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WO2019002655A1 - Procédé permettant de mettre au point une thérapie personnalisée pour un individu qui est atteint d'un cancer du poumon résistant au cisplatine - Google Patents

Procédé permettant de mettre au point une thérapie personnalisée pour un individu qui est atteint d'un cancer du poumon résistant au cisplatine Download PDF

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Publication number
WO2019002655A1
WO2019002655A1 PCT/ES2018/070463 ES2018070463W WO2019002655A1 WO 2019002655 A1 WO2019002655 A1 WO 2019002655A1 ES 2018070463 W ES2018070463 W ES 2018070463W WO 2019002655 A1 WO2019002655 A1 WO 2019002655A1
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Prior art keywords
treatment
cisplatin
pgc
gene
expression
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English (en)
Spanish (es)
Inventor
Mariano PROVENCIO PULLA
Alberto CRUZ BERMÚDEZ
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FUNDACION PARA LA INVESTIGACION BIOMEDICA DEL HOSPITAL UNIVERSITARIO PUERTA DE HIERRO MAJADAHONDA
Universidad Autonoma de Madrid
Original Assignee
FUNDACION PARA LA INVESTIGACION BIOMEDICA DEL HOSPITAL UNIVERSITARIO PUERTA DE HIERRO MAJADAHONDA
Universidad Autonoma de Madrid
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Publication of WO2019002655A1 publication Critical patent/WO2019002655A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to an in vitro method for designing personalized therapy for an individual suffering from cisplatin-resistant lung cancer comprising determining the level of expression of the PGC-1alpha gene before and after treatment with cisplatin, wherein if the level of expression of said gene is higher after than before treatment, then therapy after treatment with cisplatin comprises the administration of a compound directed to decrease OXPHOS function. Therefore, the present invention is encompassed within the field of cancer treatment, more specifically, in the treatment of lung cancer.
  • Lung cancer is the most widespread cancer in the world in terms of incidence and is the main cause of death due to cancer.
  • cisplatin-based chemotherapy is the standard treatment.
  • resistance often develops during the treatment, limiting the clinical usefulness of this drug.
  • the mechanisms of resistance of cisplatin are complex, since they involve various strategies and metabolic pathways.
  • an increase in oxidative metabolism has been suggested as the common mechanism of action for many cancers and resistance to treatment.
  • NSCLC non-small cell lung cancer
  • Mitochondria are organelles known to be the cell's powerhouse, responsible for oxidative phosphorylation or OXPHOS (from their initials in English, oxidative phosphorilation).
  • OXPHOS oxidative phosphorylation
  • acetyl CoA from glycolysis or the oxidation of fatty acids feeds the cycle of tricarboxylic acids (TCA) that generates reduced cofactors.
  • TCA tricarboxylic acids
  • ROS reactive oxygen species
  • MIMP mitochondrial inner membrane potential
  • the inventors have found that, in lung cell lines, the expression levels of the PGC-1alpha gene are increased in response to treatment with cisplatin, causing the cell to become resistant to this drug, but, surprisingly, the viability of this cell decreases after the administration of a compound aimed at decreasing OXPHOS function, such as metformin, rotenone or an interfering RNA. PGC-1alpha gene, even reversing said resistance to cisplatin, making the cell sensitive to this drug.
  • a compound aimed at decreasing OXPHOS function such as metformin, rotenone or an interfering RNA.
  • this discovery opens a new therapeutic window to the treatment of lung cancer, in particular, lung cancer that is resistant to treatment with cisplatin, because it allows to direct the treatment to the special characteristics of the individual, avoiding the administration of ineffective therapies.
  • the present invention relates to an in vitro method for designing a personalized therapy to an individual suffering from cisplatin-resistant lung cancer comprising
  • step (b) comparing the level of expression obtained in step (a) before treatment with the level of expression after treatment,
  • a level of PGC-1alpha expression after treatment greater than the level of expression of PGC-1 alpha before treatment is indicative that therapy after treatment with cisplatin comprises the administration of a compound aimed at decreasing OXPHOS function .
  • “therapy” or “treatment” means the administration to an individual of a compound, or combination of compounds, for the purpose of inhibiting a disease or pathological condition, that is, stopping its development; alleviate a disease or pathological condition, that is, cause the regression of the disease or the pathological condition; and / or stabilize a disease or pathological condition in an individual.
  • the disease or pathological condition is lung cancer, in particular, non-small cell lung cancer (NSCLC of its acronym in English non-small cell lung cancer).
  • NSCLC non-small cell lung cancer
  • lung cancer is metastatic, more in particular, NSCLC in the metastatic state.
  • lung cancer is understood to mean that disease, or group of diseases, resulting from the uncontrolled growth of the cells of the respiratory tract, in particular, of the lung tissue.
  • cancer also includes the concept of "tumors", I understand these as an aggregate set of cells resulting from the uncontrolled proliferation of a single cell.
  • carcinomas that is, malignant tumors that arise from epithelial cells.
  • lung carcinoma There are two forms of lung carcinoma, categorized by the size and appearance of the malignant cells seen histopathologically under a microscope: the tumors of non-small cells and those of small cells.
  • examples of lung cancer include, without limitation, adenocarcinomas, squamous cell carcinomas, large cell carcinomas and small cell carcinomas.
  • bronchoalveolar carcinomas and several mixed forms.
  • lung cancer can be metastatic or non-metastatic.
  • metastatic lung cancer is understood to mean that stage of cancer development in which cancerous respiratory tract cells, in particular, cancerous lung cells, invade the individual's bloodstream, reaching the lymph nodes , and colonizing other tissues than the tissue of origin.
  • the therapy is considered to be "personalized” when the compound (s) to be administered to the individual to treat a disease is specially adapted to the genotypic and phenotypic characteristics of the individual to be treated, avoiding with it the loss of time in ineffective therapies.
