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WO2025023834A1 - Composés ciblant les isoformes tpm1.8 et/ou tpm1.9 pour la prévention et/ou le traitement d'un cancer - Google Patents

Composés ciblant les isoformes tpm1.8 et/ou tpm1.9 pour la prévention et/ou le traitement d'un cancer Download PDF

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WO2025023834A1
WO2025023834A1 PCT/NL2024/050410 NL2024050410W WO2025023834A1 WO 2025023834 A1 WO2025023834 A1 WO 2025023834A1 NL 2024050410 W NL2024050410 W NL 2024050410W WO 2025023834 A1 WO2025023834 A1 WO 2025023834A1
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cancer
carcinoma
isoforms
cells
compound
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Riccardo Fodde
Peter William GUNNING
Edna Christine HARDEMAN
Nicole Sarah BRYCE
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NewSouth Innovations Pty Ltd
Erasmus University Medical Center
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NewSouth Innovations Pty Ltd
Erasmus University Medical Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates to the use of compounds targeting the alternative splicing isoforms TPM1.8 and/or TPM1.9 of Tropomyosin 1 (TPM1) in a method of preventing and/or treating cancer. Further, the present invention relates to a method for identifying further compounds which target TPM1.8 and/or TPM1.9 isoforms and are for use in a method of preventing and/or treating cancer.
  • TPM1 Tropomyosin 1
  • Phenotypic plasticity defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET).
  • EMT Epithelial-to-mesenchymal
  • MET mesenchymal-to-epithelial transitions
  • AS alternative splicing
  • Metastatic disease or the movement of cancer cells from one site to another, is a complex process requiring dramatic remodelling of the cell cytoskeleton.
  • the various components of the cytoskeleton, actin (microfilaments), microtubules (MTs) and intermediate filaments, are highly integrated and their functions are well orchestrated in normal cells.
  • Ovarian cancer is the most lethal gynecological cancer but early stages of ovarian cancer do not show any noteworthy symptoms.
  • tropomyosins play an important role in metastatic disease and are commonly downregulated upon cell transformation and dedifferentiation in cancer development.
  • AS of tropomyosins may play a role in regulating EMT/MET in cancer metastasis.
  • the tropomyosin 1 (TPM1) gene encodes various AS isoforms.
  • the present inventors set out to investigate if alternative splicing isoforms of TPM1 could be therapeutic targets for the treatment of cancer.
  • the present invention solves the problem of providing novel compounds for use in preventing and/or treating cancer, such as ovarian cancer.
  • Tpm1.8 and Tpm1.9 of the Tropomyosin 1 gene provide a novel and promising target for preventing and/or treating cancer, such as ovarian cancer.
  • Tpm1.8 and/or Tpm1.9 represent specific isoforms which are involved in EMT activation through multiple signaling pathways including Wnt, TGF- ⁇ , Hedgehog, and Notch.
  • the Tpm1.8/9 isoforms are likely to facilitate dissemination from the primary tumor to the intra-abdominal cavity.
  • Tpm1.8/9 isoforms are a promising target for preventing and/or treating cancer, such as ovarian cancer.
  • the compounds of the present invention targeting Tpm1.8 and/or Tpm1.9 isoforms are for use in preventing and/or treating cancer, such as ovarian cancer.
  • the inventors further found up- and downregulation of RBM4 and ESRP1, respectively, in CD44 hi EpCAM lo cells in immortalized high-grade serous ovarian cancer (HGSOC) cell lines OV90, SKOV3, COV504, and CAOV3 by FACS with CD44 and EpCAM antibodies.
  • CD44 hi EpCAM lo cells showed a mesenchymal-like morphology, in contrast with the epithelial appearances of their CD44 hi EpCAM hi counterparts, whereby CD44 hi EpCAM lo cells showed increased migration and invasion capacity.
  • the TPM1 alternative slicing (AS)-pattern observed in ovarian cancer involved exons 1a/2a, which earmark the Tpm1.6/7 isoforms upregulated in CD44 hi EpCAM hi cells, and exon 1b, featuring the Tpm1.8/9 isoforms upregulated in CD44 hi EpCAM lo cells as validated both by RT-qPCR and western blot analysis.
  • HGSOC cell lines exclusively encompassing CD44 hi EpCAM hi cells solely expressed the Tpm1.6/7 isoforms, whereas CD44 hi EpCAM lo cells solely expressed the Tpm1.8/9 isoforms.
  • alternative splicing driven by RBM24 and ESRP1 up- and down-regulation, respectively is a main regulator of EMT in ovarian cancer cells.
  • the compounds of the present invention target the N-terminus of Tpm1.8 and/or Tpm1.9 at residues 4 – 16.
  • the compounds of the present invention targeting Tpm1.8 and/or Tpm1.9 isoforms may counteract chemoresistance, preferably chemoresistance against taxane- and/or platinum-based chemotherapy.
  • the compounds of the present invention targeting Tpm1.8 and/or Tpm1.9 isoforms has an activity in a low micromolar range, preferably wherein the compound has an activity in a range of from 5 – 50 ⁇ M, more preferably wherein the compound has an activity in a range of from 5 – 20 ⁇ M.
  • the compounds of the present invention targeting Tpm1.8 and/or Tpm1.9 isoforms may prevent Tpm1.8 and/or Tpm1.9 isoform enrichment in the lamellipodium.
  • the present invention provides in a first aspect a compound that is an inhibitor of Tropomyosin 1 alternative splicing isoforms Tpm1.8 and/or Tpm1.9, wherein the compound is selected from - a small molecule inhibitor selected from 3-(2-Methyl-indol-1-yl)-propylamine (PubChem CID 6494468) and 1-Phenylmethyl-1h-indole-2-methanol (PubChem CID 18973468), and pharmaceutically acceptable salts, hydrates, derivates, solvates or prodrugs thereof; - an antisense polynucleotide which decreases the expression of Tropomyosin 1 isoform 1.8 and/or 1.9 (Tpm1.8 or 1.9), and - an antibody which decreases Tpm1.8 and/or 1.9 polypeptide levels and/or Tpm1.8 or 1.9 polypeptide activity.
  • a small molecule inhibitor selected from 3-(2-Methyl-indol-1-yl)
  • the present invention provides the compound according to the invention as indicated above for use in a method of preventing and/or treating cancer.
  • the cancer expresses TPM1 alternative splicing isoforms, more preferably the cancer expresses TPM1 alternative splicing isoforms of TPM1 Exon 1b.
  • the cancer is selected from acute myeloid leukemia (AML), adrenocortical carcinoma, bladder urothelial carcinoma, brain lower grade carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney chromophobe, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, mesothelioma, ovarian cancer in particular ovarian serous cancer, pancreatic adenocarcinoma, rectum adenocarcinoma, sarcoma, skin cutaneous melanoma, stomach adenocarcino
  • the cancer which is treated by the use of the compounds of the invention is ovarian cancer, preferably epithelial ovarian cancer (EOC), more preferably wherein the cancer is high-grade serous ovarian cancer.
  • the compound is administered in combination with an additional anti-cancer therapeutic agent.
  • the compound is administered in combination with an additional anti-cancer therapy.
  • the additional anti- cancer therapy is selected from a group consisting of chemotherapy, targeted therapy such as immunotherapy, stem cell therapy, hormone therapy, radiation therapy and surgery, and a combination thereof.
  • the additional anti-cancer therapy is a chemotherapy, for example a taxane- and/or platinum-based chemotherapy, such as a chemotherapy comprises cisplatin- and/or paclitaxel.
  • a chemotherapy comprises cisplatin- and/or paclitaxel.
  • the administration of said compound to a subject may occur at a dose of between 0.1 and 100 mg/kg.
  • the embodiments directed to the compounds of the present invention and its medical use in treating cancer also apply to a pharmaceutical composition comprising the compound.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
  • the embodiments directed to the use of compounds and the pharmaceutical compositions also apply to methods of inhibiting, suppressing, preventing and/or treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of said compound or said pharmaceutical composition.
  • the present invention provides a method of identifying a compound targeting Tpm1.8 and/or Tpm1.9 isoforms for inhibiting, suppressing, preventing and/or treating proliferation, motility, invasion, migration, metastasis and/or chemo-resistance of a cancer, comprising: a) contacting a cancer cell that expresses Tpm1.8 and/or Tpm1.9 isoforms with a test compound, and b) measuring in said cancer cell the inhibition of Tpm1.8 and/or Tpm1.9 expression and/or activity, wherein the finding of an inhibitory effect identifies the test compound as a compound for use in a method of inhibiting, suppressing, preventing and/or treating cancer.
  • the present invention provides an antisense polynucleotide which decreases the expression of Tropomyosin1 isoform 1.8 or 1.9 (Tpm1.8 or 1.9) or an antibody which decreases Tpm1.8 or 1.9 polypeptide levels and/or Tpm1.8 or 1.9 polypeptide activity, for use in a method of inhibiting, suppressing, preventing or treating proliferation, motility, invasion, migration, metastasis or chemo-resistance of a cancer.
