WO2013029116A1 - Méthode de prédiction de réactivité à un traitement - Google Patents

Méthode de prédiction de réactivité à un traitement Download PDF

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WO2013029116A1
WO2013029116A1 PCT/AU2012/001034 AU2012001034W WO2013029116A1 WO 2013029116 A1 WO2013029116 A1 WO 2013029116A1 AU 2012001034 W AU2012001034 W AU 2012001034W WO 2013029116 A1 WO2013029116 A1 WO 2013029116A1
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grhl3
cas
squamous cell
mirna
level
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Stephen M. Jane
Charbel DARIDO
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Monash University
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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
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to methods for predicting if a squamous cell neoplasm will be susceptible to treatment with phosphatidylinositol 3-kinase inhibitors and use of
  • phosphatidylinositol 3-kinase inhibitors in methods of treating susceptible squamous cell neoplasms.
  • Squamous cell neoplasms are among the most common cancers in humans and other animals. They usually arise from the outer layers of skin and mucous membrane cells, known as ectodermal or endodermal cells. For this reason, they can develop in a large number of organs and tissues, including skin, lips, mouth, esophagus, urinary bladder, prostate, lung, vagina and cervix, among others. Approximately ninety percent of cases of head and neck cancer (cancer of the mouth, nasal cavity, nasopharynx, throat and associated structures) are due to squamous cell neoplasms.
  • squamous cell neoplasms can spread to lymph nodes (under the armpits, in the groin or in the neck, for example) or via the bloodstream to other parts of the body. For this reason squamous cell neoplasms should be treated promptly and the removal should be carried out by a specialized medical practitioner trained in the complete surgical removal of the cancer. Surgical removal usually involves removal of the cancer under local anesthetic, or if the cancer is larger, it may need to be removed with specialized technique involving a skin graft placed over the wound. Radiotherapy, which uses X-rays to kill the cancer cells, is an alternative in selected patients, after assessment by a dermatologist. If the cancer is fou nd to have spread to lymph nodes, these may also need to be removed, or treated with radiotherapy to delay further spread.
  • squamous cell neoplasms arising in different body sites can show tremendous differences in their presenting signs and symptoms, natural history, prognosis, and response to treatment. Due to their primarily superficial nature, squamous cell neoplasms are often treated surgically. However, such treatment may leave behind some cancerous cells which may proliferate and spread over time. Also, the remnant cells may give rise to more aggressive and invasive forms of the cancer.
  • Ras signaling in concert with inhibition of nuclear factor kappa-light-chain-enhancer of activated B cell (NF- ⁇ ) function, is sufficient for malignant transformation of keratinocytes.
  • Activated Ras stimulates multiple effectors including the Raf/MEK/ERK pathway, the phosphatidylinositol 3-kinase
  • PI3K guanine nucleotide exchange factors
  • PTEN phosphatase and tensin homolog
  • PI3K inhibitors Since activation of the PI3K pathway is a recurrent feature in many human neoplasms, PI3K inhibitors have been identified as promising candidates for new cancer treatments. However, there is a high degree of variability in the susceptibility of squamous cell neoplasms to treatment with PI3K inhibitors. Consequently, there is a need for a reliable method for predicting whether a squamous cell neoplasm will be susceptible to treatment with a PI3K inhibitor.
  • a first aspect provides a method for predicting if a squamous cell neoplasm in a subject will be susceptible to treatment with a phosphatidyl inositol 3-kinase (PI3K) inhibitor, said method comprising:
  • RNA-21 micro-RNA-21
  • Grhl3 Grainy head-like 3
  • a second aspect provides a method for selecting a subject with a squamous cell neoplasm for PI3K inhibitor treatment, said method comprising:
  • the subject for PI3K inhibitor treatment if the tissue from the squamous cell neoplasm has an increased level of miRNA-21 and/or a decreased level of Grhl3 as compared to the control.
  • the methods of the first and second aspects may be carried out in vitro.
  • a third aspect provides a method for treating a squamous cell neoplasm in a subject, said method comprising: a) determining the level of miRNA-21 and/or Grhl3 in tissue from the squamous cell neoplasm;
  • An alternative form of the third aspect involves use of a PI3K inhibitor for treating a squamous cell neoplasm in a subject, wherein the squamous cell neoplasm has an increased level of miRNA-21 and/or a decreased level of Grhl3, compared to a control level.
  • Another alternative form of the third aspect involves use of a PI3K inhibitor in the manufacture of a medicament for treating a squamous cell neoplasm in a subject, wherein the squamous cell neoplasm has an increased level of miRNA-21 and/or a decreased level of Grhl3, compared to a control level.
  • a fourth aspect provides a method for preventing a squamous cell neoplasm in a subject, said method comprising applying a topical composition comprising inositol to skin of the subject.
  • An alternative form of the fourth aspect involves use of a topical composition comprising inositol for preventing a squamous cell neoplasm in a subject.
  • Another alternative form of the fourth aspect involves use of a topical composition comprising inositol in the manufacture of a medicament for preventing a squamous cell neoplasm in a subject.
