WO2014013231A1 - Materials and methods for treating pten mutated or deficient cancer - Google Patents

Abstract

Materials and methods are disclosed for treating PTEN mutated or deficient cancer using Nemo-Like kinase (NLK) inhibitors, and to methods of screening for agents for treating PTEN mutated or deficient cancer. Optionally, the PTEN mutated or deficient cancer is also FOXO1 proficient.

Description

Materials and Methods for Treating PTEN Mutated or Deficient

Cancer

Field of the Invention

The present invention relates to materials and methods for treating PTEN mutated or deficient cancer using Nemo-Like kinase (NLK) inhibitors, and to methods of screening for agents for treating PTEN mutated or deficient cancer.

Background of the Invention

Each year, the majority of new cancer drug approvals are directed against existing targets, whereas only two or three compounds are licensed against novel molecules . Rather than suggesting a limiting number of targets, this reflects the difficulty, time and cost involved in the identification and validation of proteins that are crucial to disease pathogenesis. The result is that many key proteins remain undrugged, and as a consequence opportunities to develop novel therapies are lost. This

situation could be improved by using approaches that identify the key molecular targets that underlie the pathways that are associated with disease development. For example, techniques such as gene targeting, in which a gene can be selectively inactivated or knocked-out, can be powerful. However, such approaches are limited by their cost and low throughput. Moreover, it is often the case that the current approaches to cancer treatment group together similar clinical phenotypes regardless of the differing molecular pathologies that underlie them. A consequence of this molecular heterogeneity is that individuals frequently exhibit vast differences to drug

treatments. As such, therapies that target the underlying molecular biology of individual cancers are increasingly becoming an attractive approach.

Brough et al . (Current Opinion in Genetics & Development 2011, 21:34-41) describe how the biological concept of synthetic sickness/lethality (SSL) may be used to identify new therapeutic approaches and targets in models from yeast through to human cells. It is known to target genes that are overexpressed in particular forms of cancer. However, despite these successes, developing drugs that selectively kill cancer cells without harming normal cells remains a considerable challenge. This process is particularly problematic when considering how to target a protein such as a dysfunctional tumour suppressor that is either largely inactive or even completely absent. If one gene in an SSL relationship is a tumour suppressor gene, then its synthetic lethal partner becomes a candidate therapeutic target that could be used in tumour cells with a defined tumour

suppressor gene dysfunction. SSL can occur between genes acting in the same biochemical pathway or in distinct but compensatory pathways .

We have previously identified SSL relationships to suggest that Tankyrase 1 inhibitors may be used for treating BRCA-associated cancers (WO2009/027650 ) and that DNA polymerase POLp inhibitors may be used for treating DNA mismatch repair (MMR) deficient cancers (WO2009/027641 ) . However, it remains a problem in the art in identifying further SSL relationships that may be used to improve the identification of groups of patients having the correct clinical phenotype to benefit from any new or existing cancer therapy.

Summary of the Invention

Broadly, the present invention is based on work concerning PTEN {Phosphatase and tensin homolog) , a tumour suppressor gene commonly defective in human cancer, and which is thus a

potentially important therapeutic target. Targeting tumour suppressor loss-of-function is possible by exploiting the genetic concept of synthetic lethality (SL) . By combining the use of isogenic models of PTEN deficiency with high-throughput RNA interference (RNAi) screening, we have identified Nemo-Like Kinase {NLK) inhibition as being synthetically lethal with PTEN deficiency. This SL is likely mediated by the transcription factor FOXOl (Forkhead box 01), an NLK substrate, as the

selectivity of NLK gene silencing for PTEN deficient cells can be reversed by FOXOl knockdown. In addition, we provide evidence that PTEN defective cells targeted by NLK gene depletion undergo senescence, suggesting that NLK function is critical for the continued proliferation of PTEN deficient cells. Taken together, these data provide new insight into the potential of targeting of NLK to treat a range of tumourigenic conditions characterised by PTEN deficiency. This also suggests that the present invention will be particularly applicable to the treatment of cancer cells that are FOXOl proficient, e.g. in which the activity of FOXOl is preferably at least 50% normal wild-type activity, more

preferably at least 75% wild-type activity and most preferably at least 90% wild-type activity.

Phosphatase and tensin homolog ("PTEN") is a gene that has been identified as a tumor suppressor through the action of its phosphatase product and is mutated in a large number of cancers at high frequency. The protein encoded this gene is a

phosphatidylinositol-3 , , 5-trisphosphate 3-phosphatase . It contains a tensin like domain as well as a catalytic domain similar to that of the dual specificity protein tyrosine

phosphatases. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates . It negatively regulates intracellular levels of phosphatidylinositol-3 , 4 , 5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating AKT/PKB and mTOR signalling pathways. The HUGO Gene Symbol report for PTEN can be found at

http : / /ww . genenames . org/data/hgnc data . hp?hgnc id=9588 , which provides links to the PTEN nucleic acid and amino acid sequences, as well as reference to the homologous murine and rat proteins . The amino acid sequence of human PTEN is set out in SEQ ID NO: 3.

Nemo-Like kinase (NLK) , inhibitors of which can be used in accordance with the present invention for the treatment of PTEN mutated or deficient cancer according to the present invention, is a 527 amino acid. The HUGO Gene Symbol report for NLK can be found at : http : //¾¾'¾ . genenames . org /data,/hgnc data . php?hgnc id=29658 , which provides links to the NLK nucleic acid and amino acid sequences, as well as reference to the homologous murine and rat proteins . A discussion of the properties of NLK and assays for determining its activity are provided in Ishtani et al, MBoC, Volume 22: 266- 277, January 15, 2011. The amino acid sequence of human NLK is set out in SEQ ID NO: 1.

The HUGO gene symbol report for FOXOl can be found at:

http: / /www . genenames . org/data/hgnc data . php?hqnc id=3819, which provides links to the FOXOl nucleic acid and amino acid

sequences. The amino acid sequence of human FOXOl is set out in SEQ ID NO: 2.

According, in a first aspect the present invention provides an inhibitor of a Nemo-Like kinase (NLK) for use in a method of treating an individual having cancer, wherein the cancer is a Phosphatase and Tensin Homolog (PTEN) mutated or deficient cancer .

In a further aspect, the present invention provides the use of a Nemo-Like kinase (NLK) inhibitor in the preparation of a

medicament for the treatment of an individual having a PTEN mutated or deficient cancer.

In a further aspect, the present invention provides a method of treating an individual having a PTEN mutated or deficient cancer, the method comprising administering to the individual a

therapeutically effective amount of a Nemo-Like kinase (NLK) inhibitor .

In a further aspect, the present invention provides a method of screening for agents useful in the treatment of a PTEN mutated or deficient cancer, the method employing first and second cell lines, wherein the first cell line is PTEN and the second cell line is PTEN proficient, the method comprising:

(a) contacting the first and second mammalian cell lines with at least one candidate agent;

(b) determining the amount of cell death in the first and second cell lines; and

(c) selecting a candidate agent which is synthetically lethal in the first cell line.

In a further aspect, the present invention provides a method of screening for agents useful in the treatment of PTEN mutated or deficient cancer, the method comprising:

(a) contacting a Nemo-Like kinase (NLK) with at least one candidate agent;

(b) determining an effect of the at least one candidate agent on an activity of the Nemo-Like kinase (NLK) ; and

(c) selecting a candidate agent that inhibits the activity of the Nemo-Like kinase (NLK) as being useful for the treatment of the PTEN mutated or deficient cancer.

In some aspects, the present invention provides a method which comprises having identified a candidate agent useful for the treatment of a PTEN mutated or deficient cancer according to a method as described herein, the further step of manufacturing the compound in bulk and/or formulating the agent in a pharmaceutical composition .

Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures. However various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Brief Description of the Figures

Figure 1. PTEN synthetic lethality screening

(A) High-throughput screen (HTS) schematic. PTEN proficient and deficient HCT116 cells (Horizon Discovery) were maintained in McCoy's 5A (Invitrogen) supplemented with 10% (v/v) foetal calf serum (FBS), glutamine and antibiotics and reverse transfected with a siRNA library (final siRNA concentration per well, 50 nM) using RNAiMAX (Invitrogen) . The following day media was changed and cells cultured for five subsequent days, at which point cell viability was assessed using CellTiterGlo Luminescent Cell Viability Assay (Promega) . Example heatmaps from luminescence measurements in 96 well plates are shown. For screening, a siGENOME SMARTPool library (Dharmacon) targeting 779 kinases and kinase-related genes .