  • the characteristic that determines the therapy that is going to be administered to the individual is the expression level of the PGC-1alpha gene.
  • lung cancer is "resistant to cisplatin" when the number of tumor (or carcinogenic) cells does not decrease in response to the administration of cisplatin as a consequence of the fact that the tumor cells have become progressively resistant to this compound due to the continued administration thereof to the individual suffering from lung cancer.
  • the criterion followed in clinical practice is also contemplated, where it is considered that an individual presents a lung cancer "resistant to cisplatin" when the individual does not respond to the treatment with cisplatin as established using the response criteria to the treatment of solid tumors (Response Evaluation Criteria In Solid Tumors, RECIST), three-dimensional measurement methods, or both.
  • the RECIST criteria are widely known in the state of the art and are routine practice for the expert in the field.
  • the term "individual” is equivalent to the term “subject”, so both terms can be used interchangeably throughout the present description. It is understood by “individual”, any animal belonging to any species. However, in a particular embodiment, the subject is a mammal, preferably a primate, more preferably, a human being of any race, sex or age.
  • the individual subject of the present invention suffers from lung cancer and will be treated with cisplatin as a first treatment.
  • the method of the invention comprises in a first step, step a), determining the level of expression of the PGC-1alpha gene before and after treatment with cisplatin in an isolated biological sample from said individual.
  • PGC-1 alpha gene is a gene that, in humans, is located on chromosome 4p15.2.
  • Alternative names that may be used in the scientific literature to refer to the PGC-1alpha gene include, but are not limited to, "peroxisome proliferative activated receptor, gamma, coactivator 1", “peroxisome proliferative activated receptor, gamma, coactivator 1, alpha”, “peroxisome proliferator-activated gamma receptor, coactivator 1 alpha”, “PPARG coactivator 1 alpha”, PPARGC1A, LEM6, PGC-1v, PGC1, PGC1A and PPARGC1.
  • the PGC-1alpha gene comprises a nucleotide sequence with a sequence identity of at least 80, 85, 90, 95, 96, 97, 98, 99% with the nucleotide sequence SEQ ID NO: 1 (access number to GenBank AF106698 version AF106698.1).
  • the PGC-1 alpha gene comprises a nucleotide sequence with a sequence identity of 100% with the nucleotide sequence SEQ ID NO: 1.
  • sequence identity is understood to be the degree of similarity between two nucleotide (or amino acid) sequences obtained by aligning the two sequences.
  • BLAST Altschul SF et al. Basic local alignment search tool. J Mol Biol. 1990 Oct. 5; 215 (3): 403-10].
  • the BLAST programs for example, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are public domain on the website of The National Center for Biotechnology Information (NCBI).
  • the PGC-1alpha gene encodes the PPAR gamma coactivator-1 protein.
  • the protein encoded by the PGC-1alpha gene comprises an amino acid sequence with a sequence identity of at least 80, 85, 90, 95, 96, 97, 98, 99% with the sequence SEQ ID NO: 2 (access number to GenBank NP_037393 version NP_037393.1).
  • the protein encoded by the PGC-1 alpha gene comprises an amino acid sequence with a sequence identity of 100% with the sequence SEQ ID NO: 2.
  • sequence identity has been previously defined.
  • the terms "expression” and “gene expression” include the transcription and / or translation of the nucleic acid. Therefore, the quantification of the expression of the PGC-1alpha gene can be determined from the nucleic acid of the PGC-1alpha gene or from the protein encoded by said gene, that is, from the PPAR gamma coactivator-1 protein.
  • the determination of the expression levels of the PGC-1alpha gene comprises measuring the level of cDNA, or a fragment thereof, the level of mRNA, or a fragment thereof, and / or the level of the protein encoded by said gene, or a fragment of said protein.
  • mRNA fragment or "cDNA fragment” is understood to mean the nucleotide sequence of the PGC-1alpha gene comprising one or more nucleotides absent from the 3 'and / or 5' ends with respect to the sequence of complete nucleotides of the PGC-1alpha gene.
  • the fragment of the PGC-1alpha gene is a fragment of the sequence SEQ ID NO: 1.
  • fragment of the PPAR gamma coactivator-T protein is understood to be the amino acid sequence of the PPAR gamma coactivator-1 protein comprising one or more amino acids absent from its amino terminal or carboxyl terminal end with with respect to the complete amino acid sequence of the PPAR gamma coactivator-1 protein
  • the fragment of the PPAR gamma coactivator-1 protein is a fragment of the sequence SEQ ID NO: 2.
  • the biological sample can be treated to physically or mechanically disintegrate the structure of the tissue or the cell, releasing the intracellular components in an aqueous or organic solution to isolate and prepare the nucleic acids. aen by methods known to the person skilled in the art and commercially available (Sambroock, J., et al. 2012, "Molecular cloning: a Laboratory Manual", 4th ed., Cold Spring Harbor Laboratory Press, NY, Vol.
  • mRNA levels of said gene can be quantified by the use of conventional methods, for example, methods comprising the amplification of the mRNA and the quantification of the amplification product of said MRNA, such as electrophoresis and staining, or alternatively, by means of Southern blot and use of appropriate probes, northern blot and use of specific probes of the mRNA of the gene of interest (PTGDR gene) or of its corresponding cDNA, mapping with nuclease S1, RT -PCR, hybridization, microarrays, etc.
  • the levels of the cDNA corresponding to said mRNA of the PGC-1alpha gene can also be quantified by the use of conventional techniques; in this case, the method of the invention includes a step of synthesizing the corresponding cDNA by reverse transcription (RT) of the corresponding mRNA followed by amplification and quantification of the amplification product of said cDNA.
