WO2016116602A1

WO2016116602A1 - Treatment of cancer

Info

Publication number: WO2016116602A1
Application number: PCT/EP2016/051345
Authority: WO
Inventors: Keith Mikule; Zebin Wang
Original assignee: Astrazeneca Ab
Priority date: 2015-01-23
Filing date: 2016-01-22
Publication date: 2016-07-28

Abstract

The present invention relates to methods of treating cancer by administering a TIPARP inhibitor to a patient in need thereof. The invention also relates to the use of agents capable of inhibiting TIPARP for the treatment of cancer. The invention also relates to the use of an agent that inhibits the catalytic activity of TIPARP or an agent that disrupts the zinc-finger interaction of TIPARP for the treatment of cancer. The invention also relates to methods of selecting a treatment regimen for a subject with cancer, and in particular selecting treatment regimens comprising the provision of an agent that inhibits TIPARP activity where appropriate. The methods and medical uses are particularly suited to the treatment of lung squamous cell carcinoma.

Description

TREATMENT OF CANCER

The present invention relates to methods of treating cancer in subjects in need thereof. The invention also relates to the use of compounds as defined herein for the treatment of cancer. The use of an agent that inhibits the catalytic activity of TIPARP or an agent that disrupts the zinc-finger interaction of TIPARP for the treatment of cancer. The invention also relates to methods of selecting a treatment regimen for a subject with cancer, and in particular selecting treatment regimens comprising the provision of an agent that inhibits TIPARP activity where appropriate. The methods and medical uses are particularly suited to the treatment of lung squamous cell carcinoma.

INTRODUCTION

The poly(ADP-ribose) polymerases (PARPs) are a 17-member family of enzymes that catalyse the transfer of mono- or poly-ADP-ribose units onto substrate proteins. While the biological activities of some members of the family, such as PARP1 and PARP2, are well understood, many of the other family members are poorly characterized.

The seventeen members of the PARP were identified in the human genome on the basis of homology within their catalytic domains. That said, the activity catalysed is not shared by all family members. As referred to above, some PARP family members are able to catalyse the transfer of mono- ADP-ribose units onto their substrates, while others catalyse the transfer of poly-ADP-ribose units onto substrates. Depending on the units that they cause to be transferred, PARPs may be classified as either MAR-generating PARPs (in the case of those causing mono-ADP ribosylation) or PAR-generating PARPs (in the case of those causing poly- ADP-ribosylation). A further class of catalytic-inactive PARPs have also been identified.

PARP7 also known as TIPARP (2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible PARP), is a mono-ADP-ribosyltransferase. This is in contrast to the best-characterised members of the PARP family; PARP1 and PARP2, both of which are poly-ADP- ribosyltransferases.

PAR-generating PARPs, such as PARP1 , PARP2, tankyrasel , tankyrase2, and vPARP, have all been reported to contribute to the repair of damaged DNA. PAR-generating PARPs are involved in the repair of single strand breaks within DNA through the base excision repair mechanism, establishing a negatively charged PAR chain at the site of the break which recruits enzymes that then repair the DNA. Base excision repair is the main mechanism by which single strand breaks, which are the most frequent form of damage sustained by DNA, are repaired. In contrast, double stranded breaks within the DNA tend to be repaired via homologous recombination.

Redundancy in the DNA repair system means that a reduction in PARP1 or PARP2 activity (and hence a reduction in repair of single strand DNA breaks) is seldom lethal. Unrepaired single stranded breaks lead to the formation of double stranded breaks at replication forks, however, these can then be repaired by the homologous recombination pathway.

However, studies have shown that if both the base excision repair and homologous recombination mechanisms within the same cell are defective, then this failure of the two repair mechanisms leads to cell death.

Cancer cells undergo division at a much faster rate than non-cancerous cells within the body. As a consequence they acquire DNA damage more frequently than other cells, and so are dependent upon repair of such damage for their survival. Certain types of cancer, such as those including mutations of BRCA-1 or BRCA-2, are known to have deficient homologous recombination. Thus inhibition of base excision repair in such cancers will cause cell death. This has led to considerable interest in the use of inhibitors of PAR-generating PARPs, and especially inhibitors of PARP1 and PARP2, in the treatment of cancers that have a deficiency in homologous recombination.

It is the ability of PAR-generating PARPs to cause poly-ADP ribosylation, and thus generate a negatively charged PAR chain at the site of single stranded DNA damage, that underpins the involvement in the DNA repair mechanism. The skilled person will recognise that the use of PARP inhibitors, such as inhibitors of PARP1 or PARP2, will only be expected to cause death of cancer cells that have a homologous recombination deficiency, since cells with functional homologous recombination apparatus will be expected to compensate for the loss of single strand DNA break repair. SUMMARY OF THE INVENTION

In a first aspect the invention provides, a method of treating cancer in a subject in need thereof, the method comprising providing the subject with a therapeutically effective amount of an agent that inhibits TIPARP activity.

In a second aspect the invention provides an agent that inhibits the catalytic activity of TIPARP or an agent that disrupts the zinc-finger interaction of TIPARP for use in the treatment of cancer.

The cancer to be treated by a method of the first aspect of the invention or a medical use of the second aspect of the invention may be lung squamous cell carcinoma.

In a third aspect the invention provides a method of selecting a treatment regimen for a subject with cancer, the method comprising:

• assaying a sample representative of gene expression within the cancer of the subject to determine the amount of a target molecule representative of expression of TIPARP present;

• comparing the determined amount with a reference value; and

• selecting a treatment regimen comprising provision of an agent that inhibits TIPARP activity in the event that the determined amount is larger than the reference value.

In a fourth aspect the invention provides a method of selecting a treatment regimen for a subject with cancer, the method comprising:

• assaying a sample representative of gene expression within the cancer of the subject to determine the amount of a target molecule representative of expression of EGFR present;

• comparing the determined amount with a reference value; and

selecting a treatment regimen comprising provision of an agent that inhibits TIPARP activity in the event that the determined amount is larger than the reference value.

The methods of the third and fourth aspects of the invention may optionally further comprise providing an agent that inhibits TIPARP activity to the subject in the event that the determined amount of the target molecule is larger than the reference value. In a fifth aspect the invention provides a method of screening an agent for anti-cancer activity, the method comprising assessing the ability of the agent to inhibit TIPARP activity, wherein an ability to inhibit TIPARP activity indicates that the agent possesses anti-cancer activity.

DESCRIPTION OF THE DRAWINGS

Figure 1 sets out results illustrating that TIPARP is involved in RTK/Ras/MAPK signaling activation. Fig.1 A, Percentage of AP-1 reporter inhibition by different gene silencing. AP1 -bla- ME180 cells were silenced for 72 hours with two or three different oligos for each gene in the presence of 0.5% FBS. Cells were then stimulated with 10ng/ml EGF for 5h. Each bar represents the average of quadruplicate samples (n=4). Percentage of inhibition was normalized by mock transfected samples before and after EGF stimulation. Beta-lactamase is recorded using LiveBlazer FRET B/G substrate. Beta-lactamase silencing is used as a negative control. Fig. 1 B, Cell viability following 72 hours TIPARP silencing with different oligos measured by CellTiter-Glo in NCI H596 cell(blue) and NCI-H647 cell(red). Fig. 1 C, TIPARP mRNA expression following 72 hours silencing. Fig. 1 D, Relative mRNA expression of PARP family members at 5 hours after EGF stimulation in AP1 -bla-ME180 cells.

