WO2023080210A1 - Identification of treatment target in aggressive nk leukemia - Google Patents

Abstract

The present invention addresses the problems of identifying a molecule that can serve as a potential drug target in a tumor, providing a method for suppressing the proliferation of tumor cells through knockdown or knockout of the molecule, and providing a screening method for tumor therapeutic drugs that target the identified molecule. Provided according to the present invention is a method for suppressing the proliferation of tumor cells, the method including knockdown or knockout of the functions of at least one gene selected from the group consisting of genes that encode GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes in tumor cells. Also provided is a screening method for tumor therapeutic drugs, the method including: (i) bringing a candidate substance into contact with tumor cells; (ii) measuring the expression level of at least one gene selected from the group consisting of genes that encode GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes in tumor cells, or a protein of the at least one gene; and (iii) selecting, as a therapeutic drug for the tumor, a candidate substance that decreased the expression level to a greater extent than when contact with the candidate substance did not occur.

Description

Identification of therapeutic targets for fulminant NK leukemia

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from Japanese Patent Application No. 2021-180891 filed on November 5, 2021, the entire disclosure of which is specifically incorporated herein by reference.
The present invention comprises knocking down or knocking out the function of at least one gene selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes in tumor cells, The present invention relates to a method for inhibiting tumor cell proliferation. Further, the present invention provides a method for selecting from the group consisting of (i) contacting a candidate substance with a tumor cell, (ii) genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes in tumor cells and (iii) selecting a candidate agent that reduces said expression level as a therapeutic agent for the tumor compared to when the candidate agent was not contacted. The present invention relates to a method of screening for tumor therapeutic agents, comprising:

Because the number of cases of rare cancers is small and it is difficult to proceed with clinical research and trials, it is often difficult to establish a standard treatment. For example, fulminant NK cell leukemia (Aggressive NK cell Leukemia, hereinafter referred to as ANKL) is a rare type of leukemia with a low incidence and unknown pathology, and therefore no standard treatment has been established. be.

ANKL is a systemic neoplastic proliferation of NK cells, almost always associated with Epstein-Barr virus (EBV), an aggressive clinical course, and a poor prognosis malignancy with a median survival of less than 2 months (JKC Chan et al., Aggressive NK-cell leukemia in WHO classification of tumors of haematopoietic and lymphoid tissues ^4th edition 2008, edited by Steven H. Swerdlow et al., (World Health Organization classification of tumors) International Agency for Research on Cancer: pp 276-277; the entire description of which is specifically incorporated herein by reference). In addition, it is much more common in Asians than in other ethnic groups, and in Japan, the onset peak is in the young and middle ages, including the AYA generation.

Mutations in STAT3 and RAS-MAPK pathway genes and mutations in DDX3X and epigenetic modifiers have been identified as the molecular etiology of ANKL, but with different sites of onset extranodal NK/T-cell lymphoma, nasal type (ENKL) (Dufva, O., Kankainen, M., Kelkka, T. et al. Aggressive natural killer-cell leukemia mutational landscape and drug profiling highlight JAK-STAT signaling as therapeutic target. Nat Commun 9, 1567 (2018). It has also been reported that macrophages play an important role in the tumor microenvironment in EBV-infected humanized lymphoproliferative disease mice (Hiroshi Higuchi, Natsuko Yamakawa, Ken-Ichi Imadome et al. Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma. Blood. 2018 Jun 7;131(23):2552-2567. doi: 10.1182/blood-2017-07-794529; .

Because ANKL is a rare disease, the pathophysiology has not been elucidated, and the prognosis is poor. and the development of therapeutics are important. In addition, although the frequency is even lower in Europe and the United States, there are reports of onset in East Asia and South America, so there is a possibility that many patients are affected worldwide. Therefore, there is a need to identify molecules that can serve as drug discovery targets for ANKL, and to develop therapeutic methods that improve life prognosis.

The present invention provides a method for suppressing tumor cell growth by identifying a molecule that can be a drug discovery target for tumors and knocking it down or knocking it out. An object of the present invention is to provide a drug screening method. It is also another object of the present invention to provide new approaches that enable the treatment of tumors.

