WO2013015744A1 - Mixed lineage leukemia 5 isoform is a potential biomarker and therapeutic target for hpv-associated cervical cancer - Google Patents

Abstract

A novel isoform of mixed lineage leukemia 5 (MLL5), MLL5β, is described, as is the nucleic acid encoding MLL5β. Methods of inhibiting expression or activity of MLL5β; methods of treating cervical cancer; methods of identifying compounds useful for treating cervical cancer; as well as methods for assessing cervical cells for the presence of cervical cancer and diagnosing cervical cancer, are described. siRNA useful for inhibiting expression of MLL5β are also described.

Description

MIXED LINEAGE LEUKEMIA 5 ISOFORM IS A POTENTIAL BIOMARKER AND THERAPEUTIC TARGET FOR HPV-ASSOCIATED CERVICAL CANCER

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/511 ,373, filed on July 25, 2011. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Approximately 160 million Pap tests are performed annually worldwide, approximately 55 million of which are performed in the U.S., 5 million in Canada, 45 million in the EU, 14 million in Japan and 41 million in the rest of the world. In the major world markets - North America, Europe and Japan - Pap screening has all but erased the threat of death from cervical cancer. This is not the case in the rest of the world. The World Health Organization estimates that cervical cancer affects nearly 500,000 women around the world every year and kills more than 300,000, of which 85% are in developing countries. More importantly, generally only about 5% of women in developing countries are screened for cervical disease, compared to 40 - 50%, in the developed world. In the United States, six percent (3.6 million) of the Pap smears performed are abnormal and require follow-up. This follow-up usually involves a test for HPV (human papillomavirus). It is generally accepted that cervical cancer is predominantly caused by sexually transmitted HPV. There are more than 200 types of HPV, most of which are harmless. However, several strains cause genital warts (HPV-6 and HPV-11); high risk types can cause cervical dysplasia, which if left untreated, can progress to cancer. Of particular concern are types HPV- 16 and HPV- 18 that cause more than 70% of all cervical cancers.

SUMMARY OF THE INVENTION

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. The invention pertains to methods to inhibit the expression or activity of mixed lineage leukemia 5 isoform β (ΜΙΧ5β) in a cervical cancer cell by providing the cell with an effective amount of at least one ΜΙΧ5β antagonist that inhibits production of ΜΙΧ5β protein, activity of ΜΙ 5β protein, or a combination thereof. Representative ΜίΧ5β antagonists include nucleic acids (e.g., an antisense oligonucleotide, an siRNA, an shRNA, a microRNA, an aptamer or a ribozyme); or antibodies In certain embodiments, the nucleic acid is an antisense oligonucleotide, such as one that selectively hybridizes to ΜΙΧ5β mRNA, or an siRNA, such as one that targets a 26 base pair sequence of interest in ΜΙ 5β. In some embodiments, the ΜΙ 5β antagonist interferes with or inhibits the association between ΜΙΧ5β and AP-1 binding site and its associated components at nucleotide 7326 of distal region of HPV18 LCR.

In addition, 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 β (ΜΙ 5β) antagonist that inhibits production of ΜΙΤ_,5β protein, activity of ΜΙΧ5β 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 β (ΜΙΧ5β) antagonist that the MLL5p antagonist inhibits production of ΜΙΧ5β protein, activity of ΜΙΧ5β 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 β (MLL5p), as well as to an isolated nucleic acid that encodes mixed lineage leukemia 5 isoform β (ΜΙΧ5β).

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 β (ΜΙΧ5β) in the cells following exposure of the cells to the test compound; and (c) selecting a compound that inhibits the expression or activity of ΜΙΧ5β in the cells exposed to the test compound relative to expression or activity of ΜΙΧ5β in control cervical cancer cells that were not exposed to the test compound. Inhibition of the expression or activity of ΜΙ 5β in the cervical cancer cells that have been exposed to the test compound relative to expression or activity of ΜΙΧ5β 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 β (ΜΙΧ5β), wherein the presence of ΜΙ 5β in the sample is indicative of the presence of cervical cancer cells. Cervical cancer can be diagnosed in an individual using such an assessment.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

Figure 1. MLL5 knockdown leads to down-regulations of E6 and E7 oncoproteins in HPV16/18-positive cell lines. (A) 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. (B) Figure IB 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. A marked reduction of E6 and E7 mRNA in HeLa, CaSki and SiHa cells after transfected with MLL5-siRNA#l for 72 hours but not for MLL5-siRNA#4. An internal reference gene GAPDHwas used for normalization.

