GB2530470A

GB2530470A - Methods

Info

Publication number: GB2530470A
Application number: GB1406991.8A
Authority: GB
Inventors: Maciej Adam Kaliszczak; Eric Ofori Aboagye
Original assignee: Imperial Innovations Ltd
Current assignee: Ip2ipo Innovations Ltd
Priority date: 2014-04-17
Filing date: 2014-04-17
Publication date: 2016-03-30
Also published as: GB201406991D0; WO2015159097A3; WO2015159097A2

Abstract

A method for identifying a subject with a disease or condition characterised by the presence of unwanted cells as being likely to respond to treatment comprising administration of a class of histone deactylase inhibitor in the cytoplasm ie a lysine deacetylase class 6 inhibitor (KDAC6) inhibitor having the structure of Formula I, wherein said method comprises determining whether the subject's unwanted cells have or are likely to have an increased dependency on autophagy for survival, and wherein Formula I comprises: The invention also relates to a therapy based on the above method and treatments for viral and diseases and cancer.

Description

METHODS

The present invention relates to subject assessment and therapy using particular lysine deacetylase 6 (KDAC6) inhibitors of Formula I, as defined herein.

KDACs (lysine deacetylases) are a family of proteins that remove acetyl groups from lysine residues on proteins, including both histones and non-histone proteins. They have been categorised into four classes, classes I, II, Ill and lv, and are reviewed in Brandl etaI2009.

Class I KDACs (eg KDACs 1 2, 3 and 8) are widely expressed and are mainly located in the nucleus where their substrates are largely histones. Class II KDAC5 (eg KDAC5 4, 5, 6, 7, 9 and 10) are differently expressed and regulated, and are split into two groups: class IA which consists of KDACs 4, 5, 7 and 9, and class llB which consists of KDACs 6 and 10. Class IIA KOACs are located in the nucleus and cytoplasm, while class IIB KDACs are located mainly in the cytoplasm and have little effect on histones. Class Ill KDACs belong to the sirtuin family of proteins, some of which deacetylate p53 and ct-tubulin, Class IV KDAC proteins are considered an atypical class of KDACs, classified as such based on DNA sequence similarity to the other classes.

KDAC6 is a class II KDAC. Its structure, function, and selective inhibitors thereof are reviewed in Yang et a! 2013. KDAC6 is highly expressed in heart, liver, kidney and pancreatic tissue. Given its mostly cytoplasmic location, KDAC6 has limited activity towards histones. Rather, its main targets are tubulin, HSP9O and cortacin, and it is involved in regulating cell adhesion, motility and chaperone function. KDAC6 can also deacetylate peroxiredoxin, which is involved in regulating redox reactions in viva Similar to KDAC6, KDAC class Ill isoform Sirt2 can also use tubulin as a substrate, and regulate the balance of tubulin acetylation and deacetylation.

In addition to its deacetylase activity, KDAC6 has a ubiquitin binding domain through which it binds to ubiquitinated misfolded proteins targeted for destruction. Misfolded proteins are toxic to the cell and their degradation or neutralisation is essential for survival. In this way, KDAC6 has an important role in aggregating ubiquitinated proteins to form an aggresome, a non-toxic aggregation of proteins that can then be degraded via the lysosomal/autophagy pathway.

KDAC6 has previously been implicated in tumorigenesis and metastasis. For example, the ubiquitin binding activity of KDAC6 has been implicated in various functions of cancer cells including aggresome formation and cargo protein transport. Also, the use of KDAC6 inhibitors in combination with other known anti-carcinogens has been shown to have a synergistic activity towards cancer (eg Hideshima at a! 2005). KDAC6, along with other KOACs, has thus been an attractive target for the development of anti-cancer therapies (Marks eta! 2005, Richon eta! 2002).

KDACB inhibitors currently in development are reviewed in Yang eta! 2013 and appear to derive their utility mainly from inhibition of deacetylase activity. For instance, HPOB is able to inhibit the catalytic activity of KDACG (as shown by an increase of acetylation of a-tubulin) but not its ubiquitin-binding activity (eg it does not prevent the increase of LC3 io induced by trehalose, an autophagy inducer tool compound), and it does not impair the autophagy pathway (Lee eta! PNAS 2012).

Further inhibitors of KDACs are described in WO 20081050125, as is their use in the treatment of diseases such as cancer.

Tumours are well known to exhibit phenotypic heterogeneity, being highly variable in terms of their gene expression, protein expression and other biological phenotypes. This can result in differential responses to anti-cancer therapies between subjects, whereby one group of subjects respond to a therapy positively and another group of subjects show no effect or even respond negatively. Simply put, it is appreciated that there is no one-size-fits-all approach, but that therapies need to be tailored to the individual in question. In order to do so, efforts are aimed at understanding how drugs operate at the molecular level such that subject populations can be stratified and therapies can become more targeted.

Surprisingly and unexpectedly, the inventors have now found that tumours with a high dependency on the autophagy pathway respond especially well to particular KDAC6 inhibitors. For example, Example 1 shows that cells exhibiting high levels of Myc protein are more sensitive to CIA (a particular KDAC6 inhibitor having the structure of Formula I).

The Myc status of a given cell is thought to be highly predictive of a cell's dependency on autophagy for survival since oncogenic activation of c-Myc during transformation inflicts ER stress and elicits the Unfolded Protein Response (UPR), which, in turn, induces autophagy (Hart eta! JCI 2012). Similarly, Examples 2 and 3 show that CiA (a particular KDAC6 inhibitor having the structure of Formula I) by blocking autophagy, synergises with proteasome inhibitors (which increase a cell's dependency on autophagy), and Example 4 shows that CiA inhibits the fusion of autophagosomes and lysosomes, providing further evidence that CiA inhibits KDAC6 via inhibition of autophagy.

Without wishing to be bound by any theory the inventors believe that KDAC6 binds to ubiquitinated misfolded/damaged proteins and also binds to the cytoskeleton. In this way, KDACG acts as a scaffold protein which brings together ubiquitinated proteins and the cytoskeleton to form a cytoprotective aggresome. This then aggregates with other aggresomes to form the autophagosome in which the damaged proteins are degraded.

Inhibiting the ubiquitin binding function of KDAC6 is therefore believed to inhibit the autophagy pathway. It follows, that any cells that have a high dependency on the autophagy pathway for survvaI, are expected to be particularly sensitive to inhibitors of the ubiquitin binding activity of KDAC6. Such cells include cancer cells expressing high levels of Myc-protein, or cancers that otherwise have an increased dependency on autophagy. In addition to cancerous tissue, virally infected cells also rely on autophagy for their survival and propagation. For example, Kyei eta! JOB 2009 show that inhibition of autophagy resulted in a 3-fold decrease in infectious HIV virions while Ke eta! J Olin lnv 2011 report that loss of autophagy decreases hepatitis 0 RNA replication and infectious virus production. Essentially, any disease or condition characterised by the presence of unwanted cells, which cells have a high dependency on autophagy for survival, are expected to be particularly sensitive to the particular KDAO6 inhibitors described herein.

WO 2012/088067 describes methods for predicting and/or determining responsiveness to a histone deacetylase (KDAO) inhibitor. However, there is no mention of any inhibitor of KDAC6 in particular and certainly no mention of, or characterisation of, the KDAO6 inhibitors defined herein.

Wa 2013/1 58984 describes biomarkers to identify subjects that will respond to treatment and treating such subjects. However, there is no mention of, or characterisation of, the KDAO6 inhibitors defined herein.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

A first aspect of the invention provides a method for identifying a subject with a disease or condition characterised by the presence of unwanted cells as being likely to respond to treatment comprising administration of a KDAOB inhibitor having the structure of Formula I, wherein said method comprises determining whether the subject's unwanted cells have or are likely to have an increased dependency on autophagy for survival, and wherein Formula I comprises: R:)NzxLx2] [XLX4}XX6oH (R3)a wherein:

S

Ria represents C1-4 alkyl (which latter group is optionally substituted by one or more substituents selected from halogeno and aryl), aryl or (CH2)2-L1; R22 represents H, C14 alkyl (which latter group is optionally substituted by one or more substituents selected from halogeno and aryl), aryl or (CH2)2-L3; L', L2 and L3 each represents, independently at each occurrence, a leaving group: R3 represents halogeno or C14 alkyl; a represents, independently at each occurrence, an integer from 0 to 4; X1-X2 represents C(O)-CH(Y1), C(H)=C(Y), CH2-CH(Y1), NH-CH(Y1), CH2-C(O), NH-C(O) or CH(Y1); b represents 0 or 1; X3-X4 represents CH=C(Y2), O-CH(Y2), NH-CH(Y2), O-C(O) or NH-C(O); c represents an integer from 0 to 10; Z represents -SO2. NH-or-NH.-S02-; d represents 0 or 1; X5-X6 represents CH2-CH2, CH=CH or O-CH2; and Y1 and 12 independently represent, at each occurrence, H or C alkyl; or a pharmaceutically acceptable derivative thereof, provided that at least one of the following is the case: (a) R18 represents (CH2)2-L1; (b) R2 represents (CH2)2-L3.

The term "leaving group", when used herein, includes references to halogeno (eg Cl, Br, I) and OS(O)2R4 groups wherein R4 is C18 alkyl (optionally substituted by one or more fluoro atoms) or aryl (optionally substituted by one or more substituents selected from C1 alkyl, C4 alkoxy, NO2 and halogeno).

Unless otherwise specified, alkyl groups and alkoxy groups as defined herein may be straight-chain or, when there is a sufficient number (ie a minimum of three) of carbon atoms be branched-chain, and/or cyclic. Further, when there is a sufficient number (ie a minimum of four) of carbon atoms, such alkyl and alkoxy groups may also be part cyclic/acyclic.

Such alkyl and alkoxy groups may also be saturated or, when there is a sufficient number (/e a minimum of two) of carbon atoms, be unsaturated and/or interrupted by one or more oxygen and/or sulfur atoms. Unless otherwise specified, alkyl and alkoxy groups may also be substituted by one or more halogeno, and especially fiuoro, atoms.

When used herein, the term "aryl" includes references to C610 aryl groups such as phenyl, naphthyl and the like. Unless otherwise specified, aryl groups are optionally substituted by one or more substituents selected from halogeno, C14 alkyl and C14 alkoxy. When substituted, aryl groups are preferably substituted by one to five (eg one to three) Pharmaceutically acceptable derivatives include salts and solvates. Salts which may be mentioned include acid addition salts.

Processes for preparing the KDAC6 inhibitors of Formula I are described in detail in W02008/0501 25, which is herein incorporated by reference, particularly pages 10-16. An example of a process for the preparation of a KDAC6 inhibitor of Formula I comprises: for compounds of Formula I in which L1, L2 and/or L3 represents halogeno, halogenation of a corresponding compound of Formula II: :: xx2] b [xLx}xxey0H Formula II wherein R represents C14 alkyl (which latter group is optionally substituted by one or more substituents selected from halogeno and awl), awl, (Cl-t2)2-OH or the structural fragment: ( ).

RO

wherein RX represents H or N(R)R2d, R1' and R2d independently represent C14 alkyl (which latter group is optionally substituted by one or more substituents selected from halogeno and aryl), awl or (CI-12)2-OH, is R represents H, Ci-4 alkyl (which latter group is optionally substituted by one or more substituents selected from halogeno and aryl), aryl or (CH2)2-OH, and R, R3, X1 to X6, a, b and c are as hereinbefore defined, provided that at least one of R, R2G, Rid and R2d represents (CH2)2-OH, for example under conditions known to those skilled in the art (eg reaction of the compound of Formula II under suitable conditions with a halogenating agent such as a hydrohalic acid (eg concentrated hydrochloric or hydrobromic acid, optionally in the presence of a suitable catalyst, such as a zinc halide or hexamethylphosphoramide), a phosphorous halide or oxyhalide (eq PCI3, PCI5, PBr3 or P(O)C13), a thionyl halide (eq SOCI2 or SOBr2), a mixture of trialkyl or triaryl phosphine and halogen (eq a mixture of Ph3P with Cl2, Br2 or 12), Me2SBr2 (prepared from Me2S and Br2) or a mixture of triphenylphosphine and CCL).

The subject may be any individual, for example a human or mammalian individual, such as a horse, dog, pig, cow, sheep, rat, mouse, guinea pig or primate. Preferably, the subject is a human individual.

A disease or condition characterised by the presence of unwanted cells refers to any disease or biological or medical condition or disorder in which at least part of the pathology is mediated by the presence of unwanted cells and which the removal or destruction of those particular cells is advantageous to the subject.

The disease or condition may be caused by the presence of the unwanted cells or else the presence of the unwanted cells may be an effect of the condition. Examples of particular conditions include tumours (benign or malignant), autoimmune conditions, allergic disease (eg asthma), transplantation subjects and infectious diseases, such as viral diseases. It will be appreciated that the agent also has utility in regenerative medicine (eg laboratory grown organs or tissues).

The unwanted cell may be any cell whose presence in a host is undesired. Thus, the cell may be a tumour cell (benign or malignant), a cell from a tumour microenvironment such as tumour fibroblasts or tumour blood vessels, a virally infected cell, a cell introduced as part of gene therapy, or a normal cell which one wishes to destroy for a particular reason.

For instance, it may be desirable to eliminate a subpopulation of immune cells such as T lymphocytes in autoimmune disease or such as B lymphocytes in allergic disease.

In one embodiment, the disease or condition characterised by the presence of unwanted cells is a viral disease wherein destruction of the virally infected cells may be advantageous. Examples include hepatitis B (Wang et a! Hepatology 2005), hepatitis C (Benali-Fure eta! Oncogene 2005), hepatitis D (Huang et a! J Gen Virol 2006), flavivirus (Vu eta! J Virol 2336), Borna disease (Williams et al J Virol 2335) or HIV (Ueno eta! J Cell Biol 2009). Bacterial and fungal infections are also encompassed by the invention, wherein the destruction of the bacterial cells or fungal cells would be advantageous to the subject.

In a preferred embodiment, the disease or condition characterised by the presence of unwanted cells is cancer and the unwanted cells are cancerous cells, Thus, the subject with a disease or condition characterised by the presence of unwanted cells may be a subject with cancer.

