EP2945646A1 - Inhibiteurs d'un récepteur des strogènes - Google Patents

Inhibiteurs d'un récepteur des strogènes

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Publication number
EP2945646A1
EP2945646A1 EP14740515.3A EP14740515A EP2945646A1 EP 2945646 A1 EP2945646 A1 EP 2945646A1 EP 14740515 A EP14740515 A EP 14740515A EP 2945646 A1 EP2945646 A1 EP 2945646A1
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EP
European Patent Office
Prior art keywords
era
cells
bhpi
upr
positive
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EP14740515.3A
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German (de)
English (en)
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EP2945646A4 (fr
Inventor
David J. Shapiro
Neal D. Andruska
Mathew M. Cherian
Lily MAHAPATRA
Mao CHENGJIAN
William HELFERICH
Xujuan YANG
Xiaobin ZHENG
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Publication of EP2945646A4 publication Critical patent/EP2945646A4/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4535—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom, e.g. pizotifen
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the hormone estrogen binds to a protein called the estrogen receptor (ER).
  • ER estrogen receptor
  • the complex of estrogen and the estrogen receptor bind to specific DNA sequences in the cell nucleus causing or blocking the copying of the nearby DNA and stimulating or decreasing the production of the RNA blueprints that specify the production of proteins that stimulate cell division and migration and cell death.
  • the estrogen-ERa complex plays a role in the growth and spread (metastases) of many cancers and in endometriosis. Although the roles of estrogens and estrogen receptor are best understood in breast cancer, estrogens and estrogen receptor are known to play important roles in ovarian, uterine/endometrial, cervical, liver and lung cancer.
  • ERa In several other cancers, including, colon pancreatic and brain, ERa is often present, but a direct role of estrogens has not been demonstrated.
  • the important role of estrogens in breast cancer is illustrated by the widespread therapeutic use of aromatase inhibitors that block estrogen production and the selective estrogen receptor modulators tamoxifen and Faslodex/fulvestrant/ICI 182,780 (ICI: ICI 182,780 (Imperial Chemical Industries 182,780; also known as Faslodex and fulvestrant) that work by competing with estrogens for binding to estrogen receptor a (ERa).
  • ICI ICI 182,780 (Imperial Chemical Industries 182,780; also known as Faslodex and fulvestrant) that work by competing with estrogens for binding to estrogen receptor a (ERa).
  • endometrial cells which normally line the uterus, detach from the uterus, attach at sites outside the uterus, including the ovaries, pelvic lining, bowel and rectum and proliferate in response to estrogen binding to ERa, leading to pain and infertility. 5- 10% of premenopausal women in the United States have symptom of endometriosis. Current therapies for endometriosis aim at reducing estrogen levels.
  • E2-ERa can increase or decrease expression of a gene.
  • E2 a potent estrogen
  • ERa dimerizes and binds to DNA sequences called estrogen response elements (EREs) and closely related sequences.
  • E2-ERa can also bind to ERE half sites near SP1 and AP1 sites and be brought to DNA by tethering through other proteins bound at SP1 and AP1 sites.
  • E2-ERa On DNA, E2-ERa exhibits a conformation that enables the recruitment of coactivators.
  • the bound coactivators help assemble a multi-protein complex that facilitates both chromatin remodeling and formation of an active transcription complex, (ii)
  • the E2-ERa complex can also rapidly activate several plasma membrane-associated protein kinase-based signaling pathways.
  • E2- ERa was not known to act at the endoplasmic reticulum to induce efflux of calcium from the lumen of the endoplasmic reticulum into the cytosol and activate the unfolded protein response.
  • the main sensor system for response to cell stress is the endoplasmic reticulum sensor system, the unfolded protein response (UPR).
  • the UPR is activated in response to diverse cell stresses including accumulation of unfolded protein, altered redox potential, metabolic stress, and some drugs.
  • the UPR consists of three main branches that together balance the synthesis of new proteins with the availability of chaperones and other proteins to help fold and transport proteins within cells. Moderate and transient activation of the UPR is protective, while extensive and sustained UPR activation induces cell death.
  • the transmembrane kinase PERK protein kinase RNA-like endoplasmic reticulum kinase
  • autophosphorylation protein kinase RNA-like endoplasmic reticulum kinase
  • P-PERK phosphorylates eukaryotic initiation factor 2a (elF2a), resulting in inhibition of protein synthesis.
  • the other arms of the UPR initiate with activation of the transcription factor ATF6a (activating transcription factor 6 a), leading to increased protein folding capacity and activation of the splicing factor IRE1 a (inositol-requiring protein 1 a), which alternatively splices the transcription factor XBP1 , resulting in production of active spliced (sp)-XBP1 and increased protein folding capacity.
  • Activation of the UPR is usually transient, and the UPR is turned off by production of specific proteins that reverse activation of the PERK arm of the UPR and dephosphorylate elF2a, and by production of chaperone proteins, such as BiP/GRP78 (binding immunoglobulin protein; also known as 78 kiloDalton glucose-regulated protein).
  • chaperone proteins such as BiP/GRP78 (binding immunoglobulin protein; also known as 78 kiloDalton glucose-regulated protein).
  • Endoplasmic reticulum contains a high calcium concentration compared to the cytosol. Release of this Ca 2+ from the lumen into the cytosol activates the UPR. The increased cytosol Ca 2+ is a
  • E2-ERa was not known to directly activate the UPR. Until now, hyperactivation of the UPR through use of an ERa inhibitor had not been described or proposed as a drug strategy. [019] AMPK and Protein Synthesis
  • AMPK adenosine monophosphate kinase
  • Phosphorylation activates AMPK.
  • Activated AMPK is a protein kinase that phosphorylates diverse targets resulting in inhibition of pathways that use energy and stimulation of pathways that produce energy. Cell proliferation is one important process that uses energy.
  • eEF2 protein eukaryotic elongation factor 2
  • eEF2 kinase is the only protein known to be phosphorylated by eEF2 kinase.
  • eEF2 kinase is the only protein known to be phosphorylated by eEF2 kinase.
  • the present disclosure provides a method for the killing of an ERa-containing cell comprising: exposing the cell to an effect amount of BHPI, a derivative thereof, or a pharmaceutically acceptable salt thereof.
  • the present disclosure provides, a method of inhibiting growth of an ERa-containing cell comprising contacting said cell with an effective amount of BHPI, a derivative thereof, or a pharmaceutically acceptable salt thereof.
  • the cell is a cancer cell. In another embodiment, the cell is a cancer cell.
  • the cancer cell is a human cancer cell. In another embodiment, the cancer cell is in a human patient. In another embodiment, the cancer cell is one or more of ovarian, uterine/endometrial, cervical, lung and liver cancer.
  • the present disclosure provides a method of treating cancer comprising administering to a patient in need thereof an effective amount of BHPI, derivative thereof, or pharmaceutically acceptable salt thereof.
  • the patient in need of treatment is a human patient.
  • the cancer is one or more of ovarian, uterine/endometrial, cervical, lung and liver cancer.
  • the disclosure provides a method for the killing of an ERa-containing cell comprising: exposing the cell to an effect amount of the compound of the formula:
  • X can be hydrogen, alkyl, halogen, or CF3, -CHF2, -CCI3, -CHCI2, -CBr3, -CHBr2; Y ean be hydrogen or hydroxyl; and Z can be hydrogen, alkyl, halogen, or -CF3, -CHF2, - CCI3, -CHCI2, -CBr3, -CHBr2.
  • the halogen can be one or more of fluorine, bromine, or chlorine.
  • the alkyl can be methyl.
  • the disclosure provides a method of inhibiting growth of an ERa-containing cell comprising contacting said cell with an effective amount the compound of the formula of Structure A or a pharmaceutically
  • X can be hydrogen, alkyl, halogen, or CF3, -CHF2, -CCI3, -CHCI2, -CBr3, -CHBr2; Y ean be hydrogen or hydroxyl; and Z can be hydrogen, alkyl, halogen, or -CF3, -CHF2, -CCI3, -CHCI2, - CBr3, -CHBr2.
  • the halogen can be one or more of fluorine, bromine, or chlorine.
  • the alkyl can be methyl.
  • the disclosure relates at least in part to certain inhibitors and methods.
  • the inhibitors are small molecule inhibitors that inhibit growth of cells.
  • the cells are cancer cells.
  • the inhibition is by targeting a novel estrogen receptor-dependent pathway.
  • the present disclosure provides a composition comprising any feature described, either individually or in combination with any feature, in any configuration.
  • the disclosure provides a pharmaceutical formulation comprising a composition of the disclosure.
  • the disclosure provides a pharmaceutical formulation of a compound described herein.
  • the disclosure provides a method of synthesizing a composition of the disclosure or a pharmaceutical formulation thereof.
  • a pharmaceutical formulation comprises one or more excipients, carriers, and/or other components as would be understood in the art is provided.
  • the components meet the standards of the National Formulary ("NF"), United States Pharmacopoeia (“USP”), or Handbook of Pharmaceutical
  • an effective amount of a composition of the disclosure can be a therapeutically effective amount.
  • Salts include any salts derived from the acids of the formulas herein which acceptable for use in human or veterinary applications.
  • esters refers to hydrolysable esters of compounds including diphosphonate compounds of the formulas herein. Salts and esters of the compounds of the formulas disclosed herein can include those which have the same therapeutic or pharmaceutical (human or veterinary) general properties as the compounds of the formulas herein.
  • a composition of the disclosure is a compound or salt or ester thereof suitable for pharmaceutical formulations.
  • Prodrugs of the compounds of the disclosure are useful in embodiments including compositions and methods. Any compound that will be converted in vivo to provide a biologically, pharmaceutically or therapeutically active form of a compound of the disclosure is a prodrug.
  • a prodrug such as a pharmaceutically acceptable prodrug can represent prodrugs of the compounds of the disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use.
  • Prodrugs of the disclosure can be rapidly transformed in vivo to a parent compound of a compound described herein, for example, by hydrolysis in blood or by other cell, tissue, organ, or system processes.
  • the disclosure contemplates pharmaceutically active compounds either chemically synthesized or formed by in vivo
  • the disclosure provides a method for inhibiting growth of a cell comprising any method described, in any order, using any modality.
  • composition comprising any feature described, either individually or in combination with any feature, in any configuration.
  • Figure 1 is a schematic representation of the scheme for high throughput screening and characterization of "hits”.
  • Figure 2 shows the structure and chemical name of the ERa inhibitor, BHPI (3,3,bis(4-hydroxyphenyl)-7-methyl-1 ,3,dihydro-2H-indol-2-one).
  • Figure 3 shows the results of a dose-response study of BHPI inhibition of the ERE-luciferase reporter gene.
  • Figure 3A shows the results of a dose-response study of the effect of estrogen on expression of an estrogen response element-luciferase reporter.
  • Figure 3B shows the results of a dose-response study of the effect of BHPI on expression of an estrogen response element-luciferase reporter and an androgen response element-luciferase reporter.
  • Figure 4 shows the effect of BHPI on expression of an estrogen- regulated gene in the presence of low and high concentrations of estrogen.
  • Figure 5 shows the structures of BHPI (Figure 5A) and of an inactive control compound (Figure 5B).
  • Figure 6 shows studies of BHPI interaction with ERa.
  • Figure 6A shows the effect of BHPI and a control compound on the fluorescence emission spectrum of full-length ERa.
  • Figure 6B shows the effect of BHPI on protease sensitivity of ERa ligand binding domain (LBD) using proteinase K analyzed by SDS polyacrylamide gel electrophoresis.
  • Figure 6C shows the effect of BHPI on protease sensitivity of ERa ligand binding domain (LBD) using chymotrypsin analyzed by SDS polyacrylamide gel electrophoresis.
  • Figure 7 shows qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of mRNAs in several cell lines.
  • Figure 7A-1 is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of pS2 mRNA in MCF-7 human breast cancer cells.
  • Figure 7A-2 is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of GREB-1 mRNA in MCF-7 cells.
  • Figure 7A-3 is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of SDF-1 mRNA in MCF-7 cells.
  • Figure 7B-1 is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of pS2 mRNA in T47D human breast cancer cells.
  • Figure 7B-2 is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of GREB-1 mRNA in T47D cells.
  • Figure 7B-3 is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of SDF-1 mRNA in T47D cells.
  • Figure 7C-1 is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of pS2 mRNA in BG-1 human ovarian cancer cells.
  • Figure 7C-2 is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of GREB-1 mRNA in BG-1 cells.
  • Figure 7C-3 is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of SDF-1 mRNA in BG-1 cells.
  • Figure 8 is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated down-regulation of an mRNA.
  • Figure 9A is a Western blot analysis of the effect of BHPI on ERa levels.
  • Figure 9B is a Western blot analysis of the effect of BHPI on ERa subcellular localization.
  • Figure 10A is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of pS2 mRNA in MCF-7 cells.
  • Figure 10B is a qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of GREB-1 mRNA s in MCF-7 cells.
  • Figure 10C is a chromatin immunoprecipitation (ChIP) study of the effect of BHPI on recruitment of E2-ERa and RNA polymerase to the estrogen regulated pS2 gene.
  • Figure 10D is a chromatin immunoprecipitation (ChIP) study of the effect of BHPI on recruitment of E2-ERa and RNA polymerase to estrogen regulated GREB-1 genes.
  • Figure 1 1 is a qRT-PCR analysis of the effect of increased ERa expression on BHPI inhibition of E2-ERa induction of an mRNA.
  • Figure 12 shows dose-response studies of the effect of BHPI on proliferation of ERa-positive and ERa-negative cancer cells.
  • the cell lines used were:
  • Figure 12A-1 - ERa-positive MCF-7 human breast cancer cells [068] Figure 12A-1 - ERa-positive MCF-7 human breast cancer cells.
  • Figure 12B-1 - ERa-positive BG-1 human ovarian cancer cells [070] Figure 12B-1 - ERa-positive BG-1 human ovarian cancer cells.
  • Figure 12B-2 - ERa-negative ES2 human ovarian cancer cells.
  • Figure 12C-2 - ERa-negative HeLa human cervical cancer cells.
  • Figure 12D-1 - ERa-positive PC-3 human prostate cancer cells [074] Figure 12D-1 - ERa-positive PC-3 human prostate cancer cells.
  • Figure 12D-2 - ERa-negative DU145 human prostate cancer cells.
  • Figure 13 shows MTS assays analyzing effects of different
  • BHPI concentrations of BHPI on proliferation of ERa-positive and ERa-negative cancer cells, showing that BHPI inhibits proliferation in diverse ERa-positive cancer cell lines and has no effect on cell growth in ERa-negative cell lines.
  • the cell lines used were:
  • Figure 1 3A - ERa-positive MCF-7 human breast cancer cells.
  • Figure 1 3B - ERa-positive T47D human breast cancer cells.
  • Figure 1 3C - ERa-positive kBluc-T47D human breast cancer cells.
  • Figure 1 3D - ERa-positive HCC1 500 human breast cancer cells.
  • Figure 1 3F - - ERa-positive BT-474 human breast cancer cells.
  • Figure 1 3G - ERa-positive MCF1 OAERINQ human breast cancer cells.
  • Figure 1 3H - ERa-positive MCF7ERaHA human breast cancer cells.
  • Figure 1 3K - ERa-positive BG-1 human ovarian cancer cells.
  • Figure 1 3L - - ERa-positive OVCAR-3 human ovarian cancer cells.
  • Figure 1 3M - ERa-positive CAOV-3 human ovarian cancer cells.
  • Figure 1 3N - ERa-negative ES2 human ovarian cancer cells.
  • Figure 1 30 - ERa-negative IGROVE-1 human ovarian cancer cells.
  • Figure 1 3P - ERa-positive ECC-1 human endometrial cancer cells.
  • Figure 1 3R - ERa-negative HeLa human cervical cancer cells.
  • Figure 1 3S - ERa-positive PC-3 human prostate cancer cells.
  • Figure 1 3T - - ERa-negative DU 145 human prostate cancer cells.
  • Figure 1 3U - ERa-negative 201 T human lung cancer cells.
  • Figure 1 3V - ERa-negative 273T human lung cancer cells.
  • Figure 1 3Y - ERa-negative HepG2 human hepatoma (liver) cancer cells.
  • Figure 1 3Z - - ERa-negative nonmalignant MEF Mouse embryo fibroblasts.
  • Figure 14A contains the structure of the chemical scaffolding of BHPI and related chemical structures.
  • Figure 14B is a table listing preferred substitutions for compounds with the chemical structure shown in Figure 14A.
  • Figure 14C is a dose-response study showing the effect of BHPI on proliferation of ERa-positive T47D, human breast cancer cells.
  • Figures 14D-L show dose-response studies comparing the effect of each of 9 compounds structurally related to BHPI on proliferation of ERa-positive T47D, human breast cancer cells.
  • Figure 15A shows the effect of BHPI and antiestrogens on EGF- stimulated cell proliferation of T47D human breast cancer cells.
  • Figure 15B shows the effect of BHPI and antiestrogens on EGF- stimulated cell proliferation of BG-1 human ovarian cancer cells.
  • Figure 16 shows dose-response studies of the effect of BHPI on proliferation of ERa positive cancer cell lines resistant to current therapies.
  • the cell lines used were:
  • Figure 17 shows the effect of BHPI on anchorage-independent growth of ERa positive human cancer cells.
  • Figure 17A is a photomicrograph of anchorage-independent growth of ERa positive human cancer cells treated with DMSO vehicle.
  • Figure 17B is a photomicrograph of anchorage -independent growth of ERa positive human cancer cells treated with E2.
  • Figure 17C is a photomicrograph of anchorage-independent growth of ERa positive human cancer cells treated with E2 and BHPI.
  • Figure 17D shows the quantitation of colonies formed after treatment with vehicle, E2, and E2 and BHPI.
  • Figure 18 shows the effect of BHPI on tumor size in a mouse xenograft model of estrogen-dependent cancer.
  • Figure 19 shows the effect of BHPI on human breast tumors in a mouse xenograft model showing tumor size (Figure 19A); tumor weight (Figure 19B); mouse body weight (Figure 19C); and mouse food intake (Figure 19D).
  • Figure 20 is a Western blot analysis showing levels of ERa levels in different cell lines and the effect of BHPI incorporation of 35 S-methionine into protein in those cell lines.
  • Figure 21 A shows the effect of BHPI on incorporation of S-methionine into protein in ERa-positive and ERa-negative cells.
  • Figure 21 B is a comparison of the effects of BHPI and antiestrogens on incorporation of 35 S-methionine into protein in ERa-positive and ERa-negative cells.
  • Figure 22A shows the effect of BHPI on incorporation of 35 S-methionine into protein after knockdown of ERa.
  • Figure 22B shows the effect of BHPI on incorporation of 35 S-methionine into protein after degradation of ERa with the antiestrogen ICI.
