WO2010150159A1 - Anticancer agent specific for brain tumors with mechanism of recq1 suppression - Google Patents

Abstract

The present invention refers to the use of an anticancer agent specific for brain tumors, in particular for glioblastoma, comprising at least one compound which suppresses the expression of RECQ1 gene, more particularly a siRNA (small interfering RNA).

Description

Anticancer agent specific for brain tumors with mechanism of RECQ1 suppression

Field of invention

The present invention relates to genetic engineering and its applications in the medical field.

In particular, the present invention relates to the use of a specific antitumor agent for brain tumors, particularly glioblastoma, including at least one compound that suppresses RECQ1 gene expression, more particularly a siRNA (small interfering RNA), and to a pharmaceutical composition comprising said antitumor agent.

Background

RecQ helicases are a ubiquitous family of DNA unwinding enzymes involved in the maintenance of chromosome stability. Five members of the RecQ family have been found in human cells: BLM, RECQ1 (also known as RECQL or RECQL1), RECQ4, RECQ5, and WRN (Hickson, I. D. RecQ helicases: caretakers of the genome. Nat Rev Cancer 3, 169-78 (2003); Opresko, P. L., Cheng, W.H. & Bohr, V.A. Junction of RecQ helicase biochemistry and human disease. J Biol Chem 279, 18099-102 (2004); Wu, L. & Hickson, I. D. DNA helicases required for homologous recombination and repair of damaged replication forks. Annu Rev Genet 40, 279- 306 (2006)). Mutations in the genes of three human RecQ family members, are linked to defined genetic disorders associated with genomic instability, tumor predisposition and features of premature ageing; namely, Bloom's syndrome (BLM gene mutations), Werner's syndrome (WRN), and Rothmund-Thomson syndrome, RAPADILINO and Baller-Gerold syndrome (all caused by mutation of RECQ4) (Ellis, N.A. et al. The Bloom's syndrome gene product is homologous to RecQ helicases. Cell 83, 655-66 (1995); Yu, CE. et al. Positional cloning of the Werner's syndrome gene. Science 272, 258-62 (1996); Kitao, S., Lindor, N. M., Shiratori, M., Furuichi, Y. & Shimamoto, A. Rothmund-thomson syndrome responsible gene, RECQL4: genomic structure and products. Genomics 61 , 268-76 (1999); Siitonen, H.A. et al. Molecular defect of RAPADILINO syndrome expands the phenotype spectrum of RECQL diseases. Hum MoI Genet 12, 2837-44 (2003); Van Maldergem, L. et al. Revisiting the craniosynostosis-radial ray hypoplasia association: Baller-Gerold syndrome caused by mutations in the RECQL4 gene. J Med Genet 43, 148-52 (2006)). Mutations in the RECQ1 and RECQ5 genes may be responsible for additional tumor predisposition disorders, but this remains to be proven. In this regard, interesting candidates are patients with a phenotype similar to that of RTS individuals who do not carry any mutations in the RECQ4 gene. Moreover, recent studies have linked a single nucleotide polymorphism present in the RECQ1 gene to a reduced survival in patients with pancreatic tumor (Li, D. et al. Single nucleotide polymorphisms of RECQ1 , RAD54L, and ATM genes are associated with reduced survival of pancreatic cancer. J Clin Oncol 24, 1720-8 (2006); Li, D. et al. Significant effect of homologous recombination DNA repair gene polymorphisms on pancreatic cancer survival. Cancer Res 66, 3323-30 (2006)).

Biochemical studies have demonstrated that RecQ helicases unwind DNA with a 3' to 5' polarity and, although with some differences, are capable of unwinding a variety of DNA structures other than standard B-form DNA duplexes. Consistent with an ability to unwind various DNA structures, several cellular functions have been attributed to RecQ proteins, including roles in stabilization and repair of damaged DNA replication forks, telomere maintenance, homologous recombination, and DNA damage checkpoint signaling (Bachrati, CZ. & Hickson, I. D. RecQ helicases: guardian angels of the DNA replication fork. Chromosoma 1 17, 219-33 (2008); Bohr, V.A. Rising from the RecQ-age: the role of human RecQ helicases in genome maintenance. Trends Biochem Sci (2008); Ouyang, K. J., Woo, L. L. & Ellis, N.A. Homologous recombination and maintenance of genome integrity: cancer and aging through the prism of human RecQ helicases. Mech Ageing Dev 129, 425-40 (2008); Sharma, S., Doherty, K.M. & Brosh, R. M., Jr. Mechanisms of RecQ helicases in pathways of DNA metabolism and maintenance of genomic stability. Biochem J 398, 319-37 (2006)).

Biochemical and genetic studies support roles for all five human RecQ helicases in DNA replication, DNA recombination and in response to DNA damage, although the specific role of each single human RecQ helicase is not yet well defined, and many questions concerning the different functions of the five human RecQ helicases remain unanswered. Therefore, understanding how these enzymes operate and regulate their activities of DNA processing is important if we want to shed light on the mechanisms that control the genome integrity, and to prevent tumor and aging.

Regarding RECQ1 , a specific role of this helicase in maintaining genome integrity is supported by the analysis of embryonic fibroblasts from RECQ1 -deficient mice that are hypersensitive to ionizing radiation and show an increased level of DNA damage and sister chromatid exchanges (Sharma, S. et al. RECQL, a member of the RecQ family of DNA helicases, suppresses chromosomal instability. MoI Cell Biol 27, 1784-94 (2007)).

