Biomarker for diagnosing BCR-ABL-independent tyrosine kinase inhibitor resistance, including FAM167A, and composition for preventing or treating chronic myeloid leukemia that targets FAM167A

Abstract

The present invention relates to a biomarker composition for diagnosing resistance to BCR-ABL-independent tyrosine kinase inhibitors (TKIs), including FAM167A, and a composition for preventing or treating chronic myeloid leukemia that targets FAM167A. More specifically, the present invention provides a composition, kit, and method for diagnosing BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients by confirming the mRNA expression level of the FAM167A protein, a fragment thereof, or a gene encoding the protein, as well as a pharmaceutical composition for preventing or treating chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance that targets FAM167A, and a pharmaceutical composition for inhibiting BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients.

Description

[Technical field]

[0001] The present invention relates to a biomarker composition for diagnosing BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance, which comprises FAM167A, and a composition for preventing or treating chronic myeloid leukemia which targets FAM167A. More specifically, the present invention provides a composition, kit, and method for diagnosing BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients by confirming the expression level of the FAM167A protein, a fragment thereof, or the mRNA of a gene encoding the protein, as well as a pharmaceutical composition for preventing or treating chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance by targeting FAM167A, and a pharmaceutical composition for inhibiting BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients. [Background technology]

[0002] Chronic myeloid leukemia (CML) arises from 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 upregulated domain of ABL1 during the translocation. The development of BCR-ABL-targeted tyrosine kinase inhibitors (TKIs), such as imatinib, has revolutionized the treatment of CML patients. However, resistance to TKIs remains a major obstacle to successful CML treatment.

[0003] TKI resistance occurs in two ways: BCR-ABL-dependent and BCR-ABL-independent. BCR-ABL-dependent resistance occurs mainly due to mutations in the BCR-ABL kinase domain, but 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 disease. In contrast, BCR-ABL-independent TKI resistance cannot be resolved by second-generation TKIs. Although various factors, including alterations in signal pathway activity, mutations in epigenetic regulators, microenvironmental factors, and leukemia stem cell activity, are potential contributing factors to BCR-ABL-independent TKI resistance, the underlying reasons for such types of resistance have not been fully understood. Therefore, identifying the basic mechanisms and developing effective treatments are major goals.

[0004] Nuclear factor-κB (NF-κB) family members are inducible transcription factors that regulate various genes related to immune responses, inflammation, and cancer. There are two major NF-κB activation signaling pathways: canonical and non-canonical. The canonical NF-κB pathway is mainly activated through the degradation of inhibitor of κB protein (IκB) and nuclear translocation of p50-containing dimers. In contrast, the central signaling protein of the non-canonical NF-κB pathway is NF-κB-inducing kinase (NIK). Non-canonical NF-κB activation depends on NIK accumulation through blocking ubiquitination-dependent NIK degradation. Accumulated NIK induces the processing of p100 to p52, followed by nuclear translocation of p52-containing dimers. The NF-κB pathway contributes to TKI resistance, and inhibition of such pathway restores the sensitivity of CML to TKIs. However, factors associated with direct activation of the NF-κB pathway in TKI-resistant CML are still unknown.

[0005] In this context, we have demonstrated that NF-κB is the most highly activated transcription factor tested through the analysis of in silico activated transcription factors and is responsible for differential gene expression in BCR-ABL-independent TKI-resistant CML cells. We also uncovered a critical function for FAM167A, a previously uncharacterized protein in the non-canonical NF-κB pathway in BCR-ABL-independent TKI-resistant CML. Specifically, FAM167A expression was upregulated by CD34 in patients with BCR-ABL-independent TKI resistance. + FAM167A was highly upregulated in CML cells (similar to the results in CML cell lines), indicating that FAM167A is responsible for BCR-ABL-independent TKI resistance. Neutralization of FAM167A reversed TKI resistance in cultured cells and mouse tumor models.

[0006] These findings describe a novel and important role for FAM167A in the non-canonical NF-κB pathway in association with BCR-ABL-independent TKI resistance mechanisms and present it as a promising target for overcoming TKI-resistant CML.

[0007] In relation to FAM167A, Non-Patent Document 1 has identified FAM167A-BLK as a systemic sclerosis susceptibility gene confirmed by CGA, but the role of FAM167A as a target for diagnosing BCR-ABL-independent TKI resistance and treating TKI-resistant CML is unknown. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Ota Y, Kuwana M. Updates on genetics in systemic sclerosis.Inflamm Regen.2021 Jun 15;41(1):17. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been devised to solve the above-mentioned problems, and aims to discover a novel target for diagnosing BCR-ABL-independent TKI resistance and treating TKI-resistant CML, and to provide a technology for diagnosing BCR-ABL-independent TKI resistance in CML patients, treating BCR-ABL-independent TKI-resistant CML, and inhibiting BCR-ABL-independent TKI resistance by using the target as a biomarker. [Means for solving the problem]

[0010] In order to solve the above-mentioned problems, the present invention provides a biomarker composition for diagnosing BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia patients, comprising a FAM167A protein or a gene encoding the same.

[0011] The present invention also provides compositions and kits for diagnosing BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients, comprising a preparation for measuring the expression level of FAM167A protein, a fragment thereof, or the mRNA of a gene encoding said protein.

[0012] In the present invention, the preparation for measuring the expression level of the FAM167A protein or a fragment thereof may contain an antibody or an antigen-binding fragment thereof, or an aptamer, which specifically binds to the FAM167A protein or a fragment thereof.

[0013] In the present invention, a preparation for measuring the mRNA level of the gene encoding the FAM167A protein may contain sense and antisense primers or probes that bind complementarily to the mRNA of the gene.

[0014] In the present invention, the kit may include an RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit, a DNA chip kit, a microarray kit, a SAGE (Serial Analysis of Gene Expression) kit, a gene chip kit, an ELISA (Enzyme linked immunosorbent assay) kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0015] The present invention also provides an informative method for diagnosing BCR-ABL-independent TKI resistance in a patient with chronic myeloid leukemia comprising the steps of: (a) measuring the mRNA expression level of FAM167A protein, a fragment thereof or a gene encoding said protein in a biological sample isolated from a patient with chronic myeloid leukemia; and (b) comparing the expression level measured in step (a) with the mRNA expression level of the same protein, a fragment thereof, or a gene encoding said protein measured in a biological sample isolated from a chronic myeloid leukemia patient who is not resistant to a BCR-ABL-independent tyrosine kinase inhibitor.

[0016] In the present invention, the method for providing information may further include a step of (c) determining that the patient exhibits resistance to a BCR-ABL-independent TKI if the mRNA expression level of the FAM167A protein, a fragment thereof, or a gene encoding the protein measured in a biological sample isolated from the chronic myeloid leukemia patient in step (a) is 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 not resistant to a BCR-ABL-independent tyrosine kinase inhibitor in step (b).

[0017] In the present invention, the biological sample may comprise blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymphatic fluid, respiratory tract fluid, intestinal fluid, urogenital fluid, breast milk, lymphatic system fluid, semen, cerebrospinal fluid, tracheal system fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof.

[0018] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance, and a pharmaceutical composition for inhibiting BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients, which comprises as an active ingredient a FAM167A protein inhibitor; an inhibitor of the expression of the gene encoding the FAM167A protein; or a mixture thereof.

[0019] In the present invention, the BCR-ABL-independent TKI-resistant chronic myeloid leukemia can have increased NF-κB activity compared to TKI-sensitive chronic myeloid leukemia.

[0020] In the present invention, the pharmaceutical composition may be administered in combination with a TKI.

[0021] In the present invention, the TKI is crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, ibrutinib, cabozantinib, These may include abozantinib, gefitinib, erlotinib, lapatinib, vandetanib, afatinib, osimertinib, ruxolitinib, tofacitinib, axitinib, lenvatinib, nintedanib, pazopanib, regorafenib, sorafenib or sunitinib.

[0022] The present invention also provides a method for screening for a therapeutic agent for CML exhibiting BCR-ABL-independent TKI resistance or a BCR-ABL-independent TKI resistance inhibitor, comprising the steps of: (a) treating a cell line or animal model exhibiting BCR-ABL-independent TKI resistance with a candidate agent; and (b) determining whether or not there is inhibition of FAM167A protein or inhibition of expression of the gene encoding said protein in a cell line or animal model treated with said candidate substance;

[0023] In the present invention, the screening method may further include a step of (c) determining that the candidate substance is a therapeutic agent for CML exhibiting BCR-ABL-independent TKI resistance or a BCR-ABL-independent TKI resistance inhibitor if the candidate substance inhibits FAM167A protein or inhibits expression of a gene encoding the protein.

[0024] Furthermore, the present invention provides a method for treating chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance, comprising administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of a gene encoding a FAM167A protein, or a mixture thereof to a patient with chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance.

[0025] The present invention also provides a method for inhibiting BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient, comprising administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of a gene encoding a FAM167A protein, or a mixture thereof to a chronic myeloid leukemia patient exhibiting BCR-ABL-independent TKI resistance.

[0026] The present invention also provides a method for diagnosing BCR-ABL-independent TKI resistance in a patient with chronic myeloid leukemia and treating chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance, comprising the steps of: (a) measuring the mRNA expression level of FAM167A protein, a fragment thereof or a gene encoding said protein in a biological sample isolated from a patient with chronic myeloid leukemia; (b) diagnosing the presence of BCR-ABL-independent TKI resistance when the expression level measured in step (a) is increased relative to the mRNA expression level of the same protein, a fragment thereof, or a gene encoding said protein measured in a biological sample isolated from a chronic myeloid leukemia patient who does not have BCR-ABL-independent TKI resistance; and (c) administering to a patient diagnosed in step (b) as exhibiting BCR-ABL-independent TKI resistance a therapeutically effective amount of a FAM167A protein inhibitor, an inhibitor of expression of a gene encoding a FAM167A protein, or a mixture thereof.

[0027] The present invention also provides a method for diagnosing BCR-ABL-independent TKI resistance and inhibiting BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient, comprising the steps of: (a) FAM167A protein in biological samples isolated from patients with chronic myeloid leukemia; measuring the mRNA expression level of the gene encoding the fragment or said protein; (b) diagnosing the presence of BCR-ABL-independent TKI resistance when the expression level measured in step (a) is increased relative to the mRNA expression level of the same protein, a fragment thereof, or a gene encoding said protein measured in a biological sample isolated from a chronic myeloid leukemia patient who does not have BCR-ABL-independent TKI resistance; and (c) administering to a patient diagnosed in step (b) as exhibiting BCR-ABL-independent TKI resistance a therapeutically effective amount of a FAM167A protein inhibitor, an inhibitor of expression of a gene encoding a FAM167A protein, or a mixture thereof. Effect of the Invention

[0028] In the present invention, FAM167A has been discovered as a new biomarker that can be used as an indicator of TKI resistance in CML that shows BCR-ABL-independent TKI resistance in which there is no mutation in the BCR-ABL kinase domain, and it has been confirmed that TKI resistance is reversed when FAM167A is inhibited. This provides the advantage that TKI resistance can be used to diagnose TKI resistance in CML patients who have not been treated with existing second-generation TKIs, and that TKI resistance can be reversed in CML patients who show BCR-ABL-independent TKI resistance, thereby enabling effective treatment of TKI-resistant patients. [Brief description of the drawings]