  • Figure 22C is a Western blot of ERa in cells treated with BHPI and the antiestrogen ICI.
  • Figure 23A is a Western blot analysis showing levels of ERa in cells overexpressing ERa.
  • Figure 23B is a dose-response study of the effect of increasing levels of ERa on BHPI inhibition of the incorporation of 35 S-methionine into protein.
  • Figure 24 is a comparison of the effect of BHPI and UPR activators on protein synthesis measured by incorporation of 35 S-methionine into protein.
  • Figure 25 shows the effect of BHPI and the UPR activator thapsigargin on intracellular calcium measured using the calcium sensing dye Fluo-4.
  • Figure 25A is a photomicrograph of the effect of a low concentration of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium.
  • Figure 25B-1 is a photomicrograph of the effect of a high concentration of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium
  • Figure 25B-2 is a graphical representation of the effect of a high concentration of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium.
  • Figure 25C-1 is a photomicrograph of the effect of the UPR activator thapsigargin (THG) on intracellular calcium in MCF-7 cells.
  • Figure 25C-2 is a graphical representation of the effect of the UPR activator thapsigargin (THG) on intracellular calcium in MCF-7 cells.
  • Figure 26 is a comparison of the effect of BHPI on intracellular calcium levels in ERa-positive and ERa-negative cancer cells.
  • Figure 26A is a photomicrograph of the effect of a high concentration of BHPI or thapsigargin (THG) on intracellular calcium in BG-1 cells with and without extracellular calcium.
  • Figure 26B-1 is a photomicrograph of the effect of a high concentration of BHPI or thapsigargin (THG) on intracellular calcium in HeLa cells without extracellular calcium.
  • Figure 26B-2 is a graphical representation of the effect of the BHPI and thapsigargin on intracellular calcium in HeLa cells.
  • Figure 27A shows the effect of inhibitors of calcium channel opening on intracellular calcium levels after BHPI treatment.
  • Figure 27B shows the effect of inhibitors of calcium channel opening on protein synthesis measured by incorporation of 35 S-methionine into protein.
  • Figure 28 is a model for activation of the three arms of the UPR.
  • Figure 29A is a Western blot analysis showing the effect of BHPI on phosphorylation and levels of PERK and elF2a.
  • Figure 29B-1 is a Western blot analysis showing the effect of RNAi knockdown of PERK on phosphorylation and level of elF2a.
  • Figure 29B-2 shows the effect of RNAi knockdown of PERK on protein synthesis measured by incorporation of 35 S-methionine into protein.
  • Figure 29C is a qRT-PCR analysis of the effect of RNAi knockdown on PERK mRNA levels.
  • Figure 29D is a Western blot analysis showing the effect of RNAi knockdown on PERK on PERK protein levels.
  • Figure 30A shows incorporation of 35 S-methionine into protein as a function of time after addition of BHPI.
  • Figure 30B contains Western blots of the effect of BHPI on
  • the cell lines used were MCF-7 cells in Figure 30B-1 ; BG-1 cells in Figure 30B-2; T47D cells in Figure 30B-3; and MCF-7 cells in Figure 30B-4.
  • Figure 30C is a Western blot analysis of the effect of BHPI on phosphorylation and level of elF2a in ERa-negative cancer cells.
  • Figure 30D is a qRT-PCR analysis of mRNA levels of UPR-related mRNAs in MCF-7 ERa-positive cancer cells treated with BHPI.
  • Figure 30E is a qRT-PCR analysis of mRNA levels of UPR-related mRNAs in BG-1 ERa-positive cancer cells treated with BHPI.
  • Figure 31 is a qRT-PCR analysis of spliced and unspliced UPR-related mRNAs in ERa-positive cancer cells treated with BHPI.
  • Figure 31 A is a qRT-PCR analysis of unspliced XBP-1 mRNA in ERa- positive cancer cells treated with BHPI and no estrogen.
  • Figure 31 B is a qRT-PCR analysis PCR of spliced XBP-1 mRNA in ERa-positive cancer cells treated with BHPI and no estrogen.
  • Figure 31 C is a qRT-PCR analysis of unspliced XBP-1 mRNA in ERa- positive cancer cells treated with BHPI with and without estrogen.
  • Figure 31 D is a qRT-PCR analysis of spliced XBP-1 mRNA in ERa- positive cancer cells treated with BHPI with and without estrogen.
  • Figure 32 is a Western blot analysis of the level of full length and spliced ATF6a in ERa-positive cancer cells treated with BHPI using MCF-7 cells in Figure 32A and T47D cells in Figure 32B.
  • Figure 33A is a Western blot analysis showing the effect of BHPI on phosphorylated and unphosphorylated eEF2.
  • Figure 33B is a Western blot analysis showing the effect of BHPI on phosphorylated and unphosphorylated eEF2K.
  • Figure 33C is a Western blot analysis showing the effect of BHPI on phosphorylated and unphosphorylated AMPK.
  • Figure 33D-1 is a qRT-PCR analysis of p58 mRNA levels in BHPI- treated cells.
  • Figure 33D-2 is a Western blot analysis showing the effect of BHPI on levels of BiP and p58 IPK .
  • Figure 34 is a Western blot analysis showing the effect of BHPI on phosphorylated and unphosphorylated eEF2 in T47D ERa-positive cancer cells in Figure 34A and in HeLa ERa-negative cancer cells in Figure 34B.
  • Figure 35A is a Western blot analysis showing the effect of THG (thapsigargin) on phosphorylated and unphosphorylated elF2a.
  • Figure 35B shows incorporation of 35 S-methionine into protein as a function of time in cells treated with THG.
  • Figure 35C is a Western blot analysis showing the effect of TUN (tunicamycin) on phosphorylated and unphosphorylated elF2a.
  • Figure 35D is a qRT-PCR analysis of CHOP mRNA levels in TUN (tunicamycin) treated cells.
  • Figure 35E is a Western blot analysis showing the effect of TUN (tunicamycin) on levels of BiP protein.
  • Figure 35F is a Western blot showing the effect of TUN (tunicamycin) on levels of p58 IPK .
  • Figure 36A-1 are photomicrographs showing the effect of estrogen on intracellular calcium levels in T47D breast cancer cells visualized using the dye Fluo- 4.
  • Figure 36A-2 is a graphical representation of the effect of estrogen on intracellular calcium levels T47D breast cancer cells visualized using the dye Fluo-4.
  • Figure 36B-1 are photomicrographs showing the effect of estrogen on intracellular calcium levels in PEO4 ovarian cancer cells visualized using the dye Fluo-4.
  • Figure 36B-2 is a graphical representation of the effect of estrogen on intracellular calcium levels PEO4 ovarian cancer cells visualized using the dye Fluo-4.
  • Figure 37A-1 are photomicrographs showing the effect of estrogen on intracellular calcium levels in cells treated with calcium channel blockers visualized using the dye Fluo-4.
  • Figure 37A-2 is a graphical representation of the effect of estrogen on intracellular calcium levels in cells treated with calcium channel blockers visualized using the dye Fluo-4.
  • Figure 37B is a Western blot analysis showing the effect of calcium channel blockers and E2 on levels of phosphorylated and unphosphorylated elF2a.
  • Figure 38A are photomicrographs showing the effect of RNAi knockdown of ERa on intracellular calcium levels visualized using the dye Fluo-4.
  • Figure 38B is a graphical representation of the effect of RNAi knockdown of ERa on intracellular calcium levels visualized using the dye Fluo-4.
  • Figure 39A is a qRT-PCR analysis of the effect of E2 on the level of spliced XBP1 mRNA.
  • Figure 39B is a qRT-PCR analysis of the effect of E2 on the levels of SERP1 and ERDJ mRNAs.
  • Figure 39C is a qRT-PCR analysis of the effect of E2 and
  • Figure 39D is a qRT-PCR analysis of the effect of RNAi knockdown of ERa on the level of spliced XBP1 mRNA.
  • Figure 39E is a Western blot analysis of the effect of E2 and antiestrogens on the level of full-length and spliced ATF6a protein in T47D breast cancer cells.
  • Figure 39F is a Western blot analysis of the effect of E2 on the level of full-length and spliced ATF6a protein in BG-1 ovarian cancer cells.
  • Figure 39G is a Western blot analysis of the effect of E2 on the level of full-length and spliced ATF6a protein in PE04 ovarian cancer cells.
  • Figure 39H is a qRT-PCR analysis of the effect of E2 on the level of BiP mRNA in ERa containing cancer cells.
  • Figure 39I is a Western blot analysis of the effect of E2 on the level of BiP protein in MCF-7 cells.
  • Figure 39J is a qRT-PCR analysis of the effect of RNAi knockdown of ERa and E2 on the level of BiP mRNA.
  • Figure 40A is a Western blot analysis showing the effect of E2 on levels of phosphorylated and unphosphorylated PERK.
  • Figure 40B is a Western blot analysis showing the effect of E2 on levels of phosphorylated and unphosphorylated elF2a.
  • Figure 40C shows incorporation of 35 S-methionine into protein in cells treated with E2 and the antiestrogen ICI 1 82,780 (ICI).
  • Figure 41 A shows the effect of the calcium chelator BAPTA-AM on E2- ERa stimulated cell proliferation.
  • Figure 41 B shows the effect of calcium channel blockers on E2-ERa stimulated cell proliferation in in MCF-7 breast cancer cells.
  • Figure 41 C shows the effect of calcium channel blockers on E2-ERa stimulated cell proliferation in BG-1 ovarian cancer cells.
  • Figure 41 D shows the results of luciferase assays analyzing the effect of calcium channel blockers on E2-ERa stimulated expression of an ERE-luciferase reporter gene.
  • Figure 41 E is a qRT-PCR analysis of the effect of effect of calcium channel blockers on E2-ERa regulated expression of cellular pS2 and GREB1 mRNAs.
  • Figure 42A is a qRT-PCR analysis showing the effects of E2-ERa over time on mRNAs for each UPR arm in MCF-7 cells.
  • Figure 42B shows the effect of estrogen on growth of MCF-7 tumors in athymic mice.
  • Figure 42C is a qRT-PCR analysis showing the levels of GREB-1 and pS2 mRNAs in mouse tumors with and without E2.
  • Figure 42D is a qRT-PCR analysis showing the levels of UPR-related mRNAs in mouse tumors with and without E2.
  • Figure 42E is an analysis of publically available patient microarray data showing levels of estrogen-regulated mRNAs in normal breast epithelium from normal patients, normal breast epithelium in patients with invasive ductal carcinoma of the breast, and invasive ductal carcinoma tissue.
  • Figure 42F is an analysis of publically available patient microarray data showing levels of UPR-related mRNAs in normal breast epithelium from normal patients, normal breast epithelium in patients with invasive ductal carcinoma of the breast, and invasive ductal carcinoma tissue.
  • Figure 43 is a model for E2-ERa regulation of the UPR.
  • Figure 44 shows the effect of prior activation of the UPR by E2 and by TUN on subsequent cell proliferation in cells later treated with TUN.
  • Figure 45 is a table showing the genes that comprise the UPR gene index used in bioinformatics studies.
  • Figure 46 is a bioinformatic analysis of publically available microarray data from ERa positive breast cancer cohorts.
  • Figure 46A is a bioinformatic analysis of data from two microarray chips (U133A in Figure 46A-1 and U133B in Figure 46A-2) showing Kaplan-Meier survival plots comparing time of relapse-free survival in breast cancer patients expressing high and low levels of UPR index genes.
  • Figure 46B is a bioinformatic analysis of data from two microarray chips (U133A) and (U133B) showing time to relapse in 277 breast cancer patients, hazard ratio, and p-Values for individual components of the UPR gene index.
  • Figure 46C is a bioinformatic analysis of data from microarray chips using the UPR gene signature alone (univariate analysis) comparing time to relapse in breast cancer patients using the UPR gene signature and current prognostic markers (multivariate analysis).
  • Figure 46D is a bioinformatic analysis of microarray data showing time to relapse in 474 breast cancer patients, hazard ratio, and p- Value for individual components of the UPR gene index. Microarray analysis was performed prior to initiation of tamoxifen therapy.
  • Figure 46E is a bioinformatic analysis of microarray data from two microarray chips (U133A) and (U133B) showing time to relapse in 236 breast cancer patients; shown are hazard ratio and p-Values for individual components of the UPR gene index.
  • Figure 47 is a bioinformatic analysis of microarray data from ERa positive breast cancer patients comparing expression of classical estrogen-regulated genes and UPR index components.
  • Figure 48 is a bioinformatic analysis of publically available microarray data from ERa positive breast cancer cohorts.
  • Figure 48A shows Kaplan-Meier plots of time of relapse-free survival for patients grouped by level of expression (low, medium and high) of the UPR gene index using bioinformatic analysis of microarray data.
  • Figure 48B shows Kaplan-Meier plots of time of overall survival for patients grouped by level of expression of the UPR gene index using bioinformatic analysis of microarray data.
  • Figure 48C is a bioinformatic analysis of data from microarray chips using the UPR gene signature alone (univariate analysis) comparing time of relapse- free survival and overall survival in breast cancer patients using the UPR gene signature and current prognostic markers (multivariate analysis).
  • Figure 49 is a bioinformatic analysis of publically available microarray data from ovarian cancer patients with early stage and highly malignant tumors.
  • Figure 50 shows a Kaplan-Meier plot of time of relapse-free survival in ovarian cancer patients grouped by high and low expression of UPR genes using publically available microarray data.
  • a small molecule inhibitor that blocks ERa action is described.
  • the compound can have the following structure:
  • X can be hydrogen, alkyl, halogen, or CF3, -CHF2, -CCI3, -CHCI2, - CBr3, -CHBr2; Y ean be hydrogen or hydroxyl; and Z can be hydrogen, alkyl, halogen, or -CF3, -CHF2, -CCI3, -CHCI2, -CBr3, -CHBr2.
  • the halogen can be one or more of fluorine, bromine, or chlorine.
  • the alkyl can be methyl.
  • the small molecule inhibitor of Structure A is BHPI, 3,3,bis(4-hydroxyphenyl)-7-methyl-1 ,3,dihydro-2H-indol-2-one ( Figure 14A), or a derivative thereof, or a salt thereof, or a pharmaceutical formulation thereof.
  • the small molecule inhibitor does not work by competing with estrogens for binding to ERa.
  • This small molecule inhibits protein synthesis in cancer cells that contain ERa.
  • the small molecule potently inhibits estrogen-ERa mediated gene expression. The compound works by distorting the previously unknown ability of estrogen-ERa to activate the unfolded protein response (UPR).
  • this compound targets a previously not described interaction of estrogen-ERa with components of the unfolded protein response, it represents a new therapeutic target.
  • the compound is effective in ERa containing breast, ovarian, and endometrial cancer cells even when the cells do not require estrogen for growth and are resistant to the currently used drugs, tamoxifen and fulvestrant, and it works in multidrug resistant cell lines.
  • This compound has a novel mode of action fundamentally distinct from current small molecule therapeutics that target ERa.
  • compounds of the class 3,3-bis(4- hydroxyphenyl)-1 ,3-dihydro-2H-indol-2-one (also known as: 3,3-bis(4- hydroxyphenyl)-2-oxindoles) were identified as compounds that stop the growth of estrogen-ERa-containing cancer cells by inhibiting estrogen-ERa-regulated gene expression and mRNA production, and by a novel mechanism; they inhibit the synthesis of new proteins in cancer cells that contain estrogen receptor a (ERa). Since estrogen-ERa regulated gene expression and synthesis of new proteins are each required for cells to proceed through the cell cycle, the compounds rapidly and completely stop the growth of the ERa-containing cancer cells, and the cells eventually die. As exemplified by BHPI, these compounds have an exceptionally attractive set of properties that make them excellent candidates for targeting ERa positive cancers and other diseases such as endometriosis.
  • BHPI is effective in numerous ERa-containing breast, ovarian, and endometrial cancer cells tested. At 100 nM, BHPI inhibits estrogen-ERa regulated gene expression, protein synthesis, and cell proliferation in ERa-containing cancer cell lines. In contrast, at 1 0,000 nM (a 100 fold higher concentration), BHPI has no detectable effect on growth in all tested cell lines that do not contain ERa. This is a much larger therapeutic window than most existing drugs and other ERa inhibitors.
  • BHPI By inhibiting ERa action, BHPI targets the entire spectrum of pathologies associated with ERa. These include, but are not limited to, breast, ovarian, endometrial, liver cancer, and endometriosis.
  • _BHPI is effective in widely used breast cancer models resistant to the major ER inhibitors used clinically to treat cancer, such as tamoxifen and fulvestrant. It is also effective in a widely used multidrug resistant ovarian cancer cell line resistant to adriamycin/doxorubicin, cisplatin, taxol and other standard chemotherapy agents. BHPI is fully effective in several ERa-containing cell lines in which estrogen does not stimulate cell growth.
  • BHPI is fairly low molecular weight and is simple to synthesize.
  • the BHPI family is unrelated to known inhibitors of ERa. BHPI does not act by competing with estrogens for binding in the ligand-binding pocket of ERa. Thus, it is completely different both in structure and site of action from current Selective Estrogen Receptor Modulators (SERMS) which include, but are not limited to, tamoxifen, Falsodex/fulvestrant/ICI 182,780 and raloxifene. A few noncompetitive ER inhibitors have been described. Pyrimidines, guanyl hydrazones and amphipathic benzenes have been reported as potential inhibitors of the binding of coactivators to ERa. These compounds are structurally distinct from the BHPI family of compounds and have not been shown to act as specific inhibitors of ERa- dependent growth of cancer cells.
  • SERMS Selective Estrogen Receptor Modulators
  • BHPI and its family members are inhibitors of both known and novel actions of ERa. That inhibition has important implications for treatment of ERa-dependent human pathologies and represents a novel method of use for BHPI and structurally related compounds.
  • BHPI and the related small molecules described in this application represent a new class of therapeutic agents for estrogen receptor a-dependent ovarian, endometrial/uterine, and breast cancers, and for endometriosis.
  • BHPI is effective in in ERa-containing cancer cells that are resistant to current therapeutics that target ERa and that are resistant to widely used chemotherapy agents including adriamycin/doxorubicin, cisplatin, and taxol.
  • chemotherapy agents including adriamycin/doxorubicin, cisplatin, and taxol.
  • Estrogens action of binding to estrogen receptor a plays a key role in the growth and metastases of cancers of the reproductive system including breast, ovarian, uterine/endometrial. Liver cancers are also fueled by estrogens binding to ERa.
  • existing therapies that focus on small molecules that inhibit the synthesis of estrogens or on competing with estrogens for binding to ERa are initially effective, the tumors eventually become resistant. This is due to the inability of existing therapies to completely inhibit tumor growth resulting in outgrowth of genetic variants that no longer require estrogen or ERa for growth. Many of these resistant tumors contain ERa, but they no longer need it to grow and are therefore resistant to current therapies.