(B) Results from data analysis of combined triplicate screens, represented as a scatter plot of Normalised Percent Inhibition

(NPI) values from HCT116 P TEN^~/~ and HCT116 PTEN^{+ +} screens. Blue dots corresponded to siPLK effects and red dots corresponded to siCON negative control effects. NPI values below the trend line shown were considered as candidate synthetic lethalities .

(C) Validation of PTEN synthetic lethal hits from the HTS using multiple siRNAs for each gene. Cells were transfected with siRNA as per the HTS and surviving fractions calculated from

CellTiterGlo luminescence measurements five days later. Survivim fraction data from HCT116 PTEN^~/~ and HCT116 P TEN^{+ +} cells transfected with each siRNA are shown.

(D) Western blot showing silencing effects of each siRNA. Cells were transfected with siRNA and cell lysates generated 48 hours later. Cell lysates were western blotted as shown. Whole-cell protein extracts were prepared from cells lysed with RIPA buffer

(Upstate) supplemented with protease inhibitor cocktail tablets (Roche). Protein concentrations were measured using Bio-rad Protein Assay Reagent. For western blot analysis, lysates were electrophoresed on Novex 4-12% gradient bis-tris pre-cast gels

(Invitrogen) and immunoblotted overnight at 4°C with antibodies targeting the following epitopes: NLK (C-20: sc-8210, Santa Cruz Biotechnology), PLK4 (#3258, Cell Signaling), TTK (C-19: sc-540, Santa Cruz Biotechnology) and β-Actin (119: sc-1616, Santa Cruz Biotechnology) . Incubation with primary antibody was followed by incubation with a horseradish peroxidase-conjugated secondary antibody and chemi-luminescent detection of proteins (Amersham Pharmacia, Cardiff, UK) .

Figure 2. Validation of the PTEN/NLK synthetic lethality

(A) Validation of PTEN synthetic lethal hits in additional isogenic models. Isogenic HCT116, DLD1 and HEC1A PTEN proficient and deficient models (Horizon Discovery) were transfected with siRNA as in Figure 1 and surviving fractions calculated five days later. Surviving fraction data from HCT116, HEC1A and DLD1 models is shown. * p value <0.05 Student's t test

(B) Western blot showing expression of each candidate gene in three PTEN isogenic models .

( C ) Cell inhibitory effect of siRNA targeting NLK or PLK4 in a panel of 24 human tumour cell lines (11 PTEN proficient models and 13 PTEN deficient models - see Table 1) .

(D ) Box plots illustrating surviving fraction data for PTEN proficient and deficient groups shown in (C) . p-values were calculated with Student's t-test.

Figure 3. PTEN/NLK synthetic lethality is abrogated by FOXOl silencing

(A) Survival analysis of HCT116 cells transfected with NLK and/or FOXOl siRNA. HCT116 PTEN^~/~ and HCT116 PTEN^{+ +} cells were

transfected with siRNA targeting NLK and FOXOl as shown and surviving fractions determined after five days. The p value (*) was calculated using Student's t test.

(B) Nuclear localisation of FOXOl is enhanced m PTEN deficient cells upon NLK silencing. HCT116 isogenic cells were co- transfected with GFP-tagged FOXOl cDNA (Origene) in addition to control (non targeting) siRNA (siCON) or NLK siRNA. Two days later cells were fixed in 4% (w/v) para-formaldehyde, after whi nuclei were stained with 4 ' , 6-diamidino-2-phenylindole (DAPI; D21490, Invitrogen) . DAPI and GFP signals from cells were captured using an inverted confocal Zeiss LSM710 microscope. Green signal represents GFP-FOXOl and blue signal represents nuclear DAPI staining. Arrows indicate cells with nuclear localisation of FOXOl.

(C) Senescence is increased by NLK siRNA m PTEN deficient cells Bar chart of relative relative senescence levels caused by NLK silencing are shown. HCT116-derived PTEN isogenic cell lines wer reversed transfected with a pool of two validated siRNAs against NLK, as well as siCON pool#2 (Dharmacon) as non-targeting control, using RNAiMAX (Invitrogen). Seven days after

transfection cells were fixed in PBS containing 2% (w/v) formaldehyde and 0.2% (v/v) glutaraldehyde , rinsed and incubated overnight at 37°C in a solution containing X-gal according to manufacturer instructions (Senescence β-Galactosidase Staining Kit, Cell Signaling Technology) . After aspirating staining solution, cells were overlaid with 70% (v/v) glycerol and stored at 4°C until reading absorbance at 595 nm (A₅₉₅ ) in a Victor X5 plate reader (Perkin Elmer) . A₅₉₅ values were normalised to the A₅₉₅ signal in PTEN proficient cells transfected with siCONpool#2 transfected cells to give relative senescence levels in each experimental sample as shown. siRNA targeting SKP2 was used a positive control (17),

(D ) Representative images for β-Galactosidase staining of PTEN deficient cells. Blue staining indicates β-Galactosidase . Detailed Description

Inhibitors

Compounds which may be employed or screened for use as Nemo-Like kinase (NLK) inhibitors in accordance with the present invention for treating PTEN mutated or deficient cancer. Examples of inhibitors may be found by the application of screening

technologies to these targets.

Small molecule inhibitors

By way of example, inhibitors of NLK may be identified by the use of high throughput screening strategies. Accordingly, the present invention extends to the use of small molecule inhibitors found in the screening disclosed herein and to Derivatives which are compounds of similar structure and functionality to the compounds found in the high throughput screen, but with one or more modifications, are expected to have similar physiological effects to these compounds and could therefore also be of use in the treatment of PTEN mutated or deficient cancer. The screening methods of the invention may be used to screen libraries of such derivatives to optimise their activity, if necessary.

Derivatives may be designed, based on a lead compound, by modifying one or more substituents or functional groups compared to the lead compound, for example by replacing these with alternative substituents or groups which are expected to have the same or improved physiological effect. The use of derivatives having such modifications is well known to those in the art.

Antibodies

Antibodies may be employed in the present invention as an example of a class of inhibitor useful for treating PTEN mutated or deficient cancer, and more particularly as Nemo-Like kinase (NLK) inhibitors . They may also be used in the methods disclosed herein for assessing an individual having cancer or predicting the response of an individual having cancer, in particular for determining whether the individual has PTEN mutated or deficient cancer that might be treatable according to the present

invention . As used herein, the term "antibody" includes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein comprising an antibody binding domain. Antibody fragments which comprise an antigen binding domain are such as Fab, scFv, Fv, dAb, Fd; and diabodies. It is possible to take monoclonal and other

antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP 0 184 187 A, GB 2,188,638 A or EP 0 239 400 A.

Antibodies can be modified in a number of ways and the term "antibody molecule" should be construed as covering any specific binding member or substance having an antibody antigen-binding domain with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP 0 120 694 A and EP 0 125 023 A.

It has been shown that fragments of a whole antibody can perform the function of binding antigens . Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv) , wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al Science, 242; 423-426, 1988; Huston et al, PNAS USA, 85: 5879- 5883, 1988); (viii) bispecific single chain Fv dimers (WO

93/11161) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO 94/13804; Holliger et al, P.N.A.S. USA, 90: 6444-6448, 1993); (x) immunoadhesins (WO 98/50431) . Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al, Nature Biotech, 14: 1239-1245, 1996). Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al, Cancer Res., 56: 3055-3061, 1996).

Preferred antibodies used in accordance with the present invention are isolated, in the sense of being free from

contaminants such as antibodies able to bind other polypeptides and/or free of serum components. Monoclonal antibodies are preferred for some purposes, though polyclonal antibodies are within the scope of the present invention.

The reactivities of antibodies on a sample may be determined by any appropriate means . Tagging with individual reporter molecules is one possibility. The reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently . Linkage via a peptide bond may be as a result o recombinant expression of a gene fusion encoding antibody and reporter molecule. One favoured mode is by covalent linkage of each antibody with an individual fluorochrome, phosphor or laser exciting dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine .

Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyse

reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with

biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.

Antibodies according to the present invention may be used in screening for the presence of a polypeptide, for example in a test sample containing cells or cell lysate as discussed, and may be used in purifying and/or isolating a polypeptide according to the present invention, for instance following production of the polypeptide by expression from encoding nucleic acid. Antibodies may modulate the activity of the polypeptide to which they bind and so, if that polypeptide has a deleterious effect in an individual, may be useful in a therapeutic context (which may include prophylaxis) .

Peptide fragments

Another class of inhibitors useful for treating PTEN mutated or deficient cancer includes peptide fragments that interfere with the activity of a Nemo-Like kinase (NLK) . Peptide fragments may be generated wholly or partly by chemical synthesis that block the catalytic sites of the kinase. Peptide fragments can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California), or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.