  • a fifth aspect provides a kit for detecting if a squamous cell neoplasm in a subject will be susceptible to treatment with a PI3K inhibitor, said kit comprising:
  • a sixth aspect provides a kit for treating a squamous cell neoplasm in a subject, said kit comprising: a) reagents for determining the level of miRNA-21 and/or Grhl3 in tissue from the squamous cell neoplasm;
  • the reagents for determining the level of miRNA-21 and/or Grhl3 comprise one or more of Q-RT-PCR reagents, array reagents, Northern blot reagents, Western blot reagents, and ELISA reagents.
  • the work described herein demonstrates that miRNA-21 and Grhl3 are predictive biomarkers and that elevated miRNA-21 and/or decreased Grhl3 in tissue from the squamous cel l neoplasm, compared to the control level, indicates that the squamous cell neoplasm will be susceptible to treatment with a PI3K inhibitor.
  • the inventors have established that tissue-specific control of PTEN expression plays a pivotal role in the suppression of squamous cell neoplasms.
  • PTEN expression is largely controlled by the transcription factor Grainy head-like 3 (Grhl3), a member of a highly conserved family of transcription factors critical for epidermal development and homeostasis across a wide range of species.
  • Grhl3 transcription factor Grainy head-like 3
  • the inventors found that deletion of Grhl3 in adult epidermis evokes loss of expression of PTEN, a direct GRHL3 target, resulting in activation of PI3K/(AKT)/mammalian target of rapamycin (mTOR) signaling and inducing aggressive squamous cell neoplasms.
  • Grhl3 The integral function of Grhl3, coupled with its role in maintaining the balance between keratinocyte differentiation and proliferation defines Grhl3 as a critical innate surveillant to prevent skin cancer.
  • its function is similar to inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKct), which also induces keratinocyte terminal differentiation and prevents squamous cell neoplasms.
  • IKKct nuclear factor kappa-B kinase subunit alpha
  • miRNA-21 targets PTEN in human cancers, but the inventors have shown that it also targets Grhl3.
  • This synchronous targeting of GRHL3 and PTEN by miRNA-21 establishes a proto- oncogenic network with amplification of PI3K/AKT/mTOR signaling and induction of squamous cell carcinoma in humans.
  • both GRHL3 and PTEN are reduced through upregulation of miR-21, explaining the long-standing paradox of loss of PTEN expression in the absence of genetic or epigenetic alterations to the gene.
  • the co-ordinate targeting of both tumor suppressors by miR-21 provides an example of miRNA-dependent amplification of signaling cascades that are evident in both normal and cancerous tissues.
  • the synchronous regulation of the pathway inhibitor (PTEN) and its transcriptional regulator (GRHL3) by a solitary miR (miR-21) establishes a proto-oncogenic network involving enhanced PI3K/AKT/mTOR signaling in these tumors. This appears to be the first example of coordinate miRNA-mediated regulation of both transcription factor and its direct target gene for signaling amplification.
  • Grhl3 and/or PTEN This data establishes that reduced levels of Grhl3 and/or PTEN and/or increased levels of miRNA-21 can be used to accurately predict which squamous cell neoplasms will be susceptible to treatment with compounds that inhibit PI3K/AKT/mTO signaling, or administration of Grhl3 and/or PTEN.
  • the data also defines the Grhl3 gene as a potent suppressor of squamous cell carcinoma of the skin in mammals, acting through the direct transcriptional regulation of PTEN. Supporting this conclusion are phylogenetic, biochemical, and expression data coupled with functional rescue studies in vitro in a human system, and in vivo in the mouse.
  • subject means any organism susceptible to developing a squamous cell neoplasm.
  • the subject is a mammal.
  • the mammal may be a human, or may be a domestic, zoo, or companion animal. While it is particularly contemplated that the methods of the invention are suitable for humans, they are also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as felids, canids, bovids, and ungulates and for use on laboratory anaimals including rats, mice, monkeys and apes. More preferably, the subject is a human.
  • tissue includes any tissue containing epithelial cells, such as skin tissue or mucous membrane tissue.
  • tissue from a squamous cell neoplasm as used herein means one or more cells from a squamous cell neoplasm. The cells could be isolated from the neoplasm by methods known in the art. Such methods include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, large core biopsy, open surgical biopsy, shave biopsy, punch biopsy and elliptical biopsy.
  • the tissue includes tumor blood or lymphatic vessel tissue and the miRNA-21 and/or Grhl3 is measured in vessel tissue.
  • the tissue includes fluid from the tumor and the miRNA-21 and/or Grhl3 is measured in the fluid.
  • the tissue comprises blood, and the miRNA-21 and/or Grhl3 is measured in the blood, or in plasma or serum from the blood.
