Treating Cancer

Field of the Invention

The present invention relates cyclin dependent kinase 4/ 6 inhibitors for use in methods of treating an individual with a cancer

characterised by CREB-binding protein deficiency and/or up- regulation of F0XM1 expression and to corresponding methods of selecting and treating patients for treatment.

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.

CDK4/ 6 inhibitors are a proven therapeutic strategy for oestrogen receptor positive (ER+) breast cancers (BC) . However the molecular determinants of sensitivity and resistance to CDK4/6 inhibition are unclear . Summary of the Invention

Broadly, the present invention rises from work to identify further patient populations who would benefit from treatment with CDK4/6 inhibitors, such as Palbociclib. Recent next generation sequencing studies have comprehensively mapped the genetic landscape of breast cancers and revealed that only a small number of genes are

recurrently mutated. However, a number of lower frequency mutations are bona-fide drivers. The present invention utilized a functional genomics approach silencing the 200 most frequently mutated genes in breast cancer in 3 dimensional (3D) spheroid cultures to more accurately recapitulate in vivo-like conditions of solid tumours, using the MCF10A progression series cell line panel as a model of disease progression. Several genes were identified whose silencing impacted growth in two or more cell lines. Furthermore, a second targeted validation screen showed that silencing of a cohort of these genes, including the histone acetyltransferase CREBBP, promoted growth in 3D but had limited effect under traditional 2D culture conditions in both the MCF10 progression series and a larger panel of triple negative cell line models.

Investigation of TCGA and METABRIC datasets showed that up to a third of TNBCs display haploinsufficiency either through mutations or copy number loss of CREBBP, which resulted in the up-regulation of the pro-proliferative transcription factor FOXM1. Furthermore, CREBBP-deficient cells displayed up-regulation of a conserved FOXM1- driven transcriptional programme in multiple CREBBP-altered solid tumours including lung, oesophageal, bladder and endometrial cancers. Screening of cancer cell line spheroids with drugs that targeted FOXM1 activity uncovered multiple compounds that were synthetically lethal with CREBBP status. As FOXM1 is a downstream target of CDK4 and CDK6 (Anders, Ke et al . 2011) sensitivity of CREBBP-null cells to the CDK4/6 inhibitors palbociclib and

abemaciclib in short term spheroid assays was assessed. This resulted in a clear enhancement of sensitivity in CREBBP- null cells compared to wild-type. The observed increased sensitivity was further enhanced in combination with paclitaxel, suggesting a Gl/S- G2/M blockade may provide benefit in this model. Sensitivity to palbociclib was not observed in 2D culture, suggesting that hypoxic and nutrient gradients influence the spheroid response .

Concordantly, short-term spheroid cultures of CREBBP-deficient Hs578T showed enhanced sensitivity to palbociclib. These data support our hypothesis that certain TNBC cells maybe sensitive to CDK4/6 perturbation.

The present inventors found that CREBBP is a bona fide tumour suppressor in up to a third of triple negative breast cancers

(TNBCs), as well as a wide range of other solid tumours. In preferred embodiments, CREBBP-altered tumours display up-regulation of F0XM1, which alters cancer cell metabolism under nutrient stress conditions. CREBBP-altered tumours are selectively sensitive to inhibitors that target F0XM1 activity, suggesting that this maybe a viable therapeutic approach for CREBBP altered cancers. CREBBP- altered tumours display FOXM1 up-regulation and were selectively sensitive to CDK4/6 inhibition, therefore suggesting that either CREBBP -deficiency and/or FOXM1 up-regulation could be biomarkers for response to CDK4/6 inhibition.

In the present invention, references to CREBBP denote CREB binding protein having the HGNC ID: 2348. The HUGO Gene Symbol report for CREBBP can be found at: http://www.genenames.org/cgi- bin/gene symbol report?hgnc id=2348 which provides links to the CREBBP nucleic acid and amino acid sequences, as well as reference to the homologous murine and rat proteins .

In the present invention, references to F0XM1 denote Forkhead Box Ml protein having the HGNC ID: 3818. The HUGO Gene Symbol report for F0XM1 can be found at: http://www.genenames.org/cgi- bin/gene symbol report?hgnc id=3818which provides links to the CREBBP 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 cyclin dependent kinase 4/6 (CDK4/6) inhibitor for use in a method of treating an individual having a cancer characterised by CREB- binding protein (CREBBP) deficiency and/or up-regulation of FOXM1 expression .

