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WO2012168720A1 - Substances et procédés pour traiter un cancer positif aux récepteurs des oestrogènes α (er) - Google Patents

Substances et procédés pour traiter un cancer positif aux récepteurs des oestrogènes α (er) Download PDF

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Publication number
WO2012168720A1
WO2012168720A1 PCT/GB2012/051289 GB2012051289W WO2012168720A1 WO 2012168720 A1 WO2012168720 A1 WO 2012168720A1 GB 2012051289 W GB2012051289 W GB 2012051289W WO 2012168720 A1 WO2012168720 A1 WO 2012168720A1
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Prior art keywords
inhibitor
cancer
acdk2
adck2
treatment
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Alan Ashworth
Christopher James LORD
Rachel BROUGH
Jessica FRANKUM
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Institute of Cancer Research Royal Cancer Hospital
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Institute of Cancer Research Royal Cancer Hospital
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes

Definitions

  • the present invention relates to materials and methods for treating estrogen receptor a (ER) positive cancer, and in particular ER positive breast cancer.
  • ER estrogen receptor a
  • tumor cells generally exhibit anywhere between 10 4 and 10 5 genetic changes compared to germline DNA but theoretical estimates suggest that only a few of these mutations (probably less than 10) are critical drivers of tumor formation and survival (reviewed in (1) ) ⁇
  • SSL synthetic sickness/lethality
  • SS L can occur between genes acting in the same biochemical pathway or in distinct but compensatory pathways .
  • the present invention is based on work which attempts to comprehensively define the genetic dependencies for a set of potentially “druggable” genes in a wide range of breast tumor cell line models. In doing so, we not only reaffirm the impact of Pl3-kinase and ERBB2 signalling in the disease but importantly identify novel genetic dependencies for genetically defined subsets of the disease.
  • the present invention is based on the experiments described herein in which the genetic dependency on ADCK2 is shown in endocrine receptor positive cancer .
  • references to ADCK2 denote aarF domain containing protein kinase 2 having the HGNC ID: 19039.
  • the HUGO Gene Symbol report for ESR1 can be found at:
  • ESR1 estrogen receptor 1
  • ERa which provides links to the ESR1 nucleic acid and amino acid sequences, as well as reference to the homologous murine and rat proteins.
  • the expression of ERa has been implicated in breast cancer, ovarian cancer, colon cancer, prostate cancer and endometrial cancer.
  • the present invention provides an ACDK2 inhibitor for use in a method of treating an individual having estrogen receptor (ER) positive cancer, and in particular for the treatment of ER positive breast cancer.
  • ER estrogen receptor
  • the present invention provides the use of an inhibitor of ADCK2 in the preparation of a medicament for the treatment of an individual having estrogen receptor (ER) positive breast cancer.
  • the present invention provides a method of treating an individual having estrogen receptor (ER) positive breast cancer, the method comprising administering to the individual a therapeutically effective amount of an ADCK2 inhibitor .
  • ER estrogen receptor
  • the present invention provides a method of screening for agents useful in the treatment of estrogen receptor (ER) positive breast cancer, the method comprising:
  • the present invention provides a method which comprises having identified a candidate agent useful for the treatment of estrogen receptor (ER) positive breast cancer according to a method as described herein, the further step of manufacturing the compound in bulk and/or formulating the agent in a pharmaceutical composition.
  • ER estrogen receptor
  • the present invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or is stated to be expressly avoided.
  • FIG. 1 PIK3CA and ERBB2 oncogene addiction effects in breast cancer confirmed by functional viability profiling, a. Heat map showing the results of a supervised clustering of siRNA Z scores. Breast tumour cell lines were clustered according to PIK3CA gene mutation status and differential effects between PIK3CA mutant and wild type groups identified using the median permutation test. Statistically significant effects (p ⁇ 0.05) are shown. For each siRNA, p values and the p ⁇ 0.05 threshold are shown to the right of the heat map. siRNA targeting PIK3CA is marked by the arrow, b. Waterfall and box/whiskers plots of PIK3CA siRNA Z scores across the breast tumour cell line panel. In the
  • ADCK2 silencing by multiple ADCK2 siRNA species reduces the expression of ER-target gene as shown by western blotting of MCF7 total cell lysates generated after siRNA transfection .