Figure 2. Identification of a novel MLL5 isoform. (A) Figure 2A is a characterization of the novel isoform ΜΙ 5β compared to full-length MLL5.

Shaded box indicated the inserted 26-bp which introduced a stop codon. Filled triangles indicate the locations of the sequences used for the antigens of antibody a- MLL5-227 while opened triangle indicates that used for the antigen of antibody a- MLL5-1 157. (B) Figure 2B is a characterization of a portion of Μ∑∑5β mRNA compared to MLL5. A region of MLL5 is shown (SEQ ID NO: 1), as is a region of Μ115β (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. (A) 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. (B) 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. Identification of the interacting partner of ΜΙΧ5β in HP VI 8- LCR. (A) 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. (B) 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. (C) 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.

Figure 6. A proposed model for the molecular mechanism of ΜΙΧ5β 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 ΜΙΧ5β. 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 β 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 8. Sequence information for ΜΙΧ5β. Figure 8A depicts the mRNA encoding MLL5p (SEQ ID NO:7). The ΜΙΧ5β 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 ΜΙΧ5β 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.

Figure 11. MLL5P knockdown induces apoptosis in HeLa cells. Figure 1 1 demonstrates that ΜΙΧ5β 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 ΜΙΧ5β 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).

Figure 14. Downregulation of MLL5 β 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.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

As described in Example 1 below, a novel isoform of mixed lineage leukemia 5 (MLL5) has been identified. Named isoform β (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. Furthermore, eight human primary cervical carcinoma specimens tested for the presence of MLL5(3 isoform with a MLL5p gene-specific primer were all positive for ΜΙ 5β . As a result of this discovery, specific methods relating to diagnosis and treatment of cervical cancer are now available.

In one embodiment of the invention, the expression or activity of Μ1Χ5β 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.

In one embodiment, to inhibit the expression or activity of ΜΙΧ5β in a cervical cancer cell, the cell is exposed to or treated with an effective amount of at least one ΜΙΧ5β antagonist. A "ΜΙΧ5β antagonist," as used herein, is an agent that interferes with ΜΙΧ5β either directly or indirectly such that ΜΙΧ5β 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 (RNAi), 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.

In certain embodiments, the nucleic acid is an antisense oligonucleotide, such as an antisense oligonucleotide that selectively hybridizes to ΜΙΧ5β mRNA. In certain other embodiments, nucleic acid is an siRNA, such as an siRNA that targets the 26 base pair sequence of interest in ΜΙΧ5β. The "26 base pair sequence of interest in ΜΙΧ5β," as used herein, refers to the 26 base pair sequence that is present in isoform ΜΙΧ5β but absent in MLL5. For example, the nucleic acid sequence shown in Figure 8A is an mRNA encoding ΜΙΧ5β; the 26 base pair sequence of interest in this sequence is underlined.

In certain other embodiments, the ΜΙ 5β antagonist is an antibody. As used herein, the term "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. Thus, 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

Laboratory, 1988).

In additional embodiments of the invention, the MLL5 antagonist is an agent that interferes with or inhibits the association between ΜΙ 5β and AP-1 binding site and its associated components in the distal region of HPV18 long control region (LCR). For example, because the distal region from nucleotides 7018 to 7305 of LCR contains elements involved in the activation of HPV18-LCR by MLL5 (as described in Example 1, below), 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.

In further embodiment of the invention, cervical cells can be sensitized towards gamma radiation by knocking down MLL5 . For example, cervical cancer cells can be sensitized by providing the cells with an effective amount of at least one mixed lineage leukemia 5 isoform β (MLL5p) antagonist, as described above.

Similarly, 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. .. .. ..

Furthermore, compounds (e.g., other MLL5P antagonists) useful for treating cervical cancer can be identified using methods described herein. For example, 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.

Diagnostic methods are all now available as well. Because of the specific presence of MLL5 in cervical cancer cells, the presence of cervical cancer cells in a sample of cervical 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 ΜΙΧ5β is present in the cells. The presence of ΜΙΧ5β 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 ΜΙΧ5β in a sample of cervical cells from the individual is diagnostic for cervical cancer in the individual.

In a further embodiment, the invention pertains to an isolated nucleic acid encoding mixed lineage leukemia 5 isoform β (ΜΙΧ5β). For example, a nucleic acid encoding ΜΙ 5β 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 ΜΙΧ5β. For example, Figure 8 depicts a 26 base pair sequence of interest (underlined) and its relative position in a coding sequence for ΜΙ 5β.

