WO2024037910A1 - Syk inhibitors for use in the treatment of cancer - Google Patents

Syk inhibitors for use in the treatment of cancer Download PDF

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Publication number
WO2024037910A1
WO2024037910A1 PCT/EP2023/071793 EP2023071793W WO2024037910A1 WO 2024037910 A1 WO2024037910 A1 WO 2024037910A1 EP 2023071793 W EP2023071793 W EP 2023071793W WO 2024037910 A1 WO2024037910 A1 WO 2024037910A1
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syk
cells
isoform
optionally substituted
expression
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PCT/EP2023/071793
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French (fr)
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Piona Dariavach
Pierre Martineau
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Institut National de la Santé et de la Recherche Médicale
Institut Régional Du Cancer De Montpellier
Université De Montpellier
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Publication of WO2024037910A1 publication Critical patent/WO2024037910A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4166—1,3-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators

Definitions

  • the present invention is in the field of medicine, in particular oncology.
  • Spleen tyrosine kinase is a 72 kDa non receptor tyrosine kinase that contains two tandem Src homology 2 domains at the NH2 terminus and a kinase domain at the COOH terminus.
  • Syk has two alternatively splice isoforms: full length Syk (L) and short form Syk (S) that lacks a 69-nucleotide exon.
  • Syk (L) is present in both the cytoplasm and the nucleus of the cells due to a nuclear localization signal present in the 23 residues of interdomain B encoded by exon 9, missing in Syk (S). Consequently, Syk (S) is located exclusively in the cytoplasm of the cells.
  • Syk is widely expressed in hematopoietic cells where it plays a key role in the activation of the cells following the stimulation of antigen and Fc Receptors. Following receptor engagement and clustering, Syk is recruited to the receptor through the binding of its SH2 domains to a double phosphorylated immunoreceptor tyrosine-based activation motif or IT AM. This activates Syk through auto- and trans-tyrosine phosphorylation. Activated Syk then catalyzes the phosphorylation of protein substrates primarily on tyrosines, however Syk also has the ability to phosphorylate some proteins on serine.
  • IT AM double phosphorylated immunoreceptor tyrosine-based activation motif
  • Syk plays also an important role in epithelial solid cancers where its expression is a marker predicting either poor or favorable outcome (3). Indeed, the contribution of Syk in tumor biology depends on cancer types and it can be of three origins: 1) pro-survival role by stabilizing anti-apoptotic proteins (MCL-l/BCL-2 family) (4,5) and by suppressing c-JUN expression (6). This activity underlies many of the tumor promoting activities of the kinase; 2) negative regulator of EMT by enhancing cell-cell and cell -matrix adhesion (7) (8). This activity underlies many of the tumor suppressive activities of the kinase; and 3) regulator of mitotic progression through its centrosomal kinase activity associated with y-tubuline (9) and by controlling the cell cycle G2-M phase progression (10).
  • MCL-l/BCL-2 family anti-apoptotic proteins
  • Syk As an oncogene is frequently reported in squamous carcinomas of head and neck (SCCHN) (16) where its expression enhances cell migration. Interestingly, high expression of Syk is significantly associated with recurrence and shorter survival in SCCHN patients. In ovarian cancer, the expression of Syk increases with tumor grade, and the silencing of Syk expression inhibits anchorage-independent growth and induces apoptosis in ovarian cancer cells (6). A similar pro-survival role for Syk is seen in retinoblastoma cells, where both the treatment with Syk inhibitors and knockdown of Syk expression result in a dramatic increase in apoptosis of retinoblastoma cells (17).
  • Syk (L) and Syk (S) isoforms suggest an active but opposing role of Syk (L) and Syk (S) isoforms on the growth properties of cancer cells, possibly due to different biologic functions for the two isoforms.
  • Wang and co-workers (18) showed that in primary breast tumors, Syk (L) was present in both tumor and matched normal mammary gland, and that ectopic expression of Syk (L) in a cellular model suppressed cell invasiveness. They also showed that Syk (S) was expressed in cancerous tissues and not in normal mammary gland, and Syk (S) ectopic expression did not reduce cell invasiveness in vitro.
  • Syk (L) is downregulated in 69% of tumor tissue samples compared to the adjacent non-cancerous tissue, the expression of Syk (S) remains stable, suggesting that Syk (L) but not Syk (S) is associated with tumor suppressing activities.
  • Coebergh et al (22) studied the prognostic role of Syk in a cohort of 160 chemonaive lymph node negative colon cancer patients, and they found that Syk (S) high mRNA expression correlates with metastatic relapse with hepatic lesions. However, they did not observe any association between mRNA expression of the splice variants and tumor grade, nor any evidence supporting a tumor suppressor role for Syk (L).
  • the present invention is defined by the claims.
  • the invention relates to Syk inhibitors for use in the treatment of cancer.
  • the goal of the Inventors was to evaluate the role of Syk alternative spliced isoforms in cancer biology of colorectal carcinomas. For this purpose, they used two approaches: i) short hairpin RNA (shRNA) tools to target the global expression of Syk as well as its alternative splicing variant Syk (L); ii) the use of compound C-13, an original non-enzymatic inhibitor of Syk isolated in their group (23).
  • shRNA short hairpin RNA
  • C-13 an original non-enzymatic inhibitor of Syk isolated in their group (23).
  • Their functional studies demonstrated that CRC cell lines depend on Syk long isoform for their survival, since Syk (L) expression silencing affected the viability and induced the apoptosis of the cells.
  • the application of compound C-13 phenocopied Syk (L) expression silencing in vitro, and its oral and intraperitoneal administration reduced the tumor growth of CRC DLD-1 cells xenografts in Nude mice in
  • a first aspect of the present invention relates to a Syk inhibitor for use in the treatment of a cancer in a subject in need thereof.
  • the Syk inhibitor is a non- enzymatic inhibitor of Syk.
  • the term “subject” refers to any mammals, such as a rodent, a feline, a canine or a primate.
  • the subject is a human afflicted with or susceptible to be afflicted with a cancer.
  • the subject is a human afflicted with or susceptible to be afflicted with a colorectal cancer.
  • the subject suffers from a cancer wherein Syk plays the role of an oncogene.
  • the subject bears G13D, G12D, G12V or G12C activating mutation in K-Ras gene.
  • the subject bears G13D activating mutation in K-Ras gene.
  • Activating mutations in the KRAS gene are seen in 30-50% of CRCs. Such mutations prevent the use of anti-EGFR targeted therapies.
  • the most common somatic mutations observed in KRAS anomalies in CRC are found in codons 12 and 13. Notably, the four most frequent mutations of all CRCs in terms of incidence are G12D (11%), G13D (7%), G12V (7%), and G12C (4%). This missense mutation aberrantly activates KRAS and results in activation of downstream signaling pathways.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses both primary and metastatic cancers. Examples of cancers include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the cancer is a colorectal cancer. In some embodiments, the cancer is a K-Ras mutated cancer.
  • K-Ras Ki-Ras2 Kirsten rat sarcoma viral oncogene homolog
  • K-Ras is a pro-oncogenic small GTPase. It belongs to Ras protein family (monomeric G protein). It transmits signals from growth hormone receptors and in particular the EGF receptor, which receives the signal from outside the cell.
  • K-Ras gene is mutated.
  • the K-Ras gene mutation is an activating mutation.
  • a K-Ras activating mutation leads to a constitutive activation of the RAS-RAF-MEK-ERK pathway.
  • the subject bears G13D, G12D, G12V or G12C activating mutation in K-Ras gene.
  • the activating mutation is G13D mutation.
  • treating refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • the present invention relates to a method of treating a cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a Syk inhibitor.
  • administering refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor of Syk) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • a disease, or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • the expression "therapeutically effective amount” is meant a sufficient amount of the active ingredient (e.g. Syk inhibitor) for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • Syk or “spleen tyrosine kinase” refers to is a 72 kDa non receptor tyrosine kinase that contains two tandem Src homology 2 domains at the NH2 terminus and a kinase domain at the COOH terminus. Syk has two alternatively splice isoforms: full length Syk (L) and short form Syk (S) that lacks a 69-nucleotide exon. Syk is encoded by SYK gene (Gene ID: 6850; Ensembl : ENSG00000165025; Uniprot : P43405).
  • Syk inhibitor refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of Syk. In some embodiments, the Syk inhibitor does not affect Syk kinase activity. In some embodiments, the Syk inhibitor does not inhibit the catalytic activity of Syk. In some embodiments, the Syk inhibitor does not target Syk catalytic site. In some embodiments, the Syk inhibitor binds to the protein tyrosine kinase Syk to prevent its interaction with partners. In some embodiments, the Syk inhibitor is a Syk (L) inhibitor. In some embodiments, the Syk inhibitor is a Syk (S) inhibitor.
  • the inhibitor according to the invention is a low molecular weight compound, e. g. a small organic molecule (natural or not).
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
  • Syk inhibitors include, but are not limited to Syk inhibitors as described in WO2009/133294.
  • the Syk inhibitors are selected from the molecules having the following formulas (I), (II), (III) or (IV): where:
  • R1 is an optionally substituted aromatic group, or an optionally substituted heterocycle comprising at least one S, O or N atom
  • R2 is an optionally substituted aromatic group, an optionally substituted heterocycle, an optionally saturated carbon chain, comprising an amine group, an optionally saturated carbon chain comprising an optionally substituted aromatic group or an optionally saturated carbon chain comprising an optionally substituted heterocycle comprising at least one S, O or N atom
  • R4 is an optionally saturated carbon chain comprising 1 to 5 carbon atoms, optionally substituted with an aromatic group
  • R5 is an optionally substituted aromatic group or an optionally substituted amine group
  • R6 is a hydrogen atom, alkoxy group, alkyl group or halogen
  • R7 is a hydrogen atom, alkoxy group, alkyl group or halogen
  • R8 is a hydrogen atom, alkoxy group, alkyl group or halogen
  • m 0, 1 or 2
  • R9 is a hydrogen atom and RIO is an optionally substituted phenyl group, or R9 and RIO are part of the same optionally substituted heterocycle, or R9 and RIO are part of the same optionally substituted aromatic group;
  • Rll is a hydrogen atom, alkoxy group or alkyl group
  • R12 is a hydrogen atom, alkoxy group or alkyl group
  • R13 is a hydrogen atom or an alkyl or alkoxy group
  • R14 is a hydrogen atom or an alkyl or alkoxy group
  • A is an oxygen or sulphur atom
  • R15 is an optionally saturated carbon chain comprising 1, 2 or 3 carbon atoms, optionally substituted by an optionally substituted aromatic group, an optionally substituted heterocycle or an amine group belonging to optionally substituted heterocycle;
  • R16 is a hydrogen atom, halogen or alkoxy group
  • R17 is a hydrogen atom, alkoxy group or acetoxy group.
  • the Syk inhibitor is (methyl 2- ⁇ 5-[(3-benzyl-4-oxo-2-thioxo-l,3- thiazolidin-5-ylidene)methyl]-2-furyl ⁇ benzoate) (also named C-13); 4-(4-chloro benzoyl)-3- hydroxy-5-(3-phenoxy phenyl)- 1 -(3 -pyridinyl methyl)-l,5-dihydro-2H-pyrrol-2-one; 5-(2,4- dimethoxy phenyl)-3 -hydroxy- 1- [3 -(1 H-imidazol-l-yl)propyl]-4-(2 -thienyl carbonyl)- 1,5- dihydro-2H-pyrrol-2-one; acide ⁇ 4-bromo-2-[3-(ethoxy carbonyl)-2-methyl-5-oxo-4,5- dihydro-lH-indeno[l,2-b]pyridin-4
  • the Syk inhibitor is C- 13.
  • C-13 refers to (methyl 2- ⁇ 5-[(3-benzyl-4-oxo-2-thioxo-l,3-thiazolidin-5-ylidene)methyl]-2- furyl (benzoate). This molecule has the following formula and structure in the art C23H17NO4S2:
  • Syk inhibitors includes as example BAY-613606, R112, R343, R406, R788, piceatannol, NVP-QAB205, AB8779, ER 27319, GSK143.
  • the inhibitor according to the invention is an antibody.
  • the antibody according to the invention is an antibody directed against Syk-(L) isoform.
  • the antibody according to the invention is an antibody directed against Syk-(S) isoform.
  • Antibodies directed against Syk can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against Syk can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-Syk single chain antibodies.
  • Anti-Syk antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to Syk.
  • Humanized anti-Syk antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies” are forms of nonhuman (e.g., rodent) chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the antibody according to the invention is a single domain antibody directed against Syk.
  • the antibody according to the invention is a single domain antibody directed against Syk-(L) isoform.
  • the antibody according to the invention is a single domain antibody directed against Syk-(S) isoform.
  • sdAb single domain antibody
  • VHH single domain antibody
  • VHH refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3.
  • CDR complementarity determining region
  • the term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.
  • the VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation.
  • the VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.
  • VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2.
  • Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
  • immobilized antigen e.g., antigen coated onto the plastic surface of a test tube
  • biotinylated antigens immobilized on streptavidin beads or membrane proteins expressed on the surface of cells.
  • VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations.
  • the high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B- cells in the lymphoid organs of immunized animals.
  • VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations).
  • VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies.
  • VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells.
  • the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695).
  • the “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).
  • the compound according to the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA.
  • each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then, for this invention, neutralizing aptamers of Syk are selected.
  • the compound according to the invention is a polypeptide.
  • the polypeptide is an antagonist of Syk and is capable to prevent the function of Syk.
  • the polypeptide can be a mutated Syk protein or a similar protein without the function of Syk.
  • the polypeptide is an antagonist of Syk-(L) isoform.
  • the polypeptide is an antagonist of Syk- (S) isoform.
  • the polypeptide of the invention may be linked to a cellpenetrating peptide” to allow the penetration of the polypeptide in the cell.
  • cellpenetrating peptides are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).
  • the polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention.
  • the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
  • the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell.
  • Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others.
  • Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown.
  • a common, preferred bacterial host is E coli.
  • polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy.
  • modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution.
  • the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
  • adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
  • a strategy for improving drug viability is the utilization of water-soluble polymers.
  • Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
  • water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
  • Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
  • PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
  • Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.).
  • the PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages.
  • Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain.
  • the reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules.
  • These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer.
  • the molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading).
  • increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate.
  • the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).
  • linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue.
  • tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology.
  • Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
  • the Syk inhibitor according to the invention is an inhibitor of Syk gene expression.
  • the Syk inhibitor according to the invention is a nucleic acid targeting a mRNA encoding Syk.
  • the Syk inhibitor according to the invention is a nucleic acid targeting a mRNA encoding Syk- (L) isoform.
  • the Syk inhibitor according to the invention is a nucleic acid targeting a mRNA encoding Syk-(S) isoform.
  • Small inhibitory RNAs can also function as inhibitors of Syk expression for use in the present invention.
  • Syk gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that Syk gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT.
  • the siRNA is directed against Syk-(L) isoform. In some embodiments, the siRNA is directed against Syk-(S) isoform.
  • Short hairpin RNA can also function as inhibitors of Syk expression for use in the present invention.
  • ShRNA refers to a segment of RNA that is complementary to a portion of a target gene and has a stem-loop (hairpin) structure.
  • the Syk inhibitor is a shRNA.
  • the shRNA is directed against Syk-(L) isoform.
  • the shRNA is directed against Syk-(L) isoform and comprises an amino acid sequence having at least 90% of identity with SEQ ID NO:3 or SEQ ID NO:4.
  • the shRNA is directed against Syk-(L) isoform and comprise the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:4.
  • the shRNA is directed against Syk-(S) isoform.
  • Ribozymes can also function as inhibitors of Syk gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of Syk mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of Syk gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing Syk.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno- associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • Another aspect of the invention relates to a pharmaceutical composition comprising a Syk inhibitor for use in the treatment of a cancer in a subject in need thereof.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • pharmaceutically acceptable excipients e.g. a Syk inhibitor
  • sustained-release matrices such as biodegradable polymers
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • the pharmaceutical compositions of the invention can be formulated for a topical, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
  • dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • compositions of the present invention may comprise a further therapeutic active agent.