  • squamous cell neoplasm includes a number of morphological sub-types and variants, including Bowen's disease, Marjolin's ulcer, Erythroplasia of Queyrat, head and neck cancer, papillary carcinoma, verrucous squamous cell carcinoma, papillary squamous cell carcinoma, cervical squamous cell carcinoma, squamous cell carcinoma, large cell keratinizing squamous cell carcinoma, small cell keratinizing squamous cell carcinoma, small cell squamous cell carcinoma, adenoid/pseudoglandular squamous cell carcinoma, intraepidermal squamous cell carcinoma, lymphoepithelial carcinoma, basaloid squamous cell carcinoma, clear cell squamous cell carcinoma, keratoacanthoma, signet ring cell squamous cell carcinoma and spindle cell squamous cell carcinoma.
  • Head and neck cancer includes mouth cavity, nasal cavity, sinus, lip, tongue, salivary gland, nasopharynx, larynx and throat cancer.
  • PI3K inhibitor includes any compound capable of inhibiting PI3K/AKT/mTOR signaling.
  • Examples of such compounds include A66 (C 17 H23 502S2; CAS No.: 1166227-08-2); AS 252424 (5-[l-[5-(4-Fluoro-2-hydroxy-phenyl)-furan-2-yl]-meth-(Z)-ylidene]-thiazolidine-2,4-dione); AS-605240 (5-(6-Quinoxalinylmethylene)-2,4-thiazolidine-2,4-dione; C 12 H 7 N 3 0 2 S; CAS No.: 648450- 29-7); AZD6482 (C 22 H 24 N 4 0 4 ; CAS No.: 1173900-33-8); BAG956 (2-methyl-2-[4-(2-methyl-8-pyridin-3- ylethynyl-imidazo[4,5-c]quinolin-l-yl)-phenyl]-propionitrile); BBD130 (2-Methyl-2-[4-(3
  • PIK75 N'-[(lE)-(6-bromoimidazo[l ; 2-a]pyridin-3-yl)methylene]-N ; 2-dimethyl-5- nitrobenzenesulfonohydrazide hydrochloride); PIK90 (N-(7,8-Dimethoxy-2,3-dihydro-imidazo[l,2- c]quinazolin-5-yl)-nicotinamide; C 18 H 17 N 5 0 3 ; CAS No.: 677338-12-4); PIK93 (C 14 H 16 CIN 3 0 4 S 2 ; CAS No.: 593960-11-3); PKI-587 (C 32 H 41 N 9 0 4 ; CAS No.: 1197160-78-3); PP-121 (l-Cyclopentyl-3-(lH- pyrrolo[2 ; 3-b]pyridin-5-yl)-lH-pyrazolo
  • the determining of the level of mi NA-21 and/or Grhl3 may be achieved by any methods known in the art for determining the level of nucleic acids and/or proteins.
  • Methods for determining the level of a nucleic acid include both direct and indirect methods, such as, quantitative real time polymerase chain reaction (Q-RT-PCR), array and Northern blot.
  • Methods for determining the level of a protein include Western blot and ELISA.
  • the measuring of miRNA-21 and/or Grhl3 can occur after a cancer diagnosis has been made and prior to in initiation of a standard of care cancer therapy (e.g., surgery and/or chemotherapy). In some embodiments, the measuring of miRNA-21 and/or Grhl3 occurs after a cancer has become resistant to a standard of care therapy. These embodiments are not mutually exclusive.
  • the level of miRNA-21 and/or Grhl3 in tissue can be subjected to one or more of various comparisons. In general, it can be compared to: (a) miRNA-21 and/or Grhl3 level(s) in normal tissue from the organ in which the cancer originated; (b) miRNA-21 and/or Grhl3 levels in a collection of comparable cancer tissue samples; (c) miRNA-21 and/or Grhl3 level in a collection of normal tissue samples; or (d) miRNA-21 and/or Grhl3 level in an arbitrary standard.
  • the identifying or selecting step of the screening methods described herein optionally comprises comparing the measurement of miRNA-21 and/or Grhl3 to a reference measurement of miRNA-21 and/or Grhl3, and scoring the miRNA-21 and/or Grhl3 measurement from the tissue as elevated or decreased based on statistical analysis or a ratio relative to the reference measurement.
  • the reference measurement comprises at least one of the following (a) a measurement of miRNA-21 and/or Grhl3 from healthy tissue of the subject of the same tissue type as the neoplastic tissue; (b) a database containing multiple miRNA-21 and/or Grhl3 measurements from healthy or cancerous tissues from other subjects; or (c) a reference val ue calculated from multiple miRNA-21 and/or Grhl3 measurements from healthy or cancerous tissues from other subjects, optionally further including statistical distribution information for the multiple measurements, such as standard deviation.
  • a miRNA-21 measurement of at least 1.0 standard deviation greater than a median miRNA-21 measurement in corresponding healthy tissue is scored as elevated miRNA-21.
  • a miRNA-21 measurement that is statistically significantly greater than miRNA- 21 measurements in corresponding healthy tissue, with a p-value less than 0.1, or less than 0.05, or less than 0.01, or less than 0.005, or less than 0.001 is scored as elevated miRNA-21.
  • a Grhl3 measurement of at least 1.0 standard deviation less than a median Grhl3 measurement in corresponding healthy tissue is scored as decreased Grhl3.
