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WO2013015744A1 - L'isoforme de leucémie de lignage mixte 5 est potentiellement un biomarqueur et une cible thérapeutique pour le cancer cervical associé à hpv - Google Patents

L'isoforme de leucémie de lignage mixte 5 est potentiellement un biomarqueur et une cible thérapeutique pour le cancer cervical associé à hpv Download PDF

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WO2013015744A1
WO2013015744A1 PCT/SG2012/000266 SG2012000266W WO2013015744A1 WO 2013015744 A1 WO2013015744 A1 WO 2013015744A1 SG 2012000266 W SG2012000266 W SG 2012000266W WO 2013015744 A1 WO2013015744 A1 WO 2013015744A1
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μιχ5β
cervical cancer
cells
sirna
mll5
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PCT/SG2012/000266
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English (en)
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Lih Wen DENG
Chow Wenn YEW
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National University Of Singapore
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3061—Blood cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate

Definitions

  • HPV human papillomavirus
  • the present invention pertains to the discovery of an isoform of mixed lineage leukemia 5 (MLL5), referred to herein as MLL5P, and the association between MLL5P and cervical cancer.
  • MLL5P mixed lineage leukemia 5
  • the invention pertains to methods to inhibit the expression or activity of mixed lineage leukemia 5 isoform ⁇ ( ⁇ 5 ⁇ ) in a cervical cancer cell by providing the cell with an effective amount of at least one ⁇ 5 ⁇ antagonist that inhibits production of ⁇ 5 ⁇ protein, activity of ⁇ 5 ⁇ protein, or a combination thereof.
  • ⁇ 5 ⁇ antagonists include nucleic acids (e.g., an antisense oligonucleotide, an siRNA, an shRNA, a microRNA, an aptamer or a ribozyme); or antibodies
  • the nucleic acid is an antisense oligonucleotide, such as one that selectively hybridizes to ⁇ 5 ⁇ mRNA, or an siRNA, such as one that targets a 26 base pair sequence of interest in ⁇ 5 ⁇ .
  • the ⁇ 5 ⁇ antagonist interferes with or inhibits the association between ⁇ 5 ⁇ and AP-1 binding site and its associated components at nucleotide 7326 of distal region of HPV18 LCR.
  • the invention pertains to methods to sensitize a cervical cancer cell towards gamma radiation, by providing the cell with an effective amount of at least one mixed lineage leukemia 5 isoform ⁇ ( ⁇ 5 ⁇ ) antagonist that inhibits production of ⁇ _,5 ⁇ protein, activity of ⁇ 5 ⁇ protein, or a combination thereof, as described above.
  • Methods described are similarly available for treating cervical cancer in an individual in need thereof, by administering to the subject an effective amount of at least one mixed lineage leukemia 5 isoform ⁇ ( ⁇ 5 ⁇ ) antagonist that the MLL5p antagonist inhibits production of ⁇ 5 ⁇ protein, activity of ⁇ 5 ⁇ protein, or a combination thereof.
  • the invention further pertains to siRNA molecules which knock down expression of a nucleic acid that encodes mixed lineage leukemia 5 isoform ⁇ (MLL5p), as well as to an isolated nucleic acid that encodes mixed lineage leukemia 5 isoform ⁇ ( ⁇ 5 ⁇ ).
  • the invention additionally encompasses methods of identifying a compound useful for treating cervical cancer, by (a) exposing cervical cancer cells to a test compound; (b) determining the expression or activity of mixed lineage leukemia 5 isoform ⁇ ( ⁇ 5 ⁇ ) in the cells following exposure of the cells to the test compound; and (c) selecting a compound that inhibits the expression or activity of ⁇ 5 ⁇ in the cells exposed to the test compound relative to expression or activity of ⁇ 5 ⁇ in control cervical cancer cells that were not exposed to the test compound. Inhibition of the expression or activity of ⁇ 5 ⁇ in the cervical cancer cells that have been exposed to the test compound relative to expression or activity of ⁇ 5 ⁇ in control cervical cancer cells that were not exposed to the test compound, indicates that the test compound is useful for treating cervical cancer.
  • Diagnostic methods are also included in the present invention.
  • the methods include methods of assessing a sample of cervical cells for the presence of cervical cancer cells, by assessing the sample for the presence of mixed lineage leukemia 5 isoform ⁇ ( ⁇ 5 ⁇ ), wherein the presence of ⁇ 5 ⁇ in the sample is indicative of the presence of cervical cancer cells.
