53BP1/RIF1/REV7/Shieldin 1/2/3 is a protein complex that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini, thus antagonising HR and promoting NHEJ (non-homologous end joining). This function requires interactions of 53BP1 with RIF1 (also known as MAD2L2), and subsequent recruitment of other components of this protein complex REV7/Shieldin 1/2/3 (Sylvie M Noordermeer et al. Nature, 560(7716): 117-121 , 2018; Mirman Z., et al. Nature, 560(7716): 112-116, 2018; Ghezraoui H., Nature, 560(7716): 122-127, 2018).

Inactivating mutations in one or more BRRES proteins (listed below) results in partial restoration of DNA resection-dependent DSB repair, and BRCA-deficient tumours becoming resistant to PARP inhibitors (Sylvie M Noordermeer et al. Nature,

560(7716):117-121 , 2018; Mirman Z., et al. Nature, 560(7716):112-116, 2018;

Ghezraoui H., Nature, 560(7716): 122-127, 2018). However, this dependency on the restoration of resection-dependent mechanism (back up) for DSB repair in BRCA- deficient tumours, and by extension survival, could be exploited therapeutically by targeting the back-up repair/DNA pathway in order to prevent development or resistance and/or accelerate killing of cancer cells.

Cancers

There are many cancers that are known to have a deficiency in a member of the HR pathway, including: breast cancer, prostate cancer, pancreatic cancer, liver cancer, ovarian cancer, testicular cancer, endometrium cancer, cervical cancer, thyroid cancer, parathyroid cancer liver cancer, stomach cancer, adrenal cancer, multiple endocrine neoplasia 1 , and multiple endocrine neoplasia 2.

The present invention can be employed for use in a patient with a sub-set of a cancer selected from breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, gastric cancer, colorectal cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, melanoma, bone cancer, oesophageal cancer, bladder cancer, cervical cancer, endometrial cancer or other cancers of tissue organs and cancers of the blood cells such as lymphomas and leukaemia.

By“subset” we mean that not all cancers within this group (e.g. breast cancer) will be treatable or suitable, rather as will be appreciated from the disclosure herein, it will be those cancers that possess cells which either have a deficiency in one or more members of HR pathway.

A cancer may be identified as a HR dependent DNA DSB repair deficient cancer, for example, by determining the activity of the HR dependent DNA DSB repair pathway in one or more cancer cells from a sample obtained from the individual or by determining the activity of one or more components of the pathway. Activity may be determined relative to normal (i.e. non-cancer) cells, preferably from the same tissue. In some embodiments, a cancer may be identified as deficient in an HR dependent DNA DSB repair pathway by determining the presence in cancer cells from the individual of one or more variations, for example, polymorphisms or mutations, in a nucleic acid encoding a polypeptide which is a component of the HR dependent DNA DSB repair pathway.

In a particular embodiment of the first aspect of the invention the cancer is selected from a BRCA 1 or BCRCA2 mutant cancer.

In certain embodiments, the cancer is a BRCA1 or BRCA2 mutant cancer selected from the group consisting of: breast cancer, prostate cancer, pancreatic cancer, liver cancer, ovarian cancer, testicular cancer, endometrium cancer, cervical cancer, thyroid cancer, parathyroid cancer liver cancer, stomach cancer, adrenal cancer, multiple endocrine neoplasia 1 , and multiple endocrine neoplasia 2.

In particular embodiments, the cancer is selected from the group consisting of:

breast, ovarian, prostate and pancreatic cancer.

In particular embodiments, the cancer has been identified as possessing a BRCA1 mutation that causes the BRCA1 deficiency or as being BRCA1 -deficient.

In particular embodiments, the cancer has been identified as possessing a BRCA2 mutation that causes the BRCA2 deficiency or as being BRCA2-deficient.

In particular embodiments, the cancer has been identified as also being EXD2 wild type.

In particular embodiments, the cancer has been identified as being resistant to a PARP inhibitor, such as one selected from the group consisting of: selected from the group consisting of: olaparib, rucaparib, niraparib and talazoparib.

In particular embodiments, the resistance to PARP inhibition is the result of the cancer (cancer cells) acquiring mutation in one or more member of the

In one embodiment of any of the aspects above, the patient has not received a prior therapy for the cancer, i.e. the EXD2 inhibitor is to be administered as first line treatment. In another embodiment of any of the aspects above, the patient has received one or more prior treatments for the cancer, i.e. the EXD2 inhibitor is to be administered as second-line, third-line etc. treatment.

In embodiments of any of the aspects above, the method of treatment further comprises administering a DNA damaging chemotherapeutic agent such as PARP inhibitors, ATR inhibitor, ATM inhibitors, X and gamma radiation, crosslinking agents (e.g. cisplatin, oxiplatin and their derivatives) or a replication inhibitor (e.g.

gemcitabine and derivatives).

In one embodiment, the EXD2 inhibitor is administered in combination with another anti-cancer agent, such as a PARP inhibitor.

In particular embodiments, the PARP inhibitor is selected from: olaparib, niraparib and rucaparib.

When a patient presents with a putative cancer the cancer cells are typically tested to see if they are deficient in one or more members of HR pathway. This testing can be part of a standard panel of tests for key mutations (such as in KRas, P53, MEK etc.) using gene sequencing or other mutation detection test or may be carried out using immunohistochemistry on a biopsy sample from the patient’s cancer to look for protein deficiencies (Stover et al. , Clinical Cancer Research, September 27, 2016, doi: 10.1158/1078-0432. CCR-16-0247; Hoppe et al., J Natl Cancer Inst. 110(7):704- 713, 2018. doi: 10.1093/jnci/djy085). Testing for the presence of inactivating mutations in one or more member of the 53BP1/RIF1/REV7/Shieldin protein complex, such as RIF1 may be carried out separately or at the same time.

In practice, it is likely that the patient will present to the physician. One or more tumour/cancer biopsy samples or blood samples will be taken, and these will be sent off to a testing laboratory (e.g. hospital or other clinical pathology laboratory) for testing.

As used herein, the term“sample” typically refers to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; cell free circulating tumour DNA; sputum; saliva; urine;

cerebrospinal fluid, peritoneal fluid; pleural fluid; faeces; lymph; gynaecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or bronchioalveolar lavages; aspirates; scrapings;

secretions, and/or excretions; and/or cells therefrom. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a“primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, ascites, faeces etc.), etc. In some embodiments, as will be clear from context, the term“sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a“processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification (e.g. polymerase chain reaction) or reverse transcription of mRNA, isolation and/or purification of certain components, etc.

In some embodiments, the sample may be a liquid, solid, or mixed biological sample obtained from a subject having, or suspected of having, a cancer with a particular deficiency. Suitable tissue samples include cancer tissue samples including those that may be obtained by a biopsy or following surgical resection of the cancer, surrounding tissues, and/or distant tissues in which metastasis are known or are suspected.

EXD2 inhibitors

The inventor has demonstrated that inhibition of EXD2 using siRNA causes a reduction in survival and/or proliferation of cells which have a deficiency in FIR.

Recognising that EXD2 is a druggable target, the person skilled in the art would appreciate that any agent that could inhibit EXD2, for example at the nucleic acid (e.g. mRNA) or protein level, would have utility in the present invention. EXD2 is an exonuclease required for double-strand breaks resection and efficient homologous recombination. It plays a key role in controlling the initial steps of chromosomal break repair. It is recruited to chromatin in a damage-dependent manner and functionally interacts with the MRN complex to accelerate resection through its 3'-5' exonuclease activity, which efficiently processes double-stranded DNA substrates containing nicks.

EXD2 is also referred to in the art as EXDL2 or C14orf114.

The 3-5’ exonuclease domain of EXD2 is located from amino acids 62-262 of the native protein. SEQ ID NO: 1 provides the amino acid sequence of the isoform that has been chosen as the 'canonical' EXD2 protein sequence (see NM_001193360.1 or Q9NVH0-1 ). The EXD2 gene sequence (as in NM_001193360.1 ) is disclosed in SEQ ID NO: 2.

In particular embodiments, the EXD2 inhibitor is selected from a polypeptide, polynucleotide, antibody, peptide, nucleic acid, small molecule, an RNA inhibitory molecule (RNAi), an antisense oligonucleotide (ASO) or any other suitable chemical. In particular embodiments of any of the aspects disclosed herein, the EXD2 inhibitor is a small molecule compound or a large molecule biologic.

Suitably, the EXD2 inhibitor is selected from the group consisting of: an antibody, a peptide, a nucleic acid, a small molecule compound, an RNA inhibitory molecule (RNAi) and an antisense oligonucleotide (ASO).

RNAi and ASO molecules are particularly suitable for inhibiting the expression of EXD2. The use of these approaches to down-regulate gene expression is now well- established in the art.

In a particular embodiment, the EXD2 inhibitor is an RNAi.

In a particular embodiment, the EXD2 inhibitor is a small molecule compound.

In a particular embodiment, the EXD2 inhibitor is an ASO.

A "small molecule" as used herein, is an organic molecule that is less than about 5 kilodaltons (KDa) in mass. In some embodiments, the small molecule is less than about 3 KDa, or less than about 2 KDa, or less than about 1.5 KDa, or less than about 1 KDa. Most small molecule compounds are less than around 800 daltons (Da). In some embodiments, the small molecule is less than about 800 Da, less than about 600 Da, less than about 500 Da, less than about 400 Da, less than about 300 Da, less than about 200 Da, or less than about 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and/ or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups. Small molecules often comprise one or more cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups.

By“specific for EXD2”, we mean that the inhibitor (agent) does not significantly inhibit the other DnaQ family exonucleases with the conserved DEDD motif selected from TREX1 and TREX2; in other words is 10- fold , 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold or more specific for inhibiting EXD2 than any TREX1 or TREX2. Thus, in another embodiment, the EXD2 inhibitor is specific for EXD2.

In a particular embodiment, the EXD2 inhibitor is specific for EXD2 and blocks its exonuclease activity, such as by targeting the 3-5’ exonuclease domain from amino acids 62-262), by disrupting the metal ion binding site (located within the sequence from amino acids 108 - 242) [numbering according to SEQ ID NO: 1 ], by binding to the DNA substrate and/or steric effect/hindrance Thus, the protein is impeded in its ability to process (degrade) DNA or RNA substrate.