Figure 2 sets out results illustrating that TIPARP gain-of-function enhances anchorage- independent growth. Fig. 2A, Distribution of TIPARP mRNA expression in patients categorized by copy number. -1 : heterozygous deletion; 0: diploid; 1 : low-level gain 2: high-level amplification. Fig. 2B, Quantification of colony numbers. Colony numbers were counted from triplet using GelCount System (Oxford Optronix). In NCI-H520 cells, colonies with diameter above 150μΜ were counted

Figure 3 shows data illustrating the involvement of catalytic and zinc-finger domain in the regulation of EGFR. Fig. 3A, TIPARP immunoblot of enriched parsylated proteins from NCI- H647 cell lines lysates stably expressing control, TIPARP, TIPARP H532A and TIPARP Y564A mutants after macrodomain pull-down. GST expression is used as loading control. Fig. 3B, Quantification of colony numbers. Fig. 3C, Immunoblot of EGFR, EGFR-pY1068, and TIPARP in NCI-H647 cell lines stably expressing control, wildtype and mutant TIPARP. Fig. 3D, Protein expression of NCI-H2170 control and TIPARP overexpressing cells following EGF stimulation. Lysates were collected at 0, 5 and 20 mins after 10ng/ml EGF stimulation following 48h serum starvation. Fig. 3E. Immunoblot of TIPARP and EGFR in stable cell lines. ZFM: zinc finger domain cysteine to alanine mutant; ZFDel: zinc finger domain deletion mutant. Figure 4 illustrates that compound 1 is a competitive inhibitor of TIPARP. Fig. 4A. Chemical structure of compound 1 . Fig. 4B. Protein expression of EGFR, EGFR pY1068 in NCI-H647 doxycycline inducible cell lines following 72h treatment of doxycycline w/o compound 1 at indicated concentration. Fig. 4C. Quantification of soft agar colonies of NCI-H2170 cells stably expressing vector or TIPARP, continuously treated with DMSO or compound 1 . Fig. 4D. Quantification of soft agar growth of PC9 cells continuously treated with DMSO, compound 1 , gefitinib or olaparib at 50nM, 10OnM and 500nM after two weeks from initial seeding. Fig. 4E. Quantification of soft agar colonies of NCI-H520 and NCI-H226 continuously treated with DMSO or compound 1 at 50nM, 10OnM and 500nM.

DETAILED DESCRIPTION OF THE INVENTION

Evidence presented in this specification indicates that the methods of treatment or medical uses of the invention may be particularly useful in the treatment of lung squamous cell carcinoma. Lung squamous cell carcinoma has long lacked suitable treatments, since it does not exhibit the mutations generally targeted by cancer therapies.

Many currently available treatments for lung cancer, such as non-small cell lung cancer, target the activating mutations in the epidermal growth factor receptor (EGFR). Such cancers are generally susceptible to treatment with tyrosine kinase inhibitors. Other cancer treatments, target cancers, such as breast cancers associated with BRCA-1 or BRCA-2 mutations, in which defective homologous recombination renders the cancer susceptible to PARP inhibition (as discussed in the introduction).

The inventors' finding that agents that inhibit TIPARP activity can be used in the treatment of cancer, such as lung squamous cell carcinoma, is surprising in light of what is known of the role of PARPs in DNA repair, and PARP inhibitors in cancer therapy. TIPARP, which is a MAR generating PARP, rather than as PAR generating PARP has not previously been associated with a role in DNA repair, and so inhibition of TIPARP would not be expected to prevent the repair of single strand DNA breaks. Similarly, lung squamous cell carcinoma is not generally associated with defective homologous recombination, and so even if treated with agents that inhibit PAR-generating PARPs, it would be expected that homologous recombination on the part of cells of this cancer would prevent lethality by repairing DNA once double stranded breaks were formed. In view of the above, without the data provided by the inventors, there would be no reason for the skilled person to expect that agents that inhibit TIPARP activity would be able to effective increase death of lung squamous cell carcinoma cells, and thereby treat this disease.

Certain terms used in the present disclosure will now be further defined, in order to assist the understanding of the present invention.

Definitions

"Cancer"

The present disclosure contains many references to "cancer" in the context of the methods of treatment, medical uses, and methods of selecting a treatment regimen of the invention. Cancer refers to a number of disorders caused by uncontrolled division of abnormal cells within the body. Cancers may be classified with reference to a number of different factors, including the type of cells involved, the type of tissue involved, the body site or genotype.

As referred to above, lung squamous cell carcinoma represents a particularly suitable cancer for treatment in accordance with the various aspects and embodiments of the invention. However, except for where the context requires otherwise, the cancers that may be treated by the methods of treatment, medical uses, or by selected treatment regimens should be construed more broadly than being limited to this particular form of cancer.

Merely by way of example, a suitable cancer for treatment by the methods or uses of the invention, or by a treatment regimen selected by a method of the invention, may be selected from the group consisting of: a cancer associated with elevated expression of TIPARP; a cancer associated with elevated expression of EGFR; a cancer in which EGFR is wild type; and a cancer harbouring activating mutations, such as in the EGFR tyrosine kinase domain.

Merely by way of example, when seeking to identify whether a cancer of interest does, or does not, have elevated expression of TIPARP or EGFR, expression of the selected protein in cancerous tissue can be compared to the expression of that same protein in non-cancerous tissues, such as adjacent non-cancerous tissues. This level of expression of TIPARP or EGFR in non-cancerous tissue thus provides a base line for determining whether the cancerous tissue has elevated expression of the protein in question. Expression of these proteins may be investigated with reference to a suitable target molecule representative of such expression, as discussed further elsewhere in the specification.

In a suitable embodiment a cancer for treatment by the methods or uses of the invention, or by a treatment regimen selected by a method of the invention, may be a cancer that is not associated with a homologous recombination deficiency. The cancer may be a cancer that is wild type for BRCA-1 and BRCA-2.

In a suitable embodiment a cancer for treatment by the methods or uses of the invention, or by a treatment regimen selected by a method of the invention, may be selected from the group consisting of: lung cancer, such as lung squamous cell cancer; and cervical cancer.

A suitable cancer for treatment by the methods or uses of the invention may be a squamous cell carcinoma. Suitably such as a squamous cell carcinoma may be selected from the group consisting of: lung squamous cell carcinoma; head and neck squamous cell carcinoma; and cervical squamous cell carcinoma.

The methods and medical uses of the invention may be employed in the treatment of ovarian serous cystadenocarcinoma.

A suitable cancer for treatment by the methods or uses of the invention, or by a treatment regimen selected by a method of the invention, may be a cancer other than a cancer in the group consisting of: breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer.

"Treatment of cancer"

It is to be appreciated that references to "treating" or "treatment" include prophylaxis as well as the alleviation of established symptoms of a condition. "Treating" or "treatment" of a state, disorder or condition therefore includes: (1 ) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms. "An agent that inhibits TIPARP activity"

An agent that inhibits TIPARP activity, for the purposes of the present invention, should be taken as encompassing any agent that is able to partially or completely reduce TIPARP activity. Suitably an agent that inhibits TIPARP activity may achieve such a partial or complete reduction in TIPARP activity when provided to a subject. Alternatively or additionally, a suitable agent that inhibits TIPARP activity may is one that is able to reduce partially or completely TIPARP activity as assessed in vitro. Suitably TIPARP activity may be reduced generally within the subject, or may be reduced at localised sites, such as sites of cancer. In embodiments in which it is desired to reduced TIPARP activity at a localised site, a suitable agent that inhibits TIPARP may be provided to the requisite site (such as a site of cancer to be treated).

