Compounds targeting TPM1.8 and / or TPM1.9 isomers for prevention and / or treatment of cancer

Abstract

The present invention addresses the problem of providing novel and improved therapeutic compounds for the prevention and / or treatment of cancer, such as ovarian cancer. The present invention provides compounds that target the TPM1 selective splicing variants Tpm1.8 and / or Tpm1.9. The compounds or pharmaceutical compositions comprising at least one compound according to the invention counteract chemotherapeutic resistance, such as chemotherapeutic resistance to taxane and / or platinum based chemotherapeutic agents. Furthermore, the present invention includes methods of identifying compounds targeting Tpm1.8 and / or Tpm1.9 isomers, which compounds have therapeutic effects in inhibiting, suppressing, preventing and / or treating cancer.

Description

Technical Field

[0001] This invention relates to the use of compounds that target the selective splicing isomers TPM1.8 and / or TPM1.9 of tropomyosin 1 (TPM1) in methods for the prevention and / or treatment of cancer. Furthermore, this invention relates to a method for identifying other compounds that target the TPM1.8 and / or TPM1.9 isomers and for use in methods for the prevention and / or treatment of cancer. Background Technology

[0002] Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exhibit different phenotypes upon exposure to specific environmental signals, represents a hallmark of cancer cells' involvement in the process from primary lesions to distant organ sites where metastatic colonization occurs. Phenotypic plasticity is driven by a wide range of epigenetic mechanisms that allow for the reversibility of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET).

[0003] Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are considered the basis for local spread and distant metastasis in most epithelial malignancies, including ovarian cancer, where the lack of a physical barrier between the primary and metastatic sites suggests the possible existence of alternative shedding mechanisms. EMT / MET is regulated by a wide range of epigenetic mechanisms involving chromatin remodeling due to histone methylation / acetylation, non-coding RNA, promoter DNA methylation, and post-transcriptional mechanisms such as alternative splicing (AS).

[0004] Metastasis is the leading cause of cancer death. Metastatic disease, or the movement of cancer cells from one site to another, is a complex process requiring a rapid remodeling of the cytoskeleton. The various components of the cytoskeleton, actin (microfilaments), microtubules (MTs), and intermediate filaments, are highly integrated, and their functions are well coordinated in normal cells.

[0005] Ovarian cancer is the deadliest gynecological cancer, but its early stages often don't show any noticeable symptoms. Most ovarian cancer patients are diagnosed with metastatic disease, and most are treated for metastasis, which is one of the main reasons for the poor prognosis of ovarian cancer. A better understanding of the process of cancer metastasis is needed.

[0006] Tropomyosin, as an actin-binding protein, plays a crucial role in metastatic diseases and is typically downregulated during cell transformation and dedifferentiation in tumor development. Among various epigenetic mechanisms, the tropomyosin atherosclerosis (AS) may play a role in regulating EMT / MET in cancer metastasis. The tropomyosin 1 (TPM1) gene encodes various AS isoforms. The inventors set out to investigate whether alternative splicing isoforms of TPM1 could serve as therapeutic targets for cancer treatment. This invention addresses the problem of providing novel compounds for the prevention and / or treatment of cancers such as ovarian cancer. Summary of the Invention

[0007] Unexpectedly, the inventors of this invention have discovered that two isoforms of the tropomyosin 1 gene (TPM1), Tpm1.8 and Tpm1.9, provide novel and promising targets for the prevention and / or treatment of cancers such as ovarian cancer. Without being bound by theory, it is believed that Tpm1.8 and / or Tpm1.9 represent specific isoforms involved in EMT activation through multiple signal transduction pathways, including Wnt, TGF-β, Hedgehog, and Notch. Furthermore, due to their location on plate-like pseudopodia and their functional role in cell motility, the Tpm1.8 / 9 isoforms may promote the spread from primary tumors to the peritoneal cavity. The examples and figures below particularly illustrate that the Tpm1.8 / 9 isoforms are promising targets for the prevention and / or treatment of cancers such as ovarian cancer. Moreover, compounds of this invention targeting the Tpm1.8 and / or Tpm1.9 isoforms are shown to be useful for the prevention and / or treatment of cancers such as ovarian cancer.

[0008] The inventors further discovered that by using CD44 and EpCAM antibodies for FACS, CD44 in immortalized high-grade serous ovarian cancer (HGSOC) cell lines OV90, SKOV3, COV504, and CAOV3 can be effectively inhibited. 高 EpCAM 低 In cells, RBM4 and ESRP1 are upregulated and downregulated, respectively. This is similar to CD44... 高 EpCAM 低 The cells exhibit a mesenchymal-like morphology, corresponding to CD44. 高 EpCAM 高 The contrast between the epithelial appearance of the cells and CD44 thus... 高 EpCAM 低 Cells exhibited increased migration and invasion capabilities. Furthermore, as verified by RT-qPCR and Western blot analysis, the TPM1 alternative splicing (AS) pattern observed in ovarian cancer involved exons 1a / 2a and 1b, with exon 1a / 2a marking the presence of CD44... 高 EpCAM 高The upregulated Tpm1.6 / 7 isoform in cells, exon 1b is CD44 高 EpCAM 低 The cells were characterized by an upregulated Tpm1.8 / 9 isoform. As further validation of these results, only CD44 was included. 高 EpCAM 高 The HGSOC cell line expressed only the Tpm1.6 / 7 isoform, while CD44... 高 EpCAM 低 Cells expressed only the Tpm1.8 / 9 isoform. Further analysis revealed that alternative splicing, driven by upregulation and downregulation of RBM24 and ESRP1, respectively, is a major regulator of EMT in ovarian cancer cells.

[0009] The compounds of the present invention target residues 4 through 16 of the N-terminus of Tpm1.8 and / or Tpm1.9. The compounds of the present invention targeting the Tpm1.8 and / or Tpm1.9 isomers can counteract chemotherapy resistance, preferably against chemotherapy resistance based on taxanes and / or platinum-based chemotherapy. Furthermore, the compounds of the present invention targeting the Tpm1.8 and / or Tpm1.9 isomers are active in the low micromolar range, preferably in the range of 5 μM to 50 μM, more preferably in the range of 5 μM to 20 μM. The compounds of the present invention targeting the Tpm1.8 and / or Tpm1.9 isomers can prevent the accumulation of Tpm1.8 and / or Tpm1.9 isomers in platypopodia.

[0010] Therefore, in a first aspect, the present invention provides a compound that is an inhibitor of the tropomyosin 1 selective splicing isoforms Tpm1.8 and / or Tpm1.9, wherein the compound is selected from...

[0011] - Small molecule inhibitors selected from 3-(2-methyl-indole-1-yl)-propylamine (PubChem CID 6494468) and 1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468), and their pharmaceutically acceptable salts, hydrates, derivatives, solvates or prodrugs;

[0012] - Antisense polynucleotides that reduce the expression of tropomyosin 1 isoforms 1.8 and / or 1.9 (Tpm1.8 or Tpm1.9), and

[0013] - Antibodies that reduce the levels and / or activity of Tpm1.8 and / or Tpm1.9 peptides.

[0014] In another aspect, the present invention provides the compounds according to the invention as described above, used in methods for preventing and / or treating cancer.

[0015] In a preferred embodiment of the invention, the cancer expresses a TPM1 selective splicing isoform, more preferably a TPM1 selective splicing isoform of exon 1b. Optionally, the cancer is selected from acute myeloid leukemia (AML), adrenocortical carcinoma, urothelial carcinoma of the bladder, low-grade brain carcinoma, invasive breast carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, lymphoid tumors, diffuse large B-cell lymphoma, mesothelioma, ovarian cancer, especially ovarian serous carcinoma, pancreatic adenocarcinoma, rectal adenocarcinoma, sarcoma, skin melanoma, gastric adenocarcinoma, testicular germ cell tumor, thymoma, thyroid cancer, uterine carcinosarcoma, uterine endometrial cancer, uveal melanoma, or combinations thereof. The cancer treated by using the compounds of the present invention is ovarian cancer, preferably epithelial ovarian cancer (EOC), and more preferably high-grade serous ovarian cancer.

[0016] In another embodiment of the invention, the compound is administered in combination with other anticancer therapeutic agents.

[0017] In another embodiment of the invention, the compound is administered in combination with an additional anticancer therapy. Preferably, the additional anticancer therapy is selected from chemotherapy, targeted therapy (e.g., immunotherapy), stem cell therapy, hormone therapy, radiotherapy, and surgery, and combinations thereof. More preferably, the additional anticancer therapy is chemotherapy, such as taxane- and / or platinum-based chemotherapy, such as chemotherapy containing cisplatin and / or paclitaxel.

[0018] In embodiments of the present invention, the compound may be administered to a subject at a dose ranging from 0.1 mg / kg to 100 mg / kg.

[0019] The embodiments relating to the compounds of the present invention and their medical use in treating cancer are also applicable to pharmaceutical compositions comprising the compounds. In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0020] Furthermore, embodiments relating to the use of compounds and pharmaceutical compositions are also applicable to methods of inhibiting, suppressing, preventing, and / or treating cancer, including administering a therapeutically effective amount of the compound or pharmaceutical composition to a subject in need of it.

[0021] In another aspect, the present invention provides a method for identifying compounds targeting Tpm1.8 and / or Tpm1.9 isomers, said compounds being used to inhibit, suppress, prevent, and / or treat cancer proliferation, motility, invasion, migration, metastasis, and / or chemotherapy resistance, the method comprising:

[0022] a) Contacting cancer cells expressing the Tpm1.8 and / or Tpm1.9 isomers with the test compound, and

[0023] b) Measure the inhibition of Tpm1.8 and / or Tpm1.9 expression and / or activity in the cancer cells.

[0024] The discovery of inhibitory effects will identify the tested compounds as compounds for use in methods of inhibiting, suppressing, preventing and / or treating cancer.

[0025] On the other hand, the present invention provides an antisense polynucleotide that reduces the expression of tropomyosin 1 isoforms 1.8 or 1.9 (Tpm1.8 or Tpm1.9), or provides an antibody that reduces the level and / or activity of Tpm1.8 or Tpm1.9 peptides, wherein the antisense polynucleotide or the antibody is used in methods for inhibiting, suppressing, preventing or treating cancer proliferation, movement, invasion, migration, metastasis or chemotherapy resistance.

[0026] In another aspect, the present invention provides a method for inhibiting, suppressing, preventing, or treating the proliferation, movement, invasion, spread, migration, metastasis, or chemotherapy resistance of cancer, the method comprising administering to a subject in need a therapeutically effective amount of an antisense polynucleotide that reduces the expression of tropomyosin 1 isoform 1.8 or 1.9 (Tpm1.8 or Tpm1.9) or an antibody that reduces the level of Tpm1.8 or Tpm1.9 peptides and / or the activity of Tpm1.8 or Tpm1.9 peptides.

[0027] In another aspect, the present invention provides a method for determining the presence of epithelial-mesenchymal transition (EMT) in cancer cells, comprising determining the expression of Tpm1.8 and / or Tpm1.9 in the cells, wherein an increase in expression relative to control expression indicates the presence of EMT in the cells.

[0028] The present invention also provides the use of compounds targeting the Tpm1.8 and / or Tpm1.9 isomers in the preparation of medicaments for the prevention and / or treatment of cancer in the subject.

[0029] The present invention also provides RBM24 and / or ESRP1 and / or Tpm1.8 / 9 as biomarkers for determining cancer development, particularly for determining and / or indicating whether cancer cells (preferably ovarian cancer cells) are in epithelial-mesenchymal transition, whether they are metastatic cells, whether they (are) capable of causing metastasis, or whether they are metastasizing, or whether they are resistant to taxane- and / or platinum-based therapies. Attached Figure Description

[0030] Figure 1RBP ESRP1 and RBM24 synergistically regulate TPM1 selective splicing.

[0031] (A) RT-qPCR (histogram) and Western blot analysis (below) of ESRP1, RBM24, and TPM1 isoform expression in RBM24-OE (overexpression) and shESRP1-KD (knockdown) OV90 and COV504 ovarian cancer cell lines (mean ± standard error (SEM), n=3). β-actin was used as a loading control for Western blot. (B) RT-qPCR (histogram) and Western blot analysis (below) of ESRP1, RBM24, and TPM1 isoform expression in ESRP1-OE (overexpression) and shRBM24-KD (knockdown) OV90 and COV504 ovarian cancer cell lines (mean ± SEM, n=3). β-actin was used as a loading control for Western blot.

[0032] Figure 2 Ectopic expression of the Tpm1.6 / 7 and Tpm1.8 / 9 isoforms led to increased cell migration and invasion as well as decreased cell proliferation.

[0033] (A) RT-qPCR analysis of OV90, COV504, PEA1, and PEA2 ovarian cancer cell lines transduced to ectopically express the Tpm1.6 / 7-OE and Tpm1.8 / 9-OE isoforms (mean ± SEM, n=3). P-values ​​are relative to parental cell lines. (B) Western blot analysis of OV90, COV504, PEA1, and PEA2 ovarian cancer cell lines transduced to ectopically express the Tpm1.6 / 7-OE and Tpm1.8 / 9-OE isoforms. β-actin was used as a loading control. (C) Proliferation assay of OV90, COV504, PEA1, and PEA2 ovarian cancer cell lines transduced to ectopically express the Tpm1.6 / 7-OE and Tpm1.8 / 9-OE isoforms. Optical density values ​​(OD values) from day 1 to day 6 are shown (mean ± SEM, 697 n=3). P-values ​​are relative to parental cell lines. (D) Transwell migration assays of OV90, COV504, PEA1, and PEA2 ovarian cancer cell lines transduced to ectopically express Tpm1.6 / 7-OE and Tpm1.8 / 9-OE isoforms. 5 × 10 4Cells were plated on a TC-coated membrane and incubated statically overnight. The number of cells migrating to the submembrane side was counted and plotted (mean ± SEM, n=3). P-values ​​are relative to parental cell lines. (E) Confocal images of OV90 and COV504 parental cells and Tpm1.6 / 7 / 8 / 9-OE cells seeded on collagen layers and incubated for 6 days. As indicated by the arrows, Tpm1.8 / 9-OE cells appear to collectively invade the collagen layer in the form of narrow linear bands with “leading” and “following” cells. Scale bar: 250 μm. The number of cells invading collagen was quantified and plotted (mean ± SEM, n=3). P-values ​​are relative to parental cell lines. Plots relative to PEA1 and PEA2 ovarian cancer cell lines were also calculated (below). (F) Immunofluorescence analysis was performed on OV90, COV504, PEA1, and PEA2 parental cells using antibodies against ARP2, Tpm1.6 / 7, and Tpm1.8 / 9. Cell nuclei were visualized by DAPI staining of DNA. Scale bar: 5 μm.

[0034] Figure 3 RNA sequencing analysis showed that the TPM1 isoform plays a role in the Wnt pathway and facilitates in vivo transport.

[0035] (A) Characterized pathways based on gene set enrichment analysis (GSEA) of parental cells and Tpm1.6 / 7 / 8 / 9-OE OV90 cells. Heatmaps include only significantly altered pathways, NES>1, P<0.05. (B) Volcano plot showing differentially expressed genes between Tpm1.6 / 7-OE (left, green) and Tpm1.8 / 9-OE (pink, right) OV90 cells (absolute LFC>1.5, P<0.01). (C) TOP-Flash luciferase reporter gene analysis of Tpm1.6 / 7 / 8 / 9-OE and Wnt signaling activity after siRNA knockdown of Tpm1.6 / 7 and Tpm1.8 / 9 in OV90 cells (top histogram). P values ​​are relative to comparisons with parental cell lines (mean ± SEM, n=3).

[0036] Figure 4 The Tpm1.8 / 9 isomer is enriched in malignant ascites from ovarian cancer cells and confers resistance to platinum- and taxane-based therapies.

