WO2012140414A1 - Variants de rhbdf2 et affections malignes ou inflammatoires - Google Patents

Variants de rhbdf2 et affections malignes ou inflammatoires Download PDF

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WO2012140414A1
WO2012140414A1 PCT/GB2012/050777 GB2012050777W WO2012140414A1 WO 2012140414 A1 WO2012140414 A1 WO 2012140414A1 GB 2012050777 W GB2012050777 W GB 2012050777W WO 2012140414 A1 WO2012140414 A1 WO 2012140414A1
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polypeptide
acid sequence
seq
cancer
nucleic acid
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David KELSELL
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Queen Mary University of London
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Queen Mary and Westfiled College University of London
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)

Definitions

  • the present invention relates to variant polypeptides whose expression in vivo is associated with tylosis and/or predisposition to develop oesophageal cancer.
  • the invention also relates to a method for diagnosing cancer by detecting the presence of a variant polypeoptide or its encoding gene.
  • the invention also relates to a method for screening a test compound for use in treating cancer and inflammatory conditions based on its capacity to bind to the variant polypeptide and/or affect its downstream activities.
  • Oesophageal cancer (predominantly squamous oesophageal cancer) is the seventh leading cause of cancer related deaths worldwide: there are 462,000 cases diagnosed, with 386,000 patients dying each year. Of these diagnosed cases, 85% are diagnosed as squamous oesophageal carcinoma, which have a survival rate of only 8-10% after five years. Cure rates are low and the prognosis of patients is often poor, particularly when the disease is not localised. Pathogenesis of oesophageal squamous carcinoma is still poorly understood and so there are few targeted drugs to effectively treat the condition. Current treatment involves surgery, radiotherapy and chemotherapy.
  • the present inventors have identified the single genetic cause of tylosis, a familial form of susceptibility to oesophageal cancer.
  • the identification of an inherited cancer gene implicated in oesophageal cancer opens up an entirely new approach for diagnosis, risk stratification and target development for this and other related cancers.
  • the present invention provides a polypeptide having an amino acid sequence based on the sequence shown as SEQ ID No. 1 but comprising one or more mutations compared that sequence, whose expression in vivo is associated with tylosis and/or predisposition to develop cancer, for example oesophageal cancer.
  • the polypeptide may have a reduced capacity to regulate the protease activity of RHBDL2 (SEQ ID No. 3) than the wild-type polypeptide.
  • the polypeptide may comprise one or more mutations in the region 170-200 with reference to the amino acid sequence shown as SEQ ID No. 1.
  • the polypeptide may comprise a variation in the amino acid at position 186 and/or 189 with reference to the amino acid sequence shown as SEQ ID No. 1 , for example the variation lle186Thr and/or Pro189Leu.
  • the present invention provides a nucleic acid sequence capable of encoding a polypeptide according to the first aspect of the invention.
  • the nucleic acid sequence may comprise one or more mutations with reference to the sequence shown as SEQ ID No 2.
  • the nucleic acid sequence may comprise one or more mutations in the region 500- 600 with reference to the sequence shown as SEQ ID No. 2.
  • the nucleic acid sequence may comprise a variation in the base at position 557 and/or 566 with reference to the nucleic acid sequence shown as SEQ ID No 2, for example the variation 557T>C and/or the variation 5660T.
  • the present invention provides a vector comprising a nucleic acid sequence according to the second aspect of the invention.
  • the present invention provides a host cell comprising a nucleic acid sequence according to the second aspect of the invention or vector according to the third aspect of the invention.
  • the present invention provides a screening method for determining whether a test compound may be useful in the treatment of a cancer or inflammatory conditions or in the promotion of wound healing, which method comprises investigating the capacity of the test compound to;
  • RHBDL2 (SEQ ID No. 3);
  • the present invention provides a method for diagnosing a cancer in a subject, which comprises the step of investigating:
  • the sequence of the gene or polypeptide may investigated in a sample isolated from the subject, such as a blood sample or cheek swab.
  • a finding that the gene encodes a polypeptide according to the first aspect of the invention; or that the gene comprises a nucleic acid sequence according to the second aspect of the invention may be indicative that the subject has, or is predisposed to contracting cancer.
  • the present invention provides an antibody which specifically binds a polypeptide according to the first aspect of the invention.
  • the antibody may be incapable of binding specifically to the wild-type version of the polypeptide having the amino acid sequence shown as SEQ ID No. 1.
  • the present invention provides a nucleic acid probe which specifically binds to nucleic acid sequence according to the second aspect of the invention but which does not specifically bind to a nucleic acid encoding the wild- type version of the polypeptide having the amino acid sequence shown as SEQ ID No. 1 .
  • the present invention provides a kit for diagnosing cancer which comprises an antibody which binds specifically to a polypeptide having the amino acid sequence shown as SEQ ID No. 1 ; and/or an antibody according to the seventh aspect of the invention.
  • the present invention provides a kit for diagnosing cancer which comprises a nucleic acid probe which binds specifically to a nucleic acid sequence encoding the wild-type version of the polypeptide having the amino acid sequence shown as SEQ ID No. 1 ; and/or a nucleic acid probe according to the eighth aspect of the invention.
