Pharmaceutical composition for adult T-cell leukemia, method for selecting a pharmaceutical composition, and treatment method.

Abstract

This invention provides a pharmaceutical composition for adult T-cell leukemia (ATL). [Solution] This is a pharmaceutical composition for the treatment of adult T-cell leukemia. It contains a selective inhibitor that selectively inhibits the expression of a hub gene selected based on protein-protein interactions from among genes that are highly expressed in cancer cells of adult T-cell leukemia patients compared to T cells of healthy individuals. The hub gene may be the SAP30 gene. The selective inhibitor may contain either SAP30 siRNA or OTSSP167.

Description

[Technical Field]

[0001] The present invention relates particularly to a pharmaceutical composition for adult T-cell leukemia, a method for selecting a pharmaceutical composition, and a treatment method. [Background technology]

[0002] The retrovirus HTLV-1 has a long incubation period (several decades) and causes adult T-cell leukemia (ATL) in approximately 3-5% of infected individuals. HTLV-1 is found in South America, the Caribbean, Africa, and in Kyushu, Okinawa, and Hokkaido in Japan, with approximately 10-20 million people infected worldwide. The incidence of ATL among HTLV-1 infected individuals is 0.6-0.7 cases per 1,000 people per year, and outside of Japan, it is relatively common in Central Africa and Central and South America.

[0003] ATL is recognized as a distinct disease in the WHO classification system, and its etiology and course, particularly as a T-cell tumor caused by a virus, set it apart from other hematological malignancies such as leukemia and lymphoma. For example, although multi-drug chemotherapy is used to treat ATL, it often quickly becomes resistant to treatment. Therefore, allogeneic hematopoietic stem cell transplantation is performed on eligible patients with the aim of achieving a cure, but only about one-third of patients are able to receive a transplant. Furthermore, due to complications and relapses, the long-term prognosis is poor, with a 5-year survival rate of only about 30%. Relapses are frequent, and the prognosis is very poor. In particular, the 5-year survival rate for the acute and lymphoma forms is thought to be around 10%.

[0004] It has been found that one reason for this is that ATL tumor cells can exhibit a genotype similar to regulatory T cells (Tregs), allowing them to evade the immune system's attack after hematopoietic stem cell transplantation. Thus, ATL is completely different in nature from other B-cell lymphomas, acute myeloid leukemia, and acute lymphoblastic leukemia, even though they are all blood cancers, and specialized drugs have been needed. In recent years, mogamulizumab (anti-CCR4 humanized antibody, CAS number 1159266-37-1) was developed as a molecularly targeted drug and showed promise, but long-term treatment results showed survival rates of only about 20%. Furthermore, when mogamulizumab was used as pre-transplant therapy, contrary to expectations, it actually increased complications and decreased survival.

[0005] Furthermore, referring to Patent Document 1, OTSSP167, a molecular targeted drug (MELK inhibitor) primarily used for breast cancer, is described. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Special Publication No. 2015-513313 [Non-patent literature]

[0007] [Non-Patent Document 1] K. Kawamura et al., “Development of a Unique T Cell Receptor Gene-Transferred Tax-Redirected T Cell Immunotherapy for Adult T Cell Leukemia,” 2020, Biol Blood Marrow Transplant, 26, p.1377-1385 [Overview of the project] [Problems that the invention aims to solve]

[0008] As mentioned above, ATL is a disease with a completely different phenotype from common leukemias and lymphomas, and there was a need for a specialized pharmaceutical composition that could be more effective than conventional ATL-specific pharmaceutical compositions such as mogamulizumab. Here, regarding OTSSP167 described in Patent Document 1, although paragraph

[0108] mentions "chronic myeloid leukemia (CML)" as an example of application, it was not known to those skilled in the art whether MELK inhibitors act on ATL, and therefore it was unclear whether it could be used as a pharmaceutical composition for ATL.

[0009] This invention has been made in view of these circumstances, and aims to resolve the above-mentioned problems. [Means for solving the problem]

[0010] The pharmaceutical composition for ATL of the present invention is a pharmaceutical composition for the treatment of ATL, characterized in that it contains a selective inhibitor that selectively inhibits the expression of hub genes selected based on protein-protein interactions from among genes that are highly expressed in cancer cells of ATL patients compared to T cells of healthy individuals. The pharmaceutical composition for ATL of the present invention is characterized in that the hub gene is the SAP30 gene. The pharmaceutical composition for ATL of the present invention is characterized in that the selective inhibitor comprises either SAP30 siRNA or OTSSP167. The pharmaceutical composition for ATL of the present invention is characterized by being used in combination with a pharmaceutical composition for Tax-targeted T cell immunotherapy. The present invention provides a method for selecting a pharmaceutical composition for the treatment of ATL, characterized by selecting genes that are highly expressed in cancer cells of ATL patients compared to T cells of healthy individuals, selecting a hub gene from the selected highly expressed genes based on protein-protein interactions, and selecting a selective inhibitor that selectively inhibits the expression of the selected hub gene. The present invention relates to a treatment method for ATL, characterized by selectively inhibiting the expression of hub genes selected based on protein-protein interactions from among genes that are highly expressed in cancer cells of ATL patients compared to T cells of healthy individuals. [Effects of the Invention]

[0011] According to the present invention, by including a selective inhibitor that selectively inhibits the expression of hub genes selected based on protein-protein interactions among genes highly expressed in cancer cells of ATL patients compared to normal human T cells, a pharmaceutical composition for treating ATL can be provided that can be more effective than conventional pharmaceutical compositions dedicated to ATL.

Brief Description of Drawings

[0012] [Figure 1] It is a diagram showing the difference in gene expression between tumor cells of patients according to an example of the present invention and normal human CD4-positive T cells. [Figure 2A] It is a graph showing the results of small molecule compound screening (1 μM) for ATL according to an example of the present invention. [Figure 2B] It is a graph showing the results of small molecule compound screening (100 nM) for ATL according to an example of the present invention. [Figure 2C] It is a graph showing the relationship between the concentration of OTSSP167 and cell viability in small molecule compound screening for ATL according to an example of the present invention. [Figure 2D] It is a graph showing the cell viability when OTSSP167 was added to normal human PBMC in small molecule compound screening for ATL according to an example of the present invention. [Figure 2E] It is a graph showing the results of flow cytometry analysis of ATL patient samples in small molecule compound screening for ATL according to an example of the present invention. [Figure 2F] It is a graph showing the results of flow cytometry analysis of ATL patient samples in small molecule compound screening for ATL according to an example of the present invention. [Figure 3] It is a graph showing the change in the expression level of gene expression changes induced by OTSSP167 according to an example of the present invention. [Figure 4A] It is a graph showing the anti-ATL effect (expression level of SAP30) by siRNA of SAP30 according to an example of the present invention. [Figure 4B]It is a graph showing the anti-ATL effect (cell viability) by siRNA of SAP30 according to an example of the present invention. [Figure 4C] It is a graph showing the change in gene expression of SAP30 by a small molecule compound according to an example of the present invention. [Figure 5A] In the evaluation of drug efficacy in an animal model by OTSSP167 according to an example of the present invention, it is a conceptual diagram showing the protocol of the ATL cell line ATN-1 transplantation model. [Figure 5B] In the evaluation of drug efficacy in an animal model by OTSSP167 according to an example of the present invention, it is a photograph evaluating the tumor volume after subcutaneous administration of the ATL cell line ATN-1 by IVIS. [Figure 5C] In the evaluation of drug efficacy in an animal model by OTSSP167 according to an example of the present invention, it is a graph evaluating the tumor volume after subcutaneous administration of the ATL cell line ATN-1 by IVIS. [Figure 5D] In the evaluation of drug efficacy in an animal model by OTSSP167 according to an example of the present invention, it is a photograph showing the tumor after subcutaneous administration of the ATL cell line ATN-1. [Figure 5E] In the evaluation of drug efficacy in an animal model by OTSSP167 according to an example of the present invention, it is a graph showing the change in body weight over time in the ATL cell line ATN-1 transplantation model. [Figure 6A] In the evaluation of drug efficacy in an animal model by OTSSP167 according to an example of the present invention, it is a graph showing the inhibitory effects of OTSSP167 and OTSSP167 hydrochloride on the ATL cell line MT2 in vitro. [Figure 6B] In the evaluation of drug efficacy in an animal model by OTSSP167 according to an example of the present invention, it is a conceptual diagram showing the protocol of the ATL cell line MT2 transplantation model. [Figure 6C] In the evaluation of drug efficacy in an animal model by OTSSP167 according to an example of the present invention, it is a photograph evaluating the tumor volume after subcutaneous administration of the ATL cell line MT2 by IVIS. [Figure 6D]This graph shows the tumor volume evaluated by IVIS after subcutaneous administration of ATL cell line MT2 in an animal model drug efficacy evaluation using OTSSP167 according to an embodiment of the present invention. [Figure 6E] These are graphs and photographs showing the cumulative incidence of skin ulcers after subcutaneous administration of ATL cell line MT2 in an animal model efficacy evaluation using OTSSP167 according to an embodiment of the present invention. [Figure 6F] This is a photograph illustrating the method for measuring tumor diameter by CT evaluation of the ATL cell line MT2 in the evaluation of drug efficacy in an animal model using OTSSP167 according to an embodiment of the present invention. [Figure 6G] This graph shows the time course of tumor volume in the ATL cell line MT2 during the efficacy evaluation of an animal model using OTSSP167 according to an embodiment of the present invention. [Figure 7A] This is a conceptual diagram showing the protocol for toxicity assessment in an animal model using OTSSP167 according to an embodiment of the present invention. [Figure 7B] This graph shows the change in body weight over time in an animal model toxicity evaluation test using OTSSP167 according to an embodiment of the present invention. [Figure 7C] This graph shows the results of the hematological toxicity evaluation test conducted on an animal model using OTSSP167 according to an embodiment of the present invention. [Figure 8A] This graph shows the effect of MELK knockdown according to an embodiment of the present invention. [Figure 8B] This is a photograph of a Western blot analysis of the protein levels obtained by knockdown of MELK according to an example of the present invention. [Figure 8C] This graph shows the results of an apoptosis assay in MELK knockdown according to an embodiment of the present invention. [Figure 8D] This is a conceptual diagram showing the structure of the MELK gene targeted by the MELK knockout gRNA according to an embodiment of the present invention. [Figure 8E] This graph shows the change in MELK expression levels during MELK knockout according to an embodiment of the present invention. [Figure 8F]This is a photograph of a Western blot analysis of the MELK protein level in a MELK knockout according to an embodiment of the present invention. [Figure 8G] This graph shows a comparison of cell proliferation rates in MELK knockout according to an embodiment of the present invention. [Figure 8H] This graph shows the sensitivity of cells to OTSSP167 in MELK knockout according to an embodiment of the present invention. [Figure 9A] This graph shows an example of cell sorting of ATL patient samples in integrated RNA sequencing analysis according to an embodiment of the present invention. [Figure 9B] This figure shows the gene expression changes in ATL patients and healthy individuals determined in an integrated RNA sequencing analysis according to an embodiment of the present invention. [Figure 9C] This graph shows the increase or decrease in gene expression in ATL patients compared to healthy individuals in an integrated RNA sequencing analysis according to an embodiment of the present invention. [Figure 9D] This is a PPI network diagram of the top 100 DEGs in the integrated RNA sequencing analysis according to an embodiment of the present invention, showing genes whose expression was elevated in ATL. [Figure 9E] This figure shows the GO analysis of the top 100 DEGs in an integrated RNA sequencing analysis according to an embodiment of the present invention. [Figure 10A] This figure shows the gene expression changes in ATL cells treated with OTSSP167 in an integrated analysis of RNA sequencing according to an embodiment of the present invention. [Figure 10B] This graph shows the increase or decrease in gene expression in ATL cells treated with OTSSP167 in an integrated analysis of RNA sequencing according to an embodiment of the present invention. [Figure 10C] This is a PPI network diagram of genes whose expression was reduced by OTSSP167 treatment among the top 200 DEGs in the integrated analysis of RNA sequencing according to an embodiment of the present invention. [Figure 10D] This figure shows the GO analysis of the top 400 DEGs in an integrated RNA sequencing analysis according to an embodiment of the present invention. [Figure 11A] This is a scatter plot of genes that were elevated in ATL patients and decreased in expression after OTSSP167 treatment in an integrated RNA sequencing analysis according to an embodiment of the present invention. [Figure 11B] This figure shows the GO analysis of genes that were upexpressed in ATL patients and downexpressed after OTSSP167 treatment, in an integrated RNA sequencing analysis according to an embodiment of the present invention. [Figure 11C] This is a discriminant diagram showing the top 10 genes that were elevated in ATL patients and decreased in expression after OTSSP167 treatment in an integrated RNA sequencing analysis according to an embodiment of the present invention. [Figure 12A] This graph shows the tumor volume of Tax-siCTL±OTSSP167 in ATN-1 transplanted mice in a combination therapy of cell therapy and OTSSP167 administration according to an embodiment of the present invention. [Figure 12B] This graph shows the results of a killing assay of Tax-siCTL±OTSSP167 in ATN-1 transplanted mice in a combination therapy of cell therapy and OTSSP167 administration according to an example of the present invention. [Figure 12C] This graph shows the results of a killing assay of Tax-siCTL±OTSSP167 in ATN-1 transplanted mice in a combination therapy of cell therapy and OTSSP167 administration according to an example of the present invention. [Figure 12D] This photograph shows the results of immunostaining with Tax only MT-2 and ATN-1 as controls in a combination therapy of cell therapy and OTSSP167 administration according to an embodiment of the present invention. [Figure 12E] This photograph shows the results of immunostaining with Tax on MT-2 and ATN-1 cells administered with OTSSP167 in a combination therapy of cell therapy and OTSSP167 administration according to an embodiment of the present invention. [Modes for carrying out the invention]

