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WO2023167544A1 - Biomarqueur comprenant la fam167a pour le diagnostic de la résistance contre l'inhibiteur de la tyrosine kinase indépendante de bcr-abl, et composition ciblant la fam167a pour prévenir ou traiter la leucémie myéloïde chronique - Google Patents

Biomarqueur comprenant la fam167a pour le diagnostic de la résistance contre l'inhibiteur de la tyrosine kinase indépendante de bcr-abl, et composition ciblant la fam167a pour prévenir ou traiter la leucémie myéloïde chronique Download PDF

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WO2023167544A1
WO2023167544A1 PCT/KR2023/002947 KR2023002947W WO2023167544A1 WO 2023167544 A1 WO2023167544 A1 WO 2023167544A1 KR 2023002947 W KR2023002947 W KR 2023002947W WO 2023167544 A1 WO2023167544 A1 WO 2023167544A1
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abl
bcr
fam167a
independent
protein
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Korean (ko)
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박성규
양태우
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SNU R&DB Foundation
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Seoul National University R&DB Foundation
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • 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
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Definitions

  • the present invention relates to a biomarker composition for diagnosing BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance including FAM167A and a composition for preventing or treating chronic myelogenous leukemia targeting FAM167A, in more detail Specifically, a composition, kit, and method for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myelogenous leukemia by confirming the expression level of FAM167A protein, a fragment thereof, or a gene encoding the protein, and a BCR-targeting FAM167A A pharmaceutical composition for preventing or treating chronic myelogenous leukemia showing ABL-independent TKI resistance and a pharmaceutical composition for inhibiting BCR-ABL-independent TKI resistance in a chronic myelogenous leukemia patient are provided.
  • TKI tyrosine kinase inhibitor
  • Chronic myeloid leukemia is caused by a characteristic t(9;22)(q34;q11) reciprocal translocation of the BCR-ABL Philadelphia chromosome.
  • the BCR-ABL fusion gene encodes a constitutively active BCR-ABL oncoprotein due to loss of the upstream regulatory domain of ABL1 during translocation.
  • TKIs BCR-ABL-targeted tyrosine kinase inhibitors
  • imatinib has revolutionized the treatment of CML patients.
  • resistance to TKIs remains a major obstacle to successful CML treatment.
  • TKI resistance occurs in two ways: BCR-ABL dependent and BCR-ABL independent.
  • BCR-ABL-dependent resistance is primarily caused by mutations in the BCR-ABL kinase domain, many CML patients without mutations in this domain also exhibit TKI resistance.
  • Second-generation TKIs such as nilotinib and dasatinib are effective treatments for BCR-ABL-dependent resistant diseases.
  • BCR-ABL-independent TKI resistance cannot be resolved with second-generation TKIs.
  • Several factors, including changes in signaling pathway activity, mutations in epigenetic regulators, microenvironmental factors and leukemia stem cell activity are potential contributors to BCR-ABL-independent TKI resistance, but the underlying reasons for this type of resistance are not fully understood. did not Therefore, identifying the underlying mechanisms and developing effective therapies are major goals.
  • NF- ⁇ B nuclear factor- ⁇ B
  • I ⁇ B inhibitor of the ⁇ B protein
  • NIK nuclear translocation of p50-containing dimers
  • Non-canonical NF- ⁇ B activation depends on NIK accumulation through blocking of ubiquitination-dependent NIK degradation.
  • NIK Accumulated NIK induces processing of p100 to p52, followed by nuclear translocation of p52-containing dimers.
  • the NF- ⁇ B pathway contributes to TKI resistance, and inhibition of this pathway restores the sensitivity of CML to TKIs.
  • the factors involved in the direct activation of the NF- ⁇ B pathway in TKI-resistant CML are still unknown.
  • NF- ⁇ B is the most highly activated transcription factor among those tested through in silico activated transcription factor analysis and differential gene expression in BCR-ABL-independent TKI-resistant CML cells. stated that they were in charge.
  • FAM167A a previously uncharacterized protein, in the non-canonical NF- ⁇ B pathway of BCR-ABL-independent TKI-resistant CML.
  • the expression of FAM167A was highly upregulated in CD34 + CML cells of patients with BCR-ABL-independent TKI resistance (similar to the results in CML cell lines), suggesting that FAM167A is responsible for BCR-ABL-independent TKI resistance. show Neutralization of FAM167A reversed TKI resistance in cultured cells and mouse tumor models.
  • Non-Patent Document 1 has identified FAM167A-BLK as a systemic sclerosis susceptibility gene identified by CGA, but is a target for diagnosis of BCR-ABL-independent TKI resistance and treatment of TKI-resistant CML It is not known about the role of FAM167A as a
  • Non-Patent Document 0001 Ota Y, Kuwana M. Updates on genetics in systemic sclerosis. Inflamm Regen. 2021 Jun 15;41(1):17.
  • the present invention was made to solve the above problems, discovering a new target for diagnosis of BCR-ABL-independent TKI resistance and treatment of TKI-resistant CML, and using the target as a biomarker to detect BCR-ABL in CML patients.
  • An object of the present invention is to provide a technique for diagnosing ABL-independent TKI resistance, treating BCR-ABL-independent TKI-resistant CML, and suppressing BCR-ABL-independent TKI resistance.
  • the present invention is a biomarker composition for diagnosing BCR-ABL independent tyrosine kinase inhibitor (TKI) resistance in patients with chronic myelogenous leukemia, including FAM167A protein or a gene encoding the same provides
  • the present invention also provides a composition and kit for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myelogenous leukemia, including an agent for measuring the expression level of FAM167A protein, a fragment thereof or a gene encoding the protein to provide.
  • the agent for measuring the expression level of the FAM167A protein or fragment thereof may include an antibody or antigen-binding fragment thereof, or an aptamer that specifically binds to the FAM167A protein or fragment thereof.
  • the agent for measuring the mRNA level of the gene encoding the FAM167A protein may include sense and antisense primers or probes that complementarily bind to the mRNA of the gene.
  • kits are RT-PCR kit, competitive RT-PCR kit, real-time RT-PCR kit, DNA chip kit, microarray kit, SAGE (Serial Analysis of Gene Expression) kit, gene chip kit, ELISA (Enzyme linked immunosorbent assay kits, protein chip kits, rapid kits, or multiple reaction monitoring (MRM) kits.
  • the present invention also provides an information providing method for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myelogenous leukemia, comprising the following steps:
  • the expression level measured in step (a) is the same protein measured in a biological sample isolated from a chronic myelogenous leukemia patient without BCR-ABL-independent tyrosine kinase inhibitor resistance, a fragment thereof, or a gene encoding the protein. Comparing with the mRNA expression level of .
  • the information providing method is (c) the mRNA expression level of the FAM167A protein, a fragment thereof, or a gene encoding the protein measured in a biological sample isolated from a chronic myeloid leukemia patient in step (a), When higher than the mRNA expression level of the same protein, fragment thereof, or gene encoding the protein measured in a biological sample isolated from a chronic myelogenous leukemia patient without BCR-ABL-independent tyrosine kinase inhibitor resistance in step (b), BCR- It may further include the step of determining that it exhibits resistance to ABL-independent TKI.
  • the biological sample is blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, genitourinary tract fluid, breast milk, lymphatic body fluid, and semen. , cerebrospinal fluid, intraorgan system fluids, ascites, cystic tumor fluids, amniotic fluid, or combinations thereof.
  • the present invention provides a FAM167A protein inhibitor; an inhibitor of expression of the gene encoding the FAM167A protein; Or a pharmaceutical composition for preventing or treating chronic myelogenous leukemia exhibiting BCR-ABL-independent TKI resistance containing a mixture thereof as an active ingredient and a pharmaceutical composition for suppressing BCR-ABL-independent TKI resistance in chronic myelogenous leukemia patients.
  • chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance may have increased NF- ⁇ B activity compared to chronic myeloid leukemia exhibiting TKI sensitivity.
  • the pharmaceutical composition may be administered in combination with a TKI.
  • the TKI is crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib ( imatinib), nilotinib, ponatinib, ibrutinib, cabozantinib, gefitinib, erlotinib, lapatinib, bande vandetanib, afatinib, osimertinib, ruxolitinib, tofacitinib, axitinib, lenvatinib, nintedanib ( nintedanib), pazopanib, regorafenib, sorafenib, or sunitinib.
  • the present invention also provides a method for screening a CML therapeutic agent or a BCR-ABL independent TKI resistance inhibitor exhibiting BCR-ABL independent TKI resistance, comprising the following steps:
  • the screening method (c) when the candidate substance inhibits the FAM167A protein or inhibits the expression of the gene encoding the protein, the candidate substance is a CML therapeutic agent showing BCR-ABL-independent TKI resistance or A step of determining the BCR-ABL-independent TKI resistance inhibitor may be further included.
  • the present invention is BCR-ABL, comprising administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of a gene encoding the FAM167A protein, or a mixture thereof to a chronic myelogenous leukemia patient exhibiting BCR-ABL-independent TKI resistance.
  • a method for treating chronic myelogenous leukemia exhibiting independent TKI resistance is provided.
