WO2017085473A1

WO2017085473A1 - Agent to prevent the emergence of resistance to a therapeutic agent

Info

Publication number: WO2017085473A1
Application number: PCT/GB2016/053569
Authority: WO
Inventors: Jonathan HOUSELEY
Original assignee: Babraham Institute
Priority date: 2015-11-17
Filing date: 2016-11-16
Publication date: 2017-05-26

Abstract

The invention relates to the use of a histone acetyltransferase inhibitor to prevent the emergence of mutations causing resistance to a therapeutic agent. The invention also relates to the use of an mTOR inhibitor to prevent the emergence of mutations causing resistance to a therapeutic agent. The invention also relates to novel pharmaceutical compositions comprising a histone acetyltransferase inhibitor or an mTOR inhibitor in combination with a therapeutic agent and to the uses of said compositions in the treatment of disease, such as cancer.

Description

AGENT TO PREVENT THE EMERGENCE OF RESISTANCE TO A

THERAPEUTIC AGENT

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The emergence of resistance is a consequence of evolution and is observed in populations of cells or organisms subject to strong selective pressure through drug, toxin, or chemical exposure. The likelihood of resistance emerging is generally understood to be a property of the mutation rate of cells within the population and the size of that population. In other words, large populations of cells with a high mutation rate (such as tumours) are very likely to produce at least one cell with a suitable mutation to provide resistance to any given drug. Although the drug may eradicate the vast proportion of the target population, this simply provides an unoccupied niche into which resistant cells can expand, yielding a new, drug resistant population. In organisms that cause infectious disease, this new drug resistant population can then be communicated and endemic drug resistance follows through the routine use of antibiotics in healthcare, rendering those antibiotics ineffective (Blair, J. et al (2015) Nature Reviews Microbiology 13, 42-51).

Drug resistance is a massive problem in the treatment of infectious diseases, necessitating complex chemotherapy regimens and often rendering diseases chronic or fatal. Drug resistance is generally considered as a problem of prokaryotic infections, however, drug resistance is frequently encountered in the treatment of eukaryotic parasites including yeasts such as Candida albicans and Cryptococcus neoformans, protozoa such as Plasmodia, trypanosomes and leishmania, and nematodes such as ascarids, filarias, hookworms, pinworms and whipworms. Plasmodia, the causative agent for malaria, causes massive mortality - in excess of half a million deaths per year (WHO estimate) - but yeast infections are the primary cause of death in hundreds of thousands of AIDS patients (Polvi, E. J. et al (2015) Cell. Mol. Life Sci. 72, 2261-2287), and morbidity and mortality caused by these other protozoa and nematodes is substantial, particularly in the third world. An example of drug resistance in this context was reported in 2012 when drug-resistant strains of Plasmodium falciparum led to a resurgent threat of Malaria in South East Asia and sub-Saharan Africa (Phyo, A. P. et a/ (2012) The Lancet ZIS, 1960-1966).

In addition, acquired resistance to the drugs used in chemotherapy has also become common as new targeted therapies for cancer are introduced (Sun, Y. et al (2012) Nature Medicine 18, 1359-1368 and Sakai, W. et a/ (2008) Nature 451 , 11 16-1 120). Drug- resistance associated with chemotherapy affects patients with a variety of blood cancers and solid tumours, including breast, ovarian, lung, and gastrointestinal tract cancers. Although chemotherapies provide temporary respite by initially eradicating most tumour cells, the rapid re-growth of rare drug resistant cells leads to relapse and the occurrence of a new tumour against which the chemotherapy is no longer effective.

Drug resistant mutations can be present at low frequency in wild populations, particularly for drugs in common use, and prokaryotic pathogens can exchange genetic material encoding drug resistance genes through horizontal gene transfer. In contrast, eukaryotic pathogens rarely exchange genetic material and drug resistance mutations can arise de novo during the course of the infection and treatment (Andersonn T. J. C, and Roper, C. (2005) Acta Tropica, 94, 269-280, and Ford, C. B. et al (2015) eLIFE 4, e00662). Similarly, mutations which confer drug resistance on tumor cells almost invariably arise de novo. The de novo mutations underlying drug resistance in both cancer and eukaryotic pathogen infections are assumed to occur through random mutation and, under this hypothesis, drug resistance is almost unavoidable. However, it is conceivable that the mechanisms by which de novo mutations form may in and of themselves be suppressed by drug treatment, providing a strategy for suppressing the emergence of seemingly unavoidable resistance mutations.

Combination therapy can be highly effective in suppressing some cancers and infections by forcing target cells to simultaneously possess resistance mutations in two or more pathways. However, multidrug resistance is a well-characterised mechanism by which cancers develop resistance to chemotherapy drugs (Presidis, A. (1999) Nature Biotechnology 17, 94-95), and so combination approaches do not provide a complete solution to the problem of drug resistance.

Overall, there is a desperate need to elucidate the underlying sources of mutations that bestow drug resistance, and to develop strategies which improve the effectiveness of existing therapeutics by suppressing the emergence of these mutations.

SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided the use of a histone acetyltransferase inhibitor to prevent the emergence of mutations causing resistance to a therapeutic agent. According to a further aspect of the invention, there is provided the use of an mTOR inhibitor to prevent the emergence of mutations causing resistance to a therapeutic agent.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a histone acetyltransferase inhibitor and a therapeutic agent for use in therapy.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising an mTOR inhibitor and a therapeutic agent for use in therapy. According to a further aspect of the invention, there is provided the use of a histone acetyltransferase inhibitor for the treatment of a disease or condition mediated by an increase in copy number variation.

According to a further aspect of the invention, there is provided the use of an mTOR inhibitor for the treatment of a disease or condition mediated by an increase in copy number variation.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Copy number variation at CUP1. Copy number amplification of the CUP1 locus, encoding a copper-sequestering metallothionein, which confers increased tolerance to the antimicrobial agent copper sulfate on budding yeast S. cerevisiae.

Figure 2: Map of pJH266. Three copies of the complete CUP1 repeat (marked "Repeat 1", "Repeat 2" and "Repeat 3"), and ADE2 marker (marked "ADE2 homology") and CUP1 flanking regions (marked "RL . nk" and "Le... nk") in pBlueScript SK- backbone (marked "pUC ori", "bla" and "f1... I"). Structural features are shown on outer circle of annotation, open reading frames on the inner circle. Small arrows in Repeats 1-3 represent the CUP1 open reading frames.