  • ADCK2 E2-dependent increase in ADCK2 expression is shown, e. Western blot of ERa immunoprecitated material from MCF7 cells, indicating an ERa- ADCK2 interaction, f. Tumour ADCK2 mRNA expression in ER+ve breast tumours classified according to luminal A or B status. Transcript expression data from two independent studies was used 10,21 and luminal A or B status determined according to Sorlie et al 12. p values were calculated using Student's t test.
  • ADCK2 inhibitors may be identified by the use of high throughput screening strategies Accordingly, the present invention also extends to the use of small molecule inhibitors found in the screening disclosed herein and to Derivatives which are compounds of similar structure and functionality to the compounds found in the high throughput screen, but with one or more modifications, are expected to have similar physiological effects to these compounds and could therefore also be of use in the treatment of estrogen receptor (ER) positive breast cancer.
  • the screening methods of the invention may be used to screen libraries of such derivatives to optimise their activity, if necessary.
  • Derivatives may be designed, based on a lead compound, by modifying one or more substituents or functional groups compared to the lead compound, for example by replacing these with alternative substituents or groups which are expected to have the same or improved physiological effect.
  • the use of derivatives having such modifications is well known to those in the art.
  • Antibodies may be employed in the present invention as an example of a class of inhibitor useful for treating estrogen receptor
  • ADCK2 estrogen receptor
  • ER estrogen receptor
  • antibodies include an immunoglobulin whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein comprising an antibody binding domain.
  • Antibody fragments which comprise an antigen binding domain are such as Fab, scFv, Fv, dAb, Fd; and diabodies. It is possible to take monoclonal and other
  • antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP 0 184 187 A, GB 2,188,638 A or EP 0 239 400 A.
  • CDRs complementarity determining regions
  • Antibodies can be modified in a number of ways and the term "antibody molecule" should be construed as covering any specific binding member or substance having an antibody antigen-binding domain with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP 0 120 694 A and EP 0 125 023 A.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S.
  • Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al, Nature Biotech, 14: 1239-1245, 1996) .
  • Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al, Cancer Res., 56: 3055-3061, 1996) .
  • Monoclonal antibodies are preferred for some purposes, though polyclonal antibodies are within the scope of the present invention.
  • the reactivities of antibodies on a sample may be determined by any appropriate means. Tagging with individual reporter
  • the reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals.
  • the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently . Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.
  • One favoured mode is by covalent linkage of each antibody with an individual fluorochrome, phosphor or laser exciting dye with spectrally isolated absorption or emission characteristics.
  • Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red.
  • Suitable chromogenic dyes include diaminobenzidine .
  • reporter include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyse
  • reactions that develop or change colours or cause changes in electrical properties for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with
  • Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.
  • Antibodies according to the present invention may be used in screening for the presence of a polypeptide, for example in a test sample containing cells or cell lysate as discussed, and may be used in purifying and/or isolating a polypeptide according to the present invention, for instance following production of the polypeptide by expression from encoding nucleic acid. Antibodies may modulate the activity of the polypeptide to which they bind and so, if that polypeptide has a deleterious effect in an individual, may be useful in a therapeutic context (which may include prophylaxis) .
  • Peptide fragments may be used in screening for the presence of a polypeptide, for example in a test sample containing cells or cell lysate as discussed, and may be used in purifying and/or isolating a polypeptide according to the present invention, for instance following production of the polypeptide by expression from encoding nucleic acid. Antibodies may modulate the activity of the polypeptide to which they bind and so, if
  • Another class of inhibitors useful for treating estrogen receptor (ER) positive breast cancer includes peptide fragments that interfere with the activity of ADCK2.