  • the present invention is chronic myeloid leukemia, comprising administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of a gene encoding the FAM167A protein, or a mixture thereof to a chronic myelogenous leukemia patient exhibiting BCR-ABL-independent TKI resistance.
  • a method for inhibiting BCR-ABL independent TKI resistance in a patient is provided.
  • the present invention provides a method for diagnosing BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient and treating chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance, comprising the following steps:
  • the expression level measured in step (a) is the same protein measured in a biological sample isolated from a chronic myelogenous leukemia patient without BCR-ABL-independent TKI resistance, a fragment thereof, or an mRNA of a gene encoding the protein If the expression level is higher than that, diagnosing as showing BCR-ABL independent TKI resistance; and
  • step (c) administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of a gene encoding the FAM167A protein, or a mixture thereof to a patient diagnosed as exhibiting BCR-ABL-independent TKI resistance in step (b).
  • the present invention provides a method for diagnosing BCR-ABL-independent TKI resistance and suppressing BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients, comprising the following steps:
  • the expression level measured in step (a) is the same protein measured in a biological sample isolated from a chronic myelogenous leukemia patient without BCR-ABL-independent TKI resistance, a fragment thereof, or an mRNA of a gene encoding the protein If the expression level is higher than that, diagnosing as showing BCR-ABL independent TKI resistance; and
  • step (c) administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of a gene encoding the FAM167A protein, or a mixture thereof to a patient diagnosed as exhibiting BCR-ABL-independent TKI resistance in step (b).
  • FAM167A was discovered as a novel biomarker that can be used as an indicator of TKI resistance in CML showing BCR-ABL-independent TKI resistance without mutations in the BCR-ABL kinase domain, and when inhibited, TKI resistance was reversed.
  • it is possible to effectively treat TKI-resistant patients by diagnosing TKI resistance in CML patients who are not treated with existing second-generation TKIs and reversing the TKI resistance of CML patients who exhibit BCR-ABL-independent TKI resistance. provides an advantage
  • 1A shows a strategy for the selection of genes contributing to BCR-ABL independent TKI resistance in CML.
  • FIGS. 1b and 1c show the results of analyzing the viability of K562S and K562R cells after treatment with imatinib (FIG. 1b) and nilotinib (FIG. 1c) at the indicated concentrations for 3 days, respectively.
  • FIGS. 1d and 1e show The results of flow cytometry analysis of annexin V-stained cells to evaluate apoptosis in K562S and K562R cells after treatment with imatinib (FIG. 1d) and nilotinib (FIG. 1e) at the indicated concentrations for 3 days, respectively.
  • Data represent 3 independent experiments (error bars, s.d. of triplicate (FIGS. 1B and 1C) samples) of 3 (FIGS.
  • K562S TKI-sensitive K562 cell line
  • K562R TKI-resistant K562 cell line without BCR-ABL kinase domain mutation.
  • 1F is a Venn diagram showing upregulated and downregulated genes (fold change ⁇ 2) in K562R cells examined by microarray and RNA-seq analysis.
  • Figure 1g shows a heat map analysis of the expression levels of differentially expressed genes in K562R cells.
  • FIGS. 1i to 1k show transcription factors involved in the regulation of differentially expressed genes in both microarray and RNA-seq analyzes (Fig. 1h) and 3 control genes (each group contains 500 randomly selected genes). ) (FIGS. 1i to 1k) shows the in silico analysis results. Size and color indicate the number of genes associated with transcription factors.
  • Figure 1l shows the enrichment score for transcription factors, showing the degree of association with differentially expressed genes compared to randomly selected genes.
  • A shows the ratio of genes targeted by the indicated transcription factor among genes differentially expressed between K562S and K562R
  • B shows the ratio of genes targeted by the indicated transcription factor among 500 randomly selected genes.
  • Figure 1n shows NF- ⁇ B and AP-1 luciferase reporter activities in K562S and K562R cells and K562R cells treated with 1 ⁇ M imatinib for 24 hours.
  • Figure 1o shows the results of quantitative reverse transcription PCR (qRT-PCR) analysis of mRNA levels of 7 genes in K562S and K562R cells and K562R cells treated with 1 ⁇ M imatinib for 24 hours.
  • IMA imatinib.
  • Data represent 3 (Figs. 1n and 1o) independent experiments (error bars, s.d. of duplicate (Fig. 1o) or triplicate (Fig. 1l and Fig. 1n) samples). unpaired two-tailed t-test; *P ⁇ 0.05, **P ⁇ 0.01.
  • NS no significance
  • K562S TKI sensitive K562 cell line
  • K562R TKI resistant K562 cell line without BCR-ABL kinase domain mutation.
  • Figure 2a shows NF- ⁇ B luciferase reporter activity in K562S cells 24 hours after transfection of plasmids encoding the indicated genes, and in K562R cells and K562R cells treated with 1 ⁇ M imatinib for 24 hours.
  • Figure 2b shows the viability of K562S cells transfected with plasmids encoding the indicated genes after treatment with or without imatinib (IMA, 1 ⁇ M) for 2 days. Data represent 2 independent experiments (error bars, s.d. of triplicate samples). unpaired two-tailed t-test; ***P ⁇ 0.001.
  • Figure 2c A is in silico secretion of human, mouse, rat and zebrafish FAM167A and HA-tagged human FAM167A by SecretomeP tool (http://www.cbs.dtu.dk/services/SecretomeP/) As a prediction, proteins with a NN score of 0.5 or higher are predicted to be secreted.
  • Figure 2c (b) shows the results of immunoblot analysis of FAM167A in cell lysates and culture supernatants of K562R cells. Medium was used as a control supernatant. Supernatant proteins were concentrated by acetone precipitation. A cytoplasmic protein, Erbin, was also analyzed as a control, confirming the absence of cytoplasmic protein in the culture supernatant. Data represent 3 independent experiments (Fig. 2C, B).
  • Figure 2d shows the NF- ⁇ B luciferase reporter activity for 24 hours in K562S cells after treatment with recombinant FAM167A
  • Figure 2e shows the NF- ⁇ B luciferase reporter activity in K562R cells after 24 hours of treatment with anti-FAM167A neutralizing antibody
  • Figure 2f shows NF- ⁇ B luciferase reporter activity in K562R cells after treatment with 50 ⁇ g/ml SN50, 10 ng/ml LPS or both for 24 hours
  • Figure 2g shows dominant-negative NIK (NIK-DN) NF- ⁇ B luciferase reporter activity was shown in K562R cells 24 hours after transfection of a plasmid encoding
  • FIGS. 2h and 2i show immunity against NIK (FIG. 2h) and p100/p52 (FIG. 2i) in K562S and K562R cells. It shows the result of blot analysis.
  • Figure 2j shows the result of electrophoretic mobility shift assay (EMSA) of nuclear extracts isolated from K562S and K562R cells showing supershift with NF- ⁇ B probe and anti-p52 antibody.
  • ESA electrophoretic mobility shift assay
  • 2K shows NF- ⁇ B luciferase reporter activity in K562S cells 24 hours after treatment with 100 ng/ml recombinant FAM167A and transfection of a plasmid encoding NIK-DN.
  • Figure 2l shows the results of immunoblot analysis of NIK in K562R cells after treatment with 2 ⁇ g/ml anti-FAM167A neutralizing antibody or isotype control for 12 hours.
  • Fig. 2m shows the immunoblot analysis results of p100/p52 in K562S and K562R cells after treatment with the indicated concentrations of recombinant FAM167A for 12 hours.
  • GAPDH was used as an internal standard (FIGS. 2h, 2i, 2l and 2m).
  • Data represent 3 (Fig. 2a, 2d-2g and Fig. 2i-2m) or 5 (Fig. 2h) independent experiments (error bars, triplicate (Fig. 2a, Fig. 2d-2g and Fig. 2i-2h) 2m) or s.d.) of five (Fig. 2h) samples.
  • FIG 3a shows the surface FAM167A receptor (FAM167AR) staining results of K562S and K562R cells using Myc-tagged FAM167A and Alexa Fluor 488-conjugated anti-Myc antibodies.
  • Figure 3b shows the experimental design for FAM167A receptor identification.
  • FIG. 3C shows silver staining results of immunoprecipitation samples prepared from K562R cells using Myc-tagged FAM167A and anti-Myc antibody bound to Protein G Sepharose® beads.
  • Figures 3d to 3h show the MS/MS spectrum and the peptide sequences of the indicated bands from Figure 3c.
  • Figure 3i shows the results of co-immunoprecipitation assay between FAM167A and DSG1 in K562R cells.
  • Figure 3j shows the results of qRT-PCR analysis of DSG1 mRNA levels in K562S and K562R cells and K562R cells treated with 1 ⁇ M imatinib for 24 hours.
  • 3K shows immunoblot analysis of DSG1 expression in K562S and K562R cells.
  • Figure 3l shows the results of surface staining of K562S and K562R cells with anti-DSG1 antibody.
  • Figure 3m shows immunofluorescence microscopic analysis of DSG1 expression in K562S and K562R cells. Nuclei were visualized using Hoechst 33342. Scale bar, 10 ⁇ m. GAPDH was used as an internal standard (Fig. 3g). IP: immunoprecipitation. MFI: mean fluorescence intensity. Data represent 3 independent experiments (error bars, s.d. of duplicate (FIG. 3F) or triplicate (FIGS. 3A and 3H) samples) of 3 (FIGS. 3A, 3C and 3E-3I). unpaired two-tailed t-test; **P ⁇ 0.01, ***P ⁇ 0.001. NS: not significant.
