KR102615053B1 - A biomarker for diagnosing BCR-ABL-independent tyrosine kinase inhibitor resistance comprising FAM167A and a composition for preventing or treating chronic myeloid leukemia targeting FAM167A - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance, including FAM167A, and a composition for preventing or treating chronic myeloid leukemia targeting FAM167A, in more detail. Compositions, kits, and methods for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myeloid leukemia by confirming the mRNA expression level of the FAM167A protein, fragments thereof, or genes encoding the protein, and BCR-ABL targeting FAM167A Provided are a pharmaceutical composition for preventing or treating chronic myeloid leukemia that exhibits TKI-independent resistance, and a pharmaceutical composition for suppressing BCR-ABL-independent TKI resistance in patients with chronic myeloid leukemia.

Description

A biomarker for diagnosing BCR-ABL-independent tyrosine kinase inhibitor resistance comprising FAM167A and a composition for preventing or treating chronic myeloid leukemia targeting FAM167A {A biomarker for diagnosing BCR-ABL-independent tyrosine kinase inhibitor resistance comprising FAM167A and a composition for preventing or treating chronic myeloid leukemia targeting FAM167A}

The present invention relates to a biomarker composition for diagnosing BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance, including FAM167A, and a composition for preventing or treating chronic myeloid leukemia targeting FAM167A, in more detail. Specifically, compositions, kits, and methods for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myeloid leukemia by determining the expression level of FAM167A protein, fragments thereof, or mRNA of the gene encoding the protein, and BCR-ABL targeting FAM167A. Provided are a pharmaceutical composition for preventing or treating chronic myeloid leukemia showing ABL-independent TKI resistance and a pharmaceutical composition for suppressing BCR-ABL-independent TKI resistance in patients with chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is caused by the characteristic t(9;22)(q34;q11) reciprocal translocation of the BCR-ABL Philadelphia chromosome. The BCR-ABL fusion gene encodes the constitutively active BCR-ABL oncoprotein due to loss of the upstream regulatory domain of ABL1 during translocation. The development of BCR-ABL-targeted tyrosine kinase inhibitors (TKIs), such as imatinib, has revolutionized the treatment of patients with CML. However, resistance to TKIs remains a major obstacle to successful CML treatment.

TKI resistance occurs in two ways: BCR-ABL dependent and BCR-ABL independent. BCR-ABL-dependent resistance is primarily caused by mutations in the BCR-ABL kinase domain, 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 diseases. In contrast, BCR-ABL-independent TKI resistance cannot be resolved with second-generation TKIs. Several factors, including changes in signaling pathway activity, mutations in epigenetic regulators, microenvironmental factors, and leukemia stem cell activity, are potential contributors to BCR-ABL-independent TKI resistance, but the underlying reasons for this type of resistance are not fully understood. didn't Therefore, identifying the underlying mechanisms and developing effective treatments is a major goal.

Nuclear factor-κB (NF-κB) family members are inducible transcription factors that regulate a variety of genes involved in immune responses, inflammation, and cancer. There are two major NF-κB activation signaling pathways: canonical and non-canonical. The canonical NF-κB pathway is activated primarily through 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 is dependent on NIK accumulation through blockade of ubiquitination-dependent NIK degradation. Accumulated NIK induces the processing of p100 into p52, followed by nuclear translocation of p52-containing dimers. The NF-κB pathway contributes to TKI resistance, and inhibition of this pathway restores the sensitivity of CML to TKIs. However, the factors involved in direct activation of the NF-κB pathway in TKI-resistant CML are still unknown.

Against this background, we show that NF-κB is the most highly activated transcription factor tested through in silico activated transcription factor analysis and shows differential gene expression in BCR-ABL-independent TKI-resistant CML cells. He announced that he was in charge. Additionally, we revealed an important function of FAM167A, a previously uncharacterized protein, in the noncanonical NF-κB pathway in BCR-ABL-independent, TKI-resistant CML. Specifically, the expression of FAM167A was highly upregulated in CD34 ⁺ CML cells from patients with BCR-ABL-independent TKI resistance (similar to the results in CML cell lines), suggesting that FAM167A is responsible for BCR-ABL-independent TKI resistance. It shows. Neutralization of FAM167A reversed TKI resistance in cultured cells and mouse tumor models.

These findings illustrate a novel and important role for FAM167A in the non-canonical NF-κB pathway in relation to the mechanism of BCR-ABL-independent TKI resistance and present it as a promising target to overcome TKI-resistant CML.

Regarding FAM167A, Non-Patent Document 1 has identified FAM167A-BLK as a systemic sclerosis susceptibility gene confirmed by CGA, but it is also used as a target for diagnosis of BCR-ABL-independent TKI resistance and treatment of TKI-resistant CML. As such, nothing is known about the role of FAM167A.

Ota Y, Kuwana M. Updates on genetics in systemic sclerosis. Inflamm Regen. 2021 Jun 15;41(1):17.

The present invention was created to solve the above-mentioned problems, to discover new targets for diagnosing BCR-ABL-independent TKI resistance and treating TKI-resistant CML, and using the targets as biomarkers to detect BCR-ABL in CML patients. The purpose is to provide technologies for diagnosing ABL-independent TKI resistance, treating BCR-ABL-independent TKI-resistant CML, and suppressing BCR-ABL-independent TKI resistance.

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 patients with chronic myeloid leukemia, comprising the FAM167A protein or the gene encoding it. provides.

The present invention also provides compositions and kits for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myeloid leukemia, comprising an agent for measuring the expression level of FAM167A protein, fragments thereof, or mRNA of the gene encoding the protein. to provide.

In the present invention, the agent for measuring the expression level of the FAM167A protein or fragment thereof may include an antibody, an antigen-binding fragment thereof, or an aptamer that specifically binds to the FAM167A protein or fragment thereof.

In the present invention, an agent for measuring the mRNA level of the gene encoding the FAM167A protein may include sense and antisense primers or probes that bind complementary to the mRNA of the gene.

In the present invention, the kit includes 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, and an ELISA (Enzyme Analysis) kit. It may include a linked immunosorbent assay (linked immunosorbent assay) kit, protein chip kit, rapid kit, or multiple reaction monitoring (MRM) kit.

The present invention also provides an informative method for diagnosing BCR-ABL independent TKI resistance in a patient with chronic myelogenous leukemia, comprising the following steps:

(a) measuring the mRNA expression level of FAM167A protein, a fragment thereof, or a gene encoding the protein in a biological sample isolated from a patient with chronic myeloid leukemia; and

(b) The expression level measured in step (a) is the same protein, fragment thereof, or gene encoding the protein measured in a biological sample isolated from a chronic myeloid leukemia patient without BCR-ABL-independent tyrosine kinase inhibitor resistance. Comparing with the mRNA expression level of.

In the present invention, the information provision method is (c) the mRNA expression level of the FAM167A protein, its fragment, or the gene encoding the protein measured in a biological sample isolated from the chronic myeloid leukemia patient in stage (a), If it is higher than the mRNA expression level of the same protein, fragment thereof, or gene encoding the protein measured in a biological sample isolated from a patient with chronic myeloid leukemia without BCR-ABL-independent tyrosine kinase inhibitor resistance at stage (b), BCR- An additional step of determining whether the drug is resistant to an ABL-independent TKI may be additionally included.

In the present invention, the biological sample includes blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, urogenital fluid, breast milk, lymphatic body fluid, and semen. , cerebrospinal fluid, fluid within an organ system, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof.

Additionally, the present invention provides a FAM167A protein inhibitor; An inhibitor of the expression of the gene encoding the FAM167A protein; Or a pharmaceutical composition for preventing or treating chronic myeloid leukemia showing BCR-ABL-independent TKI resistance, comprising a mixture thereof as an active ingredient, and a pharmaceutical composition for suppressing BCR-ABL-independent TKI resistance in patients with chronic myeloid leukemia.

In the present invention, chronic myeloid leukemia showing BCR-ABL-independent TKI resistance may have increased NF-κB activity compared to chronic myeloid leukemia showing TKI sensitivity.

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

In the present invention, the TKI includes crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, and imatinib ( imatinib, nilotinib, ponatinib, ibrutinib, cabozantinib, gefitinib, erlotinib, lapatinib, bande Vandetanib, afatinib, osimertinib, ruxolitinib, tofacitinib, axitinib, lenvatinib, nintedanib ( It may include nintedanib, pazopanib, regorafenib, sorafenib, or sunitinib.

The present invention also provides a method for screening for a therapeutic agent for CML or a BCR-ABL independent TKI resistance inhibitor exhibiting BCR-ABL-independent TKI resistance, comprising the following steps:

(a) treating a candidate material to a cell line or animal model showing BCR-ABL-independent TKI resistance; and

(b) Confirming whether the FAM167A protein is inhibited or the expression of the gene encoding the protein is inhibited in the cell line or animal model treated with the candidate material.

In the present invention, the screening method is (c) when the candidate substance inhibits the FAM167A protein or suppresses the expression of the gene encoding the protein, the candidate substance is used as a treatment for CML showing BCR-ABL independent TKI resistance or An additional step of determining that the inhibitor is a BCR-ABL independent TKI resistance inhibitor may be included.

In the present invention, we discovered FAM167A as a new biomarker that can be used as an indicator of TKI resistance in CML, which shows BCR-ABL-independent TKI resistance without mutations in the BCR-ABL kinase domain, and showed that TKI resistance is reversed when it is inhibited. As confirmed, this can be used to diagnose TKI resistance in CML patients not treated with existing second-generation TKIs and reverse TKI resistance in CML patients showing BCR-ABL-independent TKI resistance, thereby effectively treating TKI-resistant patients. provides an advantage.