  • anti-cancer agents may be added to the pharmaceutical composition as described below.
  • Anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluor
  • additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.
  • Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
  • Other additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.
  • the further therapeutic active agent can be an antiemetic agent.
  • Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron.
  • the further therapeutic active agent can be a hematopoietic colony stimulating factor.
  • Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
  • the other therapeutic active agent can be an opioid or nonopioid analgesic agent.
  • opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene.
  • Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
  • the further therapeutic active agent can be an anxiolytic agent.
  • Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
  • the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.
  • the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin-like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7- Hl).
  • CTL4 cytotoxic T lymphocyte-associated protein 4
  • PDCD1 programmed cell death 1
  • NK cells like various members of the killer cell immunoglobulin-like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7- Hl).
  • the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Targeting of Syk alternative spliced isoforms.
  • A The relative expression of Syk (L), Syk (S) and total Syk (L+S) in colorectal carcinoma cell lines was detected by qPCR; HPRT was used as an internal control. The Y-axis of the plot is expressed in a base-2 log scale. The figure is representative of three independent experiments.
  • B The expression of total Syk was evaluated by western blot. The panel represents the densitometric quantification of immunoblot analyses of Syk expression over control.
  • shRNAs targeting the pre-mRNAs encoding Syk proteins evaluated by RNA expression monitored by (C) qPCR; HPRT was used as an internal control, and (D) western blot, compared to mock transduced (shLUC) HCT-116, DLD-1 and DIFI CRC cells.
  • C qPCR
  • HPRT was used as an internal control
  • D western blot
  • HCT-116, DLD-1 and DIFI CRC cells was used as an internal control
  • shRNA LUC with Luciferase sequence was used as negative control.
  • DLD-1 cells were transduced with Syk shGl and shL2 shRNAs or shLUC (control shRNA), and the cell cycle distribution of cells one-week post-infection was analyzed using flow cytometry.
  • the accumulation of transduced cells in G0-G1, S and G2-M and > 4N phases of each cell line is normalized to its control and is shown as a bar graph.
  • the data are an average of three independent transduction experiments and error bars represent the mean ⁇ SD (*, P ⁇ 0.05; **, P ⁇ 0.01).
  • DLD-1 cells (5x10 ) transduced with shRNAs targeting global Syk (shGl) & long- isoform-specific Syk (shL2) or unrelated-transduced shRNA (shLUC), and (B) cells pretreated with Piceatannol or vehicle (DMSO) were serum starved, labelled with CellTracker dye, and
  • HCT-116 and DLD-1 cells (5x10 ) transduced with Syk shRNAs or control shRNA were serum starved, labelled with CellTracker dye and seeded on Matrigel-coated Transwell inserts of 8 pm pore size (BD Falcon). FCS was used as chemoattractant in the Transwell lower part. After 24 hours at 37°C, migration was observed and counted microscopically. Images were obtained and total number of cells per field of view was measured using ImageJ software. Error bars represent the mean number of migrated cells in triplicate wells, representative of three independent experiments (*, P ⁇ 0.05).
  • Syk (L) isoform expression is associated with tumorigenesis in human CRC tissues.
  • A Western blot analysis of the protein extracts of primary colorectal tumors of 13 untreated patients. T: Tumor tissue and N: normal adjacent tissue of the same patient. The panel represents the densitometric quantification of immunoblot analyses of Syk expression in tumor tissues over the corresponding normal tissues. The arrows indicate the tumor samples in which the ratio of Syk expression is at least two folds higher compared to the normal tissue.
  • FIG. 6 Treatment of CRC cell lines with C-13, an original non-enzymatic inhibitor of Syk.
  • A Cells were exposed for 2 hours to various concentrations of C-13 and the impact of C-13 on cellular phenotype evaluated 72 hours post-treatment by: cell proliferation, viability and apoptosis, and normalized against DMF (vehicle)-treated HCT-116, DLD-1 and DIFI CRC cells. Data are the mean ⁇ SD (error bars) of at least three independent experiments (*, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; compared with control).
  • D DLD- 1 cells pre-treated with the indicated concentrations of compound C-13 or DMF (vehicle) for 2 hours, were incubated with the recombinant human EGF (50 ng/ml) or a combination of EGF (50 ng/ml) + Cetuximab (50 pg/ml) for 10 minutes. Protein extracts from cell lysates were analyzed by western blot using the indicated antibodies.
  • the panel represents the densitometric quantification of immunoblot analyses of phospho-Erk (+EGF) versus phospho-Erk (-EGF) over control and phospho- Akt (+EGF) versus phospho- Akt (-EGF) over control.
  • C-13 inhibits tumor growth of xenografts in Athymic Nude mice.
  • B Kaplan-Meier survival curves of the mice described in B.
  • D Kaplan- Meier survival curves of the mice described in C.
  • Syk non-enzymatic inhibitor C-13 was purchased from ChemBridge, Inc (San Diego, CA) (ID number 6197026) and was dissolved to a final concentration of 10 mM in dimethylformamide (DMF).
  • Syk catalytic inhibitor R406 was from Santa Cruz Biotechnology and was dissolved to a final concentration of 10 mM in dimethyl sulfoxide (DMSO).
  • Syk inhibitor Piceatannol was from Sigma-Aldrich and was dissolved to a final concentration of 40 mM in DMSO.
  • Recombinant human EGF (1 mg/ml) was from Sigma- Aldrich. Cetuximab (ERBITUX®) (5 mg/ml) was purchased from Merck (Darmstadt, Germany). All stock solutions were aliquoted and stored at -20C. All reagents unless otherwise mentioned were from Sigma (St Louis, Mo).
  • EGF induction experiments cells were serum-starved for 12 hours, and they were subsequently treated with the recombinant human EGF (50 ng/ml) or with EGF (50 ng/ml) + Cetuximab (50 pg/ml) in serum free culture medium, for 10 minutes.
  • Cell propagation and passaging were as recommended by the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • cells were detached with TrypLE Express Enzyme (Gibco), and they were counted using Z1 Particle Counter (Beckman Coulter). The cell lines were tested and authenticated by short-tandem repeat profiling (LGC Standards and Eurofins Genomics). All experiments were performed at least three times.
  • shRNA silencing by small hairpin RNA To design long isoform-specific and global Syk specific shRNA, we used siRNA sequences from Pinos et al (6). Four different shRNA expressing pSIREN vectors with the puromycin selection marker and specific to Syk were used. shRNA Gl, G2 inhibit global gene expression and shRNA LI, L2 are alternative exon-specific. shRNA LUC with Luciferase sequence was used as negative control.
  • Lentiviral production was performed by cotransfecting IxlO 6 293T cells (for lentiviral packaging) with 3 pg of pSiren vector in which anti-Syk shRNAs where cloned, 1 pg of the packaging vector gagpol, and 1 pg of envelope vector, using JetPRIME (Polyplus), according to the manufacturer's instructions. Lentiviral particles were harvested, filtered and then used to infect IxlO 6 cells for 48 hours. Following shRNA transduction, cells were selected with 1 «g/ml puromycin for one week and stable clones were pooled. shRNA target sequences.
  • shSykGl 5' GCAGATGGTTTGTTAAGAG 3' (SEQ ID NO:1); shSykGl: 5' GTCGAGCATTATTCTTATA 3' (SEQ ID NO:2); shSykLl: 5' GTTCCCATCCTGCGACTTG 3' (SEQ ID NO:3); shSykLl: 5' GGTCAGCGGGTGGAATAAT 3' (SEQ ID NO:4); shLUC: 5’ TTACGCTGAGTACTTCGA 3’ (SEQ ID NO:5).
  • RNA extraction and quantitative PCR were performed from 0.5 to LIO 6 cells using the ZR RNA MiniPrep Kit (Zymo Research) as recommended by the manufacturer. Reverse transcription was performed on 1 pg RNA using PrimeScript Reverse Transcriptase (Takara Bio.), SYBR Premix Ex Taq 2X master mix (Takara Bio.) and 5 pmol of both forward and reverse primers. Quantitative PCR reactions were performed on 1 ng cDNA. HPRT was amplified as internal control. qPCR primer sequences.
  • Syk L for 5' TCAGCGGGTGGAATAATCTC 3' (SEQ ID NO:6); rev 5' TGCAAGTTCTGGCTCATACG 3' (SEQ ID NO:7)
  • Syk L+ S for 5' GAAGCCATATCGAGGGATGA 3' (SEQ ID NO:10); rev 5' CCACATCGTATGTCCAGCAC 3' (SEQ ID NO: 11)
  • Cell growth inhibition assay 2D assay.
  • Cell growth was evaluated using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to manufacturer’s recommendations. Briefly, 3.000 cells/well were seeded in 96-well plates. After 24 hours, drugs were added in serial dilution. Cells were incubated for 96 hours, after which, a volume of CellTiter-Glo® reagent equal to the volume of cell culture medium was added to each well. The plate was shaked on an orbital shaker for 15 minutes at room temperature to induce cell lysis. The luminescence was recorded using a Thermo Fisher Scientific Multiskan EX plate reader. The IC50 was determined graphically from the cell growth curves.
  • DLD-1 or HCT-116 cells were serum starved for 16 h in a 1%-FBS- medium. Cells were detached with TrypLE Express Enzyme (Gibco). 5xl0 4 cells were resuspended in a 1%-FBS-medium and they were seeded on Fluoroblok 24-well plate permeable inserts, with 8 pm pores (Corning). Following a 20-22h incubation with chemoattractant (10 % FBS in RPMI medium), the inserts were transferred in a 2 nd 24-well plate containing a 4 pg/mL Calcein AM solution (Sigma Aldrich). The fluorescence was read on an inverted fluorescence microscopy and migrated cells were counted with ImageJ software.
  • Apoptosis assay A total of 2.5xl0 5 cells were plated, and after 24 hours, they were incubated with the indicated concentrations of compound C-13 for 2 hours. ShRNA-tranduced cells were plated following one week puromycin selection, and grown for 48 hours. Cells were stained with APC-labeled Annexin V and 7-aminoactinomycin D (Biolegend). For apoptosis determination, Annexin V- and 7-ADD-positive cells were quantified using a Gallios Cytometer (Beckman Coulter) and the Kaluza Software (Beckman Coulter).
  • cells were washed with ice-cold PBS, and they were fixed and permeabilized with ice-cold methanol (Sigma Aldrich) for 15 minutes at -20°C. Cells were incubated in PBS containing 2% BSA for 30 minutes at room temperature. Cells were incubated with the primary and the fluorescent antibodies for Ih at room temperature.
  • the primary antibodies used were: rabbit polyclonal Syk (1 : 100) (sc-1077, Santa Cruz Biotechnology); mouse monoclonal anti-beta Tubulin (1 :200) (T4026, Sigma).
  • Tissue samples From these 40 CRC patients we obtained expression data for at least one tissue of 28 patients. This represented 18 normal colon mucosas (CN), 20 colon primary tumors (CT) and 19 hepatic metastases (HM). Most of the primary tumors were located in the left colon and histological analysis showed a well or moderate differentiation state. Among those 57 analyzed tissues, both CT and HM were collected in 13 patients. These 13 pairs of samples were used to study the protein expression pattern of Syk expressed in CT versus the corresponding CN.
  • CN normal colon mucosas
  • CT colon primary tumors
  • HM hepatic metastases
  • tumor tissue samples from patients were directly grinded in lysis buffer (150mM NaCl, lOmM Tris pH 7.4, ImM EDTA, ImM EGTA, 1% SDS, 1% Triton X-100, 0.5% NP-40, 2mM PMSF, lOOmM NaF, lOmM sodium orthovanadate, one cocktail protease inhibitor tablet for 10 ml) using a Mixer Mill® MM 300 unit (Qiagen, Valencia, CA). Protein concentration was determined with the Bradford assay (Pierce Coomassie Plus Protein Assay).
  • Membranes were washed 5 times (10 min. each) in TBST and appropriate HRP-conjugated secondary antibodies were diluted in blocking solution and hybridized 1 hour at room temperature with gentle shaking. After 5 washes in TBST, the proteins were detected by chemiluminescence generated with Western Lightning Chemiluminescence Reagent Plus (GE Healthcare) and detected on PXi Syngene Chemiluminscence system. For reprobing, membranes were incubated at room temperature for 10 min in stripping solution (100 mM Glycine pH 2.8; 0.1% Igepal and 1 % SDS) with gentle shaking. Membranes were then washed (3 times, 5 min. each) with TBST and blocked as described previously, before rehybridization.
  • stripping solution 100 mM Glycine pH 2.8; 0.1% Igepal and 1 % SDS
  • DLD-1 cells were serum starved, then separated in two batches. One batch was incubated with EGF (50 ng/ml) for 10 minutes at 37°C. After washing with ice- cold PBS containing phosphatase inhibitors (100 mM sodium fluoride, 5 mM sodium orthovanadate), cells were lysed for 15 minutes on ice with lysis buffer (PBS supplemented with 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS). Cell lysates were clarified by centrifugation for 15 minutes at 4°C at 16,000 g. The total protein content of the soluble fraction was quantified using the BCA assay kit (Interchim, France).
  • the total ion chromatogram acquisition was made in information-dependent acquisition (IDA) mode using the Analyst TF v.1.7 software (Sciex). Positive ion profiling was performed from m/z 350-1,500, followed by a MS/MS product ion scan from m/z 100-1,500 with the abundance threshold set at more than 100 cps.
  • the accumulation time for ions was set at 250 ms for MS scans, and 100 ms for MS/MS scans. Target ions were excluded from the scan for 10s after detection.
  • the IDA advanced “rolling collision energy (CE)” option was employed to automatically ramp up the CE value in the collision cell as the m/z value was increased. A maximum of 25 spectra were collected from candidate ions per cycle.
  • TCGA gene and protein expression matrix were recovered for the COAD and READ cohorts using API GDC portal with curl function. They were merged together to generate the CRC (colorectal) dataset based on 622 individuals. About 18 thousand genes were expressed in more than 5% of the samples. Only 52 patients had RNAseq data for the tumor and the normal tissue. Fifty tumor samples had both mRNA and protein expression values. Splicing event concerning the exon 9 of Syk were recovered from TCGA SpliceSeq database as PSI value from the web interface (24). PSI values were obtained for all samples recovers from TCGA. We assigned NA PSI value to zero in the present analysis.
  • Tumor samples were separated based on Syk exon 9 PSI value between low ( ⁇ 0.25), medium and high (>0.75). Differential gene expression among tumor samples was calculated using edgeR. We kept genes that were differentially expressed between Low and High PSI tumors. Analysis was performed on the 10.000 most variable genes. Variation was calculated on the loglO of gene expression values. Graphs were generated using R version 3.6.2 and ggplot2 library. Complex heatmap library was used to draw heatmaps. Clinical data were recovered from integrated Pancancer resource published by Liu et al. (25). Three CRC samples had no clinical information. MSI features were obtained from Bonneville et al. (26). Information was missing for 43 patients. Kras status of CRC patients was downloaded at cbioportal.
  • CMS labels were matched using CMS subtyping calls (https://github.com/Sage-Bionetworks/crc-cms- kras/blob/master/020717/cms_labels_public_all.txt).
  • mice IxlO 6 DLD-1 tumor cells were injected subcutaneously in the left flank of 6-week-old female athymic nu/nu mice (Charles River Laboratories). Tumors were detected by palpation and measured with a caliper three times per week. Mice were euthanized by cervical dislocation when the tumor volume reached 1.500 mm 3 .
  • health monitoring of mice was performed to ensure their health, including monitoring animals from external sources as well as animals kept in the experimental unit; housing and hygienic monitoring, pathogen detection, diagnostic measures to enable disease control and to maintain the health status of mice.
  • tumor growth (a maximum size of 1500 mm3) instead of survival endpoint.
  • sample collection methods and instituted species-specific husbandry refinements in order to attenuate anxiety and stress.
  • the number of animals used was reduced to the absolute minimum necessary, based on appropriate statistical sample size determination. Ethical approvals were obtained by the local ethics committee (Ethics Committee approved by the French Ministry, animal facility approval D34- 172-27, and protocol approval CEEA-LR-10830 12018032611365978#! 0830).