Previous studies have shown that RECQ1 , BLM, WRN and RECQ4 are poorly expressed in resting cells, while are expressed at high levels in cells whose proliferation was increased by transformation with viruses (see Kawabe, T., Tsuyama, N.,Kitao, S., Nishikawa, K., Shimamoto, A., Shiratori, M., Matsumoto, T., Anno, K., Sato, T., Mitsui, Y., Seki, M., Enomoto, T., Goto, M., Ellis, N.A., Ide, T., Furuichi, Y., and Sugimoto, M., "Differential regulation of human RecQ family helicases in cell transformation and cell cycle", Oncogene, 2000, Vol. 19, No. 41 , pp. 4764- 4772). Moreover, the expression of RECQ1 , BLM, WRN and RECQ4 is induced by the addition of the TPA tumor promoter to resting cells together with an induction of cell division (see Kawabe, T., Tsuyama, N., Kitao, S., Nishikawa, K., Shimamoto, A., Shiratori, M., Matsumoto, T., Anno, K., Sato, T., Mitsui, Y., Seki, M., Enomoto, T., Goto, M., Ellis, N.A., Ide, T., Furuichi, Y, and Sugimoto, M., "Differential regulation of human RecQ family helicases in cell transformation and cell cycle.", Oncogene, (2000), Vol. 19, No. 41 , pp. 4764-4772).

Furthermore, the expression levels of genes belonging to the RecQ family of DNA helicases are markedly higher in tumor cells (Patent Application Publ. No. EP1625853). Collectively, these findings suggest that RecQ DNA helicases are important for cell proliferation.

In line with these observations, previous studies demonstrated that BLM is highly expressed in tumor cells of both lymphoid and epithelial origin and that this reflects the greater fraction of proliferating cells that are present in tumors relative to the normal tissues of the same origin (Turley, H., Wu, L., Canamero, M., Gatter, K. C. & Hickson, I. D. The distribution and expression of the Bloom's syndrome gene product in normal and neoplastic human cells. Br J Cancer 85, 261-5 (2001 )).

Similarly, WRN was also recently suggested to be involved in the promotion of tumor cell growth (Opresko, P. L., Calvo, J. P. & von Kobbe, C. Role for the Werner syndrome protein in the promotion of tumor cell growth. Mech Ageing Dev 128, 423-36 (2007)).

Taken together, these findings suggest that DNA helicase RecQ family may be potential target molecules for tumor therapy.

In particular, RECQ1 can be considered as a suitable new target for the development of tumor therapies aimed at the elimination of tumor proliferating cells, although its role as a factor related to oncogenes has not been described yet.

Regarding this, a specific role of RECQ1 in tumors is supported by two recent reports showing that RECQ1 silencing in tumor cells results in mitotic catastrophe and local and systemic administration of RecQL1-siRNA mixed with polyethyleneimine polymer or cationic liposomes prevents tumor cell proliferation in vivo in mouse models (Futami, K. et al. Induction of mitotic cell death in cancer cells by small interference RNA suppressing the expression of RecQLI helicase. Cancer Sci 99, 71-80 (2008); Futami, K. et al. Anticancer activity of RecQLI helicase siRNA in mouse xenograft models. Cancer Sci 99, 1227-36 (2008)). Patent Application Publication No. EP1816194 describes cell proliferation inhibitors specific for tumors, including in particular siRNAs able to suppress RECQ1 gene expression.

Patent application Publication No. EP1625853 describes apoptosis inducers for tumor cells, including in particular siRNAs that suppress the expression of the genes of the RecQ DNA helicase family.

In the above mentioned patent applications it is stated that compounds against RECQ1 can be used in the treatment of various tumors. However, RECQ1 is normally present also in the correspondent healthy tissue and so, although the compounds described in the above patents are defined as specific for tumor cells, data regarding the selectivity of the described compounds in respect of tumor cells are not so accurate as to make compounds described safe and free of hazards and side effects. In particular, the fact that in the above mentioned patent applications TIG3 mouse cells have been used as a model of normal cells is not sufficient to ensure safety use of these compounds in humans. In this regard, the WRN knock-out mouse showed no phenotype associated with the syndrome of WRN (while a phenotype similar to WRN syndrome was observed in mice only in the case of double knock-out of WRN and p53), emphasizing that there are some significant differences between the human and mouse model (Lebel M. and Leder, P. A deletion within murine WRN syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity. Proc Natl. Acad. Sci. USA 95, 13097-14012; Lombard, D. B. et al. Mutations in the WRN gene in mice accelerated mortality in a p53-null background. MoI. Cell Biol. 20, 3286-3291 ; Lebel, M. et al. Tumorigenicity effect of nonfunctional p53 or p21 in mice mutant in the Werner syndrome helicase. Cancer Res. 61 , 1816-1819).

In view of what is described in the state of the art, the person skilled in the art would not be confident that RECQ1 inhibitors, particularly as described in the above mentioned EP1816194 and EP1625853, have enough selectivity between tumoral and normal tissue surrounding said tumor to allow a treatment with limited or at least acceptable side effects.

Then it is still felt the need for compounds to be used in tumor therapy, which possess a selective toxicity to tumor cells, and do not damage healthy tissue surrounding the tumor.

This need is felt especially for those tumors with difficult treatment or highly aggressive.

In this regard, glioblastoma is the most common and aggressive histotype of brain tumor with a very poor prognosis (Mrugala, M. M. & Chamberlain, M. C. Mechanisms of disease: temozolomide and glioblastoma-look to the future. Nat Clin Pract Oncol 5, 476-86 (2008); Stummer, W. et al. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomized controlled multicentre phase III trial. Lancet Oncol 7, 392-401 (2006)). The average survival rate is only 10-14 months despite an interdisciplinary therapeutic effort that includes surgery, radiotherapy and chemotherapy (Sathornsumetee, S. & Rich, J.N. New treatment strategies for malignant gliomas. Expert Rev Antitumor Ther 6, 1087-104 (2006)).

Very complicated protocols have been developed to treat brain tumors, as for example, a method of two-stage perioperative radioimmunotherapy including an intraoperative locoregional stage in which an agent with tumoral tropism is introduced and a second post-operative systemic stage in which the dose of antitumor agent with affinity for the first agent is administered (U.S. Patent Application 2006-0251579 A1 ). Thus, new therapeutic protocols are highly needed.

