Field of the Invention

The present invention relates a poly ADP ribose polymerase (PARP) inhibitor for use in a method of treating an individual with cancer which is mutated or deficient in the SF3B1 gene.

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 consequently 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.

Regulated splicing of the cellular transcriptome is essential for normal growth and development. It involves removal of intronic DNA from pre-mRNA transcripts via the activity of a common set of small nuclear RNAs (snRNAs) and associated proteins which assemble together with into a complex known as the spliceosome. Aberrant expression of splicing patterns has been linked to oncogenic processes in cancer that arise from mutations in pre- mRNA splice sites or in the protein components of the splicing machinery. Recent massively parallel sequencing studies have identified novel mutations in components of the RNA splicing machinery, such as SF3B1 in 15% of chronic lymphocytic leukaemias (CLL) , 10% of uveal melanomas, 4% of pancreatic cancers and 2% of breast cancers. These mutations have been shown to impact RNA splicing events in CLLs and uveal melanomas and breast cancers, and it is proposed that mis-spliced pre-mRNAs encode proteins that promote tumorigenesis and that these mutations result in neomorphic functions, and in some cases are associated with poor prognosis. It is unknown however, how these mutations contribute to tumorigenesis and by which mechanisms, in particular given recent studies suggesting that the inhibition of mutant SF3B1 does not inhibit proliferation. Inhibition of the spliceosome complex is a recently described way of targeting aberrant splicing and other oncogenic processes in cancer and is an area of intensive therapeutic research. Moreover spliceosome

targeting represents a new paradigm in cancer therapy as it provides an important counterpart to targeting classical single gene drivers .

However, current spliceosome inhibitors in clinical trials can have toxic side effects. Indeed a recent phase 1 clinical trial (NCT00459823) of the SF3B1 inhibitor E7107 has been suspended, suggesting that additional therapeutic approaches are warranted. Ultimately, a deeper understanding of the role of these mutations on tumour progression is needed and there remains a need in the art for new therapeutic strategies that target SF3B1 mutated cancers .

Summary of the Invention

Broadly, the present invention is based on work showing that mutations in SF3B1 are involved in RNA splicing and have been found to occur at relatively high frequencies in several tumour types including myleodysplastic syndrome (MDS) , chronic lymphocytic leukaemia (CLL) , pancreatic cancer, uveal melanoma, mucosal melanoma and breast cancer and also occur at low

frequency in many additional tumour types (Figure 1A) . Hotspot mutations occur most frequently at amino acids R625, R626, H662, K666 and K700 (Figure IB) . Mutations in these hotspot regions have been postulated as bona-fide drivers. The present inventors investigated whether dysfunction in RNA splicing is implicated in SF3B1 mutant cancers and whether tumours harbouring these mutations could be therapeutically targeted. Massively parallel RNA-sequencing of primary breast cancers identified consistent patterns of aberrant splicing in SF3B1 mutant tumours that validated in a re-analysis of RNA-sequencing data from The Cancer Genome Atlas (TCGA) . Furthermore, the present inventors observed a core set of transcripts that are consistently aberrantly spliced in SF3B1 mutant tumours irrespective of tumour type, providing a surrogate or biomarker of mutation status.

The present inventors further investigated whether SF3B1 mutant cells could be exploited therapeutically by performing a 5-day high-throughput drug cell viability screen utilising K562 isogenic cell lines harbouring the SF3B1 K700E hotspot mutation that was confirmed by qRT-PCR to show the conserved signature of alternative splicing (Figure 3A-C) . This identified that the PARP inhibitor BMN-673 (Talozaparib) selectively kills K700E mutant cells (Figure 3D) . Further validation in long-term (14 day) clonogenic assays confirmed that SF3B1 K700E mutant cells are selectively sensitive to inhibition with a number of

different PARP inhibitors including BMN-673, Olaparib (Lynparza) and Niriparib (Figure 3E-F) . Furthermore, the conserved

signature of alternative splicing was perturbed upon treatment with PARP inhibitors. The association with BMN-673 sensitivity and SF3B1 mutations, was also confirmed in an additional isogenic model NALM6 with K700E and K666N and H662Q mutations in long-term clonogenic assays (Figure 3H) . Moreover, in a non-isogenic panel of cells with and without SF3B1 hotspot mutations, the present inventors observed that mutant cells were significantly sensitive to BMN-673 treatment, with a comparable SF80 (dose where 80% of cells are alive) to BRCA1 mutant SUM149 breast cancer cells p=0.0134, t-test, (Figure 31) . Furthermore, in K562 and NALM6 SF3B1 mutant and wild-type isogenic cells, 24 hours post

radiation treatment, mutant cells showed a significant increase in the number of gamma-H2AX foci compared to wild-type cells (p=0.001 K562, and p=0.0005 NALM6) , indicative of an impaired ability to repair DNA upon damage (Figure 4) .

These findings indicate that mutations in SF3B1 are potentially therapeutically tractable in cancers using PARP inhibitors and show an impaired response to DNA damage.

In the present invention, references to SF3B1 denote Splicing Factor 3b Subunit 1 having the HGNC ID: 10768. The HUGO Gene Symbol report for SF3B1 can be found at:

http : / /www . genename s . org/ cgi- bin/gene symbol report?hgnc id=HGNC: 10768 which provides links to the SF3B1 nucleic acid and amino acid sequences, as well as reference to the homologous murine and rat proteins .