  • Cervical cancer can be diagnosed in an individual using such an assessment.
  • Figure 1A depicts a schematic representation of MLL5 and the regions that MLL5-siRNAs target to. Arrows mark the position of each target region of the four siRNAs used. MLL5-siRNA#l and #2 target at the N-terminal region, MLL5-siRNA#3 targets the central region and MLL5-siRNA#4 targets the C-terminal region of MLL5 mRNA.
  • FIG. 1B shows the results of an experiment in which HeLa, CaSki and SiHa cells were knocked down for 72-hrs using different siRNAs before RNA was extracted and used as template for RT-PCR.
  • An internal reference gene GAPDH was used for normalization.
  • Figure 2A is a characterization of the novel isoform ⁇ 5 ⁇ compared to full-length MLL5.
  • FIG. 1 A region of MLL5 is shown (SEQ ID NO: 1), as is a region of ⁇ 115 ⁇ (SEQ ID NO:2). MLL5fi is truncated at the exon 14 at 2034 bp and the sequence of the 26-bp insert is shown.
  • Figure 3 Effects of MLL5p-knockdown on p53 protein and E6/E7 mRNA level.
  • Figure 3 depicts RT-PCR analysis of HeLa, CaSki and SiHa after 72- hrs of MLL5P-siRNA knockdown. Consistent with the Western blot results, MLL5P-siRNA knockdown does not affect full-length MLL5 mRNA level but decreases the level of E6/E7 mRNA.
  • Figure 4 MLL5p interacts with HPV18-LCR to activate transcription.
  • Figure 4 A shows a genetic map of HPV18 with various ORF of viral proteins denoted by arrows. E6 and E7 ORF is located immediately downstream adjacent to LCR of HPV18.
  • Figure 4B depicts results of a dual luciferase assay using different fragments of HPV18-LCR cloned into pGL3 vector. 293T cells were co- transfected with pGL3 vector with different HP VI 8-LCR fragment and pEGFP- MLL5p or pEGFP-MLL5-CT (C-terminal) as negative control, along with pRLTK as an internal control.
  • Line 1 (nucleotide 7018 to 7239), 2 (nucleotide 7168 to 7350) and 3 (nucleotide 7378 to 7576) represent the amplicons of primer sets used in ChIP.
  • Figure 5 A depicts how HPV18 LCR (nucleotide 7168 to 7350) is further fragmentized to narrow down possible interacting partners. Fragments were cloned into pGL-3 vector and dual-luciferase assay was carried out as described earlier. No decrease in relative luciferase activity except fragment A3, suggesting that A3 is the shortest fragment that contains the putative interacting sites.
  • Figure 5B shows DNA sequences of AP-1 and SP-1 binding sites in HP VI 8-LCR. Only the sense strand of the DNA is shown for ease of presentation. Mutants are indicated by letter 'M' and mutated sequences are indicated in bold.
  • Binding motifs are shown by boxes. 18SP1 (SEQ ID NO:3), 18SP1M (SEQ ID NO:4), 18AP1 (SEQ ID NO:5), and 18AP1M (SEQ ID NO:6) are shown. A decrease in relative luciferase activity by AP-1 mutant but not SP-1 mutant indicated that AP-1 is likely to be the interacting partner.
  • Figure 5C depicts results of dual-luciferase assays with inactivated SET domain mutant. The decrease in relative luciferase activity corresponds to an increase in the concentration of the SET mutant vector used.
  • FIG. 6 A proposed model for the molecular mechanism of ⁇ 5 ⁇ in regulating E6/E7 gene activation.
  • Full-length MLL5 (1858 amino acid) consists of N-terminal region (NT), central domain (CD) and C-terminal region (CT). Both PHD and SET domains can be found within the N-terminal region of full-length MLL5 and the N-terminal truncated isoform ⁇ 5 ⁇ .
  • LCR region of HPV18 is located between the LI and E6/E7 ORF and it can be divided into three regions which consist of a distal region (dashed area), a central region (grey area) and a proximal region (chequered area).
  • Figure 7 Knockdown of MLL5 ⁇ makes HPV-positive cervical cancer more sensitive to gamma irradiation.