  • BHPI and related compunds have a fundamentally different mode of action that makes this class of compounds a far more versatile drug.
  • BHPI targets the interaction of ERa with the UPR and with a system that regulates elongation and independently inhibits ERa-mediated gene expression.
  • the key proteins in the pathways it targets that lead to inhibition of protein synthesis are overexpressed in many cancers.
  • BHPI works when ERa is present, it does not require that estrogen be present, and it works in cancer cells that do NOT require ERa to grow.
  • BHPI blocks cell growth and works in breast cancer cells that are resistant to tamoxifen and fulvestrant, the current mainstream ERa-targeting therapies.
  • BHPI is effective in cell lines derived from most types of reproductive cancers including breast, ovarian, and endometrial. BHPI has potential effectiveness in a wide range of advanced cancers that are not effectively targeted by current therapies.
  • BHPI completely inhibits protein synthesis, it both blocks cell growth and eventually kills the cells.
  • Current agents targeting ERa tamoxifen, fulvestrant
  • ERa prevent ERa from working, but usually do not kill the cancer cells.
  • BHPI induces rapid regression of large pre-existing ERa positive cancers in a mouse xenograft model.
  • BHPI and structural analogues of this family have the following structural elements. 1 an oxindole ring, which is an indoline ring derivative containing a carbonyl at the 2-position of the nitrogen ring; and two o-phenol (4-hydroxy phenol) rings emerging from the 3-position of the nitrogen ring. The aggregate of these components yields the compound, 3,3-diphenyloxindole.
  • a class of compounds useful for killing ERa- positive cells is provided, the complounds having the formula:
  • X can be hydrogen, alkyl, halogen, or CF3, -CHF2, -CCI3, -CHCI2, - CBr3, -CHBr2; Y can be hydrogen or hydroxyl; and Z can be hydrogen, alkyl, halogen, or -CF3, -CHF2, -CCI3, -CHCI2, -CBr3, -CHBr2.
  • the disclosure provides a method of inhibiting growth of an ERa-containing cell comprising contacting said cell with an effective amount the compound of Structure A or a pharmaceutically acceptable salt thereof wherein, each independently, X can be hydrogen, alkyl, halogen, or -CF 3 , -CHF 2 , - CCI 3 , -CHCI2, -CBr 3 , -CHBr 2 ; Y can be hydrogen or hydroxyl; and Z can be hydrogen, alkyl, halogen, or -CF 3 , -CHF 2 , -CCI3, -CHCI2, -CBr 3 , -CHBr 2 .
  • the halogen can be one or more of fluorine, bromine, or chlorine.
  • the alkyl can be methyl.
  • Compounds of the general structure of Structure A are useful in the treatment of diseases related to the function of estrogen receptor a or diseases in cells containing estrogen receptor a. These include but are not limited to cancer of the breast, ovary, uterus and cervix, liver, colon, lung, and endometriosis.
  • any type of estrogen receptor a containing cell may be treated, including but not limited, to cells of the breast, ovary, uterine endometrium, cervix, liver, colon, lung and prostate.
  • a compound is used for treatment of cells in which estrogen binding to estrogen receptor a stimulates growth of the cells.
  • a compound is used to treat cells in which the presence of estrogen receptor is sufficient to stimulate growth of the cells.
  • a compound is used to treat cells that contain estrogen receptor and in which estrogen and estrogen receptor do not themselves stimulate growth of the cells.
  • the disclosed compound acts through estrogen receptor a to inhibit the ability of estrogen, bound to estrogen receptor a, to increase or decrease the expression of specific genes, and the compound acting through estrogen receptor a activates the unfolded protein response pathway.
  • a compound of Structure A acting through estrogen receptor a activates the unfolded protein response pathway. Activation of one arm of this pathway inhibits synthesis of new protein. The compound may also activate the AMPK pathway and causes inhibition of elongation to further inhibit protein synthesis. This long-term inhibition of protein synthesis leads to cessation of cell growth and death of many target cells.
  • Examples of cells in which this pathway of estrogen receptor a action can be used therapeutically to inhibit cell growth include, but are not limited to, breast cancer, ovarian cancer, uterine endometrial cancer, uterine endometrial cells in the disease endometriosis, cervical cancer, liver cancer (hepatocellular carcinoma), colorectal cancer lung cancer, and prostate cancer. Any type of cell containing estrogen receptor a may be treated including, but not limited to, breast, gynecological including ovarian, uterine endometrial, cervical, and vulval cells, liver, colon, lung, and prostate cells.
  • Compounds different from the structural class of BHPI and related compounds may also act through estrogen receptor a to activate the PERK arm of the unfolded protein response and inhibit protein synthesis and, therefore, reduce or inhibit growth and, in some cases, kill cells containing both estrogen receptor a and unfolded protein response components.
  • estrogen receptor a include, but are not limited to, breast cancer, ovarian cancer, uterine endometrial cancer, uterine endometrial cells in the disease endometriosis, cervical cancer, liver cancer (hepatocellular carcinoma), colorectal cancer, lung cancer, and prostate cancer.
  • a compound not related to the class of compounds of Structure A is provided for targeting the estrogen receptor a to activate the PERK arm of the unfolded protein response and inhibit protein synthesis and, therefore, reduce or inhibit growth and, in some cases, kill cells containing both estrogen receptor a and unfolded protein response components.
  • a compound of the general formula of Structure A as defined herein for use as a medicament, specifically, the use of a compound of the general formula of Structure A for the preparation of a medicament for the treatment of cancer, endometriosis, and other estrogen receptor related diseases in a mammal.
  • a medicament may be used in combination therapy with one or more other chemotherapeutic agents.
  • the disclosed compounds may be present as racemic mixtures or the individual isomers, such as diasteriomers or enantiomers.
  • the formulation ecompasses each and every possible enantiomer and diasteriomer as well as racemates and mixtures that may be enriched in one of the possible steriosiomers.
  • forms in which the compound may be present as salt including pharmaceutically acceptable acidic and basic salts are provided.
  • Compounds of the general formula of Structure A are suitably formulated as pharmaceuticals with a composition appropriate to the most suitable route of administration.
  • the route of administration may be any desirable route that leads to a concentration in the target tissue or blood that is therapeutically effective.
  • Administration routes that may be applicable include but are not limited to, oral, subcutaneous, intravenous, parenteral, vaginal. The choice of route of administration depends on the physical and chemical properties of the compound in the
  • the compound may represent any portion of the total in a
  • composition will generally be in the range of 1 -95% by weight of the total weight of the composition.
  • the dosage form will be suitable to the method of administration.
  • the composition may be in the form of powders, granules, emulsions, suspensions, gels, ointments, creams injectables, sprays, and any other suitable form.
  • compositions will follow accepted pharmaceutical practice. This will involve a pharmaceutically acceptable carrier and may involve composition with other agents. Pharmaceutically acceptable compositions may be formulated to release the active compound immediately or nearly immediately after administration or to release the active over a predetermined time period. These compositions are referred to as timed release or controlled release formulations. Controlled release formulations involve formulations designed to produce a substantially constant concentration of the drug in the target tissue and/or in the blood over an extended period of time.
  • an effective compound is combined with the anticancer drug paclitaxel or with other taxanes.
  • Paclitaxel activity is stimulated by calcium.
  • BHPI and related active compounds increase intracellular calcium levels by opening an endoplasmic reticulum calcium channel. Therefore BHPI is expected to increase the effectiveness of paclitaxel.
  • any compound that acts through estrogen receptor a to open a calcium channel is provided.
  • the compound is combined with paclitaxel and/or other taxanes to increase their activity as anticancer drugs.
  • BHPI and related compounds may be combined with paclitaxel and other taxanes to treat estrogen receptor a containing ovarian cancer and other cancers in which taxanes are used therapeutically.
  • the compound may be combined with paclitaxel and other taxanes, with other chemotherapeutic agents that inhibit estrogen synthesis including Letrozole,
  • a composition of Formula A may be used in estrogen receptor alpha containing cells to open the endoplasmic reticulum calcium channel and release calcium into the cytoplasm of the cell. This includes opening the endoplasmic reticulum IP3R calcium channel, the ryanodine calcium channel, both the IP3R calcium channel and the ryanodine calcium channel and other endoplasmic reticulum calcium channels.
  • any compound that acts through estrogen receptor a to open the endoplasmic reticulum IP3R calcium channel, the ryanodine calcium channel, both the IP3R calcium channel and the ryanodine calcium channel, and other endoplasmic reticulum calcium channels is provided.
  • a compound of Formula A may be used in estrogen receptor a containing cells to activate the PERK arm of the UPR resulting in inhibition of protein synthesis.
  • a method for identifying cancer patients in which therapy using BHPI or a compound of Structure A is likely to be most effective is provided.
  • UPR is elevated in resistant ERa positive tumors ( Figures 46-50).
  • evaluating tumors using the UPR index and the level of estrogen receptor a, and determining those tumors with the highest levels of UPR index genes and ERa it is possible to identify tumors most susceptible to treatment with BHPI.
  • BHPI Breast cancers with very high levels of ERa are resistant to tamoxifen therapy.
  • Use of BHPI or a related compound is especially effective in inhibiting protein synthesis in cells that contain very high levels of ERa ( Figure 23B).
  • the use of BHPI as a cancer therapy is expected to be especially effective in the subclass of therapy-resistant cancers that express very high levels of ERa.
  • a method of treating ERa containing cancers resistant to current cancer therapies including antiestrogens, such as tamoxifen, aromatase inhibitors such as letrozole and taxanes such as paclitaxel is provided.
  • Antiestrogens such as tamoxifen
  • aromatase inhibitors such as letrozole
  • taxanes such as paclitaxel
  • Therapy-resistant ERa containing tumors overexpress the UPR index ( Figures 46, 48-50).
  • these tumors are especially susceptible to BHPI therapy.
  • a method of inducing death of ERa containing breast cancer cells resistant to current therapy is provided. Because therapy resistant cancer cells overexpress the genes of the UPR index, these ERa containing cells are especially sensitive to BHPI. They do not just stop growing; they rapidly die ( Figures 16A and 16B).
  • a method for treating ERa containing breast cancer cells whose growth is stimulated by epidermal growth factor and epidermal growth factor receptors is provided. This includes, but is not limited to the Her2/NEU positive class of breast cancers and ovarian cancer cells (see Figure 1 5A and 1 5B).
  • a method for treating ERa containing cancers that are resistant to therapy because they overexpress multidrug resistant resistance proteins, including, but not limited to, multidrug resistance protein 1 (MDR1 ) is provided.
  • OVCAR-3 cells overexpress MDR1 and are resistant to therapeutically relevant concentrations of at least eight anti-cancer drugs, but theyrespond to BHPI ( Figure 16D).
  • chemotherapeutics including paclitaxel and other taxanes and/or cisplatin is provided
  • Caov-3 cells are resistant to ICI, the active form of tamoxifen (4-
  • BHPI BHPI.
  • a method for inhibiting growth of uterine fibroids in patients with endometriosis is provided.
  • the fibroids that cause endometriosis use estrogen and ERa to stimulate growth and are expected to have their growth inhibited by BHPI.
  • a method for determining whether a tumor is a candidate for therapy with BHPI or a compound of Formula A comprises removing all or part of the tumor by biopsy or surgery; analyzing the tumor for ERa (usually done using an ELISA), extracting RNA from the tumor; performing microarray analysis to determine levels of UPR index genes; where elevated levels of UPR index genes indicate that the tumor is a good candidate for therapy with BHPI or a compound of Formula A.
  • live tumor cells may be inserted into an orthologous mouse model and directly testing the tumor for therapeutic response using BHPI or a compound of Formula A. Tumor size can be measured with calipers or using imaging.
  • BHPI elicits three effects in ER a positive cells including inhibition of estrogen ERa-regulated gene expression (which was a previously known action of ERa); activation of the UPR; and activation of AMPK.
  • a screen for small molecules that carry out any two of these three effects identifies a unique small molecule that targets these pathways. Such a screen can be used to identify addition therapeutic compounds.
  • a screen for small molecules that activate the unfolded protein response only in ERa positive cells is provided.
  • Typical cell lines would be ERa positive cells, such as MCF-7 or T47D cells, compared to ERa negative cells from the same tissue type, such as MDA-MB-231 cells.
  • the readout After treatment with a test compound, the readout would be either rapid inhibition of protein synthesis only in the ERa positive cells, or activation of any of the arms of the UPR only in the ERa positive cells.
  • These readouts can be formation of phospho-elF2 alpha, formation of spliced XBP-1 , formation of spliced ATF6 alpha, and others readouts. These can be monitored using Western blots, ELISA, qRT-PCR for spliced XBP1 , or other methods.
  • a kit for treating cancer with BHPI or a structurally related compound includes an ELISA assay for determing whether ERa is present; materials for RNA extraction; and a microarray for identifying the level of UPR gene index expression.
  • the microarray may be a commercially available microarray capable of testing for expression levels of many cell genes, or a custom microarray for testing only genes in the UPR index.
  • BHPI or a related compound alone as the active ingredient, or combined with another drug as a medicine for oral delivery or subcutaneous injection would be provided.
  • Compounds of the general formula of Structure A are suitably formulated as pharmaceuticals with a composition appropriate to the most suitable route of administration.
  • the route of administration may be any desirable route that leads to a concentration in the target tissue or blood that is therapeutically effective.
  • Administration routes that may be applicable include but are not limited to, oral, subcutaneous, intravenous, parenteral, vaginal. The choice of route of administration depends on the physical and chemical properties of the compound in the
  • the compound may represent any portion of the total in a
  • composition will generally be in the range of 1 -95% by weight of the total weight of the composition.
  • the dosage form will be suitable to the method of administration.
  • the composition may be in the form of powders, granules, emulsions, suspensions, gels, ointments, creams injectables, sprays, and any other suitable form.
  • compositions will follow accepted pharmaceutical practice. This will involve a pharmaceutically acceptable carrier and may involve composition with other agents. Pharmaceutically acceptable compositions may be formulated to release the active compound immediately or nearly immediately after administration or to release the active over a predetermined time period. These compositions are referred to as timed release or controlled release formulations. Controlled release formulations involve formulations designed to produce a substantially constant concentration of the drug in the target tissue and/or in the blood over an extended period of time.
  • the pharmaceutical composition is in unit dosage form by weight of the compound.
  • each unit dosage typically consists of 1 -100 mg administered daily.
  • the compound is generally administered in the range of 0.01 -2 mg per kg body weight daily.
  • daily dosages in the ranges of about 0.01 to about 2 mg per kg body weight; about 0.01 to about 0.2 mg per kg body weight;.about 0.2 to about 0.4 mg per kg body weight; about 0.4 to about 0.6 mg per kg body weight; about 0.6 to about 0.8 mg per kg body weight; about 0.8 to about 1 .0 mg per kg body weight; about 1 .2 to about 1 .4 mg per kg body weight; about 1 .4 to about 1 .6 mg per kg body weight; about 1 .6 to about 1 .8 mg per kg body weight; about 1 .8 to about 2 mg per kg body weight; about 0.2 to about 1 .8 mg per kg body weight; about 0.4 to about 1 .6 mg per kg body weight; about 0.6 to about 1 .4 mg per kg body weight; about 0.8 to about 1 .2 mg per kg body weight are provided.
  • the dosage of the compound to prevent or treat diseases is typically about 1 to about 100 mg dose administered daily.
  • the compound may be administered once or twice daily at a dose of about 1 to about 100 mg.
  • the pharmaceutical composition is in unit dosage form by weight of the compound.
  • each unit dosage typically consists of 1 -100 mg administered daily.
  • the compound is generally administered in the range of 0.01 -2 mg per kg body weight daily.
  • daily dosages in the ranges of about 0.01 to about 2 mg per kg body weight; about 0.01 to about 0.2 mg per kg body weight;_about 0.2 to about 0.4 mg per kg body weight; about 0.4 to about 0.6 mg per kg body weight; about 0.6 to about 0.8 mg per kg body weight; about 0.8 to about 1 .0 mg per kg body weight; about 1 .2 to about 1 .4 mg per kg body weight; about 1 .4 to about 1 .6 mg per kg body weight; about 1 .6 to about 1 .8 mg per kg body weight; about 1 .8 to about 2 mg per kg body weight; about 0.2 to about 1 .8 mg per kg body weight; about 0.4 to about 1 .6 mg per kg body weight; about 0.6 to about 1 .4 mg per kg body weight; about 0.8 to about 1 .2 mg per kg body weight are provided.
  • the dosage of the compound to prevent or treat diseases is typically about 1 to about 100 mg dose administered daily.
  • the compound may be administered once or twice daily at a dose of about 1 to about 100 mg.
  • compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Compound X'.
  • Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and
  • proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest.
  • AlkyI groups include straight-chain, branched and cyclic alkyl groups. AlkyI groups include those having from 1 to 30 carbon atoms. Alkyl groups include small alkyl groups having 1 to 3 carbon atoms. Alkyl groups include medium length alkyl groups having from 4-10 carbon atoms. AlkyI groups include long alkyl groups having more than 10 carbon atoms, particularly those having 10-30 carbon atoms. Cyclic alkyl groups include those having one or more rings. Cyclic alkyl groups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 1 0-member carbon ring and particularly those having a 3-, 4-, 5-, 6-, or 7-member ring.
  • the carbon rings in cyclic alkyl groups can also carry alkyl groups.
  • Cyclic alkyl groups can include bicyclic and tricyclic alkyl groups.
  • Alkyl groups are optionally substituted.
  • Substituted alkyl groups include among others those which are substituted with aryl groups, which in turn can be optionally substituted.
  • alkyl groups include methyl, ethyl, n-propyl, iso- propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, and cyclohexyl groups, all of which are optionally substituted.
  • Substituted alkyl groups include fully halogenated or semihalogenated alkyl groups, such as alkyl groups having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms.
  • Substituted alkyl groups include fully fluorinated or semifluorinated alkyl groups, such as alkyl groups having one or more hydrogens replaced with one or more fluorine atoms.
  • An alkoxy group is an alkyl group linked to oxygen and can be represented by the formula R-O.
  • Aryl groups include groups having one or more 5- or 6-member aromatic or heteroaromatic rings.
  • Aryl groups can contain one or more fused aromatic rings.
  • Heteroaromatic rings can include one or more N, O, or S atoms in the ring.
  • Heteroaromatic rings can include those with one, two or three N, those with one or two O, and those with one or two S, or combinations of one or two or three N, O or S.
  • Aryl groups are optionally substituted.
  • Substituted aryl groups include among others those which are substituted with alkyl or alkenyl groups, which groups in turn can be optionally substituted.
  • aryl groups include phenyl groups, biphenyl groups, pyridinyl groups, and naphthyl groups, all of which are optionally substituted.
  • Substituted aryl groups include fully halogenated or semihalogenated aryl groups, such as aryl groups having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms.