  • Tpm1.8 or 1.9 Tropomyosin1 isoform 1.8 or 1.9
  • an antibody which decreases Tpm1.8 or 1.9 polypeptide levels and/or Tpm1.8 or 1.9 polypeptide activity
  • the present invention provides a method of inhibiting, suppressing, preventing or treating proliferation, motility, invasiveness, dissemination, migration, metastasis or chemo-resistance of a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antisense polynucleotide which decreases the expression of Tropomyosin 1 isoform 1.8 or 1.9 (Tpm1.8 or 1.9) or an antibody which decreases Tpm1.8 or 1.9 polypeptide levels and/or TPM1.8 or 1.9 polypeptide activity.
  • an antisense polynucleotide which decreases the expression of Tropomyosin 1 isoform 1.8 or 1.9 (Tpm1.8 or 1.9) or an antibody which decreases Tpm1.8 or 1.9 polypeptide levels and/or TPM1.8 or 1.9 polypeptide activity.
  • the present invention provides a method for determining the presence of epithelial-to-mesenchymal transition (EMT) in cancer cells, comprising determining Tpm1.8 and or Tpm1.9 expression in said cells, whereby an increased expression relative to a control expression indicates EMT in said cells.
  • EMT epithelial-to-mesenchymal transition
  • the invention also provides a use of a compound targeting Tpm1.8 and/or Tpm1.9 isoforms for the manufacture of a medicament for preventing and/or treating cancer in a subject.
  • the invention further provides use of RBM24 and/or ESRP1 and/or Tpm1.8/9 as markers for determining cancer development, in particular for determining if and/or indicating that a cancer cell, preferably an ovarian cancer cell, is in epithelial-to- mesenchymal transition, is a metastatic cell, is (capable of) causing metastases, or is metastasizing, or is resistant against taxane- and/or platinum-based therapies.
  • DESCRIPTION OF THE DRAWINGS Figure 1 The RBPs ESRP1 and RBM24 synergistically regulate TPM1 alternative splicing.
  • (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 (Means ⁇ SEM, n 3). P values are relative to the comparison with the parental cell lines.
  • (C) Proliferation assays 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. O.D. values are shown from day 1 to 6 (Means ⁇ SEM, 697 n 3). P values are relative to the comparison with the parental cell lines.
  • (C) TOP-Flash luciferase reporter analysis of Wnt signaling activity in Tpm1.6/7/8/9-OE (upper histogram) and upon knockdown by siRNA of Tpm1.6/7 and Tpm1.8/9 in OV90 cells. P values are relative to the comparison with the parental cell lines (Means ⁇ SEM, n 3).
  • Tpm1.8/9 isoforms are enriched in malignant ascites from ovarian cancer cells and confer resistance to platinum- and taxane-based therapies.
  • TPM1 is a gene (geneID:7168 in humans) that is a member of the tropomyosin family of highly conserved, widely distributed actin-binding proteins involved in the contractile system of striated and smooth muscles and the cytoskeleton of non-muscle cells.
  • Tropomyosin is composed of two alpha-helical chains arranged as a coiled-coil. It is polymerized end to end along the two grooves of actin filaments and provides stability to the filaments.
  • the encoded protein is one type of alpha helical chain that forms the predominant tropomyosin of striated muscle, where it also functions in association with the troponin complex to regulate the calcium- dependent interaction of actin and myosin during muscle contraction.
  • TPM1 is known to have 39 transcripts (splice variants).
  • Variants Tpm1.8 (formerly known as Tm5a) and Tpm1.9 (formerly known as Tm5b) contain alternate in-frame exons in the 5' and 3' coding region compared to variant Tpm1.1.
  • 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 that are of the same length (248 aa), but have distinct protein sequences (Geeves, et al., 2015 J Muscle Res Cell Motil 36, 147-153). The accession numbers are from July 2023 and for full-length human, mouse, and rat sequences that have been documented at the nucleic acid level. Table 1.
  • inhibitor refers to a compound targeting Tpm1.8 and/or Tpm1.9 isoforms, wherein the Tpm1.8 and/or Tpm1.9 isoforms may either be in the form of DNA, mRNA or protein, and whereby the inhibitor decreases the transcriptional expression of Tropomyosin 1 isoforms Tpm1.8 and/or Tpm1.9 and/or decreases Tpm1.8 and/or Tpm1.9 polypeptide levels and/or Tpm1.8 and/or Tpm1.9 polypeptide activity.
  • An inhibitor that decreases Tpm1.8 and/or Tpm1.9 polypeptide activity may for example be a small molecule inhibitor, preferably a small molecule inhibitor as disclosed herein.
  • Another exemplary inhibitor that decreases Tpm1.8 and/or Tpm1.9 polypeptide activity may be a Tpm1.8 and/or Tpm1.9 antibody.
  • An inhibitor that decreases Tpm1.8 and/or Tpm1.9 mRNA or polypeptide levels may for example be an antisense polynucleotide.
  • Suitable antisense polynucleotides include synthesized single- stranded nucleic acids (20 to 30 nucleotides in length, e.g. oligonucleotides) in the form of antisense oligonucleotides (ASO), or small interfering RNA (siRNA) as the cleavage product of double-stranded RNA (dsRNA).
  • siRNA target sequence for human Tpm1.8/9 is 5’-CGAGAGGAAGCTGAGGGAGAC-3’ (codons 38-44 of Tpm1.8/9 in exon 1b). It is within the competence of one of skill in the art to produce such antisense polynucleotides for post-transcriptional gene silencing.
  • tumor includes reference to an abnormal growth of tissue that may be benign, pre-cancerous, malignant, or metastatic.
  • the tumor is preferably malignant, i.e., a cancer.
  • cancer refers to any malignant neoplasm resulting from the undesired growth, the invasion, and under certain conditions metastasis of impaired cells in an organism.
  • cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, such as ovarian epithelial cancer, cervical cancer, skin cancer, pancreatic cancer, spleen cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia such as acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma, lymph nodes cancer, bone marrow cancer, lung cancer, stomach cancer, eye cancer and the like.
  • treatment and “treating”, as used herein, include reference to the application of a form of therapy to a subject, with the object of e.g.
  • prevention includes reference to the application of a form of therapy to a subject, with the object to hinder the outbreak of a disease, such as cancer.
  • the terms refer to the application of a form of therapy to a subject to prevent and/or reduce the likelihood of an occurrence of a disease, such as cancer.
  • the terms include proactive and prophylactic treatment and/or application of an active component in order to hinder outbreak of a disease, such as cancer and/or to immunize the subject.
  • compound active compound
  • active component active component
  • subject refers to a human or animal suffering from and/or susceptible to cancer.
  • subject includes reference to a recipient of a compound or a pharmaceutical composition targeting Tpm1.8 and/or Tpm1.9 as described herein, i.e.
  • a subject that is suffering, or suspected of suffering, from cancer such as ovarian cancer.
  • “subject” includes subjects in which a cancer is prevented.
  • the subject is a mammal, more preferably a human.
  • the terms “patient” and “subject” can be used interchangeably herein.
  • the subject is for example a human, i.e., a human having cancer and/or at risk getting cancer.
  • the subject is for example older or younger than 50 years old.
  • the subject is older than 50 years old.
  • the subject is human suffering from cancer.
  • the subject is male or female.
  • the subject is a female human suffering from ovarian cancer such as epithelial ovarian cancer.
  • the subject is a human suffering from breast cancer.
  • resistance includes reference to the ability of cancer cells to at least partially withstand one or more anti-cancer therapies, particularly one or more chemotherapies.
  • resistance refers inter alia to a reduced efficacy of an anti-cancer therapeutic agent to treat a subject having a cancer.
  • the anti- cancer therapeutic agent is a chemotherapeutic agent
  • the resistance is referred to as “chemoresistance”.
  • chemoresistance When reference to a resistance of a cancer is made, it preferably refers to a resistance of cancer cells of said cancer to said anti-cancer therapeutic agent. For example, chemoresistance against cisplatin- and/or paclitaxel.
  • restoring includes reference to at least partial, such as complete, restoration of chemosensitivity of cancer to an anti-cancer therapeutic agent it was previously at least partially insensitive to.
  • terapéuticaally effective amount means that the amount of active ingredients administered is of sufficient quantity to achieve the intended purpose, such as, in this case, to prevent and/or treat cancer, such as ovarina cancer. This means for example to prevent proliferation, motility, invasion, migration, metastasis or chemo-resistance.
  • administration includes reference to the application of a substance, compound, and/or pharmaceutical composition to a subject. Main routes of administration are parenteral administration, enteral or gastrointestinal administration and topical administration.
  • parenteral includes reference to any form of administration that is not via the application onto the skin or via the gastrointestinal tract.
  • parenteral administration include epidural, intracerebral, intracerebroventricular, epicutaneous, sublingual, extra- amniotic, nasal, intra-arterial, intra-articular, intracardiac, intracavernous, intradermal, intralesional, intramuscular, intraocular, intraosseous, intraperitoneal, intrathecal, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, rectal or intratumoral administration.