Accordingly, in a first aspect, the present invention provides a poly ADP ribose polymerase (PARP) inhibitor for use in a method of treating an individual with cancer, which is mutated or deficient in the SF3B1 gene.

In a further aspect, the present invention provides a poly ADP ribose polymerase (PARP) inhibitor for use in a method of treating an individual with cancer which is mutated or deficient in the SF3B1 gene, the method comprising:

(a) determining in a sample obtained from the individual whether the cancer has one or more mutations or deficiencies in the SF3B1 gene; and

(b) administering a therapeutically effective amount of a

PARP inhibitor to the individual with cancer which is mutated or deficient in the SF3B1 gene.

Examples of types of cancer in which there are known include mutations in the SF3B1 gene include myleodysplastic syndrome (MDS) , chronic lymphocytic leukaemia (CLL) , pancreatic cancer, uveal melanoma and breast cancer.

In a further aspect, the present invention provides a method of selecting an individual having cancer for treatment with a poly ADP ribose polymerase (PARP) inhibitor, the method comprising:

(a) determining in a sample obtained from the individual whether the cancer is mutated or deficient in the SF3B1 gene;

(b) selecting the individual for treatment with the PARP inhibitor where the cancer has one or more gene mutations or deficiencies in the SF3B1 gene; and

(c) providing a PARP inhibitor suitable for administration to the individual .

Optionally, the method may further comprises administering a therapeutically effective amount of the PARP inhibitor to the individual .

In a further aspect, the present invention provides a method for treating an individual having cancer with a poly ADP ribose polymerase (PARP) inhibitor, the method comprising:

(a) determining in a sample obtained from the individual whether the cancer is mutated or deficient in the SF3B1 gene;

(b) selecting the individual for treatment with the PARP inhibitor where the cancer has one or more gene mutations or deficiencies in the SF3B1 gene;

(c) providing a PARP inhibitor suitable for administration to the individual; and

(d) administering a therapeutically effective amount of the PARP inhibitor to the individual .

In a further aspect, the step of testing the sample to determine whether the cancer is mutated or deficient in SF3B1 gene is performed on nucleic acid sequences obtained from an individual's cancerous or non-cancerous cells Suitable techniques are well known in the art and include the use of direct sequencing, hybridisation to a probe, restriction fragment length polymorphism (RFLP) analysis, single-stranded conformation polymorphism (SSCP) , PCR amplification of specific alleles, amplification of DNA target by PCR followed by a mini-sequencing assay, allelic discrimination during PCR, Genetic Bit Analysis, pyrosequencing, oligonucleotide ligation assay, analysis of melting curves, testing for a loss of heterozygosity (LOH) , next generation sequencing (NGS) techniques, single molecule

sequencing techniques and nanostring nCounter Technology.

In other embodiments, the test is performed on RNA sequences obtained from an individual's cancerous or non-cancerous cells. In yet further embodiments, the test is performed on proteins obtained from an individual's cancerous or non-cancerous cells.

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. Recurrent SF3B1 mutations in cancer. A) Histogram of frequency of SF3B1 somatic mutations in different cancer types from a pan-cancer analysis of TCGA. B) Lollipop plot showing distribution and frequency of SF3B1 mutations along the SF3B1 protein in a pan-cancer analysis of 157 studies from TCGA

(cbioportal accessed 31st May 2017) . Note hotspot mutations at amino acids R625, N626, K666 and K700 are observed (red) . Figure 2. SF3B1 mutations are associated with signatures of differential splicing. A) Comparison of genes alternatively spliced in papillary SF3B1 K700E breast tumours, TCGA unselected breast cancer tumors and uveal melanoma SF3B1 mutants identifies a splicing program of key genes independent of tumour type. B) Plots of normalized RNA-sequencing reads for CRNDE and TMEM14C in SF3B1 wild-type (blue) and SF3B1 mutant (red) tumors. Schematic representations of the exon structures are shown above the graph with exons represented by boxes. Differentially spliced exon bins are indicated by lighter colored shading.

Figure 3. Identification of novel therapeutic vulnerabilities in SF3B1 mutant cancer. A) Sanger sequencing traces of the

heterozygous SF3B1 K700E mutation in isogenic wild-type and K700E mutant K562 cells. B) Bar plots showing relative expression of alternatively spliced isoforms/canonical isoforms of ANKHD1 and ABCC5 in K562 wild-type and K700E and NALM6 wild-type and H662Q isogenic cell lines. C) Water-fall plot of a 5 day high- throughput single agent drug screen in K562 SF3B1 K700E mutant and wild-type cells. Drug effects are ranked by the effect in mutant versus wild-type cells. Cell viability was measured with Cell Titre Glo. D) Dose response curves of screen data in K562 SF3B1 K700E mutant (red) and wild-type (black) cells, showing sensitivity of mutant cells to the PARP1/2 inhibitor BMN673 Cell viability was measured with Cell titre Glo. E) Dose response curves of 14 day clonogenic assay of K562 K700E mutant (red) and wild-type (black) cells to PARP inhibitors BMN-673, Niriparib and Olaparib, showing increased sensitivity in mutant cells. Colony numbers were quantified with ImageXpress. F) Representative phase contrast images of K562 wild-type (WT) and mutant (K700E) cells treated with increasing concentrations of BMN-673. G) Bar-plots of ratio of alternative spliced isoforms/canonical isoform of CRNDE, ABCC5 and ANKHD1H in K562 SF3B1 wild-type and K700E mutant cells) . Dose response curves of 14 day clonogenic assay of NALM6 mutant (K700E, H662Q and K666N) and wild-type (blue) cells to the PARP inhibitor BMN-673, showing increased sensitivity in mutant cells. With different hotspot mutations I) Scatter plot

depicting the SF80 (drug dose that kills 20% of cells) in short- term (5 day) assays in a panel of non-isogenic cancer cell lines ( ild-type= HEC59, HEC1A, HEC1B, MP41; Mutant= PANC05.04, ESS-1, Mel202) , BRCA1 mutant= SUM149) showing mutant cells are more sensitive to BMN-673. Cell viability was measured with Cell Titre Glo.

