Therapeutic agent for ZFTA-fused ependymoma

Abstract

The present invention provides a pharmaceutical composition for treating ZFTA-fused ependymoma, the pharmaceutical composition comprising a YAP1 inhibitor.

Description

Therapeutic drug for ZFTA fusion ependymoma

[0001] The present invention relates to a therapeutic agent for ZFTA-fusion ependymoma.

[0002] Ependymomas are thought to be tumors of the ependymal cells that line the surface of the ventricles, and occur in the cerebral hemispheres, cerebellum, brainstem, and spinal cord. They account for 4-6% of primary pediatric brain tumors (ages 0-19), and are known to be intractable. Traditionally, like other brain tumors, ependymomas have been classified by their location and appearance (pathological analysis). However, they can now be broadly divided into supratentorial ependymomas, which occur on the cerebral side, and infratentorial ependymomas, which occur in the cerebellum or brainstem, across the thick dura mater (tentorium) that separates the cerebrum and cerebellum. Recently, detailed analysis of DNA methylation status and gene expression has led to classification of ependymomas into nine groups: ZFTA (Zinc Finger Translation Associated) fusion gene-positive supratentorial ependymomas, YAP1 fusion gene-positive supratentorial ependymomas, other ependymomas, posterior fossa ependymomas group A, posterior fossa ependymomas group B, other posterior fossa ependymomas, spinal ependymomas, MYCN-amplified spinal ependymomas, myxopapillary ependymomas, and subependymomas.

[0003] RELA-type ependymomas account for approximately 70% of supratentorial ependymomas and are a highly malignant group. In the tumor area of ​​RELA-type ependymomas, a gene in which the RELA gene and the ZFTA gene are fused (also called the ZFTA-RELA fusion gene) has been frequently observed. Furthermore, recent research has reported that the ZFTA-RELA fusion gene has carcinogenic activity, and the 2016 WHO pathological classification established a category called "RELA fusion-positive ependymomas."

[0004] However, even when a patient is diagnosed with RELA fusion-positive ependymoma based on DNA methylation status analysis, the RELA gene is not always positive, suggesting the existence of an unknown carcinogenic mechanism. The present inventors conducted further research on brain tumor specimens in which the DNA methylation diagnosis and RELA gene expression results were inconsistent. As a result, in addition to the ZFTA-RELA fusion gene (Type 1), they discovered new fusion genes, including another ZFTA-RELA fusion gene (Type 2), a ZFTA-RELA fusion gene (Type 8), ZFTA-MAML2, ZFTA-MAML3, ZFTA-NCOA1, and ZFTA-NCOA2 (see Non-Patent Document 2). These genes were called "ZFTA fusion genes" because they commonly consist of a gene sequence in which a zinc finger DNA-binding domain and a transcriptional activation domain are fused together. Furthermore, in experiments on model animals using ZFTA fusion proteins lacking the zinc finger DNA-binding domain of these ZFTA fusion genes, no tumor formation was observed (see Non-Patent Document 4).

[0005] PajtlerK.W. et al. Cancer Cell 2015, 27, 728-743.Zhenget al. Cancer Discov. 2021; 11: 2230-47.LarrewT. et al. Cancers 2021, 13, 6218.Kupp R.et al. Cancer Discov. 2021; 11: 2216-29.Mack, SC et al. Nature 553, 101-105(2018)Knott, MML et al. Cancer Metastasis Rev. 2019, 38:625-642.

[0006] Because effective chemotherapy has not been established, surgery or radiation therapy is mainly used to treat ependymoma. Therefore, the prognosis of ependymoma is easily influenced by the results of surgical procedures. Furthermore, most supratentorial ependymomas are classified as YAP1 fusion ependymomas or ZFTA fusion ependymomas. While YAP1 inhibitors have been suggested to be effective against YAP1 fusion ependymomas, no effective chemotherapeutic agents are known for patients with ZFTA fusion ependymoma, and the prognosis is often poor. Therefore, an object of the present invention is to provide a new therapeutic agent for ZFTA fusion ependymoma.