The invention additionally pertains to siR A molecules, such as those that can serve as ΜΙ 5β antagonists as described above, (e.g., siRNA molecule which knocks down expression of a nucleic acid that encodes ΜΙΧ5β).

EXAMPLE 1: Identification of a novel isoform of mixed lineage leukemia 5 (MLL5), isoform β (MLL5P), and its association with cervical cancer

Materials and Methods

Cell culture and transfection

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 μg of the DNA plasmid and 9 μΐ of DNA Transfection reagent were added into 500 μΐ of serum-free medium before transfected to cells in a 60mm² plate. The transfection mixture was incubated at room temperature for 15 minutes before addition.

Rapid amplification of cDNA ends

Putative MLL5 isoform was cloned using SMARTer™ 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.

Plasmid construction

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). To generate constructs for luciferase assays for the LCR promoter activity, 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).

siRNA transfection

The siRNAs transfection was carried out using Lipofectamine RNAiMAX (Invitrogen). Four MLL5 specific siRNA duplexes (#1, #2, #3, #4) targeting nucleotide positions at 1063, 1 147, 5215 and 6807 respectively, from the

transcription starting point (NCBI Reference Sequence: NM l 82931.2), were used and are shown in Table 1 below. Scrambled siRNA (sense: 5'- UUCUCCGAACGUGUCACGUdTdT (SEQ ID NO:l 1); antisense: 5'- ACGUCACACGUUCGGAGAAdTdT (SEQ ID NO: 12)) was used as control. All the siRNAs were generated from l st-BASE Singapore.

Western blot analysis

A total of 0.4 million cells were loaded in each lane. 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 ΜΙΧ5β (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).

cDNA synthesis and quantitative Real-time PCR (qPCR)

Total RNA was extracted using TRIzol (Invitrogen). The RNA was treated with DNase (Ambion, #2222) prior to cDNA synthesis using the first strand cDNA synthesis kit with oligo-dT primer (Invitrogen). Gene expression was measured using the iQ5 qPCR (BioRad) with the SYBR Green PCR Master Mix (BioRad) and in-house designed primers. Primers used include MLL5. forward (5'- CCACCACAAAAGAAAAAGGTTTCTC -3') (SEQ ID NO: 13), MLL5.reverse (5'- GTGTTGGTAAAGGTAGGCTAGC - 3') (SEQ ID NO:14), MLL5p.forward ( ' -G AAAACCC AG AGTGCCCTGTTCTA-3 ' ) (SEQ ID NO: 15), MLL5p.reverse (5 ' -C AAT A ACGCGAG ACT AGTCTT-3 ' ) (SEQ ID NO: 16), HPV18E6.forward (5'- GTGCCAGAAACCGTTGAATCC - 3') (SEQ ID NO: 17), HPV18 E6.reverse (5'- CGACGCCAGCTATGTTGTGAAATCGTCG -3') (SEQ ID NO:18), HPV18 E7.forward (5'-CGTCGCAACATTTACCAGCCCGACG -3') (SEQ ID NO: 19), HPV18 E7.reverse (5'- GAATGCTCGAAGGTCGTCTGC -3') (SEQ ID NO:20), HPV16 E6.forward (5'- CTGCAATGTTTCAGGACCCA -3') (SEQ ID NO:21), HPV16 E6.reverse (5'- TC ATGTATAGTTGTTTGC AGCTCTGT-3 ') (SEQ ID NO:22), HPV16 E7.forward (5'-AAGTGTGACTCTACGCTTCGGTT -3') (SEQ ID NO:23), HPV16 E7.reverse (5'-GCCCATTAACAGGTCTTCCAAA -3') (SEQ ID NO:24), GAPDH.forward (5'- GTGAAGGTCGGAGTC AACG-3 ') (SEQ ID NO:25), GAPDH.reverse (5'- TG AGGTCAATGAAGGGGTC-3 ') (SEQ ID

NO:26). Tissue specimens

Primary tumor specimens were pre-treated biopsies from women with cervical carcinoma of cervix treated in Singapore General Hospital between

December 2010 to February 2011. The samples were obtained by Gynecologist Dr Tay Sun Kuie. Patients were informed and agreed to the use of their biological sample for scientific research in accordance with Singapore regulations. Total 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).

Dual luciferase assay

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.

Chromatin Immunoprecipitation

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.