In a suitable embodiment, the agent is one that has been identified as a TIPARP inhibitor or is known to inhibit TIPARP.

In a further suitable embodiment, the agent is a selective TIPARP inhibitor, such as an agent known to selectively inhibit TIPARP. Various selective nucleic acid inhibitors of TIPARP activity, such as suitable siRNA molecules are discussed below.

In a suitable embodiment, an agent that inhibits TIPARP activity may be an agent that decreases expression of TIPARP. In a suitable embodiment, an agent that inhibits TIPARP activity may be an agent that decreases catalytic activity of TIPARP. In a suitable embodiment, an agent that inhibits TIPARP activity may be an agent that disrupts the zinc- finger interaction of TIPARP.

Agents that decrease expression of TIPARP may include nucleic acid agents. Merely by way of example, a suitable embodiment of an agent that decreases expression of TIPARP may be selected from the group consisting of: agents for use in RNA interference (RNAi), including small interfering RNA (siRNA) molecules that inhibit TIPARP expression; antisense oligonucleotides that inhibit TIPARP expression; and ribozymes that inhibit TIPARP expression. Suitable antisense oligonucleotides may be designed with reference to the previously published sequences of nucleic acids, such as mRNA, that encode TIPARP. Suitable examples of siRNA molecules that may be used as agents that decrease expression of TIPARP, and which may be employed in the methods or uses of the invention, may be commercially available siRNA molecules that inhibit TIPARP expression. Suitable examples of such commercially available siRNA molecules include those available from Life Technologies. Merely by way of example, suitable siRNA molecules may be selected from the group consisting of commercially available siRNA molecules sold by Life Technologies under the catalogue numbers: S24856 (AAUCGAAUGACAGACUCGGga - SEQ ID NO:1 ); S24857 (UCAGUACUCAGCUUAUCACtg - SEQ ID NO:2); and S25858 (AGCAGUAUAAAACAGGAGCgg - SEQ ID NO:3).

In a suitable embodiment an agent that decreases catalytic activity of TIPARP is a compound, such as compound 1 , as defined herein. Compound 1 represents an example of a small molecule compound that is particularly suitable for use in the methods or medical uses of the invention.

Certain classes of agents that inhibit TIPARP activity may be able to inhibit such activity via more than one route. Anti-TIPARP antibodies that inhibit TIPARP activity constitute suitable examples of such a class of agents. Merely by way of example, anti-TIPARP antibodies may be able to inhibit activity of TIPARP by binding to TIPARP in such a way that function of the enzyme's catalytic domain is reduced or prevented. Alternatively, or additionally, anti-TIPARP antibodies may be able to bind to TIPARP such that the interaction of the enzyme's zinc finger domain is disrupted. A further method of action by which anti-TIPARP antibodies may reduce TIPARP activity is by binding to the enzyme in such a way as to promote its clearance from the subject

"A therapeutically effective amount"

A therapeutically effective amount of an agent that inhibits TIPARP activity may be any amount of such an agent that is able to bring about a reduction in TIPARP activity sufficient to cause the death of cancer cells, and thereby provide treatment of cancer. A therapeutically effective amount of an agent that inhibits TIPARP activity may be provided by a single incidence of administration of the agent, or by the administration of the agent in a number of incidences.

The inventors have found that compound 1 is able to inhibit TIPARP activity when present at concentrations in the low nanomolar range. This is far lower than the effective inhibitory concentrations of previously reported agents. Accordingly, in an embodiment of the invention employing compound 1 a suitable therapeutically effect amount of this agent may be an amount capable of giving rise to a cellular concentration of between about 10 and 100nM and preferably to a cellular concentration in the region of 30nM. Means by which suitable doses of a chosen agent providing for such cellular concentrations may be selected will be apparent to the skilled person, and will make reference to factors such as the uptake and clearance of the agent in question within the body of the subject.

It will be appreciated that a nucleic acid agent that inhibit TIPARP activity may be provided to a subject requiring treatment by provision of such agent, or by expression of the agent by the subject's cells. In embodiments of the invention employing such a nucleic acid agent, references to therapeutically effective amounts of the agent of the invention may be interpreted accordingly, either with respect to the amount of such an agent administered to a subject, or the amount of such an agent expressed by the cells of the subject.

"Dosing regimens" and "routes of administration"

The methods of treatment and medical uses of the invention may employ any suitable dosing regimen by which an agent that inhibits TIPARP activity may be provided to a subject such that a therapeutically effective amount is achieved, and cancer cells are killed such that a subject's cancer treated. Administration may be repeated as often as necessary so that treatment is achieved. A clinician with responsibility for care of the subject will be able to determine a sufficient period of time over which the agent should be provided.

The methods of treatment and medical uses of the invention may employ any suitable route of administration by which the agent that inhibits TIPARP activity may be provided to a subject. Merely by way of example, an agent that inhibits TIPARP activity may be provided by one or more routes of administration selected from the group consisting of: oral administration; parenteral administration; administration by injection, including intravenous, subcutaneous, or intramuscular injection; and administration by inhalation.

Routes of administration include, but are not limited to, oral (e.g, by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.

"A method of selecting a treatment regimen for a subject with cancer"

The third or fourth aspects of the invention both provide methods by which an appropriate treatment regimen may be selected for a subject with cancer. The methods are based upon assaying samples representative of gene expression within the cancer of the subject to determine the amount of target molecules present. If the relevant target molecules, discussed further below, are determined to be present in an amount larger than a reference value, then this indicates that the subject will benefit from a cancer treatment regimen comprising administration of an agent that inhibits TIPARP activity.

The skilled person will readily be able to determine suitable reference values with respect to which the amount of the appropriate target molecule 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 assessed on a protein level for example by immunohistochemistry or on a DNA level for example by fluorescence in situ hybridization, or on a RNA level, by quantitative real-time PCR.

Methods in accordance with either the third or fourth aspects of the invention may optionally further comprise providing an agent that inhibits TIPARP activity to the subject in the event that the determined amount of the target molecule is larger than the reference value. Suitable agents that inhibit TIPARP activity may be selected from those referred to in connection with the first or second aspects of the invention.

In embodiments of the methods of the third or fourth aspects of the invention that comprise providing an agent that inhibits TIPARP activity to a subject that identified as able to benefit from such a form of treatment, the provision of the agent may be through any dosing regimen, or in any therapeutically effective amount, suitable to treat the subject's cancer. Guidance may be taken from the considerations set out elsewhere in this specification regarding suitable dosing regimens and therapeutically effective amounts. "A sample representative of gene expression within the cancer of a subject"

The methods of both the third or fourth aspects of the invention involve assaying samples that are representative of gene expression within the cancer of the subject for whom a treatment regimen is to be selected. In a suitable embodiment, such a sample may comprise cells of the cancer, such as in the form of a biopsy sample. Alternatively, in a suitable embodiment a sample may comprise material, such as nucleic acids, proteins, or the like, secreted by or extracted from the cancer. Such material may be present in bodily fluids, such as blood.

