WO2014155268A2 - Fgf-r tyrosine kinase activity inhibitors - use in diseases associated with lack of or reduced snf5 activity - Google Patents

Abstract

FGF-R tyrosine Kinase Activity Inhibitors useful in Case of Lack of SNF5 Activity. The invention relates to the use of FGF-R inhibitors in the (prophylactic and/or therapeutic) treatment of proliferative diseases that depend on at least partial lack of SNF5 activity, the use of said inhibitors for the preparation of a pharmaceutical composition (medicament) for said use, said inhibitors for use in a method of (therapeutic and/or prophylactic) treatment of a proliferative disease depends on at least partial lack of SNF5 activity, a method of (prophylactic and/or therapeutic) treatment of a proliferative disease that depends on at least partial lack of SNF5 activity and a pharmaceutical composition (medicament) for use in the (prophylactic and/or therapeutic) treatment of a proliferative disease that depends on at least partial lack of SNF5 activity, as well as related invention embodiments.

Description

FGF-R tyrosine Kinase Activity Inhibitors - Use in Diseases Associated with Lack of or Reduced SNF5 Activity

Summary of the Invention

The invention relates to the use of FGF-R inhibitors in the treatment of diseases associated with at least partial lack of SNF5 activity; the use of said inhibitors for the preparation of a pharmaceutical composition or as medicament for the treatment of diseases associated with at least partial lack of SNF5 activity; a method of treating a disease associated with at least partial lack of SNF5 activity and a pharmaceutical composition (medicament) for use in the (prophylactic and/or therapeutic) treatment of a disease associated with at least partial lack of SNF5 activity, as well as to related invention embodiments.

Background of the Invention.

There are a number of diseases or disorders, especially proliferative diseases or disorder, that show decreased, insufficient or otherwise inadequate SNF5 activity when compared with healthy subjects, e.g.. rhabdoid tumors (bi-allelic inactivating events in the SNF5 region , e.g.

deletion, missense, nonsense and/or frameshift mutations, or a combination of those, or other somatic alterations), familiar schwannomatosis (homozygous inactivation e.g. due to truncating mutations), Small-cell hepatoblastoma (homozygous inactivation, e.g. based on translocations and homozygous deletion of 22q1 1 .2), extraskeletal myxoid chondrosarcomas (homozygous inactivation e.g. due to frameshift and homozygous deletion), undifferentiated sarcomas (e.g. due to haploinsufficiency and homozygous inactivation e..g. based on homozygous deletion and intragenic mutation), epitheloid sarcomas (homogenous inactivation e.g. due to homozygous deletion), meningiomas (homozygous inactivation, e.g. due to missense mutation with loss of the second allele) or poorly differentiated chordomas (based on homozygous inactivation, e.g. loss of 22q1 1 .2).

For example, malignant rhabdoid tumors (MRTs) are very aggressive, pediatric tumors arising from kidney or extra-renal sites such as brain or soft tissues. MRTs are characterized by loss of function of the tumor suppressor SNF5 due to inactivating mutations or deletions of the SNF5 gene. SNF5, also known as SMARCB1 , I N 11 or BAF47, is a core component of the SWI/SNF chromatin remodeling complex, which mediates nucleosome repositioning along the DNA in an ATP- dependent fashion. In particular, SNF5 has a critical function in cell cycle control and affects the pRb tumor suppressor pathway by inducing expression of p16^INK4A and repression of Cyclin D1 . Thus, abrogation of SNF5 function leads to hyperphosphorylation of pRb and E2F-mediated cell cycle activation. In addition, inactivation of SNF5 results in the upregulation of multiple oncogenic pathways, such as Hedgehog and Aurora A signaling and the induction of the Polycomb gene Ezh2.

However, to date no targeted therapy exists to specifically inhibit aberrantly activated oncogenic signaling in MRTs and current chemotherapeutic approaches are largely inefficient. In consequence, MRTs have a poor prognosis and the majority of patients die within the first year of diagnosis.

General Description of the Invention

Surprisingly, it has now been found that the above mentioned diseases, associated with at least partial lack of SNF5 activity, have increased FGF-R activity. The diseases can therefore be treated by drug substances that decrease the activity of FGF-R. Thus the present invention relates to a completely novel approach of treating the mentioned diseases and other diseases associated with at least partial lack of SNF5 activity with an FGF-R tyrosine kinase inhibitor, alone or with concomitant other treatment, that is as sole or as adjuvant therapy.

We describe a novel repressive regulatory function of SNF5 in the transcriptional regulation of FGF-Rs. SNF5 is directly recruited to the FGFR2 promoter. Loss of SNF5 leads to the upregulation of FGF-Rs, which, without being bound by the theory, causes a significant proliferative activation and inhibition of FGFRs with a small molecule kinase inhibitor of the FGFRs causes a significant inhibition of proliferation.

These findings highlight FGF-R inhibition as a novel therapeutic option for the treatment of malignant rhabdoid tumors (MRTs) and other diseases that at least partially lack ©f-SNF5 activity. This notion is supported by in vitro and in vivo data showing that the malignant cell lines lacking SNF5 activity were sensitive to the inhibition of a FGFR inhibitor or a shRNA of FGFR. Detailed Description of the Invention

The invention relates to the use of drug compounds that inhibit FGF-R tyrosine kinase activity, also interchangeably termed as "FGFR inhibitor" in this application, in the treatment of diseases or disorders, such as proliferative diseases, associated with at least partial lack of SNF5 activity.

The term "at least partial lack of SNF5 activity", as used herein, especially refers to the level of wild type SNF5 activity either undetectable or reduced to at most 70%, at most 50%, at most 30% or at most 10% in comparison to an average activity in healthy individuals or to a control tissue or a control cell line harboring only SNF5 wild type gene or expressing normal level of SNF5 protein, when the same method to determine SNF5 activity is applied to the test sample and the control sample. Many alterations can contribute to the condition of at least partial lack of SNF5 activity. Such alterations include but are not limited to the lack of or reduced level of SNF5 protein expression or SNF5 gene mutations, e.g. deletion, missense, nonsense and/or frameshift mutations), loss of, translocation of or partially deletion of 22q1 1 .2. Particularly the term "at least partial lack of SNF5 activity" refers to the situation of SNF5 gene mutations.

Methods to determine SNF5 include but are not limited to karyotyping analyses, PCR- single-strand conformation polymorphism (SSCP), loss of heterozygosity (LOH), DNA sequencing of the SNF5 region, particularly SNF5 exons, RNA sequencing, multiplex ligation dependent probe amplification (MLPA), oligonucleotide based single nucleotide polymorphism [SNP], aCGH, molecular inversion probe analysis, genomic quantitative PCR, FISH, CISH, real-time quantitative RT-PCR analysis to determine gene expression levels, or

immunohistochemistry or immunoblotting. or other means to detect genetic or epigenetic alterations leading to at least partial lack of SNF5 activity.

The treatment may include the determination of the activity of SNF5 in patients with a disease suspected to be caused by too low SNF5 activity, this lowered activity such serving as biomar- ker for accessibility to treatment.

In one aspect the invention relates to the use of drug compounds that inhibit FGF-R tyrosine kinase activity in the treatment of a disease or disorder selected from the group consisting of Rhabdoid tumors , familiar schwannomatosis, Small-cell hepatoblastoma, extraskeletal myxoid chondrosarcomas, undifferentiated sarcomas, epitheloid sarcomas, meningiomas or poorly differentiated chordomas.

In one aspect, the invention relates to the use of an FGF-R tyrosine kinase activity inhibitor, or a pharmaceutically acceptable salt thereof, in the treatment of a disease, including a proliferative disease, associated with at least partial lack of SNF5 activity.