  • Figure 3n shows immunofluorescence microscopic analysis of DSG1 and FAM167A in K562R cells. Nuclei were visualized using Hoechst 33342. Scale bar, 10 ⁇ m.
  • FIG. 3O shows the results of NF- ⁇ B luciferase reporter activity in K562S cells 24 hours after transfection of a plasmid encoding FAM167A and treatment with the indicated concentrations of anti-FAM167A neutralizing antibody.
  • Data represent 2 independent experiments (error bars, s.d. of triplicate (Fig. 3n) samples) of 2 (Fig. 3n and Fig. 3o). unpaired two-tailed t-test; *P ⁇ 0.05.
  • Figure 4a shows surface DSG1 staining of K562R cells after DSG1 knockdown using recombinant lentiviruses encoding two different DSG1-specific shRNAs together with anti-DSG1 antibodies.
  • 4B shows NF- ⁇ B luciferase reporter activity in K562R cells following DSG1 knockdown using recombinant lentiviruses encoding two different DSG1-specific shRNAs.
  • Figure 4c shows the results of immunoblot analysis of p100/p52 in K562R cells after DSG1 knockdown.
  • 4D shows NF- ⁇ B luciferase reporter activity in K562R cells 24 hours after transfection of plasmids encoding DSG1 or DSG1 and Erbin with or without treatment with 2 ⁇ g/ml anti-FAM167A neutralizing antibody.
  • Figure 4e shows the results of qRT-PCR analysis of Erbin mRNA in K562S, K562R and K562R cells treated with 1 ⁇ M imatinib for 24 hours.
  • Figure 4f shows the results of immunoblot analysis for Erbin in K562S and K562R cells. Data represent 3 independent experiments (error bars, s.d. of triplicate (Fig. 4e) samples) of 3 (Fig. 4a, Fig. 4e and Fig. 4f). unpaired two-tailed t-test; NS: not significant.
  • Figure 4g shows the results of co-immunoprecipitation analysis of DSG1, Erbin and NIK in K562S and K562R cells.
  • Figure 4h shows the results of co-immunoprecipitation assay of DSG1 and NIK in K562R cells after treatment with 2 ⁇ g/ml of anti-FAM167A neutralizing antibody or isotype control for 3 hours.
  • Figure 4I shows immunoblot analysis of NIK ubiquitination using anti-ubiquitin antibody after NIK immunoprecipitation from K562R cells treated with 2 ⁇ g/ml anti-FAM167A neutralizing antibody or isotype control and 10 ⁇ M MG132 for 3 hours. .
  • Figure 4j A shows the results of co-immunoprecipitation analysis of DSG1, Erbin, NIK, TRAF3, CHIP and c-cbl in K562S and K562R cells
  • B shows the results of K562S cells using anti-DSG1 and anti-TRAF3 antibodies.
  • DSG1, NIK and TRAF3 co-immunoprecipitation assay results are shown
  • C shows the results of DSG1, NIK and CHIP co-immunoprecipitation assay results in K562S cells using anti-DSG1 and anti-CHIP antibodies
  • D is The results of co-immunoprecipitation of DSG1, NIK and c-cbl in K562S cells using anti-DSG1 and anti-c-cbl antibodies are shown.
  • GAPDH was used as an internal standard (Fig. 4j, A). Data represent 3 independent experiments (Fig. 4j A-D).
  • Figure 4K shows a schematic model of the FAM167A-induced non-canonical NF- ⁇ B pathway.
  • GAPDH was used as an internal standard (FIGS. 4C and 4G-4I).
  • Ub ubiquitination.
  • Data are presented in duplicate (Fig. 4c) or triplicate (Fig. 4b, Fig. 4d, Fig. 4g-4i) independent experiments (error bars, Fig. 4b, Fig. 4d, Fig. 4g). and Fig. 4h) s.d.) of the sample. unpaired two-tailed t-test; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • 5A is a schematic diagram showing FAM167A-induced inhibition of the non-canonical NF- ⁇ B pathway.
  • Figure 5b shows the viability of K562R cells transfected with a plasmid encoding NIK-DN after treatment with imatinib or nilotinib (Nilo) for 3 days.
  • Figure 5c shows the results of the viability assay of K562R cells after treatment with 2 ⁇ g/ml of anti-FAM167A neutralizing antibody or isotype control in the presence of imatinib or nilotinib for 3 days.
  • Figure 5D shows the apoptotic population of K562R cells after treatment with 2 ⁇ g/ml anti-FAM167A neutralizing antibody or isotype control in the presence of imatinib or nilotinib for 3 days.
  • Data are representative of 3 (FIGS. 5B-5D) independent experiments (error bars, s.d. of triplicate (FIGS. 5B-5D) samples).
  • Figure 6a is an experimental schedule and schematic diagram showing the mouse model.
  • Figures 6b and 6c show the volume and mice of established K562R tumors in mice treated with vehicle or 10 mg/kg/day imatinib with or without anti-FAM167A neutralizing antibody or isotype control (2 mg/kg/3 days), respectively. represents the weight of
  • Fig. 6d shows photographs of tumors collected from mice in each group on the 10th day of treatment
  • Fig. 6e shows the tumor weight on the 10th day of treatment.
  • 6F shows the results of hematoxylin and eosin (HE) staining, immunohistochemical staining of tumor sections using anti-Ki-67 and anti-NIK antibodies, and TUNEL staining. Scale bar: 100 ⁇ m. Data represent 3 (FIG. 6B-6F) independent experiments (error bars, s.d. of 5 (FIGS. 6B, 6C, 6E and 6F) mice or samples). unpaired two-tailed t-test; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001. NS, not significant.
  • Figure 7 shows the results of qRT-PCR analysis of FAM167A mRNA in KCL22S, KCL22R and KCL22R cells treated with 1 ⁇ M imatinib for 24 hours. Data represent 3 independent experiments (error bars, s.d. of triplicate samples). unpaired two-tailed t-test; **P ⁇ 0.01, ***P ⁇ 0.001.
  • MFI BCR- Relative surface expression of DSG1
  • Figure 8c is a proposed model showing the involvement of FAM167A-induced non-canonical NF- ⁇ B pathway in BCR-ABL-independent TKI resistance. unpaired two-tailed t-test; *P ⁇ 0.05, ***P ⁇ 0.001. NS: not significant.
  • BCR-ABL-independent TKI resistance cannot be treated even with second-generation TKIs, and thus, it is necessary to discover novel biomarkers that can be used for diagnosis and treatment of BCR-ABL-independent TKI-resistant CML.
  • the present invention reveals an important function of FAM167A, a previously uncharacterized protein, in the non-canonical NF- ⁇ B pathway of CML that exhibits BCR-ABL-independent TKI resistance, and neutralization of FAM167A reduces TKI resistance in cultured cells and mouse tumor models.
  • the first aspect of the present invention relates to a biomarker composition for diagnosing BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance in patients with chronic myeloid leukemia, comprising the FAM167A protein or a gene encoding the same will be.
  • TKI BCR-ABL-independent tyrosine kinase inhibitor
  • the FAM167A also known as C8orf13, is a 214 amino acid protein belonging to the FAM167 (SEC) family.
  • the gene encoding FAM167A maps to human chromosome 8, which consists of approximately 146 million base pairs.
  • the FAM167A protein or fragment thereof may include the amino acids of NCBI Reference Sequence: NP_444509.2, but may include any amino acid sequence having substantially the same or equivalent efficacy as the protein without limitation.
  • the FAM167A protein may also include an isoform or a precursor thereof.
  • the amino acid sequence of a representative FAM167A protein is shown in SEQ ID NO: 1.
  • the mRNA of the gene encoding the FAM167A protein may include the nucleotide sequence of NCBI Reference Sequence: NM_053279.3, but may include without limitation any nucleotide sequence exhibiting substantially the same or equivalent efficacy as the gene.
  • the mRNA sequence of a gene encoding a representative FAM167A protein is shown in SEQ ID NO: 2.
  • biomarker protein The specific amino acid sequence and nucleotide sequence of the biomarker protein and the gene encoding it can be found in a known database such as NCBI.
  • biomarker refers to a molecule quantitatively or qualitatively associated with the presence of a biological phenomenon
  • the biomarker of the present invention is a protein that is a standard for determining BCR-ABL-independent TKI resistance or encoding the same. refers to genes.
  • a biomarker can be derived from a genomic nucleotide sequence or from an expressed nucleotide sequence (eg, from RNA, nRNA, mRNA, cDNA, etc.), or from an encoded polypeptide.
  • the term includes nucleic acid sequences complementary to or flanking a biomarker sequence, such as nucleic acids used as probes or primer pairs capable of amplifying the biomarker sequence.
  • the "expression” means that a protein or nucleic acid is produced.
  • Protein is used interchangeably with “polypeptide” or “peptide” and refers to a polymer of amino acid residues, eg, as commonly found in proteins in nature.