Summary of the invention

It has now surprisingly been found that RECQ1 expression level increases dramatically in human brain glioblastoma relative to control brain tissues suggesting that this helicase would be an ideal target for new brain tumor therapies.

It is therefore an object of the present invention the use of antitumor agent specific for brain tumors, particularly glioblastoma, wherein said agent comprises at least one compound that suppresses RECQ1 gene expression.

Description of the invention

The present inventors found that only in the case of brain tumor, high expression of RECQ1 is coupled with an almost complete absence of that protein in perilesional and normal tissues. Therefore, RECQ1 is an ideal target for chemotherapy, especially in the case of brain tumors, because its depletion by RNAi, or its inhibition by selective compounds would affect only tumor cells, thereby preventing the possible negative effects on normal cells.

Object of the present invention is the use of a specific anticancer agent for brain tumors, particularly glioblastoma, where said agent comprises at least one compound that suppresses RECQ1 gene expression. Therefore object of the present invention is an agent comprising at least one compound that suppresses RECQ1 gene expression for use as an anticancer agent specific for brain tumors, in particular for glioblastoma.

In a first preferred embodiment of the invention said compound is a siRNA with RNA interference activity against RECQ1.

In a second preferred embodiment of the invention, said siRNA is a dsRNA comprising:

a. a sense RNA with a sequence homologous to an arbitrary sequence of 19-25 nucleotides of messenger RNA of RECQ1 gene and

b. an antisense RNA having complementary sequence to said sense RNA and optionally

c. having one or two nucleotide overhangs at both 3' ends.

In a further embodiment of the present invention said siRNA is formed after transcription from a plasmid or exogenous synthesis, such as in vitro enzymatic synthesis by using appropriate transcription enzymes.

In the embodiment of the present invention that provides the formation of siRNA from a plasmid, said siRNA may be a short hairpin RNA formed after transcription from a single promoter of this plasmid or may be a short dsRNA formed after transcription by two flanking convergent promoters on this plasmid.

In one embodiment of the present invention said siRNA can be generated after cleavage with nuclease from longer dsRNA.

In a further embodiment said siRNA is chemically synthesized.

Preferably, one of the two strands of said siRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 1-4:

δ'-GAGCUUAUGUUACCAGUUA-S' (SEQ ID NO: 1),

δ'-CUACGGCUUUGGAGAUAUA-S' (SEQ ID NO: 2),

δ'-GAUUAUAAGGCACUUGGUA-S' (SEQ ID NO: 3),

δ'-GGGCAAGCAAUGAAUAUGA-S' (SEQ ID NO: 4). Another object of the present invention is a method for treating a subject affected by a brain tumor, in particular glioblastoma, comprising administering to said subject a therapeutical amount of the above compound that suppresses RECQ1 gene expression, preferably in the form of a pharmaceutical composition.

The present invention will now be described in detail also by means of examples and figures.

Figures:

Figure 1. RECQ1 expression in human brain glioblastoma tissue. (A) lmmunostaining assay of tumoral and perilesional tissues performed using the anti-RECQ1 RQ-FL antibody and the Benchmark automatic device (Ventana Medical System, Tucson, AZ, USA). The nuclei positive for the protein are in black and the tissues were counterstained with hematoxylin (O. M. 40Ox). (B) RECQ1 expression in autoptic brain tissues, lmmunostaining assay was made manually using the anti-RECQ1 RQ-FL antibody. The nuclei positive for the protein are in black and the tissues were counterstained with hematoxylin (A: O. M. 20Ox, B: O. M. 40Ox). (C) Western blot analyses of proteins extracted from glioblastoma perilesional tissue (lines 2, 4, 6, 8), glioblastoma multiforme (lines 1 , 3, 5, 7, 10), and autoptic brain tissue (9, 1 1) were performed using the anti-RECQ1 RQ-FL antibody. Direct immunoblotting against α-tubulin was used as an internal control to guarantee that the amount of proteins loaded on each well was the same.

Figure 2. RECQ1 expression in tumors of different origins. Figure 2a: representative micrographies of immunohistochemical analysis on tissues from different types of tumors: multiform glioblastoma, lung tumor, thyroid tumor and colon carcinoma. On the left, the perilesional zone and, on the right, the tumoral one. lmmunostaining was made using the anti- RECQ1 RQ-FL antibody. The nuclei positive for the protein are in black and the tissues were counterstained with hematoxylin. (O. M 200X). Figure 2b: the histograms represent the percentage of nuclei positive for RECQ1 in perilesional and lesional tissues of the indicated tumors.

Figure 3. Proliferative capacity of RECQ1 -depleted glioblastoma cells. A) Clonogenic assays were performed in T98G, U-87, and IMR-90 cell lines where RECQ1 was depleted by siRNA treatment. Pictures show colonies formed after seeding 800 cells. B) Graphs showing the plating efficiencies expressed as colony forming capacity. Values represent the average ratio of the number of formed colonies to the number of cells seeded, expressed as percentage.

Figure 4. Proliferative capacity of T98G cells stably transfected with a shRNA against RECQ1. (A) Clonogenic assay performed in either control cells (left plate) and in clones silenced for RECQ1 (right plate). The figure shows colonies formed after plating 100, 200, 400, 800, 1600 and 3200 cells (well 1 to well 6, respectively). (B) Graph showing the plating efficiencies expressed as colony forming capacity. Values represent the average ratio of the number of formed colonies to the number of cells seeded, expressed as percentage.

Figure 5. Downregulation of RECQ1 triggers spontaneous γ-H2AX foci accumulation. A) Western blot analysis of T98G and U-87 cell lines transiently transfected with a RECQ1 -specific siRNA duplex. B) The pictures show the immunostaining of endogenous RECQ1 and endogenous γ-H2AX on T98G cells after treatment with anti-RECQ1 siRNA or control siRNAs. C) The graphs show the percentage of cells that contain a defined number of γ-H2AX foci per cell.

