WO2014187856A1

WO2014187856A1 - Nucleoli disorganisation by knocking down specific alu-repeat containing rna sequences

Info

Publication number: WO2014187856A1
Application number: PCT/EP2014/060435
Authority: WO
Inventors: Maiwen CAUDRON-HERGER; Teresa PANKERT; Karsten Rippe
Original assignee: Deutsches Krebsforschungszentrum
Priority date: 2013-05-21
Filing date: 2014-05-21
Publication date: 2014-11-27

Abstract

The present invention relates to an inhibitor of nucleolus organisation, wherein said inhibitor of nucleolus organisation is a polynucleotide. Moreover, the present invention relates to a vector and to a host cell comprising said inhibitor of nucleolus organisation. The present invention further relates to the inhibitor of nucleolus organisation for use in medicine and for use in treating cancer, as well as to a device and a kit comprising the inhibitor of nucleolus organisation. Also, the present invention relates to a method of inhibiting cancer cell proliferation, comprising contacting a cancer cell with an effective amount of inhibitor of nucleolus organisation.

Description

Nucleoli disorganisation by knocking down specific Alu-repeat containing RNA sequences

Alu elements are the most abundant transposable elements in primate genomes, having similarity to the 7SL RNA. From these Alu elements, non-coding aluRNAs are generally transcribed by RNA polymerase III, which are suspected to have the function of binding to RNA polymerase II and preventing the formation of pre-initiation complexes under environmental stress. Alternatively, aluRNAs can be found in introns of genes and transcribed by RNA polymerase II as part of large pre-mRNAs (Berger et al. "Multiple Roles of Alu- Related Noncoding RNAs". Book chapter. Long Non-Coding RNAs, Progress in Molecular and Subcellular Biology 51, DOI 10.1007/978-3-642- 16502-3_6. Springer- Verlag, 2011). In humans, three major families of Alu elements have been identified, based on sequence similarity, namely the AluS, AluY, and AluJ families.

The nucleolus is a substructure within the nucleus of eukaryotic cells comprising specific nucleic acids and proteins. It has been assigned the functions of ribosomal RNA (rRNA) transcription and of sensing and integrating cellular stress signals (reviewed in Burger et al. (2013), "Functional ribosome biogenesis is a prerequisite for p53 destabilisation: impact of chemotherapy on nucleolar functions and RNA metabolism", Biol Chem, epub ahead of print, PMID: 23640940). From this, it has been deduced that assembly of functional nucleoli is vital for the cell. In corroboration, disruption of nucleolar integrity, e.g. by compounds inhibiting ribosome biosynthesis, has been shown to induce p53 activation and inhibition of cancer cell proliferation (ibd; Rubbi & Milner (2003), EMBO J 22(22):6068; Bywater et al. (2012), Cancer Cell 22:51 ; Boulon et al. (2010), Mol Cell 40:216). It has, therefore, been proposed that inhibition of ribosome biogenesis might be a promising approach for cancer therapy. Research, however, so far has focused on protein targets and the use of small molecule inhibitors (Burger et al., ibd).

Despite the fact that anti-cancer drugs targeting ribosome biogenesis are available, compounds directly targeting the structural integrity of nucleoli are still lacking. There is, thus, a need in the art for means and methods to comply with the aforementioned needs. The technical problem underlying the invention can be seen as the provision of means and methods which allow for inhibiting nucleolar organisation and for treatment of diseases susceptible to inhibition of nucleolar organisation. The technical problem is solved by the embodiments characterized in the claims and herein below.

Accordingly, the present invention relates to an inhibitor of nucleolus organisation, wherein said inhibitor of nucleolus organisation is a polynucleotide.

The term "nucleolus organisation", as used herein, relates to the process of assembling and/or maintaining structural integrity of nucleoli in the nucleus of a cell. Preferably, the term relates to the process of assembling and/or maintaining structural integrity of functional nucleoli in the nucleus of a cell. Methods of assessing structural integrity and functionality of nucleoli are detailed herein in the Examples.

According to this specification, the term "inhibitor" relates to a compound reducing the rate at which a specific process (the inhibited process) occurs or which prevents said process from progressing or from occurring. Thus, an "inhibitor of nucleolus organisation" is a compound reducing the rate at which nucleoli are organized by the cell, or, preferably, preventing nucleolus organisation from progressing or from occurring. More preferably, the inhibitor of nucleolar organisation causes the cell to assemble structurally abnormal and dysfunctional nucleoli. Preferably, the inhibitor of nucleolus organisation inhibits nucleolus organisation by at least 25%, more preferably by at least 50%, still more preferably by at least 75%, or, most preferably, by at least 90%. Preferably, the inhibitor of nucleolus organisation is specific, i.e. specifically has the effect of inhibiting nucleolus organisation, more preferably without modulating cellular processes other than the ones described in the present specification to a detectable extent. Preferably, the inhibitor of nucleolus organisation inhibits nucleolus organisation when brought into contact with a cell. More preferably, the inhibitor of nucleolus organisation inhibits nucleolus organisation when provided in the medium surrounding a cell.

Preferably, the inhibitor of nucleolar organisation is a compound inhibiting expression and/or causing degradation of polynucleotides comprising at least 12 contiguous nucleotides of at least one of SEQ ID NO: 1 to 10. More preferably, the inhibitor of nucleolar organisation is a compound inhibiting expression and/or causing degradation of polynucleotides comprising at least 15 or at least 18 contiguous nucleotides of at least one of SEQ ID NO: 1 to 10. Even more preferably, the inhibitor of nucleolar organisation is a compound inhibiting expression and/or causing degradation of polynucleotides comprising at least 20 contiguous nucleotides of at least one of SEQ ID NO: 1 to 10. Most preferably, the inhibitor of nucleolar organisation is a compound inhibiting expression and/or causing degradation of aluRNAs.

In a preferred embodiment, the inhibitor of nucleolar organisation is a compound inhibiting expression and/or causing degradation of polynucleotides comprising at least 12 contiguous nucleotides of at least one of SEQ ID NO: 103 to 105. In a more preferred embodiment, the inhibitor of nucleolar organisation is a compound inhibiting expression and/or causing degradation of polynucleotides comprising at least 15 or at least 18 contiguous nucleotides of at least one of SEQ ID NO: 103 to 105. In an even more preferred embodiment, the inhibitor of nucleolar organisation is a compound inhibiting expression and/or causing degradation of polynucleotides comprising at least 20 contiguous nucleotides of at least one of SEQ ID NO: 103 to 105.