Figure 3: Synthetic system for study of CUP1 amplification. A) Detection of copy number amplified CUP1 alleles by southern blot after exposure to 300μΜ copper sulfate for 10 generations. Quantification of amplified alleles for multiple biological replicates is shown, p value calculated by t test. B) Copper adaptation of 3xCUP1 cells pre-exposed 300μΜ copper. Tolerance to high copper concentrations (>500μΜ) is dramatically enhanced by pre- growth in 300μΜ copper. Error bars ±1 SD.

Figure 4: Suppression of rDNA amplification by rapamycin. Cells with low rDNA copy number (-35 repeats) lacking Fob1 are induced to amplify over 60 generations by introduction of FOB1 on a plasmid. Rapamycin (RAP) completely abrogates amplification. Quantification of rDNA copy number in multiple clones is shown, p values calculated by oneway A NOVA.

Figure 5: Rapamycin represses adaptation through CNV at CUP1. A) Cells with

3xCUP1 copies were exposed to 300μΜ copper in presence or absence of rapamycin for 10 generations. Quantification of amplified alleles for multiple biological replicates is shown, p values calculated by one-way ANOVA. B) Adaptation to high copper is repressed by rapamycin, error bars ±1 SD.

Figure 6: Loss of H3K56 HDACs Hst3 and Hst4 accelerates adaptive CUP1 amplification. A) hst3A hst4A mutants undergo hyper-amplification of CUP1 in response to 300μΜ copper treatment. Quantification of amplified alleles for multiple biological replicates is shown, p values calculated by one-way ANOVA. B) Increased CUP1 amplification leads to enhanced copper adaptation, error bars ±1 SD.

Figure 7: Loss of H3K56 acetyltransferase Rtt109 inhibits adaptive CUP1 CNV.

A) Growth of rtt109h cells for 10 generations in the presence of 300μΜ CuS0₄ does not yield a sub-population of Cl/P7-amplified cells detectable by southern blot. Quantification of amplified alleles for multiple biological replicates is shown, p values calculated by one-way

ANOVA. B) Decreased CUP1 amplification leads to impaired copper adaptation, error bars

±1 SD.

Figure 8: Rapamycin inhibits de novo CNV events. Cells carrying a single copy formaldehyde-copper resistance cassette were grown in the absence of selection before plating on CuS0₄ / formaldehyde which selects for cells with a de novo random cassette duplication. The majority of duplications occur through loss of heterozygosity (all events, left), while a minority occur through chromosomal rearrangements (right). Both are effectively repressed by growth in rapamycin. Error bars ±1 SD, p-values calculated by paired t-test.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided the use of a histone

acetyltransferase inhibitor to prevent the emergence of mutations causing resistance to a therapeutic agent. According to a particular aspect of the invention which may be mentioned, there is provided the use of a histone acetyltransferase inhibitor to prevent resistance to a therapeutic agent. The term "histone acetyltransferase inhibitor" as used herein, refers to a modulator such as an inhibitor (i.e. competitive, non-competitive or un-competitive inhibitor) or antagonist (i.e. competitive, non-competitive or un-competitive antagonist) that decreases the rate or activity of a histone acetyltransferase protein or histone acetyltransferase pathway. In one embodiment, the histone acetyltransferase inhibitor inhibits a histone H3 lysine 56 (H3K56) acetyltransferase. In a further embodiment, the histone acetyltransferase inhibitor inhibits Rtt109. In one embodiment, the histone acetyltransferase inhibitor inhibits a p300/CBP histone acetyltransferase. Methods of identifying compounds which inhibit H3K56

acetyltransferases are known in the art, for example as disclosed in WO2012/178036.

It will be appreciated that other suitable histone acetyltransferase inhibitors may be used. Suitable examples include chemical compounds, antibodies which specifically bind to histone acetyltransferases or fragments thereof, histone acetyltransferase substrates, histone acetyltransferase product analogs or natural inhibitors. Further suitable examples include histone acetyltransferase inhibitors such as curcumin, C646, anacardic acid, CPTH2, garcinol and MB-3, as well as the inhibitors described in WO2012/178036. In one embodiment, the histone acetyltransferase inhibitor is curcumin or C646.

The data shown herein confirms that decreased activity of the histone acetyltransferase pathway has been linked with a decrease in copy number variation (CNV) associated with de novo mutations associated with resistance, therefore, an inhibitor or antagonist of histone acetyltransferase pathway finds great utility in the invention. Thus, in one embodiment, the histone acetyltransferase inhibitor leads to a decrease in CNV.

According to a further aspect of the invention, there is provided the use of an mTOR inhibitor to prevent the emergence of mutations causing resistance to a therapeutic agent. According to a further particular aspect of the invention which may be mentioned, there is provided the use of an mTOR inhibitor to prevent resistance to a therapeutic agent.

The use described herein has significant advantages, such as the ability to prevent the emergence of mutations which may cause, or result in, acquired resistance to a therapeutic agent emerging in response to treatment with said therapeutic agent.

The term "mTOR" as used herein, refers to the mechanistic target of rapamycin, also known as mammalian target of rapamycin (mTOR) or FK506-binding protein 12-rapamycin- associated protein 1 (FRAP1), further homologues of target of rapamycin (TOR) and associated signaling pathways, along with orthologous pathways in diverse eukaryotes. The mTOR signalling pathway is also associated with the PI3K/AKT pathway, or axis, and comprises two distinct multiprotein complexes, mTORCI and mTORC2. These two complexes have a separate network of protein partners, feedback loops, substrates, and regulators. mTOR complex 1 (mTORCI), is composed of mTOR, Raptor, ml_ST8, PRAS40, and DEPTOR and mTOR complex 2 (mTORC2), is composed of mTOR, Rictor, ml_ST8, mSinl , PRR5/Protor, and DEPTOR. The term "mTOR signaling pathway" as used herein, refers to one or more biological components that participates in or is part of the rapamycin-sensitive mTORCI complex or any component upstream or downstream of said participating component. Furthermore, it will be accepted in the art that reference herein to "mTOR pathway" comprises further orthologues of the TOR pathway; the core mTORCI complex is functionally conserved from yeast (Tor1/Tor1 , Kog1/Las24, Lst8 and Tco89) to mammals (mTOR, Raptor, ml_ST8, PRAS40, DEPTOR), and exists in all studied eukaryotes excepting some parasitic microsporidia (Shertz et al BMC Genomics (2010), 1 1 , 510, Loewith and Hall, Genetics, (201 1), 189, 1 177-1201). TOR signalling has previously been described to be determinant of cell survival in response to DNA damage (Shen et al (2007) Molecular and Cellular Biology 27(20), 7007-7017) and the TOR pathway has previously been described to regulate ribosomal DNA amplification (Jack et al (2015) PNAS 1 12(31), 9674-9679).