  • Substituted aryl groups include fully fluorinated or semifluorinated aryl groups, such as aryl groups having one or more hydrogens replaced with one or more fluorine atoms.
  • Arylalkyl groups are alkyl groups substituted with one or more aryl groups wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted.
  • Specific alkylaryl groups are phenyl-substituted alkyl groups, e.g., phenylmethyl groups.
  • Alkylaryl groups are alternatively described as aryl groups substituted with one or more alkyl groups wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted.
  • Specific alkylaryl groups are alkyl-substituted phenyl groups such as methylphenyl.
  • Substituted arylalkyl groups include fully halogenated or semihalogenated arylalkyl groups, such as arylalkyl groups having one or more alkyl and/or aryl having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms.
  • Optional substitution of any alkyl and aryl groups includes substitution with one or more of the following substituents: halogens, -CN, -COOR, -OR, COR, - OCOOR, CON(R)2 , -OCON(R)2, -N(R)2, -NO2, -SR, -SO2R, -SO2N(R)2 or -SOR groups.
  • Optional substitution of alkyl groups includes substitution with one or more alkenyl groups, aryl groups or both, wherein the alkenyl groups or aryl groups are optionally substituted.
  • Optional substitution of alkenyl groups includes substitution with one or more alkyl groups, aryl groups, or both, wherein the alkyl groups or aryl groups are optionally substituted.
  • Optional substitution of aryl groups includes substitution of the aryl ring with one or more alkyl groups, alkenyl groups, or both, wherein the alkyl groups or alkenyl groups are optionally substituted.
  • Optional substituents for alkyl and aryl groups include among others: - COOR where R is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which are optionally substituted;-COR where R is a hydrogen, or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted; -CON(R)2 where each R,
  • R independently of each other R, is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted; R and R can form a ring which may contain one or more double bonds; -OCON(R)2 where each R, independently of each other R, is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted; R and R can form a ring which may contain one or more double bonds; -N(R)2 where each R, independently of each other R, is a hydrogen, or an alkyl group, acyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl or acetyl groups all of which are optionally substituted;
  • Specific substituted alkyl groups include haloalkyl groups, particularly trihalomethyl groups and specifically trifluoromethyl groups.
  • Specific substituted aryl groups include mono-, di-, tri, tetra- and pentahalo-substituted phenyl groups; mono-, di , tri-, tetra-, penta-, hexa-, and hepta-halo-substituted naphthalene groups; 3- or 4- halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenyl groups, 3- or 4- alkoxy-substituted phenyl groups, 3- or 4-RCO-substituted phenyl, 5- or 6-halo- substituted naphthalene groups.
  • substituted aryl groups include acetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenyl groups, particularly 3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups, particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenyl groups, particularly 4-methylphenyl groups, and methoxyphenyl groups, particularly 4- methoxyphenyl groups.
  • any of the above groups which contain one or more substituents it is understood, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
  • the compounds of this disclosure include all stereochemical isomers arising from the substitution of these compounds.
  • isotopic variants of compounds disclosed herein are intended to be encompassed by the disclosure.
  • any one or more hydrogens in a molecule disclosed can be replaced with deuterium or tritium.
  • Isotopic variants of a molecule are generally useful as standards in assays for the molecule and in chemical and biological research related to the molecule or its use. Methods for making such isotopic variants are known in the art. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently.
  • the disclosed compounds may be present as racemic mixtures or the individual isomers, such as diasteriomers or enantiomers.
  • the formulation ecompasses each and every possible enantiomer and diasteriomer as well as racemates and mixtures that may be enriched in one of the possible steriosiomers.
  • forms in which the compound may be present as salt including pharmaceutically acceptable acidic and basic salts are provided.
  • MCF-7 (MCF-7: Michigan Cancer Foundation-7), T47D, T47D-kBluc, HCC-1500 (HCC-1500: human carcinoma cells-1 500), ZR-75-1 , MCF1 OA (MCF 1 0A: Michigan Cancer Foundation 10A), MDA MB-231 , CaOV-3 (CAOV-3: Cancer Ovarian-3), OVCAR-3 (OVCAR-3: Ovarian Carcinoma 3), IGROV-1 , ES2, ECC-1 (ECC-1 : endometrial carcinoma cells-1 ), HeLa, PC-3 (PC-3: prostate cancer cells-3), DU145, H1793, A549, MEF (MEF: mouse embryo fibroblast) and HepG2 (HepG2: hepatoma G2) cells were obtained from the ATCC.
  • MCF10A E RIN9 MCF10A E RIN9: Michigan Cancer Foundation 10A estrogen receptor in (positive) 9
  • Dr. R. Schiff provided BT-474 cells
  • MCF7ERaHA MCF-7 estrogen receptor a hemagglutinin
  • the small molecule libraries screened were (1 ) the -150,000 small molecule Chembridge MicroFormat small molecule library; (2) the -1 0,000 small molecule University of Illinois Marvel library developed by Drs. K. Putt and P. Hergenrother (Putt K.S., Hergenrother P.J., A nonradiometric, high-throughput assay for poly(ADP- ribose) glycohydrolase (PARG): application to inhibitor identification and evaluation. Anal Biochem. 2004;333(2):256-64); and the -2,000 small molecule NCI diversity set obtained from N IH (National Institutes of Health).
  • Reporter gene assays were carried out, as previously described in Andruska, N., et al., Evaluation of a luciferase-based reporter assay as a screen for inhibitors of estrogen-ERalpha-induced proliferation of breast cancer cells. J Biomol Screen. 2012; 1 7(7):921 -32. Briefly, for the primary screen in 384-well plates, cells were harvested at a density of 1 -million cells/ml in RPMI-1640, plated at a density of 10,000 cells/well by pipetting 10 ⁇ of cells into each well using a Matrix Wellmate dispenser. The final concentration of test compounds in each well was 7.14 ⁇ .
  • the screening medium contained 0.1 % (v/v) EtOH, 0.07% (v/v) DMSO (DMSO: dimethyl sulfoxide), and 10 nM E2. Plates were centrifuged for 2 minutes at 500 rpm, and incubated for 24 hours. For follow-on testing in 96-well plates, cells were switched to 10% CD-FBS (FBS: fetal bovine serum) for four days prior to experiments, and plated at a density of 50,000 cells/well in 96-well plates. The medium was replaced the next day with medium containing the test compounds, with or without hormone, incubated for 24 hours and luciferase (luc: luciferase) assays were performed using Bright Glow reagent (Promega, Wl).
  • FBS fetal bovine serum
  • MCF-7 cells were depleted of estrogens by 3 days of culture in 5% CD- FBS. Cells were pretreated with 1 ⁇ BHPI or DMSO (0.1 %) as a control for 105 minutes, and then were treated with either 10 nM E2 or an ethanol-vehicle control (0.1 %) for 45 minutes. ChIP was carried out as described in Cherian, M.T., et al., A competitive inhibitor that reduces recruitment of androgen receptor to androgen- responsive genes. J Biol Chem. 2012; 287(28):23368-80.
  • siRNA (short interfering RNA) knockdowns were performed using ON- TARGETplus SMARTpools, each containing a mixture of 4 siRNAs (Dharmacon, CO). Transfections were performed using DharmaFECTI Transfection Reagent (Dharmacon, CO). To knockdown ERa, MCF1 0A E RIN9 cells were treated for 16 hours with either human ERa SMARTpool (SMARTpool by Dharmacon) (ESR1 ) siRNA or Non-targeting Control Pool siRNA.
  • ESR1 human ERa SMARTpool by Dharmacon
  • DMEM/F12 Dulbecco's Minimum Essential Medium/Hams Medium F1 2
  • CD-FBS CD-FBS
  • ERa knockdown at the mRNA and protein level was assessed every 24 hours following transfection.
  • the effects of BHPI on protein synthesis following ERa knockdown were assessed 3-days post-knockdown by treating cells with either 0.1 % DMSO loading control or 1 00 nM BHPI for the indicated times, and protein synthesis was then assessed by measuring 35 S-methionine incorporation.
  • MCF-7 cells were maintained in MEM (MEM: minimal essential medium) containing 5% CD-FBS for 4 days prior to plating cells in serum-free MEM.
  • MEM minimal essential medium
  • EIF2AK3 siRNA ON-TARGETplus Human PERK
  • TARGETplus Non-targeting Control Pool siRNA Cells were treated with transfection complexes for 1 6 hours and medium was replaced with MEM, supplemented with 10% CD-calf serum. To assess PERK knockdown at the mRNA and protein level, mRNA and protein samples were collected every 24 hours post-transfection. Since E2-ERa induces PERK (see Figure 5b), knockdown experiments were carried out in the absence of estrogen. The effects of BHPI on protein synthesis following PERK knockdown were assessed 3-days post-knockdown by treating cells with either 1 % DMSO loading control or 250 nM BHPI for the indicated times and protein synthesis was then assessed by measuring 35 S-methionine incorporation. [0308] Immune-blotting
  • Phospho-p44/42 MAPK (#4370; Cell Signaling Technology, MA), p44/42 MAPK (#4695; Cell Signaling Technology, MA), Phospho-PERK (#31 79; Cell Signaling Technology, MA), PERK (#5683; Cell Signaling Technology, MA), ATF6a (Imgenex, CA), Phospho-AMPKa (#2535, Cell Signaling Technology, MA), AMPKa (adenosine monophosphate kinase a (subunit)) (#2603, Cell Signaling Technology, MA), Phospho- ⁇ ( ⁇ : adenosine monophosphate kinase ⁇ 1 (subunit)) (#4181 , Cell Signaling Technology, MA), ⁇ 1 /2 (#4150, Cell Signaling
  • Bound antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and chemiluminescent
  • MCF-7 cells were pre-treated with 1 ⁇ BHPI or DMSO (0.1 %) for 30 minutes, followed by 2 hours with or without E2. Nuclear and cytoplasmic extraction was carried out on ⁇ 6 million cells/treatment using a NE-PER Nuclear and
  • Protein synthesis rates were evaluated by measuring incorporation of 35 S-methionine into newly synthesized protein.
  • Cells were plated at a density of 10,000 cells/well in 96-well plates. Cells were incubated for 30 minutes with 3 ⁇ iC ⁇ of S-methionine (PerkinElmer, MA) per well at 37°C. Cells were washed two times with PBS, and lysed using 30 ⁇ _ of RIPA buffer. Cell lysates were collected in microfuge tubes and clarified by centrifugation at 13,000 x g for 1 0 minutes at 4°C.
  • Samples were normalized to total protein, and the appropriate volume of sample was spotted onto Whatman 540 filter paper discs and immersed in cold 10% TCA (trichloroacetic acid). The filters were washed once in 1 0% TCA and 3 times in 5% TCA and air dried Trapped protein was then solubilized and the filters were counted.
  • TCA trifluoroacetic acid
  • Cytoplasmic Ca 2+ concentrations were measured using the calcium- sensitive dye, Fluo-4 AM (Fluo-4: 2- ⁇ [2-(2- ⁇ 5-[bis(carboxymethyl)amino]-2- methylphenoxy ⁇ ethoxy)-4-(2,7-difluoro-6-hydroxy-3-oxo-3H-xanthen-9- yl)phenyl](carboxymethyl)amino ⁇ acetic acid).
  • Fluo-4 AM Fluo-4: 2- ⁇ [2-(2- ⁇ 5-[bis(carboxymethyl)amino]-2- methylphenoxy ⁇ ethoxy)-4-(2,7-difluoro-6-hydroxy-3-oxo-3H-xanthen-9- yl)phenyl](carboxymethyl)amino ⁇ acetic acid).
  • the cells were grown on 35 mm- fluorodish cell culture plates (Cat#FD35-1 00, World Precision Instruments) for two days prior to imaging experiments.
  • the cells were washed three times with HEPES buffer to remove extracellular Fluo-4-AM dye and incubated with either 2 mM CaCI 2 or 0 mM CaCI 2 for 10 minutes to complete de-esterification of the dyes. Confocal images were obtained for one minute to determine basal fluorescence intensity, and then the appropriate treatment was added.
  • ERa LBD (N304-S554) containing an N-terminal 6-His tag, was purified and stored in Tris-HCI buffer (50 mM Tris-HCI pH 8.0, 10% glycerol, 2 mM DTT (dithiothreitol), 1 mM EDTA, and 1 mM sodium orthovanadate). Purified ERa LBD protein (10 ⁇ 9) was incubated with 500 nM E2 for 20 minutes at 37 °C. Subsequently, control DMSO vehicle, BHPI (1 ⁇ ) or inactive Compound 88 (1 ⁇ ) and incubated for 20 minutes at 37 °C.
  • the binding mixture was added with/without protease K at a concentration of 7.5 ng protease K per ⁇ g protein. After a 10 minute incubation at 22 °C, the digestions were terminated by addition of SDS sample buffer. The denatured samples were analyzed on a 15% SDS-PAGE gel and visualized by coomassie blue staining.
  • MCF-7 cell mouse xenograft model is described in detail in Ju, Y.H., et al., Dietary genistein negates the inhibitory effect of tamoxifen on growth of estrogen-dependent human breast cancer (MCF-7) cells implanted in athymic mice. Cancer research. 2002; 62(9):2474-7. At least 1 2 animals, usually with 4 tumors per animal, were required per experimental group to maintain significant statistical power to detect >25% difference in tumor growth rates. Briefly, estrogen pellets (1 mg: 19 mg estrogen: cholesterol) were implanted into 60 athymic female OVX mice, which were 7 weeks of age.
  • E2 pellets were removed and a lower dose of E2 in sealed silastic tubing (1 :31 estrogen: cholesterol, 3 mg total weight) was implanted in the same site.
  • NC no treatment control
  • E2 silastic tubes in the NC group were removed, while E2 silastic tubes in the E2, B 1 0, and B 1 /B 15 groups were retained.
  • the E2 and NC group received intraperitoneal injection every other day with 1 0 ml/kg vehicle (2% DMSO, 10% Tween-20, and 88% PBS).
  • the B_10 group received 1 0 mg/kg BHPI by intraperitoneal injection every other day.
  • the B 1 /B 1 5 group received 1 mg/kg BHPI by intraperitoneal injection every other day for 14 days. Since this extremely low BHPI dose had no effect, (average tumor cross-sectional area -45 mm 2 ) they then received 15 mg/kg BHPI every day for another 10 days.
  • a "UPR Gene Signature” was constructed to carry out risk prediction analysis.
  • the UPR gene signature was evaluated for its ability to predict: (i) tumor relapse in 261 early-stage ERa+ breast cancers (GSE6532) (see, Loi, S., et al., Definition of clinically distinct molecular subtypes in estrogen receptor-positive breast carcinomas through genomic grade. J Clin Oncol. 2007; 25(10):1239-46); (ii) tumor relapse in 474 ERa+ patients receiving solely tamoxifen therapy for 5 years (GSE6532, GSE17705), (see, Loi, S., et al.,
  • Microarray data analysis was performed using BRB ArrayTools (version 4.2.1 ) and R software version 2.13.2. Gene expression values from CEL files were normalized by use of the standard quantile normalization method.
  • Univariate and multivariate hazard ratios were estimated using Cox regression analysis. Covariates statistically significant in univariate analysis were further assessed in multivariate analysis. A patient was excluded from multivariate analysis if data for one or more variables were missing. Risk prediction using the UPR gene signature was carried out using the supervised principle components method (Bair, E., and Tibshirani, R., Semi-supervised methods to predict patient survival from gene expression data. PLoS biology. 2004; 2(4):E108) and visualized using Kaplan-Meier plots and compared using log-rank tests. [0326] Statistical Analysis
  • High throughput screening (HTS) and follow-up testing for specificity, toxicity, and potency were performed.
  • the screening was a cell-based high throughput screen of approximately 1 50,000 small molecules for inhibitors of E2-ERa regulated gene expression.
  • Candidate compounds were then "filtered” through additional tests for specificity, toxicity, potency, and site of action.
  • a schematic representation of the screening process is shown in Figure 1 .
  • Several "filtering” assays were carried out. Preliminary "hits” were re-screened to eliminate most inhibitors that might act in the same way as tamoxifen and other selective estrogen receptor modulators (SERMs) by competing with estrogens for binding to ERa.
  • SERMs selective estrogen receptor modulators
  • MDA-MB-231 breast cancer cells. These are triple negative breast cancer cells and do not contain ERa. MDA-MB-231 cells are a stringent system in which to test for non-specific toxicity. MDA-MB-231 cells are highly sensitive to growth inhibition by non-specific small molecules. (Kretzer, N.M., et al., A noncompetitive small molecule inhibitor of estrogen-regulated gene expression and breast cancer cell growth that enhances proteasome-dependent degradation of estrogen receptor alpha. J Biol Chem.
  • Example 2 - BHPI and structurally related compounds selectively inhibit estrogen-dependent cell proliferation and E2-ERg mediated gene expression.
  • BHPI specifically inhibits estrogen-ERa induced expression of an estrogen response element-luciferase reporter gene with no effect on androgen
  • a dose-response study of BHPI inhibition of the ERE-luciferase reporter gene was performed.
  • the cell maintenance and luciferase assay were carried out as follows. Five to six days before the experiment, T47D-KBIuc cells (ATCC: CRL-2865) were subcultured and plated at high density (-30-40%
  • FIG. 3A shows the dose-response study of E2 induction of the ERE-luciferase reported gene.
  • Figure 3B shows the dose-response study of BHPI inhibition of E2-ERa induction of the ERE-luciferase reporter gene.
  • BHPI and structurally related compounds selectively inhibit estrogen-dependent cell proliferation and E2-ERa mediated gene expression.
  • Figure 3B shows results from dose response studies of the effect of BHPI on 1 73-estradiol (E2) induction of ERE- luciferase activity in ERa positive T47D-kBluc breast cancer cells (black bars) and for dihydrotestosterone-androgen receptor (DHT-AR) induction of prostate specific antigen (PSA)-luciferase in ERa negative Hel_aA6 cells (open bars).
  • BHPI strongly inhibited E2-ERa induction of an estrogen response element (ERE)-luciferase reporter and had no effect on androgen induction of an androgen response element (ARE)-luciferase reporter.
  • Example 3 - BHPI is not a competitive inhibitor for binding to ERa
  • BHPI is not a competitive inhibitor, and it does not act by competing with estrogens for binding to ERa.
  • Figure 4 shows the effect of BHPI on expression of an estrogen-regulated gene in the presence of low and high concentrations of estrogen.
  • ERa positive MCF-7 human breast cancer cells were maintained in 5% CD-FBS for 4 days to deplete the medium of endogenous estrogens. Cells were harvested in 10% CD-CS, and plated into 6-well plates at a density of 450,000 cells per well. The following day, the medium was replaced with fresh 10% CD-CS.
  • BHPI interacts with ERa and inhibits E2-ERa-regulated gene expression.
