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WO2006031194A1 - Procede pour cribler un compose presentant une activite antitumorale en utilisant des cellules eucaryotes surexprimant au moins un gene myc et/ou presentant une activite de regulation de mnt deficiente - Google Patents

Procede pour cribler un compose presentant une activite antitumorale en utilisant des cellules eucaryotes surexprimant au moins un gene myc et/ou presentant une activite de regulation de mnt deficiente Download PDF

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WO2006031194A1
WO2006031194A1 PCT/SE2005/001348 SE2005001348W WO2006031194A1 WO 2006031194 A1 WO2006031194 A1 WO 2006031194A1 SE 2005001348 W SE2005001348 W SE 2005001348W WO 2006031194 A1 WO2006031194 A1 WO 2006031194A1
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myc
cells
myra
compound
mnt
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Marie Arsenian Henriksson
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Karolinska Innovations AB
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Karolinska Innovations AB
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

  • the present invention relates to a method of identifying compounds having anti-tumour activity.
  • the myc family of cellular protooncogenes encodes three highly related nuclear phosphoproteins (c-Myc, N-Myc, and L-Myc).
  • c-Myc transcription factor is a key regulator of proliferation, cell growth, differentiation, apoptosis, angiogenesis, and transformation (1, 2).
  • It is a basic-helix-loop-helix-leucine zipper (bHLHZip) protein that carries out its functions through dimerization with Max, another bHLHZip protein, and binding to and activating promoters containing a specific DNA sequence CACGTG, called E- box (3, 4).
  • Myc Transactivation by Myc involves recruitment of histone acetyltransferase (HATs) to target genes through adaptor proteins, such as TREAP (5-7).
  • HATs histone acetyltransferase
  • Myc has also the ability to bind to the Miz-1 protein at initiator elements and thereby repress the transcriptional activation of Miz-1 mediated target genes such as the cyclin-dependent kinase (cdk) inhibitor pl5INK4b (8-11).
  • cdk cyclin-dependent kinase
  • Another Myc family protein, Mnt is expressed in both proliferating and differentiating cells in all tissues analyzed and has been suggested to function as a Myc antagonist (12-14).
  • c-Myc was identified as an oncogene and its role in tumorigenesis in vivo was confirmed by studies in transgenic mice in which c-Myc was constitutively expressed (15). It was demonstrated that tumorigenesis was enhanced by introduction of anti-apoptotic proteins such as Bcl-2 or BCI-X L , by failure to suppress these proteins (16, 17), or by an impaired p53 pathway (18-21).
  • anti-apoptotic proteins such as Bcl-2 or BCI-X L
  • Myc an attractive target for tailored cancer therapy.
  • the Myc protein is expressed only in cycling cells, it activates both proliferation and apoptosis (2, 30-32), and is deregulated in a wide spectrum of human tumours, accompanied by poor differentiation and aggressiveness (33-35).
  • Numerous efforts using different approaches have been made to interfere with c-Myc in order to reverse or eliminate malignant cells (reviewed in 36-38).
  • Two groups have performed screening assays using libraries of low molecular weight compounds. Based on an in vitro FRET-assay Berg et al. reported the identification of four compounds that interfered with Myc/Max dimerization (39).
  • N-myc is another member of the myc family of proto-oncogenes involved in initiation and progression of tumors. Cancers where N-myc overexpression is involved are e.g. neuroblastoma. Neuroblastoma is the most common extracranial solid childhood tumor and is derived from the symphathetic nervous system. It is highly heterogenous and is lethal in approximately 50% of the patients. Amplification of the N-myc gene is correlated to advanced stages of the disease and is found in 40-50% of the tumors with poor prognosis thereby making it one of the most important prognostic markers for neuroblastoma (63).
  • neuroblastoma cells are frozen at an early stage of differentiation and cell lines established from tumors can be used as model systems for neuronal differentiation in vitro since treatment with the phorbolester TPA or all-trans retinoic acid induce differentiation. This is correlated with downregulation of N-Myc expression, decreased or inhibited proliferation and upregulation of neuronal markers (64). It has also been shown that ectopic expression of N-Myc inhibits differentiation. Taken together, these data indicate that N-Myc amplification has an important impact on the development and maintenance of neuroblastoma.
