WO2015159097A2 - Méthodes - Google Patents

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WO2015159097A2
WO2015159097A2 PCT/GB2015/051163 GB2015051163W WO2015159097A2 WO 2015159097 A2 WO2015159097 A2 WO 2015159097A2 GB 2015051163 W GB2015051163 W GB 2015051163W WO 2015159097 A2 WO2015159097 A2 WO 2015159097A2
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autophagy
inhibitor
dependency
kdac6
protein
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PCT/GB2015/051163
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WO2015159097A3 (fr
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Maciej Adam KALISZCZAK
Eric Ofori Aboagye
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Imperial Innovations Limited
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Publication of WO2015159097A3 publication Critical patent/WO2015159097A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/37—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C07C311/38—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton
    • C07C311/44—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7023—(Hyper)proliferation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7023—(Hyper)proliferation
    • G01N2800/7028—Cancer

Definitions

  • the present invention relates to subject assessment and therapy using particular lysine deacetylase 6 (KDAC6) inhibitors of Formula I, as defined herein.
  • KDAC6 lysine deacetylase 6
  • KDACs lysine deacetylases
  • KDACs lysine deacetylases
  • Class I KDACs eg KDACs 1 , 2, 3 and 8
  • Class II KDACs eg KDACs 4, 5, 6, 7, 9 and 10
  • class MA which consists of KDACs 4, 5, 7 and 9, and class IIB which consists of KDACs 6 and 10.
  • Class IIA KDACs are located in the nucleus and cytoplasm, while class IIB KDACs are located mainly in the cytoplasm and have little effect on histones.
  • Class III KDACs belong to the sirtuin family of proteins, some of which deacetylate p53 and a-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 al 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, HSP90 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 vivo. Similar to KDAC6, KDAC class III isoform Sirt2 can also use tubulin as a substrate, and regulate the balance of tubulin acetylation and deacetylation.
  • KDAC6 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.
  • KDAC6 inhibitors in combination with other known anti-carcinogens has been shown to have a synergistic activity towards cancer (eg Hideshima er al 2005). KDAC6, along with other KDACs, has thus been an attractive target for the development of anti-cancer therapies (Marks ef a/ 2005, Richon er a/ 2002).
  • KDAC6 inhibitors currently in development are reviewed in Yang et al 2013 and appear to derive their utility mainly from inhibition of deacetylase activity.
  • HPOB is able to inhibit the catalytic activity of KDAC6 (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 induced by trehalose, an autophagy inducer tool compound), and it does not impair the autophagy pathway (Lee et al PNAS 2012).
  • KDACs are described in WO 2008/050125, 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.
  • Example 1 shows that cells exhibiting high levels of Myc protein are more sensitive to C1A (a particular KDAC6 inhibitor having the structure of Formula I).
  • C1A 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 et al JCI 2012).
  • UTR Unfolded Protein Response
  • Examples 2 and 3 show that C1A (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 C1A inhibits the fusion of autophagosomes and lysosomes, providing further evidence that C1 A inhibits KDAC6 via inhibition of autophagy.
  • C1A a particular KDAC6 inhibitor having the structure of Formula I
  • Example 4 shows that C1A inhibits the fusion of autophagosomes and lysosomes, providing further evidence that C1 A inhibits KDAC6 via inhibition of autophagy.
  • KDAC6 binds to ubiquitinated m ' isfolded/damaged proteins and also binds to the cytoskeleton.
  • KDAC6 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 survival, 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.
  • WO 2012/088067 describes methods for predicting and/or determining responsiveness to a histone deacetylase (KDAC) inhibitor.
  • KDAC histone deacetylase
  • WO 2013/158984 describes biomarkers to identify subjects that will respond to treatment and treating such subjects. However, there is no mention of, or characterisation of, the KDAC6 inhibitors defined herein.
  • 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 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: wherein:
  • R la represents C1-4 alkyl (which latter group is optionally substituted by one or more substituents selected from halogeno and aryl), aryl or (CH2) 2-L 1 ;
  • R 2a represents H, C1-4 alkyl (which latter group is optionally substituted by one or more substituents selected from halogeno and aryl), aryl or (CH2)2-L 3 ;
  • L ⁇ L 2 and L 3 each represents, independently at each occurrence, a leaving group
  • R 3 represents halogeno or C1-4 alkyl
  • a represents, independently at each occurrence, an integer from 0 to 4;
  • Y 1 and Y 2 independently represent, at each occurrence, H or C1-4 alkyl; or a pharmaceutically acceptable derivative thereof,
  • R 1a represents (CH 2 ) 2 -L 1 ;
  • R 2a represents (CH 2 ) 2 -L 3 .
  • leaving group when used herein, includes references to halogeno (eg CI, Br, I) and OS(0) 2 R 4 groups wherein R 4 is Ci-s alkyl (optionally substituted by one or more fluoro atoms) or aryl (optionally substituted by one or more substituents selected from Ci- 4 alkyl, Ci- 4 alkoxy, N0 2 and halogeno).
  • 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.