  • a Grhl3 measurement that is statistically significantly less than Grhl3 measurements in corresponding healthy tissue, with a p-value less than 0.1, or less than 0.05, or less than 0.01, or less than 0.005, or less than 0.001 is scored as decreased Grhl3.
  • the level of miRNA-21 and/or Grhl3 in a subject is compared to a predetermined "cut-off" concentration of miRNA-21 and/or Grhl3 that has been determined from observations to represent an predictive measure of miRNA-21 and/or Grhl3 that is predictive of efficacy of PI3K therapy. Determination of a suitable cut-off is made using, e.g., statistical analysis of miRNA-21 and/or Grhl3 level data collected from multiple healthy and/or cancer patients. If a "cut-off value is employed, the cut-off concentration preferably is statistically determined to have optimal discriminating value for subjects who benefit from PI3K therapy (e.g., to have maximum sensitivity and specificity). It will be appreciated that statistical analysis of a dataset will permit clinicians to make informed decisions based on concentrations other than the optimal discriminating concentration (e.g., above or below the optimal discriminating concentration).
  • the level of Grhl3 in the squamous cell neoplasm is decreased by 60% or more as compared to the control.
  • the level of Grhl3 in the squamous cell neoplasm may be decreased by more than 61%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% as compared to the control.
  • the screening methods described herein further comprise measuring the expression of at least one additional marker, such as PTEN in the tissue.
  • Standard multivariate statistical analysis tools are used to optimize the predictive value of miRNA-21 and/or Grhl3 in combination with one or more of these additional markers.
  • susceptible to treatment with a PI3K inhibitor means that the PI3K inhibitor is effective against such a cell.
  • the cell may cease to grow and/or proliferate or may die following treatment with a PI3K inhibitor.
  • treating a squamous cell neoplasm means inhibiting the growth and proliferation of neoplastic cells, and/or causing the death of neoplastic cells.
  • the treatment involves the administration of a therapeutic amount of the therapeutic compound.
  • a therapeutic amount of a therapeutic compound refers to an amount of the compound that is sufficient to inhibit, halt or eradicate the condition being treated when the compound is administered alone or in conjunction with another agent.
  • the treatment may involve the co-administration of more than one therapeutic compound. Co-administration may be simultaneous or sequential.
  • Treating” or “treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the aim is to prevent, ameliorate or slow down (lessen) squamous cell neoplasm.
  • Preventing”, “prevention”, “preventative” or “prophylactic” refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of a condition, disease, disorder, or phenotype, including an abnormality or symptom. A subject in need of prevention may be prone to develop the condition.
  • ameliorate or “amelioration” refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.
  • a subject in need of treatment may already have the condition, or may be prone to have the condition or may be in whom the condition is to be prevented.
  • administering refers to contacting a subject with a compound. Administering may be achieved by any means by which the inhibitor can be delivered to the site to be treated.
  • Suitable types of administration include both systemic and localized forms of administration, such as intravenously, intraperitoneally, intranasally, transdermally, topically, via implantation, subcutaneously, parentally, intramuscularly, orally and via adsorption.
  • the invention also provides a kit comprising reagents for carrying out the methods described herein.
  • a kit may include reagents selected from Q-RT-PCR reagents, array reagents, Northern blot reagents, Western blot reagents and ELISA reagents necessary to carry out the described methods.
  • the kit may also include instructions for carrying out the methods.
  • Q-RT-PCR reagents as used herein means the reagents required to carry out a Q-RT-PCR reaction for analysis of a tissue. Such reagents include primers, deoxynucleotides, buffers and enzymes.
  • Array reagents as used herein means the reagents required to carry out an array reaction for analysis of a tissue. Such reagents include primers, deoxynucleotides, buffers and enzymes.
  • Inositol induces the expression of Grhl3 which in turn induces the expression of PTEN.
  • the inventors have shown that miRNA-21 and Grhl3 are predictive biomarkers and that elevated miRNA- 21 and/or decreased Grhl3 in tissue from the squamous cell neoplasm, compared to the control level, indicates that the squamous cell neoplasm will be susceptible to treatment with a PI3K inhibitor.
  • the inventors propose that inducing expression of GRHL3 using inositol, can cause suppression of squamous cell neoplasms.
  • Topical composition as used herein means a composition that is specifically formulated to be applied to the skin.
  • a topical composition may comprise a therapeutic compound that is to be delivered to the skin.
  • Topical compositions include transdermal compositions, liquids, lotions, creams, gels, ointments, powders and sprays.
  • Figure 1 A - G shows hyperproliferation of Grhl3-nu ⁇ keratinocytes (A and B) Histology and PCNA IHC on skin from E18.5 wild type (WT) and Gr ) ⁇ 3 ⁇ ' ⁇ (KO) embryos.
  • C, D and E Cell numbers, appearance, and soft agar colony numbers of cultured keratinocytes from WT and KO E18.5 embryos.
  • keratinocyte cultures 2.6 X 10 4 cells were seeded at Day 0.