Figure 6. Downregualation of RECQ1 triggers spontaneous RAD51 foci accumulation. A) Western blot analysis of T98G and U-87 cell lines transiently transfected with a RECQ1 -specific siRNA duplex. B) The pictures show the immunostaining of endogenous RAD51 and BrdU incorporation on T98G cells after treatment with anti-RECQ1 siRNA or control siRNAs. C) The graphs show the percentage of cells that contain a defined number of RAD51 foci per cell.

Figure 7. RECQ1 downregulated glioblastoma cells are hypersensitive to TMZ treatment. The graphs show the dose dependent curves of T98G and U-87 cellular proliferation expressed as cellular surviving fraction at different doses of temozolomide in control and RECQ1 -depleted glioblastoma cells. Surviving fraction values are the mean ± SEM from three independent experiments.

Modes of realization of the invention

According to the present invention, the term "compound that suppresses the expression of RECQ1 gene" means a compound which, acting at different levels of gene expression, prevents the formation of RECQ1 protein.

According to the present invention, the expression "preventing the formation of RECQ1 protein" means that the protein expression is absent or reduced to a significant level from the clinical point of view.

As said above, high expression of RECQ1 in human brain glioblastoma is coupled with an almost complete absence of the same protein in perilesional and normal tissues. According to the present invention, with the expression "the almost complete absence" of RECQ1 in perilesional and normal tissues is intended a quantity not significantly detectable of the protein or a quantity such as, at therapeutic doses of the agent that suppresses RECQ1 gene expression, there are no negative effects towards the perilesional and normal tissues.

Therefore, the use of an anti-tumor agent specific for brain tumors, particularly for glioblastoma, wherein said agent comprises at least one compound that suppresses RECQ1 gene expression represent a solution to the problem of the specific treatment of brain tumors, particularly glioblastoma, without toxic and side effects to healthy tissue.

This aspect also satisfies that part of the general problem of the tumor therapy, which is still affected by toxicity events and weak tolerance by the patient.

In a particular embodiment of the present invention, the specific antitumor agent for brain tumors, particularly glioblastoma, comprises siRNAs having RNA interference activity against RECQ1.

The term "RNAi" or "RNA interference" refers to a sequence-specific post-transcriptional silencing phenomenon, mediated by "small interfering RNA" (small interfering RNA, siRNA)

(Fire et at., Nature, 391 , 806-11 (2001)) where the expression of the target gene is inhibited by inducing mRNA degradation of the target gene. This degradation is caused by the introduction in the cells of a double-stranded RNA (dsRNA) that includes a) a sense RNA having a sequence homologous to a sequence of messenger RNA of a target gene and b) an antisense RNA comprising a sequence complementary to sense RNA. While the exact mechanism of the

RNAi is still not entirely clear, it is thought that an enzyme called DICER (a nuclease RNase III family member) contacts the double-stranded RNA, and degrades it in small fragments called

"small interfering RNA" or "siRNA". The siRNAs derived from the activity of DICER are typically of about 21-23 nucleotides in length and include a duplex region comprising about 19 base pairs. The double stranded RNA of the present invention, including the effects of RNAi, preferably relates to these siRNAs.

The response of the RNAi is also characterized by an endonuclease complex containing a siRNA, commonly called RISC (RNA-induced silencing complex) that mediates the cutting of single-stranded RNA with a sequence complementary to the antisense strand of the siRNA duplex (Elbashir et al. Nature, 41 1 , 494-498 (2001).

The siRNA may be formed in vitro using synthetic oligonucleotides or appropriate transcription enzymes or in vivo using appropriate transcription enzymes or expression vectors. In the present invention, the antitumor agent specific for brain tumors and in particular for glioblastoma, is not limited to siRNAs but may include other agents, such as microRNAs with post-transcriptional silencing activity against RECQ1 , comprising a strand of RNA with a sequence partially complementary to an arbitrary sequence of 19-25 nucleotides of the mRNA of the RECQ1 gene.

The person skilled in the art can easily obtain information on the RECQ1 gene nucleotide sequences of the present invention by public gene database (eg GenBank).

RECQ1 genes of the present invention typically include, but are not limited to, those derived from animals, preferably those derived from mammals and more preferably those derived from humans.

The present invention relates to the use of double stranded RNA that are RNA (siRNA) comprising a sense RNA with a sequence homologous to an arbitrary sequence of 19-25 nucleotides of messenger RNA of the RECQ1 gene and an antisense RNA having sequence complementary to said sense RNA.

In general, since the double-stranded RNA with a 3' protruding of some nucleotides on a termination have strong effects of RNAi, the double stranded RNA used in the present invention preferably include a protruding of some nucleotides on a termination. The length of nucleotides forming the protruding as well as the sequence is not particularly limited. These protrudings may be DNA or RNA protruding ends. For example, the protruding comprises two nucleotides. Both strands of the siRNA may include a protruding on a termination, which length may be the same or different for each strand. Preferably, this protruding may be present on both strands of siRNA. The protrudings can also be stabilized against degradation, including purine nucleotides, or replacing pyrimidine nucleotides by modified analogues.

The RNA duplex portion of the siRNA can be part of a short hairpin RNA (shRNA). Thus, shRNA can also be used in the present invention. The hairpin structure can also contain protruding portions on 3'or 5'.

Thus, molecules that can form a double-stranded intramolecular RNA structure may be used in the present invention.

The RNA used to obtain RNAi should not be necessarily completely identical (homologous) to the helicase RECQ1 gene or to a portion of it, but it is preferable to use an RNA of this type. The siRNAs having RNAi activity against the RECQ1 genes used in the present invention can be suitably produced by the person skilled in the art starting from the nucleotide sequences of RECQ1. If one of the strand was determined, the nucleotide sequence of the other strand (the complementary strand) can be easily determined by the skilled person. The siRNAs used in present invention can be produced in a suitable way by the skilled person using commercially available nucleic acid synthesizers. Common customized synthesis services can also be used to synthesize the requested RNA. The siRNAs can be synthesized as two separate, complementary RNA molecules or as a single RNA molecule with two complementary regions.