The EXD2 inhibitor could be an antibody or an antibody fragment. In a particular embodiment, the EXD2 inhibitor is a monoclonal antibody. In a particular

embodiment, the EXD2 inhibitor is a monoclonal antibody fragment. In a particular embodiment, the EXD2 inhibitor is a polyclonal antibody. In a particular embodiment, the EXD2 inhibitor is an intrabody.

In particular embodiments, the EXD2 inhibitor is an oligonucleotide aptamer or peptide aptamer.

Nucleic acid aptamers (or oligonucleotide aptamers) can be generated from nucleic acid random-sequence using a systematic evolution of ligands by exponential enrichment (SELEX) technology ( Tuerk and Go/d. Science.

249(4968):5Q5-1Q, 1990). SELEX is a process of effectively selecting aptamers from different targets. To date, using SELEX technology has successfully- generated thousands of aptamers, which bind to specific targets including small molecules, metal ions, proteins, peptides, bacteria, virus, and live cells (e.g. see Ciesiolka et al. RNA 1 , 538-550, 1995; Stoltenburg et al., J. Ana!. Meth.

Chem. 2012, 415697, 2012; or Chen et al. Precision Oncology 1 ,

Article number: 37, 2017).

Peptide aptamers are short, 5-20 amino acid residues long sequences, typically embedded as a loop within a stable protein scaffold (e.g. see Colas et

al., Nature. 380:548-550, 1996). Peptide aptamers can be produced and selected in vivo through yeast two hybrid and similar techniques.

A new approach to protein inhibition called proteolysis targeting chimeras (PROTAC) involving the use of small bi-functional molecules that can inhibit a protein via the induction of its degradation (see Crew et al., J. Medicinal Chemistry.

DOI: 10.1021/acs.jmedchem.7b00635, 2017). Thus, in one particular embodiment, the EXD2 inhibitor for use in the invention is a PROTAC molecule that inhibits EXD2 via the induction of its degradation.

Nucleic acid-based therapeutic agents, such as RNAi or antisense oligonucleotides are well-known.

In particular embodiments, the EXD2 inhibitor for use in the invention is a nucleic- acid based therapeutic that comprise nucleic acid or nucleotides. By way of example, said nucleic acid therapeutic could be or comprises a dsRNA molecule, a RNAi molecule, a miRNA molecule, a ribozyme, a shRNA molecule, an antisense oligonucleotide (ASO), a guide RNA (gRNA) or a siRNA molecule. The EXD2 inhibitor for use in the invention could also be a nucleic acid-based molecule, such as one capable of inhibiting generation of mRNA of EXD2. Thus, according to another embodiment, the EXD2 inhibitor is or comprises a nucleic acid molecule capable of inhibiting mRNA of EXD2.

With knowledge of the EXD2 gene sequence (including mRNA sequence) the person of skill in the art is able to design suitable nucleic-acid based molecules to target the EXD2 gene (including mRNA) and inhibit the EXD2 protein. Suitable molecules include CAGAGGACCAGGUAAUUUA (SEQ ID NO: 3) the siEXD2 molecule utilised in the Examples herein, or GAACAAGGAGUCAAAUUUA (SEQ ID NO: 4; (Broderick et al. , Nat Cell Biol 18, 271 -280, 2016)) are the sequences of individual siRNA molecules targeting human EXD2. Thus, according to particular embodiments, the nucleic acid molecule comprises a sequence disclosed in SEQ ID Nos: 3 or 4.

There are many different types of nucleic acid-based molecules that can inhibit translation of an mRNA and/or decrease the stability of the RNA. Such an RNA inhibitor is preferably an RNAi molecule specific for EXD2 mRNA; shRNA molecule specific for EXD2 mRNA; or an antisense oligonucleotide (AON) specific for EXD2 mRNA.

In other embodiments, the agent that inhibits EXD2 is a nucleic acid or peptide- based aptamer.

The invention also provides an isolated oligonucleotide having 12-40 bases, wherein the oligonucleotide comprises a continuous stretch of at least 7 bases that is complementary to and capable of hybridizing to a continuous stretch of at least 7 bases that is complementary to and capable of hybridizing to EXD2 mRNA. In particular embodiments, the isolated oligonucleotide comprises a sequence disclosed in any of SEQ ID Nos: 3 or 4,

According to particular embodiments, the EXD2 inhibitor is selected from the group consisting of small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, PROTAC molecules, peptide aptamers, antibodies, such as Fab, scFv, VH H, natural single domain antibodies, nanobodies, affibodies, affibody-antibody chimeras, heavy-chain only antibodies (FICAbs), and non-immunoglobulins. Certain of these agents are discussed in Muyldermans (Ann Rev Biochem. 82:775- 797, 2013). Functional inhibitor agents can be identified based on their ability to induce a down-regulation or inhibition of gene expression and/or down- regulation or inhibition of the activity of a transcriptional or translational product thereof (i.e.

EXD2/ EXD2). The expression is, for example, reduced or down-regulated to less than 90%, such as less than 80% such as less than 70% for example less than 60%, for example less than 50%, such as less than 40%, such as less than 30% such as less than 20% for example less than 10%, for example less than 5%, for example less than 1 %, such as completely inhibited (0%) relative to the expression or activity in the absence of the agent that inhibits EXD2.

The EXD2 inhibitor could also be a large molecule biologic, such as an antibody.

An antibody is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable domain of the immunoglobulin molecule.

An "intact antibody" typically refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Each light chain is composed of one variable domain (VL) and one constant domain (CL). Each heavy chain comprises one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD antibodies, comprises three domains termed CH1 , CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In IgG, IgA, and IgD classes the CH1 and CH2 domains are separated by a flexible hinge region, which is a proline and cysteine rich segment of variable length (from about 10 to about 60 amino acids in various IgG subclasses). The variable domains in both the light and heavy chains are joined to the constant domains by a "J" region of about 12 or more amino acids and the heavy chain also has a "D" region of about 10 additional amino acids. Each class of antibody further comprises inter-chain and intra-chain disulfide bonds formed by paired cysteine residues. The heavy chain variable region (YH) and light chain variable region (YL) can each be further subdivided into regions of hypervariability,

termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each YH and YL, comprises three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the

immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical

complement system.

As used herein, the term“antibody” includes, by way of example, both naturally occurring and non-naturally occurring intact antibodies, such as polyclonal, multiclonal or monoclonal antibodies, as well as chimeric antibodies, humanized and primatized antibodies, CDR grafted antibodies, human antibodies, intrabodies, multi specific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies and synthetic antibodies, but also, unless otherwise specified, any antigen-binding portion thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen-binding portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.

It has been shown that the antigen-binding function of an antibody can be performed by portions of a full-length antibody. Antigen-binding portions of an antibody (also called an "antigen-binding fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., EXD2) bound by the whole antibody.

Antigen-binding portions include, for example, Fab, Fab', F(ab')2, F(ab') fragments, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), portions including complementarity determining regions (CDRs), single chain variable fragment antibodies (e.g. scFv, scFvFc and bis-scFv), minibodies, maxibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes (i.e. , isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (subtypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known. Unless dictated otherwise by contextual constraints the term further comprises all classes and subclasses of antibodies. Heavy-chain constant domains that correspond to the different classes of antibodies are typically denoted by the corresponding lower-case Greek letter a, d, e, g, and m, respectively. Light chains of the antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (l), based on the amino acid sequences of their constant domains.

Although the two domains of the Fv portion, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)); see e.g., Bird et al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993; Poljak et al., Structure. 2:1121 -1123, 1994).

The antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human or humanized antibody. A non-human antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e. , antibody molecules whose primary sequences are essentially identical. A Mab is highly specific, being directed against a single antigenic site/epitope. A mAb is an example of an isolated antibody. MAbs may be produced by hybridoma, recombinant, transgenic or other techniques known to those skilled in the art. The modifier

"monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein (Nature 256:495, 1975) or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al. ,

(Nature 348:552-554, 1990), for example.

A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germ line

immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo). Flowever, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibodies and "fully human" antibodies are used synonymously. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.

As used herein, a "humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In some embodiments, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. The humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences but are included to further refine and optimize antibody performance. In one embodiment of a humanized form of an Ab, some, most or all the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible provided they do not abrogate the ability of the antibody to bind to a particular antigen. A "humanized" antibody retains an antigenic specificity similar to that of the original antibody.

A "chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody or vice versa. The term also encompasses an antibody comprising a V region from one individual from one species (e.g., a first mouse) and a constant region from another individual from the same species (e.g., a second mouse).

An“intrabody” refers to an antibody that has been designed to be expressed intracellularly and can be directed to a specific target antigen present in various subcellular locations including the cytoplasm, nucleus and endoplasmic reticulum through in frame fusion with intracellular localization peptide sequences. It has been identified as a new class of therapeutic molecule (Chen et al. , Human Gene

Therapy. 5 (5): 595-601 , 1994). Although intrabodies can be expressed in different forms, the most commonly used format is a scFv due to their mall size. Antibody fragments, typically in scFv format, are cloned into a specific targeting vector allowing expression of the intrabody in the nucleus, cytoplasm or ER. The intrabody gene is expressed inside the target cell after transfection with an expression plasmid or viral transduction with a recombinant virus. It has been found that the usual vector-, promoter- and transfection systems for heterologous expression can be employed to express the intrabody in the cell of interest.

The term "antigen (Ag)" refers to the molecular entity used for immunization of an immunocompetent vertebrate to produce the antibody (Ab) that recognizes the Ag or to screen an expression library (e.g., phage, yeast or ribosome display library, among others). Herein, Ag is termed more broadly and is generally intended to include target molecules that are specifically recognized by the Ab, thus including portions or mimics of the molecule used in an immunization process for raising the Ab or in library screening for selecting the Ab. Thus, for antibodies of the disclosure binding to EXD2, full-length EXD2 from mammalian species (e.g., human, monkey, mouse and rat EXD2), as well as truncated and other variants of EXD2, can represent the antigen.