Results

Knockdown of MLL5 in human HPV16/18-positive cervical cancer cell lines reduces the expression level of E6 and E7 oncoproteins

We noted a marked accumulation of p53 protein in HPV18-positive HeLa cells upon knockdown of MLL5 (25). Since E6 oncoprotein has been known to target p53 for degradation via formation of complex with E3 ubiquitin-protein ligase E6AP, this led us to¹ speculate that knockdown -of MLL5 may have an effect on the expression of E6 protein. Indeed, E6 protein level in HeLa was found to decrease in a time-dependent manner (data not shown). Decrease in E6 protein expression upon knockdown of MLL5 was less likely to be a result of the disruption of E6-E6AP-p53 complex because the protein level of E6AP remained at a similar level regardless of changes in MLL5 levels. Interestingly, a significant decrease in E7 protein and an increase in hypo-phosphorylated form of Rb were also detected in HeLa when MLL5 was knocked down (Figure 1 A). Similar results were observed in two other HPV- 16 positive human cervical cancer cell lines CaSki and SiHa (data not shown). Restoration of p53 protein only occurs in HeLa cells treated with siRNA targeting to the N-terminal region but not the central or C-terminal region of MLL5 mRNA

To rule out the possibility of an off-target effect, besides the MLL5- siRNA#l, three other siRNAs targeting different sites spanning across MLL5 mRNA were employed (Figure 1). Surprisingly, massive p53 accumulation occurred only in the presence of MLL5-siRNA#l and #2, even though MLL5 was knocked down with similar efficacy with all four siRNAs (data not shown). Consistent with the previous data, massive p53 restoration only occurred in MLL5-siRNA#l but not MLL5-siRNA#4 in all three HPV-positive cell lines (data not shown). Nonetheless, a small but detectable amount of p53 protein was observed in MLL5-siRNA#4- knocked down samples, which could be attributed to the effect of full-length MLL5 knockdown as seen in our previous observations (data not shown). Next, we...

attempted to discern the effect of MLL5-knockdown with MLL5-siRNA#l and #4 on both E6 and E7 mRNA levels. A marked down-regulation of both E6 and E7 mRNA was observed in all three HPV-positive cell lines when MLL5-siRNA#l was used but not MLL5-siRNA#4 despite the comparable knockdown efficiency of both siRNAs on MLL5 mRNA (Figure IB). The role of E2 which is a known E6/E7 repressor was disregarded, since E2 was shown to be absent in these HPV-positive cells due to the disruption of E2 gene upon the integration of HPV DNA (28). These observations led us to speculate that a putative MLL5 isoform comprising the N- terminal region but lacking the central and C-terminal regions may be present.

Characterization of the novel MLL5 isoform

To validate our hypothesis, 5' and 3' Rapid Amplification of cDNA Ends (RACE) were carried out to identify the isoform. For 5' RACE, a PCR product of approximately 1000 bp was amplified from all six cell lines used (data not shown). The amplicon was sequenced and the data showed no difference as compared to the full-length MLL5, indicating that the putative MLL5 isoform has the same 5' end as the full-length MLL5. Subsequently, 3' RACE was carried out with the extension time set to seven minutes, allowing the complete amplification for full-length MLL5. All cell lines tested showed a 6-kb band which corresponds to the full-length MLL5 (data not shown). Interestingly, an additional 1.6-kbp amplicon was observed exclusively in HPV16/18-positive cell lines, CaSki, SiHa and HeLa. Upon sequencing and alignment with MLL5 gene, the 1.6-kbp band has the same sequence as the full-length MLL5 from the start-codon up to part of exon 14 (GenBank accession number: NM 182931), where it was truncated by a 26-bp sequence that introduced a stop codon and followed by a poly-A tail (Figure 2A and 2B). We denoted this novel MLL5 isoform (503 amino acid) as ΜΙΧ5.β to differ it from the one previously reported by Fujiki et al.

The full length mRNA sequence for ΜΙΧ5β is shown in Figure 8A. The protein sequence is shown in Figure 8B.

Next, a wider panel of human cell lines was used to test whether ΜΙΧ5β is exclusively present in HPV16/18-positive cell lines. Consistent with our previous result, MLL5fi was only detected in HPV16/18-positive human cervical cancer cell lines, but not in other human cancer cell lines (data not shown). It is worthy to - mention that ΜΙΧ5β was not seen in the normal diploid cell line WI-38 and HPV- negative human cervical cancer cell line C33A. Furthermore, eight human primary cervical carcinoma specimens were tested for the presence of ΜΙΧ5β isoform. Using ΜΙΧ5β gene-specific primer for cDNA synthesis, ΜΙΧ5β was successfully detected in all eight human primary cervical carcinoma samples (data not shown). ΜΙΧ5β isoform is responsible for the restoration of p53 protein level through down- regulation of E6 and E7 transcripts