According to the present invention, the following inventions are provided.
[1] A tumor comprising knocking down or knocking out the function of at least one gene selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes in tumor cells. A method for inhibiting cell proliferation.
[2] The method of [1], wherein the tumor cells are solid tumor or hematologic tumor cells.
[3] The method of [2], wherein the hematological tumor is selected from leukemia including fulminant NK cell leukemia, malignant lymphoma, or multiple myeloma.
[4] A method of screening for an antitumor drug comprising the following (i) to (iii):
(i) contacting the tumor cells with the candidate substance;
(ii) measuring the expression level of at least one gene or protein thereof selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes in tumor cells; and
(iii) Selecting the candidate substance with reduced expression level as a therapeutic agent for the tumor compared to when the candidate substance is not contacted.
[5] The method of [4], wherein the tumor cells are solid tumor or hematologic tumor cells.
[6] The method of [5], wherein the hematological tumor is selected from leukemia including fulminant NK cell leukemia, malignant lymphoma, and multiple myeloma.
[7] The method of any one of [4] to [6], wherein the tumor is leukemia including fulminant NK cell leukemia.
[8] NK cells in which the function of at least one gene selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes is knocked down or knocked out.
[9] A method for suppressing ANKL cell proliferation, comprising inhibiting intracellular and/or extracellular GGT1 activity.

FIG. 1 shows the growth inhibitory effect on ANKL cells by the gene defect of Example 1. FIG. FIG. 2 shows ANKL cell viability in various amino acid-free media. Results in cysteine-free medium are shown with lines. ANKL1 and ANKL3 in the graph represent samples taken from the same patient at different times. FIG. 3 shows the viability of GGT1-deficient ANKL cells in a medium supplemented with 20 kinds of amino acids including cysteine. FIG. 4 shows changes in viability of GGT1-deficient NK92 cell lines. FIG. 5A is a graph showing viability of the NK92 cell line. "inhibitor" indicates the NK92 cell line to which the GGT1 inhibitor was added, and "control" is the negative control. 1 was assigned to the control. FIG. 5B is a schematic diagram of the GGT1 fluorescent probe, gGlu-HMRG. FIG. 5C shows the fluorescence intensity of probes degraded by GGT1 in each sample to produce fluorescence. A probe was added to the sample, reacted for 24 hours, and fluorescence intensity was measured. "Probe" is a sample with probe only, "Probe+cell" is a sample with probe added to cells, and "Probe+cell+GGstop" is a sample with probe and GGT1 inhibitor GGsTop (registered trademark) added to cells. be. FIG. 5D is a graph showing the ratio of inside and outside cells to GGT1 activity. ANKL1 and ANKL3 in the graph represent samples derived from ANKL obtained from different patients. "out (%)" is the percentage of GGT1 activity extracellularly (on the cell membrane surface), and "in (%)" is the percentage of intracellular GGT1 activity.

Although the description of the present invention described below may be made based on representative embodiments and specific examples, the present invention is not limited to such embodiments. In the present specification, a numerical range represented by "-" means a range including the numerical values before and after "-" as lower and upper limits.

The present inventors identified GGT1, IL-10R, CD16, RPM-1, and cysteine as drug discovery targets, as described in the examples below. Each molecule is described below.

The enzyme encoded by the GGT1 (gamma glutamyltransferase 1) gene catalyzes reactions that transfer the glutamyl moiety of glutathione to various amino acid and dipeptide acceptors. The enzyme is composed of heavy and light chains and is derived from a single precursor protein. It is expressed in tissues involved in absorption and secretion and is thought to be involved in the pathogenesis of diabetes and other metabolic disorders. Multiple alternatively spliced variants have been identified. 5'UTR transcriptional variants of type I genes have been identified in various tissues and cancer cells (NCBI Gene: 267, Entrez Gene: GGT1 gamma-glutamyltransferase 1). In addition, GGT1, a cell surface enzyme involved in intracellular glutathione homeostasis, has been reported to be overexpressed in several human tumors, including cervical and ovarian cancers (Urano, Yasuteru et al. "Rapid cancer detection by topically spraying a γ-glutamyltranspeptidase-activated fluorescent probe." Science translational medicine vol. 3,110 (2011): 110ra119. doi:10.1126/scitranslmed.3002823; Disclosure (incorporated as

IL-10R (interleukin 10 receptor) is a type II cytokine receptor. This receptor is a tetramer consisting of two α subunits and two β subunits. The α subunit is expressed in hematopoietic cells (T cells, B cells, NK cells, mast cells, dendritic cells, etc.), and the β subunit is ubiquitously expressed (Y Liu et al., The Journal of Immunology February 15, 1994, 152(4) 1821-1829; Kotenko, S V et al. “The EMBO journal vol. 16,19 (1997): 5894-903; IL-10R has been proposed as a therapeutic target for diffuse large B-cell lymphoma (DLBCL) (Beguelin W et al., Leukemia. 2015 Aug;29(8):1684-94; the entire description of which is expressly incorporated herein by reference.) IL-10RA and IL-10RB refer to interleukin 10 receptor alpha subunit and interleukin 10 receptor beta subunit, respectively.