Generally, the term "epitope" refers to the area or region of an antigen to which an antibody specifically binds, i.e., an area or region in physical contact with the antibody. Thus, the term "epitope" refers to that portion of a molecule capable of being recognized by and bound by an antibody at one or more of the antibody's antigen-binding regions. Typically, an epitope is defined in the context of a molecular interaction between an "antibody, or antigen-binding portion thereof (Ab), and its corresponding antigen. Epitopes often consist of a surface grouping of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. In some embodiments, the epitope can be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. A "nonlinear epitope" or "conformational epitope" comprises non-contiguous polypeptides (or amino acids) within the antigenic protein to which an antibody specific to the epitope binds. The term

"antigenic epitope" as used herein, is defined as a portion of an antigen to which an antibody can specifically bind as determined by any method well known in the art, for example, by conventional immunoassays.

An antibody that "specifically binds" to an epitope is a term well understood in the art, and methods to determine such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. For example, an antibody that specifically binds to an EXD2 epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other EXD2 epitopes or non-EXD2 epitopes. It is also understood by reading this definition, for example, that an antibody which specifically binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" does not necessarily require (although it can include) exclusive binding.

In a particular embodiment, the antibody specifically binds to the 3-5’ exonuclease domain of EXD2 which is located from amino acids 62-262 (according to the sequence in SEQ ID NO: 1 ).

A variety of assay formats may be used to select an antibody or peptide that specifically binds a molecule of interest. For example, solid-phase ELISA

immunoassay, immunoprecipitation, Biacore™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescence-activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA) and Western blot analysis are among many assays that may be used to identify an antibody that specifically reacts with an antigen or a receptor, or ligand binding portion thereof, that specifically binds with a cognate ligand or binding partner. Typically, a specific or selective reaction will be at least twice

the background signal or noise, more typically more than 10 times background, even more typically, more than 50 times background, more typically, more than 100 times background, yet more typically, more than 500 times background, even more typically, more than 1000 times background, and even more typically, more than 10,000 times background. Also, an antibody is said to "specifically bind" an antigen when the equilibrium dissociation constant (KD) is < 7nM.

The term "binding affinity" is herein used as a measure of the strength of a non- covalent interaction between two molecules, e.g., an antibody or antigen-binding portion thereof and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules may be quantified by determination of the dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation using, e.g., the surface plasmon resonance (SPR) method (Biacore). Other standard assays to evaluate the binding ability of antibodies towards target antigens are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis, and other assays exemplified elsewhere herein. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a Biacore™ system, or KinExA.

In some embodiments, the antibody may bind to EXD2 with a KD of about 1 x 10¹⁰M or greater. For example, the antibody may bind to hEXD2 with a KD of about 9 x 10¹¹M or greater. In some embodiments, the antibody may bind to hEXD2 with a KD of about 8 x 10¹¹M or greater. In some embodiments, the antibody may bind to hEXD2 with a KD of about 7 x 10¹¹M or greater. In some embodiments, the antibody may bind to hEXD2 with a KD of about 6 x 10¹¹M or greater. In some embodiments, the antibody may bind to hEXD2 with a KD of about 5.00 x 10¹¹M or greater. These amounts are not meant to be limiting and increments between the recited values are specifically envisioned as part of the disclosure.

An antibody that specifically binds its target may bind its target with a high affinity, that is, exhibiting a low KD, and may bind to other, non- target molecules with a lower affinity. For example, the antibody may bind to non- target molecules with a KD of 1 x 10⁶M or more, in some embodiments, 1 x 10⁵M or more, in some

embodiments, 1 x 10⁴M or more, in some embodiments, 1 x 10³M or more, in some embodiments, 1 x 10²M or more. An antibody specific for hEXD2 is in some embodiments capable of binding to hEXD2 molecule (e.g. protein/polypeptide) with an affinity that is at least two-fold, 10-fold, 50-fold, 100-fold 200-fold, 500-fold, 1 ,000- fold or 10,000-fold or greater than its affinity for binding to another non-hEXD2 molecule. These amounts are not meant to be limiting and increments between the recited values are specifically envisioned as part of the disclosure.

An antibody to EXD2, for example, may be made by any method known in the art. General techniques for production of human and mouse antibodies are known in the art and/or are described herein. For example, see Flarlow and Lane (1988) “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NJ.

In some embodiments, antibodies may be made recombinantly and expressed using any method known in the art. In some embodiments, antibodies may be prepared and selected by phage display technology. See, for example, U.S. Patent Nos.

5,565,332; 5,580,717; 5,733,743; and 6,265, 50; and Winter et al. , (Annu. Rev.

Immunol. 12:433-455, 1994). Alternatively, the phage display technology (McCafferty et al. , Nature 348:552-553, 1990) can be used to produce human antibodies and antibody portions in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

In some embodiments, antibodies may be made using hybridoma technology.

Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler and Milstein (Nature 256:495-497, 1975) or as modified by Buck et al., (In Vitro, 18:377-381 , 1982). Available myeloma lines, including but not limited to X63- Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, may be used in the hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After the fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine- aminopterin-thymidine (HAT) medium, to eliminate unhybridized parent cells. The hybridomas or other immortalized B-cells are expanded and sub-cloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that produce the desired antibody may be grown in vitro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulphate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired.

In some embodiments, fully human antibodies may be obtained by using

commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are HuMAb- Mouse™ and TC Mouse™ from Medarex, Inc. (Princeton, NJ) and Xenomouse™ from Abgenix, Inc. (Fremont, CA).

Antibodies may be made recombinantly by first isolating the antibodies and antibody producing cells from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (e.g., CHO cells). Methods for recombinantly expressing antigen-binding portions of antibodies, e.g., domain, single chain, etc. are also well known in the art.

In particular embodiments, the antibody for use in the invention is selected from: a monoclonal, human, humanised, Fab, Fab', F(ab')2, F(ab'), Fd, Fv, dAb, intrabody, scFV and VHH antibody.

Antibody and nucleic-acid technology molecules (such as RNAi and ASO) technologies are suitably advanced that the person skilled in the art would be able to make an antibody or antibody-derived molecule or a nucleic-acid technology molecule that could inhibit EXD2.

Method of screening

EXD2 is an exonuclease involved in DNA repair. Many nuclease inhibitors have been identified, particularly towards other enzymes involved in DNA repair

(Bartosova and Krejci. FEBS Lett. 588:2446-56, 2014; Atsushi Shibata et al.

Molecular Cell, Volume 53, Issue 1 , 2014, Pages 7-18), so it is expected that EXD2 will be druggable and that suitable inhibitors against EXD2 can be generated. In support of this the inventor has conducted a preliminary screen for EXD2 inhibitors and a number of compounds of diverse chemical type were identified as putative inhibitor molecules (see example 2)

Techniques for screening for inhibitors of a protein (such as EXD2) or its encoding nucleic acid (such as EXD2) are known to those skilled in the art. For example, the “nuclease detection and control” assay available from IDT,

(https://www.idtdna.com/pages/products/reagents-and-kits/nuclease-detection-and- control), which are fluorescent-quenched oligonucleotide probes that emit a signal only after nuclease degradation could be employed. General methods for such screening approaches have been described (see Assay Guidance Manual,

Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004).

According to the second aspect of the invention there is provided a method of screening for a compound potentially suitable for use in the treatment of cancer deficient in HR, comprising determining the ability of the compound to inhibit EXD2 protein, wherein if the compound inhibits EXD2 protein it is identified as one that is potentially suitable for use in the treatment of cancer deficient in HR.

According to the third aspect of the invention there is provided a method of screening for a compound suitable for use in the treatment of cancer comprising cells deficient in HR, comprising the steps of:

a) obtaining purified or recombinant EXD2;

b) contacting the EXD2 in step (a) with one or more test compounds; and

c) selecting those compounds that demonstrate a reduction in EXD2 activity; wherein a compound that causes a reduction in EXD2 activity is suitable for use in the treatment of cancer comprising cells deficient in HR.

In a particular embodiment of this third aspect of the invention, a reduction in activity of EXD2 is measured in vitro by a reduction in the ability of the EXD2 nuclease to cleave a suitable substrate. A suitable substrate is any DNA or RNA substrate that can be degraded or cleaved by EXD2. One example of this approach is EXD2- dependent degradation of DNA fork like structures (Example 2, Figure 1 ), which is carried out in a buffer containing 20 mM HEPES-KOH, pH 7.5, 50 mM KCI, 0.5 mM DTT, 10 mM MnCI2, 0.05% Triton-X, 0.1 mg ml-1 BSA, 5% glycerol, and EXD2 protein. The reaction is initiated by adding substrate and incubated at 37 °C for the indicated amounts of time. Reactions are stopped by addition of EDTA to a final concentration of 20 mM and 1/5 volume of formamide.

Using this approach, the inventor was able to demonstrate that ATA - (Aurintricarboxylic acid) inhibits EXD2 activity with an IC50= 2mM.

Survival analysis using DLD1 BRCA2 deficient and BRCA2 complemented DLD1 cells confirmed the killing efficacy of this compound (approach) in vivo (IC50=2.385 uM).

Pharmaceutical compositions and therapy

The active agents disclosed herein for the treatment of cancer may be administered alone, but it is generally preferable to provide them in a pharmaceutical composition suitable for administration. Such composition typically comprises the

agent/compound and a pharmaceutically acceptable excipient. The term

“pharmaceutically-acceptable excipient” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human. The term“excipient” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Types of suitable excipient are salts, buffering agents, wetting agents, emulsifiers, preservatives, compatible carriers, diluents, carriers, vehicles, supplementary immune potentiating agents such as adjuvants and cytokines that are well known in the art and are available from commercial sources for use in pharmaceutical preparations (see, e.g. Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20^th Ed. Mack Publishing; Kibbe et al. , (2000) Handbook of Pharmaceutical Excipients, 3^rd Ed., Pharmaceutical Press; and Ansel et al., (2004) Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^th Ed., Lippencott Williams and Wilkins). Optionally, the pharmaceutical compositions contain one or more other therapeutic agents or compounds. Suitable pharmaceutically acceptable excipients are relatively inert and can facilitate, for example, stabilisation, administration, processing or delivery of the active compound/agent into preparations that are optimised for delivery to the body, and preferably directly to the site of action.

The pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.

When administered, the pharmaceutical compositions of the present invention are administered in pharmaceutically acceptable preparations/compositions.

Administration may be topical, i.e. , substance is applied directly where its action is desired, enteral or oral, i.e., substance is given via the digestive tract, parenteral, i.e., substance is given by other routes than the digestive tract such as by injection.

