WO2017009644A1 - Kinase inhibitors for use in the treatment of fascioscapulohumeral dystrophy - Google Patents

Abstract

An inhibitor of a receptor tyrosine kinase, in particular selected from a compound of formula (I), (II) or (III) as defined in the specification for use in the treatment of fascioscapulohumeral dystrophy (FSHD)wherein formula (I) is exemplified by (N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl- H-pyrrole-3-carboxamide(sunitinib),formula (II) is exemplified by (N-(1,1-dimethylethyl)-3-[[5-methyl-2-[[4-(4-methyl-1-piperazinyl)phenyl]amino]-4- pyrimidinyl]amino]-benzenesulfonamide (TG101209) and formula (III) is exemplified 10 by N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine)(Vandetanib).

Description

KINASE INHIBITORS FOR USE IN THE TREATMENT OF FASCIOSCAPULOHUMERAL DYSTROPHY

The present invention relates to compounds for use in the treatment of fascioscapulohumeral dystrophy (FSHD), to dosage forms for use in these methods and to methods of treating the disease in patients.

Background of the Invention

Muscle dystrophies are a group of disorders characterised by skeletal muscle weakness and wasting. Fascioscapulohumeral dystrophy (FSFID) is a degenerative muscle disease that leads to progressive muscle weakness, initially affecting muscles in the face, shoulders and hips, usually from teens to adulthood. It is associated with a genomic mutation on chromosome 4q35 that changes the number of repeat D4Z4 units in a macrosatellite. There is no clear consensus on how this contraction causes disease. However, these D4Z4 units contain an open reading frame encoding a transcription factor called DUX4. This gene is not normally expressed in muscle cells. Specific reductions to the number of these D4Z4 repeat units that occur in FSHD patients result in expression of DUX4 on a haplotype supplying a poly-adenylation signal. Recently, FSHD2 has been show to be caused by mutations in DNA modifying enzymes that likely result in hypomethylation of D4Z4 units, even without a contraction. Thus it is thought that DUX4 expression underlies pathology in both FSHDl and FSHD2 (Tawil et al, Skelet Muscle 2014. 10;4: 12).

In muscle of humans and mammals, a population of muscle stem cells are found on muscle fibres and these are important and necessary for repair of muscle after injury. These so-called satellite cells (SCs) are activated in response to injury and will differentiate to form new muscle fibres or repair damaged fibres. It is thought that in muscle dystrophies, this population of SCs are deregulated, becoming unable to effectively maintain and repair muscle. Muscle progenitor cells extracted from FSHD patients show a compromised ability to form muscle. This is thought to reflect sporadic expression of the DUX4 gene. Thus, it is thought that the sporadic expression of DUX4 in muscle causes muscle weakness and wasting, and results in a reduced capacity of muscle repair by SC, leading to compromised muscle function. One difficulty in understanding how DUX4 induces a disease phenotype in muscle and muscle stem cells is that it seems to only be expressed in a few cells at low levels at any one time in FSHD patients. The molecular basis for DUX4-induced defects is unclear. Several reports suggest that a general de-regulation of a number of genes/signalling pathways occur as a consequence of DUX4 expression (Banerji et al 2015, J R Soc Interface. 2015 Jan 6; 12(102):20140797), but no clear mechanism has been identified that explains the pathology of muscle and of myoblasts derived from FSHD patients. DUX4 often inhibits muscle formation by SCs and several reports have shown pro-apoptotic functions, but low levels of DUX4 does not promote rapid, overt apoptosis in mouse or in man (Vanderplanck et al, PLoS One. 2011; 6(10):e26820). There is therefore no clear route for devising pharmacological interventions to overcome DUX4-induced pathologies as there is no single mammalian model that encompasses the genetic and pathophysiological spectrum of FSFID (Lek et al. 2015; Trends in molecular medicine 21, 295-306).

Current non-clinical trials to design a treatment for FSFID have included using anti-sense oligonucleotides to knock down DUX4 gene expression (WO2013120038; Vanderplanck et al, PLoS One. 2011; 6(10):e26820) and anti-oxidant treatments Bosnakovski et al, Skelet Muscle. 2014 Feb 1;4(1):4. doi: 10.1186/2044-5040-4-4; Passerieux et al, Free Radic Biol Med. 2014 Sep 19. pii: S0891-5849(14)00433-X) to try and promote a more normal/less pathogenic phenotype in muscle.

A problem with the antisense oligonucleotide approach is that DUX4 expression is considered to be sporadic in muscle. Any knockdown of DUX4 by this approach would only be transient and so not be able to compensate for prior DUX4- induced changes to muscle stem cells. Another limitation is delivery, as the

oligonucleotides would have to penetrate the affected muscle in order to reach the muscle fibres and muscle stem cells and this would likely require high doses to be applied systematically and often.

Strategies to compensate or avoid oxidative stress are the focus of a number of groups, but these are currently at early stages. There are no treatments for FSHD. Palliative measures to cope with FSHD are available, but these are not considered to have significant ameliorative effects on the progressive muscle weakness.

Summary of the Invention

The applicants have identified key DUX4 target genes that contribute to the pathogenic phenotype of FSHD and found that inhibitors of the receptor tyrosine kinases, in particular small molecule inhibitors, can be useful in the treatment of the FSHD phenotype. In particular, they may ameliorate FSHD-associated pathologies in muscle.

According to the present invention there is provided an inhibitor of a receptor tyrosine kinase for use in the treatment of fascioscapulohumeral dystrophy (FSHD).

Inhibitors of receptor tyrosine kinases have been found to rescue muscle differentiation in cells containing DUX4, in a dose dependent manner and therefore, may promote enhanced muscle function in FSHD patients.

Such inhibitors are suitably small molecule inhibitors and examples are known in the art. Some such inhibitors are approved for use in the treatment of proliferative diseases such as cancer. The inhibitors may act on one or more typical single-pass type I receptor tyrosine kinases including, but not limited to, RET, PDGFRa/b, VEGFRl-3 or FLT3 and related proteins. In this context, 'related proteins' may include proteins from similar receptor subfamilies. Thus the inhibitors may not specifically target structurally atypical receptor tyrosine kinases such as ALK-4, ALK-5, JAK1, JAK2, JAK3 and TYK2. Furthermore, the inhibitor may inhibit a number of related RTKs including c-KIT and CSF-IR, but in those cases, it suitably also inhibits other receptor tyrosine kinases, in particular at least one of RET, PDGFRa/b, VEGFRl-3 or FLT3.

In a particular embodiment, the receptor tyrosine kinase is RET, PDGFRa/b, VEGFRl-3 and FLT3. In particular the inhibitor is principally considered to be a RET inhibitor, where RET is the receptor tyrosine kinase encoded by the RET oncogene.

Particular examples of small molecule inhibitors include indolidinone derivatives such as pyrrole substituted indolinone derivatives as described for example in WO01/60814, pyridine derivatives such as bi-aryl meta pyridine derivatives for instance as described in WO2007/053452 or quinazoline derivatives such as aryl- amino quinazoline derivatives as described for example in WOO 1/32651. The content of the above-mentioned references are incorporated herein by reference.

Thus for example, the small molecule may be a pyrrole substituted indolinone of formula (I)

where R¹ is selected from the group consisting of hydrogen, halo, alkyl, cyclkoalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, -(CO)R¹⁵, - R¹³R¹⁴, -(CH₂)_rR¹⁶ and - C(0) R⁸R⁹;

R² is selected from the group consisting of hydrogen, halo, alkyl, trihalomethyl, hydroxy, alkoxy, cyano,-NR¾¹⁴,- R¹³C(0)R¹⁴,-C(0)R¹⁵, aryl, heteroaryl, and - S(0)₂ R¹ R¹⁴;

R³ is selected from the group consisting of hydrogen, halogen, alkyl, trihalomethyl, hydroxy, alkoxy, -(CO)R¹⁵, - R¹³R¹⁴, aryl, heteroaryl,-NR¹³S(0)₂R¹⁴ -S(0)₂ R¹ R¹⁴, - R¹³C(0)R¹⁴, - R¹³C(0)OR¹⁴ and-SO₂R²⁰(wherein R²⁰ is alkyl, aryl, aralkyl, heteroaryl and heteroaralkyl);

R⁴ is selected from the group consisting of hydrogen, halogen, alkyl, hydroxy, alkoxy and - R¹³R¹⁴;

R⁵ is selected from the group consisting of hydrogen, alkyl and -C(0)R^K);

R⁶ is selected from the group consisting of hydrogen, alkyl and -C(0)R¹⁰;

R⁷ is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, -C(0)R¹⁷ and -C(0)R¹⁰; or

R⁶ and R⁷ may combine to form a group selected from the group consisting of-(CH₂)4-, -(CH₂)₅- and -(CH₂)₆-; with the proviso that at least one of R⁵, R⁶ or R⁷ must be - C(0)R¹⁰;

R⁸ and R⁹ are independently selected from the group consisting of hydrogen, alkyl and aryl;

R¹⁰ is selected from the group consisting of hydroxy, alkoxy, aryloxy, -N(R^u)(CH₂)_nR¹² and- R¹³R¹⁴;

R¹¹ is selected from the group consisting of hydrogen and alkyl; R¹² is selected from the group consisting of-NR¹ R¹⁴, hydroxy, -C(0)R¹⁵, aryl, heteroaryl, -N⁺(0 )R¹³R¹⁴,- N(OH)R¹³, and -NHC(0)R^a (wherein R^a is unsubstituted alkyl, haloalkyl, or aralkyl); R and R are independently selected from the group consisting of hydrogen, alkyl, Ci-4alkyl substituted with hydroxyalkylamino, cyanoalkyl, cycloalkyl, aryl and heteroaryl; or

R¹³ and R¹⁴ may combine to form a heterocyclo group;

R¹⁵ is selected from the group consisting of hydrogen, hydroxy, alkoxy and aryloxy; R¹⁶ is selected from the group consisting of hydroxy, -C(0)R¹⁵, - R¹³R¹⁴ and - C(0) R¹³R¹⁴;

R¹⁷ is selected from the group consisting of alkyl, cycloalkyl, aryl and heteroaryl; R²⁰ is alkyl, aryl, aralkyl or heteroaryl; and n and r are independently 1, 2, 3, or 4; or a pharmaceutically acceptable salt thereof.

As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon groups including straight chain or branched chains , which unless otherwise stated, may contain from 1 to 20 carbon atoms, in particular from 1 to 10 carbon atoms.

"Cycloalkyl" refers to alkyl groups as defined above which comprise one or more rings which may be fused. "Aryl"refers to aromatic structures comprising carbon monocyclic or fused-ring polycyclic groups such as phenyl, naphthalenyl and anthracenyl. "Heteroaryl" refers to a aromatic monocyclic or fused rings which contain at least one heteroatom such as N, O, or S. "Heteroalicyclic" refers to a monocyclic or fused ring group having in the ring (s) of 5 to 9 ring atoms in which one or two ring atoms are heteroatoms selected from N, O, or S (O) n (where n is an integer from 0 to 2), the remaining ring atoms being C. The rings may also have one or more double bonds.

"Heterocycle or heterocyclo" refers to saturated cyclic groups of from 3 to 8 ring atoms which contain at least one heteroatom selected from N, O, or S (O) n (where n is an integer from 0 to 2). "Alkoxy" refers to both -O-alkyl or O-cycloalkyl groups and "aryloxy" refers to both an-0-aiyl and an-0-heteroaiyl group, as defined herein. "Aralkyl" groups are alkyl groups substituted with an aryl group such as benzyl; and "heteroaralkyl" groups are alkyl groups substituted with a heteroaryl group as defined above.

Particular examples of compounds of formula (I) and their preparation are described in WOOl/60814, the content of which is incorporated herein by reference.

For instance, the compound of formula (I) may be a compound in which:

R¹ is selected from hydrogen, Ci_-4alkyl, -(CH₂)_rR¹⁶ and -C(0) R⁸R^9; R² is selected from hydrogen, halogen, aryl and -S(0)₂ R¹³R¹⁴;

R³ is selected from hydrogen, (C i- 4)alkyl, (C i- 4)alkoxy, aryl, heteroaryl, and - C(0)R¹⁵;

R⁴ is hydrogen;

R ⁵ is selected from hydrogen and (C i-4)alkyl ;

R⁶ is -C(0)R¹⁰;

R⁷ is selected from hydrogen, (C i-4)alkyl and aryl;

R ⁸ and R⁹ are independently selected from hydrogen, alkyl and aryl;

R¹⁰is -N(R^u)(CH₂)nR ¹², wherein n is 1, 2 or 3, R¹¹ is hydrogen and R¹²is selected from hydroxy, (C₁-₄)alkoxy, -C(0)R¹⁵, heteroaryl and - R¹³R¹⁴;

R¹³ and R¹⁴ are independently selected from the group consisting of hydrogen. (C 1-C

4)alkyl, cycloalkyl, aryl and heteroaryl; or

R¹³ and R¹⁴ may combine to form a heterocyclo group;

R¹⁵ is selected from the group consisting of hydrogen, hydroxy, (C 1-C 4)alkoxy and aryloxy;

R¹⁶ is selected from hydroxy and -C(0)R¹⁵; and

r is 2 or 3;

and wherein:

alkoxy and aryloxy are as defined above;

heteroaryl refers to a monocyclic or fused ring group of 5 to 12 ring atoms containing one, two or three ring heteroatoms selected from N, O or S, the remaining ring atoms being C;

heterocyclo group refers to a saturated cyclic radical of 3 to 8 ring atoms in which one or two ring atoms are heteroatoms selected from N, O or S(0)_n where n is an integer from 0 to 2, the remaining ring atoms being C, where one or two C atoms are optionally replaced by a carbonyl group;

the alkyl, alkoxy and cycloalkyl groups are unsubstituted;

the aryl and heteroaryl groups are optionally substituted with one or two substituents independently selected from halo, (Ci-4)alkyl, trihalo(Ci-4)alkyl, hydroxy, mercapto, cyano, N-amido, mono- or di(Ci-4)alkylamino, carboxy and N-sulfonamido;

the heterocyclo group is optionally substituted with one or two substituents independently selected from halo, -(Ci-4)alkyl, -(Ci-4)alkyl-carboxy, -(Ci-4)alkyl-ester, hydroxyl and mono- or di(Ci-4)alkylamino. Particular examples of groups R and R are independently selected from hydrogen, (Ci₄)alkyl, heteroaryl and, combined, -(CH₂)₄-, -(CH₂)₅-, -(CH₂)₂-0-(CH₂)₂- and -(CH₂)₂N(CH₃)(CH₂)₂-, and in particular (Ci_-4)alkyl.