[0029] [Figure 1a] FIG. 1a shows the strategy for the selection of genes that contribute to BCR-ABL-independent TKI resistance in CML. [Figure 1b]Figure 1b and 1c show the results of analyzing the viability of K562S and K562R cells after treatment for 3 days with the indicated concentrations of imatinib (Figure 1b) and nilotinib (Figure 1c), respectively, and Figure 1d and 1e show the results of flow cytometry of Annexin V stained cells to evaluate apoptosis in K562S and K562R cells after treatment for 3 days with the indicated concentrations of imatinib (Figure 1d) and nilotinib (Figure 1e), respectively. Data are shown from three (Figure 1b-Figure 1e) independent experiments (error bars, s.d. of triplicate (Figure 1b and Figure 1c) samples). Independent samples two-tailed t-test; ***P<0.001. K562S: TKI-sensitive K562 cell line; K562R: TKI-resistant K562 cell line lacking BCR-ABL kinase domain mutation. [Figure 1c] Figure 1b and 1c show the results of analyzing the viability of K562S and K562R cells after treatment for 3 days with the indicated concentrations of imatinib (Figure 1b) and nilotinib (Figure 1c), respectively, and Figure 1d and 1e show the results of flow cytometry of Annexin V stained cells to evaluate apoptosis in K562S and K562R cells after treatment for 3 days with the indicated concentrations of imatinib (Figure 1d) and nilotinib (Figure 1e), respectively. Data are shown from three (Figure 1b-Figure 1e) independent experiments (error bars, s.d. of triplicate (Figure 1b and Figure 1c) samples). Independent samples two-tailed t-test; ***P<0.001. K562S: TKI-sensitive K562 cell line; K562R: TKI-resistant K562 cell line lacking BCR-ABL kinase domain mutation. [Figure 1d]Figure 1b and 1c show the results of analyzing the viability of K562S and K562R cells after treatment for 3 days with the indicated concentrations of imatinib (Figure 1b) and nilotinib (Figure 1c), respectively, and Figure 1d and 1e show the results of flow cytometry of Annexin V stained cells to evaluate apoptosis in K562S and K562R cells after treatment for 3 days with the indicated concentrations of imatinib (Figure 1d) and nilotinib (Figure 1e), respectively. Data are shown from three (Figure 1b-Figure 1e) independent experiments (error bars, s.d. of triplicate (Figure 1b and Figure 1c) samples). Independent samples two-tailed t-test; ***P<0.001. K562S: TKI-sensitive K562 cell line; K562R: TKI-resistant K562 cell line lacking BCR-ABL kinase domain mutation. [Figure 1e] Figure 1b and 1c show the results of analyzing the viability of K562S and K562R cells after treatment for 3 days with the indicated concentrations of imatinib (Figure 1b) and nilotinib (Figure 1c), respectively, and Figure 1d and 1e show the results of flow cytometry of Annexin V stained cells to evaluate apoptosis in K562S and K562R cells after treatment for 3 days with the indicated concentrations of imatinib (Figure 1d) and nilotinib (Figure 1e), respectively. Data are shown from three (Figure 1b-Figure 1e) independent experiments (error bars, s.d. of triplicate (Figure 1b and Figure 1c) samples). Independent samples two-tailed t-test; ***P<0.001. K562S: TKI-sensitive K562 cell line; K562R: TKI-resistant K562 cell line lacking BCR-ABL kinase domain mutation. [Figure 1f] FIG. 1f is a Venn diagram showing up- and down-regulated genes (fold change ≧2) in K562R cells examined by microarray and RNA-seq analysis. [Figure 1g] FIG. 1g shows a heatmap analysis of the expression levels of differentially expressed genes in K562R cells. [Figure 1h]Figure 1h and Figure 1i-k show the in silico analysis of transcription factors involved in differentially expressed gene regulation in both microarray and RNA-seq analyses (Figure 1h) and three control group genes (each group contains 500 randomly selected genes) (Figure 1i-k). Size and color indicate the number of genes associated with the transcription factor. [Figure 1i] Figure 1h and Figure 1i-k show the in silico analysis of transcription factors involved in differentially expressed gene regulation in both microarray and RNA-seq analyses (Figure 1h) and three control group genes (each group contains 500 randomly selected genes) (Figure 1i-k). Size and color indicate the number of genes associated with the transcription factor. [Figure 1j] Figure 1h and Figure 1i-k show the in silico analysis of transcription factors involved in differentially expressed gene regulation in both microarray and RNA-seq analyses (Figure 1h) and three control group genes (each group contains 500 randomly selected genes) (Figure 1i-k). Size and color indicate the number of genes associated with the transcription factor. [Figure 1k] Figure 1h and Figure 1i-k show the in silico analysis of transcription factors involved in differentially expressed gene regulation in both microarray and RNA-seq analyses (Figure 1h) and three control group genes (each group contains 500 randomly selected genes) (Figure 1i-k). Size and color indicate the number of genes associated with the transcription factor. [Figure 1l] FIG. 1l shows the enrichment scores for transcription factors, indicating the degree of association with differentially expressed genes compared to randomly selected genes. [Figure 1m]Figure 1m, A, shows the percentage of genes targeted by the indicated transcription factors among genes differentially expressed between K562S and K562R, and B, shows the percentage of genes targeted by the indicated transcription factors among 500 randomly selected genes. [Figure 1n] FIG. 1n shows NF-κB and AP-1 luciferase reporter activities in K562S and K562R cells and K562R cells treated with 1 μM imatinib for 24 hours. [Figure 1o] Figure 1o shows quantitative reverse transcription PCR (qRT-PCR) analysis results for mRNA levels of seven genes in K562S and K562R cells and K562R cells treated with 1 μM imatinib for 24 h. IMA: imatinib. Data represent three (Figure 1n and Figure 1o) independent experiments (error bars, s.d. of duplicate (Figure 1o) or triplicate (Figure 1l and Figure 1n) samples). Independent samples two-tailed T-test; *P<0.05, **P<0.01. NS: not significant; K562S: TKI-sensitive K562 cell line; K562R: TKI-resistant K562 cell line lacking BCR-ABL kinase domain mutation. [Figure 2a] FIG. 2a shows NF-κB luciferase reporter activity in K562S cells 24 h after transfection with plasmids encoding the indicated genes, and in K562R cells and K562R cells treated with 1 μM imatinib for 24 h. [Figure 2b] Figure 2b shows the viability of K562S cells transfected with plasmids encoding the indicated genes after treatment with or without imatinib (IMA, 1 μM) for 2 days. Data represent two independent experiments (error bars, s.d. of triplicate samples). Independent samples two-tailed t-test; ***P<0.001. [Figure 2c]Figure 2cA shows in silico prediction of human, mouse, rat and zebrafish FAM167A and HA-tagged human FAM167A secretion by the SecretomeP tool (http: / / www.cbs.dtu.dk / services / SecretomeP / ). Proteins with an NN score of 0.5 or higher are predicted to be secreted. Figure 2cB shows immunoblot analysis of FAM167A in cell lysates and culture supernatants of K562R cells. Culture medium was used as a control for the supernatants. Supernatant proteins were concentrated by acetone precipitation. Erbin, a cytoplasmic protein, was also analyzed as a control to confirm the absence of cytoplasmic proteins in the culture supernatants. Data are representative of three independent experiments (Figure 2cB). [Figure 2d] FIG. 2d shows NF-κB luciferase reporter activity in K562S cells after treatment with recombinant FAM167A for 24 hours; FIG. 2e shows NF-κB luciferase reporter activity in K562R cells after treatment with anti-FAM167A neutralizing antibody for 24 hours; FIG. 2f shows NF-κB luciferase reporter activity in K562R cells after treatment with 50 μg / ml SN50, 10 ng / ml LPS or both for 24 hours; FIG. 2g shows NF-κB luciferase reporter activity in K562R cells 24 hours after transfection with a plasmid encoding dominant negative NIK (NIK-DN); and FIG. 2h and FIG. 2i show immunoblot analysis for NIK (FIG. 2h) and p100 / p52 (FIG. 2i) in K562S and K562R cells. [Figure 2e]FIG. 2d shows NF-κB luciferase reporter activity in K562S cells after treatment with recombinant FAM167A for 24 hours; FIG. 2e shows NF-κB luciferase reporter activity in K562R cells after treatment with anti-FAM167A neutralizing antibody for 24 hours; FIG. 2f shows NF-κB luciferase reporter activity in K562R cells after treatment with 50 μg / ml SN50, 10 ng / ml LPS or both for 24 hours; FIG. 2g shows NF-κB luciferase reporter activity in K562R cells 24 hours after transfection with a plasmid encoding dominant negative NIK (NIK-DN); and FIG. 2h and FIG. 2i show immunoblot analysis for NIK (FIG. 2h) and p100 / p52 (FIG. 2i) in K562S and K562R cells. [Figure 2f] FIG. 2d shows NF-κB luciferase reporter activity in K562S cells after treatment with recombinant FAM167A for 24 hours; FIG. 2e shows NF-κB luciferase reporter activity in K562R cells after treatment with anti-FAM167A neutralizing antibody for 24 hours; FIG. 2f shows NF-κB luciferase reporter activity in K562R cells after treatment with 50 μg / ml SN50, 10 ng / ml LPS or both for 24 hours; FIG. 2g shows NF-κB luciferase reporter activity in K562R cells 24 hours after transfection with a plasmid encoding dominant negative NIK (NIK-DN); and FIG. 2h and FIG. 2i show immunoblot analysis for NIK (FIG. 2h) and p100 / p52 (FIG. 2i) in K562S and K562R cells. [Figure 2g]FIG. 2d shows NF-κB luciferase reporter activity in K562S cells after treatment with recombinant FAM167A for 24 hours; FIG. 2e shows NF-κB luciferase reporter activity in K562R cells after treatment with anti-FAM167A neutralizing antibody for 24 hours; FIG. 2f shows NF-κB luciferase reporter activity in K562R cells after treatment with 50 μg / ml SN50, 10 ng / ml LPS or both for 24 hours; FIG. 2g shows NF-κB luciferase reporter activity in K562R cells 24 hours after transfection with a plasmid encoding dominant negative NIK (NIK-DN); and FIG. 2h and FIG. 2i show immunoblot analysis for NIK (FIG. 2h) and p100 / p52 (FIG. 2i) in K562S and K562R cells. [Figure 2h] FIG. 2d shows NF-κB luciferase reporter activity in K562S cells after treatment with recombinant FAM167A for 24 hours; FIG. 2e shows NF-κB luciferase reporter activity in K562R cells after treatment with anti-FAM167A neutralizing antibody for 24 hours; FIG. 2f shows NF-κB luciferase reporter activity in K562R cells after treatment with 50 μg / ml SN50, 10 ng / ml LPS or both for 24 hours; FIG. 2g shows NF-κB luciferase reporter activity in K562R cells 24 hours after transfection with a plasmid encoding dominant negative NIK (NIK-DN); and FIG. 2h and FIG. 2i show immunoblot analysis for NIK (FIG. 2h) and p100 / p52 (FIG. 2i) in K562S and K562R cells. [Figure 2i]FIG. 2d shows NF-κB luciferase reporter activity in K562S cells after treatment with recombinant FAM167A for 24 hours; FIG. 2e shows NF-κB luciferase reporter activity in K562R cells after treatment with anti-FAM167A neutralizing antibody for 24 hours; FIG. 2f shows NF-κB luciferase reporter activity in K562R cells after treatment with 50 μg / ml SN50, 10 ng / ml LPS or both for 24 hours; FIG. 2g shows NF-κB luciferase reporter activity in K562R cells 24 hours after transfection with a plasmid encoding dominant negative NIK (NIK-DN); and FIG. 2h and FIG. 2i show immunoblot analysis for NIK (FIG. 2h) and p100 / p52 (FIG. 2i) in K562S and K562R cells. [Figure 2j] FIG. 2j shows the results of electrophoretic mobility assays (EMSA) of nuclear extracts isolated from K562S and K562R cells showing a supershift with an NF-κB probe and an anti-p52 antibody. [Figure 2k] FIG. 2k shows NF-κB luciferase reporter activity in K562S cells treated with 100 ng / ml recombinant FAM167A and 24 hours after transfection with a plasmid encoding NIK-DN. [Figure 2l] FIG. 2l shows immunoblot analysis of NIK in K562R cells after treatment with 2 μg / ml of anti-FAM167A neutralizing antibody or isotype control for 12 hours. [Figure 2m] Figure 2m shows immunoblot analysis of p100 / p52 in K562S and K562R cells after treatment with recombinant FAM167A at the indicated concentrations for 12 h. GAPDH was used as an internal control (Figure 2h, Figure 2i, Figure 2l, and Figure 2m). Data are representative of three (Figure 2a, Figure 2d-G, and Figure 2i-M) or five (Figure 2h) independent experiments (error bars, s.d. of triplicate (Figure 2a, Figure 2d-G, and Figure 2i-M) or five (Figure 2h) samples). Independent samples two-tailed t-test; *P<0.05, **P<0.01, ***P<0.001. [Figure 3a] FIG. 3a shows surface FAM167A receptor (FAM167AR) staining of K562S and K562R cells using Myc-tagged FAM167A and Alexa Fluor 488-conjugated anti-Myc antibody. [Figure 3b] FIG. 3b shows the experimental design for the identification of the FAM167A receptor. [Figure 3c] FIG. 3c shows silver staining results of immunoprecipitation samples prepared from K562R cells using Myc tag FAM167A and anti-Myc antibody bound to Protein G Sepharose® beads. [Figure 3d] 3d-h show the MS / MS spectra and peptide sequences of the bands displayed from FIG. 3c. [Figure 3e] 3d-h show the MS / MS spectra and peptide sequences of the bands displayed from FIG. 3c. [Figure 3f] 3d-h show the MS / MS spectra and peptide sequences of the bands displayed from FIG. 3c. [Figure 3g] 3d-h show the MS / MS spectra and peptide sequences of the bands displayed from FIG. 3c. [Figure 3h] 3d-h show the MS / MS spectra and peptide sequences of the bands displayed from FIG. 3c. [Figure 3i] FIG. 3i shows the results of co-immunoprecipitation analysis between FAM167A and DSG1 in K562R cells. [Figure 3j] FIG. 3j shows qRT-PCR analysis of DSG1 mRNA levels in K562S and K562R cells and in K562R cells treated with 1 μM imatinib for 24 hours. [Figure 3k] FIG. 3k shows immunoblot analysis of DSG1 expression in K562S and K562R cells. [Figure 3l]FIG. 3l shows the results of surface staining of K562S and K562R cells using anti-DSG1 antibody. [Figure 3m] Figure 3m shows immunofluorescence microscopy analysis of DSG1 expression in K562S and K562R cells. Hoechst 33342 was used to visualize nuclei. Scale bar: 10 μm. GAPDH was used for internal control (Figure 3g). IP: immunoprecipitation. MFI: mean fluorescence intensity. Data represent three (Figure 3a, Figure 3c and Figure 3e-3i) independent experiments (error bars, s.d. of duplicate (Figure 3f) or triplicate (Figure 3a and Figure 3h) samples). Independent samples two-tailed t-test; **P<0.01, ***P<0.001. NS: not significant. [Figure 3n] Figure 3n shows immunofluorescence microscopy of DSG1 and FAM167A in K562R cells. Nuclei were visualized using Hoechst 33342. Scale bar: 10 μm. [Figure 3o] Figure 3o shows NF-κB luciferase reporter activity results in K562S cells 24 hours after transfection with a plasmid encoding FAM167A and treatment with the indicated concentrations of anti-FAM167A neutralizing antibody. Data represent two (Figure 3n and Figure 3o) independent experiments (error bars, s.d. of triplicate (Figure 3n) samples). Independent samples two-tailed t-test; *P<0.05. [Figure 4a] FIG. 4a shows surface DSG1 staining results of K562R cells after DSG1 knockdown using recombinant lentiviruses encoding two different DSG1-specific shRNAs together with anti-DSG1 antibodies. [Figure 4b] FIG. 4b shows NF-κB luciferase reporter activity in K562R cells after DSG1 knockdown using recombinant lentiviruses encoding two different DSG1-specific shRNAs. [Figure 4c] FIG. 4c shows the results of immunoblot analysis of p100 / p52 in K562R cells after DSG1 knockdown. [Figure 4d]FIG. 4d shows NF-κB luciferase reporter activity in K562R cells 24 hours after transfection with plasmids encoding DSG1 or DSG1 and Erbin with or without treatment with 2 μg / ml anti-FAM167A neutralizing antibody. [Figure 4e] FIG. 4e shows qRT-PCR analysis of Erbin mRNA in K562S, K562R and K562R cells treated with 1 μM imatinib for 24 hours. [Figure 4f] Figure 4f shows the results of immunoblotting analysis for Erbin in K562S and K562R cells. Data are representative of three (Figure 4a, Figure 4e and Figure 4f) independent experiments (error bars, s.d. of triplicate (Figure 4e) samples). Independent samples two-tailed t-test; NS: not significant. [Figure 4g] FIG. 4g shows the results of co-immunoprecipitation analysis of DSG1, Erbin and NIK in K562S and K562R cells. [Figure 4h] FIG. 4h shows the results of co-immunoprecipitation analysis of DSG1 and NIK in K562R cells after treatment with 2 μg / ml of anti-FAM167A neutralizing antibody or isotype control for 3 hours. [Figure 4i] FIG. 4i shows the results of immunoblot analysis of NIK ubiquitination using anti-ubiquitin antibody after NIK immunoprecipitation from K562R cells treated with 2 μg / ml of anti-FAM167A neutralizing antibody or isotype control and 10 μM MG132 for 3 hours. [Figure 4j]A of FIG. 4j shows the results of immunocoprecipitation analysis of DSG1, Erbin, NIK, TRAF3, CHIP and c-cbl in K562S and K562R cells, B shows the results of immunocoprecipitation analysis of DSG1, NIK and TRAF3 in K562S cells using anti-DSG1 and anti-TRAF3 antibodies, C shows the results of immunocoprecipitation analysis of DSG1, NIK and CHIP in K562S cells using anti-DSG1 and anti-CHIP antibodies, and D shows the results of immunocoprecipitation analysis of DSG1, NIK and c-cbl in K562S cells using anti-DSG1 and anti-c-cbl antibodies. GAPDH was used as an internal control (A of FIG. 4j). Data are shown from three independent experiments (A to D of FIG. 4j). [Figure 4k] Figure 4k shows a schematic model of FAM167A-induced non-canonical NF-κB pathway. GAPDH was used for internal control (Figure 4c and Figure 4g-i). Ub: ubiquitination. Data represent two (Figure 4c) or three (Figure 4b, Figure 4d, Figure 4g-i) independent experiments (error bars, s.d. of duplicate (Figure 4c) or triplicate (Figure 4b, Figure 4d, Figure 4g and Figure 4h) samples). Independent samples two-tailed t-test; *P<0.05, **P<0.01, ***P<0.001. [Figure 5a] FIG. 5a is a schematic diagram showing FAM167A-induced inhibition of the non-canonical NF-κB pathway. [Figure 5b] FIG. 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] FIG. 5c shows the results of viability analysis of K562R cells after treatment with 2 μg / ml of anti-FAM167A neutralizing antibody or isotype control for 3 days in the presence of imatinib or nilotinib. [Figure 5d]Figure 5d shows the cell death population of K562R cells after treatment with 2 μg / ml of anti-FAM167A neutralizing antibody or isotype control for 3 days in the presence of imatinib or nilotinib. Data represent three independent experiments (error bars, s.d. of triplicate samples). Independent samples two-tailed t-test; *P<0.05, **P<0.01, ***P<0.001. [Figure 6a] FIG. 6a is an experimental timeline and schematic diagram showing the mouse model. [Figure 6b] Figures 6b and 6c show the volume of K562R tumors established in mice treated with vehicle or 10 mg / kg / day imatinib in the presence or absence of anti-FAM167A neutralizing antibody or isotype control (2 mg / kg / 3 days), respectively, and mouse body weight. [Figure 6c] Figures 6b and 6c show the volume of K562R tumors established in mice treated with vehicle or 10 mg / kg / day imatinib in the presence or absence of anti-FAM167A neutralizing antibody or isotype control (2 mg / kg / 3 days), respectively, and mouse body weight. [Figure 6d] FIG. 6d shows tumor photographs collected from mice in each group on day 10 of treatment, and FIG. 6e shows tumor weights on day 10 of treatment. [Figure 6e] FIG. 6d shows tumor photographs collected from mice in each group on day 10 of treatment, and FIG. 6e shows tumor weights on day 10 of treatment. [Figure 6f] Figure 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 μm. Data represent three (Figures 6b-6f) independent experiments (error bars, s.d. of five (Figures 6b, 6c, 6e, and 6f) mice or samples). Independent samples two-tailed t-test; *P<0.05, **P<0.01, ***P<0.001. NS: not significant. [Figure 7]Figure 7 shows qRT-PCR analysis of FAM167A mRNA in KCL22S, KCL22R and KCL22R cells treated with 1 μM imatinib for 24 hours. Data represent three independent experiments (error bars, sd of triplicate samples). Independent samples two-tailed t-test; **P<0.01, ***P<0.001. [Figure 8a] Figure 8a shows qRT-PCR analysis of FAM167A mRNA expression in CD34+ stem / progenitor cells from imatinib-responsive CML patients (S) at diagnosis (n=26) and follow-up (n=26) in imatinib-resistant CML patients (R) without BCR-ABL mutations at diagnosis (n=12) and follow-up (n=11) and in imatinib-resistant CML patients with BCR-ABL mutations at diagnosis (n=10) and follow-up (n=12). Imatinib resistance and BCR-ABL mutation status were determined at follow-up. FAM167A levels were normalized to GAPDH levels. [Figure 8b] Figure 8b shows the relative surface expression of DSG1 (MFI) in CD34+ stem / progenitor cells from imatinib-responsive CML patients at diagnosis (n=8) and follow-up (n=8) and from imatinib-resistant CML patients without BCR-ABL mutations at diagnosis (n=10) and follow-up (n=7), measured by flow cytometry. [Figure 8c] Figure 8c is a proposed model showing the involvement of FAM167A-induced non-canonical NF-κB pathway in BCR-ABL-independent TKI resistance. Independent samples two-tailed t-test; *P<0.05, ***P<0.001. NS: not significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention will now be described in more detail.

