WO2009027650A1 - Matières et procédés pour exploiter la létalité synthétique dans des cancers associés à brca - Google Patents

Matières et procédés pour exploiter la létalité synthétique dans des cancers associés à brca Download PDF

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WO2009027650A1
WO2009027650A1 PCT/GB2008/002866 GB2008002866W WO2009027650A1 WO 2009027650 A1 WO2009027650 A1 WO 2009027650A1 GB 2008002866 W GB2008002866 W GB 2008002866W WO 2009027650 A1 WO2009027650 A1 WO 2009027650A1
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brca
cancer
tankyrase
cell line
cells
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PCT/GB2008/002866
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Nuala Mccabe
Christopher James Lord
Alan Ashworth
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The Institute Of Cancer: Royal Cancer Hospital
Breakthrough Breast Cancer
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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 present invention relates to materials and methods for exploiting synthetic lethality in BRCA-associated cancers, including the treatment of cancer and screening candidate compounds for use in treating cancer.
  • Germline mutations in either BRCAl or BRCA2 strongly predispose individuals to cancers of the breast and also to malignancies in the ovaries, pancreas and prostate gland. Tumours arising in women carrying a single germline mutant BRCA allele exhibit loss of heterozygosity at the BRCA locus, losing the wild-type allele and retaining the mutant copy of the gene, suggesting that BRCAl and BRCA2 act as tumour suppressors (1) .
  • BRCAl or BRCA2 deficient cells have defects in the repair of DNA double- strand breaks (DSBs) by the conservative, error-free pathway of homologous recombination (HR) , leading to cellular sensitivity to specific DNA damaging agents (1) .
  • the present invention is based on novel therapeutic approaches to the treatment of BRCA-associated cancers based on the use of complimentary gene-function and drug screening synthetic lethality approaches for designing therapies for the treatment of cancers where loss of tumour suppressor function has occurred. These results are based on exemplary experiments involving Tankyrase 1. Furthermore, methods for identifying compounds suitable for use in the treatment of BRCA-associated cancer are provided, that can, for example, be used in high- throughput screening of compound libraries. The role of Tankyrase 1 in telomere elongation is described in Seimiya, Br. J. Cancer, 2006, 94, 341-345.
  • Tankyrase 1 (TRFl- interacting ankyrin-related ADP-ribose polymerase 1) was originally identified as a TRFl-binding protein by using a yeast two-hybrid screen.
  • This 140-kDa protein consists of four characteristic domains: the N-terminus is known as the HPS domain, containing homopolymeric runs of histidine, proline, and serine.
  • the C-terminal PARP domain of Tankyrase 1 catalyses poly (ADP-ribosyl) ation of acceptor proteins using NAD as a substrate .
  • the present invention provides the use of an inhibitor of Tankyrase 1 for the preparation of a medicament for the treatment an individual having a BRCA- associated cancer.
  • the present invention provides the use of an agent that induces an increase in centrosome amplification in cancer cells for the preparation of a medicament for the treatment of an individual having a BRCA-associated cancer.
  • the induction of an increase in centrosome amplification may be determined using assays well known in the art, such as the immunofluorescence assay the use of which is exemplified herein, and is generally by reference to the basal level in untreated cells .
  • the present invention provides an inhibitor of Tankyrase 1 for treating an individual having a BRCA- associated cancer.
  • the present invention provides a method of. treating an individual having a BRCA-associated cancer, the method comprising administering a therapeutically effective amount of an inhibitor of Tankyrase 1 to the individual.
  • the individual treated for a BRCA- associated cancer may have a mutation in a BRCAl and/or BRCA2 gene.
  • BRCAl and BRCA2 are known -tumour suppressors whose wild- type alleles are frequently lost in tumours of heterozygous carriers .
  • Germline deleterious mutations in the BRCAl (MIM 113705) and BRCA2 (MIM 600185) genes convey a significantly elevated risk of developing breast and ovarian cancer and of developing these cancers at earlier ages. Such mutations are considered to be responsible for approximately 40% of familial breast cancer and for the majority of familial ovarian cancers (Martin et al., Germline mutations in BRCAl and BRCA2 in breast-ovarian families from a breast cancer risk evaluation clinic, J. Clin. Oncol., 19, 2247-2253, 2001; and Szabo & King, Population genetics of BRCAl and BRCA2, Am. J. Hum.
  • the BRCA-associated cancer may be characterised by defects or inactivation of BRCAl and/or BRCA2 genes that are associated with the cancer cells as opposed to the patient's non-cancerous cells, and in particular patients whose tumours exhibit a defect in homologous recombination, the DNA repair mechanism controlled by BRCAl and BRCA2.
  • Methods for determining the presence of one or more mutations in BRCA nucleic acid sequences obtained from an individuals cancerous or non-cancerous cells are well known in the art and include the use of direct sequencing, hybridisation to a probe, restriction fragment length polymorphism (RFLP) analysis, single- stranded conformation polymorphism (SSCP) , PCR amplification of specific alleles, amplification of DNA target by PCR followed by a mini-sequencing assay, allelic discrimination during PCR, Genetic Bit Analysis, pyrosequencing, oligonucleotide ligation assay, analysis of melting curves or testing for a loss of heterozygosity (LOH) .