In a further aspect, the present invention provides a cyclin dependent kinase 4/6 (CDK4/6) inhibitor for use in a method of treating an individual with a cancer characterised by CREB-binding protein (CREBBP) deficiency and/or up-regulation of FOXM1

expression, the method comprising:

(a) determining in a sample obtained from the individual whether the cancer is characterised by loss of CREBBP gene

expression or activity and/or by up-regulation of FOXM1 expression; and

(b) administering a therapeutically effective amount of a CDK4/ 6 inhibitor to the individual with CREB-binding protein

(CREBBP) deficient cancer.

In a further aspect, the present invention provides the use of a cyclin dependent kinase 4/ 6 (CDK4/ 6) inhibitor in the manufacture of a medicament for the treatment of an individual with a cancer characterised by CREB-binding protein (CREBBP) deficiency and/or up- regulation of FOXM1 expression.

In a further aspect, the present invention provides a kit comprising a cyclin dependent kinase 4/6 (CDK4/6) inhibitor and instructions for using the CDK4/6 inhibitor for treating an individual with a cancer characterised by CREB-binding protein (CREBBP) deficiency and/or up-regulation of FOXM1 expression.

In a further aspect, the present invention provides a method of selecting an individual having cancer for treatment with a CDK4/6 inhibitor, the method comprising:

(a) determining in a sample obtained from the individual whether the individual has a cancer characterised by CREB-binding protein (CREBBP) deficiency and/or up-regulation of FOXM1

expression;

(b) selecting the individual for treatment with the CDK4/6 inhibitor where the individual has a cancer characterised by CREB- binding protein (CREBBP) deficiency and/or up-regulation of FOXM1 expression; and

(c) providing a CDK4/6 inhibitor suitable for administration to the individual .

In some embodiments, the method further comprises administering a therapeutically effective amount of the CDK4/ 6 inhibitor to the individual .

In a further aspect, the present invention provides a method of treating an individual having cancer, the method comprising:

(a) determining in a sample obtained from the individual whether the individual a cancer characterised by CREB-binding protein (CREBBP) deficiency and/or up-regulation of FOXM1

expression;

(b) selecting the individual for treatment with the CDK4/6 inhibitor where the individual has a cancer characterised by CREB- binding protein (CREBBP) deficiency and/or up-regulation of FOXM1 expression;

(c) providing a CDK4/6 inhibitor suitable for administration to the individual; and

(d) administering a therapeutically effective amount of the CDK4/6 inhibitor to the individual.

In the present invention, the step of testing the sample to determine whether the cancer is mutated or deficient in CREBBP gene is performed on nucleic acid sequences obtained from an individual' 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 nanostrmg 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 : Functional genomics screen identifies a novel tumour suppressor gene CREBBP whose silencing impacts growth in a 3D- specific manner. (A) Heat-map displaying z-scores of the biological effect of gene ablation in the MCF10 progression series of spheroids (MCFlOa, (MCFlO)NeoT, (MCFIO)ATI, (MCF10 ) DCI S . com, (MCFIO)CAIA, (MCFIO)CAID and (MCFIO)CAIH) (light grey is increasing z-score, dark grey is decreasing z-score) . (B) Correlation of screen of top 50 hits from ATI ( TP53 wild-type (WT) ) and CA1H ( TP53 mutant) cells rescreened in 2D and spheroids, showing spheroid specific hits.

Killing controls, PLKl and UBB and potential tumour suppressors CREBBP and KMT2C are highlighted. (C) Representative DCIS.com spheroids +/- siRNA mediated silencing of CREBBP showing increased size and proliferation (Ki67) immunohistochemsitry (IHC) . (D)

Heatmap of spheroid siRNA screen in TNBC lines indicating CREBBP as the top hit, whereby CREBBP silencing leads to increased spheroid growth in multiple TNBC spheroids. Note the majority of cell lines harbour TP53 mutations (CAL-51= TP53 WT, COSMIC) (E) HAP1 CREBBP isogenic cells display increased spheroid size and cell viability only in 3D (barchart) .