[0031] All technical terms used in the present invention are used in the same manner as commonly understood by those of ordinary skill in the art unless otherwise defined. In addition, although preferred methods and samples are described in the present specification, similar or equivalent methods and samples are also included in the scope of the present invention.

[0032] As mentioned above, BCR-ABL-independent TKI resistance cannot be treated even with second-generation TKIs, and it is necessary to discover new biomarkers that can be used for the diagnosis and treatment of BCR-ABL-independent TKI-resistant CML.

[0033] Therefore, in the present invention, we sought to solve the above-mentioned problems by revealing the important function of FAM167A, a previously uncharacterized protein in the non-canonical NF-κB pathway in BCR-ABL-independent TKI-resistant CML, demonstrating that neutralization of FAM167A has the effect of reversing TKI resistance in cultured cells and mouse tumor models, and confirming that FAM167A may be a promising target for overcoming TKI-resistant CML.

[0034] Thus, a first aspect of the present invention relates to a biomarker composition for diagnosing BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia patients, comprising a FAM167A protein or a gene encoding the same.

[0035] The FAM167A, also known as C8orf13, is a 214 amino acid protein belonging to the FAM167 (SEC) family. The gene encoding FAM167A is mapped to human chromosome 8, which is composed of approximately 146 million base pairs. In humans, the FAM167A protein or a fragment thereof may include the amino acids of NCBI Reference Sequence: NP_444509.2, but may include any amino acid sequence that has substantially the same or corresponding efficacy as the protein, without limitation. The FAM167A protein may also include an isoform or a precursor thereof. In the present invention, the amino acid sequence of a representative FAM167A protein is shown in SEQ ID NO:1.

[0036] MSVPQIHVEE VGAEEGAGAA APPDDHLRSL KALTEKLRLE TRRPSYLEWQ ARLEEHTWPF PRPAAEPQAS LEEGERGGQE PLLPLREAGQ HPPSARSASQ GARPLSTGKL EGFQSIDEAI AWLRKELTEM RLQDQQLARQ LMRLRGDINK LKIEHTCRLH RRMLNDATYE LEERDELADL FCDSPLASSF SLSTPLKLIG VTKMNINSRR FSLC (SEQ ID NO: 1)

[0037] The mRNA of the gene encoding the FAM167A protein may include the nucleotide sequence of NCBI Reference Sequence: NM_053279.3, but may include any nucleotide sequence that is substantially identical to or has a corresponding function, without limitation. In the present invention, a representative mRNA sequence of the gene encoding the FAM167A protein is shown in SEQ ID NO:2.

[0038] gagaattcac cggagctcgg ggaggcgagg cgggagccct gagctgaggc cgggcagcgc ggacccgcgc aggagaggc ggcggggcaca ccggcgagtc cccagcaggg cccctgggct cccggaaggg tctccagcca cctgcccttc ccgagaagag agagctctgg gccttcttcc tgccagtctg gtcttcagt gcgttcagga cagatagacc ttggcacagg ctgccccgag attcctgcga cgctgtctgt tcctgccttc tgtggagcat ggcacccaca ggcttccagg acggatcata gacccgagcc tccaggaggg cgccctgtgc ggctcactgc gcggtccctc tcagctcccc tgccacccag cctctgaggc cggctcctgt cccggaaatg ccctcgggca cctggatgag gccatcccag agcgggaccc aactgtgcca cccaccaagc ctccccctgc gccccccgtg ccctcgcatg tccggctgcc agggcagact gtagaatgtc tgtgccccag atccacgtgg aagaagtggg tgcagaagag ggggcgggag cagccgcacc acccgatgac cacctccgga gcctgaaggc cctcaccgag aaactgaggc tggagacccg caggccctcc tacctggaat ggcaggccag gctggaggag catacctggc ccttcccgag gccggctgc gagccacagg cgagcttgga ggagggggag cgtggggggc aggagccctt gctccccctg agagaggctg ggcagcaccc cccttctgcc aggagtgcca gccaaggtgc cagacccctg tccactggca agctggagg ctttcagagc atcgatgag ctatagcctg gctcaggag gaactgacg g agatgcggct gcaggaccag caactggcca gacagctcat gcgcctgcgt ggcgacatca acaagctgaa aatcgaacac acctgccgcc tccacaggag gatgctcaac gatgccacct acgagctgga ggagcgggat gagctggccg acctcttctg tgactcccct cttgcctcct ccttcagcct ctccacacca ctcaagctta ttggcgtgac caagatgac atcaactctc ggaggttctc tctctgctga ggagccctca gactgggcgg aggggctgga gcggagggct tgggctggag gggtgtcaga ggaagctgag gccaagttac tccagtgggt ctcccggagg caggggtccc tgggactggc gactcaaggg ccccaggacc tattcagtgg tgctctccca cccaggggcc ctgggtgtgg atgccagtgt ctctgtgact ggctcttgct tactacccaa agagctctgc agaagggccg ctccaaccaa gatgttaaag gagacctggg ttcccaccat aatccatccc tccacggtca cgttcctgtt tcctggaatc actggtgcta tgaactggga ttcccaaagg gaggcccccc aacaaagctg tcatttttgc agaaggctgt cccgcaaggg ccttgggggga aattaggcat gtcagatgtg cctgtctcac gtgctgttgc tgtcccttaa gtattgtctc aaattcaccc taggtacatg actcagcaac attgacaggg agctactagg aagggaaaat cgaaaggcat gacaaatggg cacttggggga cgcagcccca gtggctggca gccagtgtct ctggtgagcc tgacactata aggctgtgta aattgtaaat tctggcgtgt gctgggacat gtgatggggg cactagcgta gcttgggtgc aacaagcaca gatgtcccca ttgtctcccc tggccacatg catctccaaa gagcctcttc actgccaccc acaccccagg gtgacagcct ggggaccac tggtgactga accaggcagg tcctgaaagc attttccata actgaattct cctgcagggg cgtgaccggg gcctcctggt ggattctggt ggtgtcacct tactgccctc tctggaaaga caatctaggg agcccagagg cccatcctga gcctcctctg agattttgtg cctgacctaa acaactagtt ttaataagac tgttactgat gtgttgttca cttgttagta actgattttt gtccaaatgc ggaagccact tgtgtaggtc aactacagtg cgtaggattt gattttaaga gtttctccct cccaacaggc ttgaggatca gcaagttaag accccagcag gttagggagg tcagtctggg gtcatacggc atggcagggg tccctcggcc agacccgtag aatcctgaga taaggagtgt ttctgacctt tggtgtcatc tagtcgagtc ctctcattag taaaggagca aagtgaaacc tgggggagga gaaggacttc cctcaggttg cacagctgtt taggctatag atattgatg tgtgaacca ttattgataa tgcctagtag atcacatgtc aatgaacttg tgg aacccagcag atat ctgccaaccc ctctactctc cacctcagcc cccacccaca tctcccagag tattgcaatt cagaacatt gggtcaggt ggagcaagc actgacagtg gcccacagg gcatgtgtca ctaatcactg tcccatg gtc tacgcacggc atctggctgc tctgtctact gtgactctt cctgtgtaat ctcagtgggg cccgtgtcca cccacacacaca gtgacccaca tagggag gttgctttc tttgtgggc tgagagtagg acaatgcaaa tgaatgatct ctagtagaca gaaagaact tggtcttt tttaaaattt caagagcca gaagttctat gcctccttca aagtaggcag aaccaacaccag tgctctgtgc aatgaagtct gcaggcctga ggaccatgta ctgctgtcct tcctcagagc tctgcacaaa cactgccaag tcctgagac gcattccttt cctgccaacc tcttccaga taagcccttg aggctcgc caccacaccacaca caccaca spider randomg ccataaacat ccttgaaccc atgcaggaaa gcccatccca tattctgaaa aaatgccaaa ttaggttttt ctttcttttt ggaaatcagt cattacagta accgaaacca ttgggttcag cgaaaatgga aagatttagc tgaatgtagt cagtccaatt aagttggatg caactgagtg atttagttgc ttgggtaacc cagtgcttgc ttgctttctt cattctctgg gtggaaacta agatcaagac acatgtttgg ggataagtta aatgtctgag ctattttgct cggtttatcc taagagaact ttattatggg atgaggaggt gacccaagat gagaagtgga gggggacagc gatgttttct aaacatcgtc cagtgttgac tggcttcctt actttgcaca gtgaacacaa ctaaccacat taattcagct ttgtgaagtc cctgctctct gtgggtctat gagtcagcag caacattggc ctaacctccg tcccagcctc ctggctcacc acatgtgtac agtgctgttt gcagttgtac tcattatcca tccatctctc tgccatcccc aagcatcgct gggtgtaaaa cgcaaactct ccaccgacac tgccatgcgt ggtcatgtct tgatgccttc aggggctcag tagctatcaa agaggcctgg agggcctggg caggcttgac gatgcctgac cgagttcaag acccacaccc tgtagcaata ccaagtgcta ttacataatc aatggacgat ttatactttt attttttatg attatttgtt tctatattgc tgttagaaaa agtgaaataa aaatacttca aaagaagata tccatataaa aataaaa(SEQ ID NO: 2)