Figure 4. SF3B1 mutant cells show an impaired response to DNA damage. Representative immunofluorescence and quantification of γΗ2ΑΧ foci in A) K562 K700E and B) NALM6 H662Q isogenic cells +/- 4Gy irradiation, showing that SF3B1 mutant cells show impaired response to DNA damage, seen by the presence of γΗ2ΑΧ foci at 24 hours post radiation.

Figure 5. Validation of PARP inhibitor sensitivity in vivo. A)

Dose response curves in PARP knockout K562 SF3B1 parental and SF3B1 K700E mutant cells to BMN-673 showing that in both mutant and wild-type cells, knock out of PARPl protein renders cells resistant to PARP inhibitors. B) Western blot of K562 SF3B1 parental and SF3B1 K700E mutant PARPl knockout cells. C) Dose response curves of T47D isogenic SF3B1 parental and K700E mutant breast cancer cells to BMN-673 showing increased sensitivity to PARP inhibitors in mutant cells. D) In vivo growth kinetics of NALM6 isogenic K700K cells treated with PARP inhibitor BMN-673 and vehicle showing no difference in growth E) In vivo growth kinetics of NALM6 isogenic SF3B1 mutant H662Q cells treated with PARP inhibitor BMN-673 and vehicle showing a significant

difference in growth on treatment with BMN-673. F) In vivo growth kinetics of a gall bladder patient derived xenograft harboring an SF3B1 R625H mutation treated with PARP inhibitor BMN-673 and vehicle showing a significant difference in growth on treatment with BMN-673. Detailed Description

Cancers with defects (mutations, loss of expression) in the following genes associated with SF3B1 mutations In the present invention, references to cancers which are mutated or deficient in the SF3B1 gene, SF3B1 denote Splicing Factor 3b Subunit 1 having the HGNC ID: 10768. The HUGO Gene Symbol report for XXX can be found at: http://www.genenames.org/cgi- bin/gene symbol report?hgnc id=HGNC: 10768 which provides links to the SF3B1 nucleic acid and amino acid sequences, as well as reference to the homologous murine and rat proteins .

Regulated splicing of the cellular transcriptome is essential for normal growth and development. It involves removal of intronic DNA from pre-mRNA transcripts via the activity of a common set of small nuclear RNAs (snRNAs) and associated proteins which assemble together with into a complex known as the spliceosome [ 1 ] (Figure 1) . Aberrant expression of splicing patterns has been linked to oncogenic processes that arise from mutations in pre- mRNA splice sites or in the protein components of the splicing machinery themselves in multiple malignancies [2-4 ] .

Dysregulation of splicing factors have been implicated in chemoresistance [ 5 ] and endocrine resistance [ 6 ] in breast cancer. Recent evidence has highlighted that known oncogenes such as MYC impart a functional dependency on the spliceosome and govern tumour growth[7, 8]. The characterisation of novel splicing signatures in cancer as well as the identification of original signalling networks involving RNA splicing regulators should allow for novel oncogenic mechanisms to be elucidated.

Heterozygous mutations in components of the RNA splicing

machinery, underpin a number of haematologic malignancies with hotspot mutations most frequently found at amino acids K700 (K700E mutational change) [3, 4, 9, 10] . SF3B1 hotspot mutations are also frequently found in solid cancers including, 10% of uveal melanomas mainly the R625 hotspot [11], 4% of pancreatic cancers, mainly the K700 hotspot [12] and 3% of breast cancers K700 hotspot [ 13-15 ] . SF3B1 mutations have been associated with poor prognosis [ 16, 17] and impact RNA splicing events. SF3B1 mutations are proposed to result in neomorphic functions [ 18 , 19] and that mis-spliced pre-mRNAs encode proteins that promote tumorigenesis [ 20 ] , however, evidence also suggests that SF3B1 hotspot mutations induce aberrant 3' splice site selection, leading to nonsense mediated decay (NMD) and subsequent down- regulation of about half of the aberrant mRNAs [21-24] .

Spliceosomal gene mutations are known to drive hematopoietic stem-progenitor cell expansion in vivo[24, 25], however, it is unknown how these mutations contribute to tumorigenesis in solid cancers, given that inhibition of mutant SF3B1 does not inhibit proliferation [26] . Examples of types of cancer in which there are known include mutations in the SF3B1 gene include

myleodysplastic syndrome (MDS) , AML (acute myeloid leukaemia) , chronic lymphocytic leukaemia (CLL) , pancreatic cancer, uveal melanoma, breast cancer, mucosal melanoma, cutaneous melanoma, adenoid cystic carcinoma, acral melanoma, endometrial cancer, adenoid cystic carcinomas of the breast and salivary gland, bladder cancer, colorectal cancer, prostate cancer, lung cancer, medulloblastoma, ampullary carcinoma, hepatocellular carcinoma, Diffuse Large B-Cell Lymphoma, renal clear cell carcinoma, stomach adenocarcinoma, Cholangiocarcinoma, multiple myeloma, Esophagogastric Cancer, low grade glioma, Cervical cancer, colon adenocarcinoma, glioblastoma, mesothelioma, thymoma, and gall bladder cancer.