B 경로의 관여를 보여주는 제안된 모델이다. 독립표본 양측 t-테스트; *P < 0.05, ***P < 0.001. NS: 유의하지 않음.Figure 1A shows a strategy for selection of genes contributing to BCR-ABL-independent TKI resistance in CML.
Figures 1b and 1c show the results of analyzing the viability of K562S and K562R cells after treatment with the indicated concentrations of imatinib (Figure 1b) and nilotinib (Figure 1c) for 3 days, respectively, and Figures 1d and 1e The results of flow cytometry analysis of Annexin V-stained cells to evaluate apoptosis in K562S and K562R cells after treatment with the indicated concentrations of imatinib (Figure 1d) and nilotinib (Figure 1e) for 3 days are shown. Data represent three (Figures 1B to 1E) independent experiments (error bars, s.d. of triplicate (Figures 1B and 1C) samples). independent samples two-tailed t-test; ***P<0.001. K562S: TKI-sensitive K562 cell line, K562R: TKI-resistant K562 cell line without BCR-ABL kinase domain mutation.
Figure 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 shows heatmap analysis of the expression levels of genes differentially expressed in K562R cells.
Figures 1H and 1I show transcription factors involved in regulating genes differentially expressed in both microarray and RNA-seq analyzes (Figure 1H) and three control genes (each group contains 500 randomly selected genes) (Figure 1I). ) shows the in silico analysis results. Size and color indicate the number of genes involved in the transcription factor.
Figure 1j shows the enrichment score for transcription factors and shows the degree of association with differentially expressed genes compared to randomly selected genes.
In Figure 1K, A shows the ratio of genes targeted by the indicated transcription factors among genes differentially expressed between K562S and K562R, and B shows the ratio of genes targeted by the indicated transcription factors among 500 randomly selected genes. will be.
Figure 1l 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 1m shows the results of quantitative reverse transcription PCR (qRT-PCR) analysis of the mRNA levels of seven genes in K562S and K562R cells and K562R cells treated with 1 μM imatinib for 24 hours. IMA: Imatinib. Data are representative of three independent experiments (Figure 1L and Figure 1M) (error bars, s.d. of duplicate (Figure 1M) or triplicate (Figure 1J and Figure 1L) 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 without BCR-ABL kinase domain mutation.
Figure 2A shows NF-κB luciferase reporter activity in K562S cells 24 hours after transfection of plasmids encoding the indicated genes and in K562R cells treated with 1 μM imatinib for 24 hours.
Figure 2B shows the survival rate 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, A, in silico of human, mouse, rat, and zebrafish FAM167A and HA-tagged human FAM167A secretion by the SecretomeP tool (http://www.cbs.dtu.dk/services/SecretomeP/). As a prediction, proteins with a NN score of 0.5 or higher are predicted to be secreted. Figure 2C B shows the results of immunoblot analysis of FAM167A in cell lysates and culture supernatants of K562R cells. The medium was used as control supernatant. Supernatant proteins were concentrated by acetone precipitation. Erbin, a cytoplasmic protein, was also analyzed as a control to confirm that there was no cytoplasmic protein in the culture supernatant. Data are representative of three independent experiments (B in Figure 2C).
Figure 2d shows NF-κB luciferase reporter activity in K562S cells for 24 hours after treatment with recombinant FAM167A, and Figure 2e shows NF-κB luciferase reporter activity in K562R cells after treatment with anti-FAM167A neutralizing antibody for 24 hours. Figure 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, and Figure 2g shows dominant-negative NIK (NIK-DN) NF-κB luciferase reporter activity is shown in K562R cells 24 hours after transfection of the plasmid encoding , and Figures 2h and 2i show immunity against NIK (Figure 2h) and p100/p52 (Figure 2i) in K562S and K562R cells. This shows the results of blot analysis.
Figure 2j shows electrophoretic mobility shift assay (EMSA) results of nuclear extracts isolated from K562S and K562R cells showing supershift with NF-κB probe and anti-p52 antibody.
Figure 2k shows NF-κB luciferase reporter activity in K562S cells 24 hours after treatment with 100ng/ml recombinant FAM167A and transfection of the plasmid encoding NIK-DN.
Figure 2l shows the results of NIK immunoblot analysis in K562R cells after treatment with 2 μg/ml anti-FAM167A neutralizing antibody or isotype control for 12 hours.
Figure 2m shows the results of immunoblot analysis of p100/p52 in K562S and K562R cells after treatment with the indicated concentrations of recombinant FAM167A for 12 hours. GAPDH was used as an internal standard (Figure 2H, Figure 2I Figure 2L and Figure 2M). Data are representative of three (Figure 2A, Figures 2D-2G and Figures 2I-2M) or five (Figure 2H) independent experiments (error bars, triplicate (Figures 2A, Figures 2D-2G and Figures 2I-2I). s.d. of five (Figure 2m) or five (Figure 2h) samples. independent samples two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001
Figure 3a shows the results of surface FAM167A receptor (FAM167AR) staining of K562S and K562R cells using Myc-tagged FAM167A and Alexa Fluor 488-conjugated anti-Myc antibody.
Figure 3b shows the experimental design for identification of the FAM167A receptor.
Figure 3C shows silver staining results of immunoprecipitation samples prepared from K562R cells using Myc-tagged FAM167A and anti-Myc antibody coupled to Protein G Sepharose® beads.
Figure 3d shows the MS/MS spectrum and the peptide sequence of the indicated band from Figure 3c.
Figure 3e shows the results of co-immunoprecipitation analysis between FAM167A and DSG1 in K562R cells.
Figure 3f shows the results of qRT-PCR analysis of DSG1 mRNA levels in K562S and K562R cells and K562R cells treated with 1 μM imatinib for 24 hours.
Figure 3g shows immunoblot analysis of DSG1 expression in K562S and K562R cells.
Figure 3h shows the results of surface staining of K562S and K562R cells using anti-DSG1 antibody.
Figure 3i shows pictures of immunofluorescence microscopy analysis of DSG1 expression in K562S and K562R cells. Nuclei were visualized using Hoechst 33342. Scale bar, 10 μm. GAPDH was used as an internal standard (Figure 3g). IP: immunoprecipitation. MFI: mean fluorescence intensity. Data represent three independent experiments (Error bars, s.d. of duplicate (FIG. 3F) or triplicate (FIGS. 3A and 3H) samples) from three independent experiments (FIGS. 3A, 3C, and FIGS. 3E-3I). independent samples two-tailed t-test; **P < 0.01, ***P < 0.001. NS: Not significant.
Figure 3j shows pictures of immunofluorescence microscopy analysis of DSG1 and FAM167A in K562R cells. Nuclei were visualized using Hoechst 33342. Scale bar, 10 μm.
Figure 3k shows the results of NF-κB luciferase reporter activity in K562S cells 24 hours after transfection of the plasmid encoding FAM167A and treatment with the indicated concentrations of anti-FAM167A neutralizing antibody. Data represent two (Figure 3J and Figure 3K) independent experiments (error bars, s.d. of triplicate (Figure 3J) samples). independent samples two-tailed t-test; *P<0.05.
Figure 4a shows the results of surface DSG1 staining of K562R cells after DSG1 knockdown using recombinant lentiviruses encoding two different DSG1-specific shRNAs together with anti-DSG1 antibodies.
Figure 4b shows NF-κB luciferase reporter activity in K562R cells after DSG1 knockdown using recombinant lentiviruses encoding two different DSG1-specific shRNAs.
Figure 4c shows the results of immunoblot analysis of p100/p52 in K562R cells after DSG1 knockdown.
Figure 4d shows NF-κB luciferase reporter activity in K562R cells 24 hours after transfection of plasmids encoding DSG1 or DSG1 and Erbin with or without treatment with 2 μg/ml anti-FAM167A neutralizing antibody.
Figure 4e shows the results of qRT-PCR analysis of Erbin mRNA in K562S, K562R, and K562R cells treated with 1 μM imatinib for 24 hours.
Figure 4f shows the results of immunoblot analysis for Erbin in K562S and K562R cells. Data are representative of three independent experiments (Figures 4A, 4E, and 4F) (error bars, s.d. of triplicate (Figure 4E) samples). independent samples two-tailed t-test; NS: Not significant.
Figure 4g shows the results of co-immunoprecipitation analysis of DSG1, Erbin, and NIK in K562S and K562R cells.
Figure 4h shows the results of co-immunoprecipitation analysis of DSG1 and NIK in K562R cells after treatment with 2 μg/ml anti-FAM167A neutralizing antibody or isotype control for 3 hours.
Figure 4i shows the results of immunoblot analysis of NIK ubiquitination using an anti-ubiquitin antibody after NIK immunoprecipitation from K562R cells treated with 2 μg/ml anti-FAM167A neutralizing antibody or isotype control and 10 μM MG132 for 3 hours. .
Figure 4j, A shows the results of co-immunoprecipitation analysis of DSG1, Erbin, NIK, TRAF3, CHIP and c-cbl in K562S and K562R cells, and B shows the results of co-immunoprecipitation analysis in K562S cells using anti-DSG1 and anti-TRAF3 antibodies. C shows the results of co-immunoprecipitation analysis of DSG1, NIK, and TRAF3, C shows the results of co-immunoprecipitation analysis of DSG1, NIK, and CHIP in K562S cells using anti-DSG1 and anti-CHIP antibodies, and D shows the results of co-immunoprecipitation analysis of DSG1, NIK, and CHIP in K562S cells using anti-DSG1 and anti-CHIP antibodies. The results of co-immunoprecipitation analysis of DSG1, NIK, and c-cbl in K562S cells using anti-DSG1 and anti-c-cbl antibodies are shown. GAPDH was used as an internal standard (A in Figure 4j). Data are representative of three independent experiments (Figure 4J, A to D).
Figure 4K shows a schematic model of the FAM167A-induced non-canonical NF-κB pathway. GAPDH was used as an internal standard (Figures 4C and 4G to 4I). Ub: Ubiquitination. Data are from two (Figure 4C) or three (Figures 4B, 4D, Figures 4G to 4I) independent experiments (error bars, in duplicate (Figure 4C) or triplicate (Figures 4B, 4D, Figure 4G). and Figure 4h) shows the sd) of the samples. independent samples two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5A is a schematic diagram showing FAM167A-induced inhibition of the non-canonical NF-κB pathway.
Figure 5b shows the survival rate of K562R cells transfected with a plasmid encoding NIK-DN after treatment with imatinib or nilotinib (Nilo) for 3 days.
Figure 5c shows the results of viability analysis of K562R cells after treatment with 2 μg/ml anti-FAM167A neutralizing antibody or isotype control in the presence of imatinib or nilotinib for 3 days.
Figure 5D shows apoptotic populations of K562R cells after treatment with 2 μg/ml anti-FAM167A neutralizing antibody or isotype control for 3 days in the presence of imatinib or nilotinib. Data represent three (Figures 5B-5D) independent experiments (error bars, s.d. of triplicate (Figures 5B-5D) samples). independent samples two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001
Figure 6A is an experimental schedule and schematic diagram showing the mouse model.
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. It represents the weight of .
Figure 6d shows photographs of tumors collected from mice in each group on the 10th day of treatment, and Figure 6e shows the tumor weight on the 10th day of treatment.
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 are representative of three (Figures 6B-6F) independent experiments (error bars, s.d. of 5 (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 shows the results of 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, s.d. of triplicate samples). independent samples two-tailed t-test; **P<0.01, ***P<0.001.
Figure 8A shows patients with imatinib-resistant CML without BCR-ABL mutations (R) at diagnosis (n = 12) and at follow-up (n = 11), and at diagnosis (n = 10) and at follow-up (n = 12). qRT-PCR of FAM167A mRNA expression in CD34 ⁺ stem/progenitor cells of imatinib-responsive CML patients (S) at diagnosis (n = 26) and at follow-up (n = 26) in imatinib-resistant CML patients with BCR-ABL mutations This shows the analysis results. Imatinib resistance and BCR-ABL mutation status were determined at follow-up. The levels of FAM167A were normalized to the levels of GAPDH.
Figure 8B shows BCR-reactivity in patients with imatinib-responsive CML at diagnosis (n = 8) and follow-up (n = 8) and at diagnosis (n = 10) and follow-up (n = 7), as measured by flow cytometry. Relative surface expression of DSG1 (MFI) on CD34 ⁺ stem/progenitor cells from imatinib-resistant CML patients without ABL mutation.
Figure 8C shows FAM167A-induced non-canonical NF- in BCR-ABL-independent TKI resistance.

