WO2023143474A1 - Methods for treating gastric cancer - Google Patents

Methods for treating gastric cancer Download PDF

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Publication number
WO2023143474A1
WO2023143474A1 PCT/CN2023/073471 CN2023073471W WO2023143474A1 WO 2023143474 A1 WO2023143474 A1 WO 2023143474A1 CN 2023073471 W CN2023073471 W CN 2023073471W WO 2023143474 A1 WO2023143474 A1 WO 2023143474A1
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genes
therapeutic compound
listed
subject
gastric cancer
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PCT/CN2023/073471
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French (fr)
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Zhiwei Chen
Jianbing FAN
Weimei RUAN
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Anchordx Medical Co., Ltd.
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Publication of WO2023143474A1 publication Critical patent/WO2023143474A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/154—Methylation markers

Definitions

  • GC GC
  • GC patients are treated with neoadjuvant/perioperative chemotherapies, followed by adjuvant chemotherapy or adjuvant chemoradiotherapy.
  • surgical options for GC include primarily subtotal or total gastrectomy.
  • none of the standard cares yield satisfactory clinical results, resulting in a 5-year overall survival rate as low as less than 30%.
  • a method for treating a subject having or suspected of having gastric cancer comprising administering to said subject a therapeutic compound effective to regulate one or more genes, wherein the one or more genes are directly or indirectly regulated by a methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7.
  • the methylation site is listed in Table 3.
  • the methylation site is listed in Table 4.
  • the one or more genes are selected from the group consisting of ARPC1B, BHLHE23, BTBD11, CALN1, CBLN4, CCDC166, CDH4, COL9A3, CPSF1, DLEU1, DPP10, ECHDC2, EPS8L1, F7, FCGBP, FENDRR, FOXI2, GNAS, HOOK2, HS3ST2, IGFBP3, TNS3, IRF2BP1, IRF2BP101, IRX1, IRX2, IRX6, KCNS1, KIAA1211L, KRT7, KRT81, LMBR1, NOM1, LPAR5, MAT2B, LOC101927835, MDGA2, MIR548Y, MEIG1, OLAH, NCAM2, PCDHGB7, PEG3, PRICKLE1, SLC13A5, SLC35F1, SLCO5A1, SLIT2, TBX18, UNC80, ZBTB7A,
  • the methylation site is listed in Table 5.
  • the one or more genes are selected from the group consisting of ARPC1B, BTBD11, CALN1, CBLN4, CDH4_1, COL9A3, DPP10, EPS8L1, F7, FCGBP, GNAS, HS3ST2, KCNS1, KRT7, KRT81, LPAR5, NCAM2, PCDHGB7, SLC13A5, SLC35F1, SLCO5A1, SLIT2, and UNC80.
  • the methylation site is listed in Table 6.
  • the one or more genes are selected from the group consisting of BHLHE23, CDH4_1, CPSF1, FOXI2, HS3ST2, IRF2BP1, IRF2BP101, NCAM2, SLC13A5, SLC35F1, UNC80, and ZBTB7A.
  • the methylation site is listed in Table 7.
  • the one or more genes are selected from the group consisting of CDH4_1, HS3ST2, NCAM2, SLC13A5, SLC35F1, and UNC80.
  • the one or more genes are regulated by suppressing transcription level and/or protein level thereof.
  • the therapeutic compound is a small-molecule inhibitor, an antisense oligonucleotide, an RNAi agent, a therapeutic peptide, a non-naturally occurring or engineered inducible CRISPR-Cas system, and/or an antagonistic antibody.
  • the therapeutic compound is an antagonistic antibody.
  • the one or more genes are regulated by elevating transcription level and/or protein level thereof.
  • the therapeutic compound is modified mRNA of the one or more genes, encapsuled with or without lipid nanoparticles.
  • the method further comprises detecting the transcription and/or protein expression of the one or more genes prior to the administration of the therapeutic compound. In other embodiments, the method further comprises detecting the transcription and/or protein expression of the one or more genes after the administration of the therapeutic compound.
  • the subject has or is suspected of having T1 stage of gastric cancer. In other embodiments, the subject has or is suspected of having lymph node metastasis. In other embodiments, the subject does not have lymph node metastasis
  • FIG. 1 depicts a heatmap of 1366 methylation sites among 12 pairs of GC with lymph node metastasis and adjacent cancer (s) and 11 pairs of GC without lymph node metastasis or adjacent cancer (s) .
  • a panel of DNA methylation sites are identified, thereby provided herein are one or more genes that are regulated by these DNA methylation sites. Further provided herein are one or more genes serve as drug target (s) in treating GC. Specifically provided herein are one or more genes serve as drug target (s) in treating GC by preventing lymph node metastasis. Further provided herein are therapeutic compounds that regulate the identified one or more genes.
  • the method described herein is used to treat patients having or suspected of having gastric cancer from different stages defined with American Joint Committee on Cancer (AJCC) TNM system.
  • the method described herein is used to treat patients having or suspected of having AJCC stage 0, including Tis, N0, and M0 (with T category describing the extent of the main (primary) tumor, including how far it has grown into the layers of the stomach wall and if it has reached nearby structures or organs, N category describes any cancer spread to nearby lymph nodes, and M category describes any spread (metastasis) to distant parts of the body, such as the liver or lungs) .
  • the method described herein is used to treat patients having or suspected of having AJCC stage IA, including T1, N0, and M0.
  • the method described herein is used to treat patients having or suspected of having AJCC stage IB, including T1, N1, and M0, or T2, N0, and M0. In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IIA, including T1, N2, M0; T2, N1, and M0, and T2, N0, and M0. In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IIB, including T1, N3a, and M0; T2, N2, and M0; T3, N1, and N0;T4a, N0, and M0.
  • the method described herein is used to treat patients having or suspected of having AJCC stage IIIA, including T2, N3a, and M0; T3, N2, and M0; T4a, N1, and M0; T4a, N2, and M0; T4b, N0, and M0.
  • the method described herein is used to treat patients having or suspected of having AJCC stage IIIB, including T1, N3b, and M0; T2, N3b, and M0; T3, N3a, and M0; T4a, N3a, and M0; T4b, N1, and M0; T4b, N2, and M0.
  • the method described herein is used to treat patients having or suspected of having AJCC stage IIIC, including T3, N4b, and M0; T4a, N3b, and M0; T4b, N3a, and M0; T4b, N3b, and M0. In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IV, including any T, any N, and M1.
  • the method described herein is used to treat patients having or suspected of having gastric cancer without metastasis. In some specific embodiments, the method described herein is used to treat patients having or suspected of having gastric cancer without lymph node metastasis. In other embodiments, the method described herein is used to treat patients having or suspected of having gastric cancer with metastasis. In some specific embodiments, the method described herein is used to treat patients having or suspected of having gastric cancer with lymph node metastasis.
  • the method described herein comprises administering to the subject a therapeutic compound with a certain route.
  • the therapeutic compound is administered parenterally (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) .
  • the therapeutic compound is orally (e.g., ingestion, buccal, or sublingual) , inhalation, intradermally, intracavity, intracranially.
  • the therapeutic compound is transdermally (topical) , transmucosally or rectally.
  • the therapeutic compound is administered within gastric cancer tumor tissue.
  • the one or more genes described herein are directly or indirectly regulated by a methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below.
  • the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 is located are the upstream region of the one or more genes.
  • the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 is located are the downstream region of the one or more genes.
  • the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the exonic region of the one or more genes.
  • the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the intronic region of the one or more genes. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the 3 UTR region of the one or more genes. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the 5’ UTR region of the one or more genes. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the intergenic of two genes.