The term "conserved", as used herein, relates to the preservation of nucleic acid sequences. Conserved sequences are nucleic acid sequences preserved in at least two variants of a sequence. The degree of conservation is typically assessed by aligning two or more sequences, preferably using algorithms known in the art (e.g. MultAlin (F. Corpet (1988), "Multiple sequence alignment with hierarchical clustering"; Nucl. Acids Res., 16 (22): 10881- 10890; or Clustal Omega (Goujon et al. ((2010), "A new bioinformatics analysis tools framework at EMBL-EBI"; Nucl. Acids Res. 38 Suppl: W695-9) and is characterized by at least two parameters: (i) The first parameter characterizing the degree of conservation is the degree of conservation between a given number of sequence variants, relating to the fraction of sequences in which the respective nucleotide is conserved. It is understood by the skilled person that when a large number of sequence variants is aligned, most nucleotide positions will only be conserved within a fraction of said sequence variants. Thus, the term "conserved between x sequences" relates to the fraction of sequence variants within which the nucleotide is conserved. Thus, e.g. a nucleotide conserved in 90% of sequences is a nucleotide identical in 9 out of 10 sequence variants, (ii) The second parameter characterizing the degree of conservation is the degree of sequence conservation, i.e. the number or fraction of nucleotides preserved within a specific stretch of nucleic acid sequence. As used herein, the term "n% conserved" relates to a degree of conservation where n nucleotides out of 100 are present in the specified number of sequence variants analyzed. Thus, e.g., the term "70% conserved" relates to a degree of conservation wherein 7 nucleotides out of 10 are identical in the sequences aligned. In consequence, the term "70% conserved between at least 90% of Alu sequences" relates to a sequence wherein 7 nucleotides out of 10 are identical within at least 9 out of 10 sequence variants aligned. According to the present invention, nucleotide insertions and deletions, leading to gaps or extensions in alignments, respectively, are not taken into account while assessing sequence conservation.

According to the present invention, the inhibitor of nucleolar organisation is a polynucleotide. The term "polynucleotide", as used herein, relates to a polynucleotide comprising a nucleic acid sequence having the biological activity of inhibiting nucleolus organisation as specified herein above. Preferably, the polynucleotide is a polynucleotide comprising or having a nucleotide sequence corresponding to the reverse complement of a stretch of 10 nucleotides at least 70% conserved between at least 90% of Alu sequences of the AluS family of sequences, of a stretch of 10 nucleotides at least 70% conserved between at least 90% of Alu sequences of the AluY family of sequences, or of a stretch of 10 nucleotides at least 70% conserved between at least 90% of Alu sequences of the AluJ family of sequences. More preferably, the polynucleotide is a polynucleotide comprising or having a nucleotide sequence corresponding to the reverse complement of a stretch of 10 nucleotides at least 80% conserved between at least 90% of Alu sequences of the AluS family of sequences, of a stretch of 10 nucleotides at least 80% conserved between at least 90% of Alu sequences of the AluY family of sequences, or of a stretch of 10 nucleotides at least 80% conserved between at least 90% of Alu sequences of the AluJ family of sequences. Even more preferably, the polynucleotide is a polynucleotide comprising or having a nucleotide sequence corresponding to the reverse complement of a stretch of 10 nucleotides at least 80% conserved between at least 95% of Alu sequences of the AluS family of sequences, of a stretch of 10 nucleotides at least 80% conserved between at least 95% of Alu sequences of the AluY family of sequences, or of a stretch of 10 nucleotides at least 80% conserved between at least 95% of Alu sequences of the AluJ family of sequences. In a preferred embodiment, the polynucleotide is a polynucleotide comprising or having a nucleotide sequence corresponding to the reverse complement of at least one of the aforesaid polynucleotides, i.e. preferably, of the respective sequence in the original, more preferably, sense, orientation.

In a more preferred embodiment, the inhibitor of nucleolus organisation comprises or consists of a reverse complement of a nucleotide sequence corresponding to a stretch of 10 nucleotides at least 70% conserved between at least 90% of Alu sequences of the AluS family of sequences and/or a nucleotide sequence being at least 70% identical thereto, wherein the members of the AluS family are the Alu sequences located on the human Chromosome 1 at nucleotide positions 39624-39924, 169374-169679, 101056-101352, 247373-247669, 144606-144899, 174518-174820, 101822-102122, 167495-167792, 76893-77201, 129999- 130313, 102976-103280, 249293-249604, 175915-176246, 111081-111386, 149396-149703, 163848-164153, 80805-81096, 165007-165310, 164359-164695, and 168485-168786; or the inhibitor of nucleolus organisation comprises or consists of a reverse complement of a nucleotide sequence corresponding to a stretch of 10 nucleotides at least 70% conserved between at least 90% of Alu sequences of the AluY family of sequences and/or a nucleotide sequence being at least 70% identical thereto, wherein the members of the AluY family are the Alu sequences located on the human Chromosome 1 at nucleotide positions 51585-51880, 90921-91213, 229525-229825, 237249-237544, 341759-342052, 413868-414187, 526833- 527143, 539503-539801, 863492-863792, 944780-945081, 1069183-1069489, 1125213- 1125510, 1128425-1128725, 1191981-1192291, 1212929-1213223, 1215563-1215862, 1324471-1324759, 1325059-1325348, 1338227-1338535, and 1346612-1346918; or the inhibitor of nucleolus organisation comprises or consists of a reverse complement of a nucleotide sequence corresponding to a stretch of 10 nucleotides at least 70% conserved between at least 90% of Alu sequences of the AluJ family of sequences and/or a nucleotide sequence being at least 70% identical thereto, wherein the members of the AluJ family are the Alu sequences located on the human Chromosome 1 at nucleotide positions 31436-31733, 101404-101690, 124568-124870, 140495-140784, 141668-141970, 144235-144525, 144987- 145294, 146229-146522, 146776-147047, 147626-147917, 148792-149093, 152276-152566, 153430-153735, 247721-248006, 319131-319439, 322409-322708, 458553-458841, 582950- 583233, 676219-676503, and 677387-677689. In a preferred embodiment, the polynucleotide is a polynucleotide comprising or having a nucleotide sequence corresponding to the reverse complement of at least one of the aforesaid polynucleotides, i.e. preferably, of the respective sequence in the original, more preferably, sense, orientation.

In an also preferred embodiment, the inhibitor of nucleolus organisation comprises or consists of a reverse complement of a nucleotide sequence corresponding to a stretch of 10 nucleotides at least 80% conserved between at least 90% of Alu sequences of the AluS family of sequences and/or a nucleotide sequence being at least 70% identical thereto, wherein the members of the AluS family are the Alu sequences located on the human Chromosome 1 at nucleotide positions 39624-39924, 169374-169679, 101056-101352, 247373-247669, 144606-144899, 174518-174820, 101822-102122, 167495-167792, 76893-77201, 129999- 130313, 102976-103280, 249293-249604, 175915-176246, 111081-111386, 149396-149703, 163848-164153, 80805-81096, 165007-165310, 164359-164695, and 168485-168786; or the inhibitor of nucleolus organisation comprises or consists of a reverse complement of a nucleotide sequence corresponding to a stretch of 10 nucleotides at least 80% conserved between at least 90% of Alu sequences of the AluY family of sequences and/or a nucleotide sequence being at least 70% identical thereto, wherein the members of the AluY family are the Alu sequences located on the human Chromosome 1 at nucleotide positions 51585-51880, 90921-91213, 229525-229825, 237249-237544, 341759-342052, 413868-414187, 526833- 527143, 539503-539801, 863492-863792, 944780-945081, 1069183-1069489, 1125213- 1125510, 1128425-1128725, 1191981-1192291, 1212929-1213223, 1215563-1215862, 1324471-1324759, 1325059-1325348, 1338227-1338535, and 1346612-1346918; or the inhibitor of nucleolus organisation comprises or consists of a reverse complement of a nucleotide sequence corresponding to a stretch of 10 nucleotides at least 80% conserved between at least 90% of Alu sequences of the AluJ family of sequences and/or a nucleotide sequence being at least 70% identical thereto, wherein the members of the AluJ family are the Alu sequences located on the human Chromosome 1 at nucleotide positions 31436-31733, 101404-101690, 124568-124870, 140495-140784, 141668-141970, 144235-144525, 144987- 145294, 146229-146522, 146776-147047, 147626-147917, 148792-149093, 152276-152566, 153430-153735, 247721-248006, 319131-319439, 322409-322708, 458553-458841, 582950- 583233, 676219-676503, and 677387-677689. In a preferred embodiment, the polynucleotide is a polynucleotide comprising or having a nucleotide sequence corresponding to the reverse complement of at least one of the aforesaid polynucleotides, i.e. preferably, of the respective sequence in the original, more preferably, sense, orientation.