This is a proposed model showing the involvement of the B pathway. independent samples two-tailed t-test; *P < 0.05, ***P < 0.001. NS: Not significant.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention.

As described above, BCR-ABL-independent TKI resistance cannot be treated even with second-generation TKIs, so there is a need to discover new biomarkers that can be used for diagnosis and treatment of BCR-ABL-independent TKI-resistant CML.

Accordingly, in the present invention, we reveal the important function of FAM167A, a previously uncharacterized protein, in the non-canonical NF-κB pathway of CML that exhibits BCR-ABL-independent TKI resistance, and demonstrate that neutralization of FAM167A promotes TKI resistance in cultured cells and mouse tumor models. By confirming that FAM167A can be a promising target for overcoming TKI-resistant CML by confirming that it exhibits a reversing effect, a solution to the above-mentioned problem was sought.

Accordingly, the first aspect of the present invention relates to a biomarker composition for diagnosing BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance in patients with chronic myeloid leukemia, comprising the FAM167A protein or the gene encoding it. will be.

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

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)

The mRNA of the gene encoding the FAM167A protein may include the nucleotide sequence of NCBI Reference Sequence: NM_053279.3, but may include without limitation any nucleotide sequence that is substantially the same as that of the gene or shows a corresponding efficacy. In the present invention, the mRNA sequence of the gene encoding the representative FAM167A protein is shown in SEQ ID NO: 2.

gagaattcac cggagctcgg ggaggcgagg cgggagccct gagctgaggc cgggcagcgc ggacccgcgc aggaggaggc ggcggggaca ccggcgagtc cccagcaggg cccctgggct cccggaaggg tctccagcca cctgcccttc ccgagaagag agagctctgg gccttcttcc tgccagtctg gt cttcgagt 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 c cttcccgag gccggctgcg gagccagg cgagcttgga ggagggggag cgtggggggc aggagccctt gctccccctg agagaggctg ggcagcaccc cccttctgcc aggagtgcca gccaaggtgc cagacccctg tccactggca agctggaagg ctttcagagc atcgatgaag ctatagcctg gctcagg aag gaactgacgg agatgcggct gcaggaccag caactggcca gacagctcat gcgcctgcgt ggcgacatca acaagctgaa aatcgaacac acctgccgcc tccacaggag gatgctcaac gatgccacct acgagctgga ggagcgggat gagctggccg acctcttctg tgactcccct cttgcctcct ccttcagcct ctccacacca ctcaagctta ttggcgtgac caagatgaac atcaactctc ggaggttctc tctctgctga ggagccctca gactgggcgg aggggctgg a gcggagggct tgggctggag gggtgtcaga ggaagctgag gccaagttac tccagtgggt ctcccggagg caggggtccc tgggactggc gactcaaggg ccccaggacc tattcagtgg tgctctccca cccaggggcc ctgggtgtgg atgccagtgt ctctgtgact ggctcttgct tactacccaa agagct ctgc agaagggccg ctccaaccaa gatgttaaag gagacctggg ttcccaccat aatccatccc tccacggtca cgttcctgtt tcctggaatc actggtgcta tgaactggga ttcccaaagg gaggcccccc aacaaagctg tcatttttgc agaaggctgt cccgcaaggg ccttggggga aattaggcat gtcagatgtg cctgtctcac gtgctgttgc tgtcctctaa gtattgtctc aaattcaccc taagtacatg actcagcaac attgacaggg agctactagg aagggaaa at cgaaaggcat gacaaatggg cacttgggga cgcagcccca gtggctggca gccagtgtct ctggtgagcc tgacactata aggctgtgta aattgtaaat tctggcgtgt gctgggacat gtgatggggg cactagcgta gcttgggtgc aacaagcaca gatgtcccca ttgtctcccc tggccacatg cat ctccaaa gagcctcttc actgccaccc acaccccagg gtgacagcct gggagaccac tggtgactga accaggcagg tcctgaaagc attttccata actgaattct cctgcagggg cgtgaccggg gcctcctggt ggattctggt ggtgtcacct tactgccctc tctggaaaga caatctaggg agcccagagg cccatcctga gcctcctctg agattttgtg cctgacctaa acaactagtt ttaataagac tgttactgat gtgttgttca cttgttagta actgattttt gt ccaaatgc ggaagccact tgtgtaggtc aactacagtg cgtaggattt gattttaaga gtttctccct cccaacaggc ttgaggatca gcaagttaag accccagcag gttagggagg tcagtctggg gtcatacggc atggcagggg tccctcggcc agacccgtag aatcctgaga taaggagtgt ttctgacctt tggtgt catc tagtcgagtc ctctcattag taaaggagca aagtgaaacc tgggggagga gaaggacttc cctcaggttg cacagctgtt taggctatag aatattgatg tgtgaaacca ttattgataa tgcctagtag atcacatgtc aatgaacttg aaccccaaag atggtcgtga tgctttgcca aacccgcaca ctgccaaccc ctctactctc cacctcagcc cccacccaca tctcccagag tattgcaatt cagaacattt gggtcaaggt ggagcaaggc actgacagtg gccccacagg g catgtgtca ctaatcactg tcccatggtc tacgcacggc atctggctgc tctgtctact gtgacttctt cctgtgtaat ctcagtgggg cccgtgtcca cccacacatc gtgacccaca taggggagag gttgcttttc ttttgtgggc tgagagtagg acaatgcaaa tgaatgatct ctagtagaca gaaaagaact tggtctcttt tttaaaattt caaagagcca gaagttctat gcctccttca aagtaggcag aacaacgcag ccaagatcta ctgtctgcca tgctctgtgc aatgaagtct gcaggcctga ggaccatgta ctgctgtcct tcctcagagc tctgcacaaa cactgccaag tcctgaagac gcattccttt cctgccaacc tctttccaga taagcccttg aggtctcggg ctgacctaca cacacacaca cacacacaca cacacacacc cccacacaca cacacacgac agagaacatg ccata aacat ccttgaaccc atgcaggaaa gcccatccca tattctgaaa aaatgccaaa ttaggttttt ctttcttttt ggaaatcagt cattacagta accgaaacca ttgggttcag cgaaaatgga aagatttagc tgaatgtagt cagtccaatt aagttggatg caactgagtg atttagttgc ttgggtaacc cag tgcttgc 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 t cccagcctc ctggctcacc acatgtgtac agtgctgttt gcagttgtac tcattatcca tccatctctc tgccatcccc aagcatcgct gggtgtaaaa cgcaaactct ccaccgacac tgccatgcgt ggtcatgtct tgatgccttc aggggctcag tagctatcaa agaggcctgg agggcctgg g caggcttgac gatgcctgac cgagttcaag acccacaccc tgtagcaata ccaagtgcta ttacataatc aatggacgat ttatactttt attttttatg attatttgtt tctatattgc tgttagaaaa agtgaaataa aaatacttca aaagaagata tccatataaa aataaaa (SEQ ID NO: 2)

The specific amino acid sequence and nucleotide sequence of the biomarker protein and the gene encoding it can be confirmed in known databases such as NCBI.

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 that serves as a standard for determining BCR-ABL-independent TKI resistance or a protein encoding the same. refers to genes. Biomarkers may be derived from genomic nucleotide sequences or expressed nucleotide sequences (e.g., from RNA, nRNA, mRNA, cDNA, etc.), or from encoded polypeptides. The term includes nucleic acid sequences complementary to or flanking a biomarker sequence, such as nucleic acids used as probes or primer pairs capable of amplifying said biomarker sequence.

In the present invention, “expression” means producing a protein or nucleic acid. “Protein” is used interchangeably with “polypeptide” or “peptide” and refers to a polymer of amino acid residues, e.g., as commonly found in proteins in their native state. “Polynucleotide” or “nucleic acid” refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single- or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included. “mRNA” refers to RNA that transfers genetic information (gene-specific base sequence) from a specific gene to a ribosome that specifies the amino acid sequence during protein synthesis.

In relation to the first aspect, the present invention provides a method for diagnosing BCR-ABL-independent TKI resistance in patients with chronic myeloid leukemia, comprising an agent for measuring the expression level of FAM167A protein, fragments thereof, or mRNA encoding said protein. A composition and a kit containing the same are provided.

In the present invention, the term "diagnosis" refers to determining a subject's susceptibility to a specific disease or condition, determining whether a subject currently has a specific disease or condition, and determining whether a subject currently has a specific disease or condition. Determining the prognosis of an affected individual, or therametrics (e.g., monitoring the individual's condition to provide information about the efficacy of treatment).

In the present invention, the term “resistance” is also referred to as “resistance” and means that the drug does not respond sensitively to the drug in question and thus the drug does not work.

In the present invention, "an agent for measuring the mRNA expression level of the FAM167A protein, its fragment, or the gene encoding the protein" refers to the mRNA expression level of the biomarker protein of the present invention, its fragment, or the gene encoding the protein. refers to a molecule that can be used to identify, preferably an antibody or antigen-binding fragment thereof, or an aptamer that specifically binds to the protein or fragment thereof, or a sense that binds complementary to the mRNA of the gene and antisense primers or probes, but are not limited thereto.

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

In the present invention, the term “antibody” is a term known in the art and refers to a specific protein molecule directed to an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to the above-mentioned biomarker, and such antibody is obtained by cloning each gene into an expression vector according to a conventional method to obtain the protein encoded by the biomarker gene. , can be manufactured from the obtained protein by conventional methods. This also includes partial peptides that can be made from the above proteins, and the partial peptides of the present invention include at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited, and as long as it is a polyclonal antibody, a monoclonal antibody, or a portion thereof that has antigen binding properties, it is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies. The antibody against the protein encoded by the biomarker gene of the present invention can be any antibody that can be produced by methods known in the art. For example, antibodies used for detection of the biomarkers described above may include intact forms with two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody molecule. The functional fragment of the antibody molecule refers to a fragment that possesses at least an antigen-binding function and may be Fab, F(ab'), F(ab')2, Fv, etc., but is not particularly limited thereto.

In the present invention, the term "aptamer" refers to a single-stranded oligonucleotide, which has a similar function to an antibody and is also called a chemical antibody, and refers to a nucleic acid molecule that has binding activity to a given target molecule. The aptamer may have various three-dimensional structures depending on its base sequence and may have high affinity for a specific substance, such as in an antigen-antibody reaction. An aptamer can inhibit the activity of a certain target molecule by binding to it.