  • mice were randomized in groups of 7 to 10 animals, and they were treated three times per week for 3 weeks with: (i) 0.2 mL of 0.9% sodium chloride solution by intraperitoneal injection of 0.35 mg/kg C- 13; (ii) 0.2 mL of 0.9% sodium chloride solution by intraperitoneal injection of DMF (vehicle, control group), (iii) 100 mg/kg C-13 dissolved in 0.1% carboxymethylcellulose solution by gavage; (iv) DMF dissolved in 0.1% carboxymethylcellulose solution (vehicle; gavage control group). Mice were euthanized by cervical dislocation when the tumor volume reached 1500 mm3.
  • Syk Long isoform is implicated in CRC cell lines survival and mitosis.
  • shRNA Short hairpin RNA
  • RNAi RNA interference
  • shRNA can be integrated into genomic DNA for long-term or stable expression, leading to a longer knockdown of the target mRNA and a durable down-regulation of protein expression, thus allowing the evaluation of protein knockdown effects on cellular functions.
  • shRNA-based isoform-specific silencing was used to investigate the effects of Syk splicing modulation on cell growth and cell survival of HCT-116, DLD-1 and DIFI colorectal cell lines.
  • Global shRNAs (shGl, shG2) equally inhibited the expression of long and short splice isoforms of Syk.
  • Long Isoform specific shRNAs inhibited the accumulation of the long splice isoform while proportionally reducing the overall gene expression level.
  • Syk inhibition increases chemotaxis and Matrigel engagement.
  • HCT-116 and DLD-1 cell lines were tested in a Fluoblok chemotaxis assay.
  • Piceatannol a hydroxystilbene derivative of resveratrol, preferentially inhibits the kinase activity of Syk in in vitro assays and is widely used as a Syk-selective inhibitor.
  • Piceatannol increased FCS-induced chemomigration of both HCT-116 and DLD-1 cell lines to the same manner than shRNA-based Syk global knockdown.
  • Cell attachment and spreading on matrix proteins is a key step in invasion. When cultured on Matrigel, many tumor cells attach and elongate, and this, together with motility, results in formation of cordlike structures and ultimately invasion.
  • Syk expression is required for the activation of PI3K-Akt survival pathway.
  • the epidermal growth factor receptor (EGFR) signaling pathway is commonly activated in colorectal cancer. Activation of this pathway occurs after ligand binding to EGFR, which leads to EGFR phosphorylation and oligodimerization at the plasma membrane. This in turn triggers a chain of downstream signaling events that include activation of the Ras/mitogen- activated protein kinase (MAPK) and phosphoinositide 3 -kinase (PI3K)-Akt pathways that are crucial mediators of growth factor-induced proliferation and cell survival, respectively.
  • MAPK Ras/mitogen- activated protein kinase
  • PI3K phosphoinositide 3 -kinase
  • HCT-116 and DLD-1 cells transduced with shRNAs that target Syk global and long-isoform specific expression or firefly luciferase (shLUC; control) were serum starved, then treated with EGFR ligand, EGF. Cell lysates were subsequently analyzed by immunoblotting using activation state-specific antibodies for Erk and Akt (data not shown).
  • HCT-116 cells display constitutively active Erkl/2 and Akt proteins, shown by their phosphorylation state, which remained comparable between untreated and EGF-treated cells.
  • EGF treatment induced the phosphorylation of Erkl/2 and Akt in DLD-1 cell line (p- Erk; p-Akt; shLUC: -/+ EGF).
  • shRNA-mediated Syk depletion did not affect the phosphorylation levels of Erkl/2 which remained comparable between EGF-treated and untreated cells (p-Erk; shGl, shL2: -/+ EGF).
  • Akt constitutive phosphorylation of Akt in HCT-116 cells
  • EGF EGF-induced phosphorylation of Akt in both cell lines
  • Cetuximab is a chimeric IgGl monoclonal antibody that targets the extracellular domain of EGFR, blocking ligand binding to the receptor and leading to the inhibition of EGFR signaling.
  • EGF exerts opposite effects on Syk alternative splicing.
  • Syk (L) isoform in CRC cell survival and cell cycle regulation
  • Syk downstream of EGFR signaling we performed Syk “pull-down assays” on lysates of serum-starved and EGF-stimulated DLD-1 cells, and we analyzed the components of the captured complexes by high-resolution mass spectrometry to identify Syk interactomes.
  • PSI values for exon 9 of Syk gene which correspond to the ratio of Syk (L) isoform expression over the total Syk transcripts (data not shown). Since Syk (L) isoform results from the inclusion of exon 9 in Syk gene, the PSI value reflects the proportion of Syk (L) isoform expression in patient’s tissue samples.
  • Compound C-13 attenuates tumor growth in CRC xenograft mice model.

Abstract

Spleen tyrosine kinase (Syk) expression have been both positively and negatively associated with tumorigenesis. The goal of the Inventors was to evaluate the contribution of Syk and its two splice variants, full length Syk (L) and short isoform Syk (S), in the tumor biology of colorectal cancer cells (CRC). The analysis of Syk expression in primary human colorectal tumors, as well as the analysis of TCGA database, revealed a high Syk mRNA expression score in colorectal cancer tumors, suggesting a tumor promotor role of Syk in CRC. Their analysis showed that Syk (L) isoform is highly expressed in the majority of the tumor tissues and that it remains expressed in tumors in which global Syk expression is downregulated, suggesting the dependence of tumors to Syk (L) isoform. They also identified a small cluster of tumor tissues, which express a high proportion of Syk (S) isoform. This specific cluster is associated with overexpressed genes related to translation and mitochondria, and down regulated genes implicated in the progression of mitosis. For their functional studies, they used short hairpin RNA tools to target the expression of Syk in CRC bearing the activating K-Ras (G13D) mutation. Their results showed that while global Syk knock down increases cell proliferation and cell motility, Syk (L) expression silencing affects the viability and induces the apoptosis of the cells, confirming the dependence of cells on Syk (L) isoform for their survival. Finally, they report the promising potential of compound C-13, an original non-enzymatic inhibitor of Syk isolated in their group. In vitro studies showed that C-13 exerts cytotoxic effects on Syk-positive CRC cells by inhibiting their proliferation and their motility, and by inducing their apoptosis, while Syk-negative cell lines viability was not affected. Moreover, the oral and intraperitoneal administration of C-13 reduced the tumor growth of CRC DLD-1 cells xenografts in Nude mice in vivo.

Description

SYK INHIBITORS FOR USE IN THE TREATMENT OF CANCER
FIELD OF THE INVENTION:
The present invention is in the field of medicine, in particular oncology.
BACKGROUND OF THE INVENTION:
Spleen tyrosine kinase (Syk) is a 72 kDa non receptor tyrosine kinase that contains two tandem Src homology 2 domains at the NH2 terminus and a kinase domain at the COOH terminus. Syk has two alternatively splice isoforms: full length Syk (L) and short form Syk (S) that lacks a 69-nucleotide exon. Syk (L) is present in both the cytoplasm and the nucleus of the cells due to a nuclear localization signal present in the 23 residues of interdomain B encoded by exon 9, missing in Syk (S). Consequently, Syk (S) is located exclusively in the cytoplasm of the cells.
Syk is widely expressed in hematopoietic cells where it plays a key role in the activation of the cells following the stimulation of antigen and Fc Receptors. Following receptor engagement and clustering, Syk is recruited to the receptor through the binding of its SH2 domains to a double phosphorylated immunoreceptor tyrosine-based activation motif or IT AM. This activates Syk through auto- and trans-tyrosine phosphorylation. Activated Syk then catalyzes the phosphorylation of protein substrates primarily on tyrosines, however Syk also has the ability to phosphorylate some proteins on serine. These phosphorylations lead to the activation of signaling cascades that include the PI3K/Akt, Ras/Erk, PLCy/NFAT, and IKK/NFKB pathways. The critical involvement of Syk in the activation of immune cells has made it a popular target for anti-inflammatory therapeutics directed against diseases such as allergic asthma, rheumatoid arthritis, lupus erythematosus and thrombocytopenic purpura (reviewed in refs (1) (2)).
Syk plays also an important role in epithelial solid cancers where its expression is a marker predicting either poor or favorable outcome (3). Indeed, the contribution of Syk in tumor biology depends on cancer types and it can be of three origins: 1) pro-survival role by stabilizing anti-apoptotic proteins (MCL-l/BCL-2 family) (4,5) and by suppressing c-JUN expression (6). This activity underlies many of the tumor promoting activities of the kinase; 2) negative regulator of EMT by enhancing cell-cell and cell -matrix adhesion (7) (8). This activity underlies many of the tumor suppressive activities of the kinase; and 3) regulator of mitotic progression through its centrosomal kinase activity associated with y-tubuline (9) and by controlling the cell cycle G2-M phase progression (10).
Many studies have investigated the overall expression of Syk as well as the expression of the splicing variants of Syk and their contribution to tumorigenesis. Coopman and coworkers were the first group reporting the role of Syk as a suppressor of tumorogenesis in breast cancer cells (11). In this model, the loss of Syk expression is due to the hypermethylation of Syk gene promoter, and it is frequently observed in primary breast tumors, while the unmethylated promoter is found in adjacent normal breast tissue (12). The loss of Syk expression is also found in gastric cancer (13), hepatocellular carcinoma (HCC) (14) and melanoma (15).
On the opposite, the role of Syk as an oncogene is frequently reported in squamous carcinomas of head and neck (SCCHN) (16) where its expression enhances cell migration. Interestingly, high expression of Syk is significantly associated with recurrence and shorter survival in SCCHN patients. In ovarian cancer, the expression of Syk increases with tumor grade, and the silencing of Syk expression inhibits anchorage-independent growth and induces apoptosis in ovarian cancer cells (6). A similar pro-survival role for Syk is seen in retinoblastoma cells, where both the treatment with Syk inhibitors and knockdown of Syk expression result in a dramatic increase in apoptosis of retinoblastoma cells (17).
Accumulating evidences suggest an active but opposing role of Syk (L) and Syk (S) isoforms on the growth properties of cancer cells, possibly due to different biologic functions for the two isoforms. Wang and co-workers (18) showed that in primary breast tumors, Syk (L) was present in both tumor and matched normal mammary gland, and that ectopic expression of Syk (L) in a cellular model suppressed cell invasiveness. They also showed that Syk (S) was expressed in cancerous tissues and not in normal mammary gland, and Syk (S) ectopic expression did not reduce cell invasiveness in vitro. On the opposite, Prinos et al (6) reported that changing the Syk alternative splicing pattern by decreasing Syk (L) and increasing Syk (S) expression in an ovarian cancer cell line induced apoptosis and altered cell survival and mitotic progression. A differential expression pattern of Syk was also found in HCC where Syk (L) mRNA expression was downregulated in 38% of the tumor samples while Syk (S) mRNA expression was detectable in 40% of the tumor samples and none in the normal liver tissue samples (19). Moreover, patients with a Syk (S)-positive HCC had a worse overall survival compared to patients with a Syk (S)-negative HCC.
Little is known on the contribution of Syk and its spicing variants in the tumor biology of colorectal cancer. Yang et al (20) showed a loss of overall Syk mRNA expression in half of CRC patients due to the hypermethylation of the Syk promoter region, which was associated with a higher tumor stage and reduced five-year overall survival in a heterogeneous group of stage I-IV colon and rectum carcinoma. Ni et al. (21) investigated the expression levels of the splice variants of Syk in 26 pairs of CRC and adjacent non-cancerous tissues. Their study showed that while Syk (L) is downregulated in 69% of tumor tissue samples compared to the adjacent non-cancerous tissue, the expression of Syk (S) remains stable, suggesting that Syk (L) but not Syk (S) is associated with tumor suppressing activities. Coebergh et al (22) studied the prognostic role of Syk in a cohort of 160 chemonaive lymph node negative colon cancer patients, and they found that Syk (S) high mRNA expression correlates with metastatic relapse with hepatic lesions. However, they did not observe any association between mRNA expression of the splice variants and tumor grade, nor any evidence supporting a tumor suppressor role for Syk (L).
SUMMARY OF THE INVENTION:
The present invention is defined by the claims. In particular, the invention relates to Syk inhibitors for use in the treatment of cancer.
DETAILED DESCRIPTION OF THE INVENTION:
The goal of the Inventors was to evaluate the role of Syk alternative spliced isoforms in cancer biology of colorectal carcinomas. For this purpose, they used two approaches: i) short hairpin RNA (shRNA) tools to target the global expression of Syk as well as its alternative splicing variant Syk (L); ii) the use of compound C-13, an original non-enzymatic inhibitor of Syk isolated in their group (23). Their functional studies demonstrated that CRC cell lines depend on Syk long isoform for their survival, since Syk (L) expression silencing affected the viability and induced the apoptosis of the cells. Moreover, the application of compound C-13 phenocopied Syk (L) expression silencing in vitro, and its oral and intraperitoneal administration reduced the tumor growth of CRC DLD-1 cells xenografts in Nude mice in vivo.
A first aspect of the present invention relates to a Syk inhibitor for use in the treatment of a cancer in a subject in need thereof. In some embodiments, the Syk inhibitor is a non- enzymatic inhibitor of Syk.
As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine or a primate. In some embodiments, the subject is a human afflicted with or susceptible to be afflicted with a cancer. In a more particular embodiment, the subject is a human afflicted with or susceptible to be afflicted with a colorectal cancer. In some embodiments, the subject suffers from a cancer wherein Syk plays the role of an oncogene. In some embodiments, the subject bears G13D, G12D, G12V or G12C activating mutation in K-Ras gene. In some embodiments, the subject bears G13D activating mutation in K-Ras gene. Activating mutations in the KRAS gene are seen in 30-50% of CRCs. Such mutations prevent the use of anti-EGFR targeted therapies. The most common somatic mutations observed in KRAS anomalies in CRC are found in codons 12 and 13. Notably, the four most frequent mutations of all CRCs in terms of incidence are G12D (11%), G13D (7%), G12V (7%), and G12C (4%). This missense mutation aberrantly activates KRAS and results in activation of downstream signaling pathways.
As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In some embodiments, the cancer is a colorectal cancer. In some embodiments, the cancer is a K-Ras mutated cancer. As used herein, the term “K-Ras” (Ki-Ras2 Kirsten rat sarcoma viral oncogene homolog) denotes a protein encoded by the K-Ras gene (Entrez: 3845; Ensembl: ENSG00000133703; Uniprot: P01116). K-Ras is a pro-oncogenic small GTPase. It belongs to Ras protein family (monomeric G protein). It transmits signals from growth hormone receptors and in particular the EGF receptor, which receives the signal from outside the cell. This protein has a GTPase activity when it is activated by the EGF receptor. In some embodiments, K-Ras gene is mutated. In some embodiments, the K-Ras gene mutation is an activating mutation. Typically, a K-Ras activating mutation leads to a constitutive activation of the RAS-RAF-MEK-ERK pathway. In some embodiments, the subject bears G13D, G12D, G12V or G12C activating mutation in K-Ras gene. In some embodiments, the activating mutation is G13D mutation.
As used herein, the terms “treating”, “treatment” or “therapy” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
In some embodiments, the present invention relates to a method of treating a cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a Syk inhibitor.
As used herein, the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor of Syk) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.
As used herein, the expression "therapeutically effective amount" is meant a sufficient amount of the active ingredient (e.g. Syk inhibitor) for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
As used herein, the term “Syk” or “spleen tyrosine kinase” refers to is a 72 kDa non receptor tyrosine kinase that contains two tandem Src homology 2 domains at the NH2 terminus and a kinase domain at the COOH terminus. Syk has two alternatively splice isoforms: full length Syk (L) and short form Syk (S) that lacks a 69-nucleotide exon. Syk is encoded by SYK gene (Gene ID: 6850; Ensembl : ENSG00000165025; Uniprot : P43405).
