WO2006109091A2 - Sensitisation of cancer cells to dna replication inhibitors - Google Patents

Abstract

This invention relates to the characterisation of the activity of the RECQL4 protein and the recognition that deficiencies in RECQL4 activity sensitise cancer cells to drugs which inhibit DNA replication. Methods of identifying RECQL4 deficient cancers which are sensitive to drugs which inhibit DNA replication and methods of treating RECQL4 deficient cancers using such drugs are provided.

Description

Sensitisation of Cancer Cells to DNA replication Inhibitors

This invention relates to the treatment of cancer and, in particular to the sensitisation of cancer cells to chemotherapeutic drugs .

The protein encoded by the human RECQL4 gene possesses a helicase domain characteristic of the RecQ family, but it lacks many of the conserved features present in other human RecQ helicases, such as domains involved in DNA binding (Hickson,

2003) . Although it is apparently indispensable for normal cell growth, the biological functions of RECQL4 are not understood. Human diseases connected with i?ECQL4-mutations appear distinct in their clinical phenotypes from Bloom or Werner's syndrome, which are caused by inactivation of the RecQ helicases BLM or WRN, respectively (Ellis, 1997) .

RECQL4-mutations occur in some cases of Rothmund-Thornson syndrome (RTS) , which is a rare genetic disorder characterized by chromosome fragility, developmental abnormalities, and predisposition to cancers including osteogenic sarcoma (Kitao et al., 1998; Kitao et al . , 1999; Vennos and James, 1995). Distinct RECQL4 mutations also occur in RAPADILINO syndrome, where they are associated with skeletal malformations but not cancer predisposition (Siitonen et al . 2003) . Extensive disruption of the murine homologue of RECQL4 by gene targeting results in early embryonal lethality accompanied by defective cell proliferation (Ichikawa et al . 2002), whilst deletion of a single exon in the murine gene causes growth retardation, developmental anomalies and impeded cell division in vitro (Hoki et al. 2003) .

The present inventors have cloned the X. laevis homolog, xRTS, of RECQL4 and characterized the role of this protein and its mammalian orthologues in the initiation of DNA replication. Furthermore, inhibition or depletion of RECQL4 activity is shown to have an anti-proliferative effect and to increase sensitivity to replication blocking drugs. These findings have various applications in the treatment of cancer.

One aspect of the invention provides a method of treating a RECQL4 deficient cancer condition in an individual comprising administering a DNA replication inhibitor to the individual.

Related aspects of the invention provide the use of a DNA replication inhibitor in the manufacture of a medicament for use in a method of treating a RECQL4 deficient cancer condition in an individual, and; a DNA replication inhibitor for use in a method of treating a RECQL4 deficient cancer condition in an individual .

A RECQL4 deficient cancer condition is a cancer condition in which cancer cells are deficient in the level or activity of RΞCQL4 polypeptide i.e. the cells have a reduced or abrogated RECQL4 activity relative to normal cells.

The wild-type human RECQL4 nucleic acid sequence has the database number AB026546.1 GI: 6440968 and the RECQL4 amino acid sequence has the database number: BAA86899.1, GI: 6440969.

Cancer cells in general are characterised by abnormal proliferation relative to normal cells and typically form clusters or tumours in an individual having a cancer condition.

In some embodiments, the individual has a condition in which both non-cancer cells and cancer cells are deficient in RECQL4, such as Rothmund-Thornson syndrome or related conditions .

In other embodiments, the individual does not have a condition associated with RECQL4 deficiency, such as Rothmund-Thomson syndrome. Non-cancer cells from the individual have a normal phenotype and cancer cells from the individual are deficient in RECQL4 i.e. healthy cells from the individual have normal RECQL4 activity.

The expression and/or activity of RECQL4 polypeptide may be reduced or abolished in RECQL4 deficient cancer cells, for example by means of mutation, polymorphism or hypermethylation of the encoding nucleic acid, or by means of mutation, polymorphism or hypermethylation in a regulatory region or a gene encoding a regulatory factor. In some embodiments, a RΞCQL4 deficient cancer may be derived from a cell lineage that has low RECQL4 expression or activity.

A cancer cell that is RECQL4 deficient may be heterozygous or homozygous for a mutation or polymorphism in the nucleic acid encoding the gene or its regulatory elements .

An RECQL4 deficient cancer may be any type of solid cancer or malignant lymphoma and especially osteogenic sarcoma, leukaemia, skin cancer, bladder cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, oesophageal cancer, pancreas cancer, renal cancer, stomach cancer and cerebral cancer. In some preferred embodiments, the RECQL4 deficient cancer may be osteogenic sarcoma or skin cancer (e.g. squaemous cell carcinoma) .

In the methods described herein, a cancer condition in an individual may have been previously identified as an RECQL4 deficient cancer or a method may comprise the step of identifying a cancer condition in an individual as being RECQL4 deficient . A cancer condition may be identified as a RECQL4 deficient cancer by determining the presence or level of a nucleic acid, for example triRNA, encoding a RECQL4 polypeptide in one or more cancer cells obtained from the individual. Low levels or absence of a nucleic acid encoding a RECQL4 polypeptide relative to controls is indicative that the cancer is a RECQL4 deficient cancer.

Various methods are available for determining the presence or absence or level in a sample of cells obtained from an individual of a particular nucleic acid sequence, for example a nucleic acid encoding an RECQL4 polypeptide. Many suitable methods are known in the art and described in Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook & Russell (2001) Cold Spring Harbor Laboratory Press NY and Current Protocols in

Molecular Biology, Ausubel et al . eds . John Wiley & Sons (1992), including, for example, Northern blotting, RT-PCR.

A cancer condition may be identified as a RECQL4 deficient cancer by determining the presence or level of one or more variant forms of a RECQL4 nucleic acid in one or more cancer cells obtained from the individual. The presence of a variant form of the nucleic acid may be indicative that the cancer is RECQL4 deficient.

The presence of a variant of a RECQL4 nucleic acid may determined by detecting the presence of the variant nucleic acid sequence or by detecting the presence of a variant polypeptide which is encoded by the variant nucleic acid sequence .

A variant nucleic acid sequence may include one or more mutations or polymorphisms, such as deletions, insertions or substitutions of one or more nucleotides, relative to the wild- type nucleotide sequence. In some embodiments, the variation may be a gene amplification or an increase or decrease in methylation. 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 activity of the polypeptide. In other words, 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, two, three, four or more mutations or polymorphisms relative to the wild-type sequence.