[0037] (A) Example of IHC (left panel) and ISH (BaseScope; right panel) analysis of patient-derived ovarian cancer using Tpm1.6 / 7 and Tpm1.8 / 9 isoform-specific antibodies (IHC) and oligonucleotide probes (ISH). Ovarian cancer tissue was obtained from primary tumors and metastases (without chemotherapy). Scale bars: 100 μm and 15 μm for IHC (inset); 20 μm and 5 μm for ISH (inset). (B) RT-qPCR analysis of Tpm1.6 / 7 and Tpm1.8 / 9 expression in OV90 cells exposed to cisplatin and paclitaxel (mean ± SEM, n=3). (C) Dose-response curves of Tpm1.6 / 7 / 8 / 9-OE cells grown in the presence of different concentrations of paclitaxel and cisplatin (x-axis and y-axis are logarithmic scale and cell viability, respectively). IC50 values ​​were calculated by triplicate for each experiment (mean ± SEM, n=3). (D) RT-qPCR (left histogram) and Western blot (right) analysis of TPM1 isoform expression in siTpm1.6 / 7 and siTpm1.8 / 9 knocked-down OV90 cells (mean ± SEM, n=3). β-actin was used as a loading control for Western blot. (E) Dose-response curves of siTpm1.6 / 7 and siTpm1.8 / 9 knocked-down OV90 cells cultured in the presence of different concentrations of paclitaxel (left) and cisplatin (right) (x-axis and y-axis are logarithmic scale and cell viability, respectively). IC50 values ​​were calculated by three technical replicates for each experiment (mean ± SEM, n=3).

[0038] Figure 5 Small molecule inhibitors targeting the Tpm1.8 / 9 isoform antagonize its effects on EMT, Wnt signaling, and chemotherapy resistance.

[0039] (A) Top: RT-qPCR analysis of TPM1 isoform and EMT-related gene expression in OV90 parental cells cultured for 24 h in the presence of 0 μM, 2 μM, 5 μM, and 10 μM 3-(2-methyl-indole-1-yl)-propylamine (PubChem CID 6494468) (denoted as compound-1 (Cp-1)) and 1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468) (denoted as compound-3 (Cp-3)). Values ​​were calculated by normalization to untreated cells. P-values ​​<0.05 are shown in red bars, while gray bars indicate lower values ​​(mean ± SEM, n=3). The figure below shows Western blot analysis of TPM1 isoform expression in OV90 parental cells cultured for 24 h in the presence of 0 μM, 2 μM, 5 μM, and 10 μM 3-(2-methyl-indol-1-yl)-propylamine (PubChem CID 6494468) (represented as compound-1) and 1-phenylmethyl-1H-indol-2-methanol (PubChem CID 18973468) (represented as compound-3). β-actin was used as a loading control for the Western blot. (B) OV90 EpCAM cultured for 24 h in the presence of 0 μM, 2 μM, 5 μM and 10 μM 3-(2-methyl-indole-1-yl)-propylamine (PubChem CID 6494468) (represented as compound-1) and 1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468) (represented as compound-3). 低 RT-qPCR analysis of TPM1 isoform and EMT-related gene expression in cells. Values ​​were calculated by normalization with untreated cells. P-values ​​<0.05 are shown in red bars, while gray bars represent lower values ​​(mean ± SEM, n=3). Below: OV90 EpCAM cells cultured for 24 h in the presence of 0 μM, 2 μM, 5 μM, and 10 μM of 3-(2-methyl-indole-1-yl)-propylamine (PubChem CID6494468) (represented as compound-1) and 1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468) (represented as compound-3). 低Western blot analysis of TPM1 isoform expression in cells. β-actin was used as a loading control for Western blot analysis. (C) Dose-response curves of parental OV90 cells treated with 3-(2-methyl-indol-1-yl)-propylamine (PubChem CID 6494468) (represented as compound-1) and 1-phenylmethyl-1H-indol-2-methanol (PubChem CID 18973468) (represented as compound-3) in the presence of different concentrations of paclitaxel and cisplatin. IC50 values ​​(mean ± SEM, n=3) were calculated by three technical replicates for each experiment. (D) OV90 EpCAM treated with 3-(2-methyl-indole-1-yl)-propylamine (PubChem CID6494468) (represented as compound-1) and 1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468) (represented as compound-3) in the presence of different concentrations of paclitaxel and cisplatin. 低 Cell dose-response curves. IC50 values ​​(mean ± SEM, n=3) were calculated by three technical replicates for each experiment. (E) OV90 parental cells (left panel) and EpCAM cells treated with 3-(2-methyl-indole-1-yl)-propylamine (PubChem CID6494468) (represented as compound-1) and 1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468) (represented as compound-3). 低 TOP-Flash luciferase reporter gene analysis of Wnt signal transduction activity in cells (right figure) (mean ± SEM, n=3).

[0040] Figure 6 Small molecule inhibitors targeting the Tpm1.8 / 9 isomer antagonize chemotherapy resistance in breast cancer cell lines.

[0041] (A) Dose-response curves of MCF-7 breast cancer cells treated with 1-phenylmethyl-1H-indole-2-methanol (PubChemCID 18973468) (denoted as 189-3) in the presence of different concentrations of paclitaxel. Compound 189-3 was added at concentrations of 5 μM and 10 μM. (B) Combined dose-response curves for all three conditions are shown in (A). IC50 values ​​were calculated by triplicate for each experiment. The addition of compound 189-3 resulted in increased sensitivity of MCF-7 cells to paclitaxel. Detailed Implementation

[0042] As used herein, the term "TPM1" refers to the gene (gene ID: 7168 in humans) of the tropomyosin family, a highly conserved and widely distributed actin-binding protein involved in the contractile systems of skeletal and smooth muscle, as well as the cytoskeleton of non-muscle cells. Tropomyosin consists of two α-helical chains arranged in a coiled spiral. It aggregates end-to-end along the two channels of the actin filament and provides stability to the actin filament. The encoded protein is an α-helical chain of the major tropomyosin that forms skeletal muscle, where it also functions in conjunction with the troponin complex to regulate the calcium-dependent interactions between actin and myosin during muscle contraction. Alternative splice transcript variants encoding a number of isoforms have been described in smooth muscle and non-muscle cells. Thirty-nine transcripts (sponge variants) of TPM1 are known. Variants Tpm1.8 (formerly Tm5a) and Tpm1.9 (formerly Tm5b) contain alternative in- and out-of-frame units in the 5' and 3' coding regions compared to variant Tpm1.1. A schematic diagram of the intron / exon organization of the mammalian TMP1 gene is provided, with reference to Schevzov et al., 2011 (Bioarchitecture 1(4):135–164), which is incorporated herein by reference, and especially to the diagram shown therein. Figure 1 These variants encode the (protein) isoforms Tpm1.8cy and Tpm1.9cy, which have different N-termini and C-termini and are shorter than the isoform Tpm1.1st. Variants Tpm1.8 (human: NP_001288218.1, mouse: NP_001157724.1, rat: NP_001029245.1) and Tpm1.9 (human: NP_001317273.1, mouse: NP_001157725.1, rat: NP_001029246.1) encode TPM1 isoforms of the same length (248 aa) but with different protein sequences (Geeves et al., 2015 J Muscle Res Cell Motil 36, 147-153). Accession numbers, effective July 2023, apply to full-length human, mouse, and rat sequences already documented at the nucleic acid level.

[0043] Table 1. Protein and mRNA sequences of Tpm1.8 and Tpm1.9.

[0044]

[0045]

[0046] The terms “Tpm1.8 and / or Tpm1.9 isoforms” and “Tpm1.8 / 9 isoforms” are used interchangeably in this document and refer to the alternative splicing products of the TPM1 gene, and may refer to its nucleic acid transcripts ((m)RNA) or polypeptide (protein) translation products.

[0047] As used herein, the term "inhibitor" refers to a compound that targets the Tpm1.8 and / or Tpm1.9 isoforms, wherein the Tpm1.8 and / or Tpm1.9 isoforms may be in the form of DNA, mRNA, or protein, and thereby the inhibitor reduces the transcriptional expression of the tropomyosin 1 isoforms Tpm1.8 and / or Tpm1.9, and / or reduces the levels of Tpm1.8 and / or Tpm1.9 peptides, and / or reduces the activity of Tpm1.8 and / or Tpm1.9 peptides. Inhibitors that reduce the activity of Tpm1.8 and / or Tpm1.9 peptides may be, for example, small molecule inhibitors, preferably the small molecule inhibitors disclosed herein. Another exemplary inhibitor that reduces the activity of Tpm1.8 and / or Tpm1.9 peptides may be Tpm1.8 and / or Tpm1.9 antibodies. Inhibitors that reduce the levels of Tpm1.8 and / or Tpm1.9 mRNA or peptides may be, for example, antisense polynucleotides. Suitable antisense polynucleotides comprise synthetic single-stranded nucleic acids (20 to 30 nucleotides in length, such as oligonucleotides) in the form of antisense oligonucleotides (ASOs), or small interfering RNA (siRNA) as a cleavage product of double-stranded RNA (dsRNA). An exemplary siRNA target sequence for human Tpm1.8 / 9 is 5'-CGAGAGGAAGCTGAGGGAGAC-3' (codons 38 to 44 of Tpm1.8 / 9 in exon 1b). Generating such antisense polynucleotides for posttranscriptional gene silencing is within the capabilities of those skilled in the art.

[0048] As used herein, the term "tumor" encompasses the abnormal growth of the tissue in question, which can be benign, precancerous, malignant, or metastatic. A tumor is preferably malignant, i.e., cancer.

[0049] As used herein, the term "cancer" refers to any malignant tumor resulting from the unintended growth, invasion, and, under certain conditions, metastasis of damaged cells in an organism. Examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer (e.g., epithelial ovarian cancer), cervical cancer, skin cancer, pancreatic cancer, spleen cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia (e.g., acute myeloid leukemia (AML)), Hodgkin lymphoma, non-Hodgkin lymphoma, lymph node cancer, bone marrow cancer, lung cancer, stomach cancer, eye cancer, etc.

[0050] As used herein, the term "treatment" includes the application of therapeutic forms to a subject with the aim of, for example, curing a patient's disease (e.g., cancer), stopping or slowing the progression of a disease (e.g., cancer), prolonging the subject's life, or alleviating the pain of a subject suffering from a disease (e.g., cancer). Preventive treatments, therapies aimed at preventing the induction or onset of a disease (e.g., cancer), are also understood to be part of the term "treatment."

[0051] As used herein, the terms “prevention” and “preventing” include the application of therapeutic forms to a subject with the aim of preventing the onset of a disease (e.g., cancer). The term refers to the application of therapeutic forms to a subject to prevent and / or reduce the likelihood of a disease (e.g., cancer) occurring. The term includes active and preventative treatment and / or the application of active ingredients to prevent the onset of a disease (e.g., cancer) and / or to immunize the subject.

[0052] As used herein, the terms “compound,” “active compound,” and “active component” refer to an ingredient, drug, and / or molecule (e.g., a small molecule) that can trigger, exert, and / or enhance therapeutic effects in a subject.

[0053] As used herein, the term "subject" refers to a person or animal that has cancer and / or is susceptible to cancer. As used herein, the term "subject" includes the recipient of the compound or pharmaceutical composition targeting Tpm1.8 and / or Tpm1.9 mentioned herein, i.e., a person who has or is suspected of having cancer (e.g., ovarian cancer). For example, "subject" includes a person in which cancer is prevented. For example, a subject is a mammal, more preferably a human. The terms "patient" and "subject" are used interchangeably herein. A subject is, for example, a person, i.e., a person who has cancer and / or is at risk of developing cancer. A subject is, for example, a person who is older or younger than 50 years of age. For example, a subject is older than 50 years of age. For example, a subject is a person who has cancer. For example, a subject is a man or a woman. For example, a subject is a woman with ovarian cancer (e.g., epithelial ovarian cancer). For example, a subject is a person with breast cancer.

[0054] As used herein, the term "drug resistance" includes the ability of cancer cells to at least partially tolerate one or more anticancer therapies, particularly one or more chemotherapeutic agents. In other words, drug resistance specifically refers to a reduced efficacy of anticancer treatment agents in treating a subject with cancer. When the anticancer treatment agent is a chemotherapeutic agent, drug resistance is referred to as "chemotherapy resistance." When referring to drug resistance in cancer, it preferably refers to the resistance of the cancer cells of said cancer to said anticancer treatment agent. For example, chemotherapy resistance to cisplatin and / or paclitaxel.

[0055] As an alternative to using the term "resistance" (e.g., "chemotherapy resistance") and counteracting it in the medical uses / methods of this invention, it may alternatively refer to restoring sensitivity to a therapy, such as restoring chemotherapy sensitivity. Therefore, the phrase "counteracting (chemotherapy) resistance" can be used interchangeably with the phrase "restoring (chemotherapy) sensitivity".

[0056] As used in this article, the term “recovery” in relation to the recovery of chemotherapy sensitivity includes at least a partial recovery (e.g., complete recovery) of the chemotherapy sensitivity of the cancer to anticancer treatment agents that were previously at least partially insensitive.

[0057] As used herein, the term "therapeuticly effective amount" refers to an amount of active ingredient applied that is sufficient to achieve the intended purpose, such as, in this case, the prevention and / or treatment of cancer (e.g., ovarian cancer). This means, for example, preventing proliferation, movement, invasion, migration, metastasis, or chemotherapy resistance.

[0058] As used herein, the term "application" includes references to the administration of a substance, compound, and / or pharmaceutical composition to a subject. The primary routes of administration are parenteral, enteral or gastrointestinal, and topical administration. As used herein, the term "parenteral" includes any form of administration not applied to the skin or via the gastrointestinal tract. Non-limiting examples of parenteral administration include epidural, intracerebral, intraventricular, epidermal, sublingual, extraamniotic, nasal, intra-articular, intra-articular, intracardiac, intracavitary, intradermal, intralesional, intramuscular, intraocular, intraocular, intraocular, intraocular, intraosseous, intraperitoneal, intrasheath, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, rectal, or intratumoral administration. As used herein, the term "intravenous" includes any parenteral administration route in which the substance or composition is injected into a vein of the subject, for example, using a core syringe. Intravenously administered substances or compositions will directly reach the bloodstream of the subject. As used herein, the term "intratumoral" includes, for example, the direct application of a substance or composition to a tumor using a hollow needle. Tumors in which intratumoral application occurs may be treated prior to application, for example, to improve tumor visibility. Intratumoral application can be used, for example, for the administration of anticancer therapeutic agents.

[0059] As used herein, the term "chemotherapy agent" includes any anticancer drug mentioned that is used as part of chemotherapy (a type of cancer treatment). Chemotherapy agents have, for example, cytotoxic and / or cell-inhibiting effects, and are used, for example, to cure a subject's cancer, alleviate a subject's symptoms, or prolong a subject's life. Non-limiting examples of chemotherapy agents include cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine, nitrosoureas, temozolomide, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, cabazitaxel, larotaxel, ortataxel, tesetaxel, paclitaxel, docetaxel, albumin-bound paclitaxel, taxotere, and epochhilone.A) Epomycin B) Epomycin C) Epomycin D) Epomycin E) Epomycin F) Vorinostat, Romidepsin, Irinotecan, Topotecan, Etoposide, Teniposide, Tafluposide, Bortezomib, Erlotinib, Gefitinib, Imatinib, Vemurafenib, Vismodegib, Azacitidine, Azathiopril ne), Capecitabine, Cytarabine, Doxifluridine, Fluorouracil, Gemcitabine, Hydroxyurea, Mercaptopurine, Methotrexate, Tioguanine, Bleomycin, Actinomycin, Carboplatin, Cisplatin, Oxaliplatin, Nedaplatin, TriplatinTetranitrate, Phenanthriplatin, Picoplatin, Satraplatin, Tretinoin, Alitretinoin, Bexarotene, Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, Aminopterin, Pemetrexed, Pralatrexate, Raltitrexed ( Raltitrexed, Pentostatin, Cladribine, Clofarabine, Fludarabine, Nelarabine, Carmofur, Floxuridine, Tegafur, Cytarabine, Gemcitabine, Decitabine, Hydroxycarbamide, Belotecan, Camptothecin, Cositecan, Etirinotecanpegol, exatecan, gimatinecan, lurtotecan, rubitecan, silatecan, aclarubicin, amrubicin, pirarubicin, zorubicin, mitoxantrone, loseoxantrone, pixantrone, bendamustine, chlorpromazine, ifosfamide, trofosfamide, prednimustine, uramustine, carmustine, formustine stine, lomustine, semustine, nimustine, ranimustine, streptozocin, mannosulfan, Treosulfan, carboquone, thiotepa, triaziquone, triethylene melamine, tertretamine, procarbazine, mitobronitol, pipobroman, dacarbazine, temozolomide, dactinomycin, bleomycin, mitomycins, plicamycin, aminolevulinic acid acid), efavirenzal, methyl aminolevulinate, padeliporfin, porphyrin sodiumsodium, Talaporfin, Temoporfin, Verteporfin, Tipifarnib, Abemaciclib, Alvocidib, Palbociclib, Ribociclib, Seliciclib, Bortezomib, Carfilzomib, Oprozomib, Ixazomib, Anagrelide, Tiazofurin, Masoprocol, Niraparib, Olaparib, Rucaparib, Belinostat Entinostat, Panobinostat, Romidesin, Vorinostat, Pi3K, Alpelisib, Copanlisib, Duvelisib, Idelalisib, Umbralisib, Atrasentan, Bexarotin, Testolactone, Amsacrine, Arsenic trioxide, Asparaginase, Pegaspargase, Belzutifan, Celecoxib, Demecolcine, Elesclomol, Elsamitrucin, Eribulin, Estramustine phosphate Combinations of chemotherapy agents, such as phosphate, etoglucid, lonidamine, lucanthone, mitoguazone, mitotane, oblimersen, ometaxine mepesuccinate, trabectedin, retinoic acid, bexarotin, tretinoin, veliparib, and venetoclax, are used, for example, to treat cancer. Combinations can be established or ad hoc by the treating physician. Established combinations are called regimens, which in some cases also include dosage and administration intervals.