As used herein, the term “Syk inhibitor” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of Syk. In some embodiments, the Syk inhibitor does not affect Syk kinase activity. In some embodiments, the Syk inhibitor does not inhibit the catalytic activity of Syk. In some embodiments, the Syk inhibitor does not target Syk catalytic site. In some embodiments, the Syk inhibitor binds to the protein tyrosine kinase Syk to prevent its interaction with partners. In some embodiments, the Syk inhibitor is a Syk (L) inhibitor. In some embodiments, the Syk inhibitor is a Syk (S) inhibitor.
In one embodiment, the inhibitor according to the invention is a low molecular weight compound, e. g. a small organic molecule (natural or not). The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
Syk inhibitors include, but are not limited to Syk inhibitors as described in WO2009/133294. Thus, in some embodiments, the Syk inhibitors are selected from the molecules having the following formulas (I), (II), (III) or (IV):
Figure imgf000010_0001
where:
R1 is an optionally substituted aromatic group, or an optionally substituted heterocycle comprising at least one S, O or N atom; R2 is an optionally substituted aromatic group, an optionally substituted heterocycle, an optionally saturated carbon chain, comprising an amine group, an optionally saturated carbon chain comprising an optionally substituted aromatic group or an optionally saturated carbon chain comprising an optionally substituted heterocycle comprising at least one S, O or N atom; R3 is an optionally substituted phenyl, 2-pyridinyl, 3-pyridinyl or 4-pyridinyl group;
Figure imgf000010_0002
where n=0 or 1; n'=0 or 1;
R4 is an optionally saturated carbon chain comprising 1 to 5 carbon atoms, optionally substituted with an aromatic group;
R5 is an optionally substituted aromatic group or an optionally substituted amine group;
R6 is a hydrogen atom, alkoxy group, alkyl group or halogen; R7 is a hydrogen atom, alkoxy group, alkyl group or halogen; R8 is a hydrogen atom, alkoxy group, alkyl group or halogen;
Figure imgf000011_0001
where m=0, 1 or 2; R9 is a hydrogen atom and RIO is an optionally substituted phenyl group, or R9 and RIO are part of the same optionally substituted heterocycle, or R9 and RIO are part of the same optionally substituted aromatic group;
Rll is a hydrogen atom, alkoxy group or alkyl group;
R12 is a hydrogen atom, alkoxy group or alkyl group; R13 is a hydrogen atom or an alkyl or alkoxy group;
R14 is a hydrogen atom or an alkyl or alkoxy group;
Figure imgf000011_0002
where
A is an oxygen or sulphur atom; R15 is an optionally saturated carbon chain comprising 1, 2 or 3 carbon atoms, optionally substituted by an optionally substituted aromatic group, an optionally substituted heterocycle or an amine group belonging to optionally substituted heterocycle;
R16 is a hydrogen atom, halogen or alkoxy group;
R17 is a hydrogen atom, alkoxy group or acetoxy group.
In some embodiments, the Syk inhibitor is (methyl 2-{5-[(3-benzyl-4-oxo-2-thioxo-l,3- thiazolidin-5-ylidene)methyl]-2-furyl}benzoate) (also named C-13); 4-(4-chloro benzoyl)-3- hydroxy-5-(3-phenoxy phenyl)- 1 -(3 -pyridinyl methyl)-l,5-dihydro-2H-pyrrol-2-one; 5-(2,4- dimethoxy phenyl)-3 -hydroxy- 1- [3 -(1 H-imidazol-l-yl)propyl]-4-(2 -thienyl carbonyl)- 1,5- dihydro-2H-pyrrol-2-one; acide {4-bromo-2-[3-(ethoxy carbonyl)-2-methyl-5-oxo-4,5- dihydro-lH-indeno[l,2-b]pyridin-4-yl]phenoxy} acetique; 3 -hydroxy-5 -(3 -methoxy phenyl)-
4-(4-methyl benzoyl)-l-[3-(4-morpholinyl)propyl]-l,5-dihydro-2H-pyrrol-2-one; 4-benzoyl-
5 -(2, 5 -dimethoxy phenyl)-3 -hydroxy- 1 - [3 -( 1 H-imidazol- 1 -yl)propyl] - 1 , 5 -dihy dro-2H-pyrr 01-
2-one; ethyl 4-[3-benzoyl-2-(2,4-dimethoxy phenyl)-4-hydroxy-5-oxo-2,5-dihydro-lH- pyrrol-l-yl] benzoate; 4-(2, 5 -dimethyl benzoyl)-3-hydroxy-5-(2-methoxy phenyl)- 1-[2-(4- morpholinyl)ethyl]-l,5-dihydro-2H-pyrrol-2-one; 5-(3-bromo-4-hydroxy-5-methoxy phenyl)-
3 -hydroxy- 1 -(2- phenylethyl)-4-(2 -thienyl carbonyl)- 1,5 -dihy dro-2H-pyrrol -2-one; 4-(4-fluoro benzoyl)-3-hydroxy-5-(3-phenoxy phenyl)- 1 -(3 -pyridinyl methyl)- 1 ,5-dihy dro-2H-pyrrol-2- one; ethyl 2-[3-(4-fluorobenzoyl) -4-hydroxy-2-(4-methyl phenyl)-5-oxo-2,5-dihydro-lH- pyrrol-l-yl]-4-methyl-l,3-thiazole-5-carboxylate; 5 -(2, 5 -dimethoxy phenyl)-3 -hydroxy -4-(4- methyl benzoyl)- 1 -(3 -pyridinyl methyl)- 1,5 -dihy dro-2H-pyrrol -2-one; ethyl 2-[3-benzoyl-4- hydroxy-2-(4-methoxy phenyl)-5-oxo-2,5-dihydro-lH-pyrrol-l-yl]-4-methyl-l,3-thiazole-5- carboxylate; 3-[2-(2,4-dimethoxy phenyl)-2-oxoethyl]-3-hydroxy-l-(l-naphthyl methyl)-l,3- dihydro-2H-indol -2-one; 3 -hydroxy -4-(4-methoxy-2-methyl benzoyl)-l-[2-(4- morpholinyl)ethyl]-5-(3-pyridinyl)-l,5-dihydro-2H-pyrrol-2-one; 2-methoxy-N-(4-{4-methyl- 5-[(2-oxo-2- phenylethyl) thio]-4H-l,2,4-triazol-3-yl} phenyl) benzamide; methyl 2-[3- benzoyl-4-hydroxy-2-(4-methyl phenyl)-5-oxo-2,5-dihydro-l H-pyrrol-l-yl]-4-methyl-l,3- thiazole-5-carboxylate; 4-[(4-benzyl-l-piperidinyl) methyl]-N-(2-methoxy-5-methyl phenyl) benzamide; 4-{[N-[(4-methoxy phenyl) sulfonyl]-N-(2- phenyl ethyl)glycyl] amino} benzamide ; 4-(4-fluoro benzoyl)-3-hydroxy-5-(4-isopropyl phenyl)-l-[3-(4- morpholinyl)propyl] - 1 , 5 -dihy dro-2H-pyrrol-2-one; N,N'- 1 , 5 -naphthalenediylbi s [2-(3 -methyl phenoxy) acetamide]; N-[4-({[4-(acetyl amino) phenyl] sulfonyl} amino)-2, 5 -dimethoxy phenyl] benzamide; 7,7-dimethyl-l-(4-methyl phenyl)-2,5-dioxo-N-(2,2,6,6-tetramethyl-4- piperidinyl)-l,2,5,6,7,8-hexahydro-3-quinoline carboxamide; acide 4-(benzyl{[l- phenyl-3-(2- thienyl)-lH-pyrazol-4-yl]methyl} amino)-4-oxo butanoique; 4-(4-fluoro benzoyl)-3 -hydroxy - 5-(3-methoxy phenyl)- l-[3-(4-morpholinyl)propyl]-l,5-dihydro-2H-pyrrol-2-one; 4-(4-chloro benzoyl)-3-hydroxy-l-[3-(4-morpholinyl)propyl]-5-(3,4,5-trimethoxy phenyl)- 1,5-dihydro- 2H-pyrrol -2-one; 4-benzoyl-3-hydroxy-5-(4-isopropyl phenyl)- 1 -[2-(4-morpholinyl)ethyl]- l,5-dihydro-2H-pyrrol-2-one; 4-(4-chloro benzoyl)-5-(3,4-dimethoxy phenyl)-3-hydroxy-l- [2-(4-morpholinyl)ethyl]-l,5-dihydro-2H-pyrrol -2-one; l-[2-(dimethyl amino)ethyl]-5-(2,5- dimethoxy phenyl)-3 -hydroxy -4-(4-methyl benzoyl)- l,5-dihydro-2H-pyrrol -2-one; 2-fluoro- N-[(5-{[(4-oxo-3,4-dihydro-2-quinazolinyl)methyl]thio}-4- phenyl-4H-l,2,4-triazol-3- yl)methyl] benzamide; 2-{[4-(l,3-dioxo-l,3-dihydro-2H-isoindol-2-yl)butanoyl] amino}-N- (tetrahydro-2-furanyl methyl)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide; 5-(3,4- dimethoxy phenyl)-4-(4-fluoro benzoyl)-3-hydroxy-l-[2-(4-morpholinyl)ethyl]-l,5-dihydro- 2H-pyrrol -2-one; 4-({4-hydroxy-l-[2-(4-morpholinyl)ethyl]-5-oxo-2- phenyl-2,5-dihydro-lH- pyrrol-3-yl} carbonyl)-N,N-dimethyl benzene sulfonamide; 5-(2,4-dimethoxy phenyl)-4-(4- fluorobenzoyl) -3-hydroxy-l-[2-(4-morpholinyl)ethyl]-l,5-dihydro-2H-pyrrol-2-one; 3- hydroxy-5-(4-methoxy phenyl)-4-(4-methyl benzoyl)- l-[2-(4-morpholinyl)ethyl]- 1,5- dihydro-2H-pyrrol-2-one; 5-(3,4-dimethoxy phenyl)-4-(2-furoyl)-3 -hydroxy- 1-[2-(4- morpholinyl)ethyl]-l,5-dihydro-2H-pyrrol-2-one; 4-(4-chloro benzoyl)-5-(3,4-dimethoxy phenyl)-3-hydroxy-l-[2-(4-morpholinyl)ethyl]-l,5-dihydro-2H-pyrrol -2-one; ethyl 2-[3 - benzoyl-4-hydroxy-2-(4-methoxy phenyl)-5-oxo-2,5-dihydro-lH-pyrrol-l-yl]-4-methyl-l,3- thiazole-5-carboxylate; 5-(3,4-dimethoxy phenyl)-3-hydroxy-l-[2-(4-morpholinyl)ethyl]-4- (2-thienyl carbonyl)-l,5-dihydro-2H-pyrrol-2-one; 4-(4-chloro benzoyl)-3-hydroxy-5-(4- methoxy phenyl)-l-[2-(4-morpholinyl)ethyl]-l,5-dihydro-2H-pyrrol-2-one; N-[2-(4-benzyl-l- piperazinyl)-2-oxoethyl]-N-(3,5-dimethyl phenyl) benzene sulfonamide; l-methyl-2-(4- methyl phenyl)-2-oxoethyl 2-(3-chloro-4-methyl phenyl)-l,3-dioxo-5-isoindoline carboxylate; N~2 — [(3,4-dimethoxy phenyl) sulfonyl]-N~l — (2-methoxy-5-methyl phenyl) - N~2 — (4-methyl phenyl) glycinamide; 5 -(2, 5 -dimethoxy phenyl)-4-(4-fluoro benzoyl)-3- hydroxy-l-[3-(4-morpholinyl)propyl]-l,5-dihydro-2H-pyrrol-2-one; 4-(4-chlorobenzoyl )-5- (2-fluoro phenyl)-3 -hydroxy- 1 - [3 -(4-morpholinyl)propyl] - 1 , 5 -dihy dro-2H-pyrrol -2-one; 4-(4- fluoro benzoyl)-3-hydroxy-5-(4-isopropyl phenyl)-l-[2-(4-morpholiny/)ethyl]-l,5-dihydro- 2H-pyrrol-2-one; 4-{[2-(4-morpholinyl)ethyl]amino}-3-(4-morpholinyl sulfonyl)-N- phenylbenzamide; 2-chloro-N-{4-[4-methyl-5-({2-oxo-2-[(tetrahydro-2-furanyl methyl) amino] ethyl}thio)-4H-l,2,4-triazol-3-yl] phenyl} benzamide; N-[2-(4-benzyl-l-piperazinyl)- 2-oxoethyl]-N-(3,4-dimethyl phenyl)-4-methyl benzene sulfonamide; acide 2-[(4-{[(4- isopropyl phenoxy) acetyl]amino}-3-methyl benzoyl) amino] benzoique; 4-({2-(3,4-dichloro phenyl)- 1 - [3 -(dimethyl amino)propyl] -4-hy droxy-5 -oxo-2, 5 -dihydro- 1 H-pyrrol-3 -yl } carbonyl)-N,N-dimethyl benzene sulfonamide; 4-(l,3-benzodioxol-5-yl carbonyl)-l-[3- (diethyl amino)propyl]-3-hydroxy-5-(3-pyridinyl)-l,5-dihydro-2H-pyrrol-2-one; l-[2- (dimethyl amino)ethyl]-3-hydroxy-4-(5-methyl-2-furoyl)-5-(3,4,5-trimethoxy phenyl)-l,5- dihydro-2H-pyrrol-2-one; 3 -hydroxy-5 -(3 -methoxy phenyl)-4-(5-methyl-2-furoyl)-l-[2-(4- morpholinyl)ethyl]-l,5-dihydro-2H-pyrrol-2-one; ethyl 4-({[(5,6-di-2-furyl-l,2,4-triazin-3- yl)thio]acetyl}amino) benzoate; ethyl 5-cyano-4-(2-furyl)-6-({2-[(3-methoxy phenyl) amino] -
2-oxoethyl} thio)-2- phenyl- l,4-dihydro-3 -pyridine carboxylate; 5 -(2, 3 -dimethoxy phenyl)-4- (2,5-dimethyl benzoyl)-3-hydroxy-l-[3-(4-morpholinyl)propyl]-l,5-dihydro-2H-pyrrol-2-one;
3-(6-amino-5-cyano-3- phenyl- 1,4-dihydro pyrano[2,3-c]pyrazol-4-yl) phenyl 2-furoate; 1,4- bis [(mesityloxy)acetyl] piperazine; N-(4-chloro phenyl)-2-{[4-(2- phenyl ethyl)-5-(3,4,5- trimethoxy phenyl)-4H-l,2,4-triazol-3-yl]thio} acetamide; acide {[3-(ethoxy carbonyl)-2- phenyl-l-benzofuran-5-yl]oxy} ( phenyl) acetique; ethyl 4-({[l-(4-chloro phenyl)-5-oxo-3-(3- pyridinyl methyl)-2-thioxo-4-imidazolidinyl]acetyl} amino) benzoate; 5,5'-oxybis [2-(tetra hydro-2-furanyl methyl)-l H-isoindole-l,3(2H)-dione]; 7-acetyl-6-[3-(benzyloxy) phenyl]-3- (methylthio)-6,7-dihydro [1,2,4] triazino[5,6-d][3,l] benzoxazepine; 3 -hydroxy- 1- [3 -(1 H- imidazol-l-yl)propyl]-4-[(7-methoxy-l-benzofuran-2-yl) carbonyl]-5-(2-pyridinyl)-l,5- dihydro-2H-pyrrol-2-one; 4-{[4-hydroxy-l-[3-(4-morpholinyl)pr opyl]-5-oxo-2-(3-pyridinyl)- 2,5-dihydro-l H-pyrrol-3-yl]carbonyl}-N,N-dimethyl benzene sulfonamide; N-{ l-[4-allyl-5- ({2-[(3-methoxy phenyl) amino] -2-oxoethyl} thio)-4H-l,2,4-triazol-3-yl]ethyl} benzamide; N-(2-hydroxy- 1,1 -dimethylethyl) -5-{4-[(3-hydroxy phenyl) amino]-l-phthalazinyl}-2- methyl benzene sulfonamide; 4-(l,3-benzodioxol-5-ylcarbonyl)-5-(2 -fluoro phenyl)-3- hydroxy-l-[3-(4-morpholinyl)propyl]-l,5-dihydro-2H-pyrrol-2-one; N-(2,4-dimethoxy phenyl)-2-{[3-(2-furylmethyl)-4-oxo-3,4-dihydro-2-quinazolinyl] thio} acetamide; N-[(2- hydroxy-7-methyl-3-quinolinyl) methyl]-3-methoxy-N-(2-methoxy phenyl) benzamide; 4-(4- chlorobenzoyl)-3-hydroxy-5-(3-methoxy phenyl)- l-[2-(4-morpholinyl)ethyl]- 1,5-dihydro- 2H-pyrrol -2-one; 2-(4-methoxy phenoxy)-N-[2-methyl-5-(3-methyl-4-oxo-3,4-dihydro-l- phthalazinyl)benzyl] acetamide; N-{4-[({[4-(4-methoxy phenyl) tetrahydro-2H-pyran-4-yl] methyl} amino) carbonyl] phenyl }-2-furamide; 4-benzoyl-5-(2,3-dimethoxy phenyl)-3- hydroxy-l-[3-(4-morpholinyl)propyl]-l,5-dihydro-2H-pyrrol-2-one; isopropyl 3-({[(4-allyl-5- {[(3-methyl benzoyl) amino] methyl}-4H-l,2,4-triazol-3-yl) thio]acetyl) amino) benzoate; 2- {5-[l-(4-morpholinyl)cyclohexyl]-lH-tetrazol-l-yl}ethyl 1-naphthyl carbamate; methyl 4- ({[3-(l,3-benzodioxol-5-ylmethyl)-2,5-di oxo-1- phenyl-4-imidazol idinyl]acetyl}amino) benzoate; 4-(3,4-dihydro-2(lH)-isoquinolinylm ethyl)-N-[2-(l-pyrrolidinyl carbonyl) phenyl] benzamide; 9-{3-chloro-4-[(4-methyl benzyl)oxy] phenyl}-10-ethyl-3,4,6,7,9,10-hexahydro- l,8(2H,5H)-acridine dione; ethyl l-(4-{[(3,4-dimethyl phenyl) (methyl sulfonyl) amino] methyl} benzoyl)-4-piperidine carboxylate; methyl 4- {[N-(3 -methoxy phenyl)-N-( phenyl sulfonyl) glycyl] amino} benzoate; N-{[5-({2-[(4-bromo-2,3-dimethyl phenyl) amino]-2- oxoethyl}thio)-4-ethyl-4H-l,2,4-triazol-3-yl]methyl}-4-chloro benzamide; 4-(2,3-dihydro- l,4-benzodioxin-6-yl carbonyl)-3-hydroxy-l-[3-(4-morpholinyl)propyl]-5-(4-pyridinyl)-l,5- dihydro-2H-pyrrol-2-one; N-(4-ethoxy phenyl)-2-{ l-(4-m ethoxy phenyl)-3-[2-(4- morpholinyl)ethyl]-5-oxo-2-thioxo-4-imidazol idinyl} acetamide; 5-(3-bromo phenyl)-3- hydroxy-4-[(7-methoxy- 1 -benzo furan-2-yl) carbonyl]- 1 -[2-(4-morpholinyl)ethyl]- 1,5- dihydro-2H-pyrrol-2-one; l-[3-(diethyl amino)propyl]-3-hydroxy-4-[(7-methoxy-l- benzofuran-2-yl) carbonyl]-5-(2-pyridinyl)-l,5-dihydro-2H-pyrrol-2-one or l-(4-{[(4,6- dimethyl-2-pyrimidinyl)thio]acetyl}-l-piperazinyl)-4-(4-methyl phenyl) phthalazine.