The aptamer of the present invention may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be linear or circular in shape, but is not limited to these. An aptamer having binding activity to each of the three types of biomarker proteins can be easily produced by a person skilled in the art according to a known method by referring to each base sequence.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3' terminal hydroxyl group, which can form a base pair with a complementary template and is used for copying the template. A short nucleic acid sequence that serves as a starting point. In the present invention, BCR-ABL-independent TKI resistance in CML patients can be diagnosed by determining whether the desired product is produced by performing PCR amplification using the sense and antisense primers of the aforementioned biomarker polynucleotide. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundreds of bases that can bind specifically to mRNA, and is labeled. The presence or absence of a specific mRNA can be confirmed. Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc. In the present invention, hybridization is performed using a probe complementary to the above-described biomarker polynucleotide, and BCR-ABL-independent TKI resistance of a patient with chronic myeloid leukemia can be diagnosed based on hybridization. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.

Primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These 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 a native nucleotide with one or more homologs, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoro). amidates, carbamates, etc.) or charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).

Considering mutations with biologically equivalent activity, the base sequence of the agent that measures the expression level of the biomarker gene used in the present invention shows substantial identity with the sequence that specifically binds to the biomarker gene. It is interpreted to include sequences. The term "substantial identity" refers to an identity of at least 60% when a specific sequence is aligned with any other sequence to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art. , more specifically refers to a sequence exhibiting 70% identity, even more specifically 80% identity, and most specifically 90% identity.

The kit of the present invention includes 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, and an ELISA (Enzyme linked immunosorbent assay). ) kit, protein chip kit, rapid kit, or MRM (multiple reaction monitoring) kit, but is not limited thereto. As a specific example, the kit may be a kit containing essential elements required to perform RT-PCR. For example, an RT-PCR kit requires, in addition to each primer specific for a biomarker gene, a test tube or other suitable container, reaction buffer (of varying pH and magnesium concentration), deoxynucleotides (dNTPs), and dideoxynucleotides. (ddNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water, sterilized water, etc. Additionally, it may include a primer pair specific for DNA or RNA used as a quantitative control.

The kit of the present invention may include a kit for extracting nucleic acids (e.g., total RNA), a fluorescent substance for labeling, enzymes and media for nucleic acid amplification, instructions for use, etc.

The kit of the present invention is a device for measuring biomarkers for pancreatic cancer in which the nucleic acid is bound or attached to a solid phase, for example. Examples of solid materials include plastic, paper, glass, silicon, etc., and plastic is a preferable solid material due to ease of processing. The shape of the solid phase is arbitrary, for example, square, circular, rectangular, film-shaped, etc.

The kit may further include a program capable of data analysis or statistical processing. The program may analyze the expression level of genes or proteins. The data analysis or statistical processing program may be one or more selected from Skyline Software, ProteoWizard Software, SCIEX OS Software, SPSS Statistics Software, MedCalc Software, MultiQuant Software, MasterView Software, or Cliquid Software, but is not limited thereto.

In the present invention, we identified FAM167A, a previously uncharacterized protein, that is upregulated in cells showing BCR-ABL-independent TKI resistance, and analysis of clinical samples also showed that FAM167A was observed in BCR-ABL ratio not only after TKI treatment but also at the time of CML diagnosis. As it was confirmed to be upregulated in the cells of patients with dependent TKI resistance, the biomarker protein can be used to diagnose BCR-ABL-independent TKI resistance in CML patients.

Accordingly, the second aspect of the present invention relates to a method of providing information for diagnosing BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients using the FAM167A biomarker.

As used herein, the term “method for providing information” refers to a method of providing information regarding BCR-ABL-independent TKI resistance in CML patients, including biomarker proteins and fragments thereof according to the present invention. Alternatively, it refers to a method of obtaining information on BCR-ABL-independent TKI resistance when the mRNA expression level of the gene encoding the protein is increased compared to CML without BCR-ABL-independent TKI resistance or, alternatively, in TKI-sensitive CML. do.

Specifically, the information provision method for diagnosing BCR-ABL-independent TKI resistance in chronic myeloid leukemia patients according to the present invention may include the following steps:

(a) measuring the mRNA expression level of FAM167A protein, a fragment thereof, or a gene encoding the protein in a biological sample isolated from a patient with chronic myeloid leukemia; and

(b) The expression level measured in step (a) is the mRNA of the same protein, fragment thereof, or gene encoding the protein measured in a biological sample isolated from a patient with chronic myeloid leukemia without BCR-ABL-independent TKI resistance. Step to compare expression level.

The information provision method of the present invention is (c) the mRNA expression level of the FAM167A protein, a fragment thereof, or a gene encoding the protein measured in a biological sample isolated from a chronic myeloid leukemia patient in stage (a), (b) BCR-ABL-independent, when higher than the level of mRNA expression of the same protein, fragment thereof, or gene encoding the protein measured in biological samples isolated from patients with chronic myeloid leukemia without tyrosine kinase inhibitor resistance. An additional step of determining whether the drug is resistant to the TKI may be additionally included.

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

In the information provision method of the present invention, the "biological sample" used for analysis is a CML patient without BCR-ABL-independent TKI resistance, or a BCR-ABL-independent TKI-resistant CML patient that can be distinguished from a TKI-sensitive CML patient. It includes a biological sample capable of confirming the mRNA level of a diagnostic-specific biomarker protein, a fragment thereof, or a gene encoding the protein. For example, the biological sample may refer to a biological sample containing CD34 ⁺ stem cells/progenitor cells isolated from a CML patient for which BCR-ABL-independent TKI resistance is to be determined, for example, blood, plasma. , serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymphatic fluid, airway fluid, serous fluid, urogenital fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, intra-organ fluid, ascites, cystic tumor fluid , amniotic fluid, or a combination thereof, but is not limited thereto.

In the information provision method of the present invention, the expression level of the biomarker can be measured at the mRNA expression level of the FAM167A protein, its fragment, or the gene encoding the protein, and the separation of the protein or mRNA from the biological sample is a known process. It can be performed using .

In the present invention, the term "protein level measurement" refers to a process of confirming the presence and expression level of the biomarker protein or fragment thereof in a sample, using an antibody that specifically binds to the protein or fragment thereof. This allows you to check the amount of protein. Analysis methods for this include western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, and rocket immunization. Rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, mass spectrometry ( mass spectrometry, FACS analysis (fluorescence activated cell sorter analysis), protein chip technology, etc., but are not limited thereto.

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

For example, the mRNA expression level of the biomarker gene can be measured by hybridization or gene amplification method. The hybridization method may confirm the presence of a gene using a probe.

When the information provision method of the present invention is implemented using a microarray method among hybridization methods, the above-described probe is used as a hybridizable array element and is immobilized on a substrate. Preferred substrates include rigid or semi-rigid supports, such as membranes, filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The hybridization array elements are arranged and immobilized on the substrate. This immobilization is carried out by a chemical bonding method or a covalent bonding method such as UV. For example, the hybridized array elements can be bonded to a glass surface modified to include epoxy compounds or aldehyde groups, and can also be bonded by UV to a polylysine coated surface. Additionally, the hybridization array elements can be coupled to substrates through linkers (eg, ethylene glycol oligomers and diamines).

Meanwhile, sample DNA applied to the microarray of the present invention can be labeled and hybridized with array elements on the microarray. Hybridization conditions can be changed in various ways. Additionally, detection and analysis of the degree of hybridization can be performed in various ways depending on the labeling substance.

The label of the probe can provide a signal to detect hybridization, which can be linked to an oligonucleotide. Suitable labels include fluorophores (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), terminal deoxynucleotidyl transferase (TdT), chromophore, and chemiluminescence. However, magnetic particles, radioactive isotopes (P32 or S35), mass labels, electron-dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (e.g. gold), and It includes, but is not limited to, haptens with specific binding partners such as antibodies, streptavidin, biotin, digoxigenin, and chelating groups. Labeling can be performed using various methods commonly practiced in the art, such as the nick translation method, the random priming method (Multiprime DNA labeling systems booklet, "Amersham" (1989)), and the kination method (Maxam & Gilbert, Methods). in Enzymology, 65:499 (1986)). Labels provide signals that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, high-frequency hybridization, and nanocrystals.

When using a probe, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined through a series of processes through an optimization procedure. These procedures are performed as a series by those skilled in the art to establish protocols for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as the length of the probe, the amount of GC, and the target nucleotide sequence.

After the hybridization reaction, the hybridization signal produced through the hybridization reaction is detected. Hybridization signals can be obtained in various ways, for example, depending on the type of label bound to the probe. For example, if the probe is labeled with an enzyme, hybridization can be confirmed by reacting the enzyme's substrate with the hybridization reaction product. Enzyme/substrate combinations that can be used include peroxidase (e.g., horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, and lucigenin (bis-N-methylacridinium nitrate). rate), resorufin benzyl ether, luminol, Amplexred reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2, 2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol/pyronine; Alkaline phosphatase and bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; These include glucose oxidase, t-NBT (nitroblue tetrazolium), and m-PMS (phenzaine methosulfate). If the probe is labeled with gold particles, it can be detected by silver staining using silver nitrate.

In the present invention, we confirmed that neutralizing FAM167A restores TKI sensitivity in cells with BCR-ABL-independent TKI resistance and reverses TKI resistance in a mouse tumor model, indicating that the BCR-ABL ratio targeting the FAM167A protein biomarker A treatment for CML showing dependent TKI resistance and a BCR-ABL independent TKI resistance inhibitor can be provided.

Accordingly, a third aspect of the invention is a FAM167A protein inhibitor; An inhibitor of the expression of the gene encoding the FAM167A protein; It relates to a pharmaceutical composition for preventing or treating CML showing BCR-ABL-independent TKI resistance, and a pharmaceutical composition for suppressing BCR-ABL-independent TKI resistance in CML patients, comprising a mixture thereof as an active ingredient.

In the present invention, the FAM167A protein inhibitor may include an agent that reduces the expression, function or activity of the FAM167A protein.

In the present invention, the expression inhibitor of the gene encoding the FAM167A protein may include an agent that reduces mRNA expression of the FAM167A gene.

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

Specifically, the FAM167A protein inhibitor may include 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 the FAM167A protein, It is not limited to this.

The expression inhibitor of the gene encoding the FAM167A protein may be an inhibitor that binds to the gene itself and interferes with transcription, or binds to mRNA transcribed from the gene and interferes with translation of the mRNA. For example, the expression inhibitor of the gene may include, but is not limited to, any one or more selected from the group consisting of antisense nucleotides, siRNA, and shRNA that bind complementary to the mRNA of the FAM167A gene.

In the present invention, the term "siRNA" refers to a double-stranded RNA that induces RNA interference through cleavage of the mRNA of the target gene, and includes an RNA strand of a sense sequence having the same sequence as the mRNA of the target gene and a sequence complementary thereto. It consists of an RNA strand with an antisense sequence.