  • Peptide fragments may be generated wholly or partly by chemical synthesis that block the catalytic sites of ADCK2.
  • Peptide fragments can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in
  • candidate compounds for inhibiting ADCK2 may be based on modelling the 3-dimensional structure of these enzymes and using rational drug design to provide candidate compounds with
  • a candidate inhibitor for example, may be a "functional analogue" of a peptide fragment or other compound which inhibits the component.
  • a functional analogue has the same functional activity as the peptide or other compound in question. Examples of such analogues include chemical compounds which are modelled to resemble the three dimensional structure of the component in an area which contacts another component, and in particular the arrangement of the key amino acid residues as they appear.
  • Another class of inhibitors useful for treatment of estrogen receptor (ER) positive breast cancer includes nucleic acid inhibitors of ADCK2, or the complements thereof, which inhibit activity or function by down-regulating production of active polypeptide. This can be monitored using conventional methods well known in the art, for example by screening using real time PCR as described in the examples. may be
  • Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of the base excision repair pathway component so that its expression is reduced or completely or substantially completely prevented.
  • anti-sense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the anti-sense oligonucleotides can interfere with expression control sequences.
  • the construction of anti-sense sequences and their use is described for example in Peyman & Ulman, Chemical Reviews, 90:543-584, 1990 and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, 1992.
  • Oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo within cells in which down-regulation is desired.
  • double-stranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA
  • the complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain.
  • the complete sequence corresponding to the coding sequence in reverse orientation need not be used.
  • fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon.
  • a suitable fragment may have about 14-23 nucleotides, e.g., about 15, 16 or 17 nucleotides.
  • RNAi RNA interference
  • RNA interference is a two-step process.
  • dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs ( ⁇ 2nt) .
  • siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore, Nature Structural Biology, 8, 9, 746-750, 2001.
  • RNAi may also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3 ' -overhang ends
  • siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologeous genes in a wide range of mammalian cell lines
  • nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific site and therefore also useful in influencing gene expression, e.g., see Kashani-Sabet & Scanlon, Cancer Gene
  • Small RNA molecules may be employed to regulate gene expression. These include targeted degradation of mRNAs by small interfering RNAs (siRNAs), post transcriptional gene silencing (PTGs), developmentally regulated sequence-specific translational repression of mRNA by micro-RNAs (miRNAs) and targeted
  • Double- stranded RNA (dsRNA) -dependent post transcriptional silencing also known as RNA interference (RNAi)
  • RNAi Double- stranded RNA
  • RNAi RNA interference
  • a 20-nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA.
  • RNA sequences are termed “short or small interfering RNAs” (siRNAs) or “microRNAs” (miRNAs) depending on their origin. Both types of sequence may be used to down- regulate gene expression by binding to complimentary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein.
  • siRNA are derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin.
  • miRNA are examples of interfering RNAs
  • siRNA and miRNA can inhibit the
  • siRNA ligands are typically double stranded and, in order to optimise the effectiveness of RNA mediated down-regulation of the function of a target gene, it is preferred that the length of the siRNA molecule is chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of the mRNA target and so that the siRNA is short enough to reduce a host response.
  • miRNA ligands are typically single stranded and have regions that are partially complementary enabling the ligands to form a hairpin.
  • miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. A DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse
  • RNA ligands intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogues thereof) , more preferably between 17 and 30
  • ribonucleotides more preferably between 19 and 25
  • ribonucleotides and most preferably between 21 and 23
  • the molecule may have symmetric 3 ' overhangs, e.g. of one or two (ribo) nucleotides, typically a UU of dTdT 3' overhang.
  • ribo ribo nucleotides
  • the skilled person can readily design suitable siRNA and miRNA sequences, for example using resources such as Ambion's siRNA finder, see http://www.ambion.com/techlib/misc/siRNA finder.html.
  • siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors) .
  • the siRNA is synthesized synthetically.
  • Longer double stranded RNAs may be processed in the cell to produce siRNAs (e.g. see Myers, Nature Biotechnology, 21: 324- 328, 2003) .
  • the longer dsRNA molecule may have symmetric 3' or 5' overhangs, e.g. of one or two ( ribo ) nucleotides , or may have blunt ends.
  • the longer dsRNA molecules may be 25 nucleotides or longer.
  • the longer dsRNA molecules are between 25 and 30 nucleotides long. More preferably, the longer dsRNA molecules are between 25 and 27 nucleotides long.
  • the longer dsRNA molecules are 27 nucleotides in length.
  • dsRNAs 30 nucleotides or more in length may be expressed using the vector pDECAP (Shinagawa et al . , Genes and Dev., 17: 1340-5, 2003) .
  • shRNAs are more stable than synthetic siRNAs.
  • a shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target.
  • the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression.
  • the shRNA is produced endogenously (within a cell) by transcription from a vector.
  • shRNAs may be produced within a cell by
  • RNA polymerase III promoter such as the human HI or 7SK promoter or a RNA polymerase II promoter.
  • the shRNA may be synthesised exogenously (in vitro) by transcription from a vector.
  • the shRNA may then be introduced directly into the cell.
  • the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length.
  • the stem of the hairpin is preferably between 19 and 30 base pairs in length.
  • the stem may contain G-U pairings to stabilise the hairpin structure.
  • the siRNA, longer dsRNA or miRNA is produced endogenously (within a cell) by transcription from a vector.
  • the vector may be introduced into the cell in any of the ways known in the art.
  • expression of the RNA sequence can be regulated using a tissue specific promoter.
  • the siRNA, longer dsRNA or miRNA is produced
  • siRNA molecules may be synthesized using standard solid or solution phase synthesis techniques, which are known in the art.
  • Linkages between nucleotides may be phosphodiester bonds or alternatives, e.g., linking groups of the formula P(0)S, (thioate); P(S)S, (dithioate) ; P(0)NR'2; P(0)R'; P(0)OR6; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through-O-or-S- .
  • Modified nucleotide bases can be used in addition to the
  • siRNA molecules containing them may confer advantageous properties on siRNA molecules containing them.
  • modified bases may increase the stability of the siRNA molecule, thereby reducing the amount required for
  • modified bases may also provide siRNA molecules, which are more, or less, stable than unmodified siRNA.
  • Modified nucleotide base encompasses nucleotides with a covalently modified base and/or sugar.
  • modified nucleotides include nucleotides having sugars, which are
  • modified nucleotides may also include 2 ' substituted sugars such as 2 ' -O-methyl- ; 2-0- alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2 ' -fluoro- ; 2 ' -halo or 2; azido-ribose , carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars and sedoheptulose .
  • 2 ' substituted sugars such as 2 ' -O-methyl- ; 2-0- alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2 ' -fluoro- ; 2 ' -halo or 2; azido-ribose , carbocyclic sugar analogues a-anomeric
  • Modified nucleotides include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles . These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4,N4- ethanocytosine , 8-hydroxy-N6-methyladenine , 4-acetylcytosine , 5- (carboxyhydroxylmethyl ) uracil, 5 fluorouracil , 5-bromouracil , 5- carboxymethylaminomethyl-2-thiouracil , 5-carboxymethylaminomethyl uracil, dihydrouracil , inosine, N6-isopentyl-adenine, 1- methyladenine, 1-methylpseudouracil, 1-methylguanine, 2,2- dimethylguanine, 2methyladenine, 2-methylguanine, 3- methylcytosine , 5-methylcytosine , N
  • the present invention is concerned with methods of screening candidate compounds to determine whether one or more candidate agents are likely to be useful for the treatment of estrogen receptor (ER) positive cancer.