  • a third member of the myc family is the L-myc gene. This gene is normally only expressed during development but it is frequently found amplified in human lung cancer.
  • the Myc network of transcriptional regulators also includes the negative regulator, Mnt which like Myc forms heterodimers with Max and bind to the consensus (CACGTG) E-Box element. Transcriptional repression by Mnt is mediated through association with mSin3 whereas deletion of the mSin3-interacting domain (SID) converts Mnt to a transcriptional activator (12, 14).
  • the mSin3 adaptor molecule recruits histone deacetylases (HDACs) and co-repressors to Mnt/Max binding sites leading to changes in chromatin structure and thereby affecting expression of specific genes (65).
  • Mnt is co-expressed with Myc in a number of proliferating cell types and has been suggested to be a modulator of Myc function. It was recently shown that Mnt-knockout mice die at birth. Furthermore, mouse embryo fibroblasts from these mice showed disrupted cell cycle control and could be transformed by Ras alone, traits resembling cells with Myc overexpression (66). The finding that Mnt is expressed in parallel with Myc in proliferating cells and can affect the same promoters, albeit with opposite effect, has led to the suggestion that Mnt is a modulator of Myc function.
  • WO 03/105759 discloses a method for identifying compounds that interfere with the association of c-Myc and Max proteins, comprising the step of performing a yeast two-hybrid assay in which the target and bait are, interchangeably, portions of c-Myc and Max proteins in the presence of the compound. They disclose treating eukaryotic cells with defined compounds and analysis of effects on proliferation but do not find any differences in inhibition of cell growth between cells with normal versus Myc overexpression upon treatment of cells.
  • a primary object of the invention is to provide a method of identifying compounds suitable for use as anti-tumor agents. This object is achieved in accordance with the invention by a method allowing for the identification of low molecular weight compounds that induce apoptosis in a Myc-dependent manner and inhibit transformation driven by Myc protein.
  • the method of the invention permits to identify compounds that affect cells with high Myc expression levels but that do not in any substantial way affect cells with an essentially normal Myc level. Consequently, by use of the screening method of the invention, compounds can be found and developed into antitumour drugs suitable for e.g. systemic administration substantially without adverse effects on normal cells. Since myc is an important oncogene that is activated in a wide variety of human tumors, the method of the invention will be a very useful tool in the search for new and efficacious anti-tumour agents.
  • the method of the invention may be used for the identification of a compound useful in the treatment of a cancer such as lymphoma, leukaemia, myeloma, neuroblastoma, lung cancer, breast cancer, ovarian cancer, head and neck cancer, osteosarcoma, rhabdomyosarcoma.
  • a cancer such as lymphoma, leukaemia, myeloma, neuroblastoma, lung cancer, breast cancer, ovarian cancer, head and neck cancer, osteosarcoma, rhabdomyosarcoma.
  • the present invention relates to a method of screening for a compound having antitumor activity by the steps of
  • the method furthermore comprises the steps of
  • the degree of apoptosis or inhibition of cell proliferation is compared between the cells overexpressing at least one myc gene, and/or having a deficient Mnt regulator activity, on the one hand, and the cells having wild type expression of at least one myc gene and/or having null-expression of at least one myc gene and/or having a functional Mnt regulator activity, on the other hand.
  • comparison is made of cells overexpressing a myc gene, on the one hand, and cells having wild type expression of this myc gene, on the other hand.
  • comparison is made of cells overexpressing a myc gene, on the one hand, and cells having wild type expression and/or having null-expression of the myc gene, on the other hand.
  • the myc gene is the myc gene that presently has been generally recognized as the most important proto-oncogene
  • the myc gene according to the invention may be selected from the c-myc, the N-myc and the L-myc genes.
  • the myc gene is c-myc
  • the method of the invention is then useful in the detection of compounds having an activity against cancers where c-myc overexpression is involved, e.g leukemia, breast, stomach, and lung cancer.
  • the myc gene is N-myc
  • the method of the invention is then useful in the detection of compounds having an activity against cancers where N-myc overexpression is involved, e.g. neuroblastoma.