The siRNA may include the siRNA itself synthesized in vitro or a form expressed by inserting the base sequence encoding the siRNA into an expression vector.

In the present invention, the “vector” refers to a gene construct containing foreign DNA inserted into the genome encoding a polypeptide.

The vector related to the present invention is a vector in which a nucleic acid sequence that suppresses the gene is inserted into the genome, and examples of these vectors include DNA vectors, plasmid vectors, cosmid vectors, bacteriophage vectors, yeast vectors, or viral vectors.

In addition, the antisense has a sequence complementary to all or part of the mRNA sequence transcribed from the FAM167A gene or fragment thereof, and can bind to the mRNA to inhibit the expression of the FAM167A gene or fragment.

In addition, the shRNA (short hairpin RNA) can be produced according to a conventional method by targeting the common base sequence region of human or mouse shRNA.

In the present invention, chronic myeloid leukemia showing BCR-ABL-independent TKI resistance may have increased NF-κB activity compared to CML showing TKI sensitivity.

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

In the present invention, the term “combined administration” means that different ingredients are administered together to a subject. That different components are administered together means 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.

Accordingly, the pharmaceutical composition of the present invention can be administered simultaneously with the TKI, separately, or sequentially.

In the present invention, the TKI includes crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, and imatinib ( imatinib, nilotinib, ponatinib, ibrutinib, cabozantinib, gefitinib, erlotinib, lapatinib, bande Vandetanib, afatinib, osimertinib, ruxolitinib, tofacitinib, axitinib, lenvatinib, nintedanib ( It may include, but is not limited to, nintedanib, pazopanib, regorafenib, sorafenib, or sunitinib.

In the present invention, the terms “prevention” and/or “treatment” refer to any act that suppresses or delays the onset of a disease or condition, any act that improves or beneficially changes the condition of a disease or condition, and delays the progression of a disease or condition. , means any act of stopping or reversing.

The pharmaceutical composition of the present invention contains suitable carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, and surfactants commonly used in the preparation of pharmaceutical compositions. It may further include one or more additives selected from the group consisting of activators, binders, and lubricants.

Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, not determined. Quality cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. It can be prepared by mixing. Additionally, in addition to simple excipients, lubricants such as magnesium styrate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

From another perspective, the carrier included in the pharmaceutical composition of the present invention may be ion exchange resin, alumina, aluminum stearate, lecithin, serum protein (e.g., human serum albumin), buffering material (e.g., various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium These include, but are not limited to, trisilicates, polyvinylpyrrolidone, cellulose-based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, or wool paper.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogenatin, etc. can be used.

The pharmaceutical composition according to the present invention can be prepared as an aqueous solution for parenteral administration, preferably using a buffered solution such as Hank's solution, Ringer's solution, or physically buffered saline. . Aqueous injection suspensions can be supplemented with substances that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

In addition, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, emulsifiers, suspending agents, or preservatives in addition to the above components.

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

The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required to achieve the effect of preventing, suppressing, or alleviating a disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form and the patient's age, weight, general health condition, gender and diet, administration time, administration route and composition. It can be adjusted depending on a variety of factors, including secretion rate, duration of treatment, and concurrent medications.

In relation to the third aspect, the present invention includes administering a therapeutically effective amount of a FAM167A protein inhibitor, an expression inhibitor of the gene encoding the FAM167A protein, or a mixture thereof to a CML patient showing BCR-ABL independent TKI resistance. A method of treating CML showing BCR-ABL-independent TKI resistance and a method of suppressing BCR-ABL-independent TKI resistance are provided.

In the present invention, the FAM167A protein inhibitor or the expression inhibitor of the gene encoding the FAM167A protein used in the above method and the method of administration thereof are as described above, and therefore, their description is omitted to avoid excessive complexity of the specification.

Meanwhile, the subject of the CML patient showing BCR-ABL-independent TKI resistance is the same as the subject of the patient described in the information provision method, and thus the description thereof is omitted to avoid excessive complexity of the present specification.

In the present invention, it was confirmed that neutralizing FAM167A restores TKI sensitivity in BCR-ABL-independent TKI-resistant cells and reverses TKI resistance in a mouse tumor model. As a result, it was confirmed that BCR-ABL inhibition depends on whether or not the FAM167A protein biomarker is inhibited. It provides criteria for judging whether it is a treatment for CML that exhibits TKI-independent resistance or as an inhibitor of BCR-ABL-independent TKI resistance.

Accordingly, the fourth aspect of the present invention relates to a method of screening for a treatment for CML or a BCR-ABL independent TKI resistance inhibitor showing BCR-ABL-independent TKI resistance using the FAM167A biomarker.

Specifically, the screening method of the present invention may include the following steps:

(a) treating a candidate material to a cell line or animal model showing BCR-ABL-independent TKI resistance; and

(b) Checking whether the FAM167A protein is inhibited and/or the expression of the gene encoding the protein is inhibited in the cell line or animal model treated with the candidate material.

In the present invention, the candidate material is estimated to have the potential as a substance that inhibits transcription and translation from the FAM167A gene base sequence to mRNA and protein, or as a medicine that inhibits the function or activity of the FAM167A protein, according to a conventional selection method. Alternatively, it may be a randomly selected individual nucleic acid, protein, peptide, other extract, natural product, compound, etc.

Thereafter, the expression level, protein level, or activity of the gene can be measured in a biological sample isolated from the cell line or animal model treated with the candidate material, and as a result of the measurement, the expression level, protein level, or activity of the gene is decreased. If this is measured, the candidate substance can be judged as a treatment for CML showing BCR-ABL-independent TKI resistance and an inhibitor of BCR-ABL-independent TKI resistance.

Therefore, the screening method of the present invention is (c) when the candidate material inhibits the FAM167A protein or suppresses the expression of the gene encoding the protein, the candidate material is used as a treatment for CML showing BCR-ABL independent TKI resistance or An additional step of determining that the inhibitor is a BCR-ABL independent TKI resistance inhibitor may be included.

In the present invention, the method for measuring gene expression, protein level, or activity can be performed through various methods known in the art, and is the same as described in the information provision method, so the excessive amount of the present specification To avoid complexity, the description is omitted.

In the present invention, the candidate substance acts as a leading compound in the development process of a treatment for CML showing BCR-ABL-independent TKI resistance and a BCR-ABL-independent TKI resistance inhibitor. By modifying and optimizing the structure to have the effect of suppressing the function of the FAM167A gene or the protein expressed therefrom, new treatments for CML and TKI resistance inhibitors can be developed.

Hereinafter, the present invention will be described in more detail through examples. However, since the present invention can make various changes and take various forms, the specific embodiments and descriptions described below are only intended to aid understanding of the present invention and do not limit the present invention to the specific disclosed form. That's not what I'm trying to do. The scope of the present invention should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope of the present invention.

Identification of differentially expressed genes and transcription factors that regulate them in TKI-resistant CML cells

1-1. cell culture

The human CML cell line K562S was obtained from the Korea 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 TKI. All cell lines were maintained at 37°C and 5% CO ₂ in a humidified cell culture incubator and incubated at Roswell Park Memorial Institute (RPMI) supplemented with 10% fetal bovine serum (HyClone), 100 U/ml penicillin, and 100 μg/ml streptomycin. ) were cultured in 1640 medium (HyClone). To maintain TKI resistance, K562R and KCL22R cells were cultured in medium supplemented with 1 μM imatinib.

1-2. Microarray and RNA-seq analysis

TKI resistance that does not involve mutations in the BCR-ABL kinase domain (i.e., BCR-ABL independent TKI resistance) is not well understood. Accordingly, the present inventors attempted to find genes that play a potential role in BCR-ABL-independent TKI resistance by identifying genes differentially expressed in TKI-resistant CML cells (Figure 1a). Therefore, K562R (FIGS. 1b to 1e), a TKI-resistant CML cell line without a mutation in the BCR-ABL kinase domain, was generated as in Example 1-1, and then TKI-sensitive K562S cells, K562R cells, and K562R treated with imatinib. Gene expression in cells was analyzed, and both microarray and RNA sequencing (RNA-seq) analyzes were performed to eliminate false positives due to the analysis method.

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 the Human HT-12 v4 Expression BeadChip (Illumina), and the array was 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 the NovaSeq 6000 system (Illumina). Genes showing a fold change ≥ 2 are defined as differentially expressed and displayed in the heatmap generated in the Multiple Experiment Viewer.

789 differentially expressed (i.e. fold change ≥ 2) genes between K562S and K562R were rigorously selected (Figure 1f and 1g).

1-3. In silico ( in silico ) analyze

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, transcription factors associated with each binding site in differentially expressed or randomly selected gene promoters from the GeneCards database (https://www.genec ards.org/). Information was obtained, and plots were created using Cytoscape. Enrichment scores for transcription factors were 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) × (number of differentially expressed genes associated with transcription factors).

In silico analysis of transcription factors involved in differential gene expression in BCR-ABL-independent TKI-resistant CML cells identified AP-1 and NF-κB as regulators of differentially expressed genes compared to randomly selected genes. It was found to be the most abundant transcription factor (Figures 1h to 1k). AP-1 and NF-κB showed significantly higher enrichment scores compared to other transcription factors, and only the scores for these two transcription factors were more than 3 standard deviations from the mean.

1-4. Luciferase reporter assay

To determine 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 determine the effect of TKI treatment. investigated.

For this, K562S and K562R cells were grown using Lipofectamine 2000 (Invitrogen) as described in Park SG, Schulze-Luehrman J, Hayden MS, Hashimoto N, Ogawa W, Kasuga M, et al. The kinase PDK1 integrates T cell antigen receptor and CD28 coreceptor signaling to induce NF-kappaB and activate T cells. Nat Immunol. 2009;10(2):158-66.] or the Renilla luciferase vector and NF-κB dependent reporter construct (pBIIx-luc) described in [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 co-transfected with the AP-1-dependent reporter construct (AP-1-luc) and other plasmids, and then processed as indicated in each document. 24 hours after transfection, luciferase activity was measured using the Dual-luciferase Reporter Assay Kit (Promega) and normalized to Renilla luciferase activity.

The results showed that NF-κB activity, but not AP-1, was significantly higher in K562R cells than in K562S cells. Additionally, there was no significant difference in AP-1 activity, although this activity was much higher in K562R cells treated with imatinib (Figure 1L). This suggests that NF-κB contributes to BCR-ABL-independent TKI resistance by regulating gene expression. Additionally, to identify genes associated with increased NF-κB activity in K562R cells, we used gene expression profiling data from K562S, K562R, and K562R cells treated with imatinib and identified seven genes that showed similar expression patterns with patterned NF-κB activity. Differentially expressed genes (FAM167A, AIF1L, ACSS1, S100A4, LY6G6D, CYP11A1, and NR5A1) were selected.