Figure 2 : CREBBP expression is altered in triple negative breast cancers resulting in increased dependency on the pro-proliferative factor FOXMl . (A) Distribution of CREBBP-deficiency (mutations and copy number loss of CREBBP) in TNBC from The Cancer Genome Atlas (TCGA) and METABRIC. 32% of TNBC's from TCGA and 25% TNBC s in METABRIC show CREBBP-deficiency . (B) CREBBP mRNA expression in TNBC patients from TCGA, showing significant lower levels of CREBBP in deficient cells. (C) Kaplan Meier curve showing significant

association of low CREBBP mRNA and relapse free survival in Basal- like tumours as a surrogate of TNBC. This was significant in multivariate analyses. (D) Barchart from the analysis of reverse phase protein array data from TCGA comparing CREBBP deficient (n=45) versus WT(n=94) TNBC's identified increased expression of FOXMl and a number of its downstream targets (proliferation related, Cyclin El, Cyclin Bl, Cyclin E2), DNA repair related (XRCC1, BRAC2 ) (FDR corrected p values) . (E) Dose response curve depicting selective sensitivity of CREBBP mutant HAP1 cells to thiostrepton (an

inhibitor of FOXMl function) , indicating mutant cells are dependent on FOXMl activity for sustained growth. (F) Representative

micrographs and proportions of cases from IHC of CREBBP and FOXMl in primary TNBC from a tissue microarray (n=67) highighting a

reciprocal relationship with CREBBP and FOXMl expression.

Figure 3: CREBBP deficient cells are sensitive to CDK4/6 inhibition.

(A) Short term 5 day dose response curves in HAP1 CREBBP mutant and WT cells showing increased sensitivity to palbociclib (survival fraction, SF50 of mutant cells = 348uM) and Abemaciclib in spheroid cultures. (B) Barchart depicting relative spheroid viability

(assessed with cell-titre glo) of combination treatment of palbocicib with paclitaxel as standard of care of TNBC, showing significant decreased cell viability in CREBBP mutant cells. (C) Short term 5 day dose response curves of a panel of TNBC spheroids to palbocicib. Note CREBBP deficient Hs578T TNBC cells (dark grey) are more sensitive than MCF7 (mid grey) and wild-type CREBBP TNBC cells (light grey) in the short-term assay, having a SF of 62nM. Hs578T are known to express low levels of CREBBP mRNA (Z score <-2 TCGA RNA-seq data) . Note all cell lines have functional RB. Figure 4: CREBBP/FOXMl axis is altered in multiple solid tumours.

(A) Frequency of alterations in CREBBP in multiple solid tumours

(TCGA) . Heterozygous deletion of the CREBBP locus is highlighted in light grey, homozygous is highlighted in dark grey and mutations are highlighted in mid grey. (B) Recurrent CREBBP alterations in Uterine corpus endometrial cancers (TCGA) show reciprocal alterations in FOXM1 , Cyclin Bl and Cyclin El protein expression (reverse phase protein array, RPPA) . The percentage (%) of total alterations per dataset is displayed. Mutations are highlighted in mid grey

(missense) and black (truncating) . Deletion of one copy of the CREBBP locus is highlighted in mid grey, loss of all copies is highlighted in dark grey. (C) Overall survival of endometrial cancer patients stratified on CREBBP status is also shown. (D) Recurrent CREBBP alterations in bladder cancer, stomach cancer and lung cancer

(TCGA) show reciprocal alterations in FOXM1 , Cyclin Bl and Cyclin El protein expression (RPPA) . The percentage (%) of total alterations per dataset is displayed. Mutations are highlighted in mid grey

(missense) and black (truncating) . Deletion of one copy of the CREBBP locus is highlighted in light grey, loss of all copies is highlighted in dark grey.

Figure 5: CREBBP is altered in other tumour types. CREBBP protein expression was assessed in multiple solid tumours including

endometrial, ovarian, bladder and squamous cell lung cancers utilising tissue microarrays. Barplot represents the proportion of cases with no (absent) , low, medium and high expression using the

Allred scoring system. B) Representative immunohistochemistry images are shown. Figure 6: CREBBP mutant cells are sensitive to CDK4/61 in vivo.