  • the myc gene is L-myc
  • the method of the invention is then useful in the detection of compounds having an activity against cancers where L-myc overexpression is involved, e.g. lung cancer.
  • the cellular screen of the invention advantageously enables simultaneous selection of substances having the desired anti-tumor activity and that can penetrate the cell membrane and enter the cell and which furthermore are essentially not toxic to the patient being treated.
  • cellular screens are made, focusing on more than one of the myc genes and the expression products thereof.
  • the screening method is performed using cells having differing expression patterns of c-myc and/or N-myc and/or L-myc.
  • the series of steps (i)-(iv) and optionally (v)-(ix) are performed at least twice, whereby the at least one myc gene is independently selected for each series of steps; and the results from the at least two series are compared.
  • double cellular screens may be made focused on N-Myc and c-Myc.
  • the rationale would be that N-Myc might be sensitive to compounds that are not affecting c-Myc, or vice versa.
  • the screening is performed on cells having a subnormal or null expression of the Mnt protein, e.g. mnt-/- cells or cells that lack the mnt gene.
  • the screening method is performed on cells with a subnormal or null expression of the Mnt protein in combination with cells having differing expression patterns of c-myc and/or N-myc and/or L-myc.
  • the compound to be tested may be brought into contact with any suitable eukaryotic cells overexpressing c-myc and/or N-myc and/or L-myc and/or having deficient Mnt protein function and the apoptosis and/or inhibition of cell proliferation is determined.
  • the compound to be tested may be brought into contact with any suitable eukaryotic cells having normal and/or null expression of c-myc and/or N-myc and/or L-myc and/or having normal Mnt protein function, and the apoptosis and/or inhibition of cell proliferation is determined.
  • the apoptosis and/or inhibition of cell proliferation data thus obtained using cells having different myc and/or mnt expression patterns will provide valuable information on the selectivity and efficacy of the tested compound as an anti-tumour agent.
  • a screen is made in human tumor cells. This is advantageous since such cells may have a different sensitivity window compared to the experimental mouse cells.
  • the screening method of the invention may be performed in parallel or successively in different cells, e.g. first in rodent cells and then in human tumour cells.
  • Figure 1 illustrates the identification of MYRAs (My-C pathway Response Agents) in a cellular screening assay.
  • Tet-Myc cells were treated with doxycycline (Dox) at the indicated concentrations (in ⁇ g/ml) and total cell extracts were analyzed for Myc expression by Western blot after 18, 42 and 66 hours, ⁇ -actin was used as a loading control (A).
  • Dox doxycycline
  • Cell viability of Tet-Myc cells was measured by crystal violet staining after treatment with 12.5 ⁇ M of MYRA-1, 25 ⁇ M of MYRA-2, or
  • Figure 2 illustrates the induction of c-Myc dependent apoptosis by MYRAs in Rat-1 cell lines.
  • Rat-1 cells with different endogenous c-Myc expression were treated with 3 ⁇ M MYRA-I or MYRA-2, or 40 ⁇ M MYRA-3 and then analyzed for cell viability and apoptosis. Relative cell viability was measured by crystal violet staining (A). Phase contrast pictures of control and MYRA-treated Rat-1 cells (B). Hoechst staining of Rat-1 cells treated with MYRAs. Apoptotic nuclei are indicated by arrows (C). Induction of apoptosis was analyzed by Cell Death Detection ELISA PLUS kit after MYRA treatment (D). The mean of at least three independent experiments in duplicate with standard deviation are shown in (A) and (D).
  • Figure 3 illustrates the interference with DNA binding of Myc/Max and Mnt/Max by MYRA 5 ⁇ g of total cell extracts from HL-60 cells (A) or Mnt/Max-transfected COS cells (B) were preincubated with 100, 50, 25 or 12.5 ⁇ M MYRA-I and MYRA-2 or 400, 200 or 100 ⁇ M MYRA-3 before addition of the labelled CMD-oligonucleotide.
  • the Mnt/Max, USF, Myc/Max and Max/Max DNA-protein complexes were identified by antibody super shifts and are indicated to the left.