  • intravenous includes reference to a parenteral route of administration wherein a substance or composition is injected into the vein of a subject, for example using a hollow needle.
  • Intratumoral includes reference to administration of a substance or composition directly into a tumor, for example using a hollow needle.
  • the tumor wherein intratumoral administration takes place may be treated prior to administration, for example in order to improve visibility of the tumor.
  • Intratumoral administration may for example be used for the administration of anti-cancer therapeutic agents.
  • chemotherapeutic agent includes reference to an anti- cancer drug that is used as part of a chemotherapy, which is a type of cancer treatment.
  • a chemotherapeutic agent has for example a cytotoxic effect and/or a cytostatic effect, and it is for example used to cure a subject from cancer, to reduce symptoms in a subject, or to prolong the life of a subject.
  • a chemotherapeutic agent include Cyclophosphamide, Mechlorethamine, Chlorambucil, Melphalan, dacarbazine, Nitrosoureas, Temozolomide, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Cabazitaxel, Larotaxel, Ortataxel, Tesetaxel, Paclitaxel, Docetaxel, Abraxane, Taxotere, Epothilone A, Epothilone B, Epothilone C, Epothilone D, Epothilone E, Epothilone F, Vorinostat, Romidepsin, Irinotecan
  • Combinations of chemotherapeutic agents are for example used for the treatment of cancer. Combinations may be established or improvised by the treating physician. Established combinations are known as regimens, wherein in some cases also dose and administration interval are included.
  • the chemotherapeutic agent as disclosed herein is for example an (i) alkylating agent (such as Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa or Trabectedin), (ii) an nitrosoureas (such as Carmustine, Lomustine or Streptozocin), (iii) an antimetabolite (Azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), Capecitabine (Xeloda
  • the term “combination” or “combination therapy”, as used herein, includes reference to using a compound targeting Tpm1.8 and/or Tpm1.9 as disclosed herein and an anti-cancer therapeutic agent as disclosed herein in the same medical treatment.
  • the compound targeting Tpm1.8 and/or Tpm1.9 and the anti-cancer therapeutic agent as disclosed herein are for example administered together at the same time (such as in the form of a single pharmaceutical composition), separately of each other at the same time (for instance in the form of separate pharmaceutical compositions) or separately of each other staggered in time. Simultaneous, separate and sequential administration of a compound targeting Tpm1.8 and/or Tpm1.9 and an anti-cancer therapeutic agent as disclosed herein in the same treatment schedule are expressly envisaged.
  • the time between administration of the compound targeting Tpm1.8 and/or Tpm1.9 and the anti-cancer therapeutic agent is for example at least one minute, at least fifteen minutes, at least sixty minutes, at least four hours, at least one day, at least one week or at least one month or at least one year, or anywhere in between such as between one minute and one year.
  • the compound targeting Tpm1.8 and/or Tpm1.9 is administered prior to administration of said anti-cancer therapeutic agent.
  • the anti-cancer therapeutic agent is administered together with, or after, administration of the compound targeting Tpm1.8 and/or Tpm1.9.
  • the anti-cancer therapeutic agent which is combined with the compound targeting Tpm1.8 and/or Tpm1.9 is for example a chemotherapy.
  • the chemotherapy which is combined 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 is for example combined with cisplatin- and/or paclitaxel.
  • pharmaceutical combination includes reference to e.g. a kit of parts containing multiple containers that hold the different active ingredients.
  • the compounds of the present invention target the N-terminal sequences of exon 1b of TPM1 (see Figures 1 and 6 of Schevzov et al.2011. Bioarchitecture 1(4): 135–164 to which reference is made for intron/exon organization of the TPM1 gene, and which is incorporated by reference herein).
  • the compounds targeting Tpm1.8 and/or Tpm1.9 isoforms target the N-terminus of Tpm1.8 and/or Tpm1.9 at residues 4 – 16.
  • the compound is a small molecule that perturbs the activity of Tpm1.8 and/or Tpm1.9 isoforms in a dose dependent manner.
  • the compounds targeting Tpm1.8 and/or Tpm1.9 isoforms preferably inhibit Tpm1.8 and/ Tpm1.9 activity in a concentration of 5 – 50 ⁇ M, 15 – 30 ⁇ M, or 10 – 20 ⁇ M.
  • the compound targeting Tpm1.8 and/or Tpm1.9 isoforms is selected from 3-(2- Methyl-indol-1-yl)-propylamine (PubChem CID 6494468), 1-Phenylmethyl-1h-indole-2- methanol (PubChem CID 18973468), or a combination thereof.
  • PubChem CID 6494468 targets both Tpm1.8/9 and Tpm4.2
  • PubChem CID: 18973468 only targets Tpm1.8/9.
  • PubChem CID 6494468 is a preferred compound.
  • PubChem CID 18973468 is a preferred compound.
  • a compound targeting Tpm1.8 and/or Tpm1.9 isoforms includes for example pharmaceutical acceptable salts, hydrates, derivates and/or solvates of 3-(2-Methyl-indol-1-yl)-propylamine (PubChem CID 6494468) and/or 1- Phenylmethyl-1h-indole-2-methanol (PubChem CID 18973468).
  • compounds targeting Tpm1.8 and/or Tpm1.9 isoforms according to the present invention include any compound which is identified by the method of identifying compounds targeting Tpm1.8 and/or Tpm1.9 as disclosed herein.
  • Compounds targeting Tpm1.8 and/or Tpm1.9 isoforms according to the present invention also include hydrates and solvates. Solvates are complexes formed by association of molecules of a solvent with a compound of the present invention.
  • the compound targeting Tpm1.8 and/or Tpm1.9 isoforms of the present invention is for example in the form of pharmaceutically acceptable salts.
  • the compound targeting Tpm1.8 and/or Tpm1.9 isoforms of the present invention also extends to include all derivatives with physiologically cleavable leaving groups that can be cleaved in vivo.
  • Compounds of the present invention further include antisense polynucleotides which decreases the expression of Tpm1.8 and/or Tpm1.9 isoforms in cancer cells.
  • Compounds of the present invention further include antibodies which decreases Tpm1.8 and/or 1.9 polypeptide levels and/or Tpm1.8 or 1.9 polypeptide activity.
  • Antisense polynucleotides and antibodies of the invention may be used in a method of inhibiting, suppressing, preventing or treating proliferation, motility, invasion, migration, metastasis or chemo-resistance of a cancer.
  • Pharmaceutical compositions A compound targeting Tpm1.8 and/or Tpm1.9 isoforms, or other compounds of the invention as disclosed herein, may be used as sole active ingredient of a pharmaceutical composition of the invention, or it may be combined with other compounds targeting the same or different targets. For example, a first compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein may be combined with a second compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein.
  • a compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein is preferably in a pharmaceutical composition that may further comprise a pharmaceutically acceptable excipient (or carrier).
  • pharmaceutically acceptable excipient or carrier includes reference to any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.
  • compositions suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Pharmaceutical compositions can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • compositions suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound targeting Tpm1.8 and/or Tpm1.9 isoforms suspended in diluents, such as water or saline; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • liquid solutions such as an effective amount of the compound targeting Tpm1.8 and/or Tpm1.9 isoforms suspended in diluents, such as water or saline
  • diluents such as water or saline
  • capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin
  • suspensions in an appropriate liquid and (d) suitable emulsions.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the compound targeting Tpm1.8 and/or Tpm1.9 isoforms, carriers known in the art.
  • Pharmaceutical compositions can be encapsulated, e.g., in liposomes, or in a formulation that provides for slow release of the active ingredient.
  • the pharmaceutical composition comprising a compound targeting Tpm1.8 and/or Tpm1.9 isoforms can be administered via liposomes, microparticles, or microcapsules.
  • the present invention provides an agent pack or kit comprising one or more containers filled with one or more compounds, compositions or medicaments of the invention.
  • information indicating approval for manufacture, use, or sale for administration to a human by a government agency regulating the manufacture, use, or sale of medicaments or biological products can be appended to such a container in a stipulated form.
  • the formulation procedure for the compound targeting Tpm1.8 and/or Tpm1.9 isoforms as a medicament or the like is known in the art.
  • the procedure is described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopeia, pharmacopeia of other countries, or the like. Thus, those skilled in the art can determine the embodiment such as the amount to be used without undue experimentation from the descriptions herein.
  • Administration and treatment methods The present invention relates to a compound targeting Tpm1.8 and/or Tpm1.9 isoforms for use in a method of preventing and/or treating cancer in a subject.
  • the administration of at least one compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein is for example through administration of a pharmaceutical composition comprising a compound targeting Tpm1.8 and/or Tpm1.9 isoforms.
  • compositions can be administered in any suitable way, such as enterally, parenterally, topically, by inhalation and the like.
  • Parenteral administration is for example epidural, intracerebral, intracerebroventricular, epicutaneous, sublingual, extra-amniotic, nasal, intra-arterial, intra-articular, intracardiac, intracavernous, intradermal, intralesional, intramuscular, intraocular, intraosseous, intraperitoneal, intrathecal, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, intratumoral or a combination thereof.