  • BHPI is a non-competitive ERa inhibitor.
  • E2 173-estradiol
  • BHPI does not compete with estrogens for binding to ERa in vitro.
  • Radioligand competition assays comparing the ability of increasing concentrations of unlabeled E2 and BHPI to compete with 0.2 nM [ 3 H]- estradiol for binding to ERa show that BHPI is at least 10,000 fold weaker competitor than E2.
  • Example 4 - BHPI binds directly to ERa and appears to change its shape
  • FIG. 5 shows the structures of BHPI (Figure 5A) and of an inactive related compound, termed Compound 8 ( Figure 5B).
  • Figure 6A shows the effect of BHPI and a control compound on the
  • fluorescence emission spectrum of full-length ERa Fluorescence emission spectra of full-length ERa in the presence of E2 and (i) DMSO; (ii) 500 nM BHPI; or
  • BHPI could alter the sensitivity of purified ERa ligand-binding domain (LBD) to protease digestion was also tested.
  • ERaLBD was subjected to protease digestion in the presence of DMSO or BHPI.
  • Figure 6B and Figure 6C show the effect of BHPI on protease sensitivity of the ERa ligand binding domain (LBD) analyzed by SDS polyacrylamide gel electrophoresis. Bands were visualized by Coomassie staining.
  • Figure 6B shows the protease digestion pattern after cleavage with proteinase K.
  • Example 5 - BHPI inhibits ER-requlated gene expression
  • Example 5a - BHPI inhibits induction of E2-ERg induced genes in breast and ovarian cancer cells
  • BHPI is an ERa-dependent inhibitor of protein synthesis.
  • the ability of BHPI to inhibit E2-ERa induction of endogenous gene expression independent of its ability to inhibit protein synthesis in cells that contain ERa was tested. Cycloheximide inhibition of protein synthesis was used as a control in these experiments. If E2-ERa induction of an mRNA was not inhibited by cycloheximide, then inhibition resulting from BHPI is due to its ability to inhibit E2-ERa mediated gene expression, not its ability to inhibit protein synthesis in ERa containing cells. Cycloheximide did not inhibit E2-ERa induction of pS2, SDF-1 , or GREB1 mRNAs ( Figure 7).
  • qRT-PCR quantitative reverse transcriptase polymerase chain reaction
  • RT-PCR quantitative RT-PCR (RT-PCR: reverse transcriptase-polymerase chain reaction) were carried out.
  • the level of each mRNA in the presence of ethanol vehicle without E2 was set equal to 1 .
  • the data represent the average of 3 independent sets of cells, each assayed in triplicate. Data are reported as mean + S.E.M.
  • Example 5b - BHPI inhibits E2-ERg-down-requlation of IL1 -R1 mRNA in ERg positive T47D breast cancer cells
  • BHPI but not cycloheximide, inhibits E2-ERa-down-regulation of IL1 - R1 mRNA in ERa positive T47D breast cancer cells.
  • ERa positive T47D human breast cancer cells were maintained in 10% cd-FBS for 4 days to deplete the medium of endogenous estrogens.
  • Cells were harvested in 10% cd-CS, and plated into 6-well plates at a density of 400,000 cells per well. The following day, the medium was replaced with fresh 1 0% cd-CS. The next day wells were treated with either an ethanol vehicle, 10 nM E2, 1 0 nM E2 + 10 ⁇ CHX, or 1 0 nM E2 + 1 ⁇ BHPI.
  • BHPI inhibited E2-ERa induction of pS2, GREB1 and CXCL2 mRNAs in ERa+ MCF-7, T47D and BG-1 cells ( Figure 7) and blocked E2-ERa down- regulation of IL1 -R1 mRNA ( Figure 8).
  • the ability of BHPI to inhibit E2-ERa induction and repression of gene expression indicates that BHPI acts at the level of ERa and not by a general inhibition or activation of transcription.
  • Example 5c - BHPI does not inhibit E2-ERa-requlated gene expression by reducing ERa protein levels or by excluding E2-ERa from the nucleus of the cell
  • FIG. 9 is a Western blot analysis of the effect of BHPI on ERa levels (Figure 9A) and subcellular localization (Figure 9B).
  • Figure 9A shows the effects of BHPI treatment on ERa protein levels.
  • MCF-7 cells were maintained in 5% cd-FBS serum + MEM for 4 days prior to cell plating in order to deplete cells of estrogen. On day 5, cells were harvested in 10% cd-CALF + MEM and plated in 6-well plates at a density of 250,000 cells per well. The medium was replaced on day 6.
  • cells were pre-treated for 30 minutes with a 0.1 % DMSO-vehicle control (-E2 and +E2 samples) or 1 ⁇ BHPI (+E2, BHPI and -E2, BHPI samples), followed by treatment for 2 hours with either a 0.1 % ethanol-vehicle control (-E2 and -E2, BHPI samples) or 1 0 nM 1 73-Estradiol (+E2 and +E2, BHPI samples).
  • Cell lysates were collected, and nuclear and cytoplasmic fractions of each lysate were separated using a NE-PER Nuclear and Cytoplasmic Extraction Kit (ThermoScientific).
  • Chromatin immunoprecipitation showed that BHPI strongly inhibited E2-stimulated recruitment of ERa and RNA polymerase II to the pS2 and GREB1 promoter regions.
  • Tunicamycin is a well- established indirect inhibitor of protein synthesis through activation of the UPR, and cycloheximide is a well-established direct inhibitor of protein synthesis.
  • Cells were then treated with or without 10 nM E2 for 2 hours. Data represent the average of 3 independent sets of cells, each assayed in triplicate. Data is reported as mean + S.E.M.
  • Figure 10A shows qRT-PCR analysis of the effect of BHPI on E2-ERa- mediated induction of pS2 mRNA.
  • Figure 10B shows qRT-PCR analysis of the effect of BHPI on E2- ERa- mediated induction of GREB-1 mRNAs.
  • BHPI RNA polymerase II
  • FIG. 10 The effects of BHPI on ERa and RNA polymerase II (RNAP) recruitment to the promoters of the pS2 and GREB-1 genes are shown in Figure 10.
  • Cells were maintained in 5% CD-FBS for 3 days to deplete the media of endogenous estrogens. Cells were pre-treated with a 0.1 % DMSO-vehicle control or 1 ⁇ BHPI for 75 minutes, before treating cells with either 0.1 % ethanol vehicle control or 10 nM E2 for 45 minutes. Cells were then treated with formaldehyde to cross-link DNA- protein complexes. ChIP were performed.
  • Figure 10C shows the ChIP study of the effect of BHPI on recruitment of E2-ERa and RNA polymerase to the estrogen regulated pS2 gene.
  • Figure 10D shows the ChIP study of the effect of BHPI on recruitment of E2-ERa and RNA polymerase to estrogen regulated GREB-1 genes.
  • ERa is shown with black bars;
  • RNA polymerase II is shown with hatched bars.
  • Data represents the average of 3 independent sets of cells, each assayed in triplicate. Date reported as mean + S.E.M.
  • Example 5e - BHPI inhibits binding of E2-ERg to gene regulatory regions and ovexpression of ERa abolishes BHPI inhibition of E2-ERg mediated gene expression
  • BHPI inhibits binding of E2-ERa to gene regulatory regions and ovexpression of ERa abolishes BHPI inhibition of E2-ERa mediated gene expression ( Figure 1 1 ). This shows that BHPI reduces recruitment of E2-ERa to regulatory elements by reducing the affinity of E2-ERa for these DNA regions.
  • MCF7ERaHA cells which are MCF-7 cells stably transfected to express a Doxycycline (Dox)-inducible ERa, were estrogen-deprived in CD-FBS for 4-days prior to harvesting cells in 1 0% CD-calf serum.
  • Dox Doxycycline
  • the MCF- 7ERaHA cells were treated with 0.25 ⁇ g/mL doxycycline (DOX) for 24 hours.
  • Cells were then treated with either 0.1 % DMSO (-E2; +E2) or 1 ⁇ BHPI (+E2, BHPI) for 30 minutes, followed by treatment with either 0.1 % ethanol (-E2) or 1 0 nM E2 (+E2; +E2, BHPI) for 4 hours.
  • BHPI did not alter ERa protein levels or nuclear localization ( Figure 9). Chromatin immunoprecipitation (ChIP) showed that BHPI strongly inhibited E2-stimulated recruitment of ERa and RNA polymerase II to the pS2 and GREB1 promoter regions ( Figure 10). If BHPI induces an ERa conformation exhibiting reduced affinity for gene regulatory regions, expressing high concentrations of ERa might provide sufficient ERa to still bind to regulatory regions, preventing inhibition by BHPI. Ten-fold overexpression of ERa in MCF7ERaHA cells abolished BHPI inhibition of induction of GREB1 mRNA
  • Example 6 - BHPI inhibits proliferation of ERa containing cancer cells
  • Example 6a - BHPI inhibits proliferation of ERa containing breast ovarian, endometrial, and prostate cancer cells
  • E2-ERa stimulates proliferation of most breast cancers and many ovarian, endometrial cervical, uterine, and liver cancers and likely several other types of cancer.
  • BHPI inhibits proliferation of ERa-containing cancer cells.
  • BHPI selectively inhibits growth of ERa positive breast cancer cells.
  • BHPI fully blocks proliferation of ERa positive MCF-7 breast cancer cells at 100 nM ( Figure 12A-1 ), but has no effect on ERa negative MDA MB-231 breast cancer cells at 10,000 nM ( Figure 12A-2).
  • Figure 12B BHPI selectively inhibits growth of ERa positive ovarian cancer cells.
  • BHPI fully blocks proliferation of ERa positive BG-1 ovarian cells at 1 00 nM ( Figure 12B-1 ), but has no effect on ERa negative ES2 ovarian cancer cells at 10,000 nM ( Figure 12B- 2).
  • BHPI selectively inhibits growth of ERa positive endometrial cancer cells.
  • BHPI fully blocks proliferation of ERa positive ECC-1 endometrial cancer cells at 1 00 nM ( Figure 1 2C-1 ), but has no effect on ERa negative HeLa cervical cancer cells at 10,000 nM ( Figure 1 2C-2).
  • Figure 12D BHPI selectively inhibits growth of ERa positive prostate cancer cells.
  • BHPI blocks proliferation of ERa positive PC-3 prostate cells at 100 nM ( Figure 12D- 1 ), but has no effect on ERa negative DU145 prostate cancer cells at 10,000 nM ( Figure 12D-2).
  • MTS (3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium)
  • Cell number was determined using a standard curve of cell number versus absorbance based on plating a known number of cells from each cell line and assaying using MTS as described in Kretzer, N.M., et al., A noncompetitive small molecule inhibitor of estrogen-regulated gene expression and breast cancer cell growth that enhances proteasome-dependent degradation of estrogen receptor alpha. J Biol Chem. 2010; 285(53):41863-73.
  • Each data point is the average of at least 6 independent samples of cells, and is reported as mean ⁇ SEM.
  • Example 6b - BHPI inhibits proliferation in diverse ERa positive cancer cell lines and has no effect on cell growth in ERa negative cell lines
  • Figure 13 shows the results of MTS assays analyzing the effects of different concentrations of BHPI on proliferation of ERa-positive and ERa-negative cancer cells.
  • Cell proliferation was evaluated as described for Figure 1 2 in Example 6a.
  • the effects of BHPI on cell proliferation in 14 ERa positive (black bars) and 12 ERa negative (grey bars) cell lines is shown in Figure 1 3.
  • Example 6c Effects of BHPI and structurally related compounds on growth of ERa positive human breast cancer cells
  • BHPI is a structure-selective inhibitor of ERa. Inhibition of proliferation is a structure-selective effect of BHPI acting through ERa and not a non-specific toxic effect.
  • a number of compounds closely related to BHPI were studied with some being effective inhibitors of cell proliferation and others only very weak inhibitors. Study of these related compounds provides information about which chemical substitutions on this chemical scaffold result in compounds effective for the inhibition of proliferation in ERa-containing cancer cells.
  • Figure 14A shows the chemical scaffold of the BHPI-related compounds.
  • Figure 14B is a table showing preferred substitutions at each site on the scaffold.
  • Example 6d - BHPI is effective in EGF-resistance models
  • EGF Epidermal growth factor
  • BHPI inhibits EGF-stimulated cell proliferation that is resistant to current antiestrogens.
  • the effects of BHPI and antiestrogens on EGF- dependent and E2-dependent growth of T47D breast cancer cells is shown in Figure 15A.
  • Cells were maintained in 10% cd-FBS for 4-days prior to experiment. Cells were plated at a density of 2,000 cells/well, the medium was changed the following day, and the appropriate treatments were added. Plates were incubated for 4 days prior to assaying using MTS with a standard curve for cell number.
  • fulvestrant (I I) (100 nM) and BHPI (100 nM) on EGF-stimulated (20 ng/ml) and E2-stimulated (100 pM) cell growth of ERa(+) BG-1 ovarian cancer cells is shown in Figure 15B.
  • Cells were maintained in 5% cd-FBS for 4-days prior to experiment. Cells were plated at a density of 200 cells/well, treated the following day, and allowed to grow for 6-days prior to assaying using MTS with a standard curve for cell number.
  • BHPI was effective in epidermal growth factor (EGF) stimulated T47D breast cancer cells that are resistant to 4-OHT, ICI1 82,780, and ralixofene (Ral).
  • Example 6e - BHPI inhibits proliferation of ERa positive breast and ovarian cancer cells resistant to current therapies
  • Figure 16 shows the dose-response studies of the effect of BHPI on proliferation of ERa positive cancer cell lines resistant to current therapies.
  • Results from tamoxifen- and ICI-resistant BT-474 human breast cancer cells are shown in Figure 16A.
  • Results from tamoxifen- and ICI-resistant ZR-75-1 human breast cancer cells are shown in Figure 16B.
  • Results from cis-platin resistant Caov-3 human ovarian cancer cells are shown in Figure 1 6C.
  • Results from multi-drug resistant OVCAR-3 human ovarian cancer cells are shown in Figure 1 6D.
  • Cells were maintained in 1 0% CD-FBS for 4 days prior to experiments. Cells were plated at a density of 2,000 cells/well.
  • the medium was changed the following day, and the cells were treated with 1 0 nM E2 and/or BHPI, or with 10 nm E2 + ICI or OHT (hatched bars). Plates were incubated for 4 days, with a medium change on day 2, prior to assaying using MTS. Cell number was determined using MTS from a standard curve of absorbance versus cell number for each cell line. The dot (" ⁇ ") denotes cell number at day 0. The hatched bars denote traditional antiestrogens (4-OHT and ICI). Data represents the average of 6 independent sets of cells, and is reported as mean + S.E.M.
  • BHPI is effective in cancer cells that contain ERa and are resistant to conventional chemotherapy or to antiestrogens such as fulvestrant/Faslodex/ICI 182,780.
  • Targeted therapies for ovarian cancer are largely unavailable.
  • NIH-OVCAR-3 (NIH OVCAR-3: National Institutes of Health ovarian carcinoma-3; OVCAR-3) cells are a widely-used model for resistance to chemotherapy agents.
  • NIH-OVCAR-3 cells are resistant to therapeutically relevant concentrations of the DNA intercalator
  • NIH-OVCAR-3 cells contain ERa.
  • BHPI effectively inhibited growth of the NIH-OVCAR-3 cells ( Figure 16D). Exposure to BHPI for longer periods of time (about a week) results in cell death. In the 5-day experiment using NIH-OVCAR-3 cells, 100 nM BHPI induced death of some of the cells and 1 ⁇ BHPI induced death of more of the cells ( Figure 16D).
  • OVAR-3 cells are also resistant to 5 ⁇ ICI ( Figure 16D). 1 ⁇ BHPI blocked proliferation of both CaOV-3 cells and OVCAR-3 cells ( Figure 16C and Figure 1 6D).
  • BHPI was also tested in breast cancer cells that contain ERa and are resistant to estrogen-based therapies.
  • BT-474 are human breast cancer cells containing amplified HER2 (the target for herceptin) and the ERa coregulator amplified in breast cancer (AIB1 ).
  • BT-474 cells are fully resistant to tamoxifen and are resistant to fulvesterant/Faslodex/ICI 1 82,780 (ICI).
  • ICI fulvesterant/Faslodex/ICI 1 82,780
  • BHPI effectively inhibited growth of BT-474 cells (Figure 16A).
  • ZR-75-1 cells are often considered partially resistant to antiestrogens. These cells showed no increase in proliferation in the presence of E2.
  • the ZR-75-1 cells were completely resistant to 4-hydroxytamoxifen (OHT; the active form of tamoxifen) and partially resistant to fulvestrant/ICI182,780 (ICI).
  • OHT 4-hydroxytamoxifen
  • ICI fulvestrant/ICI182,780
  • Example 6f - BHPI inhibits anchorage-independent growth of ERa positive cancer -7 cells in soft agar
  • Anchorage independent growth is a hallmark of cancer cells. This is often tested by evaluating growth in soft agar. BHPI blocks anchorage-independent growth of ERa positive cancer cells. The ability of BHPI to inhibit colony formation of MCF-7 human breast cancer cells was tested.
  • MCF-7 cells were plated into top agar. Cells were treated with medium containing DMSO (vehicle) and either, 10 nM 173-estradiol (E2) or ethanol (vehicle), or 1 ⁇ BHPI and 1 0 nM E2. Medium was changed every 3 days. After 21 days, colonies were counted and photographed at 5x magnification. The bar graph represents the average of the total number colonies per well with a diameter
  • Example 6g - BHPI induces regression of breast cancer in a mouse xenograft model
  • estrogen pellets (1 mg: 19 mg estrogen: cholesterol) were implanted into 60 athymic female OVX mice which were 7 weeks of age.
  • E2 pellets were removed and a lower dose of E2 in sealed silastic tubing (1 :31 estrogen: cholesterol, 3 mg total weight) was implanted in the same site.
  • mice were divided into 4 groups with tumor size normalized: E2 group, no treatment control (NC) group, B 10 group and B 1 /B 15 group.
  • E2 silastic tubes in the NC group were removed, while E2 silastic tubes in the E2, B 10, and B 1 /B 15 groups were retained.
  • the E2 and NC group received intraperitoneal injection every other day with 10 ml/kg vehicle (2% DMSO, 10% Tween-20, and 88% PBS).
  • the B_10 group received 1 0 mg/kg BHPI by intraperitoneal injection every other day.
  • the B 1 /B 1 5 group received 1 mg/kg BHPI by intraperitoneal injection every other day for 14 days. Since this extremely low BHPI dose had no effect, (average tumor cross-sectional area -45 mm 2 ) they then received 15 mg/kg BHPI every day for another 10 days.
  • Figure 18 shows the percent change in tumor size over the course of the experiment, (days 14-24) for each tumor.