In another embodiment, n is 2 or 3 and R¹² is - R¹³R¹⁴ wherein R¹³ and R¹⁴ are independently (C i-4)alkyl, such as ethyl. Alternatively, n is 2 or 3 and R¹² is - R¹³R¹⁴ wherein R¹³ and R¹⁴combine to form a group selected from -(CH₂)4-, -(CH₂) 5-, - (CH₂)₂-0-(CH₂)₂- or - (CH₂)₂N(CH₃ )(CH₂)₂-.

In particular in the compounds of formula (I), R² is halo and in particular is fluoro. Suitably R¹, R³ and R⁴ are all hydrogen.

Suitably R⁵ and R⁷ are methyl and R⁶ is a group -C(0)R¹⁰.

In a particular embodiment, R¹⁰ is a group -N(R^u)(CH₂)_nR¹² where R¹¹ is hydrogen, n is 2 and R¹² is a group R¹³R¹⁴. In particular R¹³ and R¹⁴ are both alkyl groups and in particular ethyl groups.

A particular example of a compound of formula (I) is sunitinib (N-(2- diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-lH-indol-3-ylidene)methyl]-2,4-dimethyl- lH-pyrrole-3-carboxamide) or a pharmaceutically acceptable salt thereof including an L-malate salt.

In an alternative embodiment, the small molecule ret inhibitor is a bi-aryl metapyridine derivative, for example as described in WO2007/053452. Thus the inhibitor may be a compound of formula (II)

(Π)

wherein:

X^b is selected from a group consisting of a bond, O, C=0, S0₂, and CH₂; Y^b is selected from a group consisting of a bond or R^9b; or X^b and Y^b taken together is a bond; each of R^lb and R^2b is independently selected from a group consisting of H, C 1-6 substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; or R^lb and R^2b taken together is a bond; or R^lb and R^2b taken together form a moiety selected from a group consisting of (CH₂)_m', (CH₂)r<-S-(CH₂)m<, (CH₂)_r<-SO-(CH₂)_m<, (CH₂)_r<-S0₂-(CH₂)_m<, (CH₂)_r- R^b-(CH₂)_m<, and (CH₂)_r>-0-(CH₂)_m>;

each of p', q', r', n', m' is independently an integer having the value between O and 6, R^9b is selected from a group consisting of H, Ci-C₆ alkyl, Ci-C₆ cycloalkyl, Ci-C₆ branched alkyl, Ci-C₆ substituted alkyl, Ci-C₆ aminoalkyl, and Ci-C₆ hydroxyalkyl; Go is selected from a group consisting of N, O, H, and CH,

with the proviso that if Go is N, then:

each of R^3b and R^4b is independently selected from a group consisting of H, Ci-C₆ alkyl, Ci-C₆ substituted or unsubstituted hydroxyalkyl or aminoalkyl, Ci-C₆ substituted or unsubstituted branched alkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, or R^3b and R^4b taken together form a moiety selected from a group consisting of (CH₂)_m>, (CH₂)_r>-S-(CH₂)_m>, (CH₂)_r>-SO-(CH₂)_m>, (CH₂)_r-S0₂- (CH₂)_m<, (CH₂) - R^9b-(CH₂)_m<, and (CH₂)-0-(CH₂)_m<;

with the additional proviso that if Go is N, then: one of the following apply

R^lb and R^9b taken together form a moiety selected from a group consisting of ((CH₂)_m', (CH₂)_r<-S-(CH₂)_m<, (CH₂)_r<-SO-(CH₂)_m<, (CH₂)_r<-S0₂-(CH₂)_m<, (CH₂) - R^9b-(CH₂)_m<, and (CH₂) -0-(CH₂)_m'; or R^lb and R^4b taken together forms a moiety selected from a group consisting of (CH₂)_m>, (CH₂)_r>-S-(CH₂)_m>, (CH₂)_r -SO-(CH₂)_m (CH₂)_r>-S0₂- (CH₂)_m (CH₂) - R^9b-(CH₂)m', and (CH₂) -0-(CH₂)_m'; or R^9b and R^4b taken together form a moiety selected from a group consisting of (CH₂)_m', (CH₂)_r -S-(CH₂)_m', (CH₂)_r<-SO-(CH₂)_m<, (CH₂)r -S0₂-(CH₂)_m (CH₂) - R^9b-(CH₂)_m and (CH₂) -O-

(CH₂)m'; or R^3b and R^4b taken together form a moiety selected from a group consisting of (CH₂)_m<, (CH₂)_r<-S-(CH₂)_m<, (CH₂)_r<-SO-(CH₂)_m<, (CH₂)_r -S0₂-(CH₂)_m (CH₂) - R^9b-(CH₂)_m and (CH₂) -0-(CH₂)_m';

with the further proviso that if Go is O, then:

R^3b is selected from a group consisting of H, Ci-C6alkyl and Ci-C₆ substituted or unsubstituted hydroxyalkyl or aminoalkyl, substituted or unsubstituted branched alkyl, substituted or unsubstituted cycloalkyl, substituted heterocyclic connected through carbon or nitrogen, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl connected through carbon or nitrogen, with no group R ; R and R taken together form a moiety selected from a group consisting of (CH₂)_m', (CH₂)_r'-S-(CH₂)_m', (CH₂)r^<-SO-(CH₂)m^<, (CH₂)_r ^<-S0₂-(CH₂)_m ^<, (CH₂) - R^9b-(CH₂)_m and (CH₂) -O- (CH₂)m'; or R^lb and R^3b taken together form a moiety selected from a group consisting of (CH₂)_m<, (CH₂)_r<-S-(CH₂)_m<, (CH₂)_r<-SO-(CH₂)_m<, (CH₂)_r -S0₂-(CH₂)_m (CH₂) - R^9b-(CH₂)m<, and (CH₂) -0-(CH₂)_m'; or R^9b and R^3b taken together form a moiety selected from a group consisting of (CH₂)_m<, (CH₂)_r<-S-(CH₂)_m<, (CH₂)_r<-SO-(CH₂)_m<, (CH₂)r -S0₂-(CH₂)_m (CH₂) - R^9b-(CH₂)_m and (CH₂) -0-(CH₂)_m';

with the further proviso that if Go is CH, then each of R^3b and R^4b is independently selected from a group consisting of H, Ci-C₆ alkyl, Ci-C₆ substituted or unsubstituted hydroxyalkyl or aminoalkyl, Ci-C₆ substituted or unsubstituted branched alkyl, substituted or unsubstituted aryl, Ci-C₆ substituted or unsubstituted heterocycle connected through carbon or nitrogen, and substituted or unsubstituted heteroaryl connected through carbon or nitrogen, or R^3b and R^4b taken together form a moiety selected from a group consisting of (CHR^9b)_r ^< -(CHR^9b)_m ^<-(CHR^9b)_p ^<, (CHR^9b)_r ^<-S- (CHR⁹V, (CHR⁹VSO-(CHR⁹V, (CHR^9b)_r<-S0₂ (CHR^9b)_m<, (CHR^9b)_r' - R^9b- (CHR^9b)_m and (CHR^9b)_r -0-(CHR^9b)_m';

G is N or CR^6b, and each G is independent of each other G, with the further proviso that not more than two groups G can be N, with the further proviso that for each CR^6b, each R^6b is independent of each other group R^6b;

wherein each of R^6b, R^7b, R^8b is independently selected from a group consisting of H, Ci-C₆ substituted or unsubstituted alkyl, Ci-C₆ substituted or unsubstituted alkenyl, Ci- C₆ substituted or unsubstituted alkynyl, Ci-C₆ substituted or unsubstituted

hydroxyalkyl or aminoalkyl, Ci-C₆ substituted or unsubstituted branched alkyl, Ci-C₆ substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl connected through carbon or a heteroatom, substituted or unsubstituted heteroaryl connected through carbon or a heteroatom, Ci-C₆ alkoxy, a halogen, CF₃, -OCF₃, CHR^3bR^4b, SR³, SOR^3b, S0₂R^3b, S0₂ R^3bR^4b, S0₃R^3b, POR^3b, P0₂R^3b, P0₂ R^3bR^4b, P0₂CR^3bR^4b, P0₃CR^3bR^4b, P0₃R^3b, R^3bR^4b, N0₂, CN, OH, C0 R^3bR^4b, COR^3b, COOR^3b,

R^3bCOR^4b, R^3bCO R^3bR^4b, OCO R^3bR^4b, CS R^3bR^4b, CSR^3b, R^3bCS R^3bR^4b, SCO R^3bR^4b, SCS R^3bR^4b, and SCS R^3bR^4b; or any of R^6b and R^7b taken together, or R^7b and R^8b taken together, or R^6b and R^8b taken together form a moiety independently selected from a group consisting

of -HN-CH=CH-, -HN-N=CH-, -HN-N=N-, -0(CH₂)_nO-, -S(CH₂)_nS-,

-N=CH-S, -CH=N-0, -CH=N-S-, -N=CH-0-, -C=N-0-, -C=N-0-,

-CH=CH-CH=CH- -N=CH-CH=CH-, -CH=N-CH=CH-, -0-CH=CH, and

-S-CH=CH-; or R^3b and R^4b taken together form a moiety selected from a group consisting of (CHR⁹V(CHR^9b)_m<-(CHR^9b)_p<, (CHR^9b)_r<-S-(CHR^9b)_m<, (CHR^9b)_r -SO- (CHR⁹V, (CHR^9b)r -S0₂ (CHR^9b)_m (CHR^9b)_r -NR^9b-(CHR^9b)_m and (CHR^9b)_r<-0-

A is selected from a group consisting of O, R^3b, CR^3bR^4b, S, SO, and S0₂;

Gi is selected from a group consisting of CH, N, H, S, and O;

G₂ is selected from a group consisting of CR^7b, N, NH, S, and O, with each group R^7b being independent of every other group R^7b ;

with the further proviso that X or Go includes at least one heteroatom included with X and selected from O, S and N, or Go comprises at least four non-hydrogen atoms, inclusive of the heteroatom, and R^3b and R^4b, or R^lb and R^9b, or R^lb and R^4b, or R^9b and R^4b taken together form an aromatic, heteroaromatic, cyclic or heterocyclic ring system, or if a noncyclic system is present, then more than one heteroatom is present, and if A is NR^3b, then any of R^6b, R^7b or R^8b, or any combination thereof independently includes at least two non-hydrogen substituents, or if A is NR^3b, then Q forms a fused ring from R^6b to R^7b, or from R^7bto R^8b,

or pharmaceutically acceptable salts, hydrates, solvates, crystal forms, N-oxides, and individual diastereomers of the compound (A).

Particular examples of compounds of formula (II) and their preparation are described in WO2007/053452, the content of which is incorporated herein by reference. In particular, any substituents listed above are those described in this reference.

Suitable examples of compounds of formula (II) are compounds of formula II wherein:

X^b is selected from a group consisting of a bond, O, S0₂, and CH₂; Y^b is selected from a group consisting of a bond or NR^9b; or X^b and Y^b taken together is a bond;

each of R^lb and R^2b is independently selected from a group consisting of H, Ci-6 alkyl, cycloalkyl; or R^lb and R^2b taken together is a bond; or R^lb and R^2b taken together form a moiety selected from a group consisting of (CH₂)_m', (CH₂)_r'-S-(CH₂)_m', (CH₂)_r -SO- (CH₂)_m% (CH₂)r^<-S02-(CH₂)m^<, (CH₂)_r -NR⁹-(CH₂)_m and (CH₂)_r -0-(CH₂)_m';

each of p', q', r', n', m' is independently an integer having the value between 0 and 6, R^9b is selected from a group consisting of H, Ci-6alkyl, Ci-6cycloalkyl, Ci-6 branched alkyl, Ci-6 aminoalkyl, and Ci-6 hydroxyalkyl;

Go is selected from a group consisting of N, O, H, and CH,

G is CH or C when bonded to X;

R^5b is methyl;

each of R^6b and R^7b is independently selected from a group consisting of Ci-6 alkenyl, Ci-6 alkynyl, Ci-6 hydroxyalkyl or aminoalkyl, Ci-6 cycloalkyl, Ci-6 alkoxy, a halogen, CF₃, OCF₃, S0₂H, S0₂(Ci-6 alkyl), S0₂-heterocycle, S0₂-cycloalkyl, S0₂N(Ci_-6 alkyl)H, S0₂N(Ci_-6 alkyl)(Ci-₆alkyl), S0₂ H(Ci-₆cycloalkyl), S0₂ H-heterocycle, (S0₂N(Ci-6 branched alkyl)H, N0₂, CN, OH, CO H₂, CO-(Ci_-6 alkyl), COOH, COO- (Ci-6 alkyl), and HCO-(Ci_-6 alkyl), and

R^8b is independently selected from the group consisting of H, Ci-6 alkenyl, Ci-6 alkynyl, Ci-6 hydroxyalkyl or aminoalkyl, Ci-6 cycloalkyl, halogen, CF₃, OCF₃, S0₂H ,S0₂(ci-6 alkyl), S0₂-heterocycle, S0₂-cycloalkyl, S0₂N(Ci_-6 alkyl)H, S0₂N(Ci_-6 alkyl)(Ci-₆ alkyl), S0₂ H(Ci_-6 cycloalkyl), S0₂ H-heterocycle, (S0₂N(Ci_-6 branched alkyl)HN0₂, CN, OH, CONH₂, CO-(Ci_-6 alkyl), COOH, COO-(Ci_-6 alkyl), and NHCO-(Ci-6 alkyl);

A is selected from a group consisting of NH, and N-(Ci-6 alkyl);

Gl is CH;

G2 is CR^7b, with each group R^7b independent of every other group R^7b;

and R^3band R^4b, taken together form a heterocyclic ring system,

or a pharmaceutically acceptable salt thereof.