[0013] <Embodiment> ATL is a disease distinct from other hematological malignancies such as leukemia and lymphoma. In recent years, mogamulizumab was developed as a treatment for ATL and was met with high expectations, but it did not achieve sufficient therapeutic effects. Furthermore, ATL has been problematic because conventional chemotherapy does not provide sufficient therapeutic effects, and the number of patients who can benefit from allogeneic hematopoietic stem cell transplantation is limited. However, a problem with drug development was that it was extremely time-consuming and labor-intensive to narrow down target molecule candidates through genetic analysis of cell lines and analysis of abnormal protein expression, and then to search for inhibitory molecules. Furthermore, the genes expressed as targets in cell lines may not correspond to those in actual patients, requiring the integration of patient sample information with drug efficacy evaluations in cell lines. Therefore, there was a need for efficient identification of drug targets and selection of drug candidates.

[0014] Therefore, the inventors decided to integrate comprehensive gene expression analysis using actual patient samples with small molecule compound screening and comprehensive gene expression changes induced by drug administration, without being bound by conventionally known perspectives, in order to simultaneously obtain new target molecules and drug candidates in ATL. To this end, they comprehensively identified genes that could serve as therapeutic targets by comparing gene expression levels in patient tumor cells and CD4-positive T cells of healthy individuals. From these genes, hub genes were selected based on protein-protein interactions. Then, molecular targeted drugs (selective inhibitors) that selectively inhibit the expression of the selected hub genes were selected, and their efficacy was evaluated.

[0015] Specifically, the inventors diligently conducted the following experiments and discovered a novel pharmaceutical composition for ATL: (A) Exploration of differences in gene expression between patient tumor cells and healthy CD4-positive T cells. (B) Screening of small molecule compounds against ATL (C) Gene expression changes induced by candidate small molecule compound (OTSSP167) (D) Confirmation of anti-ATL effects by siRNA and knockdown of the target candidate gene (SAP30) (E) Evaluation of drug efficacy in animal models using candidate small molecule compound (OTSSP167) In this way, the present invention was completed. The following describes in detail the pharmaceutical composition for ATL according to this embodiment, the method for selecting the pharmaceutical composition, and the treatment method.

[0016] [Pharmaceutical composition for ATL] The pharmaceutical composition for ATL (medical composition, pharmaceutical, therapeutic composition) of this embodiment is a pharmaceutical composition for the treatment of ATL, characterized in that it contains a selective inhibitor that selectively inhibits the expression of hub genes selected based on protein-protein interactions from among genes that are highly expressed in cancer cells of ATL patients compared to T cells of healthy individuals.

[0017] In this embodiment, the selective inhibitors can be compounds that inhibit products related to the hub gene both in vivo and in vivo, and compounds that promote the degradation of products related to the hub gene. These selective inhibitors in this embodiment can be analogs of the hub gene, and various compounds that affect the transcription, splicing, and other pathways related to mRNA maturation, translation, metabolism, and degradation of the hub gene.

[0018] Specifically, the selective inhibitors according to this embodiment may be, for example, peptides containing antibodies, proteins, nucleic acids, synthetic polymers such as PNA, and small molecule compounds that target nucleic acids (nucleotides) such as DNA and RNA, proteins such as various enzymes (hereinafter referred to as "target proteins") that regulate transcription and modification in the biosynthesis pathway from the hub gene. Of these, the nucleic acid may be antisense DNA or RNA, siRNA, shRNA, ribozyme, etc.

[0019] Alternatively, each selective inhibitor according to this embodiment may be a composition that reduces the transcription or expression level of normal mRNA of a hub gene, prevents the formation of mRNA by splicing, interferes with normal processing, interferes with maturation after processing, interferes with translation, prevents post-translational maturation or interaction with other molecules, or inhibits transport signals. In other words, it is possible to use selective inhibitors that suppress pathways related to hub genes, including these.

[0020] Furthermore, as selective inhibitors of the translation products of the hub gene, various compositions can be used that increase the translation of proteins antagonizing the hub gene, promote their degradation, cause them to be released into the nucleus or extracellular space, or transport them to intracellular structures. In addition, as selective inhibitors according to this embodiment, it is also possible to use synthesis promoters or inhibitors, or degradation promoters or inhibitors that target the same pathway as gene expression regulation by the hub gene.

[0021] Furthermore, compositions that act on genes, gene products, agonists / antagonists, or other pathways targeted for regulation of hub gene expression can also be used as selective inhibitors.

[0022] Furthermore, if the selective inhibitor according to this embodiment is a small molecule compound, it may inhibit the action of the hub gene by binding to or degrading the translation product.

[0023] Furthermore, as a selective inhibitor according to this embodiment, a composition relating to gene therapy mediated by the action of the hub gene on the genes or pathways targeted for expression regulation may be used. This gene therapy may involve introducing a medium that inhibits the expression of the hub gene or removes or inactivates it from the genome through artificial chromosome introduction, genome editing, or other knockout (knockdown) methods.

[0024] For example, plasmids or viral vectors may be used as the medium. These viral vectors may be composed of viruses common to those skilled in the art, such as adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, and other DNA or RNA viruses. In addition, proteins, lipid nanoparticles (LNPs), and drug delivery systems (DDS) such as liposomes may be used in these mediums as needed.

[0025] Such selective inhibitors can be identified from the amino acid sequence, mRNA sequence, genomic DNA sequence, etc., of the target protein and can be manufactured by methods easily accessible to those skilled in the art.

[0026] In this embodiment, the hub gene includes the SAP30 (Sin3A-associated protein 30) gene. The translation product of the human SAP30 gene is a 30 kDa (220 amino acid) Sin3-histone deacetylase complex (HDAC)-associated protein. Although histone acetylation is important in regulating gene expression in eukaryotes, SIN3A has not been previously known to be associated with ATL.

[0027] In other words, in this embodiment, it is particularly preferable to use SAP30 as the hub gene and an inhibitor of SAP30 as the selective inhibitor. That is, the pharmaceutical composition for ATL according to this embodiment is characterized by containing an inhibitor of SAP30. In this embodiment, OTSSP167 can be used as an example of a selective inhibitor of SAP30. Although OTSSP167 was known as a MELK inhibitor, as described in Patent Document 1, it was not known to those skilled in the art whether or not it was effective against ATL. In fact, as shown in the examples described later, MELK knockdown and knockout (KO) did not inhibit the proliferation of ATL cells.

[0028] OTSSP167 is thought to exert its therapeutic effect on ATL through its action as a selective inhibitor of the SAP30 gene, as shown in the examples described later. Furthermore, as shown in the examples described later, OTSSP167 may also exert its effect on ATL through its action on gene pathways other than MELK and SAP30, and can be suitably selected as an example of a pharmaceutical composition for the therapeutic use of ATL.

[0029] More specifically, as shown in the GO analysis of the examples described later, OTSSP167 reduces the expression of SAP30 as an epigenetic modifier. The majority of ATL patients have widespread epigenetic abnormalities, and these abnormalities may be associated with the onset and pathogenesis of ATL; therefore, targeting SAP30 could lead to a new therapeutic agent for ATL.