A) Cancer cell line spheroids were treated with increasing

concentrations of Palbociclib for five days. Spheroid viability was determined with CellTitre Glo. B) NCI-H520 cells were engrafted into mice. Once tumours had reached 200mm ³ , mice were randomised into vehicle and treatment arms and treated with Palbociclib (lOOmg/kg) daily and tumour sizes measured every 2-3 days. C)

Immunohistochemical straining of representative tumours harvested from B) for Ki67 as a marker of proliferation and H&E. D) Barplots showing quantification of mitotic counts and proliferative index showing a significant reduction in mitotic count in Palbociclib treated tumours.

Detailed Description

CDK4/6 Inhibitors

In the present invention, CDK4/6 inhibitors refer to compounds or substances that inhibit the expression levels or a biological activity of cyclin dependent kinase (CDK) 4 and 6. Some inhibitors are known and further examples may be found by the application of screening technologies to these targets.

Small Molecule Inhibitors

Examples of CDK4/6 inhibitors that may be used in accordance with the present invention include Palbociclib, Abermaciclib (LY2835219 ) , Ribociclib (LEE011), Trilaciclib (G1T28), Voruciclib (P1446A-05), NSC 625987, PD 0332991 isethionate or Ryuvidine .

In the present invention, references to Palbociclib denote 6-Acetyl- 8-cyclopentyl-5-methyl-2- { [5- ( 1-piperazinyl ) -2- pyridinyl ] amino }pyrido [ 2 , 3-d] pyrimidin-7 ( 8H) -one, having the

ChemSpider ID: 4487437. The ChemSpider report for Palbociclib, as well as its structure, can be found at:

http : //www . chemspider . com/Chemical-Structure .4487437. html . In the present invention, references to Abermaciclib denote

N- { 5- [ ( 4 -Ethyl-1-piperazinyl ) methyl ] -2 -pyridinyl }-5-fluoro-4- (4- fluoro-l-isopropyl-2 -methyl- lH-benzimidazol-6-yl ) -2-pyrimidinamine having the ChemSpider ID: 29340700. The ChemSpider report for Abermaciclib, as well as its structure, can be found at:

http : // w . chemspider . com/Chemical -Structure .29340700. html . In the present invention, references to Ribociclib denote

7 -Cyclopentyl-N, -dimethyl -2 - { [5- (1-piperazinyl) -2-pyridinyl ] amino } - 7H-pyrrolo [ 2 , 3-d] pyrimidine- 6-carboxamide having the ChemSpider ID: 30798107. The ChemSpider report for Abermaciclib, as well as its structure, can be found at: http://www.chemspider.com/Chemical- Structure.30798107.html.

In the present invention, references to Trilaciclib (G1T28) , denote 2 ' - { [5- ( 4 -Methyl -1-piperazinyl ) -2-pyridinyl ] amino } -7 ' , 8 ' -dihydro- 6 ' H-spiro [cyclohexane-1, 9 ' -pyrazino [1 ' , 2 ' : 1, 5] pyrrolo [2,3- d] pyrimidin] -6 ' -one having the ChemSpider ID: 58825997. The

ChemSpider report for Trilaciclib (G1T28) , as well as its structure, can be found at: http://www.chemspider.com/Chemical- Structure.58825997.html. In the present invention, references to Voruciclib (P1446A-05) denote 2- [2-Chloro-4- (trifluoromethyl) phenyl] -5, 7-dihydroxy-8- [ (2R, 3S) -2- (hydroxymethyl ) -l-methyl-3-pyrrolidinyl ] -4H-chromen-4-one having the ChemSpider ID: 29165867. The ChemSpider report for Voruciclib (P1446A-05) , as well as its structure, can be found at: http : //www . chemspider . com/Chemical -Structure .29165867. html .

In the present invention, references to NSC 625987 denote 1,4- Dimethoxy-9 (ΙΟίί) -acridinethione having the ChemSpider ID: 2274471. The ChemSpider report for NSC 625987, as well as its structure, can be found at: http://www.chemspider.com/Chemical-

Structure .2274471. html ?rid=bcfeca7a-48b4-4690-8b62- 66a7dcebc68b .

In the present invention, references to PD 0332991 isethionate or Palbociclib isethionate denote 6-acetyl-8-cyclopentyl-5-methyl-2- [ [5- (1-piperazinyl) -2-pyridinyl] amino ] pyrido [ 2 , 3-d] pyrimidin-7 (8H) - one isethionate salt having the ChemSpider ID: 9653501. The

ChemSpider report for PD 0332991 isethionate, as well as its structure, can be found at: http://www.chemspider.com/Chemical- Structure.9653501.html ?rid=a5539fb8-ccle- 4559- 958c- 3afb2b81d5d9&page num=0.