Large biologic molecules are typically administered by injection.

Pharmaceutical compositions for parenteral administration (e.g. by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g. solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise provided (e.g. in a liposome or other microparticulate). Such liquids may additionally contain one or more pharmaceutically acceptable carriers, such as anti-oxidants, buffers, stabilisers, preservatives, suspending agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended patient. In particular embodiments, the composition may be lyophilised to provide a powdered form that is ready for reconstitution as and when needed. When

reconstituted from lyophilised powder the aqueous liquid may be further diluted prior to administration. For example, diluted into an infusion bag containing 0.9% sodium chloride injection, USP, or equivalent, to achieve the desired dose for administration. In particular embodiments, such administration can be via intravenous infusion using an intravenous (IV) apparatus.

Suitably, the active agent and optionally another therapeutic or prophylactic agent are formulated in accordance with routine procedures as pharmaceutical

compositions adapted for intravenous administration to human beings. Typically, the active agents for IV administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions can also include a solubilizing agent.

Compositions for IV administration can optionally include a local anaesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule. Where the active compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile

pharmaceutical grade water or saline. Where the active compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavouring agents such as peppermint, oil of wintergreen, or cherry; colouring agents; and preserving agents, to provide a pharmaceutically palatable preparation. A time delay material such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable excipients. Thus, the active agent and optionally another therapeutic or prophylactic agent and their physiologically acceptable salts and solvates can be formulated into pharmaceutical compositions for administration by inhalation or insufflation (either through the mouth or the nose) or oral, parenteral or mucosal (such as buccal, vaginal, rectal, sublingual) administration. In one aspect, local or systemic parenteral administration is used.

For oral administration, the compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pre-gelatinised maize starch,

polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with

pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.

The pharmaceutical compositions of the invention are for administration in an effective amount. An“effective amount” is the amount of a composition that alone, or together with further doses, produces the desired response.

In certain embodiments, the compound/agent that inhibits EXD2 can be administered as a pharmaceutical composition in which the pharmaceutical composition

comprises between 0.1 -1 mg, 1 -10 mg, 10-50mg, 50-1 OOmg, 100-500mg, or 500mg to 5g of the active agent. In particular embodiments, the EXD2 inhibitor will be administered at approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/Kg body weight per dose. Other embodiments comprise the administration of the EXD2 inhibitor at about 200, 300, 400, 500, 600, 700, 8000, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 mg/Kg body weight dose. Using the teaching herein, one of skill in the art can determine the effective dose and dosing schedule/regime of the specific EXD2 inhibitor based on preclinical and clinical studies and standard medical and biochemical measurements and techniques.

Patient Selection

The therapeutic treatments described herein are applicable to patients who possess cancer comprising cells with a deficiency in one or more members of HR pathway. Accordingly, the invention also provides for methods for identifying such patients and/or selecting such patients for therapeutic treatment.

According to the fourth aspect of the invention there is provided a method of selecting an individual having a cancer condition for treatment comprising:

determining if one or more cancer cells from the individual are deficient in HR, wherein if the one or more cancer cells are deficient in the HR the individual is selected for treatment with a EXD2 inhibitor.

According to the fifth aspect of the invention there is provided a method of determining the responsiveness of a subject having a cancer to an EXD2 inhibitor, the method comprising determining whether the cancer comprises cells deficient in HR, wherein the presence of said deficiency indicates that the subject is likely to be responsive to an EXD2 inhibitor.

In one embodiment, determining whether the cancer comprises cells deficient in HR is determined by the presence of one or more amino acid or nucleic acid mutations in the sequence of a member of the HR pathway. The presence of particular mutations will dictate whether the cell will be deficient in HR.

In a particular embodiment, the determining of the cancer cells is carried out on a biological sample from the patient. In particular embodiments, the biological sample is a tissue sample or a biological fluid sample, such as a sample comprising: blood, plasma, serum, sputum, needle aspirate, urine or ascites

In one embodiment, the patient’s cancer cells are obtained from the individual.

In one embodiment, the patient’s cancer cells have been previously obtained from the individual.

In another embodiment, cancer cell nucleic acid is obtained from the individual.

Cancer cell nucleic acid can be obtained from the cancer cells of from circulating free DNA in blood.

In one embodiment, the patient’s cancer cell nucleic acid is obtained from the individual.

In one embodiment, the patient’s cancer cells nucleic acid has been previously obtained from the individual.

As noted herein, determining whether the cells possess a deficiency in HR can be done by directly detecting for the particular HR pathway member (protein detection) or by detecting surrogates of such proteins, e.g. mRNA or other encoding nucleic acid etc.

In particular embodiments, the patient’s cancer cells are also evaluated to determine whether the cells are deficient in the 53BP1/RIF1/REV7/Shieldin protein

complex due to one or more mutations in a gene, or the absence of or defective expression of a gene encoding a protein from the 53BP1/RIF1/REV7/Shieldin protein complex selected from the group consisting of: 53BP1 , RIF1 , REV7, Shieldin 1 , Shieldin 2 and Shieldin 3.

Mutation detection

The identification of a cancer as being deficient in HR can be done using various means.

One of the most appropriate mans is to detect the presence of de-activating mutations in a gene encoding a protein of the HR pathway. By“de-activating mutation” we mean a mutation (such as one or more base substitutions, insertions or deletions) which alters the activity or function of the protein. The mutation could cause a frameshift resulting in expression of an altered protein, which cannot function properly (e.g. cannot bind in the complex, cannot trigger signalling etc.), or a stop codon resulting in a premature protein which also cannot function properly. Alternatively, the mutation could result in the production of a protein with one or more amino acid substitutions relative to the wild-type protein and the presence of such substitutions could change the three-dimensional configuration of the protein and/or interfere with complex binding or some other activity of the protein. The mutation(s) 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 HR dependent DNA DSB repair pathway. 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.

“De-activating” includes but is not limited to completely defunct or missing protein. It includes proteins that function but at a significantly reduced amount, such as by at least 50%, at least 60%, at least 70%, at least 80%, and least 90%, at least 95%, and least 98% and at least 99%, as compared to wild-type protein.

The presence of such a mutation can be detected using conventional techniques such as gene sequencing or using polymerase chain reaction applications to detect the presence of a particular mutation, such as allele-specific amplification on a suitable nucleic acid containing sample.

The 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 by comparing the sequence obtained with the database sequence of the component, as set out above. In particular, the presence of one or more polymorphisms or mutations that cause abrogation or loss of function of the polypeptide component, and thus the HR dependent DNA DSB repair pathway as a whole, may be determined.

Sequencing and specific mutation detection may be performed using any one of a range of standard techniques. In some embodiments, the individual is heterozygous for one or more variations, such as mutations and polymorphisms, in BRCA1 and/or BRCA2 or a regulator thereof.

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.

As noted elsewhere herein, various forms of cancer possess mutations in one or more genes encoding proteins of the HR pathway.

Protein detection

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.

As noted above, the presence of an HR pathway protein can be detected in the cells, including the cell nuclei, using any of a variety of techniques. In particular

embodiments, the presence of an HR pathway protein is detected using

immunohistochemistry, immunofluorescence, Western blotting, capillary

electrophoresis, or ELISA. Furthermore, these methods can be employed using an antibody or digital barcoded antibody to the HR pathway protein. A digital barcoded antibody is an antibody whereby DNA barcodes are attached to the antibody.

Multiple barcoded antibodies can then be assayed in parallel and subsequently analysed by DNA sequencing (E.g. see Agasti et al. J Am Chem Soc.

134(45): 18499-18502, 2012).

In general, the level of an HR pathway protein can be assessed using any of a variety of methods. In many embodiments, the level of expression of an HR pathway protein is assessed by determining the level of the HR pathway gene product in a sample obtained from a tumour. The HR pathway protein level can also be

determined using a surrogate of the HR pathway protein, such as for example mRNA encoding the HR pathway protein. Optionally the mRNA is detected directly or measured after conversion to cDNA which may optionally be amplified (e.g. by reverse transcriptase PCR). The skilled person will readily be able to determine suitable reference values with respect to which the amount of the appropriate target molecule (e.g. an HR pathway protein) may be compared. Merely by way of example, expression of target molecule in cancerous tissue can be compared to expression of that same molecule in non- cancerous tissue, such as adjacent non-cancerous tissue. Expression can be assessed on a protein level for example by immunohistochemistry or on a DNA level for example by fluorescence in situ hybridization, or on an RNA level, for example by quantitative real-time PCR.

Protein measurement

In general, any suitable method for measuring proteins can be used to measure the level of an HR pathway protein/polypeptide in a sample.

In many embodiments, an immunological method or other affinity-based method is used. In general, immunological detection methods involve detecting specific antibody-antigen interactions in a sample such as a tissue section or cell sample.

The sample is contacted with an antibody that binds to the target antigen of interest. The antibody is then detected using any of a variety of techniques.

In some embodiments, the antibody that binds to the antigen (primary antibody) or a secondary antibody that binds to the primary antibody has been tagged or conjugated with a detectable label. In some embodiments, a label-free detection method is used. A detectable label may be, for example, a fluorescent dye (e.g., a fluorescent small molecule) or quencher, colloidal metal, quantum dot, hapten, radioactive atom or isotope, or enzyme (e.g., peroxidase). It will be appreciated that a detectable label may be directly detectable or indirectly detectable. For example, a fluorescent dye would be directly detectable, whereas an enzyme may be indirectly detectable, e.g., the enzyme reacts with a substrate to generate a directly detectable signal. Numerous detectable labels and strategies that may be used for detection, e.g., immunological detection, are known in the art.

Exemplary immunological detection methods include, e.g., immunohistochemistry (IHC); enzyme-linked immunosorbent assay (ELISA), flow cytometry, protein microarrays, surface plasmon resonance assays, immunoprecipitation, immunoblot (Western blot), etc. IHC generally refers to immunological detection of an antigen of interest (e.g., a cellular constituent) in a tissue sample such as a tissue section. As used herein, IHC is considered to encompass immunocytochemistry (ICC), which term generally refers to the immunological detection of a cellular constituent in isolated cells that essentially lack extracellular matrix components and tissue microarchitecture that would typically be present in a tissue sample. Traditional ELISA assays typically involve use of primary or secondary antibodies that are linked to an enzyme, which acts on a substrate to produce a detectable signal (e.g., production of a coloured product) to indicate the presence of antigen or another analyte.