  • Figure 7 depicts the cytotoxicity effect of MLL5P-siRNA with combination of gamma irradiation on human cervical cancer cell lines (HeLa, CaSki and SiHa) and normal diploid cells (WD 8) were examined. MLL5 knockdown was observed to sensitize the cancer cells towards the killing effect of gamma irradiation but not in WI38.
  • Figure 8A depicts the mRNA encoding MLL5p (SEQ ID NO:7).
  • the ⁇ 5 ⁇ mRNA is identical with full length MLL5 up till exonl4. However, in the middle of exon 14, there is an additional of 26-bp in the MLL5p-mRNA, leading to a stop codon and hence the truncated product of MLL5p.
  • the ATG start codon and the TAG stop codon are in bold.
  • the 26-bp sequence of interest is underlined.
  • Figure 8B depicts the ⁇ 5 ⁇ protein sequence (SEQ ID NO:8).
  • Figure 9 Trypan blue exclusion assay.
  • Figures 9A and 9B provide results of a trypan blue exclusion assay to quantitate the amount of surviving cells in various cell lines after different siRNA treatment.
  • MLL5P knockdown affects the HPV positive cells but not HPV-negative cells C33A and normal human diploid cells WI38 after both 72 (Fig. 9A) and 96 (Fig. 9B) hours.
  • Figure 10 Mouse in vivo xenograft study to assess the anti-tumor effect of MLL5p-siRNA.
  • Figure 10 A depicts the effect of different siRNAs on the size of -the tumor induced by HeLa cells on immuno-deficient mice. MLL5P-siRNA significantly reduced the size of the tumor comparing to other siRNAs treatment.
  • Figure 10B compares the weight of the tumors excised after completing 20 days of treatment.
  • FIG 11. MLL5P knockdown induces apoptosis in HeLa cells.
  • Figure 1 1 demonstrates that ⁇ 5 ⁇ knockdown induces senescence in HeLa cells.
  • Figure 12 Effect of different concentration of cisplatin drug followed by gamma irradiation treatment on various cell lines.
  • Figure 12 indicates that only HPV-positive cancer cell lines showed a sensitization effect towards gamma irradiation.
  • Figure 13 Effect of various siRNA treatment followed by gamma irradiation treatment on various cell lines.
  • Figure 13 demonstrates that HPV- positive cell lines treated with ⁇ 5 ⁇ and E6-siRNAs showed a sensitization effect towards gamma irradiation.
  • E6-siRNA-induced sensitization effect was HPV strain- specific.
  • HPV 18 E6 siRNA induced the sensitization effect on HeLa (HPV18- positive) but not in SiHa (HPV16-positive).
  • FIG. 14 Downregulation of MLL5 ⁇ with Cisplatin
  • Figure 14 depicts RT-PCR results that indicated that MLL5P level was downregulated upon cisplatin treatment in both HeLa and SiHa cells. Rescue experiments validated that the cisplatin-induced sensitization effect is MLL5p dependent. Over-expression of MLL5P in cisplatin treated HeLa cells prevented the sensitization effect.
  • MLL5P mixed lineage leukemia 5
  • MLL5P mixed lineage leukemia 5
  • MLL5P this protein was determined to be associated with cervical cancer: MLL5 was detected in HPV16/18-positive human cervical cancer cell lines, and was not found in a normal diploid cell line or in a HPV-negative human cervical cancer cell line.
  • eight human primary cervical carcinoma specimens tested for the presence of MLL5(3 isoform with a MLL5p gene-specific primer were all positive for ⁇ 5 ⁇ .
  • the expression or activity of ⁇ 1 ⁇ 5 ⁇ in a cervical cancer cell is inhibited.
  • the terms, "inhibit” or “decrease,” as used herein, indicate values that are relative to a baseline measurement, such as a measurement in the same or a comparable sample prior to administration of an agent as described herein, or in a control sample (or multiple control samples) in the absence of administration of an agent as described herein.
  • ⁇ 5 ⁇ antagonist is an agent that interferes with ⁇ 5 ⁇ either directly or indirectly such that ⁇ 5 ⁇ activity and/or expression is inhibited and/or decreased. It can be, in certain embodiments, a nucleic acid, such as an antisense oligonucleotide, a small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA (miRNA), and aptamer, and a ribozyme.
  • RNA interference a natural cell process by which specific niRNAs are targeted for degradation by complementary small interfering RNAs (siRNAs), enables the specific silencing of a single gene at the cell level.