"A target molecule representative of expression of TIPARP"

The methods of selecting a treatment regimen of the third aspect of the invention involve assaying a sample, representative of gene expression in the subject in respect of whom the treatment regimen is to be employed, to determine the amount of a target molecule representative of expression of TIPARP that is present in the sample. It will be appreciated that the skilled person may use any of a number of different target molecules of this sort. The following provides some considerations regarding such target molecules, and the selection of an appropriate example to put the methods of the invention into practice.

The target molecule may be any molecule the presence of which is representative of expression of TIPARP. A suitable target molecule may be selected with reference to the nature of the sample that has been obtained from the subject.

For example, in the case of a sample containing biological cells, and particularly cells of the subject's cancer, a suitable target molecule may comprise a nucleic acid. In the case of a sample that is substantially free from cells of the subject's cancer, such as a blood or interstitial fluid sample, a suitable target molecule may comprise a protein. Thus, the target molecule may be selected from the group consisting of: a nucleic acid target molecule; and a protein target molecule.

In a suitable embodiment, a target molecule representative of expression of TIPARP may be a nucleic acid representative of expression of the TIPARP gene. Suitably such a nucleic acid may be mRNA encoding TIPARP (for example, GenBank accession: NM 001 184717.1 .). It will be appreciate that there are many assays by which the presence of such nucleic acids in a sample, may be determined, and, if desired, quantified. Merely by way of example, a suitable method for assaying a sample for the presence of a nucleic acid representative of expression of TIPARP and determining the amount of such a nucleic acid present may employ in situ hybridisation; or reverse transcription PCR (rt-PCR); or RNA sequencing (RNAseq).

A target molecule representative of expression of TIPARP may be the TIPARP protein itself. When embodiments of this sort are to be employed, a suitable sample may be treated in a manner that maintains proteins within the sample. The presence of TIPARP protein in a sample representative of gene expression in the subject may be determined by any suitable assay, such as by immunolabelling or immunoblotting with an antibody that recognises TIPARP. In a suitable embodiment the presence of TIPARP protein within a sample may be determined by assaying for the activity of TIPARP (in particular the catalytic activity of TIPARP) in the sample. In such cases the sample may be treated in a manner that maintains TIPARP catalytic activity within the sample.

In a suitable embodiment a target molecule representative of expression of TIPARP may be an intracellular signalling molecule activated on expression of TIPARP. The inventors have found that expression of TIPARP is associated with activation of the receptor tyrosine kinase (RTK), Ras, MAPK intracellular pathway. Thus forms of these proteins that have been activated as a result of TIPARP expression may represent suitable target molecules for use in embodiments of this aspect of the invention.

In a further suitable embodiment, a target molecule representative of expression of TIPARP may be a gene expression of which is up-regulated by TIPARP expression, or a protein encoded by such a gene. Examples of such genes are disclosed in the table below include EGFR; NDRG1 S330; p21 ; pSmd 1 5 8 S463; Src Family Y416; ERK 1 2; Notch 1 ; NF kappa B p65 S536; Acetyl CoA Carboxylase S79; Catenin beta; p38 MAP kinase; c Raf S259; and Cdk9, such a mRNAs or the proteins encoded by these genes, may be employed as target molecules representative of expression of TIPARP.

Gene symbol Fold change p-value

EGFR 8.4 1 .209E-08

NDRG1 S330 2.8 4.379E-05

P21 2.0 3.207E-06

pSmd 1 5 8 S463 1 .9 4.451 E-05

Src family Y416 1 .8 9.251 E-06

ERK 1 2 1 .6 0.0004468 Notch 1 1 .6 0.0003816

NK kappa B p65 S536 1 .6 5.079E-05

Acetyl CoA Carboxylase 1 .6 4.907E-06

S79

Catenin beta 1 .6 0.0107197

P38 MAP kinase 1 .5 3.419E-05

C Raf S259 1 .5 2.941 E-05

Cdk9 1 .5 0.000138

As demonstrated in the table, and elsewhere in the Examples, the inventors have found that increased expression of TIPARP causes a particularly notable increase in expression of the receptor tyrosine kinase EGFR. Accordingly, EGFR may represent a suitable example of a target molecule representative of expression of TIPARP. In such embodiments, determination that the amount of EGFR in the sample exceeds the reference value indicates that a treatment regimen comprising provision of an agent that inhibits TIPARP activity should be selected.

"A target molecule representative of expression of EGFR"

The methods of the fourth aspect of the invention involve assaying a sample representative of gene expression within the cancer of a subject to determine the amount of a target molecule representative of expression of EGFR that is present. In the event that the determined amount is larger than a reference value, a treatment regimen comprising provision of an agent that inhibits TIPARP activity should be employed.

References in the methods of the invention to target molecules representative of expression of EGFR should be interpreted in much the same manner as for the target molecules indicative of TIPARP referred to above.

Thus, in a suitable embodiment a target molecule representative of expression of EGFR may be a nucleic acid representative of expression of the gene encoding EGFR. Merely by way of example, an example of a suitable nucleic acid may comprise mRNA encoding EGFR (see for example, GenBank accession: NM 005228.3). The presence of a nucleic acid, such a nucleic acid encoding EGFR, in a sample may be determined, or quantified, by the methods set out above (e.g. by in situ hybridisation; or by rt-PCR). Optional modifications of the methods of treatment or medical uses of the invention

The methods of treatment of the first aspect of the invention, or medical uses of the second aspect of the invention may also be modified to incorporate steps of assessing a sample from a subject to determine whether or not indicia are present that indicate that the subject has cancer that would benefit from such methods of treatment or medical uses.

For example, a method of treatment of the invention may involve further additional steps as follows:

• assaying a sample representative of gene expression within the cancer of the subject to determine the amount of a target molecule representative of TIPARP and/or EGFR present;

• comparing the determined amount with a reference value; and

• practising the method of treatment only if the determined amount is larger than the reference value.

Similarly, a medical use of the invention may provide the use of an agent that inhibits TIPARP activity, such as compound 1 , for use in the treatment of cancer in a subject selected by a method comprising:

• comparing the determined amount with a reference value; and

• selecting the subject to receive the agent only if the determined amount is larger than the reference value.

Embodiments of the methods of treatment or medical uses of the invention embodying these optional modifications may provide advantages in terms of their ability to stratify subjects that will benefit from such methods of treatment or medical uses.

Assays for inhibition of TIPARP activity

As referred to above, the fifth aspect the invention provides a method of screening an agent for anti-cancer activity, in which the ability of the agent to inhibit TIPARP activity is assessed. In the case that the agent is able to inhibit TIPARP activity, then this indicates that the agent possesses anti-cancer activity.

It will be appreciated that such assays may also be used in the identification of suitable agents which inhibit TIPARP activity, and which may therefore be employed in the methods of treatment or medical uses of the invention.

The inventors have determined that suitable assays for the assessment of the ability to inhibit TIPARP activity may be based upon the tendency of such TIPARP inhibitors to cause a change in the localization pattern of TIPARP within cells. In particular, inhibition of TIPARP activity may be indicated by the ability of an agent to cause a change from a pattern of localization in which the distribution of TIPARP is primarily punctate and/or perinuclear, to a pattern of diffuse localization within the nucleus.

Suitably such assays may use cells that have been modified such that they over-express TIPARP. Such modifications may utilise inducible over-expression, such as over-expression induced by doxycycline. Suitably cells may be modified in this manner by introduction of a doxycycline-inducible lentiviral transduction expression system into the cells. Suitable cells include NCI H647 cells.