  • RT reverse transcription
  • Conventional methods of quantifying expression levels can be found, for example, in Sambrook et al. cited ad supra.
  • the quantification of the expression levels is carried out by means of a quantitative polymerase chain reaction (PCR), an array of DNA or RNA, or RNA-Seq or Mass Sequencing applied to the study of RNA.
  • the level of expression of the mRNA of the PGC-1alpha gene is determined by qRT-PCR using the Taqman® gene expression assay (Taqman® gene expression assay) with the probe Hs01016719_m1. If the quantification of PGC-1 alpha gene expression is to be performed from the protein encoded by the PGC-1alpha gene, ie the PPAR gamma coactivator-1 protein, then the biological sample isolated from the subject has to be treated to extract the proteins. Methods for extracting or isolating proteins are known to the person skilled in the art and are commercially available (Sambroock, J., et al., 2012, cited supra).
  • the levels of the PPAR gamma coactivator-1 protein can be quantified by any conventional method that allows to detect and quantify said protein in a sample of a subject.
  • the levels of said protein can be quantified, for example, by the use of antibodies capable of binding to the PPAR gamma coactivator-1 protein and the subsequent quantification of the formed complexes.
  • Antibody means a glycoprotein of the gamma globulin type that forms part of the humoral immune system that binds specifically to an antigen.
  • Antibodies specific for the protein encoded by the PGC-1alpha gene are commercially available. Examples of such antibodies include, but are not limited to, antibodies 3G6 # 2178 from Cell Signaling Company, ab54481 from Abcam and H-300 sc-13067 from Santa Cruz Biotechnology. Also, the methods for producing antibodies are widely known in the state of the art.
  • label refers to a composition capable of producing a detectable signal indicative of the presence of the labeled molecule.
  • suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like.
  • a trademark is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • antibodies or reagents capable of binding said PPAR gamma coactivator-1 protein include, but are not limited to, polyclonal sera, supernatants of hybridomas or monoclonal antibodies, antibody fragments, Fv, Fab, Fab 'and F (ab') 2, scFv, diabodies, triabodies, tetrabodies and humanized antibodies.
  • antibodies against PPAR gamma coactivator-1 protein that can be used in the context of the present invention, such as those cited above.
  • biological sample refers to any biological material that can be obtained from the individual, such as a biopsy, a tissue, a cell or a fluid (serum, saliva, semen, sputum, tears). , mucus, sweat, milk, brain extracts and the like), and that can hold information about the characteristic genetic endowment of a person.
  • the biological sample is blood, serum or tissue from a biopsy.
  • isolated implies that the biological sample has been separated or extracted from the rest of the components that accompany it naturally. Techniques for obtaining biological samples from an individual are well known in the state of the art, and any of them can be employed in the practice of the present invention.
  • step (a) comprising compare the levels of expression obtained between them. If the level of expression of PGC-1alpha after treating the individual with cisplatin is greater than the level of expression of PGC-1 alpha before treatment with cisplatin, then the therapy to be administered to the individual comprises the administration of a compound directed to decrease the OXPHOS function. Otherwise, the administration of a compound aimed at decreasing the OXPHOS function is not necessary.
  • one level of expression is “greater” than another when one value of the level of expression is higher than another.
  • an expression level is “higher”, when the expression levels of the PGC-1alpha gene after treatment with cisplatin are at least 1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more with respect to the expression levels of the PGC-1alpha gene before treatment with cisplatin.
  • the therapy to be administered to the individual comprises the administration of a compound directed to diminish the function OXPHOS.
  • compound aimed at decreasing OXPHOS function is understood as the compound that decreases the ability to generate energy in the form of ATP by the OXPHOS system (the oxidative phosphorylation system that through a series of protein complexes). that act in chain finishes producing molecules of ATP), or through to diminish the contribution of reduced cofactors, the transport of electrons, the activity or quantity of some of their complexes or to dissipate the proton gradient generated.
  • OXPHOS system the oxidative phosphorylation system that through a series of protein complexes. that act in chain finishes producing molecules of ATP
  • the transport of electrons the activity or quantity of some of their complexes or to dissipate the proton gradient generated.
  • the compound directed to decrease the OXPHOS function is an inhibitor of the expression of the PGC-1alpha gene, or of the protein encoded by said gene, or is a compound that inhibits the complex I of the mitochondrial electron transport chain , preferably, a biguanide or rotenone.
  • a biguanide or rotenone are compounds that inhibit the complex I or NADH dehydrogenase or NADH: ubiquinone oxidoreductase of the mitochondrial electron transport chain.
  • inhibitor of PGC-1 alpha gene expression is understood as that compound capable of reducing, blocking or preventing the transcription and / or translation of the PGC-1 alpha gene.
  • inhibitor of the protein encoded by the PGC-1 alpha gene is understood to mean that compound that is able to reduce, block or prevent the activity or function of the protein encoded by said gene, that is, the protein PPAR gamma coactivator-1.
  • the function of PGC-1alpha is to act as a transcriptional cofactor that favors the transcription of various genes related to the antioxidant response and the OXPHOS function.
  • Examples of compounds capable of inhibiting the protein encoded by the PGC-1alpha gene include, but are not limited to, anti-PPAR gamma coactivator-1 antibodies as described above.
  • An assay to determine whether a given compound is an inhibitor of the activity or function of the protein encoded by the PGC-1 alpha gene is, for example, and without excluding others, the measurement of expression levels of genes regulated by PGC -1alpha and involved in the antioxidant response and OXPHOS function, such as the genes SOD2, UCP-2, Prx5, TFAM, TOM20, and any of the 13 mitochondria, such as MT-C01, MT-C02, MT-C03, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-ATP6, MT-ATP8 and MT-CYTB, among others.
  • the inhibitor of PGC-1alpha gene expression is an interfering RNA.