  • the nucleic acid is an antisense oligonucleotide, such as an antisense oligonucleotide that selectively hybridizes to ⁇ 5 ⁇ mRNA.
  • nucleic acid is an siRNA, such as an siRNA that targets the 26 base pair sequence of interest in ⁇ 5 ⁇ .
  • the "26 base pair sequence of interest in ⁇ 5 ⁇ ,” as used herein, refers to the 26 base pair sequence that is present in isoform ⁇ 5 ⁇ but absent in MLL5.
  • the nucleic acid sequence shown in Figure 8A is an mRNA encoding ⁇ 5 ⁇ ; the 26 base pair sequence of interest in this sequence is underlined.
  • the ⁇ 5 ⁇ antagonist is an antibody.
  • antibody is intended to encompass both whole antibodies and antibody fragments (e.g., antigen-binding fragments of antibodies, for example, Fv, Fc, Fd, Fab, Fab', F(ab'), and dAb fragments).
  • Antibody refers to both polyclonal and monoclonal antibodies and includes naturally-occurring and engineered antibodies.
  • the term “antibody” includes, for example, human, chimeric, humanized, primatized, veneered, single chain, and domain antibodies (dAbs). (See e.g., Harlow et al. , Antibodies A Laboratory Manual, Cold Spring Harbor
  • the MLL5 antagonist is an agent that interferes with or inhibits the association between ⁇ 5 ⁇ and AP-1 binding site and its associated components in the distal region of HPV18 long control region (LCR).
  • LCR HPV18 long control region
  • an MLL5 antagonist can be an agent that targets that region in order to inhibit or otherwise interfere with the interaction between MLL5P and its binding site, thereby reducing or eliminating activation of HPV18-LCR.
  • cervical cells can be sensitized towards gamma radiation by knocking down MLL5 .
  • cervical cancer cells can be sensitized by providing the cells with an effective amount of at least one mixed lineage leukemia 5 isoform ⁇ (MLL5p) antagonist, as described above.
  • MLL5p mixed lineage leukemia 5 isoform ⁇
  • an individual in need of cervical cancer treatment can be treated by administering to the individual an effective amount of at least one MLL5P antagonist as described above. .. . ..
  • compounds useful for treating cervical cancer can be identified using methods described herein.
  • cervical cancer cells can be exposed to a test compound, and the expression or activity of MLL5P can then be quantitated in the cells following exposure of the cells to the test compound.
  • Compounds that inhibit the expression or activity of MLL5P in the cells exposed to the test compound, relative to expression or activity of MLL5 in control cervical cancer cells that were not exposed to the test compound, can be useful for treating cervical cancer.
  • cervical cancer cells can be determined. For example, a sample of cervical cells (e.g., from a Pap smear or a biopsy sample) can be assessed to determine whether ⁇ 5 ⁇ is present in the cells. The presence of ⁇ 5 ⁇ in the sample of cervical cells is indicative of the presence of cervical cancer cells. Thus, cervical cancer can be diagnosed in an individual, as the presence of ⁇ 5 ⁇ in a sample of cervical cells from the individual is diagnostic for cervical cancer in the individual.
  • a nucleic acid encoding ⁇ 5 ⁇ can comprise a nucleic acid encoding MLL5 with the insertion of a 26 base pair sequence of interest.
  • the "26 base pair sequence of interest”, as used herein, refers to an insertion of 26 base pairs into a nucleic acid encoding MLL5, in which the insertion results in an alteration of the coding sequence so that the encoded protein is ⁇ 5 ⁇ .
  • Figure 8 depicts a 26 base pair sequence of interest (underlined) and its relative position in a coding sequence for ⁇ 5 ⁇ .
  • the invention additionally pertains to siR A molecules, such as those that can serve as ⁇ 5 ⁇ antagonists as described above, (e.g., siRNA molecule which knocks down expression of a nucleic acid that encodes ⁇ 5 ⁇ ).
  • siR A molecules such as those that can serve as ⁇ 5 ⁇ antagonists as described above, (e.g., siRNA molecule which knocks down expression of a nucleic acid that encodes ⁇ 5 ⁇ ).
  • EXAMPLE 1 Identification of a novel isoform of mixed lineage leukemia 5 (MLL5), isoform ⁇ (MLL5P), and its association with cervical cancer
  • Human cervical carcinoma SiHa (HPV16+), HeLa (HPV18+) and C33A (HPV negative), embryonic kidney cells HEK 293T, colorectal carcinoma HCT116 and osteosacoma U20S were cultured as monolayer in Dulbecco's Modified Eagles Medium (DMEM, Gibco) while human cervical carcinoma Caski in Roswell Park Memorial Institute 1640 (RPMI, Gibco) respectively.