  • RFLP restriction fragment length polymorphism
  • SSCP single- stranded conformation polymorphism
  • PCR amplification of specific alleles amplification of DNA target by PCR followed by a mini-sequencing assay
  • allelic discrimination during PCR allelic discrimination during PCR
  • the BRCA-associated cancer may be characterised by the cancer cells exhibiting epigenetic inactivation of BRCAl or BRCA2 or loss of BRCAl or BRCA2 function, for example by promoter hypermethylation that may be determined by methylation specific PCR to detect silencing of BRCA genes .
  • BRCA-associated cancer examples include female and male breast cancer, ovarian cancer, pancreatic cancer and prostate cancer.
  • the present invention provides a method of screening for agents useful in the treatment of BRCA-associated cancer, the method employing first and second cell lines, wherein the first cell line is a BRCA-deficient cell line and the second cell line is a BRCA-proficient cell line, the method comprising:
  • the first and second cells lines are isogenically matched. It is also preferred that the cell lines are cancer cell lines, for example a mammalian cell line such as HCT116 or HCT75 used in the examples. The use of human cell lines or those from animal models (e.g. murine or rat) are preferred.
  • the present invention provides a method of screening for agents useful in the treatment of BRCA-associated cancer, the method comprising:
  • candidate agents identified using a method of screening according to the present invention may be the subject of further development to optimise their properties, to determine whether they work well in combination with other chemotherapy or radiotherapy, to manufacture the agent in bulks and/or to formulate the agent as a pharmaceutical composition.
  • FIG. 1 Inhibition of Tankyrase 1 is lethal with BRCA deficiency
  • a Western blots demonstrating Tankyrase 1 knock-down by short- hairpin (shRNA) .
  • shRNA short- hairpin
  • Whole cell extracts from HCT116-control shRNA and HCT116-Tankyrase-1 shRNA cells were subjected to Western blot analysis with indicated antibodies.
  • Molecular mass markers (kDa) are indicated.
  • b Reduced Tankyrase 1 expression is selectively lethal with knock-down of BRCAl and BRCA2.
  • HCT116-control shRNA and HCT116-Tankyrase-1 shRNA were transfected with pSUPER-SCRAMBLED (control) , pSUPER- BRCAl or pSUPER-BRCA2 constructs, that silence BRCAl and BRCA2 expression, respectively. Blasticidin resistant colonies were counted and quantified. Results show the mean of three independent experiments and error bars are equal to one standard deviation around the mean.
  • FIG. 1 Expression of deletion mutant constructs of Tankyrase 1 is lethal in the context of BRCA deficiency a. Schematic views and western blot analysis of Tankyrase 1 deletion mutant constructs. All constructs contain a FLAG epitope tag and a nuclear localization signal (NLS) at the N- termini (not shown) . The numbers indicate the positions of amino acid residues.
  • NLS nuclear localization signal
  • HPS region containing homopolymeric runs of His, Pro, and Ser
  • ANK ankyrin domain
  • SAM multimerization domain homologous to the sterile alpha motif
  • PARP PARP catalytic domain
  • ARC ANK repeat cluster working as an independent TRFl binding site
  • Bridge above two adjacent ANK repeats indicates presence of a conserved histidine, presumably contributing to inter-repeat stabilization.
  • HTC75 cells were infected with empty pLPC (MOCK) or with the indicated FN- Tankyrase 1 retroviruses. Whole cell extracts were subjected to western blot analysis with anti-Tankyrase 1 antibody. Coomassie brilliant blue stain of loaded proteins is shown as quantitative control. b.
  • ARCV mutant Tankyrase is selectively lethal with knock-down of BRCAl and BRCA2.
  • Cell lines stably expressing mutant Tankyrase 1 plasmids or an empty vector control were transfected with pSUPER- BRCAl, pSUPER-BRCA2 or pSUPER-SCRAMBLED and selected with blasticidin for 16 days. Surviving colonies were stained and counted. Results represent the mean of three independent experiments and error bars are equal to one standard deviation around the mean.
  • Fig 3. Loss of Tankyrase 1 function exacerbates centrosome amplification in BRCA deficient cells
  • a Centrosome amplification in cells with loss of BRCAl/2 and Tankyrase 1. Confocal images showing centrosome amplification in. cells with loss of function of Tankyrase 1 and silenced BRCAl or BRCA2. Blue represents nuclear TO-PRO 3 staining and red represents centrosomes as visualised by ⁇ -tubulin staining.
  • Tankyrase 1 TRFl-interacting ankyrin-related ADP-ribose polymerase 1
  • HPS domain containing homopolymeric runs of histidine, proline and serine. The functional significance of the HPS domain is unknown.
  • Tankyrase 1 is also includes an ANK domain placing the protein within the ANK family of proteins that is composed of a long stretch of 24 ANK repeats, providing a platform for protein-protein interactions. Unlike the ANK of ankyrins, the ANK domain of Tankyrase 1 is further divided into five well-conserved subdomains . Each subdomain, designated as ARC (ANK repeat cluster) I-V, works as an independent TRFl- binding site. TRFl recognition by the most C-terminal subdomain ARC V is the most important for the telomeric function of Tankyrase 1. The sterile alpha motif (SAM) domain, adjacent to the ARC V domain, is believed to contribute to multimerization of Tankyrase 1.