  • 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 (ie a minimum of two) of carbon atoms, be unsaturated and/or interrupted by one or more oxygen and/or sulfur atoms.
  • alkyl and alkoxy groups may also be substituted by one or more halogeno, and especially fiuoro, atoms.
  • aryl includes references to Ce-io 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, Ci- 4 alkyl and Ci -4 alkoxy. When substituted, aryl groups are preferably substituted by one to five (eg one to three) substituents.
  • KDAC6 inhibitors 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 WO2008/050125, 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 L 1 , L 2 and/or L 3 represents halogeno, halogenation of a corresponding compound of Formula II: Formula II wherein R lc represents Ci -4 alkyl (which latter group is optionally substituted by one or more substituents selected from halogeno and aryl), aryl, (CH2)2-OH or the structural fragment:
  • R x represents H or N(R ld )R 2d ,
  • R ld and R 2d independently represent Ci- 4 alkyl (which latter group is optionally substituted by one or more substituents selected from halogeno and aryl), aryl or (CH2)2-OH
  • R 2c represents H
  • Ci- 4 alkyl which latter group is optionally substituted by one or more substituents selected from halogeno and aryl
  • R y , R 3 , X 1 to X 6 , a, b and c are as hereinbefore defined, provided that at least one of R'°, R 2c , R ld and R 2d 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
  • 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.
  • 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.
  • tumours benign or malignant
  • autoimmune conditions e.g asthma
  • allergic disease e.g asthma
  • transplantation subjects e.g., transplantation subjects
  • infectious diseases such as viral diseases.
  • infectious diseases such as viral diseases.
  • 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.
  • 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.
  • a subpopulation of immune cells such as T lymphocytes in autoimmune disease or such as B lymphocytes in allergic disease.
  • 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.
  • a viral disease wherein destruction of the virally infected cells may be advantageous.
  • examples include hepatitis B (Wang et al Hepatology 2005), hepatitis C (Benali-Fure et al Oncogene 2005), hepatitis D (Huang et al J Gen Virol 2006), flavivirus (Yu et al J Virol 2006), Borna disease (Williams et al J Virol 2005) or HIV (Ueno et al 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.
  • the disease or condition characterised by the presence of unwanted cells is cancer and the unwanted cells are cancerous cells.
  • the subject with a disease or condition characterised by the presence of unwanted cells may be a subject with cancer.
  • 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.
  • 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 17p.
  • the cancer is a solid tumour.
  • the solid tumour may be metastatic.
  • 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 17p 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.
  • 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.
  • a complete response or complete remission of disease or condition for example cancer
  • 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.
  • the response to treatment with a KDAC6 inhibitor having the structure of Formula I 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.
  • RECIST Response Evaluation Criteria In Solid Tumors
  • 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.
  • 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.
  • 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.
  • 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 et al 2002.
  • Hook et al 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.
  • 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.
  • 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.
  • 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.
  • the molecule may bind to the ubiquitin binding domain and bind to other sites on the protein that may cause conformational restructuring.
  • 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.
  • the KDAC6 inhibitors are considered to bind to a Zn 2+ 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.
  • 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.
  • the KDAC6 inhibitor inhibits the ubiquitin binding activity of KDAC6 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.
  • the KDAC6 inhibitor inhibits the ubiquitin binding activity of KDAC6 to an undetectable level.
  • 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 et al, PNAS 2012).
  • LC3-1 1 microtubule associated protein IA/IB-light chain 3-II
  • the level of LC3-II can be measured by immunoblotting with an antibody raised against LC3-II or to a tag conjugated to LC3-II using techniques readily available in the art.
  • 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-II.
  • the KDAC6 inhibitor having the structure of Formula I may be one that is capable of inhibiting aggresome formation and therefore autophagy.
  • 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.
  • the KDAC6 inhibitor having the structure of Formula I may also inhibit the deacetylase activity of KDAC6 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 CS1010- 1 KT). The inhibition of the catalytic activity of KDAC6 can also be assessed by monitoring acetylation of a-tubulin.
  • the KDAC6 inhibitor is one that has the structure of Formula I wherein R la and R 2a represents halogeno species. More preferably, the KDAC6 the inhibitor is C1 A or C1 B:
  • C1 A and C1 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 WO2008050125, which are incorporated herein by reference.
  • a KDAC6 inhibitor having the structure of Formula I we also include the meaning of prodrugs thereof.
  • 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.
  • 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.
  • prodrug 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 enzymaticaliy 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, Harbor 1986) and V. J. Stella et al. "Prodrugs: A Chemical Approach to Targeted Drug Delivery” Directed Drug Delivery R.
  • 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.
  • the unwanted cell is one in which autophagy is critical for its survival.
  • the unwanted cell has no alternative pathways to perform the function served by autophagy if autophagy is inhibited.
  • such cells are considered to be in a 'stressed' state, and such stresses may 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 100 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.
  • 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.
  • 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.
  • 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 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 s , 10 9 , or 10 10 cells.
  • 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.
  • 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.
  • 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.
  • control cells are normal cells that are not characteristic of the disease or condition in question.
  • the one or more control cells may be a normal non-cancer cell.
  • 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.