In one aspect, the invention relates to the use of an FGF-R tyrosine kinase activity inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition or medicament for use in a method of treating of a disease, including a proliferative disease, associated with at least partial lack of SNF5 activity

In another aspect, the invention relates to an FGF-R tyrosine kinase activity inhibitor, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease, including a proliferative disease, associated with at least partial lack of SNF5 activity.

In a further aspect, the invention relates to a method of treating of a disease, including a proliferative disease, associated with at least partial lack of SNF5 activity, comprising administering an FGF-R tyrosine kinase activity inhibitor, or a pharmaceutically acceptable salt thereof, especially in a prophylactically and/or therapeutically active amount, e.g. to an individual, e..g. a human patient, in need of such treatment.

In still a further aspect, the invention relates to a pharmaceutical composition for use in the treatment of a disease, including a proliferative disease, associated with at least partial lack of SNF5 activity, said pharmaceutical composition comprising an FGF-R tyrosine kinase activity inhibitor.or a salt thereof.

The general expressions used hereinbefore and hereinafter can, as appropriate and expedient, be replaced (individually, by two or more or all) in any invention embodiment by the following more specific definitions, thus defining more specific invention embodiments:

An FGF-R (or FGFR) inhibitor, as used herein, refers to a compound capable of inhibition of said FGFR activity directly or indirectly. Typically inhibition means direct inhibition, by which the inhibitor binds to FGFR and thereby effects inhibition, e.g. prevents FGFR from binding to its ligand or from taking the active form or from activating its downstream molecules. Indirect inhibition refers to all other ways than direct inhibition, in which the overall FGFR activity is reduced, for example inhibitors of FGFR downstream molecules, inhibitors, such as shRNA, for the trans- criptional/translational machinery of FGFR etc.

Normally an FGFR inhibitor has an IC₅₀ values below 1000 nm, below 100nm, below 10nm. For example an FGFR inhibiting antibody has an IC₅₀ normally in the range from 0.001 to 500 nM, preferably below below 100nm, or below 10nm. A small molecule FGFR inhibitor has an IC₅₀ normally in the range from 0.1 to 500 nM, preferably below 100nm, below 10nm in the proliferation test system according to Example 1 and Fig. 1 below. The term "a small molecule FGFR inhibitor", as used here, refers to a chemical compound having molecular weight normally below 2000 Dalton, usually below 1500 Dalton, usually below 1000 Dalton. Typically and preferably, a small molecule FGFR inhibitor is produced through chemical synthesis process, not through a bioengineering process. The term "a FGFR inhibitor" as used here is intended to encompass all FGFR inhibitors currently known or which will be developed in the future.

In one embodiment, the FGFR inhibitor is a small molecule FGFR inhibitor. One preferred compound is 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1 -{6-[4-(4-ethylpiperazin-1 -yl)-phenylamino]- pyrimidin-4-yl}-1 -methyl-urea (also named BGJ398 in the present disclosure) having . the formula

any (whereever used meaning any one or more) pharmaceutically acceptable salts, and/or any active metabolites or a N-oxide thereof. An active metabolite is an active form of a drug after it has been processed by the body. Another kind of active metabolite is when a drug is metabolized, especially broken down by the body into a modified form which continues to produce effects in the body. Usually these effects are comparable or similar to those of the parent drug or may be weaker, although they can still be significant or further even stronger. Formula (b) represents an N-oxide of BGJ398 which was disclosed in WO2006/000420, Example 145. Most preferred is the monophosphate salt of BGJ398 disclosed in

WO201 1/071821 A1 , which can be characterized,- not showing the protonation/deprotonation of BGJ398 and phosphoric acid but merely showing these in a uncharged form in a summary way -, by the formula:

Formula (b) represents an N-oxide of BGJ398:

Another preferred FGFR inhibitor is 1-(2,6-dichloro-3,5-dimethoxy-phenyl)-3-{6-[4-(4-ethyl- piperazin-1 -yl)-phenylamino]-pyrimidin-4-yl}-urea, which has the formula (c)

In another preferred embodiment the FGFR inhibitor has formula (d)

Further preferred FGFR tyrosine kinase inhibitors, and/or any of its pharmaceutically acceptable salts, are: AZD-4547 (N-[5-[2-(3,5-dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3,5- dimethylpiperazin-1 -yl)benzamide) with the formula:

PD 173074 (N-[2-[[4-(diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin- 7-yl]-N'-(1 , 1 -dimethylethyl)urea).

In one preferred embodiment, the FGFR inhibitor is TKI258 (amino-5-fluoro-3-[6-(4- methylpiperazin-1-yl)-1 H-benzimidazol-2-yl]quinolin-2(1 H)-one) of the formula

or an active metabolite thereof and/or its tautomer 4-amino-5-fluoro-3-[5-(4-methylpiperazin-1 - yl)-1 H-benzimidazol-2-yl]quinolin-2(1 H)-one, or a pharmaceutically acceptable salt thereof, respectively. TKI258 is a pan-tyrosine kinase inhibitor with inhibiting effect of FGFRs.

Other less specific FGFR tyrosine kinase inhibitors of the invention include include, but are not limited to intedanib, brivanib (especially the alaninate), cediranib, masitinib, orantinib, ponatinib and E-7080 (4-[3-Chloro~4~(A '-cydopropylureido)phenoxy]-7-methoxyquinoline-6-carboxamide);

In one embodiment the FGFR inhibitor is an FGFR inhibiting antibody.

The term "antibody" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, humanized antibodies, human-origin antibodies, multi specific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

"Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')z, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

In one preferred embodiment, the antibody is selected from the group consisting of:

HGS1036/FP-1039 ( J. Clin. Oncol. 28:15s, 2010): soluble fusion protein consisting of the extracellular domains of human FGFR1 linked to the Fc region of human Immunoglobulin G1 (lgG1 ), designed to sequester and bind multiple FGF ligands and lock activation of multiple FGF receptors; MFGR1877S: monoclonal antibody; AV-370 : humanized antibody; GP369/AV-396b : FGFR-lllb-specific antibody; and HuGAL-FR21 : monoclonal antibody specific to FGFR2.

In one embodiment, the FGFR inhibitor is an SiRNA or ShRNA of FGFR.

The present invention embodiments also include pharmaceutically acceptable salts of the compounds (active ingredients, drug substances) useful according to the invention described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.

The compounds useful according to the invention, as well as their pharmaceutically acceptable salts or prodrugs, can also be present as tautomers, N-oxides or solvates, e.g. hydrates. All these variants, as well as any single one thereof or combination of two or more to less than all such variants, are encompassed and to be read herein where a compound included in the inventive products, e.g. an FGF-R tyrosine kinase activity inhibitor, is mentioned.

The FGF-R tyrosine kinase inhibitors can be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g.., orally, e.g. as solid dosage forms e.g. in the form of tablets or capsules, or parenterally, e.g. in the form of liquid formulations for injection or infusion. The corresponding pharmaceutical compositions, for the purpose/use according to the invention also as such forming part of the invention, comprise the active ingredient in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent.

In general, the active ingredients will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. In general, satisfactory results are indicated to be obtained sys- temically at daily dosages of from about 0.01 to 100 mg/kg body weight, e.g. 0.03 to 2.5mg/kg or 0.05 to 25 mg/kg body weight, respectively. An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.5 mg to about 200 mg, conveniently administered, e.g. in divided doses up to four times a day or in retard form. Suitable unit dosage forms for oral administration comprise from ca. 0.5 to 200 mg active ingredient. For antibody based therapy, dosages can e.g. range from about 0.1 to about 10 mg/kg active ingredient applied in intervals of about 0.25 to about 8 treatments per month.