[0030] Alternatively, the ATL pharmaceutical composition according to this embodiment may also use other low-molecular-weight compounds besides OTSSP167 as inhibitors of SAP30, synthesized and combined as part of the pharmaceutical composition.

[0031] Specifically, potential targets for OTSSP167 include MAP2K7, mTOR, NOTCH1, and CDK1. These pathways, in addition to SAP30, may also be targets of OTSSP167. As shown in the examples described later, when the clinically targetable genes were expanded to the top 200 genes, NOTCH2NLB, which activates the NOTCH signaling pathway, was suppressed after OTSSP167 exposure. Therefore, it is also possible to target these genes and their gene products using small molecule compounds, siRNAs, antibodies, etc., that exert an effect on ATL.

[0032] Furthermore, the pharmaceutical composition for ATL according to this embodiment may also include medical compositions that promote apoptosis of ATL cancer cells related to the hub gene, induce the cancer immune system, or realize gene modification methods such as genome editing.

[0033] Furthermore, the ATL pharmaceutical composition according to this embodiment may be provided as various salts. These salts may be produced by reacting a low molecular weight compound with an acid or base that can be used in the manufacture of pharmaceuticals. For example, the ATL inhibitory effects of OTSSP167 and OTSSP167 hydrochloride according to this embodiment are equivalent, as shown in the examples described later, so either can be used.

[0034] Furthermore, the pharmaceutical composition for ATL according to the embodiments of the present invention also includes a prodrug. Here, a prodrug refers to a derivative that, after administration, is decomposed and converted under physiological conditions, such as specific pH conditions or the action of enzymes. In addition, although the prodrug may be inactive when administered to the patient, it is converted into an active therapeutic molecule in the body.

[0035] Furthermore, the pharmaceutical composition according to the embodiment of the present invention may contain any pharmaceutically acceptable carrier. That is, the ATL pharmaceutical according to this embodiment can be formulated using a pharmaceutically acceptable carrier known in the art in a dosage form suitable for parenteral or oral administration. This carrier may be, for example, a liposome carrier, colloidal gold particles, polypeptides, lipopolysaccharides, polysaccharides, lipid membranes, etc. Among these, it is preferable to use a carrier that enhances the expression-modulating effect of selective inhibitors. For example, it is preferable to use liposomes, particularly cationic liposomes.

[0036] Furthermore, pharmaceutically acceptable carriers may include, for example, physiological saline, isotonic solutions containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. It can also be administered with a suitable solubilizer, such as alcohol, specifically ethanol, polyalcohols, such as propylene glycol, polyethylene glycol, or nonionic surfactants, such as polysorbate 80(TM), HCO-50, etc. Appropriate excipients may also be included.

[0037] Furthermore, the pharmaceutical composition according to this embodiment may contain a suitable pharmaceutically acceptable carrier in order to prepare a pharmaceutically acceptable carrier. This carrier may include biocompatible materials such as silicone, collagen, and gelatin. The carrier may also be provided as an emulsion. In addition, it may contain any or any combination of pharmaceutical additives such as diluents, fragrances, preservatives, excipients, disintegrants, lubricants, binders, emulsifiers, and plasticizers.

[0038] The administration route of the pharmaceutical composition according to this embodiment is not particularly limited, and it can be administered parenterally or orally. Parenteral administration can include, for example, intravenous, intra-arterial, subcutaneous, intradermal, intramuscular, intraperitoneal, or direct administration to organs such as bone marrow or spleen.

[0039] When using the pharmaceutical composition according to this embodiment for the above-mentioned treatment, the administration interval and dosage can be appropriately selected and changed according to various conditions such as the disease status and the patient's condition.

[0040] The single dose and number of doses of the pharmaceutical composition according to this embodiment can be appropriately selected and changed depending on the purpose of administration, as well as various conditions such as the patient's age and weight, symptoms, and the severity of the disease. The number and duration of administration may be limited to a single dose, or it may be administered once to several times a day for several weeks, while monitoring the disease state. Depending on the state, further or repeated administration may be necessary.

[0041] In addition, the pharmaceutical composition according to this embodiment can be used in combination with other compositions. That is, it can be used in combination with other therapeutic compositions such as conventional chemotherapy agents and anticancer drugs. Furthermore, the pharmaceutical composition according to this embodiment may be administered simultaneously with other compositions, or it may be administered at intervals, but the order of administration is not particularly limited.

[0042] Specifically, the pharmaceutical composition according to this embodiment may be used in combination with a pharmaceutical composition for Tax-targeted T-cell immunotherapy. Tax is a protein necessary for the replication of the HTLV-1 virus and has carcinogenic properties. Therefore, as a pharmaceutical composition for Tax-targeted T-cell immunotherapy, it is possible to use the pharmaceutical composition according to this embodiment in combination with cells that target Tax, vaccine preparations, proteins including antibodies, nucleic acids, small molecule compounds, etc. This makes it possible to further suppress the proliferation of ATL cells by using Tax-siCTL, which is a Tax-specific cytotoxic T cell (CTL), in combination with OTSSP167 according to this embodiment, as shown as an example in the examples described later. In this case, as shown in the examples described later, by using it in combination with cell therapy using the pharmaceutical composition for Tax-targeted T-cell immunotherapy, it is possible to suppress the proliferation of ATL cells even in Tax-low-expressing strains.

[0043] Furthermore, in this embodiment, the period during which the disease improves or is alleviated is not particularly limited; it may be a temporary improvement or alleviation, or it may be an improvement or alleviation for a certain period of time.

[0044] Furthermore, the pharmaceutical composition according to this embodiment may also be used with the addition of other modifiers, such as those that suppress the conversion of normal T cells into cancer cells. Furthermore, the therapeutic agent of this embodiment may be used in combination with various tumor adhesion inhibitors, tumor angiogenesis inhibitors, etc., to suppress the adhesion of cancer cells and angiogenesis in ATL-derived lymphoma, thereby enhancing its effectiveness.

[0045] Furthermore, various viral vectors used in anti-cancer medicine can be added. Examples of these viral vectors include adenovirus vectors, lentivirus vectors, and retrovirus vectors.

[0046] [Treatment method] The therapeutic method according to an embodiment of the invention is a therapeutic method for ATL, characterized by selectively inhibiting the expression of hub genes selected based on protein-protein interactions from among genes that are highly expressed in cancer cells of ATL patients compared to T cells of healthy individuals.

[0047] Specifically, the pharmaceutical composition for ATL according to this embodiment can be used in veterinary therapy to treat ATL and related viruses in animals. Although ATL is a disease of humans (Homo sapiens), similar or related retroviruses that cause leukemia and lymphoma can be used to induce similar mechanisms of action.

[0048] This animal includes various mammals. Specifically, it may include rodents such as mice, rats, ferrets, hamsters, guinea pigs, or rabbits, dogs, cats, sheep, pigs, cattle, horses, or non-human transgenic primates. In other words, the ATL pharmaceutical according to the embodiment of the present invention can be used not only for the treatment of humans, but also for the treatment of various animals, and for promoting the growth of livestock, etc.

[0049] Furthermore, in the treatment method according to this embodiment, the expression level of the hub gene in the patient's ATL cancer cells may be measured, and then the selective inhibitor according to this embodiment may be applied. In other words, in order to determine whether or not to administer the ATL drug according to this embodiment, the patient's ATL cells may be obtained, the expression level of the hub gene may be measured, and the drug may be administered if it is higher than a specific unit, thus providing "personalized" medical care.

[0050] More specifically, by measuring the expression level of the SAP30 gene in a patient's ATL cells using methods such as Northern blotting, microarrays, QCM sensor measurement, or real-time PCR, and comparing it with the expression level of the SAP30 gene in normal cells, it is possible to determine whether selective SAP30 inhibitors are highly effective as a treatment.

[0051] The pharmaceutical composition for ATL according to the embodiment of the present invention can be used in combination with other therapeutic compositions such as conventional chemotherapy agents and anticancer drugs. It can also be used in combination with other cancer treatments and methods such as radiation therapy, particle beam therapy, and surgery. This may provide an effective treatment for ATL, which has a poor prognosis. In addition to the therapeutic agent of this embodiment, therapeutic compositions that alleviate pain or improve quality of life may be added to the treatment. The composition of the present invention may be administered simultaneously with other compositions, or at intervals, but the order of administration is not particularly important.

[0052] Furthermore, in the embodiments of the present invention, the period during which the disease is improved or alleviated is not particularly limited; it may be a temporary improvement or alleviation, or it may be an improvement or alleviation for a certain period of time. It can also be used for a preventative effect against the disease.

[0053] [Method for selecting pharmaceutical compositions for ATL] A method for selecting a pharmaceutical composition for ATL according to an embodiment of the present invention is a method for selecting a pharmaceutical composition for the treatment of ATL, characterized by selecting genes that are highly expressed in cancer cells of ATL patients compared to T cells of healthy individuals, selecting a hub gene from the selected highly expressed genes based on protein-protein interactions, and selecting a selective inhibitor that selectively inhibits the expression of the selected hub gene.

[0054] Specifically, an example of the steps for the selection method of the ATL pharmaceutical composition according to this embodiment is as follows: (Step S1) Patient tumor cells (CD4+CADM1+T cells) and healthy individual CD3+CD4+T cells are separated using a cell sorter. (Step S2) After RNA extraction, comprehensive gene expression analysis is performed using RNA-seq with next-generation sequencing. (Step S3) Extract the top 400 genes from among those significantly elevated in the patient's ATL paraphylaxis. (Step S4) Perform protein-protein-interaction (PPI) analysis and identify the hub gene group. (Step S5) Next, select a gene from the group of hub genes for which a selective inhibitor can be obtained by purchase or other means. (Step S6) Perform inhibitor administration experiments using ATL cell lines. (Step S7) As validation in an animal model, ATL cell lines into which reporter genes have been introduced are administered to immunodeficient mice, etc. After confirming engraftment, each inhibitor is introduced and changes in skin ulcer formation and tumor volume during the course are compared.

[0055] Furthermore, the ATL selection method of this embodiment may be executed using a program, a computer-based device, and a system for analyzing the diagnostic results, judging the test results, processing the data, and visualizing them. Such devices and systems can be developed by those skilled in the art using general techniques and methods. Such devices and systems enable high-throughput testing and facilitate patient diagnosis. In addition, the ATL pharmaceutical composition selection method according to this embodiment can be used for drug discovery applications.