  • 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.
  • 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 et a/, 2002).
  • the HCT1 16 colon cancer cell line can also be used as an appropriate control cell and can be grown in DMEM containing 10% FBS or RPMI with 10% FBS.
  • DLD1 and LOVO colonic carcinoma cell lines have no c-Myc amplification and thus are also appropriate control cells (Okuyama et al 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.
  • 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.
  • the dependency can also be compared to a mean level of dependency on autophagy for survival of cells in a healthy population.
  • 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 KDAC6 inhibitor having the structure of Formula I).
  • 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 or 8 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).
  • the assessment should preferably be done using the same sample, cell type or tissue.
  • 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.
  • detectable markers include proteins indicative of an increased dependency on autophagy for survival of a cell and various other molecules as described further below.
  • 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.
  • 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.
  • the 'normal individuals' have no or few risk factors for the development of unwanted cells, for example cancer.
  • 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 or indirectly determining the level of dependency on autophagy for survival in the unwanted cells from the subject.
  • 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.
  • 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.
  • determining whether a subject's unwanted cells have or are likely to have an increased dependency on autophagy for survival may comprise:
  • 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.
  • 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 effectors 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.
  • the level of autophagy or markers of the level of autophagy eg an upstream or downstream marker
  • 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.
  • 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.
  • the number of aggresomes within a given cell can be assayed using immunofluorescence, staining for, for example, ⁇ -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 et al 2003, Biederbick et al 1995).
  • 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 qPCR/immunoblotting of particular genes, for example CHOP, IRE1 , GADD45, Herp, Bip/GRP78, GADD153, XBP-1 , PTEN, TSC1 , TSC2, P53, DRAM or p97.
  • 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 PERK/elF2a/ATF4 axis (Hart et a/ 2012).
  • 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 Myc oncogene in cancer is well established.
  • the Myc family of proto-oncogenes (c-Myc, l-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 et al 2012 Cell 151 : 68-79) which leads to an increased requirement for degradation via the proteasome.
  • 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.
  • KDAC6 inhibitor having the structure of Formula I since such cells are highly dependent upon aggresome function and downstream protein degradation via autophagy.
  • the demand for the aggresome to eliminate misfolded polyubiquitinated proteins is greater.
  • 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.
  • Myc protein we include any one or more subtypes of Myc protein such as any one or more of c-Myc, n-Myc or l-Myc or b-Myc or s-Myc or v-Myc.
  • humans have at least three subtypes of Myc, namely c-Myc (c- MYC_HUMAN, P01106), n-Myc (n-MYC_HUMAN, P04198) and l-Myc (l-MYC_HUMAN, P12524), 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 l-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 l-Myc.
  • c-Myc c- MYC_HUMAN, P01106
  • n-Myc n-MYC_HUMAN, P04198
  • l-Myc l-MYC_HUMAN, P12524
  • 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).
  • b-Myc SEQ ID Nos: 4 and 7
  • s-Myc SEQ ID Nos: 5 and 8
  • v-Myc SEQ ID Nos: 6 and 9
  • 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.
  • the method 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.
  • the subject is human, the amount or activity of human Myc protein is measured, and so on.
  • determining whether the subject's unwanted cells have or are likely to have, an increased dependency on autophagy for survival 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.
  • 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.
  • the unwanted cells are a haematological cancer
  • 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.
  • 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.
  • the sample is one that comprises unwanted cells taken from the subject.
  • 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 plasma cells from a subject with magnetic beads coated with anti-CD138 antibody to enrich for B cells, or isolate peripheral blood mononuclear cells.
  • 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.
  • 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.
  • 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.
  • 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 survival.
  • 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, ie a positive or negative indication.
  • 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.
  • 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.
  • 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,
  • the amount of a protein indicative of an increased dependency on autophagy for survival 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).
  • 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, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 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).
  • the assessment should preferably be done using the same sample, cell type or tissue.
  • 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) available (eg RT-PCR for example by taq-man, or RNA-seq).
  • 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.
  • the 'normal individuals' have no or few risk factors for unwanted cells, for example cancer cells.
  • 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 (eg blood plasma or serum) but which nevertheless contain markers that are correlated with the dependency on autophagy for survival of the cells.
  • 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.
  • 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, 4, 5, 10, 20, 50 or 100 times more than the level of that protein in a control cell.
  • a protein indicative of an increased dependency on autophagy for survival eg Myc
  • the protein indicative of an increased dependency on autophagy for survival is Myc.
  • the control cell may be one described above.
  • 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 (eg 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 (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.
  • a normal cell with a normal dependency on autophagy for survival eg a non-cancer cell
  • 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 (eg Myc)
  • a cancerous cell that is known to have an increased level of a protein indicative of an increased dependency on autophagy for survival but not to the extent that it renders the cancer more responsive to treatment by a KDAC6 inhibitor
  • 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 (eg a non-cancer cell).
  • 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).
  • 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, 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).
  • a reagent which can identify the protein indicative of an increased dependency on autophagy for survival eg Myc protein
  • 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 include antibodies and peptides, for example those selected from a combinatorial or phage display library.