Other candidate compounds for inhibiting a Nemo-Like kinase (NLK) may be based on modelling the 3-dimensional structure of these enzymes and using rational drug design to provide candidate compounds with particular molecular shape, size and charge characteristics. A candidate inhibitor, for example, may be a "functional analogue" of a peptide fragment or other compound which inhibits the component. A functional analogue has the same functional activity as the peptide or other compound in question. Examples of such analogues include chemical compounds which are modelled to resemble the three dimensional structure of the component in an area which contacts another component, and in particular the arrangement of the key amino acid residues as they appear .

Nucleic acid inhibitors

Another class of inhibitors useful for treatment of PTEN mutated or deficient cancer includes nucleic acid inhibitors of a Nemo- Like kinase (NLK) , or the complements thereof, which inhibit activity or function by down-regulating production of active polypeptide. This can be monitored using conventional methods well known in the art, for example by screening using real time PCR as described in the examples. of Nemo- -L

or RNAi t

ate gene e

Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of the base excision repair pathway component so that its expression is reduced or completely or substantially completely prevented. In addition to targeting coding sequence, anti-sense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the anti-sense oligonucleotides can interfere with expression control sequences. The construction of anti-sense sequences and their use is described for example in Peyman & Ulman, Chemical Reviews, 90:543-584, 1990 and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, 1992.

Oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo within cells in which down-regulation is desired. Thus, double-stranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA

transcribed from the sense strand of the target gene. The complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain.

However, it is established fact that the technique works.

The complete sequence corresponding to the coding sequence in reverse orientation need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A suitable fragment may have about 14-23 nucleotides, e.g., about 15, 16 or 17 nucleotides.

An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression (Angell & Baulcombe, The EMBO Journal 16 ( 12 ) : 3675-368 , 1997 and Voinnet & Baulcombe, Nature, 389: 553, 1997). Double stranded RNA (dsRNA) has been found to be even more effective in gene silencing than both sense or antisense strands alone (Fire et al, Nature 391, 806-811, 1998). dsRNA mediated silencing is gene specific and is often termed RNA interference (RNAi) . Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and mammals are known in the art (Fire, Trends Genet., 15: 358-363, 19999; Sharp, RNA interference, Genes Dev. 15: 485-490 2001; Hammond et al . , Nature Rev. Genet. 2: 110-1119, 2001; Tuschl, Chem. Biochem. 2: 239-245, 2001; Hamilton et al., Science 286: 950-952, 1999;

Hammond, et al . , Nature 404: 293-296, 2000; Zamore et al . , Cell, 101: 25-33, 2000; Bernstein, Nature, 409: 363-366, 2001; Elbashir et al, Genes Dev., 15: 188-200, 2001; WO01/29058; W099/32619, and Elbashir et al, Nature, 411: 494-498, 2001). RNA interference is a two-step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs (~2nt) . The siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore, Nature Structural Biology, 8, 9, 746-750, 2001.

RNAi may also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3 ' -overhang ends

(Zamore et al, Cell, 101: 25-33, 2000) . Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologeous genes in a wide range of mammalian cell lines

(Elbashir et al, Nature, 411: 494-498, 2001) .

Another possibility is that nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific site and therefore also useful in influencing gene expression, e.g., see Kashani-Sabet & Scanlon, Cancer Gene Therapy, 2(3): 213-223, 1995 and Mercola & Cohen, Cancer Gene Therapy, 2(1): 47-59, 1995.

Small RNA molecules may be employed to regulate gene expression. These include targeted degradation of mRNAs by small interfering RNAs (siRNAs), post transcriptional gene silencing (PTGs), developmentally regulated sequence-specific translational repression of mRNA by micro-RNAs (miRNAs) and targeted

transcriptional gene silencing.

A role for the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci has also been demonstrated. Double- stranded RNA ( dsRNA) -dependent post transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. It acts as a signal to promote degradation of mRNA with sequence identity. A 20-nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA.

In the art, these RNA sequences are termed "short or small interfering RNAs" (siRNAs) or "microRNAs" (miRNAs) depending on their origin. Both types of sequence may be used to down- regulate gene expression by binding to complimentary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNA are derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin. Micro-interfering RNAs (miRNA) are

endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the

translation of mRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences. The siRNA ligands are typically double stranded and, in order to optimise the effectiveness of RNA mediated down-regulation of the function of a target gene, it is preferred that the length of the siRNA molecule is chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of the mRNA target and so that the siRNA is short enough to reduce a host response. miRNA ligands are typically single stranded and have regions that are partially complementary enabling the ligands to form a hairpin. miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. A DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse

complement. When this DNA sequence is transcribed into a single- stranded RNA molecule, the miRNA sequence and its reverse- complement base pair to form a partially double stranded RNA segment. The design of microRNA sequences is discussed in John et al, PLoS Biology, 11(2), 1862-1879, 2004.

Typically, the RNA ligands intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogues thereof) , more preferably between 17 and 30

ribonucleotides, more preferably between 19 and 25

ribonucleotides and most preferably between 21 and 23

ribonucleotides. In some embodiments of the invention employing double-stranded siRNA, the molecule may have symmetric 3 ' overhangs, e.g. of one or two (ribo) nucleotides, typically a UU of dTdT 3' overhang. Based on the disclosure provided herein, the skilled person can readily design suitable siRNA and miRNA sequences, for example using resources such as Ambion's siRNA finder, see http://www.ambion.com/techlib/misc/siRNA finder.html. siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors). In a preferred embodiment the siRNA is synthesized synthetically. Longer double stranded RNAs may be processed in the cell to produce siRNAs (e.g. see Myers, Nature Biotechnology, 21: 324- 328, 2003) . The longer dsRNA molecule may have symmetric 3' or 5' overhangs, e.g. of one or two ( ribo ) nucleotides , or may have blunt ends. The longer dsRNA molecules may be 25 nucleotides or longer. Preferably, the longer dsRNA molecules are between 25 and 30 nucleotides long. More preferably, the longer dsRNA molecules are between 25 and 27 nucleotides long. Most

preferably, the longer dsRNA molecules are 27 nucleotides in length. dsRNAs 30 nucleotides or more in length may be expressed using the vector pDECAP (Shinagawa et al . , Genes and Dev., 17: 1340-5, 2003) .

Another alternative is the expression of a short hairpin RNA molecule (shRNA) in the cell. shRNAs are more stable than synthetic siRNAs . A shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target. In the cell the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression. In a preferred embodiment the shRNA is produced endogenously (within a cell) by transcription from a vector. shRNAs may be produced within a cell by

transfecting the cell with a vector encoding the shRNA sequence under control of a RNA polymerase III promoter such as the human HI or 7SK promoter or a RNA polymerase II promoter.

Alternatively, the shRNA may be synthesised exogenously (in vitro) by transcription from a vector. The shRNA may then be introduced directly into the cell. Preferably, the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length. The stem of the hairpin is preferably between 19 and 30 base pairs in length. The stem may contain G-U pairings to stabilise the hairpin structure.

In one embodiment, the siRNA, longer dsRNA or miRNA is produced endogenously (within a cell) by transcription from a vector. The vector may be introduced into the cell in any of the ways known in the art. Optionally, expression of the RNA sequence can be regulated using a tissue specific promoter. In a further embodiment, the siRNA, longer dsRNA or miRNA is produced

exogenously {in vitro) by transcription from a vector.

Alternatively, siRNA molecules may be synthesized using standard solid or solution phase synthesis techniques, which are known in the art. Linkages between nucleotides may be phosphodiester bonds or alternatives, e.g., linking groups of the formula P(0)S, (thioate); P(S)S, (dithioate ) ; P(0)NR'2; P(0)R'; P(0)OR6; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through-O-or-S- .

Modified nucleotide bases can be used in addition to the

naturally occurring bases, and may confer advantageous properties on siRNA molecules containing them.

For example, modified bases may increase the stability of the siRNA molecule, thereby reducing the amount required for

silencing. The provision of modified bases may also provide siRNA molecules, which are more, or less, stable than unmodified siRNA.

The term Modified nucleotide base' encompasses nucleotides with a covalently modified base and/or sugar. For example, modified nucleotides include nucleotides having sugars, which are

covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3 'position and other than a

phosphate group at the 5 'position. Thus modified nucleotides may also include 2 ' substituted sugars such as 2 ' -O-methyl- ; 2-0- alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2 ' -fluoro- ; 2 ' -halo or 2; azido-ribose , carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars and sedoheptulose .