[0056] By configuring it as described above, the following effects can be obtained. Traditionally, ATL has been recognized as a distinct disease in the WHO classification system, and its etiology and course, particularly as a T-cell tumor caused by a virus, set it apart from other hematological malignancies such as leukemia and lymphoma. Mogamulizumab was developed as a treatment for ATL and was met with high expectations, but long-term treatment results showed that the survival rate remained at around 20%, and it was known that when used as pre-transplant therapy, it actually increased complications and decreased survival. Furthermore, ATL presented problems because conventional chemotherapy did not provide sufficient therapeutic effects, and the number of patients who could benefit from allogeneic hematopoietic stem cell transplantation was limited. Therefore, there was a need for pharmaceutical compositions that combined high efficacy with low toxicity. In other words, efficient identification of therapeutic targets and selection of drug candidates were desired.

[0057] In contrast, the ATL pharmaceutical composition according to the embodiment of the present invention uses a selective inhibitor that selectively inhibits the expression of hub genes selected based on protein-protein interactions among genes that are highly expressed in tumor cells (cancer cells) of ATL patients compared to T cells of healthy individuals. This allows for the selection and provision of a pharmaceutical composition that combines high efficacy and low toxicity for ATL. Specifically, by comprehensively interpreting comprehensive gene expression changes due to small molecule compound screening and drug administration, in addition to comprehensive gene expression analysis of tumor cells in actual patient samples, it is possible to identify target molecules that become new hub genes in ATL, and to provide inhibitors that inhibit these molecules as pharmaceutical compositions. Furthermore, as shown in the examples described later, the ATL pharmaceutical composition according to the embodiment of the present invention has low toxicity to normal cells and is suitable for use.

[0058] Furthermore, it was previously unknown which genes play a crucial role in the tumor growth of ATL.

[0059] In contrast, the pharmaceutical composition for ATL according to this embodiment is characterized in that at least one of its hub genes is the SAP30 gene. In this embodiment, as shown in the examples described later, the expression of SAP30 is upregulated in tumor cells of actual ATL patients, indicating that it is one of the genes important for tumor growth. Therefore, the ATL pharmaceutical composition according to this embodiment can reduce the survival rate of ATL tumor cells and suppress tumor growth by using a selective inhibitor that induces a decrease in SAP30 expression. In other words, ATL can be treated by inhibiting SAP30 expression with the selective inhibitor according to this embodiment.

[0060] Furthermore, when developing pharmaceutical compositions, the process of narrowing down target molecule candidates through gene expression analysis and then searching for inhibitory molecules was extremely time-consuming and labor-intensive. Additionally, the targeted gene expression in cell lines sometimes did not correspond to those in actual patients, necessitating the integration of patient sample information with drug efficacy evaluations in cell lines.

[0061] In contrast, OTSSP167, obtained from the small molecule compound screening results, as a pharmaceutical composition for ATL according to this embodiment, can exert its therapeutic effect against ATL not through the original MELK inhibition-induced antitumor effect, but by inhibiting SAP30 expression. As a result, inhibition of SAP30 expression or function becomes a novel therapeutic target for ATL.

[0062] Furthermore, as shown in the examples described later, no significant toxicity was observed with OTSSP167 administration. Therefore, its low drug toxicity may make it widely acceptable to all ATL patients, regardless of age or general condition. Even if hemoglobin levels decrease with OTSSP167 administration, anemia can be managed in the same way as with blood transfusions or other antitumor drugs. Due to its low toxicity, OTSSP167 may be able to maintain low tumor burden and be used as long-term maintenance therapy.

[0063] Furthermore, in other embodiments of the present invention, it is possible to use selective inhibitors with a hub gene other than SAP30 as pharmaceutical compositions for ATL. Examples of such hub genes include CEBPA, AURKB, EZH2, CADM1, WEE1, PLK1, MELK (Maternal embryonic leucine zipper kinase), and BIRC5.

[0064] Furthermore, other small molecule compounds besides OTSSP167 can be used as pharmaceutical compositions for ATL according to other embodiments of the present invention. Such small molecule compounds can be selected as candidates by methods such as bioinformatics and protein dynamics, targeting SAP30, using methods common to those skilled in the art.

[0065] Furthermore, the pharmaceutical composition for ATL can be used in combination with other compositions. As described above, it is possible to use cells, proteins including antibodies, nucleic acids, low molecular weight compounds, etc. The composition of the present invention may be administered, sprayed, or applied simultaneously with other compositions.

[0066] In addition, compositions that act on genes, gene products, agonists / antagonists, or other pathways targeted for SAP30 expression regulation, which are used as pharmaceutical compositions for ATL, can also be used as selective inhibitors.

[0067] Furthermore, as a pharmaceutical composition for ATL, SAP30 can also be used for gene therapy through its action on genes and pathways targeted for expression regulation.

[0068] In addition, selective inhibitors of SAP30 can be used for non-pharmaceutical applications. This makes them suitable for drug discovery purposes, such as experiments and models to study the mechanism of action in ATL in animals. [Examples]

[0069] Below, a pharmaceutical composition for ATL according to an embodiment of the present invention will be described in more detail as an example, based on specific experiments. However, this example is merely one example and is not limiting.

[0070] [Materials and Methods] (Exam design) This study aimed to identify novel therapeutic options for ATL. First, a compound library containing 50 compounds with diverse mechanisms was constructed and screened from the perspective of cell proliferation inhibition. The anti-leukemic properties of the selected compounds were validated in immunodeficient (NOD / Shi-scid, IL-2RγKO Jic; hereinafter referred to as "NOG") mice using two luciferase-expressing ATL sublines. The hematological toxicity of these compounds was evaluated after a total of 15 injections into NOG mice lacking ATL cells. Next, the mechanisms of action of the compounds against ATL were evaluated using siRNA-induced mRNA knockdown and CRISPR / Cas9-induced functional knockout. Furthermore, RNA sequencing was performed to further elucidate the target genes of the compounds, and GEP was compared between actual ATL patients and healthy donors, as well as between ATL cell lines treated with OTSSP167 and untreated ATL cell lines. Finally, the study focused on upregulated genes in ATL patients and genes downregulated after OTSSP167 exposure. The anti-ATL effect by inhibiting the gene was evaluated using siRNA-induced mRNA knockout.

[0071] (Patient specimen) Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from 15 ATL patients (12 with acute type and 3 with chronic type) and 11 healthy donors who provided written informed consent at Saitama Medical Center, Jichi Medical University. The cells were cryopreserved at -80°C until use. ATL subpopulations were classified according to standard criteria. This study was approved by the Institutional Review Board of Saitama Medical Center, Jichi Medical University. Written informed consent was obtained from all patients in accordance with the Declaration of Helsinki.

[0072] (Flow cytometry analysis and sorting) Patient cells were labeled with appropriate antibodies at room temperature in the dark for 20 minutes. After washing, they were isolated and analyzed using BD FACSVerse® (BD Biosciences). Anti-human CD3 PE-Cy7 (BioLegend, #300420), anti-human CD3 APC (BioLegend, #317318), anti-human CD4 APC-Cy7 (BioLegend, #300518), anti-human CD7 APC (BioLegend, #395606), anti-human CD25 FITC (BioLegend, #302604), anti-human CD25 PE-Cy7 (BioLegend, #356108), anti-SynCAM (TSLC / CADM1) PE (MBL, #CM004-5), 7-AAD (BioLegend, #420404), anti-Annexin V APC (BioLegend, #640920), anti-anti-human CD14 FITC (BioLegend, #325604) and anti-human CD19 FITC (BioLegend, #302206) were used in combination.

[0073] (RNA sequencing analysis) ATL cells were isolated from the CD4+CD14-CD19-CADM1+ subpopulation of PBMCs from 15 ATL patients using a BD FACS Aria II® (BD Biosciences) cell sorter. Control normal T cells (comparative example) were isolated from the CD3+CD4+ subpopulation of PBMCs from 11 healthy donors.

[0074] ATL cell lines MT-2, MT-4, TL-Su, and ATN-1 were incubated with 100 nM OTSSP167 (MedChemExpress, #HY-15512A) for 8 hours, after which viable cells were isolated. The viable cells were isolated from the 7-AAD subpopulation.

[0075] After sorting, RNA was extracted and libraries constructed using either the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio, #634888) or the RNeasy Plus Mini Kit (QIAGEN, #74134). Sequencing was outsourced to Takara Bio and performed using NovaSeq 6000 (Illumina). FASTQ files were aligned to Genome Research Consortium human (GRCh)37 / hg19 or GRCh38 / hg38 (GENCODE v. 35 or v. 39), and the sequencing data was analyzed using the DRAGEN Bio-IT platform (v.3.6.3 or v. 3.9.3, Illumina). Difference analysis of the sequencing data was performed using the Bioconductor package edgeR (v.3.32.1).

[0076] (Cell lines and cultures) MT-2, MT-4, TL-Su, and ATN-1 were used as ATL and HTLV-1 infected tumor cell lines. MT-2, MT-4, and TL-Su are human leukocyte cell lines established through co-culture with human ATL cells. MT-2 and MT-4 were purchased from the Japan Cell Line Bank (Osaka, Japan). TL-Su was subcultured from cells provided by Dr. Kannagi, formerly of Tokyo Medical and Dental University. ATN-1 was purchased from the RIKEN BioResource Center (Tsukuba, Japan). These cell lines were cultured at 37°C in a humidified environment of 5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque Co., Ltd.) supplemented with 10% fetal bovine serum.

[0077] (In vitro cell viability luminescence assay and small molecule compound screening) For each target cell, 2-4.5 × 10 4The cells were plated three times (triplicate seeding) in 100 μl of culture medium in a flat-bottomed 96-well plate. The cells were cultured for 48 hours with 50 different small molecule compounds (drugs) at various concentrations (manufactured by CEREC Biotechnology Co., Ltd.). These 50 drugs are shown in Table 1 below:

[0078] [Table 1]

[0079] Cell viability was measured using Varioscan LUX (Thermo Scientific) and Cell Counting Kit-8 (Dojin Chemical Laboratory) according to the manufacturer's recommended protocol.