  • Polynucleotide or “nucleic acid” refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single or double stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are included.
  • mRNA refers to RNA that transmits genetic information (gene-specific base sequence) to ribosomes that specify amino acid sequences from specific genes during protein synthesis.
  • the present invention is for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myelogenous leukemia, including an agent for measuring the expression level of FAM167A protein, a fragment thereof, or an mRNA encoding the protein.
  • Compositions and kits containing them are provided.
  • the term "diagnosis” refers to determining a subject's susceptibility to a specific disease or disorder, determining whether a subject currently has a specific disease or disorder, or determining a specific disease or disorder determining the prognosis of an individual suffering from the disease, or therametrics (eg, monitoring the condition of an individual to provide information about the efficacy of a treatment).
  • the term “resistance” is also referred to as “resistance”, and means that the efficacy of the drug does not work because it does not react sensitively to the corresponding drug.
  • an agent for measuring the mRNA expression level of a FAM167A protein, a fragment thereof, or a gene encoding the protein is a biomarker protein of the present invention, a fragment thereof, or an mRNA expression level of a gene encoding the protein.
  • the fragment of the FAM167A protein may be an immunogenic fragment, preferably a fragment having at least one epitope that can be recognized by an antibody against the protein.
  • an antibody is a term known in the art and refers to a specific protein molecule directed against an antigenic site.
  • an antibody refers to an antibody that specifically binds to the aforementioned biomarker, and such an antibody is cloned into an expression vector according to a conventional method to obtain a protein encoded by the biomarker gene. , It can be prepared from the obtained protein by a conventional method.
  • This also includes partial peptides that can be made from the protein, and the partial peptides of the present invention include at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids.
  • the form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or any antibody having antigen-binding properties is also included in the antibody of the present invention, and all immunoglobulin antibodies are included.
  • the antibodies of the present invention include special antibodies such as humanized antibodies.
  • Antibodies to the proteins encoded by the biomarker genes of the present invention may be any antibodies that can be produced by methods known in the art.
  • an antibody used for detection of the aforementioned biomarkers may include a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains.
  • the functional fragment of the antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2, Fv, etc., but is not particularly limited thereto.
  • the term "aptamer” refers to a single-stranded oligonucleotide, which has a function similar to that of an antibody and is also called a chemical antibody, and refers to a nucleic acid molecule having binding activity to a predetermined target molecule.
  • the aptamer may have various three-dimensional structures depending on its base sequence, and may have high affinity for a specific substance, such as an antigen-antibody reaction.
  • An aptamer can inhibit the activity of a given target molecule by binding to the given target molecule.
  • the aptamer of the present invention may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be linear or ring in shape, but is not limited thereto.
  • An aptamer having binding activity to each of the three biomarker proteins can be easily prepared by a person skilled in the art according to a known method by referring to each nucleotide sequence.
  • the term "primer” is a nucleic acid sequence having a short free 3' terminal hydroxyl group, capable of forming a base pair with a complementary template, and used for copying the template.
  • PCR amplification is performed using the above-described sense and antisense primers of the biomarker polynucleotide, and BCR-ABL-independent TKI resistance in CML patients can be diagnosed through whether a desired product is produced. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.
  • the term "probe” refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundreds of bases as short as possible to achieve specific binding with mRNA, and is labeled The presence or absence of a specific mRNA can be confirmed.
  • the probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like.
  • hybridization is performed using the aforementioned biomarker polynucleotide and a complementary probe, and BCR-ABL-independent TKI resistance in chronic myelogenous leukemia patients can be diagnosed through hybridization. Selection of suitable probes and hybridization conditions can be modified based on those known in the art.
  • Primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods.
  • Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more natural nucleotides with homologues, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoro amidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).
  • nucleotide sequence of the agent for measuring the expression level of the biomarker gene used in the present invention shows substantial identity with the sequence specifically binding to the biomarker gene, considering mutations with biologically equivalent activity. It is to be construed as to include sequences as well.
  • substantially identity refers to at least 60% identity when a specific sequence and any other sequence are aligned so as to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art. , more specifically a sequence exhibiting 70% identity, still more specifically 80% identity, and most specifically 90% identity.
  • kits of the present invention include RT-PCR kit, competitive RT-PCR kit, real-time RT-PCR kit, DNA chip kit, microarray kit, SAGE (Serial Analysis of Gene Expression) kit, gene chip kit, ELISA (Enzyme linked immunosorbent assay) ) kit, protein chip kit, rapid kit, or multiple reaction monitoring (MRM) kit, but is not limited thereto.
  • the kit may be a kit including essential elements required to perform RT-PCR.
  • an RT-PCR kit may contain, in addition to each primer specific for a biomarker gene, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like.
  • dNTPs deoxynucleotides
  • ddNTPs dideoxynucleotides
  • enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like.
  • a primer pair specific for DNA or RNA used as a quantitative control may be included.
  • the kit of the present invention may include a kit for extracting nucleic acid (eg, total RNA), a fluorescent material for labeling, an enzyme and medium for amplifying nucleic acid, instructions for use, and the like.
  • nucleic acid eg, total RNA
  • fluorescent material for labeling
  • enzyme and medium for amplifying nucleic acid
  • the kit of the present invention is a device for measuring a biomarker for pancreatic cancer in which the nucleic acid is bound or attached to, for example, a solid phase.
  • a solid phase examples of the material of the solid phase are plastic, paper, glass, silicon, and the like, and a material of the solid phase preferable from the viewpoint of ease of processing is plastic.
  • the shape of the solid phase is arbitrary, and is, for example, square, circular, rectangular, or film-like.
  • the kit may further include a program capable of data analysis or statistical processing.
  • the program may analyze the expression level of a gene or protein.
  • the data analysis or statistical processing program may be one or more selected from Skyline Software, ProteoWizard Software, SCIEX OS Software, SPSS Statistics Software, MedCalc Software, MultiQuant Software, MasterView Software, or Cliquid Software, but is not limited thereto.
  • the present invention also, in preparing a kit for diagnosing BCR-ABL independent TKI resistance in CML patients, the expression level of FAM167A protein, fragment thereof, or mRNA of a gene encoding the protein It relates to the use of the agent to be measured.
  • FAM167A a previously uncharacterized protein that is up-regulated in BCR-ABL-independent TKI-resistant cells
  • clinical sample analysis results showed that FAM167A was found to increase the BCR-ABL ratio not only after TKI treatment but also at the time of CML diagnosis.
  • the biomarker protein can be used to diagnose BCR-ABL-independent TKI resistance in CML patients.
  • the second aspect of the present invention relates to a method for providing information for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myelogenous leukemia using the FAM167A biomarker.
  • the term "method for providing information” refers to a method for providing information on BCR-ABL-independent TKI resistance in CML patients, including a biomarker protein according to the present invention, and a fragment thereof. Or, when the mRNA expression level of the gene encoding the protein is increased compared to CML without BCR-ABL independent TKI resistance, or otherwise TKI-sensitive CML, it means a method of obtaining information on BCR-ABL independent TKI resistance. do.
  • the method for providing information for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myelogenous leukemia may include the following steps:
  • step (b) the expression level measured in step (a), the same protein measured in a biological sample isolated from a chronic myelogenous leukemia patient without BCR-ABL-independent TKI resistance, a fragment thereof, or mRNA of a gene encoding the protein Comparing with the expression level.
  • the information providing method of the present invention (c) the mRNA expression level of the FAM167A protein, fragment thereof, or gene encoding the protein measured in a biological sample isolated from the chronic myelogenous leukemia patient in step (a), When higher than the mRNA expression level of the same protein, a fragment thereof, or a gene encoding the protein measured in a biological sample isolated from a chronic myeloid leukemia patient without BCR-ABL-independent tyrosine kinase inhibitor resistance at step BCR-ABL-independent It may further include the step of determining that it exhibits resistance to TKI.
  • the chronic myelogenous leukemia patient is a subject for whom resistance to BCR-ABL-independent TKIs is to be diagnosed, and the subject of the patient is a human, primates including chimpanzees, pets such as dogs and cats, and cattle. , It is interpreted as meaning including mammals such as livestock animals such as horses, sheep, and goats, and rodents such as mice and rats.
  • the "biological sample” used for analysis is a CML patient without BCR-ABL independent TKI resistance, or a BCR-ABL independent TKI resistance that can be distinguished from a TKI-sensitive CML patient.
  • a biomarker protein specific for diagnosis, a fragment thereof, or a biological sample capable of confirming the mRNA level of a gene encoding the protein is included.
  • the biological sample may refer to a biological sample containing CD34 + stem/progenitor cells isolated from a CML patient to determine BCR-ABL-independent TKI resistance, for example, blood or plasma.
  • lymph fluid airway fluid, serous fluid, genitourinary tract fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, intratracheal fluid, ascites, cystic tumor fluid , amniotic fluid, or combinations thereof, but is not limited thereto.
  • the expression level of the biomarker can be measured at the mRNA expression level of the FAM167A protein, a fragment thereof, or a gene encoding the protein, and separation of protein or mRNA from a biological sample is a known process. This can be done using
  • protein level measurement is a process of confirming the presence and expression level of the biomarker protein or fragment thereof in a sample, using an antibody or the like that specifically binds to the protein or fragment thereof. You can check the amount of protein by doing this.