  • the control group which was treated with E2 released from silastic implants and received injections of vehicle, but not BHPI, is represented by the white bars.
  • the size change of the tumors in the experimental group treated with E2 from silastic implants and injected with 15 mg/kg of BHPI daily is shown in black bars.
  • two of its 4 tumors decreased in size by -50% and 2 tumors decreased in size by just over 30%. Tumor size at day 14 is set to 0% change.
  • Example 6h - BHPI inhibits growth of human breast cancers in a mouse xenograft model
  • Example 7 - BHPI is an ERg-dependent inhibitor of protein synthesis
  • Example 7a - BHPI potently inhibits protein synthesis in ERg positive cancer cells
  • BHPI is an ERg-dependent inhibitor of protein synthesis.
  • breast, ovarian, cervical, lung, prostate, and liver cancer cells tested. Cells were estrogen-deprived for 4 days in cd-FBS prior to experiments.
  • the top panels of Figure 20 show Western blots for ERg in each cell line. Cells were plated at a density of 1 0,000 cells/well. The medium was replaced with the appropriate treatment medium the following day, and cells were treated for 24 hours before adding 5 ⁇ / ⁇ of 35 S-methionine. Cell lysates were collected, centrifuged at 13,200 rpm for 10 minutes at 4°C, and sample supernatants were transferred to Whatman filter paper. Radiolabeled protein was isolated via TCA-precipitation of labeled protein in the filters. Free amino acids are not retained in the filters.
  • Figure 20 shows a comparison of ERg protein levels and the effects of BHPI treatment on protein synthesis.
  • the number of samples was too large to run on a single gel and the data is from 3 identically processed gels.
  • Protein synthesis was determined by incorporation of 35 S-methionine into protein. Incorporation with no added BHPI was set to 100%.
  • protein synthesis in cells expressing moderate or high levels of ERg was robustly inhibited by 1 00 nM BHPI (hatched bars), while 10,000 nM BHPI (striped bars), the highest concentration tested, had very little or no effect on protein synthesis in ERg negative cells.
  • BHPI potently inhibits protein synthesis in MCF10A E RIN9 breast cells which contain ERg but has no effect in MCF10A cells which lack ERg.
  • Figure 21 A shows the effect of BHPI on protein synthesis in ERg-positive MCF1 OAERINQ breast cells and in the parental ERg-negative MCF-10A cells.
  • Figure 21 B shows the effects of the current generation ER inhibitors TPSF, Fulvestrant/faslodex/ICI 182,780 and 4-hydroxytamoxifen on protein synthesis in MCF10A E RIN9 cells and MCF1 OA cells.
  • Cells were maintained in 2% DMEM/F12 including 10 ⁇ g/ml insulin, 0.1 ⁇ g/ml cholera toxin, 0.5 ⁇ g/ml hydrocortisone, and 20 ng/ml EGF. Cells were plated at a density of 1 0,000 cells/well in 1 % CD-FBS + DMEM/F12 without supplements. Medium was replaced with the appropriate treatment medium and the indicated inhibitors the following day. Cycloheximide was at 1 0 ⁇ g/ml. Cells were treated for 24 hours before adding 3 ⁇ / ⁇ of 35 S-methionine radiolabel.
  • FIG. 21 B shows the effects of known ERg inhibitors on protein synthesis in ER(+) MCF10A E RIN9 and ER(-) MCF1 0A mammary cells. Data is the mean ⁇ S.E.M. for at least 3 sets of cells.
  • RNAi RNA interference knockdown of ERg abolishes BHPI inhibition of protein synthesis.
  • N 9 cells treated with non-coding (NC) siRNA or ERg siRNA SmartPool followed by 1 00 nM BHPI is shown in
  • FIG. 22A Protein synthesis in MCF1 0A ER
  • FIG 22B In each case there were 4 samples. Note that Figure 22C shows that ICI, a competitive inhibitor of estrogen, depleted the ERg protein. This complements the data showing that RNA interference knockdown of the mRNA leading to disappearance of the ERg protein abolishes inhibition of protein synthesis. [0408] Example 7d - Overexpression of ERa increases BHPI inhibition of protein synthesis
  • Figure 23A is a Western blot analysis showing levels of ERa in cells overexpressing ERa for each sample. Data is mean ⁇ S.E.M.
  • BHPI is an ERa-dependent inhibitor of protein synthesis.
  • Expression of ERa is necessary to make a cell that is not responsive to BHPI inhibition of protein synthesis sensitive to BHPI inhibition of protein synthesis ( Figure 21 ).
  • Current generation antiestrogens do not inhibit protein synthesis in these cells ( Figure 21 ).
  • Knockdown of the ERa abolishes sensitivity of the cells to BHPI inhibition of protein synthesis ( Figure 22).
  • Overexpression of ERa increases BHPI inhibition of protein synthesis ( Figure 23).
  • BHPI nearly abolished protein synthesis in ERa positive cancer cells. If BHPI inhibits protein synthesis through ERa, it should only work in ERa positive cells, and its potency should be related to ERa levels. BHPI inhibited protein synthesis in all 14 ERa positive cell lines and had no effect on protein synthesis in all 12 ERa negative cell lines. BHPI does not inhibit protein synthesis in ERa negative MCF-10A cells, but gains the ability to inhibit protein synthesis when ERa is stably expressed in isogenic MCF10A E RIN9 cells.
  • BHPI loses the ability inhibit protein synthesis when the ERa in the stably transfected cells is knocked down with siRNA or degraded by ICI 1 82,780. Increasing the level of ERa in MCF7ERaHA cells stably transfected to express doxycycline-inducible ERa progressively increased BHPI inhibition of protein synthesis. Thus, BHPI is likely to be especially effective against the subset of highly lethal breast cancers that contain very high levels of ERa and are often refractory to tamoxifen therapy. BHPI does not work by activating the estrogen binding protein GPR30.
  • BHPI has no effect on cell proliferation or protein synthesis in HepG2 cells that contain functional GPR30, and activating GPR30 with a selective activator, G1 , did not inhibit protein synthesis. Thus, ER is necessary and sufficient for BHPI inhibition of protein synthesis.
  • Example 8 - BHPI activates the endoplasmic reticulum stress sensor, the unfolded protein response (UPR) in ERg positive cancer cells
  • Example 8a - UPR activators inhibit protein synthesis seen with BHPI
  • Example 8b - BHPI depletes endoplasmic reticulum calcium in ERg positive breast cancer cells and activates all three arms of the UPR
  • the calcium sensitive dye Fluo-4 AM was used to monitor intracellular calcium in order to test whether BHPI alters intracellular Ca 2+ . Treating ERg positive MCF-7 and BG-1 cells with 1 ⁇ BHPI produced a large and sustained increase in intracellular calcium in the presence of extracellular Ca 2+ and a transient increase in intracellular calcium in the absence of extracellular calcium.
  • BHPI rapidly activates the UPR by depleting endoplasmic reticulum Ca 2+ and increasing cytosol Ca 2+ .
  • MCF-7 cells were treated with BHPI in the absence or presence of extracellular calcium, and averaged data for each of 10 cells at each time point was taken.
  • Figure 25 shows the effect of BHPI and the UPR activator thapsigargin on intracellular calcium measured using the calcium sensing dye Fluo-4.
  • Figure 25A is a photomicrograph of the effect of a low concentration (1 ⁇ ) of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium.
  • Figure 25B-1 is a photomicrograph of the effect of a high concentration (10 ⁇ ) of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium.
  • Figure 25B-2 is a graphical representation of the effect of a high concentration (10 ⁇ ) of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium.
  • Figure 25C-1 is a photomicrograph of the effect of the UPR activator thapsigargin (2 ⁇ ) on intracellular calcium in MCF-7 cells.
  • Figure 25C-2 is a graphical representation of the effect of the UPR activator thapsigargin on intracellular calcium in MCF-7 cells.
  • Example 8c - BHPI depletes endoplasmic reticulum calcium, in ERa positive cells but not in ERa negative cells
  • FIG. 26 shows the effect of BHPI on intracellular calcium levels in ERa positive BG-1 ovarian cancer cells but not in ERa negative HeLa endometrial cells.
  • Figure 26 shows the effect of BHPI on intracellular calcium levels in ERa positive BG-1 ovarian cells (Figure 26A) and ERa negative HeLa cervical cells (Figure 26B).
  • Figure 26A is a photomicrograph of the effect of a high concentration of BHPI or thapsigargin (THG) on intracellular calcium in ERa positive BG-1 cells with and without extracellular calcium.
  • Figure 26B is a photomicrograph of the effect of a high concentration of BHPI or thapsigargin (THG) on intracellular calcium in ERa negative HeLa cells without extracellular calcium.
  • Example 8d - BHPI acts by opening the endoplasmic reticulum IP3R channel
  • BHPI acts by opening the endoplasmic reticulum IP3R (IP3R: inositol 3-phosphate receptor) channel. Locking the channel closed with the inhibitor 2-APB prevents release of calcium into the cytosol. Inhibiting opening of the endoplasmic reticulum IP3R Ca 2+ channel abolished BHPI release of intracellular calcium and inhibition of protein synthesis.
  • Figure 27A shows the effects of inhibitors of calcium channel opening on intracellular calcium levels after BHPI treatment.
  • 2-APB which Inhibits opening of the endoplasmic reticulum IP3R Ca 2+ channel, abolished BHPI release of intracellular calcium.
  • Figure 27B shows the effect of inhibitors of calcium channel opening on protein synthesis measured by
  • Figure 28 presents a model of the activation of the unfolded protein response (UPR).
  • Endoplasmic reticulum (EnR) stress activates the three arms of the UPR.
  • BHPI activates all 3 arms of the UPR.
  • PERK arm of the UPR By activating the PERK arm of the UPR, BHPI induces phosphorylation of elF2a and inhibits protein synthesis.
  • RNAi knockdown of PERK reduces BHPI-stimulated phosphorylation of elF2a ( Figure 29).
  • Figure 30A is well correlated with the increase in phosphorylation of elF2a ( Figure 30B).
  • BHPI does not induce phosphorylation of elF2a in ERa negative cells ( Figure 30C).
  • CHOP C/EBP homology protein
  • GADD34 Growth arrest and DNA damage-inducible protein 34
  • EnR stress induces the oligomerization and phospho-activation of the transmembrane kinase PERK.
  • P-PERK phosphorylates eukaryotic initiation factor 2a (elF2a), leading to inhibition of protein synthesis and a reduction in the endoplasmic reticulum protein folding load.
  • Reduced protein synthesis increases levels of the transcription factor, ATF4 (ATF4: activating transcription factor 4), which induces the transcription factor CHOP, which induces GADD34 and several pro-apoptotic genes.
  • ATF4 ATF4: activating transcription factor 4
  • Activated IRE1 a removes an intron from full-length XBP1 (fl-XBP1 : full length X-box binding protein 1 ) mRNA, producing spliced (sp)-XBP1 mRNA, which is subsequently translated into sp-XBP1 protein (sp-XBP1 : spliced X-box binding protein 1 ).
  • sp-XBP1 increases the protein-folding capacity of the EnR and turnover of misfolded proteins by inducing EnR resident-chaperone protein genes (BiP, HEDJ, SERP1 ) (SERP1 : stress-associated endoplasmic reticulum protein 1 ) (HEDJ: heat shock protein 40 co-chaperone domain J), EnR-associated degradation (ERAD) genes and alters mRNA decay and translation.
  • EnR resident-chaperone protein genes BoP, HEDJ, SERP1
  • SERP1 stress-associated endoplasmic reticulum protein 1
  • HEDJ heat shock protein 40 co-chaperone domain J
  • ESD EnR-associated degradation
  • EnR stress promotes the translocation of the transmembrane protein, ATF6a, from the EnR to the Golgi Apparatus, where it encounters proteases that liberate the N-terminal fragment of ATF6a (sp-ATF6a: spliced activating transcription factor 6a).
  • sp-ATF6 increases the protein-folding capacity of the EnR by inducing EnR-resident chaperones, including BiP and GRP94 (GRP94: glucose regulated protein 94 kilo Daltons; also known as HSP90B1 ).
  • BHPI inhibits protein synthesis by activating the unfolded protein response (UPR).
  • E2-ERa elicits transient anticipatory activation of the endoplasmic reticulum stress sensor, the UPR.
  • the possibility that BHPI elicits sustained near- quantitative inhibition of protein synthesis by distorting the normal ability of E2-ERa to induce transient activation of the UPR was tested.
  • Moderate and transient activation of the UPR is usually protective, while extensive and sustained UPR activation induces cell death.
  • the UPR is activated by multiple mechanisms, including release of Ca 2+ from the lumen of the EnR into the cytosol. This activates the transmembrane kinase PERK by autophosphorylation.
  • P- PERK phosphorylates eukaryotic initiation factor 2a (elF2a), inhibiting translation of most mRNAs ( Figure 28A).
  • the other arms of the UPR initiate with activation of the transcription factor ATF6 ( Figure 28C), leading to increased protein folding capacity and activation of the splicing factor IRE1 a, which alternatively splices the
  • transcription factor XBP1 transcription factor 1 , resulting in production of active spliced (sp)-XBP1 and increased protein folding capacity (Figure 28B).
  • Example 9b - BHPI induces RAPID phosphorylation and activation of PERK, and PERK knockdown prevents BHPI from rapidly inhibiting protein synthesis
  • Figure 29A is a Western blot analysis showing the effect of BHPI on protein phosphorylation and levels of PERK and elF2a.
  • Figure 29B-1 is a Western blot analysis showing the effect of RNAi knockdown of PERK on
  • Figure 29B-2 shows the of RNAi knockdown of PERK on protein synthesis measured by incorporation of 35 S-methionine into protein.
  • Figure 29C shows the qRT-PCR results of the effect of RNAi knockdown on PERK mRNA levels.
  • Figure 29D is a Western blot analysis showing the effect of RNAi knockdown on PERK on PERK protein level.
  • BHPI induces phosphorylation of PERK 30 minutes following BHPI treatment.
  • Western blot analysis using ERa positive MCF-7 breast cancer cells was carried out. Blots were probed using an antibody that only detects phosphorylated and activated PERK, and antibodies for total protein levels of PERK, and ⁇ -actin.
  • siRNA knockdown of PERK reduces the ability of BHPI to inhibit protein synthesis.
  • ERa positive MCF-7 cancer cells were maintained for 4 days in 5% cd-FBS + MEM. Cells were harvested in 10% cd-calf serum + MEM without antibiotics, and plated in 96-well plates at a density of 7,500 cells/well. On day 5, cell medium was replaced with antibiotic-free medium
  • Figure 30A shows the incorporation of 35 S-methionine into protein as a function of time after addition of BHPI.
  • the time course of BHPI inhibition of protein synthesis parallels the time course of increased
  • FIG. 30A shows the time course of BHPI inhibition of protein synthesis.
  • ERa positive MCF-7, T47D, and BG-1 cells were incubated for the indicated times in 1 ⁇ BHPI.
  • 35 S-methionine incorporation into protein was reduced by approximately 50%.
  • Figure 30B shows the Western blots of the effect of BHPI on phosphorylation and level of elF2a in different cell types as a function of time after addition of BHPI.
  • BHPI increases P-elF2a at 30 minutes.
  • BHPI increases P-elF2a in ERa positive MCF-7 cells ( Figure 30B-1 ), BG-1 cells ( Figure 30B-2), and T47D cells ( Figure 30B-3).
  • BHPI increases elF2a phosphorylation in ERa positive MCF-7 cells ( Figure 30B-4).
  • Figure 30C contains Western blots showing the effect of BHPI on phosphorylation and level of elF2a in ERa negative cancer cells.
  • BHPI does not increase P-elF2a in ERa negative MDA MB-231 cells ( Figure 30C). Since the UPR activator tunicamycin (TUN) increased P-elF2a in these cells, the absence of BHPI induced phosphorylation of elF2a in the MDA MB-231 cells was not due to the inability of UPR activation to induce elF2a phosphorylation.
  • Phospho-elF2a was visualized by Western blotting using a phosphospecific antibody, which detected phosphorylation at Ser-51 . Immunoblotting used antibodies for phospho-elF2a, elF2a and ⁇ -actin as an internal standard.
  • Figure 30D and Figure 30E show the results of qRT-PCR of mRNA levels of UPR-related mRNAs in ERa-positive cancer cells treated with BHPI.
  • the induction of CHOP and GADD34 mRNAs in MCF-7 cells ( Figure 30D) and CHOP mRNA in BG-1 cells ( Figure 30E) following treatment with 1 ⁇ BHPI are shown.
  • Example 9d - BHPI induces activation of the IRE1 a-branch of the UPR in MCF-7 cells and blocks E2-ERg induction of XBP1 mRNA
  • UPR activation results in translocation of ATF6a from the endoplasmic reticulum to the Golgi where ATF6a protein is cleaved to yield active sp-ATF6a.
  • the sp-ATF6a then moves to the nucleus where it is a transcription factor that helps increase transcription of genes that encode chaperones that help fold proteins.
  • BHPI induces activation of the IRE1 a-branch of the UPR in MCF-7 cells, and blocks E2-ERa induction of XBP1 mRNA. 10 nM E2 induces XBP1 mRNA which is blocked by treatment with BHPI.
  • ERa positive MCF-7 human breast cancer cell lines were maintained in 5% cd-FBS + MEM for 4 days to deplete cells of endogenous estrogens.
  • Cells were harvested in 10% cd-CS and plated into 6-well plates at a density of 450,000 cells per well. On day 5, the medium was replaced with fresh 10% cd-CS.
  • the cells were pre-treated with either 0.1 % DMSO vehicle control (-E2, +E2 samples) or 1 ⁇ BHPI for 30 minutes prior to treating cells with either 10 nM E2 (+E2 and +E2, BHPI samples) or a 0.1 % ethanol-vehicle control (-E2 and -E2, BHPI samples).
  • FIG. 31 A shows the results of qRT-PCR of unspliced XBP-1 mRNA in MCF-7 ERa-positive cancer cells treated with BHPI and no estrogen.
  • Figure 31 B shows the results of qRT-PCR of spliced XBP-1 mRNA in MCF-7 ERa positive cancer cells treated with BHPI and no estrogen.
  • Figure 31 C shows the results of qRT-PCR of unspliced XBP-1 mRNA in MCF-7 ERa-positive cancer cells treated with BHPI with and without estrogen.
  • Figure 31 D shows the results of qRT-PCR of spliced XBP-1 mRNA in MCF-7 ERa- positive cancer cells treated with BHPI with and without estrogen.
  • 10 nM E2 or 1 ⁇ BHPI can activate the IRE1 a-branch of the UPR as indicated by increased levels of spliced-XBP1 mRNA (sp-XBP1 ) ( Figure 31 B and Figure 31 D), but co-treatment of cells with both 10 nM E2 and 1 ⁇ BHPI blocks IRE1 a activation ( Figure 31 D).