  • the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the exonic region of ncRNA. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the intronic region of ncRNA. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located remotely from the one or more genes that the methylation sites exert their regulation allosterically.
  • the one or more genes described herein serving as drug targets for treating gastric cancer are directly regulated by methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below.
  • the one or more genes described herein serving as drug targets for treating gastric cancer are indirectly regulated by a methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below.
  • the one or more genes described herein serving as drug targets for treating gastric cancer are regulated by a cascade of factors, one of which includes a methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below.
  • the one or more genes regulated by methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below are overexpressed in the subject having or suspected of having gastric cancer, compared to a control subject.
  • the overexpression described herein is on the transcription level. In some specific embodiments, the overexpression described herein is on the protein level.
  • the therapeutic compound described herein for treating gastric cancer regulate the one or more genes by suppressing transcription level and/or protein level thereof.
  • the therapeutic compound described herein is a small-molecule inhibitor.
  • the therapeutic compound described herein is an antisense oligonucleotide.
  • the therapeutic compound described herein is an RNAi agent.
  • the therapeutic compound described herein is a therapeutic peptide.
  • the therapeutic compound described herein is a non-naturally occurring or engineered inducible CRISPR-Cas system.
  • the therapeutic compound described herein is an antagonistic antibody.
  • the therapeutic compound described herein is a combination of all or part of the above.
  • the therapeutic compound described herein is an antisense oligonucleotide comprising a sequence that is at least 90%identical to the one or more genes described herein.
  • the antisense oligonucleotide is modified by one or more phosphorothioate linkages.
  • the antisense oligonucleotide contains at least one modified sugar moiety, nucleobase, or pharmacokinetic-enhancing moiety.
  • the antisense oligonucleotide is nuclease resistant.
  • the therapeutic compound described herein comprises the antisense oligonucleotide together with a pharmaceutically acceptable excipient.
  • the therapeutic compound described herein is a therapeutic peptide.
  • the therapeutic peptide comprises a binding region that specifically binds to the protein product (s) of the one or more genes described herein.
  • the therapeutic peptide further comprises a Fc region.
  • the therapeutic peptide comprises 1 to 5 amino acid substitutions, deletions, or insertions without losing the binding kinetics to the protein product (s) of the one or more genes described herein.
  • the therapeutic compound described herein comprises the therapeutic peptide together with a pharmaceutically acceptable carrier.
  • the therapeutic compound described herein is a non-naturally occurring or engineered inducible CRISPR-Cas system, which comprises a Cas protein or a polynucleotide construct encoding the Cas protein, and one or more guide RNAs.
  • the Cas protein is linked to one or more nuclear localization signals.
  • the therapeutic compound described herein is selected from the group consisting of synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies) , human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc. ) , camelized antibodies, Fab fragments, F (ab’ ) fragments, disulfide-linked Fvs (sdFv) , anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • the therapeutic compound described herein comprises complementarity determining regions (CDRs) that specifically bind to the protein products of the one or more genes described herein.
  • the one or more genes regulated by methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below are suppressed in the subject having or suspected of having gastric cancer, compared to a control subject.
  • the suppression described herein is on the transcription level.
  • the suppression described herein is on the protein level.
  • the therapeutic compound described herein is a modified mRNA of the one or more genes, encapsuled with or without lipid nanoparticle.
  • the method described herein further comprises a step of detecting the transcription and/or protein expression of the one or more genes.
  • the detecting step is performed prior to the administration of the therapeutic compound. In some specific embodiments, the detecting step is performed after the administration of the therapeutic compound.
  • the samples used herein were from patients who met the following requirements: 1) Early GC patients diagnosed as T1 stage; 2) No distant metastasis or family history; 3) No neoadjuvant treatment; 4) Lymphoma, multiple tumors, stump cancer and intraepithelial cases of neoplasia are excluded; 5) At least three gastrointestinal pathologists evaluated the stage of the tumor and the status of lymph node metastasis. In the end, included in this example were 12 pairs of tissue samples from GC with lymph node metastasis and adjacent cancer (s) and 11 pairs of tissue samples from GC without lymph node metastasis or adjacent cancer (s) , totaling 46 samples, all of which were fresh frozen tissue samples.
  • Tissue genomic DNA was isolated from fresh frozen and FFPE tissue samples using the Qiagen DNeasy Blood &Tissue Kit (Qiagen, Cat#69504) and QIAamp DNA FFPE Tissue Kit (Qiagen, Cat#56404) , respectively.
  • Bisulfite conversion was performed using the Zymo Lightning Conversion Reagent (Zymo Research, Cat#D5031) according to the manufacturer’s protocol. Following the procedures for DNA bisulfite conversion, running through Zymo-Spin TM IC Column, washing and desulphonation, the bisulfite-converted DNA was eluted twice using M-Elution buffer to a final volume of 17 ⁇ L.
  • gDNA For tissue samples, 2 ug of gDNA was fragmented into about 200 bp (peak size) by a M220 Focused-ultrasonicator (Covaris, Inc. ) following the manufacturer’s instructions and 800ng of purified fragmented genomic DNA was used for the following bisulfite conversion. After bisulfite conversion, the purified bisulfite-converted DNA was quantified at A260 by NanoDrop (Thermo Fisher Scientific) . In the next step, 100 and 150 ng of the bisulfite-converted products were applied to library preparation for fresh frozen and FFPE tissue samples, respectively.
  • Sample genomic DNA was individually constructed into genome-wide methylation library using Methyl Capture EPIC Library Prep Kit (Illumina, San Diego, CA, USA) , by following the corresponding instructions.
  • FIG. 1 illustrates a heatmap of these 1366 methylation sites in 12 pairs of GC with lymph node metastasis and adjacent cancer (s) and 11 pairs of GC without lymph node metastasis or adjacent cancer (s) .
  • the methylation sites had a significant difference in terms of methylation level between lymph node metastasis and non-metastasis in GC.
  • the CT value of each methylated area obtained by the qPCR detection is corrected by the internal reference CT value.
  • the difference and discriminant diagnostic performance AUC of the co-methylation degree of the 60 methylated regions of the 99 tissue DNA samples in the population without lymph node metastasis and population with lymph node metastasis and the discriminant diagnostic performance AUC are shown in Table 3.
  • genes shown in Table 3 46 genes were shown to have consistent methylation data between NGS data and qPCR cohorts (see Table 4) . These genes include ARPC1B, BHLHE23, BTBD11, CALN1, CBLN4, CCDC166, CDH4, COL9A3, CPSF1, DLEU1, DPP10, ECHDC2, EPS8L1, F7, FCGBP, FENDRR, FOXI2, GNAS, HOOK2, HS3ST2, IGFBP3, TNS3, IRF2BP1, IRF2BP101, IRX1, IRX2, IRX6, KCNS1, KIAA1211L, KRT7, KRT81, LMBR1, NOM1, LPAR5, MAT2B, LOC101927835, MDGA2, MIR548Y, MEIG1, OLAH, NCAM2, PCDHGB7, PEG3, PRICKLE1, SLC13A5, SLC35F1, SLCO5A1, SLIT2, TBX18, UNC80, ZBT
  • genes express their protein products on the cell membrane, which serve as the lead candidates for drug targets for treating GC (see Table 5) .
  • These genes include: ARPC1B, BTBD11, CALN1, CBLN4, CDH4_1, COL9A3, DPP10, EPS8L1, F7, FCGBP, GNAS, HS3ST2, KCNS1, KRT7, KRT81, LPAR5, NCAM2, PCDHGB7, SLC13A5, SLC35F1, SLCO5A1, SLIT2, and UNC80.