By a subject with cancer we include the meaning of subjects who have received a positive diagnosis of cancer, for example from a physician. The cancer may be benign or metastatic, it may be a primary cancer or a secondary cancer. The cancer may be a solid tumour or a blood borne tumour. The cancer may relate to diseases of skin tissues, organs, blood, and vessels, such as cancers of the bladder, bone, blood, brain, breast, cervix, chest, colon, endrometrium, esophagus, eye, gastrointestinal tract, head, kidney, liver, lymph nodes, lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis, throat, and uterus.

In certain embodiments the cancer is a blood borne cancer. The blood borne cancer may be metastatic. Examples of blood borne cancers include Hodgkin's and Non-Hodgkin's Lymphoma, Burkitt's lymphoma, myeloma or lymphomas, Leukemia and plasma cell neoplasm. Chromosome 17p genetic diseases also include blood borne cancers such as promyelocytic leukaemia which has a translocation at lip.

In other embodiments, the cancer is a solid tumour. The solid tumour may be nietastatic.

An example of a solid tumour includes inflammatory breast cancer, neuroblastoma, uterine corpus, mature b-cell neoplasm, endocervical carcinoma, endocervicitis, sinus cancer, sclerosing adenosis of breast, maxillary sinus cancer, bronchiolo-alveolar adenocarcinoma, vulva basal cell carcinoma, diffuse large b-cell lymphoma of the central nervous system, chromosome lip deletion diseases, cerebral primitive neuroectodermal tumor, medullomyoblastoma, large-cell, immunoblastic, primitive neuroectodermal tumor, osteosarcoma, somatic childhood medulloblastoma, plasma cell neoplasm, hepatic angiomyolipoma, Retinoblastoma, melanoma, small cell lung cancer and lung cancer myeloma.

By being likely to respond to treatment, we include the meaning that the subject is likely to benefit clinically as a result of the treatment with the KDAC6 inhibitor defined herein. By treatment, we include the meaning of management, treatment and/or amelioration of the disease or condition, for example cancer, A clinical benefit includes a complete remission, a partial remission, a stable disease (without progression), progression-free survival, disease free survival, improvement in the time-to-progression (of the disease or condition, for example cancer), improvement in the time-to-death, or improvement in the overall survival time of the subject from or as a result of the treatment with the KDAC6 inhibitor having the structure of Formula I. There are criteria for determining a response to therapy and those criteria allow comparisons of the efficacy to alternative treatments (Slapak and Kufe, Priniciples of Cancer Therapy, in Harrison's Principles of Internal Medicine, 13th Edition, eds Isselbacher et a!, McGraw-Hill Inc). For example, a complete response or complete remission of disease or condition, for example cancer, is the disappearance of all detectable malignant disease. A partial response or partial remission of disease or condition, for example cancer, may be, for example, an approximately 50 percent decrease & in the product of the greatest perpendicular diameters of one or more lesions or where there is not an increase in the size of any lesion or the appearance of new lesions.

It will be appreciated that the response to treatment with a KDAC6 inhibitor having the structure of Formula may be variable. The response may be mild, or it may be dramatic.

Any level of response is included in the scope of the present invention. However, it will be appreciated that the level of response will be more positive in a subject in which the unwanted cells that characterise the disease or condition have an increased dependency on autophagy for survival than in a subject in which the unwanted cells that characterise the disease or condition do not have an increased dependency on autophagy for survival.

The monitoring of a response to treatment is standard practice in the art, and there are guidelines and rules to measure responsiveness of cancers to treatment. For example, Response Evaluation Criteria In Solid Tumors (RECIST) is a set of published rules that define when tumours in cancer subjects improve ("respond"), stay the same ("stabilize"), or worsen ("progress") during treatments. Responsiveness may be measured by assessing parameters such as target-tumour shrinkage or clearance, non-target tumour shrinkage or clearance, and reduction or prevention of disease progression or metastasis.

In one embodiment, the method of the invention is an in vitro screening method, by which we include the meaning that the method is carried out in isolation of the subject's body, (eg human or animal body) to which it pertains, on a sample pre-obtained from the body, as is described further below, By a KDAC6 inhibitor having the structure of Formula I, we include the meaning of any compound having the structure of Formula I above wherein the compound inhibits the ubiquitin binding activity of KDAC6. By ubiquitin binding activity of KDAC6, we include the meaning of the ability of KDAC6 to bind to ubiquitinated proteins (eg mono-ubiquitinated proteins or poly-ubiquitinated proteins) or to free ubiquitin.

It will be appreciated that the KDAC6 inhibitor having the structure of Formula I may inhibit the ubiquitin binding activity of KDAC6 by binding to the ubiquitin binding domain of KDAC6, for example as shown in Figure 5 and as discussed in Hook eta! 2002. Hook et a/identified a ubiquitin binding domain localised in the C-terminus (the C-terminal 105 amino acids) of KDAC6. This domain did not, however, show significant similarity to other known ubiquitin binding domains, and as the domain contains a zinc finger, it was termed a Polyubiquitin Associated Zinc finger, or a PAZ domain. Thus, in one embodiment, the KDAC6 inhibitor of Formula I is one that binds to C terminus of KDAC6 (eg within the C-terminal 105 amino acids), and preferably one that binds to the PAZ domain of KDAC6.

However, it will be understood that binding of molecules to proteins can induce conformational changes at sites distant from their binding, and so it may not be necessary to bind to the ubiquitin binding domain for the molecule to be able to inhibit ubiquitin binding activity. For example, a molecule may bind to a site remote from the ubiquitin binding domain but can nevertheless cause a modification to that site and therefore affect its activity. In some cases, the molecule may bind to the ubiquitin binding domain and bind Ia to other sites on the protein that may cause conformational restructuring.

It is known that KDAC6 binds to polyubiquitin via the zinc finger motif and so it is particularly preferred if the KDAC6 inhibitor inhibits the ability of KDAC6 to bind to polyubiquitinated proteins.

In certain preferred embodiments, the KDAC6 inhibitor with the structure of Formula I comprises a metal binding moiety, preferably a zinc-binding moiety such as a hydroxamate. Hydroxamates are potent inhibitors of KDAC6 activity. Without wishing to be bound by theory, it is believed that the potency of these hydroxamates is due, at least in part, to the ability of the compounds to bind zinc. For example, the KDAC6 inhibitors are considered to bind to a Zn2 ion within the active site pocket of KDAC6, thereby enabling a mesomeric or positive inductive transfer of electron to the nitrogen mustard group of the inhibitor. In this way, there is an synergistic interaction between the reaction of the hydroxyamic acid moiety with the Zn2 of the KDAC6 enzyme and the nitrogen mustard group that results in a formation of an aziridine which can alkylate the KDAC6 enzyme and lead to irreversible inactivation of KDAC6. Therefore the compounds are considered to have similar in vitro KDAC6 potency but greatly prolonged duration of action in biological systems. Hence the compounds are considered to have a good therapeutic index and require a reduced frequency of administration to the subject.

In.silico modelling may be used to identify those compounds that inhibit ubiquitin binding activity.

Typically, the KDAC6 inhibitor, inhibits the ubiquitin binding activity of KDAG6 at least 1.15, 1.25, 2, 2.5, 5, 10, 15 and 20 times more than it inhibits the ubiquitin binding activity of another ubiquitin binding protein or any other protein. Preferably, the KDAC6 inhibitor inhibits the ubiquitin binding activity of KDAC6 to an undetectable level. Preferably, the inhibitor does not interfere with the ubiquitin binding activity of another ubiquitin binding protein or any other protein.

Ubiquitin binding activity, and inhibition thereof, can be readily tested without undue burden by, for example, assessing the ability of KDAC6 to bind to ubiquitinated proteins (eg mono-ubiquitinated proteins or poly-ubiquitinated proteins) or to free ubiquitin. Typically, this is done by assessing the binding of KDAC6 to polyubiquitin or to polyubiquitinated proteins, either those through K63 linkages, or those through K48 linkages, amongst others, in the presence and absence of the candidate inhibitor having the structure of Formula I. Binding can be assessed by any suitable technique in the art, for example by immunoprecipitation and western blotting (Lee eta!, PNAS 2012). Another way of assessing ubiquitin binding is by monitoring levels of LC3-1 1 (microtubule associated protein INIB-light chain 3-Il).

For example, the level of LC3-ll can be measured by immunoblotting with an antibody raised against LC3-ll or to a tag conjugated to LC3-ll using techniques readily available in is the art. Thus, it will be appreciated that, in addition to measuring the ability of KDACB to bind to polyubiquitin or ubiquitinated proteins directly, ubiquitin binding activity inhibition may be assessed indirectly by investigating the effect of the KDAC6 inhibitor having the structure of Formula I on markers known to correlate with ubiquitin binding activity of KDAC6, for example autophagy markers, such as LC3-ll.

The inventors believe that by inhibiting ubiquitin binding of KDAC6, the KDAC6 inhibitor having the structure of Formula I will interfere with the formation of aggresomes and inhibit autophagy. Thus, the KDAC6 inhibitor having the structure of Formula I may be one that is capable of inhibiting aggresome formation and therefore autophagy. In this way, the KDAC6 inhibitors defined herein may also be termed aggresome inhibitors, autophagy inhibitors or ubiquitin binding inhibitors rather than histone deacetylease inhibitors to reflect the fact that different modalities of the KDAC may be targeted. The KDAC6 inhibitors defined herein are considered to have little effect on transcription.

It will be appreciated that the KDAC6 inhibitor having the structure of Formula I may also inhibit the deacetylase activity of KDACC as well as the ubiquitin binding activity of KDAC6, or may only inhibit the ubiquitin binding activity of KDAC6 alone. Generally, it also inhibits the deacetylase activity of KDAC6. Deacetylase activity can be assayed using standard techniques in the art, for example by using commercially available kits (eg Sigma CS1 010-as 1 KT). The inhibition of the catalytic activity of KDAC6 can also be assessed by monitoring acetylation of o-tubulin.

Preferably, the KDAC6 inhibitor is one that has the structure of Formula I wherein R1 and R28 represents halogeno species. More preferably, the KDAC6 the inhibitor is CiA or C1B:

CIA

N OH Cl

Chemical Formula; C22H25C12N3045 Molecular Weight; 498.42

CIB NOH )

Chemical Formula: C22H27N304S Molecular Weight: 429.53 CiA and Cl B can be prepared according to standard chemical methods in the art including those described above. Specific methods are described in Examples 3 and 4 of W02008050125, which are incorporated herein by reference.

For the avoidance of doubt, by a KDAC6 inhibitor having the structure of Formula I, we also include the meaning of prodrugs thereof. For example, the KDAC6 inhibitor may be administered as a prodrug which is metabolised or otherwise converted into its active form once inside the body of a subject. Thus, the method of the invention includes a method of identifying a subject with a disease or condition characterised by the presence of unwanted cells as being likely to respond to treatment comprising administration of a prodrug of a KDAC6 inhibitor having the structure of Formula I. The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to unwanted cells (eg tumour cells) compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form (see, for example, D. E. V. Wilman "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions 14, 375-382 (615th Meeting, Belfast 1986) and V. J. Stella et at "Prodrugs: A Chemical Approach to Targeted Drug Delivery Directed Drug Delivery R. Borchardt eta/(ed.) pages 247-267(Humana Press 1985)).

By the phrase "increased dependency on autophagy for survival", we include the meaning of any increase in the dependency of unwanted cell on autophagy for survival. For example, the unwanted cell may be one that has a higher turnover of expressed proteins such that it has a higher level of misfolded proteins that need to be disposed of in a safe manner and/or a deficiency or downregulation of one or more mechanisms (eg proteasome pathway) that normally act to dispose of misfolded proteins in a safe manner. The unwanted cell may have a higher level of autophagy. Typically, the unwanted cell is one in which autophagy is critical for its survival. In other words, the unwanted cell has no alternative pathways to perform the function served by autophagy if autophagy is inhibited.

Normally, such cells are considered to be in a stressed' state, and such stresses may is include hypoxia, nutrient deprivation, oxidative stress and an increase in proliferation. The dependency on autophagy for survival may be at least 1.25, 1.5, 2, 3, 4, 5, 10, 20, 50 or times more than the dependency on autophagy for survival of a control cell, eg a cell that is known not to have an increased dependency on autophagy for survival. Preferably, by increased dependency on autophagy for survival we include the meaning that a unwanted cell is 1.25, 1.5, 2, 3, 4, 5, 10, 20, 50 or 100 times more susceptible to cell death by an inhibitor of autophagy than a control cell, eg a cell that is known not to have an increased dependency on autophagy for survival. For example, the inhibitor of autophagy may be the KDAC6 inhibitor of the invention or any other known autophagy inhibitor, and/or the control cell may be a normal cell under non-stressful conditions, such as a non-cancer cell.

Typically, the comparison is made between the dependency on autophagy for survival of a population of unwanted cells and the dependency on autophagy for survival of a population of control cells. The population may comprise at least 102, 1O, 10, 10, 106, 10, 108, 108, 10, or 1010 cells. Conveniently, the two populations are prepared as samples, and markers of dependency of autophagy analysed in each sample, and compared as described further below. It may be desirable to lyse the cells prior to analysis.

Also, it will be appreciated that the dependency on autophagy for survival of a population of unwanted cells and the dependency on autophagy for survival of a population of control cells, may be compared based on samples that do not contain the cells (eg blood plasma or serum) but which nevertheless contain markers that are correlated with the dependency on autophagy for survival of the cells. For example, when the unwanted cells are cancer cells, the cancer cells may release a protein indicative of an increased dependency on autophagy for survival of the cancer cells into the blood (eg Myc), and the level of this protein in the blood may be compared with the level of the protein in the blood of a normal individual.

It is preferred if the control cells are normal cells that are not characteristic of the disease or condition in question. Thus, when the unwanted cells are cancer cells, the one or more control cells may be a normal non-cancer cell. However, it will be appreciated that the one or more control cells may be characteristic of the disease or condition, but are ones that are known not to have an increased dependency on autophagy for survival. Thus, when the unwanted cells are cancer cells, the one or more control cells may be cancer cells that do not have an increased dependency on autophagy for survival or not to the extent that it renders them more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I. When the unwanted cells are cancer cells, the one or more control cells may be HeLa cells, for example as grown in DMEM containing 10% fetal bovine serum, lysed in buffer and sonicated (Tworkowski eta!, 2002). The HCT116 colon cancer cell line can also be used as an appropriate control cell and can be grown in DMEM containing 10% FBS or RFMI with 10% FBS. Additionally, DLD1 and LOVO colonic carcinoma cell lines have no c-Myc amplification and thus are also appropriate control cells (Okuyama eta! 2010). The control cell could be any cell in which the Myc gene is not highly or over-expressed (eg not constitutively expressed), or wherein Myc protein is inactive at basal levels.