In one aspect, the present invention relates to a pharmaceutical combination comprising or consisting essentially of, or consisting of, the first active ingredient being an FGFR inhibitor and the second active ingredient, being each in a separate dosage form or as a combination product and optionally at least one pharmaceutically acceptable carrier, where e.g. each combination partner may also be, independently of the other combination partner, in the form or a phar- maceutically acceptable salt. In one further aspect, the present invention relates to said pharmaceutical combination for use in a method of the prophylactic and/or therapeutic treatment of a disease or disorder associated with at least partially lack of SNF5 activity. In one preferred embodiment the disease or disorder is selected from a group consisting of rhabdoid tumors; familiar schwannomatosis, Small-cell hepatoblastoma, extraskeletal myxoid chondrosarcomas,, undifferentiated sarcomas , epitheloid sarcomas, meningiomas or poorly differentiated chordomas.

"Combination" refers especially to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where an FGFR tyrosine kinase inhibitor and further combination partner (e.g. an other drug as explained below, also referred to as "co-agent") may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative (= joint), e.g. additive or synergistic effect. The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration and/or at the same time.

The term "combination product" as used herein means a pharmaceutical product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients (which may also be combined).

Thus, the invention relates in a further embodiment to a combination, e.g. a combination product, particularly a pharmaceutical composition, comprising a therapeutically effective amount of (i) an FGF-R tyrosine kinase activity inhibitor, or a pharmaceutically acceptable salt thereof, respectively, and (ii) at least one further therapeutically active agent (co-agent), e.g. another compound (i) or a different co-agent. The additional co-agent is preferably selected from the group consisting of anti-proliferative (e.g. anti-cancer) agents; and/or anti-inflammatory agents.

In this case, the combination partners forming a corresponding product according to the invention may be mixed to form a fixed pharmaceutical composition or they may be administered separately or pairwise (i.e. before, simultaneously with or after the other drug substance(s)).

A combination, e.g. combination product, according to the invention can besides or in addition be administered especially for antiproliferative therapy in combination with chemotherapy, radio- therapy, immunotherapy, surgical intervention, photodynamic therapy, implants, hormones or a combination of any two or more of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments are therapy to maintain the patient's status after tumor regression, or even chemopre- ventive therapy, for example in patients at risk.

Possible anti-proliferative (e.g. anti-cancer) agents (e.g. for antiproliferative therapy, e.g. chemotherapy) as co-agents include, but are not limited to aromatase inhibitors; antiestrogens; topo- isomerase I inhibitors; topoisomerase II inhibitors; microtubule active compounds; alkylating compounds; histone deacetylase inhibitors; compounds which induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antineoplastic antimetabolites; platin compounds; compounds targeting/decreasing a protein or lipid kinase activity; anti-angio- genic compounds; compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase; gonadorelin agonists; anti-androgens; methionine aminopeptidase inhibitors; bis- phosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasome inhibitors; compounds used in the treatment of hematologic malignancies; compounds which target, decrease or inhibit the activity of Flt-3; Hsp90 inhibitors; kinesin spindle protein inhibitors; MEK inhibitors; EDG binders; antileukemia compounds; ribonucleotide reductase inhibitors; S-adenosylmethionine decarboxylase inhibitors; angiostatic steroids; corticosteroids; other chemotherapeutic compounds (as defined below); and/or photosensitizing compounds.

The term "aromatase inhibitor" as used herein relates to a compound which inhibits the estrogen production, i.e. the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively. The term includes, but is not limited to steroids, especially atame- stane, exemestane and formestane and, in particular, non-steroids, especially aminogluteth- imide, roglethimide, pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole, fadro- zole, anastrozole and letrozole.

The term "antiestrogen" as used herein relates to a compound which antagonizes the effect of estrogens at the estrogen receptor level. The term includes, but is not limited to tamoxifen, ful- vestrant, raloxifene and raloxifene hydrochloride. The term "anti-androgen" as used herein relates to any substance which is capable of inhibiting the biological effects of androgenic hormones and includes, but is not limited to, bicalutamide (CASODEX), which can be formulated, e.g. as disclosed in US 4,636,505.

The term "gonadorelin agonist" as used herein includes, but is not limited to abarelix, goserelin and goserelin acetate. The term "topoisomerase I inhibitor" as used herein includes, but is not limited to topotecan, gimatecan, irinotecan, camptothecian and its analogues, 9-nitrocampto- thecin and the macromolecular camptothecin conjugate PNU-166148 (BGJ3981 in W099/ 17804).

The term "topoisomerase II inhibitor" as used herein includes, but is not limited to the anthracyc- lines such as doxorubicin (including liposomal formulation, e.g. CAELYX), daunorubicin, epirubi- cin, idarubicin and nemorubicin, the anthraquinones mitoxantrone and losoxantrone, and the podophillotoxines etoposide and teniposide.

The term "microtubule active compound" relates to microtubule stabilizing, microtubule destabilizing compounds and microtublin polymerization inhibitors including, but not limited to taxanes, e.g. paclitaxel and docetaxel, vinca alkaloids, e.g., vinblastine, especially vinblastine sulfate, vincristine especially vincristine sulfate, and vinorelbine, discodermolides, cochicine and epo- thilones and derivatives thereof, e.g. epothilone B or D or derivatives thereof.

The term "alkylating compound" as used herein includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU or Gliadel).

The term "histone deacetylase inhibitors" or "HDAC inhibitors" relates to compounds which inhibit the histone deacetylase and which possess antiproliferative activity. This includes compounds disclosed in WO 02/22577, especially N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1 H-indol-3- yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, N-hydroxy-3-[4-[[[2-(2-methyl-1 - -indol-3-yl)- ethyl]-amino]methyl]phenyl]-2£-2-propenamide and pharmaceutically acceptable salts thereof. It further especially includes Suberoylanilide hydroxamic acid (SAHA). Compounds which target, decrease or inhibit activity of histone deacetylase (HDAC) inhibitors such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA) inhibit the activity of the enzymes known as histone deacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228 (formerly FR901228), Trichostatin A and compounds disclosed in US 6,552,065, in particular, A/-hydroxy-3-[4-[[[2-(2- methyl-1 - -indol-3-yl)-ethyl]-amino]methyl]phenyl]-2£-2-propenamide, or a pharmaceutically acceptable salt thereof and A/-hydroxy-3-[4-[(2-hydroxyethyl){2-(1 - -indol-3-yl)ethyl]-amino]- methyl]phenyl]-2£-2-propenamide, or a pharmaceutically acceptable salt thereof, especially the lactate salt.

The term "antineoplastic antimetabolite" includes, but is not limited to, 5-Fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylating compounds, such as 5-azacytidine and deci- tabine, methotrexate and edatrexate, and folic acid antagonists such as pemetrexed.

The term "platin compound" as used herein includes, but is not limited to, carboplatin, cis-platin, cisplatinum and oxaliplatin.