  • said interference RNA comprises the sequence CCUGUUUGAUGACAGCGAAtt (SEQ ID NO: 3).
  • compound that inhibits complex I of the mitochondrial electron transport chain is understood to mean that compound that is capable of blocking, decreasing, preventing or stopping mitochondrial electron transport at the level of complex I or NADH dehydrogenase, so that the cell is impaired or hindered its ability to produce ATP.
  • assays useful for determining whether a given compound is capable of inhibiting complex I of the electron transport chain include, without limitation, the measurement of mitochondrial membrane potential or the production of ATP.
  • biguanide is understood to mean that compound classified within category A10B of the ATC code (Anatomical Classification System, Therapeutics, Chemistry).
  • the biguanide is selected from the group consisting of phenformin, metformin and buformin.
  • the compound directed to decrease the OXPHOS function is metformin.
  • rotenone is understood to mean that compound with insecticidal activity, isolated from plants of the genus Lonchocarpus, which inhibits mitochondrial electron transport at the level of complex I or NADH dehydrogenase.
  • the administration of a compound directed to diminish the OXPHOS function can be carried out by any of the forms of administration that exist in the state of the art. Examples of routes of administration include, but are not limited to, oral, cutaneous, parenteral, nasal and intraperitoneal. Also within the present invention is the possibility that the compound directed to decrease the OXPHOS function is carried out in combination with a new administration of cisplatin.
  • the administration of the compound directed to decrease the OXPHOS function is carried out in combination with a new administration of cisplatin.
  • the administration of both compounds can be carried out at the same time or separately.
  • the administration of the compound directed to decrease the OXPHOS function is carried out sequentially, simultaneously or separately with the new administration of cisplatin.
  • the administration is "sequential" when the cisplatin and the compound directed to decrease the OXPHOS function are administered to the individual at different moments in time, so that first one is administered and subsequently another is administered.
  • the order in which cisplatin and the compound aimed at decreasing OXPHOS function are administered is indifferent.
  • the administration is "simultaneous" when the cisplatin and the compound directed to decrease the OXPHOS function are part of the same composition and are administered together, ie at the same time to the individual.
  • the administration is “separate” when the cisplatin and the compound directed to decrease the OXPHOS function are part of different compositions, each of them being administered individually.
  • the "separate” administration can be the same “simultaneous” if both compositions are administered at the same time.
  • the method of the invention may comprise additional steps aimed at determining other parameters useful for designing personalized therapy for an individual suffering from lung cancer and to be treated with cisplatin.
  • these parameters are the internal mitochondrial membrane potential (MIMP), ROS levels and / or mitochondrial mass.
  • the method of the invention further comprises measuring the MIMP before and after the treatment with cisplatin in an isolated biological sample from said individual, wherein a MIMP after the treatment is greater than the MIMP before the treatment. is indicative that therapy after treatment with cisplatin involves the administration of a compound aimed at decreasing OXPHOS function.
  • the MIMP can be measured, for example, by the use of DiOC6 (3) dye by flow cytometry.
  • the method of the invention further comprises measuring ROS levels before and after treatment with cisplatin in a biological sample isolated from said individual, wherein ROS levels after treatment are greater than the level of ROS before treatment is indicative that therapy after treatment with cisplatin involves the administration of a compound aimed at decreasing OXPHOS function.
  • ROS levels can be measured, for example, by determining catalase activity or quantifying malondialdehyde, among others.
  • the method of the invention further comprises measuring the mitochondrial mass before and after treatment with cisplatin in a biological sample isolated from said individual, wherein a mitochondrial mass after the treatment is greater than the mitochondrial mass before the Treatment is indicative that therapy after treatment with cisplatin involves the administration of a compound aimed at decreasing OXPHOS function.
  • the reagents, conditions and methods for measuring MIMP, ROS levels and mitochondrial mass are widely known in the state of the art.
  • the use of expression levels of the PGC-1alpha gene is also contemplated to design personalized therapy for an individual suffering from a cisplatin-resistant lung cancer.
  • the present invention relates to the in vitro use of the expression level of the PGC-1 alpha gene to design personalized therapy for an individual suffering from a lung cancer resistant to cisplatin.
  • this inventive aspect will be referred to as "use of the invention”. How to use the level of expression of the PGC-1alpha gene to design such personalized therapy has been explained in previous paragraphs.
  • therapy after treatment with cisplatin comprises administration of a compound aimed at decreasing the OXPHOS function.
  • the use of the invention further comprises the use of MIMP, ROS levels, and / or mitochondrial mass to design personalized therapy for an individual suffering from cisplatin-resistant lung cancer.
  • a MIMP, ROS levels and / or a mitochondrial mass after treatment with cisplatin greater than the MIMP, ROS levels and / or the mitochondrial mass before said treatment are indicative that the Therapy after treatment with cisplatin comprises a compound aimed at decreasing OXPHOS function. All these terms and expressions have been explained in the above aspect of the invention.
  • the compound directed to decrease the OXPHOS function is an inhibitor of the expression of the PGC-1alpha gene, or of the protein encoded by said gene, or is a compound that inhibits the complex I of the mitochondrial electron transport chain , preferably, a biguanide or rotenone.
  • the inhibitor of PGC-1alpha gel expression is an interfering RNA, which in another still more particular embodiment, comprises the sequence SEQ ID NO: 3.
  • the biguanide is selected from group consisting of phenformin, metformin and buformin.
  • the administration of the compound directed to decrease the OXPHOS function is carried out in combination with a new administration of cisplatin.
  • this new administration of cisplatin can be carried out sequentially, simultaneously or separately.
  • the new administration of cisplatin in combination with the compound aimed at decreasing the OXPHOS function is carried out sequentially, simultaneously or separately.