It will be appreciated that the one or more control cells may be cells from the same subject that is to be assessed or may be cells from one or more different subjects.

In addition to comparing the dependency on autophagy for survival of a subject's unwanted cells to control cells, the dependency can also be compared to a mean level of dependency on autophagy for survival of cells in a healthy population.

For example, it will be appreciated that the dependency on autophagy for survival is increased to a level such that the subject's unwanted cells are more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I. This level may vary depending on the type of individual (eg human, horse, dog and so on). The dependency on autophagy for survival may be determined by comparing the dependency in individuals that are known to have cells with high dependency on autophagy for survival, and which are responsive to treatment by a KDAC6 inhibitor having the structure of Formula I, with those levels in cells of normal individuals (eg those with no sign or symptoms of unwanted cells, for example cancer, or individuals with cancer wherein the dependency on autophagy is not increased or not increased to a level which makes the subject more responsive to treatment by a KDACG inhibitor having the structure of Formula I). Typically, the dependency on autophagy for survival which is indicative of the unwanted cells being more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I is a level which is greater than 1 standard deviation above the mean level of dependency on autophagy of cells from a population of normal individuals, for example greater than 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5 orB standard deviations above the mean level of dependency on autophagy for survival of cells from a population of normal individuals (eg those with no sign or symptoms of unwanted cells, for example cancer, or individuals with unwanted cells (eg cancer) wherein the dependency on autophagy of cells is not increased or not increased to a level which makes the subject more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I). It will be appreciated that when comparing level of dependency on autophagy for survival of cells between individuals, the assessment should preferably be done using the same sample, cell type or tissue. As explained above, it will be appreciated that the sample may not contain the cells themselves but nevertheless contains a detectable marker that is correlated with the dependency on autophagy for survival of the cells. Examples of such detectable markers include proteins indicative of an increased dependency on autophagy for survival of a cell and various other molecules as described further below. Preferably, the population comprises at least 5, 10, 50, 100, 200, 300, 400 or 500 individuals and more preferably at least 1000 individuals. It is preferred if the normal individuals are assessed using the most sensitive and specific detection techniques for dependency on autophagy for survival available. Preferably, the normal population is a population of individuals that have been shown not to have unwanted cells, for example cancer, with increased dependency on autophagy for survival, and thereafter have been shown not to develop such unwanted cells, for example cancer, using the same techniques, for example, for at least 6 months, or 1, 2, 3, 4 or 5 years, or more. In addition, it is preferred if the normal individuals' have no or few risk factors for the development of unwanted cells, for example cancer.

By determining whether the subject's unwanted cells have or are likely to have an increased dependency on autophagy for survival, we include the meaning of both directly as or indirectly determining the level of dependency on autophagy for survival in the unwanted cells from the subject.

In one embodiment, determining whether the subject's unwanted cells have an increased dependency on autophagy for survival involves directly determining the level of dependency on autophagy for survival in the unwanted cells from the subject. The most direct method to determine a cell's dependency on autophagy for survival involves taking a sample of cells from a subject and contacting them in vitro with an Inhibitor of autophagy, for example the KDAC6 inhibitor having the structure of Formula I described herein, and monitoring their survival compared to control cells known to have a normal dependency on autophagy for survival. If the cells from the subject have an increased dependency on autophagy for survival than do the control cells, they will die at a lower dose of drug than the dose at which control cells die, for example at least 2, 3, 4, 5, 10, 20 or 50 fold lower than the dose of drug at which control cells die. This technique may routinely be carried out in a clinical lab using components of the invention described herein.

Thus, determining whether a subject's unwanted cells have or are likely to have an increased dependency on autophagy for survival may comprise: a) contacting a sample of the subject's unwanted cells with an inhibitor of autophagy; b) assessing the effect of the inhibitor on the survival of the unwanted cells: and c) comparing the effect of the inhibitor on the survival of the unwanted cells to the effect of the inhibitor on the survival of control cells that have a normal dependency on autophagy; optionally, wherein the inhibitor of autophagy is the KDAC6 inhibitor having the structure of Formula I. The effect of the inhibitor on the survival of control cells may already be known, or be tested at the same time as the effect of the inhibitor on the survival of the subject's unwanted cells.

In another embodiment, the dependency of a given cell on autophagy for survival is measured indirectly, for example, by monitoring the levels of biological molecules or processes known to be responsible for increased dependency on autophagy for survival, or by monitoring the levels of biological molecules or processes known to be eftectors of increased dependency on autophagy for survival. A positive reading in this instance, indicates that a cell is likely to have increased dependency on autophagy for survival.

Examples of indirect measurements of a cell's dependency on autophagy for survival are an increase in the level of autophagy itself, an increase in the level of a biological molecule or process known to cause an increased dependency on autophagy for survival (for example the level of a Myc protein), or an increase in the amount of autophagic components, all of which are likely to indicate the instigation of a cell survival mechanism, suggesting that that particular cell is employing autophagy to survive and hence has an increased dependency on autophagy for survival. Thus, by measuring the level of autophagy or markers of the level of autophagy (eg an upstream or downstream marker), one can conveniently assess whether a cell is likely to have an increased dependency on autophagy for survival.

In one embodiment, by an increased dependency on autophagy for survival we include the meaning that the level of autophagy in a cell is increased by at least 2 fold, 5 fold, 10 fold, 20 fold or 50 fold times the level of autophagy in a control cell. The control cell is typically a normal cell under non-stressful conditions, such as a non-cancer cell.

Biological processes known to result in an increased level of autophagy include an increased rate of protein synthesis or an increased frequency of mistranslation. An increased rate of protein synthesis is known to overwhelm the proteasome resulting in an increased dependency on autophagy for survival, and can be measured directly, by, for example, using metabolic labelling. An increased rate of protein synthesis can also be inferred, for example, from the level of Myc protein or Myc protein activity within the cell.

Cells with high levels of Myc protein or Myc protein activity are known to have a high rate of protein synthesis and a high dependency on the autophagic pathway for protein clearance. Thus, the assessment of a cell's Myc protein amount or activity is a convenient, and particularly preferred, measure of that cell's dependency on autophagy for survival, and is described in more detail below.

The physical process of autophagy can also be monitored directly, and can be used to assess a cell's dependency on autophagy for survival. For example, the number of aggresomes within a given cell can be assayed using immunofluorescence, staining for, for example, y-tubulin/ubiquitin, and analysed by microscopy eg ubiquitin related stains for P62 (a ubiquitin-binding scaffold protein that co-localises with ubiquitinylated protein aggregates) and LC3 II (a ubiquitin-like autophagy cascade protein residing in the phagophore membrane), or monodansylcadaverine (an autofluorescent compound that accumulates in autophagic vacuoles (Knizhnik eta! 2003, Biederbick eta/i 995).

Similarly, the amount of endoplasmic reticulum can be monitored with fluorescence microscopy or electron microscopy. An increase in endoplasmic reticulum stress (also known to result in increased autophagy) can be monitored, for example by qPCRlimmunoblotting of particular genes, for example CHOP, IRE1, GADD45, Herp, BipIGRPJS, GADD153, XBP-1, PTEN, TSC1, TSC2, P53, DRAM or p97. Alternatively, lysosome formation can be monitored, for example by assaying Lamp2 by immunofluorescence. UPR activation can also be monitored as a marker for an increased dependency on autophagy, for example UPR activation can be assayed by monitoring the activation of PERKIeIF2a/ATF4 axis (Hart et a! 2012).

In a particularly preferred embodiment, assessing whether a cell has or is likely to have an increased dependency on autophagy for survival comprises assessing whether that cell has an increased level of Myc protein or an increased level of Myc activity. Myc protein and Myc protein activity closely correlate with protein synthesis rate, which potentiates an increased dependency on autophagy for survival, and so, the level of Myc protein or the level of Myc protein activity can accurately predict a cell's dependency on autophagy for survival.

The importance of the Myc oncogene in cancer is well established. The Myc family of proto-oncogenes (c-Myc, 1-Myc, n-Myc) is activated in 25-35% of all human cancers (Vita and Henrickson 2006 Seminars in Cancer Biol 16:318-330). Translocations of c-Myc to the immunoglobulin locus are also common in certain cancers. Increased Myc activity leads to increased global protein synthesis (Nie eta! 2012 Cell 151: 68-79) which leads to an increased requirement for degradation via the proteasome. In some cases, the proteasome cannot adequately manage the high amount of protein requiring turnover and so misfolded or damaged proteins are directed for destruction by autophagy, via the aggresome pathway, the components of which are often upregulated in such cells, This increased burden makes the cells dependent on autophagy for survival since accumulations of misfolded and damaged proteins are toxic to the cell and can otherwise cause apoptosis.

Without wishing to be bound by any theory, the inventors believe that unwanted cells which have an increased amount of a Myc protein are particularly responsive to treatment by a KDAC6 inhibitor having the structure of Formula I since such cells are highly dependent upon aggresome function and downstream protein degradation via autophagy. In cells that have high Myc levels, and therefore, a high protein translation, the demand for the aggresome to eliminate misfolded polyubiquitinated proteins is greater. The inventors believe that KDAC6 binds to ubiquitinated proteins and is responsible for their recruitment to the cytoskeleton in readiness for their destruction by the aggresome. The KDAC6 inhibitors having the structure of Formula I for use in the present invention are considered to target the interaction between KDAC6 and ubiquitinated proteins, thereby inhibiting the formation of the aggresome and thus autophagy, leading to cell death. Therefore, any cell which exhibits an increased dependency on autophagy for survival, be it through having increased Myc protein expression or otherwise, is believed to be a suitable target for the KDAC6 inhibitor having the structure of Formula I of the present invention.

Whether or not a subject's unwanted cells have an increased amount of a Myc protein can be assessed by determining the level of a Myc protein in the unwanted cell, directly.

However, as is described further below, there are also indirect ways of assessing whether the amount of Myc protein or a Myc protein activity in a subject's unwanted cells is likely to be increased. In either case, it will be appreciated that the determination may then be is used to assess whether the subject is likely to respond to treatment comprising administration of a KDAC6 inhibitor having the structure of Formula I. For the avoidance of doubt, by Myc protein, we include any one or more subtypes of Myc protein such as any one or more of c-Myc, n-Myc or 1-Myc or b-Myc or s-Myc or v-Myc.

For example, humans have at least three subtypes of Myc, namely c-Myc (c-MYC_HUMAN, P01106), n-Myc (n-MYC_HUMAN, P04198) and 1-Myc (1-MYC_HUMAN, P1 2524), whose protein sequences are listed in Figure 6 (SEQ ID Nos: 1-3).Thus, it may be desirable to assess the amount of any one of c-Myc, n-Myc, or 1-Myc individually, or it may be desirable to assess the amount of any two of c-Myc, n-Myc or l-Myc in combination with each other, or it may be desirable to assess the amount of all three of c-Myc, n-Myc and 1-Myc. Other subtypes of Myc found in rats and mice are b-Myc (SEQ ID Nos: 4 and 7) and s-Myc (SEQ ID Nos: 5 and 8), and another subtype found in birds and cats is v-Myc (SEQ ID Nos: 6 and 9). Preferably, it is assessed whether a subject's unwanted cells have or are likely to have, an increased amount of c-Myc.

It will be appreciated that the provided sequences serve only to identify the relevant gene or protein and that any natural variation and polymorphisms of the sequences provided are encompassed by the invention. The invention also includes Myc in other species that have orthologous sequences to those in Figure 6, for example Myc proteins from horse, dog, pig, cow, sheep, rat, mouse, guinea pig or a primate. It will be appreciated, that when the method is used to assess a particular subject's likelihood of responding to treatment, the method preferably comprises determining whether the subject's unwanted cells (eg cancer cells), have, or are likely to have, an increased amount or activity of Myc protein of the same species as the subject. Thus, when the subject is human, the amount or activity of human Myc protein is measured, and so on.

Conveniently, determining whether the subject's unwanted cells have or are likely to have, an increased dependency on autophagy for survival, for example an increased amount or activity of a Myc protein, involves either directly or indirectly determining the dependency on autophagy for survival in a sample obtained from the subject. The sample from the subject may be any suitable sample. In particular embodiments of the invention, a suitable sample is obtained from the subject who is to be assessed (eg optimised for a particular therapy regime), and this sample is provided for the direct or indirect analysis of the dependency on autophagy for survival, for example via assessment of the amount or activity of a Myc protein.

The sample may be a fluid sample and it may be blood, serum, plasma, urine or saliva.

For example, where the unwanted cells are a haematological cancer, it may be particularly desirable to assess, either directly or indirectly, the dependency of the unwanted cells on autophagy from the blood, for example by measuring the amount or activity of Myc protein in the blood, for example from peripheral blood mononuclear cells. Alternatively, the sample may be a tissue sample such as a biopsy sample or excision material taken from the subject, for example buccal scrapings. The tissue may be a biopsy of the unwanted cells, for example a biopsy of a cancer taken from the subject, in which case the dependency on autophagy for survival in the cancer may be monitored by assessing the amount or activity of Myc protein in the sample and which amount or activity of Myc protein may be measured directly or indirectly. However, it will be appreciated that the tissue may be a biopsy of another tissue in the subject, from which the dependency on autophagy for survival of the unwanted cells can be indirectly assessed, for example by assessing Myc protein amounts or activity.

so Preferably, the sample is one that comprises unwanted cells taken from the subject. It will be appreciated that before assessing the dependency on autophagy for survival, it may be appropriate to process the sample that comprises the unwanted cells. For instance, it may be desirable to enrich the sample for the unwanted cells using standard enrichment techniques known in the art. In the case of a B cell lymphoma, for example, one can treat as plasma cells from a subject with magnetic beads coated with anti-CD138 antibody to enrich for B cells, or isolate peripheral blood mononuclear cells.

Thus, the invention includes a method for identifying a subject with a disease or condition characterised by the presence of unwanted cells as being likely to respond to treatment comprising administration of a KDAC6 inhibitor having the structure of Formula I, wherein said method comprises: (a) obtaining or providing a sample from the subject; and (b) assaying the sample to determine whether the subject's unwanted cells have or are likely to have an increased dependency on autophagy for survival, Preferably, the sample is assayed to determine whether the unwanted cells (eg cancer cells) have or are likely to have an increased amount or activity of Myc protein.