The siRNAs used in the present invention can result from longer dsRNA that can be processed to shorter siRNAs, for example in the presence of the DICER enzyme.

A preferred embodiment of the present invention uses siRNAs in which one of the strands includes the nucleotide sequence of any one of SEQ ID NOs: 1-4:

δ'-GAGCUUAUGUUACCAGUUA-S' (SEQ ID NO: 1),

δ'-CUACGGCUUUGGAGAUAUA-S' (SEQ ID NO: 2),

δ'-GAUUAUAAGGCACUUGGUA-S' (SEQ ID NO: 3),

δ'-GGGCAAGCAAUGAAUAUGA-S' (SEQ ID NO: 4).

It is also included in the present invention the use of DNA allowing the expression of siRNAs used here. Specifically, the present invention relates to DNA (plasmids or vectors) that allow the expression of double stranded RNA used in the present invention. In one embodiment the plasmid is designed to include a coding sequence for the sense strand and one for the antisense strand of the double stranded RNA. The coding sequences may be the same sequence, e.g. flanked by reversed promoters (see patent application WO01 /77350), or may be two separate sequences each under transcriptional control of separate promoters. After the coding sequence is transcribed, the complementary RNA transcripts match to form a dsRNA.

The person skilled in the art can easily prepare a DNA as described above by using conventional techniques of genetic engineering. More specifically, the expression vectors used in the present invention can be prepared by properly inserting the encoding DNA and the RNA used in the present invention in various known expression vectors.

Said DNA is introduced into host cells by transfection with any procedure useful for entry into a particular cell, for example biological or physical methods, able to produce a cell having the

Il recombinant DNA stably integrated into its genome or existing as episomal element, so that DNA molecules used in the present invention are expressed by the cell host. Physical methods to introduce a double-stranded DNA or RNA in a host cell include but are not limited to precipitation with calcium phosphate, lipofection, DEAE-dextran, particle bombardment, microinjection, electroporation, immunoliposomes, lipids, cationic lipids, phospholipids or liposomes or similar.

The skilled person will understand that any method can be used to transport DNA or RNA duplex.

Viral vectors such as retrovirus, adenovirus, and Sendai virus and non-viral vectors as liposomes can be used to administer the DNAs expressing the siRNAs used in the present invention in living subjects to be treated. The administration includes for example, in-vivo and ex-vivo methods. Said subjects to be treated are preferably human, but are not particularly limited to, and they can also be animals.

The present invention provides a pharmaceutical composition comprising as active ingredient a compound that suppresses RECQ1 gene expression in an amount sufficient to downregulate the expression of said gene.

The pharmaceutical agents of the present invention can be provided as a mixture with a pharmaceutically acceptable carrier. These pharmaceutically acceptable carriers may include but are not limited to, for example, detergents, excipients, colorings, flavorings, preservatives, stabilizers, buffers, suspensions, isotonizer, binders, disintegrating, lubricants, fluidifying and corrective. Other conventional vehicles can also be used appropriately.

The preparation of pharmaceutical compositions of the present invention falls within the general knowledge of the expert in the field and do not require particular description. Examples of general knowledge are contained in Remington's Pharmaceutical Sciences, Mack Pub., latest edition.

In the specific case of siRNA, it is possible to refer to literature cited as well, for example, to WO 2005/097207 and WO 2006/039253.

In general, administration to individuals can be done through methods known in the art.

The pharmaceutical agent or pharmaceutical composition doses disclosed in the present invention can be determined properly by a physician considering the type of dosage form, method of administration, the age of the patient, weight, the symptoms etc. Even this activity falls within the normal activities of person skilled in the art, for example following the guidelines of the regulatory authorities for drug development, such as the FDA or the EMEA.

The following example further illustrates the invention.

Example

Materials and Methods:

Antibodies

The rabbit polyclonal antibody against the C-terminal region of RECQ1 (RQ-CT) was prepared by Sigma Genosys using a 16 amino acid peptide corresponding to residues 634-649 of RECQ1 (C-SGSKNTGAKKRKIDDA) with an N-terminal cysteine conjugated to a carrier protein called the keyhole limpet hemocyanin (KLH). The rabbit polyclonal antibody against the full- length RECQ1 helicase (RQ-FL) was made in the laboratory of the inventor by injecting the rabbits with full-length protein expressed and purified from insect cells according to the previously described protocol (Cui, S. et al. Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A. Nucleic Acids Res 32, 2158-70 (2004)). Antibodies against RECQ1 (sc-25547), and RAD51 (sc-8349) were purchased from Santa Cruz Biotechnology. Anti-γ-H2AX pSer139 (05-636), anti-BrdU (347580) and anti-α- Tubulin (T6074) antibodies were purchased from Upstate, BD Bioscience and Sigma, respectively. The secondary antibodies conjugated with Alexa 488 and Alexa 59 fluorophores and Toto-3 iodide were obtained from Invitrogen Molecular Probes.

Cells lines

Glioblastoma human (T98G (ATCC, CRL-1690) and U-87 (ATCC, HTB-14)) and normal human fibroblast (IMR-90) (ATCC, CCL-186)) cell lines were maintained in Dulbecco's modified Eagle's Medium (DMEM) and MEM media supplemented with Glutamax (Life Tecnologies, Inc.) and 10% (v/v) fetal bovine serum (FBS; Life Technologies, Inc.), respectively.

