WO2016128744A1 - Senescence - Google Patents

Abstract

The invention provides an inhibitor of a Tec family tyrosine kinase and uses thereof. The invention also extends to methods of treating, ameliorating or preventing senescence or an age-related disorder or a solid tumour in a subject. The invention also extends to Tec family tyrosine kinase gene-silencing molecules, pharmaceutical compositions, methods of making the compositions and compound screening assays.

Description

SENESCENCE

The present invention relates to senescence, and particularly, although not exclusively, to the treatment or prevention of senescence and/or age-related pathologies and/ or cancer. The invention also extends to pharmaceutical

compositions for use in treating such conditions, and to methods of treatment. The invention further extends to reducing senescence in biological cells.

The tumour suppressor p53 mediates cellular responses to DNA damage. It is a transcription factor capable of up-regulating a series of target genes that stop transformed cells by triggering mechanisms such as cell cycle arrest or apoptosis. Several factors cooperate to determine which of these responses will be induced by P53 after different stresses, although not all of them are known. A part from its antineoplastic activity, p53 has also been involved in ageing through the induction of senescence, a permanent cell cycle arrest in which cells remain metabolically active and adopt characteristic morphological changes. Senescent cells often appear multinucleated and large, and exhibit a spindle shape and vacuolization features. The establishment of this phenotype may be the result of telomere shortening after a number of cell divisions (replicative senescence) or a response to stress stimuli (stress-induced premature senescence, SIPS). Expression of oncogenes, such as Ras, cyclin E, E2f3 or Raf can also trigger it (oncogene-induced senescence, OIS), which underscores the role of senescence in tumour suppression. Indeed, senescent cells in vivo are often observed in pre-malignant and early stages of solid cancers, after which they may disappear. This suggests that the senescent barrier needs to be overcome in order to progress into full malignancy.

Senescence has long been associated with age-dependent organismal changes, and the accumulation of senescent cells with time has been shown to contribute to the functional impairment of different organs typically seen in ageing. These cells are typically eliminated by a mechanism known as immune surveillance. However, a progressive reduction in the rates of senescent cell clearance leads to their increase in many tissues. Senescent cells secrete growth factors, chemokines and cytokines, known together as the senescence-associated secretory phenotype (SASP), which have the effect of increasing angiogenesis as well as cell proliferation and

transformation. All this led to the hypothesis that senescence is an antagonistically pleiotropic process, with beneficial effects in the early decades of life of the organism (as a tumour suppressor mechanism) but detrimental to fitness and survival in later stages, due to promotion of ageing and tumourigenesis, mainly through SASP. Consistent with this view, it has been recently shown that clearing senescent cells from tissues has a protective effect against ageing. Using a mouse model genetically engineered to induce apoptosis in cells as they senesce, it has been demonstrated that the onset of age-related pathologies can be delayed when senescent cells are eliminated from the organism. This experiment is the first in vivo evidence of amelioration of the symptoms associated with old age through the suppression of senescent cell accumulation in tissues.

The effectors and modulators of senescence are not completely understood and the molecular mechanisms involved in the process remain to be fully elucidated. The

P53"P2i and/or pi6-RB pathways seem to participate in the induction of all forms of senescence, regardless of the triggering stimulus. In vivo suppression of p53 and/or its upstream regulator ARF is enough to prevent senescence in some models.

However, other cell types rely primarily on pi6 for triggering senescence. p2i, a p53 target gene, has often been considered critical for establishing senescence, whereas pi6 could be more involved in the maintenance of the phenotype, in coordination with an increase in intracellular Reactive Oxygen Species (ROS). There are many other genes known to be up-regulated in senescent cells, such as PPPiA, Smurf2 and PGM.

There is therefore a need to provide novel and improved therapies for preventing the accumulation of senescent cells in tissues. This could have a great impact on age- related disorders, cancer, fibrosis, diabetes and other diseases and conditions in which senescent cells may increase.

To this end, and using mass spectrometry on previously reported models of cellular senescence, the inventors have determined a number of proteins highly expressed in association with the plasma membranes of human senescent cells, but not in their dividing counterparts. Among them, they identified a number of non-receptor tyrosine kinases, for example the Bruton's tyrosine kinase (BTK), which is a nonreceptor tyrosine kinase that participates in the B-cell Receptor (BCR) signalling pathway. BTK belongs to the TEC family of kinases, which share a similar domain structure, including SRC homology domains SH2 and SH3, a catalytic region, an amino terminal PH domain and a proline rich region that contains a finger motif. BTK has a role in development, survival and differentiation of B-cell lineages, and it has been found to be mutated in the inherited immunodeficiency X-linked agammaglobulinaenia (XLA). It is highly expressed in different types of leukaemias and lymphomas and, because of this, BTK inhibitors have been recently approved for treating B-cell malignancies.

The inventors have now uncovered a previously unknown role for BTK in P53- mediated responses to stress. They have shown that it is induced after P53 expression and that it contributes to maintaining the levels of p53 protein through a positive feedback loop, likely including activation of DNA damage signals. Moreover, blocking BTK resulted in a decrease in P53 levels and the abrogation of senescence, with chemical inhibition of BTK surprisingly prolonging lifespan and fitness in vivo in an invertebrate model. They conclude that BTK is involved in organismal ageing through its enhancement of the p53 signal in cellular senescence and that its inhibition can ameliorate age-related symptoms in vivo. The inventors believe that this inhibitory effect is not limited to inhibition of BTK, but to any Tec family tyrosine kinase. They also believe that inhibition of Tec family tyrosine kinases, such as BTK, can be used to treat solid tumours, in which the accumulation of senescent cells plays an important role enhancing progression.

Therefore, in a first aspect of the invention, there is provided an inhibitor of a Tec family tyrosine kinase, for use in the treatment, amelioration or prevention of senescence or an age-related disorder or a solid tumour.

In a second aspect, there is provided a method of treating, ameliorating or preventing senescence or an age-related disorder or a solid tumour in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of an inhibitor of a Tec family tyrosine kinase.

Surprisingly, the inventors have demonstrated that Tec family tyrosine kinases (preferably Bruton's Tyrosine Kinase, BTK) were capable of reducing the

accumulation of senescent cells in an invertebrate model, which resulted in increased mobility and weight as well and up to 20% lifespan extension, confirming the in vivo relevance of their findings. The inventors have shown that, despite its role as a driver of B cell malignancies, BTK as well as other related kinases have tumour suppressor functions in solid cancers. Their inhibition blocks p53-induced senescence and thus prolongs lifespan in vivo, and can therefore be used to treat any age-related disorder.

The inventors propose that Tec tyrosine kinase inhibitors, which are already being used clinically, could also be useful as adjuvant therapies in early-stage solid tumours or after chemo/radio therapy, as well as to prevent age-dependent fitness loss.

Inhibitors of Tec family tyrosine kinases have not been used in the context of treating solid tumours because, until now, there was no rationale to believe that they would have any effect. The data provided herein surprisingly suggests, however, that such inhibitors would not be directed against and kill the cancer cells in solid tumours (as they do in leukaemias), but to the microenvironment around the cancer cells (i.e. the senescent cells and the factors secreted thereby), and would prevent the

accumulation of senescent cells. The result of this would be prevention of solid tumour growth. Accordingly, the invention described herein is a novel use of the Tec family tyrosine kinase inhibitors, because they are targeting different cells, i.e. not the cancer cells per se, but the cells that would help the cancer cells grow instead) and this has never been demonstrated before. It would not have been an obvious application of Tec family tyrosine kinase inhibitors, because without the data described herein, the skilled person would never have thought these inhibitors could be employed to treat solid tumours.

The Tec family of non-receptor tyrosine kinases consists of six proteins

designated Tec, namely: Emt (NCBI Accession Number: 3702; also known as Itk or Tsk); Btk (NCBI Accession Number: 695; previously known as Atk,

BPK or Emb); Bmx (NCBI Accession Number: 660); Txk (NCBI Accession Number: 7294; also known as Rlk) and Dsrc28C (NCBI Accession Number: 34132).

As such, preferably the Tec family tyrosine kinase inhibitor used in the invention inhibits Tec, Emt, Btk, Bmx, Txk or Dsrc28C. Preferably, however, the Tec family tyrosine kinase inhibitor inhibits Btk.