  • Table 7 shows the candidate leads that meet the following criteria: (1) consistent methylation data between NGS data and qPCR cohorts; (2) protein products are located on the cell membrane; and (3) hypo-methylation in the upstream regions or hyper-methylation in the exonic regions.
  • tissue FFPE samples are from 20 GC patients with lymph node metastasis and 20 GC patients without lymph node metastasis.
  • RNA is extracted from FFPE samples with RNeasy FFPE kit (QIAGEN GmbH, ) , according to manufacturers recommendations. Depending on the tissue samples, 1 to 4 sections of 10 ⁇ m thickness are used per preparation.
  • RT-PCR primers are designed for each target gene.
  • One-step RT-PCR is performed using the QIAGEN OneStep RT-PCR Kit (QIAGEN GmbH) according to manufacturer's recommendations. The thermal cycling conditions: 45°C for 60 minutes (RT step) and 94°C for 15 minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minutes, and a final extension step for 10 minutes at 72°C) .
  • RT-PCR products is analyzed based on delta Ct value, with GAPDH as the reference gene, to compare mRNA expression level between groups.
  • Western-blot assays together with Immunohistochemistry (IHC) assays are used to validate the over-expression of proteins of candidate genes identified in Tables 1, 3, 4, 5, 6, or 7 in the tumor tissue samples.
  • Frozen tissue samples are from 20 GC patients with lymph node metastasis and 20 GC patients without lymph node metastasis.
  • Proteins are extracted from FFPE samples with 500uL ice-cold lysis buffer (Cell Signaling Technology) .
  • the lysis buffer is mixed with about 5mg tissue sample, and samples are homogenized using electric homogenizer. After centrifugation, supernatants are collected.
  • Western-blot assays are carried out with primary HRP-conjugated antibodies purchased for each target from major suppliers. Western-blot assays are performed using western blot kit (Bio-rad) to compare protein expression level between groups.
  • Example 3 Modification of gastric cancer cell line to validate potential therapeutic candidates.
  • in-vitro functional assays are performed in gastric cancer cell lines, including N-87 &MKN-7 &MKN-28.
  • siRNA or CRISPR tools are used to knock down targeted candidate genes and evaluate the effects on tumor cell lines in proliferation/senescence assay and in-vitro metastasis assay.
  • siRNA, antisense oligonucleotide (ASO) or CRISPR tools are used to transfect small nucleotides into the gastric cancer cell lines to knock down target gene based on manufacture protocol.
  • ASO antisense oligonucleotide
  • CRISPR tools Thermal Fisher
  • the siRNA, ASO, and single-guide RNA (sgRNA) from the CRISPR-Cas system are designed to comprise nucleotide sequences that are complementary to regions of top candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7.
  • mRNA level will be analyzed to validate the knock-down effect, only the modified cell lines with equal to or more than 60%knock-down efficiency are used into next steps.
  • Proliferation assays are carried out with Immuno fluorescent staining with Ki67 antibody (Abcam) to determine the proliferation rate for different groups.
  • Senescence assays are carried out with CellEventTM Senescence Green Detection Kit (Thermo Fisher) to determine the senescence rate for different groups.
  • In-vitro metastasis assay is performed with the Boyden Chamber assay based on manufacture protocol (Sigma) .
  • cells are allowed to migrate through a cell monolayer or ECM protein mixture which is pre-seeded onto a semi-permeable membrane cell culture insert with chemo-attractants added below the membrane.
  • Migrated cells are quantified by staining cells with DNA dyes such as Calcein-AM or CyQUANT GR Dyes.
  • the Millipore’s QCM TN cell migration and invasion assays are performed to provide a quantitative determination on cell migration speed and efficiencies between different groups.
  • Selected small-molecule inhibitors and blocking antibodies purchased from major suppliers for top candidates or designed in house, are used to inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7. Effects are evaluated on tumor cell lines in proliferation/senescence assay and in-vitro metastasis assay.
  • the blocking antibodies comprises complementarity-determining regions (CDRs) in the healvy chains and light chain that confer the specificity in binding to the protein products from the targeted candidate gene.
  • Selected small-molecule inhibitors specifically bind and inhibit target protein products with effective IC 50 .
  • Proliferation assays are carried out with Immuno fluorescent staining with Ki67 antibody (Abcam) to determine the proliferation rate for different groups.
  • Senescence assays are carried out with CellEventTM Senescence Green Detection Kit (Thermo Fisher) to determine the senescence rate for different groups.
  • In-vitro metastasis assay is performed with the Boyden Chamber assay based on manufacture protocol (Sigma) . Briefly, cells are allowed to migrate through a cell monolayer or ECM protein mixture which is pre-seeded onto a semi-permeable membrane cell culture insert with chemo-attractants added below the membrane. Migrated cells are quantified by staining cells with DNA dyes such as Calcein- AM or CyQUANT GR Dyes. In parallel, the Millipore’s QCM TM cell migration and invasion assays are performed to provide a quantitative determination on cell migration speed and efficiencies between different groups
  • Xenograft assays are carried out with 10 control mice and 10 experimental mice.
  • the experimental mice are immunodeficient mice subcutaneously injected with gastric cancer primary cells treated with siRNA, CRISPR, small-molecule inhibitors, or blocking antibodies.
  • the control mice are immunodeficient mice subcutaneously injected with gastric cancer primary cells with negative control treatments.
  • the siRNA, ASO, and sgRNA from the CRISPR-Cas system are designed to comprise nucleotide sequences that are complementary to regions of top candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7.
  • Blocking antibodies are designed to comprises complementarity-determining regions (CDRs) in the healvy chains and light chain that confer the specificity in binding to inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7.
  • CDRs complementarity-determining regions
  • Selected small-molecule inhibitors specifically bind and inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7 with effective IC 50 .
  • the growth rate of the tumor is monitored by measuring tumor size on day 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 after injection.
  • siRNA, CRISPR, small-molecule inhibitors, or blocking antibodies are injected via tail vein with corresponding gastric cancer primary cells treated with siRNA, CRISPR, small-molecule inhibitors, or blocking antibodies, versus negative control treatments.
  • siRNA, ASO, and sgRNA from the CRISPR-Cas system are designed to comprise nucleotide sequences that are complementary to regions of top candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7.
  • Blocking antibodies are designed to comprises complementarity-determining regions (CDRs) in the healvy chains and light chain that confer the specificity in binding to inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7.
  • Selected small-molecule inhibitors specifically bind and inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7 with effective IC 50 .5 of experimental mice and 5 of control mice are dissected after 1 month, and the other 5 of experimental mice and the other 5 of control mice are dissected after 2 months.
  • the metastasis scale of the tumor is examined by analyzing metastasis situation in important organs, such as liver, lung, bones and other.
  • GC mouse model (INS-GAS mice) , at 2-month-old, are treated with small-molecule inhibitors, or blocking antibodies for top candidates in 10 experimental mice, or treated with sham in 10 control mice.
  • Blocking antibodies are designed to comprises complementarity-determining regions (CDRs) in the healvy chains and light chain that confer the specificity in binding to inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7.
  • CDRs complementarity-determining regions
  • Selected small-molecule inhibitors specifically bind and inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7 with effective IC 50 .
  • Mice are dissected 2 weeks or 1 month after the treatment, and tumor burden for each mouse is evaluated, including the size of tumors and their, metastasis stasis at each time point.

