  • AML-OCI3 AML-OCI3
  • DR D2-family and D1-family exert opposing actions on intracellular signaling leading to differential biological effects (Self et al., 1996).
  • Treatment with a DR D1-family agonist, SKF38393, resulted in a significant reduction in AML cell number confirming that D2-family signaling is necessary for AML cell survival ( FIG. 16 d ).

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