The genomic DNA sequence of the Btk gene, which encodes human BTK (NCBI Accession Number: 695) is provided herein as SEQ ID No:i: 1 aactgagtgg ctgtgaaagg gtggggtttg ctcagactgt ccttcctctc tggactgtaa

61 gaatatgtct ccagggccag tgtctgctgc gatcgagtcc caccttccaa gtcctggcat

121 ctcaatgcat ctgggaagct acctgcatta agtcaggact gagcacacag gtgaactcca

181 gaaagaagaa gctatggccg cagtgattct ggagagcatc tttctgaagc gatcccaaca

241 gaaaaagaaa acatcacctc taaacttcaa gaagcgcctg tttctcttga ccgtgcacaa 301 actctcctac tatgagtatg actttgaacg tgggagaaga ggcagtaaga agggttcaat

361 agatgttgag aagatcactt gtgttgaaac agtggttcct gaaaaaaatc ctcctccaga

421 aagacagatt ccgagaagag gtgaagagtc cagtgaaatg gagcaaattt caatcattga

481 aaggttccct tatcccttcc aggttgtata tgatgaaggg cctctctacg tcttctcccc

541 aactgaagaa ctaaggaagc ggtggattca ccagctcaaa aacgtaatcc ggtacaacag 601 tgatctggtt cagaaatatc acccttgctt ctggatcgat gggcagtatc tctgctgctc

661 tcagacagcc aaaaatgcta tgggctgcca aattttggag aacaggaatg gaagcttaaa

721 acctgggagt tctcaccgga agacaaaaaa gcctcttccc ccaacgcctg aggaggacca

781 gatcttgaaa aagccactac cgcctgagcc agcagcagca ccagtctcca caagtgagct

841 gaaaaaggtt gtggcccttt atgattacat gccaatgaat gcaaatgatc tacagctgcg 901 gaagggtgat gaatatttta tcttggagga aagcaactta ccatggtgga gagcacgaga 961 taaaaatggg caggaaggct acattcctag taactatgtc actgaagcag aagactccat 1021 agaaatgtat gagtggtatt ccaaacacat gactcggagt caggctgagc aactgctaaa 1081 gcaagagggg aaagaaggag gtttcattgt cagagactcc agcaaagctg gcaaatatac 1141 agtgtctgtg tttgctaaat ccacagggga ccctcaaggg gtgatacgtc attatgttgt 1201 gtgttccaca cctcagagcc agtattacct ggctgagaag caccttttca gcaccatccc 1261 tgagctcatt aactaccatc agcacaactc tgcaggactc atatccaggc tcaaatatcc 1321 agtgtctcaa caaaacaaga atgcaccttc cactgcaggc ctgggatacg gatcatggga 1381 aattgatcca aaggacctga ccttcttgaa ggagctgggg actggacaat ttggggtagt 1441 gaagtatggg aaatggagag gccagtacga cgtggccatc aagatgatca aagaaggctc 1501 catgtctgaa gatgaattca ttgaagaagc caaagtcatg atgaatcttt cccatgagaa 1561 gctggtgcag ttgtatggcg tctgcaccaa gcagcgcccc atcttcatca tcactgagta 1621 catggccaat ggctgcctcc tgaactacct gagggagatg cgccaccgct tccagactca 1681 gcagctgcta gagatgtgca aggatgtctg tgaagccatg gaatacctgg agtcaaagca 1741 gttccttcac cgagacctgg cagctcgaaa ctgtttggta aacgatcaag gagttgttaa 1801 agtatctgat ttcggcctgt ccaggtatgt cctggatgat gaatacacaa gctcagtagg 1861 ctccaaattt ccagtccggt ggtccccacc ggaagtcctg atgtatagca agttcagcag 1921 caaatctgac atttgggctt ttggggtttt gatgtgggaa atttactccc tggggaagat 1981 gccatatgag agatttacta acagtgagac tgctgaacac attgcccaag gcctacgtct 2041 ctacaggcct catctggctt cagagaaggt atataccatc atgtacagtt gctggcatga 2101 gaaagcagat gagcgtccca ctttcaaaat tcttctgagc aatattctag atgtcatgga 2161 tgaagaatcc tgagctcgcc aataagcttc ttggttctac ttctcttctc cacaagcccc 2221 aatttcactt tctcagagga aatcccaagc ttaggagccc tggagccttt gtgctcccac 2281 tcaatacaaa aaggcccctc tctacatctg ggaatgcacc tcttctttga ttccctggga 2341 tagtggcttc tgagcaaagg ccaagaaatt attgtgcctg aaatttcccg agagaattaa 2401 gacagactga atttgcgatg aaaatatttt ttaggaggga ggatgtaaat agccgcacaa 2461 aggggtccaa cagctctttg agtaggcatt tggtagagct tgggggtgtg tgtgtggggg 2521 tggaccgaat ttggcaagaa tgaaatggtg tcataaagat gggaggggag ggtgttttga 2581 taaaataaaa ttactagaaa gcttgaaagt c

[SEQ ID NO:l]

The amino acid sequence of human BTK (Accession Number: NP_000052.i) is provided herein as SEQ ID No: 2, as follows.

MAAVILESIFLKRSQQKKKTSPLNFKKRLFLLTVHKLSYYEYDF

ERGRRGSKKGSIDVEKITCVETVVPEKNPPPERQIPRRGEESSEMEQISIIERFPYPF QWYDEGPLYVFSPTEELRKRWIHQLKNVIRYNSDLVQKYHPCFWIDGQYLCCSQTAK NAMGCQILENRNGSLKPGSSHRKTKKPLPPTPEEDQILKKPLPPEPAAAPVSTSELKK WALYDYMPMNANDLQLRKGDEYFILEESNLPWWRARDKNGQEGYIPSNYVTEAEDSI EMYEWYSKHMTRSQAEQLLKQEGKEGGFIVRDSSKAGKYTVSVFAKSTGDPQGVIRHY WCSTPQSQYYLAEKHLFSTIPELINYHQHNSAGLISRLKYPVSQQNKNAPSTAGLGY GSWEIDPKDLTFLKELGTGQFGVVKYGKWRGQYDVAIKMIKEGSMSEDEFIEEAKVMM NLSHEKLVQLYGVCTKQRPIFIITEYMANGCLLNYLREMRHRFQTQQLLEMCKDVCEA MEYLESKQFLHRDLAARNCLVNDQGWKVSDFGLSRYVLDDEYTSSVGSKFPVRWSPP EVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYERFTNSETAEHIAQGLRLYRPHLASE KVYTIMYSCWHEKADERPTFKILLSNILDVMDEES

[SEQ ID NO: 2] Thus, preferably the inhibitor prevents or reduces the expression of Btk gene or activity of BTK protein, wherein the BTK protein comprises an amino acid sequence substantially as set out in SEQ ID No: 2, or a functional variant or fragment thereof, or wherein BTK is encoded by the nucleic acid substantially as set out in SEQ ID No: l, or a functional variant or fragment thereof. Since senescence is an antagonistic pleiotropic process, with beneficial effects early in life as a tumour suppressor but detrimental for ageing and tumour progression when senescent cells accumulate in tissues, there is currently a debate regarding the use of therapies focused on senescence. Whereas it would be interesting to enhance the senescent pathway in order to stop cancer cell progression, this would likely lead to even more senescent cell accumulation and an undesirable increase of its side effects. There would therefore be a higher health impact if the accumulation of senescent cells is pharmacologically prevented instead. Thus, the inventors propose the use of Tec kinase inhibitors (such as a BTK inhibitor) to eliminate the negative effects of senescent cell accumulation in the contexts of solid tumour progression and ageing. For instance, these inhibitors could be given simultaneously to treatments such as chemo or radiotherapy, both known to induce senescence.

Hence, preferably the inhibitor is used to treat, ameliorate or prevent senescence. The inventors have determined that the effects of BTK inhibitors require the presence of a functional p53. More preferably, therefore, the inhibitor is used to treat, ameliorate or prevent p53-induced senescence.

In a third aspect, there is provided use of an inhibitor of a Tec family tyrosine kinase, for preventing or reducing senescence in a biological cell.

Preferably, the use is carried out on the cell in vitro or ex vivo.

Preferably the inhibitor is used to treat, ameliorate or prevent an age-related disorder. Examples of age-related disorders which may be treated include progeria, frailty, diabetes, liver fibrosis, neurodegenerative diseases, sarcopenia and wrinkles.

Preferably, the inhibitor is used to treat, ameliorate or prevent growth of a solid tumour. For example, the solid tumour may be selected from a group consisting of: nevus; melanoma; adenoma; colon adenoma; dermal neurofibroma; and prostate intraepithelial neoplasis. Each of these has been shown to have high levels of senescent cells. It will be appreciated that chemotherapy and radiotherapy should increase the number of senescent cells in any responsive solid tumour. Preferably, however, the tumour is not blood-borne. Preferably, leukaemias and lymphomas do not constitute solid tumours, and are not treated using the inhibitors described herein. Surprisingly, the inventors have shown that p53-induced senescence can be bypassed by using either chemical inhibitors or RNAi, which resulted in senescing cells escaping the permanent arrest and resuming proliferation. BTK and other related tyrosine kinases have not been implicated before in pathways that stop the cell cycle. The inventor's hypothesis is that its effects on senescence are mediated through the positive feedback loop established by p53, as their results suggest. In the absence of BTK, for example, the signals that lead to p53 stabilization would not be reinforced and its functions in the senescent pathway would be compromised. Inhibitors capable of decreasing the biological activity of the Tec family tyrosine kinase may achieve their effect by a number of means. For instance, such inhibitors may:-

(a) bind to the Tec family tyrosine kinase to reduce its biological activity;

(b) decrease the expression of the gene encoding the Tec family tyrosine kinase; or

(c) inhibit translocation of the Tec family tyrosine kinase from the

nucleus to the cytosol.

In one embodiment, the inhibitor may directly interact with the Tec family tyrosine kinase, e.g. (a) above. Preferred inhibitors for use according to the invention may comprise small molecule inhibitors, which may be identified as part of a high throughput screen of small molecule libraries.

As described in the Examples, the inventors have also shown that the Tec family tyrosine kinase, preferably BTK, can be inhibited by Ibrutinib and CGI-1746, and there are other inhibitors which could also be used. The Tec family tyrosine kinase inhibitor may be selected from a group consisting of Ibrutinib (available under the trade name Imbruvica™), GDC-0834, RN-486, CGI-560, CGI-1746, ACP-196, HM- 71224, CC-292 (AVL-292), ONO-4059 (ONO-WG-307), CNX-774 and LFM-A13. Currently, the only one that is commercially available is Ibrutinib, and so none of the others have trade names yet. Although they cannot usually be bought through regular sources, it is possible to get them custom synthesized, for example by MedChem Express or MedKoo. Preferably, the Tec family tyrosine kinase inhibitor, which is preferably a BTK inhibitor, is Ibrutinib or CGI-1746. In another embodiment, the inhibitor may comprise an antibody raised against the Tec family tyrosine kinase, i.e. a Tec family tyrosine kinase neutralising antibody. The antibody maybe polyclonal or monoclonal. Conventional hybridoma techniques may be used to raise the antibodies, and are well-known in the art.