Conveniently, determining whether the subject's unwanted cells have or are likely to have an increased dependency on autophagy for survival, comprises determining the amount or activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc) in the unwanted cells from the subject, and assessing whether the level is increased or decreased relative to the level in control cells. Thus, in one embodiment, the method of the invention comprises: (a) obtaining a sample from the subject; and (b) assaying the amount or activity of a protein indicative of an increased dependency on autophagy for su rvi va I. It will be understood that the amount or activity of a protein indicative of increased dependency on autophagy for survival may be positively or negatively correlated with the dependency on autophagy for survival, lea positive or negative indication. Generally, the amount or activity of a protein indicative of increased dependency on autophagy for survival is one that is positively correlated with the dependency on autophagy for survival, in which case an increase in the amount or activity of that protein is indicative of an increased dependency on autophagy for survival. Typical extents of increase of amount or activity of a protein indicative of an increased dependency on autophagy for survival include those described below. However, in some instances, the amount or activity of a protein indicative of increased dependency on autophagy for survival may be one that is negatively correlated with the dependency on autophagy for survival, in which case a decrease in the amount or activity of that protein is indicative of an increased dependency on autophagy for survival. In this case, it will be appreciated that the extents of decrease of the protein that is indicative of an increased dependency on autophagy for survival may correspond to the extents of increase of the protein that is indicative of an increased dependency on autophagy for survival, as outlined in the sections below, It will be appreciated that the amount of a protein indicative of an increased dependency on autophagy for survival (eg Myc) may be increased to a level of the protein such that the subject's unwanted cells (eg cancer cells) are more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I. This level of protein may vary depending on the type of individual (eg human, horse, dog and so on). The level of the protein may be determined by comparing the levels of the protein indicative of an increased dependency on autophagy for survival (eg Myc) in individuals that are known to have unwanted cells (eg cancer cells) with increased levels of the protein and which are responsive to treatment by a KDAC6 inhibitor having the structure of Formula I, with those levels of the protein in normal individuals (eg those with no sign or symptoms of unwanted cells, for example cancer, or individuals with unwanted cells wherein the levels of the protein are either not increased or not increased to a level which makes the subject more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I). Typically, the level of the protein (eg Myc) which is indicative of increased dependency on autophagy for survival is a level of that protein which is greater than 1 standard deviation above the mean level of that protein in a healthy population, for example greater than 1.25, 1.5, 2, 2.5, 3, 3.5, 4, is 4.5, 5, 5.5, 6, 6.5, 7, 7.5 orB standard deviations above the mean level of that protein in a healthy population, that is a population of normal individuals (eg those with no sign or symptoms of unwanted cells (eg cancer cells) or individuals with unwanted cells wherein the levels of the protein indicative of an increased dependency on autophagy for survival (eg Myc) are either not increased or not increased to a level which makes the subject more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I). It will be appreciated that when comparing levels of the protein indicative of an increased dependency on autophagy for survival (eg Myc) between individuals, the assessment should preferably be done using the same sample, cell type or tissue, Preferably, the population comprises at least 5, 10, 50, 100, 200, 300, 400 or 500 individuals and more preferably at least 1000 individuals. It is preferred if the normal individuals are assessed using the most sensitive and specific detection techniques for the protein indicative of an increased dependency on autophagy for survival (eg Myc) avaiiable (eg RT-PCR for example by taq-man, or RNA-seq). Preferably, the healthy population is a population of individuals that have been shown not to have unwanted cells, for example cancer, with increased levels of a protein indicative of an increased dependency on autophagy for survival (eg Myc) and thereafter have been shown not to develop such unwanted cells, for example cancer, using the same techniques, for example, for at least 6 months, or 1, 2, 3, 4 or 5 years, or more, In addition, it is preferred if the normal individuals' have no or few risk factors for unwanted cells, for example cancer cells.

Also, it will be appreciated that the amount of a protein indicative of an increased dependency on autophagy for survival of a population of unwanted cells and the amount of a protein indicative of an increased dependency on autophagy for survival of a population of control cells, may be compared based on samples that do not contain the cells (og blood plasma or serum) but which nevertheless contain markers that are correlated with the dependency on autophagy for survival of the cells. For example, when the unwanted cells are cancer cells, the cancer cells may release a protein indicative of an increased dependency on autophagy for survival of the cancer cells into the blood (eg Myc), and the level of this protein in the blood may be compared with the level of the protein in the blood of a normal Individual.

ic Typically, unwanted cells having an increased dependency on autophagy for survival have a level of a protein indicative of an increased dependency on autophagy for survival (eg Myc) that is at least 1.25, 1.5,2, 3,41 5, 10,20, 50 or 100 times more than the level of that protein in a control cell. Preferably, the protein indicative of an increased dependency on autophagy for survival is Myc. The control cell may be one described above. For example, the control cell may be a normal cell with a normal dependency on autophagy for survival (eg a non-cancer cell), or a cancerous cell from a subject that is known not to have an increased level of a protein indicative of an increased dependency on autophagy for survival (eq Myc), or a cancerous cell that is known to have an increased level of a protein indicative of an increased dependency on autophagy for survival (eq Myc) but not to the extent that it renders the cancer more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I. In a preferred embodiment when the unwanted cells are cancer cells, a subject's cancer is more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I when the level of a protein indicative of an increased dependency on autophagy for survival (eg Myc) in the cancer cell is at least 1.25, 1.5, 2, 3,4, 5, 10, 20,50 or 100 times more than the level of protein indicative of an increased dependency on autophagy for survival (eg Myc) in the control cell (eq a non-cancer cell). Conveniently, the comparison is made between a sample obtained from the subject (eq one comprising cancer cells) and a control sample (eq one comprising control cells such as non-cancer cells).

It will be appreciated that determining the level of a protein indicative of an increased dependency on autophagy for survival can be done directly, for example by measuring the amount of protein itself 1 or indirectly, for example by measuring levels of mRNA encoding the protein or the copy number of the gene encoding the protein or levels of other proteins or markers known to be correlated with the protein indicative of an increased dependency on autophagy for survival.

Assessing the levels of protein is standard practice in the art and any suitable method may be used. For example, ELISA, immunofluorescence, HPLC, gel electrophoresis and capillary electrophoresis, followed by UV or fluorescent detection, may be used to detect and quantify a protein indicative of an increased dependency on autophagy for survival (eg Myc). Methods of measuring the levels of compounds by mass spectrometry are well known in the art and any suitable form of mass spectrometry may be used (see, for example, Mass Spectrometry Principles and Applications, E. De Hoffmann, J. Charette, V. Stroobant, Wiley & Sons, New York, NY, 1996). Western blotting, immunoprecipitation, immunohistochemistry on paraffin, irnmunofluorescence, fluorescence in situ hybridisation and flow cytometry may also be used.

One convenient way of measuring the level of a protein indicative of an increased dependency on autophagy for survival, is to make use of a reagent which can identify the protein indicative of an increased dependency on autophagy for survival (eg Myc protein).

Typically, the reagent is one which binds to the protein indicative of an increased dependency on autophagy for survival, for example a reagent that binds to a Myc protein, but it may be any other type of suitable reagent. Reagents which bind to a protein indicative of an increased dependency on autophagy for survival (eg Myc) include antibodies and peptides, for example those selected from a combinatorial or phage display library. Thus, the reagent may be an antibody and the level of a protein indicative of an increased dependency on autophagy for survival (eg Myc) in a sample may be measured using an immunoassay. The antibody selective for a protein indicative of an increased dependency on autophagy for survival (eg Myc), may itself be labelled, for example with a radioactive label or a fluorescence label or with an enzyme. Alternatively, and preferably, it is detected with a secondary antibody, which binds the antibody selective for a protein indicative of an increased dependency on autophagy for survival, for example a Myc protein, which is labelled. It is particularly convenient if the immunoassay is an ELISA or a radioimmunoassay, Immunoassays are well known in the art (see, for example, Immunoassays: A practical approach. James P. Gosling (ed), Oxford University Press, 2000, ISB4 0-19-963710-5). Commercially available ELISA kits can be used to detect levels of Myc protein, for example Novex Life Technologies Corporation KH02041.

By the term "antibodies", we include whole antibodies which bind a protein indicative of an increased dependency on autophagy for survival (eg Myc), but also fragments of antibodies which bind lysozyme such as Fv, Fab and F(ab)2 fragments as well synthetic antibodies or antibody fragments such as single chain Fv (scFv) molecules and domain antibodies (dAbs). The antibody fragments and synthetic antibodies retain antigen binding activity (and usually contain some or all of the complementarity determining regions (CDRs) of a parent antibody molecule). Antibodies that bind a protein indicative of an increased dependency on autophagy for survival (eg Myc), selectively are known in the art and are commercially available. It will be appreciated that the antibody (eg a Myc antibody) is typically one which has been raised to or selected using human protein indicative of an increased dependency on autophagy for survival (eg Myc). An example of a Myc antibody is the c-Myc Antibody (Santa Cruz GEl 0). This is a mouse monoclonal IgGi that binds to an epitope corresponding to amino acids 408-439 within the carboxy terminal domain of human c-Myc, and can be used to detect c-Myc p67 of mouse, rat, human and monkey origin by western blotting, irnmunoprecipitation, immunofluorescence, IHC(P)(immunohistochemistry-paraffin), FCM (flow cytometry) and ELISA. It is also reactive with c-Myc from additional species, including feline and canine c-Myc. Another example of a Myc antibody is the N-Myc Antibody (Santa Cruz 2). This is a mouse monoclonal lgG2a that binds to an epitope mapping at residues 327-339 of human N-Myc p67, and can be used to detect N-Myc p67 of mouse and human origin by western blotting, immunoprecipitation, immunofluorescence. Yet another example of a Myc antibody is the L-Myc Antibody (Santa Cruz C-20). This is a rabbit polyclonal lgG that binds to an epitope mapping within the C-terminus of L-Myc of human origin, and can be used to detect L-Myc of mouse, rat and human origin by western blotting, immunoprecipitation, immunofluorescence and ELISA. It is also reactive with L-Myc from additional species, including equine, canine, bovine and porcine. Any such antibodies may be used.

Furthermore, antibodies for a protein indicative of an increased dependency on autophagy for survival, for example a Myc protein, may be made using well known technology such as the hybridoma method for making monoclonal antibodies, and phage display techniques for making synthetic antibody fragments. Suitable methods for the production and use of antibodies are described and referred to in Using antibodies: A laboratory manual", Ed Harlow and David Lane, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1999. 3°

Preferably, antibodies which bind selectively to a protein indicative of an increased dependency on autophagy for survival, for example a Myc protein, as opposed to other molecules in the sample from the individual (eg blood or biopsy sample) are used.

Preferably, the antibody has at least a 10 fold-higher affinity for a protein indicative of an as increased dependency on autophagy for survival, for example a Myc protein, than for any other component in the sample and more preferably at least a 50 fold, 100 fold, 500 fold, 1000 fold or 10000 fold-higher affinity for a protein indicative of an increased dependency on autophagy for survival.

Determining the amount of a protein indicative of an increased dependency on autophagy S for survival (eg Myc) in an unwanted cell (eg a cancer cell) may comprise assessing the level of mRNA encoding that protein. Assessing mRNA levels may be done by any suitable technique in the art such as any of RNA-seq, EXOME sequencing, GENOME sequencing, Northern blot analysis, nuclease protection assays (NPA), in situ hybridization, and reverse transcription-polymerase chain reaction (RT-FCR).

It will be appreciated that the level of mRNA encoding the protein indicative of an increased dependency on autophagy for survival (eg Myc), is increased to a level of mRNA such that the subject's unwanted cells (eg cancer cells) are more responsive to treatment by a KDACB inhibitor having the structure of Formula I. This level of mRNA may vary depending on the type of individual (eg human1 horse, dog and so on). The level of mRNA may be determined by comparing the levels of mRNA encoding the protein, in individuals that are known to have unwanted cells (eg cancer cells) with increased levels of mRNA encoding the protein indicative of an increased dependency on autophagy for survival (eg Myc), and which are responsive to treatment by a KDAC6 inhibitor having the structure of Formula I, with those levels of mRNA encoding the protein in normal individuals (eg those with no sign or symptoms of unwanted cells (eg cancer cells), or individuals with unwanted cells wherein the levels of mRNA encoding the protein indicative of an increased dependency on autophagy for survival (eg Myc) are either not increased or not increased to a level which makes the subject more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I). Typically, the level of mRNA encoding the protein which is indicative of increased dependency on autophagy for survival is a level of mRNA which is greater than 1 standard deviation above the mean level of mRNA encoding the protein indicative of an increased dependency on autophagy for survival (eg Myc protein), in a healthy population, for example greater than 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 standard deviations above the mean level of mRNA encoding that protein (eg Myc protein), in a healthy population, that is a population of normal individuals (eg those with no sign or symptoms of unwanted cells, for example cancer, or individuals with unwanted cells wherein the levels of a protein indicative of an increased dependency on autophagy for survival (eg Myc) are not increased or not increased to a level which makes the subject more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I). It will be appreciated that when comparing levels of mRNA encoding a protein indicative of an increased dependency on autophagy for survival, for example a Myc protein, between individuals, the assessment should preferably be done using the same sample, cell type or tissue. Preferably, the population comprises at least 5, 10, 50, 100, 200, 300, 400 or 500 individuals and more preferably at least 1000 individuals. It is preferred if the normal individuals are assessed using the most sensitive and specific detection techniques for mRNA encoding a protein indicative of an increased dependency on autophagy for survival (eg Myc) available (eg RT-PCR for example by taq-man, or RNA-seq). Preferably, the healthy population is a population of individuals that have been shown not to have unwanted cells, for example cancer, with increased levels of a protein indicative of an increased dependency on autophagy for survival (eg Myc), and thereafter have been shown not to develop such unwanted cells, for example cancer, using the same techniques, for example, for at least 6 months, or 1, 2, 3, 4 or 5 years, or more. In addition, it is preferred if the normal individuals' have no or few risk factors for unwanted cells, for example cancer cells.

