Field of the Invention The invention relates to the use of bcr-abl inhibitors, such as imatinib, dasatinib and nilotinib, in the treatment of ABL overexpressing B-cell lymphoma, in particular mucosa- associated lymphoid tissue (MALT) lymphoma.

Background Art

Mucosa-associated lymphoid tissue (MALT) lymphoma account for 50% of all primary gastric lymphoma. The development of gastric MALT lymphoma is tightly linked to chronic infection with the human bacterial pathogen Helicobacter pylori; consequently, antibiotic eradication therapy is now the first line treatment for this malignancy. MALT

lymphomagenesis is initiated by H. py/or/ ^' -associated chronic inflammation and the subsequent accumulation of gastric organized lymphoid tissue ("MALT"), from which individual neoplastic clones may grow out and invade the adjacent epithelium, forming the lympho-epithelial lesions that are a hallmark of low grade MALT lymphoma (Sagaert X. et al., Nat Rev Gastroenterol Hepatol 2010, 7(6): 336-346).

It has recently been shown that MALT lymphoma tumor immunoglobulins are clonal, somatically hypermutated and polyreactive, i.e. they bind to a variety of unrelated self- and foreign antigens (Craig V.J. et al., Blood 1 15:581-591 (2010)). The same panel of antigens that are recognized by MALT lymphoma surface immunoglobulin induce proliferation of explanted tumor cells, supporting the notion that early low grade gastric MALT lymphoma is an antigen-dependent malignancy. Low grade gastric MALT lymphoma are infiltrated by large numbers of T-cells, which are polarized to produce Th2 cytokines such as interleukin-4. The depletion of T-cells prevents the proliferation of tumor cells ex vivo and induces tumor regression in a mouse model of gastric MALT lymphoma, implying a synergistic role for T-cell-derived signals and B-cell receptor-mediated antigen recognition during early MALT lymphoma pathogenesis.

MALT lymphoma are a sub-group of ABL overexpressing B-cell lymphoma. Other such lymphoma of the stomach are, for example, diffuse large B cell lymphoma (DLBCL). MicroRNAs are well-conserved, 18-25 nucleotide long non-coding RNAs with pivotal roles in post-transcriptional gene regulation. miRNA expression patterns correlate with particular cancer types and are predictive of clinical outcome. Over 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites. Many miRNAs are known to function as tumor suppressors, regulating the expression of oncoproteins such as RAS and c-MYC.

Bcr-abI fusion protein is an oncogene fusion protein consisting of BCR (breakpoint cluster region) and ABL (ABL1 , Abelson proto-oncogene 1). It is associated with Philadelphia chromosome, a reciprocal translocation between chromosome 22 and 9, and is often found in chronic myeloid leukemia patients, but also connected with acute lymphoblastic leukemia. The bcr-abl fusion protein encodes an unregulated cytoplasm-targeted tyrosine kinase that allows the cells to proliferate without regulation by cytokines. Recently efficient bcr-abl inhibitors have been developed. Imatinib, also known as

GleevecO/Glivec® (Novartis), dasatinib (Sprycel®, Bristol-Myers Squibb) and nilotinib (Tasigna®, Novartis). Further bcr-abl inhibitors are in development. Imatinib is a very efficient drug for the treatment of chronic myelogenous leukemia (CML) and

gastrointestinal stromal tumors (GIST). The other approved bcr-abl inhibitors are of use, if imatinib resistance is observed, and have a different side effect profile.

Summary of the Invention

The invention relates to bcr-abl inhibitors, such as imatinib, dasatinib and nilotinib, for use in the treatment of ABL overexpressing B-cell lymphoma, in particular mucosa-associated lymphoid tissue (MALT) lymphoma, and to compositions further comprising an antibiotic and/or proton-pump inhibitor for such use. Likewise it relates to bcr-abl inhibitors, such as imatinib, dasatinib and nilotinib, for use in the preparation of a medicament for the treatment of ABL overexpressing B-cell lymphoma, in particular mucosa-associated lymphoid tissue (MALT) lymphoma, and for a method of treatment of ABL overexpressing B-cell lymphoma, in particular mucosa-associated lymphoid tissue (MALT) lymphoma, comprising administering a bcr-abl inhibitor, such as imatinib, dasatinib and nilotinib. Brief Description of the Figures

Figure 1. miR-203 and its target ABL1 are dysregulated in MALT lymphoma.