  • ER estrogen receptor
  • the present invention also includes methods of screening that employ ADCK2 as protein targets for the screening of candidate compounds to find ADCK2 inhibitors. Accordingly, methods of screening may be carried out for identifying candidate agents that are capable of inhibiting an activity of ADCK2, for
  • ADCK2 estrogen receptor
  • the candidate agent may be a known inhibitor of one of the protein targets di sclosed herein, an antibody, a peptide, a nucleic acid molecule or an organic or inorganic compound, e.g. molecular weight of less than 100 Da.
  • an antibody e.g. an antibody, a peptide, a nucleic acid molecule or an organic or inorganic compound, e.g. molecular weight of less than 100 Da.
  • candidate agents that are compounds is preferred.
  • any type of candidate agent for any type of candidate agent,
  • combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a target protein.
  • libraries and their use are known in the art .
  • the present invention also specifically envisages screening candidate agents known for the treatment of other conditions, and especially other forms of cancer. This has the advantage that the patient or disease profile of known therapeutic agents might be expanded or modified using the screening techniques disclosed herein, or for
  • the agent in question may be tested to determine whether it is not lethal to normal cells or otherwise is suited to therapeutic use. Following these studies, the agent may be manufactured and/or used in the preparation of a medicament, pharmaceutical composition or dosage form.
  • peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
  • the physical properties e.g. stereochemistry, bonding, size and/or charge
  • data from a range of sources e.g. spectroscopic techniques, X-ray diffraction data and NMR.
  • Computational analysis, similarity mapping
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can be selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • synthesise is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • the present invention provides methods and medical uses for the treatment of estrogen receptor (ER) positive cancer.
  • a cancer may be identified as estrogen receptor (ER) positive cancer by carrying out one or more assays or tests on a sample of cells from an individual.
  • the sample will generally be a sample of cancer cells.
  • Activity may be determined relative to a control, for example in the case of defects in cancer cells, relative to non-cancerous cells, preferably from the same tissue.
  • Examples of known types of ER positive cancer include breast cancer, ovarian cancer, prostate cancer, and endometrial cancer.
  • the determination of the ER positive status of a patient may be carried out using a number of approaches known in the art.
  • the presence of ER protein may be determined by using techniques well known in the art such as Western blot analysis, immunohistology, chromosomal abnormalities, enzymatic or DNA binding assays and plasmid-based assays.
  • rhe determination of ER gene expression may involve determining the presence or amount of ER mRNA in a sample. Methods for doing this are well known to the skilled person. By way of example, they include determining the presence of ER mRNA; and/or (ii) using PCR involving one or more primers based on an ER nucleic acid sequence to determine whether the ER transcript is present in a sample.
  • the probe may also be immobilised as a sequence included in a microarray.
  • detecting ER mRNA is carried out by extracting RNA from a sample of the tumour and measuring ER expression
  • the expression of ER could be assessed using RNA extracted from a tumour sample using microarray analysis, which measures the levels of mRNA for a group of genes using a
  • determination of endocrine receptor status can be carried out by analysis of ER protein expression.
  • results provided herein demonstrate that reduced levels of ERa protein expression are correlated with an increased likelihood of developing resistance to endocrine therapy for breast cancer.
  • the presence or amount of ERa protein may be any suitable substance.
  • the presence or amount of ERa protein may be any suitable substance.
  • a binding agent capable of specifically binding to the ER protein, or fragments thereof is an antibody capable of specifically binding the ER or fragment thereof.
  • the antibody may be labelled to enable it to be detected or capable of detection following reaction with one or more further species, for example using a secondary antibody that is labelled or capable of producing a detectable result, e.g. in an ELISA type assay.
  • a labelled binding agent may be employed in a western blot to detect ER protein.
  • the method for determining the presence of ER protein may be carried out on tumour samples, for example using immunohistochemical (IHC) analysis.
  • IHC analysis can be carried out using paraffin fixed samples or frozen tissue samples, and generally involves staining the samples to highlight the presence and location of ER protein.