  • SAM sterile alpha motif
  • Tankyrase 1 The most striking feature of Tankyrase 1 is the C- terminal PARP domain, which catalyses poly (ADP-ribosyl) ation of acceptor proteins using NAD as a substrate. This post- translational modification provides significant negative charges to the acceptor proteins and often disrupts interactions between the acceptor proteins and the DNA.
  • Tankyrase 1 inhibitor An example of a small molecule compound which is a Tankyrase 1 inhibitor and which may be used in accordance with the invention is 3-aminobenzamide (3AB) (Seimiya et al . , Cancer Cell., Jan 7(1), 25-37, 2005.
  • the inhibitors of Tankyrase 1 may inhibit one or more activities of the polypeptide. In one activity of
  • Tankyrase 1 the C-terminal PARP domain of Tankyrase 1 catalyses poly (ADP-ribosyl) ation of acceptor proteins using NAD as a substrate.
  • the ankyrin repeat clusters (ARCs) of Tankyrase 1 that are believed to be essential for its function and most likely act as protein-interaction domains could be employed, for example by screening for candidate agents that interfere with ARC function.
  • the methods of screening disclosed herein may include the step of test candidate agents for binding to Tankyrase 1 using assays well known in the art.
  • Antibodies may be employed in the present invention as an example of a class of inhibitor useful for treating a BRCA-associated cancer, and more particularly as inhibitors of Tankyrase 1. They may also be used in the methods disclosed herein for assessing an individual having cancer or predicting the response of an individual having -cancer, in particular for determining whether - the individual has a BRCA-associated cancer that might be treatable according to the present invention.
  • the term "antibody” includes 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. Such techniques 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.
  • 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 CHl domains; (ii) the Fd fragment consisting of the VH and CHl 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).
  • Preferred antibodies used in accordance with the present invention are isolated, in the sense of being free from contaminants such as antibodies able to bind other polypeptides and/or free of serum components .
  • 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 molecules is one possibility.
  • 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.
  • Other reporters 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 biosensors. 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 Another class of inhibitors useful for treating a BRCA-associated cancer includes peptide fragments that interfere with the activity of Tankyrase 1.
  • Peptide fragments may be generated wholly or partly by chemical synthesis that block the catalytic sites of Tankyrase 1.
  • 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 M. Bodanzsky and A.
  • Bodanzsky The Practice of Peptide Synthesis, Springer Verlag, New York (1984) ; and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California
  • they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof .
  • 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 a BRCA- associated cancer includes nucleic acid inhibitors of Tankyrase 1 (NM_003747.2) , 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 .
  • Tankyrase 1 may be inhibited using anti-sense or RNAi technology.
  • anti-sense or RNAi technology The use of these approaches to down-regulate gene expression is now well-established in the art.
  • 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 1 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 transcribed from the sense strand of the target gene .
  • 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. However, it is established fact that the technique works.
  • 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 (Zamore et al, Cell, 101: 25-33, 2000) .
  • Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologeous genes in a wide range of mammalian cell lines (Elbashir et al , Nature, 411: 494-498, 2001) .
  • 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 Therapy, 2(3) : 213-223, 1995 and Mercola & Cohen, Cancer Gene Therapy, 2(1): 47-59, 1995.
  • 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 transcriptional gene silencing.
  • siRNAs small interfering RNAs
  • PTGs post transcriptional gene silencing
  • miRNAs micro-RNAs
  • targeted transcriptional gene silencing targeted transcriptional gene silencing.
  • Double- stranded RNA (dsRNA) -dependent post transcriptional silencing also known as RNA interference (RNAi)
  • 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 in 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.
  • Micro-interfering RNAs are endogenousIy encoded small non-coding RNAs, derived by processing of short hairpins . Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully- complementary sequences.
  • 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 complement. When this DNA sequence is transcribed into a single- stranded RNA molecule, the miRNA sequence and its reverse- complement base pair to form a partially double stranded RNA segment.
  • the design of microRNA sequences is discussed on John et al, PLoS Biology, 11(2), 1862-1879, 2004.
  • the 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 ribonucleotides.
  • the molecule may have symmetric 3' overhangs, e.g. of one or two (ribo) nucleotides, typically a UU of dTdT 3' overhang.
  • siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors) .
  • 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. Most preferably, 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 transfecting the cell with a vector encoding the shRNA sequence under control of a RNA polymerase III promoter such as the human Hl 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 exogenously (in vitro) by transcription from a vector.
  • 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(O)S, (thioate) ; P(S)S, (dithioate) ; P(O)NR'2; P(O)R 1 ; P(O)ORS; CO; or CONR 1 2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through-0-or-S- .
  • Modified nucleotide bases can be used in addition to the naturally occurring bases, and 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 silencing.
  • the provision of 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 covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3 'position and other than a phosphate group at the 5 'position.
  • modified nucleotides may also include 2 ' substituted sugars such as 2 ' -0-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 ' -0-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
  • 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, N6-methyladenine, 7-
  • 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 BRCA-associated cancer.