The term "mTOR inhibitor" as used herein, refers to a modulator such as an inhibitor (i.e. competitive, non-competitive or un-competitive inhibitor) or antagonist (i.e. competitive, non- competitive or un-competitive antagonist) that decreases the rate or activity of the mTOR protein or mTOR pathway. In one embodiment, the mTOR inhibitor is rapamycin.

It will be appreciated that other suitable mTOR inhibitors may be used. Suitable examples include chemical compounds, antibodies which specifically bind to mTOR or fragments thereof, mTOR substrates, mTOR product analogs or natural inhibitors. Further suitable examples include first generation mTOR inhibitors, including rapalogs such as temsirolimus (CCI-779), everolimus (RAD001) and ridaforolimus (AP-23573), and second generation mTOR inhibitors. The data shown herein confirms that decreased activity of the TOR pathway has been linked with a decrease in copy number variation (CNV) associated with de novo mutations associated with resistance, therefore, an inhibitor or antagonist of the mTOR signaling pathway finds great utility in the invention. Thus, in one embodiment, the mTOR pathway inhibitor leads to a decrease in CNV.

The term "resistance" as used herein refers to a state of reduced effectiveness of a therapeutic agent. In one embodiment, said resistance is associated with one or more genetic variations selected from: mutations (e.g. point mutations), substitutions, deletions, insertions, single nucleotide polymorphisms (SNPs), haplotypes, chromosome abnormalities (e.g. chromosomal rearrangements), copy number variation (CNV), and DNA inversions. In a further embodiment, said resistance is associated with an increase in CNV.

Eukaryotic genomes contain abundant multi-copy sequences, from low-copy segmental duplications to the giant tandem arrays found at key functional regions such as centromeres and the ribosomal DNA (rDNA) (Richard, G. F. et al (2008) Microbiology and Molecular Biology Reviews : MMBR 72, 686-727). Therefore, the term "copy number variation" as used herein, will be understood by one in the art to refer to a form of structural variation where alterations of the DNA of a genome results in the cell having an abnormal or, for certain genes, a normal variation in the number of copies of one or more sections of the DNA.

Landmark studies established that CNV is widespread in humans (Sabat, J. (2004) Science 305, 525-528 and lafrate, A. J. et al (2004) Nature Genetics 36, 949-951) with 5-10% of the reference genome showing CNV between normal individuals (Zarrei, M. et al (2015) Nature Reviews Genetics 16, 172-183). The pathological effects of CNV show that copy number impacts on gene expression and directly influence RNA processing (Craddock, N. et al (2010), Nature 464,713-720; Stankiewicz, P. & Lupski, J. R. (2010), Annu Rev Med , 437- 455; Cruz, C. and Houseley, J., (2014) eLife 3, e01581).

CNVs are divided into recurrent events that occur in multiple, independent populations, and non-recurrent events that are observed only once. The classic mechanism for CNV, nonallelic homologous recombination (NAHR), involves DNA repair using a homologous sequence template at a non-allelic site, leading to the duplication or deletion of large tracts of sequence (Liu, P. et al (2012) Current Opinion in Genetics and Development 22, 211-220 and Hastings, P. et al (2009) Nature Reviews Genetics 10, 551-564). NAHR is particularly efficient in tandem repeat sequences, forming a common source of recurrent CNV (George, C. M. et al (2012) Critical Reviews in Biochemistry and Molecular Biology 47, 294-313), but NAHR can also form non-recurrent CNVs through recombination between widespread homologous sequences such as retrotransposons. Alternatively, non-recurrent CNVs can arise through recombination between apparently non-homologous sequences utilising only a few basepairs of microhomology at the breakpoints. Repair of many different genomic lesions can give rise to CNV events through NAHR and microhomology-mediated pathways, although efficient mechanisms have evolved to ensure that CNV remains rare in normal cells (George, C M. et al (2012) Critical Reviews in Biochemistry and Molecular Biology 47, 294- 313). CNV can be beneficial, detrimental or phenotypically neutral to the cell. CNV is often considered in the context of human disease, but non-recurrent CNVs emerge under selection in microorganisms and recurrent CNVs control environmental adaptation at some loci. Therefore in a further embodiment, the increase in CNV is at loci prone to de novo mutations. It will be known to those skilled in the art that the term "de novo mutation" as used herein, refers to an alteration in a gene that is present for the first time.

It is widely considered that both de novo and recurrent mutations including CNV occur at random, and therefore that adaptation of cells or organisms to challenging environments occurs through the action of natural selection on random mutations that are the inevitable result of normal cellular function. Under such a model, although adaptation may be accelerated by raising the mutation rate using a chemical mutagen, suppression of adaptation should be impossible. In contrast to this assumption, we provide data in Figure 5 showing that the acquisition of resistance to the anti-microbial compound copper sulfate can be completely suppressed by co-administration of rapamycin. We also demonstrate that random CNV events of single copy loci are effectively suppressed by rapamycin (see Figure 8).

Furthermore, we demonstrate that the rate at which CNV events occur can be dependent upon a single histone modification, acetylation of Histone H3 lysine 56 (H3K56). H3K56 acetylation by histone acetyltransferase Rtt109 is required for DNA damage tolerance, particularly with regard to lesions that block replication. Acetylation of H3K56ac required for appropriate activation of the S-phase checkpoint and recombination-mediated repair (Thaminy et al. (2007) J Biol Chem 282, 37805-37814, Munoz-Galvan et al. (2013) PLoS Genet 9:e1003237). Remarkably, the ability of rapamycin to prevent CNV at the yeast ribosomal DNA is in part mediated through H3K56 acetylation (Jack et al. (2016) PNAS 1 12,9674-9679), linking TOR signaling to damage repair. The role of H3K56 acetylation in genome stability is conserved to higher eukaryotes, where acetylation is performed by CBP and p300 (Das et al. (2009) Nature 459, 1 13-1 17 and Vempati et al. (2010) J Biol Chem 285, 28553-28564).