[0060] The chemotherapy agents disclosed herein include, for example: (i) alkylating agents (e.g., hexamethylmelamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, nitrogen mustard, melphalan, oxaliplatin, temozolomide, thiotepa, or trabectedin); (ii) nitrosoureas (e.g., carmustine, lomustine, or streptozocin); (iii) anti-myolytic agents. Drugs (azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda), cladribine, clofarabine, cytarabine (Ara-C), decitabine, fluorouridine, fludarabine, gemcitabine (Gemzar), hydroxyurea, methotrexate, nerabine, pemetrexed (Alimta), pentostatin, pralatrexate, thioguanine or) (iv) Trifluridine / tipiridine combination; (v) anthracyclines (e.g., daunorubicin, doxorubicin (doxorubicin), doxorubicin liposome, epirubicin, idarubicin, or penrubicin); (vi) topoisomerase I or II inhibitors (e.g., irinotecan, irinotecan liposome, topotecan, etoposide (VP-16), mitoxantrone, or teniposide); (vi) taxanes (cabatasoxetine, docetaxel, albumin-bound). (vii) Vinca alkaloids (e.g., vinblastine, vincristine, vincristine liposomes, or vinorelbine); (viii) corticosteroids (e.g., prednisone, methylprednisolone, or dexamethasone); or (ix) all-trans retinoic acid, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixaprone, mitotane, omastacin, pegaspargase, procarbazine, romidesin, or vorinostatin.

[0061] As used herein, the term "combination" or "combination therapy" includes the use of compounds targeting Tpm1.8 and / or Tpm1.9 as disclosed herein and anticancer agents as disclosed herein in the same medical treatment. The compounds targeting Tpm1.8 and / or Tpm1.9 and the anticancer agents as disclosed herein may be administered simultaneously (e.g., as a single pharmaceutical composition), simultaneously separately (e.g., as separate pharmaceutical compositions), or separately and interleaved in time. It is explicitly envisioned that compounds targeting Tpm1.8 and / or Tpm1.9 and anticancer agents as disclosed herein may be administered simultaneously, separately, and sequentially in the same treatment regimen. For example, the time interval between the administration of compounds targeting Tpm1.8 and / or Tpm1.9 and anticancer agents may be, for example, at least 1 minute, at least 15 minutes, at least 60 minutes, at least 4 hours, at least 1 day, at least 1 week, at least 1 month, or at least 1 year, or any time in between (e.g., from 1 minute to 1 year). Preferably, the compound targeting Tpm1.8 and / or Tpm1.9 is administered before the anticancer therapeutic agent. Alternatively, the anticancer therapeutic agent may be administered together with or after the compound targeting Tpm1.8 and / or Tpm1.9. The anticancer therapeutic agent in combination with the compound targeting Tpm1.8 and / or Tpm1.9 is, for example, a chemotherapy drug. Chemotherapy in combination with the compound targeting Tpm1.8 and / or Tpm1.9 is, for example, taxane- and platinum-based chemotherapy. The compound targeting Tpm1.8 and / or Tpm1.9 may be, for example, in combination with cisplatin and / or paclitaxel.

[0062] As used herein, the term "drug combination" includes, for example, a kit containing multiple containers holding different active ingredients.

[0063] Throughout the specification and claims, unless the context otherwise requires, the word “comprising” and its variations such as “including” or “comprise” shall be understood to imply inclusion of the said member, integer or step, or group of members, integers or steps, but not to exclude any other member, integer or step, or group of members, integers or steps.

[0064] The terms “a”, “an”, and “the”, as well as similar references, used in the context of describing the invention (particularly in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by the context.

[0065] The description of the range of values ​​in this document is intended only as a shorthand reference for each individual value falling within that range. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were listed separately herein.

[0066] All methods described herein may be performed in any suitable order unless otherwise stated herein or clearly contradicted by the context. Any examples and all example or exemplary language (e.g., “for example”, “e.g.”) are intended only to better illustrate the invention and do not constitute a limitation on the scope of the otherwise claimed invention. No language in the specification should be construed as indicating any unclaimed element essential to the practice of the invention.

[0067] All documents cited or referenced herein (“Documents Cited herein”), and all documents cited or mentioned in the Documents Cited herein, together with any manufacturer’s specifications, descriptions, product specifications, and product tables of any products mentioned herein or in any document incorporated herein by reference, are incorporated herein by reference and may be used in the practice of this invention. More specifically, all references are incorporated by reference to the extent that each individual document is specifically and individually indicated to be incorporated by reference.

[0068] The features of the invention will be described in more detail below. It should be understood that embodiments can be combined in any manner and in any number to create additional embodiments. The examples and embodiments described are not to be construed as limiting the invention to the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed features. Furthermore, unless the context otherwise requires, any permutation and combination of all features described in this application should be considered as disclosed by the description of this application.

[0069] compound

[0070] The compounds according to the invention target the Tpm1.8 and / or Tpm1.9 isomers. For example, the compounds of the invention target the N-terminal sequence of exon 1b of TPM1 (see Schevzov et al., 2011. Bioarchitecture 1(4): 135-164). Figures 1 to 6(This reference includes the intron / exon structure of the TPM1 gene, and is incorporated herein by reference). For example, compounds targeting the Tpm1.8 and / or Tpm1.9 isomers target the N-terminus of residues 4 through 16 of Tpm1.8 and / or Tpm1.9. For example, such compounds are small molecules that interfere with the activity of the Tpm1.8 and / or Tpm1.9 isomers in a dose-dependent manner. Compounds targeting the Tpm1.8 and / or Tpm1.9 isomers preferably inhibit the activity of Tpm1.8 and / or Tpm1.9 at concentrations of 5 μM to 50 μM, 15 μM to 30 μM, or 10 μM to 20 μM. For example, compounds targeting the Tpm1.8 and / or Tpm1.9 isomers are selected from 3-(2-methyl-indol-1-yl)-propylamine (PubChem CID 6494468), 1-phenylmethyl-1H-indol-2-methanol (PubChem CID 18973468), or combinations thereof. PubChem CID 6494468 targets Tpm1.8 / 9 and Tpm4.2, while PubChem CID 18973468 targets only Tpm1.8 / 9. In some embodiments of this invention, PubChem CID 6494468 is a preferred compound. In some embodiments of this invention, PubChem CID 18973468 is a preferred compound. In addition, compounds targeting the Tpm1.8 and / or Tpm1.9 isomers include pharmaceutically acceptable salts, hydrates, derivatives and / or solvates of, for example, 3-(2-methyl-indole-1-yl)-propylamine (PubChem CID 6494468) and / or 1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468).

[0071]

[0072] Formula I: 3-(2-methyl-indol-1-yl)-propylamine (PubChem CID 6494468)

[0073]

[0074] Formula II: 1-Benzyl-1H-indole-2-methanol (PubChem CID 18973468)

[0075] Furthermore, the compounds targeting Tpm1.8 and / or Tpm1.9 isomers according to the present invention include any compound identified by the methods for identifying compounds targeting Tpm1.8 and / or Tpm1.9 as disclosed herein.

[0076] The compounds of the present invention targeting the Tpm1.8 and / or Tpm1.9 isomers further include hydrates and solvates. The solvates are complexes formed by the association of solvent molecules with the compounds of the present invention. The compounds of the present invention targeting the Tpm1.8 and / or Tpm1.9 isomers are, for example, in the form of pharmaceutically acceptable salts. The compounds of the present invention targeting the Tpm1.8 and / or Tpm1.9 isomers are also extended to include all derivatives having physiologically cleavable leaving groups that can be cleaved in vivo.

[0077] The compounds of the present invention also include antisense polynucleotides that reduce the expression of Tpm1.8 and / or Tpm1.9 isoforms in cancer cells.

[0078] The compounds of the present invention also include antibodies that reduce the levels and / or activity of Tpm1.8 and / or Tpm1.9 peptides.

[0079] The antisense polynucleotides and antibodies of the present invention can be used in methods for inhibiting, suppressing, preventing or treating cancer proliferation, movement, invasion, migration, metastasis or chemotherapy resistance.

[0080] Pharmaceutical Composition

[0081] Compounds targeting the Tpm1.8 and / or Tpm1.9 isomers, or other compounds of the invention as disclosed herein, may be used as the sole active ingredient in the pharmaceutical compositions of the invention, or may be used in combination with other compounds targeting the same or different targets. For example, a first compound targeting the Tpm1.8 and / or Tpm1.9 isomer as disclosed herein may be combined with a second compound targeting the Tpm1.8 and / or Tpm1.9 isomer as disclosed herein.

[0082] Compounds targeting the Tpm1.8 and / or Tpm1.9 isomers as disclosed herein are preferably included in pharmaceutical compositions that may also contain pharmaceutically acceptable excipients (or carriers). As used herein, pharmaceutically acceptable excipients or carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption retardants, salts, preservatives, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and similar materials and combinations thereof, as known to those skilled in the art. Unless any conventional carrier is incompatible with compounds targeting the Tpm1.8 and / or Tpm1.9 isomers, its use in pharmaceutical compositions is considered. Acceptable excipients, carriers, or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in standard textbook references, such as Remington's Pharmaceutical Sciences (e.g., Mack Publishing Co., edited by AR Gennaro, 1985).

[0083] Pharmaceutical compositions suitable for parenteral administration (e.g., via intramuscular, intraperitoneal, subcutaneous, or direct injection into the target organ) include aqueous and non-aqueous isotonic sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Pharmaceutical compositions may be provided in single-dose or multi-dose sealed containers (e.g., ampoules and vials). Injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0084] Pharmaceutical compositions suitable for oral administration may consist of: (a) a liquid solution, such as a compound suspended in an effective amount of the Tpm1.8 and / or Tpm1.9 isomers in a diluent (e.g., water or saline); (b) capsules, pods, or tablets, each containing a predetermined amount of the active ingredient, in liquid, solid, granular, or gelatinous form; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, tragacanth gum, microcrystalline cellulose, gum arabic, gelatin, colloidal silica, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharmaceutically compatible carriers. The tablet form may contain the active ingredient in a flavoring agent (typically sucrose and gum arabic or tragacanth gum), or a soft tablet containing the active ingredient in an inert matrix (e.g., gelatin and glycerin or sucrose and gum arabic emulsions, gels, etc.), and may also contain a carrier known in the art, in addition to the compound targeting the Tpm1.8 and / or Tpm1.9 isomers. The pharmaceutical composition may be encapsulated, for example, in liposomes or in a formulation providing sustained release of the active ingredient. Thus, in one specific embodiment, a pharmaceutical composition containing a compound targeting the Tpm1.8 and / or Tpm1.9 isomer can be administered via liposomes, microparticles, or microcapsules. In various embodiments of the invention, using such a composition to achieve sustained release of the compound targeting the Tpm1.8 and / or Tpm1.9 isomers may be useful.

[0085] In one specific embodiment, the present invention provides a reagent kit or reagent kit comprising one or more containers filled with one or more compounds, compositions, or drugs of the present invention. Optionally, information indicating that the drug or biological product has been approved for manufacture, use, or sale for human administration by a government agency regulating the manufacture, use, or sale of such product may be affixed to such container in a prescribed form.

[0086] Methods for formulating compounds targeting the Tpm1.8 and / or Tpm1.9 isomers as drugs are known in the art. For example, such procedures are described in the Japanese Pharmacopoeia, the United States Pharmacopoeia, and other national pharmacopoeias. Therefore, those skilled in the art can determine the implementation method (e.g., the amount to be used) based on the description herein without excessive experimentation.

[0087] Application and treatment methods

[0088] This invention relates to compounds targeting the Tpm1.8 and / or Tpm1.9 isomers in methods for preventing and / or treating cancer in subjects. Administration of at least one compound targeting the Tpm1.8 and / or Tpm1.9 isomers as disclosed herein is, for example, by administering a pharmaceutical composition comprising a compound targeting the Tpm1.8 and / or Tpm1.9 isomer. The pharmaceutical composition can be administered in any suitable manner (e.g., enteric, parenteral, topical, by inhalation, etc.). Parenteral administration includes, for example, epidural, intracerebral, intraventricular, epidermal, sublingual, extraamniotic, nasal, intraarticular, intraarticular, intracardiac, intracavitary, intradermal, intralesional, intramuscular, intramuscular, intraocular, intraocular, intraocular, intraperitoneal, intrasheathal, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, intratumoral, or combinations thereof. Administration is, for example, enteric, oral, or rectal administration.

[0089] A second compound targeting the Tpm1.8 and / or Tpm1.9 isomers, and optional other compounds, may be used in one aspect of the invention. For example, the second compound targeting the Tpm1.8 and / or Tpm1.9 isomers may be administered together with the first compound targeting the Tpm1.8 and / or Tpm1.9 isomers, for example, simultaneously (e.g., in the form of separate pharmaceutical compositions), simultaneously separately (e.g., in the form of separate pharmaceutical compositions), or separately and interleaved in time.

[0090] It is explicitly envisioned that compounds targeting Tpm1.8 and / or Tpm1.9 isomers, as disclosed herein, be administered simultaneously, separately, and sequentially in the same treatment regimen. A second compound targeting Tpm1.8 and / or Tpm1.9 isomers may be administered, for example, parenteral or enteral. Parenteral administration may include, for example, epidural, intracerebral, intraventricular, epidermal, sublingual, extraamniotic, nasal, intraarticular, intraarticular, intracardiac, intracavitary, intradermal, intralesional, intramuscular, intraocular, intraocular, intraocular, intraocular, intramuscular, intraocular, intraocular, intraocular, intraosseous, intraperitoneal, intrasheathal, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, rectal, intratumoral, or combinations thereof. Administration may include, for example, intravenous or intratumoral administration, or oral or rectal administration. Third, fourth, fifth, or other compounds targeting Tpm1.8 and / or Tpm1.9 isomers may be used, for example, in aspects of the present invention. The third, fourth, fifth or other compounds targeting the Tpm1.8 and / or Tpm1.9 isomers may be administered, for example, together with the first and second compounds targeting the Tpm1.8 and / or Tpm1.9 isomers as described above.

[0091] The present invention also includes co-administration of the anticancer therapeutic agents described herein with compounds targeting the Tpm1.8 and / or Tpm1.9 isomers. The compounds targeting the Tpm1.8 and / or Tpm1.9 isomers, and optionally second, third, fourth, fifth, and / or other compounds targeting the Tpm1.8 and / or Tpm1.9 isomers, are administered together with one or more anticancer therapeutic agents as described herein.

[0092] The anticancer therapeutic agent can be administered, for example, via parenteral administration. Parenteral administration includes, for example, epidural, intracerebral, intraventricular, epidermal, sublingual, extraamniotic, nasal, intra-articular, intra-articular, intracardiac, intracavitary, intradermal, intralesional, intramuscular, intraocular, intraocular, intraosseous, intraperitoneal, intrasheath, intrauterine, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, intratumoral, or combinations thereof. For example, administration may be intravenous or intratumoral.

[0093] For example, the same route of administration may be chosen for compounds targeting the Tpm1.8 and / or Tpm1.9 isomers, and optionally for second, third, fourth, fifth, and / or other compounds targeting the Tpm1.8 and / or Tpm1.9 isomers, as well as one or more anticancer therapeutic agents as described herein. However, compounds targeting the Tpm1.8 and / or Tpm1.9 isomers and anticancer therapeutic agents may also be administered via different routes of administration. For example, anticancer therapeutic agents may be administered parenterally, and compounds targeting the Tpm1.8 and / or Tpm1.9 isomers may be administered orally. Furthermore, as an example, a (first) compound targeting the Tpm1.8 and / or Tpm1.9 isomer may be administered orally, and a second or other compound targeting the Tpm1.8 and / or Tpm1.9 isomer may be administered parenterally.