  • soft agar 3.4 X 10 5 cells were seeded at Day 0.
  • Figure 3 A - F shows PTEN is a direct transcriptional target gene of GRHL3
  • C ChIP analysis of endogenous GRHL3 on the PTEN promoter. Chromatin from the human keratinocyte line (HaCAT) was immunoprecipitated using antisera to GRH L3, and amplified with PTEN primers. Pre-immune sera (IgG) and the muscle-specific MyoD promoter were used as negative controls, and the input chromatin is shown.
  • HaCAT human keratinocyte line
  • Figure 4 A - E shows PTEN is the critical GRHL3 target gene in dysregulated cell growth and PI3K/AKT activation
  • Figure 5 A - E shows skin and tumors from Grhl3 A/ ⁇ /K14Cre+ mice exhibit PI3K pathway activation
  • A Immunoblot of lysates from epidermis, papillomas, and squamous cell carcinomas from wild type and Grhl3 / ⁇ /K14Cre+ mice probed with PTEN and p-S6 antibodies. Actin served as the loading control.
  • Figure 6 A - H shows miR-21-induced loss of GR L3 and PTEN expression in human squamous cell carcinoma
  • A Quantification of GRHL3 and PTEN expression levels by Q-RT-PCR in human squamous cell carcinomas isolated by LCM, normalised to expression in the adjacent non-tumor containing tissue.
  • B Heat map of the relative expression of miRNAs predicted to target GRHL3 in two human squamous cell carcinoma and adjacent normal epidermis. The fold change in tumor (T) versus normal (N ) is shown.
  • Figure 7 shows that the mi -21/GRHL3/PTEN proto-oncogenic network is evident in human SCC cell lines from different tissue origins.
  • Q-PCR was performed on RNA from the listed SCC cell lines from head and neck (SCC4 and SCC9), skin (SCC 13) and cervix (A431) using primers for miR-21, Grhl3 and Pten.
  • Figure 8 shows inhibition of PI3K/mTOR pathway and reduced cellular proliferation in human SCC cell lines treated with BEZ235.
  • A The listed cell lines were treated with BEZ235 at the stated concentrations for 48 hours and cell lysates prepared and analysed by immunoblot for PI3K signaling.
  • B Growth curves for the same lines treated for varying times with differing
  • Figure 9 shows prevention of SCC development in BEZ235 treated Grhl3/K14Cre+ mice.
  • A Schematic of BEZ235 and DMBA/TPA treatment.
  • B Percentage of mice without tumors in vehicle or BEZ235 treated mice exposed to the DMBA/TPA protocol.
  • C Tumor scoring of mice treated with BEZ235 and DM BA/TPA protocol.
  • a fragment for the 3' homology arm extending 4 kb 3' from exon 4 from the mouse Grhl3 genomic locus isolated from a 129/SV/J genomic library was PCR amplified.
  • a 4.9 kb PCR fragment extending from 5' of exon 2 to 3' of exon 4 was generated with a loxP sequence and Hind ⁇ site at its 3' end, a Kpn ⁇ site at its 5' end, and flanked by Xba ⁇ sites. This was cloned into TOPO 2.1 and sequenced.
  • the "loxP" arm was then subcloned into TOPO 2.1 containing the 3' arm as an Xba ⁇ fragment and the two arms were released as a Kpn ⁇ fragment and subcloned into the Frt-PGKNeo- Frt-loxP vector (Meyers et al., (1998) PNAS 95, 13513-13518).
  • the 5.5 kb 5' homology arm was PCR amplified with flanking SacW sites and a 5' Bam ⁇ site introduced, sequenced and subcloned as a SacW fragment to generate the final construct.
  • the vector was linearized with Not ⁇ and electroporated into W9.5 embryonic stem cells.
  • G418-resistant clones in which the targeting vector had recombined with the endogenous Grhl3 gene were identified using Bam ⁇ digested genomic DNA probed with the 5' probe, which distinguished between the endogenous (8.4 kb) and targeted (6.5 kb) alleles. Recombination was confirmed with a 3' probe using a Hind ⁇ ⁇ digest that distinguished between the endogenous (15 kb) and targeted (4.8 kb) alleles.
  • Two ES cell clones were used to generate mouse lines on a C57BL/6 background.
  • Grhl3 fl + (where // is the undeleted "floxed" allele) heterozygous mice were identified by hybridising SomHI-digested genomic DNA with the 5' probe.
  • mice were genotyped by PCR using genomic DNA prepared from tail biopsies or embryonic tissues.
  • Products of 634 bp were generated from the wild type and 801 bp from the targeted Grhl3 alleles.
  • the size of the undeleted ⁇ flox) band was 425 bp and the deleted ( ⁇ ) band was 282 bp.
  • the primer sequences utilized in the genotyping and deletion efficiency assessment were:
  • Genotyping antisense 5'- TG CACCCATATCCACATG CA-3'
  • PCR conditions were 94°C for 2 minutes followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute with a final 5 minutes extension at 72°C.