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Abstract

Disclosed herein are methods for treating a subject having or suspected of having gastric cancer, and a therapeutic compound effective to regulate one or more genes, wherein the one or more genes are directly or indirectly regulated by a methylation sites disclosed herein.

Description

METHODS FOR TREATING GASTRIC CANCER
CROSS-REFERENCE
This application claims the benefit of PCT/CN2022/074112 filed January 26, 2022, which application is incorporated herein by reference in its entirety.
1. Background
GC (GC) is a malignant tumor and one of the most common malignant tumors worldwide. According to the global cancer statistics in 2020, the incidence and mortality of GC rank fifth and third respectively, with the incidence rate of men twice that of women.
Currently in clinical practice, GC patients are treated with neoadjuvant/perioperative chemotherapies, followed by adjuvant chemotherapy or adjuvant chemoradiotherapy. Alternatively, surgical options for GC include primarily subtotal or total gastrectomy. However, none of the standard cares yield satisfactory clinical results, resulting in a 5-year overall survival rate as low as less than 30%.
Therefore, there is an urgent need to develop more effective therapeutics to treat GC. The disclosure described herein satisfy this need and provide related advantages.
2. Summary
In one aspect, provided herein is a method for treating a subject having or suspected of having gastric cancer, said method comprising administering to said subject a therapeutic compound effective to regulate one or more genes, wherein the one or more genes are directly or indirectly regulated by a methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7.
In some embodiments, the methylation site is listed in Table 3.
In some embodiments, the methylation site is listed in Table 4. In some specific embodiments, the one or more genes are selected from the group consisting of ARPC1B, BHLHE23, BTBD11, CALN1, CBLN4, CCDC166, CDH4, COL9A3, CPSF1, DLEU1, DPP10, ECHDC2, EPS8L1, F7, FCGBP, FENDRR, FOXI2, GNAS, HOOK2, HS3ST2, IGFBP3, TNS3, IRF2BP1, IRF2BP101, IRX1, IRX2, IRX6, KCNS1, KIAA1211L, KRT7, KRT81, LMBR1, NOM1, LPAR5, MAT2B, LOC101927835, MDGA2, MIR548Y, MEIG1, OLAH, NCAM2, PCDHGB7, PEG3, PRICKLE1, SLC13A5, SLC35F1, SLCO5A1, SLIT2, TBX18, UNC80, ZBTB7A, ZNF704, and PAG1.
In some embodiments, the methylation site is listed in Table 5. In some specific embodiments, the one or more genes are selected from the group consisting of ARPC1B,  BTBD11, CALN1, CBLN4, CDH4_1, COL9A3, DPP10, EPS8L1, F7, FCGBP, GNAS, HS3ST2, KCNS1, KRT7, KRT81, LPAR5, NCAM2, PCDHGB7, SLC13A5, SLC35F1, SLCO5A1, SLIT2, and UNC80.
In some embodiments, the methylation site is listed in Table 6. In some specific embodiments, the one or more genes are selected from the group consisting of BHLHE23, CDH4_1, CPSF1, FOXI2, HS3ST2, IRF2BP1, IRF2BP101, NCAM2, SLC13A5, SLC35F1, UNC80, and ZBTB7A.
In some embodiments, the methylation site is listed in Table 7. In some specific embodiments, the one or more genes are selected from the group consisting of CDH4_1, HS3ST2, NCAM2, SLC13A5, SLC35F1, and UNC80.
In some embodiments, the one or more genes are regulated by suppressing transcription level and/or protein level thereof. In some specific embodiments, the therapeutic compound is a small-molecule inhibitor, an antisense oligonucleotide, an RNAi agent, a therapeutic peptide, a non-naturally occurring or engineered inducible CRISPR-Cas system, and/or an antagonistic antibody. In one specific embodiment, the therapeutic compound is an antagonistic antibody.
In some embodiment, the one or more genes are regulated by elevating transcription level and/or protein level thereof. In some specific embodiments, the therapeutic compound is modified mRNA of the one or more genes, encapsuled with or without lipid nanoparticles.
In some embodiments, the method further comprises detecting the transcription and/or protein expression of the one or more genes prior to the administration of the therapeutic compound. In other embodiments, the method further comprises detecting the transcription and/or protein expression of the one or more genes after the administration of the therapeutic compound.
In some embodiments, the subject has or is suspected of having T1 stage of gastric cancer. In other embodiments, the subject has or is suspected of having lymph node metastasis. In other embodiments, the subject does not have lymph node metastasis
Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
3. Incorporation by Reference
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
4. Brief Description of the Drawing
The patent or application file contains one drawing executed in color. Copies of this patent or patent application publication with a color drawing will be provided by the Office upon request and payment of the necessary fee. The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawing of which.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawing (also “Figure” and “FIG. ” herein) , of which:
FIG. 1 depicts a heatmap of 1366 methylation sites among 12 pairs of GC with lymph node metastasis and adjacent cancer (s) and 11 pairs of GC without lymph node metastasis or adjacent cancer (s) .
5. Detailed Description
While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
As described herein, a panel of DNA methylation sites are identified, thereby provided herein are one or more genes that are regulated by these DNA methylation sites. Further provided herein are one or more genes serve as drug target (s) in treating GC. Specifically provided herein are one or more genes serve as drug target (s) in treating GC by  preventing lymph node metastasis. Further provided herein are therapeutic compounds that regulate the identified one or more genes.
The method described herein is used to treat patients having or suspected of having gastric cancer from different stages defined with American Joint Committee on Cancer (AJCC) TNM system. In some embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage 0, including Tis, N0, and M0 (with T category describing the extent of the main (primary) tumor, including how far it has grown into the layers of the stomach wall and if it has reached nearby structures or organs, N category describes any cancer spread to nearby lymph nodes, and M category describes any spread (metastasis) to distant parts of the body, such as the liver or lungs) . In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IA, including T1, N0, and M0. In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IB, including T1, N1, and M0, or T2, N0, and M0. In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IIA, including T1, N2, M0; T2, N1, and M0, and T2, N0, and M0. In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IIB, including T1, N3a, and M0; T2, N2, and M0; T3, N1, and N0;T4a, N0, and M0. In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IIIA, including T2, N3a, and M0; T3, N2, and M0; T4a, N1, and M0; T4a, N2, and M0; T4b, N0, and M0. In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IIIB, including T1, N3b, and M0; T2, N3b, and M0; T3, N3a, and M0; T4a, N3a, and M0; T4b, N1, and M0; T4b, N2, and M0. In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IIIC, including T3, N4b, and M0; T4a, N3b, and M0; T4b, N3a, and M0; T4b, N3b, and M0. In other embodiments, the method described herein is used to treat patients having or suspected of having AJCC stage IV, including any T, any N, and M1.
In some embodiments, the method described herein is used to treat patients having or suspected of having gastric cancer without metastasis. In some specific embodiments, the method described herein is used to treat patients having or suspected of having gastric cancer without lymph node metastasis. In other embodiments, the method described herein is used to treat patients having or suspected of having gastric cancer with metastasis. In some specific embodiments, the method described herein is used to treat patients having or suspected of having gastric cancer with lymph node metastasis.
The method described herein comprises administering to the subject a therapeutic compound with a certain route. In some embodiments, the therapeutic compound is administered parenterally (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) . In some embodiments, the therapeutic compound is orally (e.g., ingestion, buccal, or sublingual) , inhalation, intradermally, intracavity, intracranially. In some embodiments, the therapeutic compound is transdermally (topical) , transmucosally or rectally. In some embodiments, the therapeutic compound is administered within gastric cancer tumor tissue.
The one or more genes described herein are directly or indirectly regulated by a methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below. In some embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 is located are the upstream region of the one or more genes. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 is located are the downstream region of the one or more genes. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the exonic region of the one or more genes. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the intronic region of the one or more genes. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the 3 UTR region of the one or more genes. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the 5’ UTR region of the one or more genes. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the intergenic of two genes. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the exonic region of ncRNA. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located in the intronic region of ncRNA. In other embodiments, the methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 are located remotely from the one or more genes that the methylation sites exert their regulation allosterically.