1-5. Quantitative reverse transcription PCR (qRT-PCR)

qRT-PCR was performed to confirm the expression level of selected genes. Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen), and cDNA was prepared with TOPscript RT Drymix (Enzynomics). qRT-PCR was performed using SYBR Green qPCR 2x Premix (Enzynomics) on a Stratagene Mx3000P (Agilent Technologies). Results were normalized to GAPDH levels. Primer sequences used in qRT-PCR are shown in Table 1, and qRT-PCR results are shown in Figure 1m.

primer order sequence number FAM167A-F 5′-GCA CAG TGA ACA CAA CTA ACC-3′ 3 FAM167A-R 5′-CTT GGG GAT GGC AGA GAG AT-3′ 4 S100A4-F 5′-TCT TGG GGA AAA GGA CAG ATG-3′ 5 S100A4-R 5′-CAT TTC TTC CTG GGC TGC TTA-3′ 6 CYP11A1-F 5′-TTC CTG CCA AGA CAC TGG TG-3′ 7 CYP11A1-R 5′-GAT CCG CCG TCC CAG ACA-3′ 8 ACSS1-F 5′-TTG GAG GTC TGG ATC CAG TC-3′ 9 ACSS1-R 5′-GAC AAA CTC TCC CTC CCC TA-3′ 10 LY6G6D-F 5′-TAC CTG GAA ACC CCC CAG T-3′ 11 LY6G6D-R 5′-TGC CTG CAG GGG CCA CAT-3′ 12 NR5A1-F 5′-TTC AGC CTG GAT TTG AAG TTC-3′ 13 NR5A1-R 5′-CTT GTG GTA CAG GTA CTC C-3′ 14 AIF1L-F 5′-AGG GTC TCA AGA GTT GTC CC-3′ 15 AIF1L-R 5′-ATA CTT GGC AGT CCT CAC GTT-3′ 16 GAPDH-F 5′-GGA GCG AGA TCC CTC CAA AAT-3′ 17 GAPDH-R 5′-GGC TGT TGT CAT ACT TCT CATG-3′ 18

FAM167A role in the non-canonical NF-κB pathway

2-1. Luciferase reporter assay of selected genes

To screen selected genes, NF-κB activity was confirmed by luciferase reporter assay in K562S cells transfected with plasmids encoding the indicated genes. The plasmid was prepared by cloning HA tags S100A4, CYP11A1, ACSS1, LY6G6D, NR5A1, AIF1L, and FAM167A into the MigR1 vector.

Among the seven selected genes, only FAM167A significantly increased NF-κB activity when expressed ectopically (Figure 2A). Additionally, compared with other genes, ectopic expression of FAM167A increased the viability of K562S cells in the presence of imatinib (Figure 2b). Although FAM167A does not have a signal sequence for secretion, FAM167A had a high score (NN) in bioinformatic analysis using SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP/), which predicts non-classical protein secretion. -score = 0.905) (A in Figure 2c).

2-2. Cell fraction preparation and immunoblot analysis

Secretion of FAM167A was analyzed by immunoblot analysis of K562R cell lysates and culture supernatants.

Cell lysates were extracted by boiling the cells in SDS sample buffer. Proteins in the culture supernatant were concentrated through precipitation using acetone and then extracted by boiling in SDS sample buffer.

Next, for immunoblot analysis, proteins were separated by SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis on an 8-15% gel and then transferred to a polyvinylidene difluoride membrane. Membranes were probed with anti-FAM167A antibody (sc-393999, Santa Cruz Biotechnology) or Erbin antibody (NBP2-13968, Novus Biologicals) as indicated. As a result of the analysis, it was confirmed that FAM167A was secreted by these cells (Figure 2C, B).

2-3. Confirmation of the NF-κB activation function of FAM167A

To directly test NF-κB activation function, recombinant FAM167A protein (MBS1363522, MyBioSource) and a neutralizing antibody specific for FAM167A (sc-393999, Santa Cruz Biotechnology) were used to increase and decrease FAM167A levels during cell culture, respectively. , NF-κB luciferase reporter assay was performed.

Consistent with the results of the bioinformatic analysis, treatment of K562S cells with soluble recombinant FAM167A increased NF-κB activity (Figure 2D), whereas neutralizing FAM167A in K562R cell cultures decreased NF-κB activity (Figure 2E). . This suggests that the secreted FAM167A protein functions as an inducer of the NF-κB pathway.

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 inhibits the non-canonical NF-κB pathway. After this, NF-κB luciferase reporter analysis was performed. For transient expression of NIK-DN, the method described in Park SG, Ryu HM, Lim SO, Kim YI, Hwang SB, Jung G. Interferon-gamma inhibits hepatitis B virus-induced NF-kappaB activation through nuclear localization of NF-kappaB- inducing kinase. Gastroenterology. pFLAG-NIK-DN described in 2005;128(7):2042-53.] was used.

SN50 treatment, which inhibits the canonical NF-κB pathway, did not significantly reduce NF-κB activity in K562R cells, but SN50 treatment blocked further lipopolysaccharide (LPS)-stimulated increase in NF-κB activity ( Fig. 2F ). In contrast, transient expression of dominant-negative NIK (NIK-DN), which inhibits 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).

2-5. Immunoblot analysis for NIK and p100/p52

Non-canonical NF-κB activation is dependent on NIK accumulation through blockade of ubiquitination-dependent NIK degradation, and accumulated NIK induces processing of p100 to p52, followed by nuclear translocation of p52-containing dimers. Therefore, we confirmed whether the non-canonical NF-κB pathway is activated in K562R cells through immunoblot analysis for NIK and p100/p52 in K562S cells and K562R cells.

Membranes were probed with anti-NIK (4994, Cell Signaling Technology), anti-p100/p52 ((sc-7386 Density measurements were performed using ImageJ software using as an internal standard.

As a result of the analysis, the level and activity of NIK, which processes p100 into p52, were higher in K562R cells than in K562S cells (Figures 2h and 2i).

2-6. Electrophoretic mobility shift assay (EMSA)

The nuclear fraction was extracted as follows. First, cells were suspended in hypotonic buffer (10mM HEPES, pH7.9; 10mM KCl; 1.5mM MgCl2; 0.5mM DTT; 0.5mM PMSF) and incubated on ice for 15 minutes. Then, 0.05% Nonidet P-40 was added and the lysate was passed through a 25-gauge needle five times. After centrifugation (1000 × g, 5 min, 4 °C) to precipitate the nuclei, the supernatant was collected and separated by high-speed centrifugation (16,000 × g, 5 min, 4 °C). The resulting supernatant was stored at -80°C as a cytosolic fraction. The pellet containing cell nuclei was washed with hypotonic buffer and incubated in hypertonic buffer (20mM HEPES, pH7.9; 420mM NaCl; 1.5mM MgCl ₂ ; 25% glycerol; 0.2mM EDTA; 0.5mM DTT; 0.5mM PMSF; 1μg/mL). It was suspended in mL leupeptin, 1 μg/mL aprotinin, and 1 μg/mL pepstatin A), and then incubated on ice for 30 minutes while vortexing every 10 minutes. The nuclear lysate was centrifuged (16,000Хg, 5 minutes, 4°C), and the resulting supernatant was stored at -80°C as the nuclear fraction.

For supershift analysis, nuclear fractions were incubated in binding buffer (5mM Tris, pH7.5; 25mM KCl; 0.5mM EDTA; 2.5% glycerol; 0.5mM DTT; 0.1μg/μl poly(dI/dC), 0.5mg/ml BSA). After incubation with anti-p52 antibody (4882, Cell Signaling Technology) or isotype control (H9658, Sigma-Aldrich) for 20 minutes at room temperature, biotinylated double-stranded NF- Additional incubation was performed with the κB probe (SEQ ID NO: 23: 5'-AGTTGAGGGGACTTTCCCAGG-3'). Reaction samples were separated through a 6% non-denaturing polyacrylamide gel and transferred to a nylon membrane. Probes on the membrane were visualized using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific) and quantified with ImageJ.

Supershift analysis performed in EMSA also showed higher NF-κB activity and p52 supershift levels in K562R cells (Figure 2J).

2-7. Confirmation of the non-canonical NF-κB pathway activation effect of FAM167A

Based on the above results, it was confirmed whether FAM167A is responsible for the activation of the non-canonical NF-κB pathway.

First, we confirmed NF-κB luciferase reporter activity in K562S cells 24 h after treatment with 100 ng/ml recombinant FAM167A and transfection of the plasmid encoding NIK-DN. Interestingly, NF stimulated by recombinant FAM167A -κB activity was reduced in cells with transient expression of NIK-DN ( Fig. 2K ).

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

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

Therefore, the above data indicate that FAM167A activates the non-canonical NF-κB pathway to a greater extent in K562R cells than in K562S cells.

Identification of FAM167A binding receptor

3-1. flow cytometry

As processing and neutralization of secreted FAM167A effectively regulates the non-canonical NF-κB pathway, we hypothesized that a receptor for FAM167A may be expressed on the cell surface. To assess the expression of potential receptors for FAM167A, K562S and K562R cells were first labeled with Myc-tagged recombinant FAM167A, followed by fluorescent dye-conjugated anti-Myc antibody staining and flow cytometry.

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

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

The results of the analysis suggested that the receptor for FAM167A was expressed on the cell surface, and the level in K562R cells was higher than that in K562S cells (Figure 3a).

After confirming the presence of the receptor on the cell surface, the receptor was immunoprecipitated from K562R cells using Myc-tagged recombinant FAM167A and an anti-Myc antibody coupled to protein G Sepharose® beads to isolate the receptor for identification (Figure 3B ).

Silver staining of proteins immunoprecipitated with Myc-tagged FAM167A was performed using the Pierce Silver Stain Kit (24612, Thermo Fisher Scientific). Silver staining revealed two distinct bands compared to background, which was also detected in control samples immunoprecipitated without FAM167A ( Fig. 3C ). LC-MS/MS analysis identified one band (approximately 30 kDa) as recombinant FAM167A and the other band (approximately 120 kDa) as DSG1, a known cell adhesion molecule (Figure 3d).

3-2. Co-immunoprecipitation using K562R cell lysates.

Cells were lysed in lysis buffer (20mM Tris-HCl, pH8.0; 150mM NaCl; 1% Triton ; 0.1mM PMSF) for 30 min on ice. The lysate was then centrifuged at 15,000 Protein G Sepharose® beads (GE Healthcare) were added and incubated with rotation at 4°C. After washing four times with lysis buffer, the immunoprecipitate was eluted by boiling in SDS sample buffer and then subjected to immunoblot analysis. Band intensity was analyzed with ImageJ software.

Through this, it was confirmed that FAM167A binds to DSG1 (Figure 3e).