Compositions According to a further aspect of the invention, there is provided a pharmaceutical composition comprising an mTOR inhibitor and a therapeutic agent for use in therapy.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.

Since the compounds described herein are intended for use in pharmaceutical compositions it will readily be understood that they are each preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are given on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the

pharmaceutical compositions.

In one embodiment, the pharmaceutical composition comprises one or more

pharmaceutically acceptable carrier(s), diluent(s) and/or excipient(s). The carrier, diluent and/or excipient must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

Pharmaceutical compositions of the invention can be prepared by intimately mixing the compounds with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. These procedures may involve mixing, granulating and

compressing or dissolving the ingredients as appropriate to the desired preparation.

The compounds of the invention may be administered in conventional dosage forms prepared by combining a compound of the invention with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as

appropriate to the desired preparation.

The compounds or their pharmaceutically acceptable salts may be administered by any convenient method, e.g. by oral, parenteral, buccal, sublingual, nasal, rectal or transdermal administration, and the pharmaceutical compositions adapted accordingly, for administration to mammals including humans.

The compounds or their pharmaceutically acceptable salts which are active when given orally can be formulated as liquids or solids, e.g. as syrups, suspensions, emulsions, tablets, capsules or lozenges.

The topical formulations of the present invention may be presented as, for instance, ointments, creams or lotions, eye ointments and eye or ear drops, impregnated dressings and aerosols, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

The formulations may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1 % up to about 98% of the formulation. More usually they will form up to about 80% of the formulation.

A liquid formulation will generally consist of a suspension or solution of the active ingredient in a suitable liquid carrier(s) e.g. an aqueous solvent such as water, ethanol or glycerine, or a non-aqueous solvent, such as polyethylene glycol or an oil. The formulation may also contain a suspending agent, preservative, flavouring and/or colouring agent.

Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatine, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatine, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents.

Typical parenteral compositions consist of a solution or suspension of the active ingredient in a sterile vehicle, water being preferred, or parenterally acceptable oil, e.g. polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions the compound can be dissolved in water for injection and filter-sterilised before filling into a suitable vial or ampoule and sealing.

Advantageously, agents such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilised powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilisation cannot be accomplished by filtration. The compound can be sterilised by exposure to ethylene oxide before suspending in the sterile vehicle.

Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.

Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active ingredient in a pharmaceutically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container which can take the form of a cartridge or refill for use with an atomising device. Alternatively the sealed container may be a disposable dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas e.g. air, or an organic propellant such as a fluoro-chloro-hydro-carbon or hydrofluorocarbon. Aerosol dosage forms can also take the form of pump-atomisers.

Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles where the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter. Compositions suitable for transdermal administration include ointments, gels and patches.

In one embodiment the composition is in unit dose form such as a tablet, capsule or ampoule.

Uses

In one embodiment, there is provided use of the invention in combination with said therapeutic agent.

The term "therapeutic agent" as used herein refers to a factor such as a microorganism, chemical substance, or a form of radiation, the presence or absence of which (as in deficiency diseases) results in disease or a more advanced form of disease. According to a further embodiment, said therapeutic agent is an anticancer agent.

The term "anticancer agent" as used herein, refers to radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents. Examples of anti-tumor agents include, but are not limited to, cisplatin, ifosfamide, paclitaxel, taxanes, topoisomerase I inhibitors (e. g., CPT-1 1 , topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, temodal, and taxol.

According to an alternative embodiment, said therapeutic agent is an anti-infective agent.

The term "anti-infective agent" as used herein, refers to antimicrobial drugs (such as antibiotics), antifungal drugs, antiviral drugs and antiparasitic agents.

In one embodiment, the anti-infective agent is an antiparasitic agent, such as an

antiprotozoal agent or an anthelmintic agent. Examples of suitable antiparasitic agents include: Abamectin, Anti-arthropod medications, Anticestodal agents, Arprinocid, Arsenamide, Ascaricide, Avermectin, Bephenium

hydroxynaphthoate, Bithionol, Carbadox, Ciclobendazole, Clopidol, Cymiazole, Decoquinate, Desaspidin, Dichlorophen, Diclazuril, Diethylcarbamazine, Dimetridazole, Ectoparasiticide, Emodepside, Epsiprantel, Ethopabate, Fexinidazole, Flubendazole, Halofuginone,

Hycanthone, Isometamidium chloride, Ivermectin, Lasalocid, Macrofilaricide, Malathion,

Medicinal fungi, Megazol, Melarsomine, Metrifonate, Milbemycin oxime/lufenuron, Narasin, Niridazole, Oltipraz, Oryzalin, Oxamniquine, Oxantel, Pafuramidine, Pediculicide, Permethrin, Praziquantel, Propamidine, Pyrantel pamoate, Quinapyramine, Rafoxanide, Robenidine, Salicylhydroxamic acid, Salinomycin, Selamectin, Stibophen, Streptomyces isolates, Tetraphenylporphine sulfonate, Tiabendazole and Toltrazuril. Examples of suitable antiprotozoal agents include: Acetarsol, Agents against amoebozoa, Amphotericin B, Amprolium, Antitrichomonal agent, Arsthinol, Atovaquone,

Atovaquone/proguanil, Azanidazole, Benznidazole, Broxyquinoline, Buparvaquone,

Carbarsone, Carnidazole, Chiniofon, Chlorquinaldol, Chromalveolate antiparasitics, Clazuril, Clefamide, Clioquinol, Coccidiostat, Dehydroemetine, Difetarsone, Diloxanide furoate, Diminazen, Disulfiram, Eflornithine, Emetine, Etofamide, Excavata antiparasitics, Fumagillin, Furazolidone, Glycobiarsol, Imidocarb, Ipronidazole, Maduramicin, Meglumine antimoniate, Melarsoprol, Mepacrine, Metronidazole, Miltefosine, Nicarbazin, Nifurtimox, Nimorazole, Nitarsone, Nitazoxanide, Nitrofural, Ornidazole, Paromomycin, Pentamidine, Pentavalent antimonial, Phanquinone, Phenamidine, Propenidazole, Quinfamide, Ronidazole,

Secnidazole, Sodium stibogluconate, Suramin, Teclozan, Tenonitrozole, Tilbroquinol, Tinidazole, Trimetrexate and Trypanocidal agent.