  • the respective mediums were supplemented with 10% fetal bovine serum (FBS, Hyclone), 2 mM glutamine (Gibco) and 100 units/ml penicillin/streptomycin (Gibco) at 37 °C with 5 % CO2.
  • Plasmid transfection was carried out using TranslT-LTl transfection reagent (Mirus, USA) according to the product manual. In brief, 3 ⁇ g of the DNA plasmid and 9 ⁇ of DNA Transfection reagent were added into 500 ⁇ of serum-free medium before transfected to cells in a 60mm 2 plate. The transfection mixture was incubated at room temperature for 15 minutes before addition.
  • Putative MLL5 isoform was cloned using SMARTerTM RACE cDNA Amplification kit (Clontech, USA) according to the product manual. PolyA+ mRNA was extracted using Oligotex Direct mRNA Midi Kit (Qiagen, USA) and used as templates for both 5' and 3 '-RACE. Gene-specific primers used for both 5 'and 3'- RACE recognized exon 7 of MLL5 and are as follow: 5primeM5.rev (5'- TTTCCCTTTTCCGGCGTTGT) (SEQ ID NO:9) and 3primeM5.for (5'- C A AC GC CGG A A A AGGG AA A AT) (SEQ ID NO: 10). RACE products were cloned into pCR2.1-TOPO (Invitrogen, USA) for sequencing.
  • FLAG-MLL5 CT (amino-acid 1 1 13 to 1858) was previously constructed27.
  • GFP-tagged MLL5 C-terminal vector (GFP-CT) was generated by cloning MLL5 C- terminal region into pEGFP-Cl (Clontech) in frame with Sail and BamHl.
  • MLL5P cDNA was amplified from HeLa cDNA. PCR amplicons were digested with BamHl and Notl and cloned into pEF6/V5-His vector (Invitrogen) for FLAG-MLL5p; while digested with Sail and BamHl and cloned into pEGFP-Cl vector for (GFP-MLL5p).
  • a 958 bp (nucleotides 7091 to 1 19) fragment containing the LCR and p-105 promoter was cloned into pGL3 -basic (Promega) with Xhol and HindlU sites. Deletion constructs were generated in a similar manner using appropriately designed primers. Mutant constructs were generated using the Quick Change site-directed mutagenesis kit (Stratagene).
  • siRNAs transfection was carried out using Lipofectamine RNAiMAX (Invitrogen).
  • MLL5 specific siRNA duplexes (#1, #2, #3, #4) targeting nucleotide positions at 1063, 1 147, 5215 and 6807 respectively, from the
  • siRNAs were generated from l st-BASE Singapore.
  • the antibody against the central region of MLL5 (amino acid 1 157-1170, designated as a-MLL5-l 157) (25) is used to probe for full-length MLL5.
  • a-MLL5-227 antibody raised against the N-terminal region of MLL5 (amino acid 227-241 ) was used to probe for ⁇ 5 ⁇ (24).
  • a-HPV16/18-E6 (SC-460), a-HPV18-E7 (SC-1590), a-p53 (SC-126), a-pRb (SC-50), -actin antibodies (SC-1616) were purchased from Santa Cruz Biotech (USA) and ⁇ x-p21 antibody (#2946) were from Cell Signaling (USA).
  • RNA and genomic DNA were extracted from the samples by using TRIzol and Wizard Genomic DNA Purification Kit (Promega). cDNA was generated from total RNA using MLL5p-specific primer (MLL5 .reverse).
  • a dual-luciferase reporter assay (Promega) was employed to measure the transcription activity of the promoter region in interest.
  • HEK 293T cells were co- transfected with both pGL3 and pEGFP, along with pRL-TK as the internal control. Cells were then harvested 48 hours post-transfection and each sample was read in triplicate by using a luminometer (Tecan). Normalization of luciferase reading by renilla reading was performed before comparisons were made.
  • FLAG-MLL5p or FLAG-CT were transfected into HeLa cells for 48 hours before cells were being harvested for ChIP as described previously (27) except the modification in the sonication step.