  • the active agents disclosed herein for the treatment of estrogen receptor (ER) positive cancer may be administered alone, but it is generally preferable to provide them in pharmaceutical compositions that additionally comprise with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers,
  • derivatives of the therapeutic agents includes salts, coordination complexes, esters such as in vivo hydrolysable esters, free acids or bases, hydrates, prodrugs or lipids, coupling partners.
  • Salts of the compounds of the invention are preferably
  • salts are known to those skilled in the art.
  • Compounds having acidic groups such as phosphates or sulfates, can form salts with alkaline or alkaline earth metals such as Na, K, Mg and Ca, and with organic amines such as triethylamine and Tris (2- hydroxyethyl ) amine .
  • Salts can be formed between compounds with basic groups, e.g., amines, with inorganic acids such as
  • hydrochloric acid phosphoric acid or sulfuric acid
  • organic acids such as acetic acid, citric acid, benzoic acid, fumaric acid, or tartaric acid.
  • Compounds having both acidic and basic groups can form internal salts .
  • Esters can be formed between hydroxyl or carboxylic acid groups present in the compound and an appropriate carboxylic acid or alcohol reaction partner, using techniques well known in the art.
  • Derivatives which as prodrugs of the compounds are convertible in vivo or in vitro into one of the parent compounds.
  • at least one of the biological activities of compound will be reduced in the prodrug form of the compound, and can be activated by conversion of the prodrug to release the compound or a metabolite of it.
  • Coupled derivatives include coupling partners of the compounds in which the compounds is linked to a coupling partner, e.g. by being chemically coupled to the compound or physically
  • coupling partners include a label or reporter molecule, a supporting substrate, a carrier or transport molecule, an effector, a drug, an antibody or an inhibitor.
  • Coupling partners can be covalently linked to compounds of the invention via an appropriate functional group on the compound such as a hydroxyl group, a carboxyl group or an amino group.
  • Other derivatives include formulating the compounds with liposomes.
  • pharmaceutically acceptable includes compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable
  • the active agents disclosed herein for the treatment of estrogen receptor a (ERa) positive breast cancer according to the present invention are preferably for administration to an individual in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • a prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of
  • compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.
  • formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into
  • the agents disclosed herein for the treatment of estrogen receptor a (ERa ) positive breast cancer may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose);
  • vaginal for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous,
  • intraarterial intracardiac
  • intrathecal intrathecal
  • intraspinal intraarterial
  • Formulations suitable for oral administration e.g., by
  • ingestion may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants , buffers, preservatives, stabilisers, bacteriostats , and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • Suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
  • concentration of the active compound in the solution is from about 1 ng/ml to about 10 ⁇ g/ml, for example from about 10 ng/ml to about 1 ⁇ g/ml.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs .
  • Compositions comprising agents disclosed herein for the treatment estrogen receptor (ER) positive breast cancer may be used in the methods described herein in combination with standard
  • chemotherapeutic regimes or in conjunction with radiotherapy examples include Amsacrine (Amsidine) , Bleomycin, Busulfan, Capecitabine (Xeloda) ,
  • Carboplatin Carmustine (BCNU) , Chlorambucil (Leukeran) ,
  • Cisplatin Cladribine (Leustat) , Clofarabine (Evoltra) , Crisantaspase (Er inase) , Cyclophosphamide, Cytarabine (ARA-C) , dacarbazine (DTIC) , Dactinomycin (Actinomycin D) , Daunorubicin, Docetaxel (Taxotere) , Doxorubicin, Epirubicin, Etoposide
  • Liposomal daunorubicin Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin,
  • Mitoxantrone Oxaliplatin (Eloxatin) , Paclitaxel (Taxol) ,
  • Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate
  • a suitable dose of the active compound is in the range of about 100 ⁇ g to about 250 mg per kilogram body weight of the subject per day.