Examples of suitable anthelmintic agents include: Albendazole, Amoscanate, Antinematodal agent, Antiplatyhelmintic agent, Ascaridole, Befuraline, Bitoscanate, Diatrizoic acid,

Dithiazanine iodide, Doramectin, Emodepside, Epsiprantel, Fenbendazole, Flavaspidic acid BB, Flubendazole, Hexylresorcinol, Levamisole, Mebendazole, Milbemycin, Milbemycin oxime, Moxidectin, Niclosamide, Nitroscanate, Oxfendazole, Oxyclozanide, Peganum harmala, Piperazine, Praziquantel, Pyrvinium, Suramin, Taeniacide, Tribendimidine and Triclabendazole.

In a further embodiment, said anti-infective agent is an agent which specifically targets one or more eukaryotic pathogens or parasites. The invention finds particular utility in the combination of an mTOR inhibitor or histone acetyltransferase inhibitor with an agent which specifically targets one or more eukaryotic pathogens or parasites and provides the advantage of being able to block development of drug resistance in eukaryotic pathogens or parasites.

In a yet further embodiment, said one or more eukaryotic pathogens or parasites are selected from: unicellular pathogens, such as C albicans, C neoformans, Plasmodium, trypanosomes, leishmania and toxoplasma; and parasitic worms, such as Schistosoma. In one embodiment, the therapy comprises co-therapy, adjunctive therapy or combination therapy, involving administration of an mTOR inhibitor and therapeutic agent.

In one embodiment, the therapy comprises co-therapy, adjunctive therapy or combination therapy, involving administration of a histone acetyltransferase and therapeutic agent.

As used herein, the terms "co-therapy", "adjunctive therapy" and "combination therapy" shall mean treatment of a subject in need thereof by administering an mTOR inhibitor or a histone acetyltransferase inhibitor and therapeutic agent by any suitable means, simultaneously, sequentially, separately or in a single pharmaceutical formulation.

Thus, according to a further aspect of the invention there is provided an mTOR inhibitor and therapeutic agent for use in co-therapy, adjunctive therapy or combination therapy wherein the mTOR inhibitor and therapeutic agent are administered simultaneously, sequentially, separately or in a single pharmaceutical formulation. In one embodiment of this aspect of the invention, the mTOR inhibitor and therapeutic agent are as defined hereinbefore for other aspects of the invention.

According to a further aspect of the invention there is provided a histone acetyltransferase inhibitor and therapeutic agent for use in co-therapy, adjunctive therapy or combination therapy wherein the histone acetyltransferase inhibitor and therapeutic agent are

administered simultaneously, sequentially, separately or in a single pharmaceutical formulation. In one embodiment of this aspect of the invention, the histone acetyltransferase inhibitor and therapeutic agent are as defined hereinbefore for other aspects of the invention.

When administration is sequential, either compound may be administered first. When administration is simultaneous, the combination may be administered either in the same or different pharmaceutical composition.

When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds in the art.

In one embodiment, there is provided the manufacture of a medicament as defined herein for use in the prevention of resistance to a therapeutic agent. According to a further aspect of the invention, there is provided the use of a pharmaceutical composition as defined herein in the manufacture of a medicament for the prevention of the emergence of mutations causing resistance to a therapeutic agent. According to a further particular aspect of the invention which may be mentioned, there is provided the use of a pharmaceutical composition as defined herein in the manufacture of a medicament for the prevention of resistance to a therapeutic agent.

According to a further aspect of the invention, there is provided a method of preventing the emergence of mutations causing resistance to a therapeutic agent, which comprises administering to a subject in need thereof a therapeutically effective amount of a

pharmaceutical composition as defined herein. According to a further particular aspect of the invention which may be mentioned, there is provided a method of preventing resistance to a therapeutic agent, which comprises administering to a subject in need thereof a

therapeutically effective amount of a pharmaceutical composition as defined herein.

In a further embodiment, the composition of the invention is used in the treatment of cancer.

Examples of cancers (and their benign counterparts) which may be treated (or inhibited) include, but are not limited to tumours of epithelial origin (adenomas and carcinomas of various types including adenocarcinomas, squamous carcinomas, transitional cell carcinomas and other carcinomas) such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gall bladder and biliary system, exocrine pancreas, kidney, lung (for example adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomas and mesotheliomas), head and neck (for example cancers of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands, nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (for example thyroid follicular carcinoma), adrenal, prostate, skin and adnexae (for example melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic naevus); haematological malignancies (i.e. leukemias, lymphomas) and premalignant haematological disorders and disorders of borderline malignancy including haematological malignancies and related conditions of lymphoid lineage (for example acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma

[DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas, Hodgkin's lymphomas, hairy cell leukaemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and haematological malignancies and related conditions of myeloid lineage (for example acute myelogenous leukemia [AML], chronic myelogenous leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythaemia vera, essential thrombocythaemia and primary myelofibrosis, myeloproliferative

syndrome, myelodysplasia syndrome, and promyelocytic leukemia); tumours of

mesenchymal origin, for example sarcomas of soft tissue, bone or cartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas, rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioid sarcomas, gastrointestinal stromal tumours, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumours of the central or peripheral nervous system (for example astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumours and schwannomas); endocrine tumours (for example pituitary tumours, adrenal tumours, islet cell tumours, parathyroid tumours, carcinoid tumours and medullary carcinoma of the thyroid); ocular and adnexal tumours (for example retinoblastoma); germ cell and trophoblastic tumours (for example teratomas, seminomas, dysgerminomas, hydatidiform moles and choriocarcinomas); and paediatric and embryonal tumours (for example medulloblastoma, neuroblastoma, Wilms tumour, and primitive neuroectodermal tumours); or syndromes, congenital or otherwise, which leave the patient susceptible to malignancy (for example Xeroderma Pigmentosum).

According to a further aspect of the invention, there is provided the use of a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of disease, such as cancer.

According to a further aspect of the invention, there is provided a method of treating disease, such as cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition as defined herein.