In the present invention, references to Ryuvidine denote 2-Methyl-5- [ (4-methylphenyl) amino] -1, 3-benzothiazole-4, 7-dione having the ChemSpider ID: 422633. The ChemSpider report for Ryuvidine, as well as its structure, can be found at: http://www.chemspider.com/Chemical- Structure .422633. html ?rid=7ece3dc2-33bf-427 f-b637-88a351b4b4 lb . siRNA Inhibitors

Another class of inhibitors useful for treatment of CREBBP mutated or deficient cancer/FOXMl up-regulated cancer includes nucleic acid inhibitors which inhibit activity or function by down-regulating production of active CREBBP 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 CREBBP or F0XM1 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 m 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-O-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 , 4-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 . Antibodies

Antibodies may be employed in the present invention as an example of a class of inhibitor useful for treating CREBBP gene mutated or deficient cancer, and more particularly as inhibitors of FOXMl. 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 CREBBP gene mutated or deficient cancer or cancer

characterised by FOXMl up-regulation 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 CREBBP deficient or mutated cancers and/or FOXM1 up- regulated cancers with CDK4/6 inhibitors. A CREBBP deficient or mutated cancer/FOXMl up-regulated cancer may be identified as such by testing a sample of cancer cells from an individual, for example to determine whether the CREBBP gene contains one or more mutations. Examples of cancers with CREBBP mutations include breast cancer, lung cancer, follicular lymphoma, diffuse large B cell lymphoma, cutaneous squamous cell carcinoma, bladder cancer, uterine

carcinosarcoma, colorectal cancer, stomach cancer (Figure 4A) (Iyer, Ozdag et al . 2004, Mullighan, Zhang et al . 2011, Pasqualucci,

Dominguez-Sola et al . 2011, Garcia-Ramirez , Tadros et al . 2017) . In the present invention, alternatively or additionally, the cancer may be characterised by an up-regulation of FOXMl expression. Types of cancer that may be treated include cancer triple negative breast cancer (TNBC) , i.e. breast cancer that is oestrogen receptor- negative, progesterone receptor-negative and HER2 receptor-negative , or basal breast cancer, for example where the cancer lacks ER, PR and HER2 positivity, e.g. as assessed by routine

immunohistochemistry, or by the PAM50 gene expression assay. Other types of cancer that may be CREBBP deficient include oesphogeal cancer, bladder cancer or lung cancer (Figure 4A) . The cancers treatable according to the present invention may also be

characterised by an up-regulation of FOXMl expression.

In some embodiments, the CREBBP-deficient cancer is characterised by one or more CREBBP gene mutation (s) or defects (s) occurring in somatic pre-cancerous or cancerous cells. In yet another

embodiment, the CREBBP deficient cancer is characterised by one or more CREBBP gene mutation (s) occurring in the germ line of the individual patient. The same approaches may be employed to

determine whether the cancer is characterised by up-regulation of FOXMl expression.

In some embodiments, a CREBBP 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 CREBBP gene to evaluate whether expression of the protein is absent or at a reduced level compared to normal. The same approaches may be employed to determine whether the cancer is characterised by up-regulation of FOXMl expression.

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

More generally, a cancer may be identified as a CREBBP deficient cancer and/or characterised by up-regulation of FOXMl expression by determining the activity of the CREBBP and/or FOXMl 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 CREBBP gene or the sample may be of cancer cells, e.g. where the cells forming a tumour exhibit defects in CREBBP 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 CREBBP or FOXMl genes 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 sample may be of normal cells from the individual where the individual has a mutation in the CREBBP gene or the sample may be cancer cells, e.g. where the cells forming a tumour contain one o more CREBBP gene mutations . Activity may be determined relative 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 CREBBP gene expression may involve determining the presence or amount of CREBBP 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 CREBBP gene mRNA (i) using a labelled probe that is capable of hybridising to the CREBBP gene nucleic acid; and/or (ii) using PCR involving one or more primers based on a CREBBP gene nucleic acid sequence to determine whether the CREBBP gene transcript is present in a sample. The probe may also be immobilised as a sequence included in a microarray. The same approaches may be employed to determine whether the cancer is characterised by up-regulation of FOXMl expression. In one embodiment, detecting CREBBP gene mRNA is carried out by extracting RNA from a sample of the tumour and measuring expression of one or more CREBBP gene specifically using quantitative real time RT-PCR. Alternatively or additionally, the expression of CREBBP 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. Alternatively this can be achieved using nanostring nCounter technology, which is useful for formalin fixed degraded material.