In some embodiments, the one or more genes described herein serving as drug targets for treating gastric cancer are directly regulated by methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below. In other embodiments, the one or more genes described herein serving as drug targets for treating gastric cancer are indirectly regulated by a methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below. In some specific embodiments, the one or more genes described herein serving as drug targets for  treating gastric cancer are regulated by a cascade of factors, one of which includes a methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below.
In some embodiments, the one or more genes regulated by methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below are overexpressed in the subject having or suspected of having gastric cancer, compared to a control subject. In some specific embodiments, the overexpression described herein is on the transcription level. In some specific embodiments, the overexpression described herein is on the protein level.
In some embodiments, the therapeutic compound described herein for treating gastric cancer regulate the one or more genes by suppressing transcription level and/or protein level thereof. In some specific embodiments, the therapeutic compound described herein is a small-molecule inhibitor. In other specific embodiments, the therapeutic compound described herein is an antisense oligonucleotide. In other specific embodiments, the therapeutic compound described herein is an RNAi agent. In other specific embodiments, the therapeutic compound described herein is a therapeutic peptide. In other specific embodiments, the therapeutic compound described herein is a non-naturally occurring or engineered inducible CRISPR-Cas system. In other specific embodiments, the therapeutic compound described herein is an antagonistic antibody. In other specific embodiments, the therapeutic compound described herein is a combination of all or part of the above.
In specific embodiments, the therapeutic compound described herein is an antisense oligonucleotide comprising a sequence that is at least 90%identical to the one or more genes described herein. In some embodiment, the antisense oligonucleotide is modified by one or more phosphorothioate linkages. In some embodiment, the antisense oligonucleotide contains at least one modified sugar moiety, nucleobase, or pharmacokinetic-enhancing moiety. In some embodiment, the antisense oligonucleotide is nuclease resistant. In some embodiment, the therapeutic compound described herein comprises the antisense oligonucleotide together with a pharmaceutically acceptable excipient.
In specific embodiments, the therapeutic compound described herein is a therapeutic peptide. In some embodiments, the therapeutic peptide comprises a binding region that specifically binds to the protein product (s) of the one or more genes described herein. In some embodiments, the therapeutic peptide further comprises a Fc region. In some embodiments, the therapeutic peptide comprises 1 to 5 amino acid substitutions, deletions, or insertions without losing the binding kinetics to the protein product (s) of the one or more genes described herein. In some embodiments, the therapeutic compound described herein comprises the therapeutic peptide together with a pharmaceutically acceptable carrier.
In specific embodiments, the therapeutic compound described herein is a non-naturally occurring or engineered inducible CRISPR-Cas system, which comprises a Cas protein or a polynucleotide construct encoding the Cas protein, and one or more guide RNAs. In some specific embodiments, the Cas protein is linked to one or more nuclear localization signals.
In specific embodiments, the therapeutic compound described herein is selected from the group consisting of synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies) , human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc. ) , camelized antibodies, Fab fragments, F (ab’ ) fragments, disulfide-linked Fvs (sdFv) , anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. In some embodiments, the therapeutic compound described herein comprises complementarity determining regions (CDRs) that specifically bind to the protein products of the one or more genes described herein.
In some embodiments, the one or more genes regulated by methylation sites listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7 below are suppressed in the subject having or suspected of having gastric cancer, compared to a control subject. In some specific embodiments, the suppression described herein is on the transcription level. In some specific embodiments, the suppression described herein is on the protein level. In some embodiments, the therapeutic compound described herein is a modified mRNA of the one or more genes, encapsuled with or without lipid nanoparticle.
In some embodiments, the method described herein further comprises a step of detecting the transcription and/or protein expression of the one or more genes. In some specific embodiments, the detecting step is performed prior to the administration of the therapeutic compound. In some specific embodiments, the detecting step is performed after the administration of the therapeutic compound.
6. Examples
The following is a description of various methods and materials used in the studies, and are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present disclosure. Efforts have been made to ensure  accuracy with respect to numbers used (e.g., amounts, percentages, etc. ) , but some experimental errors and deviations should be accounted for.
6.1 Example 1 –Identification of Methylation Sites and Corresponding Genes.
6.1.1 Methods
The samples used herein were from patients who met the following requirements: 1) Early GC patients diagnosed as T1 stage; 2) No distant metastasis or family history; 3) No neoadjuvant treatment; 4) Lymphoma, multiple tumors, stump cancer and intraepithelial cases of neoplasia are excluded; 5) At least three gastrointestinal pathologists evaluated the stage of the tumor and the status of lymph node metastasis. In the end, included in this example were 12 pairs of tissue samples from GC with lymph node metastasis and adjacent cancer (s) and 11 pairs of tissue samples from GC without lymph node metastasis or adjacent cancer (s) , totaling 46 samples, all of which were fresh frozen tissue samples.
Tissue genomic DNA was isolated from fresh frozen and FFPE tissue samples using the Qiagen DNeasy Blood &Tissue Kit (Qiagen, Cat#69504) and QIAamp DNA FFPE Tissue Kit (Qiagen, Cat#56404) , respectively.
Bisulfite conversion was performed using the Zymo Lightning Conversion Reagent (Zymo Research, Cat#D5031) according to the manufacturer’s protocol. Following the procedures for DNA bisulfite conversion, running through Zymo-SpinTM IC Column, washing and desulphonation, the bisulfite-converted DNA was eluted twice using M-Elution buffer to a final volume of 17 μL.
For tissue samples, 2 ug of gDNA was fragmented into about 200 bp (peak size) by a M220 Focused-ultrasonicator (Covaris, Inc. ) following the manufacturer’s instructions and 800ng of purified fragmented genomic DNA was used for the following bisulfite conversion. After bisulfite conversion, the purified bisulfite-converted DNA was quantified at A260 by NanoDrop (Thermo Fisher Scientific) . In the next step, 100 and 150 ng of the bisulfite-converted products were applied to library preparation for fresh frozen and FFPE tissue samples, respectively.
Sample genomic DNA was individually constructed into genome-wide methylation library usingMethyl Capture EPIC Library Prep Kit (Illumina, San Diego, CA, USA) , by following the corresponding instructions.
6.1.2 Results
The original sequencing data was cleaned up, processed, and analyzed. The percentage of methylated cytosine (β value) at each site was determined based on the reads. The  two sets of samples were compared and analyzed, with 1366 methylation sites screened. FIG. 1 illustrates a heatmap of these 1366 methylation sites in 12 pairs of GC with lymph node metastasis and adjacent cancer (s) and 11 pairs of GC without lymph node metastasis or adjacent cancer (s) . As shown in FIG. 1, the methylation sites had a significant difference in terms of methylation level between lymph node metastasis and non-metastasis in GC.
Through the gene annotation information of these 1366 methylation sites, the AUC of each methylation sites in 12 pairs of gastric cancer lymph node metastasis and adjacent cancer and 11 pairs of gastric cancer lymph node metastasis cancer and adjacent cancer is shown in Table 1. Among them, there were 1342 single sites with an AUC greater than 0.6, 1207 greater than 0.7, 691 greater than 0.8, and 46 greater than 0.9. This shows that these methylation sites can distinguish gastric cancer lymph node metastasis from non-metastasis well.
















