[0039] The specific amino acid sequences and nucleotide sequences of the biomarker proteins and the genes encoding them can be confirmed from publicly known databases such as NCBI.

[0040] The term "biomarker" of the present invention refers to a molecule quantitatively or qualitatively associated with the presence of a biological phenomenon, and the biomarker of the present invention refers to a protein or a gene encoding the same that is a criterion for determining the presence or absence of BCR-ABL-independent TKI resistance. A biomarker can be derived from a genomic nucleotide sequence, or an expressed nucleotide sequence (e.g., RNA, nRNA, mRNA, cDNA, etc.), or an encoded polypeptide. The term also refers to a nucleic acid sequence complementary to or flanking a biomarker sequence, such as a probe or a primer capable of amplifying the biomarker sequence. The nucleic acid used in the timer pair is included.

[0041] In the present invention, the term "expression" refers to the production of a protein or nucleic acid. "Protein" is used interchangeably with "polypeptide" or "peptide" and refers to a polymer of amino acid residues, such as those typically found in proteins in their natural state. "Polynucleotide" or "nucleic acid" refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single-stranded 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 also included. "mRNA" refers to RNA that conveys genetic information (gene-specific base sequence) from a specific gene to ribosomes that specify the amino acid sequence during protein synthesis.

[0042] In relation to the first aspect, the present invention provides a composition for diagnosing BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient, comprising a preparation for measuring the expression level of FAM167A protein, a fragment thereof, or mRNA encoding said protein, and a kit comprising the same.

[0043] In the present invention, the term "diagnosis" includes determining a subject's susceptibility to a particular disease or disorder, determining whether an individual currently has a particular disease or disorder, determining the prognosis of an individual with a particular disease or disorder, or therametrics (e.g., monitoring an individual's condition to provide information regarding the efficacy of a treatment).

[0044] In the present invention, the term "resistance" is also referred to as "resistance" and means that there is no sensitive reaction to the drug and the efficacy of the drug is not exerted.

[0045] In the present invention, "a preparation for measuring the mRNA expression level of a FAM167A protein, a fragment thereof, or a gene encoding said protein" means a molecule that can be used to confirm the mRNA expression level of a biomarker protein of the present invention, a fragment thereof, or a gene encoding said protein, and is preferably, but not limited to, an antibody or an antigen-binding fragment thereof that specifically binds to the protein or a fragment thereof, or an aptamer, or sense and antisense primers or probes that bind complementarily to the mRNA of the gene.

[0046] In the present invention, the fragment of the FAM167A protein may be an immunogenic fragment, and preferably a fragment having one or more epitopes that can be recognized by an antibody against the protein.

[0047] In the present invention, the term "antibody" is a term known in the art and means a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody means an antibody that specifically binds to the above-mentioned biomarker, and such an antibody can be produced by cloning each gene into an expression vector by a conventional method to obtain a protein encoded by the biomarker gene, and then producing the obtained protein by a conventional method. This also includes partial peptides that can be produced from the above protein, and the partial peptides of the present invention contain at least 7 amino acids, preferably 9 amino acids, and more preferably 12 amino acids or more. The form of the antibody of the present invention is not particularly limited, and the antibody of the present invention includes polyclonal antibodies, monoclonal antibodies, and any part thereof that has antigen-binding ability, and all immunoglobulin antibodies are included. Moreover, the antibody of the present invention The antibody of the present invention also includes special antibodies such as humanized antibodies. Such antibodies against the protein encoded by the biomarker gene of the present invention include all antibodies that can be produced by methods known in the art. For example, the antibody used to detect the above-mentioned biomarker may include not only an intact form having two full-length light chains and two full-length heavy chains, but also a functional fragment of the antibody molecule. The functional fragment of the antibody molecule means a fragment that has at least an antigen-binding function, and includes, but is not limited to, Fab, F(ab'), F(ab')2, Fv, etc.

[0048] In the present invention, the term "aptamer" refers to a single-stranded oligonucleotide, has a function similar to that of an antibody, is also called a chemical antibody, and refers to a nucleic acid molecule that has binding activity to a specific target molecule. The aptamer can have various three-dimensional structures depending on its base sequence, and can have high affinity to a specific substance like an antigen-antibody reaction. The aptamer can inhibit the activity of a specific target molecule by binding to the specific target molecule.

[0049] The aptamer of the present invention may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be linear or ring shaped, but is not limited thereto. Aptamers having binding activity to each of the three biomarker proteins can be easily produced by a person of ordinary skill in the art by a known method with reference to each base sequence.

[0050] In the present invention, the term "primer" refers to a short free 3' terminal hydroxyl group. A short nucleic acid sequence having a hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template. In the present invention, PCR amplification is performed using the sense and antisense primers of the above-mentioned biomarker polynucleotides, and BCR-ABL-independent TKI resistance in CML patients can be diagnosed based on the presence or absence of the desired product. The PCR conditions and the lengths of the sense and antisense primers can be modified based on those known in the art.

[0051] In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which is as short as a few bases to as long as several hundred bases and can form a specific bond with mRNA, and is labeled to confirm the presence or absence of a specific mRNA. The probe can be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, etc. In the present invention, hybridization is performed using a probe complementary to the above-mentioned biomarker polynucleotide, and BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients can be diagnosed based on the presence or absence of hybridization. The selection of an appropriate probe and hybridization conditions can be modified based on those known in the art.

[0052] The primer or probe of the present invention can be chemically synthesized using phosphoramidite solid support method or other widely known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more natural nucleotides with analogs, and internucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphorodiamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

[0053] The base sequence of the preparation for measuring the expression level of a biomarker gene used in the present invention is also interpreted as including a sequence showing substantial identity with a sequence that specifically binds to a biomarker gene, taking into consideration a mutation having biologically equivalent activity. The term "substantial identity" refers to a sequence that shows at least 60% identity, more specifically 70% identity, even more specifically 80% identity, and most specifically 90% identity, when a specific sequence is aligned with any other sequence to maximize its correspondence and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0054] The kit of the present invention may be, but is not limited to, an RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit, a DNA chip kit, a microarray kit, a SAGE (Serial Analysis of Gene Expression) kit, a gene chip kit, an ELISA (Enzyme linked immunosorbent assay) kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit. As a specific example, the kit may be a kit containing essential elements required for performing RT-PCR. For example, the RT-PCR kit may contain, in addition to each primer specific to a biomarker gene, a test tube or other suitable container, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC water (DEPC-water), sterile water, etc. It may also contain a primer pair specific to DNA or RNA used as a quantitative control group.

[0055] The kit of the present invention may include a kit for extracting nucleic acid (eg, total RNA), a fluorescent labeling substance, an enzyme and culture medium for amplifying nucleic acid, instructions for use, and the like.

[0056] 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 a solid phase. Examples of the material of the solid phase include plastic, paper, glass, silicon, etc., and the preferred material of the solid phase is plastic because of its ease of processing. The shape of the solid phase is arbitrary, and may be, for example, a square, a circle, a rectangle, a film type, etc.

[0057] The kit may further include a program capable of performing data analysis or statistical processing. The program may analyze gene or protein expression levels. The data analysis or statistical processing program may be one or more selected from, but not limited to, Skyline Software, ProteoWizard Software, SCIEX OS Software, SPSS Statistics Software, MedCalc Software, MultiQuant Software, MasterView Software, or Cliquid Software.

[0058] In relation to the first aspect, the present invention also relates to the use of a formulation for measuring the expression level of FAM167A protein, a fragment thereof or the mRNA of a gene encoding said protein in the manufacture of a kit for diagnosing BCR-ABL-independent TKI resistance in a CML patient.

[0059] All the above descriptions of the first aspect of the present invention apply equally to this application, and so will be omitted to avoid overcomplicating this specification.

[0060] In the present invention, the present invention provides a method for the treatment of BCR-ABL-related TKI-resistant cells, comprising: We have identified a previously uncharacterized protein, FAM167A, and clinical sample analysis has confirmed that FAM167A is upregulated in cells of BCR-ABL-independent TKI-resistant patients not only after TKI treatment but also at the time of CML diagnosis, and therefore the biomarker protein can be used to diagnose BCR-ABL-independent TKI resistance in CML patients.

[0061] Thus, a second aspect of the present invention relates to an informative method for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myeloid leukemia using the FAM167A biomarker.

[0062] The term "method for providing information" as used herein refers to a method for providing information regarding the presence or absence of BCR-ABL-independent TKI resistance in a CML patient, and means a method for obtaining information regarding BCR-ABL-independent TKI resistance when the mRNA expression level of the biomarker protein according to the present invention, a fragment thereof, or a gene encoding said protein is increased compared to CML without BCR-ABL-independent TKI resistance or TKI-sensitive CML.

[0063] Specifically, the informative method for diagnosing BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient according to the present invention may comprise the following steps: (a) measuring the mRNA expression level of FAM167A protein, a fragment thereof or a gene encoding said protein in a biological sample isolated from a patient with chronic myeloid leukemia; and (b) comparing the expression level measured in step (a) with the mRNA expression level of the same protein, a fragment thereof, or a gene encoding said protein measured in a biological sample isolated from a patient with chronic myeloid leukemia who does not have BCR-ABL-independent TKI resistance.

[0064] The method of providing information of the present invention may further include a step of (c) determining that the patient exhibits resistance to a BCR-ABL-independent TKI if the mRNA expression level of the FAM167A protein, a fragment thereof, or a gene encoding the protein measured in a biological sample isolated from the chronic myeloid leukemia patient in step (a) is 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 not resistant to a BCR-ABL-independent tyrosine kinase inhibitor in step (b).

[0065] In the information providing method of the present invention, the chronic myeloid leukemia patient is a subject for whom resistance to a BCR-ABL-independent TKI is to be diagnosed, and the patient subject is interpreted to mean mammals such as humans, primates including chimpanzees, pets such as dogs and cats, livestock animals such as cows, horses, sheep, and goats, and rodents such as mice and rats.

[0066] In the information providing method of the present invention, the "biological sample" used for analysis includes a biological sample in which the level of a biomarker protein, a fragment thereof, or an mRNA of a gene encoding the protein specific for diagnosis of BCR-ABL-independent TKI resistance, which can be distinguished from a CML patient without BCR-ABL-independent TKI resistance or a TKI-sensitive CML patient, can be confirmed. For example, the biological sample can be CD34 isolated from a CML patient for which the presence or absence of BCR-ABL-independent TKI resistance is to be determined. + It may refer to a biological sample that contains stem / progenitor cells, including, but not limited to, blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymphatic fluid, respiratory tract fluid, intestinal fluid, urogenital fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, tracheal fluid, ascites, cystic tumor fluid, amniotic fluid, or combinations thereof.

[0067] In the information providing method of the present invention, 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 isolation of the protein or mRNA from a biological sample can be carried out using known processes.

[0068] In the present invention, the term "protein level measurement" refers to a process of determining the presence or absence and expression level of the biomarker protein or a fragment thereof in a sample, and the amount of protein can be determined using an antibody that specifically binds to the protein or a fragment thereof. Analytical methods for this include, but are not limited to, western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, mass spectrometry, fluorescence activated cell sorter analysis (FACS analysis), and protein chip technology.