In preferred embodiments, the AluS sequences located on the human Chromosome 1 have the following sequences: 39624-39924: SEQ ID NO: 26, 169374-169679: SEQ ID NO: 27, 101056-101352: SEQ ID NO: 28, 247373-247669: SEQ ID NO: 29, 144606-144899: SEQ ID NO: 30, 174518-174820: SEQ ID NO: 31, 101822-102122: SEQ ID NO: 32, 167495-167792: SEQ ID NO: 33, 76893-77201: SEQ ID NO:34, 129999-130313: SEQ ID NO: 35, 102976- 103280: SEQ ID NO: 36, 249293-249604: SEQ ID NO: 37, 175915-176246: SEQ ID NO: 38, 111081-111386: SEQ ID NO: 39, 149396-149703: SEQ ID NO: 40, 163848-164153: SEQ ID NO: 41, 80805-81096: SEQ ID NO: 42, 165007-165310: SEQ ID NO: 43, 164359-164695: SEQ ID NO: 44, and/or 168485-168786: SEQ ID NO: 45. In also preferred embodiments, the AluY sequences located on the human Chromosome 1 have the following sequences: 51585- 51880: SEQ ID NO: 46, 90921-91213: SEQ ID NO: 64, 229525-229825: SEQ ID NO: 60, 237249-237544: SEQ ID NO: 65, 341759-342052: SEQ ID NO: 56, 413868-414187: SEQ ID NO: 59, 526833-527143: SEQ ID NO: 47, 539503-539801: SEQ ID NO: 50, 863492-863792: SEQ ID NO: 53, 944780-945081: SEQ ID NO: 51, 1069183-1069489: SEQ ID NO: 48, 1125213-1125510: SEQ ID NO: 54, 1128425-1128725: SEQ ID NO: 55, 1191981-1192291: SEQ ID NO: 49, 1212929-1213223: SEQ ID NO: 57, 1215563-1215862: SEQ ID NO: 52, 1324471-1324759: SEQ ID NO: 61, 1325059-1325348: SEQ ID NO: 62, 1338227-1338535: SEQ ID NO: 63, and/or 1346612-1346918: SEQ ID NO: 58. In also preferred embodiments, the AluJ sequences located on the human Chromosome 1 have the following sequences: 31436-31733: SEQ ID NO: 66, 101404-101690: SEQ ID NO: 80, 124568-124870: SEQ ID NO: 72, 140495-140784: SEQ ID NO: 84, 141668-141970: SEQ ID NO: 68, 144235-144525: SEQ ID NO: 74, 144987-145294: SEQ ID NO: 67, 146229-146522: SEQ ID NO: 78, 146776-147047: SEQ ID NO: 83, 147626-147917: SEQ ID NO: 75, 148792-149093: SEQ ID NO: 76, 152276-152566: SEQ ID NO: 77, 153430-153735: SEQ ID NO: 70, 247721-248006: SEQ ID NO: 81, 319131-319439: SEQ ID NO: 79, 322409-322708: SEQ ID NO: 71, 458553-458841: SEQ ID NO: 73, 582950-583233: SEQ ID NO: 82, 676219-676503: SEQ ID NO: 85, and/or 677387-677689: SEQ ID NO: 69. In a preferred embodiment, the polynucleotide is a polynucleotide comprising or having a nucleotide sequence corresponding to the reverse complement of at least one of the aforesaid polynucleotides, i.e. preferably, of the respective sequence in the original, more preferably, sense, orientation. Most preferably, the inhibitor of nucleolar organisation is a polynucleotide comprising or consisting of a nucleotide sequence of at least one of SEQ ID NO: 11 to 22. In a most preferred embodiment, the inhibitor of nucleolar organisation is a polynucleotide comprising or consisting of a nucleotide sequence of SEQ ID NO: 106 or of SEQ ID 229-237.

Moreover, the term "polynucleotide" as used in accordance with the present invention further encompasses variants of the aforementioned specific polynucleotides. The polynucleotide variants, preferably, comprise a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequences by at least one nucleotide substitution, addition and/or deletion whereby the variant nucleic acid sequence shall have the biological activity as specified above. Variants also encompass polynucleotides comprising a nucleic acid sequence, which are capable of hybridizing to the aforementioned specific nucleic acid sequences, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. A preferred example for stringent hybridization conditions are hybridization conditions in 6 x sodium chloride/sodium citrate (= SSC) at approximately 45°C, followed by one or more wash steps in 0.2 x SSC, 0.1% SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under "standard hybridization conditions" the temperature differs depending on the type of nucleic acid between 42°C and 58°C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2). If organic solvent is present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 42°C. The hybridization conditions for DNA:DNA hybrids are preferably for example 0.1 x SSC and 20°C to 45°C, preferably between 30°C and 45°C. The hybridization conditions for DNA:RNA hybrids are preferably, for example, 0.1 x SSC and 30°C to 55°C, preferably between 45°C and 55°C. The abovementioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above.

Further, variants include polynucleotides comprising nucleic acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nucleic acid sequences detailed above. The percent identity values are, preferably, calculated over the entire nucleic acid sequence region. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences as described herein above.

Preferably, not all nucleotides of an inhibitor of nucleolus organisation necessarily exhibit complete Watson-Crick base pairs in the interaction with the target RNA; the two strands may be substantially complementary. Preferably, complementarity between the inhibitor of nucleolus organisation and the target RNA is 100%, but can be less if desired, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. For example, 18 bases out of 20 bases may be base-paired.

The polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. isolated from its natural context) or in genetically modified form. The polynucleotide, preferably, is DNA including cDNA, or RNA. The term encompasses single as well as double stranded polynucleotides. Moreover, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificially modified ones such as biotinylated and/or fluorescently labelled polynucleotides. Further encompassed are polynucleotides comprising or consisting of artificial nucleotides, such as, preferably, phosphorothioates. The polynucleotides of the present invention either essentially consist of the aforementioned nucleic acid sequences or comprise the aforementioned nucleic acid sequences. Thus, they may contain further nucleic acid sequences as well.