  • Figure 4 illustrates the inhibition of luciferase activity by the treatment with MYRAs CVl cells were transiently transfected with pSP-Myc or pSP-vector together with pCMV- ⁇ - galactosidase and minM4Luc. 24 hours post transfection the cells were treated with the indicated concentrations of MYRA-I to MYRA-3. The luciferase activities shown were normalized for ⁇ -galactosidase as a control of transfection efficiencies. The mean values with standard deviation of at least three independent experiments are shown.
  • Figure 5 illustrates the inhibition of soft agar colony formation by MYRAs
  • Rat-la-myc and MR cells were seeded in soft agar and treated with different concentrations of
  • the screening method of the present invention is based on comparing effects of chemical compounds on eukaryotic cells with high and normal Myc levels, respectively.
  • the eukaryotic cells that may be used according to the invention may be selected from mammalian cells, yeast cells and insect cells.
  • the mammalian cells may be selected from e.g. mouse fibroblasts such as NIH3T3, rat fibroblasts such as Ratl, mouse B cells such as MPC cell lines, human B cells such as P493, Al, EREB, LCL, human cancer cells such as Burkitt lymphoma, neuroblastoma, lung carcinoma, and newly established human cancer cell lines.
  • mouse fibroblasts such as NIH3T3, rat fibroblasts such as Ratl
  • mouse B cells such as MPC cell lines
  • human B cells such as P493, Al, EREB, LCL
  • human cancer cells such as Burkitt lymphoma, neuroblastoma, lung carcinoma, and newly established human cancer cell lines.
  • the insect cells may be e.g. Drosophila S2 cells while the yeast cells may be e.g. from strains of Saccharomyces cerevisiae.
  • cells overexpressing the myc gene may be provided by culturing eukaryotic cells using a suitable inducible system, e.g. under conditions permitting to induce or repress overexpression of the myc gene as a function of the presence in the culture medium of a suitable chemical compound.
  • tetracycline-inducible systems Tet-On/Tet-Off may suitably be used in mammalian cells.
  • the tetracycline-inducible systems Tet-On/Tet-Off are based on two different regulatory elements from Escherichia coli.
  • the Tet repressor protein (TetR) binds to the tetracycline response elements (TRE) in tet operator sequences and negatively regulates the genes of the tetracycline-resistance operon in the absence of Tc (tetracycline).
  • TetR and TRE provide the basis of the Tet-On and Tet-Off for use in mammalian cells.
  • the regulatory plasmid contains a fusion protein of the TetR and the C-terminal domain of the Herpes simplex VP 16 activation domain.
  • VP 16 converts the TetR from a transcriptional repressor to a transcriptional activator which binds the TRE and activates the gene of interest in the absence of tetracycline.
  • the Tet-On system is similar to the Tet-Off with one exception; in this case the regulatory protein is based on a reverse Tet repressor (rTetR) that carries a four amino acid change that instead binds and activates transcription in the presence of tet or its analogue doxycycline (dox).
  • the second plasmid is the same in both systems namely, the response plasmid that carries the gene of interest under the control of the TRE.
  • Tet-On/Tet-Off inducible systems may be purchased from Clontech Laboratories, Inc.
  • Estrogen Receptor ER
  • the basis for this system is that the gene encoding the ligand binding domain of the ER receptor is fused with the gene of interest.
  • the fusion gene is stably transfected in cells and then stably expressed but the fusion protein is inactive in the absence of ligand. This ensures a very fast onset of protein activity upon ligand administration.
  • the MER consists of a mutated ER that no longer responds to estradiol but instead is activated by the antagonist 4-hydroxytamoxifen and thus allows the use of normal medium and FCS (that contains the estradiol analogue phenol red and estradiol, respectively). These systems may be purchased from Sigma-Aldrich.
  • FCS that contains the estradiol analogue phenol red and estradiol, respectively.
  • Cell proliferation may be assessed by various methods as generally known to the person skilled in the art, e.g. by staining with crystal violet or Giemsa (VWR International, Sweden), by use of the WST-I assay (Roche Diagnostics Scandinavia, Sweden) or by use of the MTT assay (Promega Corporation, USA).