  • 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.
  • 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.
  • 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 KHO2041.
  • 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).
  • CDRs complementarity determining regions
  • the 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 9E10). This is a mouse monoclonal Igd 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, immunoprecipitation, immunofluorescence, IHC(P)(immunohistochemistry-paraffin), FCM (flow cytometry) and ELISA.
  • c-Myc from additional species, including feline and canine c-Myc.
  • 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.
  • L-Myc Antibody Santa Cruz C-20.
  • 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.
  • the antibody has at least a 10 fold-higher affinity for a protein indicative of an 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 for survival (eg Myc) in an unwanted ceil (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-PCR).
  • the level of mRNA encoding the protein indicative of an increased dependency on autophagy for survival is increased to a level of mRNA 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 mRNA may vary depending on the type of individual (eg human, 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).
  • 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).
  • 1 standard deviation above the mean level of mRNA encoding the protein indicative of an increased dependency on autophagy for survival
  • the assessment should preferably be done using the same sample, cell type or tissue.
  • 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).
  • 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.
  • the 'normal individuals' have no or few risk factors for unwanted cells, for example cancer cells.
  • the level of mRNA encoding the protein indicative of an increased dependency on autophagy for survival 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.
  • the mRNA is one that encodes Myc.
  • the control cell may be one described above.
  • 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.
  • a normal cell with a normal dependency on autophagy for survival eg a non-cancer cell
  • 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)
  • 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).
  • 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).
  • 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 et al 2004; units correspond to a normalised value relative to an internal control such as 18s ribosomal RNA).
  • a sample from a subject having ovarian cancer contains 200 or more arbitrary units (eg normalised according to 18s 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.
  • gene 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.
  • 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.
  • 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.
  • 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.
  • determining the amount of a protein indicative of an increased dependency on autophagy for survival (eg Myc) in an unwanted 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.
  • 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).
  • 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.
  • 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 genome of the unwanted cells (eg cancer cell).
  • the Myc gene normally resides on chromosome 8 at band q24.
  • 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 RT-PCT. 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).
  • a protein indicative of an increased dependency on autophagy for survival eg Myc
  • 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 is controlled in part by phosphorylation at threonine 58 (T58) and serine 62 (S62).
  • Various cancers including Burkitt's lymphoma, and a range of breast cancers, are characterised by increased phosphorylation of Myc at S62 and decreased phosphorylation of Myc at T58, which underlines an increased stability of Myc (ie a lower turnover rate).
  • 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 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.
  • the phosphorylation status of residues S62 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 al (PNAS 109(8): 2790) which rely on phospho-specific antibodies.
  • Zhang et al also describe how the signaling cascade that controls phosphorylation at T58 and S62 in a Myc protein is coordinated by the scaffold protein Axinl , and so it will be understood that a further example of a protein indicative of an increased dependency on autophagy for survival is Axinl , 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).
  • determining whether the subject's unwanted cells eg cancer cells
  • determining whether the subject's unwanted cells 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.
  • the protein is Myc, and preferably, the unwanted cells are cancer cells.
  • the method is performed on a sample taken from the subject as described herein.
  • 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.
  • 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).
  • reporter gene By a reporter gene we include genes which encode a reporter protein whose activity may easily be assayed, for example ⁇ -galactosidase, chloramphenicol acetyl transferase (CAT) gene, luciferase or Green Fluorescent Protein (see, for example, Tan et al, 1996).
  • CAT chloramphenicol acetyl transferase
  • luciferase or Green Fluorescent Protein
  • Green Fluorescent Protein see, for example, Tan et al, 1996.
  • CAT chloramphenicol acetyl transferase
  • luciferase Green Fluorescent Protein
  • 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.
  • the expression of any of transferrin, LDHA, MCL1 or elF4E 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.
  • protein concentration can be studied by a range of antibody based methods including immunoassays, such as ELISAs and radioimmunoassays.
  • 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)
  • expression of that protein may be assessed by monitoring its enzymatic activity using any suitable assay known in the art for the particular enzyme.
  • 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 noninvasive ⁇ using PET imaging (eg 89 Zr-desferrioxamine transferrin ( 89 Zr-Tf)) (Holland et al 2013).
  • 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.
  • the activity of the protein indicative of an increased dependency on autophagy for survival 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 (eg 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 (eg 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 (eg 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 (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).
  • 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 (eg 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 or 8 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).
  • a protein indicative of an increased dependency on autophagy for survival eg Myc protein
  • the assessment should preferably be done using the same sample, cell type or tissue.
  • 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 available for determination of activity of the protein indicative of an increased dependency on autophagy for survival (eg Myc).
  • 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.
  • the 'normal individuals' have no or few risk factors for the presence of unwanted cells, for example for cancer.
  • 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.
  • the protein indicative of an increased dependency on autophagy for survival is Myc.
  • the control cell may be one described above.
  • 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.
  • a normal cell with a normal dependency on autophagy for survival eg a non-cancer cell
  • 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)
  • a cancerous cell that is known to have an increased activity of a protein indicative of an increased dependency on autophagy for survival but not to the extent that it renders the cancer more responsive to treatment by a KDAC6 inhibitor
  • a subject's cancer is more responsive to treatment by a KDAC6 inhibitor having the structure of Formula I 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).