Thus in a suitable embodiment, an assay that may be used in screening an agent for the ability to inhibit TIPARP activity may comprise:

• providing the agent to cells that express TIPARP; and

• assessing whether the agent causes a change of localization of TIPARP, such that the diffuse nuclear localization TIPARP is increased;

• wherein such an increase in diffuse nuclear localization of TIPARP indicates that the agent possesses the ability to inhibit TIPARP activity.

Furthermore, it will be appreciated that, an assay that may be used in screening an agent for anti-cancer activity may comprise:

• providing the agent to cells that express TIPARP; and

• wherein such an increase in diffuse nuclear localization of TIPARP indicates that the agent possesses anti-cancer activity. Suitably such assays of the sort described above may involve a step of inducing expression of TIPARP in the cells prior to provision of the agent.

Suitably localization of TIPARP may be achieved by immunolabelling, such as immunofluorescent labelling.

Examples of such assays, and the results that may be obtained from such assays, are set out in Examples 1 .9 (Materials and Methods) and 2.4 (Results).

Alternatively, or additionally, a suitable assay able to determine whether or not an agent of interest is able to inhibit TIPARP activity may be based upon the ability of recombinant TIPARP protein to parsylate histones in cell-free in vitro assays. Active TIPARP causes such parsylation in the presence of nicotinamide adenine dinucleotide (NAD). Accordingly, the ability of an agent to inhibit TIPARP activity may be indicated by the ability of this agent to reduce the degree of histone parsylation occurring.

Assays of this type have been used in the study described in 2.5 of the Examples that confirmed the ability of certain compounds to inhibit TIPARP activity.

The invention will now be further described with reference to the following Examples, and the drawings described above.

EXAMPLES

1 Materials and Methods 1.1 Cell culture

NCI-H2170, NCI-H647, NCI-H596, NCI-H520, NCI-H226 and PC9 cells were cultured in RPMI-1640. HEK-293T/17 and A549 were cultured in DMEM. All the medium was supplemented with 10% FBS, 2mmol/l of L-glutamine, 100U/ml of penicillin and 100ug/ml of streptomycin. AP1 -bla-ME180 was cultured in DMEM supplemented with 10% dialyzed FBS, 0.1 mM NEAA, 25mM HEPES pH 7.4, 100U/ml of penicillin, 100ug/ml of streptomycin and 5ug/ml of blasticidin. A549 was obtained from TCU, AP1 -bla-ME180 was obtained from Life Technologies and the other cells were obtained from ATCC.

1.2 Plasmids and generation of stable cell lines pQCXIP-TIPARP vector was constructed by amplifying TIPARP cDNA fragment with Agel and Xhol ends from cDNA (Origene SC107185) and cloning into pCR Blunt II (Invitrogen). The TIPARP fragment was released from pCRBIuntll TIPARP as an Agel/ Xhol fragment and ligated into pQCXIP (Clontech) digested with Agel and Xhol. The TIPARP mutants (H532A, Y564A, and ZnM (C243A, C251 A, C257A)) were produced via site-directed mutagenesis (QuickChange II XL, Agilent) and subcloned into pQCXIP as for the wild-type versions. pQCXIP-TIPARP-ZnDel was made by deleting amino acids 237 to 264 from the full length construct. pTRIPZ-TIPARP-CO was made by inserting codon-optimized TIPARP (GeneArt) into pTRIZP as an Agel/ EcoRI fragment. pQCXIP-RFP was made by inserting DsRed into pQCXIP as a Notl/ BamHI fragment.pET28b(+)-N-GST-mAf1521 and pET28b(+)-N-GST- mAf1521 G42E were made by ligating sequence of the macrodomain of Archaeoglobus fulgidus DSM 4304 (NC_000917.1 ) into pET28b(+) vector. All constructs were sequenced for verification. Retrovirus and lentivirus were generated by collecting and filtering viral supernatant at 48h and 72h following transfection of viral constructs and packaging plasmid into HEK293/T17 cells. Cells were transduced with viral supernatant with 8ug/ml polybrene by spin-inoculation and selected with puromycin.

1.3 siRNAs and gene silencing

Silencer-select siRNA oligonucleotides specific for human TIPARP were used to silence TIPARP gene expression. The catalog numbers for TIPARP siRNA are S24856, s24857 and S24858 and the catalog number for negative control is 4390843 (Life Technologies). 5nM of each siRNA was transfected using RNAiMax transfection reagent to achieve gene knock down following the manufacturer's protocol (Life Technologies).

1.4 Immunoblots

Cell lysates were prepared by using Triton X-100 lysis buffer consisting of 1 % Triton X-100, 20mM HEPEs pH 7.4, 150mM NaCI, 1 mM EDTA, 1 mM EGTA 10% glycerol, 2mM NaF,1 .5mM MgCI₂, 0.5M Na₃V0₄, freshly added phosphatase inhibitor cocktails (Sigma-Aldrich) and protease inhibitor cocktail (Roche). Protein concentration was measured using BCA protein assay kit (ThermoFisher). The following antibodies were used : TIPARP(Abcam ab84664), total EGFR (CST 2232), EGFR-pY1045 (CST 2237), EGFR-pY1 148 (CST 4404), EGFR- pT669 (CST 3056), EGFR-pY992 (CST 2235), EGFR-pY845 (CST 2231 ), EGFR-pY1068 (CST 2234), ERK-pT202/pY204 (CST 4370), FAK-pY925(CST 3284), total FAK(CST 3285), Alpha-tubulin (Sigma T6074). Horseradish peroxidase-conjugated secondary antibodies were used to amplify the signal from primary antibody (CST).

1.5 RNA analysis

RNA was isolated using Qiagen RNeasy Mini Kit (Qiagen). Samples were subjected to on- column Dnase treatment to remove genomic DNA. cDNA was generated using Biorad iScript cDNA synthesis kit (Biorad) following the manufacturer's protocol. 500ng RNA was used to generate 20ul cDNA. Quantification of mRNA was achieved by doing real-time qRT-PCR on ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) in 384-well optical reaction plates using 20ul final volume. The following Taqman probes were used: TIPARP (Hs00296054 ml ), EGFR(Hs01076078 ml ), GAPDH(Hs03929097_g1 ). Relative mRNA expression were quantified by normalizing to GAPDH. Genome-wide gene expression profiling was performed using the Affymetrix Human Genome U133 Plus 2.0 chip and Gene Set Enrichment Analysis (GSEA) was performed using the GSEA v2.0.14 software

1.6 Soft agar assay

2x10⁴ cells were counted and plated in 12-well plates in 0.35% agarose on a 0.7% agarose bed in triplicate. Colonies were stained with P-iodonitrotetrazolium violet two to three weeks after initial plating. The number of colonies was determined using the GelCount System (Oxford Optronix).

1.7 Statistical Analysis

Statistical significance was calculated by the Student's t test (two tailed) using GraphPad Prism 5.0. Statistically significant changes were indicated with asterisks (^* p<0.05, ^** p<0.001 ).

1.8 Chemical Synthesis Compound 1 was synthesized according to the procedure described previously in US patent 7,449,464.