According to a further aspect of the invention, there is provided a pharmaceutical

composition as defined herein for use in the treatment of a disease or condition mediated by an increase in copy number variation. According to a further aspect of the invention, there is provided the use of an mTOR inhibitor for the treatment of a disease or condition mediated by an increase in copy number variation (CNV). Data is presented herein in Figure 4 which demonstrates the effectiveness of a TOR inhibitor for the inhibition of CNV. It will be accepted in the art that the homology of TOR with the mammalian target of rapamycin (mTOR) allows translation of the data presented in the Examples for use throughout eukaryotic organisms where orthologues of the TOR pathways are present, such as the mTOR signaling pathway.

According to a further aspect of the invention, there is provided the use of a histone acetyltransferase inhibitor for the treatment of a disease or condition mediated by an increase in copy number variation (CNV).

In a further embodiment there is provided the use of a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of a disease or condition mediated by an increase in copy number variation.

According to a further aspect of the invention, there is provided a method of treating a disease or condition mediated by an increase in copy number variation, which comprises administering to a subject in need thereof a therapeutically effective amount of a

pharmaceutical composition as defined herein.

The term "subject" as used herein, refers to an animal, preferably a mammal, most preferably a human adult, child or infant, who has been the object of treatment, observation or experiment. In one embodiment, the subject to be treated is a human. It will be appreciated that references herein to "treatment" extend to prophylaxis, prevention of recurrence and suppression or amelioration of symptoms (whether mild, moderate or severe) as well as the treatment of established conditions.

The term "therapeutically effective amount" as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of one or more of the symptoms of the disease or disorder being treated; and / or reduction of the severity of one or more of the symptoms of the disease or disorder being treated.

In the embodiment of co-therapy, adjunctive therapy or combination therapy, a

therapeutically effective amount shall mean that amount of the combination of agents taken together so that the combined effect elicits the desired biological or medicinal response. For example, the therapeutically effective amount of co-therapy comprising administration of an mTOR inhibitor and therapeutic agent would be the amount of an mTOR inhibitor and therapeutic agent that when taken together or sequentially have a combined effect that is therapeutically effective. In a further example, the therapeutically effective amount of co- therapy comprising administration of a histone acetyltransferase inhibitor and therapeutic agent would be the amount of a histone acetyltransferase inhibitor and therapeutic agent that when taken together or sequentially have a combined effect that is therapeutically effective.

Further, it will be recognized by one skilled in the art that in the case of co-therapy with a therapeutically effective amount, the amount of an mTOR inhibitor or histone

acetyltransferase inhibitor and therapeutic agent individually may or may not be

therapeutically effective.

Dosage

The dose of the compounds of the claimed invention, used in the treatment of the

abovementioned disorders or diseases will vary in the usual way with the particular disorder or disease being treated, the weight of the subject and other similar factors. However, as a general rule, suitable unit doses may contain from 0.1 % to 100% by weight, for example from 10 to 60% by weight, of the active material, depending on the method of administration. The composition may contain from 0% to 99% by weight, for example 40% to 90% by weight, of the carrier, depending on the method of administration. The composition may contain from 0.05 mg to 1000 mg, for example from 1.0 mg to 500 mg, of the active material, depending on the method of administration. The composition may contain from 50 mg to 1000 mg, for example from 100 mg to 400 mg of the carrier, depending on the method of administration. The dose of the compound used in the treatment of the aforementioned disorders will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and other similar factors. However, as a general guide suitable unit doses may be in the range of 50 mg to 1500 mg per day, for example 120 mg to 1000 mg per day. Such therapy may extend for a number of weeks, months or years.

It will be recognised by one of skill in the art that the optimal quantity and spacing of individual dosages of the compounds of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular mammal being treated, and that such optimums can be determined by

conventional techniques. In addition, factors associated with the particular patient being treated, including patient age, weight, diet and time of administration, will result in the need to adjust dosages. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound of the invention given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

Where the inhibitor (i.e. the mTOR inhibitor or histone acetyltransferase inhibitor) and therapeutic agent are administered in separate dosage forms, the number of dosages administered per day for each compound may be the same or different. The inhibitor and therapeutic agent may be administered via the same or different routes of administration. Examples of suitable methods of administration include, but are not limited to, oral (i.e. peroral p.o.), intravenous (iv), intramuscular (im), subcutaneous (sc), intranasal,

transdermal, and rectal. Compounds may also be administered directly to the nervous system including, but not limited to, intracerebral, intraventricular, intracerebroventhcular, intrathecal, intracisternal, intraspinal and / or peri-spinal routes of administration by delivery via intracranial or intravertebral needles and / or catheters with or without pump devices. The inhibitor and therapeutic agent may be administered according to simultaneous or alternating regimens, at the same or different times during the course of the therapy, concurrently in divided or single forms.

Advantageously, the inhibitor and therapeutic agent may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. The following non-limiting examples illustrate the invention: EXAMPLE 1

Rapamycin represses adaptation through CNV at CUP1 Background

Copper tolerance in yeast is a classic example of adaptation through CNV as copper tolerance is proportional to the copy number of the tandemly repeated CUP1 gene, which encodes a copper chelating protein (Karin, M. et al (1984) PNAS 81 , 337-341 and Fogel, S. et al (1982) PNAS 79, 5342-5346). Copper tolerance therefore forms a logical model system for determining whether drugs that modulate recombination can alter the rate of adaptation (Figure 1). Materials and methods

Construction of 3xCUP1 strain

Strain UCC5179 (Gottschling group) was transformed with pJH252 (one CUP1 repeat

[ChrVIII: 214256.. 216239] cloned into pRS316) then the entire repeated region at the chromosomal CUP1 locus [ChrVIII: 212265..216250] was replaced with a KanMX6 marker to make YSF59. A plasmid (pJH266) was subcloned containing three complete CUP1 repeats [ChrVIII: 214256.. 216239] and an ADE2 marker surrounded by CUP1 flanking sequences [ChrVIII: 21 1940.. 212265 and 216250.. 216547] (Figure 2). pJH266 was digested with Sad and transformed into YSF59 to replace the KanMX6 cassette, forming YSF61. pJH252 was then selected out of this strain using fluoroorotic acid (FOA), forming YJH913. Gene deletions encompass complete open reading frames and were created using standard methods.