3-3. qRT-PCR and immunoblot analysis for DSG1

To confirm that the upregulated surface expression of DSG1 was mediated by an increase in total DSG1 protein levels, the expression of DSG1 mRNA and protein was measured by qRT-PCR and immunoblot analysis, respectively. The sequences of primers used in qRT-PCR are shown in Table 2.

primer order sequence number DSG1-F 5′-TGC TGG AGT TGA AAG GCA TTA-3′ 19 DSG1-R 5′-AGT GCA ATG TGA AAT GGG TCT-3′ 20 GAPDH-F 5′-GGA GCG AGA TCC CTC CAA AAT-3′ 17 GAPDH-R 5′-GGC TGT TGT CAT ACT TCT CATG-3′ 18

Interestingly, there was no difference in expression of mRNA or protein between K562S and K562R cells (Figures 3f and 3g). In contrast, cell surface expression of DSG1 was higher in K562R cells (Figures 3A and 3H).

3-4. Immunofluorescence staining and NF-kB luciferase reporter assay.

For immunofluorescence staining, fixed and permeabilized cells were stained with anti-DSG1 antibody (129204, R&D Systems) and then with 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 accompanying FV10-ASW software.

Additionally, NF-κB luciferase reporter activity was analyzed in K562S cells 24 h after transfection of the plasmid encoding FAM167A and treatment with the indicated concentrations of anti-FAM167A neutralizing antibody.

Through immunofluorescence staining results and NF-κB luciferase reporter activity analysis results, it was confirmed that DSG1 was mainly localized to the surface of K562R cells, not K562S cells (Figure 3i). Additionally, FAM167A mainly colocalized with DSG1 on the surface of K562R cells (Figure 3j). Although FAM167A was ectopically expressed in K562S cells, FAM167A-induced NF-κB activation was significantly inhibited by treatment with anti-FAM167A neutralizing antibody ( Fig. 3K ), indicating that secreted FAM167A activates the non-canonical NF-κB pathway . Taken together, these results show that DSG1 is a receptor for FAM167A and its surface expression is higher in K562R cells than in K562S cells.

Confirmation of regulation of NIK ubiquitination through DSG1 in FAM167A

4-1. Confirmation of NF-κB activity in K562R cells with DSG1 knocked down

Because surface expression of DSG1 was higher in K562R cells than in K562S cells (Figures 3A and 3H), we hypothesized that DSG1 knockdown in K562R cells may reduce non-canonical NF-κB activity. To test this, K562R cells were lentivirally transduced with a DSG1-specific shRNA short hairpin RNA (shRNA) construct to knock down the expression of DSG1 (Figure 4a), followed by NF-κB luciferase reporter assay and p100/ Immunoblot analysis for p52 was performed. DSG1-specific shRNA lentiviral particles (sc-35224-V) and control shRNA lentiviral particles (sc-108080) were obtained from Santa Cruz Biotechnology.

Contrary to expectations, DSG1 knockdown increased NF-κB activity and processing of p100 to p52 in K562R cells (Figures 4B and 4C).

4-2. Confirmation of NF-κB activity in cells transiently expressing DSG1 or DSG1 and Erbin

After transient expression of DSG1 and its interacting protein Erbin in K562R cells treated with or without anti-FAM167A neutralizing antibody, NF-κB activity was measured. Transient expression of DSG1 was performed using a plasmid in which DSG was cloned into the MigR1 vector, and transient expression of Erbin was performed using pClneo-Myc-Erbin (Addgene).

NF-κB activity was reduced by transient expression of DSG1 and was 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 additional reduction in NF-κB activity (not significant, p > 0.05) ( Fig. 4D ). These data show that DSG1 and Erbin inhibit, whereas FAM167A activates non-canonical NF-κB through the same pathway.

4-3. Confirmation of expression level of Erbin

To further investigate the underlying 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. Primer sequences used for qRT-PCR are shown in Table 3, and anti-Erbin antibody (NBP2-13968, Novus Biologicals) was used for immunoblot analysis.

primer order sequence number Erbin-F 5′-CAA GTC TCG GTG TTC CCT TT-3′ 21 Erbin-R 5′-AGA TCC ATT GTT CCG TGA GG-3′ 22 GAPDH-F 5′-GGA GCG AGA TCC CTC CAA AAT-3′ 17 GAPDH-R 5′-GGC TGT TGT CAT ACT TCT CATG-3′ 18

Similar to DSG1 mRNA and protein levels (Figures 3F and 3G), the mRNA and protein levels of Erbin were not significantly different between K562S and K562R cells (Figures 4E and 4F).

4-4. Confirmation of interaction between Erbin and NIK through co-immunoprecipitation analysis

Because NIK is a key component of the non-canonical NF-κB pathway, co-immunoprecipitation analysis was performed to investigate the interaction between DSG1, Erbin, and NIK.

First, cells were lysed in lysis buffer (20mM Tris-HCl, pH8.0; 150mM NaCl; 1% Triton Statin A; 0.1mM PMSF) for 30 min on ice. The lysate was then centrifuged at 15,000 xg 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 with rotation at 4°C. After washing four times with lysis buffer, the immunoprecipitate was eluted by boiling in SDS sample buffer and then subjected to immunoblot analysis. Band intensity was analyzed with ImageJ software.

Although the binding of Erbin to DSG1 was not significantly different between K562S and K562R cells, the binding of NIK to DSG1 was shown to be reduced 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 the binding of NIK to DSG1. When FAM167A was neutralized, increased binding could be observed (Figure 4h).

4-5. Confirmation of NIK ubiquitination through immunoblot analysis

Since NIK is regulated through ubiquitination-mediated degradation, we sought to investigate NIK ubiquitination through immunoblot analysis after FAM167A neutralization in K562R cells.

Specifically, NIK immunoprecipitation from K562R cells treated with 2 μg/ml anti-FAM167A neutralizing antibody or isotype control and 10 μM MG132 (474790, Sigma-Aldrich) for 3 h followed by anti-ubiquitin antibody (sc-8017, Santa NIK ubiquitination was confirmed using Cruz Biotechnology).

Immunoblot analysis showed that ubiquitination of NIK increased when FAM167A was neutralized (Figure 4I). Ubiquitination of NIK appears to be regulated by changes in its interaction with DSG1 and Erbin (Figures 4G-4I), but this protein has no reported ubiquitin ligase function. To identify the ligase responsible for NIK ubiquitination, co-immunoprecipitation analysis was performed on candidate ubiquitin ligase components including TRAF3, CHIP, and c-cbl. The anti-TRAF3 antibody (4729), anti-CHIP antibody (2080), and anti-c-cbl antibody (2747) used at this time were purchased from Cell Signaling Technology. As a result of co-immunoprecipitation analysis, no interaction was found for these (Figure 4j).

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 leads to non-canonical NF-κB activity and cellular uptake of DSG1. The relationship between surface expression can be explained (Figure 4k).

Confirmation of FAM167A role in BCR-ABL-independent TKI resistance

5-1. Cell survival rate confirmed by inhibiting 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. For this purpose, the survival rate of K562R cells transfected with a plasmid encoding NIK-DN was confirmed after treatment with imatinib (sc-202180, Santa Cruz Biotechnology) or nilotinib (17050, BioVision) for 3 days.

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

Inhibition 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 (Figures 5A and 5B).

5-2. Cell survival rate confirmed by treatment with anti-FAM167A neutralizing antibody after TKI treatment

Additionally, cell viability assays and apoptosis analysis were performed to determine the survival of K562R cells after treatment with 2 μg/ml anti-FAM167A neutralizing antibody or isotype control in the presence of imatinib or nilotinib for 3 days.

For apoptosis analysis, K562S or K562R cells were seeded in 6-well plates and treated as indicated. After 72 hours, cells were stained with Annexin V-APC and propidium iodide using the Annexin V-APC Apoptosis Detection Kit (Thermo Fisher Scientific) and analyzed on a Guava EasyCyte HT cytometer (Millipore).

Analysis of cell viability (in WST assay) and apoptosis (measured by flow cytometry) showed that neutralization of FAM167A significantly reduced the viability of K562R cells (Figures 5A and 5C) and increased apoptosis in the presence of TKIs. (Figure 5d). Therefore, FAM167A-mediated activation of the non-canonical NF-κB pathway is important for TKI resistance.

In vivo ( in vivo ) Confirmation of the role of FAM167A in BCR-ABL-independent TKI resistance

The effect of FAM167A on TKI resistance in vivo was investigated using an established mouse xenograft model by subcutaneously injecting K562R cells into nude mice.

6-1. Establishment of mouse xenograft model

K562R cells (1Х 10 ⁷ ) were suspended in 50% (v/v) serum-free Matrigel (Corning) and transplanted subcutaneously into the right flank of 6-week-old female BALB/c(nu/nu) mice. Tumor size was measured with calipers, and tumor volume was calculated using the standard formula (width ² × length/2). When the tumor volume reached 100-200 mm ³ , 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) for 10 days. intraperitoneally), or vehicle (saline, PBS). All animal experiments were performed according to protocols approved by the Seoul National University Animal Experiment Committee (SNU-210315-6) and the Gwangju Institute of Science and Technology (GIST-2019-008).

6-2. Confirmation of CML treatment effect following treatment with anti-FAM167A neutralizing antibody

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) (Figure 6B) and the body weight of the mice (Figure 6B) Figure 6c), the size (Figure 6d) and weight (Figure 6e) of tumors collected from mice in each group on day 10 of treatment were measured. Compared with imatinib alone or the combination of imatinib with an isotype control antibody, treatment with imatinib plus FAM167A neutralization (induced by intraperitoneal injection of anti-FAM167A neutralizing antibody) significantly inhibited tumor growth without body weight loss (Figure 6B to 6e).

To investigate the basis for differences in tumor growth at the cellular level, immunohistochemical staining was performed. 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. The number of Ki-67 ⁺ or TUNEL + cells in each field was counted. NIK expression in tumor cells was scored (intensity: 0 = negative, 1 = weak, 2 = moderate, 3 = strong) and the H score was calculated using the following equation.

H score = 1 × (% of cells at intensity 1) + 2 × (% of cells at intensity 2) + 3 × (% of cells at intensity 3).

Analysis of tumor tissue sections using HE staining showed lower cell density in FAM167A-neutralized tumors. Additional immunohistochemical staining showed increased levels of the apoptosis marker TUNEL, along with reduced expression of NIK and the proliferation marker Ki-67 (Figure 6f). Taken together, these data suggest that neutralizing FAM167A restores TKI sensitivity in BCR-ABL-independent TKI-resistant cells.

Confirmation of FAM167A expression in BCR-ABL-independent TKI-resistant KCL22R cells

7-1. cell culture

In addition to K562R cells, BCR-ABL-independent TKI-resistant KCL22R cells were investigated. KCL22S and KCL22R were obtained from the American Type Culture Collection (ATCC). All cell lines were maintained at 37°C and 5% CO ₂ in a humidified cell culture incubator and incubated at Roswell Park Memorial Institute (RPMI) supplemented with 10% fetal bovine serum (HyClone), 100 U/ml penicillin, and 100 μg/ml streptomycin. ) were cultured in 1640 medium (HyClone). To maintain TKI resistance, KCL22R cells were cultured in medium supplemented with 1 μM imatinib.