CD16 is a cluster of differentiation molecules found on the surface of natural killer cells, neutrophils, monocytes, and macrophages (Janeway C (2001). "Appendix II. CD Antigens". Immunobiology (5 ed.). New York: Garland.; the entire disclosure of which is expressly incorporated herein by reference). CD16 has been identified as the Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b) involved in signal transduction. CD16, the best-studied membrane receptor involved in triggering cytolysis by NK cells, is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC). Since CD16 is expressed on neutrophils, it is a potential target for cancer immunotherapy. Furthermore, CD16 has been reported to be highly expressed in ANKL (Li C, et al., Transl Res. 2014 Jun;163(6):565-77; the full description of which is expressly disclosed herein. cited).

RPM-1 is a PHR protein. The PHR proteins are mammalian Phr1/MYCBP2, Drosophila Highwire, and nematode RPM-1. PHR proteins are conserved as intracellular signaling hubs that regulate synaptogenesis and axon termination. PHR proteins have been reported to act as ubiquitin ligases that inhibit the DLK-1 and MLK-1 MAP kinase pathways (Crawley O, et al., PLoS Genet 13(12): e1007095; the full description is in incorporated herein by reference).

Cysteine (Cys, 2-amino-3-sulfanylpropionic acid) is one of amino acids. It is naturally contained as L-cysteine in food proteins, but in humans it is biosynthesized from methionine, not from essential amino acids. It is used as a food additive, and a supplement that improves skin spots is sold. Cysteine slow-release agents are used to protect the stomach and to eliminate acetaldehyde, such as when drinking alcohol.

<Method for Suppressing Growth of Tumor Cells>
The method of suppressing tumor cell growth of the present invention comprises inhibiting the function of at least one gene selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes in tumor cells. Including knocking down or knocking out. In one aspect, the methods of inhibiting growth of tumor cells of the present invention exclude methods of treating humans or exclude medical interventions on humans. In another aspect, the method of inhibiting tumor cell proliferation of the present invention is practiced in vitro or ex vivo. In yet another aspect, the method of inhibiting tumor cell proliferation of the present invention is practiced in vivo.

By target gene herein is meant a gene that can serve as a target for tumor therapy, unless otherwise specified. As used herein, target genes refer to genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes, unless otherwise specified. A summary of these genes is given above. Further, detailed information including the base sequences of these genes can be obtained based on the following Ensembl IDs. GGT1: ENSG00000100031; IL-10RB: ENSG00000243636; CD16: ENSG00000203747; MYCBP2 (RPM-1): ENSG00000005810.
Cysteine-related enzyme genes include, but are not limited to, cysteine transporters and cysteine metabolism-related enzyme genes, including cystathionine beta synthase (CBS) and cystathionine gamma-lyase.

Genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes whose functions are knocked down or knocked out in the present invention are mammalian genes, mouse, rat or monkey genes or human genes. It is preferably a gene of, and more preferably a human gene, but is not limited thereto.

In the present specification, knockdown of gene function is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less compared to the same type of unmodified cells or cells before modification. , 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less.

Knockdown is a gene deletion, disruption, substitution, point mutation, multiple point mutation, insertion mutation, or frameshift mutation CRISPR/CAS9, or CRISPR interfering or interfering RNA, interfering mRNA or interfering aptamer applied to the above target genes. by any one or more, or by suppression of gene expression by siRNA or siRNA interference or the use of zinc finger transcription factors or zinc finger nucleases or transcription activator-like effector nucleases (TALENs), or inhibitor molecules or small molecule inhibitors such as This can be done through the use of inhibitors, such as, but not limited to, activity inhibitors of protein or enzymatic activity, and any method that can reduce gene expression or activity of the target gene can be employed.

As used herein, "knockout of gene function" means that gene expression is completely or almost completely eliminated. The expression or activity of the gene knocked out in the cell is 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, compared to the same type of unmodified cells or cells before modification. It means that it is reduced to 1% or less or to 0%, but it is not limited to this. In certain embodiments, the level of expression of the knocked-out gene in the cell is undetectable using conventional means for gene expression detection known in the art.

Knockout can be performed by, but not limited to, a known genetic recombination method (gene targeting method), genome editing, or the like. From the viewpoint of efficiency, knockout is preferably performed by genome editing using an artificial restriction enzyme such as ZFN, TALEN, CRISPR/Cas9, more preferably using CRISPR/Cas9.