PARP Inhibitors

In the present invention, PARP inhibitors refer to compounds or substances that inhibit the expression levels or a biological activity of poly ADP ribose polymerase (PARP) . Some inhibitors are known and further examples may be found by the application o screening technologies to these targets.

PARP1 is a protein that is important for repairing single-strand breaks in DNA. If such nicks persist unrepaired until DNA is replicated (which must precede cell division) , then the

replication itself can cause double strand breaks to form. In one mode of action, it is known that PARP inhibitors can cause multiple double strand breaks to form, and in tumours mutations these double strand breaks cannot be efficiently repaired, thereby leading to the death of the cells. As normal, noncancerous cells do not replicate DNA as often as cancer cells, and generally have other homologous repair pathways working, the normal cells can survive PARP inhibition. PARP inhibitors have an additional mode of action: localizing PARP proteins at sites of DNA damage, which has relevance to their anti-tumor activity. The trapped PARP protein-DNA complexes are highly toxic to cells because they block DNA replication. Small Molecule Inhibitors

Examples of PARP inhibitors that may be used in accordance with the present invention include Olaparib (AZD2281), Rucaparib (AG014699) , Niraparib (MK4827), Talazoparib (BMN-673) , Veliparib (ABT-888), Iniparib (BSI 201), E7016, CEP 9722, BGB-290, E7449, AG-14361, INO-1001, A-966492, PJ34 HC1, UPF 1069, AZD2461,

ME0328, BGP-15 2HC1, Niraparib tosylate, NU1025, NVP-TNKS 656 ,

NMS-P118, benzamide and Picolinamide .

In the present invention references to "Olaparib" (AZD2281) denote 1- (Cyclopropylcarbonyl) -4- [5- [ (3, 4-dihydro-4-oxo-l- phthalazinyl ) methyl ] -2-fluorobenzoyl ] piperazine , having the ChemSpider ID: 23343272. The ChemSpider report for Olaparib, as well as its structure, can be found at:

http : //www . chemspider . com/Chemical-Structure .23343272. html .

In the present invention references "Rucaparib" (AG014699) denote 8-Fluoro-2- { 4- [ (methylamino ) methyl ] phenyl } -1 , 3 , 4 , 5-tetrahydro-6H- azepino [5, 4, 3-cd] indol-6-one having the ChemSpider ID: 8107584. The ChemSpider report for Rucaparib, as well as its structure, can be found at: http://www.chemspider.com/Chemical- Structure .8107584.html .

In the present invention references to "Niraparib" (MK4827) denote 2-{4-[(3S)-3-Piperidinyl] phenyl } -2H-indazole-7-carboxamide having the ChemSpider ID: 24531930. The ChemSpider report for ETP-46464, as well as the structure, can be found at

http: / /www. chemspider. com/Chemical-Structure .24531930.html . Niraparib may also be used in the form of Niraparib tosylate.

In the present invention references to "Talazoparib" (BMN-673) denote (8S, 9R) -5-Fluoro-8- (4-fluorophenyl) -9- (1-methyl-lH-l, 2, 4- triazol-5-yl)-2,7,8, 9-tetrahydro-3H-pyrido [ 4 , 3 , 2-de ] phthalazin-3- one having the ChemSpider ID: 28637772. The ChemSpider report for Talazoparib, as well as the structure, can be found at:

http : // w . chemspider . com/Chemical-Structure .28637772. html , In the present invention references to "Veliparib" (ABT-888) denote 2- [ (2R) -2-Methyl-2-pyrrolidinyl ] -lH-benzimidazole-4- carboxamide having the ChemSpider ID: 10134775. The ChemSpider report for Veliparib, as well as the structure, can be found at: http : //www . chemspider . com/Chemical-Structure .10134775. html .

In the present invention references to "Iniparib" (BSI 201) denote 4-Iodo-3-nitrobenzamide having the ChemSpider ID: 7971834. The ChemSpider report for Iniparib, as well as the structure, can be found at: http://www.chemspider.com/Chemical- Structure.7971834.html.

In the present invention references to "E7016" denote the PARP inhibitor with the IUPAC name 10- ( ( 4-Hydroxypiperidin-l- yl ) methyl ) chromeno [ 4 , 3 , 2-de ] phthalazin-3 ( 2H) -one .

In the present invention references to "CEP 9722" denote 10- (Aminomethyl ) -4,5,6, 7-tetrahydro-lH-cyclopenta [a] pyrrolo [3,4- c] carbazole-1, 3 (2H) -dione having the ChemSpider ID: 8124051. The ChemSpider report for CEP 9722, as well as the structure, can be found at: http://www.chemspider.com/Chemical- Structure.8124051.html .