The term "compounds targeting/decreasing a protein or lipid kinase activity"; or a "protein or lipid phosphatase activity"; or "further anti-angiogenic compounds" as used herein includes, but is not limited to, c-Met tyrosine kinase and/or serine and/or threonine kinase inhibitors or lipid kinase inhibitors, e.g., a) compounds targeting, decreasing or inhibiting the activity of the platelet-derived growth factor-receptors (PDGFR), such as compounds which target, decrease or inhibit the activity of PDGFR, especially compounds which inhibit the PDGF receptor, e.g. a N-phenyl-2-pyrimidine- amine derivative, e.g. imatinib, SU101 , SU6668 and GFB-1 1 1 ; b) compounds targeting, decreasing or inhibiting the activity of the insulin-like growth factor receptor I (IGF-IR), such as compounds which target, decrease or inhibit the activity of IGF-IR, especially compounds which inhibit the kinase activity of IGF-I receptor, such as those compounds disclosed in WO 02/092599, or antibodies that target the extracellular domain of IGF-I receptor or its growth factors; c) compounds targeting, decreasing or inhibiting the activity of the Trk receptor tyrosine kinase family, or ephrin kinase family inhibitors; d) compounds targeting, decreasing or inhibiting the activity of the Axl receptor tyrosine kinase family; e) compounds targeting, decreasing or inhibiting the activity of the Ret receptor tyrosine kinase; f) compounds targeting, decreasing or inhibiting the activity of the Kit/SCFR receptor tyrosine kinase, e.g. imatinib; g) compounds targeting, decreasing or inhibiting the activity of the C-kit receptor tyrosine kinases - (part of the PDGFR family), such as compounds which target, decrease or inhibit the activity of the c-Kit receptor tyrosine kinase family, especially compounds which inhibit the c-Kit receptor, e.g. imatinib; h) compounds targeting, decreasing or inhibiting the activity of members of the c-Abl family, their gene-fusion products (e.g. BCR-AbI kinase) and mutants, such as compounds which target decrease or inhibit the activity of c-Abl family members and their gene fusion products, e.g. a N- phenyl-2-pyrimidine-amine derivative, e.g. imatinib or nilotinib (AMN107); PD180970; AG957; NSC 680410; PD173955 from ParkeDavis; or dasatinib (BMS-354825) i) compounds targeting, decreasing or inhibiting the activity of members of the protein kinase C (PKC) and Raf family of serine/threonine kinases, members of the MEK, SRC, JAK, FAK, PDK1 , PKB/Akt, and Ras/MAPK family members, and/or members of the cyclin-dependent kinase family (CDK) and are especially those staurosporine derivatives disclosed in US 5,093,330, e.g. midostaurin; examples of further compounds include e.g. UCN-01 , safingol, BAY 43-9006, Bryostatin 1 , Perifosine; llmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521 ;

LY333531/LY379196; isochinoline compounds such as those disclosed in WO 00/09495; FTIs; PD184352 or QAN697 (a P13K inhibitor) or AT7519 (CDK inhibitor); j) compounds targeting, decreasing or inhibiting the activity of protein-tyrosine kinase inhibitors, such as compounds which target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors include imatinib mesylate (GLEEVEC) or tyrphostin. A tyrphostin is preferably a low molecular weight (Mr < 1500) compound, or a pharmaceutically acceptable salt thereof, especially a compound selected from the benzylidenemalonitrile class or the S-arylbenzenemalonirile or bi- substrate quinoline class of compounds, more especially any compound selected from the group consisting of Tyrphostin A23/RG-50810; AG 99; Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; Tyrphostin B44; Tyrphostin B44 (+) enantiomer; Tyrphostin AG 555; AG 494; Tyrphostin AG 556, AG957 and adaphostin (4-{[(2,5-dihydroxyphenyl)methyl]amino}- benzoic acid adamantyl ester; NSC 680410, adaphostin); k) compounds targeting, decreasing or inhibiting the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4 as homo- or heterodimers) and their mutants, such as compounds which target, decrease or inhibit the activity of the epidermal growth factor receptor family are especially compounds, proteins or antibodies which inhibit members of the EGF receptor tyrosine kinase family, e.g. EGF receptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF related ligands, and are in particular those compounds, proteins or monoclonal antibodies generically and specifically disclosed in WO 97/02266, e.g. the compound of ex. 39, or in EP 0 564 409, WO 99/03854, EP 0520722, EP 0 566 226, EP 0 787 722, EP 0 837 063, US 5,747,498, WO 98/10767, WO 97/30034, WO 97/49688, WO 97/38983 and, especially, WO 96/30347 (e.g. compound known as CP 358774), WO 96/33980 (e.g. compound ZD 1839) and WO 95/03283 (e.g. compound ZM105180); e.g. trastuzumab (Herceptin™), cetuximab (Erbitux™), Iressa, Tarceva, OSI-774, CI-1033, EKB-569, GW-2016, E1 .1 , E2.4, E2.5, E6.2, E6.4, E2.1 1 , E6.3 or E7.6.3, and 7H-pyrrolo-[2,3-d]pyrimidine derivatives which are disclosed in WO 03/013541 ; and

I) compounds targeting, decreasing or inhibiting the activity of the Ron receptor tyrosine kinase; m) especially compounds targeting Aurora A kinase (see Lee et al., Cancer Res. 201 1 , 71_, 3225-3235, showing that Aurora A is a repressed effector target of the chromatin remodeling protein INA1/hSNF5 required for rhabdoid tumor survival), e.g. MLN8054 (Millenium), hesperidin (Boehringer-lngelheim), ZM-447439 (AstraZeneca), VX-680 (Vertex/Merck), AZD1 152 (AstraZe- neca), PHA-680632 (Nerviano), PHA-739358 (Nerviano), JNJ-770621 (Johnson and Johnson), CCT129202, AT9283 (Astrex Therapeutics), SU6669 (Pfizer), SNS314 (Sunesis Pharmaceuticals), CYC1 16 (Cyclacel), PF-03814735 (Pfizer), or MLN8237 (alisertib; Millenium/The Takeda Oncology Company) (cf. Dar et al., Mol. Cancer Ther. 9(2), 268 ff., 2010)..

Further anti-angiogenic compounds include compounds having another mechanism for their activity, e.g. unrelated to protein or lipid kinase inhibition e.g. thalidomide (THALOMID) and TNP-470.

The term "Compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase" includes, but is not limited to inhibitors of phosphatase 1 , phosphatase 2A, or CDC25, e.g. okadaic acid or a derivative thereof.

The term "Compounds which induce cell differentiation processes" includes, but is not limited to e.g. retinoic acid, α- γ- or δ-tocopherol or a- γ- or δ-tocotrienol.

The term "cyclooxygenase inhibitor" as used herein includes, but is not limited to, e.g. Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acid and derivatives, such as celecoxib (CELEBREX), rofecoxib (VIOXX), etoricoxib, valdecoxib or a 5-alkyl-2-arylaminophenylacetic acid, e.g. 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenyl acetic acid, lumiracoxib.

The term "bisphosphonates" as used herein includes, but is not limited to, etridonic, clodronic, tiludronic, pamidronic, alendronic, ibandronic, risedronic and zoledronic acid.

The term "mTOR inhibitors" relates to compounds which inhibit the mammalian target of rapa- mycin (mTOR) and which possess antiproliferative activity such as sirolimus (Rapamune®), everolimus (Certican™), CCI-779 and ABT578.

The term "heparanase inhibitor" as used herein refers to compounds which target, decrease or inhibit heparin sulfate degradation. The term includes, but is not limited to, PI-88.

The term "biological response modifier" as used herein refers to a lymphokine or interferons, e.g. interferon γ.

The term "inhibitor of Ras oncogenic isoforms", e.g. H-Ras, K-Ras, or N-Ras, as used herein refers to compounds which target, decrease or inhibit the oncogenic activity of Ras e.g. a "farnesyl transferase inhibitor" e.g. L-744832, DK8G557 or R1 15777 (Zarnestra).

The term "telomerase inhibitor" as used herein refers to compounds which target, decrease or inhibit the activity of telomerase. Compounds which target, decrease or inhibit the activity of telomerase are especially compounds which inhibit the telomerase receptor, e.g. telomestatin.

The term "methionine aminopeptidase inhibitor" as used herein refers to compounds which target, decrease or inhibit the activity of methionine aminopeptidase. Compounds which target, decrease or inhibit the activity of methionine aminopeptidase are e.g. bengamide or a derivative thereof.

The term "proteasome inhibitor" as used herein refers to compounds which target, decrease or inhibit the activity of the proteasome. Compounds which target, decrease or inhibit the activity of the proteasome include e.g. Bortezomid (Velcade™)and MLN 341.

The term "matrix metalloproteinase inhibitor" or ("MMP" inhibitor) as used herein includes, but is not limited to, collagen peptidomimetic and nonpeptidomimetic inhibitors, tetracycline derivatives, e.g. hydroxamate peptidomimetic inhibitor batimastat and its orally bioavailable analogue marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551 ) BMS-279251 , BAY 12- 9566, TAA21 1 , MMI270B or AAJ996.