  • the cancer may be an epithelial cancer, for example a squamous epithelial cancer.
  • the cancer may be a head and/or neck cancer.
  • the cancer may be oesophageal cancer.
  • the present invention provides a method for treating a malignant or inflammatory condition which comprises the step of administering an ADAM 17 inhibitor to a subject.
  • the present invention provides an AD AM 17 inhibitor for use in the treatment of an inflammatory or malignant condition.
  • the malignant or inflammatory condition may be a condition of the gastrointestinal tract or skin.
  • the condition may, for example, be oesophageal cancer or another epithelial cancer, psoriasis or eczema. DESCRIPTION OF THE FIGURES
  • FIG. 1 Mutations identified in tylosis patients. Sequencing of the RHBDF2 gene in two families with tylosis, revealed two heterozygous missense mutations within 9 bp of each other in exon 6: c.557T>C (p.lle186Thr), in one family (A) and c.566C>T (p.Pro189Leu) in the other family (B). Both mutations affect highly conserved amino acid residues within a block of conserved amino acids in the N- terminus of the RHBDF2 protein (C).
  • FIG. 2 Schematic illustrating the proposed mode of action of RHBDF2.
  • RHBDF2 interacts with and regulates the protease activity of RHBDL2.
  • RHBDL2 can cleave substrates, including EphrinB3, within the plasma membrane
  • the presence of missense mutations in RHBDF2 in tylotic keratinocytes is thought to interfere with the interaction of RHBDF2 with RHBDL2 and, hence have a downstream effect on the intramembrane cleavage of RHBDL2 associated substrates such as EphrinB3.
  • RHBDF2 Immunofluorescence staining of RHBDF2 in normal skin (A) and skin from tylotic patients (B).
  • RHBDF2 in normal epidermis is localised predominantly at the plasma membrane.
  • Tylotic skin shows a more cytoplasmic localisation of the protein.
  • Ephrin-B3 Immunofluorescence staining of ephrin-B3 in normal skin (A) and tylotic skin (B).
  • Ephrin-B3 appears to be located throughout the cytoplasm and plasma membrane of keratinocytes in normal epidermis, with higher levels of expression in the basal layer.
  • ephrin-B3 no longer appears localised to the plasma membrane, with cytoplasmic expression throughout the epidermis.
  • Green - anti- Ephrin-B3 staining Blue - DAP I nuclear staining.
  • FIG. 1 Western blotting of RHBDF2 and RHBDL2.
  • RHBDF2 and RHBDL2 can be detected by western blotting.
  • Endogenous RHBDF2 in HaCaT keratinocyte cells, as well as over-expression of untagged RHBDF2 and GFP tagged RHBDF2 in HEK293T cells were detected (A) and endogenous expression of RHBDL2 in HaCaT and HEK293T cells (B).
  • Figure 6 Gene sequence data showing the positions of the two identified missense mutations
  • Figure 7. Electropherograms showing the two identified heterozygous mutations a) German variant: C.5660T (p.Pro189Leu), b) UK/US variant: c.557T>C (p.lle186Thr).
  • Figure 8. A plot showing the predicted 7TM structure of RHBDF2.
  • A Western blots of lysates from control cells, K1 , and tylosis cells, TYLK1 , blotted with anti-phospho-EGFR, anti-EGFR (total) and anti- -actin loading control. Cells were grown in the presence (+) or absence (-) of exogenous EGF.
  • B MTS proliferation assay showing proliferation levels in tylosis cell lines, TYLK1 and TYLK2, and control cell lines, K1 and K16, after 72 hours culture in the presence and absence of exogenous EGF.
  • C Light microscopy images of cells 2 days post scratch in the absence of exogenous EGF.
  • control cell lines K1 and K16 and tylotic cell lines, TYLK1 and TYLK2.
  • Fig. 12 Increased ADAM 17 processing and activation in mutant iRHOM2 (TYLK) keratinocytes.
  • (iii) Increased ADAM 17 at plasma membrance in Tylotic keratinocytes compared to controls (ii).
  • (iv). Increased levels of 11-8 secretion, a substrate of ADAM 17, in tylotic keratinocytes (even without LPS stimulation) compared to control cell line
  • (v) Increased levels of Amphiregulain and TGFa secretion, a substrate of ADAM 17, in tylotic keratinocytes (without stimulation) compared to control cell line.
  • ADAM 17 siRNA leads to decreased growth factor (Amphiregulin) and IL-6 cytokine release in Tylotic (TYLK1) keratinocytes to levels comparable to normal keratinocytes. Both are substrates of ADAM 17.
  • the present invention relates to a polypeptide which is a variant of rhomboid 5 homolog 2 (RHBDF2).
  • RHBDF2 also known as iRhom2, RHBDL5 and RHBDL6
  • RHBDF2 belongs to a family of seven transmembrane spanning (7TM) proteins called rhomboids, which were first identified in Drosophila. Rhomboids have been shown to be serine intramembrane proteases, and have been linked with such processes as EGFR signalling and mitochondrial remodelling.
  • iRhom2 has been proposed, in order to clarify that it is a catalytically inactive member of the rhomboid family of intramembrane serine proteases.
  • a plot showing the predicted 7TM structure of RHBDF2 is shown as Figure 8.