  • kits with specific detection and quantitation of mono- and oligonucleosomes in the cells are provided by Roche Diagnostics Scandinavia, Sweden.
  • Other means for assessing apoptosis are e.g.
  • TJNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
  • the myc gene codes for the N-Myc protein
  • the method of the invention is then useful in the detection of compounds having an activity against cancers where N-myc overexpression is involved, e.g. neuroblastoma.
  • the screen may be performed in human neuroblastoma cells with regulated N-Myc expression. It may be expected that some compounds will score positive in screens aiming both at c-Myc overexpressing and at N-Myc expressing cells, while others will turn out to be either c- or N-Myc, or cell type specific. In one embodiment of the invention, therefore, screens may be run in different cells in order to identify specific candidates for development of novel cancer therapies.
  • the present method provides a method to screen for small compounds that selectively can suppress growth or induce death of cells in aN-Myc- dependent manner.
  • suitable neuroblastoma cells with inducible N-Myc expression (67) may be used whereby the effect of the screened compounds on the growth of these cells in the presence or absence of induced N-Myc expression is compared.
  • Growth inhibition may be scored using WST-I (Roche), a reagent that is changing color by a mitochondrial enzyme in living and growing cells. Absence of orange dye thus indicates growth inhibition or apoptosis.
  • the cells suitably are treated with the screened substances in 96-well plates and analyzed for absorbance in an ELISA reader during a time course.
  • suitable human tumor cells such as lung cancer cells.
  • Substances that score positive in the screens may be analyzed with respect to their effect(s) on different Myc functions.
  • Apoptosis may be assessed by propidium iodide and annexin V stainings followed by FACS analysis and by Tunel assay. Proliferation may be studied by growth curves using trypan blue exclusion, BrdU stainings and cell cycle analysis by FACS. Additionally, transfections and reporter gene assays may be performed in order to analyze whether Myc-mediated transactivation is affected by the selected compounds.
  • Myc responsive luciferase reporter gene constructs may be analyzed in NIH3T3, CVl and HeLa cells together with Myc expression vectors in the presence and absence of the selected compounds.
  • the candidate substances may also be tested for their effect(s) on growth/apoptosis in neuroblastoma cells with (L-A-N-I, -2, -5, SK-N-BE(2), IMR 32) and without (SH-EP, SK- N-SH, SH-SY5 Y) N-Myc amplification.
  • SK-N-BE(2) and SH-SY5 Y cells may be differentiated in vitro with retinoic acid and TPA and the impact of the identified compounds on differentiation may be scored by measuring the length of the neurites (64).
  • the candidate substances may also be analyzed in SCED mice with human tumor xenografts (Burkitt lymphoma and neuroblastoma) and experimental tumors (Myc/Ras). Having found an active compound, a series of chemical analogs thereof may be synthesized for testing in the assays of the invention as described above, e.g. to provide information concerning which chemical groups/structures of the compounds that are important for the observed effects on Myc. Structural comparison between active and non-active substances may also give important findings concerning the molecular mechanism of the effect(s) on Myc. Taken together, this information may allow improved drug design to achieve higher potency and/or specificity.
  • a screening assay was performed in accordance with the invention wherein various different compounds were tested.
  • Tet-Myc cells containing a tetracycline inducible c-myc gene (41) were cultured in IMDM supplemented with 10% fetal calf serum (FCS) lacking tetracycline (Clontech Laboratories).
  • TGR-I and HO 15.19 Rat-1 cells (42) were kind gifts from J. Sedivy (Brown University, USA).
  • H0myc3 cells (43) were a kind gift from M. Cole (Dartmouth Medical School, NH, USA).
  • HO15.19 c-myc null cells were generated from parental TGR-I cells by homologous recombination, deleting both c-myc alleles.
  • Murine c-myc was reconstituted into HO 15.19 cells generating HOmyc3 cells with c-Myc overexpression.
  • Rat- Ia cells were stably transfected with LXSN containing the wild type human c-myc gene or the empty vector generating Rat-la-myc and Rat-la-LXSN, respectively (44).
  • Rat-1 and Rat-la cells were grown in DMEM.
  • MR cells were generated by the stable transfection of primary rat embryo fibroblasts (REFs) with c-Myc and H-Ras and were cultured in IMDM (45).