[0094] In some aspects of the invention, the compound targeting the Tpm1.8 and / or Tpm1.9 isomer is applied to the object at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least twelve times, at least fourteen times, at least sixteen times, at least eighteen times, at least twenty times, at least twenty-five times, at least thirty times, at least thirty-five times, at least forty times, at least fifty times, at least sixty times, at least seventy times, at least eighty times, at least ninety times, or at least one hundred times.

[0095] Compounds targeting the Tpm1.8 and / or Tpm1.9 isomers, as disclosed herein, are used, for example, in treatment regimens involving daily, weekly, or monthly administration of compounds targeting the Tpm1.8 and / or Tpm1.9 isomers. Treatment is maintained, for example, for at least three days, at least one week, at least one month, and more preferably at least six months or at least one year (e.g., two to five years).

[0096] The compounds and / or anticancer agents targeting the Tpm1.8 and / or Tpm1.9 isomers disclosed herein are used, for example, in treatment regimens involving cyclic administration, wherein each cycle comprises repeated (e.g., daily) administration of the compounds targeting the Tpm1.8 and / or Tpm1.9 isomers for several days (e.g., at least one day, at least three days, at least one week, at least two weeks, or at least three weeks). Cyclic administration of the compounds and / or anticancer agents targeting the Tpm1.8 and / or Tpm1.9 isomers disclosed herein is, for example, repeated every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks. Cyclic administration of the compounds and / or anticancer agents targeting the Tpm1.8 and / or Tpm1.9 isomers disclosed herein is, for example, repeated after 3 to 5 weeks, or, for example, repeated after 3 weeks or 5 weeks. For example, the administration of compounds and / or anticancer agents targeting Tpm1.8 and / or Tpm1.9 isomers as disclosed herein is not repeated at fixed intervals, but rather repeated as needed by the patient. For example, the periodic administration of chelating agents and / or anticancer agents as disclosed herein may be repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, or at least ten times. For example, treatment may be maintained for at least three days, at least one week, at least one month, more preferably at least six months, or at least one year (e.g., two to five years).

[0097] The subject is administered compounds targeting Tpm1.8 and / or Tpm1.9 isomers as disclosed herein and / or anticancer therapeutic agents as disclosed herein, for example, following conventional protocols for administering such compounds, while taking into account the toxicity of the agent (if any). Therefore, in some embodiments, there is a step of monitoring for toxicity attributable to the combination therapy.

[0098] Compounds targeting the Tpm1.8 and / or Tpm1.9 isomers disclosed herein can be administered in any acceptable pharmaceutical dosage form, such as as an aqueous medium (e.g., solution, suspension, emulsion). Chelating agents can also be administered orally as pills, tablets, capsules, etc. In aspects of the invention, if a subject has cancer with cancerous cells, such as ovarian cancer, he or she can be identified as eligible for treatment. Cancer cells may exhibit drug resistance, such as chemotherapy resistance. For example, cancer cells may exhibit chemotherapy resistance to taxane- and / or platinum-based chemotherapy. For example, cancer cells may exhibit chemotherapy resistance to cisplatin and / or paclitaxel. Suitable doses of the compounds of the invention in therapeutic treatment may include doses from 0.1 mg / kg to 100 mg / kg, preferably from about 0.5 mg / kg to 20 mg / kg, and even more preferably from about 1 mg / kg to 10 mg / kg.

[0099] The treatment methods according to the invention include methods for inhibiting, suppressing, preventing, and / or treating cancer, including administering a therapeutically effective amount of a compound according to the invention or a pharmaceutical composition comprising a compound according to the invention to a subject in need. In embodiments of these methods, the compound is, for example, a compound that targets the Tpm1.8 and / or Tpm1.9 isomers as disclosed herein (e.g., a small molecule inhibitor disclosed herein), or an antisense polynucleotide or antibody that reduces the level and / or activity of the Tpm1.8 or Tpm1.9 peptide.

[0100] cancer

[0101] In this invention, cancers include, for example, those selected from non-small cell lung cancer; renal cell carcinoma; renal cell carcinoma; clear cell renal cell carcinoma; lymphoma; blastoma; sarcoma; undifferentiated carcinoma; meningioma; brain cancer; oropharyngeal carcinoma; nasopharyngeal carcinoma; cholangiocarcinoma; pheochromocytoma; islet cell carcinoma; Li-Fraumeni tumor; thyroid cancer; parathyroid carcinoma; pituitary adenoma; adrenal tumor; osteosarcoma; neuroendocrine tumor; breast cancer; lung cancer; head and neck cancer; prostate cancer; esophageal cancer; tracheal cancer; liver cancer; bladder cancer; gastric cancer; pancreatic cancer; ovarian cancer; uterine cancer; cervical cancer; testicular cancer; colon cancer; rectal cancer; skin cancer; giant cell and spindle cell carcinoma; small cell carcinoma; small cell lung cancer; papillary carcinoma; oral cavity cancer; oropharyngeal carcinoma; nasopharyngeal carcinoma; respiratory tract cancer; Urogenital tract cancer; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrointestinal cancer; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; hepatocellular carcinoma combined with cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous polyposis adenocarcinoma; familial adenomatous polyposis adenocarcinoma; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-capsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine gland adenocarcinoma; sebaceous gland adenocarcinoma; ceruminous gland adenocarcinoma; mucoepidermoid carcinoma; cystic adenocarcinoma; papillary cystic adenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Malignant thymoma; Malignant ovarian stromal tumor; Malignant theca cell tumor; Malignant granulosa cell tumor; Malignant androblastoma; Sertoli cell carcinoma; Malignant Ledich's cell tumor; Malignant lipocytoma; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Hemangiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial diffuse melanoma; Giant melanocytic nevus malignant melanoma; Malignant lentigines melanoma; Acral lentigines melanoma; Nodular melanoma; Epithelioid cell melanoma Malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner's tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone;Ewing's sarcoma; malignant odontogenic tumors; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; endocrine or neuroendocrine carcinoma or hematopoietic system carcinoma; malignant pineal tumor; chordoma; carcinoma of the central or peripheral nervous system; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; glial astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioneuroma; neuroblastoma; retinoblastoma; olfactory neurogenic tumors; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; B-cell lymphoma; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; low-grade / follicular non-Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; mantle cell lymphoma; Waldenström macroglobulinemia; other specifically defined non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; preferably, leukemia is lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; acute myeloid leukemia (AML), chronic myeloid leukemia (CML); basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; chronic lymphocytic leukemia. Chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, urothelial carcinoma of the bladder, low-grade brain carcinoma, invasive breast carcinoma, squamous cell carcinoma and endocervical adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, esophageal cancer, glioblastoma multiforme, squamous cell carcinoma of the head and neck, chromophobe renal carcinoma, clear cell renal carcinoma, papillary renal carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, lymphoid tumors, diffuse large B-cell lymphoma, mesothelioma, ovarian cancer, especially serous ovarian carcinoma, pancreatic adenocarcinoma, rectal adenocarcinoma, sarcoma, melanoma of the skin, gastric adenocarcinoma, testicular germ cell tumor, thymoma, thyroid cancer, uterine carcinosarcoma, endometrial cancer of the uterine body, uveal melanoma, and combinations thereof. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor.

[0102] Preferably, in this invention, cancer is carcinoma. Carcinoma is a malignant tumor that develops from epithelial cells.

[0103] Preferably, in aspects of the invention, the cancer is a cancer expressing a TPM1 alternative splice isoform. For example, the cancer is a cancer expressing a TPM1 alternative splice isoform containing exon 1b of TPM1. For example, the cancer is a cancer exhibiting alternative splicing because it expresses the Tpm1.8 and / or Tpm1.9 isoforms. Tpm1.8 and / or Tpm1.9 expression can be determined, for example, at the RNA or protein level. At the RNA level, Tpm1.8 and / or Tpm1.9 expression can be determined, for example, by RT-PCR (reverse transcription polymerase chain reaction) or by RNA sequencing, and at the protein level by antibody detection (e.g., Western blotting, immunofluorescence, or immunohistochemistry) or mass spectrometry. When the difference in appropriate expression level parameters (e.g., the mean PSI (Δpsi; percentage of differential splicing) as in the examples below) between the tested cancer (cells) and an appropriate control (cells) is >10%, alternative splicing (expression of the Tpm1.8 and / or Tpm1.9 isoforms) can be indicated.

[0104] The inventors have discovered that in ovarian cancer, the Tpm1.8 and / or Tpm1.9 isoforms are not expressed in the primary tumor, but are expressed within a relatively small time window during the intraperitoneal systemic spread of cells and their undergoing epithelial-mesenchymal transition (EMT) (a complex developmental process that enables cancer cells to suppress their epithelial features and transform into mesenchymal features). That is, the cells thereby acquire pseudo-mesenchymal and chemotherapeutic resistance characteristics, along with motility and invasiveness. Therefore, aspects of the present invention relate to cancers expressing the TPM1 gene, particularly cancers expressing TPM1 RNA molecules containing exon 1b (the Tpm1.8 and / or Tpm1.9 isoforms are derived from exon 1b). Many cancers express TPM1 RNA molecules containing exon 1b. Database searches (TGCA database, www.cancer.gov / ccg / research / genome-sequencing / tcga) have identified gene expression at the RNA level of the complete TPM1 gene, including TPM1 containing exon 1b. RNA molecule expression occurs in acute myeloid leukemia (AML), adrenocortical carcinoma, urothelial carcinoma of the bladder, low-grade brain carcinoma, invasive breast carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, lymphoid tumors, diffuse large B-cell lymphoma, mesothelioma, ovarian cancer, pancreatic adenocarcinoma, rectal adenocarcinoma, sarcoma, skin melanoma, gastric adenocarcinoma, testicular germ cell tumors, thymoma, thyroid cancer, uterine carcinosarcoma, endometrial cancer of the uterine body, and uveal melanoma. Therefore, aspects of the present invention can be used to treat these types of cancer.

[0105] For example, the cancer is ovarian cancer, such as epithelial ovarian cancer. For example, the cancer is high-grade serous ovarian cancer.

[0106] Epithelial ovarian cancer (EOC) is a leading cause of death among gynecological malignancies due to its high case fatality rate. EOC typically becomes apparent at advanced disease stages, when metastases have spread to pelvic organs (stage II), abdomen (stage III), or beyond the peritoneal cavity (stage IV). Based on underlying genetic defects, two main subtypes of EOC have been identified. Type I tumors grow slowly, are primarily confined to the ovary, and are thought to result from well-differentiated precursor lesions known as “borderline” tumors. They are further subdivided into low-grade serous, mucinous, clear cell, and endometrioid subtypes. Mutations in KRAS, BRAF, PTEN, and CTNNB1 (β-catenin) mark type I EOC, often accompanied by a relatively stable karyotype. High-grade serous (HGSOC) and undifferentiated carcinoma are type II EOC, typically characterized by TP53 mutations and aneuploidy. HGSOC represents the most malignant and common type of ovarian cancer, accounting for 70% of all cases with poor prognosis and survival. It is noteworthy that ovarian ovarian cancer (EOC) is the only type of cancer in which there is no physical barrier between the primary lesion and the main metastatic site (i.e., the peritoneal cavity). The spread of ovarian cancer cells causes them to adhere to intra-abdominal organs and the peritoneum, eventually leading to ascites accumulation due to lymphatic obstruction.

[0107] For example, the cancer is epithelial ovarian cancer of EOC type I and / or EPO type II. For example, epithelial ovarian cancer is selected from EOC type I, EOC type II, high-grade serous ovarian cancer (HGSOC), low-grade serous ovarian cancer (LGSOC), fallopian tube cancer, primary peritoneal cancer, and combinations thereof.

[0108] Anticancer treatment drugs

[0109] This invention relates to compounds targeting the Tpm1.8 and / or Tpm1.9 isomers in methods for treating cancer. Optionally, at least one compound targeting the Tpm1.8 and / or Tpm1.9 isomers as disclosed herein may be administered in combination with additional anticancer therapeutic agents.

[0110] The anticancer treatment agent can be any anticancer treatment agent. The anticancer treatment agent described herein is, for example, an anticancer treatment agent used in a treatment type selected from chemotherapy, targeted therapy (e.g., immunotherapy), stem cell therapy, hormone therapy, radiotherapy, and surgery, or combinations thereof; preferably, the treatment type is chemotherapy.Anticancer treatment agents include, for example, cyclophosphamide, nitrogen mustard, chlorambucil, melphalan, dacarbazine, nitrosourea, temozolomide, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pentorubicin, cabazitaxel, larotaxel, oxalitaxel, tefactaxel, paclitaxel, docetaxel, albumin-bound paclitaxel, paclitaxel, epoxantrone A, epoxantrone B, epoxantrone C, epoxantrone D, epoxantrone E, epoxantrone F, vorinostatin, romidesin, irinotecan, topotecan, etoposide, teniposide, tafluposide, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vemodilamide, azacitidine, azathioprine, capecitabine, cytarabine, deoxyfluorouracil, and fluorouracil. Gemcitabine, Hydroxyurea, Mercaptopurine, Methotrexate, Thioguanine, Bleomycin, Actinomycin, Carboplatin, Cisplatin, Oxaliplatin, Nedaplatin, Triplatinum Tetranitrate, Phenylebium Platinum, Pyripril Platinum, Sapindole, Retinoic Acid, Aliretinoic Acid, Bexarotin, Vincristine, Vincristine, Vinpocetine, Vinorelbine, Vinorelbine, Aminopterin, Pemetrexed, Prandtolexa, Raltitrexed, Pentostatin, Cladribine, Clofarabine, Fludarabine, Nerapine, Carmoflurane, Fluorouracil, Furazone, Cytarabine, Gemcitabine, Decistabine, Hydroxyurea, Belotticon, Camptothecin, Cocitecon, Polyethylene Glycol Irinotecan, Exanotecan, Gematometazone, Rutonotecan, Rubitecan, Cilatecon, Arubicin, Amarabicin, Pirarubicin, Pentonotecan Zorubicin, Mitoxantrone, Loxoantrone, Pyrantrone, Bendamustine, Nitrogen Mustard Hydrochloride, Ifosfamide, Trophosphamide, Prenimustine, Uramustine, Carmustine, Formustine, Lomustine, Semustine, Nimustine, Ramustine, Levozocin, Mannosulfan, Triosulfan, Carboquinone, Thiotepa, Triaminoquinone, Triethylenetricyanamide, Hexamethylmelamine, Procarbazine, Bromomannitol, Piperbromophenazine, Dacarbazine, Temozolomide, Dermatomycin, Bleomycin, Mitomycin, Prucalomycin, Aminolevulinic Acid, Fepipiralaldehyde, Methyl Aminolevulinate, Padriboflavin, Porphyrom sodium, Talapofen, Temopofen, Vertepofen, Tepififibrazine, Abecilibi, Avozidil, Palbociclib, Rebociclib, Celicillin Bortezomib, carfilzomib, oprotozomib, ixazomib, anagrelide, thiazofuran, masoproco, niraparib, olaparib, rucaparib, belisitol, entinostat, prabistat, romidesin, vorinostat, Pi3K, apeliximab, cupanisin, duvericide, edralani, ummburicil, atrasentan, bexarotin, testosterone, acridine, arsenic trioxide, asparaginase, pegaspargase, bezantifan, celecoxib, colchicine, eleximo, ixaloxine, eribulin, estradiol phosphate, etogluconate, clonidine, lucanzab, mitoguazoline, mitotan, olimosen, omastacin, trabectedin, retinoic acid, bexarotin, retinoic acid, veripani, and veneclax or combinations thereof.For example, combinations of anticancer drugs are known chemotherapy regimens, such as CMF (cyclophosphamide, methotrexate, 5-fluorouracil, vinorelbine), AC (doxorubicin, cyclophosphamide), DA (cytarabine, anthracycline antibiotics, daunorubicin), IA (cytarabine, anthracycline antibiotics, idarubicin), DAT (daunorubicin, cytarabine, thioguanine), FLAMSA (fludarabine, cytarabine, acridine), and FLAMSA-BU (fludarabine, cytarabine). Cytidine, acridine, busulfan), FLAMSA-MEL (fludarabine, cytarabine, acridine, melphalan), TAD (thioguanine, cytarabine, daunorubicin), CAF (cyclophosphamide, doxorubicin, fluorouracil), CLIA (cladribine, idarubicin, and cytarabine), CLIA-M (mylotarg, cladribine, idarubicin, and cytarabine), and ABVD (doxorubicin, bleomycin, vincristine, dacarbazine). For example, anthracyclines (e.g., doxorubicin), antimetabolites (e.g., 5-fluorouracil (5-FU)), and / or taxanes (e.g., Nab-paclitaxel and / or paclitaxel) are selected as anticancer therapeutic agents. The anticancer therapeutic agents disclosed herein can be administered via any acceptable delivery modality, such as liposome delivery. For example, at least one compound targeting the Tpm1.8 and / or Tpm1.9 isomers as disclosed herein may be administered in combination with taxane- and / or platinum-based chemotherapy (e.g., chemotherapy including cisplatin and / or paclitaxel).