In particular in the compound of formula (II), all G groups are CH groups.

Suitably X^b and Y^b are a bond. Suitably also, p', q' and n' are 0. In a particular embodiment, Go is N, and R^3b and R^4b together form a group selected from (CH₂)_m', (CH₂)_r-S-(CH₂)_m<, (CH₂)_r-SO-(CH₂)_m<, (CH₂)_r-S0₂-(CH₂)_m<, (CH₂) -NR^9b-(CH₂)_m<, and (CH₂) -0-(CH₂)_m'; and in particular (CH₂)_r-NR^9b-(CH₂)_m\ In this embodiment, r' and m' are both 2 and R^9b is suitably an alkyl group such as methyl.

Suitably in the compounds of formula (II), A is a group NR^3b.

In particular, R^6b, R^7b and R^8b are all hydrogen. Suitably Gi is CH. Suitably G₂ is a group CR^7b and in this case, R^7b is a group of formula S0₂NR^3bR^4b. Suitably in this case R and R are independently selected from H or Ci-6alkyl. In particular, one of R^3b or R^4b is H and the other is tert-butyl.

In a particular embodiment, the compound of formula (II) is TG101209 (N-(l, l-dimethylethyl)-3-[[5-methyl-2-[[4-(4-methyl- l-piperazinyl)phenyl]amino]-4- pyrimidinyl]amino]-benzenesulfonamide),( Ramakrishnan et al. Am J Hematol. 2010 Sep;85(9):675-86. doi: 10.1002/ajh.21785).

Alternatively, the small molecule may comprise a quinazoline derivative and in particular a compound of formula III)

(III)

wherein : m is an integer from 1 to 3 ;

R^la represents halo or Ci-3alkyl;

R^2a is selected from one of the following three groups :

1) Ci-5alkylR^3a (wherein R^3a is piperidin-4-yl which may bear one or two substituents selected from hydroxy, halogeno, Ci-4alkyl, Ci-4hydroxyalkyl and Ci-4alkoxy ;

2) C2-₅alkenylR^3a( wherein R^3a is as defined above) ;

3) C2-₅alkynylR^3a (wherein R^3a is as defined herein) ; and wherein any alkyl, alkenyl or alkynyl group may bear one or more substituents selected from hydroxy, halogeno and amino ; or a salt thereof.

In a particular embodiment, m is 2 and each R^la group is a halo group.

In a further particular embodiment, R^2a is a group (1) above, and in particular, is [(1- methyl)piperidin-4-yl]methyl.

A particular example of a compound of formula (III) is vandetanib (N-(4- bromo-2-fluorophenyl)-6-methoxy-7-[(l-methylpiperidin-4-yl)methoxy]quinazolin-4- amine), which may be available under the trade names Caprelsa or ZACTIMA

(AstraZeneca).

In a particular embodiment, the small molecule inhibitor is one which will produce a dose related inhibitory response such as Sunitinib or TGI 01209 but in particular is Sunitinib. In a further aspect, the invention provides a method for treating

fascioscapulohumeral dystrophy (FSHD), said method comprising administering to a patient in need thereof, an effective amount of an inhibitor of a receptor tyrosine kinase.

Suitably, the receptor tyrosine kinase is selected from the group consisting of

RET, PDGFRa/b, VEGFR1-3 and FLT3 and related proteins, and in particular is RET. Inhibitors are suitably small molecule inhibitors such as those listed above.

In order to treat patients in the method of the invention, the inhibitor is suitably administered in the form of a pharmaceutical composition. Such compositions form a further aspect of the invention.

Suitable pharmaceutical compositions will be in either solid or liquid form. They may be adapted for administration by any convenient route, such as parenteral, oral or topical administration or for administration by inhalation or insufflation. The pharmaceutical acceptable carrier may include diluents or excipients which are physiologically tolerable and compatible with the active ingredient.

Parenteral compositions are prepared for injection, for example either subcutaneously or intravenously. They may be liquid solutions or suspensions, or they may be in the form of a solid that is suitable for solution in, or suspension in, liquid prior to injection. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like.

Oral formulations will be in the form of solids or liquids, and may be solutions, syrups, suspensions, tablets, pills, capsules, sustained-release formulations, or powders. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

Topical formulations will generally take the form of suppositories or intranasal aerosols. For suppositories, traditional binders and excipients may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient.

The amount of inhibitor administered will vary depending upon factors such as the precise nature of the inhibitor, the size and health of the patient, the nature of the condition being treated etc. in accordance with normal clinical practice. Typically, a dosage for an adult would be in the range of 5-100mg daily, for example a daily dosage of from lC^g-10mg/Kg such as from 5C^g-2mg/Kg would be expected to produce a suitable effect.

One or more such receptor tyrosine kinase inhibitors may be combined in a dosing regime for treatment of FSHD.

A screen to identify targets of DUX4 in mouse satellite cells ( SCs) was carried out as described below and showed that some tyrosine kinase receptors are highly upregulated by DUX4, including the RET receptor. Ret was found to be detectable in proliferating mouse SCs, but then is down-regulated during differentiation. To then identify routes for overcoming DUX4-induced phenotypes in satellite cells (SCs), the applicants used the inhibition of muscle differentiation as a readout of DUX4 function and investigated whether this could be rescued by genetic inhibition of the Ret gene. They could show that knockdown of Ret in SCs expressing DUX4 lead to a recovery of the cells ability to differentiate. Subsequently, they showed that over-activation of the RET gene using a constitutively active version of RET could inhibit muscle differentiation in an opposite manner.

The applicants then assessed the ability of small molecule inhibitors Sunitinib (Pfizer)(N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-lH-indol-3-ylidene)methyl]- 2,4-dimethyl-lH-pyrrole-3-carboxamide), Vandetanib (AstraZeneca)( N-(4-bromo-2- fluorophenyl)-6-methoxy-7-[(l-methylpiperidin-4-yl)methoxy]quinazolin-4-amine) and TG101209 (Ramakrishnan et al, supra)(N-(l,l-dimethylethyl)-3-[[5-methyl-2-[[4- (4-methyl-l-piperazinyl)phenyl]amino]-4-pyrimidinyl]amino]-benzenesulfonamide) in a screen to overcome the inhibitory effects on muscle differentiation of a constitutively active version of RET in mouse muscle progenitor cells. It was found that these inhibitors were able to improve/rescue muscle differentiation in these cells. Certain inhibitors, in particular, Sunitinib, could rescue muscle differentiation in these cells in a dose dependent manner. In SCs that expressed DUX4, the applicants then showed that application of Sunitinib was able to rescue muscle differentiation at specific doses in a significant manner, but did not affect muscle differentiation of cells that did not express DUX4.

Subsequently, these findings were replicated using a previously published immortalised human muscle progenitor cell line extracted from a mosaic FSHD patient. Two cell lines were used: one with a disease genotype (possessing the D4Z4 contraction) and one without (Krom et al, Am J Pathol. 2012 Oct; 181(4): 1387-401. doi: 10.1016/j.ajpath.2012.07.007).

Three measurements were taken and used to compare the two cells lines (proliferation, cell shape, muscle differentiation). The statistical difference of these 3 measurements between these two cell lines was then assessed when they were exposed to different doses of Sunitinib. This revealed an optimal dose that led to statistically insignificant differences between the two cell lines, indicative of a 'normalisation' of the disease-state cell line. Sunitinib rescued proliferation and MyoD expression in DUX4-expressing human cell lines but did not rescue proliferation in DUX4 expressing murine cells.

The results obtained from the human cells with Sunitinib is believed to result from the inhibition of receptor tyrosine kinase (s), which may include, but not limited to RET, PDGFRa/b, VEGFR1 -3 , and FLT3. A typical target of RET and other RTKs is the intracellular kinase ERK1 and ERK2. In murine iDUX4-C2C12 cells over- expressing DUX4, ERK activity is increased (Figure 7). The applicants have found that application of certain receptor tyrosine kinase inhibitors (specifically Sunitinib) does not affect ERK activity in cells that do not express DUX4, but does reduce ERK activity in DUX4-expressing cells after 20 hours of treatment (Figure 7 hereinafter ). As illustrated hereinafter, treatment of murine myoblasts expressing DUX4 with

Sunitinb resulted in significantly diminished phospho-ERK relative to total ERK, but there was no apparent change to phospho-Akt relative to total Akt. In immortalised human myoblasts derived from a FSHD patient and carrying the FSHD-associated truncated D4Z4 repeat sequence, Sunitinib treatment resulted in reduced Akt activity, although there was no statistically significant change to ERK activity

Furthermore, work carried out in vivo in mice supports the invention in that Sunitinib improved the myogenic capacity of engrafted FSHD myoblasts (paper in press).

These results indicate that such inhibitors are a novel and useful intervention for promoting enhanced muscle function in FSHD patients.

Detailed Description of the Drawings

Figure 1 : A) Mechanism by which Ret signals B) QPCR of C2C12 myoblasts containing a DOX-controllable DUX4 cassette were induced with Doxycycline, or un-induced (controls). Messenger RNA was extracted after 0, 6, 12, 24 and 48 hours and compared to un-induced controls at each time-point. The induction of DUX4 resulted in an increased transcription of Ret from 12 hours. C) QPCR of SC-derived myoblasts infected with DUX4 or DUX4C encoding retrovirus for 24 and 48 hours. Only DUX4 samples significantly increased Ret after 48 hours (p<0.05).

Figure 2: Knockdown of Ret rescues DUX4-mediated inhibition of myogenic differentiation. (A) Immunolabelling of satellite cell-derived myoblasts infected with control or DUX4 retrovirus (eGFP+) and transfected with Control or Ret siRNA following culture for 24 hours in mitogen poor differentiation media. (A) Infected cells were detected by eGFP and examined for MyHC labelling to identify terminally differentiated myoblasts. (B) The fusion index (the number of nuclei detected in cells with MyHC expression/ total nuclei) was calculated for cells expressing control or DUX4 retrovirus and transfected with control or Ret siRNA. Expression of DUX4 reduced the fusion index. However, blocking Ret expression in DUX4-expressing myoblasts increased the fusion index enough such that it was not significantly different to the control fusion index. (C) Plot of probability that a cell has MyHC

immunoreactivity derived from Binomial models described in Table 1. Error bars represent 95% confidence intervals.

Four replicates were counted for each condition and repeated using 3 mice.

Figure 3 : Small molecules inhibitors block the Ret-induced phenotype in murine myoblasts. (A) Immunolabelling with antibodies to eGFP and MyHC of C2C12 myoblasts infected with control or CA RET51 expressing retroviruses and induced to differentiate for 60 hours. Cells were treated with either 1 μg/ml Sunitinib or DMSO. Lower panels show anti-GFP labelling only. (B-D) Fusion index of C2C12 myoblasts expressing control (red) or CA RET51 expressing retrovirus (blue) and treated with Sunitinib, Zactima and TG101209 at varying doses is shown as the -log of odds. The - log of odds (-log(ratio (1-ratio)) is calculated from the fusion index (ratio of MyHC+ nuclei/ total nuclei). (E-G) Quantification of the number of C2C12 cells expressing control (red) or CA RET51 expressing retrovirus (blue) when treated with Sunitinib, Zactima and TG101209 at varying doses. (H) Immunolabelling of C2C12 cells to detect MyHC following treatment with varying doses of Sunitinib. All quantification represents an average of three independent experiments. Statistical significance assessed using a mixed Binomial model (B-D) or a quasi-Poisson model (E-G). Figure 4: Sunitinib treatment enables differentiation of DUX4-expressing myoblasts. (A-D) Quantification of SC-derived myoblasts infected with control or DUX4 retrovirus and treated with either 250ng/ml Sunitinib or DMSO vehicle. Cells were cultured at low density and incubated in (A, B) proliferation or (C, D) differentiation medium for 24 hours prior to immunolabelling to detect eGFP and a specific label: EdU (A), MyoD (B), Myogenin (C) and MyHC (D). All values are represented as the ratio of the labelled GFP+ cells to all GFP+ cells and are derived from 3 independent replicate platings from 3 mice. E) Control or DUX4-expressing myoblasts treated with varying concentrations Sunitinib or DMSO vehicle control and incubated in differentiation medium at high density for 24 hours before immunolabelling with antibodies against eGFP and MyHC.

(F) Quantification of fusion of SC-derived myoblasts uninfected (red) or infected with DUX4-expressing retrovirus (blue) and cultured in differentiation medium in the presence of varying concentrations of Sunitinib. Values for the fusion index are presented as the -log of odds and represent 3 replicates from 3 mice. (G) Fusion index of SCs expressing DUX4 or control retrovirus and exposed to DMSO (red) or 250ng/ ml Sunitinib (blue). Values for the fusion index are presented as the -log of odds and represent 3 replicates from 3 mice.

Figure 5: RET is co-expressed with PAX7 in human myoblasts extracted from muscle. (A) Relative expression of RET from QPCR using TATA Binding Protein (TBP) to normalise during proliferation (prolif) and differentiation at days 1, 2, 3 and 4 (DM1- 4). (B) PAX7 expression from the same cells.

Figure 6: Sunitinib improves the phenotype of FSHD cells. (A) Immunolabelling of mosaic FSHD patient-derived myoblast cell lines 54.6 (control) and 54.12 (containing the FSHD D4Z4 contraction) exposed to DMSO or 500ng/ ml Sunitinib. Tubulin and EdU were labelled in cells after EdU incorporation (A); MyHC was detected in cells grown at high density in differentiation medium (B). EdU incorporation was measured in cells exposed to different doses of Sunitinib and represented as a proportion of total labelled cells (C). Cell shape (eccentricity) was assessed and plotted relative to

Sunitinib concentration (B). The -log of odds of fusion was plotted relative to

Sunitinib concentration. All quantification employed 3 independent replicates.