[0080] (Transfection of ATL cells using siRNA) Cells were transfected with siRNA by electroporation using the Neon transfection system (Thermo Scientific). The procedure followed the manufacturer's protocol. Specifically, semi-confluent ATL cell lines were counted and centrifuged at 300 × g for 5 minutes. The cell pellet was resuspended and 3 × 10⁶ cells were added. 5 The individual cells were transferred to a tube and centrifuged again. 60 pmol of siRNA was added to the pellet, diluted with 3 μl of RNase-free water and 22 μl of R buffer. One-third (10 μl) of this mixture was aspirated into a Neon tip and used.

[0081] The parameters used for electroporation were 1200V, 20ms, and 2 pulses for MT-2 and ATN-1; 1300V, 10ms, and 3 pulses for MT-4; and 1300V, 30ms, and 1 pulse for TL-Su.

[0082] Immediately after electroporation, cells were released from the Neon-Tip into a 24-well plate already filled with culture medium and cultured. Transfection efficiency was confirmed by quantitative RT-PCR between 24 and 72 hours. Cell viability was measured between 72 and 120 hours after transfection.

[0083] All siRNAs used were commercially available: SAP30 (Thermo Scientific, siRNA ID; s16820), MELK (Thermo Scientific, siRNA ID; s386), and a negative control (Thermo Scientific, #4390843).

[0084] (Quantitative RT-PCR) Total RNA was extracted from each sample 24 and 72 hours after transfection using the RNeasy Plus Mini Kit (QIAGEN, #74134). Complementary DNA (cDNA) was generated using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, #1708891). Quantitative RT-PCR was performed using QuantStudio 12 K Flex (Applied Biosystems), TaqMan Gene Expression Master Mix (Thermo Scientific, #4369016), cDNA from each sample, and gene-specific probes. The probes used were MELK (Thermo Scientific, Assay ID: Hs01106438_m1), SAP30 (Thermo Scientific, Assay ID: Hs01009153_g1), and ACTB (Thermo Scientific, #4333762T). The data were collected in 2 -ΔΔCt The analysis was performed using a standard method and normalized for ACTB expression.

[0085] (Establishment of MELK knockout clones using CRISPR / Cas9) Two sets of guide RNAs (Thermo Scientific, Assay ID; CRISPR637268_SGM and CRISPR720420_SGM) and Cas9 protein (Thermo Scientific, #A50577) were transfected into the ATN-1 cell line using the Neon transfection system (Thermo Scientific). Before mixing with the ATN-1 cells, 7.5 pmol of Cas9 protein and 3.75 pmol of each guide RNA (maintaining a 2:1:1 ratio) were mixed in R buffer (Thermo Scientific, #MPK1096R) at room temperature for 15 minutes, and electroporation was performed. Electroporation was carried out according to the protocol described above. The transfected cells were a clonal population expanded from a single cell. These cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% MEM non-essential amino acid solution (Wako Pure Chemical Industries, #139-15651).

[0086] (Xenotransplant mouse model) Six-week-old female NOG mice (In-Vivo Science) were used for the in vivo experiments. All animal procedures were approved by the Institutional Animal Care and Concerns Committee of Jichi Medical University, and all animal care was provided in accordance with the committee's guidelines.

[0087] For tumor evaluation, we created luciferase-expressing cell lines of ATN-1 and MT-2. These were suspended in Isocove's Modified Dulbecco's Medium (IMDM) (Gibco, #12440053), and 5 × 10⁶ cells were produced per cell. 5Individual cells were subcutaneously injected into the back. The treatment group received OTSSP167 hydrochloride (MedChemExpress, #HY-15512A) dissolved in DMSO, which was then diluted with D-PBS (Dulbecco's phosphate-buffered saline) before use to adjust the final DMSO concentration to less than 10%. The test doses of OTSSP167 hydrochloride were 0 μg, 200 μg, and 400 μg. This corresponds to 0 mg / kg, 10 mg / kg, and 20 mg / kg for mice weighing 20 g, respectively. Subsequently, 200 μl of each concentration was injected into the tail vein three times a week. Mice's body weight and appearance were monitored twice a week to assess general health and ulcerative lesions. Ulcerative lesions were defined as ulcers at the tumor injection site.

[0088] Tumor evaluation was performed using an in vivo imaging system (IVIS Spectrum CT) (Caliper Life Science). Specifically, mice were intraperitoneally injected with 375 μg / kg of luciferin (Yeda Chemical Co., Ltd., #LU-1), and the luminescence signal was evaluated weekly. Graft survival was evaluated one week after infection. When the luminescence signal saturated, tumor progression was evaluated using CT. Images were analyzed using Living Image software v4.7.3 (PerkinElmer). Tumor volume was calculated using the formula "4 / 3π × (width / 2) × (length / 2) × (height / 2)". Blood samples were collected from the jugular vein of deeply anesthetized mice. White blood cells (WBC), hemoglobin (Hb), and platelets were measured using a blood analyzer, Celltac α (Nihon Kohden Corporation).

[0089] (Proliferation analysis) Place the cells in a 12-well plate, 1 x 10⁶ cells per well. 5 The cells were plated at a specific density and incubated for 72–96 hours. The cells were counted and measured at 1 × 10⁶. 5 The cells were replated in fresh culture medium. The cells were passaged five times, and the cumulative population was calculated at each passage.

[0090] (Western blot analysis) Cells were lysed with radioimmunoprecipitation (RIPA) buffer, and the lysate was quantified using a protein assay dye reagent (Bio-Rad Laboratories, #5000006). The same amount of protein was denatured and loaded onto an 8% SDS-PAGE gel (Thermo Scientific, #NW00080BOX), and the protein was transferred to a polyvinylidene difluoride (PVDF) membrane using the dry transfer method (Thermo Scientific, #21001). The membrane was blocked with blocking buffer for 30 minutes (Thermo Scientific, #37542). For immunodetection, an iBind Flex device (Thermo Scientific, #SLF20002PK, #SLF2010) was used. For the primary antibodies, anti-MELK antibody (MBL, #D373-3) and anti-beta-actin (ACTB) antibody (Thermo Scientific, #MA5-15739) were used at a 1:1000 dilution. For the secondary antibody, HRP-conjugated goat anti-mouse IgG (Thermo Scientific, #31430) was used at a 1:5000 dilution. After incubation for 2.5 hours, the membranes were washed and developed with Western BLoT Hyper HRP Substrate (Takara Bio, #T7103A). The membranes were imaged using the ChemiDoc XRS+ system (Bio-Rad Laboratories). MELK and ACTB were detected (Takara Bio, #T7135A) after the membranes were detached following the initial protein detection.

[0091] (Construction of protein-protein interaction (PPI) networks and gene ontology (GO) analysis) The PPI network was constructed using Cytoscape (ver. 3.91) and STRING (Search Tool for the Retrieval of Interacting Genes / Proteins). GO analysis was performed using the Cytoscape plugin applications ClueGO and CluePedia.

[0092] (statistical analysis) Differences between two groups were analyzed using Student's t-test. One-way analysis of variance (ANOVA) with Bonferroni correction was used for the statistical analysis of the three groups, either as a post-hoc test or repeated measures ANOVA. The trend of decreasing ATL cells in patients was assessed using flow cytometry analysis with the Jonckheere-Terpstra test (JT test). The cumulative incidence of ulcerative lesions was compared using a Bonferroni-corrected log-rank test as a post-hoc test. A two-sided p-value < 0.05 was considered statistically significant. In each figure of this example, "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001. All statistical analyses were performed using EZR v1.68 (Jichi Medical University Saitama Medical Center), the graphical user interface for R.

[0093] 〔result〕 (Differences in gene expression between tumor cells of patients and CD4-positive T cells of healthy individuals) First, tumor cells (CD4+CADM1+T cells) from actual patients (n=12) and CD3+CD4+T cells from healthy individuals (n=11) were separated using a cell sorter. After RNA extraction, comprehensive gene expression analysis was performed using RNA-seq with next-generation sequencing. This extracted gene groups that showed significantly altered expression in patient ATL cells. Then, key genes and pathways were identified using Protein-Protein-Interaction / GO analysis and other methods.

[0094] Figure 1 shows the results of comparing gene expression changes between CD4+CADM1+ATL cells from actual patients and CD3+CD4+T cells from healthy individuals. Figure 1(a) is a summary of the genes that showed increased (Up), decreased (Down), or not significant (NotSig). Figure 1(b) is a map of the identified genes and pathways.

[0095] As a result, increased expression of genes such as CEBPA, AURKB, EZH2, CADM1, WEE1, PLK1, MELK, SAP30, and BIRC5 was observed. Focusing on the top 300 genes with increased expression, many genes involved in pathways such as the cell cycle, cell division, and epigenetics were identified.

[0096] (Screening of small molecule compounds for ATL) We screened the efficacy of 50 different pathway inhibitors (small molecule compounds) targeting various pathways, including molecules and pathway targets whose gene expression changes were identified as described above, for inhibiting cell proliferation. In this example, in addition to inhibitors of cell cycle and epigenetic pathways and molecules whose expression is upregulated, we used 50 drugs listed in Table 1 above that target representative pathways such as angiogenesis, apoptosis, cytoskeltal signaling, DNA damage / repair, JAK / STAT, MAPK, metabolism, NF-κB, PI3K / Akt / mTOR, and TGF-beta / Smad.

[0097] Specifically, four types of ATL cell lines, MT-2, MT-4, and TL-SuATN1, were used in inhibitor administration experiments (2.25 × 10⁻¹⁰). 4 We performed cell-well, 48-hour culture to identify small molecule compounds that could be candidates for therapeutic drugs.

[0098] Figure 2A shows the results of a cell viability luminescence assay screening in the ATL cell lines (a) MT-2, (b) MT-4, (c) TL-Su, and (d) ATN-1, after introducing each drug at a concentration of 1 μM. The drug that showed a strong effect in all cell lines was OTSSP167 (drug number 50, the drug on the far right). In addition, several other drugs showed efficacy in several cell lines. These were Vorinostat and Paclitaxel, which are FDA-approved drugs for the treatment of primary cutaneous T-cell lymphoma, and Tazemetostat, which is approved in Japan for the treatment of follicular lymphoma.

[0099] Figure 2B shows the results of a cell viability luminescence assay in which the same drug was introduced at a concentration of 100 nM into the following cell lines: (a) MT-2, (b) MT-4, (c) TL-Su, and (d) ATN-1. Even when introduced at a low concentration of 100 nM, OTSSP167 (drug number 50, the drug on the far right) showed nearly half cytotoxicity in all cell lines.