Nucleic acid 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 by comparing the sequence obtained with the known sequence of the polypeptide, for example as set out in sequence databases. In particular, the presence of one or more polymorphisms or mutations that cause abrogation or loss of function of the RECQL4 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.

Having sequenced nucleic acid of an individual or sample, the sequence information can be retained and subsequently searched without recourse to the original nucleic acid itself. Thus, for example, scanning a database of sequence information using sequence analysis software may identify a sequence alteration or mutation.

A cancer may also be identified at the protein level as a RECQL4 deficient cancer (i.e. having an RECQL4 deficient phenotype) , for example by determining the level of RECQL4 polypeptide in one or more cancer cells obtained from the individual. Low levels or absence of the RECQL4 polypeptide relative to controls may be indicative of an RECQL4 deficient cancer. In some embodiments, a cancer may be identified as a RECQL4 deficient cancer by detecting the presence of a variant (i.e. a mutant or allelic variant) RECQL4 polypeptide. The presence of the variant form may be indicative of an RECQL4 deficient cancer.

The level or presence of a RECQL4 polypeptide or variant may be determined by contacting a sample with an antibody directed against the RECQL4 polypeptide or variant, and determining binding of the antibody to the sample . Binding of the antibody to the sample may be indicative of the presence or level of the RECQL4 polypeptide or variant thereof in a cell within the sample .

Many suitable methods for determining the binding of the antibody to the sample are known in the art . Tagging with individual reporter molecules is one possibility. A reporter molecule may be linked to the primary antibody that binds to the target molecule or to a secondary antibody that binds to the primary antibody. The reporter molecule 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 binding molecule (e.g. antibody) and reporter molecule. One favoured mode is by covalent linkage of a binding member with an individual fluorochrome, phosphor or laser 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 that 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. Further examples are horseradish peroxidase and chemiluminescence .

The mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.

An antibody which specifically binds to a RECQL4 polypeptide or a variant thereof may not show any significant binding to antigens in mammalian cells other than the RECQL4 polypeptide or variant, for example the antibody may show no significant binding to other RecQ helicases.

An antibody that specifically binds to a RECQL4 polypeptide or a variant thereof may be generated using techniques conventional in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with a target polypeptide or a peptide fragment of the target. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al . , (1992) Nature 357, 80-82). Alternatively or additionally, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.

Samples to be subjected to contact with the antibody may be prepared using any available technique that allows the antibody to bind to bind to cellular polypeptides in the sample.

A cancer may also be identified as a RECQL4 deficient cancer (i.e. having an RECQL4 deficient phenotype) by determining the activity of RECQL4 polypeptide in one or more cancer cells obtained from the individual . Low levels of activity relative to controls may be indicative of an RECQL4 deficient cancer.

Activity may be determined relative to normal (i.e. non-cancer) cells, preferably from the same tissue.

A sample obtained from an individual may be a tissue sample comprising one or more cells, for example a biopsy from a cancerous tissue as described above, or a non-cancerous tissue, for example for use as a control.

A DNA replication inhibitor may include any chemotherapeutic agent which inhibits or abolishes replication in mammalian cells. Many suitable compounds are known in the art and are used in the treatment of cancer, including for example, alkylating agents, such as busulfan, carmustine, chlorambucil, chlormethine-Hcl , cyclophosphamide, estramustine, ifosfamide, lomustine, melphalan, thiotepa and treosulfan; cytotoxic antibiotics (anthracyclins) such as doxorubicin, alcarubicin, idarubicin, daunorubicin, mitoxantrone, bleomycin, dactinomycin and mitomycin; antimetabolites, such as methotrexate, cytarabine, fludarabine, cladribine, gemcytabine, 5-FU, raltitrexed, mercaptopurine, tegafur, thioguanine, capecitabine,- etoposide, Amsacrine, dacarbazine, temozolamide, hydroxycarbamide (hydroxyurea) , pentostatin, cyclophosphamide- methotrexate-5-fluorouracil, and 5-fluoracil-epirubicin- cyclophosphamide; platinum compounds, such as cisplatin, carboplatin and oxaliplatin, topoisomerase I inhibitors such as irinotecan, rubitecan, topotecan and camptothecin, antineoplastics such as vinorelbine and vinblastine, and analogues, derivatives or salts of any of these.

In some preferred embodiments, gemcytabine (Gemzar™, Eli Lilley) may be employed.

Preferably, the DNA replication inhibitor is used in a dosage or formulation that is not lethal to cells which are not RECQL4 deficient. Suitable dosages and regimens for DNA replication inhibiting chemotherapeutic agents are well known to medical practitioners.

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.

In general, a suitable dose of the active compound is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day. Where 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.

While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g., formulation) comprising at least one active compound, as defined above, together 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.

Pharmaceutical compositions comprising a DNA replication inhibitor, for example an inhibitor admixed together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein, may be used in the methods described herein.

The term "pharmaceutically acceptable" as used herein pertains to 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. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990. 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 .

Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols .

The inhibitor or pharmaceutical composition comprising the inhibitor 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) ; rectal; 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, subcutaneousIy or intramuscularly.

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 (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal) , 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. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/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.

Another aspect of the invention provides a method of identifying and/or obtaining a compound that sensitises cells to DNA replication inhibition or a method of identifying and/or obtaining an anti -cancer compound comprising: contacting an RECQL4 polypeptide with a test compound; and, determining the interaction of said compound with said polypeptide .

Interaction of the compound and the RECQL4 polypeptide is indicative that the compound that sensitises cells to DNA replication inhibition.

In some embodiments, interaction may be determined by determining the binding of the compound to the RECQL4 polypeptide. Binding of the test compound to the polypeptide may be indicative that the compound sensitises cells to replication inhibition.

In other embodiments, interaction may be determined by determining the activity of the RECQL4 polypeptide in the presence of the test compound. A decrease in activity in the presence relative to the absence of the test compound may indicative that the compound sensitises cells to replication inhibition.

The RECQL4 polypeptide may be comprised in a cell or may be contacted with a test compound in vitro in a cell free system.

Suitable RECQL4 polypeptides include a polypeptide having the wild type human RECQL4 amino acid sequence (database number: BAA86899.1, GI: 6440969) or a variant or fragment thereof.