We employed anti-MLL5-227 antibody which was raised against the N- terminal region of MLL5 (amino acid 227-241) to probe for the ΜΙ_Χ5β (24). A protein band migrating at approximate 70-kDa position was successfully knocked down by both MLL5-siRNA#l and #2 but not by the MLL5-siRNA#3 and #4 (data not shown). Importantly, knockdown of the 70-kDa ΜΙΧ5β protein corresponded to the restoration of p53 levels. Next, a new siRNA duplex that specifically targets the 26-bp sequence exclusively found in MLL5p mRNA, designated as MLL5P-siRNA, was synthesized (see Table 1, below, for sequence). ΜΙΧ5β 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 ΜΙ 5β- siRNA was used (Figure 3). Subsequently, we asked whether exogenous expression of ΜΙΧ5β 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).

ΜΙ 5β activates HPV18 E6/E7 transcription through the regulation of LCR

Previous studies have shown that proteins such as epidermal growth factor and interferon regulator factor-2 are able to regulate E6/E7 gene transcription through regulatory region, termed long control region (LCR) or upstream regulatory region (URR) (29, 30). The position of the LCR is shown in the genomic map of HPV18, along with the viral genes (Figure 4 A). The regulatory region is rich in cis- regulatory elements, often acting as binding sites for various transcription factors, and has been established to play important roles in HPV gene expression and replication (31). Therefore, a dual-luciferase assay was carried out to address whether MLLSP is involved in the transcription regulation of bicistronic E6/E7 expression through the LCR. Relative luciferase activity was significantly increased when GFP-tagged MLL5p was overexpressed, providing evidence that indeed MLL5p was able to activate the transcription of the LCR (Figure 4B). Next, we constructed various fragments of HPV18-LCR fused with pGL3 vector to dissect the region that is responsible for LCR activation through association with LL5p.

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 ΜΕΕ5β.

To demonstrate that MLLP would interact with HPV18-LCR to regulate its transcriptional activity, chromatin immunoprecipitation (ChIP) experiment was set up. ΡίΑΟ-1νΕ 5β 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. Significant enrichment of amplicon using primer pair 2 was observed in the oc-FLAG-MLL5p pulled-down eluates but not in FLAG-MLL5-CT (data not shown), an indication of an interaction between ΜΙΧ5β and HPV18-LCR at nucleotide 7168 to 7350. Thus, AP-1 transcription factor binding site is essential in the MLL5p-mediated activation of HPV18-LCR.

Since there is no conserved DNA-binding motif found on MLL5 and a recent study has shown that MLL5 or its isoform (MLL5a, 608 amino acids) can form complex with other proteins to exert its H3K4 activity (13, 18, 19), we hence hypothesized that one or more transcription factors might be involved in the

MLL5 -mediated E6/E7 gene activation. An in silico study using PROMO (32) virtual laboratory was first conducted to screen for putative transcription factor binding sites on the HPV18-LCR fragment amplified by the primer pair 2. Four putative transcription factor binding sites with high matching scores were identified (Figure 5 A). Further fragments of HPV18-LCR were designed according to the distribution of the four transcription factor binding sites and cloned into pGL3 -basic vector for dual-luciferase assays. Results showed no significant reduction in relative luciferase activity except for fragment A3, suggesting that the region of nucleotides 7310 to 7350 may comprise of candidate transcription factors associating with ΜΙΧ5β for activation of LCR.

Two highly-matched transcription factor binding sites can be found in the fragment A3, namely AP-1 and SP-1. To verify the involvement of these

transcription factors in MLL5p-mediated activation, we generated pGL3 vectors carrying HPV18LCR with mutated AP-1 and SP-1 binding sites. AP-1 mutant showed a significant reduction in relative luciferase activity but not SP- 1 mutant, suggesting that AP-1 may participate in MLL5P-mediated E6/E7 activation (Figure 5B). To investigate whether the ΜΙΧ5β interacts with AP-1, immunoprecipitation was carried out. 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). Relative luciferase activity was found to decrease up to two fold when SET mutant was used (Figure 5C). Nonetheless, only a marginal decrease in the luciferase activity was observed when the concentration of exogenously-introduced SET mutant vector was increased.