  • the active compound is a salt, an ester, prodrug, or the like, the amount administered is
  • Each well in this library contained a SMARTpool of four distinct siRNA species targeting different sequences of the target transcript. Each plate was supplemented with siCONTROL
  • Antibodies targeting the following were used as per manufacturers instructions: ACTIN and C-MYC (Santa Cruz Biotech), TTK, ER, PR, ERBB2, CYCLIN Dl , TFF1, FOXOl, C-JUN and ADCK2 (AbCam) . All secondary antibodies used for western blot analysis were HRP conj ugated .
  • Z scores were calculated using the Median Absolute Deviation (MAD) of all effects in each cell line (5) .
  • MAD Median Absolute Deviation
  • siRNA screens were carried out in triplicate and comparison of Z score data from replica screens of each cell line showed the screening process to be highly robust, as demonstrated by Spearman's r2 values approaching 1 for all comparisons.
  • Raw luminescence values and processed data are now deposited on the ROCK Breast Cancer
  • Heatmaps of Z scores that separated different phenotypes as defined by a median difference permutation test were created to show the significant genes ranked row-wise according to their median difference and ordered column-wise by phenotype.
  • Protein lysates were prepared using RIPA lysis buffer (50 nM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.1% DOC, 1% TritonX-100, 50 mM NaF, 1 mM Na 3 V0 4 and protease inhibitors) . lOOmg of total cell lysate was loaded onto prefabricated 4-12% Bis-Tris gels
  • RNAi gene silencing was determined by western blotting and by viability assays of silencing effects with individual oligos.
  • Cells were transfected with individual ERBB2 , ADCK2, ESR1, PIK3CA or TTK siGenome oligos ( Dharmacon) . Protein lysates were collected 48 hours following transfection for western blot analysis. Cell viability was measured using
  • HCT116 PTEN wt or PTEN null cells were treated with colcemide (lOng/ml, Sigma) and MG132 (20uM, Sigma) for 1 hour. Cells were then lysed in hypotonic solution (0.03M Sodium Citrate) for 20 minutes at 37°C and fixed in methanol/acetic acid (3:1) . Two or three drops of suspended cells were applied to glass slides and chromosomes were stained with DAPI .
  • LSI PTEN (10q23, red/orange) /chromosome 10 centromere (CEP 10, green) Dual Colour Probe (Abbott Molecular, IL, US) was
  • Transcript profiling RNA was extracted from cell lines with Trizol and
  • Biotin-labelled cRNA was produced by means of a linear amplification kit (IL1791; Ambion, Austin, TX, http://www. ambion.com) using 250 ng of quality-checked total RNA as input. Chip hybridisations, washing, Cy3-streptavidin
  • Genomic DNA was extracted from cell lines using the QIAamp DNA Blood Mini Kit (51104, Qiagen) , according to manufacturer's instructions.
  • Microarray-based CGH analysis was performed on an in-house 32K tiling path BAC array platform as previously described (29) .
  • CBS circular binary segmentation
  • AWS smoothed log2 ratio values ⁇ -0.12 were categorised as losses, those >0.12 as gains, and those in between as unchanged. Amplifications were defined as smoothed log2 ratio values >0.4 (29) .
  • siRNA Z score Correlation of siRNA Z score with gene expression and aCGH data The correlation between siRNA Z score and normalised gene expression was examined for genes where siRNA caused significant loss of viability (Z ⁇ -2) . Z score was compared to normalized gene expression using Pearson correlation coefficient. A gene was taken as being significantly correlated if the Pearson
  • STR short tandem repeat
  • siRNA RNA interference/short interfering RNA
  • each cell line was transfected with a 96 well-plate arrayed siRNA library targeting 714 kinases and kinase-related genes (see Methods) .
  • cell viability in each well was estimated by use of a highly sensitive luminescent assay measuring cellular ATP levels.
  • luminescence readings from each well were log transformed and then centred by the plate median, to account for plate-to-plate variation.