Suitable variants or fragments of RECQL4 polypeptides retain the activity of the wild-type RECQL4 polypeptide. A variant may have one or more of addition, insertion, deletion or substitution of one or more amino acids in the wild-type polypeptide sequence. For example, up to about 5, 10, 15 or 20 amino acids may be altered. Such alterations may be caused by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the encoding nucleic acid. An amino acid sequence variant of a wild-type polypeptide sequence, may comprise an amino acid sequence which shares greater than 20% sequence identity with the wild-type sequence, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 55%, greater than 65%, greater than 70%, greater than about 80%, greater than 90% or greater than 95%. The sequence may share greater than 20% similarity with the wild-type sequence, greater than 30% similarity, greater than 40% similarity, greater than 50% similarity, greater than 60% similarity, greater than 70% similarity, greater than 80% similarity or greater than 90% similarity.

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. MoI. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444- 2448) , or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol. 147: 195-197), or the TBLASTN program, of Altschul et al . (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl . Acids Res. (1997) 25 3389-3402) may be used. Sequence identity and similarity may also be determined using Genomequest™ software (Gene-IT, Worcester MA USA) .

Sequence comparisons are preferably made over the full-length of the relevant sequence described herein. Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine .

In some embodiments, an RECQL4 polypeptide may comprise or consist of the xRTS polypeptide sequence shown in figure 1 or a variant thereof .

A test compound suitable for use in a method described herein may be any compound or entity, such as a small organic molecule, peptide or nucleic acid.

Suitable compounds may include peptide fragments of RECQL4.

Peptide fragments may be generated wholly or partly by chemical synthesis using the published sequences of the components. 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), or 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 . Other candidate compounds for inhibiting RECQL4 may be based on modelling the 3 -dimensional structure of a component of the RECQL4 and using rational drug design to provide candidate compounds with particular molecular shape, size and charge characteristics. 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, i.e. it may interfere with the interactions or activity of the DNA repair pathway component. 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 suitable RECQL4 inhibitors include nucleic acid encoding part or all of the amino acid sequence of RECQL4 or the complement thereof, which inhibit activity or function by down- regulating production of active RECQL4 polypeptide. For instance, expression may be inhibited using anti-sense or RNAi technology. The use of these approaches to down-regulate gene expression is well-established in the art.

A method described herein may comprise identifying a test compound as an inhibitor of RECQL4. Following identification of a compound using a method described above, the compound may be isolated and/or synthesised.

An compound identified using one or more primary screens (e.g. in a cell-free system) as having ability to interact with a

RECQL4 polypeptide may be assessed or investigated further using one or more secondary screens. Biological activity, for example, sensitisation to replication inhibition may be tested on cells in culture as described herein. The compound may be modified to optimise its pharmaceutical properties. The modified compound may be tested using the methods described herein to see whether it has the target property, or to what extent it is exhibited. Modified compounds include mimetics of the lead compound. Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.

The test compound may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, e.g. for any of the purposes discussed elsewhere herein. A method of the invention may comprise formulating the test compound or the modified test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier as discussed above.

Another aspect of the present invention provides a method of producing a pharmaceutical composition comprising; i) identifying a compound as a RECQL4 inhibitor using a method described herein; and, ii) admixing the identified compound with a pharmaceutically acceptable carrier.

The formulation of compositions with pharmaceutically acceptable carriers is described above.

Another aspect of the invention provides a method for preparing a pharmaceutical composition, for example, for the treatment of cancer or the sensitisation of cancer cells to replication inhibition, comprising; i) identifying a compound which inhibits RECQL4, ii) synthesising the identified compound, and; ii) incorporating the compound into a pharmaceutical composition. The identified compound may be synthesised using conventional chemical synthesis methodologies. Methods for the development and optimisation of synthetic routes are well known to persons skilled in this field.

Incorporating the compound into a pharmaceutical composition may- include admixing the synthesised compound with a pharmaceutically acceptable carrier or excipient .

Depletion or inhibition of RECQL4 is shown herein to inhibit the proliferation of cancer cells.

Another aspect of the invention provides a method of treating a cancer condition comprising; administering an inhibitor of a RECQL4 polypeptide to the individual .

Related aspects of the invention provide the use of a RECQL4 inhibitor in the manufacture of a medicament for the treatment of a cancer condition in an individual and a RECQL4 inhibitor for use in the treatment of a cancer condition.

The RECQL4 inhibitor may be a siRNA, antibody or small molecule as described herein. Suitable RECQL4 inhibitors include, for example, antibodies or antibody fragments which specifically bind to RΞCQ14 and sense or antisense nucleic acids which comprise or consist of part or all of the human RECQL4 nucleic acid sequence.

Another aspect of the invention provides a method of identifying a cancer condition in an individual as susceptible to treatment with a DNA replication inhibitor comprising: determining the presence of one or more RECQL4 deficient cancer cells in a sample obtained from the individual. The presence of one or more RECQL4 deficient cancer cells may be determined as described above.

The presence of one or more RECQL4 deficient cancer cells is indicative that the cancer condition in the individual is susceptible to treatment with a DNA replication inhibitor. Suitable DNA replication inhibitors are described above.

The present inventors have cloned and characterised the Xenopus RTS protein. Another aspect of the invention provides an isolated nucleic acid encoding a RTS polypeptide comprising or consisting of the amino acid sequence of Figure 1 or a variant thereof .

Variants of the amino acid sequence of Figure 1 are described above and include sequences having at least 70% sequence identity with the amino acid sequence of Figure I₇ greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 98%.

Preferably, a variant sequence retains the helicase activity of the wild-type sequence and/or the ability to recruit RPA70 polypeptide to chromatin.

The isolated nucleic acid may, for example, comprise or consist of the nucleic acid sequence shown in Figure 3.

An isolated nucleic acid as described herein may be comprised in a vector. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al . , 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al . eds . , John Wiley & Sons, 1992.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria systems. A common, preferred bacterial host is B. coli.

Another aspect of the present invention provides a host cell containing heterologous nucleic acid encoding a polypeptide as described herein. Suitable host cells include microbial host cells such as E. coli.

The nucleic acid may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques. The nucleic acid may be on an extra-chromosomal vector within the cell.

The introduction of nucleic acid into a host cell, which may (particularly for in vitro introduction) be generally referred to without limitation as "transformation", may employ any available technique. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.

Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide is produced.

A method of producing an RTS polypeptide may comprise: (a) causing expression from nucleic acid which encodes a polypeptide in a suitable expression system to produce the polypeptide recombinantly;

(b) testing the recombinantly produced polypeptide for helicase activity and/or ability to recruit RPA70 polypeptide to chromatin.