  • Analysis methods for this include western blotting, ELISA (enzyme linked immunosorbentassay), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunization Rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, mass spectrometry ( mass spectrometry, fluorescence activated cell sorter analysis (FACS), or protein chip technology, but is not limited thereto.
  • the term "measurement of mRNA level” is a process of confirming the presence and expression of mRNA of the biomarker gene of the present invention in a sample, which can be determined by measuring the amount of mRNA. Analysis methods for this include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection method, and northern blotting , Southern blotting, in situ hybridization, or DNA chip technology, but is not limited thereto.
  • the mRNA expression level of the biomarker gene can be measured by a hybridization method or a gene amplification method.
  • the hybridization method may be to confirm the presence of a gene using a probe.
  • the above probe is used as a hybridizable array element and is immobilized on a substrate.
  • Preferred substrates include rigid or semi-rigid supports such as membranes, filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the hybridization array elements are arranged and immobilized on the substrate. This immobilization is carried out by chemical bonding methods or covalent bonding methods such as UV.
  • the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bonded to a polylysine-coated surface by UV.
  • the hybridization array element may be coupled to a substrate through a linker (eg, ethylene glycol oligomer and diamine).
  • sample DNA applied to the microarray of the present invention can be labeled and hybridized with array elements on the microarray.
  • Hybridization conditions can be variously changed.
  • the degree of hybridization may be detected and analyzed in various ways depending on the labeling substance.
  • the label of the probe may provide a signal allowing detection of hybridization, which may be linked to an oligonucleotide.
  • Suitable labels include fluorophores (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), terminal deoxynucleotidyl transferase (TdT), chromophores, chemiluminescent provided, however, that magnetic particles, radioactive isotopes (P32 or S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (e.g., gold), and Antibodies, streptavidin, biotin, digoxigenin and haptens with specific binding partners such as chelating groups, but are not limited thereto.
  • Labeling is performed by various methods commonly practiced in the art, such as the nick translation method, the random priming method (Multiprime DNA labeling systems booklet, “Amersham” (1989)), and the kination method (Maxam & Gilbert, Methods in Enzymology, 65:499 (1986)).
  • the label provides a signal that can be detected by fluorescence, radioactivity, chromometry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization radiofrequency, nanocrystals.
  • the probe When using a probe, the probe is hybridized with the cDNA molecule.
  • suitable hybridization conditions can be determined in a series of steps by an optimization procedure. This procedure is carried out as a series of procedures by those skilled in the art to establish a protocol for use in a laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence.
  • Hybridization signals can be performed in various ways depending on the type of label bound to the probe, for example.
  • a probe is labeled by an enzyme
  • hybridization can be confirmed by reacting a substrate of the enzyme with a hybridization reaction product.
  • Enzyme/substrate combinations that can be used include peroxidases (e.g., horseradish peroxidase) with chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate).
  • a third aspect of the present invention is a FAM167A protein inhibitor; an inhibitor of expression of the gene encoding the FAM167A protein; Or a pharmaceutical composition for preventing or treating CML exhibiting BCR-ABL-independent TKI resistance, containing a mixture thereof as an active ingredient, and a pharmaceutical composition for suppressing BCR-ABL-independent TKI resistance in CML patients.
  • the FAM167A protein inhibitor may include an agent that reduces the expression or function or activity of the FAM167A protein.
  • the expression inhibitor of the gene encoding the FAM167A protein may include an agent that reduces mRNA expression of the FAM167A gene.
  • the FAM167A protein inhibitor may be a peptide or compound that reduces non-standard NF- ⁇ B activity by binding to the FAM167 protein. These inhibitors may be selected through the screening methods exemplified below, such as protein structure analysis, and may be designed using methods known in the art.
  • the FAM167A protein inhibitor may include at least one selected from the group consisting of small molecule compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the FAM167A protein, Not limited to this.
  • the expression inhibitor of the gene encoding the FAM167A protein may be an inhibitor that binds to the gene itself and interferes with transcription or binds to mRNA transcribed from the gene and interferes with translation of the mRNA.
  • the expression inhibitor of the gene may include at least one selected from the group consisting of antisense nucleotides, siRNA, and shRNA that complementarily bind to mRNA of the FAM167A gene, but is not limited thereto.
  • RNA refers to a double-stranded RNA that induces RNA interference through cleavage of mRNA of a target gene, and an RNA strand of a sense sequence having the same sequence as the mRNA of a target gene and a sequence complementary thereto It consists of an RNA strand of an antisense sequence with
  • the siRNA may include siRNA itself synthesized in vitro or a form expressed by inserting a nucleotide sequence encoding the siRNA into an expression vector.
  • the "vector” refers to a gene construct containing an external DNA inserted into a genome encoding a polypeptide.
  • Vectors related to the present invention are vectors in which a nucleic acid sequence that inhibits the gene is inserted into the genome, and examples of these vectors include DNA vectors, plasmid vectors, cosmid vectors, bacteriophage vectors, yeast vectors, or viral vectors.
  • the antisense has a sequence complementary to all or part of the mRNA sequence transcribed from the FAM167A gene or fragment thereof, and binds to the mRNA to suppress the expression of the FAM167A gene or fragment.
  • shRNA short hairpin RNA
  • shRNA short hairpin RNA
  • chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance may have increased NF- ⁇ B activity compared to CML exhibiting TKI sensitivity.
  • the pharmaceutical composition may be administered in combination with a TKI.
  • the term "concomitant administration” means that different components are administered to a subject together.
  • Administration of different components together means that each component can be administered at the same time or in any order or sequentially at different times to obtain the desired therapeutic effect.
  • composition of the present invention may be administered simultaneously with the TKI, separately or sequentially.
  • the TKI is crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib ( imatinib), nilotinib, ponatinib, ibrutinib, cabozantinib, gefitinib, erlotinib, lapatinib, bande vandetanib, afatinib, osimertinib, ruxolitinib, tofacitinib, axitinib, lenvatinib, nintedanib ( nintedanib), pazopanib, regorafenib, sorafenib, or sunitinib, but is not limited thereto.
  • prevention and/or “treatment” refers to all actions to inhibit or delay the onset of a disease or condition, all actions to ameliorate or beneficially change the state of a disease or condition, and delay the progression of a disease or condition , means any action that interrupts or reverses
  • the pharmaceutical composition of the present invention is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent, diluent, dispersing agent, and interface commonly used in the preparation of pharmaceutical compositions. It may further include one or more additives selected from the group consisting of activators, binders and lubricants.
  • the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Quality cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil may be used.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used.
  • Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. there is.
  • the carrier included in the pharmaceutical composition of the present invention is an ion exchange resin, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances (eg, various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (eg protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicates, polyvinylpyrrolidone, cellulosic matrices, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols, or wool, and the like, but are not limited thereto.
  • serum proteins eg, human serum albumin
  • buffer substances eg, various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories, and the like.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions.
  • As a base material of the suppository witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.
  • the pharmaceutical composition according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline may be used.
  • Aqueous injection suspensions may contain substances which may increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.
  • composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, or a preservative in addition to the above components.
  • the pharmaceutical composition of the present invention may be administered systemically or topically, and may be formulated into a dosage form suitable for such administration by known techniques.
  • it may be administered by mixing with an inert diluent or an edible carrier, sealing in a hard or soft gelatin capsule, or pressing into a tablet.
  • the active compound may be mixed with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.
  • the effective amount of the active ingredient of the pharmaceutical composition of the present invention means the amount required to achieve the effect of preventing, suppressing or alleviating a disease. Therefore, the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health condition, sex and diet, administration time, administration route and composition It can be controlled by various factors including secretion rate, duration of treatment, and drugs used concurrently.
  • the present invention comprises administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of a gene encoding the FAM167A protein, or a mixture thereof to a CML patient exhibiting BCR-ABL-independent TKI resistance.
  • a method for treating CML showing BCR-ABL-independent TKI resistance and a method for suppressing BCR-ABL-independent TKI resistance are provided.
  • the FAM167A protein inhibitor used in the method or the expression inhibitor of the gene encoding the FAM167A protein and the method of administration thereof are as described above, the description thereof is omitted to avoid excessive complexity in the present specification.
  • the present invention also provides a method for diagnosing BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients and treating chronic myelogenous leukemia exhibiting BCR-ABL-independent TKI resistance, comprising the following steps: to provide:
  • the expression level measured in step (a) is the same protein measured in a biological sample isolated from a chronic myelogenous leukemia patient without BCR-ABL-independent TKI resistance, a fragment thereof, or an mRNA of a gene encoding the protein If the expression level is higher than that, diagnosing as showing BCR-ABL independent TKI resistance; and
  • step (c) administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of a gene encoding the FAM167A protein, or a mixture thereof to a patient diagnosed as exhibiting BCR-ABL-independent TKI resistance in step (b).