The term "compounds used in the treatment of hematologic malignancies" as used herein includes, but is not limited to, FMS-like tyrosine kinase inhibitors e.g. compounds targeting, decreasing or inhibiting the activity of FMS-like tyrosine kinase receptors (Flt-3R); interferon, 1-b-D-a- rabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitors e.g. compounds which target, decrease or inhibit anaplastic lymphoma kinase.

The term "Compounds which target, decrease or inhibit the activity of FMS-like tyrosine kinase receptors (Flt-3R)" are especially compounds, proteins or antibodies which inhibit members of the Flt-3R receptor kinase family, e.g. PKC412, midostaurin, a staurosporine derivative, SU1 1248 and MLN518.

The term "HSP90 inhibitors" as used herein includes, but is not limited to, compounds targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90; degrading, targeting, decreasing or inhibiting the HSP90 client proteins via the ubiquitin proteasome pathway. Compounds targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90 are especially compounds, proteins or antibodies which inhibit the ATPase activity of HSP90 e.g., 17-allylamino,17-demet- hoxygeldanamycin (17AAG, 17-DMAG), a geldanamycin derivative; other geldanamycin related compounds; radicicol and HDAC inhibitors;! PI-504, CNF1010, CNF2024, CNF1010 from Con- forma Therapeutics; temozolomide, AUY922 from Novartis.

The term "antiproliferative antibodies" as used herein includes, but is not limited to, trastuzumab (Herceptin™), Trastuzumab-DM1 .erbitux, bevacizumab, rituximab, PR064553 (anti-CD40) and 2C4 Antibody. By antibodies is meant anx variant as defined above, e.g. intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least 2 intact antibodies, and antibodies fragments so long as they exhibit the desired biological activity.

The term "antileukemia compounds" includes, for example, Ara-C, a pyrimidine analog, which is the 2 ^'-alpha-hydroxy ribose (arabinoside) derivative of deoxycytidine. Also included is the purine analog of hypoxanthine, 6-mercaptopurine (6-MP) and fludarabine phosphate. For the treatment of acute myeloid leukemia (AML), compounds of formula (I) can be used in combination with standard leukemia therapies, especially in combination with therapies used for the treatment of AML. In particular, compounds of formula (I) can be administered in combination with, e.g., far- nesyl transferase inhibitors and/or other drugs useful for the treatment of AML, such as Dauno- rubicin, Adriamycin, Ara-C, VP-16, Teniposide, Mitoxantrone, Idarubicin, Carboplatinum and PKC412.

"Somatostatin receptor antagonists" as used herein refers to compounds which target, treat or inhibit the somatostatin receptor such as octreotide, lanreotide and pasireotide .

"Tumor cell damaging approaches" refer to approaches such as ionizing radiation. The term "ionizing radiation" referred to above and hereinafter means ionizing radiation that occurs as either electromagnetic rays (such as X-rays and gamma rays) or particles (such as alpha and beta particles). Ionizing radiation is provided in, but not limited to, radiation therapy and is known in the art. See Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, Devita et al., Eds., 4^th Edition, Vol. 1 , pp. 248-275 (1993).

The term "EDG binders" (S1 P receptor modulators) as used herein refers a class of immunosuppressants that modulates lymphocyte recirculation (homing), such as FTY720 or other propanolamine derivatives.

The term "kinesin spindle protein inhibitors" is known in the field and includes SB715992 or SB743921 from GlaxoSmithKline, pentamidine/chlorpromazine from CombinatoRx.

The term "MEK inhibitors" is known in the field and includes ARRY142886 from Array

PioPharma, AZD6244 from AstraZeneca, PD181461 from Pfizer, leucovorin.

The term "ribonucleotide reductase inhibitors" includes, but is not limited to to pyrimidine or purine nucleoside analogs including, but not limited to, fludarabine and/or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (especially in combination with ara-C against ALL) and/or pentostatin. Ribonucleotide reductase inhibitors are especially hydroxyurea or 2-hydroxy-1 H-isoindole-1 ,3-dione derivatives, such as PL-1 , PL-2, PL-3, PL-4, PL-5, PL-6, PL-7 or PL-8 mentioned in Nandy et al., Acta Oncologica, Vol. 33, No. 8, pp. 953- 961 (1994).

The term "S-adenosylmethionine decarboxylase inhibitors" as used herein includes, but is not limited to the compounds disclosed in US 5,461 ,076.

Also included are in particular those compounds, proteins or monoclonal antibodies of VEGF / VEGFR disclosed in WO 98/35958, e.g. 1 -(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceutically acceptable salt thereof, e.g. the succinate, or in WO 00/09495, WO 00/27820, WO 00/59509, WO 98/1 1223, WO 00/27819 and EP 0 769 947; those as described by Prewett et al, Cancer Res, Vol. 59, pp. 5209-5218 (1999); Yuan et al., Proc Natl Acad Sci U S A, Vol. 93, pp. 14765-14770 (1996); Zhu et al., Cancer Res, Vol. 58, pp. 3209-3214 (1998); and Mor- denti et al., Toxicol Pathol, Vol. 27, No. 1 , pp. 14-21 (1999); in WO 00/37502 and WO 94/10202; ANGIOSTATIN, described by O'Reilly et al., Cell, Vol. 79, pp. 315-328 (1994); ENDOSTATIN, described by O'Reilly et al., Cell, Vol. 88, pp. 277-285 (1997); anthranilic acid amides; ZD4190; ZD6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, e.g. rhuMAb and RHUFab, VEGF aptamer e.g. Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 lgG1 antibody, Angiozyme (RPI 4610) and Bevacizumab.

"Photodynamic therapy" as used herein refers to therapy which uses certain chemicals known as photosensitizing compounds to treat or prevent cancers. Examples of photodynamic therapy include treatment with compounds, such as e.g. VISUDYNE and porfimer sodium (examples for photosensitizing compounds).

"Angiostatic steroids" as used herein refers to compounds which block or inhibit angiogenesis, such as, e.g., anecortave, triamcinolone, hydrocortisone, 1 1 -a-epihydrocotisol, cortexolone, 17a-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone and dexamethasone.

"Corticosteroids" as used herein includes, but is not limited to compounds, such as e.g. fluocino- lone, dexamethasone; in particular in the form of implants.

Description of the Figures (the description of the figures is also part of the invention disclosure and completes the Examples given below wherein the respective Figures are cited):

Fig. 1 : FGF-R inhibition with BGJ398 impairs growth of MRT cell lines in vitro.

Proliferation assays withBGJ398 in A204 (A), G401 (B) and G402 (C) cells. Cell were plated in 96-wells and treated with BGJ398 at the indicated concentrations for 4 days. The effect on proliferation was assayed by methylene blue staining. Half maximal inhibitory concentrations (IC₅₀) for BGJ398 were calculated using XLfit and are indicated in the graphs.

Fig. 2: FGF-R-dependency of the MRT cell lines A204, G401 and G402.

(A) Immunoblot analysis of p-FRS2 and p-ERK1/2 in MRT lines treated with DMSO or BGJ398 for 40 min as indicated. Total ERK1/2 and β-Tubulin expression was used to monitor equal loading. (B) Quantitative RT-PCR (qRT-PCR) analysis of FGFR1 and FGFR2 mRNA expression in MRT cell lines and soft tissue cancer lines SKLMS1 and SKUT1 . Expression values are given as average with standard errors of the mean (SEM) (n≥3) with respect to GAPDH mRNA levels (arbitrarily set as 100). (C) qRT-PCR and immunoblot analysis of SNF5-deficiency in MRT lines. SKLMS1 and SKUT1 cells were used as positive controls for SNF5 expression. SNF5 mRNA expression is given as average with SEM (n≥3) with respect to GAPDH mRNA levels (arbitrarily set as 100). β-Tubulin expression was used to monitor equal loading.