IHC generally refers to the immunological detection of a tissue or cellular constituent in a tissue or cell sample comprising substantially intact (optionally permeabilized) cells. As used herein, the term“ELISA” also encompasses use of non-enzymatic reporters such as fluorogenic, electrochemiluminescent, or real-time PCR reporters that generate quantifiable signals. It will be appreciated that the term“ELISA” encompasses a number of variations such as“indirect”,“sandwich”,“competitive”, and“reverse” ELISA.

In some embodiments, e.g., wherein IHC is used for detecting a protein of interest, a sample is in the form of a tissue section, which may be a fixed or a fresh (e.g., fresh frozen) tissue section or cell smear in various embodiments. A sample, e.g., a tissue section, may be embedded, e.g., in paraffin or a synthetic resin or combination thereof. A sample, e.g., a tissue section, may be fixed using a suitable fixative such as a formalin-based fixative. The section may be a paraffin-embedded, formalin-fixed tissue section. A section may be deparaffinized (a process in which paraffin (or other substance in which the tissue section has been embedded) is removed (at least sufficiently to allow staining of a portion of the tissue section). To facilitate the immunological reaction of antibodies with antigens in fixed tissue or cells it may be helpful to unmask or“retrieve” the antigens through pre-treatment of the sample. A variety of antigen retrieval procedures (sometimes called antigen recovery), can be used in IHC. Such methods can include, for example, applying heat (optionally with pressure) and/or treating with various proteolytic enzymes. Methods can include microwave oven irradiation, combined microwave oven irradiation and proteolytic enzyme digestion, pressure cooker heating, autoclave heating, water bath heating, steamer heating, high temperature incubator, etc. To reduce background staining in IHC, the sample may be incubated with a buffer that blocks the reactive sites to which the primary or secondary antibodies may otherwise bind. Common blocking buffers include, e.g., normal serum, non-fat dry milk, bovine serum albumin (BSA), or gelatin, and various commercial blocking buffers. The sample is then contacted with an antibody that specifically binds to the antigen whose detection is desired (e.g., an HR pathway protein). After an appropriate period of time, unbound antibody is then removed (e.g., by washing) and antibody that remains bound to the sample is detected. After immunohistochemical staining, a second stain may be applied, e.g., to provide contrast that helps the primary stain stand out. Such a stain may be referred to as a“counterstain”. Such stains may show specificity for discrete cellular compartments or antigens or stain the whole cell. Examples of commonly used counterstains include, e.g., hematoxylin, Hoechst stain, or DAPI. The tissue section can be visualized using appropriate microscopy, e.g., light microscopy, fluorescence microscopy, etc. In some embodiments, automated imaging system with appropriate software to perform automated image analysis is used.

A suitable IHC test for detecting an HR pathway protein is used in Ross-lnnes et al, (Nature 481 (7381 ):389-393, 2012); here IHC staining was performed on metastatic tumour samples.

In some embodiments, flow cytometry (optionally including cell sorting) is used to detect expression of an HR pathway protein. The use of flow cytometry would typically require the use of isolated cells substantially removed from the surrounding tissue microarchitecture, e.g., as a single cell suspension polypeptide level could be assessed by contacting cells with a labelled probe or antibody that binds to the protein of interest (e.g. the HR pathway protein) wherein said probe or antibody is appropriately labelled (e.g., with a fluorophore, quantum dot, or isotope) so as to be detectable by flow cytometry. In some embodiments, cell imaging can be used to detect the target subunit protein of the HR pathway protein.

In some embodiments, an antibody for use in an immunological detection method, e.g., IHC, is monoclonal. In some embodiments, an antibody is polyclonal. In some embodiments, an antibody is an antigen-binding portion of an intact antibody. One of ordinary skill in the art would readily be able to generate additional antibodies suitable for use to detect an HR pathway protein/polypeptide using standard methods. In some embodiments, a ligand that specifically binds to an HR pathway protein but is not an antibody is used as an affinity reagent for detection of the HR pathway protein. For example, nucleic acid aptamers or certain non-naturally occurring polypeptides structurally unrelated to antibodies based on various protein scaffolds may be used as affinity reagents. Examples include, e.g., agents referred to in the art as affibodies, anticalins, adnectins, synbodies, etc. See, e.g., Gebauer, M. and Skerra, A., Current Opinion in Chemical Biology, (2009), 13(3): 245-255 or

W02009/140039.

In some embodiments, a non-affinity based method is used to assess the level of an HR pathway protein. For example, mass spectrometry could be used to detect and quantitate the amount of an HR pathway protein.

Normalisation

It will be understood that suitable controls and normalization procedures can be used to accurately quantify expression of an HR pathway protein, where appropriate. For example, measured values can be normalized based on the expression of one or more RNAs or polypeptides whose expression is not correlated with a phenotypic characteristic of interest. In some embodiments, a measured value can be normalized to account for the fact that different samples may contain different proportions of a cell type of interest, e.g., cancer cells, versus non-cancer cells. For example, in some embodiments, the percentage of stromal cells, e.g., fibroblasts, may be assessed by measuring expression of a stromal cell-specific marker, and the overall results adjusted to accurately reflect target mRNA or polypeptide level specifically in the tumour cells. It would also be understood that if a sample such a tissue section contains distinguishable (e.g., based on standard histopathological criteria), areas of neoplastic and non-neoplastic tissue, such as at the margin of a tumour, the level of target protein expression could be assessed specifically in the area of neoplastic tissue, e.g., for purposes of comparison with a control level, which may optionally be the level measured in the non-neoplastic tissue.

53 BP 1/RIF 1/RE V7/Shieldin protein complex

The identification of a cancer as being deficient in 53BP1/RIF1/REV7/Shieldin protein complex, or having an inactivating mutation in one or more of the

members/components of the 53BP1/RIF1/REV7/Shieldin 1 ,2 and 3 protein complex can be performed using any of the methods described above for HR, but adapted for the 53BP1/RIF1/REV7/Shieldin 1 ,2 and 3 protein complex member.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean“including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular

encompasses the plural unless the context otherwise requires. Where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, embodiments described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1 94); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991 ) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, suitable methods and materials are described herein.

The invention will be described in further detail below and with reference to the accompanying Examples and Figures.

DESCRIPTION OF THE FIGURES

Figure 1. A) Proliferation of HeLa WT and EXD2 cells upon BRCA1 knockdown (mean +/- SEM, n= 3 independent experiments).

B) Proliferation of HeLa WT and EXD2^~ cells upon BRCA2 knockdown (mean +/- SEM, n= 3 independent experiments).

C) Proliferation of U20S WT and EXD2^~ cells upon BRCA1 knockdown (mean +/- SEM, n= 4 independent experiments).

D) Proliferation of DLD1 WT and BRCA2^~ cells upon EXD2 knockdown (mean +/- SEM, n= 3 independent experiments).

E) Proliferation of SUM149 and SUM149 revertant cells upon EXD2 knockdown (mean +/- SEM, n= 3 independent experiments).

F) Proliferation of MDA-MB-436 BRCA1^~ cells (Triple Negative Breast Cancer Cell Line) upon EXD2 knockdown (mean +/- SEM, n= 3 independent experiments); left panel. Western blotting confirming the depletion of EXD2 (right panel).

G) Proliferation of RPE1 WT, EXD2- and EXD2^ND/ND cells upon BRCA1

knockdown (mean +/- SEM, n= 3 independent experiments).

H) Quantification of the HeLa WT and EXD2^~ cells upon BRCA2 knockdown showing micronuclei frequency (mean +/- SEM n= 3 independent

experiments).

I) Quantification of the relative Alt-EJ efficiency upon EXD2, MRE11 or BRCA2 knockdown as indicated (mean +/- SEM, n= 5 independent experiments, t- test).

J) Proposed model for EXD2 role in supporting genome duplication and survival of BRCA1 and BRCA2 mutant cells. Replicative stress leads to fork stalling. Stressed forks are protected by EXD2 activity in a pathway cooperating with RECQ1 , allowing for efficient fork restart and timely accomplishment of DNA replication. Loss of EXD2 leads to extensive degradation of nascent DNA at stalled forks, compromising fork restart and ultimately adversely impacting genome stability. Combined deficiency in EXD2 and BRCA1/2 results in cell death due to the absence of functional HR as well as the back-up Alt-EJ pathway underpinned by EXD2 activity.

Figure 2.

A) Western blotting confirming the depletion of BRCA1 in HeLa WT and EXD2^A cells 72h post-transfection with control siRNA (siCtrl) or siRNA targeting BRCA1 as indicated. a-tubulin serves as a loading control.

B) Western blotting confirming the depletion of BRCA2 in HeLa WT andEXD2^A cells 72h post-transfection with control siRNA (siCtrl) or siRNA targeting BRCA2 as indicated. a-tubulin serves as a loading control.

C) Western blotting confirming the depletion of BRCA1 in U20S WT and EXD2 cells 72h post-transfection with control siRNA (siCtrl) or siRNA targeting BRCA1. MCM2 acts as a loading control.

D) Western blotting confirming the depletion of EXD2 in DLD1 WT and DLD1 BRCA2 cells 72h post-transfection with control siRNA (siCtrl) or siRNA targeting EXD2. MCM2 acts as a loading control.

E) Western blotting confirming the depletion of EXD2 in SUM149 and SUM149 revertant cells 72h post-transfection with control siRNA (siCtrl) or siRNA targeting EXD2. MCM2 acts as a loading control.

F) Western blotting confirming the depletion of BRCA1 in RPE1 WT, EXD2^~ and EXD2^ND/ND cells 72h post-transfection with control siRNA (siCtrl) or siRNA targeting BRCA1. MCM2 acts as a loading control.

Figure 3.

A) Quantification of the relative alt-EJ efficiency in cells treated with control siRNA or siRNA targeting EXD2 as indicated (mean, n=2 independent experiments). Right panel - Western blotting confirming the depletion of EXD2 and MRE11 in U20S EJ2-GFP cells 72h post-transfection with control siRNA (siCtrl) or siRNA targeting EXD2 or MRE11 as indicated. PCNA serves as a loading control.