  • Tumours were induced in wild type, Grhl3 +/ ⁇ /PTEN +/+ , PTEN +/ ⁇ /Grhl3 +/+ , PTEN +/ ⁇ /Grhl3 +/ ⁇ , and Grhl3 A/ ⁇ /K14Cre+ mice through the application of 25 ⁇ g DMBA (Sigma-Aldrich) in acetone to a shaved area on the back followed one week later by twice-weekly application of TPA (7.6 nmol) in 150 ⁇ of acetone for up to 30 weeks. In some experiments TPA was applied without DMBA initiation. Skin barrier analysis was performed as previously described (Hardman et al., (1998) Development 125, 1541-1552).
  • Glutamine and 100 ⁇ g/ml penicillin/streptomycin solution.
  • Cell size in the epidermal basal layer was determined from 24 independent sections derived from six E18.5 wild type and 6r/i/3 _ ⁇ embryos using the AxioVisionLE software from Zeiss. The difference in size was statistically significant (p ⁇ 0.008) using a Student's t-test.
  • ChIP was performed as described previously (Wilanowski et al., (2008) EMBO J. 27, 886-897), with anti-GRHL3 antibodies (Aviva Systems Biology, San Diego, CA USA) and primers amplifying the PTEN promoter as detailed below.
  • EMSAs were performed using recombinant mouse GRHL3 protein, and oligonucleotides containing the conserved GRHL3 binding site in the PTEN promoter as detailed below.
  • the DNA consensus-binding site for GRHL3 was used as a cold competitor (Ting et al., (2005) Science 308, 411-413).
  • PTEN promoter sense 5'-CACCAGTTTGGGGACTCTCT-3'
  • PTEN promoter sense only 5'-GGGGCTGCTTGTGTAACCAGCTCCCCAGGCGC-3' Immunoblot Analysis and IHC
  • immunohistochemistry were: PTEN (#9559), AKT (#9272), phospho-AKT (#9271), PDK1 (#3062), phospho-PDKl (#3061), 4EBP1 (#9644), phospho-4EBPl (#9451), S6 (#2217), phospho-S6 (#2215), ERK (#9102), phospho-ERK (#9101), GAPDH (#2118) (Cell Signalling), Actin (sc-1616) and HSP70 (sc- 24) (Santa Cruz), PCNA (PC10, DAKO), and GRH L3 (Aviva Systems Biology).
  • the shRNA target oligonucleotides for Grhl3 and the scrambled (Scr) control were cloned into the pSUPER. retro. neo+GFP vector using the BglW and Hind ⁇ sites.
  • the target sequences and generation of viruses are detailed as previously described (Caddy et al., (2010) Dev. Cell 19, 138-147).
  • HaCAT cells were trans-infected over a 24-hour period and GFP-positive cells were selected by FACS, and cultured for 8 days, or harvested for preparation of lysates. Knockdown of GRHL3 expression was confirmed by immunoblot using antibodies to GRH L3 and GAPDH.
  • Surgical specimens of squamous cell carcinoma resected from patients were embedded in OCT medium and stored at -80°C.
  • the sections were stained with Histogene LCM frozen staining kit just before commencing LCM.
  • the cryosections (8 ⁇ ) were microdissected using a VeritasTM microdissection instrument (Arcturus) according to the standard protocol. Tumour tissues and normal tissues were captured onto Capsure macro LCM caps.
  • RNA extraction and amplification were performed according to the manufacturer's instructions.
  • the reagents for staining, RNA extraction and RNA amplification were obtained from Arcturus. Q-RT-PCR was performed as detailed above. Mutational Analysis of H-ras
  • PCR primers amplifying codons 12, 13, and 61 were designed on the basis of genomic DNA sequences for H-ras. M utations were detected by sequencing as previously described (Ise et al., (2000) Oncogene 19, 2951-2956). The mutations were further confirmed by restriction fragment length polymorphism (RFLP) analysis as previously detailed (Jaworski et al., (2005) Oncogene 24, 1290-1295).
  • RFLP restriction fragment length polymorphism
  • RNA was isolated from HaCaT cells, squamous cell carcinoma and normal tissue samples using Trizol (Invitrogen). The differential microRNA expression in two normal skin and two squamous cell carcinoma samples were analysed using Genechip miRNA array (Affymetrix, CA, USA) and the data were displayed using the Partek GS software.
  • Genechip miRNA array Affymetrix, CA, USA
  • RNA isolated from 10 squamous cell carcinoma and matched normal samples was reverse transcribed using the QuantiMir RT kit (System Biosciences, CA, USA) according to the manufacturer's instructions.
  • the forward primer of miR-21 was designed based on the mature microRNA sequence and custom made from Geneworks. Q-PCR was performed using Power Sybr Green Master Mix from Applied Biosystems (Foster City, CA).
  • the lentiviral vector-based microRNA precursor construct was used (System Biosciences).
  • the 3'UTR region of human GRHL3 containing the predicted site for miR-21 was subcloned into the pM IR-REPORT Luciferase vector and co-transfected with the pMIR-REPORT ⁇ -Galactosidase control plasmid (Applied Biosystems).