Modified nucleotides are known in the art and include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles . These classes of pyrimidines and purines are known in the art and include pseudoisocytosine , N4,N4- ethanocytosine , 8-hydroxy-N6-methyladenine , 4-acetylcytosine, 5- ( carboxyhydroxylmethyl ) uracil, 5 fluorouracil , 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6-isopentyl-adenine , 1- methyladenine, 1-methylpseudouracil, 1-methylguanine , 2,2- dimethylguanine, 2methyladenine, 2-methylguanine, 3- methylcytosine , 5-methylcytosine , N6-methyladenine , 7- methylguanine, 5-methylaminomethyl uracil, 5-methoxy amino methyl-2-thiouracil, -D-mannosylqueosine , 5- methoxycarbonylmethyluracil, 5methoxyuracil, 2 methylthio-N6- isopentenyladenine , uracil-5-oxyacetic acid methyl ester, psueouracil, 2-thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil,

4-thiouracil , 5methyluracil, N-uracil-5-oxyacetic acid

methylester, uracil 5-oxyacetic acid, queosine, 2-thiocytosine,

5-propyluracil , 5-propylcytosine , 5-ethyluracil, 5ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine, and

2 , 6, diaminopurine, methylpsuedouracil, 1-methylguanine, 1- methylcytosine .

Methods of screening

In some aspects, the present invention is concerned with methods of screening candidate compounds to determine whether one or more candidate agents are likely to be useful for the treatment of PTEN mutated or deficient cancer. As described herein, there are three preferred general approaches that may be used for these methods of screening, either alone or in any combination or order .

In the screening methods of the present invention, it may be advantageous to use a PTEN mutant cell line to test the

effectiveness of a candidate Nemo-Like kinase (NLK) inhibitor, full list of PTEN mutant cell lines is available for retrieved from the COSMIC database: http : / /ww . sanger . c . uk/perl/genetics/CGP/cosmic?act ion=mutations &ln=PTEN&start=l&end=404&coords=¾A:AR&page=l ) In a first approach, a method of screening may involve using cell lines to determine whether a candidate agent is synthetically lethal in a first cell line which is PTEN mutated or deficient. This method preferably also uses a second cell line that is PTEN proficient as a control and candidate agents are selected which are synthetically lethal in the first cell line and which preferably do not cause any substantial amount cell death in the second cell line and/or normal cells. Thus, in this embodiment of the invention exploits synthetic lethality in cancer cells. Two mutations are synthetically lethal if cells with either of the single mutations are viable, but cells with both mutations are inviable . Identifying synthetic lethal combinations

therefore allows a distinct approach to identifying therapeutic targets that allows selective killing of tumour cells.

Preferably, the method is carried out using cancer cell lines, e.g. mammalian or human cancer cell lines, and more specifically PTEN mutated or deficient cancer cell lines.

One preferred way of initially identifying synthetic lethal interactions involves the use of RNAi screens. Synthetic lethality describes the scenario in which two normally nonessential genes become essential when both are lost, or

inhibited. Targeting a gene that is synthetically lethal with a cancer specific mutation should selectively kill tumour cells while sparing normal cells. One of the major advantages of this approach is the ability to target cancer cells containing loss- of-function mutations, that is, mutations in tumour suppressor genes. Previously, it has been difficult to devise therapeutic strategies to target these mutations as recapitulating tumour suppressor function is technically difficult. Most

pharmacological agents inhibit rather than activate protein function and therefore cannot be used to target loss-of-function alterations in tumours . Identification of synthetic lethal relationships with tumour suppressor genes could allow cells that contain the tumour suppressor mutations to be selectively killed. The use of synthetic lethality to target cancer-specific

mutations has been demonstrated by the selective killing of PTEN mutated or deficient cancer cells using Nemo-Like kinase (NLK) inhibitors . These inhibitors showed profound selectivity, killing cells with PTEN mutations or deficiency, while normal cells were unaffected. However, with the advent of high- throughput RNAi screens it is now possible, in principle, to perform large-scale synthetic-lethal gene identification in mammalian cells, as is routinely done in yeast. Screening deletion mutants that have defects in cell-cycle checkpoint or DNA repair mechanisms in yeast has yielded synthetically lethal genes and small-molecule inhibitors. Using mammalian isogenic- paired cell lines that differ in a single genetic target, RNAi can be used to identify drug targets that when inhibited will result in the selective death of tumour cells. In some

embodiments, the cells used in the method are preferably FOXOl proficient .

Chemical screens have been performed previously on isogenic cancer cell lines for synthetic lethal interactions. However, such approaches have the significant disadvantage of having to identify the cellular targets of an active small molecule. This can be achieved by illustrating the affinity of a small molecule for a particular protein, but this is time-consuming and suffers the limitation that irrelevant proteins will bind in addition to the target. A variation on the synthetic lethality theme is to use compounds that inhibit a cancer-specific target and then screen RNAi libraries to identify targets that selectively kill the cells treated with this compound.

The present invention also includes methods of screening that employ Nemo-Like kinase (NLK) s as protein targets for the screening of candidate compounds to find Nemo-Like kinase (NLK) inhibitors. Accordingly, methods of screening may be carried out for identifying candidate agents that are capable of inhibiting a Nemo-Like kinase (NLK) for subsequent use of development as agents for the treatment of PTEN mutated or deficient cancer. Conveniently, this may be done in an assay buffer to help the components of the assay interact , and in a multiple well format to test a plurality of candidate agents. The activity of a Nemo- Like kinase (NLK) can then be determined in the presence and absence of the one or more candidate compounds to determine whether a given candidate is an inhibitor of a Nemo-Like kinase (NLK) .

By way of example, the candidate agent may be a known inhibitor of one of the protein targets disclosed herein, an antibody, a peptide, a nucleic acid molecule or an organic or inorganic compound, e.g. molecular weight of less than 100 Da. In some instances the use of candidate agents that are compounds is preferred. However, for any type of candidate agent,

combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a target protein. Such libraries and their use are known in the art. The present invention also specifically envisages screening candidate agents known for the treatment of other conditions, and especially other forms of cancer. This has the advantage that the patient or disease profile of known therapeutic agents might be expanded or modified using the screening techniques disclosed herein, or for

therapeutic agents in development, patient or disease profiles established that are relevant for the treatment of PTEN mutated or deficient cancer.

Following identification of a candidate agent for further investigation, the agent in question may be tested to determine whether it is not lethal to normal cells or otherwise is suited to therapeutic use. Following these studies, the agent may be manufactured and/or used in the preparation of a medicament, pharmaceutical composition or dosage form.

The development of lead agents or compounds from an initial hit in screening assays might be desirable where the agent in question is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g.

peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore".

Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can

conveniently be selected so that the mimetic is easy to

synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or

modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

Treatment of cancer

The present invention provides methods and medical uses for the treatment of PTEN mutated or deficient cancer. A cancer may be identified as PTEN mutated or deficient cancer by testing a sample of cancer cells from an individual, for example to determine whether the PTEN protein contains one or more mutations or to determine the expression of the PTEN gene to evaluate whether expression of the protein is absent or at a reduced level compared to normal. It is known that genetic inactivation of PTEN occurs in glioblastoma, endometrial cancer and prostate cancer and reduced expression is found in many other tumor types that include lung and breast cancer. Examples of known PTEN mutated or deficient cancers that are treatable in accordance with the present invention include cancers affecting the

autonomic ganglia, biliary tract, bone, breast, CNS, cervix, endometrium, eye, haematopoietic and lymphoid tissue, kidney, large intestine, liver, lung, meninges, oesophagus, ovary, pancreas, prostate, salivary gland, skin, soft tissue, stomach, testis, thyroid, upper aerodigestive tract, urinary tract, or vulva. Cancer associated mutations in PTEN occur across the entire coding sequence of the gene and include both premature truncating mutations and single amino acid substitutions in both the catalytic and non-catalytic domains of the protein. The frequency of monoallelic mutations in PTEN has been estimated at 50%-80% in sporadic tumors (including endometrial carcinoma, glioblastoma, and prostate cancer) and at 30%-50% in breast, colon, and lung tumors . Complete loss of PTEN is observed at highest frequencies in endometrial cancer and glioblastoma and is generally associated with advanced cancers and metastases.

Common tumor associated PTEN mutations include C.3880G,

C.6970T, C.3880T, c.800delA, c.968delA, c.517C>T,

c.968_969insA, c.518G>A, c .955_958delACTT, C.1003OT,

C.950 953delTACT. Based on the examples, the present invention is particularly applicable to the treatment of cancer cells that are also FOXOl proficient, e.g. in which the activity of FOXOl is preferably at least 50% normal wild-type activity, more preferably at least 75% wild-type activity and most preferably at least 90% wild-type activity .

The sample may be of normal cells from the individual where the individual has a mutation in the PTEN gene or the sample may be of cancer cells, e.g. where the cells forming a tumour contain one or more PTEN mutations . Activity may be determined relative to a control, for example in the case of defects in cancer cells, relative to non-cancerous cells, preferably from the same tissue.