7-2. Determination of FAM167A levels in KCL22R cells

Through sequence analysis, it was confirmed that there was no mutation in the ABL region. In addition, the mRNA expression level of FAM167A was confirmed by qRT-PCR, and KCL22R cells showed higher FAM167A levels than KCL22S cells, and imatinib treatment of KCL22R cells showed increased FAM167A expression (Figure 7).

Confirmation of FAM167A expression in CML patients

To confirm the clinical significance of the results of this study, we analyzed FAM167A levels in CD34 ⁺ stem/progenitor cells isolated from CML patients by qRT-PCR.

8-1. Primary human samples

Peripheral blood or bone marrow samples were collected from 58 patients with CML (clinopathological characteristics listed in Table 4) at Chonnam National University Hospital, Hwasun. Samples were collected at diagnosis, before treatment with TKIs (diagnosis), and again at follow-up after treatment with TKIs (follow-up). Mononuclear cells isolated from patients were stored frozen in liquid nitrogen until further use. Informed consent was obtained in accordance with the Declaration of Helsinki, and all procedures were approved by the Seoul National University Institutional 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. The mutational status of BCR-ABL in CML samples was confirmed by sequencing analysis.

patient number age gender Diagnosis Imatinib resistance ^One
Imatinib-responsive patients BCR-ABL mutation status ² One 74 F CP reactivity - 2 59 F CP reactivity - 3 68 F CP reactivity - 4 67 F CP reactivity - 5 69 M CP reactivity - 6 60 M CP reactivity - 7 57 M CP reactivity - 8 48 M CP reactivity - 9 48 M CP reactivity - 10 41 M CP reactivity - 11 56 M CP reactivity - 12 75 F CP reactivity - 13 56 M CP reactivity - 14 46 M CP reactivity - 15 69 M CP reactivity - 16 76 F CP reactivity - 17 69 F CP reactivity - 18 23 M CP reactivity - 19 69 F CP reactivity - 20 53 F CP reactivity - 21 67 M CP reactivity - 22 61 M CP reactivity - 23 70 M CP reactivity - 24 73 F CP reactivity - 25 26 M CP reactivity - 26 18 M CP reactivity - 27 52 M CP reactivity - 28 58 M CP reactivity - 29 66 M CP reactivity - 30 34 M CP reactivity - 31 79 F CP reactivity - 32 53 M CP reactivity - 33 43 M CP reactivity - 34 43 F CP reactivity - Imatinib-resistant patients without BCR-ABL mutation One 62 M CP tolerance no mutations 2 37 F CP tolerance no mutations 3 44 M CP tolerance no mutations 4 77 M CP tolerance no mutations 5 61 F CP tolerance no mutations 6 37 F CP tolerance no mutations 7 56 M CP tolerance no mutations 8 28 M CP tolerance no mutations 9 49 M CP tolerance no mutations 10 20 F AP tolerance no mutations 11 53 F CP tolerance no mutations 12 52 M AP tolerance no mutations Imatinib-resistant patients with BCR-ABL mutations One 51 M CP tolerance Y253H 2 61 F CP tolerance G250E 3 60 F AP tolerance F359I 4 40 M BP tolerance T315I 5 43 F CP tolerance P480L 6 44 M CP tolerance H396R 7 58 M CP tolerance Y215F 8 30 M CP tolerance F317L 9 34 M CP tolerance E255K 10 61 M BP tolerance E255K 11 60 F CP tolerance T315I 12 54 M CP tolerance G250E

¹ Imatinib resistance is clinically classified according to the European Leukemia Net guidelines. ² Confirmation of BCR-ABL mutation status through base sequence analysis. CP: chronic phase; AP: Accelerator; BP: blast stage.

The data show that expression of FAM167A was higher in cells from BCR-ABL-independent imatinib-resistant patients (R, no mutation) than in cells from imatinib-responsive patients or BCR-ABL-dependent imatinib-resistant patients (S and R mutations, respectively). showed (Figure 8a). Additionally, FAM167A levels were higher in cells from BCR-ABL-independent imatinib-resistant patients before receiving treatment (i.e., at diagnosis), suggesting that expression of FAM167A may predict BCR-ABL-independent TKI resistance in CML patients. suggests.

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) 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).

statistical analysis

The number of repetitions for each experiment is indicated in the figure. Data are expressed as mean ± standard deviation (s.d.). 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).

Claims (21)

A biomarker composition for diagnosing BCR-ABL-independent imatinib or nilotinib resistance in patients with chronic myeloid leukemia, comprising the FAM167A protein or the gene encoding it. Diagnosing BCR-ABL-independent imatinib or nilotinib resistance in patients with chronic myeloid leukemia, comprising an agent that measures the expression level of FAM167A protein, fragments thereof, or mRNA of the gene encoding said protein. composition for. The patient with chronic myeloid leukemia according to claim 2, wherein the agent for measuring the expression level of the FAM167A protein or fragment thereof comprises an antibody, an antigen-binding fragment thereof, or an aptamer that specifically binds to the FAM167A protein or fragment. A composition for diagnosing BCR-ABL independent imatinib or nilotinib resistance. The method of claim 2, wherein the agent for measuring the mRNA level of the gene encoding the FAM167A protein comprises sense and antisense primers or probes that bind complementary to the mRNA of the gene, and determines the BCR-ABL ratio in patients with chronic myeloid leukemia. Composition for diagnosing imatinib-dependent or nilotinib resistance. A kit for diagnosing BCR-ABL-independent imatinib or nilotinib resistance in patients with chronic myeloid leukemia, comprising the composition of claim 2. The method of 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, and an ELISA ( A kit for diagnosing BCR-ABL-independent imatinib or nilotinib resistance in patients with chronic myeloid leukemia, which is an Enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay) kit, protein chip kit, rapid kit, or MRM (multiple reaction monitoring) kit. (a) measuring the mRNA expression level of FAM167A protein, a fragment thereof, or a gene encoding the protein in a biological sample isolated from a patient with chronic myeloid leukemia; and
(b) The expression level measured in step (a) is the same protein measured in a biological sample isolated from a patient with chronic myeloid leukemia without BCR-ABL-independent imatinib or nilotinib resistance. Comparing the mRNA expression level of the fragment or the gene encoding the protein;
A method of providing information for diagnosing BCR-ABL-independent imatinib or nilotinib resistance in patients with chronic myeloid leukemia, including. The method of claim 7, wherein (c) the mRNA expression level of the FAM167A protein, its fragment, or the gene encoding the protein measured in the biological sample isolated from the chronic myeloid leukemia patient in step (a) is, step (b) The BCR-ABL ratio is higher than the mRNA expression level of the same protein, fragment thereof, or gene encoding the protein measured in biological samples isolated from patients with chronic myelogenous leukemia without BCR-ABL-independent imatinib or nilotinib resistance. A method of providing information for diagnosing BCR-ABL-independent imatinib or nilotinib resistance in a patient with chronic myeloid leukemia, further comprising the step of determining that dependent imatinib or nilotinib resistance is present. The method of claim 7, wherein the biological sample is blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, urogenital fluid, breast milk, lymphatic body fluid, An informative method for diagnosing BCR-ABL-independent imatinib or nilotinib resistance in a patient with chronic myeloid leukemia, comprising semen, cerebrospinal fluid, intra-organ fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof. A pharmaceutical composition for preventing or treating chronic myelogenous leukemia showing BCR-ABL-independent imatinib or nilotinib resistance, comprising as an active ingredient a neutralizing antibody that specifically binds to the FAM167A protein. delete delete The method of claim 10, wherein the BCR-ABL independent chronic myeloid leukemia showing imatinib or nilotinib resistance has increased NF-κB activity compared to the leukemia showing imatinib or nilotinib sensitivity. A pharmaceutical composition for preventing or treating chronic myelogenous leukemia showing imatinib or nilotinib resistance. The pharmaceutical composition for preventing or treating chronic myelogenous leukemia showing BCR-ABL-independent imatinib or nilotinib resistance according to claim 10, which is administered in combination with a tyrosine kinase inhibitor (TKI). The method of claim 14, wherein the TKI is crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, and imatinib. (imatinib), nilotinib, ponatinib, ibrutinib, cabozantinib, gefitinib, erlotinib, lapatinib, Vandetanib, afatinib, osimertinib, ruxolitinib, tofacitinib, axitinib, lenvatinib, nintedanib Chronic myelogenous leukemia with BCR-ABL-independent imatinib or nilotinib resistance, including nintedanib, pazopanib, regorafenib, sorafenib, or sunitinib Pharmaceutical composition for prevention or treatment. A pharmaceutical composition for suppressing BCR-ABL-independent imatinib or nilotinib resistance in patients with chronic myeloid leukemia, comprising as an active ingredient a neutralizing antibody that specifically binds to the FAM167A protein. The method of claim 16, wherein the BCR-ABL independent chronic myeloid leukemia showing imatinib or nilotinib resistance has increased NF-κB activity compared to the leukemia showing imatinib or nilotinib sensitivity. Pharmaceutical composition for suppressing BCR-ABL-independent imatinib or nilotinib resistance. The pharmaceutical composition for suppressing BCR-ABL-independent imatinib or nilotinib resistance in patients with chronic myeloid leukemia according to claim 16, which is administered in combination with a tyrosine kinase inhibitor (TKI). The method of claim 18, wherein the TKI is crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, and imatinib. (imatinib), nilotinib, ponatinib, ibrutinib, cabozantinib, gefitinib, erlotinib, lapatinib, Vandetanib, afatinib, osimertinib, ruxolitinib, tofacitinib, axitinib, lenvatinib, nintedanib BCR-ABL-independent imatinib or nilotinib resistance in patients with chronic myelogenous leukemia, including nintedanib, pazopanib, regorafenib, sorafenib, or sunitinib Pharmaceutical composition for inhibition. (a) treating a candidate material in a cell line or non-human animal model showing BCR-ABL-independent imatinib or nilotinib resistance; and
(b) confirming whether the FAM167A protein is inhibited or the expression of the gene encoding the protein is inhibited in a cell line or non-human animal model treated with the candidate material;
A method for screening a treatment for chronic myelogenous leukemia exhibiting BCR-ABL-independent imatinib or nilotinib resistance, or a BCR-ABL-independent imatinib or nilotinib resistance inhibitor, comprising a. The method of claim 20, (c) when the candidate inhibits the FAM167A protein or inhibits the expression of a gene encoding the protein, the candidate is used in chronic myeloid disease showing BCR-ABL-independent imatinib or nilotinib resistance. A treatment for chronic myelogenous leukemia or a BCR-ABL-independent imatinib or nilotinib-resistant treatment for leukemia or a BCR-ABL-independent imatinib or nilotinib resistance inhibitor, further comprising the step of determining: Method for screening for imatinib or nilotinib resistance inhibitors.