[0007] The present invention provides the following [1] to

[15] . [1] A pharmaceutical composition for treating ZFTA-fusion ependymoma, comprising a YAP1 inhibitor. [2] The pharmaceutical composition according to [1], wherein the YAP1 inhibitor comprises verteporfin. [3] The pharmaceutical composition according to [1] or [2], wherein the ZFTA-fusion ependymoma is a ZFTA-fusion ependymoma accompanied by expression of a ZFTA-fusion gene comprising the nucleotide sequence represented by SEQ ID NO: 1. [4] Use of a YAP1 inhibitor for treating ZFTA-fusion ependymoma. [5] The use according to [4], wherein the YAP1 inhibitor comprises verteporfin. [6] The use according to [4] or [5], wherein the ZFTA-fusion ependymoma is a ZFTA-fusion ependymoma accompanied by expression of a ZFTA-fusion gene comprising the nucleotide sequence represented by SEQ ID NO: 1. [7] A method for treating ZFTA-fusion ependymoma, comprising administering a YAP1 inhibitor to a subject diagnosed as having ZFTA-fusion ependymoma. [8] The method according to [7], wherein the YAP1 inhibitor comprises verteporfin. [9] The method according to [7] or [8], wherein the ZFTA-fusion ependymoma is a ZFTA-fusion ependymoma accompanied by expression of a ZFTA fusion gene comprising the nucleotide sequence represented by SEQ ID NO: 1.

[10] A YAP1 inhibitor for use in treating ZFTA-fusion ependymoma.

[11] The YAP1 inhibitor according to

[10] , wherein the YAP1 inhibitor comprises verteporfin.

[12] The YAP1 inhibitor according to

[10] or

[11] , wherein the ZFTA-fusion ependymoma is a ZFTA-fusion ependymoma accompanied by expression of a ZFTA fusion gene comprising the nucleotide sequence represented by SEQ ID NO: 1.

[13] Use of a YAP1 inhibitor for the manufacture of a medicament for treating ZFTA-fusion ependymoma.

[14] The use according to

[13] , wherein the YAP1 inhibitor comprises verteporfin.

[15] The use according to

[13] or

[14] , wherein the ZFTA-fusion ependymoma is a ZFTA-fusion ependymoma accompanied by expression of a ZFTA fusion gene comprising the nucleotide sequence represented by SEQ ID NO: 1.

[0008] According to the present invention, a new therapeutic agent for ZFTA fusion-type ependymoma can be provided, and a new chemotherapy option can be provided in addition to surgery and radiation therapy.

[0009] (a) is a graph showing changes in EP1NS gene expression using YAP1 shRNA, and (b) is a photograph showing bands after electrophoresis. This is a graph showing the viable cell ratio in the presence of verteporfin, dioscin, and ponatinib. This is a micrograph showing Ki67-positive nuclei and cleaved caspase 3-positive nuclei in the presence of verteporfin, and a graph showing their ratio to the total cell number. This is a graph showing the results of ChIP-seq analysis of YAP1 in human supratentorial ependymoma.

[0010] Each embodiment of the present invention will be described below.

[0011] A first embodiment of the present invention is a pharmaceutical composition for treating ZFTA-fusion ependymoma, comprising a YAP1 inhibitor. The YAP1 inhibitor is contained as an active ingredient in the pharmaceutical composition.

[0012] YAP1 (yes-associated protein 1) is a protein that functions as a transcription factor, activating the transcription of genes involved in cell proliferation and suppressing the transcription of genes involved in apoptosis. YAP1 expression is regulated by mechanical signals such as extracellular matrix stiffness, tensile stress, shear stress, and adhesive surfaces, and is dependent on the integrity of the cytoskeleton. YAP1 is also inhibited by the Hippo signaling pathway. When the Hippo signaling pathway is not activated, YAP1 / TAZ translocates into the cell nucleus and regulates the expression of genes that regulate cell proliferation and apoptosis.

[0013] As used herein, the term "YAP1 inhibitor" refers to, for example, a substance that can suppress the expression of the YAP1 gene at the transcriptional or translational level, or a substance that can bind to a functional site of YAP1 to suppress functional expression, and is preferably a substance that exhibits a cell proliferation inhibitory effect at a concentration of 1 mM. Inhibiting the functional expression of YAP1 includes inhibiting the phosphorylation of YAP1, inhibiting the nuclear translocation of YAP1, and inhibiting the interaction of YAP1 with the transcription factor TEAD.

[0014] Examples of substances capable of suppressing expression of the YAP1 gene at the transcriptional or translational level include nucleic acids that suppress YAP1 gene expression, peptides, sugars or glycoproteins, and low-molecular-weight compounds with a molecular weight of 1,000 or less. Examples of nucleic acids that suppress YAP1 gene expression include at least one selected from the group consisting of antisense oligonucleotides against the YAP1 gene, siRNA, shRNA, miRNA, and ribozymes.