For example, the recombinantly produced polypeptide may be tested for ability to overcome the inhibition of replication caused by xRTS depletion in egg extracts, as described herein.

A polypeptide may be isolated and/or purified (e.g. using an antibody) for instance after production by expression from encoding nucleic acid (for which see below) . Thus, a polypeptide may be provided free or substantially free from contaminants with which it is naturally associated (if it is a naturally-occurring polypeptide) . A polypeptide may be provided free or substantially free of other polypeptides.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety. The invention encompasses each and every combination and sub- combination of the features that are described above .

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below.

Figure 1 shows the protein sequence of xRTS . Numbers indicate codons, and signature motifs for the DNA helicase domain (I -VI) are solid underlined. Motifs characteristic of the RecQ family are dashed underlined.

Figure 2 shows an alignment of an N-terminal region of xRTS with the corresponding region from the Sld2/DRC1 protein of S pombe . Identical residues are marked with an asterisk, and conservative changes, with double or single dots, the number being proportional to the similarity of the two residues .

Figure 3 shows the xRTS cDNA sequence.

Figure 4 shows DNA replication assays using egg extracts immuno- depleted either with a control antiserum (mock depleted) or with anti-xRTS (xRTS depleted) . Nascent DNA synthesis measured in ng per μl of egg extract is plotted on the vertical axis, against time, for each experiment. As positive and negative controls, xRTS depleted extracts were supplemented either with one-twentieth volume of mock depleted (xRTS depleted + mock depleted) or xRTS depleted (xRTS depleted + xRTS depleted) extract preparations respectively.

Figure 5 shows DNA replication assays using xRTS depleted extracts supplemented either with recombinant wild-type human RTS protein (xRTS depleted + HsRTS depleted) or with the recombinant Asp605Ala mutant (xRTS depleted + HsRTS D605A) predicted to inactivate DNA helicase activity. Recombinant HsRTS and HsRTSAspβO5Ala proteins were partially purified from E. coli .

Figure 6 shows a semi-quantitative plot of the timecourse of chromatin loading derived from a typical immunoblotting experiment. The relative amount of chromatin-bound protein at each time point was determined by densitometry, taking the maximal value to be 100%, and plotted on the vertical axis against time .

Figure 7 shows DNA replication assays using M13 single-stranded (ss)DNA. Circular M13 ssDNA was used as a template for replication in xRTS-depleted or mock-depleted egg extract. Nascent DNA synthesis determined by incorporation of 32P-dATP is plotted on the vertical axis against time. Unlike chromosomal DNA, the complementary strand of M13 ssDNA can be synthesized in egg extracts by the replication machinery independent of replication origins or initiation complexes.

Figure 8 shows a hypothetical model for the function of xRTS during replication initiation. Pre-RC formation defines origins of replication. Recruitment of xRTS helicase to the origins promotes unwinding and the stabilization of ssDNA by RPA, enabling loading of the replication machinery.

Figure 9 shows shRNA-mediated depletion of RTS in primary cultures of murine cells induces proliferative failure. Growth curves for murine-embryo-fibroblasts transduced with pSUPER- retroviral vectors encoding control shRNA or RTS shRNA are shown .

Figure 10 shows the sensitivity of xRTS-depleted nuclei to the DNA replication inhibitor gemcytabine in a standard replication assay. Diamond - undepleted; triangle- mock depleted, asterix - xRTS depleted; straight cross - xRTS depleted + Gem + Mock depl; square - undepleted + gem; diag cross - mock depleted + gem; circle - xRTS depleted + gem; rectangle - xRTS depleted + gem + xRTS depleted.

Figure 11 shows 32P radiolabelling experiments which show that xRTS is phosphorylated by CDKs during DNA replication initiation.

Figure 12 shows the timing of DNA replication initiation and indicates that the onset of DNA replication coincides with xRTS phosphorylation .

Experiments Materials and Methods Cloning of xRTS

Expressed sequence tags (ESTs) encoding xRTS were identified using the TblastN algorithm from the Xenopus EST database at the Sanger Centre, Hinxton by homology with the human RECQL4 protein sequence and the S. pombe DRCl protein sequence. The predicted coding sequence was assembled from four ESTs (BE507000,

BU912021, BE025795, BE026568) and included the putative codons for translation initiation and termination. A cDNA clone encoding the entire coding sequence was isolated by reverse transcription-polymerase chain reaction (RT-PCR) performed on total RNA from Xenopus egg extract after oligo-dT primed reverse transcription with Superscript II (Gibco) . Specific PCR primers based on the EST sequences (5' TGC CCA TGG AGA TGG AGC GCT ATA ATG AGG TTA AGG 3 ' , and 5 ' ACT CTC GAG CAA CAT CCT CTG CTG CTC ACG GAC T 3') were used in PCR reactions with a proof-reading polymerase (Accuprime Pfx, Invitrogen) . The veracity of the nucleotide sequence was established in multiple independent RT- PCR reactions

Constructs Human RECQL4 cDNA was cloned by RT-PCR from HeLa cell RNA using the primers, 5' - ATA GCG GCC GCT ATG GAG CGG CTG CGG GAC GTG - 3 ' , and 5 ' - ATA TCT AGA TCA GCG GGC GAC CTG CAG GAG CTC TT - 3' . The cDNA was directionally cloned into pcDNA3.1 HisA vector using Notl and Xbal restriction sites to generate pcDNA3.1 HisA HsRecQL4 (wild-type). pET30a HsRecQL4-6xHis constructs were made by PCR amplification of the HsRecQL4 cDNA using the primers 5' - GTC GAT

CAT ATG GAG CGG CTG CGG GAC GTG - 3 ' , and 5 ' - CGT CTC GAG GCG GGC CAC CTG CAG GAG CTC TTC CGT - 3', followed by cloning between the Ndel and Xhol sites in pET30a. To construct the Asp605Ala mutant, the plasmid was subjected to site-directed mutagenesis (QuickChange XL Multi site-directed mutagenesis kit, Stratagene) , using the primer 5'-GTT GCT TTT GCC TGC ATT GCT GAG GCC CAC TGC CTC C-3' . The insert was re- cloned into pΞT30a to ensure that the vector sequence was intact. All constructs were verified by nucleotide sequencing of the inserts.

Anti-xRTS antiserum Polyclonal rabbit antiserum was raised against the N-terminal 250 residues of xRTS expressed as a Glutathione-S-transferase (GST) fusion protein in E. CoIi₁ and affinity-purified against the antigen after preadsorption of GST-reactive antibodies on a GST-Sepharose 4B column. Validation of reactivity is shown in Figure 1.