In some embodiments, the Syk inhibitor is C- 13. As used herein, the term “C-13” refers to (methyl 2-{5-[(3-benzyl-4-oxo-2-thioxo-l,3-thiazolidin-5-ylidene)methyl]-2- furyl (benzoate). This molecule has the following formula and structure in the art C23H17NO4S2:
Figure imgf000015_0001
Other Syk inhibitors includes as example BAY-613606, R112, R343, R406, R788, piceatannol, NVP-QAB205, AB8779, ER 27319, GSK143.
In one embodiment, the inhibitor according to the invention (i.e. Syk inhibitor) is an antibody. In a more particular embodiment, the antibody according to the invention is an antibody directed against Syk-(L) isoform. In a more particular embodiment, the antibody according to the invention is an antibody directed against Syk-(S) isoform. Antibodies directed against Syk can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against Syk can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-Syk single chain antibodies. Compounds useful in practicing the present invention also include anti-Syk antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to Syk. Humanized anti-Syk antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of nonhuman (e.g., rodent) chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).
In another embodiment, the antibody according to the invention is a single domain antibody directed against Syk. In a more particular embodiment, the antibody according to the invention is a single domain antibody directed against Syk-(L) isoform. In a more particular embodiment, the antibody according to the invention is a single domain antibody directed against Syk-(S) isoform. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH. The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation. VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B- cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254). In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then, for this invention, neutralizing aptamers of Syk are selected.
In one embodiment, the compound according to the invention is a polypeptide. In a particular embodiment the polypeptide is an antagonist of Syk and is capable to prevent the function of Syk. Particularly, the polypeptide can be a mutated Syk protein or a similar protein without the function of Syk. In a more particular embodiment, the polypeptide is an antagonist of Syk-(L) isoform. In a more particular embodiment, the polypeptide is an antagonist of Syk- (S) isoform. In one embodiment, the polypeptide of the invention may be linked to a cellpenetrating peptide” to allow the penetration of the polypeptide in the cell. The term “cellpenetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012). The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli. In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa). In addition, to the polymer backbone being important in maintaining circulatory half-life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
In another embodiment, the Syk inhibitor according to the invention is an inhibitor of Syk gene expression. In a more specific embodiment, the Syk inhibitor according to the invention is a nucleic acid targeting a mRNA encoding Syk. In a more specific embodiment, the Syk inhibitor according to the invention is a nucleic acid targeting a mRNA encoding Syk- (L) isoform. In a more specific embodiment, the Syk inhibitor according to the invention is a nucleic acid targeting a mRNA encoding Syk-(S) isoform.
Small inhibitory RNAs (siRNAs) can also function as inhibitors of Syk expression for use in the present invention. Syk gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that Syk gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). In some embodiments, the siRNA is directed against Syk-(L) isoform. In some embodiments, the siRNA is directed against Syk-(S) isoform.
Short hairpin RNA (shRNA) can also function as inhibitors of Syk expression for use in the present invention. ShRNA refers to a segment of RNA that is complementary to a portion of a target gene and has a stem-loop (hairpin) structure. Thus, in some embodiments, the Syk inhibitor is a shRNA. In some embodiments, the shRNA is directed against Syk-(L) isoform. In some embodiments, the shRNA is directed against Syk-(L) isoform and comprises an amino acid sequence having at least 90% of identity with SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the shRNA is directed against Syk-(L) isoform and comprise the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the shRNA is directed against Syk-(S) isoform.
Ribozymes can also function as inhibitors of Syk gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of Syk mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays. Both antisense oligonucleotides and ribozymes useful as inhibitors of Syk gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing Syk. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles are provided in Kriegler, 1990 and in Murry, 1991. Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno- associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.
Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .
In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
In order to test the functionality of a putative Syk inhibitor a test is necessary. For that purpose, to identify Syk inhibitors, many methods are available in the art. As example, the skilled person can refer to WO2009/133294.
Another aspect of the invention relates to a pharmaceutical composition comprising a Syk inhibitor for use in the treatment of a cancer in a subject in need thereof.
Any therapeutic agent of the invention (e.g. a Syk inhibitor) may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc. The pharmaceutical compositions of the invention can be formulated for a topical, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like. Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used. Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent. For example, anti-cancer agents may be added to the pharmaceutical composition as described below. Anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A.
In some embodiments, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.
Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies. Other additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.
In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron.
In another embodiment, the further therapeutic active agent can be a hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
In still another embodiment, the other therapeutic active agent can be an opioid or nonopioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
In yet another embodiment, the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent. Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin-like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7- Hl). Typically, the checkpoint blockade cancer immunotherapy agent is an antibody. In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1. Targeting of Syk alternative spliced isoforms. (A) The relative expression of Syk (L), Syk (S) and total Syk (L+S) in colorectal carcinoma cell lines was detected by qPCR; HPRT was used as an internal control. The Y-axis of the plot is expressed in a base-2 log scale. The figure is representative of three independent experiments. (B) The expression of total Syk was evaluated by western blot. The panel represents the densitometric quantification of immunoblot analyses of Syk expression over control. The impact of shRNAs targeting the pre-mRNAs encoding Syk proteins evaluated by RNA expression monitored by (C) qPCR; HPRT was used as an internal control, and (D) western blot, compared to mock transduced (shLUC) HCT-116, DLD-1 and DIFI CRC cells. Experiments were performed in three biological and three technical replicates, and an average for each cellular phenotype was calculated for each shRNA. The panel represents the densitometric quantification of Syk expression over control. shRNA Gl, G2 inhibit global gene expression & shRNA LI, L2 are alternative exon-specific. shRNA LUC with Luciferase sequence was used as negative control. The qPCR relative expression values for the overall gene expression Syk (L+S), long splice isoform (Syk L) and short splice isoform (Syk S) were normalized against HPRT used as control. Error bars represent the mean ± SD of three independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001, compared with control). For the western blots, total proteins were extracted one week posttransfection and equal amount of proteins were loaded on an 8% SDS-PAGE. The anti-Syk antibody and anti-tubulin antibodies used are described in materials & methods. Tubulin served as a loading control.
Figure 2. Changing the splicing pattern of Syk affects cell viability and induces apoptosis. The impact of shRNAs targeting global Syk (shGl) & long-isoform-specific Syk (shL2) pre-mRNAs on cellular phenotype evaluated by: cell proliferation, viability and apoptosis, and normalized to unrelated-transduced shRNA (shLUC) HCT- 116, DLD- 1 and DIFI CRC cells. Cell numbers were monitored using cell counts (Countess II, Promega), apoptosis was monitored using Annexin V-FITC marker and analyzed by flow cytometry (Gallios Beckman Coulter) and viability was monitored using CellTiter-Glo Luminescent cell viability assay (Promega). The relative changes were calculated and used to generate the bar graphs. The error bars represent the variation observed in three biological and three technical replicate (*, P<0.05; **, P<0.01; ***, P<0.001, compared with control).
Figure 3. Syk (L) isoform regulates cell-cycle progression and cytokinesis.
DLD-1 cells were transduced with Syk shGl and shL2 shRNAs or shLUC (control shRNA), and the cell cycle distribution of cells one-week post-infection was analyzed using flow cytometry. The accumulation of transduced cells in G0-G1, S and G2-M and > 4N phases of each cell line is normalized to its control and is shown as a bar graph. The data are an average of three independent transduction experiments and error bars represent the mean ± SD (*, P<0.05; **, P<0.01).
Figure 4. Syk inhibition increases chemotaxis and Matrigel engagement. (A) HCT- 4
116 and DLD-1 cells (5x10 ) transduced with shRNAs targeting global Syk (shGl) & long- isoform-specific Syk (shL2) or unrelated-transduced shRNA (shLUC), and (B) cells pretreated with Piceatannol or vehicle (DMSO) were serum starved, labelled with CellTracker dye, and
4 migrated toward FCS in Fluoroblock chambers. (C) HCT-116 and DLD-1 cells (5x10 ) transduced with Syk shRNAs or control shRNA were serum starved, labelled with CellTracker dye and seeded on Matrigel-coated Transwell inserts of 8 pm pore size (BD Falcon). FCS was used as chemoattractant in the Transwell lower part. After 24 hours at 37°C, migration was observed and counted microscopically. Images were obtained and total number of cells per field of view was measured using ImageJ software. Error bars represent the mean number of migrated cells in triplicate wells, representative of three independent experiments (*, P<0.05).
Figure 5. Syk (L) isoform expression is associated with tumorigenesis in human CRC tissues. (A) Western blot analysis of the protein extracts of primary colorectal tumors of 13 untreated patients. T: Tumor tissue and N: normal adjacent tissue of the same patient. The panel represents the densitometric quantification of immunoblot analyses of Syk expression in tumor tissues over the corresponding normal tissues. The arrows indicate the tumor samples in which the ratio of Syk expression is at least two folds higher compared to the normal tissue. (B) Analysis of Syk mRNA differential gene expression in tumor and normal tissue of N=50 patients from TCGA database COAD and READ cohorts. (C) Correlation between Syk mRNA and Syk protein expression in tumor tissues of N=52 patients. (D) Analysis of Syk (L) isoform expression over the total Syk transcripts in tumor tissues of N=50 patients. The horizontal dashed line indicates the median of PSI value in tumor samples. (E) High proportion of Syk (S) isoform are only present in tumors. The PSI values were compared in 50 patients presenting both tumor and normal tissue value. The above gray curve corresponds to the 1.5 limit of Syk(L) expression in tumor sample over normal tissue ratio, whereas the bottom gray curve is the reverse one. Dashed line figures out the 0.25 PSI limit defining Low PSI samples.
Figure 6. Treatment of CRC cell lines with C-13, an original non-enzymatic inhibitor of Syk. (A) Cells were exposed for 2 hours to various concentrations of C-13 and the impact of C-13 on cellular phenotype evaluated 72 hours post-treatment by: cell proliferation, viability and apoptosis, and normalized against DMF (vehicle)-treated HCT-116, DLD-1 and DIFI CRC cells. Data are the mean ± SD (error bars) of at least three independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001; compared with control). (B) Cells pretreated for 2 hours with C-13 or vehicle (DMF) were serum starved, labelled with CellTracker dye, and migrated toward FCS in Fluoroblock chambers. (C) Cells pretreated with C-13 or vehicle (DMF) were serum starved, labelled with CellTracker dye and seeded on Matrigel-coated Transwell inserts of 8 pm pore size (BD Falcon). FCS was used as chemoattractant in the Transwell lower part. After 24 hours at 37°C, migration was observed and counted microscopically. Images were obtained and total number of cells per field of view was measured using Imaged software. Error bars represent the mean number of migrated cells in triplicate wells, representative of three independent experiments (*, P<0.05; **, P<0.01; P<0.001; compared with control). (D) DLD- 1 cells pre-treated with the indicated concentrations of compound C-13 or DMF (vehicle) for 2 hours, were incubated with the recombinant human EGF (50 ng/ml) or a combination of EGF (50 ng/ml) + Cetuximab (50 pg/ml) for 10 minutes. Protein extracts from cell lysates were analyzed by western blot using the indicated antibodies. The panel represents the densitometric quantification of immunoblot analyses of phospho-Erk (+EGF) versus phospho-Erk (-EGF) over control and phospho- Akt (+EGF) versus phospho- Akt (-EGF) over control. (E) Syk- positive CRC cell lines DLD-1, SW620 and HCT-116; and Syk-negative breast cancer cells lines MDAMB231 and BT549 were exposed to various concentrations of C-13 for 72h; (F) HCT-116, DLD-1 and HT29 cell lines were treated in parallel with C-13 (vehicle DMF) and R406 (vehicle DMSO) for 72 h; and cell viability was measured using CellTiter-Glo Luminescent cell viability assay (Promega). The relative changes were calculated and used to generate the bar graphs. The error bars represent the variation observed in three biological and three technical replicate. (*, P<0.05; **, P<0.01; ***, P<0.001; compared with Syk-negative MDAMB23 1 cell line for panel D; compared with vehicle for panel E).
Figure 7. C-13 inhibits tumor growth of xenografts in Athymic Nude mice. (A) Effect of intraperitoneal treatment with C-13 (0.35 mg/kg) or DMF (vehicle) (3 times/week) on tumor growth in Athymic Nude mice xenografted with IxlO6 DLD-1 CRC cells (n= 10 mice/group); (B) Kaplan-Meier survival curves of the mice described in B. (C) Effect of oral administration of C-13 (100 mg/kg) or DMF (vehicle) (3 times/week) on tumor growth of Athymic Nude mice xenografted with IxlO6 DLD-1 CRC cells (n = 7 mice/group). (D) Kaplan- Meier survival curves of the mice described in C.