In yet another embodiment, the inhibitor according to the invention may comprise an inactive peptide fragment of the Tec family tyrosine kinase, which competes with endogenous Tec family tyrosine kinase and thereby reduces its activity. For instance, truncation mutants of Tec family tyrosine kinase that do not bind to nucleic acid or other transcription factors, and which inhibit the ability of the Tec family tyrosine kinase to bind nucleic acid, may also be used as inhibitors of the invention.

In a further embodiment, the inhibitor may prevent or reduce expression of the Tec family tyrosine kinase (i.e. (b) above).

As described in the Examples, the inventors have demonstrated that inhibition of BTK expression by RNAi successfully reduced the accumulation of senescent cells in an invertebrate model, which resulted in increased mobility and weight as well and up to 20% lifespan extension, confirming the in vivo relevance of their findings.

Therefore, the inhibitor according to the invention may be a gene-silencing molecule.

The term "gene-silencing molecule" can mean any molecule that interferes with the expression of the gene encoding the Tec family tyrosine kinase. Such molecules can include, but are not limited to, RNAi molecules, including siNA, siRNA, miRNA, shRNA, ribozymes and antisense molecules. The use of such molecules represents an important aspect of the invention.

Therefore, according to a fourth aspect of the present invention, there is provided a Tec family tyrosine kinase gene-silencing molecule, for use in the treatment, amelioration or prevention of senescence or an age-related disorder or a solid tumour.

Gene-silencing molecules may be antisense molecules (antisense DNA or antisense RNA) or ribozyme molecules. Ribozymes and antisense molecules may be used to inhibit the transcription of the gene encoding the Tec family tyrosine kinase, preferably BTK. Antisense molecules are oligonucleotides that bind in a sequence- specific manner to nucleic acids, such as DNA or RNA. When bound to mRNA that has a complimentary sequence, antisense RNA prevents translation of the mRNA. Triplex molecules refer to single antisense DNA strands that bind duplex DNA forming a colinear triplex molecule, thereby preventing transcription. Particularly useful antisense nucleotides and triplex molecules are ones that are complimentary to, or bind, the sense strand of DNA (or mRNA) that encodes the Tec family tyrosine kinase, preferably BTK.

The expression of ribozymes, which are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA substrates, may also be used to block protein translation. The mechanism of ribozyme action involves sequence specific hybridisation of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage, e.g. hammerhead motif ribozymes. It is preferred that the gene-silencing molecule is a short interfering nucleic acid (siNA). The siNA molecule may be double-stranded and therefore comprises a sense and an antisense strand. The siNA molecule may comprise an siDNA molecule, or an siRNA molecule or an shRNA molecule. However, it is preferred that the siNA molecule comprises an siRNA molecule. Hence, the siNA molecule according to the invention preferably down-regulates gene expression by RNA interference (RNAi).

RNAi is the process of sequence specific post-transcriptional gene-silencing in animals and plants. It uses small interfering RNA molecules (siRNA) that are double- stranded and homologous in sequence to the silenced (target) gene. Hence, sequence specific binding of the siRNA molecule with mRNAs produced by transcription of the target gene allows very specific targeted 'knockdown' of gene expression.

Preferably, the siNA molecule is substantially identical with at least a region of the coding sequence of the Tec family tyrosine kinase encoding gene (see above) to enable down-regulation of the gene. Preferably, the degree of identity between the sequence of the siNA molecule and the targeted region of the Tec family tyrosine kinase gene is at least 6o% sequence identity, preferably at least 75% sequence identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 97% identity, and most preferably at least 99% identity. The siNA molecule may comprise between approximately 5bp and 5obp, more preferably between lobp and 35bp, even more preferably between i5bp and 30 bp, and yet still more preferably, between i6bp and 25bp. Most preferably, the siNA molecule comprises less than 22 bp.

Design of a suitable siNA molecule is a complicated process, and involves very carefully analysing the sequence of the target mRNA molecule. Using considerable inventive endeavour, the inventors have chosen a defined sequence of siNA which has a certain composition of nucleotide bases, which they have shown has the required affinity and also stability to cause the RNA interference. The siNA molecule may be either synthesised de novo, or produced by a micro-organism. For example, the siNA molecule may be produced by bacteria, for example E.coli.

It will be appreciated that such siNAs may comprise uracil (siRNA or shRNA) or thymine (siDNA). Accordingly, the nucleotides U and T, as referred to above, may be interchanged. However, it is preferred that siRNA or shRNA is used.

Especially preferred siNA molecule sequences, which are adapted to down-regulate expression of the BTK gene may comprise the following sequences, i.e. the cDNA- targeted region and the sequence of the siRNA duplexes for Btk are as follows:

Btk siR A-i (accession no. NM_....000061 and L29788); targeted region (cDNA): ^l682- TTG GTAA ACG ATCA AGG AG ~^':70° [SEQ ID No:3]

sense siRNA: 5'-UUGGUAAACGAUCAAGGAGUU [SEQ ID No:4l;

antisense siRNA: UUAACCAUUUGCUAGUUCCUC-s' [SEQ ID N0.5];

Btk siRNA-2; targeted region (cD A): ^8t¾~GGGAAAGAAGGAGGTTTCA~9-3 [SEQ ID No:63;

sense siRNA: s'-GGGAAAGAAGGAGGUUUCAUU [SEQ ID No:?];

antisense siRNA: UUCCCLWCUL^TCCUCCAAAGU-5' [SEQ ID No:8];

Btk siRNA -3; targeted region (cDNA): ^G ^GCTTAAAACCTGGGAG^ [SEQ ID No: ₉];

sense siRNA: s'-GAAGCUUAAAACCUGGGAGUU [SEQ ID No:i()];

antisense siRNA: UUCUUCGAAUUUUCCACCCUC-5' [SEQ ID No:ii]; The siRNA of any of SEQ ID No. 4, 5, 7, 8, 10 or 11 maybe used as a siNA molecule for use according to the present invention.

Gene-silencing molecules used according to the invention are preferably nucleic acids (e.g. siRNA, miRNA, shRNA, antisense or ribozymes). Such molecules may (but not necessarily) be ones, which become incorporated in the DNA of cells of the subject being treated. Undifferentiated cells may be stably transformed with the gene- silencing molecule leading to the production of genetically modified daughter cells (in which case regulation of expression in the subject may be required, e.g. with specific transcription factors, or gene activators).

The gene-silencing molecule may be either synthesised de novo, and introduced in sufficient amounts to induce gene-silencing (e.g. by RNA interference) in the target cell. Alternatively, the molecule may be produced by a micro-organism, for example, E.coli, and then introduced in sufficient amounts to induce gene-silencing in the target cell.

The molecule may be produced by a vector harbouring a nucleic acid that encodes the gene-silencing sequence. The vector may comprise elements capable of controlling and/ or enhancing expression of the nucleic acid. The vector may be a recombinant vector. The vector may for example comprise plasmid, cosmid, phage or virus DNA. In addition to, or instead of using the vector to synthesise the gene-silencing molecule, the vector may be used as a delivery system for transforming a target cell with the gene-silencing sequence.

The recombinant vector may also include other functional elements. For instance, recombinant vectors can be designed such that the vector will autonomously replicate in the target cell. In this case, elements that induce nucleic acid replication maybe required in the recombinant vector. Alternatively, the recombinant vector may be designed such that the vector and recombinant nucleic acid molecule integrates into the genome of a target cell. In this case nucleic acid sequences, which favour targeted integration (e.g. by homologous recombination) are desirable. Recombinant vectors may also have DNA coding for genes that may be used as selectable markers in the cloning process.

The recombinant vector may also comprise a promoter or regulator or enhancer to control expression of the nucleic acid as required. Tissue specific promoter/enhancer elements may be used to regulate expression of the nucleic acid in specific cell types, for example, vasculature cells. The promoter maybe constitutive or inducible.

Alternatively, the gene-silencing molecule may be administered to a target cell or tissue in a subject with or without it being incorporated in a vector. For instance, the molecule may be incorporated within a liposome or virus particle (e.g. a retrovirus, herpes virus, pox virus, vaccina virus, adenovirus, lentovirus and the like).

Alternatively a "naked" siNA or antisense molecule may be inserted into a subject's cells by a suitable means e.g. direct endocytotic uptake.

The gene-silencing molecule may also be transferred to the cells of a subject to be treated by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. For example, transfer may be by: ballistic transfection with coated gold particles; liposomes containing an siNA molecule; viral vectors comprising a gene-silencing sequence or means of providing direct nucleic acid uptake (e.g. endocytosis) by application of the gene-silencing molecule directly.

In a preferred embodiment of the present invention, siNA molecules maybe delivered to a target cell (whether in a vector or "naked") and may then rely upon the host cell to be replicated and thereby reach therapeutically effective levels. When this is the case, the siNA is preferably incorporated in an expression cassette that will enable the siNA to be transcribed in the cell and then interfere with translation (by inducing destruction of the endogenous mRNA coding the kinase, preferably BTK). In another embodiment, the inhibitor may inhibit of BTK translocation from the nucleus into the cytoplasm (i.e. (c) above).