  • the terms “sequential”, “simultaneous” or “separate” have been previously defined in the present description.
  • the expression levels of the PGC-1 alpha gene can be used to design a therapy for an individual suffering from any type of cisplatin-resistant lung cancer.
  • lung cancer is metastatic.
  • lung cancer is NSCLC.
  • lung cancer is metastatic NSCLC.
  • the level of expression of the PGC-1 alpha gene comprises determining the level of cDNA, mRNA and / or protein encoded by said gene.
  • the use of a compound directed to decrease OXPHOS function in the preparation of a drug for the treatment of cisplatin-resistant lung cancer in an individual is contemplated.
  • the present invention relates to the use of a compound directed to decrease OXPHOS function in the manufacture of a drug for the treatment of cisplatin-resistant lung cancer in an individual, wherein said individual comprises a level of expression of the PGC-1alpha gene after treatment with cisplatin greater than the level of expression of the PGC-1alpha gene before treatment.
  • the present invention also relates to a compound directed to decrease OXPHOS function for use in the treatment of cisplatin-resistant lung cancer in an individual, wherein said individual comprises an expression level of the PGC-1 alpha gene after the treatment with cisplatin greater than the level of expression of the PGC-1alpha gene before treatment.
  • a method for the treatment of cisplatin-resistant lung cancer in an individual comprising the administration to said individual of a compound directed to decrease the OXPHOS function, wherein said individual comprises a level of expression of the PGC-1alpha gene after treatment with cisplatin greater than the level of expression of the PGC-1alpha gene before treatment.
  • the compound aimed at decreasing the OXPHOS function is used in combination with cisplatin in the preparation of the drug.
  • the individual also comprises a MIMP after treatment with cisplatin greater than MIMP before treatment, ROS levels after treatment with cisplatin greater than ROS levels before treatment, and / or a mitochondrial mass after treatment with cisplatin greater than the mitochondrial mass before treatment with cisplatin;
  • NSCLC NSCLC and / or metastatic
  • the level of expression of the PGC-1alpha gene corresponds to the level of cDNA, mRNA and / or the protein encoded by said gene;
  • the compound aimed at decreasing the OXPHOS function is an inhibitor of the expression of the PGC-1alpha gene, or of the protein encoded by said gene, or is a compound that inhibits the complex I of the mitochondrial electron transport chain, preferably a biguanide or rotenone.
  • the inhibitor of PGC-1 alpha gene expression is an interfering RNA.
  • the biguanide is selected from the group consisting of phenformin, metformin and buformin. All of these particular embodiments as well as the expressions and terms employed have been described in previous paragraphs, and are applicable to the present inventive aspect.
  • kits comprising the reagents necessary to determine the expression levels of the gene PGC-1alfa. Therefore, in another aspect, the present invention relates to a kit for designing a personalized therapy to an individual suffering from lung cancer and to be treated with cisplatin, hereinafter "kit of the invention", which It comprises the reagents necessary to determine the expression levels of the PGC-1alpha gene.
  • kit is understood as the product that contains the reagents necessary to carry out the method of the invention adapted to allow its transport and storage.
  • Suitable materials for packaging the kit components include, but are not limited to, polyethylene, polypropylene, polycarbonate and the like, glass, plastic, etc.
  • the kit can also include bottles, jars, paper, envelopes, etc.
  • reagent that allows determining the level of expression of a gene means a compound or a group of compounds that allows determining the level of expression of a gene, both by means of the determination of the level of cDNA or mRNA and by means of the protein level. Therefore, reagents of the first type include nucleic acids, e.g. primers and probes, capable of specifically hybridizing with the mRNA encoded by the gene involved.
  • the reagents of the second type are compounds that specifically bind to the protein encoded by the gene and, preferably, the antibodies are included although they may be specific aptamers.
  • the reagents necessary to determine the expression levels of the PGC-1alpha gene comprise
  • nucleic acid that hybridizes specifically with the PGC-1alpha gene, or with a fragment thereof, and / or
  • Example of a nucleic acid that hybridizes specifically with the PGC-1 alpha gene is, for example, a probe.
  • probe is understood to mean the nucleic acid molecule whose nucleotide sequence hybridizes specifically with the nucleotide sequence of a target gene.
  • the target gene is the PGC-1alpha gene.
  • specifically-form hybrid refers to conditions that allow the hybridization of two polynucleotides in highly or moderately rigorous conditionals.
  • High stringency conditions involve, in general: (1) low ionic strength and high temperature for washing, for example sodium chloride 0.015M / sodium citrate 0.0015M / 0, 1% sodium dodecyl sulfate at 50 ° C , (2) use during the hybridization of a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1 % polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with sodium chloride at 750 mM, 75 mM sodium citrate at 42 ° C, or (3) use of 50% formamide, 5xSSC (0.75 M) NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 times Denhardt's solution, sonicated salmon sperm DNA (50 mg / mL), 0.1 % SDS, and 10% dextran
  • Modely stringent conditions include the use of wash solution and the less stringent hybridization conditions than those described above. How to carry out the hybridization of two nucleotide sequences can be found in Sambroock, J., et al. 2012, cited ad supra.
  • the kit of the invention comprises reagents that are capable of binding specifically to said protein, such as antibodies Examples of antibodies that can specifically recognize the protein encoded by the PGC-1 alpha gene and bind to it have been described in previous paragraphs.
  • the PGC-1alpha gene comprises a nucleotide sequence with a sequence identity of at least 80, 85, 90, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 1.
  • the PGC-1alpha gene comprises a nucleotide sequence with a sequence identity of 100% with the sequence SEQ ID NO: 1.
  • the kit of the invention may comprise reagents aimed at determining other parameters useful for designing personalized therapy for an individual suffering from cisplatin-resistant lung cancer. As indicated above, among these parameters are MIMP, ROS levels and / or mitochondrial mass. Thus, in a particular embodiment, the kit further comprises reagents for measuring MIMP, ROS levels and / or mitochondrial mass.