[0111] Chemotherapy resistance

[0112] In aspects of the invention, compounds targeting the Tpm1.8 and / or Tpm1.9 isomers for the prevention and / or treatment of cancer counteract chemotherapy resistance. For example, compounds targeting Tpm1.8 and / or Tpm1.9 are used to reverse chemotherapy resistance in drug-resistant cancers (e.g., drug-resistant ovarian cancer). For example, chemotherapy resistance is natural chemotherapy resistance and / or acquired chemotherapy resistance. For example, chemotherapy resistance is acquired through one or more previously administered chemotherapy treatments. For example, chemotherapy resistance counteracted by compounds targeting Tpm1.8 and / or Tpm1.9 according to the invention is chemotherapy resistance to any chemotherapeutic agent in the art. For example, chemotherapy resistance counteracted by compounds targeting Tpm1.8 and / or Tpm1.9 according to the invention is chemotherapy resistance to taxane- and / or platinum-based chemotherapy. For example, chemotherapy resistance counteracted by compounds targeting Tpm1.8 and / or Tpm1.9 according to the invention is chemotherapy resistance to cisplatin and / or paclitaxel. For example, compounds targeting the Tpm1.8 and / or Tpm1.9 isomers are used to restore chemosensitivity to any chemotherapy in the art. For example, compounds targeting the Tpm1.8 and / or Tpm1.9 isomers are used to restore chemosensitivity to taxane- and / or platinum-based chemotherapy. For example, compounds targeting the Tpm1.8 and / or Tpm1.9 isomers are used to restore chemosensitivity to cisplatin and / or paclitaxel. For example, compounds targeting the Tpm1.8 and / or Tpm1.9 isomers according to the invention at least partially counteract chemotherapy resistance and / or restore chemosensitivity, for example, completely restoring the chemosensitivity of cancer to previously at least partially insensitive anticancer treatment agents. For example, compared to the specific IC50 of the chemotherapy agent in an untreated control, and / or compared to the specific IC50 of the chemotherapy agent prior to the use of a compound targeting the Tpm1.8 and / or Tpm1.9 isomer, the compound targeting Tpm1.8 and / or Tpm1.9 according to the present invention reduces the specific IC50 level of the chemotherapy agent by 10% to 100%, 20% to 95%, 25% to 90%, 30% to 85%, 35% to 80%, 40% to 75%, 45% to 70%, 50% to 65%, or 55% to 60%. For example, compared to an untreated control, and / or compared to the specific IC50 of a chemotherapy agent prior to the use of a compound targeting the Tpm1.8 and / or Tpm1.9 isomer, the compound targeting Tpm1.8 and / or Tpm1.9 according to the invention reduces the specific IC50 level of the chemotherapy agent by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%. For example, chemotherapy agents for which cancer has developed resistance are taxane- and / or platinum-based chemotherapy agents, such as cisplatin and / or paclitaxel.

[0113] Filtering methods

[0114] The present invention also includes methods for identifying compounds targeting the Tpm1.8 and / or Tpm1.9 isomers for use in inhibiting, suppressing, preventing, and / or treating cancer proliferation, motility, invasion, migration, metastasis, and / or chemotherapy resistance. For example, the method includes constructing a model of the N-terminus of Tpm1.8 and / or Tpm1.9, preferably human Tpm1.8 and / or Tpm1.9, containing the most diverse regions among the four TPM genes. For example, the model contains the N-terminus of residues 4 through 16.

[0115] Furthermore, methods for identification include, for example, performing computer-aided virtual docking screening to identify compounds that may bind to Tpm1.8 and / or Tpm1.9, preferably at the N-terminus of Tpm1.8 and / or Tpm1.9, more preferably at the N-terminus of residues 4 through 16 of Tpm1.8 and / or Tpm1.9. For example, computer-aided virtual docking screening can be performed on the ZINC database (Irwi et al., J. Chem. Inf. Model. 2020, 60, 12, 6065–6073), a database of commercially available compounds, or other chemical databases for lead compound discovery, using online docking programs such as AutoDock, GOLD, Deep Docking, Glide, or POSIT. Candidate compounds can be purchased from suppliers and their activity tested in vitro or in vivo.

[0116] For example, the method includes contacting human fibroblasts with a test compound. Contact may be performed for, for example, at least 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, or 52 hours. After contact, the human fibroblasts are fixed and stained, for example, using Tpm1.8 and / or Tpm1.9 specific antibodies. Furthermore, methods for identifying the compounds of the present invention include, for example, analyzing the enrichment of Tpm1.8 and / or Tpm1.9 isoforms in the lamellae of fibroblasts, wherein, compared to control fibroblasts, fibroblasts contacted with the test compound of the present invention, identified as targeting the Tpm1.8 and / or Tpm1.9 isoforms, show a reduction in the enrichment of Tpm1.8 and / or Tpm1.9 isoforms in the lamellae. Compounds that promote a reduction in the enrichment of Tpm1.8 and / or Tpm1.9 isomers in lamellae pseudopodia compared to control fibroblasts are, for example, compounds identified as being used in methods for inhibiting, suppressing, preventing, and / or treating cancer.

[0117] Methods for identifying compounds targeting the Tpm1.8 and / or Tpm1.9 isomers further include, for example, contacting cancer cells expressing the Tpm1.8 and / or Tpm1.9 isomers with the test compound. Cancer cells are, for example, ovarian cancer cells. For example, contacting cancer cells (e.g., ovarian cancer cells) with the test compound at concentrations ranging from 5 μM to 50 μM, 15 μM to 30 μM, or 10 μM to 20 μM. Furthermore, the identification methods include measuring inhibition of Tpm1.8 and / or Tpm1.9 expression and / or activity in cancer cells, wherein finding inhibition (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of an appropriate (untreated) control) identifies the test compound as a compound used in methods for inhibiting, suppressing, preventing, and / or treating cancer. For example, compared with untreated cancer cells and / or cancer cells prior to the use of compounds targeting the Tpm1.8 and / or Tpm1.9 isomers, the compounds identified by the methods used for identification inhibited the expression and / or activity of Tpm1.8 and / or Tpm1.9 by 10% to 100%, 20% to 95%, 25% to 90%, 30% to 85%, 35% to 80%, 40% to 75%, 45% to 70%, 50% to 65%, or 55% to 60%. For example, compared with untreated cancer cells, and / or with cancer cells prior to the use of compounds targeting the Tpm1.8 and / or Tpm1.9 isomers, the compounds identified by the methods used for identification inhibit the expression and / or activity of Tpm1.8 and / or Tpm1.9 by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%.

[0118] The present invention also provides a method for determining the presence of epithelial-mesenchymal transition (EMT) in cancer cells, comprising determining the expression of Tpm1.8 and / or Tpm1.9 in said cells, whereby increased expression relative to control expression indicates the presence of EMT in said cells. Control expression may be, for example, the expression of a housekeeping gene (e.g., GAPDH). Expression is preferably determined by measuring (m)RNA levels, but may also be measured by measuring proteins as described elsewhere herein.

[0119] The present invention also provides a method for determining the presence of epithelial-mesenchymal transition (EMT) in cancer cells, comprising determining the expression of RBM24 and / or ESRP1 in the cells, wherein downregulation of ESRP1 (or reduced expression relative to control expression) and upregulation of RBM24 (or increased expression relative to control expression) indicate the presence of EMT in the cells.

[0120] The present invention also provides the use of RBM24 and / or ESRP1 and / or Tpm1.8 / 9 as markers for determining cancer development, particularly for indicating that cancer cells (preferably ovarian cancer cells) are in the EMT stage.

[0121] For the purposes of clarity and concise description, the features are described herein as part of the same or separate implementations; however, it should be understood that this disclosure includes implementations having all or some of the described features in combination.

[0122] The present invention will now be illustrated by the following examples, which are for illustrative purposes only.

[0123] Example

[0124] Example 1

[0125] In high-grade serous ovarian cancer, selective splicing of broad-spectrum target genes forms the basis for epithelial-mesenchymal cell plasticity, metastatic spread, and chemotherapy resistance.

[0126] Materials and methods

[0127] Cell culture

[0128] The human ovarian cancer cell line OV90, obtained from the American Type Culture Collection (ATCC), was cultured in a 1:1 mixture of MCDB105 medium (M6395; Sigma Aldrich containing 1.5 g / L sodium bicarbonate) supplemented with 15% heat-inactivated fetal bovine serum (FBS; #16140071, Thermo Fisher Scientific) and 1% penicillin / streptomycin (Pen / Strep; penicillin: 100 U / mL, streptomycin: 100 μg / mL; 15140122 Thermo Fisher Scientific) and medium 199 (31150022; Thermo Fisher Scientific, containing 2.2 g / L sodium bicarbonate). CAOV3 (ATCC), SKOV3 [European Certified Cell Culture Collection (ECACC) of Sigma], COV504 (ECACC), and HEK293T (ATCC) cell lines were cultured in DMEM medium (11965092, Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine (200 mM; 25030081; Thermo Fisher Scientific), and 1% Pen / Strep. PEA1 (ECACC) and PEA2 (ECACC) cell lines were cultured in RPMI 1640 medium (61870036, Thermo Fisher Scientific) containing 10% FBS, 1% Pen / Strep, and 2 mM L-glutamine.

[0129] The identity of each cell line was confirmed by DNA fingerprinting using microsatellite markers (amelogenin, CSF1PO, D13S317, D16S539, D5S818, D7S820, THO1, TPOX, vWA, D8S1179, FGA, Penta E, Penta D, D18S51, D3S1358, D21S11) and compared with similar data provided by ATCC, EACC and https: / / web.expasy.org / cellosaurus / (data not shown).

[0130] Plasmid transfection and lentiviral transduction

[0131] cDNA encoding Tpm1.6, Tpm1.7, Tpm1.8, and Tpm1.9 was excised from bacterial expression vectors pGEX or pET [from PWG Schevzov et al., Tropomyosin isoforms and reagents, Bioarchitecture, 2011;1(4):135-64] and cloned into mammalian expression vector pcDNA3.1(+). Stable transfection of ESRP1 (Sino Biological plasmid #HG13708-UT) and Tpm1.6 / 7 / 8 / 9 expression vectors was performed using FuGENE HD transfection reagent (Promega, E2311) according to the manufacturer's protocol, and selection was performed using genimycin (#10131035, Thermo Fisher Scientific).

[0132] Using pDONR 233-RBM24 (Horizon, #OHS6084) as the starter plasmid, the inducible pSLIK-RBM24 vector was constructed following the Gateway Cloning instruction manual (11791-020, Thermo Fisher Scientific). The inducible shZEB1 lentiviral vector was obtained as described in our previous study (Reference 18). For all the above inducible vectors, the ESRP1 (Horizon, V3THS_335722) and shRBM24 (Horizon, V3SH11240-225117283) lentiviral constructs were packaged into HEK293T cells via psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259). The virus-containing supernatant was collected 24 h post-transfection, filtered, and used to infect OV90 and COV504 cell lines. Selection was performed using 750 ng / mL puromycin (#ant-pr-1, InvivoGen) or 800 μg / mL genimycin for 1 to 2 weeks. Ectopic expression and knockdown of the target gene were verified by qPCR and Western blotting 72 h post-transfection.

[0133] siRNA transfection

[0134] The target sequence for siRNA used in human Tpm1.6 / 7 is 5'-AAGCTGGAGCTGGCAGAGAAA-3' (codons 70 to 76 of Tpm1.6 / 7 in exon 2b), and the target sequence for siRNA used in human Tpm1.8 / 9 is 5'-CGAGAGGAAGCTGAGGGAGAC-3' (codons 38 to 44 of Tpm1.8 / 9 in exon 1b). Tpm siRNA (Horizon Discovery, Waterbeach, UK) and control siRNA (#4390843, Thermo Fisher Scientific) were transfected using Lipofectamine RNAiMAX (#13778150, Invitrogen) according to the manufacturer's instructions. Cells were collected 72 hours after siRNA transfection for RNA and protein analysis. To assess the effect of gene knockdown on drug resistance, cells were seeded at a density of 5,000 cells / well 24 hours after siRNA transfection and then incubated for 3 days in the presence of cisplatin (#PHR1624, Sigma-Aldrich) or paclitaxel (#S1150, Selleck Chemicals).

[0135] RT-qPCT and PCR analysis

[0136] Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, 15596018) according to the manufacturer's instructions, followed by reverse transcription using a high-capacity cDNA reverse transcription kit (Life Technologies, 4368814). RT-qPCR was performed using Fast SYBR Green premix (#4368708, Thermo Fisher Scientific) on an AppliedBiosystems StepOne Plus real-time thermal cycling research device, with three replicates per analysis. Relative gene expression was determined by normalizing the expression of each target gene relative to GAPDH expression. Results were analyzed using the 2-(ΔΔCt) method. RT-qPCR primers are listed in Supplementary Table 4.

[0137] Western blot analysis

[0138] Cells were lysed in 2×Laemmli buffer (4% SDS, 48% Tris 0.5M pH 6.8, 20% glycerol, 18% H2O, bromophenol blue, and 10% 1M DTT) and subjected to SDS-PAGE, followed by transfer to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). After blocking with TBS-Tween containing 5% milk, the membranes were incubated with primary antibodies against ESRP1 (1:1000, # PA5-25833, Thermo Fisher Scientific), RBM24 (1:100, #18178-1-417 AP, Proteintech), TPM1 (1:1000, #MBS127505, MyBioSource), and β-actin (1:2000, #4970, Cell Signaling). The secondary antibodies were goat anti-rabbit immunoglobulin / HRP (1:10000, #P0161, DAKO) and (1:10000, #P0448, DAKO). Detection was performed using an Amersham AI600 imaging system (GE Healthcare) via Pierce ECT protein blot substrate (#34578, Thermo Fisher Scientific).

[0139] Flow cytometry analysis and sorting

[0140] Single-cell suspensions in PBS supplemented with 1% FBS were incubated on ice for 30 min with anti-EpCAM-FITC (1:20, #GTX30708, Genetex) and anti-CD44-APC (1:20, #559250, BD Pharmingen) antibodies and analyzed on a FACSAria III cell sorter (BD Biosciences). CD44 cells were sorted. 高 EpCAM 高 and CD44 高 EpCAM 低OV90 and CAOV3 cells were incubated at 37°C in a humidified atmosphere with 5% CO2 for 3 to 5 days, and then RNA or protein was collected as described above. First, patient-derived ascites fluid was washed once or twice with 1×RBC lysis buffer [150 mM NH4Cl (#7173-51-5, Sigma Aldrich), 10 mM KHCO3 (#298-14-6, Sigma Aldrich), 100 µM EDTA (#60-00-4, Sigma Aldrich)] to remove red blood cells. The cell pellet was then treated with anti-CD90.1 (Brilliant® Violet 421, 1:20, #328122, clone: ​​5E10, BioLegend), anti-CD45-APC (1:20, #304037, clone: ​​HI30, BioLegend), and SYTOX. TM The cells were labeled with a Red (1:1000, #S34859, Thermo Fisher Scientific) antibody and sorted by flow cytometry (FACS). RNA from CD45-CD90- and CD45-CD90+ cells was directly separated after FACS sorting.