  • Administration is for example enterally, such as orally or rectally.
  • a second and optionally a further compound targeting Tpm1.8 and/or Tpm1.9 isoforms can be employed in an aspect of the invention.
  • the second compound targeting Tpm1.8 and/or Tpm1.9 isoforms is administered together with the first compound targeting Tpm1.8 and/or Tpm1.9 isoforms, e.g. together at the same time (such as in the form of a single pharmaceutical composition), separately of each other at the same time (for instance in the form of separate pharmaceutical compositions) or separately of each other staggered in time.
  • Simultaneous, separate and sequential administration of compounds targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein in the same treatment schedule are expressly envisaged.
  • the second compound targeting Tpm1.8 and/or Tpm1.9 isoforms is for example administered parenterally or enterally.
  • the parenteral administration is for example epidural, intracerebral, intracerebroventricular, epicutaneous, sublingual, extra-amniotic, nasal, intra-arterial, intra-articular, intracardiac, intracavernous, intradermal, intralesional, intramuscular, intraocular, intraosseous, intraperitoneal, intrathecal, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, rectal, intratumoral administration or a combination thereof.
  • the administration is for example intravenous or intratumoral administration, or is orally or rectally.
  • a third, fourth, fifth or further compound targeting Tpm1.8 and/or Tpm1.9 isoforms is for example used in aspects of the invention.
  • the third, fourth, fifth or further compound targeting Tpm1.8 and/or Tpm1.9 isoforms is for example administered together with the first and second compound targeting Tpm1.8 and/or Tpm1.9 isoforms as described above.
  • the present invention further comprises co-administering an anti-cancer therapeutic agent as described herein in combination with a compound targeting Tpm1.8 and/or Tpm1.9 isoforms.
  • a compound targeting Tpm1.8 and/or Tpm1.9 isoforms, and optionally a second, third, fourth, fifth and/or further compound targeting Tpm1.8 and/or Tpm1.9 isoforms is administered together with one or more anti-cancer therapeutic agents as described herein. Administration of said anti-cancer therapeutic agent is for example performed parenterally.
  • Parenteral administration is for example epidural, intracerebral, intracerebroventricular, epicutaneous, sublingual, extra-amniotic, nasal, intra-arterial, intra-articular, intracardiac, intracavernous, intradermal, intralesional, intramuscular, intraocular, intraosseous, intraperitoneal, intrathecal, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, intratumoral administration or a combination thereof.
  • the administration is intravenous or intratumoral administration.
  • the same route of administration is for example selected for a compound targeting Tpm1.8 and/or Tpm1.9 isoforms, and optionally a second, third, fourth, fifth and/or further compound targeting Tpm1.8 and/or Tpm1.9 isoforms, and one or more anti-cancer therapeutic agents as described herein.
  • the compound targeting Tpm1.8 and/or Tpm1.9 isoforms and the anti-cancer therapeutic agent are administered through different routes of administration.
  • the anti-cancer therapeutic agent can be administered parenterally, and the compound targeting Tpm1.8 and/or Tpm1.9 isoforms orally.
  • the (first) compound targeting Tpm1.8 and/or Tpm1.9 isoforms is administered orally, and the second, or further, compound targeting Tpm1.8 and/or Tpm1.9 isoforms is administered parenterally.
  • the compound targeting Tpm1.8 and/or Tpm1.9 isoforms is administered to a subject 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.
  • a compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein is for example employed in a treatment regimen that involves daily, weekly or monthly administration of the compound targeting Tpm1.8 and/or Tpm1.9 isoforms.
  • Treatment is for example maintained for at least three days, at least a week, at least a month, and more preferably at least 6 months or at least a year such as 2-5 years.
  • a compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein and/or an anti-cancer therapeutic agent is for example employed in a treatment regimen that involves administration in cycles where each cycle comprises repetitive administration, for example daily administration, of the compound targeting Tpm1.8 and/or Tpm1.9 isoforms for several days, for example for at least one day, at least three days, at least a week, at least two weeks or at least three weeks.
  • the administration of a compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein and/or an anti-cancer therapeutic agent in cycles is for example repeated every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks.
  • the administration of a compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein and/or an anti-cancer therapeutic agent in cycles is for example repeated after 3 – 5 weeks, for example after three weeks or after five weeks.
  • the administration of a compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein and/or an anti-cancer therapeutic agent is repeated at no fixed interval, but according to the patient’s need.
  • the administration of a chelating agent as disclosed herein and/or an anti-cancer therapeutic agent in cycles is repeated at least one time, at least two times, at least thee 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 10 times.
  • treatment is maintained for at least three days, at least a week, at least a month, and more preferably at least six months or at least a year, such as 2-5 years.
  • Administration of a compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein and/or an anti-cancer therapeutic agent as disclosed herein to a subject for example follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy.
  • a compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein can be administered in any acceptable pharmaceutical dosage form, for example as an aqueous medium such as a solution, suspension, emulsification.
  • a chelating agent can also be administered orally as a pill, tablet, capsule, etc.
  • the subject can be identified as eligible for therapy if he or she has a cancer with cancer cells, such as ovarian cancer.
  • the cancer cells for example exhibit a resistance such as a chemoresistance.
  • the cancer cells exhibit a chemoresistance against a taxane- and/or platinum-based chemotherapy.
  • the cancer cells exhibit a chemoresistance against cisplatin- and/or paclitaxel.
  • a suitable dosing of the compounds of the present invention in methods of treatment may include a dose in the range of 0.1- 100 mg/kg, preferably about 0.5-20 mg/kg, even more preferably about 1-10 mg/kg.
  • Methods of treatment according to this invention include methods of inhibiting, suppressing, preventing and/or treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to the present invention or of a pharmaceutical composition comprising a compound according to the present invention.
  • the compound is for example a compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein, such as a small molecule inhibitor as disclosed herein, or an antisense polynucleotide or antibody which decrease Tpm1.8 or 1.9 polypeptide levels and/or Tpm1.8 or 1.9 polypeptide activity.
  • the cancer is for example selected from non- small cell lung cancer; renal cancer; renal cell carcinoma; clear cell renal cell carcinoma; lymphoma; blastoma; sarcoma; carcinoma, undifferentiated; meningioma; brain cancer; oropharyngeal cancer; nasopharyngeal cancer; biliary cancer; pheochromocytoma; pancreatic islet cell cancer; Li-Fraumeni tumor; thyroid cancer; parathyroid cancer; pituitary tumor; adrenal gland tumor; osteogenic sarcoma tumor; neuroendocrine tumor; breast cancer; lung cancer; head and neck cancer; prostate cancer; esophageal cancer; tracheal cancer; liver cancer; bladder cancer; stomach cancer; pancreatic cancer; ovarian cancer; uterine cancer; cervical cancer; testicular cancer; colon cancer; rectal cancer; skin cancer; giant and spindle cell carcinoma; small cell carcinoma; small cell lung cancer; papillary carcinoma; oral cancer; oropharyngeal cancer;
  • said cancer is a solid tumor. In some embodiments, said cancer is a liquid tumor. Preferably, in aspects of the present invention the cancer is a carcinoma. Carcinoma is a malignancy that develops from epithelial cells. Preferably, in aspects of the present invention the cancer is a cancer that expresses TPM1 alternative splicing isoforms. For example, the cancer is a cancer that expresses TPM1 alternative splicing isoforms comprising Exon 1b of TPM1. For example, the cancer is a cancer that exhibits alternative splicing in that it expresses Tpm1.8 and/or Tpm1.9 isoforms. Tpm1.8 and or Tpm1.9 expression may for instance be determined at the level of RNA or protein.
  • Tpm1.8 and or Tpm1.9 expression may for example be determined by RT-PCR (reverse-transcription polymerase chase reaction) or by RNA-sequencing, and, at the protein level, by antibody detection (e.g. western blot, immunofluorescence, or immunohistochemistry) or mass spectrometry.
  • the presence of alternative splicing expression of Tpm1.8 and/or Tpm1.9 isoforms may be indicated when the difference of an appropriate expression-level parameter (e.g. mean PSI ( ⁇ psi; differential Percentage Spliced In) as in the examples below) between the tested cancer (cell) and an appropriate control (cell) is >10%.
  • an appropriate expression-level parameter e.g. mean PSI ( ⁇ psi; differential Percentage Spliced In) as in the examples below
  • Tpm1.8 and/or Tpm1.9 isoforms are not expressed in the primary tumors, but are expressed in a rather small time-window during the systemic dissemination of the cells in the abdominal cavity when they undergo epithelial-mesenchymal transition (EMT), a complex developmental program that enables carcinoma cells to suppress their epithelial features changing to mesenchymal ones, i.e. whereby cells acquire quasi-mesenchymal and chemo-resistant features in combination with mobility and invasiveness.