[0100] Figure 2C shows the relationship between OTSSP167 concentration and cell viability (%). OTSSP167 was added to each ATL cell line, and cell viability (%) was examined after 48 hours. The results showed that OTSSP167 inhibited the proliferation of ATL cell lines in a dose-dependent manner.

[0101] Therefore, we further investigated the toxicity of OTSSP167 in the peripheral blood of healthy individuals (n=6).

[0102] Figure 2D shows the results of examining the cell survival rate (%) after 48 hours when OTSSP167 was added to normal peripheral blood mononuclear cells (PBMCs) from healthy donors. The decrease in survival rate of healthy donor PBMCs due to OTSSP167 was less severe than that of ATL cell lines. In other words, it was confirmed that cytotoxicity was not significant (over 80% survival) at OTSSP167 concentrations of 10-100 nM.

[0103] Figure 2E shows the results of flow cytometry analysis of ATL patient samples. Specifically, peripheral blood samples from a 55-year-old male diagnosed with chronic ATL were used. CD3+CD4+ cells were separated for CD7 and CADM1. In PBMCs of ATL patients, the CD7-CADM1+ fraction, which is thought to be where ATL cells are normally distributed, was dose-dependently reduced by OTSSP167. On the other hand, the CD7+CADM1- fraction, which is thought to be where normal cells are distributed, did not show a significant reduction.

[0104] Figure 2F shows the ratio of the CD7-CADM1+ fraction among CD3+CD4+ cells in the same sample as in Figure 2E. A statistically significant monotonic decreasing trend was observed with OTSSP167. Specifically, for the decreasing trend, the Jonckheere-Terpstra test showed P < 0.001. On the other hand, normal lymphocytes seemed to be less affected in vitro.

[0105] (Changes in gene expression induced by the candidate small molecule compound (OTSSP167)) By the above screening, the drug OTSSP167, which showed an effect at a low concentration in any cell line, was selected. In this example, the results using OTSSP167 are shown below.

[0106] First, four ATL cell lines (MT-2, MT-4, TL-Su, ATN-1) were used to confirm comprehensive gene expression changes before and after OTSSP167 administration by RNA-seq. Specifically, OTS100 nM was added to 2×10 6 cells, and viable cells were collected by FACS 8 hours later. At this time, cells stained with 7-AAD were excluded. As a result, about 5×10 5 cells were collected. RNA-seq was performed on this.

[0107] Furthermore, the analysis was performed in combination with RNA-seq data examining the differences in gene expression between the tumor cells of the above patients and healthy human CD4-positive T cells. Specifically, genes that were upregulated in patient cells and whose expression decreased in cell lines by OTSSP167 administration were identified, and molecules that could be candidate targets for the anti-ATL effect induced by OTSSP167 administration were selected.

[0108] Figure 3 shows the results of investigating gene expression changes induced by the candidate small molecule compound (OTSSP167). Figure 3(a) shows the results of selecting the top 400 genes that showed gene expression in patient cells and healthy human cells, and then selecting the top 200 genes that showed gene changes after OTSSP167 administration. Figure 3(b) shows the results of narrowing down the genes that can be expressed as proteins and searching for genes in which expression was increased by 2 log fold changes (4 times) or more in patient cells and whose gene expression was decreased by 3 log fold changes or more in the cell line after OTSSP167 administration. As a result, SAP30 was identified.

[0109] (Confirmation of anti-ATL effect of siRNA against the candidate target gene (SAP30)) Although siRNA was introduced and knock-out was performed on MELK, the original target of OTSSP167, almost no change in viability was observed in ATL cell lines, and no anti-ATL effect was observed by inhibiting the expression of MELK itself. Therefore, it was considered that in ATL, OTSS167 exerted its anti-ATL effect not by inhibiting the original MELK, but by inhibiting other molecules.

[0110] Therefore, we introduced siRNA into the SAP30 gene, one of the molecules whose expression is elevated in ATL patient cells and whose expression decreases upon administration of OTSSP167, using electroporation, and evaluated the survival of ATL cell lines.

[0111] Figure 4A shows the gene expression levels of SAP30 after siRNA introduction. Figure 4A(a) shows the results for MT-2, (b) for MT-4, (c) for TL-Su, and (d) for ATN-1 cell lines. In all cases, knockdown of SAP30 significantly reduced SAP30 expression. "*" indicates P<0.05%, and "**" indicates P<0.01%.

[0112] Figure 4B shows the results for siRNA introduction and cell viability. Figure 4B(a) shows the results for MT-2 cell lines, (b) for MT-4, (c) for TL-Su, and (d) for ATN-1 cell lines. In each graph, "Negative Control" shows the negative control, and "SAP30 siRNA" shows cells introduced with SAP30 siRNA. The vertical axis shows viability (%). "**" indicates 0.01% significance, and "***" indicates 0.001% significance.

[0113] As a result, a statistically significant decrease in survival rate was observed in all ATL cell lines. This indicates that inhibition of SAP30 is a novel therapeutic target for ATL, and that the anti-ATL effect of OTSSP167 involves a pathway mediated by the suppression of SAP30 expression. In other words, SAP30 knockdown efficiently inhibited the proliferation or survival rate of ATL cells.

[0114] Figure 4C shows the results of an additional analysis in which 12 small molecule compounds that reduced the viability of ATL cells in the above analysis were selected and added to each cell line, followed by a quantitative comparison of SAP30 expression using RT-PCR (RT-qPCR). The results for OTSSP167 are enclosed in a square. In all cell lines, OTSSP167 reduced SAP30 expression the most.

[0115] (Evaluation of drug efficacy in animal models using candidate small molecule compound (OTSSP167)) Therefore, as a validation in an animal model, luciferase-transformed ATL cell line (MT2) was subcutaneously administered to immunodeficient (NOG) mice. After confirmation of engraftment, a MELK inhibitor (OTSSP167) was administered three times a week for 5 weeks (total of 15 doses: 0 mg / kg (vehicle control), 10 mg / kg, or 20 mg / kg). The mice were then observed for 5 weeks (total of 10 weeks), and changes in skin ulcer formation and tumor volume during the course were compared.

[0116] In the in vivo experiments of this example, OTSSP167 hydrochloride (MedChemExpress, #HY-15512A) was used. This is because OTSSP167 hydrochloride generally has better solubility in dimethyl sulfoxide (DMSO) compared to OTSSP167 alone, thus reducing DMSO-induced toxicity. As will be described later, the in vitro inhibitory effect on the MT-2 cell line was equivalent between OTSSP167 and OTSSP167 hydrochloride.

[0117] Figure 5A shows the protocol for the ATN-1 transplantation model. Luciferase-expressing ATN-1 cells were subcutaneously transplanted into 15 NOG mice to confirm the in vivo therapeutic effect of OTSSP167 on ATL. Specifically, on day 7 after transplantation, engraftment of ATN-1 cells was observed in 14 out of 15 mice, and one mouse that did not engraft was excluded from subsequent experiments. From day 11 to day 32, OTSSP167 and its solvent (10% DMSO + 90% D-PBS) were administered intravenously a total of 10 times, three times a week, to the OTSSP167 10 mg / kg group (n=5), the OTSSP167 20 mg / kg group (n=5), and the control group (n=4).

[0118] Figure 5B shows the results of luminescence imaging, in which tumor volume was measured using an in vivo imaging system (IVIS) in an ATN-1 transplantation model. The evaluation results are shown for 14 days (Day 14) and 34 days (Day 34) after subcutaneous administration of the ATN-1 strain, respectively. Compared to the control group, the proliferation of the ATN-1 strain was suppressed in the OTSSP167 10 mg / kg administration group and the OTSSP167 20 mg / kg administration group.

[0119] Figure 5C shows the time-course evaluation of ROI for each group. Repeated measures ANOVA was used for comparisons between the three groups. OTSSP167 showed luciferase activity of P<0.001. Post-hoc tests were performed using the Bonferroni method for comparisons between two of the three groups. Compared to the control group, ATN-1 proliferation was significantly suppressed in the OTSSP167 10 mg / kg and OTSSP167 20 mg / kg groups. In the figure, "*" indicates P<0.05 and "***" indicates P<0.001.

[0120] Figure 5D shows the macroscopic findings of subcutaneous tumors surgically removed. Compared to the control group, macroscopic findings clearly show that ATL proliferation was suppressed in the OTSSP167 10 mg / kg and OTSSP167 20 mg / kg groups. In other words, OTSSP167 significantly suppressed ATL proliferation in a dose-dependent manner, even with respect to the size of the excised tumor.

[0121] Figure 5E shows the time course of weight changes in the ATN-1 transplant model. In the control group, weight increased rapidly with tumor growth. Repeated measures ANOVA was used to compare the three groups, with P<0.001. Post-hoc tests were performed using the Bonferroni method to compare two of the three groups. From day 21 onward, the control group had significantly heavier weight compared to the OTSSP167 10 mg / kg and OTSSP167 20 mg / kg groups. In the figure, "**" indicates P<0.01 and "***" indicates P<0.001. In other words, weight (BW) increased excessively with tumor growth in the control group, while weight remained stable in the OTSSP167 groups.

[0122] Figure 6A shows a comparison of OTSSP167 alone, used outside of the animal experiments in this example, and OTSSP167 hydrochloride, used in the animal experiments. Using the MT-2 cell line, OTSSP167 and OTSSP167 hydrochloride were reacted, and the cell viability (%) after 48 hours was compared. This was because OTSSP167 hydrochloride has high solubility in DMSO, and it was thought that this could mitigate the toxicity of DMSO in the animal experiments. The in vitro growth inhibitory effect on the MT-2 cell line was comparable between OTSSP167 and OTSSP167 hydrochloride.

[0123] Figure 6B shows the protocol for the MT-2 transplantation model. To evaluate the long-term clinical course, another in vivo experiment was performed using a luciferase-expressing MT-2 strain. In this experiment, MT-2 cells expressing luciferase were subcutaneously transplanted into 15 NOG mice, and engraftment was confirmed on day 7. From day 7 to day 38, OTSSP167 or a solvent control (vehicle control, 10% DMSO + 90% D-PBS) was administered intravenously three times a week for a total of 15 times. The mice were then observed until day 70 without further intervention. Specifically, for tumor evaluation, IVIS was used until day 39, and CT was used thereafter. This is because, generally, the luminescence signal is attenuated when passing through ulcerative lesions, making it difficult to monitor tumor volume by luciferase activity after the appearance of ulcerative lesions.