It was found in the work underlying the present invention that specific cell types may show a certain preference for inhibition by polynucleotides comprising specific nucleic acid sequences according to the present invention. It is, thus, understood by the skilled person that the polynucleotides of the present invention, preferably, are used as a pool of at least two, more preferably at least three, most preferably at least five of the inhibitor of nucleolar organisation polynucleotides as specified above. Moreover, disclosed herein in the examples are methods of assessing if a specific cell is sensitive to a specific inhibitor of nucleolar organisation polynucleotide. The term "reverse complement" in relation to a nucleic acid sequence is understood by the skilled person. Since polynucleotides have a 5' to 3' orientation, a given nucleic acid sequence has to be complemented, i.e. changed to the corresponding nucleotides according to the Watson-Crick rules of base -pairing, and reversed, i.e. inverted in sequence, in order to obtain the complementary polynucleotide, i.e. the polynucleotide hybridizing to a polynucleotide comprising the given nucleic acid sequence.

It is understood by the skilled person that inhibition of expression or induction of degradation of a specific RNA can be achieved in various ways. It is also understood by the skilled person that the exact embodiment of polynucleotide being the inhibitor of nucleolar organisation of the present invention will depend on the method intended.

Preferably, the inhibitor of nucleolar organisation is a ribozyme. The term "ribozyme" as used herein refers to catalytic RNA molecules possessing a well defined tertiary structure that allows for catalyzing either the hydrolysis of one of their own phosphodiester bonds (self- cleaving ribozymes), or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome. The ribozymes envisaged in accordance with the present invention are, preferably, those, which specifically hydrolyse the target RNAs. In particular, hammerhead ribozymes are preferred in accordance with the present invention. How to generate and use such ribozymes is well known in the art (see, e.g., Hean and Weinberg, 2008).

More preferably, the inhibitor of nucleolar organisation is a polynucleotide inducing RNA interference. As used herein, "RNA interference (RNAi)" refers to sequence-specific, post- transcriptional gene silencing of a selected target gene by degradation of RNA transcribed from the target gene (target RNA). Target RNAs, preferably, are RNAs comprising the consensus sequences as specified above, more preferably, the target RNAs are aluRNAs. It is to be understood that silencing as used herein does not necessarily mean the complete abolishment of expression in all cases. RNAi, preferably, reduces expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% as compared to the expression level in a reference without RNAi.

RNAi requires in the cell the presence of dsRNAs that are homologous in sequence to the target RNAs. The term "dsRNA" refers to RNA having a duplex structure comprising two complementary and anti-parallel nucleic acid strands. The RNA strands forming the dsRNA may have the same or a different number of nucleotides, whereby one of the strands of the dsRNA can be the target RNA. It is, however, also contemplated by the present invention that the dsRNA is formed between two sequence stretches on the same RNA molecule.

RNAi may be used to specifically inhibit expression of the target RNAs of the present invention in vivo. Accordingly, it may be used for therapeutic approaches to treat cancers, causing inhibition of nucleolus organisation in cancer cells and, thereby, preferably, causing said cancer cells to enter apoptosis. For such therapeutic approaches, expression constructs for siRNA may be introduced into cancer cells of the host. Accordingly, siRNA may be combined efficiently with other therapy approaches. Methods relating to the use of RNAi to silence genes in animals, including mammals, are known in the art (see, for example, Hammond et al. (2001), Nature Rev. Genet. 2, 110-119; Bernstein et al. (2001), Nature 409, 363-366; WO 9932619; and Elbashir et al. (2001), Nature 411: 494-498).

Thus, according to the present invention, the inhibitor of nucleolar organisation, preferably is an RNAi agent. As used herein, the term "RNAi agent" refers to either a siRNA agent or a miRNA agent as specified below. The RNAi agent of the present invention is of sufficient length and complementarity to stably interact with the target RNA, i.e. it comprises at least 15, at least 17, at least 19, at least 21, at least 22 nucleotides complementary to the target RNA. By "stably interact" is meant interaction of the RNAi agent or its products produced by the cell with a target RNA, e.g., by forming hydrogen bonds with complementary nucleotides in the target RNA under physiological conditions.

The term "siRNA agent" as meant herein encompasses: a) a dsRNA consisting of at least 15, at least 17, at least 19, at least 21 consecutive nucleotides base-paired, i.e. forming hydrogen bonds with complementary nucleotides, b) a small interfering RNA (siRNA) molecule or a molecule comprising an siRNA molecule. The siRNA is a single -stranded RNA molecule with a length, preferably, greater than or equal to 15 nucleotides and, preferably, a length of 15 to 49 nucleotides, more preferably 17 to 30 nucleotides, and most preferably 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides, c) a polynucleic acid encoding a) or b), wherein, preferably, said polynucleic acid is operatively linked to an expression control sequence. Thus, the function of the siRNA agent to inhibit expression of the target gene can be modulated by said expression control sequence. Preferred expression control sequences are those which can be regulated by exogenous stimuli, e.g. the tet operator, whose activity can be regulated by tetracycline, or heat inducible promoters. Alternatively or in addition, one or more expression control sequences can be used which allow tissue- specific expression of the siRNA agent.

It is, however, also contemplated by the current invention that the RNAi agent is a miRNA agent. A "miRNA agent" as meant herein encompasses: a) a pre-microRNA, i.e. an mRNA comprising at least 30, at least 40, at least 50, at least 60, at least 70 nucleotides base-paired to a complementary sequence on the same mRNA molecule ("stem"), i.e. as a dsRNA, separated by a strech of non-base-paired nucleotides ("loop"), b) a pre-microRNA, i.e. a dsRNA molecule comprising a stretch of at least 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25 base-paired nucleotides formed by nucleotides of the same RNA molecule (stem), separated by a loop, c) a microRNA (miRNA), i.e. a dsRNA comprising at least 15, at least 17, at least 18, at least 19, at least 21 nucleotides on two separate RNA strands, d) a polynucleic acid encoding a) or b), wherein, preferably, said polynucleic acid is operatively linked to an expression control sequence as specified above.

Most preferably, the inhibitor of nucleolar organisation is an antisense oligo. The term "antisense oligo" is known to the skilled person and relates to an oligonucleotide hybridizing to a target RNA, causing the formation of a DNA/RNA hybrid. Said DNA/RNA hybrid is a substrate for RNase H, which degrades the RNA portion of said DNA/RNA hybrid. Thus, the antisense oligo comprises at least five, preferably at least seven, more preferably at least nine, or, most preferably, at least ten DNA nucleotides. Preferably, the antisense oligo has a length of at least 15 nucleotides, preferably at least 18 nucleotides, still more preferably at least 20 nucleotides. Most preferably, the antisense oligo has ten DNA nucleotides flanked by five RNA nucleotides on the 5' and the 3' side, respectively, as shown herein in the examples.