It will be appreciated that inhibitors according to the invention may be used in a medicament, which maybe used in a monotherapy, i.e. use of only an inhibitor (e.g. Ibrutinib or CGI-1746 or siNA molecule) for treating, ameliorating, or preventing a disease-related disease or a solid tumour. Alternatively, inhibitors according to the invention maybe used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing a disease-related disease or a solid tumour. The inhibitors according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition maybe in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given.

Medicaments comprising inhibitors according to the invention may be used in a number of ways. For instance, oral administration may be required, in which case the inhibitors maybe contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising inhibitors of the invention maybe administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin, for example, adjacent the treatment site, i.e. location of the senescent cells and/or solid tumour.

Inhibitors according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with inhibitors used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).

In a preferred embodiment, inhibitors and compositions according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).

It will be appreciated that the amount of the inhibitor that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the inhibitor and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the inhibitor within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular inhibitor in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease being treated. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Generally, a daily dose of between o.o^g/kg of body weight and o.5g/kg of body weight of the inhibitor according to the invention may be used for treating, ameliorating, or preventing the disease characterised by inappropriate vascular remodelling, depending upon which inhibitor is used (e.g. Ibrutinib or CGI-1746 or siNA molecule). More preferably, the daily dose of inhibitor is between o.oimg/kg of body weight and 500mg/kg of body weight, more preferably between o.img/kg and 200mg/kg body weight, and most preferably between approximately img/kg and loomg/kg body weight.

When the inhibitor is a small molecule, e.g. Ibrutinib or CGI-1746, the daily dose may be between i4omg/day to 42omg/day.

When the inhibitor is an siNA molecule, the daily dose may be between 1 μg/kg of body weight and 100 mg/kg of body weight, and more preferably, between

approximately

and 10 mg/kg, and even more preferably, between about and img/kg. When the inhibitor (e.g. antibody or siNA) is delivered to a cell, daily doses may be given as a single administration (e.g. a single daily injection).

Typically, a therapeutically effective dosage should provide about ing to

of the inhibitor per single dose, and preferably, 2ng to 50ng per dose. Antibody inhibitors may be administered in amounts between

and loomg/kg, preferably in amounts between

and lomg/kg, and more preferably may be administered at about img/kg. Such doses are particularly suitable when

administered every few (e.g. every three) days.

The inhibitor may be administered before, during or after onset of the disease characterised by inappropriate vascular remodelling. Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the inhibitor may require administration twice or more times during a day. As an example, inhibitors may be administered as two (or more depending upon the severity of the disease being treated) daily doses of between 25mg and 7000 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses of inhibitors according to the invention to a patient without the need to administer repeated doses.

When the inhibitor is a nucleic acid, conventional molecular biology techniques (vector transfer, liposome transfer, ballistic bombardment etc) may be used to deliver the inhibitor to the target tissue.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations comprising the inhibitor according to the invention and precise therapeutic regimes (such as daily doses of the inhibitor and the frequency of administration). The inventors believe that they are the first to describe a

pharmaceutical composition for treating age-related diseases or solid tumours or senescence.

Hence, in a fifth aspect of the invention, there is provided an age-related disorder or solid tumour treatment composition, comprising an inhibitor of a Tec family tyrosine kinase, and a pharmaceutically acceptable vehicle. The term "age-related disorder or solid tumour treatment composition" can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of any disease caused by cell senescence or a solid tumour in a subject.

The invention also provides in a sixth aspect, a process for making the composition according to the fifth aspect, the process comprising contacting a therapeutically effective amount of an inhibitor of a Tec family tyrosine kinase and a

pharmaceutically acceptable vehicle.

Preferably, the Tec family tyrosine kinase is BTK.

Preferably, the inhibitor is a small molecule inhibitor (e.g. Ibrutinib or CGI-1746), an antibody, or a gene-silencing molecule (e.g siNA).

A "subject" maybe a vertebrate, mammal, or domestic animal. Hence, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or maybe used in other veterinary

applications. Most preferably, however, the subject is a human being. A "therapeutically effective amount" of the inhibitor is any amount which, when administered to a subject, is the amount of medicament or drug that is needed to treat the solid tumour or age-related disease, or produce the desired effect.

For example, the therapeutically effective amount of inhibitor used may be from about o.oi mg to about 8oo mg, and preferably from about o.oi mg to about 500 mg. It is preferred that the amount of inhibitor is an amount from about 0.1 mg to about 250 mg, and most preferably from about 0.1 mg to about 20 mg. When the inhibitor is a small molecule, e.g. Ibrutinib or CGI-1746, the daily dose may be between i4omg/day to 42omg/day.

A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent (e.g. the inhibitor of a Tec family tyrosine kinase, preferably BTK) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The inhibitor according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The inhibitor may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

The inhibitors and pharmaceutical compositions of the invention may be

administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The inhibitors according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral

administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions. Knowledge of the surprising role that Tec family tyrosine kinases (preferably BTK) play in senescence and solid tumours has enabled the inventors to develop a novel screening assay for identifying whether or not test compounds can act as useful inhibitors for treating or preventing any of these diseases, for example for inclusion in the pharmaceutical compositions described herein.

Thus, according to a seventh aspect, there is provided an assay for screening a test compound to test whether or not the compound has efficacy for treating or preventing senescence or an age-related disorder or solid tumour, the assay comprising:

(i) exposing a biological system to a test compound;

(ii) detecting the activity or expression of a Tec family tyrosine kinase in the biological system; and

(iii) comparing the activity or expression of the Tec family tyrosine kinase in the biological system treated with the test compound relative to the activity or expression of the Tec family tyrosine kinase found in a control biological system that was not treated with the test compound, wherein a decreased level of activity or expression of the Tec family tyrosine kinase in the presence of the test compound relative to that detected in the control biological system is an indication of the ability of the test compound to treat or prevent senescence or an age-related disorder or a solid tumour. It will be appreciated that the assay according to the seventh aspect may be adapted such that it is used to test whether or not a test compound actually causes an age- related disorder or solid tumour.

Therefore, according to a eighth aspect of the invention, there is provided an assay for screening a test compound to test whether or not the compound causes senescence or an age-related disorder or a solid tumour, the assay comprising:

(i) exposing a biological system to a test compound;

(ii) detecting the activity or expression of a Tec family tyrosine kinase in the biological system; and

(iii) comparing the activity or expression of the Tec family tyrosine kinase in the biological system treated with the test compound relative to the activity or expression of the Tec family tyrosine kinase found in a control biological system that was not treated with the test compound, wherein an increased level of activity or expression of the Tec family tyrosine kinase in the presence of the test compound relative to that detected in the control biological system is an indication that the test compound causes senescence or an age-related disorder or a solid tumour. In the use of the third aspect, or the assays of the seventh and eighth aspects, the Tec family tyrosine kinase is preferably BTK. The use of the third aspect, and the assays of the invention are based upon the inventors' realisation that the extent of tyorise kinase expression and/or activity may be closely related to the development of senescence or an age-related disorder or a solid tumour. The screening assay of the seventh aspect is particularly useful for screening libraries of compounds to identify compounds that may be used as inhibitor used in the invention. The assay of the eighth aspect may be used to identify compounds that cause disease. Accordingly, the screen according to the seventh aspect of the invention maybe used for environmental monitoring (e.g. to test effluents from factories) or in toxicity testing (e.g. to test the safety of putative pharmaceuticals, cosmetics, foodstuffs and the like).

The term "biological system" can mean any experimental system that would be understood by a skilled person to provide insight as to the effects a test compound may have on the Tec family tyrosine kinase activity or expression in the physiological environment. The system may comprise: (a) an experimental test subject when an in vivo test is to be employed; (b) a biological sample derived from a test subject (for instance: blood or a blood fraction (e.g. serum or plasma), lymph or a cell/biopsy sample); (c) a cell line model (e.g. a cell naturally expressing a Tec family tyrosine kinase or a cell engineered to express a Tec family tyrosine kinase); or (d) an in vitro system that contains a Tec family tyrosine kinase or its gene and simulates the physiological environment such that Tec family tyrosine kinase activity or expression can be measured.

The screen preferably assays biological cells or lysates thereof. When the screen involves the assay of cells, they may be contained within an experimental animal (e.g. a mouse or rat) when the method is an in vivo based test. Alternatively, the cells may be in a tissue sample (for ex vivo based tests) or the cells may be grown in culture. It will be appreciated that such cells should express, or may be induced to express, a functional Tec family tyrosine kinase. It is also possible to use cells that are not naturally predisposed to express a Tec family tyrosine kinase provided that such cells are transformed with an expression vector. Such cells represent preferred test cells for use according to the third, seventh and eighth aspects of the invention. This is because animal cells or even prokaryotic cells may be transformed to express human Tec family tyrosine kinase and therefore represent a good cell model for testing the efficacy of candidate drugs for use in human therapy.

It is most preferred that biological cells used according to the screening assays are derived from a subject displaying one example of an age-related disease or a solid tumour.

With regards to "detecting the activity or expression of the Tec family tyrosine kinase" according to the screening assays described herein, the term "activity" can mean the detection of binding between Tec family tyrosine kinase and nucleic acid and/or other transcription factors; and translocation or determination of an end- point physiological effect.