Figure 7: shows the results of an experiment in which murine iC2C12 myoblasts were untreated, or treated with SOOng/μΙ doxycycline (Dox) to induce DUX4 and/or

250ng^l Sunitinib for 20 hours, and proteins then extracted; where (A) shows

representative images of a membrane probed with antibodies against DUX4, total

AKT, phosphorylated (p) AKT, total ERK1/2 and phosphorylated (p) ERK1/2, with

Caveolin-1 used as a loading control and (B and C) shows the quantification of the ratio between pERKl/2 : total ERK1/2 and pAKT : total AKT compared to control.

Data is represented as mean ± SEM from 3 independent experiments, where an asterisk denotes a significant difference (p<0.05) from Control, using a two-tailed unpaired

Student's t Test.

Figure 8: shows the results of an experiment in which a human immortalised myoblast clone (54.12) derived from a patient mosaic for the D4Z4 contraction that is associated with FSFID were untreated (Ctrl), or treated with either 250ng^l or 2.5 μg/μl Sunitinib for 20 hours, and proteins extracted; where (A) shows a membrane probed with antibodies against total (t) AKT, phosphorylated (p) AKT, tERKl/2 and pERKl/2, with Vinculin used as a loading control; and (B and C) are graphs showing quantification of the ratio between pERKl/2 : tERKl/2 and pAKT : tAKT in the Sunitinib -treated groups compared to control. Data is represented as mean ± SEM from 3 biological replicates, where an asterisk denotes a significant difference (p<0.05) from Control, using a two-tailed unpaired Student's t Test.

Example 1

Identification and Confirmation of Ret as Target for FSHD Therapy

To understand the molecular mechanisms of FSFID, a microarray of murine satellite cell (SC)-derived myoblasts expressing DUX4, modified DUX4 versions, or its ortholog DUX4c was prepared as described in (Banerji,J R Soc Interface. 2015 Jan 6; 12(102):20140797). A large range of possible DUX4 targets were indicated using a selection matrix. One gene that showed a significant change in expression from the SC microarray was the receptor tyrosine kinase Ret. This was up-regulated by DUX4. Ret phosphorylation activates signalling pathways such as MAP-kinase and PI3K-Akt as illustrated in Figure 1 A. Ret signalling is crucial for neural and kidney differentiation, and RET mutations are associated with Hirschsprung's disease, while gain-of-function mutations are associated with certain cancers (including medullary thyroid carcinoma, multiple endocrine neoplasias type 2A and 2B, phaeochromocytoma and parathyroid hyperplasia). Absence of RET ligands can trigger apoptosis. Ret has 2 main isoforms, Ret9 and Ret51, differing in the C-terminal region.

To identify genes affected by DUX4 expression, gene expression in SCs infected by DUX4, DUX4c (a homologue of DUX4 containing the same homeodomain DNA binding sites) and truncated DUX4 constructs was compared.

C57BL10 male mice aged between 8 and 10 weeks were used for myofibre and satellite cell-derived myoblast preparations. Muscle myofibres were isolated from the extensor digitorum longus (EDL) as previously described (Moyle et al. Methods Mol Biol, (2014). 1210: p. 63-78). Briefly, dissected EDLs were digested in DMEM

(Glutamax) (Life Technologies) containing 0.2% collagenase (Sigma Aldrich) for 2 hours in a 37°C 5% C0₂ incubator before manual disruption with a heat-polished glass pipette in 5% bovine serum albumin (BSA) (Sigma Aldrich) coated dishes. Individual washed myofibres with associated quiescent satellite cells were subsequently fixed in 4% paraformaldehyde/PBS (PFA) or cultured in non-adherent or adherent conditions. Non-adherent cultures were grown in DMEM (Glutamax) with 10% horse serum (HS) (Gibco), 0.5% chicken embryo extract (CEE) (ICN Flow), 1% penicllin/streptomycin (Sigma Aldrich) for up to 72 hours. For adherent cultures of proliferating satellite cells, myofibres were plated on lmg/ml Matrigel (Collaborative research) coated dishes in DMEM (Glutamax) with 20% foetal calf serum (FBS) Gold (PAA), 10% HS, 1%) CEE, 1/10,000 basic FGF (bFGF) for 72 hours. Subsequently, myofibres were removed by pipette agitation and satellite cells re-plated by trypsinisation in 0.25% Trypsin-EDTA and myoblasts re-plated to expand for experimentation.

Total RNA was extracted using RNeasy Kit (Qiagen). Between 500ng - ^g of RNA was used to prepare cDNA using the QuantiTect Reverse Transcription Kit with genomic DNA wipeout (Qiagen). Quantitative reverse transcription PCR (qRT-PCR) was performed on an Mx3005PQPCR system (Stratagene) with MESA Blue qPCR MasterMix Plus and ROX reference dye (Eurogentec). Primers used to measure Ret expression (both Ret9 and Ret51) were as follows:

F: 5 ' - AAGC AGGAGCC AGAC AAGAG-3 ' (SEQ ID NO 1)

R: 5'-ACACCTTCGGACTCACTGCT-3'), (SEQ ID NO 2)

Expression of Ret was increased over 3 fold in DUX4 expressing SCs, relative to controls, while expression of RET co-receptors GFRa2, GFRa3 and GFRa4 were unaltered, although GFRal expression was reduced 0.5-fold. Expression of DUX4c did not alter Ret or GFRa co-receptor expression, suggesting that Ret may not be a direct DUX4 homeodomain-binding target.

The applicants next examined whether DUX4 or DUX4c can induce Ret in inducible iC2C12 myoblasts, which contain a doxycycline-regulated DUX4 cassette. DUX4 or DUX4c were induced with 200ng doxycycline and cultured for 6, 12, 24 and 48 hours and analysed by qRT-PCR. Expression of Ret was significantly increased after 12 hours in the iC2C12-DUX4 line, compared to un-induced myoblasts, and maintained at 24 and 48 hours. Induction of DUX4c did not significantly alter Ret expression at any time-point. To confirm that Ret is a DUX4 target gene in SCs, we infected SCs with DUX4 and DUX4c. Expression of DUX4 increased Ret transcription after 48 hours relative to control cells. We then confirmed that DUX4 activation of Ret leads to the production of membrane-located RET protein by immunostaining with an antibody to RET51 in DUX4-infected cells.

Example 2

A: Ret and Ret co-receptors are expressed in satellite cells

To determine whether Ret is expressed in murine satellite cells, myofibres with their associated satellite cells were isolated and either immediately fixed, or cultured. For immunostaining, floating myofibres or plated satellite cells were fixed in 4% paraformaldehyde/PBS, permeabilised with 0.5% Triton/PBS (Sigma Aldrich) and blocked in PBS containing 5% swine serum + 5% goat serum (DAKO) for 1 hour (except for samples using goat Ret51 antibody, which were blocked in 10% swine serum). Samples were incubated in primary antibody overnight at 4°C, washed 3 times in 0.025%) Tween/PBS and visualised by incubating with AlexaFluor conjugated secondary antibodies (Life Technologies) at 1/500 dilution for 1 hour at room temperature. Nuclei were visualised by mounting in aqueous mountant containing DAPI (Vectashield). (Moyle et al. 2014 supra.). Primary antibodies used were: Ret (rabbit polyclonal, C-20), Ret51 (goat polyclonal, C-19) and pRet Tyrl062 (rabbit polyclonal) (all Santa Cruz Biotechnology), Pax7 (mouse monoclonal), Myogenin (mouse monoclonal) and MyHC (mouse monoclonal, MF-20) all DSFIB, eGFP (rabbit and chicken polyclonals) Life Technologies, phospho-Histone HI and phospho- Histone H3 (rabbit polyclonals, Upstate).

Co-immunostaining fixed myofibres at TO and T24 for Pax7 and Ret51 revealed that Ret51 was barely detectable in murine quiescent satellite cells, but expression increased after 24 hours of activation. This correlates with the expression profile of phospho-Ret Tyl062, which identifies active Ret signalling. After 72 hours of culture, a proportion of satellite cell-derived myoblasts (SCs) stop expressing Pax7 and commit to differentiation, expressing Myogenin. Ret51 was found in both Myogenin-negative uncommitted myoblasts and Myogenin-positive differentiating myoblasts.

To analyse the expression of Ret and its GFRa co-receptors during myogenic progression, SCs were isolated from 3, 8-week old C57BL/10 mice and mRNA harvested from multiple cultures in proliferation (P) or after 6, 12, 24 and 48 hours in differentiation medium (DM). Ret, GFRal, GFRal and GFRa4, but not GFRa3, were robustly expressed in SC cultures. Expression of Ret decreased in differentiating myoblasts, whereas expression of GFRal, GFRa2 and GFRa4 increased upon differentiation, indicating endogenous regulation and a potential role in satellite cell function.

B. Ret is required for SC proliferation

To test whether satellite cells require Ret for normal function, the applicants used siRNA-mediated knock-down.

Satellite cells were transfected with 20nM of a scrambled-sequence control siRNA or Ret Silencer® Select siRNA (Life Technologies) (directed at both Ret isoforms) in the presence of Lipofectamine RNAiMAX (Life Technologies) for 6 hours at 37°C, 5% CO2 in proliferation medium. The siRNA sequence 5'- GCUUGUACAUCGGGACUUATT-3' (ID: s72895) (SEQ ID NO 3) was used to knockdown murine Ret expression, and control siRNA was supplied by Life

Technologies. Gene knockdown was confirmed 48 hours after transfection. Knockdown was assessed by qRT-PCR. Cells were cultured in 6-well plates for at least 48 hours under experimental conditions and total RNA extracted and QPCR performed as described in Example 1. Primers used were as follows:

All mouse Ret isoforms:

F: 5 ' - AAGC AGGAGCC AGAC AAGAG-3 ' (SEQ ID NO 1)

R: 5'-ACACCTTCGGACTCACTGCT-3') (SEQ ID NO 2)

Mouse Ret9 isoform:

F: 5 ' -GATCC AGAGGCC AGAC AAC -3' (SEQ ID NO 3)

R: 5'- GTAGAATCTAGTAAATGCA-3 ') (SEQ ID NO 4)

MouseRet57 isoform:

F: 5' -GATCC AGAGGCC AGAC AAC-3' (SEQ ID NO 3)

R: 5'-AGGACTCTCTCCAGGCCAG-3' (SEQ ID NO 5)

Mouse GFRal:

F: 5 ' -GC AC AGCT ACGGGATGCTC-3 ' (SEQ ID NO 6)

R: 5'- CTCTGGCTGGCAGTTGGT -3' (SEQ ID NO 7)

Mouse GFRa2:

F: 5'-ACCGTGTGCCCAGCGAGTATA-3' (SEQ ID NO 8)

R: 5 ' -CGAC AGTTGGCGTGGAAGT-3 ' (SEQ ID NO 9)

Mouse GFRa4:

F: 5'-ACCCCTGCTTGGATGGTGCC-3' (SEQ ID NO 10)

R: 5'-CAGCCAGGACACCTTGGGCG-3' (SEQ ID NO 11)

Mouse Gapdh:

F: 5 ' -GTGAAGGTCGGTGTGAACG-3 ' (SEQ ID NO 12)

R: 5 ' - ATTTGATGTT AGTGGGGTCTCG-3 ' (SEQ ID NO 13)

Mouse Tbp:

F: 5 ' - ATCCC AAGCGATTTGCTG-3 ' (SEQ ID NO 14)

R: 5'-CCTGTGCACACCATTTTTCC-3' (SEQ ID NO 15)

Human RET:

F: 5'-GCTCCACTTCAACGTGTC-3' (SEQ ID NO 16)

R: 5'-GCAGCTTGTACTGGACGTT-3'(SEQ ID NO 17)

Primers for murine Pax7, MyoD and My/5 have previously been published (Collins, C.A., et al., PLoS One, 2009. 4(2): p. e4475 10.1371/journal.pone.0004475). Samples were normalised to the housekeeping genes Gapdh and Tbp (as designated per experiment) and statistical significance was measured using the paired student's T-test.

A mean knock-down efficiency of -80% was achieved. To measure how knockdown of Ret affected cell proliferation, SCs were pulsed with EDU for 2 hours with ΙΟμΜ of 5-Ethynyl-2'-deoxyuridine (EdU) before fixation and processing using with the Click-iT® EdU Alexa Fluor® Imaging Kit (Life Technologies) and immunostaining for Pax7. Knock-down of Ret significantly reduced the proportion of SCs that had incorporated EdU.

SiRNA-mediated knock-down of Ret was also associated with a decrease in the proportion of SCs expressing Pax7, from a mean+SEM of 90.7±2.2% in control myoblasts to 84.0±2.0% with Ret siRNA, which was associated with lower Pax7 mRNA levels. The reduced number of proliferating Pax7+ SCs and lower expression of Pax7 when Ret is knocked down suggests that Ret contributes to maintaining SCs in an undifferentiated state. Ret knockdown did not alter My/5 orMyoD expression.

C: Knock-down of Ret enhances myogenic differentiation of SCs

For differentiation, satellite cell-derived myoblasts were cultured in mitogen- poor medium, containing DMEM + 2% HS + penicillin/streptomycin.

SCs treated with Ret siRNA had reduced Pax7 levels and proliferation rate, implying that the cells may be differentiating prematurely. To confirm this, cultures of SCs treated with control or Ret siRNA as described in Example 2B above were plated at high density and incubated in differentiation medium for 48 hours, before immunostaining with antibodies to Myogenin and Myosin Heavy Chain (MyHC).

The applicants found that the proportion of Myogenin-positive nuclei was significantly increased in Ret siRNA-treated cultures (85.4±4.0%) relative to control siRNA-treated cells (69.1±4.0% to 85.4±4.0 with Ret siRNA). In contrast, the proportion of nuclei in MyHC-expressing multinucleate myotubes (the fusion index) was unaltered (60.0±3.0% in control to 62.7±2.2% with Ret siRNA).