Fig. 3: shRNA-mediated knockdown of FGFR1 in G402 cells

(A) Effect of FGFR1 -targeting in monolayer cell proliferation assays in G402 cells with stable integration of doxycycline-inducible shRNA expression vectors. Doxycycline (25 ng/ml) was added to the growth medium as indicated. Cell growth was monitored at the indicated days after cell seeding. Results are shown as relative growth to the corresponding non-doxycycline treated cells from triplicate experiments. (B) Efficacy of FGFR1 -knockdown was monitored after 7 days of doxycycline-induction by immunoblot.

Fig. 4: Re-expression of SNF5 in MRT lines abrogates FGF-R expression.

(A) Immunoblot analysis (left panel) of FGFR2 protein expression in G401 cells five days post retroviral transduction of SNF5. β-Tubulin expression was used to monitor equal loading.

FGFR2 mRNA levels (right panel) were determined by qRT-PCR. Expression is shown as relative levels to control infected cells. Data are given as average with SEM (n=3). FGFR2 mRNA expression values were normalized to GAPDH mRNA copies. Data were compared by unpaired Student's t test; ^* p<0.05. (B) Analysis of FGFR1 protein and mRNA expression in G402 cells upon re-expression of SNF5 as described in (A)

Fig. 5: Re-expression of SNF5 in SNF5-deficient cell lines abrogates FGF-R expression.

Effect of SNF5 re-expression on FGFR1 levels in MRT line A204 (A) and KYM1 rhabdomyosarcoma cells (B) and on FGFR2 expression in HLC1 lung adenocarcinoma cells (C). Protein expression was analyzed by immunoblot 5 days post retroviral transduction of SNF5. β-tubulin expression was used to monitor equal loading.

Fig. 6: Cell cycle inhibition does not affect FGFR1 expression in the G402 MRT line.

(A) Effect of 10 nM Staurosporine treatment on FGFR1 expression in G402 cells at the indicated times. Cleaved PARP expression was analyzed to monitor efficacy of Staurosporine treatment, β-tubulin was used to control for equal loading.

Fig. 7: SNF5 loss of function induces FGFR2 expression in human fibroblasts. (A) Effect of siRNA-mediated knockdown of SNF5 on FGFR2 expression in BJ cells. SNF5 and FGFR2 expression levels were analyzed by qRT-PCR at 72 h post siRNA transfection. Expression is shown as relative levels to cells transfected with non-targeting control siRNA and is given as average with SEM (n≥3). Expression values were normalized to GAPDH mRNA copies. (B) Immunoblot analysis of FGFR2 expression upon knockdown of SNF5 in BJ cells as described in (A). β-Tubulin expression was used to monitor equal loading. (C) Effect of siRNA- mediated knockdown of BRG1 on FGFR2 expression in BJ cells. SNF5 and FGFR2 expression levels were analyzed as described in (A).

Fig.8: SNF5 is recruited to the FGFR2 promoter in BJ cells.

(A) Schematic overview of the human FGFR2 promoter. Amplicons of primer pairs used for ChIP are shown as small squares and location is indicated relative to the transcriptional start site (TSS, +1). Exons are shown as large boxes. (B) FGFR2 promoter occupancy by SNF5 in BJ cells. Fold enrichment from chromatin immunoprecipitations (ChIP) with a SNF5-specific antibody compared to an IgG control was analyzed by qPCR using the primer pairs indicated in (A). (C) Fold enrichment of the negative control locus IGX1A and the promoter region of the known SNF5 target gene CDKN1A. Fold enrichment is given as average with SEM (n≥3). Data were compared by unpaired Student's t test with respect to the fold enrichment of the IGX1 A locus; ^* p<0.05.

Fig. 9: FGFR inhibition by BGJ398 impairs MRT growth in vivo

G401 MRT cells were grown subcutaneously in nude mice. Treatment with BGJ398 at 50 mg/kg body weight started when tumor volume reached at least 100 mm³. Mice were treated daily for 24 days. Tumor volume changes over the course of treatment are shown as average with SEM (n≥7). Statistical analysis was performed by unpaired Student's t test with respect to vehicle- treated controls (^*p < 0.05).

Examples:

The following Examples, while constituting particular embodiments of the invention, illustrate the invention without limiting the scope thereof.

Immunoblot analysis RNA purification and quantitative real-time PCR (qPCR)

RNA was purified with the RNeasy Mini kit . Random hexamer primed cDNA was synthesized with 0.5-2 μ9 RNA and MuLV reverse transcriptase. Quantitative real-time PCR was performed in an iQ5 Real-Time PCR Detection System.

Primer sequences used are

f-CCAAGGTCATCCATGACAAC (SEQ ID NO: 1)and

r-AG AG G CAG G G ATG ATGTTCT (SEQ ID NO: 2)for human GAPDH,

f-GGGACATTCACCACATCGACTA (SEQ ID NO: 3) and

r-GGGTGCCATCCACTTCACA (SEQ ID NO: 4)for human FGFR1,

f-TG AAG G AAGG ACACAG AATG G A (SEQ ID NO: 5) and

r-GCCAACAGTCCCTCATCATCA (SEQ ID NO: 6) for human FGFR2,

f-GTGATCCATGAGAACGCATC (SEQ ID NO: 7) and

r-TCAGGCGTCATCAACTTCTC (SEQ ID NO: 8) for human SNF5,

f-CTCCGAGAAGGACAAGAAGG (SEQ ID NO: 9) and

r-TGCATGATGGTGTTCATCAG (SEQ ID NO: 10) for human BRG1 .

Generation of stable cell lines with hairpin shRNAs and proliferation assays

Hairpin shRNAs were cloned in pLKO-Tet-On vector to produce replication-incompetent lentiviruses. Upon lentiviral infection, stable pools of G401 cells were generated by selection with puromycin at a concentration of 1.5 μ9/ηιΙ for 5 days. For monolayer cell proliferation assays, cells were seeded in 96-well plates and shRNAs were induced with doxycycline . Cell proliferation was evaluated by methylene blue staining.

shRNA sequences were as follows:

human FGFR1 -sh1 : GCCAAGACAGTGAAGTTCAAA (SEQ ID NO: 1 1 ),

human FGFR1-sh2: TCTTGAAGACTGCTGGAGTTA (SEQ ID NO: 12),

NT-shRNA: AGAAGAAGAAATCCGTGTGAA (SEQ ID NO: 13).

NT-shRNA is a non-targeting sequence that is used as a control. Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) with BJ cells was performed using the SimpleChIP Enzymatic Chromatin IP Kit . Primers sequences used to monitor enrichment of human FGFR2 promoter DNA were

f-AGGCTGAAAGCACACAGTTG (SEQ ID NO: 14) and r-CCTGGTCTCAGTGGGAGTTT (SEQ ID NO: 15) for -9133,

f-TGCGAAGAAAAGAGACCTCA (SEQ ID NO: 16) and

r-AAGGGCAGAAAAGCCAGTAA (SEQ ID NO: 17) for -2420,

f- AACTTAAGCACG G CTG CTC (SEQ ID NO: 18) and

r- CAACTGCACACCAAGCTGTA (SEQ ID NO: 19) for -1021,

f- AACATTTCCAAGTGGCTTCC (SEQ ID NO: 20) and

r-ACTTTAAAATGCGCCTGCTT (SEQ ID NO: 21 ) for -462,

f-CTCTGAGCCTTCGCAACTC (SEQ ID NO: 22) and

r- AAG AAAG G ACTC AG G CTTG G (SEQ ID NO: 23) for +207,

f- AGGACCACTCTTCTGCGTTT (SEQ ID NO: 24) and

r- GATTACCTTGAATGGCAACG (SEQ ID NO: 25) for +397,

f-TCTGTGGCTGCATAGGTGAT (SEQ ID NO: 26) and

r-TAGCAGAGGCAGAACTTCCA (SEQ ID NO: 27) for +639,

f-CGAACTGGACCGACTTTTTC (SEQ ID NO: 28) and

r-AATGAGCGCGCAAGTTAGA (SEQ ID NO: 29) for +1108,

f-TGCTTTTGTAGTTGCCCTTG (SEQ ID NO: 30) and

r-CTCAGATACGTGCAGCCACT (SEQ ID NO: 31 ) for +4118

This primer pair is specific for the CDKN1 A promoter region.