Production of knockout cells by CRISPR / Cas9, Cas9 is introduced into cells using lentivirus, transferred into mice to obtain Cas9-expressing cells, sgRNA is introduced into Cas9-expressing cells using a vector, and introduced into mice again. The transfected and established cells can be analyzed to obtain gene knockout cells.

sgRNA for the target gene used for CRISPR / Cas9, PAM in the exon you want to delete (proto-spacer adjacent motif; 5'-NGG-3') 5 'upstream 20 bases containing, target gene PAM upstream homologous gene It can be designed and synthesized to bind complementary sequences. This sgRNA forms a complex with Cas9 mRNA or protein, and is injected into the pronucleus or cytoplasm of mouse fertilized eggs at the same time. After complementary binding to 20 bases upstream of PAM, 3 to 4 bases upstream of PAM Cut (DSB; double strand break) at . DSB is immediately repaired after introduction, but the repair involves insertion or deletion (indel) of several to several tens of bases through a non-homologous end-joining (NHEJ) pathway. Since this indel can introduce a frameshift mutation into the target gene, it can be used for efficient gene knockout.

In the method of suppressing the growth of tumor cells of the present invention, the tumor cells may be cells of solid tumors or hematologic tumors. In the present invention, hematological tumors in which tumor cell proliferation is suppressed include, but are not limited to, leukemias including fulminant NK cell leukemia, malignant lymphomas, and multiple myeloma. Leukemias include fulminant NK-cell leukemia, acute myelogenous leukemia, acute lymphocytic leukemia/lymphoblastic lymphoma, acute promyelocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and myelodysplastic syndrome. but not limited to these. Malignant lymphomas include, but are not limited to, adult T-cell leukemia/lymphoma, Burkitt's lymphoma, primary nasal NK-cell lymphoma, Hodgkin's lymphoma, B-cell lymphoma, gastric MALT lymphoma, and the like. In the present invention, the blood tumor whose growth of tumor cells is suppressed may be an NK cell tumor, and the NK cell tumor is extranodal NK/T-cell lymphoma, nasal type. : ENKL), fulminant NK cell leukemia, chronic NK cell hyperplasia.

In the present invention, solid tumors whose growth of tumor cells is suppressed include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, mesothelioma, and Ewing. Tumor, Leiomyosarcoma, Rhabdomyosarcoma, Colon cancer, Pancreatic cancer, Breast cancer, Ovarian cancer, Prostate cancer, Gastric cancer, Lung cancer, Uterine cancer, Squamous cell carcinoma, Basal cell carcinoma, Adenocarcinoma , sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic lung cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma Cancer, seminiferous carcinoma, embryonic carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, Sarcoma and carcinoma include craniopharyngioma, ependymoma, pineocytoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. include but are not limited to:

In one embodiment, in the method of inhibiting tumor cell proliferation of the present invention, ANKL cell proliferation is inhibited.

ANKL cells are believed to be derived from mature NK cells. Neoplastic NK cells are typically CD2 ⁺ , surface CD3 ⁻ , cytoplasmic CD3ε ⁺ , CD56 ⁺ , EBV ⁺ and have a germline configuration of T-cell receptor (TCR) and immunoglobulin (Ig) genes . The exclusive expression of CD2 and CD56 and the absence of CD3 and TCR in ANKL indicates its origin from NK cells (Li C et al 2014 supra). In addition, ANKL cases are positive for cytotoxic molecules, and serum levels of CXCR1, CCR5, and soluble Fas ligand have been found to be high, suggesting that the chemokine system plays an important role in systemic infiltration of NK leukemic cells and liver dysfunction. (Makishima H et al, 2007 Leukemia Research, Volume 31, Issue 9, 2007, Pages 1237-1245; the entire disclosure of which is expressly incorporated herein by reference).

In one embodiment, tumor cells used in the present invention can be, for example, cells isolated from surgically removed organs or cells isolated from blood provided by patients. In addition, tumor cells can be obtained from ATCC and cell sales companies. For example, ANKL cells can be isolated by the following method. Separation of ANKL cells can be performed by layering patient's peripheral blood or the like on Ficoll (Ficoll (registered trademark) Paque Plus SIGMA) and centrifuging at 1000 rpm for 20 minutes) to separate mononuclear cells. In addition, ANKL cells from PDX (patient-derived xenograft) mice collected the liver, spleen, and bone marrow, crushed the tissue with a preparation, overlaid on Ficoll (Paque Plus SIGMA), and spun at 1000 rpm. , centrifugation for 20 minutes) to separate mononuclear cells.