[0141] Cell proliferation assay

[0142] To analyze cell proliferation rate, 2×10 3 Ovarian cancer parental cells and Tpm1.6 / 7 / 8 / 9-OE cells were seeded into 96-well plates and incubated at 37°C and 5% CO2. After 24 hours (Day 1), the cells were incubated for 3 hours at 37°C and 5% CO2 in medium supplemented with 0.45 mg / mL MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide; Sigma-Aldrich]. The 96-well plates were then centrifuged at 1,000 rpm for 5 min and the medium was removed. The optical density (OD) was read at 595 nm using a microplate reader (Model 550, Bio-Rad). Background measurements were subtracted from each data point. Triple replicates were performed for each individual cell line.

[0143] Cell migration and invasion assays

[0144] Using PET Transwel cells with an 8μm pore size (#353097, BD Falcon) TM ) and TC-treated multi-well cell culture plates (#353047, BD Falcon) TM Migration was measured using 5 × 10⁻⁶ particles. 4443 cells were seeded in the upper chamber containing 100 μL of serum-free medium. Medium supplemented with 10% FBS was used as a chemical attractant in the lower chamber. After 24 h, cells that had migrated to the lower chamber were fixed with 446 4% PFA (#9713.9010, VWR Chemicals), stained with 0.1% trypan blue solution (#15250061, Thermo Fisher Scientific), and counted under a microscope. Invasion assays were performed as described above, with 200 μL of Matrigel mixed with 10 μL to 20 μL of 0.01 M Tris (pH 8.0) / 0.7% NaCl solution added only to the top of the Transwell-Clear chamber, and incubated at 37°C for 2 h. After removing excess liquid, 5 × 10⁻⁶ cells were seeded. 4 450 cells were added to each well in 150 μL of serum-free medium. Then, 0.5 mL of medium supplemented with 10% FCS was added. After 24 hours, cells that had invaded the lower chamber were fixed with 4% PFA and stained with 0.1% trypan blue. Cells were counted using a microscope.

[0145] 200 μL of collagen premix [7.5% 10×PBS, 57% collagen, 0.1% 1M NaOH, 9.5% H2O, and 25% culture medium] was plated onto a scaffold and incubated at 37°C for 1 h. 5×10 4 Cells were added to the top of a solid collagen substrate and incubated at 37°C. After 1 week, the entire scaffold containing cells and collagen was fixed with 4% PFA and embedded in paraffin. 4 μm sections were mounted and counterstained with hematoxylin. The slides were dehydrated and mounted in Pertex (#00811, Histolab).

[0146] Immunohistochemistry - Cultured Cells

[0147] 0.5×10 4 Up to 1×10 4Cells were seeded into 24-well plates containing coverslips coated with 0.2% gelatin. After 6 to 24 hours, the culture medium was removed, ice-cold methanol was added to each well, and the plates were incubated at 40°C for 20 minutes. Cells were washed twice with PBS and incubated by rotation in 0.2% Triton for 20 minutes. Cells were blocked in PBS containing 2% FBS for 1 hour. Primary antibodies of Tpm1.6 / 7 (1:200, from PWG), Tpm1.8 / 9 (1:200, from PWG), and Arp2 (1:200, #ab47654, Abcam) were added, and the plates were incubated overnight at 40°C. Cells were washed twice with PBS and then supplemented with secondary antibodies [goat anti-rat Alexa Fluor 488 conjugate (1:250, #A10528, Life Technologies); goat anti-rabbit Alexa Fluor 546 conjugate (1:250, #A11035, Life Technologies); goat anti-mouse Alexa Fluor 647 conjugate (1:250, #A32728, Life Technologies)] and DAPI (#D1306, Thermo Fisher Scientific). Slides were mounted using VECTASHIELD® (#H100010, VECTOR Laboratories), and cells were imaged using an LSM-700 (Zeiss) with 20×, 40×, and 63× objectives. Images were analyzed using ImageJ.

[0148] Immunochemistry - Patient Samples

[0149] Formalin-fixed, paraffin-embedded (FFPE) tissue blocks from ovarian cancer patients were obtained from the Department of Pathology, Erasmus Medical Center, Rotterdam. The mean fixation time was 1 to 2 years. 4 μm sections were mounted on slides, dewaxed with xylene (#28979.294, VWR Chemicals), and hydrated. Antigen retrieval was performed in Tris-EDTA buffer (pH 9.0) using a pressure cooker program (25). Slides were incubated with 3% catalase (#95321, Sigma Aldrich) at room temperature for 10 min and blocked with PBS-Tween solution (#P1379, Sigma Aldrich) containing 5% milk (#115363, Millipore) for 30 min. Immunohistochemical staining was performed using antibodies against Tpm1.6 / 7 (1:100), Tpm1.8 / 9 (1:100), and mitochondria (1:100, #MAB1273, Sigma Aldrich), followed by detection using the EnVision Plus-HRP system (Dako). Slides were incubated overnight at 4°C with the primary antibody, washed twice with PBS-Tween, and incubated for 30 min with either rat EnVision+ System-HRP (#P0405, Dako) or mouse EnVision+ System-HRP (#K4007, Dako). Slides were counterstained with hematoxylin (#MHS16, Sigma Aldrich).

[0150] In vivo studies

[0151] Mouse experiments were conducted according to the guidelines for experimental animal testing in cancer research (Netherlands Health, Trade and Veterinary Public Health Inspection Agency) and the Animal Experimentation Committee (DEC). Six- to eight-week-old female NOD.Cg-PrkdcscidIl2rgtm1Wjl 485 / SzJ (NSG) mice were used. 1×10⁻⁶ mice containing the Tpm1.6 / 7 / 8 / 9 isoform overexpressing were used. 5Each mouse received an intraperitoneal (IP) injection of 50 μL PBS containing 486OV90 cells. 150 mg / kg D-luciferin (#L2916, Invitrogen) was injected intraperitoneally for bioluminescence signaling. Ten minutes after anesthesia induced by isoflurane (#B506, Zoetis), bioluminescence was measured using an IVIS spectral imaging system (Caliper Life Science, Hopkinton, MA), and analyzed using LIVINGIMAGE 4.4 software (Caliper Life Science). Ascites fluid was obtained via syringe, tumor organoids were collected, and washed with RBC lysis buffer. Mice were euthanized, and tissues were fixed in 4% PFA for further analysis.

[0152] Mouse tumor organoid culture

[0153] Ascites fluid from mice containing tumor organoids was collected. The tumor organoids were washed once or twice with RBC lysis buffer and then plated in 24-well ultra-low adsorption surface culture plates (#33019010, Corning). The tumor organoids were cultured in a 1:1 mixture of OV90 medium and advanced DMEM / F12 medium (#2322978; Thermo Fisher Scientific) containing 4% B27 (#A1895601, Life Technologies), 2% N-2 supplement (#11520536, Thermo Fisher Scientific), and 0.04% EGF (#PMG8045, Invitrogen).

[0154] BaseScope Measurement

[0155] BaseScope assays were performed according to the operating guidelines of ACD (Advanced Cell Diagnostics, Newark, California). 4 μm sections were cut onto Superfrost plus slides (#10149870, Thermo Fisher Scientific) and stored overnight at room temperature. Sections were baked at 60 °C for 1 h, then dewaxed in xylene and 100% ethanol. Sections were dried at 60 °C for 5 min, incubated in hydrogen peroxide at room temperature for 10 min, subjected to target retrieval at 100 °C for 15 min, and then treated with protease at 40 °C for 30 min. Add the BaseScope probe and incubate the slide at 40°C for 2 hours in an oven. Then add reagents AMP1 (incubate at 40°C for 30 min), AMP2 (incubate at 40°C for 30 min), AMP3 (incubate at 40°C for 15 min), AMP4 (incubate at 40°C for 30 min), AMP5 (incubate at 40°C for 30 min), AMP6 (incubate at room temperature for 15 min), AMP7 (incubate at room temperature for 30 min), and AMP8 (incubate at room temperature for 15 min). Add Solid Red A and Solid Red B to the slide and incubate at room temperature for 10 min, then counterstain with Girdle hematoxylin. Dry the slide at 60°C for 15 min, then mount it in VectaMount permanent mounting medium (H5000, Vector Labs). Image using an LSM-700 with a 40× objective lens.

[0156] Chemotherapy resistance and IC50 measurement

[0157] Cells were seeded at 5000 cells / well in 96-well plates and allowed to adhere overnight. Three technical replicates were performed for each test condition. Cisplatin (#PHR1624, Sigma-Aldrich) and paclitaxel (#S1150, Selleck Chemicals) were dissolved in DMSO (#D2650, Sigma-Aldrich). Cells were incubated with cisplatin and paclitaxel for 3 days. After removal of the chemotherapy drugs, cells were washed with PBS and allowed to regrow in standard medium for 1 day. Cell viability was assessed using the MTT assay as previously described (Sacchetti et al., eLife. 2021;10). Absolute viability values ​​were converted to a percentage of viability compared to the DMSO control treatment, and then a non-linear log(inhibitor) versus response was fitted in GraphPad Prism v7.0 to obtain IC50 values.

[0158] TOP-Flash reporter gene assay

[0159] For the β-catenin / TCF reporter gene assay (TOP-Flash reporter gene assay), cells were plated in 48-well dishes. After 48 hours, when 70% confluence was reached, cells were transfected using Fugene HD with 125 ng of TOP-Flash or FOP-Flash reporter gene construct and 25 ng of René luciferase vector for normalization purposes. Luciferase activity was measured 24 hours post-transfection using a dual-luciferase reporter gene assay system (#E1910, Promega). Luminescence intensity was measured using a GloMax luminescence detector (#9100-102, Promega).

[0160] Identification of compounds targeting Tpm1.8 / 1.9

[0161] A model of the N-terminus of human Tpm1.8 (identical to Tpm1.9) was constructed, containing the most diverse regions among the four TPM genes (residues 4 through 16). Virtual computer-aided docking screening was performed using the ZINC database, and six compounds with the best docking scores were purchased from the vendor. Human fibroblasts were exposed to each of the six compounds, or individually, for 24 h, fixed, and stained with isoform-specific antibodies against Tpm1.8 / 9 (Brayford et al., 2016. Current biology: CB. 26(10):1312-8). Control cells showed strong enrichment of Tpm1.8 / 9 in lamellae (Brayford et al., 2016). Two of the compounds, Tpm1.8 / 9-1 and Tpm-3, prevented its enrichment in lamellae at concentrations of 10 μM and 20 μM, respectively. These were selected for further investigation.

[0162] Selective splicing analysis

[0163] EpCAM high / low RNA sequencing data were obtained from ovarian cancer cell lines OV90 and CAOV3. Sequencing reads were aligned to GRCh37.p13 using STAR55 (https: / / www.gencodegenes.org / human / release_19.html). MISO56 was used to quantify AS events, with annotation information from https: / / miso.readthedocs.io / en / fastmiso / index.html#iso-centric. MISO uses selective exon reads and adjacent conserved reads to measure the percentage of transcript isoforms containing a specific exon, referred to as the splice percentage (PSI or Ψ). The PSI ranges from 0 (i.e., no isoform contains a specific selective exon) to 1 (i.e., all detected isoforms contain a selective exon). If fewer than three samples in the dataset had more than 10 informative reads for PSI calculation, surrogate events with low expression of the relevant transcript isoform were removed. Next, we compared EpCAM in the OV90 and CAOV3 ovarian cancer cell lines. 高 and EpCAM 低 PSI between groups. An AS event is defined as a differential splicing event when the mean PSI difference (Δpsi; percentage of differential splicing) between two groups is >10%.

[0164] RNA sequencing analysis of subpopulations in OV90 and CAOV3

[0165] RNA was isolated from the sorted population using Trizol reagent. Libraries were prepared using the TruSeq RNA Sample Preparation Kit v2 (Erasmus MC, Biomics). Samples were sequenced using Illumina HiSeq 2000, and adapter sequences were removed using Trimmomatic (v.0.33). Subsequently, the sequencing reads were aligned to the human reference genome hg38 using a paired-end alignment strategy with the RNA-seq alignment software STAR (v2.4.2a) (Dobin et al., Bioinformatics. 2013;29(1):15-21), and annotation information was obtained using Homo sapiens GENCODE v23. The raw reads were imported into DESeq2 (v1.36.0) and normalized using variance stable transformation (VST) (Love et al., Genome Biol. 2014;15(12):550). Differentially expressed genes were identified by comparing Epcamlow versus Epcamhigh / batch samples using absolute LogFC > 1.5 and corrected P(padj) < 0.05, and visualized using ComplexHeatmap (v2.12.1) after z-score normalization. Pathway activity was assessed using gene set enrichment analysis (fgsea v1.22.0), with the feature gene set derived from the Molecular Signature database (Korotkevich et al., 2021. bioRxiv www.doi.org / 10.1101 / 060012).

[0166] RNA sequencing analysis of TPM1 OE in OV90

[0167] Paired-end mRNA sequencing was performed using DNA nanosphere sequencing (DNBseq) technology to achieve a depth of 25M reads per sample (Shenzhen BGI Genomics). Adapter trimming and quality filtering were performed using the SOAPnuke pipeline (BGI Genomics). The cleaned fastq files were aligned with the GRCh37 reference genome using STAR alignment software (v2.7.9a) (Dobin et al., Bioinformatics. 2013;29(1):15-21) combined with RSEM (Li et al., BMC Bioinformatics. 2011;12:323). Gene-level data were imported using tximport (v1.24.0) and downstream analysis was performed using DESeq2 (v1.36.0) (Love et al., Genome Biol. 2014;15(12):550). Counts were normalized using variance stabilization transformation (VST). Gene set activity was assessed using a feature gene set from the Molecular Signature database using Gene Set Variation Analysis (GSVA, v1.44.5) (Hanzelmann et al., BMC Bioinformatics. 2013;14:7), and visualized using the ComplexHeatmap package (v2.12.1). Principal component analysis was performed using the top 500 genes with the highest row variance. Differential expression analysis was performed by comparing TPM1.6 / 7 samples with TPM1.8 / 9 samples, and the results were displayed using volcano plots with EnhancedVolcano (v1.14.0).

[0168] scRNA sequencing analysis of ovarian cancer cells

[0169] Publicly available data from Vázquez-García et al. were retrieved from the CellxGene portal (Vazquez-Garcia et al., Nature. 2022;612(7941):778-86). Downstream analysis was performed in Seurat (v4.3.0) (Hao et al., Cell. 2021;184(13):3573-87 e29). The Epcamlow tag was evaluated using AddModuleScore based on a previously identified list of upregulated genes (N=38). A threshold (>0.1) was used to annotate cells with the highest association with the Epcamlow feature (“low-expression-like cells”, representing 4% of cancer cells). Next, low-expression-like cells were visualized on the integrated UMAP dimensionality-reduced embedding map from Vázquez-García et al. (ibid.), and pathway activity of clusters containing low-expression-like cells was visualized using ComplexHeatmap. After the data transfer protocol was approved, the FASTQ file from Izar et al. (Nat Med. 2020;26(8):1271-9) was downloaded from the TerraBio repository, processed by RSEM, and aligned to the hg19 human reference genome using STAR alignment software, with isoform annotations from UCSC (Love et al., Genome Biol. 2014;15(12):550; Li et al., BMC Bioinformatics. 2011;12:323). The file was imported using tximport (v1.24.0), and cells containing at least 500 different genes (nFeature_RNA > 500) were selected. Cells were clustered according to the percentage of their respective TPM1 isoform expression (k-means, k=4), and subsequent analysis was performed in Seurat (v4.3.0), where cells expressing TPM1.7 were compared with TPM1.9 using FindMarkers. Gene set enrichment analysis was performed using a feature gene set, and pathways were filtered based on similar activity in OV90 cell lines and patient data.

[0170] Survival analysis of TPM1 in TCGA OVCA

[0171] RSEM-processed data from the TCGA cohort were downloaded from tsvDB (Sun et al., BMC Genomics. 2018;19(1):405). The data were log2 transformed and survival analysis was performed using the survival package. Based on the clinical data of overall survival, survival curves were generated using the survminer package for the entire TPM1 gene, TPM1.7 (isoform _uc002alk), and TPM1.9 (isoform _uc002alt) (Therneau & Grambsch. Modeling Survival Data: Extending the Cox Model. Springer-Verlag New York 2000).

[0172] Data availability

[0173] RNA sequencing data have been deposited into the Gene Expression Omnibus (GEO) database and can be accessed using the following accession numbers: GSE192920 (subpopulations in OV90 and CAOV3), GSE231560 (TPM1 OE in OV90). Other single-cell RNA sequencing data used in this study are publicly available, and SmartSeq2 data [GSE146026 (Izar et al., ibid.)] and 10× Genomics data from Synapse [syn25569736 (Vázquez-García et al., ibid.)] are available from GEO.