In the present invention references to BGB-290 denote the PARP inhibitor with the IUPAC name (lOaR) -2-Fluoro-5, 8, 9, 10, 10a, 11- hexahydro-10a-methyl-5, 6, 7a, 11- tetraazacyclohepta [def] cyclopenta [a] fluoren-4 (7H) -one . In the present invention, references to E7449 denotes the dual inhibitor of PARPl/2 and tankyrase 1/2 with the IUPAC name 8- ( 1 , 3-Dihydro-2H-isoindol-2-ylmethyl ) -1 , 2-dihydro-3H- pyridazino [ 3 , 4 , 5-de ] quinazolin-3-one . The ChemSpider report for E7449, as well as the structure, can be found at

http : / /www . chemspider . com/Chemical- Structure .35308197.html ?rid=09851de8-06a2-4bfc-ac86-fa76184ae7c8

In the present invention, references to AG-14361 denotes the PARP inhibitor with the IUPAC name 2-{4-

[ (Dimethylamino ) methyl ] phenyl } -5 , 6-dihydroimidazo [4,5,1- jk] [ 1 , 4 ] benzodiazepin-7 ( 4H) -one . The ChemSpider report for AG- 14361, as well as the structure, can be found at

http : / /www . chemspider . com/Chemical- Structure .8015794. html ?rid=c4058806-7e85-4dal-a330-af797ca4abbb .

In the present invention, references to INO-1001 denotes the PARP inhibitor with the IUPAC name 3-Aminobenzamide . The ChemSpider report for INO-1001, as well as the structure, can be found at http : / /www . chemspider . com/Chemical- Structure .1583.html ?rid=a407418 l-540b-433c-ba85- 656eb9386625&page_num=0.

In the present invention, references to A-966492 denotes the PARP inhibitor with the IUPAC name 2- { 2-Fluoro-4- [ (2S) -2- pyrrolidinyl ] phenyl } -ΙΗ-benzimidazole-4-carboxamide . The

ChemSpider report for A-966492, as well as the structure, can be found at http://www.chemspider.com/Chemical- Structure .17599373. html ?rid=d8fb316b-e7b7-497b-ab8a- fab20356da41&page num=0.

In the present invention, references to PJ34 HC1 denotes the PARP inhibitor with the IUPAC name N ² ,N ² -Dimethyl-N- (6-oxo-5, 6-dihydro- 2-phenanthridinyl) glycinamide hydrochloride (1:1) . The

ChemSpider report for PJ34 HC1, as well as the structure, can be found at http://www.chemspider.com/Chemical- Structure .21395828. html ?rid=7329c485-d925-47 Id- 9a84- 88715307d0a4&page_num=0.

In the present invention, references to UPF 1069 denotes the PARP inhibitor with the IUPAC name 5- (2-Oxo-2-phenylethoxy) - 1 (2H) -isoquinolinone . The ChemSpider report for UPF 1069, as well as the structure, can be found at

http : / /www . chemspider . com/Chemical- Structure .24606050. html?rid=9fbed684 -3923-46d3-96c3- f6e0947b4887&page_num=0.

In the present invention, references to AZD2461 denotes the PARP inhibitor with the IUPAC name 4- { 4-Fluoro-3- [ ( 4-methoxy-l- piperidinyl ) carbonyl ] benzyl } -1 ( 2H) -phthalazinone . The ChemSpider report for AZD2461, as well as the structure, can be found at http : //www . chemspider . com/Chemical-

Structure .29315037. html ?rid=7a61b285-ef5d-4649- 92d6-ble73bdeb0al .

In the present invention, references to ME0328 denotes the PARP inhibitor with the IUPAC name 3- (4-Oxo-l, 4-dihydro-2- quinazolinyl) -N- [ (IS) -1-phenylethyl] propanamide . The ChemSpider report for ME0328, as well as the structure, can be found at http : / /www . chemspider . com/Chemical- Structure .30774269.html?rid=192d3271-272a-4de9-8c81-dafb4c85a0c2. In the present invention, references to BGP-15 2HC1 denotes the PARP inhibitor with the IUPAC name 3-Pyridinecarboximidamide , N- [2-hydroxy-3- (1-piperidinyl) propoxy] -, hydrochloride (1:2) . The ChemSpider report for BGP-15 2HC1, as well as the structure, can be found at http://www.chemspider.com/Chemical- Structure .8060481. html ?rid=ff1 f392e-db49-4c46-a9af-47887d6b9aab .

In the present invention, references to NU1025 denotes the PARP inhibitor with the IUPAC name 8-Hydroxy-2-methyl-4 ( 1H) - quinazolinone . The ChemSpider report for NU1025, as well as the structure, can be found at http://www.chemspider.com/Chemical- Structure .56978. html ?rid=c47ef4f9-03cc-425d-bb61-bf937082 lOfc .

In the present invention, references to NVP-TNKS656 denotes the Tankyrase and PARP inhibitor with the IUPAC name N- (Cyclopropylmethyl ) -2- [4- ( 4-methoxybenzoyl ) -1-piperidinyl ] -N- [ (4- oxo-1, 5,7, 8-tetrahydro-4H-pyrano [ 4 , 3-d] pyrimidin-2- yl) methyl] acetamide . The ChemSpider report for NVP-TNKS 656 , as well as the structure, can be found at

http : / /www . chemspider . com/Chemical- Structure .30819493. html?rid=5dd982bf-87d4-4481-98c2-a8066ff25c2e.