SNF5 re-expression in MRT lines

Retroviral supernatants were collected from 293FT transfected with pBabe-SNF5 and pCL-10A1 packaging vector . For re-expression of SNF5, MRT lines were plated in 6-well plates at initial densities of 1 .2 to 2.4 x 10⁵ cell/well and retroviral supernatant was applied the following day twice for 4 h. Puromycin selection was initiated at 48 h post retroviral transduction and RNA and protein lysates or RNA were prepared at day 3 post selection.

siRNA transfection

Cells were plated in 6-well plates at initial densities of 10⁵ cells/well one day before transfection with siRNA at concentrations of 50 nM using Lipofectamine RNAiMAX transfection reagent as described by the manufacturer. Oligos were obtained from Qiagen and included assays SI00726810, SI04237457, SI04297762 for targeting of SNF5 and SI00047579, SI03098998, SI00047586 for BRG1. AllStars Negative Control oligos were used as non-silencing siRNA control. RNA or protein lysates were prepared at 72 h post transfection. Proliferation assays

For manual cell proliferation assays, cells were seeded in 96-well plates at a density of 10³ to 10⁴ cell per well in a volume of 100 μΙ. Media containing dilutions of BGJ398 or DMSO was added 24 hours thereafter. After 4 days cell density was analyzed using methylene blue staining. The concentration of compound providing 50% of proliferation inhibition (IC50) was determined using XLfit (idbs).

Cell cycle inhibition studies

For Staurosporine treatment cells were plated in 6-well plates at initial densities of 5 x 10⁵ cells/well and were analyzed after 48 h or 5 days post treatment. Lysates were prepared with M- PER lysis buffer supplemented with Complete protease inhibitors cocktail and PhosSTOP phosphatase inhibitor cocktail tablets . Cellular lysates were separated by SDS-PAGE, and transferred to PVDF membranes. Proteins were visualized using antibodies FGFR1 ((#sc- 57132, Santa Cruz), FGFR2 ((#sc-122, Santa Cruz), cleaved PARP (#9541 , Cell Signaling), appropriate horseradish peroxidase-labeled secondary antibodies and a chemiluminescence detection reagent . Anti- -tubulin was used as a loading control.

Xenograft mouse models and anti-tumor efficacy studies

Trypsinized G401 cell monolayers were washed and suspended at 5 x 10⁷ cells/ml in cold phosphate-buffered saline with 50% Matrigel . Each animal was inoculated subcutaneously in the right flank with 0.2 ml of the suspension (1 x 10⁷ cells). The tumors were periodically callipered in two dimensions to monitor growth as the mean volume approached 160-230 mm³. Eight days after tumor cell implantation, on day 1 of the study, the animals were sorted into two groups of ten mice, with treatment with BGJ398 or vehicle control was initiated. Treatment was performed once daily by oral gavage. Tumor volumes were monitored at the indicated times over the course of treatment. Tumor size, in mm³, was calculated from: Tumor Volume = 2 / w² x I; where w = width and I = length, in mm, of the tumor.

Statistical analysis

All data shown represent mean ± standard error of the mean (SEM). Statistical analyses were performed using Student's t tests (two-tailed). A significance level of p<0.05 is indicated by an asterisk (^*).

Example 1 : FGF-R inhibition with BGJ398 impairs growth of MRT cell lines in vitro Fig. 1 shows that three MRT cell lines (A204, G410 and G402) are sensitive to treatment with BGJ398. This shows that BGJ398 is active against MRT cell proliferation.

Proliferation assay with BGJ398 over four days led to impaired proliferation at half-maximal inhibitory concentrations (IC50) < 250 nM in all three cell lines, indicating a strong dependence of the MRT lines on FGF-R signaling for proliferation.

Example 2: FGF-R-dependency of the MRT cell lines A204, G401 and G402

Fig. 2 shows that the MRT cell lines mentioned in Example 1 have a constitutively active FGF-R pathway (Fig. 2A). MRT lines show high expression of FGFR1 or FGFR2 (Fig. 2B) in the absence of SNF5 (Fig. 2C). FGFR levels are high in the MRTs in comparison to SNF5- expressing control cells SKLMS1 and SKUT1 . This finding supports the involvement of FGF-R in the manifestation of MRT proliferation. Taken together, the data from Examples 1 and 2 indicate that MRT lines aberrantly activate and depend on FGF-R signaling.

Example 3: shRNA-mediated knockdown of FGFR1 in G402 cells

Fig. 3 shows that the MRT line G402 depends on FGFR1 expression for proliferation.

Proliferation of G402 cells is inhibited upon knock-down of FGFR1 expression. This finding supports the essential function of FGF-R signaling in MRT growth. Cell lines derived from parental G402 cells were established with stable integration of doxycycline-inducible shRNA- expression vectors by lentiviral infection. Those lines were used to study the growth

dependency on FGFR1 expression in this MRT model. The induction of shRNA expression led to a strong anti-proliferative effect in two lines expressing different FGFR1 -targeting shRNAs. In contrast, the expression of a non-targeting control shRNA did not affect proliferation of G402 cells (Fig. 3A). The effective knockdown of FGFR1 levels upon induction of shRNA-expression was monitored by immunoblot (Fig. 3B). This result confirms the FGFR-dependency of the MRT line observed from the proliferation studies using BGJ398 (Example 1 ). Furthermore, this shows that FGFR1 is an essential driver of proliferation in G402 cells. This example shows that knockdown of FGFR1 , induced by doxycycline, in G402 cells lead to diminished MRT growth, see Fig. 3. This further supports the general concept that MRT and related diseases are dependent on FGF-R activity. See also Description of the Figures for more details.

Example 4: (A) Re-expression of SNF5 in MRT lines abrogates FGF-R expression This example shows that re-expression of SNF5 causes a decrease in FGF-R expression in MRT lines and together with the Examples 1 -3 it supports the manifestation that absence or loss of function of SNF5 causes aberrant FGF-R expression and FGF-R pathway activation.

MRT lines are characterized by the absence of the tumor suppressor SNF5 (Fig. 2C). We thus aimed to investigate whether loss of SNF5 function would underlie the elevated FGFR expression observed in MRT lines. To this end, we re-introduced SNF5 in the MRT lines G401 and G402 by means of retroviral transduction. In both G401 and G402 cells we found that re- expression of SNF5 abrogates elevated protein expression of FGFR2 and FGFR1 , respectively, which was paralleled by a decrease of mRNA levels (Fig. 4A and B).

(B) Re-expression of SNF5 in SNF5-deficient cell lines abrogates FGF-R expression

This example shows high FGF-R expression in another MRT line and in other, non-MRT lines with absence of SNF5 (KYM1 and HLC1 ). The high expression of FGF-Rs is abrogated upon re- expression of SNF5. This is further supporting that FGF-R expression is aberrantly increased when SNF5 is absent or reduced in function in MRTs and related diseases.