  • 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 et al 2013 J Paediatr Hematol Oncol, 35(5): 209); and/or identifying the presence of point mutations in ALK such as at position 1 174 (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 C1A), or at positions F1 174L and R1275Q (gain-of-function mutations, Schonherr er a/ Oncogene 2012), for example using RNA-seq; and/or assessing the levels of 2-microglobulin in the subject.
  • ALK anaplastic
  • Avet Loiseau et a/ (Blood, 2001 , 98(10): 3082) 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 32-microglobulin levels above 3mg/L.
  • a protein indicative of an increased dependency on autophagy for survival eg Myc
  • the amount or activity of a protein indicative of an increased dependency on autophagy for survival 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 a-tubulin acetylation to provide an additional global measure of the level of inhibition, including its effect on the catalytic activity of the KDAC6 inhibitor.
  • the amount or activity of a protein indicative of an increased dependency on autophagy for survival 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.
  • KDAC6 inhibitors having the structure of Formula I are particularly effective, on their own, to treat diseases or conditions characterised by the presence of 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 et al 2005), or carfilzomib (Mishima et al 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 C1A or C1 B.
  • 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).
  • 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.
  • 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).
  • the one or more further therapeutic agents are suitable to treat the disease or condition characterised by the presence of unwanted cells.
  • the one or more further therapeutic agents are one or more anti-cancer agents.
  • the one or more anti-cancer agents may be an approved chemotherapeutic agent known in the art, such as bortezomib or farnesyl transferase inhibitors.
  • anti-cancer agents may be selected from alkylating agents including nitrogen mustards such as mechlorethamine (HlSfe), cyclophosphamide, ifosfamide, melphalan (L- sarcolysin) and chlorambucil; methylating agents such as temozolomide (believed to be especially useful for treating brain tumours, Knizhnik et al); 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 (ameth,
  • the further therapeutic agent may be one that increases this dependency and/or inducers of the aggresome/autophagy.
  • proteasome inhibitors eg bortezomib
  • mistranslation inducers eg aminoglycoside antibiotics, amino acid analogues
  • Src-inhibitors eg dasatinib
  • DNA Protein Kinase-inhibitors eg NU 7441
  • PI3 Kinase/mTOR inhibitors eg NVP-BEZ235
  • AKT-inhibitors eg K2206
  • HSP 90 inhibitors eg 17AAG, Zaarur ei a/ JBC 2008, or AUY922, SNX-5422, Ganetespib (STA- 9090)
  • TNFa-modulators Tuour 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.
  • 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.
  • 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.
  • 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).
  • the disease or condition is cancer, such as a solid cancer, a haematological cancer, inflammatory breast cancer or a neuroblastoma.
  • 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.
  • virus infection is hepatitis B, hepatitis C, hepatitis D, flavivirus, Borna disease or HIV.
  • 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.
  • the KDAC6 inhibitor having the structure of Formula I is C1A or C1 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.
  • 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.
  • the KDAC6 inhibitor having the structure of Formula I will usually be administered separately from the one or more further therapeutic agents.
  • 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.
  • 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.
  • the one or more further therapeutic agents may be administered before, at the same time, or after, the KDAC6 inhibitor of Formula I.
  • the KDAC6 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 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.
  • the KDAC6 inhibitor is C1A or C1 B.
  • 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.
  • 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 10 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.
  • 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.
  • 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.
  • 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 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 one or more further therapeutic agents.
  • the one or more further therapeutic agents 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 hours, or between 30 minutes and 9 hours, or between 35 minutes
  • the KDAC6 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 regimes provide examples of appropriate administration times.
  • 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 DAC6 inhibitor according to the method according to the first aspect of the invention.
  • 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 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.
  • 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.
  • the KDAC6 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 therapeutic agent, including those described above in relation to the first aspect of the invention.
  • 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.
  • 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.
  • 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), PI3 Kinase/mTOR inhibitors (eg NVP- BEZ235), AKT-inhibitors (eg MK2206), HSP 90 inhibitors (eg 17AAG, Zaarur et al JBC 2008, or AUY922, SNX-5422, Ganetespib (STA-9090)) and TNFa-modulators (Tumour Necrosis Factor).
  • a proteasome inhibitor optionally bor
  • 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.
  • 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.
  • 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.
  • any agent described herein is typically administered as part of a pharmaceutical composition together with a pharmaceutically acceptable excipient, diluent, adjuvant, or carrier.
  • 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.
  • the agents may be administered at varying doses.
  • 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.
  • the agents eg KDAC6 inhibitor and/or further therapeutic agents
  • 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, hydroxypropy!methylcellulose (HPMC), hydroxy-propylcellulose (HPC), 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.
  • 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 glyco
  • 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.
  • 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 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.
  • aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • 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.
  • the daily dosage level of the agents will usually be from 1 to 5000 mg per adult, administered in single or divided doses.