  • methods of screening candidate compounds to determine whether one or more candidate agents are likely to be useful for the treatment of BRCA-associated cancer.
  • there are two preferred general approaches that may be used for these methods of screening either alone or in any combination or order.
  • a method of screening may involve using cell lines to determine whether a candidate agent is synthetically lethal in a first cell line which is a BRCA deficient cell line.
  • BRCA deficient cell lines include those in which BRCA nucleic acid or polypeptide are not expressed or functional in the cell, for example because the cell has a BRCA gene having one or more mutations, or the expression of the BRCA gene in the cell is reduced or inhibited, for example by the use of RNA interference techniques.
  • Other examples include cells which are BRCA deficient because production of a BRCA polypeptide is reduced or the polypeptide is inactive. All of these cell types can be readily determine by the skilled person using techniques well known in the art and by reference to non BRCA deficient cells.
  • the method also uses a second cell line that is BRCA proficient as a control and candidate agents are selected which are synthetically lethal in the first cell line and preferably which do not cause any substantial cell death in the second cell line and/or normal cells.
  • a second cell line that is BRCA proficient as a control and candidate agents are selected which are synthetically lethal in the first cell line and preferably which do not cause any substantial cell death in the second cell line and/or normal cells.
  • synthetic lethality in cancer cells Two mutations are synthetically lethal if cells with either of the single mutations are viable, but cells with both mutations are inviable. Identifying synthetic lethal combinations therefore allows a distinct approach to identifying therapeutic targets that allows selective killing of tumour cells.
  • the method is carried out using cancer cell lines, e.g. mammalian or human cancer cell lines, and more preferably BRCAl and/or BRCA2 -deficient cancer cell lines.
  • RNAi screens One preferred way of initially identifying synthetic lethal interactions involves the use of RNAi screens.
  • Synthetic lethality describes the scenario in which two normally nonessential genes become essential when both are lost, or " inhibited.
  • Targeting a gene that is synthetically lethal with a cancer specific mutation should selectively kill tumour cells while sparing normal cells.
  • One of the major advantages of this approach is the ability to target cancer cells containing loss- of-function mutations, that is, mutations in tumour suppressor genes.
  • loss- of-function mutations that is, mutations in tumour suppressor genes.
  • Most pharmacological agents inhibit rather than activate protein function and therefore cannot be used to target loss-of-function alterations in tumours. Identification of synthetic lethal relationships with tumour suppressor genes could allow cells that contain the tumour suppressor mutations to be selectively killed.
  • RNAi screens it is now possible, in principle, to perform large-scale synthetic-lethal gene identification in mammalian cells, as is routinely done in yeast. Screening deletion mutants that have defects in cell-cycle checkpoint or DNA repair mechanisms in yeast has yielded synthetically lethal genes and small-molecule inhibitors. Using mammalian isogenic-paired cell lines that differ in a single genetic target, RNAi can be used to identify drug targets that when inhibited will result in the selective death of tumour cells.
  • a second method of screening may be employed based on the work described herein in which a protein target is identified as being synthetically lethal when their expression is inhibited in BRCA-associated cancers.
  • a protein target that may be used is Tankyrase 1.
  • methods of screening may be carried out for identifying candidate agents that are capable of inhibiting an activity of one or more of these targets, for subsequent use of development as agents for the treatment of BRCA-associated cancer.
  • the Tankyrase 1 and candidate agent are contacted in the presence of a substrate for Tankyrase 1 to determine whether the activity of Tankyrase 1 on the substrate is inhibited by the candidate agent.
  • the screening may employ the activity of the C-terminal PARP domain of Tankyrase 1 in catalyzing poly (ADP-ribosyl) ation of acceptor proteins using NAD as a substrate.
  • this may be done in an assay buffer to help the components of the assay interact, and in a multiple well format to test a plurality of candidate agents.
  • Preferred substrates produce a detectable change when they are processed by Tankyrase 1 to facilitate detection of effects of a candidate agent on Tankyrase 1.
  • the candidate agent may be a known inhibitor of one of the protein targets disclosed herein, 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 are preferred.
  • 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.
  • Such 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, i.e. non-BRCA associated 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 therapeutic agents in development, patient or disease profiles established that are relevant for the treatment of BRCA-associated cancer.
  • 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.
  • the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic or 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 BRCA-associated cancer.
  • the BRCA-associated cancer may arise because an individual has a mutation in a BRCA gene, and especially in the context of the present invention one or more mutations in BRCAl and/or BRCA2.
  • a review of mutations in the BRCAl and BRCA2 genes linked to the occurrence of cancer is provided in Wooster and Weber, Breast and ovarian cancer, N. Engl. J. Med., 348, 2339-2347, 2003
  • the BRCA-associated cancer is characterised by the cancer cells having a defect in DNA mismatch repair or the cancer cells exhibiting epigenetic inactivation of a BRCA gene, or loss of the loss of protein function.
  • the BRCA gene is BRCAl and/or BRCA2.
  • a cancer may be identified as a BRCA-associated cancer by determining the activity of the BRCA polypeptide in a sample of cells from an individual.