B) Quantification of the frequency of dicentric chromosomes from mitotic

spreads from U20S WT and EXD2^~ cells (mean +/- SEM, n=50 metaphase spreads pooled from 2 independent experiments). Right panel - Representative images of analysed metaphase spreads.

Figure 4.

A) Graphical representation of the assay for identification of EXD2 inhibitors. The DNA substrate is labelled with two dyes: a fluorophore (6-FAM) at the 3' end of the“template strand”, and a dark quencher (Iowa Black FQ) at the 5' end of the“nascent strand”. The reaction efficiency (relative digestion) is determined by measuring an increase in fluorescence emission at a wavelength of

519 nm.

B) EXD2 nuclease activity in the presence of labelled DNA substrate (240 nM).

First bar - no EXD2; second bar - EXD2 (200 nM). Enzymatically inactive EXD2 (+EXD2 ND) - third bar, serves as a negative control.

C) The nuclease activity of EXD2 was analysed in the presence of varying

concentrations of EXD2 enzyme (100-400 nM) the DNA substrate

concentration was (240 nM).

D) EXD2 nuclease activity in the presence of various concentrations of

compounds A - ATA; Aurintricarboxylic acid and B - 2-(N- Morpholino)ethanesulfonic acid sodium salt (x axis) and DNA substrate (240 nM). The IC50 observed that inhibits 50% of the EXD2 nuclease activity at a given concentration of DNA substrates is indicated with dotted line.

E) Survival assay of DLD1 WT and DLD1 BRCA2^~ cells treated with indicated concentrations of ATA (a putative EXD2 inhibitor); mean +/- SEM, n= 3 independent experiments.

Figure 5.

Phosphor imaging of 5’ radiolabelled indicated DNA substrates (labelled strand shown in red) incubated for indicated amounts of time with EXD2 WT protein.

Figure 6.

Proliferation of FleLa WT and EXD2^A cells (2 independent clones) upon RIF1 knockdown.

Figure 7. Survival analysis of SUM149 BRCA1 cell line and the revertant SUM149 (rev) expressing BRCA1 treated with PARP inhibitor Niraparib with and without EXD2 knockdown by siRNA (mean +/- SEM, n= 3 independent experiments).

Figure 8.

Schematic diagram outlining the use of EXD2 inhibitors in treatment of BRCA1/2 as well as HR deficient tumours. EXD2 inhibition lead to killing of BRCA1/2 and HR- deficient tumours by two mechanisms (i) synthetic lethal interaction with the HR pathway and (ii) suppression of development of resistance via inactivation of Alt-EJ as well as synthetic lethal interaction with RIF1 - component of the

53BP1/RIF1/REV7/ Shieldin protein complex, which is frequently inactivated leading to PARPi resistance in BRCA1 /2-deficient tumours.

EXAMPLES Example 1

EXD2 is synthetic lethal with BRCA1 and BRCA2 deficiency

Germline mutations in the BRCA1/BRCA2 genes account for up to 80% of familial breast and ovarian cancer cases (King et al. , Science 302, 643-646, 2003; Prakash et al., Cold Spring Harbor Perspectives in Biology 7, a016600, 2015). In their absence, cells are unable to repaie DNA double strand breaks by homology directed repair and in addition, nascent DNA at the stalled replication forks is extensively degraded, likely contributing to BRCA1/2 associated genome instability and sensitivity to replication stress-inducing therapies (Chaudhuri et al., Nature 539, 456, 2016; Ceccaldi, R. et al. Nature 518, 258-262, 2015).

Recently, we identified a novel factor called EXD2 and showed that EXD2 is required for efficient repair of damaged DNA. Interestingly, we also discovered that EXD2 cells show hypersensitivity to agents inducing collapse of DNA replication forks (Broderick et al., Nat Cell Biol 18, 271 -280, 2016).

To further our understanding of the role of EXD2 in genome stability and protection of stressed replication forks we analysed the genetic interaction between BRCA1 and BRCA2 and the EXD2 nuclease. With this in mind, we first analysed the proliferative capabilities of both, single and double mutants. Strikingly, combined depletion of EXD2 with BRCA1/2 in cellular model comprising cancer cell lines HeLa or U20S leads to an almost complete inhibition of cell growth (Fig. 1 A-C and Fig. 2A- C). Importantly, this phenotype was recapitulated in BRCA1 and BRCA2 -deficient patient derived cancer cell lines including relevant controls (i.e. cells expressing BRCA1 and BRCA2; Drean, A. et al. Modeling Therapy Resistance in BRCA1/2- Mutant Cancers. Molecular cancer therapeutics 16, 2022-2034 (2017)) - Fig. 1 D-E and Figure 2 D-E. Furthermore, the same interaction was also observed in triple negative breast cancer model cell line MDA-MB-436 (Fig. 1 F), which is BRCA1 deficient.

Crucially, using another cellular model system i.e. RPE1 cells that were either knockout for EXD2 or were expressing EXD2 protein carrying an inactivation mutation within the nuclease domain (this mutation renders EXD2 enzymatically inactive (Broderick et al., Nat Cell Biol 18, 271-280, 2016) and also to a great extent would mimic the action of a small molecule inhibitor of EXD2) we showed that EXD2 nuclease activity is required to promote cells survival in the context of BRCA- deficiency (Fig. 1 G and Fig. 2G). In support of this data, our analysis of the frequency of micronuclei formation (a marker of genome instability) showed a marked increase in micronuclei in the double deficient mutants as compared to the singles (Fig. 1 FI).

Since, EXD2 functionally interacts with the MRE11 nuclease (Broderick et al., Nat Cell Biol 18, 271 -280, 2016), a protein required for alternative end-joining we considered the possibility that the synthetic growth defect may reflect the loss of Alt- EJ to rescue unprotected forks. This pathway relies on limited resection of DSBs by the MRE11 nuclease to generate short stretches of homology used to bridge the break Floward et al., PLoS genetics 11 , e1004943, 2015) and recent work has implicated Alt-EJ in survival of BRCA1 /2-deficient tumours (Ceccaldi, R. et al. Nature 518, 258-262, 2015; Mateos-Gomez et al., Nature 518, 254-257, 2015). To test this, we depleted EXD2 in a U20S cell line expressing the Alt-EJ reporter construct (Gunn et al., The Journal of Biological Chemistry 286, 42470-42482, 2011 ), using BRCA2 and MRE11 knockdown as a positive and negative control, respectively. As previously reported, silencing of BRCA2 increased the level of Alt-EJ events whereas knockdown of MRE11 reduced its efficiency (Ceccaldi, R. et al. Nature 518, 258-262, 2015; Howard et al, PLoS genetics 11 , e1004943, 2015). Strikingly, we noticed that Alt-EJ was also significantly impaired in the absence of EXD2 (Fig. 1 1 and Fig. 3A). We confirmed this discovery using another approach based on the analysis of the frequency of chromosomal fusions. In support for a role of EXD2 in promoting Alt-EJ mechanism, we also noticed a dramatic deficiency of EXD2r'^~ cells to generate chromosome fusions (Fig. 3B), which are dependent on the alternative end-joining pathway (Alt-EJ) (Mateos-Gomez et al., Nature 518, 254-257, 2015).

Given that Alt-EJ is essential for survival of BRCA1/2 mutants (Ceccaldi, R. et al. Nature 518, 258-262, 2015; Mateos-Gomez et al., Nature 518, 254-257, 2015) and that EXD2 is required for Alt-EJ mechanism we showed that depletion of EXD2 is not compatible with cells survival of BRCA-deficient tumours due to an accumulation of DNA double strand breaks, which cannot be rescued by Alt-EJ back-up mechanism driving cell death, even under normal growth conditions, Model Fig. 1 J.

Inactivation of the 53BP1/RIF1/REV7/Shieldin protein complex is associated with development of resistance in BRCA-deficient tumours treated with PARP inhibitors Sylvie M. Noordermeer, Haico van Attikum, Trends in Cell Biology, Vol. 29, No. 10, 2019). Strikingly, we have also discovered that EXD2 is essential for survival of cells deficient for RIF1 (and likely other components of the same protein complex) Fig. 6. Thus, likely EXD2 inhibition will contribute to BRCA-tumours killing by at least two mechanism (i) synthetic lethal interaction with BRCA1 /2-deficiency as well as (ii) an inhibition of development of resistance to PARPi treatment in these tumours via inactivation of the Alt-EJ pathway and synthetic lethal interaction with RIF1 (or other members of BRRES protein complex), which are frequently inactivated in BRCA- deficient tumours leading to PARPi resistance (Model, Fig. 8).

Moreover, we also identified that EXD2 depletion/inhibition can re-sensitise PARPi resistant tumours (i.e. SUM149^REV) to PARPi treatment (Fig. 7).

In summary, we have made a novel and unanticipated discovery that EXD2 is essential for survival of BRCA1 and BRCA2 mutant cells as well as cells deficient for RIF1 , a protein that is part of the 53BP1/RIF1/REV7/Shieldin complex, which frequent inactivation in BRCA-tumours is associated with resistance to PARPi treatment (Noordermeer et al. Nature, 560(7716): 1 17-121 , 2018; Mirman Z., et al. Nature, 560(7716): 1 12-1 16, 2018; Ghezraoui H., Nature, 560(7716): 122-127, 2018). . We discover a novel role for EXD2 in promoting repair of DSB via alternative end joining (Alt-EJ). Thus, in the absence of both EXD2 and BRCA1 /2 proteins DNA damage (DSBs) arising in cells cannot be repaired by either canonical HR or the Alt- EJ back up mechanism.

This is an important discovery from clinical perspective, as this work identifies EXD2 and its nuclease activity as a novel target for cancers disrupted for BRCA genes and likely other genes required for HR-dependent DNA break repair.

Material and methods:

Cell Lines Generation

HeLa, U20S and RPE1 EXD2^_/_ cells were generated as previously described (Broderick et al. , Nat Cell Biol 18, 271 -280, 2016) using CRISPR/CAS9 approach. RPE1 EXD2^ND/ND (D108A/E 1 10A) were generated using following DNA oligos:

GTCTAATTCACTTCTAAGCAA (SEQ ID NO: 5) and

G ACTT GG AATT G ACT GT G AGT (SEQ ID NO: 6) cloned into pAIO-NK vector (a kind gift from Dr. A. Blackford) to generate the gRNAs; these and ssODN (single-stranded donor oligonucleotides) were used to introduce specific mutations disrupting the nuclease domain (IDT Technology)

AG G AGG C AG AGT G G GAT C AAAT C GAG C C CTTG CTT AG AT CT G AATT AG AAG ATT TT C C AGT ACTTGGTATCGCTTGTGCGTGGGT AAGTT AAAAAG C AAAAGTT AAAA AA (SEQ ID NO: 7).