  • RHBDF2 has been sequenced and full information is available at http://www.ncbi.nlm.nih.gov/gene/79651.
  • the sequence for full-length, wild-type RHBDF2 is given as SEQ ID No. 1. It is also shown in Figure 6.
  • transcript variant of the RHBDF2-encoding sequence differs in the 5' UTR and uses an alternate, in frame splice site in the 5' coding region compared to isoform 1 (the full length version). This results in a shorter protein (isoform 2), compared to isoform 1.
  • the present invention also extends to variant versions of this shorter RHBDF2 isoform, and the specific mutations (lle186Thr and Pro189Leu) may be mapped on to the shorter sequence to find their corresponding position on isoform 2.
  • polypeptide is used in the normal sense to mean a series of residues, typically L-amino acids, connected one to the other typically by peptide bonds between the -amino and carboxyl groups of adjacent amino acids
  • polypeptide analogues such as those having a non-natural backbone.
  • wild type is used to mean a polypeptide having a primary amino acid sequence which is identical with the native protein (i.e., having the sequence shown as SEQ ID No. 1 ).
  • variant or mutant is used herein to mean a naturally occurring polypeptide which differs from the wild-type sequence.
  • the variant polypeptide has a primary amino acid sequence which differs from the wild type sequence by one or more amino acid mutations.
  • the mutations may be additions or deletions, but substitutions are the most common.
  • the mutant sequence may, for example, have 1 , 2 or 3 amino acid mutations compared to the wild-type sequence. Usually a variant sequence comprises a single amino acid substitution.
  • the polypeptide of the present invention has an amino acid based on the sequence shown as SEQ ID No. 1 , but comprising one or more mutations compared to that sequence.
  • the sequence variant may be associated with tylosis (see below).
  • the sequence variant may be associated with one or more epithelial cancers such as oesophageal cancer, lung cancer or ovarian cancer.
  • the sequence variant may be associated with tylosis with oesophageal cancer (TOC).
  • TOC oesophageal cancer
  • associated with means that the variant version of the gene is detectable and/or expressed in at least one individual, more commonly a population of individuals having the disease.
  • the present inventors have identified two missense mutations associated with TOC in three separate families (one mutation was associated with a family from Germany; and one mutation was found in two separate UK families). The two mutations are thought to have arisen independently.
  • RHBDF2 RHBDF2 gene which are associated with tylosis and/or the predisposition to develop epithelial cancer, such as oesophageal cancer.
  • the present invention also provides a method for identifying a mutation associated with a disease which comprises the step of analysing the sequence of the RHBDF2 gene or the RHBDF2 protein in a subject having the disease and comparing the sequence with that of the wild type gene (SEQ ID No. 2) or protein (SEQ ID No. 1 ) in order to identify any mutations.
  • the subject may be part of a group of subjects, for example a family, which have a pre-disposition to contract the disease.
  • the disease may be tylosis or epithelial cancer, such as oesophageal, lung or ovarian cancer.
  • the mutation may affect an amino acid in RHBDF2 which is conserved between species.
  • the mutation may impair the function of RHBDF2, compared to the wild-type polypeptide.
  • the two missense mutations identified by the present inventors are at positions 186 and 189 with reference to the amino acid position numbering given in Figure 6.
  • the polypeptide of the present invention may, therefore have one or more mutations in the region 170-200, 180-190 or 185-195 with reference to the amino acid sequence shown as SEQ ID No. 1.
  • the polypeptide may comprise a variation in the amino acid at position 186 and/or 189 with reference to the amino acid sequence shown as SEQ ID No. 1 ; for example the polypeptide may comprise the variation lle186Thr and/or Pro189Leu.
  • the polypeptide of the present invention may have a reduced capacity to regulate RHBDL2 than the wild type polypeptide.
  • RHBDL2 is another member of the rhomboid-like family. Unlike RHBDF2, RHBDL2 has serine protease activity and has been shown to cleave EphrinB3, thrombomodulin and the adhesion-associated molecule CLEC14A (Freeman (2009) Seminars in Cell & Developmental Biology 20:231-239). The polypeptide of the present invention may have a reduced capacity to regulate the protease activity of RHBDL2, compared to wild type RHDBF2. RHBDL2 has been sequenced and full information is available at http://www.ncbi.nlm.nih.gov/gene/54933.
  • RHBDL2 is regulated by RHDBF2.
  • RHBDF2 is thought to associate with RHBDL2 within the plasma membrane and inhibit the protease activity of RHBDL2 (see Figure 2).
  • the polypeptide of the invention may have a reduced ability to inhibit the protease activity of RHBDL2.
  • the polypeptide of the present invention may reduce the cleavage of one or more RHBDL2 substrates, such as EphrinB3, EGF, thrombomodulin and CLEC14A.
  • the present invention also provides a nucleic acid sequence which encodes a polypeptide according to any preceding claim.
  • the nucleic acid sequence may be, for example, a naturally occurring or synthetic RNA/DNA sequence, a recombinant RNA/DNA sequence (i.e. prepared by use of recombinant DNA techniques), a cDNA sequence or a partial genomic DNA sequence, including combinations thereof.