  • Each sample was sonicated at 40% amplitude for 15 minutes, consisting of 15 cycles of 30 seconds sonication with 30 seconds cool down interval to generate DNA fragments of around 300-500 bp. Pull down was done by a-FLAG (Sigma-Aldrich) and a-mouse IgG antibodies. PCR amplicons were run on a 2% agarose gel to check for enriched region compared to IgG pulled down samples.
  • ⁇ 5 ⁇ was shown to be successfully knocked down and this corresponded to an increase in p53 protein level (data not shown). Consistent with our hypothesis, the E6/E7 levels were found to be significantly reduced in all three human cervical cancer cell lines when ⁇ 5 ⁇ - siRNA was used ( Figure 3). Subsequently, we asked whether exogenous expression of ⁇ 5 ⁇ can abrogate the MLL5 -siRNA-mediated p53 restoration. Effect of p53 restoration when knocked down with MLL5P-siRNA was found to be rescued when GFP-MLL5p was over-expressed but not GFP-tagged full-length MLL5 (data not shown).
  • LCR long control region
  • URR upstream regulatory region
  • Relative luciferase activity of fragment A showed the largest decrease, suggesting that the distal region from nucleotides 7018 to 7305 of LCR contains the essential elements required for the activation of HPV18-LCR by ⁇ 5 ⁇ .
  • chromatin immunoprecipitation (ChIP) experiment was set up. ⁇ -1 ⁇ 5 ⁇ or FLAG-MLL5-CT was over-expressed in HeLa for 48 hours before the cells were cross-linked and sonicated. Immunoprecipitation was carried out using the a-FLAG antibody or the a-mouse IgG antibody which serves as a negative control. The DNA eluates were used as the templates for PCR reactions using primer sets spanning the region of interest shown in Figure 4B.
  • MLL5a MLL5 or its isoform
  • H3K4 activity 13, 18, 19
  • GFP-MLL5 was observed to interact with FLAG- AP-1 in the presence of HP V- 18 LCR (data not shown) Although studies have shown that full-length MLL5 lacks intrinsic H3K4 methyltransferase activity (15, 17), nonetheless, it possesses a SET domain which is a well-known domain that possesses HKMT activity (33). Moreover, four other MLL protein family members were found to exert H3K4 methyltransferase activity through their SET domain (15, 16, 34). Therefore, to investigate whether MLL5p activates E6/E7 transcription through the SET domain, SET-inactivated (Y358A) MLL5p mutant was constructed and used in the dual-luciferase assay (18).
  • MLL5B as a therapeutic target for human cervical cancers
  • MLL5p knockdown can effectively sensitize cervical cancer cells towards radiation (Figure 7).
  • Figure 7 We speculated that the accumulation of p53 in cervical cancer cells after ⁇ 5 ⁇ knockdown will work synergistically with radiation treatment to 'push' the cells into apoptosis. Since MLL5P can only be detected in cancerous cells but not normal cells, MLL5P could be a novel therapeutic target for cervical cancers that offers less adverse effect.
  • ⁇ 5 ⁇ knockdown is able to decrease the expression of another key oncogene, E7 simultaneously, therefore making ⁇ 5 ⁇ knockdown a more effective treatment option.
  • Soft agar colony formation assay is commonly used to monitor anchorage- independent growth, which measures proliferation in a semisolid culture media after 3-4 weeks by manual counting of colonies.
  • Anchorage-independent growth is one of the hallmarks of cancer transformation, which is considered the most accurate and stringent in vitro assay for detecting malignant transformation of cells.
  • MLL5 isoform
  • ⁇ 5 ⁇ a novel MLL5 isoform
  • the truncated MLL5 isoform encoded a 503-amino-acid polypeptide and is exclusively present in HPV16/18-positive cervical cancer cells and can be detected in human primary cervical carcinoma.
  • ⁇ 5 ⁇ isoform associates with AP-1 binding site located at nucleotide 7326 of the distal region of HPV18 LCR, upstream of the E6 and E7 promoter site. The association with AP-1 transcriptional factor is essential for the MLL5P-mediated bicistronic E6/E7 gene expression.
  • MLL5 participates in the cell cycle regulatory network at multiple stages (24, 25).
  • Cdc2 phosphorylation of MLL5 by Cdc2 is required for mitotic progression and depletion of MLL5 induces p53 activation at active DNA replication forks (26, 35).