Tissues

The tumoral and perilesional brain tissues were provided by the Neurosurgery Unit of the "Azienda Ospedaliero-Universitaria" of Udine, Italy, while the tumoral and perilesional tissues of colon, lung, thyroid and heart tissue were provided by the ACADEM Department of the Cattinara hospital of Trieste, Italy. The tissues were obtained by surgical resection and they were fixed in formalin and paraffin embedded. In the case of the brain tumors, a portion of the biopsy tissue was frozen at -80 ⁰C for the subsequent western blot analysis. The tumor diagnosis was confirmed in each case by histopathological analysis.

Western analysis

Protein extracts from brain tissue were prepared using a buffer TNEN (5OmM Tris pH 7.5, 150 mM NaCI, 2 mM EDTA, 0.2% triton X-100 and 0.3% NP-40) following the previously described procedure (Odreman, F. et al. Proteomic studies on low- and high-grade human brain astrocytomas. J Proteome Res 4, 698-708 (2005)). T98G, U-87 and IMR-90 whole cell extracts were prepared using a HNNG buffer (15 mM Hepes pH 7.5, 250 mM NaCI, 1 % (v/v) NP-40, 10% (v/v) glycerol, 1 mM PMSF) supplemented with 0.2 mM sodium orthovandate (Sigma), 10 mM sodium glycerol-2-phosphate (Sigma), 25 mM NaF (Sigma) and protease inhibitors cocktail of Roche, lmmunoblots analyses were carried out with 2 to 10 μg of whole-cell extract. Proteins were separated on SDS-PAGE containing 12% Acrylamide gel and detected by immunoblotting using the SuperSignal West Femto Maximum Sensitivity Substrate of Pierce.

lmmunohistochemistry

For the immunohistochemistry analysis, 4 μm-thick tissue sections were cut with the microtome from formalin-fixed, paraffin-embedded samples representative of the following tumors: glioblastoma, colon tumors, thyroid tumor and lung tumor. The sections were deposited on SuperFrost® Plus slides and incubated at least for 12 hours at 37°C. The immunostaining procedures were performed automatically using the Benchmark device (Ventana Medical System, Tucson, AZ, USA) and manually, using heat-induced epitope retrieval method. The detection of RECQ1 was performed using the polyclonal antibody produced in rabbit which recognizes the full protein sequence (RQ-FL), and the antibody directed against the last 16 residues at the C-terminus (RQ-CT); the antibodies, at a 1 :150 dilution, were detected using chromogen diaminobenzidine (DAB). For the manual detection, the incubations were performed in a humidified chamber. Briefly, tissue sections, were deparaffinized, immersed in xylene for 30 min, and then hydrated in a decreasing alcohol series. Endogenous peroxidase activity was blocked by incubating the tissue sections in 0.3% H2O2 for 20 min. The sections were subject to heat-induced epitope retrieval, immersing the slides in boiling 10 mM citrate buffer, pH 6.0 for 20 min. The sections were incubated for 20 minutes with blocking serum (Vectastain Universal Elite ABC kit, Vector Laboratories, Burlingame, CA, USA) and for 60 minutes with the primary antibody directed against RECQ1 , 1 :150 diluted. The sections not incubated with the primary antibody were used as negative controls. The slides were washed for some times with PBS and 0.1 % of Triton X-100, and incubated for 60 minutes with the biotinylated secondary antibody and with Vectastain ABC system for 30 min (Vectastain Universal Elite ABC kit, Vector Laboratories, Burlingame, CA, USA); the detection was made using a solution containing DAB and H2O2 (DAB substrate kit, Vector Laboratories, Burlingame, CA, USA). The sections were counterstained with Mayer hematoxylin. Preadsorption of the antibody, using an excess of specific peptide (1 :5 at 37°C for 30 min) was performed to verify the antibody specificity.

RNA interference

In order to decrease RECQ1 expression level in the glioblastoma cell line (T98G), cells were transiently transfected with a mix of 4 siRNAs against RECQ1 (ON-TARGETplus, human RECQ1 , NM_032941 , DHARMACON) (target sequences (target): 5'- GAGCUUAUGUUACCAGUUA-3' (SEQ ID NO: 1 ), δ'-CUACGGCUUUGGAGAUAUA-S' (SEQ ID NO: 2), δ'-GAUUAUAAGGCACUUGGUA-S' (SEQ ID NO: 3), 5'- GGGCAAGCAAUGAAUAUGA-3' (SEQ ID NO: 4)) for 72 hours at 100 nM final concentration using the Hyperfect transfection system of QIAGEN and following QIAGEN guide instructions. Control experiments were performed using a siRNA against Luciferase of Dharmacon. Alternatively, an efficient reduction of the expression level of RECQ1 in the T98G cells was also obtained by transfection with a pcDNASup plasmid (hybrid plasmid obtained by combination of pcDNA3) (Invitrogen, life technologies) and pSUPER (OligoEngine) ) encoding a shRNA (short hairpin RNA) against RECQ1 mRNA (target sequence (target): 5'- GAGCUUAUGUUACCAGUUA-3' (SEQ ID NO: 1)). The T98G cell clones downregulated for RECQ1 were obtained by selection with Geneticin G418 antibiotic (0.5 mg/L) (GIBCO BRL).

Clonogenic assays

Clonogenic assays were conducted in vitro as already described (Franken, N.A., Rodermond, H. M., Stap, J., Haveman, J. & van Bree, C. Clonogenic assay of cells in vitro. Nat Protoc 1 , 2315-9 (2006)). Briefly, the assays were performed in six-well plates, with control clones produced by T98G cells transfection with empty vector or clones obtained from T98G cells transfected with the same vector containing RECQ1-shRNA. Cells were seeded at different dilutions (100, 200, 400, 800, 1600 and 3200 cells per well) in six-well plates, and the colonies formed after at least one week of growth were counted using VersaDoc 4000 imaging system (BioRad). Proliferative capacity of control T98G cells and of the RECQ1 -depleted cells was evaluated by their plating efficiency (PE) and expressed as colony forming capacity. The PE was calculated as the average ratio of the number of formed colonies versus the number of cells seeded, expressed as percentage. Cell survival assays were performed by plating transfected cells before treatment with temozolomide (TMZ: Sigma). Cells were treated overnight with different doses of TMZ (5, 50, 250 and 500 μM). Surviving fractions were calculated following a procedure already published (Franken, N.A., Rodermond, H. M., Stap, J., Haveman, J. & van Bree, C. Clonogenic assay of cells in vitro. Nat Protoc 1 , 2315-9 (2006)).