From 1366 methlyation sites disclosed in Table 1, 60 top methylated regions, which contain multiple methlyation sites, were selected for the design and develop qPCR assays for each of them, then these 60 methylated regions using qPCR were tested on 99 independent tissue samples: 40 cases of cancer and adjacent samples from people with lymph node metastasis in GC;59 cases of cancer and adjacent samples from people without lymph node metastasis; The pathological and clinical information composition of all samples is shown in Table 2.
Table 2 Pathological and clinical composition information of tissue DNA samples
Among them, the CT value of each methylated area obtained by the qPCR detection is corrected by the internal reference CT value. The relative cycle number of the target area d-CT = CT (target area) -CT (internal reference) ; If the target area is not detected, the relative number of cycles was assigned to the target area d-CT = 35.
The difference and discriminant diagnostic performance AUC of the co-methylation degree of the 60 methylated regions of the 99 tissue DNA samples in the population without lymph node metastasis and population with lymph node metastasis and the discriminant diagnostic performance AUC are shown in Table 3.









Out of genes shown in Table 3, 46 genes were shown to have consistent methylation data between NGS data and qPCR cohorts (see Table 4) . These genes include ARPC1B, BHLHE23, BTBD11, CALN1, CBLN4, CCDC166, CDH4, COL9A3, CPSF1, DLEU1, DPP10, ECHDC2, EPS8L1, F7, FCGBP, FENDRR, FOXI2, GNAS, HOOK2, HS3ST2, IGFBP3, TNS3, IRF2BP1, IRF2BP101, IRX1, IRX2, IRX6, KCNS1, KIAA1211L, KRT7, KRT81, LMBR1, NOM1, LPAR5, MAT2B, LOC101927835, MDGA2, MIR548Y, MEIG1, OLAH, NCAM2, PCDHGB7, PEG3, PRICKLE1, SLC13A5, SLC35F1, SLCO5A1, SLIT2, TBX18, UNC80, ZBTB7A, ZNF704, and PAG1.
Out of the 46 genes mentioned above, 22 genes express their protein products on the cell membrane, which serve as the lead candidates for drug targets for treating GC (see Table 5) . These genes include: ARPC1B, BTBD11, CALN1, CBLN4, CDH4_1, COL9A3, DPP10, EPS8L1, F7, FCGBP, GNAS, HS3ST2, KCNS1, KRT7, KRT81, LPAR5, NCAM2, PCDHGB7, SLC13A5, SLC35F1, SLCO5A1, SLIT2, and UNC80.
Separately, considering the abnormal overexpression of protein products are more druggable, another group of lead candidates for drug targets to treat GC are the ones not only illustrated consistent data between NGS and qPCR cohorts, but also showed hypo-methylation in the upstream regions or hyper-methylation in the exonic regions. These genes and the related methylation sites are shown in Table 6.
Furthermore, Table 7 below shows the candidate leads that meet the following criteria: (1) consistent methylation data between NGS data and qPCR cohorts; (2) protein products are located on the cell membrane; and (3) hypo-methylation in the upstream regions or hyper-methylation in the exonic regions.