  • the invention is directed to the in vitro use of the kit of the invention to design personalized therapy for an individual suffering from cisplatin-resistant lung cancer, wherein said individual comprises a level of expression of the PGC-1 alpha gene after treatment with cisplatin greater than the level of PGC-1alpha gene expression before treatment.
  • the individual further comprises a MIMP after treatment with cisplatin greater than MIMP before treatment, ROS levels after treatment with cisplatin greater than ROS levels before treatment, and / or a mitochondrial mass after treatment with cisplatin greater than the mitochondrial mass before treatment with cisplatin.
  • lung cancer is metastatic and / or lung cancer is non-small cell lung cancer.
  • the present invention relates to the use of a kit comprising a compound directed to decrease OXPHOS function in the treatment of cisplatin-resistant lung cancer in an individual, wherein said individual comprises expression levels of the PGC gene. -1 alpha after treatment with cisplatin greater than PGC-1alpha gene expression levels before treatment.
  • the compound directed to decrease the OXPHOS function is an inhibitor of the expression of the PGC-1alpha gene, or of the protein encoded by said gene, or is a compound that inhibits the complex I of the transport chain of mitochondrial electrons, preferably, a biguanide or rotenone.
  • the biguanide is selected from the group consisting of phenformin, metformin and buformin.
  • the inhibitor of PGC-1alpha gene expression is an interfering RNA.
  • Other parameters that the individual may have increased after treatment with cisplatin compared to said parameters before treatment with cisplatin are MIMP, ROS levels and / or mitochondrial mass.
  • the invention relates to an in vitro method for designing personalized therapy for an individual suffering from lung cancer and to be treated with cisplatin comprising
  • step (b) comparing the level of expression obtained in step (a) before treatment with the level of expression after treatment,
  • the method further comprises measuring the internal mitochondrial membrane potential (MIMP) before and after treatment with cisplatin in an isolated biological sample from said individual, wherein a MIMP after the treatment is greater than the MIMP before of the treatment is indicative that the therapy comprises a second administration of cisplatin in combination with a compound directed to decrease the OXPHOS function.
  • MIMP internal mitochondrial membrane potential
  • the method further comprises measuring the levels of oxygen free radicals (ROS) before and after the treatment with cisplatin in an isolated biological sample from said individual, where ROS levels after treatment are greater than the level of ROS before treatment is indicative that the therapy comprises a second administration of cisplatin in combination with a compound aimed at decreasing OXPHOS function.
  • ROS oxygen free radicals
  • the lung cancer of the method is metastatic and / or non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the measurement or quantification of the expression levels of the PGC-1alpha gene comprises measuring the level of cDNA, mRNA and / or the protein encoded by said gene.
  • the biological sample is blood, serum or tissue from a biopsy.
  • the administration of cisplatin in combination with the compound directed to decrease the OXPHOS function is carried out sequentially, simultaneously or separately.
  • the compound directed to decrease the OXPHOS function is a biguanide, preferably, the biguanide is selected from the group consisting of phenformin, metformin and buformin.
  • the invention relates to the in vitro use of the expression level of the PGC-1alpha gene to design personalized therapy for an individual suffering from lung cancer and to be treated with cisplatin.
  • the use of MIMP and / or ROS levels is also contemplated in order to design personalized therapy for an individual suffering from lung cancer and going to be treated with cisplatin.
  • an expression level of PGC-1alpha after treatment with cisplatin greater than the level of expression of PGC-1alpha before said treatment is indicative that the therapy comprises a second administration of cisplatin in combination with a compound aimed at decreasing the OXPHOS function.
  • a MIMP and / or ROS levels after treatment with cisplatin greater than MIMP and / or ROS levels prior to said treatment is indicative that the therapy comprises a second administration of cisplatin in combination with a compound aimed at decreasing the OXPHOS function.
  • the compound directed to decrease the OXPHOS function is a biguanide, preferably, the biguanide is selected from the group consisting of phenformin, metformin and buformin.
  • the administration of cisplatin in combination with the compound directed to decrease the OXPHOS function is carried out sequentially, simultaneously or separately.
  • the lung cancer is metastatic and / or non-small cell lung cancer.
  • the level of expression of the PGC-1alpha gene comprises determining the level of cDNA, mRNA and / or protein encoded by said gene.
  • combinations of the particular embodiments described herein are also included, as well as the simultaneous presence of all the particular embodiments.
  • the invention relates to the use of cisplatin combined in a sequential, simultaneous or separate manner with a compound aimed at decreasing OXPHOS function in the preparation of a drug for the treatment of lung cancer in an individual to be treated with cisplatin, wherein said individual comprises a level of PGC-1alpha expression after treatment greater than the level of expression of PGC-1 alpha before treatment.
  • the individual further comprises a MIMP after the treatment greater than the MIMP before treatment and / or ROS levels after treatment, greater than the ROS levels before treatment.
  • the lung cancer is metastatic and / or non-small cell lung cancer.
  • the expression level of the PGC-1alpha gene corresponds to the level of cDNA, mRNA and / or protein encoded by said gene.
  • the compound directed to decrease the OXPHOS function is a biguanide, preferably, the biguanide is selected from the group consisting of phenformin, metformin and buformin.
  • the invention relates to a kit for designing a personalized therapy to an individual suffering from lung cancer and to be treated with cisplatin comprising the reagents necessary to determine the expression levels of the PGC-1alpha gene.
  • the reagents of the kit comprise
  • nucleic acid that specifically hybridizes the PGC-1 alpha gene, and / or
  • the PGC-1alpha gene comprises a nucleotide sequence with a sequence identity of at least 80, 85, 90, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 1.