The term "expression" can mean the detection of the Tec family tyrosine kinase protein in any compartment of the cell (e.g. in the nucleus, cytosol, the Endoplasmic Reticulum or the Golgi apparatus); or detection of the mRNA encoding the Tec family tyrosine kinase.

Expression of the Tec family tyrosine kinase in the biological system may be detected by Western blot, immuo-precipitation or immunohistochemistry. The screening assays may also be based upon the use of cell extracts comprising the Tec family tyrosine kinase. Such extracts are preferably derived from the cells described above.

The activity or expression of Tec family tyrosine kinase maybe measured using a number of conventional techniques known to the skilled person. The test may be an immunoassay-based test. For instance, labelled antibodies may be used in an immunoassay to evaluate binding of a compound to Tec family tyrosine kinase in the sample. The Tec family tyrosine kinase may be isolated and the amount of label bound to it detected. A reduction in bound label (relative to controls) would suggest that the test compound competes with the label for binding to Tec family tyrosine kinase and that it was also a putative therapeutic compound for use in treating disease.

Alternatively, a functional activity measuring Tec family tyrosine kinase activity may be employed. Furthermore molecular biology techniques may be used to detect Tec family tyrosine kinase in the screen. For instance, cDNA may be generated from mRNA extracted from tested cells or subjects and primers designed to amplify test sequences used in a quantitative Polymerase Chain Reaction to amplify from cDNA.

When a subject is used (e.g. an animal model or even an animal model engineered to express human Tec family tyrosine kinase), the test compound should be

administered to the subject for a predetermined length of time and then a sample taken from the subject for assaying Tec family tyrosine kinase activity or expression. The sample may for instance be blood or biopsy tissue. It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms "substantially the amino acid/nucleotide/peptide sequence", "functional variant" and "functional fragment", can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the nucleotide sequence identified as SEQ ID No: i (i.e. the DNA sequence encoding BTK) or the BTK protein identified as SEQ ID No: 2. Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 50%, more preferably greater than 65%, 70%, 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90%, 92%, 95%, 97%, 98%, and most preferably at least 99% identity with any of the sequences referred to herein.

The skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g.

functional form and constants.

Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (v) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al, 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al, 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two amino

acid/ polynucleotide/ polypeptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N/T)*ioo.

Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequence shown in SEQ ID No: 1 or its complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/ 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequence shown in SEQ ID No : 2.

Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non- polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

All of the features described herein (including any accompanying claims, abstract and drawings), and/ or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/ or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which: - Figure 1 shows BTK levels increase in response to p53 up-regulation. A) Western blot showing BTK and p53 expression in EJp53 in the presence of tet (-) or 6 days after removal of tet to up-regulate P53 (+). B) Western blot of HCT116 24 hours after being treated with 4θθμΜ tBH for 2 hours. C) Kaplan-Meier survival curves of patients with CLL, breast or lung cancer, segregated according to high (red) or low (green) expression of BTK, obtained from public databases through a bioinformatics analysis using PPISURV (www.bioprofiling.de). Each graph represents a different GEO dataset.

Figure 2 shows BTK expression induces apoptosis in a p53-independent manner. A) Western blot showing BTK and p53 expression in EJp53 in the presence of tet (-), 6 days after removal of tet to up-regulate p53 (+), or transfected with a plasmid containing BTK (+) or an empty vector control (-). Transfection was performed the same day as tet removal and lysates were collected 2 days after transfection. B) Representative growth curves of the cells in A. Plot shows mean of a triplicate experiment and error bars represent standard deviation. C and D) Representative colony formation assay with the same cells, with a graph representing the mean percentage of colonies (compared to controls) of a triplciate experiments. Error bars show standard deviation. E) Representative FACS plots of the Pi-stained EJp53 cells (same conditions as in A). F) Representative FACS plots of the same Annexin V- stained EJp53 cells (same conditions as in A). Figure 3 shows BTK induces activation of DNA damaging pathways and apoptosis in HCT116. A) Western blot showing total BTK levels in lysates of HCT116 24 hours after being transfected with an empty vector (C) or a BTK vector (BTK). B) Western blot showing Caspase 3 and PARP cleavage in the same cells. C) Representative plot of PI- stained HCT116. Percentage indicate cell death (sub Gi fraction) after BTK

transfection comparing to empty vector. D) Same, with Annexin V-stained cells. E) Western blot of lysates of the same cells, showing levels of total P53, phosphorylated P53 (ser 15), ATM and γΗ2ΑΧ.

Figure 4 shows BTK contributes to p53-induced senescence. A) Western blot showing total BTK and p53 levels in lysates of EJp53 cultured in the presence of tet (- ) or 6 days after removal of tet to up-regulate P53 (+), treated with DMSO (-) or o^g Ibrutinib (+) from the beginning of the experiment. Fresh media and drugs were added very 2-3 days. B) Representative growth curves of the cells in A. Fresh media and drugs were added every time cells were counted. Graphs represent mean of a triplicate experiment and error bars represent standard deviation. C) Right:

Representative pictures of EJp53 cells cultured in the presence of tet (Control) or 6 days after tet removal to induce p53 expression (p53), treated with DMSO (-) or o^g Ibrutinib (+) from the beginning of the experiment. Left: EJp53 cells stably expressing a control shRNA (shLuci) or an shRNA against BTK (shRNA), also cultured in the presence of tet (Control) or 6 days after tet removal to induce p53 expression (p53). Magnification: ιοχ. D) and E) Colony formation assay with the same cells, plus EJp53 treated with 2 g CGI-1746 for 14 days, with a graph representing the mean percentage of colonies (compared to controls) of three independent experiments. Error bars show standard deviation.

Figure 5 shows BTK inhibtion extends lifespan and ameliorates ageing in vivo. A) Kaplan-Meier survival curves of Drosophila melanogaster treated with DMSO

(Control) or Ibrutininb (see methods for concentration). n=ioo. B) Same using P53 KO flies (p53 [5A-1-4]). C) Senescence Associated (SA)- -gal staining of heads of Drosophila melanogaster treated with Ibrutinib for 14 to 40 days, compared to DMSO treated flies (Control). D) Climbing assay to test the motor function of the flies in A. Graph shows average percentage of reached threshold in five measures, 4 to 29 days after starting the treatment. Error bars represent standard deviation. Statistical significance: **** P≤ 0.001, *** P≤ 0.001, ** P≤ 0.01, * P≤ 0.05 , ns P > 0.05. E) Wet weight of flies in A. Bars represent average weight of 6 (control) or 5 (Ibrutinib) flies and error bars represent standard deviations. Statistical significance: * P≤ 0.05.

Figure 6 shows expression of BTK correlates with another marker of senescence and good prognosis in cancer. Correlation between BTK and senescence marker EBP50 levies in clinical samples of breast and lung cancer (top) and patient survival related to co-expression of the two genes (green) or not (red) (bottom).

Figure 7 shows A) Left: Western blot showing total p53 and phospho-ERK protein levels in EJp53 cultured in the presence (-) or absence of tet (+) to induce P53, and treated with DMSO (-) or o^g Ibrutinib (+) from the beginning of the experiment. Right: EJp53 infected with an adenovirus expressing P53 (Ad) for 6 days, treated with DMSO (-) or o^g Ibrutinib (+) from the beginning of the experiment. B) Western blot showing P53 protein levels in lysate of patient-derived B cells, non- stimulated (C) or stimulated (see methods) to induce P53 (S). S+I indicates stimulated cells treated with o^g Ibrutinib simultaneously. C) Representative immunofluorescence images of EJp53 stably expressing an shRNA control (shLuci) or one against BTK (shBTK), cultured in the presence of tet (Control) or 6 days after removal of tet to up-regulate P53 (p53). Red fluorescence indicates BTK expression. Blue fluorescence indicates DAPI staining (nucleus) Magnification: 6ox. D) Representative growth curves of EJp53 treated with DMSO or 2 g CGI-1746 and cultured in the presence (Control) or absence (p53) of tet to induce P53 expression. Fresh media and drugs were added every time cells were counted. Graphs represent mean of a triplicate experiment and error bars represent standard deviation. E) Growth curves of EJp53 stably expressing a control shRNA (shLuci) or one against BTK (shBTK) and cultured in the presence or absence (+P53) of tet to induce p53 expression. F) Western blot showing BTK and p53 protein levels in lysates of EJp53 cultured in the presence (-) or absence (+) of tet for 6 days to induce P53 expression, treated with DMSO (-) or o^g Ibrutinib (+) from the beginning of the experiment, compared to EJp53 stably expressing a control shRNA (-) or one against BTK (+), also induced to express P53. G) Western blot sowing P53 protein levels in lysates of EJp53 cultured in the presence (-) or absence (+) of tet for 6 days to induce P53 expression, and then treated with o^g Ibrutinib or 2 g CGI-1746 for the specified time.

Figure 8 shows BTK inhibition prolongs lifespan in vivo. A) Kaplan-Meier survival curves of Drosophila melanogaster treated with DMSO (Control) or CGI-1746 (see methods for concentration). n=ioo. B) Survival of flies in the experiment shown in A and in Figure 5A, represented by the number of live flies present in the tubes. Graphs show averages of flies per tube (10 tubes starting with 10 flies each) and error bars represent standard deviation. C) Average of total numbers of live flies in the same experiments.

Figure 9 shows the chemical structure of Ibrutinib; and

Figure 10 shows the chemical structure of CGI-1746.