Advantageously, it was found in the work underlying the present invention that nucleolar organisation is dependent on the expression of Alu sequences in the nucleus by RNA polymerase II. It was further found that said Alu sequences comprise regions highly conserved throughout the various copies of the respective Alu family members and that these conserved sequences can be used to induce degradation and/or to prevent expression of said Alu sequences, causing the cell to fail at organizing nucleoli in the nucleus. Since nucleoli are essential for ribosome assembly and since nucleoli further act as stress sensors in the cell, cells unable to organize functional nucleoli stop proliferating and will eventually enter apoptosis. In a preferred finding of the work underlying the present invention, it was found that Alu sequences are present in the nucleoli in the sense and in the antisense orientation and that inhibition of production of Alu sequences of sense or of antisense orientation is effective in disturbing nucleolar organisation and rRNA production.

The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.

In a further embodiment, the present invention also relates to a vector comprising the inhibitor of nucleolar organisation of the present invention.

The term "vector", preferably, encompasses phage, plasmid, viral or retroviral vectors as well artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs, which allow for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below. The vector encompassing the polynucleotides of the present invention, preferably, further comprises selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art. For example, a plasmid vector can be introduced in a precipitate such as a calcium phosphate precipitate or rubidium chloride precipitate, or in a complex with a charged lipid or in carbon- based clusters, such as fullerens. Alternatively, a plasmid vector may be introduced by heat shock or electroporation techniques. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells.

More preferably, in the vector of the invention the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof. Expression of said polynucleotide comprises transcription of the polynucleotide, preferably into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known in the art. They, preferably, comprise regulatory sequences ensuring initiation of transcription and, optionally, poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Moreover, inducible expression control sequences may be used in an expression vector encompassed by the present invention. Such inducible vectors may comprise tet or lac operator sequences or sequences inducible by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. Beside elements, which are responsible for the initiation of transcription, such regulatory elements may also comprise transcription termination signals, such as the SV40- poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pBluescript (Stratagene), pCDM8, pRc/CMV, pcDNAl, pcDNA3 (InVitrogene) or pSPORTl (GIBCO BRL). Preferably, said vector is an expression vector and a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994).

Further, the present invention relates to an inhibitor of nucleolus organisation according to the present invention for use in medicine.

The present invention also relates to an inhibitor of nucleolus organisation according to any one of claims 1 to 7 for use in the treatment of cancer.

The term "treatment" refers to an amelioration of the diseases or disorders referred to herein or the symptoms accompanied therewith to a significant extent. Said treating as used herein also includes an entire restoration of the health with respect to the diseases or disorders referred to herein. It is to be understood that treating as used in accordance with the present invention may not be effective in all subjects to be treated. However, the term shall require that a statistically significant portion of subjects suffering from a disease or disorder referred to herein can be successfully treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc.. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment shall be effective for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population.

The term "cancer", as used herein, relates to a disease of an animal, including man, characterized by uncontrolled growth by a group of body cells ("cancer cells"). This uncontrolled growth may be accompanied by intrusion into and destruction of surrounding tissue and possibly spread of cancer cells to other locations in the body.

Preferably, the cancer is selected from the list consisting of acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, aids-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, brain stem glioma, breast cancer, burkitt lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, kaposi sarcoma, laryngeal cancer, medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non- small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and wilms tumor. More preferably, the cancer is a solid cancer; most preferably, the cancer is a cervix cancer or a breast cancer.

Preferably, the inhibitor of nucleolar organisation for use in medicine or for treating cancer is provided in a pharmaceutical composition. The term "pharmaceutical composition" as used herein relates to the polynucleotides of the present invention and optionally one or more pharmaceutically acceptable carrier. The polynucleotides of the present invention can be formulated as pharmaceutically acceptable salts. Acceptable salts comprise acetate, methylester, HC1, sulfate, chloride and the like. The pharmaceutical compositions are, preferably, administered systemically, or, more preferably, locally or topically. Suitable routes of administration conventionally used for drug administration are oral, intravenous, or parenteral administration as well as inhalation. However, depending on the nature of a polynucleotide and the disease to be treated, the pharmaceutical compositions may be administered by other routes as well. For example, polynucleotides may be administered in a gene therapy approach by using viral vectors or viruses or liposomes.

Moreover, the polynucleotides can be administered in combination with other drugs either in a common pharmaceutical composition or as separated pharmaceutical compositions wherein said separated pharmaceutical compositions may be provided in form of a kit of parts. The polynucleotides are, preferably, administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Preferably, the polynucleotides are combined with compounds mediating cell entry, e.g. with transfection reagents, calcium phosphate, or the like. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may be, for example, either a solid, a gel or a liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

The diluent(s) is/are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

A therapeutically effective dose refers to an amount of the polynucleotides to be used in a pharmaceutical composition of the present invention, which prevents, ameliorates or treats the symptoms accompanying a disease or condition referred to in this specification. Therapeutic efficacy and toxicity of such polynucleotides can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.

The dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above-described methods. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular polynucleotide to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. A typical dose can be, for example, in the range of 1 to 1000 μg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. However, depending on the subject and the mode of administration, the quantity of substance administration may vary over a wide range to provide from about 0.01 mg per kg body mass to about 10 mg per kg body mass, preferably.

The pharmaceutical compositions and formulations referred to herein are administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said pharmaceutical compositions may be administered more than one time, for example from one to four times daily up to a non-limited number of days.

Specific pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active polynucleotide referred to herein above in admixture or otherwise associated with a pharmaceutically acceptable carrier or diluent. For making those specific pharmaceutical compositions, the active polynucleotide(s) will usually be mixed with a carrier or the diluent, or enclosed or encapsulated in a capsule, sachet, cachet, paper or other suitable containers or vehicles. The resulting formulations are to be adapted to the mode of administration, i.e. in the forms of tablets, capsules, suppositories, solutions, suspensions or the like. Dosage recommendations shall be indicated in the prescribers or users instructions in order to anticipate dose adjustments depending on the considered recipient.

The present invention further relates to a host cell comprising the inhibitor of nucleolus organisation and/or the vector according to the present invention.

The term "host cell", as used herein, relates to any bacterial, archeal, or eukaryotic cell. Preferably the host cell is a cell naturally or artificially comprising Alu sequences and Nucleoli. Preferably, the host cell is a primate cell. More preferably, the host cell is a cell of a subject as specified herein below. Preferably, the cell is a cell maintained in vitro.

Moreover, the present invention relates to a use of an inhibitor of nucleolus organisation according to the present invention for the inhibition of cancer cell proliferation. The term "cancer cell proliferation" relates to an increase in cancer cell mass and/or number. Preferably, the term relates to the production of additional cancer cells by division of at least one parental cell.

The present invention also relates to a device comprising the inhibitor of nucleolus organisation, a vector, and/or a host cell according the present invention.

The term "device", as used herein, relates to a system of means comprising at least the inhibitor of nucleolar organisation, a vector, and/or a host cell according the present invention referred to in the claims or herein and, preferably, a means of applying said polynucleotides to a subject. Means of applying the polynucleotides of the present invention, including the polynucleotides, are well known to the skilled person and include, e.g. syringes, infusion sets, inhalers, and the like. Preferably, the aforesaid means are comprised by a single device.