6.2 Example 2 –mRNA and Protein level analysis.
RT-PCR experiment is performed to check mRNA level for candidate genes identified in Tables 1, 3, 4, 5, 6, or 7 in the tissue FFPE samples. The tissue FFPE samples are from 20 GC patients with lymph node metastasis and 20 GC patients without lymph node metastasis.
Briefly, RNA is extracted from FFPE samples with RNeasy FFPE kit (QIAGEN GmbH, ) , according to manufacturers recommendations. Depending on the tissue samples, 1 to 4 sections of 10 μm thickness are used per preparation. RT-PCR primers are designed for each target gene. One-step RT-PCR is performed using the QIAGEN OneStep RT-PCR Kit (QIAGEN GmbH) according to manufacturer's recommendations. The thermal cycling conditions: 45℃ for 60 minutes (RT step) and 94℃ for 15 minutes, followed by 35 cycles of 94℃ for 30 seconds, 55℃ for 30 seconds, and 72℃ for 1 minutes, and a final extension step for 10 minutes at 72℃) . RT-PCR products is analyzed based on delta Ct value, with GAPDH as the reference gene, to compare mRNA expression level between groups.
Furthermore, Western-blot assays together with Immunohistochemistry (IHC) assays are used to validate the over-expression of proteins of candidate genes identified in Tables 1, 3, 4, 5, 6, or 7 in the tumor tissue samples. Frozen tissue samples are from 20 GC patients with lymph node metastasis and 20 GC patients without lymph node metastasis.
Proteins are extracted from FFPE samples with 500uL ice-cold lysis buffer (Cell Signaling Technology) . The lysis buffer is mixed with about 5mg tissue sample, and samples are homogenized using electric homogenizer. After centrifugation, supernatants are collected.
Western-blot assays are carried out with primary HRP-conjugated antibodies purchased for each target from major suppliers. Western-blot assays are performed using western blot kit (Bio-rad) to compare protein expression level between groups.
6.3 Example 3 –Modification of gastric cancer cell line to validate potential therapeutic candidates.
After verification of protein products of candidate genes, in-vitro functional assays are performed in gastric cancer cell lines, including N-87 &MKN-7 &MKN-28.
SiRNA or CRISPR tools are used to knock down targeted candidate genes and evaluate the effects on tumor cell lines in proliferation/senescence assay and in-vitro metastasis assay. Briefly, siRNA, antisense oligonucleotide (ASO) or CRISPR tools (Thermal Fisher) are  used to transfect small nucleotides into the gastric cancer cell lines to knock down target gene based on manufacture protocol. The siRNA, ASO, and single-guide RNA (sgRNA) from the CRISPR-Cas system are designed to comprise nucleotide sequences that are complementary to regions of top candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7. After the transfection, mRNA level will be analyzed to validate the knock-down effect, only the modified cell lines with equal to or more than 60%knock-down efficiency are used into next steps. Proliferation assays are carried out with Immuno fluorescent staining with Ki67 antibody (Abcam) to determine the proliferation rate for different groups. Senescence assays are carried out with CellEventTM Senescence Green Detection Kit (Thermo Fisher) to determine the senescence rate for different groups. In-vitro metastasis assay is performed with the Boyden Chamber assay based on manufacture protocol (Sigma) . Briefly, cells are allowed to migrate through a cell monolayer or ECM protein mixture which is pre-seeded onto a semi-permeable membrane cell culture insert with chemo-attractants added below the membrane. Migrated cells are quantified by staining cells with DNA dyes such as Calcein-AM or CyQUANT GR Dyes. In parallel, the Millipore’s QCMTN cell migration and invasion assays are performed to provide a quantitative determination on cell migration speed and efficiencies between different groups.
Selected small-molecule inhibitors and blocking antibodies, purchased from major suppliers for top candidates or designed in house, are used to inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7. Effects are evaluated on tumor cell lines in proliferation/senescence assay and in-vitro metastasis assay. Briefly, the blocking antibodies comprises complementarity-determining regions (CDRs) in the healvy chains and light chain that confer the specificity in binding to the protein products from the targeted candidate gene. Selected small-molecule inhibitors specifically bind and inhibit target protein products with effective IC50. Proliferation assays are carried out with Immuno fluorescent staining with Ki67 antibody (Abcam) to determine the proliferation rate for different groups. Senescence assays are carried out with CellEventTM Senescence Green Detection Kit (Thermo Fisher) to determine the senescence rate for different groups. In-vitro metastasis assay is performed with the Boyden Chamber assay based on manufacture protocol (Sigma) . Briefly, cells are allowed to migrate through a cell monolayer or ECM protein mixture which is pre-seeded onto a semi-permeable membrane cell culture insert with chemo-attractants added below the membrane. Migrated cells are quantified by staining cells with DNA dyes such as Calcein- AM or CyQUANT GR Dyes. In parallel, the Millipore’s QCMTM cell migration and invasion assays are performed to provide a quantitative determination on cell migration speed and efficiencies between different groups
6.4 Example 4 –in vivo xenograph and gastric cancer mouse model to validate potential therapeutic candidates.
Xenograft assays are carried out with 10 control mice and 10 experimental mice. The experimental mice are immunodeficient mice subcutaneously injected with gastric cancer primary cells treated with siRNA, CRISPR, small-molecule inhibitors, or blocking antibodies. The control mice are immunodeficient mice subcutaneously injected with gastric cancer primary cells with negative control treatments. Briefly, the siRNA, ASO, and sgRNA from the CRISPR-Cas system are designed to comprise nucleotide sequences that are complementary to regions of top candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7. Blocking antibodies are designed to comprises complementarity-determining regions (CDRs) in the healvy chains and light chain that confer the specificity in binding to inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7. Selected small-molecule inhibitors specifically bind and inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7 with effective IC50. The growth rate of the tumor is monitored by measuring tumor size on day 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 after injection.
In parallel, another 10 experimental mice or control mice are injected via tail vein with corresponding gastric cancer primary cells treated with siRNA, CRISPR, small-molecule inhibitors, or blocking antibodies, versus negative control treatments. Briefly, the siRNA, ASO, and sgRNA from the CRISPR-Cas system are designed to comprise nucleotide sequences that are complementary to regions of top candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7. Blocking antibodies are designed to comprises complementarity-determining regions (CDRs) in the healvy chains and light chain that confer the specificity in binding to inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7. Selected small-molecule inhibitors specifically bind and inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7 with effective IC50.5 of experimental mice and 5 of control mice are dissected after 1 month, and the other 5 of experimental mice and the other 5 of control mice are dissected after 2 months. The metastasis  scale of the tumor is examined by analyzing metastasis situation in important organs, such as liver, lung, bones and other.
Separately, GC mouse model (INS-GAS mice) , at 2-month-old, are treated with small-molecule inhibitors, or blocking antibodies for top candidates in 10 experimental mice, or treated with sham in 10 control mice. Briefly, Blocking antibodies are designed to comprises complementarity-determining regions (CDRs) in the healvy chains and light chain that confer the specificity in binding to inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7. Selected small-molecule inhibitors specifically bind and inhibit protein products from the targeted candidate genes that are identified in Tables 1, 3, 4, 5, 6, or 7 with effective IC50. Mice are dissected 2 weeks or 1 month after the treatment, and tumor burden for each mouse is evaluated, including the size of tumors and their, metastasis stasis at each time point.