Typically, when a subject's unwanted cells (eg cancer cells) have increased dependency on autophagy for survival, the level of mRNA encoding the protein indicative of an increased dependency on autophagy for survival (eg Myc) is at least 1.25, 1.5, 2, 3, 4, 5, 10, 20, 50 or 100 times more than the level of mRNA encoding that protein in a control cell, and preferably at least 4 times more, Preferably, the mRNA is one that encodes Myc.

The control cell may be one described above. For example it may be a normal cell with a normal dependency on autophagy for survival (eg a non-cancer cell), or a cancerous cell from a subject that is known not to have an increased level of mRNA encoding a protein indicative of an increased dependency on autophagy for survival (eg Myc), or a cancerous cell that is known to have an increased level of mRNA encoding a protein indicative of an increased dependency on autophagy for survival (eg Myc) but not to the extent that it renders the cancer more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I. In a preferred embodiment when the unwanted cells are cancer cells, a subject's cancer is more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I when the level of mRNA encoding a protein indicative of an increased dependency on autophagy for survival (eg Myc) in the cancer cell is at least 1.25, 1.5, 2, 3, 4, 5, 10, 20, 50 or 100 times more than the level of mRNA encoding a protein indicative of an increased dependency on autophagy for survival (eg Myc) in the control cell (eg a non-cancer cell).

Conveniently, the comparison is made between a sample obtained from the subject (eg one comprising cancer cells) and a control sample (eg one comprising control cells such as non-cancer cells).

Bieche eta! (Cancer Research, 1999) assessed Myc RNA levers in tumour RNA samples, and normalised values against TBP (a component of the DNA-binding protein complex TFIID) as the endogenous RNA control. They found that values of 3 or more were indicative of Myc overexpression in tumour RNA samples. Thus if a sample from a subject having 3 or more arbitrary units of mRNA encoding Myc (eg normalised according to TBP levels), the subject can be considered as being one that is likely to respond to treatment by a KDAC6 inhibitor having the structure of Formula I. In a particular embodiment, when the protein indicative of an increased dependency on autophagy for survival is Myc, and when the unwanted cells are ovarian cancer cells, the level of mRNA encoding Myc (eg c-Myc) that is indicative of an increased dependency on autophagy for survival may be one that is equal to, or above, 200 arbitrary units, (as described in Iba eta! 2004; units correspond to a normalised value relative to an internal control such as 18s ribosomal RNA). Thus, if a sample from a subject having ovarian cancer contains 200 or more arbitrary units (eg normalised according to lBs rRNA levels) of mRNA encoding Myc (eg c-Myc), the subject can be considered as being one that is likely to respond to treatment by a KDAC6 inhibitor having the structure of Formula I. Another indirect way of determining the amount of a protein indicative of an increased dependency on autophagy for survival (eg Myc) in an unwanted cell (eg a cancer cell), is by performing a genetic analysis of the gene encoding the protein. By genetic analysis, we include the meaning of assessing the sequence integrity of the gene (eg the presence of one or more mutations in the gene compared to the naturally occurring wild type sequence), the location of the gene in the genome (eg the presence of one or more translocations involving the gene), the methylation and/or acetylation status of the gene, and the number of copies of the gene in the genome. By gene, we include the meaning of all non-transcribed regions (eg promoters and enhancers and such like) and transcribed regions of the gene (eg introns, exons, 5'-UTR, 3'-UTR and such like) that are associated with the encoding of the protein of that gene. It will also be appreciated that a genetic analysis may comprise assessing genetic markers at sites distant from the gene encoding the protein indicative of an increased dependency on autophagy for survival, but which are nevertheless in linkage disequilibrium with, or otherwise correlated with, a genetic abnormality or marker associated with an increased amount or activity of the protein indicative of an increased dependency on autophagy for survival.

Thus, in one embodiment where the protein indicative of increased dependency on autophagy for survival in an unwanted cell is Myc, the determination may be done by conducting a genetic analysis of the Myc gene (or other site distal from the Myc gene as described above), and in particular, identifying the presence of any genetic marker that is correlated with an increased amount or activity of Myc. The genetic marker may be one or more mutations in the Myc gene, one or more translocations involving the Myc gene, and/or an increased copy number of the Myc gene.

In a particular example, determining the amount of a protein indicative of an increased dependency on autophagy for survival (eg Myc) in an unwanted cell (eg a cancer cell), is by assessing the number of copies of the gene encoding that protein in the unwanted cell.

High gene copy numbers generally correspond to more translated protein encoded by that gene. Assessing gene copy number can be done by various techniques known in the art including comparative genomic hybridisation (CGH), fluorescent in-situ hybridisation (FISH), array CGH (BAC and oligonucleotide), SNP-array, representational oligonucleotide microarray analysis (ROMA), and/or PCR based methods. Thus, in one embodiment, determining whether the subject's unwanted cells has or is likely to have an increased dependency on autophagy for survival, comprises assessing the copy number of a gene that encodes a protein that is indicative of an increased dependency on autophagy for survival (eg Myc), in the subject's unwanted cells (eg cancer cells). The gene copy number will be increased to a level such that the subject's unwanted cells (eg cancer cells) are more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I. This level may be determined by comparing the gene copy numbers in individuals that are known to have unwanted cells with increased copy numbers of a gene encoding a protein indicative of increased dependency on autophagy for survival (eg Myc), and which are responsive to treatment by a KDAC6 inhibitor having the structure of Formula I, with those gene copy numbers in normal individuals (eg those with no sign or symptoms of unwanted cells (eg cancer), or individuals with unwanted cells wherein the gene copy number of a gene encoding the protein indicative of an increased dependency on autophagy for survival (eg Myc) is either not increased or not increased to a level which makes the subject more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I). Typically, the gene copy number that is indicative of a likelihood to respond to treatment by a KDAC6 inhibitor having the structure of Formula I is 2, 3, 4, 5. 6. 7. 8, 9 or 10 copies of the gene encoding a protein indicative of an increased dependency on autophagy for survival (eg Myc) per cell.

In a further example, another means of indirectly determining the dependency on autophagy for survival is by assessing the position of a gene which encodes a protein indicative of an increased dependency on autophagy for survival (eg Myc) within the genorne of the unwanted cells (eg cancer cell). For example, in the human genome, the Myc gene normally resides on chromosome 8 at band q24. However, in various cancers, such as Burkitt's lymphoma, translocations involving Myc have been identified that lead to upregulated Myc expression (Oncogene 20(4): 5595, 2001). For example, the c-Myc gene is translocated to one of the immunoglobulin loci in virtually all Burkitt's lymphomas. Thus, in one embodiment, determining whether the subject's unwanted cells have or are likely to have an increased dependency on autophagy for survival comprises assessing the presence of one or more translocations involving a gene encoding a protein indicative of an increased dependency on autophagy for survival (eg Myc). Translocations can be screened for by any suitable technique in the art, such as conventional cytogenetic analysis, FISH and RI-PCI. Where the translocation results in an increased expression of the protein indicative of an increased dependency on autophagy for survival, it is likely that the unwanted cells in the subject have an increased dependency on autophagy for survival.

Still yet a further way of indirectly determining the dependency on autophagy for survival focuses on the phosphorylation status of a protein indicative of an increased dependency on autophagy for survival (eg Myc). For example, for some proteins, expression is known, in part, to be controlled by its protein stability, which can be regulated by interdependent phosphorylation at various residues. The stability of Myc, for instance, is controlled in part by phosphorylation at threonine 58 (T58) and serine 62 (S62). Initial phosphorylation of S62 by ERK or CDK kinases in response to mitogen signaling transiently increases Myc stability, whereas subsequent phosphorylation of T58 by GSK3j3 triggers dephosphorylation of 562 by protein phosphatase 2A-B56a (PP2A-B56a), ubiquitination by the SCF-Fbw7 E3 ligase, and proteosomal degradation (Zhang et at, PNAS 109(8): 2790). Various cancers including Burkitt's lymphoma, and a range of breast cancers, are so characterised by increased phosphorylation of Myc at S62 and decreased phosphorylation of Myc at T58, which underlines an increased stability of Myc (Ic a lower turnover rate).

Hence, an indirect assessment of the dependency on autophagy for survival of an unwanted cell is by the assessment of the phosphorylation status a protein indicative of an increased dependency on autophagy for survival, wherein any phosphorylation status that ss is known to be correlated with increased protein stability is likely to be indicative of the unwanted cells (eg cancer cells) in the subject having an increased dependency on autophagy for survival. For example, when the protein is Myc, the phosphorylation status of residues 562 and T58 of Myc in an unwanted cell may be assessed, and the status compared to that in a control cell. If Myc in the unwanted cell shows increased phosphorylation at S62 and decreased phosphorylation at T58, the cell is likely to have a higher amount of Myc and thus an increased dependency on autophagy for survival. Any suitable technique may be used to assess the phosphorylation status of a protein indicative of an increased dependency on autophagy for survival (eg Myc), including, but not limited to, those described in Zhang et S (PNAS 109(8): 2790) which rely on phospho-specific antibodies. Zhang et a! also describe how the signaling cascade that controls phosphorylation at T58 and S62 in a Myc protein is coordinated by the scaffold protein Axini, and so it will be understood that a further example of a protein indicative of an increased dependency on autophagy for survival is Axini and that an assessment of Axinl, and particularly splice variants thereof, may be used as a marker for the phosphorylation status of Myc, and thereby, the amount of Myc in an unwanted cell. In particular, it is believed that the amount of Axin 1 is negatively correlated with Myc, and so one can assess Axin 1 levels as a marker or Myc (eg lower levels of Axin 1 likely correspond to higher levels of Myc). Also, one could evaluate phosphorylation status by searching for mutations at known phosphorylation sites in the protein indicative of an increased dependency on autophagy for survival, for example mutations at T58 and/or 562 in a Myc protein, which would be expected to affect phosphorylation.

It will also be appreciated that one can assess an unwanted cell's dependency on autophagy for survival by determining the activity of a protein that is indicative of an increased dependency on autophagy for survival (eg Myc), for example in a sample obtained from the subject. Thus, in one embodiment, determining whether the subjects unwanted cells (eg cancer cells) have or are likely to have an increased dependency on autophagy for survival comprises determining the activity of a protein indicative of an increased dependency on autophagy for survival, and assessing whether the activity is increased relative to the activity in control cells. Preferably, the protein is Myc, and preferably, the unwanted cells are cancer cells. Conveniently, the method is performed on a sample taken from the subject as described herein.

Also, it will be appreciated that the activity of a protein indicative of an increased dependency on autophagy for survival of a population of unwanted cells and the activity of a protein indicative of an increased dependency on autophagy for survival of a population of control cells, may be compared based on samples that do not contain the cells (eg blood plasma or serum) but which nevertheless contain markers that are correlated with the dependency on autophagy for survival of the cells.

Where the protein indicative of an increased dependency on autophagy for survival is a transcription factor such as Myc, transcription may be measured using reporter genes to assay the activity of particular promoters under the control of the protein indicative of an increased dependency on autophagy for survival (eg Myc).

By a reporter gene we include genes which encode a reporter protein whose activity may easily be assayed, for example j3-galactosidase, chloramphenicol acetyl transferase (CAT) gene, luciferase or Green Fluorescent Protein (see, for example, Tan et aI, 1996). Several io techniques are available in the art to detect and measure expression of a reporter gene which would be suitable for use in the present invention. Many of these are available in kits both for determining expression in v/tm and in viva. (eg Promega luciferase assay system).

Alternatively, the activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc) may be assayed by the analysis of downstream targets. For example, where the protein indicative of an increased dependency on autophagy for survival is a transcription factor (eg Myc), a particular protein whose expression is known to be under the control of the protein indicative of an increased dependency on autophagy for survival may be quantified. For example, when the protein indicative of an increased dependency on autophagy for survival is Myc, the expression of any of transferrin, LDHA, MCL1 or eIF4E may be assessed. Assaying protein levels in a biological sample can be done using any suitable method known in the art, such as those described above. For example, protein concentration can be studied by a range of antibody based methods including immunoassays, such as ELISAs and radioimmunoassays. Where the protein, whose expression is known to be under the control of the protein indicative of an increased dependency on autophagy for survival (eg Myc), is an enzyme, expression of that protein may be assessed by monitoring its enzymatic activity using any suitable assay known in the art for the particular enzyme. For example, downstream targets of Myc include lactate dehydrogenase, and so the activity of lactate dehydrogenase in a sample may be assessed. Myc also activates the transferrin receptor which can be monitored non-invasively using PET imaging (eg 89Zr-desferrioxamine transferrin (89Zr-Tf)) (Holland eta! 2013).

It is known that the activity of Myc is regulated to an extent by its subcellular location, and in particular by its differential localisation between the nucleus and the cytoplasm. The more Myc protein that is present in the nucleus as opposed to the cytoplasm, the more active the Myc protein is. Thus, it will be understood that assessing the dependency of unwanted cells on autophagy may comprise determining the subcellular localisation of Myc in the unwanted cells, and in particular the proportion of Myc present in the nucleus of the unwanted cells as opposed to in the cytoplasm of the unwanted cells.

It will be appreciated that the activity of the protein indicative of an increased dependency on autophagy for survival (eq Myc) is increased to a level of activity such that the subject's disease or condition characterised by the presence of unwanted cells, for example cancer, is more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I. This level of activity may vary depending on the type of individual (eq human, horse, dog and so on). The level of activity may be determined by comparing the levels of activity in individuals that are known to have unwanted cells, for example cancers, with increased levels of activity of a protein indicative of an increased dependency on autophagy for survival (eq Myc), and which are responsive to treatment by a KDAC6 inhibitor having the structure of Formula I, with those levels of activity in normal individuals (eq those with no sign or symptoms of unwanted cells, for example cancer, or individuals with unwanted cells, for example cancer cells, wherein the levels of activity of a protein indicative of an increased dependency on autophagy for survival (eq Myc) are not increased or not increased to a level which makes the subject more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I). Typically, the level of activity which is indicative of an increased dependency on autophagy for survival is a level of activity which is greater than 1 standard deviation above the mean level of activity of a protein indicative of an increased dependency on autophagy for survival (eq Myc protein) of cells from a population of normal individuals, for example greater than 1.25, 1.5, 2, 2.5, 3, 3,5, 4, 4,5, 5, 5.5, 6, 6.5, 7, 7.5 orB standard deviations above the mean level of activity of that protein, in a population of normal individuals (eg those with no sign or symptoms of unwanted cells, for example cancer, or individuals with unwanted cells, for example cancer, wherein the levels of activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc protein) are not increased or not increased to a level which makes the subject more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I). It will be appreciated that when comparing levels of activity of a protein indicative of an increased dependency on autophagy for survival, for example a Myc protein between individuals, the assessment should preferably be done using the same sample, cell type or tissue. Preferably, the population comprises at least 5, 10, 50, 100, 200, 300, 400 or as 500 individuals and more preferably at least 1000 individuals. It is preferred if the normal individuals are assessed using the most sensitive and specific detection techniques available for determination of activity of the protein indicative of an increased dependency on autophagy for survival (eg Myc). Preferably, the normal population is a population of individuals that have been shown not to have unwanted cells, for example cancer, with increased levels of activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc), and thereafter have been shown not to develop such unwanted cells, for example cancer, using the same techniques, for example, for at least 6 months, or 1, 2, 3, 4 or 5 years, or more. In addition, it is preferred if the normal individuals' have no or few risk factors for the presence of unwanted cells, for example for cancer.