  • the tablets or capsules of the compound of the invention may contain from 1 mg to 1000 mg (ie from about 60-120 mg/m 2 ) 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 can also be 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 dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2- tetrafluoroethane (HFA 134A3 or 1 ,1 , 1 ,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas.
  • a suitable propellant eg dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2- tetrafluor
  • 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 KDAC6 inhibitor and/or further therapeutic agent) for delivery to the subject.
  • an agent eg KDAC6 inhibitor and/or further therapeutic agent
  • 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.
  • the agents eg KDAC6 inhibitor and/or further therapeutic agents
  • the compounds of the invention may also be transdermal ⁇ administered, for example, by the use of a skin patch.
  • the agents eg KDAC6 inhibitor and/or further therapeutic agents
  • the 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.
  • they may be formulated in an ointment such as petrolatum.
  • the agents eg KDAC6 inhibitor and/or further therapeutic agents
  • the 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.
  • ком ⁇ онентs 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- 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.
  • oral or topical administration of the agents is the preferred route, being the most convenient.
  • the drug may be administered parenterally, eg sublingually or buccally.
  • the agent eg KDAC6 inhibitor and/or further therapeutic agents
  • the agent 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.
  • the formulation is a pharmaceutical formulation.
  • the formulation may be a veterinary formulation.
  • administration is not restricted to a one time administration.
  • 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.
  • 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.
  • a KDAC6 inhibitor having the structure of Formula I 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.
  • 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.
  • the subject may be one that had not previously been tested for suitability in responding to treating by administration of a KDAC6 inhibitor having a structure of Formula I.
  • the subject may be one who has not responded to the additional further therapeutic agents above.
  • 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.
  • 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.
  • the subject is subsequently administered a KDAC6 inhibitor having the structure of Formula I, and optionally one or more other therapeutic agents.
  • 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 DAC6 inhibitor having the structure of Formula I, and optionally one or more other therapeutic agents.
  • 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.
  • 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.
  • KDAC6 inhibitor having the structure of Formula I is C1A or C1 B.
  • 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 or 100 times more than its dependency on autophagy for survival in the absence of the agent.
  • the agent may be one that increases the cellular demand for autophagy.
  • 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.
  • the agent may be one that increases the level or production of protein including damaged proteins, such as mis-translated or mis- folded proteins.
  • 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.
  • 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 non-functional.
  • 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-
  • 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.
  • 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.
  • 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.
  • 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.
  • the KDAC6 inhibitor having the structure of Formula I eg C1A and C1 B
  • 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 (ii) a KDAC6 inhibitor having the structure of Formula I.
  • 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 (ii) 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.
  • 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.
  • the binding partner eg antibody
  • the binding partner 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 antibody binding to a primary antibody).
  • 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.
  • 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.
  • the KDAC6 inhibitor having the structure of Formula I is C1A or C1 B.
  • the kit further comprises control cells that have a normal dependency on autophagy.
  • 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.
  • 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.
  • 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.
  • 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 17AAG, AUY922, SNX-5422, and Ganetespib (STA-9090); or a TNFa-modulator.
  • a proteasome inhibitor optionally bortezomib
  • a further anti-cancer agent optionally a farnesyl transferase inhibitor such as Lonafarnib
  • an anti-viral agent such as Lonafarnib
  • Src-inhibitor optionally dasatin
  • 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.
  • the KDAC6 inhibitor having the structure of Formula I is C1A or C1 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.
  • the KDAC6 inhibitor having the structure of Formula I is C1A or C1 B, and/or the control cells are non-cancer cells.
  • the one or more control cells are selected from the group consisting of HeLa cells, HCT116 cells, DLD1 cells and LOVO cells.
  • 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 C1A or C1 B.
  • the virus eg adenovirus
  • the virus 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 al 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 C1A or C1 B, and optionally wherein the viral disease is any of hepatitis B, hepatitis C, hepatitis D, flavivirus, Borna disease or HIV.
  • 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 C1A or C1 B, and optionally wherein the viral disease is any of hepatitis B, hepatitis C, hepatitis D, flavivirus, Borna disease or HIV.
  • 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 C1 A or C1 B.
  • 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 C1A or C1 B.
  • 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.
  • FIG. 1 C1A inhibits the proliferation of cancer cells with greater sensitivity in those amplified in MYC.
  • B Growth inhibitory effect of C1A in a panel of neuroblastoma cell lines.
  • Figure 2. C1A abrogates autophagy induced by autophagy tools compounds.
  • HCT-1 16 cells were treated for 24 h with chloroquine (CQ - 50 ⁇ ), 3-MA (5 mM), bortezomib (Bort - 5 nM) alone or in combination with C1A (10 ⁇ ) and stained for LC3.
  • CQ - 50 ⁇ chloroquine
  • 3-MA 5 mM
  • bortezomib Bort - 5 nM
  • C1A C1A
  • Bob HDAC6 inhibitor tubastatin A
  • HDAC6 inhibitor C1A synergises with proteasome inhibition.
  • A Effect of 24h treatment with bortezomib (bort- 5 nM) alone or in combination with C1A (10 ⁇ ) or Tubastatin A (Tub-10 ⁇ ) on caspase activity in HCT-1 16 cells.