[0069] In the present invention, the term "mRNA level measurement" refers to a process of confirming the presence or absence of mRNA of the biomarker gene of the present invention in a sample and the degree of expression, which can be known by measuring the amount of mRNA. Analytical methods for this include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection method, northern blotting, southern blotting, in situ hybridization, and DNA chip technology.

[0070] For example, the mRNA expression levels of the biomarker genes can be measured by hybridization or gene amplification methods, which can use probes to confirm the presence of genes.

[0071] When the information providing method of the present invention is carried out in a microarray format among hybridization formats, the above-mentioned probes are used as hybridizable array elements and immobilized on a substrate. Preferred substrates are solid or semi-solid supports, including, for example, 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. Such immobilization is carried out by chemical binding methods or covalent binding methods such as UV. For example, the hybridization array elements may be bound to a glass surface modified to include an epoxy compound or an aldehyde group, or may be bound to a polylysine-coated surface by UV. The hybridization array elements may also be bound to the substrate via a linker (Y: ethylene glycol oligomer and diamine).

[0072] In addition, the sample DNA applied to the microarray of the present invention may be labeled and hybridized with the array elements on the microarray. The hybridization conditions may be varied in various ways. In addition, the detection and analysis of the degree of hybridization may be carried out in various ways depending on the labeling substance.

[0073] The label of the probe can provide a signal to detect the presence or absence of hybridization and can be attached to the oligonucleotide. Suitable labels include, but are not limited to, fluorophores (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), terminal deoxynucleotidyl transferase (TdT), chromophores, chemiluminophores, magnetic particles, radioisotopes (P32 or S35), mass labels, electron-dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (e.g., gold), and heptanes with specific binding partners such as antibodies, streptavidin, biotin, digoxigenin, and chelating groups. Labeling can be accomplished by a variety of methods commonly practiced in the art, such as nick translation (n-translation) or other methods. Labeling can be accomplished through ick translation), random priming (Multiprime DNA labelling systems booklet, "Amersham" (1989)) and kination (Maxam & Gilbert, Methods in Enzymology, 65:499 (1986)). Labeling provides signals detectable by fluorescence, radioactivity, chromogenic measurement, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, mass spectrometry, binding affinity, hybridization radio frequency, nanocrystals.

[0074] When using a probe, the probe is hybridized with cDNA molecule. In the present invention, suitable hybridization conditions can be determined in a series of steps by optimization procedures. Such procedures are carried out by those skilled in the art in a series of steps to establish a protocol for use in a laboratory. For example, the 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 the length and GC content of the probe and the target nucleotide sequence.

[0075] After the hybridization reaction, a hybridization signal generated through the hybridization reaction is detected. The hybridization signal can be detected in various ways depending on the type of label bound to the probe. For example, when the probe is labeled with an enzyme, the presence or absence of hybridization can be confirmed by reacting the hybridization reaction product with a substrate of the enzyme. Enzyme / substrate combinations that can be used include peroxidase (e.g., horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl), and the like. and pyrocatechol, TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; alkaline phosphatase and bromochloroindolylphosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrate; glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate), etc. When the probe is labeled with gold particles, it can be detected by silver staining using silver nitrate.

[0076] In the present invention, it has been confirmed that neutralization of FAM167A restores TKI sensitivity in cells with BCR-ABL-independent TKI resistance and reverses TKI resistance in a mouse tumor model, and therefore it is possible to provide a therapeutic agent for CML exhibiting BCR-ABL-independent TKI resistance and a BCR-ABL-independent TKI resistance inhibitor that targets the FAM167A protein biomarker.

[0077] Therefore, a third aspect of the present invention relates to a pharmaceutical composition for preventing or treating CML exhibiting BCR-ABL-independent TKI resistance and a pharmaceutical composition for inhibiting BCR-ABL-independent TKI resistance in CML patients, comprising as an active ingredient a FAM167A protein inhibitor; an inhibitor of expression of the gene encoding the FAM167A protein; or a mixture thereof.

[0078] In the present invention, the FAM167A protein inhibitor may include a preparation that reduces the expression or the function or activity of the FAM167A protein.

[0079] In the present invention, the expression inhibitor of the gene encoding the FAM167A protein may include a preparation that reduces the mRNA expression of the FAM167A gene.

[0080] The FAM167A protein inhibitor may be a peptide or a compound that binds to the FAM167 protein and reduces non-canonical NF-κB activity. Such inhibitors may be selected through the screening methods exemplified below, such as protein structure analysis, or may be designed using methods known in the art.

[0081] Specifically, the FAM167A protein inhibitor may include, but is not limited to, any one or more selected from the group consisting of small molecule compounds, peptides, peptide mimetics, aptamers, antibodies and natural products that specifically bind to FAM167A protein.

[0082] The expression inhibitor of the gene encoding the FAM167A protein can be an inhibitor that binds to the gene itself and inhibits transcription, or binds to the mRNA transcribed from the gene and inhibits the translation of the mRNA.For example, the expression inhibitor of the gene can include, but is not limited to, any one or more selected from the group consisting of antisense nucleotides, siRNAs and shRNAs that complementarily bind to the mRNA of the FAM167A gene.

[0083] In the present invention, the term "siRNA" refers to a double-stranded RNA that induces RNA interference through cleavage of the mRNA of a target gene, and is composed of an RNA strand with a sense sequence having a sequence similar to that of the mRNA of a target gene and an RNA strand with an antisense sequence having a complementary sequence thereto.

[0084] The siRNA may be in the form of an siRNA itself synthesized in vitro or an siRNA expressed by inserting a base sequence encoding the siRNA into an expression vector.

[0085] In the present invention, the term "vector" refers to a genetic construct containing foreign DNA inserted into a genome that encodes a polypeptide.

[0086] The vectors related to the present invention are vectors in which the nucleic acid sequence that inhibits the gene is inserted into the genome, and these vectors include DNA vectors, plasmid vectors, cosmid vectors, bacteriophage vectors, yeast vectors, and virus vectors. It is possible.

[0087] Furthermore, the antisense has a sequence complementary to the whole or part of the mRNA sequence transcribed from the FAM167A gene or a fragment thereof, and can bind to the mRNA to inhibit the expression of the FAM167A gene or a fragment thereof.

[0088] In addition, the shRNA (short hairpin RNA) may be prepared by a conventional method by targeting a common base sequence site of human or mouse shRNA.

[0089] In the present invention, the BCR-ABL-independent TKI-resistant chronic myeloid leukemia can have increased NF-κB activity compared to TKI-sensitive CML.

[0090] In the present invention, the pharmaceutical composition may be administered in combination with a TKI.

[0091] In the present invention, the term "co-administration" means that different components are administered to a subject together, meaning that each component can be administered at the same time, or in any order, or sequentially at different times to achieve the desired therapeutic effect.

[0092] Thus, the pharmaceutical compositions of the present invention may be administered simultaneously, separately or sequentially with the TKI.

[0093] In the present invention, the TKI is crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, ibrutinib, cabozantinib, gefitinib, erlotinib, lapatinib, and the like. patinib, vandetanib, afatinib, osimertinib, ruxolitinib, tofacitinib, axitinib, lenvatinib, nintedanib, pazopanib, regorafenib, sorafenib or sunitinib, but are not limited to these.

[0094] In the present invention, the terms "prevention" and / or "treatment" refer to any action that inhibits or delays the onset of a disease or condition, any action that improves or beneficially alters the disease or condition, and any action that slows, halts or reverses the progression of a disease or condition.

[0095] The pharmaceutical composition of the present invention may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0096] Specifically, the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate. Cate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil can be used.

[0097] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and can include various excipients, such as wetting agents, sweeteners, flavorings, preservatives, etc., in addition to water and liquid paraffin, which are frequently used simple diluents.

[0098] In other embodiments, carriers included in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), crosslinked silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic matrices, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycol or wool paper.

[0099] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. As base materials for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. may be used.

[0100] The pharmaceutical composition according to the present invention can be prepared in an aqueous solution for parenteral administration, preferably in a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline. Aqueous injection suspensions can contain substances capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

[0101] Furthermore, the pharmaceutical composition of the present invention may further contain, in addition to the above-mentioned components, a lubricant, a wetting agent, an emulsifying agent, a suspending agent, a preservative, or the like.

[0102] The pharmaceutical composition of the present invention can be administered systemically or locally, and can be formulated into a suitable dosage form for such administration by known techniques.For example, when administered orally, it can be mixed with an inert diluent or edible carrier, sealed into a hard or soft gelatin capsule, or compressed into a tablet.For oral administration, the active compound can be mixed with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.

[0103] 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 effective amount of the active ingredient of the pharmaceutical composition of the present invention is determined based on the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form, and the age and weight of the patient. It can be adjusted depending on various factors including general health condition, sex and diet, administration time, administration route and excretion rate of the composition, treatment period and concurrently used drugs.

[0104] In relation to the third aspect, the present invention provides a method for treating CML with BCR-ABL-independent TKI resistance and a method for inhibiting BCR-ABL-independent TKI resistance, comprising the step of administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of a gene encoding a FAM167A protein, or a mixture thereof to a CML patient with BCR-ABL-independent TKI resistance.

[0105] In the present invention, the FAM167A protein inhibitor or the expression inhibitor of the gene encoding the FAM167A protein used in the method and the method of administering the same are as described above, and therefore, the description thereof will be omitted to avoid overly complicating this specification.

[0106] In addition, the CML patient subjects exhibiting BCR-ABL-independent TKI resistance are the same as the patient subjects described in the information provision method, and therefore, description thereof will be omitted to avoid excessive complexity of this specification.

[0107] In relation to said third aspect, the present invention also provides a method for diagnosing BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient and treating BCR-ABL-independent TKI-resistant chronic myeloid leukemia comprising the steps of:

[0108] (a) measuring the mRNA expression level of FAM167A protein, a fragment thereof or a gene encoding said protein in a biological sample isolated from a patient with chronic myeloid leukemia; (b) diagnosing the presence of BCR-ABL-independent TKI resistance when the expression level measured in step (a) is increased relative to the mRNA expression level of the same protein, a fragment thereof, or a gene encoding said protein measured in a biological sample isolated from a chronic myeloid leukemia patient who does not have BCR-ABL-independent TKI resistance; and (c) administering to a patient diagnosed in step (b) as exhibiting BCR-ABL-independent TKI resistance a therapeutically effective amount of a FAM167A protein inhibitor, an inhibitor of expression of a gene encoding a FAM167A protein, or a mixture thereof.

[0109] In relation to said third aspect, the present invention also provides a method for diagnosing BCR-ABL-independent TKI resistance and inhibiting BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient, comprising the steps of: (a) measuring the mRNA expression level of FAM167A protein, a fragment thereof or a gene encoding said protein in a biological sample isolated from a patient with chronic myeloid leukemia; (b) diagnosing the presence of BCR-ABL-independent TKI resistance when the expression level measured in step (a) is increased relative to the mRNA expression level of the same protein, a fragment thereof, or a gene encoding said protein measured in a biological sample isolated from a chronic myeloid leukemia patient who does not have BCR-ABL-independent TKI resistance; and (c) administering to a patient diagnosed in step (b) as exhibiting BCR-ABL-independent TKI resistance a therapeutically effective amount of a FAM167A protein inhibitor, an inhibitor of expression of a gene encoding a FAM167A protein, or a mixture thereof.

[0110] Similarly, the FAM167A protein inhibitor or the expression inhibitor of the gene encoding the FAM167A protein used in the method and the method for administering the same are as described above, and therefore will not be described here in order to avoid overly complicating this specification.

[0111] In addition, the CML patient subjects exhibiting BCR-ABL-independent TKI resistance are the same as the patient subjects described in the information provision method, so the description thereof will be omitted to avoid excessive complexity of this specification.

[0112] In the present invention, 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, etc. are the same as those described in the pharmaceutical composition, so the description thereof will be omitted to avoid overly complicating this specification.

[0113] In relation to the third aspect, the present invention also provides the use of a FAM167A protein inhibitor; an inhibitor of expression of a gene encoding a FAM167A protein; or a mixture thereof in the manufacture of a medicament for preventing or treating CML exhibiting BCR-ABL-independent TKI resistance, or in the manufacture of a medicament for inhibiting BCR-ABL-independent TKI resistance in a CML patient.

[0114] In relation to the third aspect, the present invention also relates to the use of a FAM167A protein inhibitor; an inhibitor of expression of a gene encoding a FAM167A protein; or a mixture thereof in the manufacture of a medicament for preventing or treating chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance.

[0115] In relation to the third aspect, the present invention also relates to the use of a FAM167A protein inhibitor; an inhibitor of expression of a gene encoding a FAM167A protein; or a mixture thereof in the manufacture of a medicament for inhibiting BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient exhibiting BCR-ABL-independent TKI resistance.

[0116] All the above descriptions of the third aspect of the present invention apply equally to this application, and therefore will not be repeated to avoid overcomplicating this specification.

[0117] In the present invention, it has been confirmed that neutralization of FAM167A restores TKI sensitivity in cells with BCR-ABL-independent TKI resistance and reverses TKI resistance in a mouse tumor model, and therefore a criterion is provided for determining whether a therapeutic agent for CML exhibiting BCR-ABL-independent TKI resistance or a BCR-ABL-independent TKI resistance inhibitor is present based on the presence or absence of inhibition of the FAM167A protein biomarker.