In the examples described later, it was revealed that growth of tumor cells in which GGT1, IL-10R, CD16, or RPM-1 had been knocked out was suppressed. It was also found that tumor cells could not survive in media without cysteine. From these results, we identified GGT1, IL-10R, CD16, RPM-1, and cysteine as therapeutic targets for tumor cells, particularly ANKL.

In another aspect, the method of inhibiting tumor cell growth of the present invention comprises removing, reducing, or depleting cysteine. As shown in Example 2 below, cysteine is required for the survival of ANKL cells. Cysteine is contained in protein in foods, but is not an essential amino acid for humans, and is synthesized from methionine. It is believed that the proliferation of ANKL cells can be suppressed by a diet regimen in which a cysteine-free diet is taken, or by knocking down or knocking out cysteine-related enzymes as described above. In the present invention, removing, reducing, or depleting cysteine can be performed, for example, by ingesting a cysteine-free diet.

<Screening method>
The method of screening for tumor therapeutic agents of the present invention includes the following.
(i) contacting the tumor cells with the candidate substance;
(ii) measuring the expression level of at least one gene or protein thereof selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes in tumor cells; and
(iii) Selecting the candidate substance with reduced expression level as a therapeutic agent for the tumor compared to when the candidate substance is not contacted.

As described above, the inventors identified GGT1, IL-10R, CD16, RPM-1, and cysteine as therapeutic targets for tumor cells, particularly ANKL. These molecules can be used to screen tumor therapeutics.

Bringing the candidate substance into contact with the tumor cells means allowing the tumor cells and the candidate substance (test substance) to exist in the same reaction system or culture system. Examples include, but are not limited to, adding candidate substances to cell culture vessels, mixing cells and candidate substances, and culturing cells in the presence of candidate substances.

measuring the expression level of at least one gene or protein thereof selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes using an immunological assay; can be done. Immunological assays include, for example, ELISA, Western blot, immunoprecipitation, slot or dot blot assay, immunohistochemical staining, radioimmunoassay (RIA), fluorescence immunoassay, immunoassay using avidin-biotin or streptavidin-biotin system, and the like. Including but not limited to. Preferably, it is an ELISA, such as a sandwich ELISA. In addition, the expression level of at least one gene or protein thereof selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes can be measured as the expression level of mRNA. Northern blotting, Southern blotting, quantitative RT-PCR, real-time PCR, in situ hybridization, etc. can be performed using primers or probes according to conventional methods, but are limited to these. not.

The expression level of at least one gene or protein thereof is compared between the tumor cells contacted with the candidate substance and the tumor cells not contacted with the candidate substance, and the candidate substance whose expression level is reduced is used as a therapeutic agent for tumors. can be selected. Selected candidate substances can be subjected to further analysis.

Candidate substances or test substances can be any compound, such as proteins (e.g., antibodies and antigen-binding fragments thereof), peptides, nucleic acids, carbohydrates, lipids, high-molecular-weight or low-molecular-weight compounds other than proteins, and derivatives thereof. etc., but not limited to these.

In the screening method of the present invention, the tumor cells may be cells of solid tumors or hematologic tumors. The types of solid tumors or hematological tumors used in the screening method of the present invention are the same as described in <Method for inhibiting growth of tumor cells> above. In particular, the hematological tumor is preferably selected from leukemia including fulminant NK cell leukemia, malignant lymphoma, and multiple myeloma.

In the present invention, the tumors for which therapeutic agents are screened are the same as those described in <Method for inhibiting tumor cell proliferation> above. Especially preferred are leukemias including fulminant NK cell leukemia.

In certain embodiments, the enzymatic activity of the protein can be measured separately for intracellular and extracellular (eg, on the cell membrane surface). For example, it is possible to assess whether a candidate substance can reduce the enzymatic activity of the protein only at the cell membrane surface, or both intracellularly and extracellularly. In one aspect, candidate substances that have reduced the enzymatic activity of the protein in cells can be selected as therapeutic agents for tumors. In another aspect, a candidate substance that has decreased enzymatic activity of the protein on the cell membrane surface can be selected as a therapeutic agent for tumors.

In the examples described later, it was revealed that ANKL cells have higher intracellular GGT1 activity values than extracellular ones. Separate measurement of the expression level and activity of therapeutic target molecules intracellularly and extracellularly (for example, on the cell membrane surface) can provide clues for elucidating the molecular pathogenesis of ANKL.