EXAMPLE:
Material and Methods
Drugs and reagents. Syk non-enzymatic inhibitor C-13 was purchased from ChemBridge, Inc (San Diego, CA) (ID number 6197026) and was dissolved to a final concentration of 10 mM in dimethylformamide (DMF). Syk catalytic inhibitor R406 was from Santa Cruz Biotechnology and was dissolved to a final concentration of 10 mM in dimethyl sulfoxide (DMSO). Syk inhibitor Piceatannol was from Sigma-Aldrich and was dissolved to a final concentration of 40 mM in DMSO. Recombinant human EGF (1 mg/ml) was from Sigma- Aldrich. Cetuximab (ERBITUX®) (5 mg/ml) was purchased from Merck (Darmstadt, Germany). All stock solutions were aliquoted and stored at -20C. All reagents unless otherwise mentioned were from Sigma (St Louis, Mo).
Cell culture. All cell lines were from the ATCC and were cultured in antibiotic- and antimycotic-free medium (Gibco). The human colorectal adenocarcinoma HCT-116, HT29, SW480 and SW620 were grown in RPMI Glutamax medium, and DLD-1 cell line was grown in DMEM/F12 (50/50) Glutamax medium. All culture media were supplemented with 10% (v/v) FBS. The DIFI cell line was grown in RPMI Glutamax medium supplemented with 20% (v/v) FBS. For EGF induction experiments, cells were serum-starved for 12 hours, and they were subsequently treated with the recombinant human EGF (50 ng/ml) or with EGF (50 ng/ml) + Cetuximab (50 pg/ml) in serum free culture medium, for 10 minutes. Cell propagation and passaging were as recommended by the American Type Culture Collection (ATCC). For cell enumeration, cells were detached with TrypLE Express Enzyme (Gibco), and they were counted using Z1 Particle Counter (Beckman Coulter). The cell lines were tested and authenticated by short-tandem repeat profiling (LGC Standards and Eurofins Genomics). All experiments were performed at least three times.
Syk silencing by small hairpin RNA. To design long isoform-specific and global Syk specific shRNA, we used siRNA sequences from Pinos et al (6). Four different shRNA expressing pSIREN vectors with the puromycin selection marker and specific to Syk were used. shRNA Gl, G2 inhibit global gene expression and shRNA LI, L2 are alternative exon-specific. shRNA LUC with Luciferase sequence was used as negative control. Lentiviral production was performed by cotransfecting IxlO6 293T cells (for lentiviral packaging) with 3 pg of pSiren vector in which anti-Syk shRNAs where cloned, 1 pg of the packaging vector gagpol, and 1 pg of envelope vector, using JetPRIME (Polyplus), according to the manufacturer's instructions. Lentiviral particles were harvested, filtered and then used to infect IxlO6 cells for 48 hours. Following shRNA transduction, cells were selected with 1 «g/ml puromycin for one week and stable clones were pooled. shRNA target sequences. shSykGl: 5' GCAGATGGTTTGTTAAGAG 3' (SEQ ID NO:1); shSykGl: 5' GTCGAGCATTATTCTTATA 3' (SEQ ID NO:2); shSykLl: 5' GTTCCCATCCTGCGACTTG 3' (SEQ ID NO:3); shSykLl: 5' GGTCAGCGGGTGGAATAAT 3' (SEQ ID NO:4); shLUC: 5’ TTACGCTGAGTACTTCGA 3’ (SEQ ID NO:5).
RNA extraction and quantitative PCR. Total RNA extractions were performed from 0.5 to LIO6 cells using the ZR RNA MiniPrep Kit (Zymo Research) as recommended by the manufacturer. Reverse transcription was performed on 1 pg RNA using PrimeScript Reverse Transcriptase (Takara Bio.), SYBR Premix Ex Taq 2X master mix (Takara Bio.) and 5 pmol of both forward and reverse primers. Quantitative PCR reactions were performed on 1 ng cDNA. HPRT was amplified as internal control. qPCR primer sequences.
Syk L: for 5' TCAGCGGGTGGAATAATCTC 3' (SEQ ID NO:6); rev 5' TGCAAGTTCTGGCTCATACG 3' (SEQ ID NO:7)
Syk S: for 5' TGGCAGCTAGTCGAGCATTA 3' (SEQ ID NO:8); rev 5' CAGGGGAGGACGCAGGAT 3' (SEQ ID NO:9)
Syk L+ S: for 5' GAAGCCATATCGAGGGATGA 3' (SEQ ID NO:10); rev 5' CCACATCGTATGTCCAGCAC 3' (SEQ ID NO: 11)
HPRT: for 5’ CTGACCTGCTGGATTACA 3’ (SEQ ID NO: 12); rev 5’ GCGACCTTGACCATCTTT 3’ (SEQ ID NO: 13)
Cell growth inhibition assay (2D assay). Cell growth was evaluated using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to manufacturer’s recommendations. Briefly, 3.000 cells/well were seeded in 96-well plates. After 24 hours, drugs were added in serial dilution. Cells were incubated for 96 hours, after which, a volume of CellTiter-Glo® reagent equal to the volume of cell culture medium was added to each well. The plate was shaked on an orbital shaker for 15 minutes at room temperature to induce cell lysis. The luminescence was recorded using a Thermo Fisher Scientific Multiskan EX plate reader. The IC50 was determined graphically from the cell growth curves.
Migration. DLD-1 or HCT-116 cells were serum starved for 16 h in a 1%-FBS- medium. Cells were detached with TrypLE Express Enzyme (Gibco). 5xl04 cells were resuspended in a 1%-FBS-medium and they were seeded on Fluoroblok 24-well plate permeable inserts, with 8 pm pores (Corning). Following a 20-22h incubation with chemoattractant (10 % FBS in RPMI medium), the inserts were transferred in a 2nd 24-well plate containing a 4 pg/mL Calcein AM solution (Sigma Aldrich). The fluorescence was read on an inverted fluorescence microscopy and migrated cells were counted with ImageJ software.
Invasion. Same as migration, but the Fluoroblok insert was coated with 100 pL of 300 pg/pL BD Matrigel Matrix (BD Biosciences).
Apoptosis assay. A total of 2.5xl05 cells were plated, and after 24 hours, they were incubated with the indicated concentrations of compound C-13 for 2 hours. ShRNA-tranduced cells were plated following one week puromycin selection, and grown for 48 hours. Cells were stained with APC-labeled Annexin V and 7-aminoactinomycin D (Biolegend). For apoptosis determination, Annexin V- and 7-ADD-positive cells were quantified using a Gallios Cytometer (Beckman Coulter) and the Kaluza Software (Beckman Coulter).
Cell-cycle analysis. To determine the cell-cycle distribution, 2.5xl05 cells were plated in 25 cm2 flasks. After 48 hours, cells were washed in ice-cold PBS, fixed in 75% ethanol, and labeled with 40 mg/mL propidium iodide (Sigma- Aldrich) containing 100 mg/mL RNase A (Sigma). Cell-cycle distribution was then determined with a Gallios Cytometer (Beckman Coulter) and quantified using the Kaluza Software (Beckman Coulter). Immunofluorescence. 5xl04 cells per well were seeded on Lab-Tek II glass chambers (Thermo Fischer Scientific). After 24 hours, cells were washed with ice-cold PBS, and they were fixed and permeabilized with ice-cold methanol (Sigma Aldrich) for 15 minutes at -20°C. Cells were incubated in PBS containing 2% BSA for 30 minutes at room temperature. Cells were incubated with the primary and the fluorescent antibodies for Ih at room temperature. The primary antibodies used were: rabbit polyclonal Syk (1 : 100) (sc-1077, Santa Cruz Biotechnology); mouse monoclonal anti-beta Tubulin (1 :200) (T4026, Sigma). Primary antibodies were detected by Alexa Fluor 488-conjugated anti-mouse IgG (1 :400) (715-546-151, Jackson ImmunoResearch laboratories) and APC-conjugated anti-rabbit IgG (1 :200) (711-136- 152, Jackson ImmunoResearch laboratories) antibodies, respectively. Nuclei were stained with Hoechst 33342 dye. After washing, the slides were mounted in VECTASHIELD Mounting medium (H-1400, Vector laboratories) and were analyzed visually using Zeiss Imager M2 microscope.
Patients and Ethics Statement. Forty colorectal cancer patients with synchronous and unresectable liver metastases were enrolled in a prospective study at the ICM Cancer center from January 2000 to June 2004. Normal colon, colon cancer and hepatic metastasis samples were collected at the time of surgery, prior to chemotherapy. The study was approved by ICM (Institut du Cancer de Montpellier) CORT (Comite de Recherche Translationnelle) ethical committee and all participating patients were informed of the study and had to provide signed written informed consent before enrollment.
Tissue samples. From these 40 CRC patients we obtained expression data for at least one tissue of 28 patients. This represented 18 normal colon mucosas (CN), 20 colon primary tumors (CT) and 19 hepatic metastases (HM). Most of the primary tumors were located in the left colon and histological analysis showed a well or moderate differentiation state. Among those 57 analyzed tissues, both CT and HM were collected in 13 patients. These 13 pairs of samples were used to study the protein expression pattern of Syk expressed in CT versus the corresponding CN. Prior to western blot analysis, tumor tissue samples from patients were directly grinded in lysis buffer (150mM NaCl, lOmM Tris pH 7.4, ImM EDTA, ImM EGTA, 1% SDS, 1% Triton X-100, 0.5% NP-40, 2mM PMSF, lOOmM NaF, lOmM sodium orthovanadate, one cocktail protease inhibitor tablet for 10 ml) using a Mixer Mill® MM 300 unit (Qiagen, Valencia, CA). Protein concentration was determined with the Bradford assay (Pierce Coomassie Plus Protein Assay). Then, 50 pg of total proteins solubilized in lx Laemmli sample buffer (10 % glycerol, 5 % P -mercaptoethanol, 2.3% SDS, 62.5 mM Tris-HCl (pH 6.8), and 0.1 % bromophenol blue) were resolved by 10% SDS-PAGE. Cell culture samples. Cells were solubilized in DOC modified lysis buffer (1% Igepal, 0.25% sodium deoxycholate, 0.1% SDS in PBS buffer supplemented with protease and phosphatase inhibitors) and the protein concentration was determined (PIERCE BCA Protein Assay, Thermo Fisher Scientific). Equivalent total protein amounts (50 pg per sample) solubilized in lx Laemmli sample buffer were subjected to 8-10% SDS-PAGE.
Immunoblotting. Following electrotransfer to Amersham nitrocellulose membrane (GE Healthcare, pore size 0.45 m) using a standard protocol, the membranes were first stained with Ponceau S (Sigma), to confirm protein loading equivalence, then blocked 1 hour at room temperature with gentle shaking in TBS containing 0.1 % Tween-20 (TBST) and 5 % skimmed milk. Primary antibodies were diluted in blocking solution and hybridized overnight at 4 °C with gentle shaking. The antibodies used were: mouse monoclonal anti-Syk (1 :500) (4D10, #scl240 Santa Cruz Biotechnology); rabbit polyclonal anti-Phospho-Akt (1 :2,000) (#4060, Cell Signaling Technology) ; mouse monoclonal anti-Phospho-p44/42 MAP Kinase (1 :2,000) (#9106, Cell Signaling Technology); rabbit polyclonal anti-p44/42 MAP Kinase (1 : 1,000) (#9102, Cell Signaling Technology); mouse monoclonal anti-beta Tubulin (1 :5,000) (SAB4200715, Sigma); rabbit polyclonal anti-EGF Receptor (1 : 1000) (#2232, Cell Signaling Technology); goat polyclonal anti-Akt (1 :200) (sc-1618, Santa Cruz Biotechnology); HRP- linked anti-goat antibody (1 :5000) (sc-2033, Santa Cruz Biotechnology); HRP-linked antirabbit antibody (1 :5,000) (sc-2004, Santa Cruz Biotechnology); HRP-linked anti-mouse antibody (1 :5,000) (sc-2314, Santa Cruz Biotechnology). Membranes were washed 5 times (10 min. each) in TBST and appropriate HRP-conjugated secondary antibodies were diluted in blocking solution and hybridized 1 hour at room temperature with gentle shaking. After 5 washes in TBST, the proteins were detected by chemiluminescence generated with Western Lightning Chemiluminescence Reagent Plus (GE Healthcare) and detected on PXi Syngene Chemiluminscence system. For reprobing, membranes were incubated at room temperature for 10 min in stripping solution (100 mM Glycine pH 2.8; 0.1% Igepal and 1 % SDS) with gentle shaking. Membranes were then washed (3 times, 5 min. each) with TBST and blocked as described previously, before rehybridization.
Mass Spectrometry
Syk pull-down assays. DLD-1 cells were serum starved, then separated in two batches. One batch was incubated with EGF (50 ng/ml) for 10 minutes at 37°C. After washing with ice- cold PBS containing phosphatase inhibitors (100 mM sodium fluoride, 5 mM sodium orthovanadate), cells were lysed for 15 minutes on ice with lysis buffer (PBS supplemented with 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS). Cell lysates were clarified by centrifugation for 15 minutes at 4°C at 16,000 g. The total protein content of the soluble fraction was quantified using the BCA assay kit (Interchim, France). 5.5 mg of protein lysates from serum-starved and EGF-treated DLD-1 cells were incubated with agarose-conjugated anti-Syk 4D10 monoclonal antibody (#sc-1240, Santa Cruz Biotechnology) and agarose-conjugated mouse IgG (#sc-2343, Santa Cruz Biotechnology, control) for 2 hours at 4°C. Beads were washed 3 times in lysis buffer, then they were resuspended in lx Laemmli sample buffer and the protein contents were analyzed by SDS-PAGE.
Sample preparation. Proteins from the immunoprecipitation batches were resolved by 8% SDS-PAGE and detected with Coomassie-brilliant blue staining. Lanes were cut into five fractions. Gel pieces were subjected to in-gel alkylation and digestion with trypsin.
LC separation and MS/MS detection. Desalted peptide mixtures were fractionated on an Ekspert 425 nanoLC system (Eksigent) equipped with a C18 column (Discovery BIO Wide Pore, Supelco). The mobile phases were solvent A (water, 0.1% FA) and B (acetonitrile, 0.1% FA). Injection was performed with 98% solvent A at a flow rate of 5 pl/min. Peptides were separated at 30°C with the following gradient: 2% to 40% B for 105 min, 40- 80% B for 5 min. The column was washed with 80% solvent B for 5 min and equilibrated with 98% solvent A. Peptide separation was monitored online with the coupled TripleTOF 5600 mass spectrometer (Sciex). The total ion chromatogram acquisition was made in information-dependent acquisition (IDA) mode using the Analyst TF v.1.7 software (Sciex). Positive ion profiling was performed from m/z 350-1,500, followed by a MS/MS product ion scan from m/z 100-1,500 with the abundance threshold set at more than 100 cps. The accumulation time for ions was set at 250 ms for MS scans, and 100 ms for MS/MS scans. Target ions were excluded from the scan for 10s after detection. The IDA advanced “rolling collision energy (CE)” option was employed to automatically ramp up the CE value in the collision cell as the m/z value was increased. A maximum of 25 spectra were collected from candidate ions per cycle.
Data analysis. Combining the 5 runs per sample, peptide and protein identifications were performed in Uniprot/Swiss- Prot2016_01 database by ProteinPilotTMSoftware V 4.5 (Sciex). This software calculates a confidence percentage that reflects the probability that the hit is a false positive, meaning that at 99% confidence level (unused score>2), there is a false positive identification chance of about 1%.