MLL5B as a therapeutic target for human cervical cancers

Through pilot studies, we observed that MLL5p knockdown can effectively sensitize cervical cancer cells towards radiation (Figure 7). We speculated that the accumulation of p53 in cervical cancer cells after ΜΙΧ5β 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. Besides that, ΜΙ 5β knockdown is able to decrease the expression of another key oncogene, E7 simultaneously, therefore making ΜΙΧ5β 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. We performed soft agar assay on MLL5p knockdown HeLa cells and found that the anchor-independent growth of HeLa cells were greatly reduced (data not shown). This observation underlines the use of MLL5p-siRNA as a therapeutic target for human cervical cancer. Discussion

In this study, we identified a novel MLL5 isoform, ΜΙΧ5β, resulting from an additional 26 nucleotides that introduces a stop codon in the exon 14 of MLL5 mRNA. 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. ΜΙΧ5β 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. Knockdown of MLL5p isoform led to down-regulation of both E6 and E7 oncoproteins, resulting in the restoration of p53 protein levels and a reduction in Rb phosphorylation. Our study is the first paper to describe the presence of the novel ΜΙΧ5β isoform and to illustrate the involvement of ΜΙΧ5β in HPV16/18-related cervical cancers.

We have previously proposed that MLL5 participates in the cell cycle regulatory network at multiple stages (24, 25). We showed that phosphorylation of MLL5 by Cdc2 is required for mitotic progression and depletion of MLL5 induces p53 activation at active DNA replication forks (26, 35). Although 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). Given that fact that viruses often cause diseases when they enter host cells and 'hijack' the important cellular apparatus to redirect the cellular functions for their benefit, it is possible that the induction of ΜΤΤ5β isoform may be an invasive strategy employed by HPV to regulate E6/E7 promoter activity (Figure 6). ΜΙΧ5β 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 ΜΙΧ5β was found to play a role in the activation. However, when SET domain was inactivated, the effect of E6/E7 gene activation was not depleted completely, suggesting a possibility that besides interaction with AP-1, other proteins may be required to play a cooperative role for the E6/E7 activation. It is possible that there might be an unidentified post- translational regulation on ΜΙ 5β isoform which is required for full activation activity. When ΜΙΧ5β was depleted through transient siRNA knockdown, no ΜΙ 5β-ΑΡ-1 complex can be formed, leading to the absence of the interaction with LCR. This resulted in a down-regulation effect on both the E6/E7 transcript as revealed by RT-PCR and Western blot analysis.

Depletion of MLL5P in HPV16/18-positive cervical cancer cell lines caused a dramatic reduction in the transcription of two important oncogenes, E6 and E7 (Figure 1). Such reduction in E6/E7 expression allows for the restoration of p53 and pRb pathways, leading to the re-activation of cell cycle checkpoints and apoptotic pathway. Due to the prominent roles of oncogenic proteins E6 and E7 in cervical cancer tumourigenesis, much effort have been made to explore the therapeutic potentials of direct suppression of E6 and E7 expression through the use of siRNAs or over-expression of E2 proteins in vitro and in vivo (36-38). Given that ΜΙ 5β- siRNA was able to simultaneously reduce the E6/E7 transcripts and has, there is greater potential for ΜΙΧ5β as a novel specific therapeutic target for human cervical cancers than siRNA targeting E6 or E7 alone. Besides that, the fact that ΜΙΧ5β 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. Moreover, successful detection of ΜΙΧ5β in primary cervical tumors further implies the clinical importance of this isoform.

Our current model raised an interesting question of the possible mechanism behind ΜΙΧ5β derivation. A recent study has shown that viral infection can induce a novel isoform of endogenously expressed protein to facilitate its own survival and continuous replication through alternative splicing (39). SF2/ASF, a key splicing regulator, has been found to be up-regulated by HPV infection and this could imply that HPV infection may have an effect on normal alternative splicing machinery (40). Therefore, it is possible that ΜΙΧ5β may be a result of deregulation of splicing machinery upon HPV infection. Another possible mechanism involves genomic structure alterations at HPV insertion sites where defined cellular gene functions were disrupted by integrated HPV genome fragments. 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). We have attempted to map the 26-bp sequence that was found to insert in the exon 14 of MLL5 gene but to no avail. Since the 26-bp sequence is not found in MLL5 genome, we favour the second mechanism as a possible way of ΜΙΧ5β derivation. Such HPV genome integration is unlikely to be a totally random event as the same identical ΜΙ 5β 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.