D: Knockdown of GFRal, GFRa2 or GFRa4 recapitulates aspects of Ret knockdown

To determine which of the 3 GFRa co-receptors expressed in SCs may activate RET signalling, SCs were transfected with siRNA against GFRal, GFRa2 or GFRa4 and the expressions of Pax7, phospho-Hi stone H1/H3, Myogenin and MyHC was quantified as described above. As with Ret siRNA, knock-down of GFRa4 (but not GFRal or GFRal) was associated with fewer Pax7+ SCs in proliferation medium. However, phospho-Hi stone H1/H3 immunostaining revealed that knock-down of GFRal, GFRa2 and GFRa4 were all associated with fewer cells in the active stages of the cell cycle. After 48 hours in differentiation medium, knockdown of GFRal and GFRa4 significantly increased the number of Myogenin-positive nuclei, and knockdown of GFRa4 significantly increased the fusion index. Taken together, knockdown of all co-receptors recapitulated aspects of Ret knockdown, indicating that RET may signal through multiple co-receptors.

E: Active RET signalling drives satellite cell-derived myoblast proliferation but does not affect differentiation

The applicants next used retroviral-mediated constitutive expression to examine the effects of increased RET on SC proliferation and maintained expression throughout differentiation.

Plasmids encoding human RET9 and RET51 and constitutively active (CA) forms containing the Cys634Lys mutation (causing ligand-independent dimerisation of the RET receptor) that occurs in multiple endocrine neoplasia type 2A (MEN2A) patients were obtained¹. These coding sequences were sub-cloned into a modified pMSCV-puro vector (Clontech), in which the puromycin resistance gene was replaced with an internal ribosomal entry site (IRES) and enhanced green fluorescent protein

(eGFP) to identify infected cells, as previously published (denoted as MIG) (Zammit, P.S., et al., J Cell Sci, 2006. 119(Pt 9): p. 1824-32). Constructs were fully sequenced to ensure fidelity.

Retroviruses were produced using HEK 293 T packaging cells, by co- transfecting the expression vectors in the presence of an ectopic helper plasmid.

Replication incompetent viral particles were harvested from the culture medium and functionality confirmed by western blot and immunofluorescence.

Specifically, for blot analysis, HEK 293T cells were transfected with RET expression vectors for 24 hours and total protein extracted in the presence of complete protease inhibitor cocktail (Roche). Samples were quantified using the BioRad Protein Assay system (BioRad) and equal quantifies run on pre-cast 4-20% electrophoresis gels (Invitrogen) with 0.35μ1 dithiothreitol + bromophenol blue dye at 120V. Gels were transferred to PVDF membranes using the iBlot dry-blotting system (Invitrogen), blocked in 5% milk powder/PBS and incubated overnight at 4°C in primary antibody dissolved in PBS+ 1% milk powder. Membranes were then washed and protein bands visualised by incubating with horseradish peroxidase (HRP) conjugated secondary antibodies and immersing in enhanced chemiluminescence (ECL) reagents (GC Biolabs) before being exposed using X-ray photomicrography.

The applicants confirmed that these retroviral constructs encoded RET protein of 170kDa as expected by Western blotting and that there increased human RETmRNA 48 hours after infection. Expression of endogenous murine Ret was unaltered by human RET expression, revealing no effect of the constructs on endogenous RET signalling.

To ascertain whether RET signalling affects proliferation rate, SCs were infected with retroviruses encoding the RET isoforms and exposed to EdU for 2hrs in proliferation medium. Data was analysed using either paired student t-tests, or by statistical models fitted to the data that incorporated random effects due to mouse or between experiment variation. Quasi-Poisson models were used for evaluating significance of differences in the numbers of cells between conditions and mixed Binomial models employed likewise for ratios including the fusion index. Models tested either the linear relationship between a factor and the result, or each combination of factors as an independent parameter. The fusion index was expressed as a log of odds for binomial models in which the ratio of fusion is defined as:

nucleii, MyHC

YCLtio =

total nucleii

The log of odds is calculated by:

Statistical models and analyses were generated using the R programming language (R Development Core Team, 2010). Image analysis was performed by a customised Matlab script that quantified cell shape deviation from a sphere to give a reading of eccentricity.

Constitutive expression of wild-type RET9 and RET51 did not alter the proportion of eGFP+/EdU+ SCs compared to infection with control retrovirus.

However, the CA RET constructs significantly increased proliferation rate from 47.6±0.8% in control to 56.9±1.8% (CA RET9) and 59.2±1.7% (CA RET51) respectively. To determine whether constitutive expression of the RET constructs affected muscle differentiation, the applicants analysed expression of Myogenin and MyHC in SCs cultured in differentiation medium. After 24 hours, CA RET51 expression reduced the percentage of myoblasts that were Myogenin+ (49.5±1.2%) relative to controls (58.9±3.1%). At later stages of differentiation, there was no change to the number of Myogenin+ myoblasts or of the fusion index.

Example 3

Down-regulation of Ret rescues DUX4-mediated inhibition of myogenic

differentiation

Effective drug design to ameliorate DUX4-induced muscle pathologies requires an understanding of which DUX4 targets contribute to myoblast pathology. Our findings that Ret is a DUX4 target important for controlling SC proliferation and differentiation make it a good candidate to manipulate in order to improve DUX4- induced pathogenesis.

DUX4 also suppresses myogenic regulatory factor (MRF) gene expression and inhibits proliferation and myogenic differentiation. To determine if DUX4-induced pathology could be rescued by inhibiting RET signalling, the applicants measured the effects of siRNA mediated inhibition of Ret in DUX4-expressing SCs. The applicants initially tested whether knockdown of Ret could rescue SC proliferation rate. Murine SCs infected with control or DUX4 retrovirus were transfected with 20nM control or Ret siRNA for 48 hours in proliferation medium and treated with EdU for two hours. As expected, the proportion of myoblasts incorporating EdU was significantly reduced when DUX4 was expressed; transfection of Ret siRNA transfection also resulted in a reduced EdU uptake. Transfection of Ret siRNA into DUX4-expressing cells did not significantly alter the proportion of cells incorporating Edu relative to control siRNA treated cells. Likewise, knockdown of Ret did not change the number of Pax7+ myoblasts in the presence of DUX4. Together this suggests that Ret knockdown is unable to rescue the DUX4-mediated inhibition of proliferation in SCs.

To determine the importance of Ret in mediating DUX4 inhibition of SC differentiation, the applicants next tested whether knockdown of Ret by siRNA could rescue myoblast differentiation in the presence of DUX4 compared to cells infected with the MIG control retrovirus. There was a slight increase in the fusion index of myoblasts expressing control retrovirus and transfected with siRNA to Ret relative to a mismatch control siRNA. Myoblasts expressing DUX4 had a dramatically reduced fusion index when transfected with control siRNA; in contrast, transfection of siRNA to Ret resulted in elevated fusion of DUX4 expressing myoblasts. To assess whether there was a significant rescue, the applicants fitted the data to a binomial model to compensate for any biological variability and to identify interaction effects (Table 1). As expected, the binomial model revealed that DUX4 expression dramatically decreased the fusion index (p= 2.7 lx 10^"65). Ret knockdown had a small effect on muscle fusion in control retrovirus infected cells, increasing it slightly relative to cells transfected with control siRNA (p=3.06 x 10^"4). In contrast, in the presence of DUX4, Ret-knockdown rescued muscle fusion relative to cells expressing DUX4 and transfected with a control siRNA (p=2.15xl0^"21). Therefore, although Ret knockdown causes only a slight increase in muscle fusion, in the presence of DUX4 it is able to significantly improve myogenic differentiation. Table 1 : (a) Maximum likelihood parameters for a logistic model containing an interaction term, and a random effect term (the mouse) describing the probability of a nuclei being present in a MyHC+ cells the presence or absence of DUX4 and RET. (b) Corresponding ratios computed from the model, for all 4 tested conditions with the ratio representing the probability of a GFP+ cell expressing MyHC. In all condition but the control (Intercept), the error contribution of the baseline (Intercept) has been omitted when computing the confidence intervals (C.I.). y represents the log-of-odds of the fusion index, μ represents the intercept parameter (representing the control: MIG control retrovirus, control siRNA), β are the parameters representing the effects of each treatment, or the interaction as specified and δ indicates whether the effect is present or absent.

V— β + <¾UX4 ^■ /¾UX4 + ½ET ^■ βκΕ + #DUX4 ^■ RET "

a)

Parameter Estimate Std.err. t value P value

Intercept -0.208 0.177 -1.174 0.240

DUX4 -1.456 0.085 -17.064 1.7 x 10-65

RET 0.164 0.045 3.610 0.00031

Interaction 0.970 0.102 9.498 2.1 x 10-21

b)

Treatment Ratio Low C.I. High C.I. control+MIG 0.448 0.365 0.535

control+DUX4 0.159 0.138 0.183

RET+MIG 0.489 0.447 0.512

RET+DUX4 0.371 0.344 0.398

Example 4

Small molecule RET inhibitors rescue myogenic capacity of DUX4-expressing myoblasts

As knockdown of Ret in the presence of DUX4 improves myogenic

differentiation, this suggests that Ret induction by DUX4 is important for inhibiting muscle formation by SCs. To obtain a quantitative understanding of how RET signalling potentiates DUX4 activity, small molecular inhibitors of RET were evaluated for their ability to affect myogenesis in the presence or absence of DUX4. Three kinase inhibitors that block RET phosphorylation were tested: Zactima

(Vandetanib/ZD6474), TG101209 ² and Sunitinib (Sutent, SU11248). Zactima and Sunitinib have been clinically approved as therapeutics for treating cancers arising from over-activation of RET signalling. As these drugs also inhibit several receptor tyrosine kinases other than RET at a higher IC50, including VEGFR, EGF, MET, and c- Kit, the applicants first aimed to show whether they could overcome the effects of a specific over-activation of RET in myoblasts.

To determine the doses required to specifically inhibit RET signalling in myoblasts, different concentrations of each drug were tested on CA RET51 -expressing C2C12 myoblasts induced to differentiate for 60 hours.

C2C12 myoblasts were maintained in DMEM supplemented with 10% FBS with 1% L-Glutamine (Sigma Aldrich) and 1% pen/strep. C2C12s were plated at 70% confluency and infected with RET or DUX4-encod g retrovirus in the presence of 4μg/ml Polybrene for 6 hours at 37°C, 5% CO2 in proliferation medium, To induce differentiation, C2C12 myoblasts were cultured in mitogen-poor medium, containing DMEM with 2% HS, pen/strep and L-Glutamine, C2C12.

Crucially, in this immortalised cell line, expression of CA RET51 inhibits myotube formation, with an average fusion index of 4.4 ±2.4% compared to 52.4 ±5.0%) in cells infected by control retrovirus (Figure 3 A, C). Statistical models revealed that Sunitinib and TGI 01209 were able to significantly rescue muscle differentiation in CA RET51 -expressing C2C12s in a dose-dependent manner (Figures 3 B-D, Sunitinib: p=2xl0^"16, TG101209: p=2 x 10^"16, Zactima: p = 4.18 x 10^"14).

Crucially, this occurred without overtly affecting cell number (Figures 3E-G). Control myoblasts were unaffected by Sunitinib and TG101209 at low doses (250ng/ml), but at higher doses showed a small increase in the fusion index (Figures 3B,C,E,F, Table 2). Application of Sunitinib did not affect muscle fusion and myotubes appeared normal at all doses tested (Figure 3H). In contrast, Zactima significantly affected cell number in a non-dose dependent manner and had a strong effect on fusion in control samples (Figure 3D, G, Table 2). Table 2: Maximum likelihood parameters for a logistic model describing the effect on myoblast fusion following expression of RET51-MEN2A (RET51CA) or MIG control retrovirus in murine C2C12 cells when treated with different concentrations of Sunitinib, TG101209 or ZACTIMA (indicated by ng/ml). The model contains interaction terms for RET51-MEN2A and Sunitinib (RET51C A: Sunitinib), Ret51- ME 2A with TGI 01209 (RET51CA: TGI 01209) or Ret5-MEN2A with ZACTIMA (RET51CA:Zactima) that reveals the gradient of response of muscle fusion.

Significance of interaction effects relative to the baseline (MIG control retrovirus infected cells with no drug present) is indicated by P values, y represents the log of odds of the fusion index, μ represents the intercept parameter (representing the control treatment: MIG control retrovirus with no drug), β are the parameters representing the effects of each treatment, or the interaction as specified and δ indicates whether the effect is present or absent.

drugs

Parameter Estimate Std. Error z value Pr(>|z|) (Intercept) 0.008026 0.308986 0.03 0.979

RET51CA -2.157703 0.024436 -88.30 < 2e-16 *** Sunitinib 0.326147 0.036056 9.05 < 2e-16 ***

RET51C A: Sunitinib 1.574679 0.032506 48.44 < 2e-16 *** TG101209 -2.873471 0.342331 -8.39 < 2e-16 *** RET51CA:TG101209 3.295843 0.386403 8.53 < 2e-16 *** Zactima -0.304531 0.040307 -7.56 4.18e-14 *** RET51CA:Zactima 2.923664 0.045197 64.69 < 2e-16 ***

Sunitinib appeared to be the most effective compound tested for inhibition of RET, as it was the compound that promoted an effective rescue of fusion in the presence of CA RET51 without causing large changes to cell number. Therefore subsequent efforts focused on the use of Sunitinib.

Example 5

Sunitinib does not rescue proliferation in DUX4-expressing murine myoblasts but does rescue myogenic induction

The applicants then examined whether they could use Sunitinib in DUX4- expressing murine SCs to rescue fusion, similar to that observed using Ret siRNA (Figure 4). First, the applicants assessed how 250ng/ml Sunitinib affected SC proliferation by measuring EdU incorporation in SCs (Figure 4A). There was no significant difference in proliferation of cells treated with DMSO (43.5±2.0%) or Sunitinib 43.3±0.7%). The applicants then asked if proliferation of SCs expressing DUX4 was affected by application of Sunitinib. Sunitinib treatment did not significantly improve the proliferation rate of DUX4-expressing SCs (8.6±2.0%) compared to DUX4-expressing cells treated with DMSO (8.7 ±1.7%).