The activity of the PTEN may be determined by using techniques well known in the art such as Western blot analysis,

immunohistology, chromosomal abnormalities, enzymatic or DNA binding assays and plasmid-based assays.

The determination of PTEN gene expression may involve determining the presence or amount of PTEN mRNA in a sample. Methods for doing this are well known to the skilled person. By way of example, they include determining the presence of PTEN mRNA (i) using a labelled probe that is capable of hybridising to the PTEN nucleic acid; and/or (ii) using PCR involving one or more primers based on a PTEN nucleic acid sequence to determine whether the PTEN transcript is present in a sample. The probe may also be immobilised as a sequence included in a microarray.

Preferably, detecting PTEN mRNA is carried out by extracting RNA from a sample of the tumour and measuring PTEN expression specifically using quantitative real time RT-PCR. Alternatively or additionally, the expression of PTEN could be assessed using RNA extracted from a tumour sample using microarray analysis, which measures the levels of mRNA for a group of genes using a plurality of probes immobilised on a substrate to form the array. The determination of whether the cells are express FOXOl and hence are FOXOl proficient may be done in an analogous manner.

Alternatively or additionally, the present invention the

determination of whether a patient has a PTEN mutated or

deficient cancer can be carried out by analysis of PTEN protein expression, for example to examining whether reduced levels of PTEN protein are expressed or whether the PTEN protein contains one or more mutations. The determination of whether the cells are FOXOl proficient may be done in an analogous manner.

Preferably, the presence or amount of PTEN and/or FOXOl protein may be determined using a binding agent capable of specifically binding to the PTEN and/or FOXOl protein, or fragments thereof. A preferred type of PTEN and/or FOXOl protein binding agent is an antibody capable of specifically binding the PTEN and/or FOXOl protein or fragment thereof. The antibody may be labelled to enable it to be detected or capable of detection following reaction with one or more further species, for example using a secondary antibody that is labelled or capable of producing a detectable result, e.g. in an ELISA type assay. As an

alternative a labelled binding agent may be employed in a western blot to detect PTEN and/or FOXOl protein.

Alternatively, or additionally, the method for determining the presence of PTEN protein and/or FOXOl protein may be carried out on tumour samples, for example using immunohistochemical (IHC) analysis. IHC analysis can be carried out using paraffin fixed samples or frozen tissue samples, and generally involves staining the samples to highlight the presence and location of PTEN protein and/or FOXOl protein.

Pharmaceutical compositions

The active agents disclosed herein for the treatment of PTEN mutated or deficient cancer may be administered alone, but it is generally preferable to provide them in pharmaceutical

compositions that additionally comprise with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives,

lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.

Examples of components of pharmaceutical compositions are provided in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

Examples of small molecule therapeutics useful for treating PTEN mutated or deficient cancer via inhibition of other kinases include: BEZ235, Olaparib and GDC0941.

These compounds or derivatives of them may be used in the present invention for the treatment of PTEN mutated or deficient cancer. As used herein "derivatives" of the therapeutic agents includes salts, coordination complexes, esters such as in vivo

hydrolysable esters, free acids or bases, hydrates, prodrugs or lipids, coupling partners.

Salts of the compounds of the invention are preferably

physiologically well tolerated and non toxic. Many examples of salts are known to those skilled in the art. Compounds having acidic groups, such as phosphates or sulfates, can form salts with alkaline or alkaline earth metals such as Na, K, Mg and Ca, and with organic amines such as triethylamine and Tris (2- hydroxyethyl ) amine . Salts can be formed between compounds with basic groups, e.g., amines, with inorganic acids such as

hydrochloric acid, phosphoric acid or sulfuric acid, or organic acids such as acetic acid, citric acid, benzoic acid, fumaric acid, or tartaric acid. Compounds having both acidic and basic groups can form internal salts .

Esters can be formed between hydroxyl or carboxylic acid groups present in the compound and an appropriate carboxylic acid or alcohol reaction partner, using techniques well known in the art.

Derivatives which as prodrugs of the compounds are convertible in vivo or in vitro into one of the parent compounds. Typically, at least one of the biological activities of compound will be reduced in the prodrug form of the compound, and can be activated by conversion of the prodrug to release the compound or a metabolite of it.

Other derivatives include coupling partners of the compounds in which the compounds is linked to a coupling partner, e.g. by being chemically coupled to the compound or physically

associated with it. Examples of coupling partners include a label or reporter molecule, a supporting substrate, a carrier or transport molecule, an effector, a drug, an antibody or an inhibitor. Coupling partners can be covalently linked to compounds of the invention via an appropriate functional group on the compound such as a hydroxyl group, a carboxyl group or an amino group. Other derivatives include formulating the compounds with liposomes .

The term "pharmaceutically acceptable" as used herein includes compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable

benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

The active agents disclosed herein for the treatment of PTEN mutated or deficient cancer according to the present invention are preferably for administration to an individual in a

"prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of

administration and other factors known to practitioners.

Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, Lippincott, Williams & Wilkins. A composition may be

administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into

association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product .

The agents disclosed herein for the treatment of PTEN mutated or deficient cancer may be administered to a subject by any

convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual) ;

pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal;

parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial,

intracardiac, intrathecal, intraspinal, intracapsular,

subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal ; by implant of a depot, for example, subcutaneously or

intramuscularly. Formulations suitable for oral administration (e.g., by

ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal) , include aqueous and non-aqueous isotonic, pyrogen-free , sterile injection solutions which may contain anti-oxidants , buffers, preservatives, stabilisers, bacteriostats , and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs . Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs .

Compositions comprising agents disclosed herein for the treatment PTEN mutated or deficient cancer may be used in the methods described herein in combination with standard chemotherapeutic regimes or in conjunction with radiotherapy. Examples of other chemotherapeutic agents include Amsacrine (Amsidine) , Bleomycin, Busulfan, Capecitabine (Xeloda) , Carboplatin, Carmustine (BCNU) , Chlorambucil (Leukeran) , Cisplatin, Cladribine (Leustat) ,

Clofarabine (Evoltra) , Crisantaspase (Erwinase),

Cyclophosphamide, Cytarabine (ARA-C) , Dacarbazine (DTIC) ,

Dactinomycin (Actinomycin D) , Daunorubicin, Docetaxel (Taxotere), Doxorubicin, Epirubicin, Etoposide (Vepesid, VP-16), Fludarabine (Fludara), Fluorouracil (5-FU) , Gemcitabine (Gemzar) , Hydroxyurea (Hydroxycarbamide, Hydrea) , Idarubicin (Zavedos) . Ifosfamide (Mitoxana) , Irinotecan (CPT-11, Campto), Leucovorin (folinic acid), Liposomal doxorubicin (Caelyx, Myocet), Liposomal

daunorubicin (DaunoXome®) Lomustine, Melphalan, Mercaptopurine , Mesna, Methotrexate, Mitomycin, Mitoxantrone , Oxaliplatin

(Eloxatin) , Paclitaxel (Taxol) , Pemetrexed (Alimta) , Pentostatin (Nipent) , Procarbazine, Raltitrexed (Tomudex®) , Streptozocin (Zanosar®) , Tegafur-uracil (Uftoral) , Temozolomide (Temodal) , Teniposide (Vumon) , Thiotepa, Tioguanine (6-TG) (Lanvis),

Topotecan (Hycamtin) , Treosulfan, Vinblastine (Velbe),

Vincristine (Oncovin), Vindesine (Eldisine) and Vinorelbine (Navelbine ) .

Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate

intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

In general, a suitable dose of the active compound is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound, and so the actual weight to be used is increased proportionately.

Experimental

Loss of function mutations in the tumour suppressor gene PTEN or lack of PTEN expression have both been observed in a wide range of human tumours (1) . PTEN encodes a phosphatase whose activity antagonises PI3 kinase signaling by dephosphorylating the plasma membrane lipid phosphoinositide-3 , , 5-trisphosphate (PIP3) . The loss of PTEN phosphatase activity is thought to foster

tumourigenesis , at least in part, by causing downstream

constitutive activation of AKT (1) . In addition to this role, PTEN has been suggested to have a nuclear function in maintaining genomic stability (2).

The presence of tumour-specific loss-of-function mutations in PTEN raises the possibility of identifying PTEN synthetic lethal interactions that could be exploited therapeutically.

Previously, we have shown that PTEN deficiency is synthetically lethal with inhibition of PARP1 (3), and effect predicted from the proposed role of PTEN in maintaining genomic stability (2) . It is likely that additional SLs involving PTEN exist, and these might prove valuable candidate therapeutic approaches . High- throughput genetic screening using RNA interference (RNAi) represents a straightforward method to identifying SLs in a relatively unbiased fashion (4).