Ubiquitous transcription factors such as AP-1 and SP-1 have well- established roles in HPV transcription regulation (44-46). In general, 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). In our study, we identified the distal AP-1 binding site at nucleotide 7326 as the important partner for ΜΙΧ5β to interact with LCR. Only this AP-1 site plays an essential role in the transcription activation by ΜΙΧ5β and the activation is at least five-fold stronger than the other two previously reported AP-1 sites which is located at the central region (nucleotide 7609 and 7793) (47, 48) (Figure 4B). This could be attributed to the role of the chromatin structure in protecting the AP-1 binding sites in the central region but not the AP-1 in the distal region from exposure to the AP-1 transcription factor (50). Besides that, in silico study indicated that the AP-1 binding site at nucleotide 7326 is highly conserved in several other HPV such as HPV 16 and HPV45. These findings illustrate the identification of a novel AP-1 binding site which activates E6/E7 through formation of ΜΙ 5β-ΑΡ-1 complex.

In summary, we have successfully identified a novel MLL5 isoform

(ΜΙΧ5β) which plays a role in activating E6/E7 through the formation of a complex with AP-1 and binds to the distal region of the HPV- 18 LCR. Upon depletion of ΜΙ 5β using siRNA, E6 and E7 oncoproteins are down-regulated, leading to the restoration of p53 and hypo-phosphorylated Rb, thereby re-establishing the cell cycle checkpoint controls. Due to the simultaneous down-regulation of E6 and E7 transcripts by this isoform, ΜΙΧ5β may be a more effective therapeutic target than E6 or E7 individually. Moreover, our study provides a platform for further investigation on the novel mechanism in which isoforms may be derived upon viral infection and the possibilities of ΜΙΧ5β as a biomarker and for HPV-induced cervical cancers.

Table 1 : siRNA Sequences

EXAMPLE 2: Assessment of Targeting ΜΙΧ5β In Carcinoma Cells

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 MLL5β-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) and WI38 (human diploid cell line) were chosen to provide a panel of cell lines from different origin. Cells were first treated with various siRNA to knockdown the gene of interest and number of living cells was counted after 72 and 96 hours of siRNA treatment. The scrambled control was used as an internal control to determine the percentage of surviving cells.

As shown in Figure 9, HeLa and SiHa cells treated with MLL5P-siRNA showed a significant decrease in cell survivability in both 72 (Fig. 9A) and 96 (Fig. 9B) hours. E6 and E7-siRNAs treated HeLa and SiHa cells showed reduced cell survivability but not as significant as MLL5p-siRNA. Besides that, treatment by E6 and E7-siRNAs are strain-specific in which HPV18E6 and E7 siRNAs will not work on HPV16-positive SiHa cells. The results indicate that MLL5P-siRNA indeed exhibits death inducing and growth suppressing properties in HPV -positive human cervical cancer cell lines. It is interesting to note that MLL5 -siRNA shown a synergistic effect than individual E6 and E7-siRNA alone and it can act independent of the HPV strains.

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.

Next, since MLL5P had been found to knock down both E6 and E7, we hypothesized that 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. Indeed, 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.

Current standard therapy for human cervical cancers involves the usage of anti-cancer chemotherapy drugs, e.g. cisplatin, along with radiotherapy. Through some unknown mechanism, cisplatin is able to induce p53 accumulation in cells and thereby pushes cancer cells to go through apoptosis when used concurrently with radiotherapy. However, because cisplatin and radiotherapy target both normal and cancerous cells indiscriminately, adverse side effects are observed. In addition, there are patients who develop resistance to cisplatin, limiting its effect. Seeing that MLL5P knockdown can similarly induce p53 accumulation as described herein, we assessed whether ΜΙΧ5β could enhance the therapeutic effect of cisplatin.

To investigate the hypothesis, first a cytotoxicity assay was performed using cisplatin treated cells coupled with or without gamma irradiation. As shown in Figure 12, 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. In 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.

Since ΜΙΧ5β knockdown induces a p53 accumulation in HeLa and SiHa, we hypothesized that MLL5p knockdown would induce sensitization effect towards gamma irradiation for HeLa and SiHa cells. We carried out cytotoxicity assays using various cells knocked down by scrambled, MLL5P, E6 and E7 siRNAs. A

significant sensitization effect of HeLa and SiHa was observed when MLL5P was knocked down and coupled with gamma irradiation (Figure 13). E6 was found to downregulate p53 through degradation; E6 knockdown cells also showed a sensitization effect towards gamma irradiation. However, similar to cell survival assay in Figure 9, the sensitization effect was strain-dependent, where HPV18-E6 siRNA only worked on HPV18-positive HeLa but not HPV-16 SiHa, and vice versa. Since MLL5P is only found to be present in HPV16/18-positive cervical cancer cells, MLL5P knockdown did not affect normal WI38 cells. This will be

advantageous in minimizing the side effect caused by cancer treatment.

Lastly, we investigated whether 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. As shown in Figure 14, 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.

Summary: It was discovered that 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. 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.