DUX4 expression in SCs also leads to repression of MyoD, as previously described. Treatment with Sunitinib rescued the number of MyoD-containing myoblasts in the presence of DUX4 when analysed by a statistical model (p=3.8xl0^"4) (Figure 4B). Thus, Sunitinib is unable to rescue proliferation of murine SCs in the presence of DUX4, but can improve myogenic commitment.

Example 6

Sunitinib rescues myogenic capacity of DUX4-expressing myoblasts

To determine if Sunitinib enhances muscle fusion in murine SCs expressing DUX4, the applicants measured fusion in SCs infected with DUX4-encoding or control retroviruses. Infected cells were grown at high density in the presence of 125- 500ng/ml of Sunitinib and induced to differentiate for 24 hours, prior to the fusion index being calculated (Figure 4E). Fusion of SCs infected with the control retrovirus (MIG) was not affected by Sunitinib. Statistical models revealed that the treatment of DUX4-expressing SCs with Sunitinib significantly increased the fusion index at all doses relative to cells treated with DMSO (p<2 x 10^"16, Figure 4F, Table 3).

Table 3 : (a) Maximum likelihood parameters for a logistic model containing an interaction term to describe the effect of DUX4 expression and Sunitinib during fusion of satellite-cells grown at high density, y represents the log-of-odds of the fusion index, μ represents the intercept parameter (representing the control treatment), β are the parameters representing the effects of each treatment, or the interaction as specified and δ indicates whether the effect is present or absent, y represents the log-of-odds of the fusion index, μ represents the intercept parameter (representing the control treatment: no retrovirus, with no drug present), β are the parameters representing the effects of each treatment, or the interaction as specified and δ indicates whether the effect is present or absent, (b) Corresponding log of odds ratios computed from the model for all 4 tested conditions. y— μ + <¾UX4 ¹ /¾>UX4 +

a)

Parameter Estimate Std.err. z value P value

Intercept 0.105 0.0397 2.654 0.00794

DUX4 -0.901 0.0814 -11.065 < 2e-16

Sunitinib 0.024 0.0434 0.563 0.574

Interaction 1.767 0.0869 20.324 < 2e-16 b)

Treatment Ratio Low C.I. High C.I.

CONTROL :DMSO 0.526 0.507 0.546

DUX4:DMSO 0.311 0.282 0.342

CONTROL: SUNITINIB 0.532 0.524 0.541

DUX4: SUNITINIB 0.730 0.720 0.740

On average, the applicants found that application of Sunitinib at 125, 250 or 500 ng/μΐ to cells expressing DUX4 resulted in a fusion index of 72.8 ±10.7% relative to 31.33±1.0% in an absence of Sunitinib. To determine if Sunitinib had an interaction effect with DUX4 in SCs that affects the fusion index and promotes a recovery relative to control cells, the applicants used another model (Table 4). Table 4: (a) Maximum likelihood parameters for a logistic model containing an interaction term, and a random effect term (the mouse) that describes the fusion index of cells infected with DUX4 or MIG control retrovirus and grown at high density when exposed to Sunitinib at varying concentrations, y represents the log-of-odds of the fusion index, μ represents the intercept parameter (representing the control treatment: MIG control retrovirus with no drug), β are the parameters representing the effects of each treatment, or the interaction as specified and δ indicates whether the effect is present or absent, (b) Corresponding log of odds ratios computed from the model, for all 4 tested conditions.

y— μ + ⁽¾UX4 ¹ ¾UX4 + ^Sunitinib - ¾unitinib + <¾UX4 - ^Sunitinib - ^interaction a)

Parameter Estimate Std. Error z value Pr(>|z|)

(Intercept) 1.04035 0.04454 23.358 <2e-16 ***

DUX4 -2.03527 0.07349 -27.694 <2e-16 ***

SUNITINIB 0.13110 0.05146 2.547 0.0108 *

Interaction 1.56783 0.09772 16.045 <2e-16 ***

Recovery -0.33633 0.06507 -5.1685 2.36e-07 *** b)

Treatment Low C.I. Ratio Estimate High C.I.

CONTROL :DMSO 0.722 0.739 0.755

CONTROL: SUNITINIB 0.748 0.763 0.779

DUX4:DMSO 0.244 0.270 0.297

DUX4: SUNITINIB 0.643 0.669 0.694 This revealed that Sunitinib has an effect on fusion dependent on the presence of DUX4 (p = 2 x 10^"16) and there is significant recovery of fusion in the presence of DUX4 due to Sunitinib (p = 2.3 x 10^"7, Figure 4G) Crucially, treatment with Sunitinib at all doses did not affect the fusion index, or number of murine myoblasts in an absence of DUX4.

Example 7

Sunitinib enables DUX4-expressing SCs to differentiate independent of fusion to unaffected myoblasts

Using a retroviral expression system resulted in a DUX4 infection rate of approximately 40-50%. However, this approach does not allow the applicants to determine whether the increased rate of fusion of DUX4 expressing cells in the presence of Sunitinib was due to Sunitinib rescuing the differentiation defect directly, or simply enabling DUX4-expressing myoblasts to fuse to uninfected myoblasts. To determine whether DUX4-expressing SCs could differentiate independently of fusion, the applicants seeded SCs at low density before treating with 250ng/ml Sunitinib and inducing differentiation for 24 and 48 hours. Samples were then analysed on an individual cell basis using immunolabelling to detect Myogenenin and MyHC.

Sunitinib treatment was observed to cause only a small change in the number or myoblasts expressing Myogenin (27.4±1.1%) relative to vector control treated cells (27.6±0.9%) and this was not significant. By comparing the proportion of single DUX4 expressing myocytes that were eGFP+/Myogenin+ in the absence of Sunitinib

(1 l.OtO.6) relative to those exposed to Sunitinib (27.4±1.1%) for 24 hours, the applicants observed a significant increase (Figure 4C). The proportion of myoblasts expressing MyHC+ myoblasts was significantly reduced in the presence of DUX4 (2.4±0.6%) relative to control cells (19.4±2.5%) or cells exposed to Sunitinib

(21.1±2.1%). In the presence of Sunitinib, the proportion of eGFP+/MyHC+ cells in DUX4-infected cultures was significantly increased following 24 hours exposure (7.8±1.3) (Figure 4D). The applicants used statistical models to evaluate the importance of the interaction between Sunitinib and DUX4 on MyHC expression in SCs plated at low density. Sunitinib application had no effect on the number of cells expressing MyHC after 24 or 48 hours (p>0.05), in contrast to DUX4 (p < 2 x 10^"16). However, Sunitinib strongly affected the expression of MyHC in the presence of DUX4 at 24 hours (p = 4.21 x 10^"5) and 48 hours (p = 0.00794, Table 5).

Table 5: (a) Maximum likelihood parameters for a logistic model containing an interaction term, and a random effect term (the mouse) to describe MyHC expression in SCs at 1 or 2 days of culture when grown at low density and exposed to Sunitinib or drug vector DMSO and infected with DUX4 or MIG control retrovirus, y represents the log-of-odds of the fusion index, μ represents the intercept parameter (representing the control treatment: MIG control retrovirus with no drug), β are the parameters representing the effects of each treatment, or the interaction as specified and δ indicates whether the effect is present or absent, (b) Corresponding ratios computed from the model, for all 4 tested conditions.

<¾ay ^■ <¾UX4 ^" ¾ ,DUX4 + (1 ^~ ¾ay) ^" ¾UX4 ^" ¾,DUX4 +

<¾ay - ^Sunitinib ' βΐ, Sunitinib + (1 ^— <¾ay) ' ^Sunitinib ' βΐ, Sunitinib +

<¾ay - fcuX4 '

a)

Parameter Estimate Std. Error z value Pr(>|z|)

(Intercept) -1.42432 0.10827 -13.156 < 2e-16 *** factor(Days)2 1.38906 0.10641 13.054 < 2e-16 *** factor(Days)l :DUX -2.27886 0.23129 -9.853 < 2e-16 *** factor(Days)2:DUX4 -3.16428 0.18259 -17.330 < 2e-16 *** factor(Days)l : Sunitinib 0.10749 0.11549 0.931 0.35203 factor(Days)2: Sunitinib 0.05296 0.09415 0.563 0.57373 factor(Days)l :DUX4: Sunitinib 1.12318 0.27425 4.096 4.21e-05 *** factor(Day s)2 :DUX4 : Sunitinib 0.61641 0.23220 2.655 0.00794 ** b)

Day DUX4: Sunitinib Low C.I. Ratio Estimate High C.I.

1 control + DMSO 0.1629 0.1940 0.2293

1 control + Sunitinib 0.1788 0.2113 0.2480

1 DUX4 + DMSO 0.0156 0.0241 0.0370

1 DUX4 + Sunitinib 0.0600 0.0778 0.1002

2 control + DMSO 0.4444 0.4912 0.5381

2 control + Sunitinib 0.4574 0.5044 0.5514

2 DUX4 + DMSO 0.0277 0.0392 0.0552

2 DUX4 + Sunitinib 0.0566 0.0738 0.0956 In summary, these results show that Sunitinib is able to rescue muscle differentiation of SCs in the presence of DUX4.

Example 8

Expression of RET in human myoblasts

The applicants next investigated whether RET is co-expressed in human SCs, similar to the mouse.

Primary myoblasts were isolated from the vastus lateralis of three individuals and immediately processed for analysis as described in Boldrin, L. et al. PLoS Curr, 2011. 3: p. RRN1294. When cultured as previously described (Agley, C.C., et al., J Vis Exp, 2015(95): p. 52049), the applicants found that RET was detectable at low levels in proliferating and early differentiating myoblasts (Figure 5).

To determine whether Sunitinib improves myogenic differentiation in human myoblasts with DUX4, the applicants obtained clonal cell lines derived from a mosaic FSHD patient, described in Krom, Y.D., et al, Am J Pathol, 2012. 181(4): p. 1387- 401. Although the non-contracted control line (54.6) and the D4Z4 contracted FSFID line (54.12) both proliferate and undergo differentiation in culture, the applicants found that 54.12 myoblasts proliferate at a slower rate, have an eccentric cell shape and differentiate into myotubes of an abnormal morphology and size (as revealed by a lower fusion index), when compared to control cells. The applicants note that the reduced proliferation and muscle fusion of the 54.12 cells is similar to phenotypes observed in mouse SCs expressing DUX4.

To determine if Sunitinib was able to act similarly to rescue DUX4-associated phenotypes in the human FSFID cell line, the applicants treated both cell lines with varying concentrations of Sunitinib and grew them in either proliferation or differentiation medium. The applicants evaluated whether Sunitinib treatment affected proliferation by EdU pulse labelling, cell shape by measuring eccentricity and muscle differentiation by calculating the fusion index. There was no change in the cell shape of 54.6 cells in the presence of Sunitinib at any dose (p > 0.05, Fig 6A, D). 54.12 cells treated with lower doses of Sunitinib (125, 250 ng/ ml) showed a significant difference in cell shape relative to 54.6 cells (p <0.01); cells treated at higher doses however, showed no significant difference in cell shape compared to 54.6 cells (Figure 6D , p > 0.05, Table 6). Table 6: Maximum likelihood parameters for a linear model containing an interaction term between the cell line and Sunitinib and incorporating a random effect term (the experiment). The model is a linear model that describes the relationship between the shape of 54.6 (control) and 54.12 (FSHD) cells relative to different doses of Sunitinib. P values are approximate and are based on the t-value and represent the probability that there is a difference in cell shape at a specific concentration of Sunitnib. y represents the eccentricity, μ represents the intercept parameter (representing the control treatment: 54.6 cells with no drug), β are the parameters representing the effects of each treatment, or the interaction as specified and δ indicates whether the effect is present or absent.

^FSHD ^■ δ concentration ' ^interaction, concentration

Estimate Std. Error t value Pr(>|t|)

(Intercept) 0.8060526 0.0076921 104.79 < 2e-16 *** as.factor(Sunitinib)125:control 0.0007486 0.0061040 0.12 0.90261 as.factor(Sunitinib)250:control -0.0063433 0.0061040 -1.04 0.30099 as.factor(Sunitinib)500:control 0.0065669 0.0061040 1.08 0.28435 as.factor(Sunitinib)750:control -0.0021753 0.0061040 -0.36 0.72224 as.factor(Sunitinib)0:FSHD 0.0193053 0.0061040 3.16 0.00202 ** as.factor(Sunitinib)125:FSHD 0.0189015 0.0058850 3.21 0.00179 ** as.factor(Sunitinib)250:FSHD 0.0196323 0.0059881 3.28 0.00138 ** as.factor(Sunitinib)500:FSHD 0.0016575 0.0061040 0.27 0.78648 as.factor(Sunitinib)750:FSHD 0.0102796 0.0061040 1.68 0.09500 .