The present invention further relates to a kit comprising the inhibitor of nucleolus organisation a vector, and/or a host cell according to the present invention and an instruction manual.

The term "kit", as used herein, refers to a collection of the aforementioned components, preferably, provided separately or within a single container. The container, also preferably, comprises instructions for carrying out a method of the present invention. Examples for such the components of the kit as well as methods for their use have been given in this specification. The kit, preferably, contains the aforementioned components in a ready-to-use formulation. Preferably, the kit may additionally comprise instructions, e.g., a user's manual for applying the inhibitor of nucleolar organisation with respect to the applications provided by the methods of the present invention. Details are to be found elsewhere in this specification. Additionally, such user's manual may provide instructions about correctly using the components of the kit. A user's manual may be provided in paper or electronic form, e.g., stored on CD or CD ROM. The present invention also relates to the use of said kit in any of the methods according to the present invention.

Further, the present invention relates to a method of inhibiting cancer cell proliferation, comprising a) contacting a cancer cell with an effective amount of inhibitor of nucleolus organisation, and/or a vector according the present invention, and b) thereby inhibiting cancer cell proliferation.

The method of inhibiting cancer cell proliferation of the present invention, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to providing cancer cells for step a), or further analysis steps in step b). Moreover, one or more of said steps may be performed by automated equipment.

The present invention further relates to a method for identifying an inhibitor of nucleolus organisation, comprising a) contacting a host cell with a compound suspected to be an inhibitor of nucleolus organisation, b) detecting inhibition of nucleolus organisation, and c) thereby identifying an inhibitor of nucleolus organisation.

The method for identifying an inhibitor of nucleolus organisation of the present invention, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to providing a host cell for step a), or further analysis steps in step b). Moreover, one or more of said steps may be performed by automated equipment.

The term "compound" refers to a chemical molecule, i.e. any organic or inorganic substance. The organic molecule may belong to any known chemical class of molecules. Preferably, organic molecules are lipids, fatty acids, purines, pyrimidines, alkaloids, amino acids, peptides, polypeptides, proteins, biogenic amines, isoprenoids or steroids. The term "compound suspected to be an inhibitor of nucleolus organisation" relates to any compound for which it has not been excluded that it has the capacity to be an inhibitor of nucleolus organisation. Preferably, a compound suspected to be an inhibitor of nucleolus organisation is a compound for which a suspicion, or, more preferably, an expectation exists that it is an inhibitor of nucleolus organisation. Such suspicion or expectation may, e.g., come from molecular modeling, from the fact that said compound is a derivative of a known inhibitor of nucleolar organisation, or from the fact that said compound has been shown to have an impact on nucleolus organisation in a subject or in a host cell. The term "detecting inhibition of nucleolus organisation" relates to assessing if inhibition of nucleolus organisation has occurred in a host cell or not. Preferably, said assessing is accomplished as described herein in the examples, i.e. by visualising the nucleoli of a host cell, e.g. by staining specific components of the nucleolus. It is understood that said detecting inhibition of nucleolus organisation may also be perfomed in a two-step process, e.g. by first testing for cells entering apoptosis, and second ascertaining that nucleolus organisation was inhibited as described above.

The present invention also relates to the use of an inhibitor of nucleolus organisation and/or of a vector according to the present invention for the manufacture of a medicament; and to the use of an inhibitor of nucleolus organisation and/or of a vector according to the present invention for treating cancer.

Further, the present invention relates to a method of treating cancer in a subject afflicted with cancer, comprising a) treating said subject with an effective amount of an inhibitor of nucleolus organisation according to the present invention, and b) thereby treating cancer in a subject afflicted with cancer.

The method of treating cancer of the present invention, preferably, may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to diagnosing cancer in a subject or additional cancer treatment in step a).

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

Figure Legends

Figure 1

Confocal laser scanning microscopy images of wild-type HeLa cells after no treatment (control) or after RNA polymerase II inhibition (alpha-amanitin).

The distribution of the proteins C23 and RNA polymerase I was revealed by immunofluorescence. Scale bar, 10 μιη. Figure 2

The distribution of the protein UBF (upstream binding factor) was revealed by immunofluorescence. RNA was stained by click chemistry and DNA was labeled with 4',6- diamidin- 2'-phenylindol-dihydrochlorid (DAPI).

Scale bar, 10 mm.

Figure 3

Distribution of RNA-Seq reads of nucleoli purified RNA within the intergenic spacer of the rDNA (IGS). The X-axis (IGS position) indicates the base number within the human rDNA gene (GenBank U13369). The Y-axis indicated the number of reads mapping to each position. The dashed line shows the distribution of the reads if they are mapped first on the IGS. The continued line shows the distribution of the reads if they are mapped first on the genome (reference genome does not contain the rDNA region) and the unmapped reads on the rDNA gene.

Figure 4

Normalized distribution of RNA-Seq reads for total RNA isolated from HeLa S3 cells after no treatment (continued line), RNA polymerase I (dashed line) and II (dotted line) inhibition within the IGS. For X-axis and Y-axis description, see legend Figure 6. One particular region showing a peak sensitive to inhibition of RNA polymerase II but not to inhibition of RNA polymerase I has been selected.

Figure 5

Scheme depicting the principle of anti- sense oligo (ASO) treatment. (A) a chimeric oligonucleotide composed of RNA and DNA is designed in the reverse-complement direction of one region of a target RNA. (B) When the ASO is hybridized to the target RNA, the RNA/DNA double strand is recognized by the RNase H, which results in RNA degradation. The ASO can then bind an other target RNA fragment.

Figure 6

Confocal laser scanning microscopy images of wild-type HeLa cells after transfection with Lipofectamin only (control Mock) or Lipofectamin-ASO complexes as indicated. The distribution of the ASO and protein B23 (nucleophosmin) were revealed respectively by the signal of the Cy3-dye and immunofluorescence. RNA was stained by click chemistry and DNA was labeled with DAPI. Scale bar, 10 μιη.

Figure 7

Confocal laser scanning microscopy images of wild-type HeLa cells showing the distribution of the protein C23/nucleolin (by immunofluorescence) and the distribution of endogenous RNAs containing Alu repeat sequences (RNA FISH). The DNA was stained with DAPI. Scale bar, 10 μιη.

Figure 8

(A) Confocal laser scanning microscopy images of MCF7 cells expressing Bax-eGFP after transfection with lipofectamin only (control Mock) or Lipofectamin-ASO complexes as indicated. The fluorescence of the eGFP shows the distribution of the protein Bax. The distribution of the protein B23 was revealed by immunofluorescence. DNA was stained with DAPI. White arrows indicate cells with fragmented nucleoli but which are not apoptotic yet. Scale bar, 10 μιη. (B) The graph displays the percentage of apoptotic cells 14 hours post- transfection in the indicated samples, as revealed by the dotted distribution of the protein Bax. Error bars represent the standard deviation for, respectively, 183 and 234 cells.