  • the nucleic acid may be based on the sequence shown as SEQ ID No.2 (the wild- type RHBDF2 gene) but with one or more base pair mutations, i.e. additions, deletions or substitutions. If the mutation is a deletion and/or addition, the mutation or combination thereof may be such that the mutant sequence is still "in frame” when compared to the wild-type gene, so that only a small number, such as less than 10, amino acids are affected. The mutation or combination of mutations may produce a single amino acid substitution in the encoded protein.
  • the mutation may be in exon 6 of the RHBDF2 gene.
  • the two missense mutations identified by the present inventors are at positions 557and 566 with reference to the nucleic acid sequence numbering given in Figure 6.
  • the nucleic acid sequence may comprise one or more mutations in the region 500- 600, 520-580 or 550-570 with reference to the sequence shown as SEQ ID No. 2.
  • the nucleic acid sequence may comprise a variation in the base at position 557 and/or 566 with reference to the nucleic acid sequence shown as SEQ ID No 2.
  • the nucleic acid sequence may comprise the variation 557T>C and/or the variation 5660T.
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  • the present invention also provides a vector comprising a nucleic acid sequence in accordance with the present invention.
  • the vector may be capable of expressing the polypeptide of the present invention in a host cell.
  • an "expression vector” is a cloning vector that is engineered to allow the expression of protein from a nucleic acid sequence, i.e., its coding sequence is properly transcribed and the RNA is translated.
  • the expression vector may provide an appropriate promoter sequence for the initiation of transcription and restriction sites that allow insertion of cDNA.
  • a "plasmid,” which is a small, circular DNA molecule capable of autonomous replication in a cell, is commonly used as a cloning vector.
  • Various plasmids are available commercially, including plasmids containing a promoter, cloning sites and an antibiotic resistance gene.
  • the vector may be capable of inducing expression of the polypeptide in a prokaryotic or eukaryotic host cell.
  • Vectors suitable for transfecting or transforming mammalian cells include viral vectors, such as retroviral vectors.
  • the present invention also provides a host cell which comprises a nucleic acid sequence according to the invention.
  • the cell may be transfected or transduced with a vector as described herein.
  • the cell may express or be capable of expressing a polypeptide according to the invention under certain conditions.
  • the cell may be in vitro, ex vivo or in vivo.
  • the cell may be prokaryotic or eukaryotic.
  • the cell may be an epithelial cell, such as a keratinocyte.
  • ADAM metallopeptidase domain 17 (ADAM 17), also called TACE (tumor necrosis factor-a-converting enzyme), is a 70-kDa enzyme that belongs to the ADAM protein family of disintegrins and metalloproteases.
  • ADAM 17 is understood to be involved in the processing of tumor necrosis factor alpha (TNF-a) at the surface of the cell, and from within the intracellular membranes of the trans-Golgi network. This process, which is also known as 'shedding', involves the cleavage and release of a soluble ectodomain from membrane-bound pro-proteins (such as pro-TNF-a).
  • ADAM 17 was the first 'sheddase' to be identified, and is also understood to play a role in the release of a diverse variety of membrane-anchored cytokines, cell adhesion molecules, receptors, ligands, and enzymes.
  • ADAM 17 releases membrane bound ligands including TNFa and Desmoglein 2.
  • iRhom2 (RHBDF2), is required for TNF release in mice.
  • iRhom2 binds TACE and promotes its exit from the endoplasmic reticulum (Adrain et al (2012) Science 13;335(6065):229-32; Mcllwain et al (2012) Science 13;335(6065):229-32; Lichtenthaler (2012) Science 13;335(6065): 179-80).
  • the present inventors have shown that in Tylosis, activating mutations in iRhom2 increase ADAM 17 activity and movement through the Golgi to plasma membrane.
  • ADAM17 The amino acids sequence of ADAM17 is available at http://www.ncbi.nlm.nih.gOv/protein/AAI46659.1.
  • the present invention also provides a screening method to screen for molecules useful in the treatment of a disease, such as tylosis or cancer or an inflammatory condition, or in the promotion of wound healing.
  • the molecule may target, i.e. affect one or more steps of the RHBDF2/RHBDL2 pathway.
  • the screening method may comprise the step of investigating the capacity of a test molecule to affect the expression or activity of RHBDF2, RHBDL2 or a RHBDL2- associated substrate.
  • the method may involve investigating the capacity of a test molecule to;
  • the method may involve investigating the effect of the molecule on cellular event downstream of the RHBDL-2 associated substrate, such as EphrinB3 associated downstream signalling (e.g. via Stat3 and/or ERK phosphorylation, or decreased phosphorylation of EphA4) or EGF (e.g. via EGFR phosphorylation).
  • EphrinB3 associated downstream signalling e.g. via Stat3 and/or ERK phosphorylation, or decreased phosphorylation of EphA4
  • EGF e.g. via EGFR phosphorylation
  • the test molecule may be capable of down-regulating an aberrantly activated signalling molecule.
  • the test molecule may be a small organic molecule or a biological molecule such as an antibody (or fragment thereof), peptide, or siRNA.
  • the method may involve screening for a molecule which shows differential binding between a variant polypeptide of the present invention and the wild-type sequence shown as SEQ ID No. 1 .