  • qRT-PCR analysis was carried out using primers designed to only detect XBP1 mRNA lacking the suppressor intron, which is removed by activated IRE1 a.
  • UPR activation results in the activation of the protein sensor, IRE1 a.
  • IRE1 a is an endoribonuclease, which upon activation, removes a suppressor intron (piece of RNA sequence) from XBP1 mRNA.
  • the spliced form of XBP1 mRNA is translated into XBP1 protein. Removal of the suppressor intron produces a more potent XBP1 protein capable of initiating the gene transcription program of the UPR.
  • Analysis of spliced XBP-1 (sp-XBP1 ) serves as a downstream readout of IRE1 a activation, and thus activation of this branch of the UPR.
  • BHPI activates all 3 arms of the UPR.
  • PERK arm of the UPR By activating the PERK arm of the UPR, BHPI induces phosphorylation of elF2a and inhibits protein synthesis.
  • RNAi knockdown of PERK reduces BHPI- stimulated phosphorylation of elF2a ( Figure 29).
  • Figure 30A the time course of BHPI inhibition of protein synthesis
  • Figure 30B shows that BHPI does not induce phosphorylation of elF2a in ERa negative cells.
  • additional proof that BHPI activates the UPR is shown by the induction of the downstream factors CHOP and GADD34 (see model in Figure 28B and Figure 28C and data in Figure 30D and Figure 30E).
  • Example 9e - BHPI activates the ATF6a branch of the UPR
  • BHPI activates the ATF6a branch of the UPR.
  • Western blot analysis showing levels of full-length (fl-ATF6a) and spliced-ATF6a (sp-ATF6a) in BHPI- treated cells is shown in Figure 32.
  • MCF-7 cells Figure 32A
  • T47D cells Figure 32B
  • Cells were incubated for 4-days in 5% cd-FBS to deplete cells of estrogens and plated at a density of 250,000 cells per well in 10% cd-calf serum. Cells were incubated with 10 nM 173-estradiol or ethanol for 24 hours prior to treatment with either 1 ⁇ BHPI or a DMSO control, and protein samples were collected at the indicated times.
  • ATF6a bands signals were normalized using the signals from the appropriate ⁇ -Actin band, and then normalized to the lowest signaling intensity band (24 hours).
  • BHPI induces an increase in spliced ATF6a, 30 minutes post-treatment. Consistent with previous studies using well-establish UPR activators, BHPI also induces an acute decrease in fl-ATF6a, followed by a rebound in fl-ATF6a levels.
  • BHPI activates all 3 arms of the UPR.
  • PERK arm of the UPR By activating the PERK arm of the UPR, BHPI induces phosphorylation of elF2a and inhibits protein synthesis.
  • RNAi knockdown of PERK reduces BHPI- stimulated phosphorylation of elF2a ( Figure 29).
  • Figure 30A the time course of BHPI inhibition of protein synthesis
  • Figure 30B is well correlated with the increase in phosphorylation of elF2a
  • BHPI does not induce phosphorylation of elF2a in ERa negative cells (Figure 30C).
  • Example 10 - BHPI inhibits protein synthesis by inducing phosphorylation of eEF2
  • BHPI inhibits protein synthesis by inducing
  • Example 10a - BHPI inhibits protein synthesis through activation of AMPK. leading to phosphorylation of eEF2
  • FIG. 33A is a Western blot analysis eEF2 phosphorylation (Thr-56) over time in BHPI-treated ERa+ MCF-7 cells.
  • Figure 33B is a Western blot analysis showing the time course of decreasing eEF2K (Ser-366) phosphorylation in BHPI-treated cells. Ser-366 dephosphorylation activates eEF2K.
  • Figure 33C is a Western blot analysis of the time course of AMPKa (Thr-1 72) and ⁇ (Ser-108) phosphorylation in BHPI-treated cells.
  • Figure 33D-1 shows the results of qRT-PCR analysis showing changes in p58 IPK (p58 IPK : protein 58 kilo Dalton inhibitor of interferon protein kinase) mRNA with -E2 set to 1
  • BHPI phosphorylation of eukaryotic elongation factor 2 is a second site of BHPI inhibition of protein synthesis. After approximately 2 hours, BHPI establishes a secondary pathway for inhibition of protein synthesis in ERa positive cancer cells by phosphorylation and inactivation of eukaryotic elongation factor 2, (eEF2) ( Figure 33A, Figure 34A). In ERa negative HeLa cells, BHPI did not elicit formation of P-eEF2, but eEF2 was phosphorylated by the eEF2 kinase activators forskolin (FOR) and rotterlin (Rot) ( Figure 34B).
  • FOR eEF2 kinase activators forskolin
  • Rot rotterlin
  • eEF2 phosphorylation is regulated by a single upstream kinase, eukaryotic elongation factor 2 kinase (eEF2K).
  • eEF2K eukaryotic elongation factor 2 kinase
  • Figure 33B BHPI induced dephosphorylation of eEF2K at Ser-366 and activation of eEF2K
  • Figure 33C BHPI-induced phosphorylation and inactivation of eEF2 in ERa positive cancer cells occurs by rapid phosphorylation and activation of the metabolic sensor, AMP kinase (AMPK) ( Figure 33C).
  • AMPK AMP kinase
  • BHPI activates the pathway P-AMPKT->eEF2KT->P-eEF2i(inactive), inhibiting elongation and protein synthesis. Also, P-eEF2 is rapidly degraded, reducing eEF2 levels ( Figure 33A and Figure 35A). Since protein synthesis is inhibited at both initiation and elongation, eEF2 cannot be replenished.
  • Figure 34 is a Western blot analysis showing the effect of BHPI on phosphorylated and unphosphorylated eEF2 in ERa positive and ERa negative cancer cells.
  • Western blots of the time course of BHPI effects on phosphorylation of eEF2 (at Thr-56) in ERa positive T47D cell are shown in Figure 34A, and in ERa negative HeLa cells in Figure 34B.
  • Cells were maintained for 4-days in CD-FBS to deplete cells of estrogens, plated at 225,000 cells/well, and induced for 24 hours with E2 before treating cells for the indicated times with a DMSO control (+E2), 1 ⁇ BHPI (+E2, BHPI), or 10 ⁇ forskolin (+E2, FOR).
  • BHPI phosphorylation of eukaryotic elongation factor 2 is a second site of BHPI inhibition of protein synthesis. After approximately 2 hours, BHPI establishes a secondary pathway for inhibition of protein synthesis in ERa positive cancer cells by phosphorylation and inactivation of eukaryotic elongation factor 2, (eEF2) ( Figure 33A, Figure 34A). In ERa negative HeLa cells, BHPI did not elicit formation of P-eEF2, but eEF2 was phosphorylated by the eEF2 kinase activators forskolin (FOR) and rotterlin (Rot) ( Figure 34B). [0455] Example 10c - Conventional UPR activators induces transient elF2a phosphorylation and inhibition of protein synthesis and do not induce
  • Figure 35A is a Western blot analysis showing the time course of Thapsigargin (THG) effects on phosphorylation of elF2oc (Ser-51 ) and eEF2 (Thr-56).
  • Figure 35B shows incorporation of 35 S-methionine into protein as a function of time in cells treated with THG. Unlike BHPI, thapsigargin does not induce phosphorylation of eEF2, induces transient phosphorylation of elF2a and protein synthesis shows partial recovery after 4 hours.
  • Figure 35C is a Western blot analysis of
  • FIG. 35D shows the induction of CHOP mRNA following treatment of MCF-7 cells with 10 ⁇ g/mL of the UPR activator tunicamycin. CHOP mRNA levels were determined by qRT-PCR with the ribosomal protein 36B4 as an internal standard. CHOP, a downstream marker for activation of the PERK arm of the UPR (see Figure 28A) is induced at 4 hours after TUN treatment.
  • Figures 35E and 35F show the analysis of the time course of tunicamycin (TUN) effects on BiP and p58 IPK levels.
  • Figure 35E is a Western blot analysis showing the effect of TUN on levels of BiP protein.
  • the chaperone BiP is induced at 8 and 24 hours after TUN treatment.
  • Figure 35F is a Western blot showing the effect of TUN on levels of p58 IPK .
  • the protein p58 IPK which reverses PERK phosphorylation is also induced at 8 and 24 hours.
  • BHPI induces persistent activation of the PERK arm of the UPR as shown by elF2a phosphorylation at 24 hours (Figure 30B), inhibition of protein synthesis at 24 hours (Figure 20) and a decline in levels of BiP and p58 IPK after 8 hours (Figure 33E and Figure 33F).
  • Example 1 1 E2, acting through binding to ERa, opens the IP3R calcium channel in the endoplasmic reticulum causing an efflux of calcium from the interior of the endoplasmic reticulum into the cytosol
  • E2 acting through binding to ERa, opens the IP3R calcium channel in the endoplasmic reticulum causing an efflux of calcium from the interior of the endoplasmic reticulum into the cytosol in breast cancer cells ( Figure 36) and ovarian cancer cells. Since RNAi knockdown of ERa abolishes the calcium increase in the cytosol ( Figure 38), it is mediated through ERa. BHPI also opens the IP3R calcium channel in the endoplasmic reticulum and causes a much more massive calcium efflux than E2. This is consistent with the idea that BHPI is working by distorting a normal action of E2-ERa and converting it from protective to lethal.
  • Estrogen stimulates calcium release from the endoplasmic reticulum through IP3R Ca 2+ -channels.
  • Figure 37A shows the effects of 300 nM estrogen (E2) on cytosolic calcium levels in T47D breast cancer cells pre-treated with 2-APB, ryanodine, or ethanol-vehicle for 30 minutes in the absence of extracellular calcium (0 mM CaCI 2 ). Visualization of intracellular Ca 2+ was done using Fluo-4. The highest Ca 2+ concentrations are shown with the brightest white ( Figure 37A-1 ).
  • Figure 37A-2 shows the quantitation of cytosolic calcium levels in ERa positive T47D breast cancer cells treated with E2 in the absence of extracellular calcium, and in cells pre- treated with 2-APB or ryanodine in the absence of extracellular calcium.
  • E2 was added at 60 seconds, and fluorescence intensity prior to 60 seconds was set to 1 .
  • blocking calcium release from the endoplasmic reticulum through IP3R Ca 2+ -channels blocks E2-activation of the PERK arm of the UPR.
  • Example 1 1 c - Removing ERa from breast cancer cells prevents estrogen-induced Ca 2+ -release from the endoplasmic reticulum.
  • FIG. 38 shows the effect of estrogen on cytosolic calcium levels after ERa knock down in T47D cells.
  • Cells were treated with 50 nM non-coding (NC) siRNA or ERa siRNA SmartPool followed by 300 nM E2.
  • NC non-coding
  • E2 E2-mediated calcium release from the endoplasmic reticulum was dependent on ERa.
  • RNAi knockdown of ERa prevented E2- stimulated calcium release from ERa positive T47D cells ( Figure 38).
  • EnR calcium homeostasis is regulated by the IP3R (inositol
  • Example 1 1 d E2-ERg activates the IRE1 a and ATF6a branches of the UPR, inducing the production of the major EnR chaperone, BiP, and others
  • FIG 39A shows the results of qRT-PCR of the effect of E2 on the level of spliced XBP1 mRNA.
  • E2-ERa induces splicing of XBP1 mRNA. This indicates that E2-ERa activates the IRE1 a branch of the UPR. Activation of the IRE1 a branch of the UPR activates the nuclease activity in IRE1 a, enabling it to splice XBP-1 mRNA (model in Figure 28). Thus, formation of spliced XBP-1 mRNA serves as a readout for activation of the IRE1 a branch of the UPR.
  • FIG 39B shows the results of qRT-PCR of the effect of E2 on the levels of SERP1 and ERDJ (ERDJ: endoplasmic reticulum- (ER-) localized DnaJ) mRNAs.
  • E2-ERa stimulates induction of downstream transcriptional targets of spliced-XBP1 , SERP1 and ERDJ.
  • the increase in SERP1 and ERDJ mRNA coincides with increased splicing of XBP1 mRNA, which together indicate that E2-ERa stimulates activation of the IRE1 a of the UPR.
  • -E2 treatment set to 1 .
  • * P ⁇ 0.05, ** P ⁇ 0.01 compared with -E2 samples.
  • Figure 39C shows the results of qRT-PCR of the effect of E2 and antiestrogens on the level of spliced XBP1 mRNA.
  • qRT-PCR comparing the effect of ICI 182,780 and 4-hydroxytamoxifen (4-OHT) on E2-ERa of sp-XBP1 in T47D breast cancer cells is shown (-E2 set to 1 ).
  • Figure 39D shows the qRT-PCR results of the effect of RNAi knockdown of ERa on the level of spliced XBP1 mRNA. RNAi knockdown of ERa abolishes E2-ERa induction of sp-XBP1 .
  • E2-ERa activates the ATF6a-branch of the UPR, as indicated by increased levels of spliced ATF6a (sp-ATF6a).
  • Western blot analysis showing full- length ATF6a (fl-ATF6a) and spliced-ATF6a (sp-ATF6a) in E2-treated ERa positive cells is shown in Figure 39E, Figure 39F, and Figure 39G.
  • Figure 39E is a Western blot analysis of the effect of E2 and antiestrogens on the level of full-length and spliced ATF6a protein in T47D breast cancer cells.
  • Figure 39F is a Western blot analysis of the effect of E2 on the level of full-length and spliced ATF6a protein in BG-1 ovarian cancer cells.
  • Figure 39G is a Western blot analysis of the effect of E2 on the level of full-length and spliced ATF6a protein in PE04 ovarian cancer cells.
  • Western blot analysis was carried out using an antibody that detects the N-terminal fragment of ATF6a, in both the 90-kDA full-length ATF6a (fl-ATF6a) protein and the 50-kDa spliced or activated form of ATF6a (sp-ATF6a), and an antibody that detects ⁇ -actin.
  • FIG. 39H is an qRT-PCR analysis of the effect of E2 on the level of BiP mRNA in ERa positive MCF-7 breast cancer cells and PE04 ovarian cancer cells.
  • Figure 391 is a Western blot analysis of the effect of E2 on the level of BiP protein in MCF-7 cells.
  • Figure 39J is a qRT-PCR analysis of the effect of RNAi knockdown of ERa and E2 on the level of BiP mRNA. RNAi knockdown of ERa abolishes E2-induction of BiP.
  • Figure 39D shows that RNAi knockdown of ERa prevents E2 induction of spliced XBP-1 .
  • E2 activation of the UPR is mediated through its binding to ERa.
  • Figure 39I unlike BHPI, estrogen induces BiP chaperone at 24 hours.
  • Example 1 1 e - E2-ERg activates the PERK arm of the UPR
  • Figure 40 shows that E2-ERa activates the PERK arm of the UPR.
  • Figure 40A Western blot analysis showing the effect of E2 on levels of phosphorylated and unphosphorylated PERK.
  • Figure 40B is a Western blot analysis showing the effect of E2 on levels of phosphorylated and unphosphorylated elF2a.
  • Example 12 E2-ERa-mediated efflux of calcium from the interior of endoplasmic reticulum into the cvtosol is required for E2-ERg-stimulated proliferation of cancer cells and for E2-ERg-requlation of gene expression
  • Figure 41 shows that elevation of cytosolic calcium, mediated through Ca 2+ -release from the endoplasmic reticulum, is required for E2-ERa mediated gene expression and E2-ERa stimulated cell proliferation in breast and ovarian cancer cells. Elevation of cytosolic calcium, mediated through Ca 2+ -release from the endoplasmic reticulum, is required for E2-ERa mediated gene expression and E2- ERa stimulated cell proliferation in breast and ovarian cancer cells.
  • Figure 41 A shows the effects of the intracellular calcium chelator BAPTA-AM (BAPTA-AM: 1 ,2- Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester, intracellular calcium chelator) on E2-ERa stimulated cell proliferation.
  • BAPTA-AM 1 ,2- Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester, intracellular calcium chelator
  • E2-ERa stimulated proliferation of MCF-7 breast cancer cells (Figure 41 B) and BG-1 ovarian cancer cells ( Figure 41 C) treated with 200 ⁇ ryanodine (RyR), 200 ⁇ 2-aminoethyl diphenylborinate (2- APB), or both inhibitors (RyR + 2-APB) for 4 days.
  • Figure 41 D shows ERE-luciferase activity of kBluc-T47D breast cancer cells treated with E2 and 100 ⁇ ryanodine (RyR), 200 ⁇ 2-APB, or both inhibitors (RyR + 2-APB).
  • the dot denotes cell number at day 0. * P ⁇ 0.05; ** P ⁇ 0.01 .
  • Locking ryanodine channels with ryanodine and IP3R channels with 2-APB produced 20% and 40% declines in E2- stimulated proliferation, respectively.
  • Treatment of MCF-7 cells with 2-APB and ryanodine together blocked E2-ERa induced cell proliferation of MCF-7 breast and BG-1 ovarian cancer cells ( Figure 41 B and Figure 41 C), and strongly inhibited E2- ERa induced expression of a stably transfected ERE-luciferase reporter gene (Figure 41 D).
  • Example 13 - UPR is up-regulated in estrogen-treated tumors
  • the UPR is up-regulated in estrogen-treated tumors in human xenograft tumors in mice and by using bioinformatics in cell culture samples taken at different stages of tumor progression.
  • E2-ERa regulates the UPR in MCF-7 breast cancer cells and mouse xenograft, and elevated E2-ERa activity is correlated with increased UPR activity in patient tumor samples.
  • Figure 42B shows MCF-7 tumor growth in the presence or absence of estrogen in athymic mice. All mice were treated with estrogen to induce tumor formation. On "day 0", E2 in silastic tubes was removed from the -E2 group, while silastic tubes were retained in +E2 treatment group.
  • Figure 42C is a qRT-PCR analysis of classical E2-ERa regulated genes showing levels of GREB-1 and pS2 mRNAs in mouse tumors with and without E2.
  • Figure 42E is an analysis of publically available patient microarray data showing levels of estrogen-regulated mRNAs in normal breast epithelium from normal patients, normal breast epithelium in patients with invasive ductal carcinoma of the breast and invasive ductal carcinoma tissue.
  • Figure 42E shows relative mRNA levels of classical E2-ERa regulated genes.
  • Figure 42F is an analysis of publically available patient microarray data showing levels of UPR-related mRNAs in normal breast epithelium from normal patients, normal breast epithelium in patients with invasive ductal carcinoma of the breast and invasive ductal carcinoma tissue.
  • p-values represent comparisons to histologically normal breast epithelium from patients who underwent reduction mammoplasty. * P ⁇ 0.05; ** P ⁇ 0.01 ; *** P ⁇ 0.001 ; ns, not significant.