(A) LNA (locked nucleic acid) real-time RT-PCR validation of miR-203 expression in human (h) samples of tonsil (T) and MALT lymphoma (MALT-L). miR-203 levels were normalized to U6 snRNA expression.

(B) Expression levels of miR-203 in human gastritis (G) and MALT lymphoma (MALT-L) FFPE tissue samples as determined by LNA RT-PCR.

(C) Expression levels of miR-203 in murine (m) MALT lymphoma (MALT-L) and corresponding gastritis (G) samples as determined by LNA RT-PCR.

(D) ABL1 expression in MALT-L and corresponding gastritis material derived from eight patients. ABL1 transcript levels were quantified by real-time RT-PCR and normalized to GAPDH expression.

(E) miR-203 and ABL1 expression in four pairs of murine MALT lymphoma and corresponding gastritis from the same stomach.

Figure 2. The miR-203 promoter is specifically hypermethylated in MALT lymphoma. Bisulfite sequencing of the miR-203 promoter region in human tonsil (T), gastritis (G), MALT lymphoma (MALT-L), and gastric diffuse large B-cell lymphoma (gDLBCL, i.e. high grade MALT lymphoma) samples. Three representative samples are shown for each tissue type (four for MALT-L), and three single clones are represented for each individual sample. Black and white circles represent methylated and unmethylated CpG

dinucleotides, respectively. All 61 sequenced CpGs are indicated. Figure 3. The expression of miR-203 is regulated epigenetically and controls ABL1 expression and lymphoma cell proliferation.

(A) The DNA methylation status of the miR-203 promoter region in BL2 cells as determined by bisulfite genomic sequencing. Cells were either treated with 5 μΜ

5'-azacytidine (Aza), alone or in combination with 3 mM 4-phenylbutyric acid (Aza + PBA), or left untreated (-). Three single clones were sequenced for each treatment and the percentage of methylated CpG dinucleotides is shown. Black and white circles represent methylated and unmethylated CpG, respectively.

(B, C) Real-time RT-PCR analysis of miR-203 and ABL1 transcript levels (normalized) in BL2 cells with and without drug treatment.

(D) ABL1 protein levels of the experiment outlined in A-C as assessed by Western blot analysis with a-tubulin (aT) serving as a loading control. (E) Normalized ABL1 expression of BL2 cells transfected with either miR-203 precursor molecules (miR-203) or negative control precursor molecules (C) at 50 nM or 100 nM final concentration.

(F) ABL1 protein levels of the experiment described for E as assessed by Western blot analysis with a-tubulin (aT) serving as loading control.

(G) The proliferation of cells transfected as described for E as determined by

[ ³ H]-thymidine incorporation.

Figure 4. miR-203 re-expression and pharmacological ABL inhibition block MALT lymphoma growth in vitro and in vivo.

(A-C) Primary murine MALT lymphoma cells were transduced with lentiviruses carrying a miR-203-expressing or empty vector. miR-203 and ABL1 expression was assessed by real time RT-PCR; the proliferation of transduced cells was determined by [ ³ H]-thymidine incorporation with (dark grey) or without (light gray) stimulation with H. felis sonicate. C = control (vector alone).

(D, E) [ ³ H]-thymidine incorporation of gastric (D) and advanced splenic (E) primary murine MALT lymphoma cells stimulated with H. felis sonicate and treated with increasing concentrations of imatinib (grey) or dasatinib (white). Unstimulated cells (black) are included for comparison. Vertical bars indicate standard deviations.

(F) Gastric MALT lymphoma formation in female BALB/c mice infected for 18 months with H. felis. One group received imatinib (I) through the drinking water for months 16-18 of the experiment. Macroscopically visible tumors >1 mm in diameter are plotted. C = control, n = number of tumors per mice.