The applicants noted that proliferation of 54.6 cells was unaffected by application of Sunitinib, except at the lowest dose in which a slight increase was noted (p=0.0072). There was significantly lower proliferation of 54.12 cells relative to 54.6 cells in an absence of Sunitinib (p = 2.55 x 10^"5) or when treated with 125 ng/ml (p = 0.00045) Sunitinib. However treatment with higher Sunitinib concentrations

(250ng/ml, 500ng/ml and 750 ng/ ml) enhanced proliferation and there was consequently a lack of significant difference in proliferation of 54.12 cells relative to 54.6 cells (Figure 6A, C, p > 0.05, Table 7). Table 7: a) Maximum likelihood parameters for a logistic model containing an interaction term between the cell line and Sunitinib and incorporating a random effect term (the experiment). The model is a binomial model that tests the relationship between the proliferation of 54.6 (control) and 54.12 (FSHD) cells relative to different doses of Sunitinib. P values represent the probability of a difference in proliferation between the control cells with varying doses of Sunitinib and between control and FSHD cells at different doses of Sunitinib. y represents the proliferation index, μ represents the intercept parameter (representing the control treatment: 54.6 cells with no drug), β are the parameters representing the effects of each treatment, or the interaction as specified and δ indicates whether the effect is present or absent, (b)

Corresponding log of odds ratios computed from the model, for all 4 tested conditions. y = μ + ^FSHD ^■ _/#FSHD +

^FSHD ^■ δ concentration " /^interaction, concentration

Estimate Std. Error z value Pr(> z )

(Intercept) -0.42241 0.04249 -9.942 < 2e-16 *** as.factor(Sunitinib)125:control 0.16640 0.06193 2.687 0.007212 ** as.factor(Sunitinib)250:control 0.09586 0.05881 1.630 0.103107

as.factor(Sunitinib)500:control -0.04183 0.06189 -0.676 0.499161

as.factor(Sunitinib)750:control -0.08055 0.05961 -1.351 0.176628

as.factor(Sunitinib)0:FSHD -0.28239 0.06706 -4.211 2.55e-05 *** as.factor( Sunitinib) 125 :FSHD -0.23422 0.06669 -3.512 0.000445 *** as.factor(Sunitinib)250:FSHD -0.05570 0.06459 -0.862 0.388491

as.factor(Sunitinib)500:FSHD -0.01153 0.07004 -0.165 0.869297

as.factor(Sunitinib)750:FSHD -0.10628 0.06835 -1.555 0.119972 b)

Parameters Low C.I. Ratio Estimate High C.I.

SunitinibO : control 0. .376 0. .396 0. .416

Sunitinib 125 : control 0. .415 0. .436 0. .458

Sunitinib250:control 0. .400 0. .419 0. .439

Sunitinib 500: control 0. .365 0. .386 0. .407 Sunitinib750 : control 0.358 0.377 0.396

SunitinibO:FSHD 0.309 0.331 0.354

Sunitinib 125 :FSHD 0.357 0.380 0.403

Sunitinib250:FSHD 0.382 0.406 0.430

Sunitinib500:FSHD 0.359 0.383 0.408

Sunitinib750:FSHD 0.328 0.352 0.377

Muscle fusion of 54.6 cells was affected by Sunitinib at small, but significant levels, in a non-linear manner. In contrast, the applicants showed that Sunitinib had a strong and highly significant effect on the fusion index of 54.12 cells at all

concentrations tested (p < 2 x 10^"16). Fusion increased progressively in the presence of 125 and 250 ng/ml Sunitinib at which it had the maximum effect and fusion was nearly comparable to that of 54.6 cells (Figure 6B, E). At higher doses of Sunitinib, fusion of 54.12 cells declined (Table 8).

Table 8: a) Maximum likelihood parameters for a logistic model containing an interaction term between the cell line and Sunitinib and incorporating a random effect term (the experiment). The model is a binomial model that tests the relationship between the fusion of 54.6 (control) and 54.12 (FSFID) cells relative to different doses of Sunitinib. Estimate represents the relative change in fusion between conditions. P values represent the probability of a difference in fusion between the control cells with varying doses of Sunitinib and between control and FSFID cells at different doses of Sunitinib. y represents the log-of-odds of the fusion index, μ represents the intercept parameter (representing the control treatment: 54.6 cells with no drug), β are the parameters representing the effects of each treatment, or the interaction as specified and δ indicates whether the effect is present or absent, (b) Corresponding log of odds ratios computed from the model, for all 4 tested conditions.

^FSHD ^■ concentration ' /-'interaction, concentration a) Estimate Std. Error z value Pr(>|z|)

(Intercept) 0.74077 0.09022 8.21 < 2e-16 *** as.factor(Sunitinib)125:control 0.14834 0.02892 5.13 2.91e-07 *** as.factor(Sunitinib)250:control 0.03045 0.02857 1.07 0.287

as.factor(Sunitinib)500:control 0.43074 0.03167 13.60 < 2e-16 *** as.factor(Sunitinib)750:control 0.36837 0.02997 12.29 < 2e-16 *** as.factor(Sunitinib)0:FSHD -1.85964 0.03452 -53.88 < 2e-16 *** as.factor(Sunitinib)125:FSHD -1.07835 0.03288 -32.79 < 2e-16 *** as.factor(Sunitinib)250:FSHD -0.37114 0.03292 -11.27 < 2e-16 *** as.factor(Sunitinib)500:FSHD -1.09843 0.03513 -31.26 < 2e-16 *** as.factor(Sunitinib)750:FSHD -1.34316 0.03494 -38.44 < 2e-16 *** b)

Parameters Low C.I. Ratio Estimate High C.I

SunitinibO : control 0.637 0.677 0.715

Sunitinib 125: control 0.671 0.709 0.744

Sunitinib250 : control 0.644 0.684 0.721

Sunitinib 500: control 0.730 0.763 0.794

Sunitinib750 : control 0.717 0.752 0.784

SunitinibO:FSHD 0.214 0.246 0.281

Sunitinib 125 :FSHD 0.409 0.453 0.498

Sunitinib250:FSHD 0.555 0.599 0.641

Sunitinib500:FSHD 0.473 0.518 0.563

Sunitinib750:FSHD 0.398 0.442 0.487

The applicants showed that treatment of FSHD mosaic cell lines with Sunitinib in cells lacking the D4Z4 truncation did not result in a large effect on myoblast function, as measured by morphology, proliferation or ability to form muscle fibres. In contrast, they showed that mosaic cells containing the D4Z4 truncation are sensitive to Sunitinib and their cell shape becomes more similar to control cells lacking the truncation. Furthermore, they showed that these cells are more able to proliferate and form muscle fibres when treated with Sunitinib at an optimal dose of between 250-500 ng/ml. Example 9

Sunitinib suppresses DUX-4 mediated pERKl/2 signalling in cells

Murine iC2C12 myoblasts were untreated, or treated with SOOng/μΙ doxycycline (Dox) to induce DUX4 and/or 250ng^l Sunitinib for 20 hours, and proteins then electrophoresed onto a PVDF membrane. The membrane was probed with antibodies against DUX4, total AKT, phosphorylated (p) AKT, total ERKl/2 and phosphorylated (p) ERKl/2, with Caveolin-1 used as a loading control. Protein bands were visualised by incubating with horseradish peroxidase-conjugated secondary antibodies and visualised using clarity western ECL substrate. Protein band intensity was quantified with the ChemiDoc™ MP System and normalised to housekeeping protein Caveolin-1. The ratios of pERK:total ERK and pAk total AKT in the treated groups were subsequently compared to the ratios in the control group. Induction of DUX4 is shown by the presence of DUX4 only in iC2C12-DUX4 myoblasts stimulated with doxycycline. All bands shown were visualised on the same membrane and representative results are shown in Figure 7A. Quantification of the following ratios were calculated:

i) pERKl/2 relative to total ERKl/2

ii) pAKT relative to total AKT

Results are shown in Figures 7 B and C and show that compared to control cells with no Dox or addition of Sunitinib, Sunitinib suppresses DUX4-mediated pERKl/2 signalling.

Since it is known that RET signalling is often mediated by the ERKl/2 signalling, it seems possible that Sunitinib action to inhibit DUX4-mediated changes is through an inhibition of ERKl/2 activity.

Example 9

Sunitinib suppresses pAKT signalling in FSHD myoblasts

A human immortalised myoblast clone (54.12) derived from a patient mosaic for the D4Z4 contraction that is associated with FSHD were untreated (Ctrl), or treated with either 250ng^l or 2.5 μg/μl Sunitinib for 20 hours, and proteins extracted onto a membrane. The membrane was probed with antibodies against total (t) AKT, phosphorylated (p) AKT, tERKl/2 and pERKl/2, with Vinculin used as a loading control. Protein bands were visualised by incubating with horseradish peroxidase- conjugated secondary antibodies and visualised using clarity western ECL substrate. The results are shown in Figure 8A.

Protein band intensity was quantified with the ChemiDoc™ MP System and normalised to housekeeping protein Vinculin and the results are shown in Figures 8B and 8C.

These show that Sunitinib suppresses pAKT signalling, even at the lower dose tested.

Claims

Claims

1. An inhibitor of a receptor tyrosine kinase selected from a compound of formula (I), (II) or (III),

where R¹ is selected from the group consisting of hydrogen, halo, alkyl, cyclkoalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, -(CO)R¹⁵, - R¹³R¹⁴, -(CH₂)_rR¹⁶ and - C(0) R⁸R⁹;

R² is selected from the group consisting of hydrogen, halo, alkyl, trihalomethyl, hydroxy, alkoxy, cyano,- R¹³R¹⁴,- R¹³C(0)R¹⁴,-C(0)R¹⁵, aryl, heteroaryl, and - S(0)₂ R¹³R¹⁴;

R³ is selected from the group consisting of hydrogen, halogen, alkyl, trihalomethyl, hydroxy, alkoxy, -(CO)R¹⁵, - R¹³R¹⁴, aryl, heteroaryl,- R¹³S(0)₂R¹⁴ -S(0)₂ R¹³R¹⁴, - R¹³C(0)R¹⁴, - R¹³C(0)OR¹⁴ and-SO₂R²⁰(wherein R²⁰ is alkyl, aryl, aralkyl, heteroaryl and heteroaralkyl);

R⁴ is selected from the group consisting of hydrogen, halogen, alkyl, hydroxy, alkoxy and - R¹³R¹⁴;

R⁵ is selected from the group consisting of hydrogen, alkyl and -C(0)R¹⁰;

R⁶ is selected from the group consisting of hydrogen, alkyl and -C(0)R¹⁰;

R⁷ is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, -C(0)R¹⁷ and -C(0)R¹⁰; or

R⁶ and R⁷ may combine to form a group selected from the group consisting of-(CH₂)4-, -(CH₂)₅- and -(CH₂)6-; with the proviso that at least one of R⁵, R⁶ or R⁷ must be - C(0)R¹⁰;

R⁸ and R⁹ are independently selected from the group consisting of hydrogen, alkyl and aryl; R is selected from the group consisting of hydroxy, alkoxy, aryloxy, -N(R )(CH2)_nR and- R¹³R¹⁴;

R¹¹ is selected from the group consisting of hydrogen and alkyl; R¹² is selected from the group consisting of- R¹³R¹⁴, hydroxy, -C(0)R¹⁵, aryl, heteroaryl, -N⁺(0 )R¹³R¹⁴,- N(OH)R¹³, and - HC(0)R^a (wherein R^a is unsubstituted alkyl, haloalkyl, or aralkyl); R¹³ and R¹⁴ are independently selected from the group consisting of hydrogen, alkyl, Ci-4alkyl substituted with hydroxyalkylamino, cyanoalkyl, cycloalkyl, aryl and heteroaryl; or

R¹³ and R¹⁴ may combine to form a heterocyclo group;

R¹⁵ is selected from the group consisting of hydrogen, hydroxy, alkoxy and aryloxy; R¹⁶ is selected from the group consisting of hydroxy, -C(0)R¹⁵, - R¹³R¹⁴ and - C(0) R¹³R¹⁴;

R¹⁷ is selected from the group consisting of alkyl, cycloalkyl, aryl and heteroaryl; R²⁰ is alkyl, aryl, aralkyl or heteroaryl; and n and r are independently 1, 2, 3, or 4;

(Π)

wherein:

X^b is selected from a group consisting of a bond, O, C=0, S0₂, and CH2; Y^b is selected from a group consisting of a bond or R^9b; or X^b and Y^b taken together is a bond; each of R^lb and R^2b is independently selected from a group consisting of H,

C 1-6 substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; or R^lb and R^2b taken together is a bond; or R^lb and R^2b taken together form a moiety selected from a group consisting of (CH₂)_m',

(CH2)r^<-S-(CH₂)m^<, (CH2)r^<-SO-(CH₂)m^<, (CH₂)r^<-SC>2-(CH2)m^<, (CH2)r- R^b-(CH₂)m^<, and (CH₂)r^<-0-(CH₂)m^<;

each of p', q', r', n', m' is independently an integer having the value between O and 6, R^9b is selected from a group consisting of H, Ci-C₆ alkyl, Ci-C₆ cycloalkyl, Ci-C₆ branched alkyl, Ci-C₆ substituted alkyl, Ci-C₆ aminoalkyl, and Ci-C₆ hydroxyalkyl; Go is selected from a group consisting of N, O, H, and CH,

with the proviso that if Go is N, then:

each of R^3b and R^4b is independently selected from a group consisting of H, Ci-C₆ alkyl, Ci-C₆ substituted or unsubstituted hydroxyalkyl or aminoalkyl, Ci-C₆ substituted or unsubstituted branched alkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, or R^3b and R^4b taken together form a moiety selected from a group consisting of (CH₂)_m>, (CH₂)_r>-S-(CH₂)_m>, (CH₂)_r>-SO-(CH₂)_m>, (CH₂)_r-S0₂- (CH₂)_m<, (CH₂) - R^9b-(CH₂)_m<, and (CH₂)-0-(CH₂)_m<;

with the additional proviso that if Go is N, then: one of the following apply

R^lb and R^9b taken together form a moiety selected from a group consisting of ((CH₂)_m', (CH₂)_r<-S-(CH₂)_m<, (CH₂)_r<-SO-(CH₂)_m<, (CH₂)_r<-S0₂-(CH₂)_m<, (CH₂) - R^9b-(CH₂)_m<, and (CH₂) -0-(CH₂)_m'; or R^lb and R^4b taken together forms a moiety selected from a group consisting of (CH₂)_m>, (CH₂)_r>-S-(CH₂)_m>, (CH₂)_r>-SO-(CH₂)_m>, (CH₂)_r -S0₂- (CH₂)_m (CH₂) - R^9b-(CH₂)m', and (CH₂) -0-(CH₂)_m'; or R^9b and R^4b taken together form a moiety selected from a group consisting of (CH₂)_m', (CH₂)_r -S-(CH₂)_m', (CH₂)_r<-SO-(CH₂)_m<, (CH₂)r -S0₂-(CH₂)_m (CH₂) - R^9b-(CH₂)_m and (CH₂) -O-