[0015] Examples of substances capable of binding to a functional site of the YAP1 protein and suppressing its functional expression include anti-YAP1 antibodies or antigen-binding fragments thereof (e.g., neutralizing antibodies), anti-TAZ antibodies or antigen-binding fragments thereof, anti-YAP / TAZ antibodies or antigen-binding fragments thereof, and anti-TEAD antibodies or antigen-binding fragments thereof. These antibodies may be monoclonal or polyclonal. These antibodies may be mouse, rat, guinea pig, hamster, rabbit, monkey, dog, chimeric, humanized, or human antibodies. Specific examples of antibodies include YAP rabbit antibodies, YAP / TAZ rabbit antibodies, YAP mouse antibodies, TAZ rabbit antibodies, TAZ mouse antibodies, and TEAD1 rabbit antibodies.

[0016] Expression inhibitors that suppress the expression of the YAP1 gene can be designed and produced by methods known in the art based on information such as the genomic sequence, mRNA sequence, protein sequence, and three-dimensional structure of the protein of the YAP1 gene.

[0017] Examples of YAP1 inhibitors include (4RS,4aSR)-4,4a-dihydro-3,4-methoxycarbonyl-9-(2-methoxycarbonylethyl)-4a,8,14,19-tetramethyl-18-vinyl-23H,25H-benzo[b]porphine-13-propionic acid, (4RS,4aSR)-4,4a-dihydro-3,4-methoxycarbonyl-13-(2-methoxycarbonylethyl)-4a,8,14,19-tetramethyl-18-vinyl-23H,25H-benzo[b]porphine-9-propionic acid (verteporfin is a mixture of these), AICAR (acadesine), cytochalasin D (zygosporin A), TRULI (Lats-IN-1), K-975, VT104, VT107, and VT3989. The YAP1 inhibitor may also be a compound described in WO 2017 / 053706, WO 2018 / 185266, WO 2020 / 081572, WO 2020 / 243423, WO 2022 / 072741, WO 2022 / 159986, WO 2023 / 060227, or WO 2023 / 114984.

[0018] The pharmaceutical composition according to this embodiment may consist solely of the active ingredient, or may contain additives commonly used in the pharmaceutical technology field, such as excipients, buffers, stabilizers, antioxidants, binders, disintegrants, fillers, emulsifiers, and flow additives, in addition to the active ingredient.

[0019] The dosage form of the pharmaceutical composition according to this embodiment may be any dosage form such as powder, pills, granules, tablets, syrup, lozenges, capsules, and injections.

[0020] The pharmaceutical composition according to the present embodiment may be administered orally or parenterally. As an example of a specific dosage, when administered to a human adult male (body weight 60 kg), the daily dosage of the pharmaceutical composition according to the present embodiment is typically 0.0001 pg to 10,000 mg / day / person in terms of the amount of the active ingredient.

[0021] A second embodiment of the present invention is a method for treating ZFTA-fusion ependymoma, comprising administering a YAP1 inhibitor to a subject determined to be suffering from ZFTA-fusion ependymoma.

[0022] As used herein, the term "subject diagnosed as suffering from ZFTA fusion ependymoma" refers to a human subject who has been found to have a ZFTA fusion gene (a gene in which a zinc finger DNA-binding domain and a transcriptional activation domain are fused) among human subjects suffering from ependymoma. The zinc finger DNA-binding domain commonly has the nucleotide sequence set forth in SEQ ID NO: 1. Known ZFTA fusion genes include, for example, the ZFTA-RELA fusion gene (Type 1), the ZFTA-RELA fusion gene (Type 2), the ZFTA-RELA fusion gene (Type 8), ZFTA-MAML2, ZFTA-MAML3, ZFTA-NCOA1, and ZFTA-NCOA2.

[0023] For example, a human subject may be diagnosed as having ZFTA fusion ependymoma by genetically analyzing blood, cerebrospinal fluid, tumor cell, or other samples collected from the subject. If the expression level of the ZFTA fusion gene is significantly higher than that of a sample collected from a human subject who is not diagnosed with ZFTA fusion ependymoma, the human subject may be diagnosed as having ZFTA fusion ependymoma. Alternatively, a human subject may be diagnosed as having ZFTA fusion ependymoma if the expression level of the ZFTA fusion gene in the sample collected from the human subject is higher than a predetermined threshold. Such a threshold may be determined based on the expression level in a sample collected from a healthy adult, or may be determined based on the expression level in a sample collected from the human subject at a time when the subject was not at risk of having ZFTA fusion ependymoma.