Cell growth

Single colonies of YJH913 were grown to saturation in synthetic complete glucose media (SC), and diluted 1 : 1000 into SC containing 0 θΓ 300μΜ CuS0₄ and 0 or 25nM rapamycin. These cultures were grown to saturation at 30°C.

Copper sensitivity assay

2.5μΙ culture was diluted to 200μΙ SC in each well of a 96 well flat-bottomed cell culture plate, with concentrations of CuS0₄ between 0 and 2mM. Plates were covered with a gas permeable membrane and grown at 30°C for 3 days. Cells were re-suspended by pipetting and OD600 measured using a BD FLUOstar Omega plate reader. Southern blotting

Cells from 2ml saturated culture were washed with 50mM EDTA then spheroplasted with 250μΙ 0.34U/ml lyticase (Sigma L4025) in 1.2M sorbitol, 50mM EDTA, 10mM DTT at 37°C for 45 min. After centrifuging at 1 ,000g, cells were gently resuspended in 400μΙ of 0.3% SDS, 50mM EDTA, 100μg/ml RNase A and incubated at 37°C for 30 min. 4μΙ of 20mg/ml proteinase K was added, samples were mixed by inversion and heated to 65°C for 30 min. 160μΙ 5M KOAc was added after cooling to room temperature, samples were mixed by inversion and then chilled on ice for 30 min. After 10 min centrifuging at 20,000g, the supernatant was poured into a new tube containing 500μΙ phenol:chloroform pH8 and samples were mixed on a wheel for 30 min. Samples were centrifuged for 10 min at

10,000g, the upper phase was extracted using cut tips and precipitated with 400μΙ isopropanol. Pellets were washed with 70% ethanol, air-dried and left overnight at 4°C to dissolve in 20μΙ TE. After gentle mixing, 10μΙ of each sample was digested with 20U each of EcoRI and Xho\ for 3 hours, phenol:chloroform extracted, ethanol precipitated and separated on a 25cm 1 % TBE gel at 120V overnight. Gel was washed in 0.25N HCI for 15 min, 0.5N NaOH for 45 min and twice in 1.5M NaCI 0.5M Tris pH7.5 for 20 min before transfer to HyBond N+ membrane in 6x SSC. Membrane was probed using a random primed probe to CUP1 [ChrVIII: 214256.. 216239] in UltraHyb (Life Technologies) at 42°C and washed with 0.1x SSC 0.1 % SDS at 42°C. Bands were quantified using ImageQuant (GE) and data analysed using GraphPad. Results

Adaptation through CNV at CUP1

To study CNV during adaptation, we have generated strains with three synthetic CUP1 repeats at the endogenous locus. Growth of these cells in sub-lethal copper leads to adaptation, dramatically increasing copper tolerance as reported (Figure 3A). As a tool to study the mechanism of this process, we have optimised blotting methods to allow detection of amplified CUP1 alleles present in less than 0.1 % of the population, orders of magnitude better than can be observed using PCR, sequencing or array-based CNV detection methods. By southern blotting, we can observe and quantify rare amplified CUP1 alleles which appear in response to copper at levels significantly above controls, explaining the enhanced copper resistance of the adapted population (Figure 3B).

Suppression of rDNA amplification by rapamycin

We have previously found that ribosomal DNA (rDNA) amplification is completely repressed by the TOR inhibitor rapamycin (Figure 4), consistent with rDNA CNV being an active, controlled process (Jack, C. J. et al (2015) PNAS 1 12, 9674-9679). However, the rDNA is a highly specialized locus with specific and apparently private mechanisms for modulating recombination (Tsang and Carr (2008) DNA Repair (Amst)..7, 1613-1623), and these mechanisms absolutely require the Fob1 protein which only binds to the rDNA (Kobayashi, T., ef a/ (1998) Genes Dev. 12, 3821-3830).

Importantly, however, we find that rapamycin treatment impacts CNV at the CUP1 locus: formation of amplified CUP1 alleles through exposure to sub-lethal copper is completely repressed by rapamycin treatment (Figure 5A), and in consequence only minimal adaptation occurs in cells treated with a combination of copper and rapamycin (Figure 5B).

EXAMPLE 2 Adaptation through CNV at CUP1 is enhanced by H3K56 acetylation Background

Acetylation of histone H3 lysine 56 (H3K56ac) is known to be involved in maintaining genome stability, particularly during growth in the presence of DNA damaging agents. Our previous studies of the rDNA highlighted a mechanistic connection between TOR signaling and H3K56ac, leading us to ask whether H3K56 acetylation is important for the CNV events underlying copper adaptation. Materials and methods

Construction of further 3xCUP1 strains

For the hst3b hst4h strain, the open reading frames of HST3 and HST4 were sequentially replaced with TRP1 and URA3 markers in the 3xCUP1 strain described in Example 1. For the rtt109h strain and control, a new 3xCl/P7strain was constructed. The strategy was as in Example 1 , but the parent strain was BY4742 in which the ADE2 gene was replaced with a LEU2 marker. The 3xCUP1 plasmid was also modified such that after the introduction of the 3xCUP1 cassette, the adjacent RSC30 gene remained in-tact. Re-analysis of the effect of rapamycin on copper adaptation in this strain gave similar results to those shown in Figure 5 (data not shown). RTT109 was deleted in this strain by mobilising the rtt109::KanMX4 cassette from the commercially available gene deletion collection.

Cell growth and analysis were performed as in Example 1. Results

Loss of Hst3 and Hst4 accelerates adaptive CUP1 amplification

We mutated histone deacetylases (HDACs) Hst3 and Hst4 that target H3K56ac in the 3xCUP1 strain; we find that hst3A hst4A mutants show a massive increase in the formation of amplified alleles, from <1 % in wild type to -35% in the mutants (Figure 6A). This change substantially increased resistance to high levels of copper (1.5-2mM) after copper exposure (Figure 6B), showing that H3K56ac is a critical factor controlling the rate of adaptive CNV at the CUP1 locus.

H3 K56 acetylation enhances CUP1 amplification

We also deleted the H3K56 histone acetyltransferase Rtt109 in a 3xCUP1 strain; we find that in rtt109h mutants no formation of amplified CUP1 alleles in the presence of copper is detectable by southern blot (Figure 7A), resulting in a major decrease in copper resistance, showing that inhibition of H3K56 histone acetylation transferase activity is a viable strategy for slowing the emergence of adaptive CNV.