1.9 Screening for TIPARP Inhibitors

NCI H647 pTRIPZ-PARP7 CO cells, in which expression of TIPARP is controlled by a doxycycline-inducible lentiviral transduction expression system, were created as discussed above. The cells were plated on day 0. On day 1 , cells were treated with either DMSO (negative control), ^g/ml doxycycline (positive control) or ^g/ml doxycycline and one of the following four compounds:

Compound 1 : 6-[4-[3-[(4-oxo-3H-phthalazin-1 -yl)methyl]benzoyl]piperazin-1 -yl]pyridine-3- carbonitrile;

Olaparib: 4-[[3-[4-(cyclopropanecarbonyl)piperazine-1 -carbonyl]-4-fluoro-phenyl]methyl]-2H- phthalazin-1 -one;

Control Compound 1 : 4-[[3-[(6S)-3-(1 ,1 -difluoroethvl)-6-methyl-6,8-dihvdro-5H-

[1 ,2,4]triazolo[4,3-a]pyrazine-7-carbonyl]phenyl]-difluoro-methyl]-2H-phthalazin-1 -one; and

Control Compound 2:5-methvl-4-(3-(3-(trifluoromethyl)-5,6,7,8-tetrahvdro-[1 ,2,41triazolo[4,3- a]pyrazine-7-carbonyl)phenoxy)phthalazin-1 (2H)-one

The cells were then incubated for 3 days. On day 4, cells were fixed with 4% paraformaldehyde for 5 minutes and permealized with ice cold 1 :1 methanol: acetone for 5 minutes. Following that cells were blocked with 3% BSA in TNT buffer for 1 hour at room temperature and incubated with the primary antibody overnight. On day 5, cells were washed and incubated with a secondary antibody diluted 1 :500, for 1 hour and stained with Hoechst dye 33342 for 30 minutes. Images were collected using ImageXpress.

2 Results

2.1 TIPARP is involved in RTK/Ras/MAPK signaling activation

Bioinformatic analysis of the Cancer Cell Line Encyclopaedia Database (Broad Institute, version used - CCLE_Expression_Entrez_2012-09-29.gct) revealed a statistically significant positive association between TIPARP expression and "Rasness" signature score (r=0.4971 , p<0.0001 , n=1037), a composite score that quantitatively depicting the activation status of Ras signaling pathway(Loboda, et al. BMC Medical Genomics 3:26, 2010). The correlation suggested a potential interaction between TIPARP and Ras signaling. We then evaluated the impact of TIPARP knockdown on RTK/RAS/MAPK signalling directly utilizing CellSensor AP- 1 reporter system. Under undisturbed conditions, activation of RTK/RAS/MAPK upon EGF stimulation leads to robust elevation of AP-1 transcriptional activity. TIPARP was efficiently silenced by more than 90% using three different small-interfering oligonucleotides (siRNAs - SEQ ID NOs:1 -3) (Fig1 E), and all of the three siRNAs against TIPARP were able to inhibit AP- 1 reporter activation. The most efficient knockdown was able to inhibit the activation by up to 70%, which achieved the same level of inhibition by EGFR or MAP2K1 (MEK1 ) silencing (Fig1 A). The inventors evaluated whether TIPARP knockdown could impact cell growth of two lung cancer cell lines with relatively high TIPARP expression and consistently observed reduced cell viability following efficient TIPARP silencing (Fig1 B-C). Similar results were also observed in additional lung and breast cancer cell lines, following a longer period of growth analysis.

Interestingly, EGF-stimulation triggered robust and selective upregulation of TIPARP mRNA expression among all the PARP family enzymes, suggesting a key role for TIPARP in mediating cellular responses to growth stimuli, which differentiates its function from the other PARPs (Figl D. Previous studies demonstrated that TIPARP expression can be potently stimulated by environmental toxin TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) (Ma et al., Biochemical and Biophysical Research Communications 289:499-506, 2001 ; MacPherson et al., Nucleic Acids Research 47:1604-1621 , 2013), our results identified that growth factor stimulation constitutes a second physiological mechanism for upregulation of TIPARP expression.

2.2 TIPARP is amplified in cancers and its overexpression promotes anchorage- independent growth

By analyzing the genomic profile of TIPARP available in ONCOMINE datasets with ONCOMINE POWERTOOLS (Life Technologies), we noticed that the chromosomal 3q25 region containing TIPARP is amplified in multiple tumour types including lung squamous cell carcinoma (LUSC), cervical squamous (CS), ovarian serous cystadenocarcinoma (OV), and head and neck squamous cell carcinoma (HNSC). Approximate 6.3% LUSC, 4.0% CS, 2.3% OV, and 1 .7% HNSC patient samples had at least four copies of the TIPARP gene, see table below which lists count and frequency of patient samples with more than four copies of TIPARP:

In TCGA LUSC samples, amplification of TIPARP translates to higher average mRNA expression in amplified samples (Fig2A) (Cancer Genome Atlas Research, Nature 489:519- 525, 2012).

To investigate the effect of TIPARP overexpression, we examined the consequence of TIPARP gain-of-function by transducing multiple lung squamous cell lines with low TIPARP expression levels with retrovirus encoding TIPARP to mimic the effect of TIPARP amplification and or activation in tumours. Following stable TIPARP integration, TIPARP was expressed efficiently in NCI-H2170, NCI-H647 and NCI-H520 cells. The inventors discovered that TIPARP overexpression significantly increased the colony incidence in NCI-H2170 and NCI- H647 cells (Fig2B), neither of which at basal level display robust growth under anchorage- independent conditions. In NCI-H520 cells, which grow aggressively in soft agar, TIPARP overexpression stimulates the formation of large (colony diameter>150μm) colonies (Fig2B), indicating it is able to enhance proliferation and or survival of the NCI-H520 cells in these conditions. Together these results suggest that TIPARP upregulation confers growth advantage under anchorage-independent conditions.

To understand the molecular mechanisms that may account for enhanced tumorigenicity, we took a high-throughput approach to evaluate changes to the gene expression profile of cells with either TIPARP overexpression or silencing. Genome-wide transcriptome changes were captured using Affymetrix Human Genome U133 Plus 2.0 Arrays covering about 47,000 transcripts. Gene set enrichment analysis identified that numerous EGF/EGFR signature gene sets were highly enriched in TIPARP overexpressing cells, with FDR<0.25, NES>1 .2 Nominal p-value <0.05 as cut-off as listed below: Gene set Size ES NES NOM FDR p-val q-val

AMIT_EGF_RESPONSE_120_HELA 62 0.61 2.04 0 0.001

AMIT_EGF_RESPONSE_60_HELA 39 0.64 1 .96 0 0.003

K0BAYAHI_EGFR_SIGNALING_6HR-DN 17 0.75 1 .93 0 0.004

AMIT_EGF_RESPONSE_480_HELA 142 0.5 1.85 0 0.009

AMIT_EGF_RESPONSE_120_MCF10A 40 0.58 1.78 0.002 0.02

NAGASHIMA_EGF_SIGNALING_UP 52 0.55 1 .78 0.001 0.019

AMIT_EGF_RESPONSE_240_HELA 55 0.48 1.57 0.003 0.08

ZWANG_CLASS_1_TRANSIENTLY_INDUCED_BY_EGF 411 0.37 1 .53 0 0.101

ZWANG_CLASS_2-TRANSIENTLY_INDUCED_BY_EGF 41 0.49 1 .51 0.023 0.111

Z WAN G_C L ASS_3_T R AN S I E NTL Y_l N D U C E D_B Y EG F 189 0.36 1.37 0.013 0.199

Conversely, EGF response gene sets were downregulated by TIPARP knockdown in NCI- H2170 cells, as listed below:

These results support the positive correlation between TIPARP expression and RTK/Ras/MAPK signalling.