Furthermore, the present invention relates to a method for suppressing proliferation of ANKL cells, including inhibiting intracellular and/or extracellular GGT1 activity in NK cells, particularly ANKL cells. The present invention relates to a method for inhibiting proliferation of ANKL cells, including inhibiting intracellular GGT1 activity in NK cells, particularly ANKL cells. Examples described later revealed that inhibition of extracellular GGT1 activity had no effect on cell survival in ANKL cells (NK92). It is believed that intracellular GGT1 activity is involved in the survival of ANKL cells. In one aspect, the methods of inhibiting growth of tumor cells of the present invention exclude methods of treating humans or exclude medical interventions on humans. In another aspect, the method of inhibiting tumor cell proliferation of the present invention is practiced in vitro or ex vivo. In yet another aspect, the method of inhibiting tumor cell proliferation of the present invention is practiced in vivo.

<NK cells with gene function knocked down or knocked out>
In another aspect of the present invention, NK cells in which the function of at least one gene selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes is knocked down or knocked out provided.
In one embodiment, NK cells in which the functions of the above genes have been knocked down or knocked out can be used to analyze the etiology and pathology of NK cell tumors. In another embodiment, NK cells with gene function knocked down or knocked out can be used to treat NK cell tumors.

NK cells in which the functions of the above genes of the present invention have been knocked down or knocked out may include normal NK cells as well as neoplastic NK cells. The neoplastic NK cells may be, but are not limited to, ANKL cells.
Knockdown or knockout can be performed as described in <Method for suppressing growth of tumor cells> above.

The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples. In this specification, "%" and the like are based on mass unless otherwise specified.

The present inventors investigated whether at least 15 known molecules are involved in ANKL cell proliferation or survival in patient-derived xenograft (PDX) models and in vitro co-culture systems, gene deletion methods by CRISPR, fractionation and purification methods. was analyzed using The following examples are the results of identifying six molecules (GGT1, IL-10R, CD16, RPM-1, and cysteine) that decrease tumor survival.

The ANKL cells used in the experiments of Examples 1 to 3 were isolated from the peripheral blood of one patient (45 years old) at Tokai University Hospital. The ANKL cells were obtained by layering the patient's peripheral blood on Ficoll (Ficoll (registered trademark) Paque Plus SIGMA) and separating mononuclear cells using centrifugation at 1000 rpm for 20 minutes). All experiments were performed with patient consent after approval by an ethics review board.
A patient-derived xenograft (PDX) model was created by transferring patient tumor cells into immunodeficient mice (NOD/Shi-scid, IL-2RγKO, In vivo science).

Example 1
<RPM-1, IL-10R, CD16>
In this example, CRISPR/Cas9 was used to deplete RPM-1, IL-10R, and CD16 in ANKL cells.
CRISPR/Cas9 was introduced into ANKL cells using lentivirus. Lentivirus was prepared using HEK293T cells as follows. HEK293T cells were cultured in D-MEM (Wako, Cat #044-29765) supplemented with 10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin, and placed in a 10 cm dish with ethylenimine (PEI-MAX, Polysciences, Cat #24765). -1) was added, and 10 μg of FUGW-Cas9-Venus vector, 10 μg of pCAG-KGP1R vector, 2 μg of pCAG-4RTR2 vector, and 3 μg of pCMV-VSVG were transfected. Lentiviral supernatants were harvested 48 hours after transfection, centrifuged at 12000 g for 4 hours at 20° C., and pellets were resuspended in HBSS containing 10 mM HEPES. Concentrated lentiviral supernatants were bound to RetroNectin (Takara Bio, Cat#T100B) coated 24-well plates. ANKL cells collected from the liver of PDX mice prepared using ANKL cells collected from patients were seeded on a lentivirus-bound RetroNectin-coated plate (1-5 × 10 ⁵ cells per well), 1220 g, 32 C. for 2 hours by spin infection.

ANKL cells were harvested from the liver of ANKL PDX mice, spin-infected with the above lentivirus, washed twice, injected intravenously into NOD/Shi-scid, IL-2RγKO mice, and ANKL cells were cultured 3-4 weeks later. were harvested, sorted based on the amount of expression of the fluorescent protein Venus, and serially injected into mice. This collection, selection, and injection process was performed twice to obtain Cas9-expressing ANKL cells.

By introducing the sgRNAs listed in Table 1 into the CSII-sgRNA-mOrange vector into Cas9-expressing ANKL cells collected from the liver of PDX mice, ANKL cells deficient in each gene were established. Cells were either injected into mice immediately after transfer or cultured for 2 days in RPMI-1640 supplemented with 5% FBS, non-essential amino acid solution, 1 mM sodium pyruvate, 0.1 mM 2-mercaptothes. After adding 1 mM 2-mercaptoethanol, mOrange-positive cells were separated by FACSAria (TM) III (Nippon Becton Dickinson Co., Ltd.), and the above four types of genes were individually deficient ANKL cells or non-defective ANKL cells. were injected into 1×10 ⁴ mice each.
Fourteen days after the injection, each ANKL cell in the liver was quantified by FACSVerse (TM) (Nippon Becton Dickinson Co., Ltd.).