Xenopus egg extracts

CSF extracts were prepared essentially as stated in Murray,

1991, with minor modifications. Briefly, de-jellied Xenopus eggs were placed in a SW55 rotor, and crushed at 10,000rpm for 10 mins at 16⁰C in an ultracentrifuge (Beckmann) . The golden cytosolic fraction was carefully removed by side-puncture in the cold-room, and supplemented with protease inhibitors, cytochalasin D to 10 μg/ml and 1/20 v/v energy mix. Extracts were diluted 1/10 v/v with 2M sucrose and further clarified at 10,000 rptn for 20 minutes and the clear cytosolic fraction removed again and kept on ice hereafter until use. All experiments were performed using freshly prepared egg extracts. CSF extracts were activated and driven into interphase by the addition of CaC12 to a final concentration 0.4mM and incubated at room temperature before the addition of DNA template after 20 mins . Where necessary extracts were supplemented with cycloheximide (lOOμg/ml final) at the time of activation to prevent entry into mitosis.

Immunodepletion xRTS was immunodepleted from egg extract by 3 rounds of sequential immunodepletion. Saturating amounts of anti-xRTS (or rabbit IgG, for mock depletion) were pre-incubated with Protein A-conjugated Dynabeads (Dynal Biotech) for 90 min at 4⁰C, washed once in HEPES buffered saline, thrice in SDB (1OmM HΞPES-KOH pH7.4, 10OmM KCl, ImM MgCl₂, 15OmM sucrose, with protease inhibitors and cytochalasin D) , and divided into three equal aliquots . For each immunodepletion, one aliquot was completely resuspended in the extract by gentle pipetting, and incubated for 35-40 minutes, with gentle tapping to keep the beads suspended. Depleted extract was separated from the beads by two serial passes through a magnetic particle separator (Dynal, MPC-S) .

Replication assays

Replication assays using demebranated sperm nuclei were performed as described in Mills et al . , 1989), using 3600 nuclei/μl in the presence of cycloheximide.

Isolation of chromatin-bound proteins

Sperm nuclei were incubated at a concentration of 4000 nuclei/μl in 50 μl of egg extract for the indicated times, then diluted 10 -fold with ice-cold SDB supplemented with 0.1% Triton X-100, lOμg/ml leupeptin, pepstatin, chymostatin mix (LPC), ImM DTT, ImM orthovanadate, ImM NaF, 0.2mM PMSF, and incubated on ice for 5 min after gentle mixing. The mixture was layered carefully onto a ImI cushion of MMR/40% glycerol and spun at 6,800 g in a Eppendorf benchtop centrifuge fitted with swing-out buckets at 4°C for 20 mins . The top layer and the cushion were aspirated and the pellet of chromatin washed in the same buffer again and centrifuged for 5 mins. Pellets were resuspended in 50μl of SDS-PAGE sample buffer. Samples were subject to SDS-PAGE, transferred onto PVDF membrane (Millipore) and processed for Western blot analysis with the indicated primary and secondary antibodies .

Biotin dUTP incorporation and microscopy

Sperm nuclei were added to a final concentration of 1,000 nuclei/μl in 20μl of interphase extract in the presence of aphidocolin (50/xg/ml) . Biotin-dUTP was added at indicated times and incubated for a further 10 minutes before diluting in 500μl of MMR and 500μl of 8% formaldehyde (Agar Scientific, UK) . Mixtures were kept at room temperature for 10 minutes before spinning nuclei onto poly-L-lysine coated coverslips. Coverslips were washed in PBS, permeablised in PF buffer

(0.75 x PBS/0.1% TxlOO/0.02% SDS), blocked in PF buffer/2% BSA and labelled with anti-xRTS. Biotin was detected with Alexa568- strepavidin, and anti-xRTS, with Alexa488 -conjugated anti-rabbit (Molecular Probes) before visualization on a Zeiss LSM confocal microscope equipped with AxioVision software.

Purification of recombinant wild-type and mutant HsRTS proteins HsRTS proteins were expressed as C-terminally hexa-histidine tagged polypeptides in E.coli (Rosetta (DE3)pLysS, Novagen) from the plasmid pET30a-HsRTS-6His encoding wild-type or mutant forms. Expressed protein was purified in its native state using a two-step method. All procedures were done at 4°C. Cells were lysed in Buffer A (1OmM phosphate buffer pH8, 30OmM NaCl, 1OmM imidazole with protease inhibitors) and the soluble fraction was applied onto Ni-NTA resin (Qiagen) . After washing with Buffer A (plus 1OmM imidazole) , bound proteins were eluted in Buffer B (Buffer A with 20OmM imidazole) . Eluates were dialysed into Buffer C (1OmM phosphate buffer pH8 , 15OmM NaCl), and applied onto a HiTrap-Heparin column (Amersham) pre-equilibrated in the same buffer using the AKTA purifier 10 system. The column was washed with buffer containing 30OmM NaCl, and bound protein was eluted using a linear salt gradient extending from 0.3-1.2M NaCl. HsRTS protein typically eluted between ionic strengths of 450-75OmM NaCl. Peak fractions containing HsRTS protein were pooled and concentrated 5-10 fold using a spun-concentrator device (Vivascience) before being dialysed into HKM buffer (1OmM HEPES-KOH pH7.7 , 10OmM KCl, ImM MgC12 with 10% glycerol). Small single-use aliquots of this preparation were snap-frozen in liquid-nitrogen and kept at -80⁰C until use.

Results Features of the xRTS protein xRTS encodes a novel protein of 1,500 residues with 60% overall identity to its human counterpart. It, too, includes the ~380 amino acid helicase domain that defines it as a member of the RecQ family, but lacks domains present in other vertebrate RecQ helicases (Figure 1) . A phylogenetic dendrogram of the known eukaryotic homologs of xRTS suggests early divergence from other members of the RecQ family. Indeed, the N- terminal region of xRTS (and its homologs in the human and other eukaryotic genomes) bears a hitherto unrecognized homology to the yeast proteins Sld2/DRC1 (Figure 2) , not found in other RecQ helicases. Sld2/DRC1 are essential for establishment of DNA replication forks in yeast, but have no known homologs in vertebrates (Masumoto et al . , 2002; Noguchi et al . , 2002; Wang and Elledge, 1999) . To characterize the function of xRTS, we raised a polyclonal rabbit antiserum directed against its N-terminal 250 residues. The antiserum recognizes a single 168kDa protein in Western blot analysis of X. laevis egg extracts corresponding to the predicted molecular weight of xRTS .

xRTS is essential for chromosomal DNA replication xRTS is essential for chromosomal DNA replication in X. laevis egg extracts. Replication of de-membranated sperm chromatin is markedly reduced and delayed in extracts pre-depleted of xRTS using affinity-purified anti-xRTS antibodies coupled to Protein A-conjugated Dynabeads (Figure 4) . Control reactions performed with extracts mock-depleted with a control antibody show no such reduction. Moreover, supplementation of xRTS- depleted extracts with a small amount of mock-depleted (but not xRTS-depleted) extract restores their ability to support chromosomal DNA replication.