[0118] Therefore, a fourth aspect of the present invention relates to a method for screening for a therapeutic agent or an inhibitor of BCR-ABL-independent TKI resistance for CML exhibiting BCR-ABL-independent TKI resistance, using the FAM167A biomarker.

[0119] Specifically, the screening method of the present invention may comprise the following steps: (a) treating a cell line or animal model exhibiting BCR-ABL-independent TKI resistance with a candidate agent; and (b) determining whether or not there is inhibition of FAM167A protein and / or inhibition of expression of the gene encoding said protein in a cell line or animal model treated with said candidate substance;

[0120] In the present invention, the candidate substance may be a substance that inhibits the transcription or translation into mRNA or protein in the FAM167A gene base sequence, or may be a drug that inhibits the function or activity of the FAM167A protein, which is predicted by conventional selection methods, or may be randomly selected individual nucleic acids, proteins, peptides, other extracts, natural products, compounds, etc.

[0121] The expression level, protein level or activity of the gene can then be measured in a biological sample isolated from the cell line or animal model treated with the candidate substance, and if the measurement results show that the expression level, protein level or activity of the gene is reduced, the candidate substance can be determined to be a therapeutic agent for CML exhibiting BCR-ABL-independent TKI resistance and a BCR-ABL-independent TKI resistance inhibitor.

[0122] Therefore, the screening method of the present invention may further include a step of (c) determining that the candidate substance is a therapeutic agent for CML exhibiting BCR-ABL-independent TKI resistance or a BCR-ABL-independent TKI resistance inhibitor if the candidate substance inhibits FAM167A protein or inhibits expression of the gene encoding the protein.

[0123] In the present invention, the method for measuring the expression level, protein level or activity of the gene described above can be carried out by various methods known in the art, and is the same as that described in the information providing method, so the description thereof will be omitted to avoid overly complicating this specification.

[0124] In the present invention, the candidate substance acts as a leading compound in the process of developing a therapeutic agent for CML exhibiting BCR-ABL-independent TKI resistance and a BCR-ABL-independent TKI resistance inhibitor, and a new therapeutic agent for CML and a TKI resistance inhibitor can be developed by modifying and optimizing its structure so that the lead compound can exhibit an effect of inhibiting the function of the FAM167A gene or a protein expressed therefrom.

[0125] The present invention will be described in more detail below based on examples. However, the present invention can be modified in various ways and can have various forms, and the specific examples and explanations described below are merely intended to help understand the present invention and are not intended to limit the present invention to the specific disclosed forms. The scope of the present invention should be understood to include all modifications, equivalents, or alternatives that fall within the spirit and technical scope of the present invention. EXAMPLES

[0126] [Example 1] Identification of differentially expressed genes and transcription factors regulating them in TKI-resistant CML cells 1-1.Cell culture The human CML cell line K562S was obtained from the Korean Cell Line Bank. KCL22S was obtained from the American Type Culture Collection (ATCC). The K562R cell line was generated from K562S by treating cells with gradually increasing concentrations of TKIs. All cell lines were maintained at 37°C and 5% CO2 in a humidified cell culture incubator and cultured in RPMI (Roswell Park Memorial Institute) 1640 medium (HyClone) supplemented with 10% fetal bovine serum (HyClone), 100 U / ml penicillin, and 100 μg / ml streptomycin. To maintain TKI resistance, K562R and KCL22R cells were cultured in medium supplemented with 1 μM imatinib.

[0127] 1-2. Microarray and RNA-seq analysis TKI resistance without BCR-ABL kinase domain mutations (i.e., BCR-ABL-independent TKI resistance) is not well known. This prompted the inventors to identify genes differentially expressed in TKI-resistant CML cells and to search for genes that potentially play a role in BCR-ABL-independent TKI resistance (Figure 1a). Thus, BCR- A TKI-resistant CML cell line, K562R (Figures 1b to 1e), lacking a mutation in the ABL kinase domain was generated as described in Example 1-1 above, and gene expression was analyzed in TKI-sensitive K562S cells, K562R cells, and imatinib-treated K562R cells. Microarray and RNA sequencing (RNA-seq) analyses were also performed to eliminate false positives due to the analytical method.

[0128] Microarray and 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). Labeled cRNA was hybridized to Human HT-12 v4 Expression BeadChip (Illumina) and arrays were scanned with a Bead Array Reader confocal scanner (Illumina). For RNA-seq, libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) according to the Illumina TruSeq protocol. Paired-end sequencing was performed using a NovaSeq 6000 system (Illumina). Genes showing a fold change ≥2 were defined as differentially expressed and displayed in the heatmap generated in the Multiple Experiment Viewer.

[0129] 789 differentially expressed (i.e., fold change ≥ 2) genes were stringently selected between K562S and K562R (Fig. 1f and Fig. 1g).

[0130] 1-3. In silico analysis In addition to identifying differentially expressed genes, we sought to identify transcription factors that could specifically regulate differentially expressed genes in BCR-ABL-independent resistant CML cells. For in silico analysis of activated transcription factors, information on transcription factors associated with their respective binding sites in differentially expressed or randomly selected gene promoters was obtained from the GeneCards database (https: / / www.genecards.org / ) and plots were generated using Cytoscape. The enrichment score of transcription factors was calculated using the following equation: 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).

[0131] Through in silico analysis of transcription factors involved in differential gene expression in BCR-ABL-independent TKI-resistant CML cells, we found that AP-1 and NF-κB were the most enriched transcription factors among regulators of differentially expressed genes compared to randomly selected genes (Figure 1h-m). AP-1 and NF-κB showed significantly higher enrichment scores than other transcription factors, with only the scores for these two transcription factors falling outside the mean by 3 standard deviations.

[0132] 1-4. Luciferase reporter analysis To confirm the differences in AP-1 and NF-κB activity in TKI-resistant cell lines, AP-1 and NF-κB reporter assays were performed in K562R cells, K562S cells, and imatinib-treated K562R cells to investigate the effects of TKI treatment.

[0133] For this purpose, K562S and K562R cells were transfected with Lipofectamine 2000 (Invitrogen) as described in the literature [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.] and an NF-κB-dependent reporter construct (pBIIx-luc) as described in the literature, or an AP-1-dependent reporter construct (AP-1-luc) as described in the literature (Zhang D, Zhang G, Hayden MS, Greenblatt MB, Bussey C, Flavell RA, et al. A toll-like receptor that prevents infection by uropathogenic bacteria. Science. 2004;303(5663):1522-6.) and other plasmids were co-transfected and treated as described in each literature. Twenty-four hours after transfection, luciferase activity was measured using the Dual-luciferase Reporter Assay Kit (Promega) and normalized to Renilla luciferase activity.

[0134] The results showed that NF-κB activity, but not AP-1, was significantly higher in K562R cells than in K562S cells. This activity was also significantly higher in K562R cells treated with imatinib, whereas AP-1 activity was not significantly different (Fig. 1n). This suggests that NF-κB contributes to BCR-ABL-independent TKI resistance by regulating gene expression. To confirm the genes associated with increased NF-κB activity in K562R cells, we selected seven differentially expressed genes (FAM167A, AIF1L, ACSS1, S100A4, LY6G6D, CYP11A1, and NR5A1) that showed similar expression patterns as pattern NF-κB activity using gene expression profile data from K562S, K562R, and imatinib-treated K562R cells.

[0135] 1-5. Quantitative reverse transcription PCR (qRT-PCR) qRT-PCR was performed to confirm the expression levels of selected genes. Total cellular RNA was isolated using RNeasy Mini Kit (Qiagen) and cDNA was prepared as 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. The primer sequences used for qRT-PCR are shown in Table 1, and the qRT-PCR results are shown in Figure 1o.

[0136] [Table 1]

[0137] [Example 2] FAM167A role in the non-canonical NF-κB pathway 2-1. Luciferase reporter analysis of selected genes To screen the selected genes, NF-κB activity was confirmed by luciferase reporter assay in K562S cells transfected with plasmids encoding the indicated genes, which were prepared by cloning HA-tagged S100A4, CYP11A1, ACSS1, LY6G6D, NR5A1, AIF1L, and FAM167A into the MigR1 vector.

[0138] Among the seven selected genes, only FAM167A significantly increased NF-κB activity when ectopically expressed (Fig. 2a). Also, compared with the other genes, ectopically expressed FAM167A increased the viability of K562S cells in the presence of imatinib (Fig. 2b). Bioinformatic analysis using SecretomeP (http: / / www.cbs.dtu.dk / services / SecretomeP / ), which predicts the secretion of non-classical proteins, showed that FAM167A had a high score (NN-score = 0.905) (Fig. 2c, A), despite the absence of a signal sequence for secretion.

[0139] 2-2. Preparation of cell fractions and immunoblot analysis Secretion of FAM167A was analyzed by immunoblot analysis of K562R cell lysates and culture supernatants.

[0140] Cell lysates were extracted by boiling cells in SDS sample buffer, and proteins in the culture supernatant were concentrated through precipitation with acetone and then extracted by boiling in SDS sample buffer.

[0141] Next, for immunoblot analysis, proteins were separated by SDS-polyacrylamide gel electrophoresis on 8-15% gels and then transferred to polyvinylidene difluoride membranes. The membranes were probed with anti-FAM167A antibody (sc-393999, Santa Cruz Biotechnology) or Erbin antibody (NBP2-13968, Novus Biologicals) as indicated. The analysis confirmed that FAM167A was secreted by these cells (Figure 2cB).

[0142] 2-3. Confirmation of NF-κB activation function of FAM167A To directly test the NF-κB activation function, we used recombinant FAM167A protein (MBS1363522, MyBioSource) and a neutralizing antibody specific for FAM167A (sc-393999, Santa Cruz Biotechnology) to increase and decrease FAM167A levels, respectively, during cell culture, followed by NF-κB luciferase reporter analysis.

[0143] Consistent with the bioinformatic analysis results, treatment of K562S cells with soluble recombinant FAM167A increased NF-κB activity (Fig. 2d), whereas neutralization of FAM167A in K562R cell cultures decreased NF-κB activity (Fig. 2e), suggesting that secreted FAM167A protein functions as an inducer of the NF-κB pathway.

[0144] 2-4. Confirmation of NF-κB activation pathway To determine which NF-κB pathway is activated in K562R cells, we treated them with SN50 (481480, Sigma-Aldrich), which inhibits the canonical NF-κB pathway, or transiently expressed NIK-DN, which suppresses the non-canonical NF-κB pathway, and then performed NF-κB luciferase reporter analysis. For transient expression of NIK-DN, we used the same method as in [Park SG, Ryu HM, Lim SO, Kim YI, Hwang SB, Jung G. Interferon-gamma inhibits hepatitis We used pFLAG-NIK-DN, which is described in [B virus-induced NF-kappaB activation through nuclear localization of NF-kappaB-inducing kinase. Gastroenterology. 2005;128(7):2042-53.].

[0145] Although SN50 treatment, which inhibits the canonical NF-κB pathway, did not significantly reduce NF-κB activity in K562R cells, SN50 treatment blocked additional lipopolysaccharide (LPS)-stimulated increases in NF-κB activity (Figure 2f). In contrast, transient expression of dominant-negative NIK (NIK-DN), which suppresses the non-canonical NF-κB pathway, significantly reduced NF-κB activity in K562R cells to levels similar to those observed in unstimulated K562S cells (Figure 2g).

[0146] 2-5. Immunoblot analysis for NIK and p100 / p52 Non-canonical NF-κB activation depends on NIK accumulation through blocking ubiquitination-dependent NIK degradation, which induces the processing of p100 to p52 and subsequently to the p52-containing Nuclear translocation of the dimer follows. Therefore, we confirmed whether the non-canonical NF-κB pathway was activated in K562R cells through immunoblot analysis for NIK and p100 / p52 in K562S and K562R cells.

[0147] The membrane was stained with anti-NIK (4994, Cell Signaling Technology), anti-p100 / p52 (sc-7386X, Santa Clara, CA) as indicated. The densitometry was performed using ImageJ software using GAPDH as an internal control.

[0148] Analysis showed that the level and activity of NIK, which processes p100 with p52, was even higher in K562R cells than in K562S cells (FIGS. 2h and 2i).

[0149] 2-6. Electrophoretic mobility shift assay (EMSA) The nuclear fraction was extracted as follows. First, cells were suspended in hypotonic buffer (10 mM HEPES, pH 7.9; 10 mM KCl; 1.5 mM MgCl2; 0.5 mM DTT; 0.5 mM PMSF) and incubated on ice for 15 min. Then, 0.05% Nonidet P-40 was added and the lysate was passed five times through a 25-gauge needle. 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 the cytoplasmic fraction. The pellet containing the cell nuclei was washed with hypotonic buffer, suspended in hypertonic buffer (20 mM HEPES, pH 7.9; 420 mM NaCl; 1.5 mM MgCl2; 25% glycerol; 0.2 mM EDTA; 0.5 mM DTT; 0.5 mM PMSF; 1 μg / mL leupeptin, 1 μg / mL aprotinin, 1 μg / mL pepstatin A), and incubated on ice for 30 min with vortexing every 10 min. The nuclear lysate was centrifuged (16,000 Хg, 5 min, 4 °C) and the resulting supernatant was stored at -80 °C as the nuclear fraction.