  • full-length MLL5 (1858 amino acids) has been shown to lack intrinsic histone methyltransferase H3K4 activity (15, 17); an isoform of MLL5 comprising a total of 608 amino acids with PHD and SET domain exhibited GlcNAcylation ⁇ dependent HKMT activity in retinoic acid-induced granulopoiesis (18).
  • ⁇ 5 ⁇ interacts with AP-1 transcription factor and recognizes the AP-1 binding site on nucleotide 7326 of the distal region of LCR ( Figure 6, dashed area).
  • the SET domain in ⁇ 5 ⁇ was found to play a role in the activation.
  • ⁇ 5 ⁇ - siRNA was able to simultaneously reduce the E6/E7 transcripts and has, there is greater potential for ⁇ 5 ⁇ as a novel specific therapeutic target for human cervical cancers than siRNA targeting E6 or E7 alone.
  • ⁇ 5 ⁇ can be detected only in HPV-positive cancer cells but not normal diploid cell line suggests that the potential side-effects of MLI ⁇ -siRNA therapy can be minimized by employing MLI ⁇ -specific siRNA.
  • successful detection of ⁇ 5 ⁇ in primary cervical tumors further implies the clinical importance of this isoform.
  • HPV integration sites are found to be randomly distributed throughout the whole genome but with several preferred genomic fragile sites. One of which is located at 8q24 that contains the myc gene (41, 42). Furthermore, a novel gene APM-1 which is a fusion of the HPV and human gene has been reported, further illustrating the possible effect of HPV genome integration on human genes (43).
  • Such HPV genome integration is unlikely to be a totally random event as the same identical ⁇ 5 ⁇ mRNA were detected in all HPV16/18-positive cell lines and human primary cervical carcinoma samples we have tested. However, further studies are required before any feasible conclusion can be drawn.
  • HPV LCR Ubiquitous transcription factors such as AP-1 and SP-1 have well- established roles in HPV transcription regulation (44-46).
  • HPV LCR can be classified into three different regions based on the position of highly-conserved E2 binding sites. As shown in Figure 6, the distal region (dashed area) contains transcription termination signal while the central region (gray area) contains the majority of transcription factor binding sites which include two AP-1 binding sites (nucleotides 7609 and 7793), and was termed specific enhancer region (47, 48). The proximal region (chequered area) contains the early promoter and origin of replication (49).
  • a trypan blue exclusion assay was used to determine the number of living cells, in order to monitor the effect of MLL ⁇ -siRNA in inducing cell death and growth suppression in vitro using cell lines.
  • E6 and E7 siRNAs were included as a comparison of the effectiveness of the MLL5 ⁇ -siRNA.
  • HeLa HPV18-positive human cervical cancer cell line
  • SiHa HPV16-positive human cervical cancer cell line
  • C33A HPV-negative human cervical cancer cell line
  • WI38 human diploid cell line
  • MLL5P-siRNA After assessing the effect of MLL5p-siRNA in in vitro setting, we were interested to investigate whether MLL5P-siRNA would exhibit similar effect in in vivo setting.
  • a mouse system was established using HeLa induced tumor in immunodeficient mouse. MLL5 , E6 and E7 siRNAs were injected into the tumor every other day for 20 days and the tumor size was monitored. Consistent with the in vitro results, MLL5P-siRNA showed highest tumor suppressor ability compared to E6 and E7 alone ( Figures 10A, 10B). These results further confirm the utility of MLL5 -siRNA as a novel therapeutic target for cervical cancer.
  • MLL5p-siRNA activates both apoptosis and senescence pathways, explaining our previous observation that MLL5p-siRNA exhibits a higher killing effect on cancer cells comparing to E6 and E7-siRNAs alone.
  • MLL5p- siRNA treated HeLa cells were found to have alleviated level of cleaved-P ARP, an apoptotic marker; and they also showed a high percentage of senescence cells ( Figure 11). This confirmed that MLL5p-siRNA was able to induce both apoptosis and senescence pathways, and enables MLL5 -siRNA to have a better utility for cervical cancer treatment.
  • a cytotoxicity assay was performed using cisplatin treated cells coupled with or without gamma irradiation.
  • cisplatin induces a sensitization effect towards gamma irradiation, where the gamma irradiation treatment downstream of cisplatin treatment will cause an elevated cytotoxicity effect to the cells compared to cells without cisplatin treatment, in both HeLa and SiHa cells.
  • C33A cells with mutant p53 no sensitization effect can be observed, providing evidence that the mechanism is p53 -dependent.