We used a high-throughput screening (HTS) approach to identify novel SLs involving PTEN (Figure 1A) . Specifically, we reverse- transfected PTEN wild type and null human tumour cell lines with a short-interfering (si) RNA library arrayed in 96-well plates and measured effects of each siRNA on cell proliferation using

CellTiter-Glo® reagent (Promega) five days after transfection . The siRNA library we used comprised 779 siRNA SMART-pools

(Dharmacon) , each designed to target a specific kinase or kinase- related gene. We selected this gene subset to screen given its inherent pharmacological tractability . To maximise the possibility of identifying SLs specific to PTEN, we carried out triplicate screens in a pair of isogenically matched HCT116- derived wild-type (PTEN^+/+) and ΡΓ£-\Γ^~ colorectal tumour cell lines (5) . PTEN deficiency in this model was achieved by targeting a truncating mutation to both copies of PTEN at exon 2, with the resultant mutant alleles encoding an ostensibly dysfunctional PTEN mutant protein consisting of only the 24 N-terminal amino- acids ( 6 , 7 ) .

The sensitivity of the screen and its overall performance were monitored through a series of commonly used HTS metrics (8) .

These included: (i) PLK1 siRNA causing a reduction in viability of more than 90% in both cell types, when compared to

transfection with a non-targeting siRNA control (siCON) ; (ii) Z'- factor estimation between negative (siCON) and positive (siPLKl) control wells where a Z' ≥0.5 was set as a threshold of

acceptable dynamic range (Figure SI); and (iii) <10% inhibition in cell proliferation caused by control non-targeting siRNA compared to mock transfected wells. Transfected cell numbers were titrated to ensure equal transfection efficiency and confluency in PTEN null and wild-type cells. Following screen optimisation, each cell line was screened three times and the data from replica screens combined in the final analysis (Figure IB) .

To identify PTEN selective effects we first calculated NPI

(normalised percent inhibition) scores (9) for each siRNA pool in both PTEN^+/+ and PTEN^~7~ cell lines. To calculate NPI scores, which scale cell viability effects according to maximal and minimal effects, we defined the maximal inhibitory effect as that caused by siRNAs targeting PLK1 and the minimal effect as that caused by non-targeting control siRNAs . This procedure equalised data distributions between both lineages, allowing the comparison of NPI scores between PTEN^~'^~ and PTEN^+/+ cells . This ultimately allowed each siRNA pool to be ranked ordered according to its PTElf^ selective effect. We selected the 20 greatest PTEN^~'^~ selective effects (Table SI) for technical validation using the original screening protocol in a lower-throughput format to minimise false positive effects . This analysis highlighted three genes with clear PTEN selective effects: NLK {Nemo-Like Kinase), PLK4 {Polo-Like Kinase 4), and TTK (MPS1, Monopolar Spindle 1) (Figure 1C) . Recently, we have used functional viability profiling in non-isogenic breast tumour cell line models to identify candidate SL effects, an analysis that identified TTK as synthetically lethal with PTEN (10) . The identification of the PTEN/TTK SL in the screen described here indicated that the HTS performed on the isogenic models was able to identify bona fide effects. The NLK, PLK4 and TTK effects were then subject to further revalidation to exclude possible RNAi off-target effects. Given that a phenotype caused by two distinct siRNA species suggests a lower likelihood of an off- target effect (11), we reassayed viability effects using each of the four different siRNA species that comprise the SMARTpools used in the primary screen. Analysis using these "deconvoluted" pools resulted in the identification of at least two siRNAs for each gene that reproduced the original effects (Figure 1C) . In addition, the ability of these individual siRNAs to silence their target genes was confirmed by immunoblotting (Figure ID) .

Highly penetrant "hard" SLs that are relatively unaffected by additional genetic modifications are preferred over those that are readily abrogated by other changes in the genetic background of a cell. It seems likely that such hard synthetic lethalities are more likely to be robust in genetically heterogeneous tumours

(12) . To address this issue, we used two additional isogenic models of PTEN deficiency derived from (i) HEC1A endometrial and

(ii) DLD1 colorectal tumour cell lines. In both HEC1A and DLD1 models, homozygous mutation of PTEN was achieved using the same targeting strategy as for the HCT116 models, where truncating mutations were introduced into both copies of PTEN exon 2. Both, NLK and PLK4 SLs re-validated in the HEC1A model, whilst the PTEN/PLK4 SL failed to re-validate in the DLD1 isogenic matched pair (Figure 2A) , possibly due to reduced basal levels of PLK4 in DLD1 cells (Figure 2B)

To further assess the generality of our observations, we examined NLK and PLK4 SLs in a wide panel of genetically heterogenous PTEN deficient or proficient tumour cell lines (Table 1) . Here we used a combination of two validated siRNAs to target each gene, alongside appropriate positive (siPLKl) and negative (siCON) controls (Figure S2) . Silencing of either NLK or PLK , had a greater cell inhibitory effect in the PTEN deficient cohort when compared to the PTEN proficient group (Figure 2C, 2D), an effect especially evident for NLK silencing.

Given the generality of the NLK/PTEN SL, we went on to

investigate the mechanism of NLK action in the context of PTEN deficiency. Opposing the pro-oncogenic effects of PTEN

dysfunction are molecular networks that normally promote

quiescence, senescence or cell death (13). These include the Forkhead O-class transcription factors such as FOXOl ( ' Forkhead- box-01' isoform) (14). In the face of PTEN deficiency, FOXOl can exert tumour suppressive effects by mediating the transcription of genes such as p21, p27, and BIM that promote quiescence, senescence or cell death. However, this tumour suppressive function of FOXOl can in turn be circumvented by the AKT

activation that characterises PTEN deficiency. The

phosphorylation of FOXOl by activated AKT causes FOXOl to be excluded from the nucleus and ultimately degraded in the

cytoplasm, minimizing its ability to drive a tumour suppressive transcriptional programme (14).

One of the established functions of NLK is the AKT-independent phosphorylation of FOXOl; this phosphorylation also causes FOXOl inactivation via its nuclear exclusion (15). We hypothesised that NLK inhibition could be synthetically lethal with PTEN deficiency as it causes reactivation of FOXOl and therefore cellular senescence. To test if the PTEN/NLK synthetic lethality was FOXOl dependent, we transfected PTEN null cells with both NLK and FOXOl siRNA and assessed the effect on cell inhibition. Whilst FOXOl siRNA alone did not selectively target PTEN null cells and NLKl siRNA caused PTEN synthetic lethality (as before) , FOXOl siRNA reversed the PTEN/NLK synthetic lethality (Figure 3A, Figure S3), supporting the hypothesis that the PTEN/NLK SL is FOXOl dependent. NLK silencing also caused an increase in nuclear FOXOl localisation (Figure 3B) , again supporting the hypothesis. Furthermore, NLK silencing induced senescence in PTEN deficient cells when compared to PTEN proficient

counterparts (Figure 3C, 3D), suggesting that the instigation of a senescent programme by FOXOl could explain the PTEN/NLK synthetic lethality.

Our study demonstrates that NLK silencing inhibits PTEN deficient tumour cells. Our mechanistic dissection of this effect suggests that re-instatement of a FOXOl-driven process, possibly cellular senescence that normally suppresses oncogenesis, could explain the PTEN selective effect of NLK inhibition. As a potential route to identifying leads for cancer drug discovery, others have already identified compounds that cause the translocation FOXOl to the nucleus (16) . The data presented here suggests that inhibition of NLK could provide a similar target for drug development. In this scenario, PTEN deficiency could be used as a biomarker to identify patients likely to respond to a clinical NLK inhibitor.

Table 1. List of 24 cell lines, categorised as PTEN proficient (n=ll) or deficient (n=13) as defined by western blotting.