Immunofluorescence

For immunofluorescence assays, cells were seeded in special slides provided with single chamber (NALGENE), after transient downregulation of the level of RECQ1 expression by RNA interference for 24 hours. A second transfection with the siRNA was repeated immediately after the cells were seeded in the chamber slides. The cells were left under these conditions for 72 hours. The medium was then removed and the chamber slides were washed, fixed and immunostained by a set of specific antibodies following a protocol already described (Marcello, A. et al. Recruitment of human cyclin T1 to nuclear bodies through direct interaction with the PML protein. Embo J 22, 2156-66 (2003)). The primary antibodies used for the immunofluorescence analysis are described in the first "Material and Methods" paragraph. BrdU staining was performed after incubation of cells in 10 μM BrdU (Sigma) for 60 minutes and DNA denaturation by 2N HCI (Merck). The slides were then incubated with the secondary Alexa 488 and Alexa 594 fluorophores-conjugated antibodies (Invitrogen, Molecular Probes).

Immunofluorescence analysis was performed using a Zeiss LSM 510 Meta confocal microscope. Images were then acquired using the LSM software. The cells containing γ-H2AX and RAD51 foci were counted in at least 100 nuclei.

Results

Our immunohistochemical analysis of human brain gliomas tissues showed that RECQ1 was overexpressed in the tumor samples relative to the perilesional tissues (Figure 1A). The RECQ1 expression was confined in the nuclei and more than 90 % of cells in each tumor sample positively stained for RECQ1 using either the antibody raised against the C-terminus (RQ-CT) or the antibody against the full-length protein (RQ-FL). Conversely, only small percentage of the cells in each sample positively stained for RECQ1 in perilesional tissues and in autoptic normal brain tissues (Figure 1 B). The specificities of the two anti-RECQ1 antibodies were confirmed by pre-adsorption experiments showing that after pre-adsorption of the tissue with the recombinant RECQ1 protein only a minimal non-specific stain could be detected. The higher expression of RECQ1 in brain gliomas was then confirmed by Western blot analysis showing that the RECQ1 protein is at least 10-fold higher in the tumoral samples relative to the perilesional or normal brain samples, where RECQ1 is almost completely absent (Figure 1 C).

The same analysis was then repeated on tissues from colon carcinoma, lung and thyroid tumors (Figure 2). The result clearly showed that RECQ1 was highly abundant in all these tumors of different type. However, RECQ1 seemed to be highly expressed also in the perilesional regions of these tumoral tissues.

To test the role of RECQ1 in glioblastoma cell growth and proliferation, the colony forming capacity of two different glioblastoma cell lines (T98G, U-87), and normal human fibroblasts IMR-90 transiently transfected with a mix of 4 siRNAs against RECQ1 was compared to that of cells transfected with a luciferase siRNA duplex, as control (Figure 3). More than 80% depletion of RECQ1 was observed in all whole cell extracts upon treatment with the RECQ1 -specific siRNA duplex as compared to the control cells. Our colony forming assays demonstrated a significant reduction in both the size and number of colonies of T98G and U-87 RECQ1 depleted glioblastoma cells (Figure 3A).

In particular, the transiently downregulated RECQ1 T98G glioblastoma cells showed a reduction in their proliferative capacity in comparison with the control cells (Figure 3). However, the downregulation of RECQ1 in normal human primary fibroblasts did not significantly affect the proliferation capacity of these cells in agreement with previous findings (Futami, 2008). The same experiments were then repeated on glioblastoma cell line (T98G) stably transfected with plasmid codifying RECQ1 -specific shRNAs compared to T98G cell line transfected with a control plasmid (Figure 4). Analysis of selected G418 resistant clones that carry plasmid codifying RECQ1 shRNA compared to the control cells enabled us to assess the effect of stable

RECQ1 depletion on the colony forming ability of RECQ1 -depleted T98G cells in vitro. Even in this case, colony forming assays demonstrated a significant reduction in both size and number of colonies when T98G cells were stably downregulated for RECQ1 (Figure 4B). In particular, RECQ1 -depleted glioblastoma cells showed -10 fold reduction (5.3%) in their proliferative capacity in comparison with samples of control cells (59.4%). Thus, our data suggest a regulatory role of human RECQ1 in active proliferation in glioblastoma cells.

RecQ helicases are involved in the stabilization and repair of damaged DNA replication forks in response to endogenous stress or exogenous DNA damages. A failure to stabilize forks can lead to fork collapse and double strand breaks. Consistently, previous studies with RECQ1 deficient HeLa cells showed an increased level of DNA damages and sister chromatid exchanges (Sharma, S. et al. RECQL, a member of the RecQ family of DNA helicases, suppresses chromosomal instability. MoI Cell Biol 27, 1784-94 (2007)). To examine the role of RECQ1 in preventing and responding to DNA damages in glioblastoma cells, the extent of spontaneous γ-H2AX foci formation in control or RECQ1 siRNA transfected T98G and U-87 cells was analyzed (Figure 5). The present results indicate that RECQ1 depletion results in a dramatic increase in spontaneous γ-H2AX foci formation indicating that the reduced expression of RECQ1 is associated with defects in DNA repair. Approximately 90% of the RECQ1 depleted cells contained more than ten γ-H2AX foci per nucleus, compared to only 15% for the wild-type cells. Previous studies with other human RecQ enzymes suggested that these enzymes play an important role in homologous recombination (HR) repair at sites of chromosomal DNA damage. To investigate if also RECQ1 might have a role in this pathway, we analyzed the ability of RAD51 , a major protein involved in the strand invasion step of HR repair, to form foci in T98G control and RECQ1 depleted cells. As shown in figure 6, the RECQ1 depleted cells exhibited an increased number of spontaneous RAD51 foci relative to the control cells. However, the increase in the number of RAD51 foci is less pronounced compared with the γ-H2AX foci suggesting the double-strand breaks might not be the major form of damage that leads to γ- H2AX foci formation in the absence of RECQ1.