  • EMT epithelial-mesenchymal transition
  • TPM1 RNA molecules that comprise exon 1b There are numerous cancers that express TPM1 RNA molecules that comprise exon 1b. From a database search (TGCA database, world wide web address www.cancer.gov/ccg/research/genome-sequencing/tcga) it was determined that gene expression at the RNA level of the complete TPM1 gene including the expression of TPM1 RNA molecules that comprise exon 1b occurs in acute myeloid leukemia (AML), adrenocortical carcinoma, bladder urothelial carcinoma, brain lower grade carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney chromophobe, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, liver hepatocellular carcinoma, lung aden
  • the cancer is an ovarian cancer, such as epithelial ovarian cancer.
  • the cancer is a high-grade serous ovarian cancer.
  • Epithelial ovarian cancer (EOC) is the leading cause of death amongst gynecologic malignancies due to its high case-to-fatality ratio. EOC generally becomes manifest at advanced disease stages, i.e. when metastases have already spread to pelvic organs (stage II), the abdomen (stage III), or beyond the peritoneal cavity (stage IV). Based on the underlying genetic defects, two main EOC subtypes have been recognized.
  • Type I tumors are slow growing, mostly restricted to the ovary, and thought to arise from well- differentiated precursor lesions called “borderline” tumors. They are further subdivided into low-grade serous, mucinous, clear cell, and endometrioid subtypes. Mutations in KRAS, BRAF, PTEN, and CTNNB1 ( ⁇ -catenin) earmarks type I EOCs, often together with a relatively stable karyotype. High-grade serous (HGSOC) and undifferentiated carcinomas are type II EOCs and are frequently characterized by TP53 mutations and by aneuploidy. HGSOC represents the most malignant and common ovarian cancer type accounting for up to 70% of all cases with poor prognosis and survival.
  • EOC is the only cancer type where no physical barrier exists between primary lesion and the main metastatic site, i.e. the intraperitoneal cavity.
  • the dissemination of ovarian cancer cells results in their adhesion to intra-abdominal organs and the peritoneum eventually leading to ascites accumulation due to the obstruction of lymphatic vessels.
  • the cancer is an epithelial ovarian cancer of EOC Type I and/or a EPO Type II.
  • the epithelial ovarian cancer is selected from the group consisting of a EOC Type I, EOC Type II, high grade serous ovarian cancer (HGSOC), low-grad serous ovarian cancer (LGSOC), fallopian tube cancer, primary peritoneal cancer and combinations thereof.
  • Anti-cancer therapeutic agent The present invention relates to a compound targeting Tpm1.8 and/or Tpm1.9 isoforms for use in a method of treating cancer in a subject.
  • at least one compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein is administered in combination with an additional anti-cancer therapeutic agent.
  • An anti-cancer therapeutic agent may be any anti-cancer therapeutic agent.
  • the anti-cancer therapeutic agent as described herein is for example an anti-cancer therapeutic agent used in a treatment type selected from the group of chemotherapy, targeted therapy such as immunotherapy, stem cell therapy, hormone therapy, radiation therapy and surgery, or a combination thereof; preferably, said treatment type is chemotherapy.
  • the anti-cancer therapeutic agent is for example selected from the group comprising Cyclophosphamide, Mechlorethamine, Chlorambucil, Melphalan, dacarbazine, Nitrosoureas, Temozolomide, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Cabazitaxel, Larotaxel, Ortataxel, Tesetaxel, Paclitaxel, Docetaxel, Abraxane, Taxotere, Epothilone A, Epothilone B, Epothilone C, Epothilone D, Epothilone E, Epothilone F, Vorinostat, Romidepsin, Irinotecan, Topotecan, Etoposide, Teniposide, Tafluposide, Bortezomib, Erlotinib, Gefitinib, Imatinib, Vemurafenib,
  • a combination of anti-cancer therapeutic agents is a known chemotherapy regimen, such as CMF (Cyclophosphamide, Methotrexate, 5- fluorouracil, vinorelbine), AC (doxorubicin, cyclophosphamide), DA (cytarabine, an anthracycline antibiotic, daunorubicin), IA (cytarabine, an anthracycline antibiotic, idarubicin), DAT (daunorubicin, cytarabine, tioguanine), FLAMSA (fludarabine, cytarabine, amsacrine), FLAMSA-BU (fludarabine, cytarabine, amsacrine, busulfan), FLAMSA-MEL (fludarabine, cytarabine, amsacrine, melphalan), TAD (tioguanine, cytarabine, daunorubicin), CAF (cyclo
  • anthracycline such as a doxorubicin, an antimetabolite, such as 5-fluorouracil (5-FU), and/or a taxane, such as Nab-paclitaxel and/or Paclitaxel is for example selected as the anti-cancer therapeutic agent.
  • the anti-cancer therapeutic agent as disclosed herein can be administered by any acceptable delivery mode, such as e.g. by liposomal delivery.
  • the at least one compound targeting Tpm1.8 and/or Tpm1.9 isoforms as disclosed herein is administered in combination with a taxane- and/or platinum-based chemotherapy, such as a chemotherapy comprising cisplatin- and/or paclitaxel.
  • the compounds targeting Tpm1.8 and/or Tpm1.9 isoforms for preventing and/or treating of cancer counteracts chemoresistance.
  • the compounds targeting Tpm1.8 and/or Tpm1.9 are for use in reversing a chemoresistance of a resistant cancer, e.g. a resistant ovarian cancer.
  • the chemoresistance is a native chemoresistance and/or an acquired chemoresistance.
  • the chemoresistance is acquired by one or more previous performed chemotherapies.
  • the chemoresistance which is counteracted by a compound targeting Tpm1.8 and/or Tpm1.9 according to the present invention is a chemoresistance to any chemotherapeutic agent in the art.
  • the chemoresistance which is counteracted by a compound targeting Tpm1.8 and/or Tpm1.9 according to the present invention is a chemoresistance against taxane- and/or platinum-based chemotherapy.
  • the chemoresistance which is counteracted by a compound targeting Tpm1.8 and/or Tpm1.9 according to the present invention is a chemoresistance against cisplatin- and/or paclitaxel.
  • the compound targeting Tpm1.8 and/or Tpm1.9 isoforms is used to restore chemosensitivity for any chemotherapy in the art.
  • the compound targeting Tpm1.8 and/or Tpm1.9 isoforms is used to restore chemosensitivity for taxane- and/or platinum-based chemotherapy.
  • the compound targeting Tpm1.8 and/or Tpm1.9 isoforms is used to restore chemosensitivity for cisplatin- and/or paclitaxel.
  • the compound targeting Tpm1.8 and/or Tpm1.9 isoforms according to the present invention counteracts chemoresistance and/or restores chemosensitivity at least partially, such as complete restoration of chemosensitivity of cancer to an anti-cancer therapeutic agent it was previously at least partially insensitive to.
  • the compound targeting Tpm1.8 and/or Tpm1.9 reduces a chemotherapeutic agent’s specific IC50 level by 10 – 100%, 20 – 95%, 25 – 90%, 30 – 85%, 35 – 80%, 40 – 75%, 45 – 70%, 50 – 65% or 55 – 60 % compared to the chemotherapeutic agent’s specific IC50 in an untreated control and/or compared to the chemotherapeutic agent’s specific IC50 before the use of the compound targeting Tpm1.8 and/or Tpm1.9 isoforms.
  • the compound targeting Tpm1.8 and/or Tpm1.9 reduces a chemotherapeutic agent’s specific IC50 level by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% compared to an untreated control and/or compared to the chemotherapeutic agent’s specific IC50 before the use of the compound targeting Tpm1.8 and/or Tpm1.9 isoforms.
  • the chemotherapeutic agent to which the cancer is resistant to is a taxane- and/or platinum-based chemotherapeutic agent, such as cisplatin- and/or paclitaxel.
  • the present invention further includes a method for identifying a compound targeting Tpm1.8 and/or Tpm1.9 isoforms for inhibiting, suppressing, preventing and/or treating proliferation, motility, invasion, migration, metastasis and/or chemo-resistance of a cancer.
  • the method comprises constructing a model of the N-terminus of Tpm1.8 and/or Tpm1.9, preferably of human Tpm1.8 and/or Tpm1.9, which contains the region of greatest diversity between the four TPM genes.
  • the model comprises the N-terminus at residues 4-16.
  • the method for identifying for example comprises performing a virtual in silico docking screen for identifying compounds that potentially bind Tpm1.8 and/or Tpm1.9, preferably in the N-terminus of Tpm1.8 and/or Tpm1.9, more preferably in the N-terminus of Tpm1.8 and/or Tpm1.9 at residues 4 – 16.
  • a virtual in silico docking screen of the ZINC database of database of purchasable compounds is performed, using online available docking programs such as AutoDock, GOLD, Deep Docking, Glide, or POSIT.
  • Candidate compounds can be purchased from vendors and tested in vitro or in vivo for their activity.
  • the method comprises contacting human fibroblasts with a test compound.
  • the contacting is for example performed for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 36, 48, 52 hours.
  • the human fibroblasts are for example fixed and stained. Staining is for example performed using Tpm1.8 and/or Tpm1.9 specific antibodies.