  • a molecule which tests positive by a method according to the invention may be able to restore the function of RHBDF2 which is impaired in tylosis and/or epithelial cancer patients.
  • the molecule may be able to inhibit RHBDL2 protease activity, for example to a level equivalent to that seen in normal, healthy, individuals.
  • the molecule may be a ⁇ -lactam.
  • a series of monocyclic ⁇ -lactams have recently been identified as potent and selective inhibitors of rhomboid intramembrane proteases (Pierrat et al (201 1 ) ACS Chem. Biol. 6:325-335).
  • the molecule may be capable of down-regulating the expression of the mutant form of RHBDF2 in a patient.
  • the molecule may be able to down-regulate the cleavage of one or more RHBDL2 substrates.
  • RHBDL2 substrates include: EphrinB3, a transmembrane ligand for EphB receptor; EGF; thrombomodulin; and the adhesion-associated molecule CLEC14A.
  • CLEC14A is a C-type lectin which is overexpressed in cells of tumour vasculature
  • the molecule may down-regulate Ephrin-Eph signalling.
  • Screening for binding may be carried out by any suitable method known in the art, such as by ELISA, dot-blot, immunoassay, FACS, phage display, Western blot etc.
  • a therapeutic screening assay may be used to develop RHBDL2 inhibitors that will compensate for loss of functional RHBDF2.
  • RHBDL2 specific siRNAs may be screened to find those that will prevent the RHBDL2-intramembrane cleavage of EphrinB3 or EGF in stable cell lines expressing WT or mutant RHBDF2 and/or in tylotic patient cells.
  • EphrinB3 membrane localisation may be assessed by microscopy and Western blotting of cellular fractions.
  • Screening for activity may be carried out in cell culture.
  • Cell lines may be used which express wild-type RHBDF2 or a variant polypeptide according to the present invention.
  • Patient cells may also/alternatively be used, such as affected cells from Tylosis or epithelial cancer patients.
  • RHBDL2 The level and/or location of RHBDL2, its substrates or the resultant cleaved products may be found, for example, by microscopy or Western blotting of cellular fractions.
  • RHBDL2 or its substrates may be tagged, for example, with a radioactive, colour! metric or fluorescent label to aid detection and/or localisation.
  • the molecule may affect the expression and/or activity of ADA 17.
  • the molecule may downregulate the expression and/or activity of ADAM 17.
  • the molecule may inhibit the traffic of ADAM 17 from the endoplasmic reticulum to the Golgi and/or from the Golgi to the plasma membrane.
  • the molecule may affect shedding of pro-inflammatory cytokines such as 11-6, IL-8 and/or TNF-a.
  • the molecule may affect shedding of EGF family growth factors, such as TGFa and/or Amphiregulin. Expression of a variant version of RHBDF2 in cells such as keratinocytes causes sustained EGFR signalling and a hyperproliferative phenotype (see Figure 13).
  • the cells display an "activated" immune profile with raised levels of proinflammatory cytokines including IL8.
  • the test molecule may affect the expression and/or activity of AD AM 17 such that the dysregulated growth factor and/or immune phenotype is completely or partially reversed to the wild-type phenotype.
  • the present invention also provides method for diagnosing a cancer in a subject, which comprises the step of investigating:
  • the present invention provides a method for diagnosing a cancer in a subject, which comprises the step of investigating the sequence of the RHBDF2 gene or the RHBDF2 polypeptide in the subject.
  • a finding that the subject has a variant version of the RHBDF2 gene or expresses a variant version of the RHBDF2 polypeptide may indicate that the subject has, or is predisposed to contract tylosis or an epithelial cancer, such as oesophageal cancer.
  • sequence of a gene in an individual may be ascertained using various methods known in the art, for example direct sequencing, Northern or Southern blot or PGR.
  • Germline DNA eg. from blood
  • somatic DNA eg. from a tumour
  • the presence of a mutation in a polypeptide may be determined using an entity which shows differential binding activity between the mutant and wild-type polypeptides, such as an antibody which is selective for either the mutant or wild- type versions.
  • the presence of a mutant polypeptide may then be detected by immunological methods such as immunohistochemistry, Western blot, dot-blot, immunoassay or ELISA.
  • the present invention also provides an antibody which specifically binds to a varient polypeptide according to the first aspect of the invention.
  • the antibody may be selective for the variant polypeptide and not show significant binding activity to the wild-type RHBDF2 polypeptide.
  • the antibody may be used in the method of diagnosis or disease risk assessment described above.
  • Techniques for preparing antibodies are known in the art and include classical immunisation and phage display. In order to obtain a selective antibody, two binding selection steps could be included to select for the subset of antibodies which both bind the mutant polypeptide and do not significantly bind the wild-type polypeptide.
  • a fragment of the RHBDF2 molecule which comprises the mutation compared to the wild-type sequence, may be used, in order to focus antibody generation on the desired portion of the molecule.
  • antibody includes intact antibodies, fragments of antibodies, e.g., Fab, F(ab')2 fragments, and intact antibodies and fragments that have been mutated either in their constant and/or variable region (e.g., mutations to produce chimeric, partially humanized, or fully humanized antibodies, as well as to produce antibodies with a desired trait, e.g., enhanced IL 13 binding and/or reduced FcR binding).