(CH₂)m'; or R^3b and R^4b taken together form a moiety selected from a group consisting of (CH₂)_m<, (CH₂)_r<-S-(CH₂)_m<, (CH₂)_r<-SO-(CH₂)_m<, (CH₂)_r -S0₂-(CH₂)_m (CH₂) - R^9b-(CH₂)_m and (CH₂) -0-(CH₂)_m<;

with the further proviso that if Go is O, then:

R^3b is selected from a group consisting of H, Ci-C6alkyl and Ci-C₆ substituted or unsubstituted hydroxyalkyl or aminoalkyl, substituted or unsubstituted branched alkyl, substituted or unsubstituted cycloalkyl, substituted heterocyclic connected through carbon or nitrogen, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl connected through carbon or nitrogen, with no group R^4b; R^lb and R^9b taken together form a moiety selected from a group consisting of (CH₂)_m', (CH₂)_r -S-(CH₂)_m', (CH₂)_r<-SO-(CH₂)_m<, (CH₂)_r<-S0₂-(CH₂)_m<, (CH₂) - R^9b-(CH₂)_m<, and (CH₂) -O- (CH₂)m'; or R^lb and R^3b taken together form a moiety selected from a group consisting of (CH₂)_m<, (CH₂)_r<-S-(CH₂)_m<, (CH₂)_r<-SO-(CH₂)_m<, (CH₂)_r -S0₂-(CH₂)_m (CH₂) - R^9b-(CH₂)m', and (CH₂) -0-(CH₂)_m'; or R^9b and R^3b taken together form a moiety selected from a group consisting of (CH₂)_m<, (CH₂)_r<-S-(CH₂)_m<, (CH₂)_r<-SO-(CH₂)_m<, (CH₂)r -S0₂-(CH₂)_m (CH₂) - R^9b-(CH₂)_m and (CH₂) -0-(CH₂)_m';

with the further proviso that if Go is CH, then each of R^3b and R^4b is independently selected from a group consisting of H, Ci-C₆ alkyl, Ci-C₆ substituted or unsubstituted hydroxyalkyl or aminoalkyl, Ci-C₆ substituted or unsubstituted branched alkyl, substituted or unsubstituted aryl, Ci-C₆ substituted or unsubstituted heterocycle connected through carbon or nitrogen, and substituted or unsubstituted heteroaryl connected through carbon or nitrogen, or R^3b and R^4b taken together form a moiety selected from a group consisting of (CHR^9b)_r ^< -(CHR^9b)_m ^<-(CHR^9b)_p ^<, (CHR^9b)_r ^<-S- (CHR⁹V, (CHR⁹VSO-(CHR⁹V, (CHR^9b)_r<-S0₂ (CHR^9b)_m (CHR^9b)_r' - R^9b- (CHR^9b)_m and (CHR^9b)_r -0-(CHR^9b)_m';

G is N or CR^6b, and each G is independent of each other G, with the further proviso that not more than two groups G can be N, with the further proviso that for each CR^6b, each R^6b is independent of each other group R^6b;

wherein each of R^6b, R^7b, R^8b is independently selected from a group consisting of H, Ci-C₆ substituted or unsubstituted alkyl, Ci-C₆ substituted or unsubstituted alkenyl, Ci- C₆ substituted or unsubstituted alkynyl, Ci-C₆ substituted or unsubstituted

hydroxyalkyl or aminoalkyl, Ci-C₆ substituted or unsubstituted branched alkyl, Ci-C₆ substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl connected through carbon or a heteroatom, substituted or unsubstituted heteroaryl connected through carbon or a heteroatom, Ci-C₆ alkoxy, a halogen, CF₃, -OCF₃, CHR^3bR^4b, SR³, SOR^3b, S0₂R^3b, S0₂ R^3bR^4b, S0₃R^3b, POR^3b, P0₂R^3b, P0₂ R^3bR^4b, P0₂CR^3bR^4b, P0₃CR^3bR^4b, P0₃R^3b, R^3bR^4b, N0₂, CN, OH, C0 R^3bR^4b, COR^3b, COOR^3b,

R^3bCOR^4b, R^3bCO R^3bR^4b, OCO R^3bR^4b, CS R^3bR^4b, CSR^3b, R^3bCS R^3bR^4b, SCO R^3bR^4b, SCS R^3bR^4b, and SCS R^3bR^4b; or any of R^6b and R^7b taken together, or R^7b and R^8b taken together, or R^6b and R^8b taken together form a moiety independently selected from a group consisting

of -HN-CH=CH-, -HN-N=CH-, -HN-N=N-, -0(CH₂)„0-, -S(CH₂)„S-,

-N=CH-S, -CH=N-0, -CH=N-S-, -N=CH-0-, -C=N-0-, -C=N-0-,

-CH=CH-CH=CH- -N=CH-CH=CH-, -CH=N-CH=CH-, -0-CH=CH, and

-S-CH=CH-; or R^3b and R^4b taken together form a moiety selected from a group consisting of (CHR^9b)_r'-(CHR^9b)_m'-(CHR^9b)p (CHR^9b)_r<-S-(CHR^9b)_m<, (CHR^9b)_r<-SO- (CHR⁹V, (CHR⁹VS0₂ (CHR⁹V, (CHR^9b)_r -NR^9b-(CHR^9b)_m and (CHR^9b)_r -0-

A is selected from a group consisting of O, R^3b, CR^3bR^4b, S, SO, and S0₂;

Gi is selected from a group consisting of CH, N, H, S, and O;

G₂ is selected from a group consisting of CR , N, NH, S, and O, with each group R being independent of every other group R^7b ;

with the further proviso that X or Go includes at least one heteroatom included with X and selected from O, S and N, or Go comprises at least four non-hydrogen atoms, inclusive of the heteroatom, and R^3b and R^4b, or R^lb and R^9b, or R^lb and R^4b, or R^9b and R^4b taken together form an aromatic, heteroaromatic, cyclic or heterocyclic ring system, or if a noncyclic system is present, then more than one heteroatom is present, and if A is NR^3b, then any of R^6b, R^7b or R^8b, or any combination thereof independently includes at least two non-hydrogen substituents, or if A is NR^3b, then Q forms a fused ring from R^6b to R^7b, or from R^7bto R^8b, or

(III)

wherein : m is an integer from 1 to 3 ;

R^la represents halo or Ci-3alkyl;

R^2a is selected from one of the following three groups :

1) Ci-5alkylR^3a (wherein R^3a is piperidin-4-yl which may bear one or two substituents selected from hydroxy, halogeno, Ci-4alkyl, Ci-4hydroxyalkyl and Ci-4alkoxy ;

2) C₂-₅alkenylR^3a( wherein R^3a is as defined above) ;

3) C₂-₅alkynylR^3a (wherein R^3a is as defined herein) ; and wherein any alkyl, alkenyl or alkynyl group may bear one or more substituents selected from hydroxy, halogeno and amino ; or a pharmaceutically acceptable salt of any of these; for use in the treatment of fascioscapulohumeral dystrophy (FSHD).

2. An inhibitor according to claim 1 which is a compound of formula (I).

3. An inhibitor according to claim 2 wherein in the compound of formula (I). R¹ is selected from hydrogen, Ci_-4alkyl, -(CH₂)_rR¹⁶ and -C(0) R⁸R^9;

R² is selected from hydrogen, halogen, aryl and -S(0)₂ R¹³R¹⁴;

R³ is selected from hydrogen, (C i- 4)alkyl, (C i- 4)alkoxy, aryl, heteroaryl, and - C(0)R¹⁵;

R⁴ is hydrogen;

R ⁵ is selected from hydrogen and (C i-4)alkyl ;

R⁶ is -C(0)R¹⁰;

R⁷ is selected from hydrogen, (C i-4)alkyl and aryl;

R ⁸ and R⁹ are independently selected from hydrogen, alkyl and aryl;

R¹⁰is -N(R^u)(CH₂)nR ¹², wherein n is 1, 2 or 3, R¹¹ is hydrogen and R¹²is selected from hydroxy, (Ci_-4)alkoxy, -C(0)R¹⁵, heteroaryl and - R¹³R¹⁴;

R¹³ and R¹⁴ are independently selected from the group consisting of hydrogen. (C 1-C

4)alkyl, cycloalkyl, aryl and heteroaryl; or

R¹³ and R¹⁴ may combine to form a heterocyclo group;

R¹⁵ is selected from the group consisting of hydrogen, hydroxy, (C 1-C 4)alkoxy and aryloxy;

R¹⁶ is selected from hydroxy and -C(0)R¹⁵; and

r is 2 or 3;

and wherein:

alkoxy and aryloxy are as defined above;

heteroaryl refers to a monocyclic or fused ring group of 5 to 12 ring atoms containing one, two or three ring heteroatoms selected from N, O or S, the remaining ring atoms being C;

heterocyclo group refers to a saturated cyclic radical of 3 to 8 ring atoms in which one or two ring atoms are heteroatoms selected from N, O or S(0)_n where n is an integer from 0 to 2, the remaining ring atoms being C, where one or two C atoms are optionally replaced by a carbonyl group; the alkyl, alkoxy and cycloalkyl groups are unsubstituted;

the aryl and heteroaryl groups are optionally substituted with one or two substituents independently selected from halo, (Ci-4)alkyl, trihalo(Ci-4)alkyl, hydroxy, mercapto, cyano, N-amido, mono- or di(Ci-4)alkylamino, carboxy and N-sulfonamido;

the heterocyclo group is optionally substituted with one or two substituents independently selected from halo, -(Ci-4)alkyl, -(Ci-4)alkyl-carboxy, -(Ci-4)alkyl-ester, hydroxyl and mono- or di(Ci-4)alkylamino.

4. An inhibitor according to claim 3 which is Sunitinib.

5. A method for treating fascioscapulohumeral dystrophy (FSHD), said method comprising administering to a patient in need thereof, an effective amount of one or more inhibitors of receptor tyrosine kinases.

6. A method according to claim 5 wherein the receptor tyrosine kinase is other than ALK4, ALK5, JAK1, JAK2, JAK3 or TYR2.

7. A method according to claim 5 or claim 6 wherein when the inhibitor inhibits c-Kit, it also inhibits at least one further receptor tyrosine kinase.

8. A method according to any one of claims 4 to 7 wherein the receptor tyrosine kinase is selected from the group consisting of RET, PDGFRa/b, VEGFRl-3 and FLT3 and related proteins.

9. A method according to any one of claims 4 to 8 wherein the inhibitor is a small molecule inhibitor.

10. A method according to claim 9 wherein the small molecule inhibitor is selected a compound of formula (I), (II) or (III) as defined herein.

11. A method according to claim 10 wherein the small molecule is a compound of formula (I).

12. A method according to claim 11 wherein the compound of formula (I) is a compound of formula (I) where;

R¹ is selected from hydrogen, Ci_-4alkyl, -(CH₂)_rR¹⁶ and -C(0) R⁸R^9;

R² is selected from hydrogen, halogen, aryl and -S(0)₂ R¹³R¹⁴;

R³ is selected from hydrogen, (C i- 4)alkyl, (C i- 4)alkoxy, aryl, heteroaryl, and -

C(0)R¹⁵;

R⁴ is hydrogen;

R ⁵ is selected from hydrogen and (C i-4)alkyl ;

R⁶ is -C(0)R¹⁰;

R⁷ is selected from hydrogen, (C i-4)alkyl and aryl;

R ⁸ and R⁹ are independently selected from hydrogen, alkyl and aryl;

R¹⁰is -N(R^u)(CH₂)nR ¹², wherein n is 1, 2 or 3, R¹¹ is hydrogen and R¹²is selected from hydroxy, (Ci_-4)alkoxy, -C(0)R¹⁵, heteroaryl and - R¹³R¹⁴;

R¹³ and R¹⁴ are independently selected from the group consisting of hydrogen. (C 1-C

4)alkyl, cycloalkyl, aryl and heteroaryl; or

R¹³ and R¹⁴ may combine to form a heterocyclo group;

R¹⁵ is selected from the group consisting of hydrogen, hydroxy, (C 1-C 4)alkoxy and aryloxy;

R¹⁶ is selected from hydroxy and -C(0)R¹⁵; and

r is 2 or 3;

and wherein:

alkoxy and aryloxy are as defined above;

heteroaryl refers to a monocyclic or fused ring group of 5 to 12 ring atoms containing one, two or three ring heteroatoms selected from N, O or S, the remaining ring atoms being C;

heterocyclo group refers to a saturated cyclic radical of 3 to 8 ring atoms in which one or two ring atoms are heteroatoms selected from N, O or S(0)_n where n is an integer from 0 to 2, the remaining ring atoms being C, where one or two C atoms are optionally replaced by a carbonyl group;

the alkyl, alkoxy and cycloalkyl groups are unsubstituted;

the aryl and heteroaryl groups are optionally substituted with one or two substituents independently selected from halo, (Ci-4)alkyl, trihalo(Ci-4)alkyl, hydroxy, mercapto, cyano, N-amido, mono- or di(Ci-4)alkylamino, carboxy and N-sulfonamido;

the heterocyclo group is optionally substituted with one or two substituents independently selected from halo, -(Ci-4)alkyl, -(Ci-4)alkyl-carboxy, -(Ci-4)alkyl-ester, hydroxyl and mono- or di(Ci-4)alkylamino.

13. A method according to claim 8 wherein the compound of formula (I) is Sunitinib.

14. A method according to any one of claims 5 to 13 wherein the inhibitor(s) is administered in the form of a pharmaceutical composition.

15. A pharmaceutical composition comprising of one or more inhibitors of a receptor tyrosine kinase and a pharmaceutically acceptable carrier or excipient, for use in the treatment of fascioscapulohumeral dystrophy (FSHD).