Typically, when a subject's unwanted cells, for example cancer cells, are more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I, the activity of the protein indicative of an increased dependency on autophagy for survival (eg Myc) in the unwanted cells, for example cancer cells, is at least 1.25, 1.5, 2, 3, 4, 5, 10, 20, 50 or 100 times more than the activity of that in a control cell. Preferably, the protein indicative of an increased dependency on autophagy for survival is Myc, The control cell may be one described above. For example, the control cell may be a normal cell with a normal dependency on autophagy for survival (eg a non-cancer cell), or a cancerous cell from a subject that is known not to have an increased activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc), or a cancerous cell that is known to have an increased activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc) but not to the extent that it renders the cancer more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I. In a preferred embodiment when the unwanted cells are cancer cells, a subject's cancer is more responsive to treatment by a KDAC6 inhibitor having the structure of Formula when the activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc) in the cancer cell is at least 1.25, 1.5, 2, 3, 4, 5, 10, 20, 50 or 100 times more than the activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc) in the control cell (eg a non-cancer cell). Conveniently, the comparison is made between a sample obtained from the subject (eg one comprising cancer cells) and a control sample (eg one comprising control cells such as non-cancer cells).

Any other way of assessing an unwanted cell's dependency on autophagy for survival is included in the scope of the method of the invention, Other possibilities include identifying the presence of an ALK (anaplastic lymphoma kinase)/MYC translocation, which has been shown to lead to an aggressive variant of anaplastic large cell lymphoma (Liang eta! 2013 J Paediatr Hematol Oncol, 35(5): 209); and/or identifying the presence of point mutations in ALK such as at position 1174 (point mutations at position 1174 of ALK are present in neuroblastoma cell lines KELLY and SY-SY5Y, which express an increased amount of Myc protein, and are particularly responsive to CiA), or at positions F1174L and R12750 (gain-of-function mutations, Schonherr eta) Oncogene 2012). for example using RNA-seq; and/or assessing the levels of 32-microglobulin in the subject. In terms of 2-microglobulin levels, Avet Loiseau eta! (Blood, 2001, 98(10): 3062) describe how in human multiple myeloma tumours, high Myc levels are correlated with high serum levels of B2-microglobulin. Specifically, Myc rearrangements were observed significantly more often in subjects with serum 2-microglobulln levels above amg/L.

It will be appreciated that when determining the amount or activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc), it may be desirable to express that amount or activity of the protein as a ratio with the value of another parameter that has been determined. For example, it may be desirable to normalise the amount or is activity of protein (eg Myc) to take into account the number of cells (eg cancer cells) in the sample, and so the value of another parameter directly correlated to the number of cells may be assessed. Additionally, or alternatively, the amount or activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc or another marker or parameter that is correlated with the amount of Myc), may be used in combination with some other parameter that is known to be indicative of an increased likelihood of the particular disease or condition responding to treatment comprising administration of a KDAC6 inhibitor having the structure of Formula I. The amount or activity of Myc can be monitored in conjunction with the level of ct-tubulin acetylation to provide an additional global measure of the level of inhibition, including its effect on the catalytic activity of the KDAC6 inhibitor. Also, it will be understood that the amount or activity of a protein indicative of an increased dependency on autophagy for survival (eg Myc or another marker or parameter that is correlated with the amount of Myc) may be expressed as a ratio against another marker or parameter that is correlated with the amount or activity of a protein indicative of an increased dependency on autophagy for survival protein.

The inventors have shown that KDAC6 inhibitors having the structure of Formula I are particularly effective, on their own, to treat diseases or conditions characterised by the presence ot unwanted cells that have an increased dependency on autophagy for survival.

Other compounds are not considered to be required for an enhanced effect to be achieved.

This is in contrast to previous work, in which it was shown that an enhanced effect of a KDAC6 inhibitor was only observed when used in conjunction with further agents, such as bortezomib (Hideshima at a! 2005), or carfllzomib (Mishima at a! 2013). Accordingly, in one embodiment, a KDAC6 inhibitor having the structure of Formula I is used as the sole medicament in the treatment of the disease or condition characterised by the presence of unwanted cells. Preferably, the KDAC6 inhibitor is CiA or C1B.

In an alternative embodiment, the KDAC6 inhibitor having the structure of Formula I is used in combination with one or more further therapeutic agents (eg anti-cancer agents or anti-viral agents). In this way, the method of the invention may be used to identify a subject with a disease or condition characterised by the presence of unwanted cells, such as cancer, as being likely to respond to a treatment comprising administered a KDAC6 inhibitor having the structure of Formula I and one or more further therapeutic agents. For example, synergistic or additive effects can occur with other anti-proliferative, anti-cancer, immune-modulatory, anti-inflammatory substances or anti-viral agents. Combination therapy includes administration of a KDAC6 inhibitor having the structure of Formula I with other biologically active ingredients (such as a second anti-cancer agent) and non-drug therapies (such as surgery or radiation treatment).

Preferably, the one or more further therapeutic agents are suitable to treat the disease or condition characterised by the presence of unwanted cells.

Preferably, the one or more further therapeutic agents are one or more anti-cancer agents.

For example, the one or more anti-cancer agents may be an approved chemotherapeutic agent known in the art, such as bortezomib or farnesyl transferase inhibitors, Other examples of anti-cancer agents may be selected from alkylating agents including nitrogen mustards such as mechlorethamine (I-IN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; methylating agents such as temozolomide (believed to be especially useful for treating brain tumours, Knizhnik at a!); ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa: alkyl sulphonates such as busulphan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MF), thioguanine (6-thioguanine; IG) and pentostatin (2Ldeoxycoformycin); natural products including ymca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes; miscellaneous agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenedione such as mitoxantrone and s anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o,p'-DDD) and aminoglutethimide; taxol and analogues/derivatives; cell cycle inhibitors; proteosome inhibitors such as Bortezomib (Velcade®); signal transductase (eg tyrosine kinase) inhibitors such as Imatinib (Glivec®), COX-2 inhibitors, and hormone agonists/antagonists such as flutamide and tamoxifen.

As described above, the inventors believe that a KDAC6 inhibitor having the structure of Formula I is particularly useful to treat cancers that have an increased amount of Myc protein, due to the dependency of such cancers on the aggresome/autophagy. Thus, the further therapeutic agent may be one that increases this dependency and/or inducers of the aggresome/autophagy. Examples of such agents include proteasome inhibitors (eg bortezomib) mistranslation inducers (eg aminoglycoside antibiotics, amino acid analogues), Src-inhibitors (eg dasatinib), DNA Protein Kinase-inhibitors (eg NU 7441), P13 Kinase/mTOR inhibitors (eg NVP-6EZ235), AKT-inhibitors (eq MK2206), HSP 90 inhibitors (eg 17AAG, Zaarur eta! JBC 2008, or AUY922, SNX-5422, Ganetespib (STA- 9090)) and TNFa-modulators (Tumour Necrosis Factor).

A second aspect of the invention provides a method of treating a subject with a disease or condition characterised by the presence of unwanted cells, wherein the subject is one that has been identified as being likely to respond to administration of the KDAC6 inhibitor according to the method of the first aspect of the invention, the method comprising administering a KDAC6 inhibitor having the structure of Formula I. Optionally, the method may further comprise administering one or more further therapeutic agents as described below.

The invention thus also provides a KDAC6 inhibitor having the structure of Formula I, and optionally one or more further therapeutic agents, for use in treating a subject having a disease or condition characterised by the presence of unwanted cells, wherein the subject is one that has been identified as being likely to respond to administration of the KDAC6 inhibitor according to the method of the first aspect of the invention.

Similarly, the invention provides the use of a KDAC6 inhibitor having the structure of Formula I, and optionally one or more further therapeutic agents, in the manufacture of a medicament for treating a subject having a disease or condition characterised by the presence of unwanted cells, wherein the subject is one that has been identified as being likely to respond to administration of the KDAC6 inhibitor according to the method of the first aspect of the invention.

Preferably, the disease or condition characterised by the presence of unwanted cells is cancer (including all of those mentioned above in relation to the first aspect of the invention), but it could be any disease or condition mentioned herein, for example as outlined above in relation to the first aspect of the invention (eg a viral disease).

In an embodiment, the disease or condition is cancer, such as a solid cancer, a haematological cancer, inflammatory breast cancer or a neuroblastoma.

In an alternative embodiment, the disease or condition is a virus infection, optionally wherein the virus infection is hepatitis B, hepatitis C, hepatitis D, flavivirus, Borna disease or HIV.

Preferences for the KDAC6 inhibitor having a structure of Formula I include those described above in relation to the first aspect of the invention and also apply to all aspects below. Preferably, the KDAC6 inhibitor having the structure of Formula I is CiA or Cl B. The KDAC6 inhibitor having the structure of Formula I may be administered as a sole treatment, or in combination wIth one or more further therapeutic agents. For example, although the subject has been identified as one that is likely to respond to treatment comprising administration of a KDAC6 inhibitor having a structure of Formula I, it may nevertheless be desirable to also administer one or more further therapeutic agents to the subject.

It is appreciated that the KDAC6 inhibitor having the structure of Formula I will usually be administered separately from the one or more further therapeutic agents. In such an embodiment, the KDAC6 inhibitor and the one or more further therapeutic agents may be administered sequentially, or they may be administered substantially simultaneously, typically through distinct routes of administration.

For example, a KDAC6 inhibitor having the structure of Formula I may be administered to the subject before, at the same time as, or after, the one or more further therapeutic agents.

Similarly, the one or more further therapeutic agents may be administered before, at the same time, or after, the KDAC6 inhibitor of Formula I. Specifically, the KOAC6 inhibitor having the structure of Formula I may be administered -48, -24, -10, -9, -8, -7, -6, -5, -4, - 3, -2, -1, -0.5, -0.25, -0.16, -0.08, -0.016,0, 0.016, 0.08, 0.16, 0.25, 0.5, 1, 2, 3, 4, 5,6,7, 8, 9, 10, 24, 48 hours relative to the administration of the one or more further therapeutic agents, wherein 0 hours means that the agents are administered together, -10 hours means that the KDAC6 inhibitor having the structure of Formula I is administered 10 hours io before the administration of the one or more therapeutic agents, and wherein 10 hours means that the KDAC6 inhibitor of Formula I is administered 10 hours after the administration of the one or more therapeutic agents, and so on. Preferably, the KDAC6 inhibitor is CiA or 018.

In one example, the one or more further therapeutic agents may be administered to the subject up to 24 hours after administration of the KDAC6 inhibitor of Formula I; for example up to 8 hours after administration of the KDAC6 inhibitor of Formula I, or up to 1 hour after administration of the KDAC6 inhibitor of Formula I. For instance, the one or more further therapeutic agents may be administered to the subject immediately after up to 48 hours after administration of the KDAC6 inhibitor of Formula I, or between 1 minute and 36 hours, or between 5 minutes and 30 hours, or between 10 minutes and 24 hours, or between 15 minutes and 20 hours, or between 20 minutes and 15 hours, or between 25 minutes and hours, or between 30 minutes and 9 hours, or between 35 minutes and 8 hours, or between 40 minutes and 7 hours, or between 45 minutes and 6 hours, or between 50 minutes and 5 hours, or between 55 minutes and 4 hours, or between 1 and 3 hours, or between 1 and 2 hours, or any combination thereof, after administration of the KDAC6 inhibitor of Formula I. It is intended that the one or more further therapeutic agents should be administered at a time when the KDAC6 inhibitor of Formula I is still detectable in the subject. Appropriate timing will be determined for each drug. It will be appreciated that the timing of such administration will depend to a great extent on the pharmacodynamics and pharmacokinetics of the KDAC6 inhibitor of Formula I. Nevertheless, the above regimes provide examples of appropriate administration times.

Likewise, the KDAC6 inhibitor of Formula I may be administered to the subject up to 24 hours after administration of the one or more further therapeutic agents; for example up to 8 hours after administration of the one or more further therapeutic agents, or for example up to 1 hour after administration of the one or more further therapeutic agents. For instance, the KDAC6 inhibitor having the structure of Formula I may be administered, to the subject immediately after up to 48 hours after administration of the one or more further therapeutic agents, or between 1 minute and 36 hours, or between 5 minutes and 30 hours, or between 10 minutes and 24 hours, or between 15 minutes and 20 hours, or between 20 minutes and 15 hours, or between 25 minutes and 10 hours1 or between 30 minutes and 9 hours, or between 35 minutes and 8 hours, or between 40 minutes and 7 hours, or between 45 minutes and 6 hours, or between 50 minutes and 5 hours1 or between 55 minutes and 4 hours, or between 1 and 3 hours, or between I and 2 hours, or any combination thereof, after administration of the one or more further therapeutic agents. It is intended that the KDAG6 inhibitor of Formula I should be administered at a time when the one or more further therapeutic agents are still be detectable in the subject. Appropriate timing will be determined for each drug. It will be appreciated that the timing of such administration will depend to a great extent on the pharmacodynamics and pharmacokinetics of the one or more further therapeutic agents. Nevertheless, the above Is regimes provide examples of appropriate administration times.