  • the present invention also provides a method for diagnosing BCR-ABL-independent TKI resistance and inhibiting BCR-ABL-independent TKI resistance in a chronic myelogenous leukemia patient, comprising the following steps:
  • the expression level measured in step (a) is the same protein measured in a biological sample isolated from a chronic myelogenous leukemia patient without BCR-ABL-independent TKI resistance, a fragment thereof, or an mRNA of a gene encoding the protein If the expression level is higher than that, diagnosing as showing BCR-ABL independent TKI resistance; and
  • step (c) administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of a gene encoding the FAM167A protein, or a mixture thereof to a patient diagnosed as exhibiting BCR-ABL-independent TKI resistance in step (b).
  • the FAM167A protein inhibitor used in the method or the expression inhibitor of the gene encoding the FAM167A protein and the method of administration thereof are the same as those described above, so the description thereof is omitted to avoid excessive complexity in the present specification.
  • the FAM167A protein inhibitor, the expression inhibitor of the gene encoding the FAM167A protein, or a mixture thereof may be administered in combination with a TKI, and the combination administration and the type of TKI are the same as those described in the pharmaceutical composition. In order to avoid excessive complexity of the specification, its description is omitted.
  • the present invention also provides a medicament for preventing or treating CML exhibiting BCR-ABL-independent TKI resistance, or a medicament for inhibiting BCR-ABL-independent TKI resistance in CML patients.
  • a medicament for preventing or treating CML exhibiting BCR-ABL-independent TKI resistance or a medicament for inhibiting BCR-ABL-independent TKI resistance in CML patients.
  • an inhibitor of expression of the gene encoding the FAM167A protein or mixtures thereof.
  • the present invention also provides, in the preparation of a medicament for preventing or treating chronic myelogenous leukemia exhibiting BCR-ABL-independent TKI resistance, a FAM167A protein inhibitor; an inhibitor of expression of the gene encoding the FAM167A protein; or the use of mixtures thereof.
  • the present invention also provides, in the preparation of a medicament for inhibiting BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients exhibiting BCR-ABL-independent TKI resistance, a FAM167A protein inhibitor; an inhibitor of expression of the gene encoding the FAM167A protein; or the use of mixtures thereof.
  • a fourth aspect of the present invention relates to a method for screening a CML therapeutic agent or BCR-ABL independent TKI resistance inhibitor exhibiting BCR-ABL-independent TKI resistance using the FAM167A biomarker.
  • the screening method of the present invention may include the following steps:
  • the candidate substance is presumed to have potential as a substance that inhibits transcription or translation from the FAM167A gene sequence to mRNA or protein, or as a drug that inhibits the function or activity of the FAM167A protein according to a conventional selection method, or or randomly selected individual nucleic acids, proteins, peptides, other extracts, natural products, compounds, etc.
  • the expression level, protein level or activity of the gene can be measured in a biological sample isolated from the cell line or animal model treated with the candidate substance, and as a result of the measurement, the expression level, protein level or activity of the gene is reduced. If it is determined, the candidate substance can be determined as a therapeutic agent for CML exhibiting BCR-ABL-independent TKI resistance and a BCR-ABL-independent TKI resistance inhibitor.
  • the screening method of the present invention (c) when the candidate substance inhibits the FAM167A protein or inhibits the expression of a gene encoding the protein, the candidate substance is a CML therapeutic agent or a CML showing BCR-ABL-independent TKI resistance.
  • a step of determining the BCR-ABL-independent TKI resistance inhibitor may be further included.
  • the method for measuring the expression level, protein level or activity of the gene above can be performed through various methods known in the art, and is the same as described in the information providing method, so excessive Its description is omitted to avoid complexity.
  • the candidate substance acts as a leading compound in the development process of a CML therapeutic agent showing BCR-ABL-independent TKI resistance and a BCR-ABL-independent TKI resistance inhibitor in the future, and the leading substance is By modifying and optimizing the structure to exhibit the effect of inhibiting the function of the FAM167A gene or the protein expressed therefrom, it is possible to develop new therapeutic agents for CML and inhibitors of TKI resistance.
  • the human CML cell line K562S was obtained from Korea Cell Line Bank.
  • KCL22S was obtained from the American Type Culture Collection (ATCC).
  • the K562R cell line was generated by treating cells with progressively increasing concentrations of TKI from K562S. All cell lines were maintained at 37 °C and 5% CO 2 in a humidified cell culture incubator, RPMI (Roswell Park Memorial Institute ) cultured in 1640 medium (HyClone). To maintain TKI resistance, K562R and KCL22R cells were cultured in medium supplemented with 1 ⁇ M imatinib.
  • TKI resistance without mutation of the BCR-ABL kinase domain ie, BCR-ABL independent TKI resistance
  • the present inventors sought to find genes that play a potential role in BCR-ABL-independent TKI resistance by identifying genes that are differentially expressed in TKI-resistant CML cells (FIG. 1a). Therefore, a TKI-resistant CML cell line, K562R (Figs. 1b to 1e), having no mutation in the BCR-ABL kinase domain, was generated as in Example 1-1, and then treated with TKI-sensitive K562S cells, K562R cells, and imatinib. Gene expression was analyzed in the cells, and both microarray and RNA sequencing (RNA-seq) analyzes were performed to eliminate false positives due to the assay method.
  • RNA-seq RNA sequencing
  • RNA-seq analysis methods are as follows. Total RNA was extracted with TRI reagent (Molecular Research Center). For microarrays, synthesis of cDNA and biotinylated cRNA was performed using the Illumina TotalPrep RNA Amplification Kit (Ambion). The labeled cRNA was hybridized to Human HT-12 v4 Expression BeadChip (Illumina) and the array was scanned with a Bead Array Reader confocal scanner (Illumina). For RNA-seq, the library was prepared using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) according to the Illumina TruSeq protocol. Paired-end sequencing was performed using the NovaSeq 6000 system (Illumina). Genes exhibiting a fold change ⁇ 2 are defined as being differentially expressed and displayed in the heatmap generated in the Multiple Experiment Viewer.
  • Enrichment score (proportion of transcription factors associated with differentially expressed genes)/(proportion of transcription factors associated with randomly selected genes) x (number of differentially expressed genes associated with transcription factors).
  • Regulators of differentially expressed genes compared to randomly selected genes through in silico analysis of transcription factors involved in differential gene expression of BCR-ABL-independent TKI-resistant CML cells It was found to be the most abundant transcription factor among (Figs. 1h to 1m). AP-1 and NF- ⁇ B showed significantly higher enrichment scores than other transcription factors, and scores for only these two transcription factors were 3 standard deviations away from the mean.
  • AP-1 and NF- ⁇ B reporter assays were performed on K562R cells, K562S cells, and imatinib-treated K562R cells to determine the effect of TKI treatment. investigated.
  • K562S and K562R cells were prepared using Lipofectamine 2000 (Invitrogen) as described in [Park SG, Schulze-Luehrman J, Hayden MS, Hashimoto N, Ogawa W, Kasuga M, et al.
  • the kinase PDK1 integrates T cell antigen receptor and CD28 coreceptor signaling to induce NF-kappaB and activate T cells. Nat Immunol. 2009;10(2):158-66.] Renilla luciferase vector and NF- ⁇ B dependent reporter construct (pBIIx-luc) described or [Zhang D, Zhang G, Hayden MS, Greenblatt MB , Bussey C, Flavell RA, et al.
  • NF- ⁇ B activity was significantly higher in K562R cells than in K562S cells. In addition, this activity was much higher in K562R cells treated with imatinib, but there was no significant difference in AP-1 activity (Fig. 1n). This suggests that NF- ⁇ B contributes to BCR-ABL-independent TKI resistance by regulating gene expression.
  • gene expression profile data of K562S, K562R, and K562R cells treated with imatinib were used to identify 7 genes that showed similar expression patterns to the pattern NF- ⁇ B activity. Differentially expressed genes (FAM167A, AIF1L, ACSS1, S100A4, LY6G6D, CYP11A1 and NR5A1) were selected.
  • qRT-PCR was performed to confirm the expression level of the selected genes.
  • Total cellular RNA was isolated using RNeasy Mini Kit (Qiagen), and cDNA was prepared with TOPscript RT Drymix (Enzynomics).
  • qRT-PCR was performed using SYBR Green qPCR 2x Premix (Enzynomics) on a Stratagene Mx3000P (Agilent Technologies). Results were normalized to GAPDH levels. Primer sequences used for qRT-PCR are shown in Table 1, and qRT-PCR results are shown in FIG. 1o.
  • NF- ⁇ B activity was confirmed by a luciferase reporter assay in K562S cells transfected with plasmids encoding the indicated genes.
  • the plasmid was prepared by cloning the HA tags S100A4, CYP11A1, ACSS1, LY6G6D, NR5A1, AIF1L and FAM167A into the MigR1 vector.
  • FAM167A significantly increased NF- ⁇ B activity when ectopically expressed (FIG. 2A).
  • FAM167A ectopic expression of FAM167A increased the viability of K562S cells in the presence of imatinib (FIG. 2B).
  • FAM167A Secretion of FAM167A was analyzed by immunoblot analysis of K562R cell lysates and culture supernatants.
  • Cell lysates were extracted by boiling the cells in SDS sample buffer. Proteins in the culture supernatant were concentrated by precipitation with acetone and then extracted by boiling in SDS sample buffer.
  • proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on an 8-15% gel and then transferred to a polyvinylidene difluoride membrane.