To explore the possibility that RECQ1 might represent a suitable new target for brain tumor treatment, the sensitivity of glioblastoma cells to temozolomide (TMZ), which is a commonly used anticancer agent for the treatment of human brain tumors (Friedman, H. S., Kerby, T. & Calvert, H. Temozolomide and treatment of malignant glioma. Clin Cancer Res 6, 2585-97 (2000); Yung, W.K. et al. Multicenter phase Il trial of temozolomide in patients with anaplastic astrocytoma or anaplastic oligoastrocytoma at first relapse. Temodal Brain Tumor Group. J Clin Oncol 17, 2762-71 (1999)), was investigated. TMZ is an alkylating agent that effectively inhibits glioblastoma cell proliferation. Its toxicity is primarily due to formation of O6-methylguanine in DNA, which mispairs with thymine during DNA replication cycles and after accumulation of unrepaired DNA mismatches results in cell death (D'Atri, S. et al. Involvement of the mismatch repair system in temozolomide-induced apoptosis. MoI Pharmacol 54, 334-41 (1998); Denny, BJ. , Wheelhouse, R. T., Stevens, M. F., Tsang, L. L. & Slack, J.A. NMR and molecular modeling investigation of the mechanism of activation of the antitumor drug temozolomide and its interaction with DNA. Biochemistry 33, 9045-51 (1994)). Thus, the colony forming capacity of RECQ1 -depleted glioblastoma cell lines after treatment with TMZ was analysed (Figure 7). Cellular survival curves using increasing TMZ concentrations showed that RECQ1 depleted T98G and U87 cell lines were hypersensitive to the action of TMZ suggesting a possible role of RECQ1 in DNA repair pathways linked to DNA replication.

The resistance of glioma cells to TMZ is mainly associated with levels of the DNA repair protein O⁶-alkylguanine alkyltransferase (AGT), which removes alkyl groups at the O⁶ position of guanine. In fact, 06-benzylguanine (06-BG), an inhibitor for AGT, reduces resistance to TMZ (Wedge, S. R., Porteous, J. K. & Newlands, E. S. 3-aminobenzamide and/or 06-benzylguanine evaluated as an adjuvant to temozolomide or BCNU treatment in cell lines of variable mismatch repair status and O6-alkylguanine-DNA alkyltransferase activity. Br J Cancer 74, 1030-6 (1996)). Moreover, it has also been demonstrated that chemosensitivity of tumour cells to TMZ correlates with the inhibition of telomerase activity (Kanzawa, T. et al. Inhibition of telomerase activity in malignant glioma cells correlates with their sensitivity to temozolomide. Br J Cancer 89, 922-9 (2003)). In the present study using malignant glioma cell lines with low levels of AGT (U87-MG) and high levels of AGT (T98G), it has been found that RECQ1 suppression by RNA interference increased sensitivity to TMZ in these cells independently of AGT expression levels.

These data suggest that RECQ1 is a suitable new target for the inhibition of cell proliferation in brain tumors and support the notion that RECQ1 plays a unique role in DNA repair during DNA replication in malignant cells.

Potential Application

The present invention finds its application in the medical field, in the treatment of brain tumors, in particular of glioblastoma.

Since it was discovered that the high expression of RECQ1 in human brain glioblastoma is coupled with an almost complete absence of the same protein in perilesional and normal tissues, compounds that suppress RECQ1 gene expression can be used in the treatment of brain tumors, particularly of glioblastoma, given that there is not toxic side effects on the normal tissue.

In this respect, the present invention demonstrates that the use of compounds that suppress RECQ1 gene expression compromise significantly the proliferative ability of glioblastoma cells.

Claims

1. Use of an anticancer agent specific for brain tumors, in particular for glioblastoma, wherein said agent comprises at least one compound which suppresses RECQ1 gene expression.

2. The use according to claim 1 , wherein said compound is a siRNA having an RNA interference activity against RECQ1.

3. The use of the anticancer agent according to claim 2, wherein said siRNA is a dsRNA comprising: a. a sense RNA having a sequence homologous to an arbitrary sequence of 19-25 nucleotides of messenger RNA of RECQ1 gene and b. an antisense RNA having the sequence complementary to said sense RNA and optionally c. having one or two nucleotide overhangs at the two 3' ends.

4. The use of the anticancer agent according to claim 2 or 3, wherein said siRNA is formed after transcription from a plasmid or exogenous synthesis.

5. The use of the anticancer agent according to claim 4, wherein said siRNA is a short hairpin RNA formed after said transcription from a single promoter of said plasmid.

6. The use of the anticancer agent according to claim 4, wherein said siRNA is a short dsRNA formed after said transcription from two flanking convergent promoters on said plasmid.

7. The use of the anticancer agent according to any one of claims 2 to 6 wherein said siRNA is generated after cleavage with nucleases from longer dsRNA.

8. The use of the anticancer agent according to claim 2 or 3 wherein said siRNA is chemically synthesized.

9. The use of the anticancer agent according to any one of claims 2 to 8, wherein one of the two strands of said siRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 1 to 4: δ'-GAGCUUAUGUUACCAGUUA-S' (SEQ ID NO: 1), δ'-CUACGGCUUUGGAGAUAUA-S' (SEQ ID NO: 2), δ'-GAUUAUAAGGCACUUGGUA-S' (SEQ ID NO: 3), 5'- GGGCAAGCAAUGAAUAUGA-3' (SEQ ID NO: 4).