[0124] Figure 6C shows the results of luminescence imaging of the MT-2 transplantation model. It shows the tumor appearance on day 7 (before administration) and on day 38 (after administration). Compared to the control group, the OTSSP167 10 mg / kg and OTSSP167 20 mg / kg administration groups showed a tendency towards suppression of MT-2 proliferation.

[0125] Figure 6D shows the time-series evaluation of ROI for each group. Repeated measures ANOVA was used for comparisons between the three groups. The result was P<0.001. Post-hoc tests using the Bonferroni method were performed for comparisons between two groups. The result "*" indicates P<0.05.

[0126] Figure 6E(a) shows the timing of the appearance of ulcerative lesions in the MT-2 transplantation model. Specifically, it shows the cumulative incidence of skin ulcers after subcutaneous administration of MT2 in an animal model (n=5×3 groups). The horizontal axis represents the number of days after subcutaneous administration of MT2, and the vertical axis represents the percentage of mice that developed skin ulcers (skin lesions). In this example, ulcerative lesions were gradually observed as skin lesions at the tumor site from day 28 after MT-2 cell administration. The cumulative incidence of skin ulcerative lesions was compared using the log-rank test, with P<0.001. A comparison between the two groups was also performed using the Bonferroni method. The median time to the appearance of ulcerative lesions was 35 days in the control group, 45 days in the OTSSP167 10 mg / kg group, and 59 days in the OTSSP167 20 mg / kg group. In the figure, "*" indicates P<0.05 and "**" indicates P<0.01.

[0127] Figure 6E(b) shows the macroscopic findings of the ulcerative lesions that actually occurred. In the MT-2 transplant model, ulcerative lesions were observed in addition to nodular lesions as the disease worsened.

[0128] As a result, skin ulcer formation after subcutaneous administration of the MT2 strain appeared earliest in the control group (0 mg / kg), while no ulcers were observed in the 20 mg / kg group during OTSSP167 administration (15 times in total). Therefore, as mentioned above, since tumor volume cannot be accurately assessed using only the luminescence signal, we decided to evaluate the tumor volume by calculating it using CT.

[0129] Figure 6F shows the method for measuring tumor diameter in CT evaluation of the MT-2 transplant model. From day 38 onwards, the width, length, and height of the tumor diameter were evaluated using CT. The tumor volume was calculated using the formula 4 / 3π × (width / 2) × (length / 2) × (height / 2).

[0130] Figure 6G shows the time-course evaluation of tumor volume in each group. Compared to the control group, the OTSSP167 10 mg / kg and OTSSP167 20 mg / kg groups showed dose-dependent suppression of MT-2 proliferation. Similar to experiments using ATN-1 cells, OTSSP167 suppressed MT-2 cell proliferation in a dose-dependent manner. The statistical test result was P<0.001.

[0131] As a result, when the tumor volume of the MT2 strain was evaluated and measured using IVIS and CT, similar results were obtained as with the ATN-1 strain. In other words, OTSSP167 suppressed the proliferation of ATL even in xenograft models.

[0132] Next, we conducted an in vivo toxicity evaluation test of OTSSP167.

[0133] Figure 7A shows the toxicity assessment protocol. NOG mice that had not received ATL cell transplants were intravenously administered either OTSSP167 or a solvent control (vehicle control, 10% DMSO + 90% D-PBS solvent control) three times a week for a total of 15 doses from day 1 to day 32. Blood tests were performed on day 36.

[0134] Figure 7B shows the time course of body weight in the toxicity evaluation study. No significant difference in weight loss was observed among the control group, the OTSSP167 10 mg / kg group, and the OTSSP167 20 mg / kg group. Repeated measures ANOVA was used to compare the three groups. P=0.55.

[0135] Figure 7C shows the results of the hematological toxicity assessment of OTSSP167. OTSS, P167, and a vehicle control (10% DMSO + 90% D-PBS) were administered a total of 15 times from Day 1 to Day 32, and blood tests were performed on Day 36. White blood cell count, hemoglobin level, and platelet count were measured. No significant hematological toxicity was observed between the control group, the OTSSP167 10 mg / kg group, and the OTSSP167 20 mg / kg group. However, a slight decrease in hemoglobin levels was observed as the OTSSP167 dose increased. ANOVA was used for comparison between the three groups. White blood cell count was P=0.33, hemoglobin level was P=0.081, and platelet count was P=0.55.

[0136] (Effects of MELK knockdown and knockout on ATL cell proliferation) OTSSP167 is known as a MELK target inhibitor. Therefore, we investigated whether OTSSP167 inhibits ATL cell proliferation through the MELK pathway by knocking down MELK with siRNA or knocking it out with CRISPR / Cas9 (hereinafter also referred to as "KO"). Here, cells were transfected with MELK-targeting siRNA using electroporation with the Neon Transfection System (Thermo Scientific).

[0137] Figure 8A shows the effects of MELK knockdown in ATL cell lines. The upper column shows the expression levels of the MELK gene in MT-2, MT-4, TL-Su, and ATN-1 cell lines. In all cases, MELK expression was significantly reduced after knockdown. In the figure, "*" indicates P<0.05 and "**" indicates P<0.01. The lower column shows the survival rates of each cell line. No significant decrease in survival rate was observed after MELK knockdown.

[0138] Figure 8B shows protein levels in MT-2, MT-2, MT-4, TL-Su, and ATN-1 cell lines. "wt" indicates cells without knockdown, "KD" indicates cells with knockdown, the "MELK" image shows the detected MELK band, and "ACTB" shows the control β-actin band. MELK knockdown resulted in a definite decrease in MELK protein.

[0139] These results indicate that reducing the MELK protein through MELK knockdown does not inhibit the survival rate of ATL cells. In other words, inhibiting the expression of MELK itself does not cause ATL cells to die.

[0140] Figure 8C shows the results of the apoptosis assay. The upper section shows dot plots of flow cytometry analysis of MT-2 cells reacted with 0 nM to 500 nM OTSSP167. UL (upper left) represents apoptotic cells labeled with 7-AAD, LR (lower right) represents apoptotic cells labeled with Annexin V, and UR (upper right) represents apoptotic cells labeled with both. OTSSP167 dose-dependently increased the proportion of apoptotic cells labeled with Annexin V or 7-AAD. The lower section shows dot plots of flow cytometry analysis of ATL cell lines with MELK knocked down. Almost no apoptotic cells were observed that were labeled with Annexin V or the 7-AAD fraction.

[0141] Thus, flow cytometry analysis of MT-2 cells subjected to apoptosis assays revealed that most MT-2 cells after MELK knockdown were not labeled with annexin V and 7-AAD; in other words, they did not die by apoptosis. On the other hand, MT-2 cells treated with OTSSP167 were sufficiently labeled with annexin V and 7-AAD, and the proportion of labeled apoptotic cells increased in a dose-dependent manner.

[0142] Next, to determine whether MELK is essential for ATL proliferation, we created an ATN-1 cell line lacking the MELK protein (hereinafter referred to as "MELK-KO ATN-1 cells") using the CRISPR / Cas9 system.

[0143] Figure 8D shows the structure of the MELK gene targeted by the gRNA in the establishment of MELK-KO ATN-1 cells. MELK was knocked out using gRNAs targeting exons 5 and 13. The MELK gene was knocked out using the CRISPR / Cas9 system, and clones were expanded from single cells.

[0144] Figure 8E shows the changes in MELK expression levels in MELK-KO ATN-1 cells. Quantitative RT-PCR measurements of MELK-KO ATN-1 cells revealed a significant decrease in MELK gene expression (P<0.05).

[0145] Figure 8F shows the results of a Western blot illustrating the levels of MELK protein. "wt" indicates results from ATN-1 cells, and "KO" indicates results from MELK-KO ATN-1 cells in which MELK was knocked out. The "MELK" image shows the band of detected MELK, and "ACTB" shows the band of β-actin from the control. The MELK protein was deleted in the MELK-KO ATN-1 cells. In other words, efficient knockout of MELK was demonstrated by quantitative RT-PCR and Western blot analysis.

[0146] Figure 8G shows a comparison of the proliferation rates of normal ATN-1 cells and MELK-KO ATN-1 cells. Cell counts were counted every 72 to 96 hours to compare proliferation. The proliferation rate of MELK-KO ATN-1 cells was comparable to that of ATN-1 cells.

[0147] As a result, neither MELK knockdown nor MELK knockout inhibited the proliferation of ATL cells.

[0148] Figure 8H is a graph showing the sensitivity of MELK-KO ATN-1 cells to OTSSP167. The horizontal axis represents the concentration of OTSSP167, and the vertical axis represents cell viability (%). Thus, despite the absence of MELK, MELK-KO ATN-1 cells showed sensitivity to the MELK inhibitor OTSSP167.

[0149] These results indicated that OTSSP167 exerted its anti-ATL effect independently of MELK inhibition. In other words, the effect of OTSSP167 on ATL cells was shown to be based on a mechanism other than MELK inhibition.

[0150] (Integrated analysis of RNA sequencing) Next, an integrated RNA sequencing (RNA-seq) analysis was performed again with the aim of identifying clinically targeted genes by OTSSP167 administration to ATL cells. In this analysis, clinically targeted genes were defined not only as genes that were elevated in actual ATL patients compared to normal donors, but also as genes that were decreased by OTSSP167 administration to ATL cell lines.

[0151] First, we analyzed an abnormal subpopulation of CD4+CADM1+ T cells from 15 ATL patients, similar to Figure 1 above, and normal CD4+ T cells from 11 healthy donors, and compared their gene expression profiles (GEPs).

[0152] Figure 9A shows an example of cell sorting of samples from ATL patients. CD4+CD14-CD19-CADM1+ fractions were sorted from PBMCs or lymph nodes collected from 15 ATL patients and used for RNA sequencing analysis.

[0153] Figure 9B shows a cluster of genes with altered expression between ATL patients and healthy individuals. For 15 ATL patients, CD4+CADM1+ T cells were used (12 acute-type and 3 chronic-type). Data from lymph nodes was used for ATL04, and peripheral blood for the others. Peripheral blood data from 11 healthy donors was also used. Figure 9B shows the top 100 differentially expressed genes (DEGs) in these CD4+ T cells. The ranking of gene expression alterations was determined based on the false discovery rate (FDR).