Thus the invention includes a KDAC6 inhibitor having the structure of Formula I, for use in treating a subject with a disease or condition characterised by the presence of unwanted cells, wherein the subject is one that has been identified as being likely to respond to administration of the KDAC6 inhibitor according to the method of the first aspect of the invention, and wherein the subject is being administered a further therapeutic agent suitable for use in treating the disease or condition. The invention also include a therapeutic agent suitable for treating a subject with a disease or condition characterised by the presence of unwanted cells, for use in treating that disease or condition in a subject that is being administered a KDAC6 inhibitor having the structure of Formula I, and who has been identified as being likely to respond to administration of the KDAC6 inhibitor according to the method according to the first aspect of the invention.

Likewise, the invention also includes use of a KDAC6 inhibitor having the structure of Formula I, in the manufacture of a medicament for treating a subject with a disease or condition characterised by the presence of unwanted cells, wherein the subject is one that has been identified as being likely to respond to administration of the KDAC6 inhibitor according to the method of the first aspect of the invention, and wherein the subject is being administered a further therapeutic agent suitable for use in treating the disease or 36 condition. The invention also includes the use of a therapeutic agent suitable for treating a subject with a disease or condition characterised by the presence of unwanted cells, in the manufacture of a medicament for treating that disease or condition in a subject that is being administered a KDAC6 inhibitor having the structure of Formula I, and who has been identified as being likely to respond to administration of the KDAC6 inhibitor according to the method according to the first aspect of the invention.

However, it is appreciated that in some cases, a combined formulation could be useful, for example, if the KDAC6 inhibitor and one or more further therapeutic agent could be administered to the patient in a single infusion that would be quicker and easier to administer. Thus, in certain embodiments, the KDACS inhibitor may be part of a composition (eg a pharmaceutical composition) that further comprises one or more further therapeutic agents.

The one or more further therapeutic agents may be any other therapeuuc agent, including those described above in relation to the first aspect of the invention. Preferably, the one or more further therapeutic agents are ones that are suitable to treat the disease or condition characterised by the presence of unwanted cells. For example, where the disease or condition is cancer, the further therapeutic agent may be an anti-cancer agent (eg as described above), and when the disease or condition is a viral disease, the further therapeutic agent may be an anti-viral agent. In one embodiment, the further therapeutic agent may be any one or more of a proteasome inhibitor, optionally bortezomib; a mistranslation inducer, optionally an aminoglycoside antibiotic or amino acid analogue; a further anti-cancer agent, optionally a farnesyl transferase inhibitor, optionally wherein the farnesyl transferase inhibitor is Lonafarnib; an anti-viral agent; Src-inhibitors (eg dasatinib), DNA Protein Kinase-inhibitors (eg NU 7441), P13 Kinase/mTOR inhibitors (eg NVP-BEZ235), AKT-inhibitors (eg MK2206), HSP 90 inhibitors (eg 17MG, Zaarur et a! JBC 2008, or AUY922, SNX-5422, Ganetespib (STA-9090)) and TNFa-modulators (Tumour Necrosis Factor).

Terms such as "treating" or "treatment" or "to treat" refer to both therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject is successfully "treated" according to the methods of the present invention if the patient shows one or more of the following: a reduction In the number of or complete absence of cancer cells; a reduction in the tumour size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumour metastasis; inhibition or an absence of tumour growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of effects.

It will be appreciated that administration of any agent described herein is typically administered as part of a pharmaceutical composition together with a pharmaceutically acceptable excipient, diluent, adjuvant, or carrier. Thus, any mention of a KDACS inhibitor having the structure of Formula I (eg CiA or Cl B), and any mention of a further therapeutic agent, equally applies to a pharmaceutically acceptable composition comprising that KDAC6 inhibitor having the structure of Formula I and/or further therapeutic agent (eg a formulation).

The agents will normally be administered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and subject to be treated, as well as the route of administration, the agents may be administered at varying doses.

Preferably, the formulation is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the agent or active ingredient.

In human therapy, the agents (eg KDAC6 inhibitor and/or further therapeutic agents) can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the agents can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-or controlled-release applications. The compounds of invention may also be administered via intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC)J sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Capsules or tablets may also be enteric coated to enhance gastric stability.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules.

Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The agents (eg KDAC6 inhibitor and/or further therapeutic agents) can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral Formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the Formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The Formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

For oral and parenteral administration to human subjects, the daily dosage level of the agents (eg KDAC6 inhibitor and/or further therapeutic agents) will usually be from 1 to 5000 mg per adult, administered in single or divided doses.

Thus, for example, the tablets or capsules of the compound of the invention may contain from 1 mg to 1000mg (Ic from about 60-120 mg/m2) of active compound for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage which will be most suitable for any individual subject and it will vary with the age, weight and response of the particular subject. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.

The agents (eg KDAC6 inhibitor and/or further therapeutic agents) can also be -lo administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, eg dichlorodifluorornethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 1 34A3 or 111,1 2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, eg using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, eg sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be Formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or puff' contains at least 1 mg of an agent (eg KDACG inhibitor and/or further therapeutic agent) for delivery to the subject. It will be appreciated that he overall daily dose with an aerosol will vary from subject to subject, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the agents (eg KDAC6 inhibitor and/or further therapeutic agents) can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The compounds of the invention may also be transdermally administered, for example, by the use of a skin patch.

They may also be administered by the ocular route, particularly for treating diseases of the eye.

For ophthalmic use, the agents (eg KDAC6 inhibitor and/or further therapeutic agents) can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the agents (eg KDAC6 inhibitor and/or further therapeutic agents) can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-is octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Generally, in humans, oral or topical administration of the agents (eg KDAC6 inhibitor and/or further therapeutic agents) is the preferred route, being the most convenient. In circumstances where the recipient suffers from a swallowing disorder or from impairment of drug absorption after oral administration, the drug may be administered parenterally, eg sublingually or buccally.

For veterinary use, the agent (eg KDAC6 inhibitor and/or further therapeutic agents) is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosIng regimen and route of administration which will be most appropriate for a particular animal.

Conveniently, the formulation is a pharmaceutical formulation. The formulation may be a veterinary formulation.

It will be appreciated that the term administration is not restricted to a one time administration. The term administration is taken to cover all of, but not limited to, a single dose administration, multiple administrations over a period of time, variable dosage administrations over a period of time, variable means of administration over a period of time, administration in conjunction with one or more further therapeutic agents, Administration can be by any means known in the art and includes, but is not limited to, oral, intravenous, topically direct to the tumour, sublingually or suppository.

As explained herein, the inventors believe that the subset of subjects having a disease or condition characterised by the presence of unwanted cells, wherein those cells have an increased dependency on autophagy for survival, are likely to be responsive to treatment comprising administration of a KDAC6 inhibitor having the structure of Formula I. However, it will also be appreciated that one can effectively create such a subject by first enhancing the dependency on autophagy of unwanted cells in that subject. The subject may be one that had previously been tested and identified as being not likely to respond to treatment comprising administration of a KDAC6 inhibitor having the structure of Formula I. Alternatively, a subject may have been identified as one that is likely to respond to treatment comprising administration of a KDAC6 inhibitor having the structure of Formula I, but for whom it is desired to increase this response further by increasing the dependency of that subject's unwanted cells on autophagy for survival. As a further alternative, the subject may be one that had not previously been tested for suitability in responding to treating by administration of a KDACfi inhibitor having a structure of Formula I. In a yet further alternative, the subject may be one who has not responded to the additional further therapeutic agents above, For example, the subject may have a caner that is resistant to other anti-cancer therapies, such that it is highly desirable to engineer that subject's cancer cells so that they are likely to respond to KDAC6 inhibitors having the structure of Formula I. Accordingly, a third aspect of the invention provides a method for making an unwanted cell in a subject with a disease or condition characterised by the presence of the unwanted cells, more responsive to treatment comprising administration of a KDAC6 inhibitor having the structure of Formula I, the method comprising administering an agent that increases the dependency on autophagy of the unwanted cells.

The unwanted cells of the disease or condition may. or may not have been characterised as having an increased dependency on autophagy for survival, according to the first aspect of the invention.

In an embodiment, the subject is subsequently administered a KDACB inhibitor having the structure of Formula I, and optionally one or more other therapeutic agents.

Thus, the invention also includes a method of treating a subject with a disease or condition characterised by the presence of unwanted cells wherein the method comprises administering to the subject one or more agents to increase the dependency on autophagy for survival of the unwanted cells, prior to administering a KDAC6 inhibitor having the structure of Formula I, and optionally one or more other therapeutic agents. Similarly, the invention includes a KDAC6 inhibitor having the structure of Formula I, and optionally one or more other therapeutic agents, for use in treating a disease or condition characterised by unwanted cells wherein the subject is one who has been previously administered one or more agents to increase the dependency on autophagy for survival of the unwanted cells. Likewise, the invention includes the use of a KDAC6 inhibitor having the structure of Formula I, and optionally one or more other therapeutic agents, in the manufacture of a medicament for treating a disease or condition characterised by unwanted cells wherein the subject is one who has been previously administered one or more agents to increase the dependency on autophagy for survival of the unwanted cells.

Preferences for the unwanted cell, disease or condition, subject, KDAC6 inhibitor having the structure of Formula I, one or more other therapeutic agents, and routes of administration, and such like, for this and all subsequent aspects of the invention, include those defined above in relation to the first and second aspects of the invention. Preferably, the KDAC6 inhibitor having the structure of Formula I is CiA or Ci B. By an agent that increases a cell's dependency on autophagy for survival, we include the meaning of any agent that increases the cell's dependency on autophagy for survival at least 1.25, 1.5, 2, 3, 4, 5, 10, 20, 50 a 100 times more than its dependency on autophagy for survival in the absence of the agent. For example, the agent may be one that increases the cellular demand for autophagy. In this case, the agent may be one that induces cellular stress in the unwanted cell such as any of hypoxia, oxidative stress, nutrient deprivation and/or an increase in cellular proliferation. Also, the agent may be one that increases the level or production of protein including damaged proteins, such as mis-translated or mis-folded proteins. Additionally or alternatively, the agent may be one that reduces the activity of a biological pathway or process that performs the same or similar function to autophagy, such as one that reduces the activity of the proteasome. Examples of suitable agents include a proteasome inhibitor such as bortezomib; an agent that increases production of a normally benign protein or a protein with a property that is beneficial in treating the disease of condition characterised by the presence of unwanted cells (eg when the disease is cancer, the agent may be one that increases production of a tumour suppressor such as p53 protein or one that increases production of GM-CSF (granulocyte-macrophage colony-stimulating factor)); or an agent that increases production of a mutated or truncated version of a protein that is non4unctional. When the agent is one that increases the production of protein, the agent may be a nucleic acid encoding that protein (eg a nucleic acid encoding a benign protein in an infected cell), The nucleic acid may be incorporated into a vector such as a viral vector (eg adenovirus), or the nucleic acid may be otherwise incorporated into a virus that selectively targets the unwanted cells (eg the agent could be an oncolytic vaccinia virus, for example JX-594, which expresses GM-CSF). The agent may be a mistranslation inducer such as an aminoglycoside antibiotic or amino acid analogue. In one embodiment, the agent comprises an engineered adenovirus that selectively infects the unwanted cells and that results in the overexpression of a benign protein in the unwanted cells.

Typically, the agent to increase the dependency on autophagy for survival of the unwanted cells comprises a targeting moiety capable of targeting to the unwanted cells and a further moiety that is capable of increasing the dependency on autophagy for survival of the unwanted cells. The targeting moiety may be a binding partner (eg antibody) of an entity on the surface of, or otherwise associated with, the unwanted cell, such as a surface antigen. It would be within the skilled person's routine experimentation to select an appropriate targeting moiety for a given unwanted cell, for example by interrogating databases and scientific literature for appropriate surface antigens to target. The further moiety may be any of the agents described in the paragraph immediately above.

However, in some circumstances, the agent that increases the dependency of the unwanted cell on autophagy for survival may be one that also increases the dependency of a wanted cell on autophagy for survival. In this case, the KDAC6 inhibitor having the structure of Formula I (eg CiA and CiA) is preferentially targeted to the unwanted cells, for example by being conjugated to an appropriate targeting moiety or by local administration to the site of the unwanted cells (eg cancer cells).

It will be appreciated that the methods of the above aspects of the invention can be made easier for a clinician by the provision of various kit of parts comprising items necessary to carry out the methods. Thus, the invention also provides a range of kits of parts comprising

suitable components.

A fourth aspect of the invention provides a kit of parts comprising (i) a means for assessing whether the unwanted cells of a subject, who has a disease or condition characterised by the presence of unwanted cells, have an increased dependency on autophagy and (H) a KDAC6 inhibitor having the structure of Formula I. Such a kit may be used to identify and subsequently treat those subject's deemed as being likely to respond to treatment with a KDAC6 inhibitor having the structure of Formula I. The means for assessing whether the unwanted cells have an increased dependency on autophagy for survival can be any suitable means known to the skilled person that assesses a cell's dependency on autophagy for survival. Methods for assessing a cell's dependency on autophagy for survival are discussed above in relation to the first aspect of the invention, and any means that are used in any of those may be a suitable means for the purposes of the present kit of parts. In one embodiment, the means for assessing whether the unwanted cells have an increased dependency on autophagy for survival comprises one or more of (a) a binding partner for a protein whose amount or activity is indicative of an increased dependency on autophagy, optionally wherein the binding partner is an antibody; (b) an oligonucleotide that hybridises to a nucleic acid encoding a protein whose amount or activity is indicative of an increased dependency on autophagy; and (c) an autophagy inhibitor, optionally wherein the autophagy inhibitor is a KDAC6 inhibitor having the structure of Formula I. In ternis of (a), it will be appreciated that the binding partner (eg antibody) is preferably one that is detectable and so may comprise a detectable moiety or else is otherwise detectable by being able to bind to a further molecule (eg a secondary anlibody binding to a primary antibody). In terms of (b), the oligonucleotide may be, for example, a primer or probe that selectively hybridises to the gene or mRNA of the protein that is indicative of an increased dependency on autophagy, as is described further in relation to the genetic analyses above.

In a preferred embodiment, wherein the means for assessing whether the unwanted cells have an increased dependency on autophagy for survival comprises a reagent suitable for assessing the amount or activity of Myc in the unwanted cells. The means may be an antibody against Myc protein or an oligonucleotide (eg primer or probe) that can be used to assess a genetic abnormality or marker associated with increased amount or activity of Myc.