  • B Impact of ACY/C1A treatment (9h - both at 2 ⁇ ) alone or in combination with bortezomib (5 nM) on caspase 3/7 activity in OPM-2 cells.
  • Figure 4. HDAC6 inhibition by C1A prevents autophagosome-lysosome fusion.
  • FIG. 1 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.
  • FIG. 1 Protein sequences of human Myc proteins; c-Myc (SEQ ID No: 1), n-Myc (SEQ ID No: 2) and l-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).
  • Example 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 C1A.
  • the mean GI50 is shown in Figure 1A.
  • Cell lines known to express high levels of Myc include multiple myeloma cell lines (eg KMS-12BM express high c-Myc, U266 express high l-Myc (Holien et al 2012) (Fourney et al 1990). Levels of Myc have been obtained from literature and from Developmental Therapeutics Program NCI/NIH website (Pattern Id : MT350). RPMI-8226 cell line common to our panel and to the NCI-60 was chosen as a reference for MYC expression. C1A sensitivity was also tested across a panel of neuroblastoma cells lines, with a range of known Myc expression levels (Figure 1 B).
  • Example 2 C1 A exerts its toxic effect via abrogation of autophagy
  • the inventors investigated whether C1A exerted its toxic effect via abrogation of autophagy (Figure 2).
  • HCT-116 cells were treated for 24 h with chloroquine (CQ - 50 ⁇ ), 3-MA (5 mM), or bortezomib (Bort - 5 nM) (all of which are modulators of autophagy), alone or in combination with C1A (10 ⁇ ) 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).
  • FIG. 2A clearly shows that treatment with CQ, 3MA and Bort all increase the levels of LC3. This increase is reduced in the presence of C1A, and no LC3 is detected in the presence of C1A alone. This shows that C1A is abrogating autophagy.
  • HCT-116 cells were treated with Bortezomib (5nM), C1A (10uM), or Tubastatin A (10uM) 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 C1A 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 C1A and Bortezomib, and approximately 8 fold for the combination of Tubastatin A and Bortezomib.
  • Example 4 C1A inhibits the fusion of autophagosomes and Ivsosomes
  • LC3 is tagged with both GFP and mcherry. GFP (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 C1 A.

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Abstract

L'invention concerne une méthode permettant d'identifier un sujet atteint d'une maladie ou d'une affection caractérisée par la présence de cellules indésirables comme étant susceptible de répondre à un traitement consistant à administrer un inhibiteur de KDAC6 ayant la structure de formule (I), ladite méthode consistant à déterminer si les cellules indésirables du sujet présentent ou sont susceptibles de présenter une plus grande dépendance vis-à-vis de l'autophagie pour la survie, la formule (I) comprenant :
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008050125A1 (fr) 2006-10-24 2008-05-02 Imperial Innovations Ltd. Composés et leurs utilisations
WO2012088067A1 (fr) 2010-12-20 2012-06-28 Quintiles Transnational Corporation Procédés pour prédire et/ou déterminer la réponse à un inhibiteur d'histone désacétylase (hdac)
WO2013158984A1 (fr) 2012-04-19 2013-10-24 Acetylon Pharmaceuticals, Inc. Biomarqueurs permettant d'identifier des patients qui réagiront à un traitement, et traitement desdits patients

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8859502B2 (en) * 2010-09-13 2014-10-14 Celgene Corporation Therapy for MLL-rearranged leukemia
GB201110546D0 (en) * 2011-06-22 2011-08-03 Imp Innovations Ltd Compositions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008050125A1 (fr) 2006-10-24 2008-05-02 Imperial Innovations Ltd. Composés et leurs utilisations
WO2012088067A1 (fr) 2010-12-20 2012-06-28 Quintiles Transnational Corporation Procédés pour prédire et/ou déterminer la réponse à un inhibiteur d'histone désacétylase (hdac)