Examples

Materials and Methods

Cell Culture

EJp53 and HCT116 were maintained in DMEM supplemented with 10% fetal bovin serum (FBS, Gibco) and penicillin-streptomycin (50 unit/ml). EJp53 was also maintained in hygromycin (100 g/ml) and genticin (750 g/ml). In order to inhibit P53 expression in these cells, tetracycline (tet) was added to the medium every 3 days to a final concentration of ^g/ml. To induce P53 expression, cells were washed three times and seeded directly in culture medium in the absence of tet, following previously reported protocols64. Ibrutinib (Sellechem, PCI-32765), and CGI-1746 (Axon Medchem, 2018) were used to inhibit BTK in this study. 200 μΜ of Tert- Butylhydroperoxide (tBH, Merck) were added for 2 hours to induce DNA damage. Peripheral blood samples were obtained from patients with Chronic Lymphocytic Leukaemia (CLL) after informed consent and approval from the Local Research Ethics Committee. The patients were treatment na^'ive and had a cell count >50 x 109/L. Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density centrifugation. Heparinised whole blood was diluted 1:1 with phosphate-buffered saline (PBS) and gently layered onto 15ml Ficoll (Histopaque 1077, Sigma Aldrich) prior to centrifugation at 400g for 30mm with gentle acceleration and braking. The mononuclear cell layer was removed from the interphase, washed and resuspended in RPMI-1640 medium (Invitrogen/Life Technologies) supplemented with 10% fetal bovine serum (Lonza), L-Glutamax (2mmol/L), penicillin (50 U/ml) and streptomycin (50 mg/ml) (complete media). The isolated mononuclear cells have a CLL cell purity of more than 95% in all cases determined by flow cytometry. They were cultured at 3 x 106 cells/ml complete media supplemented with long/ml His-tagged rhCDi54 (R&D Systems), ^g/ml cross-linking His Tag monoclonal antibody (R&D Systems) and long/ ml rhIL4 (R&D Systems).

Growth curves

For measuring cell growth, 1x104 EJp53cells were plated in 6 cm plates a day before chemicals were added. Cells were counted every 4 days using a BIO-RAD TC20 automated cell counter, after which fresh media and chemicals were added to the cultures. For RNAi experiments, cells were transfected the day before 1x104 cells were plated in 6 cm plates.

Immunoblot Analysis

1 μg/ml Protease Inhibitor Cocktail Set III (Calbiochem) added to cell lysates. Protein concentrations were determined using Bradford protein assay (Fermentas). 20 g of total protein per sample were subjected to 10% or 6% SDS PAGE and transferred to Immobilon-P membranes (Millipore). An ECL detection system (Thermo Scientific) was used to visualize the results. Alternatively, an Odyssey CLx Infrared Imaging System (Li-COR) was used. Primary antibodies used were: anti-phospho-Histone 2A (ser 139, Millipore 05-636), anti-phosphoATM (S1918, Abeam ab8i292), anti- phospho-p53 (ser 15, Cell Signalling 9284), anti-p53 (DO-i, Santa Cruz, sc-129), anti- BTK (D3H, Cell Signalling 8547), anti-P-actin (abeam ab8227). An ECL detection system (Thermo Scientific) was used to visualize the results. Alternatively, an Odyssey CLx Infrared Imaging System (Li-COR) was used.

Immunofluorescence

Cells were processed as previously described46. Briefly, cells were fixed using 4% paraformaldehyde, washed t with PBS and permeabilised with 0.1% Triton X-100. Cells were then washed with PBS and blocked with 1% BSA. Coverslips were incubated with 100 μΐ i:ioo primary antibody overnight at 4°C with 1: 50 anti-BTK (D3H5, Cell Signalling 8547).The following day, coverslips were washed with PBS and incubated with 100 μΐ secondary anti-rabbit or anti-mouse antibodies (Alexa Fluor 488 and 594, Invitrogen) for 45 minutes in the dark. After incubation, coverslips were washed three times with PBS and stained with 4',6-Diamidino-2- Phenylindole, Dihydrochloride (DAPI, Invitrogen) for 10 minutes. Slides were analysed using a Nokia TE300 semi-automatic microscope.

Senescence-associated^-Galactosidase (SA-fi-Gal) staining

Cells were washed three times with PBS and fixed with 4 % formaldehyde for 5 minutes at room temperature, then stained as previously described. Flies were fixed in 4 % formaldehyde for 15 min at room temperature, then washed one with PBS and stained using the same buffer for 48 hours at 37°C.

Colony formation assay

500 cells were split into 60 mm plates in triplicates. Plates were inubated at 37°C and 5% CO2 for 14-15 days. Media was changed every 5 days. Plates were then washed with 3 ml PBS and fixed with 2 ml of 10% neutral buffered formalin (Sigma-Aldrich). Plates were incubated at room temperature for 30 min. Formalin was removed by aspiration and plates were washed twice with PBS, then allowed to completely air dry with the lids removed. 5 ml of Staining Reagent (6.4 ml of PO4 buffer (67Π1Μ), 89.6 ml of dH20, 4 ml Giemsa stain (Fluka)) was finally added to the plates and they were incubated at room temperature for 5 hours. The staining solution was poured off and the plates were rinsed gently with ddH20. Plates were allowed to air dry and the number of colonies from each plate was then counted and recorded.

BTK Gverexpression

Transfection was performed using Lipofectamine 2000 (Invitrogen). Cells were split one day before transfection and were 70-80% confluent on the day of transfection. 8μg of empty plasmid (Mission pLKO.i Empty vector control plasmid DNA, Sigma Aldrich SHCooi) or a BTK plasmid (OriGene RG211582) were diluted in 0.5 ml of serum-free media, mixed with 2θμ1 of lipofectamine 2000 and incubated following manufacturer's instructions. After incubation, the complexes were added to the plates and the cells were placed in the incubator at 37°C. Medium was changed after 5 hours and cells were left for 18-24 hours before using them for further experiments.

BTK Silencing

To generate cells with a stable downregulation of BTK, an shRNA against BTK (Santa Cruz sc-29841-sh) was used. Btk shRNA Plasmid (m) is a pool of 3 target-specific lentiviral vector plasmids each encoding 19-25 nt (plus hairpin) shRNAs designed to knock down Btk gene expression. Each plasmid contains a puromycin resistance gene for the selection of cells stably expressing shRNA. Each vial contains 20 μg of lyophilized shRNA plasmid DNA, and is suitable for up to 20 transfections. Btk siRNA (m): sc-29842 and Btk shRNA (m) Lentiviral Particles: sc-29842-V can also be used as alternative Btk gene silencing products. An shRNA against luciferase (Sigma Aldrich SHC007, Mission pLKO.ipuro Luciferase) was used as a control. The shRNA were transfected into EJp53 and HCT116 using Lipofectamine 2000 following manufacturer's protocols. 10 μΐ of Lipofectamine 2000 were mixed with 0.5 ml of media that contained ^g of shRNA. The medium in the plates was changed after an overnight incubation and 2 g/ ml puromycin added to each plate to select for transfected cells Cells were kept under selection for 2 weeks.

Propidium iodide staining

To assess the percentage cell death, a PI staining followed by a FACS analysis was performed as previously described. 10,000 events were recorded for each sample using the Beckton Dickinson FACSCanto II and FACSDiva 6.0 software (Beckton Dickinson) for acquisition and analysis.

Annexin V staining

Cells were washed with PBS and stained with the Anenxin-V-Fluos Staining kit (Boehringer Manheim, Basel, Switzerland), following instructions provided by manufacturer, and then analysed by FACS. Cells positive for Annexin V were considered apoptotic. 10,000 events were recorded for each sample using the Beckton Dickinson FACSCanto II and FACSDiva 6.0 software (Beckton Dickinson) for acquisition and analysis.

Food preparation for in vivo experiments The following recipe was used to make standard maize media: 504g Maize meal (Quaker, UK), 555g Glucose (Fisher Scientific, UK), 350g, Brewer's yeast (MP Biomedicals, UK), 62.5g Agar (Biogene, USA), 94ml Nipagen in ethanol (20%), 21ml Propanoic acid (>99-5%, Sigma-Aldrich, UK), Tap water Up to 7 litres. BTK inhibitors were administered to flies as a food additive (conditioned media). The drugs required suspension in (vehicle) DMSO which was also used as a control additive. All conditioned media (DMSO/drug) was made with a final concentration of 2% (v/v). Dmp53 null mutant (p53[5A-i-4], stock no. 6815) flies were acquired from the Bloomington stock centre, USA, and Canton-S flies were used as wild-type. All experiments were performed on male adult flies in constant light-dark cycle (12:12) at 25°C, and flies were never anesthetised.

In vivo longevity assays

The parents of Fi flies used in this assay were synchronised for age. Male Fiflies were allowed to mature for one day after embryo laying (AEL) on standard maize food prior to each experiment. Virgin flies were then transferred to conditioned media, 10 vials of 10 flies were setup and incubated at 25°C throughout the experiment. The flies were transferred to fresh vials twice a week and the number of viable flies was scored each day. Flies that advertently escaped or accidently lost, were right censored and recorded to inform the analysis. Statistical analyses were performed using the log-rank (Mantel-Cox test) test.

Climbing assay

The motor function of the flies after drug administration was tested using the climbing assay. Following the same experimental procedure as the longevity assay (see above), climbing was scored prior to transfer to new food. Flies were transferred into a tube made of two empty vials in an isolated temperature controlled room (25°C). The flies were allowed to acclimatise to the new environment for one minute. The vial was tapped firmly to bring the flies to the bottom of the vial. The climbing was scored as a percentage of flies that touch or pass an 8cm line/threshold at the top of the tube after 10 seconds. The assay was repeated five times for each vial. The difference between drug-treated and control flies was assessed using the Mann- Whitney test (data were not normally distributed) and adjusted for multiple testing using the Benjamini & Hochberg procedure.