  • SDS sodium dodecyl sulfate
  • Membranes were probed with anti-FAM167A antibody (sc-393999, Santa Cruz Biotechnology) or Erbin antibody (NBP2-13968, Novus Biologicals) as indicated.
  • anti-FAM167A antibody sc-393999, Santa Cruz Biotechnology
  • NBP2-13968 Novus Biologicals
  • FAM167A protein MMS1363522, MyBioSource
  • FAM167A-specific neutralizing antibody sc-393999, Santa Cruz Biotechnology
  • NF- ⁇ B pathway is activated in K562R cells.
  • SN50 (481480, Sigma-Aldrich) which inhibits the canonical NF- ⁇ B pathway, or transiently expressed NIK-DN, which inhibits the non-canonical NF- ⁇ B pathway.
  • NIK-DN transiently expressed NF- ⁇ B pathway.
  • the literature [Park SG, Ryu HM, Lim SO, Kim YI, Hwang SB, Jung G. Interferon-gamma inhibits hepatitis B virus-induced NF-kappaB activation through nuclear localization of NF-kappaB- inducing kinases. Gastroenterology. 2005;128(7):2042-53.] was used.
  • SN50 treatment which inhibits the canonical NF- ⁇ B pathway, did not significantly reduce NF- ⁇ B activity in K562R cells, but SN50 treatment blocked further lipopolysaccharide (LPS)-stimulated increases in NF- ⁇ B activity (Fig. 2f).
  • LPS lipopolysaccharide
  • NIK-DN dominant-negative NIK
  • Non-canonical NF- ⁇ B activation relies on NIK accumulation through blocking of ubiquitination-dependent NIK degradation, and accumulated NIK induces processing of p100 to p52, followed by nuclear translocation of p52-containing dimers. Therefore, through immunoblot analysis for NIK and p100/p52 in K562S and K562R cells, activation of the non-canonical NF- ⁇ B pathway was confirmed in K562R cells.
  • Membranes were probed with anti-NIK (4994, Cell Signaling Technology), anti-p100/p52 ((sc-7386 X, Santa Cruz Biotechnology) and GAPDH (sc-166574, Santa Cruz Biotechnology) antibodies as indicated.
  • GAPDH Densitometry was performed using ImageJ software using as an internal standard.
  • the nuclear fraction was extracted as follows. First, cells were suspended in hypotonic buffer (10 mM HEPES, pH7.9; 10 mM KCl; 1.5 mM MgCl2; 0.5 mM DTT; 0.5 mM PMSF) and incubated on ice for 15 minutes. Then, 0.05% Nonidet P-40 was added and the lysate was passed through a 25-gauge needle 5 times. After centrifugation (1000 ⁇ g, 5 min, 4° C.) to precipitate nuclei, the supernatant was collected and separated by high-speed centrifugation (16,000 ⁇ g, 5 min, 4° C.). The resulting supernatant was stored at -80 °C as a cytoplasmic fraction.
  • hypotonic buffer 10 mM HEPES, pH7.9; 10 mM KCl; 1.5 mM MgCl2; 0.5 mM DTT; 0.5 mM PMSF
  • the pellet containing cell nuclei was washed with a hypotonic buffer and washed in hypertonic buffer (20 mM HEPES, pH7.9; 420 mM NaCl; 1.5 mM MgCl 2 ; 25% glycerol; 0.2 mM EDTA; 0.5 mM DTT; 0.5 mM PMSF; 1 ⁇ g/ml mL leupeptin, 1 ⁇ g/mL aprotinin, 1 ⁇ g/mL pepstatin A), and then incubated on ice for 30 minutes with vortexing every 10 minutes. After the nuclear lysate was centrifuged (16,000 g, 5 min, 4 °C), the resulting supernatant was stored at -80 °C as a nuclear fraction.
  • hypertonic buffer 20 mM HEPES, pH7.9; 420 mM NaCl; 1.5 mM MgCl 2 ; 25% glycerol; 0.2 mM ED
  • nuclear fractions were lysed in binding buffer (5 mM Tris, pH7.5; 25 mM KCl; 0.5 mM EDTA; 2.5% glycerol; 0.5 mM DTT; 0.1 ⁇ g/ ⁇ l poly(dI/dC), 0.5 mg/ml BSA). was incubated for 20 minutes at room temperature with an anti-p52 antibody (4882, Cell Signaling Technology) or an isotype control (H9658, Sigma-Aldrich), followed by biotinylated double-stranded NF- Further incubation was performed with the ⁇ B probe (SEQ ID NO: 23: 5'-AGTTGAGGGGACTTTCCCAGG-3').
  • NF- ⁇ B luciferase reporter activity was confirmed in K562S cells 24 hours after treatment with 100 ng/ml recombinant FAM167A and transfection of a plasmid encoding NIK-DN.
  • NF stimulated by recombinant FAM167A - ⁇ B activity was reduced in cells with transient expression of NIK-DN (Fig. 2k).
  • the data indicate that FAM167A activates the non-canonical NF- ⁇ B pathway to a greater extent in K562R cells than in K562S cells.
  • FAM167A As treatment and neutralization of secreted FAM167A effectively regulates the non-canonical NF- ⁇ B pathway, it was hypothesized that the receptor for FAM167A could be expressed on the cell surface.
  • K562S and K562R cells were first labeled with Myc-tagged recombinant FAM167A, followed by fluorescent dye-conjugated anti-Myc antibody staining and flow cytometry.
  • Flow cytometry and cell sorting were performed using Guava EasyCyte HT (Millipore), FACSCanto II (BD Biosciences) or FACSAria III (BD Biosciences), and data were analyzed with FlowJo software (TreeStar).
  • the receptor was immunoprecipitated in K562R cells using Myc-tagged recombinant FAM167A coupled to protein G Sepharose® beads and an anti-Myc antibody to isolate the receptor for identification (FIG. 3B). ).
  • Cells were lysed in lysis buffer (20 mM Tris-HCl, pH8.0; 150 mM NaCl; 1% Triton X-100; 10% glycerol; 2 mM EDTA; 1 ⁇ g/ml Leupeptin; 1 ⁇ g/ml; Aprotinin; 1 ⁇ g/ml Pepstatin A). ; 0.1 mM PMSF) on ice for 30 min. Then, the lysate was centrifuged for 15 minutes at 15,000 xg and 4 °C, and the supernatant was incubated at 4 °C with rotation with anti-FAM167A (sc-393999, Santa Cruz Biotechnology).
  • lysis buffer 20 mM Tris-HCl, pH8.0; 150 mM NaCl; 1% Triton X-100; 10% glycerol; 2 mM EDTA; 1 ⁇ g/ml Leupeptin; 1 ⁇ g/ml; Aprotinin; 1 ⁇
  • Protein G Sepharose® beads (GE Healthcare) were added and incubated while rotating at 4 °C. After washing 4 times with lysis buffer, the immunoprecipitate was eluted by boiling in SDS sample buffer and subjected to immunoblot analysis. Band intensities were analyzed with ImageJ software.
  • DSG1 mRNA and protein were measured by qRT-PCR and immunoblot analysis, respectively.
  • the sequences of the primers used for qRT-PCR are shown in Table 2.
  • NF- ⁇ B luciferase reporter activity was assayed in K562S cells 24 hours after transfection of a plasmid encoding FAM167A and treatment with the indicated concentrations of anti-FAM167A neutralizing antibody.
  • DSG1-specific shRNA short hairpin RNA (shRNA) constructs were transduced with lentivirus into K562R cells to knock down the expression of DSG1 (Fig. 4a), followed by NF- ⁇ B luciferase reporter assay and p100/ Immunoblot analysis for p52 was performed.
  • DSG1-specific shRNA lentiviral particles (sc-35224-V) and control shRNA lentiviral particles (sc-108080) were obtained from Santa Cruz Biotechnology and used.
  • DSG1 knockdown increased NF- ⁇ B activity and processing of p100 to p52 in K562R cells ( FIGS. 4b and 4c ).
  • NF- ⁇ B activity was measured after transient expression of DSG1 and its interacting protein Erbin in K562R cells treated with or without anti-FAM167A neutralizing antibody.
  • Transient expression of DSG1 was performed using a plasmid cloned with DSG into MigR1 vector, and transient expression of Erbin was performed using pClneo-Myc-Erbin (Addgene).
  • NF- ⁇ B activity was reduced by transient expression of DSG1 and was further reduced by transient expression of both DSG1 and Erbin (FIG. 4D).
  • transient expression of DSG1 and Erbin in the presence of FAM167A-neutralizing antibody caused little further reduction of NF- ⁇ B activity (not significant, p > 0.05) ( FIG. 4D ).
  • NIK is a key component of the non-canonical NF- ⁇ B pathway
  • a co-immunoprecipitation assay was performed to investigate the interaction between DSG1, Erbin and NIK.
  • lysis buffer (20 mM Tris-HCl, pH8.0; 150 mM NaCl; 1% Triton X-100; 10% glycerol; 2 mM EDTA; 1 ⁇ g/ml leupeptin; 1 ⁇ g/ml; aprotinin; 1 ⁇ g/ml peptide).