  • the mutant construct (Mut21) was generated by deleting 10 bp (419 -428) from the miR-21 site in the 3'UTR of GRHL3.
  • HEK293T cells were cultured in 24-well plates, and each well transfected with 0.5 ⁇ g of either pM ⁇ R-GRHL3'UTR or pM ⁇ R-GRHL3'UTR-Mut21 Luciferase with the pMIR ⁇ -Galactosidase vector. After 15h, the miR-21 precursor construct was coinfected with and without the lentivector-based antagomir to miR-21 "miRZip-21" (System Biosciences).
  • Firefly Luciferase was measured 48h after transfection/infection using the Dual-light Chemiluminescent Reporter Gene Assay System (Applied Biosystems) as per the manufacturer's instructions and normalized to ⁇ -Galactosidase activity to control for differences in transfection efficiency.
  • mice carrying a conditionally targetable Grhl3 allele were generated, with loxP sites flanking exons 2 and 4 of the gene.
  • mice homozygous for the floxed alleles were healthy and fertile, and when crossed with Grhl3 + ⁇ mice carrying a B6-Cre transgene expressed at the two-cell stage of development, generated Grhl3 A/ ⁇ /B6-Cre+ mice (where ⁇ is the deleted floxed allele) that phenocopied the Grhl3-nu ⁇ animals (data not shown).
  • Grhl3 fl ⁇ mice were crossed with a line carrying a keratin (K) 14-driven Cre transgene.
  • Example 4 - PTEN is a Direct Transcriptional Target Gene of GRHL3
  • Loss of PTEN leads to accumulation of PIP3, and as a consequence, increased activity of the serine/threonine kinases PDK1 and AKT, with resultant activation of the mTOR kinase complex 1 (mTORCl).
  • mTORCl mTOR kinase complex 1
  • S6K1 and phosphorylation of 4EBP1 and ribosomal protein S6, which provide a robust readout of mTORCl signalling Analysis of these downstream effectors of PI3K signalling in epidermis from Grhl3-nu ⁇ embryos revealed increased levels of PDK1, S6 and 4EBP1, as well as their phosphorylated forms, p-PDKl, p-S6 and p-4EBPl ( Figure 3E).
  • Example 5 Expression of PTEN Rescues the Tumorigenic Phenotypes Induced by Loss of GRHL3
  • mice carrying heterozygous deletions of the two genes were intercrossed to generate compound heterozygotes (Grh ⁇ ' /PTEN ⁇ ' ), and these animals were compared with Grhl3 + ⁇ /PTEN + + and Grh ⁇ 3 + + /PTEN + ⁇ controls in the chemical- induced tumor model ( Figure 4A).
  • a human keratinocyte cell line (HaCAT) was generated in which the expression of GRHL3 had been knocked down using a specific shRNA containing lentivirus (GRHL3- kd) ( Figure 4D).
  • a line transduced with a scrambled control shRNA (Scr) served as the control.
  • the level of PTEN was markedly reduced in the GRHL3-kd line compared to control, and p-AKT and p-S6 levels were increased in these cells in both the presence ( Figure 4D), and absence of serum. This latter result was consistent with the resistance to growth factor and nutrient withdrawal observed with constitutive PI3K activation in tumor cell lines.
  • GRHL3 coding exons and splice donor and acceptor sites failed to detect any m utations in the tumors, and the methylation status of the CpG islands in the GRHL3 promoter was unchanged in tumors compared to normal skin (data not shown).
  • An alternate mechanism for the reduction in GRHL3 expression in the tumors could be through overexpression of a specific microRNA (miRNA), which has been shown in some contexts to function as an oncogene by targeting tumor suppressors.
  • miRNA microRNA
  • miR-21 exhibited the greatest differential between normal and tumor tissue, and its sequence aligned with nucleotides 414-436 in the GRHL3 3'UTR ( Figure 6C). Interestingly, miR-21 has previously been shown to function as an oncogene by targeting PTEN. An additional 10 squamous cell carcinomas and their matched controls were examined and they were found to demonstrate a greater than 6-fold difference in miR-21 expression between the two groups ( Figure 6D).
  • Example 8 Elevated levels of miRNA-21 and reduced levels of Grhl3 are predictive for squamous cell carcinomas that will be susceptible to PI3K inhibitors
  • samples of cells from squamous cell neoplasms having increased levels of miRNA-21 and/or reduced levels of Grhl3 are compared to samples of cells from squamous cell neoplasms having no statistically significant difference in the level of miRNA-21 and/or Grhl3 as compared to a control level.
  • FIG 8. Differential responsiveness to treatment in this context is illustrated in Figure 8. Three cell lines with the defined perturbation of the miR-21/Grhl3/Pten axis all demonstrate reduced growth with exposure to low concentrations of the dual inhibitor BEZ235. In contrast, SCC4 which lacks the gene expression signature only displays reduced growth at the highest concentrations of BEZ235 indicative of differential susceptibility of SCC depending on miR-21/Grhl3/PTEN levels.