In a preferred embodiment, the KDAC6 inhibitor having the structure of Formula I is CiA or Cl B. In one embodiment, the kit further comprises control cells that have a normal dependency on autophagy, In this way, the kit can be used to compare the dependency on autophagy for survival of the unwanted cells to a normal level of dependency in control cells, and thereby identify whether the dependency on autophagy in the unwanted cells is increased relative to that in the control cells. Preferences for the control cells include those described above in relation to the first aspect of the invention, and are typically of the same cell type or from the same tissue or organ as the unwanted cells in question.

In addition to control cells, or instead of control cells, the kit of parts may further comprise instructions for making a comparison between the dependency on autophagy of the unwanted cells and that of the control cells (if present in the kit), or between the dependency of autophagy for survival of the unwanted cells and a mean value for the of dependency on autophagy of cells from a population of normal individuals.

In a further embodiment, the kit of parts may comprise one or more therapeutic agents, such as any of those defined above in relation to the first and second aspects of the invention. For example, the one or more further therapeutic agents may be any of a proteasome inhibitor, optionally bortezomib; or a further anti-cancer agent, optionally a farnesyl transferase inhibitor such as Lonafarnib; an anti-viral agent; a Src-inhibitor, optionally dasatinib; a DNA Protein Kinase-inhibitor, optionally NU 7441; an AKT-inhibitor, optionally MK2206; a HSP 90 inhibitor such as any of 17MG, AUY922, SNX-5422, and Ganetespib (STA-9090); or a TNFa-modulator.

The KDAC6 inhibitor having the structure of Formula I may be provided in a form as described above, for example as part of a pharmaceutical composition, and may be provided as either individual doses in separate aliquots, or one large amount from which aliquots may be taken. Preferably the KDAC6 inhibitor having the structure of Formula I is CiA or Cl B. A fifth aspect of the invention provides a kit of parts comprising (i) a KDAC6 inhibitor having the structure of Formula I, and (ii) one or more control cells having a normal dependency on autophagy. Preferences for the KDAC6 inhibitor having the structure of Formula I and the one or more control cells having a normal dependency on autophagy include those defined above in relation to the first and second aspects of the invention. Preferably, the KDAC6 inhibitor having the structure of Formula I is CiA or Cl B, and/or the control cells are non-cancer cells. In an embodiment, the one or more control cells are selected from the group consisting of HeLa cells, HCT116 cells, DLD1 cells and LOVO cells. Preferably the control cells are cells from the same tissue type as the unwanted cells, taken from a normal individual, and most preferably are non-cancer cells, The control cell may be any cell in which the Myc gene is not highly or over-expressed (eg not constitutively expressed), or wherein Myc protein is inactive at basal levels.

A sixth aspect of the invention provides a kit of parts comprising (i) an engineered virus (eg adenovirus or lentivirus), engineered such that it is capable of increasing the dependency of autophagy for survival of an unwanted cell, optionally wherein it results in the overexpression of a benign protein in the unwanted cell; and (ii) a KDAC6 inhibitor having the structure of Formula I, optionally wherein the KDAC6 inhibitor having the structure of Formula I is CiA or Cl B. Preferably the virus (eg adenovirus) is one that is a conditional replicating virus, for example one which replicates under conditions found selectively in unwanted cells, or which replicates under conditions found selectively in cells with a high dependency on autophagy. Engineering of viruses is standard practice in the art and is described in the context of lentivirus in Levine et a! PNAS 2006 and in http://www.lentigen.com/products/stemcell-overview.

A seventh aspect of the invention provides a method of treating a viral disease comprising administering a KDAC6 inhibitor having the structure of Formula I, optionally wherein the KDAC6 inhibitor having the structure of Formula I is CiA or C1B, and optionally wherein the viral disease is any of hepatitis B, hepatitis C, hepatitis D, flavivirus, Borna disease or HIV. Similarly, the invention includes KDAC6 inhibitor having the structure of Formula I. for use in treating a viral disease, optionally wherein the KDAC6 inhibitor having the structure of Formula I is CiA or C1B, and optionally wherein the viral disease is any of hepatitis B, hepatitis C, hepatitis D, flavivirus, Borna disease or HIV. Optionally, the KDAC6 inhibitor having the structure of Formula I is used in combination with one or more further therapeutic agents, such as those described above in relation to the first and second aspects of the invention (eg an anti-viral agent). The one or more therapeutic agents may be administered to the subject separately from the KDAC6 inhibitor having the structure of Formula I. An eighth aspect of the invention provides a method of treating a cancer selected from the group consisting inflammatory breast cancer and a neuroblastoma, comprising administering a KDAC6 inhibitor having the structure of Formula I, optionally wherein the KDAC6 inhibitor having the structure of Formula I is CiA or CIB.

Similarly, the invention includes a KDAC6 inhibitor having the structure of Formula I, for use in treating a cancer selected from the group consisting of inflammatory breast and a neuroblastoma, optionally wherein the KDAC6 inhibitor having the structure of Formula I is CiA or C1B. Optionally, the KDAC6 inhibitor having the structure of Formula I is used in combination with one or more further therapeutic agents, such as those described above in relation to the first and second aspects of the invention (eg an anti-cancer agent). The one or more therapeutic agents may be administered to the subject separately from the KDAC6 inhibitor having the structure of Formula I. Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention.

The invention will now be described with the aid of the following figures and examples.

Figure I. CIA inhibits the proliferation of cancer cells with greater sensitivity in those amplified in MYC. (A) Mean GIso in a panel of cancer cells. The average 0150 concentration is plotted on the centre line (mean 0150 = 2.4 pM). Using a log scale, the sensitivity of a particular cell line is represented by deflections to the right from the mean.

Multiple myeloma cell lines express elevated MYC messenger (Fourney et a!, 1990) (eg KMS-12BM express high C-Myc; U266 express L-MYC -Holien eta!, 2012). (B) Growth inhibitory effect of CiA in a panel of neuroblastoma cell lines.

Figure 2. CIA abrogates autophagy induced by autophagy tools compounds. (A) HCT-116 cells were treated for 24 h with chloroquine (CQ -50 pM), 3-MA (5 mM), bortezomib (Bort -5 nM) alone or in combination with CiA (10 pM) and stained for LC3.

(B) Proteins levels from (A) were subject to ELISA quantification for levels of P62. Impact of tool compound, HDAC6 inhibitor tubastatin A (Tub) is shown for comparison.

Figure 3. HDAC6 inhibitor CIA synergises with proteasome inhibition. (A) Effect of 24h treatment with bortezomib (boil-5 nM) alone or in combination with CiA (10 pM) or Tubastatin A (Tub-b pM) on caspase activity in HCT-116 cells. (B) Impact of ACYIC1A treatment (9h -both at 2 pM) alone or in combination with bortezomib (5 nM) on caspase 317 activity in OPM-2 cells.

Figure 4. HDAC6 inhibition by CIA prevents autophagosome-lysosome fusion. (A) Diagram showing the pH-sensitive LC3 tagged plasmid with GFP and mcherry used in this study. GFP tag (green) is degraded in acidic conditions (found in lysosomes) whereas mcherry tag is stable (red). Yellow spots (mix of red and green) show lack of acidification.

(B). MEFS with different status of HDACG (WT versus 1<0) were transtected with LC3 tagged plasmid and yellow spots quantified, Comparison was done with treatment with CiA for 24 h at 10 pM.

Figure 5. Structure and function of HDAC6. (A) Functional domain organisation of HDAC6 comprising of cell localisation fragments (nuclear and cytoplasmic), a tandem catalytic domain, involved in the deacetylation of a numerous substrates (such as a-tubulin) and an ubiquitin binding motif (Zn-UBP). (B) Ubiquitin-dependent function of HDAC6.The latter mediates the transport of ubiquitin protein along microtubules, leads to the formation of aggresomes and controls the recruitment of the autophagic machinery to resorb protein aggregates.

Figure 6. Sequences of Myc. (A) Protein sequences of human Myc proteins; c-Myc (SEQ ID No: 1), n-Myc (SEQ ID No: 2) and 1-Myc (SEQ ID No: 3). (B) Protein sequences of non-human Myc proteins; rat b-Myc (SEQ ID No: 4), mouse s-Myc (SEQ ID No: 5) and feline v-Myc (SEQ ID No: 6). (C) Nucleotide sequences of non-human Myc coding regions; b-Myc (SEQ ID No: 7), s-Myc (SEQ ID No: 8) and v-Myc (SEQ ID No: 9).

Examole 1: Cells exhibiting high levels of Myc protein are more sensitive to KDAC6 inhibitor having the structure of Formula I Cancer cell lines from a range of cancer types (leukemia, multiple myeloma, prostate cancer, ovarian cancer, neuroblastoma, colon cancer, lung adenocarcinoma, epidermal carcinoma, endometrial adenocarcinoma and breast cancer) were subjected to treatment with CiA. The mean 0150 is shown in Figure 1A. The average G150 is plotted as the 0 line (mean 0150=2.4uM). Using a log scale, the sensitivity ofa particular cell line is represented by deflections to the right from the mean. Cell lines known to express high levels of Myc include multiple myeloma cell lines (eg KMS-12BM express high c-Myc, U266 express high 1-Myc (Holien eta! 2012) (Fourney eta! 1990). Levels of Myc have been obtained from literature and from Developmerita! Therapeutics Program NCI/NIH website (Pattern Id: MT350). RPMI-8225 cell line common to our panel and to the NCI-60 was chosen as a reference for MYC expression.

CiA sensitivity was also tested across a panel of neuroblastoma cells lines, with a range of known Myc expression levels (Figure 1B). Cell lines with high levels of n-Myc expression are clearly much more sensitive to treatment with CIA than cell lines with a low expression of n-Myc, and sensitivity increases across a gradient correlating with increasing n-Myc expression. This effect was observed in cells treated with CiA alone.

This is in contrast to the prior art in which KDAC6 inhibitors have only been shown to be useful in treating cancer when used in conjunction with other drugs, such as Bortezomib.

Cell lines with high levels of Myc generally tend to have high rates of protein synthesis and a higher dependency on autophagy, and so the results suggest that CiA was exerting its toxic action via inhibition of the cytoprotective autophagy.

Example 2: CIA exerts its toxic effect via abrogation of autophagy Following the discovery that the cells with high levels of Myc expression are much more sensitive to CiA than those cells with low levels of Myc expression, and the knowledge that high Myc expression levels correlate with high protein expression rates and dependency on autophagy, the inventors investigated whether CiA exerted its toxic effect via abrogation of autophagy (Figure 2).

HCT-116 cells were treated for 24 h with chloroquine (CQ -50 pM), 3-MA (5 mM), or bortezomib (Boil -5 nM) (all of which are modulators of autophagy), alone or in combination with CiA (10 pM) and stained for LC3. LC3 is a marker of both inhibition and induction of autophagy, Blockage of autophagy (eg by CQ or 3-MA) induces an accumulation of LC3, as does induction of autophagy flux (eg by bortezomib).

Figure 2A clearly shows that treatment with CQ, 3MA and Boil all increase the levels of LC3. This increase is reduced in the presence of CiA, and no LC3 is detected in the presence of CiA alone. This shows that CiA is abrogating autophagy.

Similar results are seen in Figure 2B for cells treated with CQ and Boil, in the presence and absence of CiA, and autophagy assessed by measuring P62 levels. P62 is another marker of autophagy that correlates with LC3 (P62 binds to LC3 upon autophagy). Again, the results show that CiA abrogates autophagy. A comparison with the known autophagy as inhibitor, tubastatin, confirms CiA's KDA6 inhibitory effect.

Example 3: CIA synergises with proteasome inhibitors.

Given the hypothesis that CiA exerts its action via inhibition of autophagy, it follows that agents which increase the need for autophagy (similarly to the high Myc cells in Example 1), for example, proteasome inhibitors, ought to exacerbate the action of CiA.

HCT-1 16 cells were treated with Bortezomib (SnM), CiA (1 OuM), or Tubastatin A (1 GuM) alone or in combination for 24 h and the level of caspase activity (as a measure of apoptosis) monitored (Figure 3A). It can be clearly seen that when CiA was used in combination with either Bortezomib orTubastatin A at concentrations that had no effect at all when used alone, the level of apoptosis in the cell line rose significantly, approximately 12 fold for the combination of CiA and Bortezomib, and approximately 8 fold for the combination of Tubastatin A and Bortezomib.

This provides further evidence that CiA is acting via its inhibitory effect on autophagy and additionally implies that CiA is more effective autophagy inhibitor than Tubastatin A, even more evident as the concentration of Tubastatin A was already slightly more inhibitory than CiA when used alone.

A similar result was obtained when an alternative cell line was used, OPM-2. This cell line is known to have a high level of Myc expression which further confirmed the findings from Example 1 as CiA had to be used at a fifth the concentration that used for cell line HCT- 116 to obtain a low level of inhibition when used alone (Figure 3B). The concentrations used were 2uM CiA, 5nM Bortezomib and in this experiment 2uM ACY was used rather than Tubastatin A. Again, the combination of CiA and Bortezomib produced a significantly higher rate of apoptosis than either drug alone, and the combination of Bortezomib and ACY gave a similar synergistic effect, but not as great.

Example 4: CIA inhibits the fusion of autophagosomes and lysosomes To further investigate the mechanistic action of CiA, a pH sensitive version of LC3 was used (Figure 4A). LC3 is tagged with both GFP and mcherry. GFF (green) is degraded in the acidic conditions found within lysosomes, whereas mcherry (red) is stable. The yellow spots show a lack of acidification, and the number of yellow spots indicates the level of inhibition of autophagy. Figure 4B shows fluorescent images of mouse embryonic fibroblasts carrying either the WT KDAC6, a knockout of KDAC6, and the WT KDAC6 but with the addition of CiA. It is clear that in the WT case, most of the spots are red, indicating that the lysosomes are acidified (GFP is degraded) and the fusion between the autophagosome and the lysosome is occurring. In cells with the KDAC6 knockout, there are very few red spots, indicating that most of the lysosomes are not acidified and autophagosome-lysosome fusion is not occurring, consistent with the role of KDAC6 in this process. In WI cells treated with CiA, compared to untreated cells, the number of red spots has decreased and the numbers of yellow spots has increased significantly. The numbers of yellow spots are quantified in the corresponding chart. Therefore, CiA inhibits the fusion of autophagosomes and lysosomes, providing further evidence that CiA inhibits KDAC6 in a manner which results in an inhibition of autophagy.

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