WO2013158984A1 (fr) 2012-04-19 2013-10-24 Acetylon Pharmaceuticals, Inc. Biomarqueurs permettant d'identifier des patients qui réagiront à un traitement, et traitement desdits patients

Non-Patent Citations (65)

* Cited by examiner, † Cited by third party
Title
"Immunoassays: A practical approach", 2000, OXFORD UNIVERSITY PRESS
"lmmunoassays: A practical approach", 2000, OXFORD UNIVERSITY PRESS
"Using antibodies: A laboratory manual", 1999, COLD SPRING HARBOR PRESS
AVET LOISEAU ET AL., BLOOD, vol. 1998, 2001, pages 3082 - 3086
AVET LOISEAU ET AL., BLOOD, vol. 98, no. 10, 2001, pages 3082
BENALI-FURE ET AL., ONCOGENE, 2005
BENALI-FURE ET AL., ONCOGENE, vol. 24, 2005, pages 4921 - 33
BIECHE ET AL., CANCER RESEARCH, 1999
BIECHE ET AL., CANCER RESEARCH, vol. 59, 1999, pages 2759 - 65
BIEDERBICK ET AL., EUR J CELL BIOL, vol. 66, 1995, pages 3 - 14
BOXER ET AL., ONCOGENE, vol. 20, 2001, pages 5595 - 5610
BRANDL ET AL., BIOL CELL, vol. 101, 2009, pages 193 - 205
D. E. V. WILMAN: "Prodrugs in Cancer Chemotherapy", BIOCHEMICAL SOCIETY TRANSACTIONS, vol. 14, 1986, pages 375 - 382
E. DE HOFFMANN; J. CHARETTE; V. STROOBANT: "Mass Spectrometry Principles and Applications", 1996, WILEY & SONS
ED HARLOW; DAVID LANE: "Using antibodies: A laboratory manual", 1999, COLD SPRING HARBOR PRESS
FOURNEY ET AL., DIS MARKERS, vol. 8, 1990, pages 117 - 24
HART ET AL., JCI, 2012
HART ET AL., JCI, vol. 122, no. 12, 2012, pages 4621 - 34
HIDESHIMA ET AL., PNAS, vol. 102, 2005, pages 8567 - 8572
HOLIEN ET AL., BLOOD, vol. 120, 2012, pages 2450 - 2453
HOLLAND ET AL., NATURE MEDICINE, vol. 18, 2012, pages 1586 - 91
HOOK ET AL., PNAS, vol. 21, 2002, pages 13425 - 13430
HUANG ET AL., J GEN VIROL, 2006
HUANG ET AL., J GEN VIROL, vol. 87, 2006, pages 1715 - 23
IBA ET AL., CANCER SCI, vol. 9, 2004, pages 418 - 23
KE ET AL., J CLIN INV, 2011
KE ET AL., J CLIN INV, vol. 121, 2011, pages 37 - 56
KNIZHNIK ET AL., PLOS ONE, vol. 8, 2013, pages E E55665
KYEI ET AL., JCB, 2009
KYEI ET AL., JCB, vol. 186, 2009, pages 255 - 268
LEE ET AL., PNAS, 2012
LEE ET AL., PNAS, vol. 110, 2012, pages 15704 - 15709
LEE, PNAS, 2012
LEVINE ET AL., PNAS, vol. 103, pages 17372 - 17377
LEVINE, PNAS, 2006
LIANG ET AL., J PAEDIATR HEMATOL ONCOL, vol. 35, 2013, pages 209 - 213
LIANG ET AL., J PAEDIATR HEMATOL ONCOL, vol. 35, no. 5, 2013, pages 209
MARKS ET AL., EXPERT OPINION ON INVESTIGATIONAL DRUGS, vol. 14, 2005, pages 1497 - 511
MISHIMA ET AL., ASH 2013 ANNUAL MEETING ABSTRACT 4431, 2013
NIE ET AL., CELL, vol. 151, 2012, pages 68 - 79
OKUYAMA ET AL., CANCER RESEARCH, vol. 70, 2010, pages 10213 - 10223
ONCOGENE, vol. 20, no. 4, 2001, pages 5595
RICHON ET AL., CLINICAL CANCER RESEARCH, vol. 8, 2002, pages 662 - 4
SCHONHERR ET AL., ONCOGENE, 2012
SCHONHERR ET AL., ONCOGENE, vol. 31, 2012, pages 5193 - 200
SLAPAK; KUFE ET AL.: "Harrison's Principles of Internal Medicine", MCGRAW-HILL INC, article "Principles of Cancer Therapy"
SLAPAK; KUFE ET AL.: "Harrison's Principles of Internal Medicine", MCGRAW-HILL INC, article "Priniciples of Cancer Therapy"
TAN ET AL., EMBO J., vol. 15, 1996, pages 4629 - 42
TWORKOWSKI ET AL., ONCOGENE, vol. 21, 2002, pages 8515 - 8520
UENO ET AL., J CELL BIOL, 2009
UENO ET AL., J CELL BIOL, vol. 186, 2009, pages 255 - 68
V. J. STELLA ET AL.: "Directed Drug Delivery R.", 1985, HUMANA PRESS, article "Prodrugs: A Chemical Approach to Targeted Drug Delivery", pages: 247 - 267
VITA; HENRICKSON, SEMINARS IN CANCER BIOL, vol. 16, 2006, pages 318 - 330
WANG ET AL., HEPATOLOGY, 2005
WANG ET AL., HEPATOLOGY, vol. 41, 2005, pages 761 - 70
WILLIAMS ET AL., J VIROL, vol. 80, 2006, pages 8613 - 8626
WILLIAMS, J VIROL, 2005
YANG ET AL., DRUG DISCOV THER, vol. 7, 2013, pages 233 - 242
YU ET AL., J VIROL, 2006
YU, J VIROL, vol. 80, 2006, pages 11868 - 80
ZAARUR ET AL., JBC, 2008
ZAARUR, JBC, vol. 283, 2008, pages 27575 - 27584
ZHANG ET AL., PNAS, vol. 109, 2012, pages 2790 - 2795
ZHANG ET AL., PNAS, vol. 109, no. 8, pages 2790
ZHANG, PNAS, vol. 109, no. 8, pages 2790

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