In the present invention, references to NMS-P118 denotes the PARP1 inhibitor with the IUPAC name 2- [1- (4, 4-

Difluorocyclohexyl ) piperidin-4-yl ] -6-fluoro-3-oxo-2 , 3-dihydro-lH- isoindole-4-carboxamide . See https : / / www . generon . co . uk/other- products-186/nms-pll 8-1262417-51-5--231035782. html In the present invention, references to Picolinamide denotes the PARP inhibitor with the IUPAC name 2-Pyridinecarboxamide . The ChemSpider report for Picolinamide, as well as the structure, can be found at http://www.chemspider.com/Chemical-

Structure .14342.html ?rid=484683af-467e-4ald-aa08-15a024101ba5. siRNA Inhibitors

Another class of inhibitors useful for treatment of SF3B1 mutated or deficient cancer includes nucleic acid inhibitors which inhibit activity or function by down-regulating production of active PARP 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.

Expression of PARP may be inhibited using anti-sense or RNAi technology. The use of these approaches to down-regulate gene expression is now well-established in the art.

Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of the base excision repair pathway component so that its expression is reduced or completely or substantially completely prevented. 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 ) : 3675-3684 , 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 heterologous 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'-0-methyl- ; 2-0- alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro- ; 2 ' -halo or 2; azido-ribose , carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars and sedoheptulose .

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-thiouraci

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 .

Antibodies

Antibodies may be employed in the present invention as an example of a class of inhibitor useful for treating SF3B1 gene mutated or deficient cancer, and more particularly as inhibitors of PARP . 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 the SF3B1 gene 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 of recombinant expression of a gene fusion encoding antibody and reporter molecule. One favoured mode is by covalent linkage of each antibody with an individual fluorochrome, phosphor or laser exciting dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine .

Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.

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

Treatment of cancer

The present invention provides methods and medical uses for the treatment of SF3B1 gene deficient or mutated cancers with PARP inhibitors. A SF3B1 gene deficient or mutated cancer may be identified as such by testing a sample of cancer cells from an individual, for example to determine whether the SF3B1 gene contains one or more mutations. Examples of cancers having SF3B1 deficiencies or mutations are set out in described above with reference to [3, 4, 9-20] . Thus, types of cancer in which there are known include mutations in the SF3B1 gene include

myleodysplastic syndrome (MDS) , AML (acute myeloid leukaemia) , chronic lymphocytic leukaemia (CLL) , pancreatic cancer, uveal melanoma, breast cancer, mucosal melanoma, cutaneous melanoma, adenoid cystic carcinoma, acral melanoma, endometrial cancer, adenoid cystic carcinomas of the breast and salivary gland, bladder cancer, colorectal cancer, prostate cancer, lung cancer, medulloblastoma, ampullary carcinoma, hepatocellular carcinoma, Diffuse Large B-Cell Lymphoma, renal clear cell carcinoma, stomach adenocarcinoma, Cholangiocarcinoma, multiple myeloma, Esophagogastric Cancer, low grade glioma, Cervical cancer, colon adenocarcinoma, glioblastoma, mesothelioma, thymoma, and gall bladder cancer. Aberrant expression of splicing patterns has been linked to oncogenic processes in cancer that arise from mutations in premRNA splice sites or in the protein components of the splicing machinery (Bonomi et al . , Oncogenic alternative splicing

switches : role in cancer progression and prospects for therapy, Int. J. Cell Biol. 2013. 2013: p. 962038.2) . Recent massively parallel sequencing studies have identified novel mutations in components of the RNA splicing machinery, such as SF3B1 in:

15% of chronic lymphocytic leukaemias (CLL) , see Quesada et al . , Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia. Nat. Genet., 44(1) : 47-52, 2012;

10% of uveal melanomas, see Furney et al . , SF3B1 mutations are associated with alternative splicing in uveal melanoma. Cancer Discovery, 3(10) : 1122-9, 2013;

4% of pancreatic cancers, see Biankin et al . , Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes,

Nature, 491(7424) : 399-405, 2012;

2% of breast cancers, see Ellis et al., Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature, 2012. 486(7403) : 353-60, 2012; and Network, Comprehensive molecular portraits of human breast tumours. Nature, 490(7418) : 61-70, 2012.

In some embodiments, the SF3Bl-deficient cancer is characterised by one or more SF3B1 gene mutation (s) or defects (s) occurring in somatic pre-cancerous or cancerous cells. While SF3B1 mutations are mostly believed to be somatic, there is some evidence SF3B1 mutations are associated with clonal hematopoiesis, e.g. as a result of ageing, and there may also be SF3B1 deficient cancers characterised by one or more SF3B1 gene mutation (s) occurring in the germ line of the individual patient. In some embodiments, a SF3B1 gene deficient or mutated cancer may be identified as such by testing a sample of cancer cells from an individual to determine the expression of the SF3B1 gene to evaluate whether expression of the protein is absent or at a reduced level compared to normal .

In other embodiments, the SF3B1-deficient cancer is characterised by the cancer cells having a defect in or the cancer cells exhibiting epigenetic inactivation of a SF3B1 gene, or loss of protein function.

More generally, a cancer may be identified as a SF3B1 deficient cancer by determining the activity of the SF3B1 polypeptides in a sample of cells from an individual. The sample may be of normal cells from the individual where the individual has a mutation in the SF3B1 gene or the sample may be of cancer cells, e.g. where the cells forming a tumour exhibit defects in SF3B1 activity. Activity may be determined relative to a control, for example in the case of defects in cancer cells, a relative to non-cancerous cells, preferably from the same tissue. The activity of the SF3B1 gene may be determined by using techniques well known in the art such as Western blot analysis, immunoprecipitation, immunohistology, chromosomal abnormalities, enzymatic or DNA binding assays, and plasmid-based assays.