[0024] The YAP1 inhibitor can be defined as in the first embodiment.

[0025] 1. Preparation of Human Ependymoma Cell Line (EP1NS Cells) The EP1NS human ependymoma cell line expresses the ZFTA-RELA fusion gene and exhibits a methylation status similar to that of the primary tumor. The EP1NS human ependymoma cell line was prepared and maintained in culture as described previously (Acta Neuropathol. 2011 November; 122(5): 637-650.). Specifically, primary tumor cells were isolated from the malignant ascites of a patient with supratentorial anaplastic ependymoma corresponding to WHO grade III and cultured in neurosphere medium (NSM). Neurospheres formed after 3 weeks of culture. The neurospheres were dissociated every 7–10 days. The resulting DKFZ-EP1NS cells were confirmed to be free of mycoplasma, viruses, and cellular contamination. The DKFZ-EP1NS cells were frozen and stored in liquid nitrogen using cryopreservation medium. DKFZ-EP1NS cells were cultured for up to 9 months, corresponding to more than 30 passages. Dissociated neurospheres gave rise to new neurospheres, demonstrating long-term self-renewal. Long-term self-renewing rosette-type human embryonic stem cell (hESC)-derived neural stem cells (NSCs) were cultured. DKFZ-EP1NS cells were frozen and thawed in cryopreservation medium without losing their self-renewal capacity.

[0026] 2. In vitro screening: The resulting EP1NS human ependymoma cell line was cultured in 96-well plates and individually administered drugs from a commercially available FDA-approved drug library (product name: FDA-approved Drug Library L1300, obtained from Selleckchem.com) in culture. Drugs effective at a minimum concentration of 1 mM were identified. Cell metabolism was quantified by luminescence using Cell TiterGlo 72 hours after drug administration. The drug library contains 1,194 drugs, including transmembrane transporter inhibitors, stem cell and Wnt signaling inhibitors, tyrosine kinase inhibitors, protease inhibitors, PI3K / Akt / mTOR signaling inhibitors, NF-κB inhibitors, neural signaling inhibitors, MAPK inhibitors, JAK / STAT inhibitors, immune and inflammatory therapeutic agents, GPCR and G protein inhibitors, gene expression therapeutic agents, endocrine and hormonal therapeutic agents, DNA damage therapeutic agents, cytoskeletal signaling inhibitors, cell cycle inhibitors, autophagy inhibitors, apoptosis inhibitors, and angiogenesis inhibitors.

[0027] Normal neural tissue-derived neural stem / progenitor cells (NSPCs) were prepared according to a published literature description (J. Neurosci. Res. 2002 Sep 15;69(6):869-879), and drugs were identified, focusing on those with a growth inhibitory potency of less than 10 μM on NSPCs and EP1NS cells. Large-scale screening revealed that, from a list of drugs that exhibited growth inhibitory effects at low concentrations, inhibitors of the YAP1-mediated Hippo signaling pathway have the potential to suppress the growth of ZFTA-fusion ependymoma.

[0028] 3. Changes in EP1NS Gene Expression Using YAP1 shRNA To verify whether YAP1 inhibition has an antitumor effect on ZFTA-fusion ependymoma, YAP1 shRNA and EdU (5-ethynyl-2'-deoxyuridine) were added to EP1NS cells, cultured, and the amount of EdU incorporation was examined. For the YAP1 shRNA, shYAP1 1699 represented by SEQ ID NO: 2 or shYAP1 1499 represented by SEQ ID NO: 3 was used. shScramble represented by SEQ ID NO: 4 was used as a negative control.

[0029] Figure 1(a) is a graph showing changes in EP1NS gene expression using YAP1 shRNA, and Figure 1(b) is a photograph showing bands after electrophoresis. As shown in Figure 1(a), the addition of YAP1 shRNA significantly reduced the number of EdU-positive cells. These results suggest that inhibiting YAP1 function may be a treatment for ZFTA-fusion ependymoma.

[0030] 4. Antitumor Effects of shRNA Next, based on previous literature and the mechanism of action of the drug, we estimated the proliferation signaling pathway that is effective in EP1NS cells and examined the antitumor effects of shRNA on molecules acting in that signaling pathway. Specifically, we hypothesized the following: When L1CAM binds to the cell surface-expressed protein Shootin1 in the extracellular matrix, integrin is activated and phosphorylates tyrosine 419 (Y419) of SRC. Phosphorylated (pLATS1 / 2) inhibits YAP1 phosphorylation, but phosphorylated SRC (pSRC) inhibits pLATS1 / 2, thereby suppressing YAP1 phosphorylation and allowing YAP1 to translocate into the nucleus and form a complex with TEAD.