  • fragment refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained via chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. Binding fragments include Fab, Fab', F(ab') 2, Fabc, Fd, dAb, Fv, single chains, single-chain antibodies, e.g., scFv, single domain antibodies, and an isolated complementarity determining region (CDR).
  • Antibody-like molecules include the use of CDRs separately or in combination in synthetic molecules such as SMIPs and small antibody mimetics, which comprise two CDR regions and a framework region.
  • An antibody or binding portion thereof also may be part of a larger molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • the antibody may be for use in a diagnostic method, for example an immunohistochemical method.
  • the present invention also provides a nucleic acid probe which specifically binds to a variant nucleic acid sequence, but not to the wild-type RHBDF2 gene or transcript.
  • the probe may be RNA or DNA and may be a sense or antisense sequence.
  • the probe may be labelled to aid detection, for example with a fluorescent, radioactive or colorimetric label.
  • the probe may comprise a sequence complementary to the portion of the nucleic acid which comprises the mutation.
  • the probe may be, for example, between 5 and 30 nucleotides in length.
  • the length should be such that it binds sufficiently stably to the mutant sequence to enable detection, but short enough to enable binding to the mutant sequence and binding to the wild-type sequence to be distinguished.
  • the present invention also provides a pair of probes which are complementary to sequences flanking the portion of the nucleic acid which comprises the mutation which can be used to produce a copy of the mutant sequence, when used in a method such as PCR.
  • the probes may, for example, produce a sequence fragment (e.g. PCR product) of between 30 and 1000 base pairs in length.
  • the probe or primer pair may be for use in a diagnostic method, for example in a method to investigate the sequence of the RHBDF2 gene or transcript in an individual, or in a sample from an individual.
  • the present inventors have found two naturally occurring mutations in the RHBDF2 gene from three separate sets of tylosis patients, indicating that this mutation is the single genetic cause of this condition.
  • Type A tylosis or non-epidermolytic focal plamoplantar keratoderma (NFPPK), causes oral precursor lesions ("white spongy plaques") and palmoplantar ketatoderma.
  • Tylosis is associated with a high risk of squamous cell oesophageal cancer (up to 95% by age 65).
  • the epithelial cancer may be a squamous cell carcinoma, such as a squamous cell carcinoma of the lung, oesophagus or ovary.
  • the subject undergoing diagnosis may exhibit one or more symptoms, of a disease such as tylosis or cancer, or may be asymptomatic.
  • the subject may be from a genetic background which suggests they may be at risk from contracting tylosis or cancer.
  • the subject may have a family history of developing tylosis and/or cancer or, in the case of oesophageal cancer, they may have the premalignant condition Barrett's oesophagus.
  • mutant RHBDF2 causes upregulation of the protease RHBDL2 leading to an increase in EGF release and EGFR phosphorylation.
  • EGF is known to be involved in wound healing as it stimulates fibroblasts to secrete collagenase to degrade the matrix during the remodelling phase. It also stimulates keratinocyte and fibroblast proliferation.
  • the present invention provides a method for treating a malignant or inflammatory condition which comprises the step of administering an ADAM 17 inhibitor to a subject.
  • ADAM 17 inhibitors may be used to treat a cancer or inflammatory condition of the gastrointestinal tract or skin. They may, for example, be used to treat epithelial cancers such as oesophageal cancer, psoriasis or eczema.
  • ADAM 17 inhibitors include GW280264X, TAPI-2 and GI254023X.
  • An inhibitor may be based on a TIMP molecule. TIMPs are the natural inhibitors of ADAM 17 in humans.
  • the hydroxamate inhibitor DPC333 may also be used (Le Gall et al 2010J Cell Sci. 123(Pt 22):3913-22. Epub 2010 Oct 27).
  • an siRNA approach may be used to down-regulate ADAM 17.
  • Example 1 Identification of the tylosis with oesophageal cancer (TOO gene
  • Type A tylosis (focal non-epidermolytic palmoplantar keratoderma) is associated with a high risk of squamous cell oesophageal cancer (up to 95% by age 65) in three extensive pedigrees (Ellis ei al 1994 European journal of cancer. Part B, Oral oncology. 30B(2): 102-12; Hennies ei al 1995 Genomics 29(2):537-40; Stevens et al 1996 Archives of dermatology 132(6):640-51 ). The incidence of other cancers in these families is not altered compared to the normal Caucasian population (Ellis et al (1994) and Stevens et al (1996) as above).
  • the syndrome (TOC: OMIM 148500) is inherited as an autosomal dominant with the cutaneous phenotype being fully penetrant by the age of puberty. Oral leukokeratosis and follicular hyperkeratosis are also features of the syndrome.
  • the tylosis with oesophageal cancer ( TOC) locus has been mapped by the applicants to a small region on chromosome 17q25 using linkage and haplotype analyses (Kelsell et al (1996) Human molecular genetics 5(6):857-60; Risk et al (1999) Genomics.
  • the present inventors revisited the TOC locus, making various adaptations including extending the minimal region (new distal recombination event between D17S 839 and D17S1603) after reassessing recombination and affected status of a family member. All exons from this revised minimal region included all exons (plus some non-coding sequence) was included on a targeted sequence capture array (Nimblegen). The DNA from an affected individual from the UK tylosis family was hybridised to the array and the subsequent DNA "library” was then sequenced by next-generation sequencing on a GAM sequencer (lllumina).