EXAMPLE 3

de novo CNV events and loss of heterozygosity are inhibited by rapamycin Background

Many pathological CNV events do not occur in multi-copy sequences, but rather involve the de novo amplification or deletion of sequences during gross chromosomal rearrangements (GCRs). The recombination events underlying GCRs share many similarities with those leading to CNV in multi-copy sequences, leading us to ask whether rapamycin also inhibits such events. We therefore used a published reporter for gene amplification (Zhang, H., et al. (2013) Genetics 193, 785-801) to determine whether rapamycin inhibits de novo gene amplification through chromosomal rearrangement or loss of heterozygosity (LOH).

Materials and methods

Five cultures each of JAY538 (gift from JL Argueso) were inoculated from single colonies in 4ml SC media without or with 25nM rapamycin and grown at 30°C for four days. Cells were washed twice in water and plated. 200 cells were plated on SC media to determine viability, while 1x10⁶ cells were plated on SC or SC -Trp plates containing 1.8mM formaldehyde and 150μΜ CuS0₄ as described to detect marker amplifications (Zhang, H., et al. (2013) Genetics 193, 785-801) with modifications recommended by JL Argueso (personal communication). The strain is diploid and the reporter is heterozygous, with a TRP1 marker at the equivalent location on the other copy of chromosome V. The omission of tryptophan (Trp) from media prevents growth of cells that have undergone LOH events, allowing detection of rarer chromosomal rearrangements. Plates were imaged, colonies counted and compared by a two-tailed t-test.

Results

The reporter strain developed by JL Argueso carries single copies of the SFA 1 and CUP1 genes inserted at a single locus on chromosome V, controlling formaldehyde and copper resistance respectively. During growth of the reporter strain from single colony to saturation (10-12 generations) in the absence of selection, reporter duplications emerged in -0.04% of cells, the majority of these occurring through LOH events, the minority (0.01 %) representing chromosomal rearrangements. Growth in the presence of rapamycin reduced the frequency of reporter duplications >20-fold (p<0.0001), duplications through chromosomal rearrangement being reduced >6-fold (p=0.005) (Figure 8).

Discussion

It is widely assumed that all mutations leading to drug resistance in eukaryotic cells arise through random mutation. This has led to the conclusion that such mutations are

unavoidable, however our experiments prove this not to be the case. Inhibition of TOR signaling and histone acetylation effectively reduce the rate at which mutations emerge. These strategies may target the mechanisms that initially cause resistance mutations, and/or they may inhibit DNA repair of random DNA damage. The latter case is important as most DNA damage must be processed to yield a stable genetic change, and inhibition of that processing would prevent the proliferation of the cell in which the damage had occurred. Targeting such pathways therefore provides a novel means to prevent the emergence of resistant cells in a population under treatment.

Claims

1. Use of a histone acetyltransferase inhibitor to prevent the emergence of mutations causing resistance to a therapeutic agent.

2. Use of an mTOR inhibitor to prevent the emergence of mutations causing resistance to a therapeutic agent.

3. The use as defined in claim 1 or claim 2, wherein said resistance is associated with one or more genetic variations selected from: mutations (e.g. point mutations), substitutions, deletions, insertions, single nucleotide polymorphisms (SNPs), haplotypes, chromosome abnormalities (e.g. chromosomal rearrangements), copy number variation (CNV) and DNA inversions.

4. The use as defined in any one of claims 1 to 3, wherein said resistance is caused by an increase in copy number variation.

5. The use as defined in claim 4, wherein the increase in copy number variation is at loci prone to de novo mutation.

6. The use as defined in any one of claims 1 to 5, in combination with said therapeutic agent.

7. The use as defined in claim 6, wherein said therapeutic agent is an anticancer agent.

8. The use as defined in claim 6, wherein said therapeutic agent is an anti-infective agent.

9. The use as defined in claim 8, wherein said anti-infective agent is an agent which specifically targets one or more eukaryotic pathogens or parasites.

10. The use as defined in claim 9, wherein said one or more eukaryotic pathogens are selected from: unicellular pathogens, such as C albicans, C neoformans, Plasmodium, trypanosomes, leishmania and toxoplasma; and parasitic worms, such as Schistosoma.

1 1. The use as defined in claim 2, wherein the mTOR inhibitor is rapamycin.

12. A pharmaceutical composition comprising a histone acetyltransferase inhibitor and a therapeutic agent for use in therapy.

13. A pharmaceutical composition comprising an mTOR inhibitor and a therapeutic agent for use in therapy.

14. The pharmaceutical composition for use as defined in claim 12 or claim 13, wherein said therapy comprises co-therapy, adjunctive therapy or combination therapy.

15. The pharmaceutical composition for use as defined in any one of claims 12 to 14, wherein the inhibitor and the therapeutic agent are administered simultaneously,

sequentially, separately or in a single pharmaceutical formulation.

16. The pharmaceutical composition for use as defined in claim 13, wherein the mTOR inhibitor is rapamycin.

17. The pharmaceutical composition for use as defined in any one of claims 12 to 16, wherein said therapeutic agent is an anticancer agent.

18. The pharmaceutical composition for use as defined in any one of claims 12 to 16, wherein said therapeutic agent is an anti-infective agent.

19. The pharmaceutical composition for use as defined in claim 18, wherein said anti- infective agent is an agent which specifically targets one or more eukaryotic pathogens.

20. The pharmaceutical composition for use as defined in claim 19, wherein said one or more eukaryotic pathogens are selected from: unicellular pathogens, such as C albicans, C neoformans, Plasmodium, trypanosomes, leishmania and toxoplasma; and parasitic worms, such as Schistosoma.

21. Use of a pharmaceutical composition as defined in any one of claims 12 to 20, in the manufacture of a medicament for the prevention of resistance to a therapeutic agent.

22. A method of preventing resistance to a therapeutic agent, which comprises administering to a subject in need thereof a therapeutically effective amount of a

pharmaceutical composition as defined in any one of claims 12 to 20.

23. Use of a histone acetyltransferase inhibitor for the treatment of a disease or condition mediated by an increase in copy number variation.

24. Use of an mTOR inhibitor for the treatment of a disease or condition mediated by an increase in copy number variation.