  • the method of identifying compounds according to the present invention for example comprises analyzing enrichment of Tpm1.8 and/or Tpm1.9 isoforms in the lamellipodium of the fibroblasts, wherein a fibroblast contacted with a test compound identified as compound targeting Tpm1.8 and/or Tpm1.9 isoforms according to the present invention shows reduced of enrichment of Tpm1.8 and/or Tpm1.9 isoforms in the lamellipodium compared to a control fibroblast.
  • the compound facilitating a reduced enrichment of Tpm1.8 and/or Tpm1.9 isoforms in the lamellipodium compared to a control fibroblast is for example identified as a compound for use in a method of inhibiting, suppressing, preventing and/or treating cancer.
  • the method of identifying compounds targeting Tpm1.8 and/or Tpm1.9 isoforms further comprises for example contacting a cancer cell that expresses Tpm1.8 and/or Tpm1.9 isoforms with a test compound.
  • the cancer cell is for example an ovarian cancer cell.
  • the cancer cell such as ovarian cancer cell, is contacted with a test compound in a concentration in the range of 5 – 50 ⁇ M, 15 – 30 ⁇ M, or 10 – 20 ⁇ M.
  • the method for identifying comprises measuring in the cancer cell the inhibition of Tpm1.8 and/or Tpm1.9 expression and/or activity, wherein the finding of an inhibitory effect (e.g.
  • the test compound identified by the method for identifying inhibits Tpm1.8 and/or Tpm1.9 expression and/or activity in a range of 10 – 100%, 20 – 95%, 25 – 90%, 30 – 85%, 35 – 80%, 40 – 75%, 45 – 70%, 50 – 65% or 55 – 60 % compared to an untreated cancer cell and/or compared to the cancer cell before the use of the compound targeting Tpm1.8 and/or Tpm1.9 isoforms.
  • the compound identified by the method for identifying inhibits Tpm1.8 and/or Tpm1.9 expression and/or activity by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% compared to an untreated cancer cell and/or compared to the cancer cell before the use of the compound targeting Tpm1.8 and/or Tpm1.9 isoforms.
  • the present invention further provides a method for determining the presence of epithelial-to-mesenchymal transition (EMT) in cancer cells, comprising determining Tpm1.8 and or Tpm1.9 expression in said cells, whereby an increased expression relative to a control expression indicates EMT in said cells.
  • EMT epithelial-to-mesenchymal transition
  • a control expression may for instance be an expression of a household gene such as GAPDH.
  • the expression is preferably determined by measuring the level of (m)RNA, but also protein may be measured as described elsewhere herein.
  • the present invention further provides a method for determining the presence of epithelial-to-mesenchymal transition (EMT) in cancer cells, comprising determining RBM24 and/or ESRP1 expression in said cells, whereby an ESRP1 downregulation (or decreased expression relative to a control expression) and RBM24 upregulation (or increased expression relative to a control expression) indicates EMT in said cells.
  • EMT epithelial-to-mesenchymal transition
  • the invention further provides use of RBM24 and/or ESRP1 and/or Tpm1.8/9 as markers for determining cancer development, in particular for indicating that a cancer cell is in EMT, preferably an ovarian cancer cell.
  • RBM24 and/or ESRP1 and/or Tpm1.8/9 markers for determining cancer development, in particular for indicating that a cancer cell is in EMT, preferably an ovarian cancer cell.
  • the human ovarian cancer cell line OV90 obtained from the American Type Culture Collection (ATCC), was cultured in a 1:1 mixture of MCDB 105 medium (M6395; Sigma Aldrich containing 1.5 g/L sodium bicarbonate) and Medium 199 (31150022; Thermo Fisher Scientific containing 2.2 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 ⁇ g/mL; 15140122 Thermo Fisher Scientific).
  • CAOV3 (ATCC), SKOV3 [European Collection of Authenticated Cell Cultures (ECACC) via Sigma], COV504 (ECACC), 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) with 10% FBS, 1% Pen/Strep, and 2 mM L-glutamine.
  • each cell line was confirmed by DNA fingerprinting with microsatellite markers (Amelogenin, CSF1PO, D13S317, D16S539, D5S818, D7S820, THO1, TPOX, vWA, D8S1179, FGA, Penta E, Penta D, D18S51, D3S1358, D21S11) and compared with the analogous data provided by ATCC, EACC, and https://web.expasy.org/cellosaurus/ (data not shown).
  • microsatellite markers Amelogenin, CSF1PO, D13S317, D16S539, D5S818, D7S820, THO1, TPOX, vWA, D8S1179, FGA, Penta E, Penta D, D18S51, D3S1358, D21S11
  • Plasmid transfection and lentiviral transduction cDNAs encoding Tpms 1.6, 1.7, 1.8 and 1.9 were excised from the bacterial expression vectors pGEX or pET [from P.W.G. Schevzov et al., Tropomyosin isoforms and reagents, Bioarchitecture, 2011;1(4):135-64] and cloned into the mammalian expression vector pcDNA3.1(+).
  • ESRP1 Seso Biological plasmid # HG13708- UT
  • Tpm1.6/7/8/9 expression vectors were performed with the FuGENE HD transfection reagent (Promega, E2311) according to the manufacturer’s protocol, and selected with Geneticin (#10131035, Thermo Fisher Scientific).
  • the inducible pSLIK-RBM24 vector was constructed using pDONR 233-RBM24 as entry plasmid (Horizon, #OHS6084) following Gateway Cloning instructions (11791- 020, Thermo Fisher Scientific).
  • the inducible shZEB1 lentiviral vector was obtained as described in our previous study (18).
  • ESRP1 Horizon, V3THS_335722
  • shRBM24 Horizon, V3SH11240-225117283
  • lentiviral constructs were packaged by psPAX2 (Addgene # 12260) and pMD2.G (Addgene # 12259) into HEK293T cells.
  • the virus-containing supernatant was collected 24 h after transfection, filtered, and used to infect the OV90 and COV504 cell lines. Selection was applied with 750 ng/mL puromycin (#ant-pr-1, InvivoGen) or 800 ⁇ g/mL Geneticin for 1- 2 wk.
  • siRNA transfection siRNA target sequence for human Tpm1.6/7 was 5’- AAGCTGGAGCTGGCAGAGAAA-3’(codons 70-76 of TPM1.6/7 in exon 2b) and for human Tpm1.8/9 was 5’-CGAGAGGAAGCTGAGGGAGAC-3’(codons 38-44 of Tpm1.8/9 in exon 1b).
  • Tpm siRNAs (Horizon Discovery, Waterbeach UK) and control siRNA (#4390843, Thermo Fisher Scientific) were transfected by Lipofectamine RNAiMAX (#13778150, Invitrogen) according to the manufacturer’s instructions.
  • RT-qPCR was performed using the Fast SYBR Green Master Mix (#4368708, Thermo Fisher Scientific) on an Applied Biosystems StepOne Plus Real-Time Thermal Cycling Research device, with three replicates for each analysis. Relative gene expression was determined by normalizing the expression of each target gene to that of GAPDH. Results were analyzed using the 2-( ⁇ Ct) method. RT-qPCR primers are listed in Supplementary Table 4.
  • the membranes were incubated with primary antibodies directed against ESRP1 (1:1000, # PA5-25833, Thermo Fisher Scientific), RBM24 (1:100, #18178-1-417 AP, Proteintech), TPM1 (1:1000, #MBS127505, MyBioSource) and ⁇ -actin (1:2000, #4970, Cell Signaling).
  • the secondary Ab was a goat anti-rabbit immunoglobulins/HRP (1:10000, #P0161, DAKO)(1:10000, #P0448, DAKO). Detection was by the Pierce ECT western blotting substrate (#34578, Thermo Fisher Scientific) using the Amersham AI600 imager (GE Healthcare).
  • Patient-derived ascites were first washed 1-2 times with 1 ⁇ RBC lysis buffer [150 mM NH4Cl (#7173-51-5, Sigma Aldrich), 10 mM KHCO3 (#298-14-6, Sigma Aldrich), 100 ⁇ M EDTA (#60-00-4, Sigma Aldrich)] to remove erythrocytes.
  • 1 ⁇ RBC lysis buffer 150 mM NH4Cl (#7173-51-5, Sigma Aldrich), 10 mM KHCO3 (#298-14-6, Sigma Aldrich), 100 ⁇ M EDTA (#60-00-4, Sigma Aldrich)] to remove erythrocytes.
  • the cell pellets were then labeled 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 Red (1:1000, #S34859, Thermo Fisher Scientific) antibodies, and sorted by FACS.
  • RNA from CD45- CD90- and CD45- CD90+ cells was isolated directly after FACS sorting.
  • Immunohistochemistry – cultured cells 0.5-1 ⁇ 10 4 cells were plated into 24-well plates containing glass cover slips coated with 0.2% gelatin. After 6-24 h, culture medium was removed, ice-cold methanol added to each well and plates incubated for 20 min at 40 °C. Cells were washed twice with PBS, and incubated in 0.2% Triton for 20 min with rotation. Cells were blocked in 2% FBS in PBS for 1 h. Primary antibodies for Tpm1.6/7 (1:200, from P.W.G.), Tpm1.8/9 (1:200, from P.W.G.), Arp2 (1:200, #ab47654, Abcam) were added and incubated O/N at 40 °C.