A non-target control (commissioned by GeneScript) was used to assess baseline cellular responses to the CRISPR-Cas9 component in the absence of target gene-specific sgRNA. In addition, as a comparative example, the oncogene c-myc, which shows an important role in carcinogenesis in many carcinomas but is difficult to develop drugs because its inhibition also greatly affects normal cells, was used (Table 1, it is described as MYC).

Results The survival rate of ANKL cells in mice injected with RPM-1, IL-10R, and CD16 gene-deficient ANKL cells (hereinafter referred to as tumor survival rate) was higher than the tumor survival rate in mice injected with gene-free ANKL cells. It was 50% or less against (Fig. 1).
As a comparative example, ANKL cells in which the oncogene c-myc was deficient using CRISPR were examined in the competitive PDX mouse model, and the survival rate was about 60% (Fig. 1). It was shown that the survival rate of ANKL cells deficient in RPM-1, IL-10R, and CD16 genes was lower than that of ANKL cells deficient in oncogene c-myc.

Example 2
<cysteine>
1×10 ⁶ ANKL cells were cultured in 2 ml of RPMI (FCS 10%) medium supplemented with all 19 amino acids except cysteine (Cys) in a 6 cm culture dish for 72 hours. Dead cells were stained by annexin staining and PI staining, quantified by FACSVerse (TM), and negative fractions were defined as viable cells.

In addition to media containing all 20 major amino acids, media containing none of them, essential amino acids (methionine (Met), threonine (Thr), isoleucine (Ile), histidine (His), valine (Val), lysine (Lys) ), phenylalanine (Phe), leucine (Leu), tryptophan (Trp)), non-essential amino acids (asparagine (Asn), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly)), cysteine Conditionally essential amino acids other than (glutamine (Gln), arginine (Arg), tyrosine (Tyr), serine (Ser), proline (Pro) even in the medium excluding any one amino acid, the case of removing the cysteine and ANKL cells were cultured in the same manner, dead cells were stained with annexin staining and PI staining, quantified by FACSVerse (TM), and negative fractions were defined as viable cells.

Results The group to which 20 types of amino acids containing cysteine were added had a survival rate of 100% (All in the figure), whereas the group to which only cysteine was removed had a survival rate of 0% (Fig. 2). The survival rate was lower in the group from which cysteine was removed than in the group from which essential amino acids, non-essential amino acids, and conditionally essential amino acids other than cysteine were removed.

Example 3
<GGT1>
GGT1 deficiency in ANKL cells was performed in the same manner as in Example 1. sgRNAs for GGT1 are listed in Table 1.
As in Example 1, 1×10 ⁶ ANKL cells deficient in GGT1 by CRISPR were cultured in a 6 cm culture dish in RPMI (10% FCS) culture medium supplemented with 20 amino acids including cysteine for 72 hours. Dead cells were stained by annexin staining and PI staining, quantified by FACSVerse (TM), and negative fractions were defined as viable cells.
A non-target control was used to assess baseline cellular responses to the CRISPR-Cas9 component in the absence of target gene-specific sgRNA.

Results The viability of GGT1-deficient ANKL cells was less than 20% relative to that of non-gene-deficient ANKL cells (Fig. 3).

Example 4
<GGT1-deficient NK92 cells>
In this example, the GGT1 gene was knocked out in the ANKL cell line NK92.
Methods NK92 cell line was purchased from ATCC.
GGT1 deletion in the NK92 cell line was performed as in Example 1. sgRNAs for GGT1 are listed in Table 1. As in Example 1, 1×10 ⁵ cells of the NK92 cell line deficient in GGT1 by CRISPR were cultured in a 6-well plate in Artemis-1 (Japan Techno Service Co., Ltd.) culture medium supplemented with 2% human serum.
The ratio of infection-positive cells was measured by flow cytometry every week, with the infection-positive cells 3 days after virus infection of non-specific sgRNA (denoted as non-target1 in FIG. 4) and GGT1 being 100%. Specifically, dead cells were stained by annexin staining and PI staining, quantified by FACSVerse (TM), and negative fractions were defined as viable cells.

Results Knocking out GGT1 using the CRISPR/Cas9 system by expressing sgRNA against the Cas9 and GGT1 genes by lentiviral infection rapidly killed the ANKL cell line NK92.