(G) Representative micrographs of H&E-stained (hematoxylin and eosin stained) sections of the mice shown in F. Scale bar indicates 50 μηι.

(H) H. felis colonization as determined by f/aS-specific qPCR of the mice shown in F. m = H. felis copies per stomach.

Detailed Description of the Invention

The invention relates to bcr-abl inhibitors, such as imatinib, dasatinib and nilotinib, for use in the treatment of ABL overexpressing B-cell lymphoma, in particular mucosa-associated lymphoid tissue (MALT) lymphoma. Mucosa-associated lymphoid tissue (MALT) lymphoma is a well characterized form a B- cell non-Hodgkin lymphoma. The stomach is the most commonly affected organ in which MALT lymphoma pathogenesis is clearly associated with Heliobacter pylori gastro- duodenitis (Sagaert X. et al., Nat Rev Gastroenterol Hepatol 2010, 7(6):336-346). As used herein MALT lymphoma includes both low grade MALT lymphoma and high grade MALT lymphoma, the more aggressive form of MALT lymphoma, also termed gastric diffuse large B-cell lymphoma (gDLBCL). Furthermore, MALT lymphoma comprises forms of the disease in any of the organs that can be inflicted, for example in the stomach, lung, thyroid gland, salivary gland and lacrimal gland, and comprises also its splenic and nodal counterparts. Further B-cell lymphoma overexpressing ABL are, for example, nodal or extranodal diffuse large cell B-cell lymphoma (DLBCL), for example diffuse large B-cell lymphoma of the stomach.

Imatinib is 4-[(4-methylpiperazin-1-yl)methyl]-N-[4-methyl-3-[(4-pyridin -3-ylpyrimidin-2-yl)- amino]phenyl]benzamide, and is usually applied as the mesylate.

Dasatinib is N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-pipe razinyl]-2-methyl- 4-pyrimidinyl]amino]-5-thiazole carboxamide monohydrate. Nilotinib is 4-methyl-N-[3-(4-methyl-1 H-imidazol-1-yl)- 5-(trifluoromethyl)phenyl]-3- [(4-pyridin-3-ylpyrimidin-2-yl) amino]benzamide, applied as the hydrochloride hydrate.

Further bcr-abl inhibitors considered are, for example, those listed by Valent P., Clinical Medicine Insights: Therapeutics 2010:365-380, in particular SKI-606 (bosutinib),

INNO-406 (bafetinib), AZD0530 (saracatinib), AP23464, AP23848, CGP76030, PP1 ,

PD166326, TG100598, TG101 114, ON012380, ON01910, AP24534, VX-680 (tozasertib), VE-465, PHA-739358 (danusertib), KW-2449, AT9283, XL228, AS703569, SGX-70393, DCC-2036, SGX393, and also FTY720 (fingolimod) and BAY 43-9006 (sorafenib). Whenever a "bcr-abl inhibitor" is mentioned, this expression is also used to designate salts, hydrates or other solvates. Salts are, in particular, pharmaceutically acceptable salts. For basic compounds, such as the amines imatinib, nilotinib and other bcr-abl inhibitors with related structures comprising a basic amino function, acid addition salts are primarily considered. Acid addition salts may comprise more than one acid molecule, depending on the number of basic nitrogen atoms of the free base. Such acid additions salts may be formed with organic or inorganic acids, especially with pharmaceutically acceptable acids. Suitable inorganic acids are, for example, halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organic acids are, for example, carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, oleic acid, glycolic acid, lactic acid, oxalic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or ethane- sulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1 ,2-disulfonic acid, camphor-10- sulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 1 ,5-naphthalene- disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid, nicotinic acid, orotic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N-cyclohexylsulfamic acid, N- methyl-, N-ethyl- or N-propyl-sulfamic acid, or other organic protonic acids, such as ascorbic acid.