[0154] Figure 9C shows a pie chart illustrating the increase or decrease in gene expression in ATL patient CD4+CADM1+ T cells compared to healthy donor CD4+ T cells. Of the 15,811 genes that could be analyzed, 2,526 (16.0%) were significantly elevated in ATL patient cells with an FDR < 0.05. Further investigation revealed increased expression of immune checkpoint molecules such as CTLA-4 and TIGIT in abnormal CD4+CADM1+ T cells from ATL patients.

[0155] Figure 9D shows the protein-protein interaction (PPI) network of genes whose expression was elevated in ATL among the top 100 DEGs. The intensity of the color indicates Log2 fold change; the darker the color, the stronger the expression in ATL cells compared to healthy donor CD4+ T cells. In ATL patients, the expression of CTLA4 and TIGIT, which act to suppress T cells in response to activation of the TCR / NF-κB pathway, was elevated. Along with this, the expression of EZH2 and other genes, which are thought to suppress the expression of tumor suppressor genes by trimethylating histone H3K27, was also elevated. This indicates that epigenetic changes also contribute to the proliferation of ATL. Among these genes whose expression was elevated in ATL cells is SAP30, which was the focus of this example.

[0156] Figure 9E shows the results of gene ontology (GO) analysis of immune system processes in genes whose expression was elevated in ATL among the top 100 DEGs. In ATL cells, genes involved in "leukocyte differentiation," "regulation of leukocyte activation," and "positive regulation of the immune response" were highly expressed, and CTLA4 and EZH2 commonly influenced these pathways.

[0157] Thus, RNA sequencing analysis revealed up-expression genes in ATL cells. In these processes, CTLA4 and EZH2 were shared in the GO term. Furthermore, in ATL patients, it is thought that CTLA4 enhances CD28 signaling, and that the activation of CD28, a co-stimulatory molecule, along with T cell receptor (TCR)-NF-κB signaling, enhances EZH2 expression.

[0158] Next, we compared gene expression changes between ATL cell lines treated with OTSSP167 and untreated ATL cell lines.

[0159] Figure 10A shows the top 50 genes whose expression levels changed after adding OTSSP167 to the ATL cell line. The ranking of gene expression changes was determined based on FDR.

[0160] Figure 10B shows pie charts illustrating the increase and decrease in gene expression between ATL cell lines treated with and without OTSSP167. Of the 7484 genes that could be analyzed, the addition of OTSSP167 significantly reduced the expression of 540 genes (7.2%), with an FDR < 0.05.

[0161] Figure 10C shows the PPI network of genes whose expression decreased after the addition of OTSSP167, among the top 200 DEGs. The intensity of the color indicates the Log2 fold change. Darker colors indicate a greater decrease in expression after OTSSP167 exposure (administration).

[0162] Figure 10D shows the results of GO analysis on the molecular function of genes whose expression was reduced after OTSSP167 exposure (administration) among the top 400 DEGs. Various functions, including multiple kinase activities and nucleotide binding, were suppressed. This suggests that OTSSP167 has a wide range of effects.

[0163] As a result, molecular functional analysis of the PPI network and GO enrichment revealed that OTSSP167 inhibits various pathways, including several kinase activities and nucleotide binding.

[0164] Next, we examined in detail the genes that showed increased expression in ATL patients compared to healthy donors, and the genes that showed decreased expression after OTSSP167 treatment.

[0165] Figure 11A is a scatter plot of genes with significantly altered expression, created based on the results of the integrated RNA sequencing analysis described above. The upper part of the graph indicates increased expression in ATL patients, and the rightward part indicates decreased expression after treatment with OTSSP167. The gray area in the upper right represents genes whose expression was increased in ATL patient cells compared to healthy donor cells, and whose expression was decreased after adding OTSSP167 to the ATL cell line.

[0166] Figure 11B shows the results of GO analysis on the cellular composition categories of genes that were upexpressed in ATL patients and downexpressed after OTSSP167 exposure. Here, it was found that genes constituting the "mitochondrial inner membrane," "cyclin-dependent protein kinase complex," and "Sin3 complex" were the constituent elements.

[0167] Therefore, in order to further narrow down the candidates, we individually selected the top 10 upregulated genes in ATL patients and the top 10 genes that were downregulated after OTSSP167 exposure.

[0168] Figure 11C shows the top 10 genes whose expression was upregulated in ATL and the top 10 genes whose expression was downregulated after OTSSP167 exposure. The ranking of expression changes was determined based on FDR. According to this, only the SAP30 gene was included in both groups.

[0169] In conclusion, it was considered that the anti-ATL effect of OTSSP167 is primarily induced by inhibiting the expression of SAP30. In fact, as shown in Figures 4A and 4B above, knockdown of SAP30 in ATL cell lines effectively inhibited the proliferation or survival rate of ATL cells.

[0170] (Combination therapy of cell therapy and OTSSP167 administration) The inventors had been developing Tax-restricted T-cell immunotherapy (hereinafter referred to as "cell therapy") for ATL as described in Non-Patent Literature 1. Therefore, as an alternative treatment strategy, a combination therapy of OTSSP167 and cell therapy was performed. For this purpose, Tax-siCTL, a Tax-specific cytotoxic T cell described in Non-Patent Literature 1, was used.

[0171] Figure 12A shows the results of measuring tumor volume by luminescence imaging using IVIS for ATN-1 transplanted mice administered with Tax-siCTL and ATN-1 transplanted mice administered with Tax-siCTL and OTSSP167 at 20 mg / kg. The dark color represents the group that received only Tax-siCTL after transplantation of a low-Tax-expressing ATN-1 strain (Tax-siCTL group, n=6), and the light color represents the group that received OTSSP167 at 20 mg / kg five times after ATN-1 transplantation before receiving Tax-siCTL (OTSSP167+Tax-siCTL group, n=5). The horizontal axis represents the number of days elapsed, and the vertical axis represents the tumor volume calculated by IVIS.

[0172] At the start of the experiment (Day 0), ATN-1 was subcutaneously transplanted (n=12), and engraftment was confirmed in n=11 patients on day 7. On day 11, Tax-siCTL was administered to the Tax-siCTL group. The OTSSP167+Tax-siCTL group was administered OTSSP167 from day 11 to day 19. The OTSSP167+Tax-siCTL group was administered Tax-siCTL on day 21. Follow-up observations were conducted thereafter. Due to unforeseen accidents, etc., ultimately, n=3 patients survived in the Tax-siCTL group and n=6 patients survived in the OTSSP167+Tax-siCTL group.

[0173] As a result, the combination of Tax-siCTL and OTSSP167 demonstrated efficacy against the ATN-1 strain, and no recurrence was observed during the observation period.

[0174] Figure 12B shows the results of a killing assay targeting ATN-1 strain ± OTSSP167. As an immune cell killing assay (cancer immunotherapy), ATN-1 strain transplanted mice were given Tax-siCTL, and cytotoxicity (%) was measured at 4 hours (4h) and 6 hours (6h) using fluorescence imaging with Terascan (Minervatec Co., Ltd.). Sample 1 was administered (exposed) to 200 nM of OTSSP167, while Sample 2 is a control without OTSSP167 administration (exposure). At an E / T ratio of 30, the cytotoxicity rate was approximately 30% when OTSSP167 was used in combination.

[0175] Figure 12C shows the results measured at 8 hours (8h) and 24 hours (24h) in the same killing assay as in Figure 12B. At an E / T ratio of 30, the cytotoxicity rate exceeded 30% in both cases when OTSSP167 was used in combination.

[0176] As a result, combining cell therapy with OTSSP167 increased the effectiveness against ATL.

[0177] Next, to investigate the effect of OTSSP167 on the presence or absence of Tax expression, immunohistochemical staining for Tax was performed on tissues of ATN-1 transplanted NOG mice after 12 doses of OTSSP167.

[0178] The upper panel of Figure 12D shows the results of immunostaining of NOG mice transplanted with MT-2, an ATL cell line that highly expresses Tax, without administration of OTSSP167; this is a positive control. The lower panel shows the results of immunostaining of ATN-1, which has low Tax expression; this is a negative control. The results of nuclear staining with Tax antibody and DAPI (4',6-diamidino-2-phenylindole), and the results of merging these stains are shown, respectively. In all figures, the grayscale is inverted to improve visibility. The same applies to the figures below.

[0179] The upper panel of Figure 12E shows the results of immunohistochemical staining for Tax, DAPI, and Merge in MT-2 transplanted mice and the lower panel shows the results in ATN-1 transplanted mice, both administered with OTSSP167 at 10 mg / kg. These results were performed in the same manner as in Figure 12D. Even in ATN-1, which has low Tax expression, OTSSP167 enabled tumors to stain with Tax antibodies. This suggests that OTSSP167 can promote Tax expression and enhance the efficacy of Tax-targeted cell therapies.

[0180] It goes without saying that the configuration and operation of the above embodiment are examples and can be modified as appropriate without departing from the spirit of the present invention. [Industrial applicability]

[0181] According to the present invention, a selective inhibitor of the SAP30 gene can be provided as a pharmaceutical composition used for the treatment of ATL, and is industrially applicable.

Claims

1. A pharmaceutical composition for the treatment of adult T-cell leukemia, The aforementioned selective inhibitor includes a selective inhibitor that selectively inhibits the expression of hub genes selected based on protein-protein interactions among genes that are highly expressed in cancer cells of adult T-cell leukemia patients compared to T cells of healthy individuals. A pharmaceutical composition for adult T-cell leukemia characterized by the following features.

2. The aforementioned hub gene is the SAP30 gene. The pharmaceutical composition for adult T-cell leukemia according to feature 1.

3. The selective inhibitor comprises either SAP30 siRNA or OTSSP167. The pharmaceutical composition for adult T-cell leukemia according to feature 2.

4. Used in combination with pharmaceutical compositions for Tax-targeted T-cell immunotherapy. The pharmaceutical composition for adult T-cell leukemia according to feature 1.

5. A method for selecting a pharmaceutical composition for the treatment of adult T-cell leukemia, In cancer cells from the aforementioned adult T-cell leukemia patient, genes with higher expression levels were selected compared to T cells from healthy individuals. From the selected high-expression genes, a hub gene is selected based on protein-protein interactions. Select an inhibitor that selectively inhibits the expression of the selected hub gene. A method for selecting a pharmaceutical composition characterized by the following features.

6. A treatment method for adult T-cell leukemia, In cancer cells of adult T-cell leukemia patients, the expression of hub genes selected based on protein-protein interactions, which are more highly expressed than in T cells of healthy individuals, is selectively inhibited. A treatment method characterized by the following features.

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