  • RNA interference screens were carried out in triplicate and comparison of Z score data from replica screens of each cell line showed the screening process to be highly robust.
  • This panel encapsulated each of the major breast cancer subtypes (4) and included hormone receptor (Estrogen Receptor (ER) , Progesterone Receptor (PR) ) , ERBB2 positive as well as ER, PR and ERBB2 negative models
  • FISH fluorescent in situ hybridisation
  • PIK3CA which encodes the pllO catalytic subunit of Pl3-kinase
  • Somatic mutations of PIK3CA have been shown to induce oncogenic transformation in vitro and in vivo (6).
  • PIK3CA mutations are found in 8% - 35% of human breast cancers making them one of the most common genetic aberrations in this disease (7) .
  • Supervised hierarchical clustering of functional viability data according to PIK3CA mutation status identified 30 siRNAs that caused loss of viability preferentially in the PIK3CA mutant.
  • PIK3CA siRNA gives effects of Z ⁇ -2, suggesting that PIK3CA addiction is one of the most significant effects in PIK3CA mutant tumor cells.
  • PIK3CA mutant breast cancer lines were preferentially sensitive to AKT2 and AKT3 siRNAs (Fig. lc) .
  • tumour specific PIK3CA mutations Whilst a wide variety of tumour specific PIK3CA mutations have been identified, the vast majority occur in two hotspots, either in the kinase domain (for example p.H1047R) or in the helical domain (e.g. p.E542K or p.E545K) . It has been suggested that helical and kinase domain mutants have distinct physiologic phenotypes in human cells (9, 10), and the
  • amplification status (based upon an integration with aCGH and FISH data identified a number of genes that when silenced were selectively lethal in ERBB2 amplified breast cancer lines (Fig. Id) .
  • ADCK2 Uncharacterized aarF domaincontaining protein kinase 2 is a member of a family of aarF-domain containing proteins. Other members of this protein family are localised to the mitochondria and mitochondrial membranes and have been implicated in
  • ADCK2 could target ER+ve breast tumor cells by modulating estrogen signalling.
  • sensitivity to siRNA targeting of ESR1 the gene encoding ER
  • closely correlated with the inhibitory effect of ADCK2 siRNA suggesting that ADCK2 is an important dependency in breast tumor cells with a strong
  • ADCK2 silencing reduced estrogen signalling, as measured by the expression of well established ER target genes, including ESR1 itself (Fig. 2c), estrogen stimulation stimulated ADCK2 expression, suppression of estrogen signalling (with
  • ADCK2 and estrogen receptor (ESR1) information in the breast tumour cell line panel The ADCK2 and ESR1 copy number, expression (as assessed by microarray, immunohistochemistry (IHC; for ESR1 only) and western blot) and cell line dependency for ADCK2 and ESR1 (as defined by Z score) is summarized here.
  • Ash orth A Lord CJ, Reis-Filho JS . Genetic interactions in cancer progression and treatment. Cell 2011;145:30-8.
  • Zhao L Vogt PK. Class I PI3K in oncogenic cellular
  • Musacchio A, et al Sustained Mpsl activity is required in mitosis to recruit 0-Mad2 to the Madl-C-Mad2 core complex. J Cell Biol 2010;190:25-34.

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Abstract

L'invention repose sur des expériences selon lesquelles la dépendance génétique à ADCK2 est mise en évidence dans un cancer positif aux récepteurs endocriniens. En conséquence, l'invention concerne l'utilisation d'inhibiteurs d'ACDK2 pour traiter des individus atteints d'un cancer positif aux récepteurs des oestrogènes α (ER), tels que le cancer du sein positif aux ER, le cancer de l'ovaire, le cancer de la prostate ou le cancer de l'endomètre.
PCT/GB2012/051289 2011-06-10 2012-06-08 Substances et procédés pour traiter un cancer positif aux récepteurs des oestrogènes α (er) Ceased WO2012168720A1 (fr)

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