  • the sample may be of normal cells from the individual where the individual has a mutation in a BRCA gene or the sample may be of cancer cells, e.g. where the cells forming a tumour exhibit defects BRCA activity.
  • Activity may be determined relative to a control, for example in the case of defects in cancer cells, a relative to non-cancerous cells, preferably from the same tissue.
  • the activity of the BRCA gene 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.
  • a cancer may be identified as a BRCA- associated cancer by determining the presence in a cell sample from the individual of one or more variations, for example, polymorphisms or mutations, in a nucleic acid encoding a BRCA polypeptide .
  • Sequence variations such as mutations and polymorphisms may include a deletion, insertion or substitution of one or more nucleotides, relative to the wild-type nucleotide sequence.
  • the one or more variations may be in a coding or non-coding region of the nucleic acid sequence and, may reduce or abolish the expression or function of the BRCA gene.
  • the variant nucleic acid may encode a variant polypeptide which has reduced or abolished activity or may encode a wild-type polypeptide which has little or no expression within the cell, for example through the altered activity of a regulatory element .
  • a variant nucleic acid may have one or more mutations or polymorphisms relative to the wild-type sequence.
  • the presence of one or more variations in a nucleic acid which encodes a BRCA gene may be determined by detecting, in one or more cells of a test sample, the presence of an encoding nucleic acid sequence which comprises the one or more mutations or polymorphisms, or by detecting the presence of the variant component polypeptide which is encoded by the nucleic acid sequence .
  • sequence information can be retained and subsequently searched without recourse to the original nucleic acid itself.
  • scanning a database of sequence information using sequence analysis software may identify a sequence alteration or mutation.
  • Methods according to some aspects of the present invention may comprise determining the binding of an oligonucleotide probe to nucleic acid obtained from the sample, for example, genomic DNA, RNA or cDNA.
  • the probe may comprise a nucleotide sequence which binds specifically to a nucleic acid sequence which contains one or more mutations or polymorphisms and does not bind specifically to the nucleic acid sequence which does not contain the one or more mutations or polymorphisms, or vice versa.
  • the oligonucleotide probe may comprise a label and binding of the probe may be determined by detecting the presence of the label .
  • a method may include hybridisation of one or more (e.g. two) oligonucleotide probes or primers to target nucleic acid. Where the nucleic acid is double-stranded DNA, hybridisation will generally be preceded by denaturation to produce single-stranded DNA. The hybridisation may be as part of a PCR procedure, or as part of a probing procedure not involving PCR. An example procedure would be a combination of PCR and low stringency hybridisation.
  • Binding of a probe to target nucleic acid may be measured using any of a variety of techniques at the disposal of those skilled in the art.
  • probes may be radioactively, fluorescently or enzymatically labelled.
  • Other methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using
  • PCR RN'ase cleavage and allele specific oligonucleotide probing.
  • Probing may employ the standard Southern blotting technique. For instance, DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined.
  • Suitable selective hybridisation conditions for oligonucleotides of 17 to 30 bases include hybridization overnight at 42 0 C in 6X SSC and washing in 6X SSC at a series of increasing temperatures from 42°C to 65°C.
  • Nucleic acid which may be genomic DNA, RNA or cDNA, or an amplified region thereof, may be sequenced to identify or determine the presence of polymorphism or mutation therein.
  • a polymorphism or mutation may be identified hy comparing the sequence obtained with the database sequence of the component, as set out above.
  • the presence of one or more polymorphisms or mutations that cause abrogation or loss of function of a BRCA polypeptide may be determined.
  • Sequencing may be performed using any one of a range of standard techniques. Sequencing of an amplified product may, for example, involve precipitation with isopropanol, resuspension and sequencing using a TaqFS+ Dye terminator sequencing kit. Extension products may be electrophoresed on an ABI 377 DNA sequencer and data analysed using Sequence Navigator software.
  • a specific amplification reaction such as PCR using one or more pairs of primers may conveniently be employed to amplify the region of interest within the nucleic acid sequence, for example, the portion of the sequence suspected of containing mutations or polymorphisms.
  • the amplified nucleic acid may then be sequenced as above, and/or tested in any other way to determine the presence or absence of a mutation or polymorphism which reduces or abrogates the expression or activity of the BRCA polypeptide.
  • a cancer may be identified as BRCA- associated by assessing the level of expression or activity of a positive or negative regulator of a BRCA polypeptide, such as BRCAl and/or BRCA2.
  • Expression levels may be determined, for example, by Western blot, ELISA, RT-PCR, nucleic acid hybridisation or karyotypic analysis.
  • the individual or their tumour exhibit one or more variations, such as mutations and polymorphisms, in the BRCAl and/or BRCA2 genes. Mutations and polymorphisms associated with cancer may also be detected at the protein level by detecting the presence of a variant (i.e. a mutant or allelic variant) polypeptide.
  • the active agents disclosed herein for the treatment of BRCA- associated 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, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents .
  • pharmaceutically acceptable carriers include but not limited to provide a pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents .
  • pharmaceutically acceptable carriers for the treatment of BRCA- associated cancer
  • adjuvants e.g., diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic
  • 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 physiologically well tolerated and non toxic. Many examples of 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, or 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 associated with it.