Bioinformatic analysis. TCGA gene and protein expression matrix were recovered for the COAD and READ cohorts using API GDC portal with curl function. They were merged together to generate the CRC (colorectal) dataset based on 622 individuals. About 18 thousand genes were expressed in more than 5% of the samples. Only 52 patients had RNAseq data for the tumor and the normal tissue. Fifty tumor samples had both mRNA and protein expression values. Splicing event concerning the exon 9 of Syk were recovered from TCGA SpliceSeq database as PSI value from the web interface (24). PSI values were obtained for all samples recovers from TCGA. We assigned NA PSI value to zero in the present analysis. Tumor samples were separated based on Syk exon 9 PSI value between low (<0.25), medium and high (>0.75). Differential gene expression among tumor samples was calculated using edgeR. We kept genes that were differentially expressed between Low and High PSI tumors. Analysis was performed on the 10.000 most variable genes. Variation was calculated on the loglO of gene expression values. Graphs were generated using R version 3.6.2 and ggplot2 library. Complex heatmap library was used to draw heatmaps. Clinical data were recovered from integrated Pancancer resource published by Liu et al. (25). Three CRC samples had no clinical information. MSI features were obtained from Bonneville et al. (26). Information was missing for 43 patients. Kras status of CRC patients was downloaded at cbioportal. CMS labels were matched using CMS subtyping calls (https://github.com/Sage-Bionetworks/crc-cms- kras/blob/master/020717/cms_labels_public_all.txt).
In vivo studies
Xenografts. IxlO6 DLD-1 tumor cells were injected subcutaneously in the left flank of 6-week-old female athymic nu/nu mice (Charles River Laboratories). Tumors were detected by palpation and measured with a caliper three times per week. Mice were euthanized by cervical dislocation when the tumor volume reached 1.500 mm3. During the experiments, health monitoring of mice was performed to ensure their health, including monitoring animals from external sources as well as animals kept in the experimental unit; housing and hygienic monitoring, pathogen detection, diagnostic measures to enable disease control and to maintain the health status of mice. Among the efforts to alleviate suffering, we adapted the protocol to minimize distress for the mice. Consequently, we defined tumor growth (a maximum size of 1500 mm3) instead of survival endpoint. We also refined the sample collection methods and instituted species-specific husbandry refinements in order to attenuate anxiety and stress. The number of animals used was reduced to the absolute minimum necessary, based on appropriate statistical sample size determination. Ethical approvals were obtained by the local ethics committee (Ethics Committee approved by the French Ministry, animal facility approval D34- 172-27, and protocol approval CEEA-LR-10830 12018032611365978#! 0830).
Tumor treatment. When tumors reached the volume of 40-70 mm3, mice were randomized in groups of 7 to 10 animals, and they were treated three times per week for 3 weeks with: (i) 0.2 mL of 0.9% sodium chloride solution by intraperitoneal injection of 0.35 mg/kg C- 13; (ii) 0.2 mL of 0.9% sodium chloride solution by intraperitoneal injection of DMF (vehicle, control group), (iii) 100 mg/kg C-13 dissolved in 0.1% carboxymethylcellulose solution by gavage; (iv) DMF dissolved in 0.1% carboxymethylcellulose solution (vehicle; gavage control group). Mice were euthanized by cervical dislocation when the tumor volume reached 1500 mm3.
Statistical analysis. For in vitro experiments, data were compared using the unpaired Student’s t test. For in vivo experiments, data were described using median, mean and standard deviation. Linear mixed regression models were used to determine the relationship between tumor growth and number of days after implantation. The variables included in the fixed part of the model were the number of days after implantation and the treatment group; their interaction were also evaluated. Random intercepts and random slopes were considered for time effect. The model coefficients were estimated by maximum likelihood. Statistical significance was set at the 0.05 level. Statistical analyses were conducted using the STATA 16.0 software (StataCorp, TX, USA).
Results
Syk isoforms expression in CRC cell lines.
We assessed the expression levels of total Syk (L+S) as well as Syk (L) and Syk (S) isoforms in six CRC cell lines by RT-qPCR (Figure 1A). This analysis showed that these cell lines express different levels and splicing isoform ratios of Syk: the expression levels of global Syk (L+S) and that of Syk long isoform transcript are 5 to 4000 fold higher in HT29 and DIFI cell lines compared to the four other cell lines. Among the six cell lines, DIFI expresses the higher level of Syk short isoform transcript, which is 8 to 1800 fold higher than the other cell lines. The mRNA level is well correlated with the protein expression level detected by western blot analysis (Figure IB), except for SW620 that expressed a high level of protein despite a low level of mRNA, suggesting that unknown post-translational mechanisms may affect Syk expression. However, we could not visualize at the protein level, the two Syk isoforms due to their co-migration by SDS-PAGE and the absence of suitable isoform-specific antibodies.
Syk Long isoform is implicated in CRC cell lines survival and mitosis.
Our goal was to evaluate the role of Syk alternative spliced isoforms in cancer biology of colorectal carcinomas. Short hairpin RNA (shRNA) and RNA interference (RNAi) have become preferred tools for evaluating the impact of gene expression on viability of cancer cells, and for understanding individual isoform functions. Compared to RNAi, shRNA can be integrated into genomic DNA for long-term or stable expression, leading to a longer knockdown of the target mRNA and a durable down-regulation of protein expression, thus allowing the evaluation of protein knockdown effects on cellular functions. For our purpose, we used shRNA-based isoform-specific silencing to investigate the effects of Syk splicing modulation on cell growth and cell survival of HCT-116, DLD-1 and DIFI colorectal cell lines. We compared the effects of shRNA on global and long-isoform specific expression by RT-qPCR and RT-PCR (Figure 1C and data not shown). Global shRNAs (shGl, shG2) equally inhibited the expression of long and short splice isoforms of Syk. Long Isoform specific shRNAs (shLl, shL2) inhibited the accumulation of the long splice isoform while proportionally reducing the overall gene expression level. Interestingly, Syk (L) depletion increased the expression of the short isoform to 1.6 fold in DLD-1 and to 2.8 fold in DIFI cell line. These observations were confirmed by Western blot analysis using antibodies against total Syk protein to evaluate the impact of the shRNA-induced mRNA downregulation at the protein level (Figure ID). Our results showed that among the global shRNAs, shGl and among the isoform specific shRNAS, shL2 were the most efficient ones for reducing Syk gene expression both at the mRNA and at the protein levels. Syk has been previously described as implicated in cell-survival pathways and apoptosis. We evaluated the impact of shRNA-based global and long-isoform specific silencing of Syk on the viability of CRC cells. Inhibiting the expression of the long isoform of Syk resulted in an increase of apoptotic marker-positive cells in the three cell lines (Figure 2). Accordingly, cell proliferation and the ratio of viable cells were markedly decreased by shRNA- mediated silencing of the long isoform of Syk (Figure 2). On the opposite, Syk general knock down with global shRNA shGl did not significantly affect cell apoptosis and viability. However, it increased cell proliferation of HCT-116 and DLD-1 cells, and had the opposite effect in DIFI cells where Syk general knock down reduced cell proliferation (Figure 2). Transfection of an unrelated shRNA (shLUC) did not affect any of these cellular phenotypes. We concluded that changing the splicing pattern of Syk influences CRC cell viability and apoptosis, in a manner that is independent of the overall level of gene expression. K-Ras mutations occur as an early event in about 50% of CRC cases, resulting in constitutive activation of the RAS-RAF-MEK-ERK pathway, and a subsequent resistance to anti-EGFR therapy by monoclonal antibodies Cetuximab and Panitumumab. Thus, we focused our studies on the role of Syk in HCT-116 and DLD-1 cell lines, which both bear the activating K-Ras (G13D) mutation, and which express low and high levels of Syk, respectively. Previous works reported the implication of Syk in the control of cell cycle and mitosis (9) (27) (10). To further investigate the effect of Syk alternative splicing on cell proliferation, we monitored the impact of shRNA-based Syk targeting on cell-cycle progression of HCT-116 and DLD-1 cell lines. Our data showed that changing the alternative splicing of Syk led to the accumulation of cells in the G2-M phase, consistent with a defective mitosis, and to the emergence of cells with a >4N DNA content due to polyploidization through mitotic skipping (Figure 3). This was particularly true for DLD-1 cells, which were more affected than HCT- 116 in their proliferation and survival (Figure 2). Upon the visual examination of DLD-1 cells expressing shRNA that target Syk global and isoform specific expression, we noted an increasing amount of mitotic cells with abnormal metaphases and chromosome alignment aberrations in comparison to cells expressing unrelated shRNA (data not shown). We also noted an increasing amount of “giant cells” displaying large and aberrant nuclei with supernumerary centrosomes and multipolar anaphase spindles confirming the role of Syk in mitotic exit and cytokinesis (data not shown). This mitotic arrest was particularly visible following Syk long-isoform knockdown, but was also present following Syk general knock down with global shRNA, indicating that Syk (L) controls mitotic exit and cytokinesis. Taken together, our data suggest that Syk (L) plays an important role in the cell survival and the control of the cell cycle in CRC cell lines.
Syk inhibition increases chemotaxis and Matrigel engagement.
To investigate any correlation between Syk expression and cell motility, HCT-116 and DLD-1 cell lines were tested in a Fluoblok chemotaxis assay. We monitored the impact of Syk knockdown on cell motility, using shRNA-based Syk global and isoform specific expression. Chemomigratory ability was significantly increased in cells expressing shRNA that target Syk global expression relative to cells expressing unrelated shRNA, while Syk long-isoform knockdown did not affect cell migration ability (Figure 4A). Next, we used the pharmacologic inhibition of Syk to further study its role in cell motility. Piceatannol, a hydroxystilbene derivative of resveratrol, preferentially inhibits the kinase activity of Syk in in vitro assays and is widely used as a Syk-selective inhibitor. We used a non-toxic concentration range of Piceatannol (up to 12.5 pM) commonly used in studies using human cells. As shown in Figure 4B, Piceatannol increased FCS-induced chemomigration of both HCT-116 and DLD-1 cell lines to the same manner than shRNA-based Syk global knockdown. Cell attachment and spreading on matrix proteins is a key step in invasion. When cultured on Matrigel, many tumor cells attach and elongate, and this, together with motility, results in formation of cordlike structures and ultimately invasion. We used this experimental metastasis model to evaluate the role of Syk in colorectal carcinoma invasion and metastasis. As shown in Figure 4C, DLD-1 cell lines expressing shRNA that target Syk global expression, as well as long-isoform specific shRNA, displayed increased cell invasion compared to cells expressing control shRNA. This tendency was also observed in Matrigel invasion of HCT-116 cell lines expressing both shRNA, however, it did not reach a significant statistical value because of the high variability of this experiment. Together, our data indicated that global Syk knock down increases cell proliferation and cell motility, while the absence of Syk (L) isoform affects cell viability and induces cell apoptosis.
Syk expression is required for the activation of PI3K-Akt survival pathway.
The epidermal growth factor receptor (EGFR) signaling pathway is commonly activated in colorectal cancer. Activation of this pathway occurs after ligand binding to EGFR, which leads to EGFR phosphorylation and oligodimerization at the plasma membrane. This in turn triggers a chain of downstream signaling events that include activation of the Ras/mitogen- activated protein kinase (MAPK) and phosphoinositide 3 -kinase (PI3K)-Akt pathways that are crucial mediators of growth factor-induced proliferation and cell survival, respectively. Previous studies have shown that the presence of activating PIK3CA and Ras mutations in CRC cell lines result in constitutive activation of these pathways downstream of EGFR signaling. Considering the presence of activating K-Ras (G13D) mutation in both HCT-116 and DLD-1 cell lines, as well as PIK3CA (H1047R) mutation in HCT-116 cell line, we investigated the effects of Syk targeting on EGFR ligand-induced activation of Erk and Akt. For this purpose, HCT-116 and DLD-1 cells transduced with shRNAs that target Syk global and long-isoform specific expression or firefly luciferase (shLUC; control) were serum starved, then treated with EGFR ligand, EGF. Cell lysates were subsequently analyzed by immunoblotting using activation state-specific antibodies for Erk and Akt (data not shown). Our data showed that HCT-116 cells display constitutively active Erkl/2 and Akt proteins, shown by their phosphorylation state, which remained comparable between untreated and EGF-treated cells. However, EGF treatment induced the phosphorylation of Erkl/2 and Akt in DLD-1 cell line (p- Erk; p-Akt; shLUC: -/+ EGF). In both cells lines, shRNA-mediated Syk depletion did not affect the phosphorylation levels of Erkl/2 which remained comparable between EGF-treated and untreated cells (p-Erk; shGl, shL2: -/+ EGF). However, targeting Syk global and Syk (L) isoform affected the level of constitutive phosphorylation of Akt in HCT-116 cells, as well as the level of EGF-induced phosphorylation of Akt in both cell lines (p-Akt; shGl, shL2: -/+ EGF) (data not shown). Cetuximab is a chimeric IgGl monoclonal antibody that targets the extracellular domain of EGFR, blocking ligand binding to the receptor and leading to the inhibition of EGFR signaling. The pretreatment of the cells with Cetuximab attenuated the mitogenic effect of EGF and inhibited the phosphorylation of Akt and Erkl/2 in both cell lines; however, the phosphorylation of Erkl/2 in HCT-116 cell lines was mildly affected by Cetuximab. In all cases, the expression level of EGFR in the different cell lines, analyzed by western blot (data not shown) and by FACS (data not shown) was not affected. Together, these results showed that Syk (L) expression is required for the sustained and/or growth factor- induced activation of the PI3K/Akt pathway.
EGF exerts opposite effects on Syk alternative splicing.
Growth factors can affect cell survival by regulating the activity of splicing modulators and the splicing of kinases such Syk. We investigated the effect of short-term EGF treatment on the alternative splicing of Syk in HCT-116 and DLD-1 cell lines in vitro. Our data showed that EGF exposure increases exon 9 inclusion and the expression levels of Syk (L) isoform in DLD-1 cell line, while it exerts the opposite effect in HCT-116 cell line by inducing exon skipping and the increase in Syk (S) isoform expression (data not shown). Therefore, we concluded that mitogenic signaling regulates both the amount and the ratio of Syk splicing isoforms but in a manner dependent on cell background. Considering the accumulating evidence supporting the role of Syk (L) isoform in CRC cell survival and cell cycle regulation, we further investigated the role of Syk downstream of EGFR signaling. For this, we performed Syk “pull-down assays” on lysates of serum-starved and EGF-stimulated DLD-1 cells, and we analyzed the components of the captured complexes by high-resolution mass spectrometry to identify Syk interactomes. Our data showed that upon EGF treatment, there is an increased association of Syk with ligands that are implicated in the control of apoptosis, and in the regulation of mitosis and cell cycle G2/M checkpoint, such as Nuclear death domain protein p84N5, which activates a G2/M cell cycle checkpoint prior to the onset of apoptosis (28); RAD50, which regulates mitotic progression independent of DNA repair functions (29); and cell division cycle protein 23 homolog (CDC23), a protein component of anaphase-promoting complex (APC) (30) (data not shown). These data confirm the implication of Syk in the control of the cell cycle and the apoptosis of CRC cell lines, which is enhanced under the influence of oncogenic growth factors such as EGF. Syk Long isoform expression is associated with tumorigenesis in human CRC tissues.