Claims (20)

  1. A method for treating a subject having or suspected of having gastric cancer, said method comprising administering to said subject a therapeutic compound effective to regulate one or more genes, wherein the one or more genes are directly or indirectly regulated by a methylation site listed in Table 1, Table 3, Table 4, Table 5, Table 6, or Table 7.
  2. The method of claim 1, wherein the methylation site is listed in Table 3.
  3. The method of claim 2, wherein the methylation site is listed in Table 4.
  4. The method of claim 3, wherein the one or more genes are selected from the group consisting of ARPC1B, BHLHE23, BTBD11, CALN1, CBLN4, CCDC166, CDH4, COL9A3, CPSF1, DLEU1, DPP10, ECHDC2, EPS8L1, F7, FCGBP, FENDRR, FOXI2, GNAS, HOOK2, HS3ST2, IGFBP3, TNS3, IRF2BP1, IRF2BP101, IRX1, IRX2, IRX6, KCNS1, KIAA1211L, KRT7, KRT81, LMBR1, NOM1, LPAR5, MAT2B, LOC101927835, MDGA2, MIR548Y, MEIG1, OLAH, NCAM2, PCDHGB7, PEG3, PRICKLE1, SLC13A5, SLC35F1, SLCO5A1, SLIT2, TBX18, UNC80, ZBTB7A, ZNF704, and PAG1.
  5. The method of claim 3, wherein the methylation site is listed in Table 5.
  6. The method of claim 5, wherein the genes are selected from the group consisting of ARPC1B, BTBD11, CALN1, CBLN4, CDH4_1, COL9A3, DPP10, EPS8L1, F7, FCGBP, GNAS, HS3ST2, KCNS1, KRT7, KRT81, LPAR5, NCAM2, PCDHGB7, SLC13A5, SLC35F1, SLCO5A1, SLIT2, and UNC80.
  7. The method of claim 3, wherein the methylation site is listed in Table 6.
  8. The method of claim 7, wherein the genes are selected from the group consisting of BHLHE23, CDH4_1, CPSF1, FOXI2, HS3ST2, IRF2BP1, IRF2BP101, NCAM2, SLC13A5, SLC35F1, UNC80, and ZBTB7A.
  9. The method of claim 3, wherein the methylation site is listed in Table 7.
  10. The method of claim 9, wherein the genes are selected from the group consisting of CDH4_1, HS3ST2, NCAM2, SLC13A5, SLC35F1, and UNC80.
  11. The method of preceding claims, the one or more genes are regulated by suppressing transcription level and/or protein level thereof.
  12. The method of claim 11, wherein the therapeutic compound is a small-molecule inhibitor, an antisense oligonucleotide, an RNAi agent, a therapeutic peptide, a non-naturally occurring or engineered inducible CRISPR-Cas system, and/or an antagonistic antibody.
  13. The method of claim 12, wherein the therapeutic compound is an antagonistic antibody.
  14. The method of preceding claims, the one or more genes are regulated by elevating transcription level and/or protein level thereof.
  15. The method of claim 14, the therapeutic compound is a modified mRNA of the one or more genes, encapsuled with or without lipid nanoparticles.
  16. The method of preceding claims, further comprising detecting the transcription and/or protein expression of the one or more genes prior to the administration the therapeutic compound.
  17. The method of preceding claims, further comprising detecting the transcription and/or protein expression of the one or more genes after the administration the therapeutic compound.
  18. The method of preceding claims, wherein the subject has or is suspected of having T1 stage of gastric cancer.
  19. The method of preceding claims, wherein the subject has or is suspected of having lymph node metastasis.
  20. The method of preceding claims, wherein the subject does not have lymph node metastasis.
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104630219A (en) * 2013-11-15 2015-05-20 上海交通大学医学院附属瑞金医院 saRNA (Ribonucleic Acid) molecule of cancer suppressor gene IRX1, as well as composition and application thereof
CN105624279A (en) * 2015-01-22 2016-06-01 香港中文大学深圳研究院 Stomach cancer marker as well as expression and methylation detection method, kit and application thereof
CN106434918A (en) * 2016-09-26 2017-02-22 复旦大学附属华山医院 Application of specific gene mutation in molecular diagnosis and targeted therapy of pituitary adenoma detection
CN108728539A (en) * 2018-06-05 2018-11-02 浙江大学 A kind of application of HoxD10 genes in gastric cancer
CN109963598A (en) * 2016-07-05 2019-07-02 约翰霍普金斯大学 The composition and method based on CRISPR/CAS9 for treating cancer
CN109988765A (en) * 2019-03-29 2019-07-09 中国医科大学附属盛京医院 A kind of targeted inhibition agent of FENDRR gene and application thereof
US20210332440A1 (en) * 2020-04-17 2021-10-28 Universal Diagnostics, S.L. Detection of advanced adenoma and/or early stage colorectal cancer
CN113913423A (en) * 2020-07-09 2022-01-11 上海吉凯基因医学科技股份有限公司 Application of human CFAP65 gene and related product
CN114317738A (en) * 2021-10-25 2022-04-12 广州市基准医疗有限责任公司 Methylation biomarker related to detection of gastric cancer lymph node metastasis or combination and application thereof

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104630219A (en) * 2013-11-15 2015-05-20 上海交通大学医学院附属瑞金医院 saRNA (Ribonucleic Acid) molecule of cancer suppressor gene IRX1, as well as composition and application thereof
CN105624279A (en) * 2015-01-22 2016-06-01 香港中文大学深圳研究院 Stomach cancer marker as well as expression and methylation detection method, kit and application thereof
CN109963598A (en) * 2016-07-05 2019-07-02 约翰霍普金斯大学 The composition and method based on CRISPR/CAS9 for treating cancer
CN106434918A (en) * 2016-09-26 2017-02-22 复旦大学附属华山医院 Application of specific gene mutation in molecular diagnosis and targeted therapy of pituitary adenoma detection
CN108728539A (en) * 2018-06-05 2018-11-02 浙江大学 A kind of application of HoxD10 genes in gastric cancer
CN109988765A (en) * 2019-03-29 2019-07-09 中国医科大学附属盛京医院 A kind of targeted inhibition agent of FENDRR gene and application thereof
US20210332440A1 (en) * 2020-04-17 2021-10-28 Universal Diagnostics, S.L. Detection of advanced adenoma and/or early stage colorectal cancer
CN113913423A (en) * 2020-07-09 2022-01-11 上海吉凯基因医学科技股份有限公司 Application of human CFAP65 gene and related product
CN114317738A (en) * 2021-10-25 2022-04-12 广州市基准医疗有限责任公司 Methylation biomarker related to detection of gastric cancer lymph node metastasis or combination and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN SHANG, YU YANQI, LI TAO, RUAN WEIMEI, WANG JUN, PENG QUANZHOU, YU YINGDIAN, CAO TIANFENG, XUE WENYUAN, LIU XIN, CHEN ZHIWEI, : "A novel DNA methylation signature associated with lymph node metastasis status in early gastric cancer", CLINICAL EPIGENETICS, BIOMED CENTRAL LTD, LONDON, UK, vol. 14, no. 1, 1 December 2022 (2022-12-01), London, UK, XP093080842, ISSN: 1868-7075, DOI: 10.1186/s13148-021-01219-x *

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