  • COS and CVl cells were grown in IMDM. Media was supplemented with 10% FCS, penicillin and streptomycin and cells were incubated at 37°C in an atmosphere of 5% CO 2 .
  • Antibodies Antibodies
  • Tet-Myc cells were seeded per well in 96- well plates and were incubated for 18 hours in the presence or absence of 2 ⁇ g/ml doxycycline. Cells were then treated with 12.5 or 25 ⁇ M of each of the 1990 substances from the diversity set library from the National Cancer Institute, Bethesda, Maryland (http://dtp.nci.nih.gov) in duplicate. Cell viability was analyzed after 48 hours of treatment using WST-I Cell Proliferation Agent (Roche Diagnostics) according to the manufacturer's instruction. The same samples were then fixed with 1% TCA (MERCK-Schuchardt) and stained with 0.04% crystal violet (Sigma-Aldrich).
  • the bound crystal violet was dissolved with 1% SDS and analysed at 570 nm. Compounds that affected cell viability in Myc overexpressing cells at least two fold more compared to uninduced cells were recorded as positive. All these data were verified in at least three independent experiments.
  • Cell Death Detection ELISA (Roche Diagnostics) was used to detect cytoplasmic mono- and oligocucleosom.es according to the manufacturer's instruction. Hoechst 33342 (Sigma- Aldrich) staining was used to verify the morphology of apoptotic nuclei and images were recorded on a DAS microscope Leitz DM RB with a Hamamatsu dual mode cooled CCD camera C4880 and processed in LtnageProPlus software and Adobe Photoshop.
  • Tet-Myc cells were boiled in sample buffer (40 mM Tris-HCl pH 6.8, 5% glycerol, 2.5% SDS and 0.01% Bromophenol Blue), proteins were separated by 10% SDS- PAGE and blotted to nitrocellulose membranes. After incubation with antibodies the membranes were developed by enhanced chemiluminescence (ECL) according to the manufacturer's instructions (GE Healthcare), ⁇ -actin was used as control for equal loading and transfer.
  • ECL enhanced chemiluminescence
  • 2.5xlO 5 CVl cells were seeded in each well of a 12-well plate. 24 hours later 1.0 ⁇ g pSP-Myc or pSP-vector was co-transfected with 0.2 ⁇ g of pCMV ⁇ -galactosidase and 0.2 ⁇ g of rninM4Luc with SuperFect® Transfection Reagent (QIAGEN) according to the manufacturer's instruction. 24 hours after transfection cells were treated with MYRA-I to MYRA-3 separately for 24 hours and cell extracts were analyzed for luciferase acitivity using a TD-20/20 luminometer (Turner Designs). Luciferase activity was normalized for differences in ⁇ -galactosidase levels as a control of transfection efficiency.
  • TGR-I c-myc wild type
  • HO 15.19 c-myc null
  • HOmyc3 c-myc reconstituted Rat-1 cells that overexpress Myc 2-4 times compared to TGR-I cells (43, 47).
  • MYRA treatment resulted in different effects depending on the c-Myc status of the cells with the most predominant inhibition of H0myc3 cells.
  • the relative cell survival of HOmyc3 cells was 14%, 31% and 24% after treatment of MYRA-I to MYRA-3, respectively ( Figure 2A).
  • the corresponding cell survival of treated TGR-I cells was 46%, 63% and 47%.
  • HO 15.19 myc null cells have a longer cell cycle time than the TGR-I cells (42), we treated these cells with compounds from 48 hours up to 96 hours. After 96 hours of treatment with MYRA-I to -3, the relative cell viability of HO 15.19 cells was 72%, 90% and 78%, respectively. No effects were observed after 48 hours (Fig. 2A and data not shown). As shown in Fig. 2B the MYRA-treated HOmyc3 cells were sparsely distributed compared to the corresponding TGR-I cells. In contrast, MYRA-treatment had virtually no effect on HO 15.19 cells (Fig. 2B). Taken together, these results indicated that the effect of MYRA on cell viability is c-Myc dependent. Candidate substances induce Myc-dependent apoptosis
  • Morphological changes of cells and nuclei showed that treatment with MYRAs induced apoptotic cell death in a Myc-dependent manner, which is supported by the quantitative data from the apoptosis ELISA assay.