  • Examples of 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 benefit/risk ratio.
  • a subject e.g. human
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • the active agents disclosed herein for the treatment of BRCA- associated 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 administration and other factors known to practitioners.
  • compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.
  • the 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 association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • agents disclosed herein for the treatment of BRCA-associated 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.
  • vaginal parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal ; by implant of a depot, for example, subcutaneously or intramuscularly.
  • Formulations suitable for oral administration 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 non- aqueous 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 of BRCA-associated cancer may be used in the methods described herein in combination with standard chemotherapeutic regimes or in conjunction with radiotherapy.
  • chemotherapeutic agents include inhibitors of topoisomerase I and II activity, such as camptothecin, drugs such as irinotecan, topotecan and rubitecan, alkylating agents such as temozolomide and DTIC (dacarbazine) , and platinum agents like cisplatin, cisplatin-doxorubicin-cyclophosphamide, carboplatin, and carboplatin-paclitaxel .
  • chemotherapeutic agents include doxorubicin-cyclophosphamide, capecitabine, cyclophosphamide-methotrexate-5-fluorouracil, docetaxel, 5- flouracil-epirubicin-cyclophosphamide, paclitaxel, vinorelbine, etoposide, pegylated liposomal doxorubicin and topotecan.
  • Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
  • 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 calculated on the basis of the parent compound, and so the actual weight to be used is increased proportionately.
  • tumour suppressor gene function that characterises many cancers.
  • loss of tumour suppressor function in comparison to oncogene activation, presents several problems in the design of potential therapeutic approaches that target these cancers.
  • gain of function or activity can potentially be pharmacologically inhibited.
  • synthetic lethal interactions with tumour suppressor mutations has been suggested as an attractive approach (Kaelin, 2005, Iorns et al . , 2007).
  • Two genes are synthetically lethal if a mutation in either gene alone is compatible with viability but simultaneous mutation of both causes cell death (Kaelin, 2005) .
  • the discovery of genes that are synthetically lethal with known cancer- predisposing mutations could aid the identification of novel cancer drug targets .
  • Tankyrase 1 was identified for its ability to bind TRFl, a positive regulator of telomere length (6, 7) .
  • Tankyrase 1 binding to TRFl results in poly (ADP-ribosyl) ation of TRFl and leads to TRFl release from the telomeres and its proteolytic degradation (8) .
  • over- expression of Tankyrase 1 results in telomere elongation (7) .
  • the interaction between Tankyrase 1 and TRFl requires the C- terminal region of Tankyrase 1 (7) .
  • This region contains a domain of 24 ankyrin (AMK) repeats, which, are organized in five highly conserved subdomains (ACRl-V, or ANK repeat clusters) (7, 9) .
  • ACRl-V, or ANK repeat clusters 7, 9 .
  • TRFl binds to all five ACR subdomains, only the ACRV domain is required for its poly (ADP-ribosyl) ation and release from the telomeres (7) .
  • a recent report showed that inhibition of Tankyrase 1 accentuates the ability of a telomerase inhibitor, MST-312 to induce telomere shortening (10) , indicating that Tankyrase 1 may be a potential therapeutic target.
  • Tankyrase 1 has also been localized to the Golgi and to the mitotic spindle poles (11, 12) .
  • a reduction in Tankyrase 1 expression has also been shown to cause cells to accumulate in M phase (13) and have abnormal spindle structures (14) , suggesting a role for Tankyrase 1 in spindle structure and function.
  • Retroviral pSM2c vectors for Tankyrase-1 short hairpin RNA (shRNA, cat. #RHS1764- 9688056) and control "scrambled" non-silencing shRNA (cat. #RHS1703) were obtained from Open Biosystems (Huntsville, AL,
  • HCT116 cells were infected with these retroviruses and the puromycin-resistant fractions were selected.
  • HTC75 cells expressing truncated forms of Tankyrase 1 have been previously described (7) .
  • Transfections were carried out using Fugene 6 (Roche) according to the manufacturer's instructions. JRNA Interference pSUPER-BRCAl, pSUPER-BRCA2 and pSUPER-SCRAMBLED RNA interference constructs were previously described (15) .
  • Clonogenic survival assays were performed as described in (2) .
  • HCT116 and HTC75 cells were transfected in 6 well plates with either pSUPER-BRCAl , pSUPER- BRCA2 or pSUPER-SCRAMBLED plasmids each expressing resistance to the antibiotic blasticidin (15) .
  • Twenty-four hours after transfection cells were trypsinised and re-seeded in 6-well plates.
  • Forty-eight hours post-transfection treatment with blasticidin was commenced and cells were re-fed every 3 days. After 14-16 days, cells were washed with PBS, fixed in methanol and stained with crystal violet . Colonies were counted using a ColCount machine (Oxford Optronix) .
  • HCT116 cells were transfected with either pSUPER-BRCAl, pSUPER- BRCA2 or pSUPER-SCRAMBLED. Twenty-four hours after transfection, cells were divided and then treated with blasticidin as described above. Cells were kept in selection for one week, plated onto coverslips and the next day fixed with cold (-20 0 C) methanol. Cells were stained with a 1:1000 dilution of mouse monoclonal anti- ⁇ -tubulin antibody (Sigma, USA) . After washing, the primary antibody was visualised using Alexa Fluor-555 (Invitrogen, USA) and nuclei with TO-PRO-3 iodide (Molecular Probes) .