In some embodiments, a cancer may be identified as CREBBP 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 CREBBP

deficient 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 CREBBP 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 of the nucleic acid sequence and may reduce or abolish the expression or function of the CREBBP 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, m the present invention the

determination of whether a patient has a CREBBP cancer can be carried out by analysis of CREBBP protein expression, for example by examining whether levels of CREBBP protein are supressed.

In some aspects, the presence or amount of CREBBP protein may be determined using a binding agent capable of specifically binding to the CREBBP protein, or fragments thereof. A preferred type of CREBBP protein binding agent is an antibody capable of specifically binding the CREBBP 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 CREBBP protein. The same approaches may be employed to determine whether the cancer is characterised by up-regulation of FOXMl expression by measuring the amount of FOXMl protein in a sample (Figure 2F) .

Alternatively, or additionally, the method for determining the presence of a CREBBP 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 the CREBBP protein.

In a further aspect, the present invention provides an assay comprising :

measuring or quantifying a mutation or deficiency in a CREBBP gene and/or up-regulation of FOXMl in a biological sample obtained from an individual with cancer; and

comparing the measured or quantified amount of the CREBBP gene and/or FOXMl gene expression with a reference value, and if the CREBBP gene is mutated or deficient relative to the reference value and/or if the FOXMl gene is up-regulated relative to a reference value, identifying the individual as having an increased probability of being responsive to treatment with a CDK4/6 inhibitor. As indicated above, this many also entail measuring or quantifying measuring both CREBBP and F0XM1 expression or quantifying to determine whether the cancer is also characterised by up-regulation of FOXM1 expression and a deficiency in CREBBP expression.

Methods of screening for CDK4/6 inhibitors

The present invention also includes methods of screening candidate compounds to find CDK4/6 inhibitors. Accordingly, methods of screening may be carried out for identifying candidate agents that are capable of inhibiting CDK4/6, for subsequent use of development as agents for the treatment of a cancer characterised by a CREBBP gene mutation or deficiency or up-regulation of F0XM1 expression. 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 CDK4/6 can then be determined in the presence and absence of the one or more candidate compounds to determine whether a given candidate is a CDK4/6 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 CREBBP mutated or deficient cancer/FOXMl up-regulated 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 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 ί 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.

Pharmaceutical Composi tions

The active agents herein for the treatment of CREBBP deficient/FOXMl up-regulated 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 CREBBP deficient/FOXMl up-regulated 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 CREBBP 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, wi 11 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 CREBBP 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 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 CREBBP gene mutated or deficient cancer/FOXMl up-regulated 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 CDK4/ 6 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. Examples of preferred further chemotherapeutic agents that may be employed in combination with the present invention include administration of a taxane, such as Paclitaxel, Docetaxel, Abraxane and Taxostere, eribulin, vinoebulin or a MCTl/4 inhibitor or a metabolic inhibitor or a monocarboxylase transporters

inhibitors, such as AZD3965.

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 (Vuraon) , Thiotepa, Tioguanine (6-TG) (Lanvis), Topotecan (Hycamtin) , Treosulfan, Vinblastine (Velbe) , Vincristine (Oncovin) , Vindesine (Eldisine) or 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 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

CREBBP is a novel tumour suppressor gene

Studies of genomic drivers have relied largely on 2D modelling and do not accurately recapitulate the clinical environment. Spheroid cultures of the triple-negative MCFIOA progression series were used in functional genomics screens to identify novel tumour suppressor genes. Targeting the 200 most frequently mutated genes in BC identified the histone acetyltransferase (HAT) CREBBP as a novel tumour suppressor in spheroid but not 2D culture (Figure 1) . CREBBP deficiency leads to FOXMl up-regulation