  • SCC cell lines derived from oral and skin SCC patients
  • SCC-15, SCC-25, SCC-68 and CAL-27 will be grown in specific culture media.
  • the dual pll0-PI3K and mTORCl/2 inhibitory activity of NVP-BEZ235 will be assessed in these SCC cells. Dose-response experiments are predicted to show that NVP-BEZ235 is able to inhibit the phosphorylation of AKT, S6, and 4EBP1 in these cells.
  • Viability and cell proliferation will be determined in comparison between cells with high miRNA-21 and/or reduced levels of Grhl3 (eg. SCC-9, SCC-13, SCC-15, SCC-25, SCC-68 and CAL-27), and cells having no statistically significant difference in the level of miRNA-21 and/or Grhl3 (eg. SCC-4) as compared to a control level (HaCaT cells).
  • Grhl3 eg. SCC-9, SCC-13, SCC-15, SCC-25, SCC-68 and CAL-27
  • HaCaT cells a control level
  • PI3K inhibitors protects against squamous cell neoplasm formation in genetically predisposed subjects
  • Example 10 Topical administration of inositol protects against squamous cell neoplasm formation in genetically predisposed subjects
  • Bioactive Inositol will be administered concurrently with the DMBA/TPA carcinogens treatment for a period of 15 weeks. Mice will be monitored twice weekly for tumour number, tumour size and progression. All animals in which a tumor progresses to more than 1cm X 1cm area will be immediately euthanized by cervical dislocation and their tissues will be examined histologically. Topical administration of Inositol will show its important capacity as a preventive agent against skin SCC and also demonstrate that it is possible to propose topical use of Inositol-rich cream as a prophylactic application in genetically predisposed subjects (low Grhl3).

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Abstract

L'invention concerne une méthode pour prédire si un néoplasme de cellules squameuses chez un sujet est susceptible d'être traité par un inhibiteur de la PI3K, une méthode pour sélectionner un sujet souffrant d'un néoplasme de cellules squameuses afin de le traiter par un inhibiteur de la PI3K, une méthode pour traiter un néoplasme de cellules squameuses chez un sujet et des kits relatifs à ces méthodes.
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WO2015001491A1 (fr) 2013-07-02 2015-01-08 Rhizen Pharmaceuticals Sa Inhibiteurs de protéine kinase pi3k, en particulier inhibiteurs delta et/ou gamma
ITMI20132141A1 (it) * 2013-12-19 2015-06-20 Pan Metodo per il rilevamento di un rischio aumentato di sviluppare un cancro della pelle ed un uso di una variante genotipica del gene grhl3
WO2015093998A1 (fr) 2013-12-19 2015-06-25 Instytut Biologii Doświadczalnej Im. M. Nenckiego Polskiej Akademii Nauk Procédé de détection d'un risque accru de développer un cancer de la peau et utilisation d'un variant de génotype du gène grhl3
WO2017068402A1 (fr) 2015-10-23 2017-04-27 Instytut Biologii Doświadczalnej Im. M. Nenckiego, Polskiej Akademii Nauk Procédé permettant de détecter un risque accru de cancer du rein et utilisation des variants génotypiques des gènes grhl1 et/ou grhl2 et/ou grhl3
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015001491A1 (fr) 2013-07-02 2015-01-08 Rhizen Pharmaceuticals Sa Inhibiteurs de protéine kinase pi3k, en particulier inhibiteurs delta et/ou gamma
ITMI20132141A1 (it) * 2013-12-19 2015-06-20 Pan Metodo per il rilevamento di un rischio aumentato di sviluppare un cancro della pelle ed un uso di una variante genotipica del gene grhl3
WO2015093998A1 (fr) 2013-12-19 2015-06-25 Instytut Biologii Doświadczalnej Im. M. Nenckiego Polskiej Akademii Nauk Procédé de détection d'un risque accru de développer un cancer de la peau et utilisation d'un variant de génotype du gène grhl3
US10000816B2 (en) 2013-12-19 2018-06-19 Instytut Biologii Doswiadczalnej Im. M. Nenckiego Polskiej Akademii Nauk Method for detecting an increased risk of developing skin cancer and a use of a genotype variant of the GRHL3 gene
AU2014367369B2 (en) * 2013-12-19 2020-03-26 Instytut Biologii Doswiadczalnej Im. M. Nenckiego Polskiej Akademii Nauk A method for detecting an increased risk of developing skin cancer and a use of a genotype variant of the GRHL3 gene
WO2017068402A1 (fr) 2015-10-23 2017-04-27 Instytut Biologii Doświadczalnej Im. M. Nenckiego, Polskiej Akademii Nauk Procédé permettant de détecter un risque accru de cancer du rein et utilisation des variants génotypiques des gènes grhl1 et/ou grhl2 et/ou grhl3
WO2020030708A1 (fr) * 2018-08-07 2020-02-13 Piqur Therapeutics Ag Traitement du carcinome à cellules squameuses

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