  • Tumoroids 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).
  • BaseScope Assay BaseScope assays were performed following the guidelines from ACD (Advanced Cell Diagnostics, Newark, CA). 4 ⁇ m sections were cut onto Superfrost plus slides (#10149870, Thermo Fisher Scientific) and stored O/N at RT. Sections were baked for 1 h at 60°C before deparaffinizing in xylene and 100% ethanol.
  • Sections were dried for 5 min at 60°C , incubated in hydrogen peroxide at RT for 10 min, underwent target retrieval for 15 min at 100°C, and protease treatment for 30 min at 40°C.
  • BaseScope probes were added and slides incubated in an oven for 2 h at 40°C before adding reagents AMP1 (30 min at 40°C), AMP2 (30 min at 40°C), AMP3 (15 min at 40°C), AMP4 (30 min at 40°C), AMP5 (30 min at 40°C), AMP6 (15 min at RT), AMP7 (30 min at RT) and AMP8 (15 min at RT).
  • a virtual in silico docking screen of the ZINC database was performed and 6 compounds with the best docking scores were purchased from suppliers.
  • Human fibroblasts were exposed to each of the six compounds or vehicle alone for 24 hr, fixed and stained for Tpm1.8/9 using isoform specific antibodies (Brayford et al, 2016. Current biology: CB. 26(10):1312-8).
  • Control cells show strong enrichment of Tpm1.8/9 in the lamellipodium (Brayford et al, 2016).
  • Two of the compounds, Tpm1.8/9-1 and -3 prevented enrichment in the lamellipodium at 10 and 20 ⁇ M. They were selected for further studies.
  • EpCAMhi/lo RNASeq data was obtained from the ovarian cancer cell lines OV90 and CAOV3 and the sequencing reads mapped to GRCh37.p13. genome by STAR55 (https://www.gencodegenes.org/human/release_19.html).
  • MISO56 was used to quantify AS events with annotation from https://miso.readthedocs.io/en/fastmiso/index.html#iso- centric.
  • the MISO uses the alternative exon reads and adjacent conservative reads to measure the percentage of transcript isoform with specific exon included, termed Percentage Spliced In (PSI or ⁇ ).
  • PSI Percentage Spliced In
  • no isoform includes a specific alternative exon) to 1 (i.e. all of the isoforms detected comprise the alternative exon).
  • AS events were defined as differentially spliced events when the difference of mean PSI between two groups ( ⁇ psi; differential Percentage Spliced In) was >10%.
  • RNA seq analysis of subpopulations in OV90 and CAOV3 RNA was isolated from sorted populations with Trizol reagent.
  • RNA seq analysis of TPM1 OE in OV90 Paired end mRNA sequencing was performed with the DNA Nanoball sequencing (DNBseq) technology till a depth of 25M reads per sample (BGI Genomics, Shen Zhen).
  • Adapter trimming and quality filtering was performed using the SOAPnuke pipeline (BGI Genomics). Clean fastq files were aligned to the GRCh37 reference genome with RSEM (v1.3.3)( Li et al. BMC Bioinformatics.2011;12:323) using the STAR aligner (v2.7.9a) (Dobin et al. Bioinformatics. 2013;29(1):15-21).
  • a threshold (>0.1) was used to annotate cells with the highest association to the Epcamlow signature (“low-like cells”, 4% of cancer cells).
  • low-like cells were visualized on the integrated UMAP embedding from Vázquez-Garc ⁇ a et al. (supra) and pathway activity of clusters encompassing low-like cells were visualized with ComplexHeatmap.
  • FASTQ files from Izar et al. (Nat Med.2020;26(8):1271-9) were downloaded from the TerraBio repository and processed with RSEM using the STAR aligner to the hg19 human reference genome with isoform annotation from UCSC (Love et al. Genome Biol.
  • TPM1 in TCGA OVCA RSEM processed data from the TCGA cohort was downloaded from the tsvDB (Sun et al. BMC Genomics. 2018;19(1):405). Data was log2 transformed and survival analysis was performed with the survival package. Survival curves were generated with the survminer package for the whole TPM1 gene, TPM1.7 (isoform_uc002alk) and TPM1.9 (isoform_uc002alt) based on clinical data on overall survival (Therneau & Grambsch. Modeling Survival Data: Extending the Cox Model. Springer-Verlag New York 2000).
  • RNA sequencing data has been deposited to the Gene Expression Omnibus (GEO) and can be accessed using the following identifiers: GSE192920 (subpopulations in OV90 and CAOV3), GSE231560 (TPM1 OE in OV90).
  • GEO Gene Expression Omnibus
  • Other single cell RNA sequencing data used in this study are publicly available and can be accessed from GEO for the SmartSeq2 data [GSE146026 (Izar et al., supra)] and from Synapse for the 10X Genomics data [syn25569736 (Vázquez-Garc ⁇ a et al., supra)].
  • Statistical analysis For statistical comparison, we performed unpaired t test. Statistical analyses were performed using Prism 7 software (GraphPad). Data with statistical significance are as indicated.
  • EpCAM lo cells Upon sorting and short-term culturing, EpCAM lo cells showed a mesenchymal-like morphology, in contrast with the epithelial appearances of their EpCAM hi counterparts. Accordingly, EpCAM lo cells showed increased migration and invasion capacity in trans-well assays.
  • RNAseq analysis was carried out on the EpCAM hi/lo cells sorted by FACS from the OV90 and CAOV3 lines.
  • Principal component analysis (PCA) by multidimensional scaling (MDS) revealed a clear separation of the quasi-mesenchymal EpCAM lo cells from their epithelial counterpart in the second dimension.
  • non-adnexa including omentum, peritoneum, etc.
  • EMT and inflammatory signaling pathways were significantly upregulated in EpCAM lo -like clusters, in particular in cluster #2 in ascites and metastatic lesions.
  • HGSOC cell lines encompass subpopulations of quasi-mesenchymal cells endowed with increased motility and invasive capacity, and characterized by EMT-TFs the expression of which is central to their cellular identity. Similar subpopulations of ovarian cancer cells are found in patient-derived primary and metastatic lesions, and in malignant ascites. Differential expression of RNA-binding proteins underlies alternative splicing of a subset of target genes in quasi-mesenchymal ovarian cancer cells.
  • RNA-binding proteins known to be involved in alternative splicing are differentially expressed between EpCAMlo and EpCAM hi cells in colon cancer cell lines and play significant functional roles in controlling E-to-M and M-to-E transitions and phenotypic plasticity during local dissemination and distant metastasis.
  • RBPs RNA-binding proteins
  • ESRP1 and RBM24 were respectively down- and upregulated in EpCAM lo cells in both cell lines.
  • other RBPs i.e.
  • ESRP2, RBM47, RBMS3, and QKI were differentially expressed in one of the cell lines examined.
  • Differentially spliced genes were analyzed by MISO (Mixture of Isoforms) and the results were filtered by selecting ⁇ PSI (differential Percentage Spliced In) values >10% and by comparing them with the corresponding lists of AS targets previously found in colon cancer cell lines. The large majority of ovarian cancer AS targets was not found in colon cancer.
  • TPM1 AS-pattern observed in ovarian cancer involved exons 1a/2a, which earmark the Tpm1.6/7 isoforms upregulated in EpCAM hi cells, and exon 1b, featuring the Tpm1.8/9 isoforms upregulated in EpCAM lo , as validated both by RT-qPCR and western blot analysis.
  • the PEA1 and PEA2 cell lines exclusively express RBM24 and ESRP1, respectively. Accordingly, both ESRP1 knockdown and RBM24 ectopic expression in the OV90 and COV504 cell lines resulted in the up- and downregulation of the Tpm1.8/9 and Tpm1.6/7 isoforms, respectively, both at the RNA and protein levels. Vice versa, RBM24 knockdown and ESRP1 ectopic expression resulted in the up- and downregulation of the Tpm1.6/7 and Tpm1.8/9 isoforms, respectively ( Figure 1A-B).
  • Tpm1.8/9-overexpressing (OE) ovarian cancer cells appeared to invade the collagen layer collectively, as narrow linear strands with “leader”and “follower”cells ( Figure 2E).
  • Tpm1.8/9 are specifically expressed in lamellipodia, i.e. the membrane protrusions found at the leading edge driven by branched as well as unbranched filaments composed of actin and Tpm1.8/9 that promote cell motility.
  • IF analysis with TPM1 isoform-specific antibodies confirmed the co-localization of Tpm1.8/9 and ARP2, a specific marker for lamellipodia, at the edge of ovarian cancer cells; instead, Tpm1.6/7 were mainly localized in the cytoplasm (Figure 2F).