  • Cisplatin treatment does not differentiate between cancerous and normal cells, as the cytotoxicity caused by cisplatin in normal WI38 cells is as high as other cancerous cell lines. This could explain the adverse side effect caused by cisplatin treatment.
  • MLL5P plays a role in the anti-cancer effect of cisplatin.
  • qRT-PC was performed to check the mRNA level of MLL5P, E6 and E7 in cisplatin treated HeLa cells.
  • a significant and concentration dependent downregulation of all MLL5p, E6 and E7 was observed in cisplatin treated HeLa cells.
  • a rescue experiment was carried out to further confirm the involvement of MLL5P in cisplatin-mediated anti-cancer effect, where MLL5 was exogenously introduced into cisplatin-treated HeLa cells.
  • the overexpressed MLL5P was found to inhibit the sensitization effect caused by cisplatin treatment, thereby confirming that MLL5P played a role in the cisplatin mediated anti-cancer effect.
  • MLL5p-siRNA can induce apoptosis and senescence in HPV16/18-positive cell lines and thereby inhibit the growth of cancer cells, both in vitro in a soft agar assay and in vivo in a mouse xenograft study. It was also established that MLL5p plays a role in the cisplatin-mediated anti-cancer effect. Hence, by using MLL5P as a novel therapeutic target, specific targeting of abnormal cancer cells can be enhanced, and adverse side effects caused by cisplatin treatment can be avoided.
  • MLL5 -siRNA In addition, the ability of MLL5 -siRNA to downregulate both E6 and E7 offered a synergistically increased anti-cancer effect over E6 and E7 siRNAs alone. Moreover, MLL5 -siRNA is HPV-strain independent; therefore, it works on both HPV16/18-positive cells, unlike E6 and E7 siRNAs that can only target a specific strain.
  • Singapore Research Fund R-l 83-000-268-733 to L.W.D. C.W.Y. and P.L. are the recipients of research scholarships from Yong Loo Lin School of Medicine, National University Health System, and National University of Singapore. References
  • Incassati A Patel D, McCance DJ. Induction of tetraploidy through loss of p53 and upregulation of Plkl by human papillomavirus type-16 E6. Oncogene.
  • the S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hstl, and is a meiotic-specific repressor of the sporulation gene program. Genes Dev. 2001 ;15:2991-3004.
  • ALL-1 is a histone methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Mol Cell. 2002; 10:11 19-28.
  • Cripe TP Haugen TH, Turk JP, Tabatabai F, Schmid PG, 3rd, Durst M, et al.

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Abstract

La présente invention concerne une nouvelle isoforme de leucémie de lignage mixte 5 (MLL5), MLL5β, ainsi que l'acide nucléique codant pour MLL5β. La présente invention concerne en outre des procédés d'inhibition de l'expression ou l'activité de MLL5β ; des procédés de traitement du cancer cervical ; des procédés d'identification de composés utiles pour traiter le cancer cervical ; ainsi que des procédés pour évaluer les cellules cervicales pour la présence de cancer cervical et diagnostiquer le cancer cervical. La présente invention concerne en outre de ARNsi utiles pour inhiber l'expression de MLL5β.
PCT/SG2012/000266 2011-07-25 2012-07-24 L'isoforme de leucémie de lignage mixte 5 est potentiellement un biomarqueur et une cible thérapeutique pour le cancer cervical associé à hpv WO2013015744A1 (fr)

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Citations (1)

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WO2006033664A1 (fr) * 2004-03-08 2006-03-30 Avalon Pharmaceuticals Determination de genes lies au cancer et cibles therapeutiques utilisant des techniques cytogenetiques moleculaires

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006033664A1 (fr) * 2004-03-08 2006-03-30 Avalon Pharmaceuticals Determination de genes lies au cancer et cibles therapeutiques utilisant des techniques cytogenetiques moleculaires

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANSARI K. ET AL.: "Mixed lineage leukemia: roles in gene expression, hormone signaling and mRNA processing", FEBS JOURNAL, vol. 277, 2010, pages 1790 - 1804 *
HODGE J. ET AL.: "Identifying the Molecular Signature of the Interstitial Deletion 7q Subgroup of Uterine Leiomyomata Using a Paired Analysis", GENES CHROMOSOMES CANCER, vol. 48, no. 10, 2009, pages 865 - 885 *

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