[0174] Statistical analysis

[0175] For statistical comparisons, we used unpaired t-tests. Statistical analysis was performed using Prism 7 software (GraphPad). Statistically significant data are shown in the figures. Information regarding repeatability, independent experiments, and statistical tests can be found in the descriptions of the figures.

[0176] result

[0177] In high-grade serous ovarian cancer, a subset of quasi-stromal cells coexists with epithelial cells.

[0178] Following the previously employed experimental strategy for identifying quasi-mesenchymal cell subsets in immortalized colon cancer cell lines, we analyzed HGSOC cell lines OV90, SKOV3, COV504, and CAOV3 via FACS using CD44 and EpCAM antibodies. 高 EpCAM 高 Cells and CD44 高 EpCAM 低Cells (hereafter referred to as EpCAM) 高 and EpCAM 低 Different distributions of EpCAM cells were used to label each cell line, although the percentages varied. After sorting and short-term culture, EpCAM... 低 The cells exhibited a mesenchymal-like morphology, similar to their EpCAM. 高 The epithelial appearance of the cells creates a contrast. Therefore, EpCAM 低 Cells exhibited increased migration and invasion capabilities in trans-well assays. To elucidate the global gene expression profiles of two distinct subsets of ovarian cancer cells, EpCAM cells sorted from OV90 and CAOV3 cell lines by FACS were analyzed. 高 / 低 Cells were subjected to RNA sequencing analysis.

[0179] Principal component analysis (PCA) using multidimensional scaling (MDS) revealed the quasi-interstitial EpCAM. 低 The cells are clearly separated from their epithelial cells in the second dimension. Unsupervised clustering of RNA sequencing data highlights EpCAM from both cell lines. 低 and EpCAM 高 Differentially expressed genes among subgroups. Among these genes, different EMT transcription factors (EMT-TF; ZEB 1 / 2, RUNX 2, SNAI 2), EMT target genes (CDH1, VIM), and EMT-related RBPs (ESRP1, RBM24) mark EpCAM in OV90 and CAOV3. 低 Cells. Therefore, pathway analysis (PA) of genes expressing markers in epCAM-like ovarian cancer cells revealed significant associations with EMT, KRAS signaling, and several TNF and interferon-related inflammatory pathways. To confirm the role of EMT in the establishment of epCAM... 低 The central role played by the identity, as shown in PA analysis, is the downregulation of ZEB1 expression via shRNA in OV90, SKOV3, and COV504 cell lines. As expected, FACS analysis of these cells showed that knockdown of this differentially expressed EMT-TF led to EpCAM. 低 A significant reduction in subpopulations. To demonstrate the clinical relevance of the above data obtained from immortalized cancer cell lines, publicly available single-cell RNA sequencing data from ovarian cancer derived from 42 patients were used to search for associations with EpCAM. 低 Subpopulations of quasi-mesenchymal cells. For this purpose, we utilized EpCAM obtained from the aforementioned RNA sequencing analysis of OV90 and CAOV3 cell lines. 低 Features. According to EpCAM 低After classifying the cancer cells from the patient using specific characteristics, approximately 60% were found to cluster in one of the three subpopulations of HGS ovarian cancer cells classified as EMT-like cells (labeled #2, #4, and #6) as defined in the original study by Vazquez-Garcia et al. (Ovarian cancer mutational processes drive site-specific immune invasion, Nature, 2022;612(7941:778-86). Notably, a significant proportion of EpCAM cells... 低 The presence of cells outside the three clusters indicates significant heterogeneity in cellular identity, reminiscent of a quasi-mesenchymal state within a cell line. Compared to primary tumors, cells from EpCAM... 低 Cells within the cluster were present in higher proportions in metastatic lesions and ascites (i.e., non-adnexal tissues, including the omentum, peritoneum, etc.). We then evaluated EpCAM in primary ovarian cancer (adnexal tissues), ascites, and metastatic lesions (non-adnexal tissues). 低 Module scoring of the characteristic gene set of the sample cluster. EMT and inflammatory signaling pathways (TNFα, IL6 / Jak / Stat3, IFN, and TGFβ) in EpCAM 低 It was significantly upregulated in the sample clusters, especially in cluster #2 in ascites and metastatic lesions.

[0180] In summary, these results indicate that the HGSOC cell line comprises a subset of quasi-mesenchymal cells with increased motility and invasiveness, characterized by EMT-TF expression, which is crucial to its cellular identity. Similar subsets of ovarian cancer cells are present in patient-derived primary and metastatic lesions, as well as in malignant ascites.

[0181] Differential expression of RNA-binding proteins forms the basis for selective splicing of a subset of target genes in stromal ovarian cancer cells.

[0182] Several RNA-binding proteins (RBPs) involved in alternative splicing are known to be present in EpCAM in colon cancer cell lines. 低 and EpCAM 高 Differential expression exists between cells, and these cells play important functional roles in controlling epithelial-mesenchymal transition (E-to-M) and mesenchymal-epithelial transition (M-to-E) and phenotypic plasticity during local spread and distant metastasis. Several differentially expressed RBPs may play an active role in EMT-related alternative splicing between epithelial-like and mesenchymal-like ovarian cancer cell subsets. ESRP1 and RBM24 are present in EpCAM cells in both cell lines. 低EpCAM-1 was downregulated and upregulated in cells, respectively. Furthermore, other RBPs (i.e., ESRP2, RBM47, RBMS3, and QKI) showed differential expression in one of the cell lines examined. EpCAM-1 of RBM4 and ESRP1 was also downregulated. 低 Specific upregulation and downregulation were validated by RT-qPCR and Western blot analysis of subsets sorted from OV90 and C0V504 cell lines, respectively.

[0183] Genes with differential splicing were analyzed using MISO (mixture of isomers), and the results were filtered by selecting those with a ΔPSI (percentage of differential splicing) value >10% and comparing them with a corresponding list of AS targets previously identified in colon cancer cell lines. The vast majority of ovarian cancer AS targets were not found in colon cancer. Among the ovarian-specific AS targets (n=39) with known functions in EMT, these represented different cellular components, such as the extracellular matrix, focal adhesions, and actin cytoskeleton, as well as cellular processes such as ECM organization, integrins, and TGF-β-mediated signal transduction and cell migration. Figure 3 In particular, the presence of tropomyosin 1 (TPM1) (a member of the actin-binding protein panfamily) is noteworthy due to its high ΔPSI values ​​in OV90 and CAOV3 cells and its known cellular functions (i.e., its role in the cytoskeleton of non-muscle cells and in the contractile systems of striated and smooth muscle). Although a direct causal relationship between TPM1 and EMT has not been reported, previous studies have shown that TGF-β signaling increases the expression of high molecular weight tropomyosin and the formation of actin stress fibers, thereby affecting cell motility and invasion.

[0184] Notably, the TPM1 AS pattern observed in ovarian cancer involves exons 1a / 2a and 1b, with exons 1a / 2a marking EpCAM. 高 Upregulated Tpm1.6 / 7 isoforms in cells, exon 1b marking EpCAM 低 Upregulated Tpm1.8 / 9 isoforms in cells were observed, as verified by RT-qPCR and Western blot analysis. Notably, the Western blot results for Tpm1.8 / 9 showed a clear match with RNA-based analysis, while this was less pronounced for Tpm1.6 / 7, which appeared to undergo more subtle changes at the protein level. Further validation of these results was achieved using only EpCAM. 高 (PEA2) or EpCAM 低 The HGS ovarian cancer cell line (PEA1) expressed only the TPM1.6 / 7 and TPM1.8 / 9 isoforms, respectively. To establish EpCAM using TPM1 as a model... 低The causal relationship between differential expression of ESRP1 and RBM24 in ovarian cancer cells and observed downstream AS targets was determined by RBP knockdown and overexpression assays in OV90 and COV504 cell lines. Firstly, as per its unique EpCAM... 高 / 低 As predicted by the distribution, the PEA1 and PEA2 cell lines express RBM24 and ESRP1 independently, respectively. Therefore, ESRP1 knockdown and RBM24 ectopic expression in the OV90 and COV504 cell lines led to upregulation and downregulation of the Tpm1.8 / 9 and Tpm1.6 / 7 isoforms at the RNA and protein levels, respectively. Conversely, RBM24 knockdown and ESRP1 ectopic expression led to upregulation and downregulation of the Tpm1.6 / 7 and Tpm1.8 / 9 isoforms, respectively. Figure 1 A to Figure 1 B). Notably, the observed changes in RBP expression were also accompanied by changes in EpCAM. 低 A significant increase in subpopulations was observed, as confirmed by FACS analysis. Compared to assays of gain and loss of function of individual RBPs, simultaneous RBM24 overexpression and ESRP1 knockdown not only significantly affected TPM1 isoform conversion, but more importantly, also affected EpCAM. 高 / 低 The relative percentages of the subgroups had a significant impact. The latter indicates that alternative splicing driven by RBM24 upregulation and ESRP1 downregulation is a major regulator of EMT in ovarian cancer cells.

[0185] In summary, these results indicate that HGS ovarian cancer cells in the epithelial and pseudomesenchymal subsets are differentially expressed by specific RBPs known to be involved in alternative splicing. Therefore, differential AS patterns of EMT-related target gene subsets characterize EpCAM. 低 Cellular. While some AS target genes are shared with target genes previously identified in colorectal cancer, most appear to be ovarian cancer-specific. Among them, TPM1 has attracted attention because of its function as an actin-binding cytoskeletal protein in various cell types, and its previously reported role as a tumor suppressor in breast cancer.

[0186] TPM1 isoform targeting transcriptional and functional consequences in stromal ovarian cancer cells

[0187] To assess the functional relevance of specific TPM1 isoforms, ectopic expression was induced in various ovarian cancer cell lines (OV90, COV504, PEA1, and PEA2) and validated by RT-qPCR and Western blot analysis. Figure 2 A to Figure 2 B). Cell viability and proliferation assays showed that the expression of Tpm1.6 / 7 and Tpm1.8 / 9 were significantly correlated with increasing and decreasing cell division rates, respectively. Figure 2C). Notably, ectopic expression of Tpm1.8 / 9 in OV90 and COV504 cells resulted in complete growth arrest within 6 days. Furthermore, trans-well assays clearly showed that overexpression of Tpm1.8 / 9, compared to Tpm1.6 / 7, led to significantly increased migration and invasion signatures. Figure 2 D). Tpm1.8 / 9-overexpressing (OE) ovarian cancer cells appear to collectively invade the collagen layer in the form of narrow linear chains containing “leader” and “follower” cells. Figure 2 E). The latter observation is interesting because, in non-muscle cells, Tpm1.8 / 9 is specifically expressed in lamellae (membranous projections found at the leading edge), which are driven by branching actin and Tpm1.8 / 9, which promote cell movement, as well as unbranched filaments. Immunofluorescence (IF) analysis using a TPM1 isoform-specific antibody confirmed the co-localization of Tpm1.8 / 9 and ARP2 (a specific marker of lamellae) at the edge of ovarian cancer cells; conversely, Tpm1.6 / 7 is mainly localized in the cytoplasm ( Figure 2 F).

[0188] To conduct a more comprehensive study of the transcriptional and functional consequences of ectopic expression of specific TPM1 isoforms, RNA sequencing analysis was performed on OV90-overexpressing cells. Unsupervised hierarchical clustering and principal component analysis confirmed the distinct transcriptional identities of OV90 parents, Tpm1.6 / 7-OE, and Tpm1.8 / 1.9-OE cells. Gene set enrichment analysis (GSEA) was then performed to allow for the identification of specific signal transduction pathways and gene ontology functional characteristics for each of the aforementioned sample groups. Figure 3 As shown in the heatmap of A, TPM1.8 / 9 overexpression led to significant activation of Hedgehog, Wnt / β-catenin, TGF-β, and Notch signaling pathways (i.e., pathways known to be involved in EMT induction and regulation). Therefore, EMT was also found to serve as a marker for these cells, as illustrated by the different expression patterns of several EMT-related genes between Tpm1.6 / 7- and Tpm1.8 / 9-OE samples. Figure 3 B).

[0189] To functionally validate the activation of the classical Wnt / β-catenin signaling pathway, a Top-FLASH reporter gene assay was performed. Figure 3 As shown in C, an approximately 10-fold increase in luciferase activity was observed following ectopic expression of individual Tpm1.8 and 1.9 isoforms. Similarly, Wnt signaling activity was significantly reduced after Tpm1.8 / 9 siRNA-driven knockdown.

[0190] In summary, these results indicate that TPM1 isoforms confer specific functional characteristics on ovarian cancer cells. In particular, the Tpm1.8 / 9 isoforms are closely associated with EMT induction and inflammatory signaling pathways, which likely form the basis for the transcavitary spread of ovarian cancer cells and ascites formation. However, their ectopic overexpression is also a potential source of artifacts, as EMT and MET are equally important in the formation of intraperitoneal metastases.

[0191] The Tpm1.8 / 9 isomer confers resistance to taxane- and platinum-based chemotherapy and is expressed in ascites fluid from ovarian cancer patients.

[0192] To investigate the role of TPM1 alternative splicing in female patients with ovarian cancer, tumor tissues from patients were examined using isoform-specific antibodies and oligonucleotide probes via immunohistochemistry (IHC) and in situ hybridization (ISH). In the analyzed high-grade and low-grade serous tumors (n=13), Tpm1.6 / 7 appeared to be consistently expressed in both primary and metastatic lesions. Figure 4A). Conversely, Tpm1.8 / 9 expression was barely detectable above background levels, with focal and enhancing staining observed in a few cases. While based on a generally limited number of tumors, these observations appear to suggest that although the Tpm1.6 / 7 isoforms are predominantly expressed in most epithelial tumor cells, their corresponding Tpm1.8 / 9 isoforms are rarely observed, possibly only associated with advanced disease and chemoresistant stages. Intraperitoneal ascites formation primarily occurs in stage III and IV ovarian cancer patients due to tumor cell spread to the peritoneum and lymphatic obstruction. Therefore, tumor cells derived from ascites may contain quasi-mesenchymal and chemoresistant cell types along the metastatic route during metastatic colonization of abdominal organs. Therefore, we evaluated whether the Tpm1.8 / 9 isoforms, compared to Tpm1.6 / 7, are transiently expressed in ascites from ovarian cancer patients. Ascites fluid samples were collected from n=13 patients, and cellular contents were sorted via FACS according to [CD45(Lin)- / CD90+ 233] and [CD45(Lin)- / CD90-] gating (covering immune / stromal and cancer cells, respectively). Total RNA was then extracted from the sorted cells for RT-qPCR analysis of TPM1 isoform expression. Increased Tpm1.8 / 9 expression was observed in ascites-derived cancer cells (CD90-) compared to Tpm1.6 / 7. In immune / stromal cells (CD90+ 237), Tpm1.6 / 7 expression levels were increased compared to Tpm1.8 / 9. Finally, expression profiles from the Cancer Genome Atlas (TCGA) project and the TCGA splice variant database (TSVdb; http: / / www.tsvdb.com / ) were used, and clinical follow-up data were integrated with the expression of TPM1 (whole genome) and its isoforms. Kaplan-Meier analysis showed borderline significance for the TPM1 gene and its Tpm1.6 / 7 isoforms, while the p-values ​​for Tpm1.8 / 9 were all >0.05. Given the extremely low expression levels of these low molecular weight isoforms in primary ovarian cancer from which the TGCA data were derived, the p-values ​​for Tpm1.8 / 9 being >0.05 are not surprising.

[0193] Given that these results point to the upregulation of specific Tpm1.8 / 9 in ovarian cancer ascites, particularly in advanced and recurrent stages of disease, often accompanied by chemotherapy resistance and poor prognosis, we investigated whether these identical Tpm1 isoforms could confer resistance to platinum- and taxane-based therapies commonly used in the clinical management of ovarian cancer. Therefore, OV90 parental cell lines were cultured in the presence of two different chemotherapeutic agents (cisplatin and taxane), and the expression of TPM1 isoforms in the surviving cells after treatment was analyzed. RT-qPCR analysis showed that Tpm1.6 / 7 expression was significantly downregulated with both agents compared to untreated parental cells.