Together, these controls confirm that the immunodepletion procedure itself does not impair the capacity of the extracts to support DNA replication.

The requirement for xRTS in chromosomal DNA replication can be complemented by adding recombinant human RECQL4 (HsRTS) protein, partially purified from E. coli by affinity chromatography, to xRTS-depleted egg extracts (Figure 5) . In contrast, a mutant protein (HsRTS Asp605Ala, D605A) , in which an Asp residue in the Walker B motif, critical for the helicase activity of RecQ domains has been replaced, fails to complement xRTS-depleted extracts. These findings, when taken together with the considerable level of sequence conservation in the RecQ domains of xRTS and its human ortholog, provide indication that the putative helicase activity of xRTS is necessary for its function. Chromatin association during replication initiation To identify possible functions of xRTS during replication, we studied the time-course of its association with chromatin during DNA replication in egg extracts (Figure 6) . xMCM3 and xORC2 , components of the pre-replicationcomplex (pre-RC) of proteins which bind to origins of replication during the Gl phase of cell cycle, associate with chromatin 20 min after the addition of nuclei to extract; xORC2 remains chromatin- associated throughout and xMCM3 levels decline during the course of chromosomal DNA replication.

The accumulation of xRTS on chromatin occurs after pre-RC formation, peaking around 40 min, and declining as replication proceeds to completion. The peak in recruitment of xRTS overlaps with that of xRPA70, a single strand (ss) DNA binding protein which loads at origins of replication after pre-RC formation. Blocking elongation by addition of the polymerase inhibitor aphidicolin does not prevent (but in fact, enhances) the accumulation of xRTS on chromatin. Collectively, these observations provide indication that xRTS is recruited to chromatin early during replication initiation, after pre-RC formation but before or during the establishment of active replication forks.

A role for xRTS in replication initiation Consistent with this proposal, the recruitment of xRTS to chromatin is dependent on the formation of a pre-RC at origins of replication. Geminin, an inhibitor of replication found in multi-cellular eukaryotes, prevents pre-RC formation by blocking

Cdtl, a factor essential for loading the MCM proteins onto chromatin (Wohlschlegel et al . , 2000).

DNA replication reactions were assembled (0 min) in the presence or absence of recombinant geminin (which blocks pre-RC assembly) or p21N (which blocks CDK activity) , before analysis of chromatin-bound proteins 60 min afterwards. In one reaction, geminin was added 20 min after the reaction was assembled, and analysis was carried out 40 min later. Immunoblots showed the presence or absence of xMCM3 - a component of the pre-RC complex - or xRTS . Addition of geminin but not p21N at 0 min prevents chromatin loading of xRTS (i.e. the accumulation of xRTS (as well as MCM3) on chromatin) . Addition of geminin after pre-RC assembly (20 min)} is without effect.

Taken together these experiments show that xRTS requires the presence of mature pre-RCs for binding to chromatin, confirming that the recombinant protein preparation does not contain additional factors that inhibit the process.

Transformation of the pre-RC into an active replication fork during the transition from Gl to S phase of the cell cycle is mediated by the activity of cyclin/cyclin-dependent-kinase (cdk) complexes. Cyclin/cdk complexes appear to be first required at a step that follows the chromatin loading of

MCM proteins, but occurs before Cdc45 loading and the unwinding of replication origins for recruitment of the elongation machinery (Walter J, 2000; Zou and Stillman, 2000) . Inhibition of cyclin/cdk activity by the addition of an N-terminal fragment of the cdk inhibitor p21 (p21N) to replication reactions does not prevent xRTS accumulation on chromatin, indicating that the recruitment of xRTS to chromatin follows pre-RC formation, but precedes origin unwinding and the establishment of active replication forks.

To examine the intra-nuclear localization of xRTS during replication initiation, Sperm nuclei (1000 sperm heads/μl extract) were incubated in interphase egg extract with aphidocolin (50 μg/ml) for 30 min, Incubations were pulsed with biotin-dUTP for a further 10-15 min to label sites of nascent DNA synthesis by incorporation of biotin-dUTP, before being fixed, and stained with appropriate antibodies and streptavidin conjugate. xRTS and dUTP incorporation were visualized by confocal microscopy.

Significant overlap was observed between xRTS staining and sites of biotin-dUTP incorporation, indicating that xRTS is located at sites of DNA synthesis during replication initiation. In this respect, its behaviour differs from that of the MCM complex, which performs its functions during initiation at a distance from sites of DNA synthesis (Laskey and Madine, 2003) .

Because xRTS remains associated with chromatin during replication elongation (Figure 4) , it is not possible to ascertain using chromosomal DNA templates whether xRTS is required solely for initiation, or has additional functions during elongation. However, we find that xRTS-depleted extracts are competent to replicate a circular ssDNA template derived from the M13 bacteriophage (Figure 7) . The replication of M13 ssDNA templates in egg extracts proceeds in a manner akin to DNA strand-elongation during chromosomal DNA replication (Cox and Leno, 1990; Jenkins et al . , 1992;

Mechali and Harland, 1982) . It utilizes components of the replication machinery, but occurs independently of pre-RC formation and the conventional events required to initiate chromosomal DNA replication.

Taken together, our results identify an essential role for xRTS during chromosomal DNA replication. xRTS is recruited to chromatin after pre-RC formation, but before the unwinding of replication origins and the establishment of active replication forks, providing evidence that it participates in replication initiation. Indeed, our results provide indication that xRTS is dispensable for elongation reactions, at least during the replication of ssDNA templates. Recruitment of RPA

A critical but poorly understood requirement for replication initiation is that origins of replication, defined by pre-RC formation, are made accessible for loading of the replication machinery (Walter J, 2000; Wohlschlegel et al . 2002) . This process involves the unwinding of DNA at origins, through a helicase activity that remains to be definitively identified, but which is proposed to include the MCM2-7 protein complex (Pacek and Walter, 2004) .