Weight of flies after drug administration The wet weight of the flies was measured in groups of five flies (one replicate); the number of replicates is shown. Flies under DMSO-control and experimental conditions were weighed and compared using Mann-Whitney test (data were not normally distributed).

Results

BTK is up-regulated in response to 53 and is involved in tumour suppressor pathways.

The inventors identified BTK as a protein selectively induced in senescence. They first confirmed these results in EJp53, a p53-null bladder cancer cell line with a tetracycline (tet)-regulatable p53 expression system that undergoes senescence after tet removal. As shown in Figure lA, BTK protein levels were highly elevated in EJp53 induced to express p53. Moreover, colon cancer cell line HCT116, which has wild type P53_> also showed BTK induction after P53 up-regulation by DNA damaging agents (Figure lB), indicating that BTK maybe involved in p53-mediated responses to stress.

BTK is known to have oncogenic functions in B-cell malignancies, but the results described herein suggest that, in other contexts, it may have a previously unknown role contributing to tumour suppressor mechanisms as part of the p53 pathway. To address this discrepancy, the inventors analysed survival rates in cancer patients from publicly available data using the online tool PPISURV. High expression of BTK correlated with a poor prognostic in a chronic lymphocytic leukaemia GEO datasets, but was a marker of good prognosis in lung and breast cancer datasets (Figure lC). Moreover, BTK expression in these two solid cancers strongly correlated with the expression of EBP50, a marker of senescence that has previously been shown to be linked to better prognosis as well (Figure 6). All this is consistent with BTK having tumour suppressor functions in certain malignancies, likely mediated by his involvement in the p53 pathway.

BTK expression induces cancer cell death independently of ss

To characterize the role of BTK in the p53 pathway, the inventors transfected BTK into EJp53 (Figure 2A). As shown in Figure 2B, cells in which BTK was expressed were unable to grow, even in the absence of P53. This was confirmed with a colony formation assay (Figures 2C and D). The inventors observed that this was due to a BTK-dependent induction of cell death (Figures 2E) and an Annexin staining confirmed that this was of apoptotic nature (Figures 2F). They confirmed these results in HCT116, which also showed induction of cell death after BTK transfection (Figure 3). This was due to the activation of the apoptotic pathway, as shown by Annexin staining and PARP/Caspase3 cleavage (Figures 3B and D). Of note, there was a significant increase in γ-Η2ΑΧ, ATM and p53 phosphorylation in these cells after BTK up-regulation (Figure 3E), which indicates a BTK-mediated induction of DNA damage pathways. These data together suggest a pro-apoptotic and antiproliferative role of BTK that relies on activation of the DNA damage response and suggests a possible feedback loop mechanism to increase the stabilisation of the P53 protein through phosphorylation. This is supported by the fact that BTK expression was sufficient to elevate P53 protein levels (see Figure 2A).

BTK plays a key role in p 3-induced senescence

There is a plethora of commercially available small molecule inhibitors against BTKs, one of which, Ibrutininb, has recently been approved to be used in humans to treat different forms of leukaemia, with minimal side effects. Ibrutinib irreversibly blocks the catalytic activity of BTK at cysteine-481 and abolishes the full stimulation of BTK through the suppression of tyrosine-223 autophosphorylation. Since BTK was found up-regulated in senescent cells and the inventors have shown it plays a role in P53 responses, they used Ibrutinib to determine whether BTK expression is important for P53-induced senescence. As shown in Figure 4A, Ibrutinib was not only able to suppress BTK activation but also its expression in EJp53. This was confirmed by a reduction in the phosphorylation of the ERK MAPK, a downstream event in the BTK pathway (Figure A). Of note, the levels of p53 were importantly reduced when BTK was inhibited (Figure 4A and 4 Figure 7A). To rule out a possible interference of

Ibrutinib with the tet-regulatable p53 system, they infected EJp53 with an adenovirus expressing P53. As shown in Figure 7B, Ibrutinib was also able to reduce P53 protein levels in this situation, confirming that BTK inhibition interferes with the

accumulation of P53. Moreover, on primary malignant B cells incubated with CD154 and IL4, a protocol that was observed induces P53 activation, Ibrutinib was also capable of inhibiting P53 expression (Figure 7B), confirming that this phenomenon can also be observed in other cell lines and in the physiological expression of P53. This suggests that BTK may be involved in the stabilization of p53 protein levels in different contexts, which would explain its contribution to p53 functions.

Inhibition of BTK by Ibrutinib importantly increased cell proliferation in EJp53 induced to senesce, consistent with an important role of BTK in the arrest/senescence pathway (Figure 4B). The same results were observed using CGI- 1746, a more specific inhibitor of BTK, and EJp53 stably expressing an shRNA against BTK (Figures 7C-E), both of which also inhibited p53 (Figures 7F and G). Of note, the reduction in P53 protein levels was only observed after seven days of incubation with the chemical inhibitors, suggesting that the effect of BTK on P53 protein accumulation is not immediate. The inhibition of senescence was confirmed by the fact that the morphological changes associated, which include multinucleated and large cells with a spindle shape and vacuolization features, were not evident in cells in which BTK had been blocked (Figure 4C). Finally, a colony formation assay confirmed that either genetic or chemical inhibition of BTK bypassed p53-induced senescence (Figure 4D and E). Collectively, these results show that BTK has a critical role in inducing and/ or maintaining the p53-dependent the senescent phenotype, likely through an up-regulation of DNA damaging signals that allow P53 protein levels to be stably increased. This also suggests a potential strategy to prevent accumulation of senescent cells in vivo by chemically inhibiting BTK functions, which could have an impact on the symptoms associated with ageing.

BTK inhibitors ameliorate ageing and extend lifespan in vivo

To test the hypothesis that BTK inhibition may affect organismal ageing, the inventors treated Drosophila melanogaster with either Ibrutinib or CGI-1746. Flies that were fed these drugs throughout their lifespan (ad libitum, mixed with food at different concentrations) lived longer than controls (Figure 5A and Figure 8).

Importantly, there was no change in life span when P53KO flies were fed Ibrutinib (Figure 5B), showing that the anti-ageing effects of BTK inhibition were dependent on the presence of p53 and consistent with the in vitro results. Moreover, this correlated with a reduction in staining of the flies with Senescence Associated (SA)- - gal (Figure 5C), a widely used marker of senescence, showing reduced accumulation of senescent cells. This is also consistent with the inhibition of senescence observed in human cells in vitro and indicates that BTK inhibition prolongs lifespan in

Drosophila by preventing p53-dependent senescence in vivo. In addition, a climbing assay showed an increase in mobility of Ibrutinib-treated flies all across different points in their lifespan (Figure 5D), which suggests an improvement of their fitness in the absence of BTK expression. Together with this, the inventors also observed a significant weight gain after treatment with Ibrutinib (Figure 5E), which could correlate with increased fitness. All these results together show that BTK plays an important role in organismal senescence and that its inhibition can ameliorate ageing and even prolong lifespan. Discussion

Despite the considerable knowledge accumulated in the fifty years since Leonard Hayflick first described cellular senescence, the molecular pathways involved in the establishment and maintenance of this phenotype have not been completely characterized. Through a proteomics screening, the inventors uncovered a novel link between BTK and the p53 pathway that reveals a crucial role for BTK in p53-induced cellular senescence. Importantly, we show that this correlates to an effect on improving fitness and delaying organismal ageing in flies.

BTK is a non-receptor tyrosine kinase that is critical for B-cell maturation and it has been implicated in oncogenic signalling. Its chemical inhibition is one of the most recent and most successful advances in the treatment of B-cell malignancies.

However, here the inventors show that BTK may actually have tumour suppressor functions in other contexts. BTK is induced by p53 functions and its overexpression is sufficient to trigger cell death. BTK up-regualtion can induce phosphorylation of γΗ2ΑΧ, ATM and p53 itself, which are critical events in the responses to DNA damage. This provides a mechanism to explain the observation that BTK increases the protein levels of p53. This suggests a P53-BTK positive feedback loop enhancing P53 functions that would need further elucidation. For instance, it would be interesting to determine how BTK induces the activation of several DNA damage response elements that may eventually lead to P53 up-regulation, and whether this includes direct phosphorylation of P53. Of note, overexpression of BTK can induce apoptosis in epithelial cancer cells in culture in the absence of P53. Whether this is part of physiological BTK functions in vivo will have to be investigated. However, it strongly suggests that BTK can also have p53-independent anti-tumourigenic properties. This is a striking difference from its known effects promoting blood malignancies. The most surprising finding of this report is that p53-induced senescence can be bypassed in culture in using either chemical inhibitors or RNAi. These approaches resulted in a fraction of cells escaping the permanent arrest and resuming

proliferation. BTK has not been implicated before in pathways that stop the cell cycle. The results indicate that its effects on senescence are mediated through the aforementioned positive feedback loop established with P53. In the absence of BTK, the signals that lead to P53 stabilization would not be reinforced and eventually P53 protein levels will be severely reduced. Although this may not interfere with the initial arrest induced by p53, it compromises the establishment of a permanent arrest phenotype, thus effectively bypassing senescence.