  • the kit further comprises reagents for measuring MIMP and / or ROS levels.
  • the present invention relates to the in vitro use of a kit as described in previous paragraphs, to design a personalized therapy to an individual suffering from lung cancer and to be treated with cisplatin. All of these particular embodiments as well as the expressions and terms employed have been described in previous paragraphs, and are applicable to the present inventive aspects.
  • the word "comprises” and its variants do not intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention.
  • the following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
  • FIG. 2 Metabolic reprogramming in lines resistant to CDDP.
  • A Glucose consumption of cell lines.
  • B Relationship between the growth rate in galactose and glucose of the parental cell lines and resistant to CDDP, an increase in this ratio is indicative of an increase in OXPHOS function.
  • the mitochondrial inner membrane potential (C) and the total ROS levels (D) were measured by flow cytometry with the fluorescent probes TMRE and DCFHDA respectively.
  • E OXPHOS Supercomplexes in lines H1299 and H460.
  • F and G Mitochondrial mass measured by Mitotracker Green (MTG) and TOM20 immunofluorescence.
  • H Relative levels of mRNA for the PGC-1a gene compared to the parental lines.
  • Figure 4 Resistance to Cisplatin increases the sensitivity to PGC-1 to siRNA, Metformin and Rotenone.
  • A Levels of PGC-1 a after gene silencing with a non-specific (control) siRNA or specific to the sequence of the PGC-1a gene.
  • B The interference of PGC-1 a decreases the mitochondrial mass measured with MTG.
  • C Sensitivity to interference from PGC-1 a in A549 and H1299.
  • D Treatment with metformin reduces MIMP in both the parental and resistant lines.
  • E and F Sensitivity to metformin and rotenone for the parental and resistant lines A549 and H1299.
  • the graphs represent the percentage of viable cells (Y-axis) after 48 hours of different treatments with CDDP (X-axis).
  • the data are the average of at least three different experiments and are represented as a percentage relative to untreated cells.
  • the error bars indicate the standard deviation.
  • Figure 5 Changes in the early response to CDDP treatment.
  • the CDDP-resistant and parental cell lines were treated with 5 ⁇ of CDDP for 24 hours. Changes in ROS (A), levels of PGC1-a (B), mitochondrial mass (C) and MIMP (D) were evaluated. Simultaneous treatment with 10 mM NAC is able to avoid the increase in ROS and MIMP induced by CDDP treatment at 24 hours (E).
  • Cell cycle analysis and MIMP during treatment with CDDP F).
  • FIG. 6 Treatment with ZLN005 induces an increase in mitochondrial mass and reduces sensitivity to CDDP in cell lines.
  • A Mitochondrial mass measured by MTG and TOM20 IHC in cells treated with ZLN005 for 48 hours. The bars are means of at least three different experiments and are represented as a percentage relative to cells without treatment.
  • FIG. 7 Metabolic inhibition in concomitance with CDDP in parental cells resistant to CDDP.
  • the interference of PGC-1a reduces the increase in MTG produced by CDDP (A).
  • Effect of the combined treatment of siRNA against PGC-1a and CDDP on cell viability (B).
  • Treatment with metformin reduces the increase in MIMP produced by treatment with CDDP (C).
  • Metformin and rotenone eliminate differences between parental cell lines and resistant to cisplatin treatment for A549 and H1299 cells (D and E).
  • the graphs represent the percentage of viable cells (Y axis) after 48 hours of different treatments with Metformin or Rotenone (X axis).
  • the data are the average of at least three different experiments and are represented as a percentage relative to the untreated cells.
  • the error bars indicate the standard deviation.
  • Line A549 comes from a primary tumor of non-small cell lung cancer (NSCLC or NSCLC), while lines H1299 and H460 come from NSCLC metastasis, more specifically, H1299 cells are derived from lymph node and H460 of a pleural effusion. All cells were cultured routinely in DMEM (Dulbecco's Modified Eagle's medium) supplemented with 10% fetal bovine serum, penicillin and streptomycin. Cisplatin was obtained from therapeutic surpluses of the Medical Oncology service of the Hospital Puerta de Hierro Majadahonda. Metformin was obtained from sigma.
  • DMEM Dulbecco's Modified Eagle's medium
  • 3x10 3 cells were seeded in a 96 well multi-well plate and cultured in DMEM until the next day. The next morning the medium was replaced by DMEM that contained the different treatments according to the legends of the figures. Cell viability was determined by the commercial kit Cell counting Kit-8 (CCK-8) (Dojindo EU GmBH, Kunststoff, Germany) following the manufacturer's instructions.
  • the cytoplasmic ROS were determined using 2 ', 7'-Dichlorofluorescein diacetate (H2DCFHDA).
  • the internal mitochondrial MIMP potential was determined using Tetramethylrhodamine ethyl ester (TMRE).
  • TMRE Tetramethylrhodamine ethyl ester
  • the cell cycle was determined by the use of Vybrant TM DyeCycle TM Violet Stain.
  • the mitochondrial mass was determined using Mitotracker Green FM. All fluorescent probes were purchased from Thermo Scientific. For these tests, 1x10 5 cells were cultured.
  • the cells were recovered in DMEM and analyzed immediately on a Cytomic FC500 MPL cytometer (BeckmanCoulter). The size and complexity were used to determine the viable population of the cells, and the average intensity of the fluorescence was determined with the MXP software (BeckmanCoulter). The experiments were performed in duplicate in at least three independent passes.