Origin unwinding is marked by the recruitment of RPA to the exposed ssDNA, thus stabilizing these structures to facilitate polymerase recruitment (Walter J, 2000) .

Chromosomal DNA replication reactions were assembled using mock depleted or xRTS depleted extracts as described above and chromatin-bound proteins were extracted at 20-120 min after assembly for analysis by immuno-blotting. xRTS loads onto chromatin somewhat after xMCM3 , but coincident with XRPA70, in reactions using the mock-depleted extract. xRPA — but not xMCM3 — fails to load onto chromatin after xRTS depletion, thus xMCM3 continues to be recruited to chromatin during chromosomal DNA replication while recruitment of the 7OkDa component of RPA (xRPA70) is markedly suppressed.

The hierarchy and sequential nature of the recruitment of these proteins to chromatin provides further evidence that xRTS recruitment follows pre-RC formation but precedes and is required for xRPA recruitment.

Replication reactions were assembled (0 min) as described above using mock depleted, xRTS depleted or XRPA70 depleted egg extracts. Chromatin-bound proteins were analysed by immuno- blotting 60 min afterwards. Depletion of xRTS prevents the association of xRPA but not xMCM3 with chromatin. In contrast, xRPA depletion has no effect on recruitment of either xMCM3 or xRTS .

Inhibition of DNA polymerase with aphidicolin is reported to uncouple origin-unwinding activity from the replication fork, resulting in enhanced RPA recruitment (Pacek and Walter, 2004) . That aphidicolin treatment increases the chromatin association of both xRTS and xRPA is consistent with this view. Thus, collectively, our findings implicate xRTS in a previously unrecognized, early step during the initiation of DNA replication (Figure 8) . xRTS is recruited to origins of replication after pre-RC formation, and its recruitment is in turn required for the chromatin association of xRPA. Because xRPA is proposed to act in concert with uncharacterized helicase activities, which may include the MCM complex, to unwind and stabilize DNA replication origins, our findings reveal a hitherto unrecognized role for xRTS in this process.

xRTS deficiency sensitises to the replication blocking effects of the anti-cancer drug gemcytabine .

Titration experiments were performed to find a dose of gemcytabine that clearly causes chromatin binding of xRTS (a marker of its activity) but does not significantly reduce the ability of nuclei to complete DNA synthesis. This does was determined to be approximately 0.8 μg Gemzar per μl of extract.

We then tested whether xRTS-depleted nuclei are more sensitive to gemcytabine in a standard replication assay (Figure 10) . xRTS depletion was found to reduce the ability of nuclei to complete DNA replication in the presence of gemcytabine. As positive and negative controls, we showed that this sensitivity can be overcome by the re-supply of xRTS (by the addition of 1/20^th volume of mock depleted extract) , but not by the addition of the same volume of xRTS-depleted extract. xRTS-depleted nuclei were found to be more sensitive to gemcytabine than mock-depleted nuclei. Depletion of xRTS therefore sensitizes nuclei to replication inhibition by gemcytabine .

Depletion of mammalian RTS induces proliferative failure Murine-embryo-fibroblasts were transduced with pSUPER-retroviral vectors encoding control shRNA or RTS shRNA. Growth curves for the transduced cells are shown in figure 9

shRNA-mediated depletion of RTS was observed to induce proliferative failure in these primary cultures of murine cells.

We have examined the phosphorylation of xRTS by CDKs that act during the initiation of DNA replication using the Xenopus cell- free replication system. As shown in Figure 11, calcium chloride was added to activate replication, followed 20 min later by the addition of the sperm DNA template for replication. 32P-labelled gATP was added to the reaction coincident with the addition of template DNA (lanes 1, 2), or at varying intervals from 10-30min after the addition of template DNA (lanes 3-8) . Reactions were incubated for lOmin after gATP addition, xRTS was immunoprecipitated using a specific antibody, and the immune complexes were analysed by SDS-PAGE. At each time point, p21N, an inhibitor of CDK activity, was added as a negative control.

xRTS was observed to be phosphorylated during the initiation of DNA replication (lane 7, figure 11) as marked by the incorporation of 32P radiolabel into the protein. Addition of p21N completely suppresses phosphorylation (lane 8, figure 11) , demonstrating that it is dependent on cyclin/CDK activity. xRTS phosphorylation by cyclin/CDK activity is restricted to a narrow temporal window during DNA replication initiation (compare figure 11 lanes 1, 3 and 5 with lane 7) .

In a parallel experiment showing the timing of DNA replication initiation using the same extracts (Figure 12) , the onset of measurable replication by incorporation of radiolabel into nascent DNA coincides with the time at which xRTS becomes phosphorylated by CDKs in the experiment shown in Figure 11. Again, the addition of p21N, which inhibits cyclin/CDK activity, works as a negative control in this experiment.

This work provides fresh insight into the events that initiate DNA replication in vertebrate cells. Our results provide indication that RECQL4 is essential for this process, working after pre-RC assembly but before the establishment of active replication forks in a mechanism responsible for the stabilization of unwound origins by the ssDNA-binding protein, RPA. These findings identify the RECQL4 DNA helicase as a target for the development of anti-cancer agents that synergize with Gemzar and other replication-blocking drugs, and also allow for the definition of new clinical contexts in which Gemzar and related agents may be used, with or without such novel synergistic agents. Furthermore, because depletion of mammalian RECQL4 proteins is shown herein to induce proliferative failure in its own right, inhibition of RECQL4, for example by siRNA, antibodies or small molecules, may be an effective approach for the treatment of cancer.

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Claims

Claims :

1. A method of treating a RECQL4 deficient cancer condition in an individual comprising: administering a DNA replication inhibitor to the individual

2. A method according to claim 1 wherein the DNA replication inhibitor is selected from the group consisting of topoisomerase inhibitors, cytotoxic antibiotics, alkylating agents, antimetabolites, anti-neoplasties and platinum agents.