Figure 9

Alignment of 20 AluS sequences (the name of each sequence indicates its position: chr, start, end). Positions 1 to 4 and 5 indicate larger regions (more than 12 nucleotides) showing increased homology between the various AluS sequences. The position of some ASO sequences is indicated at the bottom of the alignment. The SEQ ID NOs of the sequences are: (A) chr=l 39624-39924: SEQ ID NO: 107, chr=l 169374-169679: SEQ ID NO: 108, chr=l 101056-101352: SEQ ID NO: 109, chr=l 247373-247669: SEQ ID NO: 110, chr=l 144606- 144899: SEQ ID NO: 111, chr=l 174518-174820: SEQ ID NO: 112, chr=l 101822-102122: SEQ ID NO: 113, chr=l 167495-167792: SEQ ID NO: 114, chr=l 76893-77201: SEQ ID NO: 115, chr=l 129999-130313: SEQ ID NO: 116, chr=l 102976-103280: SEQ ID NO: 117, chr=l 249293-249604: SEQ ID NO: 118, chr=l 175915-176246: SEQ ID NO: 119, chr=l 111081-111386: SEQ ID NO: 120, chr=l 149396-149703: SEQ ID NO: 121, chr=l 163848-164153: SEQ ID NO: 122, chr=l 80805-81096: SEQ ID NO: 123, chr=l 165007- 165310: SEQ ID NO: 124, chr=l 164359-164695: SEQ ID NO: 125, and chr=l 168485- 168786: SEQ ID NO: 126. (B)-(D) chr=l 39624-39924: SEQ ID NO: 127, chr=l 169374- 169679: SEQ ID NO: 128, chr=l 101056-101352: SEQ ID NO: 129, chr=l 247373-247669: SEQ ID NO: 130, chr=l 144606-144899: SEQ ID NO: 131, chr=l 174518-174820: SEQ ID NO: 132, chr=l 101822-102122: SEQ ID NO: 133, chr=l 167495-167792: SEQ ID NO: 134, chr=l 76893-77201: SEQ ID NO: 135, chr=l 129999-130313: SEQ ID NO: 136, chr=l 102976-103280: SEQ ID NO: 137, chr=l 249293-249604: SEQ ID NO: 138, chr=l 175915-176246: SEQ ID NO: 139, chr=l 111081-111386: SEQ ID NO: 140, chr=l 149396- 149703: SEQ ID NO: 141, chr=l 163848-164153: SEQ ID NO: 142, chr=l 80805-81096: SEQ ID NO: 143, chr=l 165007-165310: SEQ ID NO: 144, chr=l 164359-164695: SEQ ID NO: 145, and chr=l 168485-168786: SEQ ID NO: 146.

Figure 10

Same as Figure 9 for 20 AluY sequences. The SEQ ID NOs of the sequences are: (A) chr=l 51585-51880: SEQ ID NO: 147, chr=l 526833-527143: SEQ ID NO: 148, chr=l 1069183- 1069489: SEQ ID NO: 149, chr=l 1191981-1192291: SEQ ID NO: 150, chr=l 539503- 539801: SEQ ID NO: 151, chr=l 944780-945081: SEQ ID NO: 152, chr=l 1215563- 1215862: SEQ ID NO: 153, chr=l 863492-863792: SEQ ID NO: 154, chr=l 1125213- 1125510: SEQ ID NO: 155, chr=l 1128425-1128725: SEQ ID NO: 156, chr=l 341759- 342052: SEQ ID NO: 157, chr=l 1212929-1213223: SEQ ID NO: 158, chr=l 1346612- 1346918: SEQ ID NO: 159, chr=l 413868-414187: SEQ ID NO: 160, chr=l 229525-229825: SEQ ID NO: 161, chr=l 1324471-1324759: SEQ ID NO: 162, chr=l 1325059-1325348: SEQ ID NO: 163, chr=l 1338227-1338535: SEQ ID NO: 164, chr=l 90921-91213: SEQ ID NO: 165, chr=l 237249-237544: SEQ ID NO: 166. (B)-(D) chr=l 51585-51880: SEQ ID NO: 167, chr=l 526833-527143: SEQ ID NO: 168, chr=l 1069183-1069489: SEQ ID NO: 169, chr=l 1191981-1192291: SEQ ID NO: 170, chr=l 539503-539801: SEQ ID NO: 171, chr=l 944780-945081: SEQ ID NO: 172, chr=l 1215563-1215862: SEQ ID NO: 173, chr=l 863492-863792: SEQ ID NO: 174, chr=l 1125213-1125510: SEQ ID NO: 175, chr=l 1128425-1128725: SEQ ID NO: 176, chr=l 341759-342052: SEQ ID NO: 177, chr=l 1212929-1213223: SEQ ID NO: 178, chr=l 1346612-1346918: SEQ ID NO: 179, chr=l 413868-414187: SEQ ID NO: 180, chr=l 229525-229825: SEQ ID NO: 181, chr=l 1324471-1324759: SEQ ID NO: 182, chr=l 1325059-1325348: SEQ ID NO: 183, chr=l 1338227-1338535: SEQ ID NO: 184, chr=l 90921-91213: SEQ ID NO: 185, chr=l 237249- 237544: SEQ ID NO: 186. Figure 11

Same as Figure 9 for 20 AluJ sequences. The SEQ ID NOs of the sequences are: (A) chr=31436-31733: SEQ ID NO: 187, chr=l 144987-145294: SEQ ID NO: 188, chr=l 141668-141970: SEQ ID NO: 189, chr=l 677387-677689: SEQ ID NO: 190, chr=l 153430- 153735: SEQ ID NO: 191, chr=l 322409-322708: SEQ ID NO: 192, chr=l 124568-124870: SEQ ID NO: 193, chr=l 458553-458841: SEQ ID NO: 194, chr=l 144235-144525: SEQ ID NO: 195, chr=l 147626-147917: SEQ ID NO: 196, chr=l 148792-149093: SEQ ID NO: 197, chr=l 152276-152566: SEQ ID NO: 198, chr=l 146229-146522: SEQ ID NO: 199, chr=l 319131-319439: SEQ ID NO: 200, chr=l 101404-101690: SEQ ID NO: 201, chr=l 247721- 248006: SEQ ID NO: 202, chr=l 582950-583233: SEQ ID NO: 203, chr=l 146776-147047: SEQ ID NO: 204, chr=l 140495-140784: SEQ ID NO: 205, and chr=l 676219-676503: SEQ ID NO: 206; (B)-(D) chr=l 31436-31733: SEQ ID NO: 207, chr=l 144987-145294: SEQ ID NO: 208, chr=l 141668-141970: SEQ ID NO: 209, chr=l 677387-677689: SEQ ID NO: 210, chr=l 153430-153735: SEQ ID NO: 211, chr=l 322409-322708: SEQ ID NO: 212, chr=l 124568-124870: SEQ ID NO: 213, chr=l 458553-458841: SEQ ID NO: 214, chr=l 144235- 144525: SEQ ID NO: 215, chr=l 147626-147917: SEQ ID NO: 216, chr=l 148792-149093: SEQ ID NO: 217, chr=l 152276-152566: SEQ ID NO: 218, chr=l 146229-146522: SEQ ID NO: 219, chr=l 319131-319439: SEQ ID NO: 220, chr=l 101404-101690: SEQ ID NO: 221, chr=l 247721-248006: SEQ ID NO: 222, chr=l 582950-583233: SEQ ID NO: 223, chr=l 146776-147047: SEQ ID NO: 224, chr=l 140495-140784: SEQ ID NO: 225, chr=l 676219- 676503: SEQ ID NO: 226.