For acid compounds, such as carboxylic acids, salts with alkali or earth alkali cations are considered, e.g. sodium, potassium, calcium or magnesium salts. Alternatively, salts with ammonia, or primary, secondary or tertiary amines are considered, for example with ethanolamine, di(2-hydroxyethyl)amine, tri(2-hydroxyethyl)amine or dimethyl-

2-hydroxyethyl-amine, also quaternary salts, such as tetramethylammonium, or salts with the basic amino acids lysine or arginine.

For the administration, the active ingredient is preferably in the form of a pharmaceutical preparation comprising a bcr-abl inhibitor as active ingredient in chemically pure form, and optionally a pharmaceutically acceptable carrier and optionally adjuvants.

The bcr-abl inhibitor is used in an amount effective against the bacterial infectious disease in humans. The dosage of the active ingredient depends upon the age, weight, and individual condition of the human being, the individual pharmacokinetic data, and the mode of administration. In the case of an individual human having a bodyweight of about 70 kg the daily dose administered of an bcr-abl inhibitor is from 0.01 mg/kg bodyweight to 100 mg/kg bodyweight, preferably from 0.1 mg/kg bodyweight to 50 mg/kg bodyweight, more preferably from 1 mg/kg to 20 mg/kg bodyweight administered as a single dose or as several doses. The bcr-abl inhibitor can be used alone or in combinations with other drugs. Pharmaceutical compositions for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular administration, are especially preferred. The pharmaceutical compositions comprise from approximately 1 % to approximately 95% active ingredient, preferably from approximately 20% to approximately 90% active ingredient.

For parenteral administration preference is given to the use of solutions of the bcr-abl inhibitor, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example, can be made up shortly before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, viscosity-increasing agents, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes.

For oral pharmaceutical preparations suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, and also binders, such as starches, cellulose derivatives and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, flow conditioners and lubricants, for example stearic acid or salts thereof and/or polyethylene glycol. Tablet cores can be provided with suitable, optionally enteric, coatings. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient. Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The capsules may contain the active ingredient in the form of granules, or dissolved or suspended in suitable liquid excipients, such as in oils.

Slow release oral pharmaceutical preparations are also considered, for example preparations containing swellable polymers, in particular compositions comprising the active ingredient, a polymeric binder, and a non-ionic, pharmaceutically acceptable surfactant. Polymeric binders considered are, for example, homo- and copolymers of N-vinylpyrrolidone, cellulose esters or ethers, high molecular weight polyalkylene oxides, polyacrylates, vinyl acetate polymers, polyvinyl alcohol and polysaccharides. Transdermal/intraperitoneal and intravenous applications are also considered, for example using a transdermal patch, which allows administration over an extended period of time, e.g. from one to twenty days.

The bcr-abl inhibitor can be administered alone or in combination with one or more other therapeutic agents, possible combination therapy taking the form of fixed combinations of the bcr-abl inhibitor and one or more other therapeutic agents known in the treatment of MALT lymphoma in humans, the administration being staggered or given independently of one another, or being in the form of a fixed combination.

Possible combination partners considered are antibiotics, particularly those known to eradicate H. pylori, for example beta-lactams such as amoxicillin, macrolides such as clarithromycin, chinolones such as levofloxacin, metronidazole, furthermore proton pump inhibitors, such as omeprazole, pantoprazole or rabeprazole.

The invention further relates to bcr-abl inhibitors, such as imatinib, dasatinib or nilotinib, for use in the preparation of a medicament for the treatment of ABL overexpressing B-cell lymphoma, in particular mucosa-associated lymphoid tissue (MALT) lymphoma.

Pharmaceutical preparations considered are mentioned above. These medicaments are manufactured by methods known in the art, especially by conventional mixing, coating, granulating, dissolving or lyophilizing. The invention further relates to a method of treatment of ABL overexpressing B-cell lymphoma, in particular mucosa-associated lymphoid tissue (MALT) lymphoma comprising administering to a patient in need thereof a therapeutically effective amount of a bcr-abl inhibitor, such as imatinib, dasatinib or nilotinib. The scientific rationale for the use of a bcr-abl inhibitor in the treatment of ABL

overexpressing B-cell is as follows: miR-203 is down-regulated in gastric MALT lymphoma due to promoter hypermethylation. The ectopic re-expression of miR-203 in a lymphoma cell line or in primary MALT lymphoma cells down-regulates the expression of the miR-203 target ABL1 and blocks tumor cell proliferation in vitro. Finally, inhibition of ABL1 tyrosine kinase activity by imatinib blocks MALT lymphoma cell proliferation ex vivo and prevents tumor formation in mice, suggesting for the first time an important oncogenic role for ABL1 in the

pathogenesis of B-cell tumors not harboring the BCR-ABL1 fusion. Patient material