Example 5
<Expression position of GGT1 in cells>
Further analysis was performed on GGT1, identified as a therapeutic target for ANKL.
First, GGsTop (registered trademark), a GGT1 inhibitor, was added to 1×10 ⁵ NK92 cell lines in Artemis-1 (Japan Techno Service Co., Ltd.) culture medium supplemented with 2% human serum in a 6-well plate. Kojunyaku Co., Ltd.) was added at a concentration of 100 μg/mL. However, GGsTop® addition had no effect on cell survival of the NK92 cell line (Fig. 5A).

(GGT1 activity on cell membrane surface)
Next, in order to examine the GGT1 activity on the surface of the cell membrane, a sample containing only a gGlu-HMRG probe (ProteoGREEN ^™ -gGlu, Goryo Chemical Co., Ltd.) that is cleaved by GGT1 to generate fluorescence, a sample containing the probe added to cells, and The sample added with the agent GGsTop (registered trademark) was allowed to react for 24 hours, and the fluorescence intensity was measured. Although the gGlu-HMRG probe does not permeate the cell membrane, it reacts with GGT on the cell membrane surface to produce HMRG that emits strong fluorescence (Fig. 5B). In the NK92 cell line probe-added sample (Probe + cell), the fluorescence intensity increased compared to the probe-only sample (Probe), but in the inhibitor-added sample (Probe + cell + GGstop), Fluorescence intensity decreased compared to probe-only samples (Fig. 5C). When the GGT1 inhibitor GGsTop (registered trademark) was added to the ANKL cell line NK92, GGT1 activity on the cell membrane surface was inhibited.

(ratio of inner and outer to activity of GGT1)
In addition, to analyze the inner and outer ratios of GGT1 activity, gGlu-HMJCR, an intracellular permeable probe, was combined with GGsTop®, a GGT1 inhibitor that does not permeate the cell membrane, to GGT1 activity was measured to determine intracellular and extracellular GGT1 activity.
Fluorescence intensities were measured for the "cell and probe sample", "cell and probe and inhibitor sample", "probe and inhibitor sample" and "probe only sample". "Cell and probe sample" is intracellular GGT1 activity + extracellular GGT1 activity + medium GGT1 activity, "cell, probe and inhibitor sample" is intracellular GGT1 activity + measurement background, "probe and inhibitor 'sample' measures the background of the measurement, and 'probe-only sample' measures the GGT1 activity of the medium. is subtracted to calculate the value of intracellular GGT1 activity + extracellular GGT1 activity, and the fluorescence intensity of the "probe and inhibitor sample" is subtracted from the fluorescence intensity of the "cell, probe and inhibitor sample" The value of GGT1 activity was calculated and ratioed.
Fluorescence intensity was measured for three types of ANKL cells, NK92 cell line, ANKL1, and ANKL3, and the intracellular and extracellular GGT1 activity values were calculated and ratioed. The results are shown in Figure 5D. A probe that permeates the cell membrane and is degraded by GGT1 to show fluorescence and the GGT1 inhibitor GGsTop (registered trademark) were added to measure the activity of intracellular and extracellular GGT1. was significantly higher.

SEQ ID NOs: 1-5: Sequences of guide RNAs

Claims (9)

1. Proliferating tumor cells comprising knocking down or knocking out the function of at least one gene selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes in tumor cells. how to suppress
2. The method of claim 1, wherein said tumor cells are cells of a solid tumor or a hematologic tumor.
3. The method of claim 2, wherein said hematological tumor is selected from leukemia, including fulminant NK-cell leukemia, malignant lymphoma, or multiple myeloma.
4. A method of screening for an antitumor drug comprising (i) to (iii) below:
   (i) contacting the tumor cells with the candidate substance;
   (ii) measuring the expression level of at least one gene or protein thereof selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes in tumor cells; and
   (iii) Selecting the candidate substance with reduced expression level as a therapeutic agent for the tumor compared to when the candidate substance is not contacted.
5. The method of claim 4, wherein said tumor cells are cells of a solid tumor or hematological tumor.
6. The method according to claim 5, wherein said hematological tumor is selected from leukemia including fulminant NK-cell leukemia, malignant lymphoma, and multiple myeloma.
7. The method according to any one of claims 4 to 6, wherein the tumor is leukemia, including fulminant NK-cell leukemia.
8. NK cells in which the function of at least one gene selected from the group consisting of genes encoding GGT1, IL-10R, CD16, RPM-1, and cysteine-related enzymes is knocked down or knocked out.
9. A method of suppressing the proliferation of ANKL cells, comprising inhibiting intracellular and/or extracellular GGT1 activity.

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