  • the glucose consumption was determined by measuring the concentration present in the cell medium over time. To do this, 3x10 5 cells were seeded in a 6-well plate. The next day, the medium was removed and 500 ⁇ _ of fresh DMEM was added. After 6 hours of incubation, the concentration present in the medium was determined. To measure the concentration of glucose in the medium, an Accu-Check Performa (Roche) glucometer was calibrated and used. Both the samples and the standard curve (500-0.15 g / L) were diluted 1 ⁇ 2 with distilled water before the measurement. The results are shown as milligrams of glucose consumed at 6 hours. At least three independent measurements were obtained to generate the mean.
  • Oxygen consumption of the intact cells was measured in DMEM at 37 ° C using a Clark type oxygen electrode (Hansatech Instruments). Basal respiration was calculated as the rate of oxygen consumption minus non-mitochondrial respiration after the addition of 1 mM KCN. At least three independent measurements were obtained to generate the mean.
  • Mitochondria were purified from cell cultures as described in Pello et al., 2008 (Hum Mol Genet 17: 4001-401 1). Protein concentrations were determined using the microBCA protein kit (Thermo Scientific). To obtain native mitochondrial proteins, the pellets were solubilized with 4 grams of digitonin (Sigma) per gram of protein in 1.5M aminocaproic acid, 50 mM Bis-Tris, pH 7.0. Native electrophoresis was performed as described in Wittig et al., 2006. NDUFA9 (Abcam, ab14713) was used for the immunodetection of supercomplexes I + II I2 + IV. SDHA (Abcam, ab14715) was used for normalization. The quantification of 4 different membranes from 2 different extracts was performed with ImageJ. 1.9 Immunofluorescence:
  • the cells were fixed with 4% paraformaldehyde, incubated overnight with TOM20 antibody (Santa Cruz Biotechnology, sc-17764, 1/50). Alexa Fluor 488 anti-mouse (Invitrogen Life Technologies, 1/500) was used as a secondary antibody.
  • the nuclei were stained with Topro-3 (Invitrogen Life Technologies, 1/1000). Five images of each sample were collected with a TCS SP5 confocal microscope (Leica Microsystems) equipped with 20 ⁇ HCX PL APO (0.7 numerical aperture).
  • RNA from the cells was extracted using the "RNeasy mini Kit with DNAse” kit (Qiagen) and the cDNA was synthesized using the "NZY First-StrandcDNASynthesis Kit” (NZYtech) kit.
  • the expression levels of PGC-1 a were assessed by qRT-PCR using the Taqman® expression assay (Hs01016719_m1).
  • the levels of TBP (Hs00427621_m1) were used as endogenous control.
  • mice Four different models of PDX were generated from four patients with NSCLC. All treatments were carried out in passage 2 mice, with palpable bilateral tumors. At least two mice per group were injected intraperitoneally with 2.5 mg / kg CDDP or vehicle twice a week on non-consecutive days. After the treatments, the animals were sacrificed and the tumors were collected for further analysis.
  • the cells were seeded in a 24-well plate in DMEM. The next day, the media were changed to DMEM or DMEM containing the different treatments (5 ⁇ CDDP and / or 20 ⁇ ZLN005). After 48 hours of treatment, the cells were harvested, washed twice in PBS and resuspended in 50 ⁇ l of annexin V binding solution diluted 10 times. 1 ⁇ _ of annexin V-FITC conjugate was added to the cells and incubated for 15 minutes at room temperature. Finally, the cells were further diluted with 150 ⁇ _ of annexin V binding solution diluted 10 times and analyzed immediately by flow cytometry. Frontal and lateral scattering were used to select the cell population. The bars on the graph represent the average percentage of annexin-positive cells from 4 different assays.
  • mitochondrial mass levels were measured using the Mitotracker Green molecular probe (MTG) ( Figure 2F), as well as through immunofluorescence with the TOM20 antibody (Figure 2G).
  • MMG Mitotracker Green molecular probe
  • Figure 2G The results show an increase in mitochondrial mass for cell lines H1299 and H460 resistant to CDDP.
  • no changes were observed for the cell line A549 resistant to CDDP.
  • this cell line also showed no increase in OXPHOS function.
  • Xenografts derived from patients (PDX) treated with cisplatin show an increase in PGC-1a and mitochondrial mass.
  • OXPHOS function can lead to an increase in the levels of superoxide and hydrogen peroxide.
  • the ROS levels measured with DCFHDA were significantly increased. Surprisingly, this increase occurs both as a stable modification in CDDP-resistant cell lines and also as an early response to exposure to cisplatin in all cell lines studied. All these results indicate a process of metabolic reprogramming towards a greater OXPHOS function against the Warburg Effect.
  • Most tumor cells preferentially use the aerobic glycolysis strategy despite their lower energy efficiency (compared to the more efficient use of the OXPHOS system) since this "Warburg metabolism" is adapted to support exponential growth.
  • the concomitant treatment of CDDP with metformin, rotenone or with the silencing of PGC-1a increases the effect of CDDP, in all cases.
  • the combined therapy of metformin or rotenone with CDDP eliminates the sensitivity differences between parent and resistant for both cell lines A549 and H1299.
  • the combined therapy of PGC-1 a and CDDP eliminates the sensitivity differences between parent and resistant for line H1299 but not for line A549.

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Abstract

La présente invention concerne un procédé in vitro permettant de mettre au point une thérapie personnalisée pour un individu qui est atteint d'un cancer du poumon résistant au cisplatine, lequel procédé consiste à déterminer le niveau d'expression du gène PGC-1alfa avant et après le traitement avec du cisplatine, puis, si le niveau d'expression dudit gène est supérieur après le traitement avec du cisplatine qu'avant le traitement avec du cisplatine, à administrer un composé conçu pour réduire la fonction OXPHOS.
PCT/ES2018/070463 2017-06-28 2018-06-28 Procédé permettant de mettre au point une thérapie personnalisée pour un individu qui est atteint d'un cancer du poumon résistant au cisplatine Ceased WO2019002655A1 (fr)

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