The sample may be of normal cells from the individual where the individual has a mutation in the SF3B1 gene or the sample may be of cancer cells, e.g. where the cells forming a tumour contain one or more SF3B1 gene 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 determination of SF3B1 gene expression may involve

determining the presence or amount of SF3B1 gene 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 SF3B1 gene mRNA (i) using a labelled probe that is capable of hybridising to the SF3B1 gene nucleic acid; and/or (ii) using PCR involving one or more primers based on a SF3B1 gene nucleic acid sequence to determine whether the SF3B1 gene transcript is present in a sample. The probe may also be immobilised as a sequence included in a microarray. It is also possible to use quantitative PCR or nanostring nCounter technology to assess the downstream consequences of mutation, i.e. in the case of SF3B1 mutations alternative splicing of the 'core' set of transcripts we have identified to be alternatively spliced.

In one embodiment, detecting SF3B1 gene mRNA is carried out by extracting RNA from a sample of the tumour and measuring

expression of one or more SF3B1 gene specifically using

quantitative real time RT-PCR. Alternatively or additionally, the expression of SF3B1 gene 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.

In some embodiments, a cancer may be identified as SF3B1

deficient or mutated by determining the presence in a cell sample from an individual's tumour of one or more chromosomal

abnormalities, for example deletions in part or loss of entire chromosomes, corresponding to gene loss. Chromosomal

abnormalities may be visualised through any karyotyping technique known in the art, including but not limited to Giemesa staining, quinacrine staining, Hoechst 33258 staining, DAPI (4'-6- diamidino-2-phenylindole ) staining, daunomycin staining, and fluorescence in situ hybridization.

In some embodiments, a cancer may be identified as a SF3B1 deficient or mutated cancer by determining the presence in a cell sample from the individual of one or more variations, for example, polymorphisms or mutations, in a nucleic acid encoding a SF3B1 polypeptide. Sequence variations such as mutations and polymorphisms may include a deletion, insertion or substitution of one or more nucleotides, relative to the wild-type nucleotide sequence. The one or more variations may be in a coding or non-coding region o the nucleic acid sequence and may reduce or abolish the

expression or function of the SF3B1 gene. In other words, the variant nucleic acid may encode a variant polypeptide which has reduced or abolished activity or may encode a wild-type

polypeptide which has little or no expression within the cell, for example through the altered activity of a regulatory element A variant nucleic acid may have one or more mutations or polymorphisms relative to the wild-type sequence.

Alternatively or additionally, in the present invention the determination of whether a patient has a SF3B1 cancer can be carried out by analysis of SF3B1 protein expression, for example by examining whether levels of SF3B1 protein are supressed.

In some aspects, the presence or amount of SF3B1 protein may be determined using a binding agent capable of specifically binding to the SF3B1 protein, or fragments thereof. A preferred type of SF3B1 protein binding agent is an antibody capable of

specifically binding the SF3B1 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 SF3B1 protein.

Alternatively, or additionally, the method for determining the presence of a SF3B1 protein may be carried out on tumour samples for example using immunohistochemical (IHC) analysis or in situ RNA-hybridisation . 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 the SF3B1 protein. In a further aspect, the present invention provides an assay comprising :

measuring or quantifying a mutation or deficiency in a SF3B1 gene in a biological sample obtained from an individual with cancer; and

comparing the measured or quantified amount of the SF3B1 gene with a reference value, and if the SF3B1 gene is mutated or deficient relative to the reference value, identifying the individual as having an increased probability of being responsive to treatment with a PARP inhibitor.

Methods of screening for PARP inhibitors

The present invention also includes methods of screening that employ PARP as a protein target for the screening of candidate compounds to find PARP inhibitors. Accordingly, methods of screening may be carried out for identifying candidate agents that are capable of inhibiting PARP, for subsequent use of development as agents for the treatment of SF3B1 gene mutated or deficient. 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 PARP can then be determined in the presence and absence of the one or more candidate compounds to determine whether a given candidate is a PARP inhibitor.

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 SF3B1 gene 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 t 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.

Pharmaceutical compositions

The active agents herein for the treatment of SF3B1 deficient or mutated 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.

These compounds or derivatives of them may be used in the present invention for the treatment of SF3B1 deficient or mutated 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 include prodrugs of the compounds which 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 SF3B1 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 SF3B1 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 non- aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. 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 mg/ml, for example from about 10 ng/ml to about 1 mg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.

Compositions comprising agents disclosed herein for the treatment of SF3B1 gene mutated or deficient cancer may be used in the methods described herein in combination with standard

chemotherapeutic regimes or in conjunction with radiotherapy. As radiotherapy also leads to DNA strand breaks, causing severe DNA damage and leading to cell death, the combination of radiotherapy with PARP inhibitors offers the potential to lead to formation of double strand breaks from the single-strand breaks generated by the radiotherapy in tumor tissue. This combination could therefore lead to either more powerful therapy with the same radiation dose or similarly powerful therapy with a lower radiation dose, potentially avoiding some of the side effects with radiotherapy.