CREBBP regulates global gene transcription by altering the acetylation state of histones and non-histones and has been associated with the progression of several tumour types (Iyer, Ozdag et al . 2004, Dietze, Bowie et al . 2005, Mullighan, Zhang et al . 2011, Pasqualucci, Dominguez-Sola et al . 2011) . We noted one third of TNBC's harbour genomic alterations in CREBBP (copy number (CN) loss and mutations) and associated with poor survival (Figure 2A-C) . TNBC's harbouring CREBBP genomic alterations ( CREBBP-deficient ) have increased abundance of FOXMl and its downstream transcriptional targets (CCNB1, CCNE1, XRCC1 and BRCA2) (Figure 2D) . Furthermore, CREBBP-null cells are selectively dependent on FOXMl activation as they showed selective sensitivity to FOXMl transcriptional binding inhibitors (Figure 2E) . Furthermore, primary breast cancers with high FOXMl protein showed loss or low levels of CREBBP protein expression (Figure 2F) .

CREBBP-deficient cells are sensitive to CDK4/6 inhibition

CREBBP-null isogenic cells show up-regulation of FOXMl and selective sensitivity to a number of CDK4/ 6 inhibitors including palbociclib and abemaciclib (Figure 3A) . Notably FOXMl is a downstream target of CDK4 and CDK6 (Anders, Ke et al . 2011) . The observed increased sensitivity was further enhanced in combination with paclitaxel (Figure 3B) , suggesting a G1/S-G2/M blockade may provide benefit in this model. Sensitivity to palbociclib was not observed in 2D culture, suggesting that hypoxic and nutrient gradients influence the spheroid response. Concordantly, short-term (5 day) spheroid cultures of CREBBP-deficient Hs578T showed enhanced sensitivity to palbociclib (SF50=62nM) , (Figure 3C) . These data support our hypothesis that CREBBP-deficient of FOXMl up-regulated cells may be sensitive to CDK4/6 perturbation.

CREBBP-deficient cells show metabolic re-wiring

CDK4/ 6 is known to activate FOXMl and supresses reactive oxygen species protecting cells from senescence. We hypothesised metabolic plasticity influenced sensitivity of CREBBP-deficient cells to palbociclib in spheroid models. Concordantly our analysis showed that CREBBP-deficient spheroids derive approximately 50% of their energetic demands from acetate, compared to below 5% for CREBBP-ΉΊ spheroids (Figure 4E) . Rescue of the phenotype by addition of acetate highlighted dependency on the FOXMl axis (Figure 4E) . These results also highlight the potential for use of acetate-based positron-emission tomography (PET) as a probe of tissue metabolism via catabolic or anabolic pathways mediated by acetyl-CoA. ^X1 C- acetate imaging has been shown to be more sensitive and accurate than glucose-based PET for prostate cancer. For example acetate- based tracers (e.g. nc-acetate) could be used as pharmacodynamic biomarkers to interrogate metabolic changes and monitor response to palbociclib in CREBBP-altered cancers.

The CREBBP/FOXMl axis is altered in multiple solid tumours

Investigation of additional tumour types identified that CREBBP is altered through copy number and mutations in many different cancers (Figure 4A) . CREBBP-deficiency is associated with a poorer overall survival in uterine carcinomas (Figure 4B) . Analysis of data from TCGA identified that CREBBP deficient cancers show up-regulation of FOXM1 and its down-stream targets akin to triple-negative breast cancer (Figure 4C) . This suggests CREBBP deficiency leads to FOXM1 up-regulation in multiple cancer types, all of which may benefit from CDK4/6 inhibitor treatment.

CREBBP protein expression was assessed in multiple solid tumours including endometrial, ovarian, bladder and squamous cell lung cancers utilising tissue microarrays. Figure 5A shows barplot representations of the proportion of cases with no (absent) , low, medium and high expression using the Allred scoring system, which Figure 5B provides representative immunohistochemistry images.

Experiments were then carried out to determine whether CREBBP mutant cells are sensitive to CDK4/6i in vivo. Cancer cell line spheroids were treated with increasing concentrations of Palbociclib for five days, as shown in Figure 5A. Figure 5B shows the results of experiments in which NCI-H520 cells were engrafted into mice. Once tumours had reached 200mm ³ , mice were randomised into vehicle and treatment arms and treated with Palbociclib (lOOmg/kg) daily and tumour sizes measured every 2-3 days. These experiments showed a significant reduction in the mitotic count of Palbociclib treated tumours . References

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

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