  • HCT116-Tankyrase 1 shRNA short-interfering hair-pin RNA
  • HCT116-Control shRNA a control cell line transfected with a plasmid encoding a non-targeting shRNA (HCT116-Control shRNA)
  • HCT116- Tankyrase 1 shRNA cells and control cells were subsequently transfected with pSUPER shRNA constructs targeting either BRCAl or BRCA2 and clonogenic survival assays were performed.
  • Loss of the ARCV domain of Tankyrase 1 has previously been shown to significantly reduce the ability of Tankyrase 1 to poly (ADP-ribosyl) ate TRFl and release TRFl from telomeres, thereby preventing telomere elongation (7) .
  • Silencing of BRCAl or BRCA2 significantly increased lethality in the ACRV- expressing cells compared to the isogenic control cell lines MOCK and TANK-I (Fig. 2b) .
  • cells stably expressing other truncated forms of Tankyrase 1, ARCl and delta ANK did not show any selective lethality when transfected with pSUPER-BRCAl or pSUPER-BRCA2 compared to the control cells.
  • TRFl is a member of the shelterin complex and functions predominantly at the telomeres (16) .
  • TIFs or telomere dysfunction induced foci DNA damage foci
  • Tankyrase 1 has also been localized to the mitotic spindle poles (11) .
  • BRCAl and BRCA2 deficient cells have also been previously shown to display centrosome amplification and furthermore BRCAl has been shown to interact with the centrosome in M phase cells (18, 19) . Therefore we investigated whether the synthetic lethality between BRCA and Tankyrase 1 was due to the exacerbation of known mitotic phenotypes associated with BRCA deficiency.
  • this phenotype was considerably exacerbated in cells with silenced BRCAl or BRCA2 in the presence of the ACRV Tankyrase 1 mutant (Fig 3a, b) . This difference was statistically significant (p ⁇ 0.05) when compared to control cells.
  • HCT116 derivatives A similar phenotype was also observed with HCT116 derivatives.
  • a higher percentage of HTC116-Tankyrase 1 shRNA cells displayed centrosome amplification when transfected with pSUPER-BRCAl or pSUPER-BRCA2 compared to cells transfected with the control plasmid (Fig 3a, c) .
  • Tankyrase 1 silenced cells have been demonstrated to accumulate in M phase (13) and have abnormal spindle structures (14) . Therefore it is possible that a combination of genomic instability (caused by BRCA deficiency) and spindle dysfunction (caused by Tankyrase 1 deficiency) causes centrosome amplification and, ultimately cell death. Regardless of the exact mechanism, the demonstration of synthetic lethality suggests that a therapeutic approach that is based on interfering with Tankyrase 1 function in patients with BRCA-associated tumours, is worthy of further investigation. Given that specific, high potency, small molecule inhibitors of PARPl and PARP2 proteins have already been developed, it seems likely that similar inhibitors may be developed for Tankyrase 1. Furthermore, it may be conceivable that such therapeutics may be exploited to treat not only BRCA associated cancers but also sporadic cancers displaying properties of "BRCAness" or those with deficiencies in the HR pathway (21) . References;
  • telomere-directed molecular cancer therapeutics Cancer Cell 2005; 7:25-37.
  • Tankyrase is a golgi-associated mitogen- activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles. J Biol Chem 2000; 275:38437-44. 13. Dynek JN, Smith S. Resolution of sister telomere association is required for progression through mitosis. Science 2004; 304:97-100.