  • RNAseq analysis was carried out on the OV90 overexpressing cells. Unsupervised hierarchical clustering and principal component analyses confirmed the distinct transcriptional identity of the OV90 parental, Tpm1.6/7-OE, and Tpm1.8/1.9-OE cells. Gene set enrichment analysis (GSEA) was then performed to allow the identification of specific signaling pathways and gene ontology functions characteristic of each of the above sample groups. As shown in the heatmap of Figure 3A, TPM1.8/9 overexpression resulted in the pronounced activation of Hedgehog, Wnt/ ⁇ -catenin, TGF- ⁇ , and Notch signaling, i.e.
  • GSEA Gene set enrichment analysis
  • TPM1 isoforms confer specific functional features on ovarian cancer cells.
  • Tpm1.8/9 isoforms are strongly associated with EMT-inducing and inflammatory signaling pathways likely to underlie ‘transcoelomic’ dissemination of ovarian cancer cells and the formation of ascites.
  • their ectopic overexpression is also a potential source of artifacts as EMT is equally essential as is MET in the formation of intra-abdominal metastases.
  • Tpm1.8/9 isoforms confer resistance to taxane- and platinum-based chemotherapy and are expressed in ovarian cancer patients-derived ascites.
  • TPM1 alternative splicing in women suffering from ovarian cancer
  • patient-derived tumor tissues were examined by immunohistochemistry (IHC) and in situ hybridization (ISH) analyses with isoform- specific antibodies and oligonucleotide probes.
  • IHC immunohistochemistry
  • ISH in situ hybridization
  • Tpm1.6/7 appeared to be consistently expressed in primary and metastatic lesions ( Figure 4A).
  • Tpm1.8/9 expression was virtually undetectable above background levels, with few cases showing patchy and enhanced staining.
  • Tpm1.8/9 isoforms are transiently expressed in ovarian cancer patient-derived ascites when compared with Tpm1.6/7.
  • Total RNA was then extracted from the sorted cells for RTqPCR analysis of TPM1 isoform expression. Increased Tpm1.8/9 expression was observed in the ascites-derived cancer cells (CD90-) when compared with Tpm1.6/7.
  • Tpm1.6/7 expression levels were increased when compared with Tpm1.8/9.
  • expression profiles from the Cancer Genome Atlas (TCGA) project were employed and the TCGA Splicing Variants Database (TSVdb; http://www.tsvdb.com/) and integrated the clinical follow-up data with the expression of TPM1 (whole gene) and its isoforms.
  • TSVdb Cancer Genome Atlas
  • Kaplan-Meier analysis was borderline significant for the TPM1 gene and its Tpm1.6/7 isoforms, whereas the p values for Tpm1.8/9 were both >0.05.
  • RTqPCR analysis revealed a dramatic downregulation of Tpm1.6/7 expression with both agents when compared with the untreated parental cells.
  • Tpm1.8/9 expression appeared to increase in cells surviving both the cisplatin- and paclitaxel-treatment ( Figure 4B).
  • siRNA assays were developed to selectively downregulate the Tpm1.6/7 and Tpm1.8/9 isoforms in parental OV90 cells and assess their chemo-resistance. As shown in Figure 4D-E, the specific downregulation of the Tpm1.8/9 isoforms, validated both at the RNA and protein levels, reduced the cisplatin- and paclitaxel-specific IC50, while the opposite was true for the siRNA-driven knockdown of Tpm1.6/7.
  • Tpm1.8/9-specific small molecule inhibitors for ovarian cancer therapy Given the newly uncovered role played by the Tpm1.8/9 isoforms in ovarian cancer and the broad spectrum of consequences at the cellular and molecular level that their specific inhibition may exert on cell motility and proliferation, EMT/MET, several oncogenic signalling pathways including Wnt, and therapy resistance, the development of small molecule antagonists may provide novel tools in the clinical management of late- stage ovarian cancer. Differences in the N-and C-termini between tropomyosin isoforms provide an opportunity to develop compounds that preferentially target specific isoforms.
  • Tpm1.8/9 docking identified 6 candidate compounds that were tested for biological activity in fibroblasts based on their ability to disperse the target Tpm1.8/9 away from actin-containing structures in the lamellipodium.
  • the two compounds 3-(2-Methyl-indol-1-yl)- propylamine (PubChem CID 6494468) and 1-Phenylmethyl-1h-indole-2-methanol (PubChem CID 18973468), showed activity in the low micro molar range.
  • their most effective concentration was determined in the 0 to 10 ⁇ M ( ⁇ 10 ⁇ M) range both on the OV90 parental cell line and on its sorted EpCAM lo subpopulation.
  • EMP epithelial-mesenchymal plasticity
  • EMT is a highly variable process with a very broad spectrum of upstream signals from the TME, intracellular regulatory mechanisms, and downstream effectors, the identity of which largely depends on the tumor type and its micro- and macro-environment. The same is true when it comes to the nature of the epigenetic mechanisms that underlie EMP. It was recently shown that the differential expression of RNA-binding proteins between the epithelial tumor bulk and subpopulations of quasi- mesenchymal colon cancer cells underlies alterative splicing at a variety of target genes known to play functional roles in EMT, metastasis, and resistance to chemotherapy. Here, a similar strategy toward the identification and functional characterization of genes whose splicing is altered during EMT/MET in ovarian cancer was applied.
  • EMT even though through distinct cellular and molecular mechanisms, EMT does play a key role in ovarian cancer metastasis and chemo- resistance.
  • Gene ontology analysis of the ovarian cancer specific AS targets revealed an extremely broad spectrum of biological processes, molecular functions, and cellular components likely to collectively contribute to the transition to quasi-mesenchymal ovarian cancer cells capable of local invasion and distant metastatic colonization.
  • AS targets likely to contribute to ovarian cancer progression have been reported such as BCL2L12 and ECM1.
  • TPM1 was selected because of its function in the regulation of cell motility through cytoskeletal modifications, and its alleged role as a tumor suppressor and even oncogene in multiple cancer types.
  • TPM1 alternative spliced isoforms are found in multiple tissues.
  • the present results establish a direct causative relation between the Tpm1.8/9 isoforms, arising due to the differential expression of several RBPs between epithelial and quasi-mesenchymal ovarian cancer cells, and EMT activation through multiple signaling pathways including Wnt, TGF- ⁇ , Hedgehog, and Notch.
  • the respective up- and down-regulation of the RBM24 and ESRP1 RNA-binding proteins earmark the epithelial-to-mesenchymal transitions in the ovarian cancer cell lines here examined.
  • Tpm1.8/9 isoforms are likely to facilitate dissemination from the primary tumor to the intra-abdominal cavity, as also shown by their enrichment in patient-derived ascites. Allegedly as a consequence of their EMT-inducing capacity, ectopic expression of the low- molecular weight TPM1 isoforms confers resistance to taxane- and platinum-based chemotherapy. Accordingly, Tpm1.8/9 are also found to be expressed at very low levels, if any, in primary ovarian cancers and their metastases albeit increased in malignant ascites.
  • Tpm1.8/9 form a relevant therapeutic target.
  • small molecule inhibitors prevents EMT and reduce cell motility, and simultaneously inhibit the activation of key signal transduction pathways such as Wnt, known to play a central role in ovarian cancer stemness, EMT, and chemoresistance.
  • Wnt key signal transduction pathways
  • Example 2 Materials and Methods Breast cancer MCF-7 cells growing in DMEM with 10 % FBS were washed in PBS, trypsinised for 3 minutes and resuspended in complete media. MCF-7 cells were counted and seeded at a density of 2x10E3 per well in a volume of 100 ⁇ L with complete media onto a 96 well plate. Cells were incubated for 24 hours prior to adding drug compounds. Paclitaxel was serially diluted from 50 nM to 0.39 nM final concentration in complete media.
  • Paclitaxel was also serially diluted from 50 nM to 0.39 nM final concentration in complete media with the addition of 189-3 compound (1-Phenylmethyl-1h-indole-2-methanol (PubChem CID 18973468)) at 5 ⁇ M or 10 ⁇ M constant.
  • Serial dilutions of either the Paclitaxel alone or with the 189-3 compound were added in triplicate on the 96 well plate. After 72 hours incubation, a 20 ⁇ L volume of MTS reagent (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay – Promega G3581) was added per well and the plate was incubated for a further 2 hours.

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Abstract

La présente invention a pour objet de nouveaux composés thérapeutiques améliorés pour la prévention et/ou le traitement d'un cancer, tel que le cancer de l'ovaire. À cet effet, la présente invention porte sur des composés ciblant les variants d'épissage alternatif de TPM1 Tpm1.8 et/ou Tpm1.9 Les composés ou une composition pharmaceutique comprenant au moins un composé selon la présente invention contrecarrent la chimiorésistance, telle que la chimiorésistance aux agents chimiothérapeutiques à base de taxane et/ou de platine. En outre, la présente invention porte sur un procédé d'identification de composés ciblant les isoformes Tpm1.8 et/ou Tpm1.9 qui ont un effet thérapeutique d'inhibition, de suppression, de prévention et/ou de traitement d'un cancer.
PCT/NL2024/050410 2023-07-24 2024-07-24 Composés ciblant les isoformes tpm1.8 et/ou tpm1.9 pour la prévention et/ou le traitement d'un cancer Pending WO2025023834A1 (fr)

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