SEQ ID NO:l amino acid sequence of human NLK:

10 20 30 40 50 60

MSLCGARANA KMMAAYNGGT SAAAAGHHHH HHHHLPHLPP PHLHHHHHPQ HHLHPGSAAA

70 80 90 100 110 120

VHPVQQHTSS AAAAAAAAAA AAAMLNPGQQ QPYFPSPAPG QAPGPAAAAP AQVQAAAAAT

130 140 150 160 170 180

VKAHHHQHSH HPQQQLDIEP DRPIGYGAFG VVWSVTDPRD GKRVALKKMP NVFQNLVSCK

190 200 210 220 230 240

RVFRELKMLC FFKHDNVLSA LDILQPPHID YFEEIYVVTE LMQSDLHKII VSPQPLSSDH

250 260 270 280 290 300

VKVFLYQILR GLKYLHSAGI LHRDIKPGNL LVNSNCVLKI CDFGLARVEE LDESRHMTQE

310 320 330 340 350 360 VVTQYYRAPE ILMGSRHYSN AIDIWSVGCI FAELLGRRIL FQAQSPIQQL DLITDLLGTP

370 380 390 400 410 420

SLEAMRTACE GAKAHILRGP HKQPSLPVLY TLSSQATHEA VHLLCRMLVF DPSKRISAKD

430 440 450 460 470 480

ALAHPYLDEG RLRYHTCMCK CCFSTSTGRV YTSDFEPVTN PKFDDTFEKN LSSVRQVKEI

490 500 510 520

IHQFILEQQK GNRVPLCINP QSAAFKSFIS STVAQPSEMP PSPLVWE

SEQ ID NO: 2 amino acid sequence of human FOXOl :

10 20 30 40 50 60

MAEAPQVVEI DPDFEPLPRP RSCTWPLPRP EFSQSNSATS SPAPSGSAAA NPDAAAGLPS

70 80 90 100 110 120

ASAAAVSADF MSNLSLLEES EDFPQAPGSV AAAVAAAAAA AATGGLCGDF QGPEAGCLHP

130 140 150 160 170 180

APPQPPPPGP LSQHPPVPPA AAGPLAGQPR KSSSSRRNAW GNLSYADLIT KAIESSAEKR

190 200 210 220 230 240

LTLSQIYEWM VKSVPYFKDK GDSNSSAGWK NSIRHNLSLH SKFIRVQNEG TGKSSWWMLN

250 260 270 280 290 300

PEGGKSGKSP RRRAASMDNN SKFAKSRSRA AKKKASLQSG QEGAGDSPGS QFSKWPASPG

310 320 330 340 350 360

SHSNDDFDNW STFRPRTSSN ASTISGRLSP IMTEQDDLGE GDVHSMVYPP SAAKMASTLP

370 380 390 400 410 420

SLSEISNPEN MENLLDNLNL LSSPTSLTVS TQSSPGTMMQ QTPCYSFAPP NTSLNSPSPN

430 440 450 460 470 480

YQKYTYGQSS MSPLPQMPIQ TLQDNKSSYG GMSQYNCAPG LLKELLTSDS PPHNDIMTPV

490 500 510 520 530 540

DPGVAQPNSR VLGQNVMMGP NSVMSTYGSQ ASHNKMMNPS SHTHPGHAQQ TSAVNGRPLP

550 560 570 580 590 600

HTVSTMPHTS GMNRLTQVKT PVQVPLPHPM QMSALGGYSS VSSCNGYGRM GLLHQEKLPS

610 620 630 640 650

DLDGMFIERL DCDMESI IRN DLMDGDTLDF NFDNVLPNQS FPHSVKTTTH SWVSG

SEQ ID NO: 3 amino acid sequence of human PTEN:

10 20 30 40 50 60

MTAIIKEIVS RNKRRYQEDG FDLDLTYIYP NIIAMGFPAE RLEGVYRNNI DDVVRFLDSK

70 80 90 100 110 120

HKNHYKIYNL CAERHYDTAK FNCRVAQYPF EDHNPPQLEL IKPFCEDLDQ WLSEDDNHVA

130 140 150 160 170 180

AIHCKAGKGR TGVMICAYLL HRGKFLKAQE ALDFYGEVRT RDKKGVTIPS QRRYVYYYSY

190 200 210 220 230 240

LLKNHLDYRP VALLFHKMMF ETIPMFSGGT CNPQFVVCQL KVKIYSSNSG PTRREDKFMY

250 260 270 280 290 300

FEFPQPLPVC GDIKVEFFHK QNKMLKKDKM FHFWVNTFFI PGPEETSEKV ENGSLCDQEI

310 320 330 340 350 360

DSICSIERAD NDKEYLVLTL TKNDLDKANK DKANRYFSPN FKVKLYFTKT VEEPSNPEAS

370 380 390 400

SSTSVTPDVS DNEPDHYRYS DTTDSDPENE PFDEDQHTQI TKV

References

The documents disclosed herein are all expressly incorporated by reference in their entirety.

1. Hollander MC, Blumenthal GM, Dennis PA PTEN loss in the continuum of common cancers, rare syndromes and mouse models Nat Rev Cancer 2011; 11: 289-301.

2. Shen WH, Balajee AS, Wang J, Wu H, Eng C, Pandolfi PP et al . Essential role for nuclear PTEN in maintaining chromosomal integrity Cell 2007; 128: 157-170.

3. Mendes-Pereira AM, Martin SA, Brough R, McCarthy A, Taylor JR, Kim JS et al . Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors EMBO Mol Med 2009; 1: 315-322.

4. Iorns E, Lord CJ, Turner N, Ashworth A Utilizing RNA interference to enhance cancer drug discovery Nat Rev Drug Discov 2007; 6: 556-568. 5. Lee C, Kim JS, Waldman T Activated PI3K signaling as an endogenous inducer of p53 in human cancer Cell Cycle 2007; 6:

394-396.

6. Lee C, Kim JS, Waldman T PTEN gene targeting reveals a radiation-induced size checkpoint in human cancer cells Cancer Res 2004; 64: 6906-6914.

7. Kim JS, Lee C, Bonifant CL, Ressom H, Waldman T Activation of p53-dependent growth suppression in human cells by mutations in PTEN or PIK3CA Mol Cell Biol 2007; 27: 662-677.

8. Lord CJ, McDonald S, Swift S, Turner NC, Ashworth A A high- throughput RNA interference screen for DNA repair determinants of PARP inhibitor sensitivity DNA Repair (Amst) 2008; 7: 2010-2019.

9. Boutros M, Bras LP, Huber W Analysis of cell-based RNAi screens Genome Biol 2006; 7: R66. 10. Brough R, Frankum JR, Sims D, Mackay A, Mendes-Pereira AM, Bajrami I et al . Functional Viability Profiles of Breast Cancer Cancer Discov 2011; 1: 260-273.

11. Echeverri CJ, Beachy PA, Baum B, Boutros M, Buchholz F, Chanda SK et al . Minimizing the risk of reporting false positives in large-scale RNAi screens Nat Methods 2006; 3: 777-779. 12. Ashworth A, Lord CJ, Reis-Filho JS Genetic interactions in cancer progression and treatment Cell 2011; 145: 30-38.

13. Nardella C, Clohessy JG, Alimonti A, Pandolfi PP Pro- senescence therapy for cancer treatment Nat Rev Cancer 2011; 11: 503-511.

14. Huang H, Tindall DJ FOXO factors: a matter of life and death Future Oncol 2006; 2: 83-89. 15. Kim S, Kim Y, Lee J, Chung J Regulation of FOXOl by TAK1- Nemo-like kinase pathway J Biol Chem 2010; 285: 8122-8129.

16. Kau TR, Schroeder F, Ramaswamy S, Woj ciechowski CL, Zhao JJ, Roberts TM et al . A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells Cancer Cell 2003; 4: 463-476.

17. Lin HK, Chen Z, Wang G, Nardella C, Lee SW, Chan CH et al . Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence Nature 2010; 464: 374-379.

Claims

Claims :

1. A Nemo-Like kinase (NLK) inhibitor for use in a method of treatment of an individual having cancer, wherein the cancer is a Phosphatase and Tensin Homolog (PTEN) mutated or deficient cancer .

2. The NLK inhibitor for use in a method of treatment according to claim 1, wherein the PTEN deficient cancer is PTEN null or the PTEN mutated cancer comprises a truncating mutation or one or more substitutions .

3. The NLK inhibitor for use in a method of treatment according to claim 1 or claim 2, wherein cancer cells that are PTEN mutated or deficient are FOXOl proficient .

4. The NLK inhibitor for use in a method of treatment according to any one of claims 1 to 3, wherein the PTEN mutant or deficient cancer affects the autonomic ganglia, biliary tract, bone, breast, CNS, cervix, endometrium, eye, haematopoietic and lymphoid tissue, kidney, large intestine, liver, lung, meninges, oesophagus, ovary, pancreas, prostate, salivary gland, skin, soft tissue, stomach, testis, thyroid, upper aerodigestive tract, urinary tract, or vulva.

5. The NLK inhibitor for use in a method of treatment according to any one of claims 1 to 4, wherein the PTEN mutant or deficient cancer is breast cancer, endometrial carcinoma, glioblastoma, prostate cancer, colon cancer or lung cancer.

6. The NLK inhibitor for use in a method of treatment according to any one of the preceding claims, wherein the NLK kinase has at least 90% amino acid sequence identity with SEQ ID NO: 1.

7. The NLK inhibitor for use in a method of treatment according to any one of the preceding claims, wherein the NLK kinase comprises the amino acid sequence identity with SEQ ID NO: 1.