Immunoblotting

Cell lysis was carried out in urea buffer (9 M urea, 50 mM Tris HCL, pH 7.3, 150 mM b-mercaptoethanol) followed by sonication using a soniprep 150 (MSE) probe sonicator. In some instances, cells were lysed in SDS loading buffer (2% SDS, 10% (v/v) glycerol, 2% 2-Mercaptoethanol and 62.5 mM Tris-HCI, pH 6.8) followed by boiling for 10 min. Samples were resolved by SDS-PAGE and transferred to PVDF or nitrocellulose. Protein concentrations were determined by Bradford assay by spectrophotometry using a NanoDrop 2000 device (Thermo Scientific). Immunoblots were carried out using the indicated antibodies: a-Tubulin (Sigma, B-5-1 -2; T5168, 1 : 100,000), BRCA1 (Millipore, OP-92, 1 : 1000), BRCA2 (Millipore, OP-95, 1 : 1000), EXD2 (Sigma, HPA005848, 1 :1000), MCM2 (Abeam, ab4461 , 1 :10,000), MRE11 (Abeam, ab214, 1 :1000), PCNA (Santa-Cruz, PC-10, 1 :500).

Cell Survival and Proliferation Assays

Alamar Blue survival assays were performed in accordance with the manufacturer’s recommendations (Life Technologies). Briefly, 500 cells per well in 96-well plates were untreated or treated with indicated doses of camptothecin or ionising radiation and incubated for 7 days. Alamar blue reagent (Life Technologies) was added to each well and fluorometric measurements taken after 2h incubation at 37°C. For proliferation assays cells were seeded at 500 cells per well and Alamar blue reagent added and measurements taken each day as indicated.

RNAi treatment siRNAs employed were as follows, siBRCAI - ACCAUACAGCUUCAUAAAUAA, siBRCA2 (ON-TARGETplus SMART pool - Cat # L-003462-00-0005, Dharmacon.), siEXD2 - CAGAGGACCAGGUAAUUUA (SEQ ID NO: 3) and Dharmacon SMART POOL (Cat # L-020899-02-0005), siMRE11 - GGAGGUACGUCGUUUCAGA, ON- TARGETplus Non-targeting Pool (D-00180-10-20, Dharmacon), or siRNA targeting luciferase - CGTACGCGGAATACTTCGA (SEQ ID NO: 8) were used as control siRNAs where appropriate. Oligonucleotides were transfected using HiPerfect reagent (Qiagen), according to the manufacturer’s protocol.

Immunofluorescence microscopy

For analysis of micronuclei cells were analysed using a protocol adapted from (Broderick et al. , Nat Cell Biol 18, 271 -280, 2016). Briefly, cells were collected by mitotic shakeoff and spun onto poly-L-Lysine coated slides at 1000 x g for 3 min. Mitotic cells were then fixed using 4% PFA in PBS for 10 min at room temperature and mounted with Vectashield containing DAPI. Images were acquired using a Zeiss LSM 710 laser scanning confocal microscope with Zen software using a 63x objective. Image analysis was carried out with FIJI (ImageJ) software.

Alt-EJ GFP reporter assay

48 hours after siRNA transfection, U20S EJ2-GFP cells (Gunn et al., The Journal of Biological Chemistry 286, 42470-42482, 2011 ) were transfected using Amaxa nucleofection with an l-Scel expression vector (pCMV-l-Scel) or a vector expressing mCherry fluorescent protein (pmCherry-C1 ). 72 hours after l-Scel transfection cells were harvested and analysed by flow cytometry (BD LSR II). 2x10⁴ cells were analysed per experimental condition. Number of GFP-positive cells per 1000 m Cherry-positive cells was determined using BD FACS DIVA software. The data were then related in each experiment to siControl treated sample set as 1. Statistical significance was determined with the Student’s f-test.

Chromosomal aberrations analysis

Sub-confluent cultures of FleLa WT and the of FleLa EXD2¹ cl.1 and cl.2 cells, grown in DMEM with 10% FBS (Gibco-BRL) plus antibiotics, at 37°C, were exposed to DMF (Sigma), 20 mM Cisplatin (Sigma) diluted in DMF for 16 h or 4 Gy X-ray irradiation. Two hours before harvest, the cells were exposed to colcemid (0.1 pg/ml) (Gibco). Cells were harvested by trypsinization (Gibco), re-suspended in culture medium and then spun down (10 minutes at 1000 rpm). Supernatant was removed, and then 0.075 M KCI (Sigma) at room temperature was added drop by drop. For hypotonic treatment, cell suspensions were incubated for 20 minutes at room temperature, and then 1 ml of fixative 3:1 methanol (Applichem GmbFI, Darmstadt, Germany)-acetic acid (Merck, Darmstadt, Germany) was added. Cells were re centrifuged for 10 minutes at 1000 rpm, supernatant was removed, fixative was added, and then re-centrifuged in fixative for another two times. Finally, chromosome preparations were dropped onto wet microscope slides and left to air-dry. For inverted DAPI chromosome banding staining, slides were mounted with 0.1 pg/ml DAPI in Vectashield antifade medium (Vector Laboratories, Burlingame, CA). Images of chromosome spreads were captured using a x63 magnification lens on a fluorescent Axio-lmager Z1 , Zeiss microscope, equipped with a MetaSystems charge-coupled device camera and the MetaSystems Isis software. Chromosome lesions were recorded as breaks per chromosome number, per metaphase, in 75 metaphase spreads per condition, pooled from 3 independent experiments.

Example 2

We have presented evidence that EXD2 knockout or inhibition of EXD2’s

exonuclease activity is synthetic lethal with the deficiency in BRCA1/2 genes suggesting that EXD2 could be an effective target in cancer therapy (Example 1 ). To facilitate this, we have developed a screen that could be used for identification of small molecule inhibitors of EXD2’s nuclease activity.

The screen entails the use of oligonucleotides to generate a specific DNA substrate that is preferentially digested by purified EXD2 in vitro (Figure 4, schematic A and Figure 5). The DNA substrate is labelled with two dyes: a fluorophore (6-FAM) at the 3'end of the“template strand”, and a dark quencher (Iowa Black FQ) at the 5' end of the“nascent strand”.

The fluorescence emitted by the 6-FAM fluorophore is quenched by FRET, but it increases when the“labelled nascent strand” is digested by EXD2 and dissociates from the“template” strand. In the initial screen, we analysed the efficiency of purified EXD2 nuclease in digesting this substrate. EXD2 nuclease efficiently cleaves the DNA substrate used here (Figure 4B) and by extension, any potential inhibitors are expected to block substrate digestion. Accordingly, this analysis showed up to a 30- fold increase in the fluorescent signal after incubating the EXD2 protein with the DNA substrate and importantly, the nuclease-dead version of EXD2 did not digest the substrate (Figure 4B). Thus, the above-described analysis provides a proof-of- principle for using our assay for a high throughput screen for small molecule inhibitors of EXD2.

In addition, we have also established a purification protocol that allows us to purify high quantities of EXD2 protein sufficient for a high-throughput screen (Material and Methods) and determined the optimal concentration of EXD2 needed to generate maximum fluorescence (Figure 4C).

As indicated above, the inhibitory activity of a compound with the potential to block EXD2 nuclease activity is determined by a reduction in fluorescence signal. We tested this by using three candidate compounds predicted to have an inhibitory activity towards DEDD-type nucleases - (Fluang et al. , J. Med. Chem., 59(17):8019- 29, 2016). In agreement with the assay principle design we identified that ATA - (Aurintricarboxylic acid) inhibits EXD2 activity with an IC50= 2mM, which was significantly more potent than the inhibition observed for 2-(N-Morpholino)

ethanesulfonic acid sodium salt (Figure 4D).

Finally, we show that ATA - which inhibits EXD2 nuclease activity in vitro, kills BRCA2-deficient cells (DLD1 ) with high efficacy also in vivo (IC50=2 uM, Figure 4E). Importantly, this compound is well tolerated by DLD1 cells complemented with BRCA1 (WT). This data substantiates our claim that inhibition of EXD2 by a small molecule provides a novel strategy to kill BRCA-mutated tumours or more broadly HR-deficient tumours and limit therapeutic-resistance (Model, Figure 1 J and Figure 8).

Material and Methods Protein Purification

GST-His- EXD2 K76-V564 was purified as described previously (Broderick et al. , Nat Cell Biol 18, 271 -280, 2016). Briefly, GST protein expression was induced with 0.1 mM IPTG (isopropyl-p-d-thiogalactopyranoside) (Sigma-Aldrich) at 16°C for 18 hours. Bacteria were harvested by centrifugation and resuspended in lysis buffer containing 50 mM phosphate pH 8.0, 300mM NaCI, 1 mM DTT, 1 % Triton X-100, 10 mM imidazol and PMSF. Lysates were sonicated and cleared by centrifugation. Supernatants were incubated with Ni resin (Qiagen) for 2 h with rotation at 4°C. Beads were washed with lysis buffer containing 20 mM imidazol, and eluted with lysis buffer containing 300 mM imidazole. Eluates were then incubated with

Glutathione HiCap Matrix (Qiagen) for 2 h with rotation at 4°C. Beads were washed with buffer containing increasing concentration of NaCI, elution buffer (50 mM Tris- HCI pH 7.0, 150 mM NaCI, 1 mM EDTA, 1 mM DTT, 0.2% Triton X-100) and resuspended in elution buffer supplemented with PreScission Protease (50 units/ml) (GE Healthcare) and incubated for 18 h with rotation at 4°C. Eluates were dialysed to buffer containing 20 mM Hepes-KOH pH7.2, 100 mM NaCI, 1 mM DTT, 10% glycerol, aliquoted and stored at -80°C.

In vitro nuclease assay

To generate 5’ end labelled substrates, the indicated ssDNA oligo was labelled using [y-³²P] dATP and PNK enzyme (New England Biolabs). To obtain fork substrates, ssDNA oligos were mixed in an equimolar ratio and annealed by heating at 100 °C for 5 min followed by gradual cooling to room temperature.