Acknowledgements

This work was supported in part by Ministry of Education Academic

Research Fund Tier 2 Grant R- 183 -000- 195-1 12 and National University of

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

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While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of inhibiting the expression or activity of mixed lineage leukemia 5 isoform β (ΜΙΧ5β) in a cervical cancer cell, comprising providing the cell with an effective amount of at least one ΜΙΧ5β antagonist, wherein the

MLL5P antagonist inhibits production of ΜΙΧ5β protein, activity of ΜΙΧ5β protein, or a combination thereof.

2. The method of Claim 1, wherein the ΜΙ 5β antagonist is a nucleic acid.

3. The method of Claim 2, wherein the nucleic acid is selected from the group consisting of an antisense oligonucleotide, an siRNA, an shRNA, a microRNA, an aptamer and a ribozyme.

4. The method of Claim 3, wherein the nucleic acid is an antisense

oligonucleotide.

5. The method of Claim 4, wherein the antisense oligonucleotide selectively hybridizes to ΜΙ 5β mRNA.

6. The method of Claim 3, wherein nucleic acid is an siRNA.

7. The method of Claim 6, wherein the siRNA targets the 26 base pair sequence of interest in ΜΙ 5β.

8. The method of Claim 1, wherein the ΜΙ 5β antagonist is an antibody.

9. The method of Claim 1 , wherein the ΜΙΧ5β antagonist interferes with or inhibits the association between ΜΙΧ5β and AP-1 binding site and its associated components at nucleotide 7326 of distal region of HPV18 LCR.

10. A method of sensitizing a cervical cancer cell towards gamma radiation, comprising providing the cell with an effective amount of at least one mixed lineage leukemia 5 isoform β (ΜΙΧ5β) antagonist, wherein the ΜΙΧ5β antagonist inhibits production of ΜΙ 5β protein, activity of ΜΙΧ5β protein, or a combination thereof.

1 1. The method of Claim 10, wherein the ΜΙΧ5β antagonist is an siRNA.

12. The method of Claim 11 , wherein the siRNA targets the 26 base pair

sequence of interest in ΜΙΧ5β.

13. A method of treating cervical cancer in an individual in need thereof,

comprising administering to the subject an effective amount of at least one mixed lineage leukemia 5 isoform β (ΜΙΧ5β) antagonist, wherein the ΜΙΧ5β antagonist inhibits production of Μ1 5β protein, activity of ΜΙΧ5β protein, or a combination thereof.

14. The method of Claim 13, wherein the Μ11Χ5β antagonist is an siRNA.

15. The method of Claim 14, wherein siRNA targets the 26 base pair sequence of interest in ΜΙΧ5β.

16. An siRNA molecule which knocks down expression of a nucleic acid that encodes mixed lineage leukemia 5 isoform β (ΜΙ 5β).

17. A method of identifying a compound useful for treating cervical cancer, comprising:

(a) exposing cervical cancer cells to a test compound;

(b) determining the expression or activity of mixed lineage leukemia 5 isoform β (ΜΙΧ5 ) in the cells following exposure of the cells to the test compound; and

(c) selecting a compound that inhibits the expression or activity of ΜΙ 5β in the cells exposed to the test compound relative to expression or activity of ΜΙΧ5β in control cervical cancer cells that were not exposed to the test compound, wherein inhibition of the expression or activity of ΜΙΧ5β in the cervical cancer cells that have been exposed to the test compound relative to expression or activity of ΜΙΧ5β in control cervical cancer cells that were not exposed to the test compound, is indicative that the test compound is useful for treating cervical cancer.

A method of assessing a sample of cervical cells for the presence of cervical cancer cells, comprising assessing the sample for the presence of mixed lineage leukemia 5 isoform β (ΜΙ 5β), wherein the presence of ΜΙ 5β in the sample is indicative of the presence of cervical cancer cells.

A method of diagnosing cervical cancer in an individual, comprising assessing a sample of cervical cells from the individual for the presence of mixed lineage leukemia 5 isoform β (ΜΙΧ5β), wherein the presence of ΜΙ 5β in the sample is diagnostic for cervical cancer in the individual.

An isolated nucleic acid encoding mixed lineage leukemia 5 isoform β (ΜΙΧ5β).

The isolated nucleic acid of Claim 20, comprising the sequence of the 26 base pair sequence of interest in ΜΙΧ5β.

The isolated nucleic acid of Claim 20, comprising the sequence shown in Figure 8A. 23. The isolated nucleic acid of Claim 20, wherein the nucleic acid encodes the protein shown in Figure 8B.