2.3 Intact MAR catalytic activity and RNA binding domain are required for TIPARP function

PARPs capable of generating poly ADP-ribose chains contain a conserved H-Y-E triad within the catalytic domain where the presence of histidine and tyrosine are essential for substrate positioning (Hottiger et al., Trends in Biochemical Sciences 35:208-219, 2010). Mono-ADP ribosyltransferases often express leucine, isoleucine or valine instead of glutamate in the triad and lack the polymer transferring and elongation activity which requires the presence of glutamate (Hottiger et al., Trends in Biochemical Sciences 35:208-219, 2010). In the case of TIPARP, it has been shown that both histidine and tyrosine are necessary for it to function as a repressor of AHR signaling. TIPARP H532A and Y564A mutants fail to automodify themselves in the auto-modification assay in vitro when incubated with NAD⁺ (MacPherson et al., Nucleic Acids Research 47:1604-1621 , 2013). However, it is unclear whether the auto- modification occurs intracellular^.

To address this, we used the macrodomain from Af 1521 , which is reported to bind both MAR- and PAR-peptides, as bait to pull-down parsylated proteins from wild-type, or various mutant TIPARP overexpressing cells. Lysates were made from cells stably overexpressing TIPARP mutants predicted to either lack catalytic activity (H532A, Y564A) or have gain-of-function poly-ADP-ribose activity (1631 E). TIPARP was only detectable in macrodomain pull-downs from wild-type and the 1631 E expressing cells, suggesting that H532A and Y564A mutants are catalytically inactive in cells (Fig3A). Interestingly, the 1631 E mutant was more efficiently pulled down than the wild-type variant indicating that PAR activity may have been conferred by this mutation. Compared with wild-type TIPARP overexpressing cells, catalytically-inactive mutants formed fewer colonies under anchorage-independent conditions and had diminished EGFR signaling (Fig3B-D), although slight EGFR induction was observed in comparison compared to control cells. Together, these results demonstrated that the catalytic activity of TIPARP plays an important role in its ability to both regulate EGFR and enhance tumorigenicity.

Given that TIPARP modulation could be occurring at the transcript level, we decided to evaluate whether TIPARP's CCCH zinc finger domain played a functional role in its activity, postulated to allow for RNA interaction. Two zinc finger domain mutants were constructed to address this question. One was a mutant whereby three cysteines within the C3H1 zinc finger domain were converted to alanine, and the second was a deletion mutant that excised the entire zinc finger domain. Elevated EGFR protein expression were absent in cells expressing either of the mutants (Fig3E), raising the possibility that EGFR regulation might happen through a TIPARP-RNA interaction.

2.4 A screening method for potential TIPARP inhibitors

We developed a method for screening potential TIPARP inhibitors as described herein. NCI H647 cells were transformed with a doxycycline inducible lentiviral transduction system that caused inducible exogenous TIPARP expression in the presence of doxycycline. Localisation of exogenous TIPARP was visualised by fluorescent immunolabelling using antibody recognizing exogenous TIPARP Hoechst dye was used to define the nuclear area.

Cells grown without doxycycline showed minimal fluorescent signal. After 72 hour incubation with doxycycline, exogenous TIPARP expression (and consequent immunolabelling) became much more prominent in both the nuclear and perinuclear area of the cells.

Four potential inhibitors were studied (Compound 1 ; the PARP-1 inhibitor Olaparib; and Control Compounds 1 and 2). The effectiveness of each agent in inhibiting TIPARP activity was assessed at two different concentrations (30nM and 1 μΜ). It was observed that Compound 1 reduced TIPARP activity most significantly, as illustrated by the change in distribution of TIPARP that occurred on treatment of cells with this compound. Treatment with Compound 1 (at either 30nM or 1 μΜ) altered the punctate localisation of TIPARP within treated cells to pan-nuclear staining, as compared to those cells treated with Olaparib, Control Compound 1 , or Control Compound 2, which failed to alter the localisation pattern of the TIPARP.

Agents that are putative inhibitors of TIPARP activity may be provided to the cells at various concentrations, and TIPARP expression quantified by immunofluorescence. This allows selection of those agents (such as Compound 1 ) which inhibit TIPARP expression to a desirable extent.

2.5 Identification of novel selective TIPARP inhibitors

This study has provided links between MAR-generating enzymes and key cancer- related functions, which encourages further investigation of clinical utility by targeting this class of enzymes in human malignancies. However, selective small molecule inhibitors capable of blocking mono-ADP ribosyltransferase activity with high potency have not been described so far, which restrict the ability to test this therapeutic strategy preclinically. To address this limitation, a subset of NAD+-mimetic compound libraries were screened in a cell-free parsylation assay using histone as substrate across panel of PARPs for small molecules that have high potency against TIPARP enzymatic activity. A number of compounds having IC50 below 1 μΜ, including Compound 1 , were identified. Compound 1 demonstrates particularly strong activity against TIPARP (IC50 33nM). To our knowledge, this is the first compound ever reported that reaches low nanomolar range activity against mammalian mono-ADP ribosyltransferase TIPARP. The chemical structure of compound 1 is shown (Fig4A). Since catalytic activity is important for TIPARP function and essential for EGFR regulation, we sought to determine whether compound 1 is able to block TIPARP activity and diminish its function intracellular^. To achieve this, we constructed an inducible NCI-H647 cell line where TIPARP expression can be temporally controlled by doxycycline. In this system, we found that compound 1 was able to effectively suppress the elevation of EGFR expression and activity upon TIPARP induction. This result confirmed the dependence of EGFR regulation on TIPARP catalytic activity and demonstrated the ability of compound 1 in inhibiting TIPARP activity (Fig4B). Additionally, continuous exposure of compound 1 blocked the induction of anchorage-independent growth in TIPARP stably overexpressing cells (Fig4C), highlighting the potential of compound 1 to reverse enhanced tumorigenicity caused by TIPARP. The efficacy of compound 1 was further assayed in PC9, a lung cancer cell line driven by mutant EGFR signalling with exonl 9 deletion. As expected, PC9 is highly sensitive to getifinib, a selective EGFR inhibitor. Interestingly, whereas olaparib which is a potent inhibitor of PARP1/2 has little effect on overall growth of PC9 cells in soft agar, compound 1 at low dose (50nM) was able to efficiently block the anchorage-independent growth by about 80%, indicating the dependence of PC9 cells on TIPARP catalytic activity rather than PARP1/2 activity for survival (Fig4D). These results clearly differentiated a role of TIPARP from PARP1/2 in regulating cellular response. Compound 1 was tested in two lung squamous cell lines NCI-H520 and NCI-H226 harbouring wildtype EGFR allele, which are not sensitive to EGFR inhibitors in adherent condition (Ware et al., PloS one. 2010;5(1 1 ):e141 17). However, growth inhibition was seen in NCI-520 cells following EGFR siRNA silencing suggesting that these cells still depends on EGFR signalling for survival (data not shown). Both cell lines grew aggressively in soft agar. We discovered that continuous treatment of compound 1 significantly impaired colony formation of both lines in a dose-dependent manner (Fig4E). Consistent with the finding that TIPARP silencing attenuated EGFR mRNA expression in several non-EGFR mutant cell lines and led to growth inhibition, these results suggested that compound 1 could be of clinical utility in treating EGFR wildtype lung cancers.