[0150] For supershift analysis, nuclear fractions were incubated with anti-p52 antibody (4882, Cell Signaling Technology) or isotype control (H9658, Sigma-Aldrich) in binding buffer (5 mM Tris, pH 7.5; 25 mM KCl; 0.5 mM EDTA; 2.5% glycerol; 0.5 mM DTT; 0.1 μg / μl poly(dI / dC), 0.5 mg / ml BSA) for 20 min at room temperature, followed by additional incubation with biotinylated double-stranded NF-κB probe (SEQ ID NO: 23: 5'-AGTTGAGGGGACTTTCCCAGG-3') for 20 min at room temperature. Reaction samples were separated through a 6% non-denaturing polyacrylamide gel and transferred to a nylon membrane. The probe on the membrane was visualized using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific) and quantified with ImageJ.

[0151] Supershift analysis performed by EMSA also revealed higher NF-κB activity and p52 supershift levels in K562R cells (Fig. 2j).

[0152] 2-7. Confirmation of the effect of FAM167A in activating the non-canonical NF-κB pathway Based on the above results, we confirmed whether FAM167A was responsible for activating the non-canonical NF-κB pathway.

[0153] First, NF-κB luciferase was induced in K562S cells 24 hours after treatment with 100 ng / ml recombinant FAM167A and transfection with a plasmid encoding NIK-DN. Interestingly, reporter activity was confirmed and NF-κB activity stimulated by recombinant FAM167A was reduced in cells with transient expression of NIK-DN (FIG. 2k).

[0154] Second, we performed immunoblot analysis of NIK in K562R cells after treatment with 2 μg / ml of anti-FAM167A neutralizing antibody or isotype control for 12 hours. Consistent with the above results, neutralization of FAM167A greatly reduced NIK levels in K562R cells (Figure 2l).

[0155] Finally, immunoblot analysis of p100 / p52 was performed in K562S and K562R cells after treatment with the indicated concentrations of recombinant FAM167A for 12 h. Treatment of K562S and K562R cells with recombinant FAM167A increased the processing of p100 to p52 (Fig. 2m).

[0156] Thus, the data indicate that FAM167A activates the non-canonical NF-κB pathway to a greater extent in K562R cells than in K562S cells.

[0157] [Example 3] Identification of FAM167A-binding receptors 3-1.Flow cytometry We hypothesized that the processing and neutralization of secreted FAM167A could effectively regulate the non-canonical NF-κB pathway, and thus the receptor for FAM167A could be expressed on the cell surface.To evaluate the expression of potential receptors for FAM167A, we first labeled K562S and K562R cells with Myc-tagged recombinant FAM167A, followed by fluorescent dye-conjugated anti-Myc antibody staining and flow cytometry.

[0158] Specifically, for staining of surface receptors, cells were incubated with Myc-tagged FAM167A (MyBioSource) and then stained with Alexa Fluor 488-conjugated anti-Myc antibody (9B11, Cell Signaling Technology).

[0159] Flow cytometry and cell sorting were performed using a Guava EasyCyte HT (Millipore), FACSCanto II (BD Biosciences) or FACSAria III (BD Biosciences), and data were analyzed with FlowJo software (TreeStar).

[0160] The analysis results suggested that the receptor for FAM167A was expressed on the cell surface and that the level in K562R cells was higher than that in K562S cells (Fig. 3a).

[0161] After confirming the presence of the receptor on the cell surface, to isolate the receptor for identification, the receptor was immunoprecipitated in K562R cells using Myc-tagged recombinant FAM167A bound to protein G Sepharose® beads and anti-Myc antibody (Figure 3b).

[0162] Silver staining of proteins immunoprecipitated with Myc-tagged FAM167A was performed using the Pierce Silver Stain Kit (24612, Thermo Fisher Scientific). The silver staining showed two distinct bands compared to the background detected in the control sample immunoprecipitated without FAM167A (Figure 3c). LC-MS / MS analysis confirmed one band (~30 kDa) with recombinant FAM167A and the other band (~120 kDa) with DSG1, a known cell adhesion molecule (Figures 3d-h).

[0163] 3-2. Co-immunoprecipitation using K562R cell lysates Cells were lysed with lysis buffer (20 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Triton X-100; 10% glycerol; 2 mM EDTA; 1 μg / ml leupeptin; 1 μg / ml aprotinin; 1 μg / ml pepstatin A; 0.1 mM PMSF) for 30 min on ice. The lysates were then centrifuged for 15 min at 15,000×g and 4°C, and the supernatants were incubated with anti-FAM167A (sc-393999, Santa Cruz Biotechnology) at 4°C with rotation. Protein G Sepharose® beads (GE Healthcare) were added and incubated at 4°C with rotation. After washing four times with lysis buffer, immunoprecipitates were eluted in SDS sample buffer by boiling, followed by immunoblot analysis. Band intensities were analyzed with ImageJ software.

[0164] Through this, we confirmed that FAM167A binds to DSG1 (Figure 3i).

[0165] 3-3.qRT-PCR and immunoblot analysis for DSG1 To confirm that the upregulated surface expression of DSG1 was mediated by increased total DSG1 protein levels, DSG1 mRNA and protein expression were measured by qRT-PCR and immunoblot analysis, respectively. The sequences of the primers used for qRT-PCR are shown in Table 2.

[0166] [Table 2]

[0167] Interestingly, there was no difference in mRNA or protein expression between K562S and K562R cells (Fig. 3j and k). In contrast, cell surface expression of DSG1 was even higher in K562R cells (Fig. 3a and l).

[0168] 3-4. Immunofluorescence staining and NF-kB luciferase reporter analysis For immunofluorescence staining, fixed and permeabilized cells were stained with anti-DSG1 antibody (129204, R&D Systems) followed by Alexa Fluor 488-conjugated anti-mouse IgG antibody (Invitrogen). Nuclei were visualized with Hoechst 33342 (Sigma-Aldrich) staining. Confocal images were acquired using an FV1000 confocal microscope (Olympus) and the accompanying FV10-ASW software.

[0169] NF-κB luciferase reporter activity was also analyzed in K562S cells 24 h after transfection with a plasmid encoding FAM167A and treatment with the indicated concentrations of anti-FAM167A neutralizing antibodies.

[0170] Immunofluorescence staining and NF-κB luciferase reporter activity assay confirmed that DSG1 was primarily restricted to the cell surface of K562R cells, but not K562S cells. (Fig. 3m). FAM167A was also colocalized with DSG1 mainly on the surface of K562R cells (Fig. 3n). Although FAM167A was ectopically expressed in K562S cells, FAM167A-induced NF-κB activation was greatly suppressed by treatment with anti-FAM167A neutralizing antibody (Fig. 3o), indicating that secreted FAM167A activates the non-canonical NF-κB pathway. Taken together, these results indicate that DSG1 is a receptor for FAM167A and its surface expression is even higher in K562R cells than in K562S cells.

[0171] [Example 4] Confirmation of FAM167A regulation of NIK ubiquitination through DSG1 4-1. Confirmation of NF-κB activity in DSG1-knockdown K562R cells Because surface expression of DSG1 was even higher in K562R cells than in K562S cells (Figures 3a and 3l), we hypothesized that DSG1 knockdown could reduce non-canonical NF-κB activity in K562R cells. To test this, we lentivirally transduced K562R cells with DSG1-specific short hairpin RNA (shRNA) constructs to knockdown DSG1 expression (Figure 4a), followed by NF-κB luciferase reporter analysis and immunoblot analysis for p100 / p52. DSG1-specific shRNA lentiviral particles (sc-35224-V) and control shRNA lentiviral particles (sc-108080) were obtained from Santa Cruz Biotechnology.

[0172] Unexpectedly, DSG1 knockdown increased NF-κB activity and p100 processing to p52 in K562R cells (FIGS. 4b and 4c).

[0173] 4-2. Confirmation of NF-κB activity in cells transiently expressing DSG1 or DSG1 and Erbin NF-κB activity was measured after transient expression of DSG1 and its interacting protein Erbin in K562R cells with or without treatment with anti-FAM167A neutralizing antibody. DSG1 was expressed using a plasmid in which DSG was cloned into the MigR1 vector, and Erbin was expressed using pClneo-Myc-Erbin (Addgene).

[0174] NF-κB activity was reduced by transient expression of DSG1 and further reduced by transient expression of both DSG1 and Erbin (Fig. 4d). Moreover, transient expression of DSG1 and Erbin in the presence of FAM167A-neutralizing antibody caused little further reduction in NF-κB activity (not significant, p>0.05) (Fig. 4d). These data indicate that DSG1 and Erbin inhibit, whereas FAM167A activates, non-canonical NF-κB through the same pathway.

[0175] 4-3. Confirmation of Erbin expression level To further explore the basic molecular mechanism of this pathway, we first confirmed the mRNA and protein expression levels of Erbin in K562S and K562R cells by qRT-PCR and immunoblot analysis, respectively. The primer sequences used for qRT-PCR are shown in Table 3, and anti-Erbin antibody (NBP2-13968, Novus Biologicals) was used for immunoblot analysis.

[0176] [Table 3]

[0177] Similar to DSG1 mRNA and protein levels (FIGS. 3f and 3g), Erbin mRNA and protein levels were not significantly different between K562S and K562R cells (FIGS. 4e and 4f).

[0178] 4-4. Confirmation of the interaction between Erbin and NIK through co-immunoprecipitation analysis Because NIK is a core component of the non-canonical NF-κB pathway, we performed co-immunoprecipitation analysis to investigate the interaction between DSG1, Erbin and NIK.

[0179] First, cells were lysed with lysis buffer (20 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Triton X-100; 10% glycerol; 2 mM EDTA; 1 μg / ml leupeptin; 1 μg / ml aprotinin; 1 μg / ml pepstatin A; 0.1 mM PMSF) on ice for 30 min. Then, the lysate was centrifuged at 15,000×g and 4°C for 15 min, and the supernatant was incubated with anti-DSG1 antibody (32-6000, Invitrogen) at 4°C with rotation. Protein G Sepharose® beads (GE Healthcare) were added and incubated at 4°C with rotation. After washing four times with lysis buffer, the immunoprecipitates were eluted by boiling in SDS sample buffer, followed by immunoblot analysis. Band intensity was analyzed with ImageJ software.

[0180] We showed that Erbin binding to DSG1 was not significantly different between K562S and K562R cells, but NIK binding to DSG1 was decreased in K562R cells (Figure 4g). To further determine whether FAM167A causes changes in NIK binding to DSG1, we neutralized FAM167A protein in K562R cells and assessed NIK binding to DSG1. When FAM167A was neutralized, increased binding could be observed (Figure 4h).

[0181] 4-5. Confirmation of NIK ubiquitination through immunoblot analysis Because NIK is regulated through ubiquitination-mediated degradation, we sought to investigate NIK ubiquitination through immunoblot analysis after FAM167A neutralization in K562R cells.

[0182] Specifically, NIK immunoprecipitation from K562R cells treated with 2 μg / ml of anti-FAM167A neutralizing antibody or isotype control and 10 μM MG132 (474790, Sigma-Aldrich) for 3 hours was performed using an anti-ubiquitin antibody (sc-8017, Santa Clara, CA). NIK ubiquitination was confirmed using a ELISA kit (Cruz Biotechnology).

[0183] Immunoblot analysis showed that NIK ubiquitination was increased when FAM167A was neutralized (Fig. 4i). NIK ubiquitination correlated with DSG1 and Erbin. Although NIK is regulated by changes in its interaction (Figure 4g-i), these proteins have no reported ubiquitin ligase function. To identify the ligase involved in NIK ubiquitination, we performed co-immunoprecipitation analysis against 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 were purchased from Cell Signaling Technology. The co-immunoprecipitation analysis did not find any interactions with these antibodies (Figure 4j).

[0184] Overall, these results suggest that FAM167A not only activates the non-canonical NF-κB pathway by regulating the binding of NIK to DSG1 and Erbin, but also activates the subsequent ubiquitination, which may explain the relationship between non-canonical NF-κB activity and cell surface expression of DSG1 ( Figure 4 k).

[0185] [Example 5] Confirmation of the role of FAM167A in BCR-ABL-independent TKI resistance 5-1. Confirmation of cell viability by suppression of non-canonical NF-κB pathway after TKI treatment To investigate whether FAM167A is responsible for BCR-ABL-independent TKI resistance, we first investigated the role of the non-canonical NF-κB pathway by checking the viability of K562R cells transfected with a plasmid encoding NIK-DN after treatment with imatinib (sc-202180, Santa Cruz Biotechnology) or nilotinib (17050, BioVision) for 3 days.

[0186] For cell viability assays, K562S or K562R cells were seeded in 96-well plates and treated as indicated before transient transfection with expression constructs. After 72 h, cell viability was measured using WST reagent (DoGenBio).

[0187] Suppression of the non-canonical NF-κB pathway by transient expression of NIK-DN effectively reduced the viability of K562R cells in the presence of TKIs (FIGS. 5a and 5b).

[0188] 5-2. Confirmation of cell viability by anti-FAM167A neutralizing antibody treatment after TKI treatment Furthermore, cell viability assays and apoptosis analysis were performed to confirm the viability of K562R cells after treatment with 2 μg / ml of anti-FAM167A neutralizing antibody or isotype control for 3 days in the presence of imatinib or nilotinib.

[0189] For apoptosis analysis, K562S or K562R cells were seeded in 6-well plates and treated as indicated. After 72 h, 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 cell analyzer (Millipore).

[0190] Analysis of cell viability (by WST assay) and apoptosis (measured by flow cytometry) showed that neutralization of FAM167A significantly reduced the viability of K562R cells (Figure 5a and 5c) and increased apoptosis in the presence of TKIs (Figure 5d). Thus, FAM167A-mediated activation of the non-canonical NF-κB pathway is critical for TKI resistance.