3. A method according to claim 1 or claim 2 wherein the DNA replication inhibitor is selected from the group consisting of busulfan, carmustine, chlorambucil, chlormethine-Hcl, cyclophosphamide, estramustine, ifosfamide, lomustine, melphalan, thiotepa, treosulfan, doxorubicin, alcarubicin, idarubicin, daunorubicin, mitoxantrone, bleomycin, dactinomycin and mitomycin, methotrexate, cytarabine, fludarabine, cladribine, gemcytabine, 5-FU, raltitrexed, mercaptopurine, tegafur, thioguanine, capecitabine,- etoposide, Amsacrine, dacarbazine, temozolamide, hydroxycarbamide (hydroxyurea) , pentostatin, cyclophosphamide-methotrexate-5-fluorouracil, 5- fluoracil-epirubicin-cyclophosphamide, cisplatin, carboplatin, oxaliplatin, irinotecan, rubitecan, topotecan, camptothecin, vinorelbine and vinblastine, and analogues, derivatives or salts of any of these.

4. A method according to claim 3 wherein the DNA replication inhibitor is gemcytabine.

5. A method according to any one of the preceding claims comprising identifying the cancer condition in an individual as being RECQL4 deficient.

6. A method according to claim 5 wherein the cancer condition is identified as a RECQL4 deficient cancer by determining the presence or level of a nucleic acid encoding a RECQL4 polypeptide in one or more cancer cells obtained from the individual

7. A method according to claim 5 wherein the cancer condition is identified as a RECQL4 deficient cancer by determining the presence or level of one or more variant forms of a RECQL4 nucleic acid in one or more cancer cells obtained from the individual

8. A method according to claim 5 wherein the cancer condition is identified as a RECQL4 deficient cancer by determining the level of RECQL4 polypeptide in one or more cancer cells obtained from the individual .

9. A method according to claim 8 wherein the level of RECQL4 polypeptide in the cancer cells is determined by contacting a sample obtained from the individual with an antibody which specifically binds the RECQL4 polypeptide, and determining binding of the antibody to the sample

10. A method according to claim 5 wherein the cancer condition is identified as a RECQL4 deficient cancer by determining the activity of RECQL4 polypeptide in one or more cancer cells obtained from the individual .

11. A method according to claim 5 wherein the cancer condition is identified as a RECQL4 deficient cancer by detecting the presence of a variant RECQL4 polypeptide in one or more cancer cells obtained from the individual.

12. A method according to claim 11 wherein the presence of a variant RECQL4 polypeptide is determined by contacting a sample obtained from the individual with an antibody which specifically binds the variant RECQL4 polypeptide, and determining binding of the antibody to the sample

13. Use of a DNA replication inhibitor in the manufacture of a medicament for use in a method of treating a RECQL4 deficient cancer condition in an individual.

14. Use according to claim 13 wherein the method comprises identifying the cancer condition in an individual as being RΞCQL4 deficient.

15. A method of identifying and/or obtaining a compound that sensitises cells to a DNA replication inhibition comprising: contacting an RECQL4 polypeptide with a test compound; and, determining the interaction of said compound with said polypeptide .

16. A method according to claim 15 wherein interaction is determined by determining the binding of the compound to the RECQL4 polypeptide, binding of the test compound to the polypeptide being indicative that the compound sensitises cells to replication inhibition.

17. A method according to claim 15 wherein interaction is determined by determining the activity of the RECQL4 polypeptide in the presence of the test compound, a decrease in activity in the presence relative to the absence of the test compound being indicative that the compound sensitises cells to replication inhibition.

18. A method according to any one of claims 15 to 17 comprising identifying the test compound as a RECQL4 inhibitor.

19. A method according to claim 18 comprising formulating the test compound into a pharmaceutical composition with a pharmaceutically acceptable excipient .

20. A method of treating a cancer condition in an individual comprising; administering an RECQL4 inhibitor to the individual.

21. A method according to claim 20 wherein the RECQL4 inhibitor is administered in combination with a DNA replication inhibitor .

22. Use of a RECQL4 inhibitor in the manufacture of a medicament for the treatment of a cancer condition in an individual

23. Use of a RECQL4 inhibitor and a DNA replication inhibitor in the manufacture of a medicament for the treatment of a cancer condition in an individual

24. A method of identifying a cancer condition in an individual as susceptible to treatment with a DNA replication inhibitor comprising: determining the presence of one or more RECQL4 deficient cancer cells in a sample obtained from the individual

25. A method according to claim 24 wherein the presence of one or more RECQL4 deficient cancer cells is indicative that the cancer condition in the individual is susceptible to treatment with a DNA replication inhibitor.

26. A method according to claim 24 or claim 25 wherein the presence of one or more RECQL4 deficient cancer cells in the sample is determined by determining the presence or level of a nucleic acid encoding a RECQL4 polypeptide in one or more cells in the sample .

27. A method according to claim 24 or claim 25 wherein the presence of one or more RECQL4 deficient cancer cells in the sample is determined by determining the presence or level of one or more variant forms of a RΞCQL4 nucleic acid in one or more cells in the sample.

28. A method according to claim 24 or claim 25 wherein the presence of one or more RECQL4 deficient cancer cells in the sample is determined by determining the level of RECQL4 polypeptide in one or more cells in the sample.

29. A method according to claim 28 wherein the level of RECQL4 polypeptide is determined by contacting a sample obtained from the individual with an antibody which specifically binds the RECQL4 polypeptide, and determining binding of the antibody to the sample

30. A method according to claim 24 or claim 25 wherein the presence of one or more RECQL4 deficient cancer cells in the sample is determined by determining the activity of RECQL4 polypeptide in one or more cells in the sample.

31. A method according to claim 24 or claim 25 wherein the presence of one or more RECQL4 deficient cancer cells in the sample is determined by detecting the presence of a variant RECQL4 polypeptide in one or more cells in the sample.

32. A method according to claim 31 wherein the presence of a variant RECQL4 polypeptide is determined by contacting a sample obtained from the individual with an antibody which specifically binds the variant RECQL4 polypeptide, and determining binding of the antibody to the sample

33. An isolated nucleic acid encoding a RTS polypeptide consisting of an amino acid sequence having at least 70% sequence identity to the sequence of Figure 1.

34. An isolated nucleic acid according to claim 33 wherein the RTS polypeptide consists of the amino acid sequence of Figure 1.

35. A vector comprising a nucleic acid according to claim 33 or claim 34.

36. A host cell comprising a vector according to claim 35.

37. An RTS polypeptide comprising an amino acid sequence having at least 70% sequence identity to the sequence of Figure 1.

38. An antibody that binds specifically to a polypeptide consisting of the amino acid sequence of figure 1.

39. A method of producing a protein comprising: expressing a nucleic acid according to claim 33 or claim 34 and, determining the helicase activity of the expression product thereof .