In a previous study, we reported a gene expression signature associated with treatment response in advanced CRC patients (31). Thirteen pairs of colon primary tumors (CT) and normal colon mucosas (CN) were collected at the time of surgery, before chemotherapy. We used these samples to determine the profile of Syk expression in primary human colorectal tumors. For this purpose, we performed western blot analysis of protein extracts from tumor tissues of these 13 untreated patients and we compared the expression of Syk protein to that of normal adjacent tissues of the same patient. Our data showed that Syk protein levels were higher in 50% of the analyzed human tumors compared to the normal tissues (Figure 5A). However, we were not able to analyze the ratio of Syk (L) versus Syk (S) isoforms due to the absence of long isoform-specific antibodies. Next, we conducted an analysis integrating Syk global and isoform-specific expression data, as well as clinical meta-data of 622 CRC patients (COAD and READ cohorts) from The Cancer Genome Atlas (TCGA). This analysis showed a high Syk mRNA expression score in CRC tumors (data not shown). Accordingly, the comparison of mRNA expression levels of global Syk in tumor and normal tissue of same patients (N=50) revealed an upregulation of Syk gene expression in two thirds of the primary tumors (Figure 5B) Furthermore, high mRNA expression was associated with high protein level in TCGA tumors but the opposite was not always true (N=52) (Figure 5C). This observation is possibly due to Syk protein turnover differences. We next analyzed Syk splicing variants expression in CRC tumors using the COAD and READ cohorts of TCGA SpliceSeq database. This database reports the PSI (percent-splice-in) values for the splice events in tumor and normal adjacent tissues (24) (data not shown). We extracted PSI values for exon 9 of Syk gene, which correspond to the ratio of Syk (L) isoform expression over the total Syk transcripts (data not shown). Since Syk (L) isoform results from the inclusion of exon 9 in Syk gene, the PSI value reflects the proportion of Syk (L) isoform expression in patient’s tissue samples. The comparison of PSI values of normal and tumor tissues of the same patients (N=50) revealed that both normal and tumor tissues present high PSI values reflecting the dominant expression of Syk (L) isoform (data not shown). Our analysis showed that most tumor samples express higher amounts of Syk (L) than Syk (S) mRNA (PSI values > 0.5, N=43/50) (Figure 5D). Moreover, the PSI value of tumor tissues in which Syk expression is down-regulated remains high, suggesting a dependence of tumors to Syk (L) isoform expression. Interestingly, 10% of the analyzed tumor tissues (N=5/50), and none of the normal tissues, have low PSI values (PSI < 0.25), reflecting the expression of a high proportion of Syk (S) isoform (Figure 5E). The level of expression of global Syk in these tumor samples is comparable to that of the adjacent normal tissue (Figure 5D). To further characterize this specific subpopulation and the consequences of Syk PSI value on tumor biology, we classified 619 CRC tumor samples based on gene expression data. We first classified samples as low PSI (PSI<0.25; 20 samples), medium PSI (452 samples) and high PSI (PSI>0.75; 147 samples) tumors. Next, we selected 1653 genes differentially expressed between the low and high PSI samples and we analyzed tumor subpopulations by hierarchical clustering (data not shown). Interestingly, we identified a small cluster of 40 samples designated as low PSI-like tumors. This cluster includes 17 low PSI, 13 medium PSI and 10 high PSI tumors. This group of 40 samples forms a specific cluster associated with a large set of 583 strongly over-expressed genes and 1070 down-regulated genes, compared to other tumor samples (REG value). The REG positive and negative groups overlap the two clusters obtained from unsupervised analysis. Our analysis showed that 495 genes from the first main cluster associated with REG positive values were related to translation and mitochondria, whereas 1158 genes from the second main cluster related to REG negative values were associated with centriole and centriolar functions (data not shown). Finally, this group of PSI-low tumors was not related to any classical CRC classification such as MSI status, CMS molecular classification, K-Ras mutational status, or TNM stage, nor to Syk total expression level. Taken together, our analysis suggests the dependence of CRC tumors to the pro-survival Syk (L) isoform, except in the new identified subgroup of low PSI tumors in which gene reprograming has taken place to overcome the effect of Syk (L) down-regulation.
Treatment of CRC cell lines with C-13, an original non-enzymatic inhibitor of Syk.
In our previous works, we developed an original approach for the identification of protein-protein interaction inhibitors of Syk (32). This led to the discovery of new classes of non-enzymatic inhibitors of Syk with improved selectivity profile and which bind to a cavity located at the interface between the SH2 domains and the linker domain of Syk (Patent No WO2009133294). Among the newly isolated scaffolds, compound C-13 showed a high potential for the inhibition of mast cell degranulation in vitro (Syk IC50 = 2 pM) and for the prevention of anaphylactic shock in mice using oral delivery route in vivo (Syk IC50 = 110 mg/kg) (23). We studied the impact of compound C-13 on the biology of CRC cell lines. We exposed a wild type (DIFI), and two K-Ras mutant (HCT-116, DLD-1) CRC cell lines to various concentrations of C-13 for 2 hours and then measured cell proliferation, viability and apoptosis 72 hours post-treatment. Our data showed that C-13 inhibits cell proliferation and induces apoptosis of the cells (Figure 6A) in a manner that mimic shRNA Syk (L) isoform- specific targeting but not total Syk targeting by shRNA (Figure 2). Moreover, C-13 inhibits FCS-induced chemomigration (Figure 6B and data not shown) and invasion (Figure 6C and data not shown) of CRC cell lines in a concentration-dependent manner. Interestingly, the cytotoxic effect of C-13 on DLD-1 cell line was higher than on the two other cell lines, and was confirmed by western blot analysis which showed that EGF-induced phosphorylation of Erkl/2 and Akt were both markedly reduced in DLD-1 cells pre-treated with C-13 (Figure 6D). FACS analysis showed that the level of membrane expression of EGFR in the cell lines was not affected by C-13 (data not shown). To demonstrate that the effect of C-13 on cells viability is indeed due to a Syk-specific inhibition and not to an off-target interaction, we used Syk-positive CRC cell lines DLD-1, SW620 and HCT-116, and Syk-negative breast cancer cells lines MDAMB231 and BT549 as control. Cells were exposed to various concentrations of C-13 for 72 h and cell viability was measured. Our data showed that C-13 specifically exerts cytotoxic effects on Syk-positive cell lines, while Syk-negative cell lines viability was unaffected (Figure 6E). Finally, we compared C-13 with R406 (the active metabolite of Fostamatinib R788), a specific, ATP-competitive inhibitor of Syk (Syk IC50 = 41 nM) that has shown efficacy in the treatment of autoimmune diseases (33) and in preclinical leukaemia studies (34,35). We compared the effects of increasing concentration of the two drugs on the survival of HCT-116, DLD-1 and HT29 CRC cell lines that express low to high levels of Syk, respectively (Figure 6F). Our data showed that despite a 300 folds lower in vitro affinity for Syk compared to R406 (4pm for C-13 (23) versus 12nM for R406 (36)), C-13 affects the viability of Syk-positive CRC cell lines in a more efficient manner than R406. Together with the effect of Piceatannol on cell migration (Figure 4B), our data demonstrate that compounds that target Syk catalytic activity are less efficient than compound C-13 on the inhibition of CRC cell lines proliferation and motility.
Compound C-13 attenuates tumor growth in CRC xenograft mice model.
We established two in vivo mice models to examine the anti -tumor effect of C-13 on CRC tumor growth. For these experiments, xenografts in athymic nude mice were established by subcutaneous injection of IxlO6 DLD-1 cells and the drug administration was initiated when tumors reached the volume of 40-70 mm3. For the intraperitoneal (IP) and oral administrations, we referred to our previous in vivo studies with C-13 in order to choose nontoxic doses of this compound and to minimize adverse effects (23). We first investigated the effect of IP administration of C-13 on tumor growth. For this purpose, two groups of 9 mice received an IP administration of compound C-13 at a dosage of 0.35 mg/kg or DMF alone (vehicle). The drug was administered three times a week over 3 weeks. The tumor sizes were measured and recorded every 2 days, and tumor growth curves analyzed for each group. At the given dose, there was a significant decrease in tumor growth in the group treated with C-13 compared to the control group treated with vehicle only (p=0.04) suggesting C-13 ’s potently inhibitory effect against tumor growth (Figure 7A). Kaplan-Meier survival analysis confirmed this observation (Figure 7B). We did not evidence any apparent toxicity of the treatment and body weight growth and physical activity of mice were similar in both groups. In a second in vivo experiment, we investigated the effect of the oral administration of C-13 on the tumor growth of DLD-1 cells xenografts in athymic nude mice. For this purpose, 100 mg/kg of C-13 or DMF alone (vehicle) were administered by gavage to two groups of 7 mice xenografted with DLD-1 cells, three times per week for 3 weeks. As shown in Figure 7C and Figure 7D, there is a decrease in tumor growth of the group of mice treated with C-13 compared to the control group, although we did not reach a significant statistical value (p=0.11). Together, our in vivo data confirm that C-13 is a promising and original Syk-specific molecule, with potential applications in the treatment of K-Ras-mutated CRC.
REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
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7. Larive RM, Urbach S, Poncet J, Jouin P, Mascre G, Sahuquet A, et al. Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell-cell adhesion. Oncogene, juin 2009;28(24):2337-47.
8. Zhang X, Shrikhande U, Alicie BM, Zhou Q, Geahlen RL. Role of the Protein Tyrosine Kinase Syk in Regulating Cell-Cell Adhesion and Motility in Breast Cancer Cells. Mol Cancer Res. mai 2009;7(5):634-44.
9. Zyss D, Montcourrier P, Vidal B, Anguille C, Merezegue F, Sahuquet A, et al. The Syk tyrosine kinase localizes to the centrosomes and negatively affects mitotic progression. Cancer Res. 1 dec 2005;65(23): 10872-80.
10. Uckun FM, Ma H, Ozer Z, Goodman P, Zhang J, Qazi S. A Previously Unknown Unique Challenge for Inhibitors of SYK ATP -Binding Site: Role of SYK as A Cell Cycle Checkpoint Regulator. EBioMedicine. nov 2014; 1(1): 16-28.
11. Coopman PJP, Do MTH, Barth M, Bowden ET, Hayes AJ, Basyuk E, et al. The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells. Nature, aout 2000;406(6797):742-7.
12. Yuan Y, Mendez R, Sahin A, Dai JL. Hypermethylation leads to silencing of the SYK gene in human breast cancer. Cancer Res. 15 juill 2001;61(14):5558-61.
13. Wang S, Ding Y-B, Chen G-Y, Xia J-G, Wu Z-Y. Hypermethylation of Syk gene in promoter region associated with oncogenesis and metastasis of gastric carcinoma. World J Gastroenterol. 15 juin 2004; 10(12): 1815-8.
14. Yuan Y, Wang J, Li J, Wang L, Li M, Yang Z, et al. Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma. Clin Cancer Res Off J Am Assoc Cancer Res. 15 nov 2006;12(22):6687-95.
15. Hoeller C, Thallinger C, Pratscher B, Bister MD, Schicher N, Loewe R, et al. The non-receptor-associated tyrosine kinase Syk is a regulator of metastatic behavior in human melanoma cells. J Invest Dermatol, juin 2005; 124(6): 1293-9.
16. Luangdilok S, Box C, Patterson L, Court W, Harrington K, Pitkin L, et al. Syk Tyrosine Kinase Is Linked to Cell Motility and Progression in Squamous Cell Carcinomas of the Head and Neck. Cancer Res. 8 aout 2007;67(16):7907-16. 17. Zhang J, Benavente CA, McEvoy J, Flores-Otero J, Ding L, Chen X, et al. A novel retinoblastoma therapy from genomic and epigenetic analyses. Nature, janv 2012;481(7381):329-34.
18. Wang L, Duke L, Zhang PS, Arlinghaus RB, Symmans WF, Sahin A, et al. Alternative splicing disrupts a nuclear localization signal in spleen tyrosine kinase that is required for invasion suppression in breast cancer. Cancer Res. 1 aout 2003;63(15):4724-30.
19. Hong J, Yuan Y, Wang J, Liao Y, Zou R, Zhu C, et al. Expression of variant isoforms of the tyrosine kinase SYK determines the prognosis of hepatocellular carcinoma. Cancer Res. 15 mars 2014;74(6): 1845-56.
20. Yang Z, Huo L, Chen H, Ni B, Xiang J, Kang L, et al. Hypermethylation and prognostic implication of Syk gene in human colorectal cancer. Med Oncol Northwood Lond Engl. 2013;30(2):586.
21. Ni B, Hu J, Chen D, Li L, Chen D, Wang J, et al. Alternative splicing of spleen tyrosine kinase differentially regulates colorectal cancer progression. Oncol Lett, sept 2016;12(3):1737-44.
22. Coebergh van den Braak RRJ, Sieuwerts AM, Kandimalla R, Lalmahomed ZS, Bril SI, van Galen A, et al. High mRNA expression of splice variant SYK short correlates with hepatic disease progression in chemonaive lymph node negative colon cancer patients. Suzuki H, editeur. PLOS ONE. 28 sept 2017;12(9):e0185607.
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35. Suljagic M, Longo PG, Bennardo S, Perlas E, Leone G, Laurenti L, et al. The Syk inhibitor fostamatinib disodium (R788) inhibits tumor growth in the Ep- TCL1 transgenic mouse model of CLL by blocking antigen-dependent B-cell receptor signaling. Blood. 2 dec 2010;116(23):4894-905.
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Claims

CLAIMS: A Syk inhibitor for use in the treatment of a cancer in a subject in need thereof. The Syk inhibitor for use according to claim 1, wherein the cancer is a colorectal cancer. The Syk inhibitor for use according to claim 1, wherein the cancer is a K-Ras mutated cancer. The Syk inhibitor for use according to claim 1, wherein the Syk inhibitor is C-13. The Syk inhibitor for use according to claim 1, wherein the Syk inhibitor is selected from the molecules having the following formulas (I), (II), (III) or (IV):
Figure imgf000049_0001
where: Rl is an optionally substituted aromatic group, or an optionally substituted heterocycle comprising at least one S, O or N atom;
R2 is an optionally substituted aromatic group, an optionally substituted heterocycle, an optionally saturated carbon chain, comprising an amine group, an optionally saturated carbon chain comprising an optionally substituted aromatic group or an optionally saturated carbon chain comprising an optionally substituted heterocycle comprising at least one S, O or N atom;
R3 is an optionally substituted phenyl, 2-pyridinyl, 3-pyridinyl or 4-pyridinyl group; or
Figure imgf000050_0001
where n=0 or 1; n'=0 or 1; R4 is an optionally saturated carbon chain comprising 1 to 5 carbon atoms, optionally substituted with an aromatic group;
R5 is an optionally substituted aromatic group or an optionally substituted amine group;
R6 is a hydrogen atom, alkoxy group, alkyl group or halogen; R7 is a hydrogen atom, alkoxy group, alkyl group or halogen;
R8 is a hydrogen atom, alkoxy group, alkyl group or halogen; or
Figure imgf000050_0002
where m=0, 1 or 2; R9 is a hydrogen atom and RIO is an optionally substituted phenyl group, or R9 and
RIO are part of the same optionally substituted heterocycle, or R9 and RIO are part of the same optionally substituted aromatic group;
Rll is a hydrogen atom, alkoxy group or alkyl group;
R12 is a hydrogen atom, alkoxy group or alkyl group; R13 is a hydrogen atom or an alkyl or alkoxy group;
R14 is a hydrogen atom or an alkyl or alkoxy group; or
Figure imgf000051_0001
where
A is an oxygen or sulphur atom;
R15 is an optionally saturated carbon chain comprising 1, 2 or 3 carbon atoms, optionally substituted by an optionally substituted aromatic group, an optionally substituted heterocycle or an amine group belonging to optionally substituted heterocycle;
R16 is a hydrogen atom, halogen or alkoxy group;
R17 is a hydrogen atom, alkoxy group or acetoxy group.
6. The Syk inhibitor for use according to claim 1, wherein the Syk inhibitor is an antibody.
7. The Syk inhibitor for use according to claim 1, wherein the Syk inhibitor is an inhibitor of Syk gene expression.
8. The Syk inhibitor for use according to claim 7, wherein the Syk inhibitor is a nucleic acid targeting a mRNA encoding Syk.
9. The Syk inhibitor for use according to claim 7 or 8, wherein the Syk inhibitor is a nucleic acid targeting a mRNA encoding Syk-(L) isoform or Syk-(S) isoform.
10. The Syk inhibitor for use according to claim 7 to 9, wherein the Syk inhibitor is a shRNA. The Syk inhibitor for use according to claim 10, wherein the shRNA is directed against Syk-(L) isoform or Syk-(S) isoform. The Syk inhibitor for use according to claim 10 or 11, wherein the shRNA is directed against Syk-(L) isoform and comprises an amino acid sequence having at least 90% of identity with SEQ ID NO:3 or SEQ ID NO:4. The Syk inhibitor for use according to claim 10 or 11, wherein the shRNA is directed against Syk-(L) isoform and comprise the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:4. A method of treating a cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a Syk inhibitor.
PCT/EP2023/071793 2022-08-17 2023-08-07 Syk inhibitors for use in the treatment of cancer WO2024037910A1 (en)

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