Claims

1 . A method of treating cancer in a subject in need thereof, the method comprising providing the subject with a therapeutically effective amount of an agent that inhibits TIPARP activity.

2. A method according to claim 1 , wherein the cancer is lung squamous cell carcinoma.

3. A method according to claim 1 or claim 2, wherein the cancer is selected from the group consisting of: a cancer associated with elevated expression of TIPARP; a cancer associated with elevated expression of EGFR; and a cancer in which EGFR is wild type.

4. A method according to any preceding claim, wherein the cancer is not associated with a homologous recombination deficiency.

5. A method according to claim 4, wherein the cancer is wild type for BRCA-1 and BRCA- 2.

6. A method according to any preceding claim, wherein the cancer is selected from the group consisting of: lung cancer, such as lung squamous cell cancer; and cervical cancer.

7. A method according to any preceding claim, wherein the cancer is a cancer other than breast cancer, ovarian cancer, prostate cancer, or pancreatic cancer.

8. A method according to any preceding claim, wherein the agent that inhibits TIPARP activity is selected from the group consisting of: an agent that decreases expression of TIPARP; an agent that decreases catalytic activity of TIPARP; and an agent that disrupts the zinc-finger interaction of TIPARP.

9. A method according to claim 8, wherein the agent that decreases expression of TIPARP is selected from the group consisting of: agents for use in RNA interference (RNAi), including small interfering RNA (siRNA) molecules that inhibit TIPARP expression; antisense oligonucleotides that inhibit TIPARP expression; and ribozymes that inhibit TIPARP expression.

10. A method according to claim 9, wherein the siRNA molecules that inhibit TIPARP expression are selected from the group consisting of commercially available siRNA molecules sold by Life Technologies under the catalogue numbers: S24856; S24857; and s 25858.

1 1 . A method according to claim 8, wherein the agent that decreases expression of TIPARP is an anti-TIPARP antibody that inhibits TIPARP activity

12. An agent that inhibits the catalytic activity of TIPARP or an agent that disrupts the zinc- finger interaction of TIPARP for use in the treatment of cancer.

13. An agent for use according to claim 12, wherein the agent is 6-[4-[3-[(4-oxo-3H- phthalazin-1 -yl)methyl]benzoyl]piperazin-1 -yl]pyridine-3-carbonitrile (Compound 1 ).

14. An agent for use according to claim 13, wherein the agent is for use in an amount capable of giving rise to a cellular concentration of between about 1 0 and 100 nM.

15. An agent for use according to any of claims 12 to 14, wherein the cancer is lung squamous cell carcinoma.

16. An agent for use according to any of claims 1 2 to 1 5, wherein the cancer is selected from the group consisting of: a cancer associated with elevated expression of TIPARP; a cancer associated with elevated expression of EGFR; and a cancer in which EGFR is wild type.

17. An agent for use according to any of claims 1 2 to 16, wherein the cancer is not associated with a homologous recombination deficiency.

18. An agent for use according to claim 17, wherein the cancer is wild type for BRCA-1 and BRCA-2.

19. An agent for use according to any of claims 1 2 to 1 8, wherein the cancer is selected from the group consisting of: lung cancer, such as lung squamous cell cancer; and cervical cancer.

20. An agent for use according to any of claims 12 to 19, wherein the cancer is a cancer other than breast cancer, ovarian cancer, prostate cancer, or pancreatic cancer.

21 . A method for selecting a treatment regimen for a subject with cancer, the method comprising:

• comparing the determined amount with a reference value; and

22. A method according to claim 21 , wherein the target molecule is selected from the group consisting of: a nucleic acid target molecule; and a protein target molecule.

23. A method according to claim 22, wherein the target molecule is a nucleic acid representative of expression of the TIPARP gene.

24. A method according to claim 23, wherein the target molecule is an mRNA encoding TIPARP.

25. A method according to claim 22, wherein the target molecule is TIPARP protein.

26. A method according to claim 21 , wherein the target molecule is an intracellular signalling molecule activated on expression of TIPARP.

27. A method according to claim 26, wherein the target molecule is a protein that has been activated as a result of TIPARP expression selected from the group consisting of: a receptor tyrosine kinase (RTK); Ras; and MAPK.

28. A method according to claim 21 , wherein the target molecule is a protein or mRNA encoded by any of the group of genes consisting of: EGFR; NDRG1 S330; p21 ; pSmd 1 5 8 S463; Src Family Y416; ERK 1 2; Notch 1 ; NF kappa B p65 S536; Acetyl CoA Carboxylase S79; Catenin beta; p38 MAP kinase; c Raf S259; and Cdk9.

29. A method of selecting a treatment regimen for a subject with cancer, the method comprising:

• comparing the determined amount with a reference value; and

30. A method according to claim 29, wherein the target molecule is selected from the group consisting of: a nucleic acid target molecule; and a protein target molecule.

31 . A method according to claim 30, wherein the target molecule is a nucleic acid representative of expression of the EGFR gene.

32. A method according to claim 31 , wherein the target molecule is an mRNA encoding EGFR.

33. A method according to claim 30, wherein the target molecule is EGFR protein.

34. A method according to any of claims 21 to 33, wherein the cancer is lung squamous cell carcinoma.

35. A method according to any of claims 21 to 34, wherein the cancer is selected from the group consisting of: a cancer associated with elevated expression of TIPARP; a cancer associated with elevated expression of EGFR; and a cancer in which EGFR is wild type.

36. A method according to any of claims 21 to 35, wherein the cancer is not associated with a homologous recombination deficiency.

37. A method according to claim 36, wherein the cancer is wild type for BRCA-1 and BRCA-2.

38. A method according to any of claims 21 to 37, wherein the cancer is selected from the group consisting of: lung cancer, such as lung squamous cell cancer; and cervical cancer.

39. A method according to any of claims 21 to 38, wherein the cancer is a cancer other than breast cancer, ovarian cancer, prostate cancer, or pancreatic cancer.

40. A method according to any of claims 21 to 39, further comprising providing an agent that inhibits TIPARP activity to the subject in the event that the determined amount of the target molecule is larger than the reference value.

41 . A method according to claim 40, wherein the agent that inhibits TIPARP activity is selected from the group consisting of: an agent that decreases expression of TIPARP; an agent that decreases catalytic activity of TIPARP; and an agent that disrupts the zinc-finger interaction of TIPARP.

42. A method according to claim 41 , wherein the agent that decreases expression of TIPARP is selected from the group consisting of: agents for use in RNA interference (RNAi), including small interfering RNA (siRNA) molecules that inhibit TIPARP expression; antisense oligonucleotides that inhibit TIPARP expression; and ribozymes that inhibit TIPARP expression.

43. A method according to claim 42, wherein the siRNA molecules that inhibit TIPARP expression are selected from the group consisting of AAUCGAAUGACAGACUCGGga (SEQ ID NO:1 ); UCAGUACUCAGCUUAUCACtg (SEQ ID NO:2); and AGCAGUAU AAA AC AG G AG Cgg (SEQ ID NO:3).

44. A method according to claim 41 , wherein the agent that decreases expression of TIPARP is an anti-TIPARP antibody that inhibits TIPARP activity.