[0194] Conversely, in cells that survived treatment with cisplatin and paclitaxel, the expression of Tpm1.8 / 9 appeared to be increased. Figure 4 B). Dose-response curves of OV90 cells expressing a single isoform ectopically confirmed that, after cisplatin treatment, Tpm1.8 / 9-OE cells (IC50 = 3.266 / 3.414 µM) showed 40-fold higher resistance than Tpm1.6 / 7 OE cells (IC50 = 0.078 / 0.043 µM). Figure 4 C). The same results were observed with paclitaxel treatment (IC50 = 1.28 / 1.66 µM for Tpm1.8 / 9-OE cells; IC50 = 0.017 / 0.015 µM for Tpm1.6 / 7-OE cells). To validate these observations, an siRNA assay was developed to selectively downregulate the Tpm1.6 / 7 and Tpm1.8 / 9 isoforms in parental OV90 cells and assess their chemotherapeutic resistance. Figure 4 D to Figure 4 As shown in E, specific downregulation of the Tpm1.8 / 9 isoform, validated at both RNA and protein levels, reduced the specific IC50 for cisplatin and paclitaxel, while siRNA-driven knockdown of Tpm1.6 / 7 had the opposite effect.

[0195] TPM1.8 / 9 specific small molecule inhibitors for ovarian cancer therapy

[0196] Given the recently discovered role of the Tpm1.8 / 9 isoforms in ovarian cancer, and the potentially wide-ranging consequences of their specific inhibition at the cellular and molecular levels on cell motility and proliferation, EMT / MET, several oncogenic signaling pathways including Wnt, and therapy resistance, the development of small molecule antagonists may provide new tools for the clinical management of advanced ovarian cancer. Differences in the N- and C-termini of tropomyosin isoforms offer opportunities for developing compounds that preferentially target specific isoforms. Compounds targeting the C-terminus of Tpm3.1 have shown inhibition of this isoform's function in vitro and in vivo by incorporating overlapping links between adjacent dimers in actin / Tpm3.1 copolymers. Compounds targeting the N-terminus have shown similar activity. Based on the differences in the N-terminal sequences of exon 1b in the TPM1, TPM3, and TPM4 genes, compounds targeting Tpm4.2 and Tpm1.8 / 9 were developed. Compounds with high docking scores were identified using computer-aided docking of compound libraries with models. Six candidate compounds were identified for Tpm1.8 / 9 docking, and their bioactivity was tested in fibroblasts based on their ability to drive the target Tpm1.8 / 9 away from actin-containing structures in plate-like pseudopodia. Two compounds, 3-(2-methyl-indole-1-yl)-propylamine (PubChem CID 6494468) and 1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468), showed activity in the low micromolar range. To functionally validate these two compounds, they were tested in the OV90 parental cell line and in EpCAM cells sorted from it. 低 The most effective concentrations of these compounds in the subgroups were determined in the range of 0 to 10 μM (≤10 μM). RT-qPCR and Western blot analysis showed that neither compound affected the expression of Tpm1.6 / 7 or Tpm1.8 / 9 at the RNA or protein level. Figure 5 A to Figure 5 B). This is not unexpected, as the translocation of Tpm1.8 / 9 from the cell's actin filaments to the soluble library does not lead to Tpm1.8 / 9 renewal. However, the expression of EMT-related genes was affected by two compounds: ZEB1 and VIM expression were suppressed, while EpCAM expression was increased, which is also consistent with the expected MET-inducing effect of Tpm1.8 / 9 antagonists. Figure 5 A to Figure 5 B). Furthermore, compared to untreated cells, treated OV90 somatic cells and EpCAM... 低 Cells showed a 2- to 5-fold reduction in the specific IC50 values ​​for cisplatin and paclitaxel. Figure 5 C to Figure 5D). Finally, compounds 3-(2-methyl-indole-1-yl)-propylamine (PubChem CID 6494468) and 1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468) significantly reduced Wnt / β-catenin signal transduction, as shown in the TopFLASH reporter assay. As expected, with most EpCAM hi (Wnt-lo) and EpCAM lo Compared to the parental OV90 cell line with coexisting cells, EpCAM... lo The cells exhibited transcriptional profiles of Wnt-hi and EMT-hi, and the inhibitory effect of the compound on Wnt was more pronounced in these cells.

[0197] In summary, these in vitro results demonstrate the potential for future development of novel therapeutic strategies for high-grade serous ovarian cancer centered around the Tpm1.8 / 9 isoform.

[0198] discuss

[0199] Epithelial-mesenchymal plasticity (EMP), the transient and reversible identity conferred upon cancer cells by the EMT / MET process, is generally considered to be the basis for tumor progression, local invasion, distant metastasis, therapy resistance, and immune evasion. EMT is a highly variable process with a very broad range of upstream signaling, intracellular regulatory mechanisms, and downstream effector factors from the TME, and its identity largely depends on the tumor type and its micro and macro environments. The same is true when it comes to the nature of the epigenetic mechanisms underlying EMP. Recent studies have shown that differential expression of RNA-binding proteins between epithelial tumor host cells and stromal colon cancer cell subsets forms the basis for alternative splicing of various target genes known to function in EMT, metastasis, and chemotherapy resistance. Here, a similar strategy was employed to identify and functionally characterize genes that undergo splicing alterations during ovarian cancer EMT / MET. Comparison of alternatively spliced ​​genes between epithelial and stromal colon cancer cells and ovarian cancer cells revealed a few common targets, while the vast majority were ovarian-specific genes, which may reflect different patterns of local spread and metastatic colonization characteristics in these cancer types. While most colorectal cancers metastasize via the hematogenous route, ovarian cancer is unique in that it involves the "cross-cavity" spread of tumor cells and the formation of ascites in the abdominal and pelvic cavities, which provides a favorable tumor microenvironment (TME) for spreading cancer cells. Nevertheless, previous research has shown that EMT does indeed contribute to ovarian cancer progression and chemotherapy resistance. Therefore, even through different cellular and molecular mechanisms, EMT does play a crucial role in ovarian cancer metastasis and chemotherapy resistance. Gene ontology analysis of ovarian cancer-specific AS targets revealed an extremely broad range of biological processes, molecular functions, and cellular components that may collectively contribute to the transformation into quasi-mesenchymal ovarian cancer cells capable of local invasion and distant metastasis.

[0200] Previously, AS targets that may lead to ovarian cancer progression, such as BCL2L12 and ECM1, have been reported. Here, TPM1 was chosen among EMT-related AS targets because of its function in regulating cell motility through cytoskeleton modification, and its associated roles as a tumor suppressor and even an oncogene in various cancer types. Alternative splicing isoforms of TPM1 are present in various tissues. The current results establish a direct causal relationship between the Tpm1.8 / 9 isoforms, arising from the differential expression of several RBPs between epithelial and pseudo-mesenchymal ovarian cancer cells, and activating EMT through multiple signal transduction pathways, including Wnt, TGF-β, Hedgehog, and Notch. Notably, the respective upregulation and downregulation of the RBM24 and ESRP1 RNA-binding proteins signify epithelial-mesenchymal transition in the ovarian cancer cell lines examined here. Due to their localization to sheet-like pseudopodia and their functional roles in cell motility, the Tpm1.8 / 9 isoforms may promote the spread from the primary tumor to the peritoneal cavity, as evidenced by their enrichment in patient-derived ascites. As a result of its EMT-inducing ability, ectopic expression of the low molecular weight TPM1 isoform leads to resistance to taxane- and platinum-based chemotherapy. Thus, even with some expression, Tpm1.8 / 9 was found to be expressed at very low levels in primary ovarian cancer and its metastases, although expression increased in malignant ascites. Nevertheless, we attempted to provide in vivo evidence of the metastatic potential of ovarian cancer cells overexpressing the Tpm1.8 / 9 isoform, but failed to show any increase compared to Tpm1.6 / 7. This seemingly contradictory result could be explained by the transient and reversible nature of EMT and the multi-step events that underlie diffusion and metastasis. While the acquisition of quasi-mesenchymal features is essential for local invasion and systemic spread, MET is equally important for colonization of distant organs. In OV90 cells overexpressing the Tpm1.8 / 9 isoform with EMT-inducing properties, MET was suppressed, negatively impacting their metastatic potential. The presence of tumor cells with severely impaired colony-forming ability in the malignant ascites of recipient mice intraperitoneally injected with transplanted Tpm1.8 / 9-OE cells supports the hypothesis that MET inhibition in these cells negatively impacts their metastatic potential. Further evidence from RNA sequencing profiling of these cells suggests that non-physiological expression levels of the TPM1 isoform in these cells may contribute to potential off-target artifacts. Although compared to its epithelial cell counterpart, EpCAM… 低 Several inflammatory pathways in ovarian cancer cells are upregulated along with Tpm1.8 / 9, but ectopic expression of Tpm1.6 / 7 also leads to the activation of similar inflammation-related pathways (such as IL6 / Jak / Stat3, IFN, and TNF) compared to Tpm1.8 / 9 OE cells.

[0201] Due to the multifunctional role of TPM1 selective splicing in ovarian cancer malignancy and therapy resistance, Tpm1.8 / 9 forms a relevant therapeutic target. As shown in this article, the development of small molecule inhibitors can prevent EMT and reduce cell motility, while also inhibiting the activation of key signaling pathways such as Wnt, which is known to play a central role in the stemness, EMT, and chemotherapy resistance of ovarian cancer. Finally, combination therapy with conventional taxane- and platinum-based chemotherapy agents may improve therapeutic efficacy by antagonizing chemotherapy resistance.

[0202] Example 2

[0203] Materials and methods

[0204] Breast cancer MCF-7 cells grown in DMEM containing 10% FBS were washed with PBS, trypsinized for 3 minutes, and resuspended in complete culture medium. MCF-7 cells were counted and divided into groups of 2 × 10⁻⁶ cells. 3 Cells were seeded at a density of 100 µL per well in a 96-well plate using complete culture medium. The cells were incubated for 24 hours, and then the drug compound was added.

[0205] Paclitaxel was serially diluted from 50 nM to a final concentration of 0.39 nM in complete medium. For combined treatments, paclitaxel was serially diluted from 50 nM to a final concentration of 0.39 nM in complete medium, and compound 189-3 (1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468)) was added at a constant concentration of 5 µM or 10 µM. Individual serial dilutions of paclitaxel or a combination thereof with serial dilutions of compound 189-3 were added in triplicate to 96-well plates.

[0206] After 72 hours of incubation, add 20 μL of MTS reagent (Cell Titer 96 Aqueous OneSolution Cell Proliferation Assay - Promega G3581) to each well and incubate the plate for another 2 hours. Read the plate at 490 nm using a Fluostar microplate reader.

[0207] Data were collected in Excel file format, and the percentage of cell proliferation for each treatment relative to the control was calculated. Figure 6 The number of cells in the control group (with only culture medium added) was set to 100%, and the percentage of control cells was normalized to the percentage of treated cells. The average value of each experimental replicate was entered into GraphPad Prism 8 to generate IC50 curves.

[0208] result

[0209] MCF-7 breast cancer cell line showed reduced cell proliferation after exposure to paclitaxel, with an IC50 of 8.5 nM. The addition of 5 µM and 10 µM of 189-3 increased the sensitivity of MCF-7 cells to paclitaxel. Both doses of 189-3 were equally effective, with 5 µM and 10 µM reducing the IC50 of paclitaxel to 4.5 nM and 4.7 nM, respectively. Figure 6 B). We conclude that compound 189-3 increases the sensitivity of MCF-7 cells to paclitaxel by 2-fold.

Claims

1. A compound that is an inhibitor of the tropomyosin 1 selective splicing isoforms Tpm1.8 and / or Tpm1.9, wherein the compound is selected from... - Small molecule inhibitors selected from 3-(2-methyl-indole-1-yl)-propylamine (PubChem CID 6494468) and 1-phenylmethyl-1H-indole-2-methanol (PubChem CID 18973468), and their pharmaceutically acceptable salts, hydrates, derivatives, solvates or prodrugs; - Antisense polynucleotides that reduce the expression of tropomyosin 1 isoforms 1.8 and / or 1.9 (Tpm1.8 or Tpm1.9), and - Antibodies that reduce the levels and / or activity of Tpm1.8 and / or Tpm1.9 peptides.

2. The compound according to claim 1, used in a method for preventing and / or treating cancer.

3. The compound for use according to claim 2, wherein the cancer-expressing TPM1 selective splice isomer, preferably the cancer-expressing TPM1 exon 1b selective splice isomer, more preferably selective splice isomers Tpm1.8 and / or Tpm1.

9.

4. The compound according to any one of the preceding claims, wherein the cancer is selected from acute myeloid leukemia (AML), adrenocortical carcinoma, urothelial carcinoma of the bladder, low-grade brain carcinoma, invasive breast carcinoma, squamous cell carcinoma and endocervical adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, esophageal cancer, glioblastoma multiforme, squamous cell carcinoma of the head and neck, chromophobe renal carcinoma, clear cell renal carcinoma, papillary renal carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, lymphoid tumor, diffuse large B-cell lymphoma, mesothelioma, ovarian cancer, especially ovarian serous carcinoma, pancreatic adenocarcinoma, rectal adenocarcinoma, sarcoma, melanoma of the skin, gastric adenocarcinoma, testicular germ cell tumor, thymoma, thyroid cancer, uterine carcinosarcoma, endometrial cancer of the uterine body, uveal melanoma, and combinations thereof.

5. The compound according to any one of the preceding claims, wherein the cancer is ovarian cancer, more preferably high-grade serous ovarian cancer.

6. The compound for use according to any one of claims 2 to 5, wherein the compound is administered in combination with an additional anticancer therapy, said additional anticancer therapy preferably selected from chemotherapy, targeted therapies such as immunotherapy, stem cell therapy, hormone therapy, radiotherapy and surgery, and combinations thereof, more preferably chemotherapy based on taxanes and / or platinum.

7. The compound for use according to any one of claims 2 to 6, wherein the use comprises administering the compound to a subject at a dose of 0.1 mg / kg to 100 mg / kg.

8. A pharmaceutical composition comprising the compound according to claim 1, preferably the composition further comprising a pharmaceutically acceptable carrier.

9. A method for inhibiting, suppressing, preventing, or treating the proliferation, movement, invasion, spread, migration, metastasis, or chemotherapy resistance of cancer, comprising administering to a subject in need a therapeutically effective amount of the compound of claim 1 or the pharmaceutical composition of claim 8.

10. The method of claim 9, wherein the cancer expresses a TPM1 selective splice isomer, preferably the TPM1 selective splice isomer of exon 1b of TPM1, more preferably selective splice isomers Tpm1.8 and / or Tpm1.

9.

11. The method according to claim 9, wherein the cancer is selected from acute myeloid leukemia (AML), adrenocortical carcinoma, urothelial carcinoma of the bladder, low-grade brain carcinoma, invasive breast carcinoma, squamous cell carcinoma and endocervical adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, esophageal cancer, glioblastoma multiforme, squamous cell carcinoma of the head and neck, chromophobe renal carcinoma, clear cell renal carcinoma, papillary renal carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, lymphoid tumors, diffuse large B-cell lymphoma, mesothelioma, ovarian cancer, especially serous ovarian carcinoma, pancreatic adenocarcinoma, rectal adenocarcinoma, sarcoma, melanoma of the skin, gastric adenocarcinoma, testicular germ cell tumor, thymoma, thyroid cancer, uterine carcinosarcoma, endometrial cancer of the uterine body, uveal melanoma, and combinations thereof, preferably wherein the cancer is ovarian cancer, more preferably high-grade serous ovarian cancer.

12. The method according to claim 10 or 11, wherein the therapeutically effective amount comprises a dose from 0.1 mg / kg to 100 mg / kg.

13. A method for identifying compounds targeting Tpm1.8 and / or Tpm1.9 isomers, said compounds being used to inhibit, suppress, prevent, and / or treat cancer proliferation, motility, invasion, migration, metastasis, and / or chemotherapy resistance, said method comprising: a) Contacting cancer cells expressing the Tpm1.8 and / or Tpm1.9 isomers with the test compound, and b) Measure the inhibition of Tpm1.8 and / or Tpm1.9 expression and / or activity in the cancer cells. The discovery of inhibitory effects identifies the test compound as a compound for use in methods of inhibiting, suppressing, preventing, and / or treating cancer.

14. The compound according to claim 1, used in a method for inhibiting, suppressing, preventing, or treating the movement, invasion, spread, migration, metastasis, or chemotherapy resistance of cancer.

15. A method for determining the presence of epithelial-mesenchymal transition (EMT) in cancer cells, comprising determining the expression of Tpm1.8 and / or Tpm1.9 in the cells, wherein an increase in expression relative to control expression indicates the presence of EMT in the cells.

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