  • MYRA-I and MYRA-2 interfered with the DNA binding not only of Myc/Max but also of Mnt/Max and Max/Max complexes. This is not surprising since Mnt and Myc share DNA binding site and regulate an overlapping set of target genes in vivo (12). In addition, loss of Mnt has been shown to induce similar effects to that of Myc overexpression such as induction of Myc target genes, accelerated proliferation and apoptosis, and transformation of primary fibroblasts in conjunction with Ras (49, 50). In contrast, neither MYRA-I nor MYRA-2 showed any effect on the DNA binding activity of USF that binds to the same E-box (51).
  • MYRA-I and MYRA-2 could possibly discriminate Myc network members from other E-box binding proteins.
  • the effective concentrations of MYRA in the EMSA were higher than those used in the cell viability assays, which is similar to the conditions reported by others (39).
  • Both MYRA-I and MYRA-2 reduced Myc transactivation in a dose-dependent manner as demonstrated in luciferase assays, hi contrast, MYRA-3 showed neither significant effect in the EMSA nor in the luciferase assay.
  • a Myc-targeting therapy should only intervene with cells with deregulated Myc and not affect the majority of normal cells in vivo. It was observed that MYRAs predominantly induced apoptosis in cells with Myc overexpression compared to cells with normal Myc levels. This in turn suggests that they play predominant roles in cells with high Myc levels. From the above, it appears that cellular screening assays according to the present invention may be used as efficient tools to identify low-molecular-weight compounds that by different mechanisms of action target the Myc pathway. These compounds may be suitable for use as anti-tumour compounds and/or may provide a starting point for medicinal chemistry and drug design.

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Abstract

La présente invention concerne un procédé pour cribler un composé présentant une activité antitumorale, qui consiste à disposer de cellules eucaryotes surexprimant au moins un gène myc et/ou présentant une activité de régulation de Mnt déficiente, à mettre ces cellules en contact avec ledit composé, à mettre en culture les cellules en présence du composé, puis à mesurer le degré d'apoptose des cellules ou l'inhibition de la prolifération cellulaire. Ces étapes sont de préférence réalisées sur des cellules présentant une expression de type sauvage et/ou une expression nulle d'au moins un gène myc et/ou une activité de régulation de Mnt fonctionnelle. Le degré d'apoptose ou l'inhibition de la prolifération cellulaire est comparé entre les cellules surexprimant au moins un gène myc et/ou présentant une activité de régulation de Mnt déficiente et les cellules présentant une expression de type sauvage et/ou une expression nulle d'au moins un gène myc et/ou une activité de régulation de Mnt fonctionnelle.
PCT/SE2005/001348 2004-09-16 2005-09-16 Procede pour cribler un compose presentant une activite antitumorale en utilisant des cellules eucaryotes surexprimant au moins un gene myc et/ou presentant une activite de regulation de mnt deficiente Ceased WO2006031194A1 (fr)

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US60/610,193 2004-09-16

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001059090A2 (fr) * 2000-02-11 2001-08-16 The Johns Hopkins University Cdk4 en tant que cible de c-myc
WO2003105759A2 (fr) * 2002-06-12 2003-12-24 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Inhibition pharmacologique de la fonction myc

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001059090A2 (fr) * 2000-02-11 2001-08-16 The Johns Hopkins University Cdk4 en tant que cible de c-myc
WO2003105759A2 (fr) * 2002-06-12 2003-12-24 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Inhibition pharmacologique de la fonction myc

Non-Patent Citations (2)

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Title
BERG T. ET AL: "Small-molecule antagonists of Myc/Max dimerization inhibit Myc-induced transformation of chicken embryo fibroblasts", PNAS, vol. 99, no. 6, 19 March 2002 (2002-03-19), pages 3830 - 3835, XP002995347 *
YIN X. ET AL: "Low molecular weight inhibitors of Myc-Max interaction and function", ONCOGENE, vol. 22, 2003, pages 6151 - 6159, XP002995346 *

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