By way of example, additional agents that might be employed in combination with the use of PARP inhibitors as disclosed herein include one or more spliceosomal inhibitors, for example agents that target components of the spliceosome, such as SF3B1, e.g. using a SF3B1 inhibitor. In particular, small molecule inhibitors of SF3B1 are known in the art, see for example

Effenberger et al . (2017), Modulating splicing with small molecular inhibitors of the spliceosome. WIREs RNA, 8: n/a, el381. doi : 10.1002/wrna.1381 and Bonnal et al . (2012) Nature Reviews Drug Discovery 11: 847-859. Spliceosomal inhibitors are being tested in clinical trials, for example the testing of H3B- 8800, see https://www.ncbi.nlm.nih.gov/pubmed/29457796 and Nature Medicine, 24: 497-504, 2018.

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) or Vinorelbine (Navelbine) , or the PARP inhibitors (Olaparib (Lynparza) , Rucaparib, Niraparib, Veliparib and Talazoparib) . 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 mg 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 Examples

Spliceosomal gene mutations result in conserved alternative splicing signatures

In a pan-cancer analysis of The Cancer Genome Atlas (Figure 1A) , we identified mutations in SF3B1 to be present in additional malignancies to that previously reported, including lung cancer, gall bladder cancer and bladder cancer. The most common

mutational hotspots among all cancer types are mutations at amino acidsR625, N626, K666 and K700. We have previously employed DEXSeq to identify a common set of alternatively spliced

transcripts in SF3B1 K700E mutant breast versus subtype matched wild-type tumours and have

shown that these can be used as a surrogate of driver mutation status. Using a phenotype 2:1 match (for example for breast cancer based on stage, ER, HER2, TP53 and PIK3CA) status of wild- type versus mutant tumours identified a core set of genes that are aberrantly spliced. Additional non-hotspot SF3B1 mutant tumours were identified and identified a common set of genes that overlap with K700E mutations, indicating a similar neomorphic function (Figure 2A-B) .

SF3B1 mutant cancers are sensitive to PARP inhibitors

We further investigated whether SF3B1 mutant cells could be exploited therapeutically by performing a 5-day high-throughput drug cell viability screen utilising K562 isogenic cell lines harbouring the SF3B1 K700E hotspot mutation that was confirmed by qRT-PCR to show the conserved signature of alternative splicing (Figure 3A-C) . This identified that the PARP inhibitor BMN-673 (Talozaparib) selectively kills K700E mutant cells (Figure 3D) . Further validation in long-term (14 day) clonogenic assays confirmed that SF3B1 K700E mutant cells are selectively sensitive to inhibition with a number of different PARP inhibitors

including BMN-673, Olaparib (Lynparza) and Niriparib (Figure 3E- F) . Furthermore, the conserved signature of alternative splicing was perturbed upon treatment with PARP inhibitors. The

association with BMN-673 sensitivity and SF3B1 mutations, was also confirmed in an additional isogenic model NALM6 with K700E and K666N and H662Q mutations in long-term clonogenic assays

(Figure 3H) . Moreover in a non-isogenic panel of cells with and without SF3B1 hotspot mutations, we observed that mutant cells were significantly sensitive to BMN-673 treatment, with a comparable SF80 (dose where 80% of cells are alive) to BRCA1 mutant SUM149 breast cancer cells p=0.0134, t-test, (Figure 31) .

SF3B1 mutant cells show defective DNA repair

K562 and NALM6 SF3B1 mutant and wild-type isogenic cells, were treated with 4gy of irradiation. 24 hours post radiation

treatment, mutant cells showed a significant increase in the number of gamma-H2AX foci compared to wild-type cells (p=0.001 K562, and p=0.0005 NALM6) , indicative of an impaired ability to repair DNA upon damage (Figure 4) . These findings indicate that mutations in SF3B1 are potentially therapeutically tractable in cancers, and show an impaired response to DNA damage.

Validation of PARP inhibitor sensitivity in vivo. A) Dose response curves in PARP knockout K562 SF3B1 parental and SF3B1 K700E mutant cells to BMN-673 showing that in both mutant and wild-type cells, knock out of PARPl protein renders cells resistant to PARP inhibitors. This shows that sensitivity to PARP inhibitors is therefore likely mediated through the

inhibitor's action on PARP trapping rather than inhibition of its enzymatic activity. B) Western blot of K562 SF3B1 parental and SF3B1 K700E mutant PARPl knockout cells showing depletion of the PARP1 protein in knockout cells. C) Dose response curves of T47D isogenic SF3B1 parental and K700E mutant breast cancer cells grown as 3D spheroids to BMN-673 showing increased sensitivity to PARP inhibitors in mutant cells. D) In vivo growth kinetics of NALM6 isogenic K700K cells treated with PARP inhibitor BMN-673 and vehicle showing no difference in growth E) In vivo growth kinetics of NALM6 isogenic SF3B1 mutant H662Q cells treated with PARP inhibitor BMN-673 and vehicle showing a significant

difference in growth on treatment with BMN-673, indicating that patients with SF3B1 mutations would benefit from PARP inhibitor treatments. F) In vivo growth kinetics of a gall bladder patient derived xenograft harboring an SF3B1 R625H mutation treated with PARP inhibitor BMN-673 and vehicle showing a significant

difference in growth on treatment with BMN-673, indicating that patients with SF3B1 mutations would benefit from PARP inhibitor treatments .

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