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Abstract

L'invention concerne des matières et des procédés pour exploiter la létalité synthétique dans des cancers associés à BRCA, y compris un traitement anticancéreux et le criblage de composés candidats anticancéreux. Les méthodes de traitement de cancers associés à BRCA, basées sur l'utilisation d'une fonction de gène complémentaire et des méthodes de criblage de médicaments par létalité synthétique, visent à concevoir des thérapies pour le traitement de cancers dans lesquels une perte de la fonction de suppresseur de tumeur a eu lieu. L'invention est basée sur des expériences exemplaires utilisant la Tankyrase 1.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110097329A1 (en) * 2009-06-26 2011-04-28 Massachusetts Institute Of Technology Compositions and methods for treating cancer and modulating stress granule formation
WO2015108986A1 (fr) * 2014-01-16 2015-07-23 Clovis Oncology, Inc. Utilisation d'inhibiteurs de parp pour traiter des patients atteints de cancer du sein ou de l'ovaire présentant une perte d'hétérozygotie
US9193689B2 (en) 2012-03-07 2015-11-24 Institute Of Cancer Research: Royal Cancer Hospital (The) 3-aryl-5-substituted-isoquinolin-1-one compounds and their therapeutic use
US9611223B2 (en) 2013-09-11 2017-04-04 Institute Of Cancer Research: Royal Cancer Hospital (The) 3-aryl-5-substituted-isoquinolin-1-one compounds and their therapeutic use

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030987A2 (fr) * 1999-10-25 2001-05-03 Rigel Pharmaceuticals, Inc. Tankyrase h, compositions intervenant dans le cycle cellulaire et procedes d'utilisation
WO2005012524A1 (fr) * 2003-07-25 2005-02-10 The University Of Sheffield Utilisation d'arni inhibiteur de l'activite parp pour la fabrication d'un medicament pour le traitement du cancer
US20060149059A1 (en) * 2003-03-12 2006-07-06 Kudos Pharmaceuticals Limited Phthalazinone derivatives

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030987A2 (fr) * 1999-10-25 2001-05-03 Rigel Pharmaceuticals, Inc. Tankyrase h, compositions intervenant dans le cycle cellulaire et procedes d'utilisation
US20060149059A1 (en) * 2003-03-12 2006-07-06 Kudos Pharmaceuticals Limited Phthalazinone derivatives
WO2005012524A1 (fr) * 2003-07-25 2005-02-10 The University Of Sheffield Utilisation d'arni inhibiteur de l'activite parp pour la fabrication d'un medicament pour le traitement du cancer

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BUTCHER ET AL: "Epigenetic inactivation of BRCA1 is associated with aberrant expression of CTCF and DNA methyltransferase (DNMT3B) in some sporadic breast tumours", EUROPEAN JOURNAL OF CANCER, PERGAMON PRESS, OXFORD, GB, vol. 43, no. 1, 10 January 2007 (2007-01-10), pages 210 - 219, XP005826119, ISSN: 0959-8049 *
DE SOTO JOSEPH A ET AL: "The inhibition and treatment of breast cancer with poly (ADP-ribose) polymerase (PARP-1) inhibitors.", INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES 2006, vol. 2, no. 4, 2006, pages 179 - 185, XP007906272, ISSN: 1449-2288 *
DYNEK J N ET AL: "Resolution of Sister Telomere Association Is Required for Progression Through Mitosis", SCIENCE 20040402 US, vol. 304, no. 5667, 2 April 2004 (2004-04-02), pages 97 - 100, XP002502779, ISSN: 0036-8075 *
GELMINI S ET AL: "Tankyrase, a positive regulator of telomere elongation, is over expressed in human breast cancer", CANCER LETTERS, NEW YORK, NY, US, vol. 216, no. 1, 8 December 2004 (2004-12-08), pages 81 - 87, XP004611752, ISSN: 0304-3835 *
KANAI MASAYUKI ET AL: "Involvement of poly(ADP-ribose) polymerase 1 and poly(ADP-ribosyl)ation in regulation of centrosome function.", MOLECULAR AND CELLULAR BIOLOGY, vol. 23, no. 7, April 2003 (2003-04-01), pages 2451 - 2462, XP007906259, ISSN: 0270-7306 *
NOTTBOHM AMANDA C ET AL: "A colorimetric substrate for poly(ADP-ribose) polymerase-1, VPARP, and tankyrase-1.", ANGEWANDTE CHEMIE (INTERNATIONAL ED. IN ENGLISH) 2007, vol. 46, no. 12, 2007, pages 2066 - 2069, XP002502780, ISSN: 1433-7851 *
SEIMIYA H: "The telomeric PARP, tankyrases, as targets for cancer therapy.", BRITISH JOURNAL OF CANCER 13 FEB 2006, vol. 94, no. 3, 13 February 2006 (2006-02-13), pages 341 - 345, XP002502777, ISSN: 0007-0920 *
SEIMIYA HIROYUKI ET AL: "Tankyrase 1 as a target for telomere-directed molecular cancer therapeutics.", CANCER CELL JAN 2005, vol. 7, no. 1, January 2005 (2005-01-01), pages 25 - 37, XP002502778, ISSN: 1535-6108 *
TRESZEZAMSKY ALEJANDRO D ET AL: "BRCA1- and BRCA2-deficient cells are sensitive to etoposide-induced DNA double-strand breaks via topoisomerase II.", CANCER RESEARCH 1 AUG 2007, vol. 67, no. 15, 1 August 2007 (2007-08-01), pages 7078 - 7081, XP002502781, ISSN: 0008-5472 *

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US20110097329A1 (en) * 2009-06-26 2011-04-28 Massachusetts Institute Of Technology Compositions and methods for treating cancer and modulating stress granule formation
US9272022B2 (en) 2009-06-26 2016-03-01 Massachusetts Institute Of Technology Compositions and methods for treating cancer and modulating stress granule formation
US9193689B2 (en) 2012-03-07 2015-11-24 Institute Of Cancer Research: Royal Cancer Hospital (The) 3-aryl-5-substituted-isoquinolin-1-one compounds and their therapeutic use
US9611223B2 (en) 2013-09-11 2017-04-04 Institute Of Cancer Research: Royal Cancer Hospital (The) 3-aryl-5-substituted-isoquinolin-1-one compounds and their therapeutic use
WO2015108986A1 (fr) * 2014-01-16 2015-07-23 Clovis Oncology, Inc. Utilisation d'inhibiteurs de parp pour traiter des patients atteints de cancer du sein ou de l'ovaire présentant une perte d'hétérozygotie

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