  • RHBDF2 (also known as RHBDL5/6) belongs to a family of seven transmembrane spanning proteins called Rhomboids which were first identified in Drosophila and shown to be serine intra-membrane proteases linked with EGFR signalling and mitochondrial remodelling (see Figure 9 and Freeman et al (1992) Development 1 16(2):335-46; Dimmer et al (2002) Molecular Biology of the Cell 13:847- 853; McQuibban et al (2003) Nature 423(6939):537-541 ).
  • human RHBDF2 has no protease activity as it lacks the conserved Ser residue in position 721.
  • RHBDF2 has been shown to regulate epithelial cancer cell growth and survival possibly via GPCR-mediated EGFR signalling and/or by interacting with TGFa ligands (Zou et al (2009) The FASEB journal:. 23(2):425-32; Nakagawa (2005) Developmental dynamics: an official publication of the American Association of Anatomists. 233(4):1315-31 ).
  • RHBDL2 has retained its protease activity following pro-enzyme activation (Lei and Li (2009) Journal of molecular biology 394(5):815-25).
  • RHBDL2 has been shown to cleave EphrinB3, a trans-membrane ligand for EphB receptor as well as other membrane associated molecules, including thrombomodulin and the adhesion-associated molecule CLEC14A (Rho et al (2010) Biochemical and biophysical research communications 404(1 ):103-108; Pascall and Brown (2004) Biochemical and biophysical research communications 317(1 ):244-52; Lohi et al (2004) Current biology 14(3):236-41 ; Rohani et al (2011 ) PLoS biology, 9(3), e1000597. Epub Mar 1 ).
  • RHBDL2 may be regulated by RHBDF2, where it can associate with RHBDL2 to inhibit its protease activity (http://www.vwfb.de/Seeon2010/Meeting Report Bor Seeon 2010.pdf).
  • RHBDF2 may be regulated by RHBDF2, where it can associate with RHBDL2 to inhibit its protease activity (http://www.vwfb.de/Seeon2010/Meeting Report Bor Seeon 2010.pdf).
  • the present inventors predict that the missense mutations in RHBDF2 impair its interaction with RHBDL2 leading to increased RHBDL2 protease activity resulting in, for example, reduction in active EphrinB3 signalling at the plasma membrane ( Figure 2).
  • Example 2 Investigating the expression of RHBDF2 and an RHBDL2-associated substrate in normal and tylotic skin
  • the present inventors produced and characterised a new polyclonal antibody directed against human RHBDF2 and used this in conjunction with a commercially available antibody (Sigma, HPA018080) to conduct immunohistochemical analysis of RHBDF2 in normal and tylotic skin.
  • RHBDF2 is thought to be involved in the regulation of the protease activity of RHBDL2 and thus may affect the intramembrane cleavage of RHBDL2-associated substrates, including EphrinB3 and CLEC14A.
  • EphrinB3 expression in tylotic skin had a dysregulated pattern of expression compared to normal skin ( Figure 4). This suggests there may be less active membrane signalling of EphrinB3 in tylotic epidermis ( Figure 2).
  • the phosphoarray data also revealed increased phosphorylation of the Insulin Receptor and Insulin-like growth factor I receptor (IGF1 R) in tylotic keratinocytes compared to control cells (Fig. 9).
  • IGF1 R Insulin-like growth factor I receptor
  • RHBDF2 and RHBDL2 can be detected by western blotting ( Figure 5).
  • the present inventors have developed constructs of RHBDF2 with and without EGFP tags. Site-directed mutagenesis is performed to introduce the two missense mutations into these constructs. After transfection in normal keratinocytes, detailed localisation studies are performed to compare the effect of each of the two missense mutations (p.lle186Thr and p.Pro189Leu) on RHBDF2 and EphrinBS localisation compared to WT RHBDF2 protein. The effect on other RHBDL2 substrates such as CLEC14A is also investigated.
  • Co-immunoprecipitation experiments are also performed using the tylotic patient cell lines and normal keratinocytes to study the interaction between RHBDF2 and RHBDL2 to determine whether the mutations in RHBDF2 affect its binding to RHBDL2. This is carried out with both endogenous proteins, and in cells overexpressing wild-type and mutant constructs with/without EGFP ( Figure 5).
  • EGF is another known substrate for RHBDL2 and EGFR activation can be regulated directly or indirectly by iRhom
  • EGFR activity was investigated and revealed an increase in EGFR phosphorylation in tylotic keratinocytes compared to control cells (Fig. 10A).
  • EphA4 has been shown to be upregulated in astrocyte gliosis after CNS injury and its subsequent small molecule inhibition led to decreased wound closure with less scarring (Parmentier-Batteur et al (201 1 ) J Neurochem). These data are consistent with our observations of the behaviour of tylotic keratinocytes after scratch injury displaying increased migration and higher levels of phospho-EphA4 compared to control keratinocytes.