Exonuclease assays - was performed as in². Briefly, reactions were carried out in a buffer containing 20 mM HEPES-KOH, pH 7.5, 50 mM KCI, 0.5 mM DTT, 10 mM MnCh, 0.05% Triton-X, 0.1 mg ml¹ BSA, 5% glycerol, and EXD2 protein and initiated by adding substrate and incubated at 37 °C for the indicated amounts of time.

Reactions were stopped by addition of EDTA to a final concentration of 20 mM and 1/5 volume of formamide. The samples were resolved on denaturing 15% or 20% polyacrylamide TBE-Urea gels. Gels were fixed, dried and visualised using a Typhoon FLA 9500 instrument (GE Healthcare).

Establishment of an assay allowing to screen for inhibitors of EXD2

The screen entailed the use of the“fork like structure” having a reporter fluorescent moiety, 6-FAM (excitation max. = 494 nm, emission max. = 519 nm) on the 3' end of the“template strand”, and the dark quenching group, Iowa Black FQ (absorption max. = 531 nm), on the 5' end of the“nascent” strand (Figure 1 ). The resection reaction was carried out as outlined above (in vitro nuclease assay). Fluorescence was measured using a SpectraMax M5 instrument (Molecular Devices) (Ex 494 nm, Em 519 nm, Cutoff 515 nm).

Test compounds:

ATA; Aurintricarboxylic acid

2-(N-Morpholino)ethanesulfonic acid sodium salt

Claims

1. An EXD2 inhibitor or a pharmaceutical composition thereof for use in a method of treatment of cancer in an individual, wherein the cancer comprises cells deficient in homologous recombination dependent double strand break repair (HRD).

2. The EXD2 inhibitor or pharmaceutical composition thereof for use according to claim 1 , wherein the cancer comprises one or more cancer cells having a reduced or abrogated ability to repair DNA double stranded breaks by homologous

recombination relative to normal cells.

3. The EXD2 inhibitor or pharmaceutical composition thereof for use according to claim 1 , wherein the cancer cells are deficient due to one or more mutations in a gene, or the absence of or defective expression of a gene encoding a protein selected from the group consisting of homologous recombination factors: BRCA1 , BRCA2, RAD51 , RAD51A, RAD51 B, RAD51 C, RAD51 D, RAD52, PALB2, BARD1 , MRE11 , ATM, ATR, WRN and BLM.

4. The EXD2 inhibitor or pharmaceutical composition thereof for use according to any one of the preceding claims, wherein the cancer cells have a deficiency in BRCA1 or BRCA2 genes.

5. The EXD2 inhibitor or pharmaceutical composition thereof for use according to any one of the preceding claims, wherein cells deficient in HRD are dependent on EXD2 for survival.

6. The EXD2 inhibitor or pharmaceutical composition thereof for use according to any one of the preceding claims, wherein the method of treatment comprises the step of identifying a cancer condition in an individual as deficient in HRD.

7. The EXD2 inhibitor or a pharmaceutical composition thereof for use according to claim 6, wherein the cancer is identified an HRD deficient cancer by:

(i) determining the HR activity of cancer cells from an individual relative to normal cells;

(ii) determining the activity of one or more components of the HR pathway in cancer cells from the individual relative to normal cells’ (iii) determining the presence in cancer cells from the individual of one or more mutations or polymorphisms in a nucleic acid sequence encoding a component of the HR pathway.

8. The EXD2 inhibitor or pharmaceutical composition thereof for use according to any one of the preceding claims, wherein the cancer cells have a BRCA1 or BRCA2 deficient phenotype.

9. The EXD2 inhibitor or pharmaceutical composition thereof for use according to any one of the preceding claims, wherein the cancer cells are resistant to treatment with a PARP inhibitor.

10. The EXD2 inhibitor or pharmaceutical composition thereof for use according to claim 9, wherein the PARP inhibitor is selected from the group consisting of:

olaparib, rucaparib, niraparib and talazoparib.

11. The EXD2 inhibitor or pharmaceutical composition thereof for use according to claim 9 or 10, wherein the resistance to PARP inhibition is the result of the cancer (cancer cells) acquiring mutation in one or more member of the

53BP1/RIF1/REV7/Shieldin protein complex selected from the group consisting of: 53BP1 , RIF1 , REV7, Shieldin 1 , Shieldin 2 and Shieldin 3.

12. The EXD2 inhibitor or pharmaceutical composition thereof for use according to any one of the preceding claims, wherein the EXD2 inhibitor is selected from the group consisting of: a small molecule compound, a peptide/polypeptide (such as an aptamer), a nucleic acid (such as an aptamer, an RNA inhibitory molecule (RNAi), a guide RNA (gRNA) or an antisense oligonucleotide (ASO)), and an antibody (such as an intrabody).

13. The EXD2 inhibitor or pharmaceutical composition thereof for use according to claim 12, wherein said antibody is a monoclonal antibody, an antibody fragment or a polyclonal antibody.

14. The EXD2 inhibitor or pharmaceutical composition thereof for use according to claim 12, wherein said nucleic acid comprises a dsRNA molecule, a RNAi molecule, a miRNA molecule, a ribozyme, a shRNA molecule, an antisense oligonucleotide (ASO), a guide RNA (gRNA) or a siRNA molecule.

15. The EXD2 inhibitor or pharmaceutical composition thereof for use according to any one of the preceding claims, wherein the EXD2 inhibitor is specific for EXD2 and blocks its exonuclease activity, such as by disrupting the metal ion binding site (located within the sequence from amino acids 108 -242), binding to the DNA substrate and/or steric effect/hindrance.

16. The EXD2 inhibitor or pharmaceutical composition thereof for use according to any one of the preceding claims, wherein the cancer is selected from breast, ovarian, prostate, lung, kidney, gastric, colorectal, testicular, head and neck, pancreas, brain, melanoma, bone, oesophageal, bladder, cervix, endometrial or other cancers of tissue organs and cancers of the blood cells such as lymphomas and leukaemia or any of the above cancers with mutations in genes within the HR pathway.

17. The EXD2 inhibitor or pharmaceutical composition thereof for use according to any one of the preceding claims, wherein the cancer is selected from the group consisting of: breast, ovarian, prostate and pancreatic cancer.

18. The EXD2 inhibitor or pharmaceutical composition thereof for use according to any one of the preceding claims, wherein the method of treatment further comprises administering a DNA damaging chemotherapeutic agent such as PARP inhibitors, ATR inhibitors, ATM inhibitors, X and gamma radiation, crosslinking agents (e.g. cisplatin, oxiplatin and their derivatives) or a replication inhibitor (e.g. gemcitabine and derivatives).

19. A method of selecting an individual having a cancer condition for treatment comprising:

20. The method according to claim 19, further comprising determining whether the individual’s cancer comprises cancer cells which are resistant to PARP inhibition and/or deficient in 53BP1/RIF1/REV7/Shieldin protein complex.

21. The method according to claim 19 or 20, wherein the determining of the cancer cells is carried out on a biological sample from the patient.

22. A method of selecting an individual having a cancer condition for treatment comprising:

determining from a biological sample from the individual whether their cancer comprises cells deficient in HR and selecting the individual for treatment with an EXD2 inhibitor if their cancer is identified as being deficient in HR.

23. The method according to claim 21 or 22, wherein the biological sample is a tissue sample or a biological fluid sample, such as a sample comprising: blood, plasma, serum, sputum, needle aspirate, urine or ascites.

24. The method according to any one of claims 16 to 23, wherein the cancer cells are identified as deficient in HR by:

(ii) determining the activity of one or more components of the HR pathway in cancer cells from the individual relative to normal cells; or

25. The method according to claim 24, wherein the component of the HR pathway is selected from the group consisting of: BRCA1 , BRCA2, RAD51 , RAD51A,

RAD51 B, RAD51 C, RAD51 D, RAD52, PALB2, BARD1 , MRE11 , ATM, ATR, WRN and BLM.

26. The method according to any one of claims 19 to 25, wherein the individual is selected for treatment with an EXD2 inhibitor if their cancer is determined to be defective in a component of the HR pathway selected from the group consisting of: BRCA1 , BRCA2, RAD51 , RAD51A, RAD51 B, RAD51 C, RAD51 D, RAD52, PALB2, BARD1 , MRE11 , ATM, ATR, WRN and BLM.

27. The method according to any one of claims 19 to 26, wherein the individual is selected for treatment with an EXD2 inhibitor if their cancer is determined to be BRCA1 or BRCA2 deficient.

28. The method according to any one of claims 19 to 27, wherein the cancer cells are resistant to PARP inhibition.

29. The method according to claim 28, wherein the resistance to PARP inhibition is due to inactivation of the RIF1/53BP1/REV7/Shieldin protein complex.

30. The method according to claim 29, wherein inactivation of the

RIF1/53BP1/REV7/Shieldin protein complex is due to the presence of mutations in one or more member of the 53BP1/RIF1/REV7/Shieldin protein complex selected from the group consisting of: 53BP1 , RIF1 , REV7, Shieldin 1 , Shieldin 2 and Shieldin 3.

31. A method of determining the responsiveness of a subject having a cancer to an EXD2 inhibitor, the method comprising determining whether the cancer comprises cells deficient in FIR, wherein the presence of said deficiency indicates that the subject is likely to be responsive to an EXD2 inhibitor.

32. The method according to claim 31 , wherein determining whether the cancer comprises cells deficient in FIR is determined by the presence of one or more amino acid or nucleic acid mutations in the sequence of member of the FIR pathway.

33. A method of screening for a compound potentially suitable for use in the treatment of cancer deficient in FIR, comprising determining the ability of the compound to inhibit EXD2 protein, wherein if the compound inhibit EXD2 protein it is identified as one that is potentially suitable for use in the treatment of cancer deficient in FIR.

34. A method of screening for a compound suitable for use in the treatment of cancer deficient in FIR, comprising the steps of:

a) obtaining purified or recombinant EXD2;

b) contacting the EXD2 in step (a) with one or more test agents; and

c) selecting those agents that demonstrate a reduction in EXD2 activity.

35. A method according to claim 34, wherein a reduction in activity of EXD2 is measured in vitro by a reduced ability of the EXD2 nuclease to cleave a substrate.