We observed a striking repression of FGFR1 levels upon re-expression of SNF5 in the MRT line A204 (Fig. 5A). Interestingly, two SNF5-deficient non-MRT lines, the rhabdomyosarcoma line KYM1 and the lung adenocarcinoma line HLC1 , also displayed high levels FGFR1 or FGFR2, respectively, which were abrogated upon reconstitution of SNF5 (Fig. 5B and C). (C) Cell cycle inhibition does not affect FGFR1 expression in the G402 MRT line

This example shows that the effect observed upon re-expression of SNF5 (Examples 4A and 4B) are specific for the function of SNF5. This supports that SNF5 has a direct repressive function on FGF-R expression and that the effects observed in Examples 4A and 4B are not unspecific side-effects of SNF5 re-expression.

Re-expression of SNF5 causes cell cycle arrest due to the activation of p16^INK4A and inhibition of the CDK4/RB/E2F pathway. To rule out an unspecific effect on FGFR expression in response to the cell cycle arrest caused by forced SNF5 re-expression in the MRT lines, we treated the MRT line G402 with the cell cycle inhibitor Staurosporine, which did not affect FGFR1 expression in these cells (Fig. 6). In summary, the data shown in Example 4 illustrates that SNF5 negatively affects expression of FGFR1 and FGFR2 and indicates that MRT cell lines and other cancer lines with absent SNF5 express elevated levels of FGF-Rs in consequence of SNF5 loss of function.

See also Description of the Figures for more details.

Example 5: SNF5 loss of function induces FGFR2 expression in human fibroblasts

This data shows that depletion of SNF5 in a SNF5-expressing cell lines causes increased expression of FGFR2. This data supports that aberrant FGF-R expression can occur when SNF5 function is lost.

In addition to the re-expression studies in SNF5-deficient cell lines, we monitored the effect of siRNA-mediated SNF5-knockdown on FGFR expression in cell lines endogenously expressing SNF5. We found that knockdown of SNF5 led to increased mRNA levels of FGFR2 in BJ cells (Fig. 7A), a non-immortalized human fibroblast line. The transcriptional induction of FGFR2 upon SNF5 knockdown was paralleled by an increase in FGFR2 protein levels (Fig. 7B). Since SNF5 is a core component of the SWI/SNF chromatin remodeling complex, we tested whether inhibition of SWI/SNF function by knockdown of the ATPase core subunit BRG1 would similarly affect FGFR2 expression in BJ cells and found that loss of BRG1 function resulted in a slight but consistent induction of FGFR2 (Fig. 7C). These results show that SNF5 represses FGFR2 transcription in a SWI/SNF-dependent fashion in human fibroblasts.

Example 6: SNF5 is recruited to the FGFR2 promoter in BJ cells

This example shows that SNF5 is bound to the FGFR2 promoter in a SNF5 expressing cell line and it supports the finding of Example 4 that SNF5 has a direct function in repressing FGF-R expression.

SNF5 has been shown to be directly recruited to target gene promoters and to suppress gene transcription by modification of the adjacent chromatin structure. To determine whether FGFR expression is controlled by SNF5 in a similar fashion we analyzed SNF5 localization to the FGFR2 promoter in BJ cells by chromatin immunoprecipitation (ChIP). To this end, we designed a series of primer pairs spanning a region from 9 kb upstream to 4 kb downstream the transcriptional start site (TSS) in the human FGFR2 promoter (Fig. 8A) and identified occupancy of the FGFR2 promoter by SNF5 with a focal peak binding site closely following the TTS (Fig. 8B). At this site, we found an approximately 10-fold enrichment of ChlP-DNA compared to the basal enrichment levels present at distal sites and the negative control locus IGX1 A, and similar to the fold enrichment observed for the known SNF5 target gene CDKN1 A (Fig. 8C). This indicates that SNF5 exerts its repressive function on FGFR2 expression via a direct interaction with the FGFR2 promoter locus.

Example 9: Pharmacological FGFR signaling inhibition by BGJ398 impairs MRT growth in vivo

This results shows that inhibition of FGF-R signaling is able to repress growth of a MRT in vivo. Together with the in vitro data from Examples 1 and 3 this supports the finding of an aberrant function of FGF-R signaling in MRTs and that FGF-R inhibition with BGJ938 is active in repressing MRT growth.

Besides the in vitro analysis of FGFR-dependence in MRT cells, we aimed to test the effect of pharmacological FGFR inhibition on MRT growth in vivo. To this end we applied the MRT line G401 as a xenograft transplantation model and found that BGJ398 treatment significant impaired tumor growth in vivo (Fig. 9). This result is in line with the anti-proliferative effect of FGFR inhibition on G401 cells in vitro and indicates that pharmacological inhibition of FGFR signaling is also able to suppress growth of MRTs in vivo.

From the Examples it follows that we identified and validated FGF-R-dependency of MRT cell models and found that generally SNF5-deficiency can be expected to correlate with elevated expression of FGFR1 or FGFR2.

These findings highlight FGF-R inhibition as a novel therapeutic option for the treatment of MRTs and other diseases involving lack of SNF5 activity.

Claims

Claims:

1. An FGFR inhibitor or a pharmaceutically acceptable salt thereof for use in a

therapeutically effective amount treating a disease associated with at least partial lack of SNF5 activity.

2. The FGFR inhibitor of claim 1 , wherein the disease is a proliferative disease.

3. The FGFR inhibitor of claim 1 , wherein the disease is selected from the group consisting of Rhabdoid tumors, familiar schwannomatosis, Small-cell hepatoblastoma, extraskeletal myxoid chondrosarcomas, undifferentiated sarcomas, epitheloid sarcomas,

meningiomas and poorly differentiated chordomas.

4. The FGFR inhibitor of claim 1 , wherein the disease is a malignant Rhabdoid tumor.

5. The FGFR inhibitor of any one of the preceding claims being a small molecule FGFR inhibitor, an FGFR inhibiting antibody or an SiRNA of FGFR.

6. The FGFR inhibitor of claim 5 having an IC50 value less than 100nm in the proliferation assay as defined in Example 1.

7. The FGFR inhibitor of any one the claims 1 to 4 selected from the group consisting of:

(1 ) BGJ398 which is 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1 -{6-[4-(4-ethylpiperazin-1 - yl)-phenylamino]-pyrimidin-4-yl}-1 -methyl-urea, a pharmaceutically acceptable salt, an active metabolite and/or an N-oxide thereof;

(2) AZD-4547 which is N-[5-[2-(3,5-dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3,5- dimethylpiperazin-1-yl)benzamide, a pharmaceutically acceptable salt, an active metabolite and/or an N-oxide thereof; and

(3) TKI258 which is amino-5-fluoro-3-[6-(4-methylpiperazin-1 -yl)-1 H-benzimidazol-2- yl]quinolin-2(1 H)-one), a pharmaceutically acceptable salt, an active metabolites and/or its tautomer 4-amino-5-fluoro-3-[5-(4-methylpiperazin-1 -yl)-1 H-benzimidazol-2- yl]quinolin-2(1 H)-one and/or a pharmaceutically acceptable salt thereof.

8. The FGFR inhibitor of claim 7 being the phosphate salt of BGJ398.

9. A pharmaceutical composition for use in treatment of a proliferative disease associated with at least partial lack of SNF5 activity.

10. A pharmaceutical combination for use in the treatment of a disease associated with at least partial lack of SNF5 activity.

1 1 . An FGFR inhibitor for use in combination with at least one another anti-proliferative

agent in the treatment of a disease associated with at least partial lack of SNF5 activity.

12. A method of treating a disease associated with at least partial lack of SNF5 activity comprising the steps of administering therapeutically effective amount of FGFR inhibitor, alone or in combination with at least another active ingredient in therapeutically effective amount, preferably an anti-proliferative amount, to said patient.

13. Method for determining whether a patient is sensitive to an FGFR inhibitor treatment, comprising the steps of a) determining whether said patient at least partially lacks of SNF5 activity by qRT-PCR, immunoblotting, sequencing or other mutation detecting methods; where b) if a) is affirmative, then said patient is sensitive to an FGFR inhibitor treatment.