  • Statin A 0.1 mM PMSF
  • Lysates were then centrifuged for 15 minutes at 15,000 xg and 4°C, and supernatants were incubated at 4°C with rotation with anti-DSG1 antibody (32-6000, Invitrogen).
  • Protein G Sepharose® beads (GE Healthcare) were added and incubated while rotating at 4 °C. After washing 4 times with lysis buffer, the immunoprecipitate was eluted by boiling in SDS sample buffer and subjected to immunoblot analysis. Band intensities were analyzed with ImageJ software.
  • anti-ubiquitin antibody sc-8017, Santa Cruz Biotechnology
  • ubiquitination of NIK increased when FAM167A was neutralized (Fig. 4i).
  • the ubiquitination of NIK appears to be regulated by changes in its interactions with DSG1 and Erbin (Fig. 4g-i), but this protein has no reported ubiquitin ligase function.
  • co-immunoprecipitation assays were performed for candidate ubiquitin ligase components including TRAF3, CHIP and c-cbl.
  • the anti-TRAF3 antibody (4729), anti-CHIP antibody (2080) and anti-c-cbl antibody (2747) used at this time were purchased from Cell Signaling Technology. As a result of co-immunoprecipitation analysis, no interaction was found for them (Fig. 4j).
  • FAM167A is responsible for BCR-ABL-independent TKI resistance.
  • imatinib sc-202180, Santa Cruz Biotechnology
  • nilotinib 17050, BioVision
  • K562S or K562R cells were seeded in 96-well plates and treated as indicated prior to transient transfection with expression constructs. After 72 hours, cell viability was measured using WST reagent (DoGenBio).
  • cell viability assays and apoptosis assays were performed to confirm the viability of K562R cells after treatment in the presence of imatinib or nilotinib for 3 days with 2 ⁇ g/ml of anti-FAM167A neutralizing antibody or isotype control.
  • K562S or K562R cells were seeded in 6-well plates and treated as indicated. After 72 hours, cells were stained with Annexin V-APC and propidium iodide using the Annexin V-APC Apoptosis Detection Kit (Thermo Fisher Scientific) and analyzed on a Guava EasyCyte HT cytometer (Millipore).
  • FAM167A The effect of FAM167A on TKI resistance in vivo was investigated using a mouse xenograft model established by subcutaneous injection of K562R cells into nude mice.
  • K562R cells (1 ⁇ 10 7 ) were suspended in 50% (v/v) serum-free Matrigel (Corning) and transplanted subcutaneously into the right flank of 6-week-old female BALB/c (nu/nu) mice. Tumor size was measured with calipers, and tumor volume was calculated using a standard formula (width 2 ⁇ length/2). When the tumor volume reached 100-200 mm 3 , mice were randomly divided into groups for 10 days with imatinib (10 mg/kg/day, intraperitoneal), anti-FAM167A antibody or isotype control (2 mg/kg/3 days, intraperitoneal). intraperitoneally), or with vehicle (saline, PBS). All animal experiments were performed according to protocols approved by the Animal Research Committee of Seoul National University (SNU-210315-6) and the Gwangju Institute of Science and Technology (GIST-2019-008).
  • H score 1 x (% of cells at intensity 1) + 2 x (% of cells at intensity 2) + 3 x (% of cells at intensity 3).
  • KCL22S and KCL22R were obtained from the American Type Culture Collection (ATCC). All cell lines were maintained at 37 °C and 5% CO 2 in a humidified cell culture incubator, RPMI (Roswell Park Memorial Institute ) cultured in 1640 medium (HyClone). To maintain TKI resistance, KCL22R cells were cultured in medium supplemented with 1 ⁇ M imatinib.
  • Peripheral blood or bone marrow samples were collected from 58 patients with CML (clinicopathologic characteristics listed in Table 4) at Hwasun Chonnam National University Hospital. Samples were taken again at diagnosis, before treatment with TKI (diagnosis), and at follow-up after treatment with TKI (follow-up). Mononuclear cells isolated from patients were stored frozen in liquid nitrogen until further use. Informed consent was obtained according to the Declaration of Helsinki, and all procedures were approved by the Seoul National University Clinical Review Board (E2103/003-007) and the Gwangju Institute of Science and Technology (20180629-BR-36-01-02). TKI responders and TKI-resistant patients were clinically classified according to European Leukemia Net guidelines. The mutational status of BCR-ABL in CML samples was confirmed by sequencing analysis.
  • Imatinib resistance is clinically classified according to the European Leukemia Net guidelines. 2 Verification of the mutational status of BCR-ABL by sequencing. CP: chronic phase; AP: accelerator; BP: blast phase.
  • FAM167A was higher in cells from BCR-ABL-independent imatinib-resistant patients (R, no mutation) than in cells from imatinib-responsive or BCR-ABL dependent imatinib-resistant patients (S and R mutations, respectively). showed (Fig. 8a).
  • FAM167A levels were higher in cells from BCR-ABL-independent imatinib-resistant patients before receiving treatment (i.e., at diagnosis), suggesting that expression of FAM167A could predict BCR-ABL-independent TKI resistance in CML patients.

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Abstract

La présente invention concerne une composition de biomarqueur comprenant la FAM167A pour le diagnostic de la résistance contre l'inhibiteur de tyrosine kinase (TKI) indépendante de BCR-ABL et une composition ciblant la FAM167A pour prévenir ou traiter la leucémie myéloïde chronique. Plus spécifiquement, par confirmation des niveaux d'expression de m RNA d'une protéine FAM167A, d'un fragment de cette dernière, ou d'un gène codant pour la protéine, la présente invention concerne : une composition, un kit et un procédé, pour le diagnostic de la résistance à un TKI indépendante de BCR-ABL d'un patient ayant une leucémie myéloïde chronique ; une composition pharmaceutique ciblant la FAM167A pour prévenir ou traiter la leucémie myéloïde chronique présentant une résistance à un TKI indépendante de BCR-ACL ; et une composition pharmaceutique pour l'inhibition d'une résistance à un TKI indépendante de BCR-ABL d'un patient ayant une leucémie myéloïde chronique.
PCT/KR2023/002947 2022-03-03 2023-03-03 Biomarqueur comprenant la fam167a pour le diagnostic de la résistance contre l'inhibiteur de la tyrosine kinase indépendante de bcr-abl, et composition ciblant la fam167a pour prévenir ou traiter la leucémie myéloïde chronique Ceased WO2023167544A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060147911A1 (en) * 2002-04-17 2006-07-06 Brandt Burkhard H Method for characterizing primary tumors
KR20180109811A (ko) * 2015-11-10 2018-10-08 에스디지노믹스 주식회사 항암 치료 내성 판단 방법 및 상기 방법에 사용되는 조성물
KR20190142746A (ko) * 2018-06-18 2019-12-27 연세대학교 산학협력단 Flt3 억제제를 유효성분으로 포함하는 만성 골수성 백혈병 약물 내성 억제용 조성물
CN112481376A (zh) * 2020-11-16 2021-03-12 徐同鹏 一种LncRNA FAM167A-AS1分子标志物及其在胃癌中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9573958B2 (en) 2012-08-31 2017-02-21 Principia Biopharma, Inc. Benzimidazole derivatives as ITK inhibitors
JP2018130113A (ja) 2017-02-15 2018-08-23 国立大学法人 東京大学 慢性骨髄性白血病（ｃｍｌ）の検査方法及び検査用キット、チロシンキナーゼ阻害剤（ｔｋｉ）耐性ｃｍｌの単離方法、並びにｃｍｌにおけるｔｋｉ耐性の低減剤及びそのスクリーニング方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060147911A1 (en) * 2002-04-17 2006-07-06 Brandt Burkhard H Method for characterizing primary tumors
KR20180109811A (ko) * 2015-11-10 2018-10-08 에스디지노믹스 주식회사 항암 치료 내성 판단 방법 및 상기 방법에 사용되는 조성물
KR20190142746A (ko) * 2018-06-18 2019-12-27 연세대학교 산학협력단 Flt3 억제제를 유효성분으로 포함하는 만성 골수성 백혈병 약물 내성 억제용 조성물
CN112481376A (zh) * 2020-11-16 2021-03-12 徐同鹏 一种LncRNA FAM167A-AS1分子标志物及其在胃癌中的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ITO IKUE, KAWAGUCHI YASUSHI, KAWASAKI AYA, HASEGAWA MINORU, OHASHI JUN, KAWAMOTO MANABU, FUJIMOTO MANABU, TAKEHARA KAZUHIKO, SATO : "Association of the FAM167A-BLK region with systemic sclerosis", ARTHRITIS & RHEUMATISM, WILEY INTERSCIENCE, US, vol. 62, no. 3, 1 March 2010 (2010-03-01), US , pages 890 - 895, XP093087811, ISSN: 0004-3591, DOI: 10.1002/art.27303 *
YANG TAEWOO, SIM KYU-YOUNG, KO GWANG-HOON, AHN JAE-SOOK, KIM HYEOUNG-JOON, PARK SUNG-GYOO: "FAM167A is a key molecule to induce BCR-ABL-independent TKI resistance in CML via noncanonical NF-κB signaling activation", JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH, vol. 41, no. 1, XP093087815, DOI: 10.1186/s13046-022-02298-1 *

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