  • Eph/Ephrin signalling is not only important in development but is also dysregulated in many cancers and has been implicated in invasion and metastasis. EphrinB3 has been shown to be strongly expressed in a high proportion of endometroid ovarian carcinomas and implicated in increased invasion of glioma cells. Furthermore dysregulated EphA4 signalling has been demonstrated in a number of cancer types including gastric, prostate and T-cell lymphomas. Towards investigating the role of RHBDF2-regulated signalling in oesophageal tumourigenesis, immunohistochemical studies were performed in squamous oesophageal tumours from both tylotic and sporadic cases.
  • pEGFR is known to be increased in many tumour types including those of the oesophagous.
  • squamous oesophageal tumours show cytoplasmic localisation of RHBDF2 and increased EphrinB3 expression (Fig. 1 1 ).
  • RHBDF2 shows moderate to strong cytoplasmic staining in a number of other tumour types including ovarian, endometrial, thyroid and gastric cancers (www.proteinatlas.org).
  • Example 7 Increased AD AM 17 processing and activation in mutant iRHOM2 (TYLK) keratinocytes Analysis of mutant iRHOM2 (TYLK) keratinocytes showed there was an increase in ADAM 17 pro-domain release compared to controls ( Figure 12(i)). There was also increased ADAM 17 at plasma membranes in Tylotic keratinocytes compared to controls ( Figure 12 (ii)). The levels of IL-8 secretion were also increased in tylotic keratinocytes, even without LPS stimulation, compared to a control cell line. IL-8 is a substrate of ADAM 17 ( Figure 12 (iii)).
  • tylotic keratinocytes showed increased levels of Amphiregulin and TGFa secretion, without stimulation, compared to a control cell line. Again, Amphiregulin and TGFa are substrates of ADAM 17. Taken together, these results indicate that the aberrant growth factor and inflammatory cytokine response seen in keratinocytes derived from Tylotic patients is at least partly due to increased ADAM17 activity mediated by the inherited "gain of function" iRHOM2 mutation (see Figure 13). Summary
  • the present inventors have identified novel missense mutations in iRHOM2 (RHBDF2), which encodes the inactive rhomboid protease iRHOM2 as the underlying cause of Tylosis with Oesophageal Cancer.
  • RHBDF2 iRHOM2
  • This is an autosomal dominant form of susceptibility to oesophageal cancer with up to 95% chance of developing the cancer by age 65.
  • Patients also present with palmoplantar keratoderma resulting from hyperproliferation of keratinocytes.
  • the functional data demonstrates that mutant iRHOM2 represents a gain-of-function allele that results in sustained EGFR signaling in the patient-derived keratinocyte cell lines leading to a hyperproliferative phenotype (Figure 13).
  • ADAM 17 is also dramatically upregulated, implicating mutant iRHOM2 in its processing and activation.
  • the increase in expression and activity of ADAM 17 is though to be at least partly responsible for the dysregulated growth factor and immune phenotype.

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Abstract

La présente invention concerne un polypeptide ayant une séquence d'acides aminés basée sur la séquence décrite en tant que SEQ ID NO: 1 mais comprenant une ou plusieurs mutations par rapport à cette séquence, dont l'expression in vivo est associée à la tylose et/ou une prédisposition au développement du cancer. L'invention concerne en outre une séquence d'acide nucléique capable de coder pour un tel polypeptide ; un procédé de criblage pour déterminer si un composé peut être utile dans le traitement d'un cancer ou d'affections inflammatoires ; et un procédé pour diagnostiquer un cancer chez un sujet, sur la base de l'expression d'un tel polypeptide.
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WO2015172143A1 (fr) 2014-05-09 2015-11-12 The Jackson Laboratory Procédés d'identification de composés qui modifient l'activité de polypeptides irhom et utilisation correspondante
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CN114480408A (zh) * 2022-02-17 2022-05-13 南开大学 Rhx6基因以及其在制备抗乳腺癌药物中的应用
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WO2014043223A1 (fr) * 2012-09-11 2014-03-20 Hospital For Special Surgery Inhibition de irhom2 dans le traitement des troubles induits par un complément
WO2015172143A1 (fr) 2014-05-09 2015-11-12 The Jackson Laboratory Procédés d'identification de composés qui modifient l'activité de polypeptides irhom et utilisation correspondante
CN106471130A (zh) * 2014-05-09 2017-03-01 杰克逊实验室 鉴定改变iRhom多肽活性的化合物的方法及其用途
EP3140418A4 (fr) * 2014-05-09 2017-12-27 The Jackson Laboratory Procédés d'identification de composés qui modifient l'activité de polypeptides irhom et utilisation correspondante
WO2017075585A1 (fr) * 2015-10-30 2017-05-04 The Jackson Laboratory Compositions et procédés associés à des analyses de tumeurs
CN108471732A (zh) * 2015-10-30 2018-08-31 杰克逊实验室 与肿瘤分析相关的组合物和方法
US12404345B2 (en) 2021-03-01 2025-09-02 Scirhom Gmbh Humanized antibodies against iRHOM2
CN114480408A (zh) * 2022-02-17 2022-05-13 南开大学 Rhx6基因以及其在制备抗乳腺癌药物中的应用
CN114480408B (zh) * 2022-02-17 2023-12-19 南开大学 Rhx6基因以及其在制备抗乳腺癌药物中的应用

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