[0191] [Example 6] Confirmation of the role of FAM167A in BCR-ABL-independent TKI resistance in vivo A mouse xenograft model was established by subcutaneous injection of K562R cells into nude mice. We then investigated the effect of FAM167A on TKI resistance in vivo.

[0192] 6-1. Establishment of mouse xenograft model K562R cells (1Х10 7 ) were suspended in 50% (v / v) serum-free Matrigel (Corning) and implanted subcutaneously into the right flank of 6-week-old female BALB / c (nu / nu) mice. Tumor size was measured with a caliper, and tumor volume was calculated using the standard formula (width x width x height). 2 The tumor volume was calculated using the following formula: × length / 2. 3 Upon reaching maturity, mice were randomly divided into groups and treated with imatinib (10 mg / kg / day, intraperitoneally), anti-FAM167A antibody or isotype control (2 mg / kg / 3 days, intraperitoneally), or vehicle (saline, PBS) for 10 days. All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Seoul National University (SNU-210315-6) and Gwangju Institute of Science and Technology (GIST-2019-008).

[0193] 6-2. Confirmation of the efficacy of anti-FAM167A neutralizing antibody treatment in treating CML We measured the volume (Figure 6b) and mouse weight (Figure 6c) 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), and the size (Figure 6d) and weight (Figure 6e) of tumors collected from mice in each group on day 10 of treatment. Compared to imatinib alone or the combination of isotype control antibody and imatinib, treatment with imatinib and FAM167A neutralization (induced by intraperitoneal injection of anti-FAM167A neutralizing antibody) significantly suppressed tumor growth without weight loss (Figure 6b-e).

[0194] Immunohistochemical staining was performed to investigate the basis for the differences in tumor growth at the cellular level. Fixed and paraffin-embedded tumor sections were stained with hematoxylin and eosin (HE), anti-Ki-67 antibody (SP6, Abcam) and anti-NIK antibody (Abcam) using standard procedures. TUNEL staining was performed with the DeadEnd Colorimetric TUNEL System (Promega). Images were captured with NIS-Elements software provided with an Eclipse Ti inverted microscope (Nikon). Quantification was performed using ImageJ software. Ki-67 in each field was stained with 100% CI, 0.01 to 0.01. + Or the number of TUNEL+ cells was counted. NIK expression was scored in tumor cells (intensity: 0=negative, 1=weak, 2=medium, 3=strong), and the H score was calculated using the following equation:

[0195] H score = 1 x (% of cells with intensity 1) + 2 x (% of cells with intensity 2) + 3 x (% of cells with intensity 3).

[0196] Analysis of tumor tissue sections through HE staining revealed lower cell density in FAM167A-neutralized tumors. Further immunohistochemical staining showed increased levels of the apoptosis marker TUNEL, along with decreased expression of NIK and proliferation marker Ki-67 (Fig. 6f). Taken together, these data suggest that neutralizing FAM167A restores TKI sensitivity in cells with BCR-ABL-independent TKI resistance.

[0197] [Example 7] Confirmation of FAM167A expression in BCR-ABL-independent TKI-resistant KCL22R cells 7-1.Cell culture In addition to K562R cells, we also investigated BCR-ABL-independent TKI-resistant KCL22R cells. KCL22S and KCL22R were obtained from the American Type Culture Collection (ATCC). All cell lines were cultured in humidified cytoplasmic culture media. The cells were maintained at 37°C and 5% CO2 in a cell culture incubator and cultured in RPMI (Roswell Park Memorial Institute) 1640 medium (HyClone) supplemented with 10% fetal bovine serum (HyClone), 100 U / ml penicillin, and 100 μg / ml streptomycin. To maintain TKI resistance, KCL22R cells were cultured in medium supplemented with 1 μM imatinib.

[0198] 7-2. Confirmation of FAM167A levels in KCL22R cells Sequence analysis confirmed that there was no mutation in the ABL region.FAM167A mRNA expression levels were also confirmed by qRT-PCR, which showed that KCL22R cells showed higher FAM167A levels than KCL22S cells, and imatinib treatment of KCL22R cells showed increased FAM167A expression (Figure 7).

[0199] [Example 8] Confirmation of FAM167A expression in CML patients To confirm the clinical significance of the results of this study, CD34 isolated from CML patients was analyzed by qRT-PCR. + FAM167A levels in stem / progenitor cells were analyzed.

[0200] 8-1. Primary human samples Peripheral blood or bone marrow samples were collected from 58 CML patients (clinicopathological characteristics listed in Table 4) at Chonnam National University Hospital, Hwasun, Korea. Samples were collected at the time of diagnosis, before treatment with TKIs (diagnosis), and again at follow-up after treatment with TKIs (follow-up). Mononuclear cells isolated from patients were frozen and stored in liquid nitrogen until further use. Prior 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 the European Leukemia Net guidelines. BCR-ABL mutation status in CML samples was confirmed by sequencing analysis.

[0201] [Table 4-1] [Table 4-2]

[0202] 1 Imatinib resistance was classified clinically according to the European Leukemia Net guidelines. 2 BCR-ABL mutation status was confirmed by sequence analysis. CP: chronic phase; AP: accelerated phase; BP: blast phase.

[0203] Data showed that expression of FAM167A was even 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). (Figure 8a). FAM167A levels were also higher in cells of BCR-ABL-independent imatinib-resistant patients before receiving treatment (i.e., at the time of diagnosis), suggesting that FAM167A expression can predict BCR-ABL-independent TKI resistance in CML patients.

[0204] Flow cytometry also showed that surface expression of DSG1 was higher in cells from BCR-ABL-independent imatinib-resistant patients after imatinib treatment (follow-up observation) than before treatment (diagnosis) (Figure 8b). Collectively, these results suggest that FAM167A and DSG1 are associated with BCR-ABL-independent TKI resistance in CML patients through activation of the non-canonical NF-κB pathway (Figure 8c).

[0205] statistical analysis The number of replicates for each experiment is indicated in the figures. Data are presented as mean ± standard deviation (sd). Two-tailed unpaired Student t-test was used to determine statistical significance. P values ​​< 0.05 were considered significant (*P < 0.05, **P < 0.01 and ***P < 0.001).

[0206] The national research and development projects that supported this invention are as follows: (1) [Project unique number] 1711130627 [Project number] 2021R1A2C3011211 [Department name] Ministry of Science, ICT and Technology [Name of issue management (specialized) organization] Korea Research Foundation [Research project name] Individual basic research (Ministry of Science, Technology, Information and Communication) (R&D) [Research title] Research into the role of gamma herpesvirus infection in neuroinflammatory diseases [Contribution rate] 1 / 2 [Name of project execution institution] Seoul National University [Research Period] 2021.03.01~2022.02.28 (2) [Project unique number] 1711170880 [Proposal number] 2022M3A9I2017587 [Department name] Ministry of Science, ICT and Technology [Name of issue management (specialized) organization] Korea Research Foundation [Research Project Name] Source Technology Development Project / Bio-Medical Technology Development [Research title] Development of a multi-omics-based platform for future novel and variant influenza mutant strains [Contribution rate] 1 / 2 [Name of project execution institution] Seoul National University [Research period] 2023.01.01~2023.12.31

Claims

1. A biomarker composition for diagnosing BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance in a patient with chronic myeloid leukemia, comprising FAM167A protein or a gene encoding the same.

2. A composition for diagnosing BCR-ABL-independent TKI resistance in a patient with chronic myeloid leukemia, comprising a preparation for measuring the expression level of FAM167A protein, a fragment thereof, or the mRNA of a gene encoding said protein.

3. The composition for diagnosing BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient according to claim 2, wherein the preparation for measuring the expression level of the FAM167A protein or a fragment thereof comprises an antibody or an antigen-binding fragment thereof, or an aptamer that specifically binds to the FAM167A protein or a fragment thereof.

4. The composition for diagnosing BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient according to claim 2, wherein the preparation for measuring the mRNA level of the gene encoding the FAM167A protein comprises sense and antisense primers or probes that complementarily bind to the mRNA of the gene.

5. A kit for diagnosing BCR-ABL-independent TKI resistance in a patient with chronic myeloid leukemia, comprising the composition according to claim 2.

6. The kit for diagnosing BCR-ABL-independent TKI resistance in a patient with chronic myeloid leukemia according to claim 5, wherein the kit is an RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit, a DNA chip kit, a microarray kit, a SAGE (Serial Analysis of Gene Expression) kit, a gene chip kit, an ELISA (Enzyme linked immunosorbent assay) kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

7. (a) measuring the mRNA expression level of FAM167A protein, a fragment thereof or a gene encoding said protein in a biological sample isolated from a patient with chronic myeloid leukemia; and (b) comparing the expression level determined in step (a) with the mRNA expression level of the same protein, a fragment thereof or a gene encoding said protein determined in a biological sample isolated from a patient with chronic myeloid leukemia who is not resistant to a BCR-ABL-independent tyrosine kinase inhibitor; An informative method for diagnosing BCR-ABL-independent TKI resistance in a patient with chronic myeloid leukemia, comprising:

8. (c) determining that a patient with chronic myeloid leukemia is resistant to a BCR-ABL-independent TKI when the mRNA expression level of the FAM167A protein, a fragment thereof, or a gene encoding said protein measured in a biological sample isolated from the patient with chronic myeloid leukemia in step (a) is higher than the mRNA expression level of the same protein, a fragment thereof, or a gene encoding said protein measured in a biological sample isolated from a patient with chronic myeloid leukemia not resistant to a BCR-ABL-independent tyrosine kinase inhibitor in step (b).

9. The method for providing information for diagnosing BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient according to claim 7, wherein the biological sample comprises blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymphatic fluid, respiratory tract fluid, intestinal fluid, urogenital fluid, breast milk, lymphatic system fluid, semen, cerebrospinal fluid, tracheal system fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof.

10. FAM167A protein inhibitor; An inhibitor of the expression of the gene encoding the FAM167A protein; or Mixtures of these; A pharmaceutical composition for preventing or treating BCR-ABL-independent TKI-resistant chronic myeloid leukemia, comprising the above compound as an active ingredient.

11. The pharmaceutical composition for preventing or treating BCR-ABL-independent TKI-resistant chronic myeloid leukemia according to claim 10, wherein the FAM167A protein inhibitor comprises at least one selected from the group consisting of low molecular weight compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to FAM167A protein.

12. The pharmaceutical composition for preventing or treating BCR-ABL-independent TKI-resistant chronic myeloid leukemia according to claim 10, wherein the expression inhibitor of the gene encoding the FAM167A protein comprises any one or more selected from the group consisting of antisense nucleotides that bind complementarily to the mRNA of the FAM167A gene, small interfering RNA (siRNA), and short hairpin RNA (shRNA).

13. The pharmaceutical composition for preventing or treating BCR-ABL-independent TKI-resistant chronic myeloid leukemia according to claim 10, wherein the BCR-ABL-independent TKI-resistant chronic myeloid leukemia has increased NF-κB activity compared to TKI-sensitive leukemia.

14. The pharmaceutical composition for preventing or treating BCR-ABL-independent TKI-resistant chronic myeloid leukemia according to claim 10, which is administered in combination with a TKI.

15. The TKIs include crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, ibrutinib, cabozantinib, gefitinib, erlotinib, lapatinib, vandetanib, and the like. The pharmaceutical composition for preventing or treating BCR-ABL-independent TKI-resistant chronic myeloid leukemia according to claim 14, comprising at least one of the following: nib, afatinib, osimertinib, ruxolitinib, tofacitinib, axitinib, lenvatinib, nintedanib, pazopanib, regorafenib, sorafenib, or sunitinib.

16. FAM167A protein inhibitor; An inhibitor of the expression of the gene encoding the FAM167A protein; or Mixtures of these; As an active ingredient, it is an inhibitor of BCR-ABL-independent TKI resistance in patients with chronic myeloid leukemia. Pharmaceutical composition for use.

17. The pharmaceutical composition for inhibiting BCR-ABL-independent TKI resistance in a patient with chronic myeloid leukemia according to claim 16, wherein the chronic myeloid leukemia exhibiting BCR-ABL-independent TKI resistance has increased NF-κB activity compared to leukemia exhibiting TKI sensitivity.

18. The pharmaceutical composition for inhibiting BCR-ABL-independent TKI resistance in a chronic myeloid leukemia patient exhibiting BCR-ABL-independent TKI resistance according to claim 16, which is administered in combination with a TKI.

19. The TKIs include crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, ibrutinib, cabozantinib, gefitinib, erlotinib, lapatinib, vandetanib, and the like. The pharmaceutical composition for inhibiting BCR-ABL-independent TKI resistance in a patient with chronic myeloid leukemia according to claim 18, comprising:

20. (a) treating a cell line or animal model exhibiting BCR-ABL-independent TKI resistance with a candidate agent; and (b) determining whether or not there is inhibition of FAM167A protein or inhibition of expression of the gene encoding said protein in the cell line or animal model treated with said candidate substance; The present invention relates to a method for screening for a therapeutic agent for CML exhibiting BCR-ABL-independent TKI resistance or a method for screening for an inhibitor of BCR-ABL-independent TKI resistance, comprising:

21. (c) determining that the candidate substance is a therapeutic agent for CML exhibiting BCR-ABL-independent TKI resistance or a BCR-ABL-independent TKI resistance inhibitor when the candidate substance inhibits FAM167A protein or inhibits expression of a gene encoding the protein.

Images