Given the strong effect that BTK inhibition has on blocking senescence in cell cultures, the inventors have reasoned that it could also have an impact on ageing. Ageing is a complex multi-process phenomenon that is triggered during the life span of an organism. It has been proposed that the accumulation of different cellular alterations determines the age-associated symptoms. For instance, telomere attrition, impairment in proteostasis and increased caloric consumption have been considered among the hallmarks of ageing. The possibility of expanding the healthy lifespan of humans by blocking these processes has been studied extensively, although no interventions have yet been clinically tested.

The results show that an invertebrate model organism, such as Drosophila, which has a BTK gene homolog, is sensitive to BTK inhibitors. Average lifespan of a fly population was significantly extended using Ibrutinib or CGI-1746 but more importantly, markers of fitness such as ability to climb or weight were increased in treated flies. This indicates that BTK inhibitors not only delay ageing but also help maintain a healthy status. The limitations with the delivery of the drug (it is difficult to quantify the concentrations achieved in vivo) make more complicated experiments in this model quite difficult to be performed. However, the inventors were able to determine that the effects of BTK inhibitors require the presence of a functional P53. This is consistent with the hypothesis of BTK acting as an enhancer of the p53 pathway in senescence and provides a mechanism to explain the observed effects. Also, it is important to note that the percentage of senescent cells in the heads of treated flies decreased, which confirms that the effects of BTK inhibitors on organismal ageing are mediated by a blocking in the senescent response, as expected.

The fact that Ibrutinib is already being used clinically suggests that it could be an important tool to ameliorate the symptoms of ageing in humans, initially in populations that are especially at risk, like those suffering of premature ageing syndromes (such as progeria) or frailty (25-50% of people older than 85 years are classified as frail). This treatment could lead to healthier ageing and perhaps even a lifespan extension of these patients.

Drugs with potential effects on ageing are currently being investigated. However, none of them has been proved yet to be a good candidate to be used in humans. For instance, the pharmacological inhibition of mTOR by rapamycin can delay aging in mice models, but the immunosuppressant effect of this compound prevents its long term use as an anti-ageing drug. In comparison, Ibrutinib has shown to have minimal side effects, and more specific BTK inhibitors currently being tested could reduce them even more.

The fact that chemical inhibition of senescence to ameliorate ageing could also have detrimental effects on tumour suppression needs to be carefully considered.

Senescence provides an actual barrier against the progression of cancers such as melanoma and it would be important to determine whether alternative mechanisms are sufficient to keep these lesions in check if senescence is bypassed. Only in vivo testing of BTK inhibitors in mammalian models will allow determination of whether the absence of BTK delays the symptoms of ageing but at the same time increases the risk of the emergence of solid tumours. It is also important to remember that large cohorts of patients are currently being given BTK inhibitors for the treatment of leukaemia, so far with no reports of increased incidence of secondary malignancies. The effects of these drugs on tissue ageing could be readily evaluated taking advantage of this situation. In summary, the inventors showed that BTK has important cellular functions besides those already known in B cell physiology and that they oppose cancer progression. Strikingly, these include a pivotal role in cellular senescence that strongly impact organismal ageing. The potential anti-ageing effects of BTK inhibition that can be inferred from the results present a novel strategy to ameliorate the symptoms of ageing in humans that needs to be further explored.

Summary

Senescence is a mechanism that prevents the emergence of transformed cells. It can be triggered by different stimuli, from DNA damaging stresses to telomere attrition. It has been shown that a build-up of senescent cells in tissues contributes to ageing as well as tumour progression. Thus, there is a rationale to devise therapies to prevent the accumulation of senescent cells in order to slow down age-related pathologies and cancer. Here, the inventors show that Bruton's Tyrosine Kinase (BTK), a nonreceptor tyrosine kinase that is part of the B-cell Receptor signalling pathway, is highly up-regulated in senescent cells. The inventors found that BTK is involved in damage responses, through the induction of histone H2AX, ATM and p53

phosphorylation, with a concomitant increase in P53 protein levels. This suggests a previously unknown positive feedback loop between BTK and p53 to enhance P53- mediated responses. Furthermore, the inventors determined that BTK can induce cell death in a p53-independent manner, which supports a possible tumour suppressor function of BTK. Consistent with this, inhibition of BTK expression blocked P53- induced senescence, underscoring its important role as a mediator of this pathway. BTK inhibitors were capable of reducing the accumulation of senescent cells in an invertebrate model, which resulted in increased mobility and weight as well and up to 20% lifespan extension, confirming the in vivo relevance of our findings. The inventors conclude that, despite its role as a driver of B cell malignancies, BTK may have tumour suppressor functions in solid cancers. Its inhibition blocks p53-induced senescence and thus prolongs lifespan in vivo. The inventors propose that BTK inhibitors, which are already being used clinically, could be useful as adjuvant therapies in early-stage solid tumours or after chemo/radio therapy, as well as to prevent age-dependent fitness loss.

Claims

Claims

1. An inhibitor of a Tec family tyrosine kinase, for use in the treatment, amelioration or prevention of senescence or an age-related disorder or a solid tumour.

2. An inhibitor for use according to claim l, wherein the Tec family tyrosine kinase inhibitor inhibits Tec, Emt, Btk, Bmx, Txk or Dsrc28C.

3. An inhibitor for use according to either claim 1 or claim 2, wherein the Tec family tyrosine kinase inhibitor inhibits Btk.

4. An inhibitor for use according to claim 3, wherein the inhibitor prevents or reduces the expression of Btk gene or activity of BTK protein, wherein the BTK protein comprises an amino acid sequence substantially as set out in SEQ ID No: 2, or a functional variant or fragment thereof, or wherein BTK is encoded by the nucleic acid substantially as set out in SEQ ID No: 1, or a functional variant or fragment thereof.

5. An inhibitor for use according to any preceding claim, wherein the age-related disorder includes progeria, frailty, diabetes, liver fibrosis, neurodegenerative diseases, sarcopenia or wrinkles.

6. An inhibitor for use according to any preceding claim, wherein the solid tumour is selected from a group consisting of: nevus; melanoma; adenoma; colon adenoma; dermal neurofibroma; and prostate intraepithelial neoplasis.

7. An inhibitor for use according to any preceding claim, wherein the inhibitor: -

(a) binds to the Tec family tyrosine kinase to reduce its biological activity;

(b) decreases the expression of the gene encoding the Tec family tyrosine kinase; or

(c) inhibits translocation of the Tec family tyrosine kinase from the nucleus to the cytosol.

8. An inhibitor for use according to any preceding claim, wherein the Tec family tyrosine kinase inhibitor is selected from a group consisting of Ibrutinib (available under the trade name Imbruvica™), GDC-0834, RN-486, CGI-560, CGI-1746, ACP- 196, HM-71224, CC-292 (AVL-292), ONO-4059 (ONO-WG-307), CNX-774 and LFM- A13.

9. An inhibitor for use according to any preceding claim, wherein the Tec family tyrosine kinase inhibitor is Ibrutinib or CGI-1746.

10. An inhibitor for use according to any preceding claim, wherein the inhibitor comprises a Tec family tyrosine kinase neutralising antibody.

11. An inhibitor for use according to any preceding claim, wherein the inhibitor is a gene-silencing molecule.

12. An inhibitor for use according to any preceding claim, wherein the gene- silencing molecule is an RNAi molecule, siNA, siRNA, miRNA, shRNA, ribozyme or antisense molecule.

13. An inhibitor for use according to claim 12, wherein the siNA molecule is SEQ ID No. 4, 5, 7, 8, 10 or 11.

14. A Tec family tyrosine kinase gene-silencing molecule, for use in the treatment, amelioration or prevention of senescence or an age-related disorder or a solid tumour.

15. An age-related disorder or solid tumour treatment composition, comprising an inhibitor of a Tec family tyrosine kinase as defined in any one of claims 1-13, and a pharmaceutically acceptable vehicle.

16. A process for making the composition according to claim 15, the process comprising contacting a therapeutically effective amount of an inhibitor of a Tec family tyrosine kinase and a pharmaceutically acceptable vehicle.

17. An assay for screening a test compound to test whether or not the compound has efficacy for treating or preventing an age-related disorder or solid tumour, the assay comprising:

(i) exposing a biological system to a test compound;

(ii) detecting the activity or expression of a Tec family tyrosine kinase in the biological system; and (iii) comparing the activity or expression of the Tec family tyrosine kinase in the biological system treated with the test compound relative to the activity or expression of the Tec family tyrosine kinase found in a control biological system that was not treated with the test compound,

wherein a decreased level of activity or expression of the Tec family tyrosine kinase in the presence of the test compound relative to that detected in the control biological system is an indication of the ability of the test compound to treat or prevent an age- related disorder or solid tumour.

18. An assay for screening a test compound to test whether or not the compound causes an age-related disorder or solid tumour, the assay comprising:

(i) exposing a biological system to a test compound;

(ii) detecting the activity or expression of a Tec family tyrosine kinase in the biological system; and

(iii) comparing the activity or expression of the Tec family tyrosine kinase in the biological system treated with the test compound relative to the activity or expression of the Tec family tyrosine kinase found in a control biological system that was not treated with the test compound,

wherein an increased level of activity or expression of the Tec family tyrosine kinase in the presence of the test compound relative to that detected in the control biological system is an indication that the test compound causes an age-related disorder or solid tumour remodelling.

19. Use of an inhibitor of a Tec family tyrosine kinase, for preventing or reducing senescence in a biological cell.

20. Use according to claim 19, wherein the use is carried out on the cell in vitro or ex vivo.