[0031] Based on this hypothesis, we evaluated the effects of verteporfin, a YAP1 inhibitor found in the screening, and dioscin and ponatinib, an SRC inhibitor, on EP1NS cell proliferation. Furthermore, since ivermectin has been reported to inhibit AKT1 and CCND1 downstream of YAP1, the importance of YAP1 signal activation is inferred.

[0032] The results are shown in Figure 2. Figure 2 is a graph showing the viable cell ratio in the presence of verteporfin, dioscin, and ponatinib. Verteporfin, a YAP1 inhibitor, exhibited an IC ５０ = 0.51 μM, and the SRC inhibitors Dioscin and Ponatinib each had an IC ５０ = 0.28 μM, 0.86 μM.

[0033] 5. Preparation of a human patient-derived tumor xenograft (PDX) model. 1 × 10 ependymoma cells were extracted from ependymoma specimens excised from human ependymoma patients. ６ Human ependymoma-derived BT165 cells were harvested and a cell suspension was prepared. Within 60 minutes of tumor resection, the cell suspension was injected (transplanted) into the cerebral cortex of adult NSG immunodeficient mice and maintained in vivo. Tumor cells were extracted from individuals showing neurological disease symptoms, isolated, and then the same number of cells were transplanted back into NSG mice for maintenance.

[0034] 6. Verification of tumor atrophy effects in an ependymoma PDX model. YAP1 forms dimers with TEAD1-4 in the nucleus, inducing cancer signaling; verteporfin was found to inhibit this interaction. Verteporfin is known to cross the blood-brain barrier and reach brain tumor cells. Therefore, using an ependymoma PDX model, the antitumor effects of the drugs identified in the above screening were verified and their tumor atrophy effects were analyzed. The drug (verteporfin) was administered to ependymoma PDX model animals, and tumor tissue was collected 7 days after administration. The collected tumor tissue was stained for DNA with DAPI or SC101, immunostained for Ki67 and cleaved caspase 3, and observed under a fluorescence microscope.

[0035] The results are shown in Figure 3. In Figure 3, the upper row represents the vehicle group (VEH), and the lower row represents the drug-administered group. (a) is a micrograph immunostained for Ki67, with DAPI staining, SC101 staining, and a magnified image thereof from the left. (b) is a micrograph immunostained for cleaved caspase-3, with DAPI staining, SC101 staining, and a magnified image thereof from the left. The white arrows in Figure 3 indicate cells stained for cleaved caspase-3. The graph in Figure 3 shows the ratio of Ki67-positive nuclei and cleaved caspase-3-positive nuclei to the total number of cells. As shown in Figure 3, in the drug-administered group, the ratio of Ki67-positive nuclei was significantly reduced, while the ratio of caspase-3-positive nuclei was increased. This confirmed that administration of a YAP1 inhibitor inhibited tumor cell proliferation and significantly increased cell death.

[0036] 7. Binding of AKT1 and CCND1 to the Promoter To analyze which genes the YAP1 gene regulates in human primary ependymoma, we performed chromatin immunoprecipitation (ChIP-seq). Tumor cells collected from patients with ZFTA-fusion ependymoma were formalin-fixed and lysed, and DNA was fragmented by enzymatic digestion. The fragmented DNA was immunoprecipitated using an anti-YAP1 antibody, and the purified DNA was used to examine whether YAP1 binds to gene regulatory regions of downstream genes involved in cell proliferation.

[0037] The results are shown in Figure 4. As shown in Figure 4, strong binding of YAP1 to the CCND1 and AKT1 promoters was observed in both YAP1-fusion-type ependymoma (YAP1-EPN) and ZFTA-fusion-type ependymoma (ZFTA-EPN).

Claims

1. A pharmaceutical composition for treating ZFTA-fusion ependymoma, comprising a YAP1 inhibitor.

2. The pharmaceutical composition of claim 1, wherein the YAP1 inhibitor comprises verteporfin.

3. The pharmaceutical composition according to claim 1 or 2, wherein the ZFTA-fusion ependymoma is a ZFTA-fusion ependymoma accompanied by expression of a ZFTA-fusion gene comprising the nucleotide sequence represented by SEQ ID NO: 1.

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