Human patient material was obtained from eight patients with H. py/or/ ^' -positive, t(77; 78)(q21 :q21)-negative gastric low grade MALT lymphoma that were part of a previously published study conducted at Philipps-University Hospital Marburg, Germany (Huynh M.Q. et al., Leuk Lymphoma 2008, 49:974-983). miRNA expression analysis was performed on archived cases of H. py/or/ ^' -positive gastritis, H. py/or/ ^' -positive gastric low grade MALT lymphoma and gastric high grade lymphoma drawn from the surgical pathology files of the Institute of Pathology at the Cantonal Hospital St. Gallen,

Switzerland. All data were blinded to guarantee patients' protection. All procedures were in agreement with the guidelines for use of human material in research issued by the participating Institutions' Ethics Committees.

Animal experimentation, cell culture, nucleoporation and lentivirus infection

Female BALB/c mice were infected orally at six weeks of age with three consecutive doses of -5x10 ⁷ H. felis (CS1 , ATCC 49179) and maintained in a University of Zurich SPF facility. For the purpose of imatinib treatment, at the 15 month infection time point mice received 1.2 mM imatinib (-75 mg/kg per day) through their drinking water for 3 months. At the 18 month infection time point, macroscopically visible tumors were collected and single cell suspensions were generated and cultured in RPMI supplemented with 10% FCS and antibiotics. Tumor cells were stimulated with 10 μg/ml Helicobacter lysate and cultured either in the presence or absence of imatinib (Novartis, Basel, Switzerland) or dasatinib (Bristol-Myers Squibb) for 3 days. Tumor cell proliferation was quantified by [ ³ H]-thymidine incorporation assay. The Burkitt's lymphoma BL2 cell line was described in Cohen J.H. et al., J Natl Cancer Inst 1987, 78:235.-242. For demethylation treatment, BL2 cells were seeded at 5x10 ⁵ cells/ml 24 hours prior to treatment with 5 μΜ 5'-azacytidine and/or 3 mM 4-phenylbutyric acid (both from Sigma-Aldrich). For the purpose of miR-203 re-expression, 1x10 ⁶ BL2 cells were nucleoporated using an Amaxa Nucleoporator (Gaithersburg, MD) with either 50 nM or 100 nM of precursor oligonucleotides (either miR-203 specific miRNA precursor or negative control precursor 1 , Ambion, Austin, TX) in a total volume of 2 ml. 48 hours after electroporation, the cells were collected and ABL1 expression was analyzed by real time RT-PCR (LightCycler 480 SYBR Green I master kit; Roche, Basel, Switzerland) or Western Blot, using antibodies against ABL1 (BD Biosciences, CA, USA) and a-tubulin (Sigma-Aldrich, St. Louis, MO). Primary MALT lymphoma cells were transduced with lentiviral particles harboring a miR-203 expression construct (Systems Biosciences, Mountain View, CA) in the presence of 8 μg/ml polybrene by spinoculation. Tumor cell proliferation was quantified by a [ ³ H]-thymidine incorporation assay. Expression of mature mir-203 was validated by real-time PCR.

Microarray-based miRNA expression profiling and bisulfite genomic sequencing miRNA microarray experiments were performed using the Agilent Human miRNA Microarray Kit version 10.0. Normalization and statistical analysis was performed using the quantile normalization implemented in the package PreprocessCore and the package genefilter, respectively. Specific oligonucleotides for methylation-specific PCR (MS-PCR) and bisulfite sequencing of the miR-203 CpG island were performed as described by Bueno M.J. et al., Cancer Cell 2008, 13:496-506.