8. The NLK inhibitor for use in a method of treatment according to any one of the preceding claims, wherein the inhibitor is a nucleic acid inhibitor, an antibody, a small molecule or a peptide .

9. The NLK inhibitor for use in a method of treatment according to any one of the preceding claims, wherein the nucleic acid inhibitor is a RNAi molecule or a siRNA molecule or an shRNA molecule .

10. The NLK inhibitor for use in a method of treatment according to any one of the preceding claims, wherein treatment with an Nemo-Like kinase (NLK) inhibitor is combined with a further anticancer therapy.

11. The NLK inhibitor for use in a method of treatment according to claim 10, wherein treatment with Nemo-Like kinase (NLK) inhibitor is used in conjunction with a further chemotherapeutic agent .

12. The NLK inhibitor for use in a method of treatment according to claim 11, wherein the further chemotherapeutic agent is

Amsacrine (Amsidine) , Bleomycin, Busulfan, Capecitabine (Xeloda) , Carboplatin, Carmustine (BCNU) , Chlorambucil (Leukeran) ,

Cisplatin, Cladribine (Leustat) , Clofarabine (Evoltra) ,

Crisantaspase (Erwinase) , Cyclophosphamide, Cytarabine (ARA-C) , Dacarbazine (DTIC) , Dactinomycin (Actinomycin D) , Daunorubicin, Docetaxel (Taxotere) , Doxorubicin, Epirubicin, Etoposide

(Vepesid, VP-16) , Fludarabine (Fludara), Fluorouracil (5-FU) , Gemcitabine (Gemzar) , Hydroxyurea (Hydroxycarbamide, Hydrea)

, Idarubicin (Zavedos). Ifosfamide (Mitoxana) , Irinotecan (CPT-11, Campto), Leucovorin (folinic acid), Liposomal doxorubicin

(Caelyx, Myocet) , Liposomal daunorubicin (DaunoXome®) Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin,

Mitoxantrone , Oxaliplatin (Eloxatin) , Paclitaxel (Taxol) ,

Pemetrexed (Alimta) , Pentostatin (Nipent), Procarbazine,

Raltitrexed (Tomudex®) , Streptozocin (Zanosar®), Tegafur-uracil (Uftoral), Temozolomide (Temodal), Teniposide (Vumon) , Thiotepa, Tioguanine (6-TG) (Lanvis), Topotecan (Hycamtin) , Treosulfan, Vinblastine (Velbe), Vincristine (Oncovin), Vindesine (Eldisine) or Vinorelbine (Navelbine) .

13. The NLK inhibitor for use in a method of treatment according to claim 11 or claim 12, wherein the further chemotherapeutic agent is 5FU, a BCL-XL inhibitor, a BCR-ABL, cKIT or PDGFR inhibitor, a CDK inhibitor, a CHK inhibitor, a COX2 inhibitor, a EGFR inhibitor, a HER2 targeted agent, a HSP inhibitor, a hTERT inhibitors, an IDO inhibitor, a MDM2 targeted agent,

Methotrexate, a mTOR inhibitor, a PARP inhibitor, a PI3K

inhibitor, a platinum salt, a proteasome inhibitor, a RAR or RXR inhibitor, a SRC inhibitor, a TGFB2 inhibitor, a topoisomerase inhibitor, a VEGF, RAF, cKIT or PDGFR inhibitor, or a SMC mimetic .

14. The NLK inhibitor for use in a method of treatment according to any one of the preceding claims, wherein the method comprises the steps of determining whether the individual has a PTEN mutated or deficient cancer and administering the NLK inhibitor to individual having a PTEN mutated or deficient cancer.

15. The NLK inhibitor for use in a method of treatment according to any one of the preceding claims, wherein determining whether the individual has PTEN mutated or deficient cancer comprises measuring PTEN protein expression in a sample obtained from the individual to determine whether the PTEN protein is mutated or deficient, and optionally whether the cancer is FOXOl proficient.

16. The NLK inhibitor for use in a method of treatment according to claim 15, wherein determining PTEN and/or FOXOl protein expression comprises one or more of determining PTEN and/or FOXOl protein expression in a tumour sample using immunohistochemistry, determining PTEN and/or FOXOOl protein expression comprises measuring PTEN protein levels in a cell lysate by ELISA or

Western blotting, and/or determining PTEN protein expression comprises using a binding agent capable of specifically binding to the PTEN or FOXOl protein, or a fragment thereof.

17. The NLK inhibitor for use in a method of treatment according to any one of the preceding claims, wherein determining whether the individual has a PTEN mutated or deficient cancer, and optionally whether the cells express FOXOl, is performed on genomic nucleic acid extracted from a sample of cells obtained from the breast cancer or from a sample of cancer cells

circulating in blood.

18. The NLK inhibitor for use in a method of treatment according to claim 17, wherein determining the expression of the PTEN and/or FOXOl gene(s) comprises extracting RNA from a sample of cancer cells and measuring expression by real time PCR and/or by using a probe capable of hybridising to PTEN and/or FOXOl RNA.

19. The NLK inhibitor for use in a method of treatment according to claim 18, wherein the probe is immobilised in a microarray.

20. The NLK inhibitor for use in a method of treatment according to any one of the preceding claims, wherein the method comprises the initial step of obtaining a sample from said individual.

21. The NLK inhibitor for use in a method of treatment according to any one of the preceding claims, wherein the sample is a tumour sample, a blood sample, a tissue sample or a cell sample.

22. Use of a Nemo-Like kinase (NLK) inhibitor in the preparation of a medicament for the treatment of an individual having a PTEN mutated or deficient cancer.

23. A method of treating an individual having a PTEN mutated o deficient cancer, the method comprising of determining whether the individual has a PTEN mutated or deficient cancer and administering a therapeutically effective amount of a Nemo-Like kinase (NLK) inhibitor to individual having a PTEN mutated or deficient cancer.

24. A method of screening for agents useful in the treatment of a PTEN mutated or deficient cancer, the method employing first and second cell lines, wherein the first cell line is PTEN mutated or deficient and the second cell line is PTEN proficient, the method comprising:

(a) contacting the first and second mammalian cell lines with at least one candidate agent;

(b) determining the amount of cell death in the first and second cell lines; and

(c) selecting a candidate agent which is synthetically lethal in the first cell line.

25. The method according to claim 24, wherein the cell lines are cancer-derived cell lines.

26. The method of claim 25, wherein the cell lines are a PTEN mutated or deficient murine stem cell lines.

27. The method according to any one of claims 24 to 26, wherein the first and second cells lines are isogenically matched.

28. The method according to any one of claims 24 to 27, wherein the PTEN mutated or deficient cell line is produced by RNA interference of both copies of the PTEN gene.

29. The method according to any one of claims 24 to 28, wherein step (c) comprises selecting candidate agents that do not cause a substantial amount of cell death in the second cell line.

30. The method of claim 29, wherein the cell line is a cancer cell line.

31. The method according to any one of claims 24 to 29, further comprising the step of determining whether a candidate agent selected in step (c) is an inhibitor of a Nemo-Like kinase (NLK) .

32. A method of screening for agents useful in the treatment of PTEN mutated or deficient cancer, the method comprising:

(a) contacting a Nemo-Like kinase (NLK) with at least one candidate agent;

(b) determining an effect of the at least one candidate agent on an activity of the Nemo-Like kinase (NLK) ; and

(c) selecting a candidate agent that inhibits the activity of the Nemo-Like kinase (NLK) as being useful for the treatment of the PTEN mutated or deficient cancer.

33. The method according to claim 32, further comprising the step of contacting a candidate agent selected in step (c) with a PTEN mutated or deficient cancer cell line to determine whether the candidate agent is cytotoxic to the cancer cell line.

34. The method according to any one of claims 24 to 33, wherein the candidate agent is a candidate nucleic acid inhibitor, a candidate antibody or a candidate small molecule or a candidate peptide .

35. The method according to claim 34, wherein the candidate agent is a compound that is part of a compound library.

36. The method according to claim 34 or claim 35, wherein the candidate compound has a molecular weight of less than 100 Da.

37. The method according to any one of claims 24 to 36, wherein the candidate agent or candidate compounds is a drug approved for use in the treatment of cancer.

38. The method according to any one of claims 24 to 37, further comprising determining whether a candidate agent is not lethal on normal cells .

39. The method according to any one of claims 24 to 38, which comprises determining the effect of combinations of two or more candidate compounds on the cell lines or protein targets .

40. A method which comprises having identified a candidate agent useful for the treatment of PTEN mutated or deficient cancer according to a method of any one of claims 24 to 39, the further step of manufacturing the compound in bulk and/or formulating the agent in a pharmaceutical composition.