  • E2-ERa action Increases expression and activation of the UPR. Since E2-ERa acts at endoplasmic reticulum to activate all 3 arms of the UPR and induces formation of sp-XBP1 , spATF6a and P-PERK ( Figure 39 and Figure 40), the effect of E2 on levels of the mRNAs encoding UPR sensors and downstream targets was investigated. E2 rapidly induced mRNAs encoding sensors for all 3 UPR arms and the chaperones BiP and GRP94 ( Figure 42A). These were early responses, which generally tapered off at later times points (i.e., 24 hours). However, estrogen produced sustained increases in resident chaperones and some component of the UPR, such as elF2a ( Figure 42A).
  • ATF6a mRNA ATF6a arm
  • PERK and p58 IPK mRNA PERK arm
  • E2-ERa activity and UPR pathway activity were compared in histologically normal breast epithelium, taken from patients either undergoing reduction mammoplasty or at the time of diagnosis of breast cancer, with carcinoma samples from patients diagnosed with invasive ductal carcinoma (IDC). IDC samples displayed higher levels of ERa mRNA; higher levels of pS2 and GREB-1 mRNA, which are classical E2-upregulated genes; and lower levels of IL1 -R1 mRNA, which is an E2-downregulated gene ( Figure 42E).
  • variable expression of ERa mRNA and protein or E2-ERa pathway activity correlates with expression of UPR genes in ERa positive cancer was assessed. Expression of several UPR genes displayed highly significant correlation with expression of ERa and ERa-target genes.
  • Figure 43 is a model of the effects of estrogen, acting through ERa, on the activation of the UPR.
  • the model illustrates the pathway identified by which E2- ERa activates the UPR and the consequences of that activation.
  • E2-ERa rapidly opens the IP3R calcium channel in the endoplasmic reticulum. This allows calcium to move from the inside of the endoplasmic reticulum, where it is present in high concentration, into the cytosol, where the calcium concentration is low. The increased calcium cytosol is required for estrogen to stimulate gene expression and cell proliferation ( Figure 41 ).
  • Example 15 - Activation of the UPR is often protective
  • Example 15a Anticipatory activation of the UPR by estrogen protects cells from exposure to higher levels of subsequent cell stress
  • FIG 44 shows the effect of prior activation of the UPR by E2 and by TUN on subsequent cell proliferation in cells later treated with TUN.
  • Anticipatory activation of the UPR by estrogen protects cells from exposure to higher levels of subsequent cell stress.
  • Weak anticipatory activations of the UPR with estrogen or tunicamycin protects cells from subsequent UPR stress.
  • Estrogen protects cells from subsequent exposure to higher levels of stress. Previous studies have demonstrated a UPR pre-conditioning phenomenon, whereby transient exposure to mild UPR stress protects cells from subsequent cell stress. (Rutkowski, D.T. and Kaufman, R.J. , That which does not kill me makes me stronger: adapting to chronic ER stress. Trends Biochem Sci. 2007; 32(10):469-76.) In one such study, treatment of cells with very low doses of the UPR activator, tunicamycin, resulted in mild UPR activation, which stimulated an adaptive response that protected cells from subsequent exposure to tunicamycin.
  • E2 should also induce an adaptive UPR response, which protect cancer cells from subsequent exposure to UPR stress.
  • tunicamycin TUN
  • E2 and tunicamycin had nearly identical effects; each elicited an approximate 10 fold increase in the concentration of tunicamycin required to induce apoptosis
  • FIG. 45 is a table showing the genes that comprise the UPR gene index used in bioinformatics studies.
  • the components of the UPR index include the 3 primary UPR sensors, direct readouts of each arm of the UPR, genes whose expression responds to activation of the UPR, chaperone protein that help fold proteins in the endoplasmic reticulum, and ERAD proteins that help degrade unfolded proteins at the endoplasmic reticulum.
  • the UPR index components represent a broad set of genes related to the UPR and the folding and destruction of unfolded proteins.
  • These UPR genes independently predictive either of relapse free or overall survival (p ⁇ 0.05) were used to construct the UPR gene signature, which was then used to carry out risk prediction analysis.
  • EROI La endoplasmic reticulum oxidoreductin 1 -like protein a
  • ER01 1_ ⁇ endoplasmic reticulum oxidoreductin 1 -like protein ⁇
  • ERAD endoplasmic reticulum associated protein degradation
  • EDEM1 endoplasmic reticulum degradation enhancer, mannosidase alpha-like 1
  • HERPUD1 homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1
  • HRD1 HMG-CoA reductase degradation protein 1 .
  • Example 15c - The UPR genomic index is a new biomarker that predicts relapse free and overall survival of breast cancer patients
  • Figure 46 is a bioinformatic analysis of publically available microarray data from ERa positive breast cancer cohorts.
  • Figure 46A-1 and Figure 46A-2 show bioinformatic analysis of data from two microarray chips (U133A) ( Figure 46A-1 ) and (U133B) ( Figure 46A-2) showing Kaplan-Meier survival plots comparing time of relapse-free survival in breast cancer patients expressing high and low levels of UPR index genes. These data show that high expression of the UPR index is associated with a shorter interval of relapse-free survival.
  • Figure 46B is a bioinformatic analysis of data from two microarray chips (U133A and U133B) showing time to relapse in 277 breast cancer patients, hazard ratio, and p- Values for individual components of the UPR gene index. Elevated expression of individual components of the UPR gene index is predictive of reduced survival.
  • Gene abbreviations appearing in Figure 46B include: HUGO: human genome organization; EIF2S1 : eukaryotic initiation factor 2 subunit 1 ; EIF2AK3: eukaryotic initiation factor 2 alpha kinase 3; DDIT3: DNA damage inducible transcript 3; DNAJC3: DnaJ homolog subfamily C member 3; HSPA5: heat shock protein A5; HSP90B1 : Heat shock protein 90 B1 ; and SYVN1 : Synovial apoptosis inhibitor 1 .
  • Figure 46C is a bioinformatic analysis of data from microarray chips (univariate analysis) comparing time to relapse in breast cancer patients using the UPR gene signature and current prognostic markers; (multivariate analysis). Testing with the UPR gene signature provides additional information about time to relapse after including information from several current prognostic markers.
  • Gene abbreviations appearing in Figure 46C include: PPP1 R1 5A: Protein Phosphatase 1 , Regulatory Subunit 15A (another name for GADD34: Growth arrest and DNA damage-inducible protein 34).
  • Figure 46D is a bioinformatic analysis of microarray data showing time to relapse in 474 breast cancer patients, hazard ratio, and p- Value for individual components of the UPR gene index.
  • Microarray analysis was performed prior to initiation of tamoxifen therapy. Since all these patients were treated with tamoxifen, elevated expression of the UPR gene index predicts the subsequent response of tumors to tamoxifen years later. Thus, the UPR gene index is a powerful predictor of the future prognosis of ERa positive cancer patients.
  • Figure 46E is a bioinformatic analysis of microarray data from two microarray chips (U133A and U133B) showing time to relapse in 236 breast cancer patients; shown are hazard ratio and p-Values for individual components of the UPR gene index. All Kaplan-Meier plots assessing UPR risk prediction were computed using leave-one-out cross-validation. UPR signature genes shown in the tables are listed with their respective univariate Cox hazard ratio and p-value testing the hypothesis if expression data is predictive of relapse or overall survival. Expression of individual components of the UPR gene index predicts overall survival of ERa positive breast cancer patients. Because data from several independent cohorts of ERa positive cancer patients was used, and each cohort produces a similar outcome, the data is especially strong and is not due to an artifact in producing data from a single patient cohort.
  • Example 15d Expression of UPR genes is positively correlated with expression of ERa and ERa-regulated target-genes
  • Figure 47 is a bioinformatic analysis of microarray data from ERa positive breast cancer patients comparing expression of classical estrogen-regulated genes and UPR index components. Correlations between the UPR and ERa protein levels (ERa), ERa mRNA levels (ESR1 ), or transcriptional activity of E2-ERa were analyzed. E2-ERa transcriptional activity was assessed using downstream target genes of E2-ERa (pS2, GREB1 ). Analysis was carried out on a cohort of 278 breast cancer patients (GSE20194), which consists of 164 ERa positive tumors and 1 14 ERa negative tumors. Quantitation of ERa protein was by IHC. Pearson correlation coefficients and parametric p-values are shown in the table, "n.s.” indicates that no significant correlation was observed. Gene abbreviations appearing in Figure 46C include: TRIB3: tribbles homolog 3.
  • UPR index While expression of UPR genes is correlated with ERa levels and expression of ERa-regulated genes, the UPR index is not simply a surrogate marker for ERa activity. In multivariate analysis, the UPR index, but not ERa, or classical ERa-regulated genes, exhibits a statistically significant increase in hazard ratio. Also, UPR index exhibits predictive power to stratify patients into high and low risk groups above ERa status. Thus, while active ERa is important for expression of the UPR signature, it's the UPR signature, not ERa level or activity, that is predictive of reduced time to recurrence and reduced survival.
  • Expression of ERa-regulated genes in the cancers provides a measure of how active ERa is in the tumors.
  • High activity of ERa, as measured by high expression of the ERa-regulated genes is associated with high expression of the UPR gene index. This helps tie the elevated expression of the UPR in the most aggressive tumors to ERa.
  • Example 15e - Expression of the UPR gene signature predicts relapse-free and overall survival in ERa positive breast tumor cohorts
  • Figure 48 is a bioinformatic analysis of publically available microarray data from ERa positive breast cancer cohorts.
  • Figure 48C is a bioinformatic analysis of data from microarray chips (univariate analysis) comparing time of relapse-free survival and overall survival in breast cancer patients using the UPR gene signature and current prognostic markers; (multivariate analysis) Testing was done to determine whether the UPR gene signature provides additional information about time of relapse-free survival and overall survival over and above information from several current prognostic markers.
  • Figure 48C shows univariate and multivariate Cox regression analysis of the UPR signature, clinical covariates, and classical estrogen-induced genes for time to recurrence and survival (n.s., not significant). Median used to classify tumors into high and low risk groups.
  • the UPR gene signature predicts clinical outcome in ERa positive breast cancer. Activation of the UPR pathway represents a novel prognostic indicator predictive of relapse and survival in ERa positive breast cancer.
  • Activation of the UPR pathway represents a novel prognostic indicator predictive of relapse and survival in ERa positive breast cancer.
  • a UPR gene signature consisting of genes encoding components of the UPR pathway and downstream targets of UPR activation was developed (Figure 45).
  • Figure 45 We next explored whether the UPR signature was a useful prognostic marker. Using data from 261 ERa positive breast cancer patients, each assigned to a high- or low- genomic UPR grade, we observed reduced time to relapse for patients
  • Example 16 - UPR expression is elevated in highly malignant ovarian cancers compared to normal ovarian cells
  • LMP low malignant potential
  • PERK arm of the UPR and TRB3 is a downstream readout of PERK activity.
  • GRP94 and BiP are chaperones and downstream readouts of the ATF6a pathway.
  • ERDJ and SERP1 are downstream readouts of sp-XBP1 and the IRE1 a pathway.
  • Elevated expression of the UPR components is associated with a reduced time to relapse in late-stage ovarian cancer patients. This confirms the advantages of using BHPI to target these tumors that overexpress genes in the UPR pathway. This also confirms the idea of using ERa to target the UPR in ovarian cancer. Because these tumors overexpress genes in the UPR pathway, they will be especially susceptible to BHPI.
  • BHPI is a novel type of ERa inhibitor that may be beneficial in all diseases in which estrogen-ERa is associated with increased cell proliferation and increased expression of the UPR occurs.
  • diseases include breast cancer and ovarian cancer in which estrogen-ERa is associated with increased cell proliferation and with increased expression of the UPR, and cervical, uterine/endometrial, vulval, and liver cancers and endometriosis in which an association between estrogen-ERa stimulated cell proliferation and the underlying pathology of the disease has been identified.
  • estrogen-ERa stimulated cell proliferation is the central feature in the pathology of endometriosis, and BHPI strongly inhibits estrogen-ERa stimulated cell proliferation, BHPI is a viable therapy for endometriosis.
  • BHPI is a unique small molecule whose non-competitive interaction with ERa elicits three effects.
  • (1 ) BHPI potently inhibits protein synthesis by activating the UPR and its PERK-elF2a arm; (2) BHPI inhibits elongation by inducing phosphorylation of eEF2; and (3) BHPI independently inhibits induction and repression of gene expression by E2-ERa.
  • the data indicate these diverse inhibitory effects of BHPI are mediated through ERa.
  • BHPI inhibits protein synthesis in all 14 ERa positive cells tested with no effect on protein synthesis in all 1 2 ERa negative cell lines.
  • ERa is sufficient to make ERa negative MCF1 OA cells sensitive to BHPI inhibition of protein synthesis, and knockdown of the ERa with siRNA, or degrading ERa with ICI 182,780, abolishes BHPI inhibition of protein synthesis.
  • Overexpression of ERa progressively increases BHPI inhibition of protein synthesis.
  • ChIP shows BHPI reduces binding of E2-ERa to gene regulatory regions, suggesting that BHPI reduces affinity for response elements, and overexpression of ERa reverses BHPI inhibition of gene expression.
  • Altered fluorescence emission spectrum and protease sensitivity demonstrate that BHPI interacts directly with ERa. With wishing to be bound by any theory, it is likely that BHPI binding alters ERa conformation, altering interactions with its many binding partners, and leading to the diverse inhibitory effect of BHPI.
  • BHPI works by opening endoplasmic reticulum calcium channels, rapidly depleting calcium stores in the lumen of the endoplasmic reticulum, strongly activating the UPR, and potently inhibiting protein synthesis.
  • the UPR plays important roles in tumorigenesis, therapy resistance, and cancer progression.
  • Moderate and transient UPR activation is protective, while strong and sustained activation triggers cell death.
  • Moderate UPR activation promotes an adaptive stress response leading to increased expression of the UPR and antiapoptic chaperones, and this protects cancer cells from subsequent exposure to higher levels of cell stress.
  • UPR targeting efforts focus on inactivating a protective stress response by inhibiting UPR components
  • UPR overexpression in cancer suggests that sustained pharmacological activation of the UPR represents a novel alternative anticancer strategy.
  • Classical UPR activators are non-specific and highly toxic.
  • BHPI selectively hyperactivates the UPR activation pathway identified for estrogen-ERa. By increasing the amplitude and duration of UPR activation, BHPI converts UPR activation from protective to lethal.
  • BHPI Unlike classical UPR activators, BHPI induces sustained activation of the UPR by severing UPR signaling through inhibition of protein synthesis at a second site. BHPI inhibits elongation through activation of the major metabolic energy sensor, AMPK, leading to phosphorylation and inactivation of eEF2. AMPK plays an important role in breast, ovarian, and endometrial cancers, and AMPK- activating drugs, such as metformin, exhibit potential as anticancer agents. AMPK- activators may have potential as a new way to target the UPR and induce sustained UPR activation in endocrine-related cancers. The ability of BHPI to target two pathways results in long-term inhibition of protein synthesis, blocking proliferation and killing cancer cells.
  • BHPI Independent of its effects on the UPR and inhibition of protein synthesis, BHPI also inhibits E2-ERa-mediated gene expression. Conventional UPR activators do not inhibit E2-ERa-mediated gene expression. Also, at early times when BHPI is fully effective, inhibition of protein synthesis does not inhibit E2-ERa regulated gene expression. Since BHPI inhibits both induction and repression of gene expression by E2-ERa, BHPI inhibition of E2-ERa-regulated gene expression is not due to non-specific toxic effects.
  • BHPI can selectively target cancer cells because its targets, ERa and the UPR, are both overexpressed in breast and ovarian cancers. Despite a role for ERa in gynecological cancers, most ovarian cancer cells show little dependence on estrogens for growth and endocrine therapy is largely ineffective. Other noncompetitive ERa inhibitors have not demonstrated effectiveness in therapy resistant ERa positive ovarian cancer cells. BHPI extends the reach of ERa inhibitors to gynecologic cancers that do not respond to current endocrine therapies and is highly effective in several drug-resistance models including: (1 ) tamoxifen resistant
  • MCF7ERaHA which overexpress ERa
  • BT-474 and ZR-75-1 breast cancer cells (2) tamoxifen-resistant BT-474 and ZR-75-1 breast cancer cells; (3) cisplatin, tamoxifen and ICI 182,780-resistant CaOV3 ovarian cancer cells; and (4) multi-drug resistant OVCAR-3 ovarian cancer cells.
  • BHPI is effective in a broad range of ERa-containing cancers, including, but not limited to breast, ovarian, and endometrial cancers.
  • BHPI is an exceptional candidate for therapeutic exploration.
  • the UPR is classically viewed as a pathway activated in response to intrinsic or extrinsic stresses, which include protein misfolding, environmental stress and drug treatment. In this "reactive mode", UPR sensors are activated in response to endoplasmic reticulum stress. An alternative "anticipatory mode" of UPR activation is observed in B-cell differentiation where UPR activation precedes the massive production and secretion of immunoglobulin by plasma cells. Because the signals responsible for anticipatory activation of the UPR were unknown, this process was not well understood.

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Abstract

L'invention concerne une méthode de traitement d'un cancer positif pour ERα, comprenant l'administration à un patient en ayant besoin d'une quantité efficace d'un inhibiteur à petites molécules ou d'un sel pharmaceutiquement acceptable de celui-ci, l'inhibiteur à petites molécules agissant par l'intermédiaire du récepteur α des œstrogènes et active un ou plusieurs des trois bras de la voie de réponse de la protéine non repliée (UPR).
EP14740515.3A 2013-01-18 2014-01-21 Inhibiteurs d'un récepteur des strogènes Ceased EP2945646A4 (fr)

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CN107164519A (zh) * 2017-06-26 2017-09-15 深圳优圣康医学检验所有限公司 一种基于荧光pcr检测esr1基因突变的引物及探针
PE20210662A1 (es) * 2018-07-03 2021-03-31 Univ Illinois Activadores de la respuesta a proteinas desplegadas
WO2021222738A1 (fr) * 2020-05-01 2021-11-04 The Board Of Trustees Of The University Of Illinois Composés pour cancers positifs au récepteur des oestrogènes
WO2022032146A1 (fr) * 2020-08-07 2022-02-10 Systems Oncology, Llc Procédés d'inhibition de la croissance de cancers era positifs
CN113237860A (zh) * 2021-05-17 2021-08-10 丹望医疗科技(上海)有限公司 检测类器官最大截面积的方法及其应用

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CA2022356A1 (fr) * 1990-07-31 1992-02-01 David J. Shapiro Methode et matieres pour la production de proteines
AU2005214338B2 (en) * 2004-02-13 2011-11-10 President And Fellows Of Harvard College 3-3-di-substituted-oxindoles as inhibitors of translation initiation
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IL240019A0 (en) 2015-09-24
AU2014207272A1 (en) 2015-07-30

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