Figure 12

Alignment of the consensus sequences (consensus_l to consensus_5) of AluS, AluY and AluJ from the selected positions 1 to 5 as indicated on Figure 9 to 11. The SEQ ID NOs of the sequences are: (A) AluS consensus_l: SEQ ID NO: 86, AluY consensus_l: SEQ ID NO: 87, AluJ consensus_l: SEQ ID NO: 88, Consensus: SEQ ID NO: 89; (B) AluS consensus_2: SEQ ID NO: 90, AluY consensus_2: SEQ ID NO: 91, AluJ consensus_2: SEQ ID NO: 92, Consensus: SEQ ID NO: 93; (C) AluS consensus_3: SEQ ID NO: 94, AluY consensus_3: SEQ ID NO: 95, AluJ consensus_3: SEQ ID NO: 96, Consensus: SEQ ID NO: 97; (D) AluS consensus_4, AluY consensus_4, AluJ consensus_4, Consensus: all: SEQ ID NO: 98; (E) AluS consensus_5: SEQ ID NO: 99, AluY consensus_5: SEQ ID NO: 100, AluJ consensus_5: SEQ ID NO: 101, Consensus: SEQ ID NO: 102. Figure 13

(A) Recruitment of GFP-tagged proteins to stably integrated lacO arrays in vivo. A schematic representation illustrates how GFP-fusion proteins are tethered to genomic integrations of lacO arrays in U20S F6B2 cells. (B) Alu-containing RNAs (aluRNA) interact with nucleolin (NCL) and nucleophosmin (NPM). Confocal laser scanning microscopy images show the localization of GBP-LacI-RFP, GFP-NCL, GFP-NPM or GFP-TIP5 (fluorescence) and aluRNA (RNA FISH). The insets emphasize one of the lacO loci. The dashed line delimits the nucleus of each cell. Scale bar, 10 μιη.

Figure 14

(A) Western blot analysis of siRNA-mediated depletion of NCL and NPM in HeLa cells. Histone H3 was used as loading control. siRNA treatments reduced the amount of the corresponding protein by more than 50% (NCL) and 80% (NPM). (B) Confocal laser scanning microscopy images show NCL and NPM distribution in HeLa cells transfected with siRNAs as indicated. The dashed line delimits the nucleus of each cell. Scale bar, 10 μιη.

Figure 15

Time course of nucleolar disruption induced by (A) a-amanitin or (B) aluRNA knockdown. CLSM images of immunostained NPM and DAPI stained DNA. HeLa cells were treated as indicated with a-amanitin or transfected with an ASO for the indicated time. Scale bars, 10 μιη.

Figure 16

(A) Example of an aluRNA sequence (SEQ ID NO: 238) and aluRNA consensus positions (see Figure 12); (B)-(G) Various cell lines were treated with ASO as indicated and the fraction of nucleolar structures (in percent) defined as "normal", "abnormal" (clear morphological aberrations) or "dispersed" (dispersion of the structure within the nucleoplasm) was evaluated via fluorescence microscopy. Nucleolin was used as nucleolar marker for this purpose. Error bars represent the 95% interval of confidence; (B) Treatment of DU145 cells (prostate cancer cells), (C) Treatment of HeLa cells (cervix cancer cells), (D) Treatment of MCF7 cells (breast cancer cells) , (E) Treatment of U20S cells (bone cancer cells), (F) Treatment of SF188 cells (pediatric glioblastoma cells), (G) Treatment of JOPACA cells (pancreatic cancer cells). (H) Treatment of HUVEC cells (human umbilical vein cells, control cells). It is noted that it was not possible to obtain a transfection efficiency above 20% in HUVEC cells with the method used for cell lines of (B)-(G). For this reason, HUVEC cells were electroporated. With this method, an efficiency of 60% is typically obtained. The error bars represent the standard deviation for about 100 cells for each sample.

Figure 17

(A) Northern blot showing 47S pre-rRNA levels in untreated, AMD-, a-amanitin- and ML- 60218-treated HeLa cells (actinomycin D, AMD, is an inhibitor of RNA polymerase I. ML- 60218 is an inhibitor of RNA polymerase III. (B) The graph shows pre-rRNA levels determined by RT-qPCR. Error bars represent the standard deviation for 6 independent measurements.

Figure 18

(A) Northern blot showing the level of 47 S pre-rRNA in mock- treated (transfection only), control ASO (ASO-IGS- 13848) and aluRNA ASO-treated (ASO-IGS-32777 rev) HeLa cells.

(B) Quantification of 47S pre-rRNA by qRT-PCRs. Error bars represent the standard deviation for 6 independent measurements.

Figure 19

Intronic aluRNA are expressed and found in nucleoli. (A) Scheme illustrating the origin of intronic aluRNA transcribed in sense or antisense orientation from RNA polymerase II primary transcripts. (B) Top: Heatmaps of read density of intronic Alu repeats expressed in sense or antisense directions in nucleolar RNA.

Figure 20

Graphs illustrating the proportion (in percent) of apoptotic cells 14 hours after the indicated treatment (transfected = Lipofectamin transfection reagent only, Ctrl ASO = transfection with ASO-IGS- 13848, aluRNA ASO = ASO-IGS-32933b) in (A) DU145 cells and (B) JOPACA cells. Error bars represent the standard deviation from 2 FACS analyses.

Figure 21

Graph illustrating the proportion (in percent) of dead cells 14 hours or 24 hours after the indicated treatment (transfected = transfection reagent only, Ctrl ASO = transfection with ASO-IGS- 13848, AluRNA ASO = ASO-IGS-32933b) in JOPACA cells. Error bars represent the standard deviation from 3 FACS analyses. Figure 22

Confocal laser scanning microscopy images of wild-type HeLa cells after 5 hours treatment with cycloheximide (CHX, protein synthesis inhibitor) or ML-60218 (RNA polymerase III inhibitor). The distribution of the proteins nucleolin (NCL) was revealed by immunofluorescence. DNA was stained with DAPI. Scale bars, 10 μιη.

The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

Example 1

The inhibition of RNA polymerase II transcription by alpha-amanitin during 5 hours resulted in the loss of nucleolar structure as depicted by the redistribution of C23 (also called nucleolin or NCL, a protein known to be associated to the nucleoli) and RNA polymerase I (Figure 1). The dispersion of the nucleoli observed here is specific for RNA polymerase II inhibition and is not observed after RNA polymerase I or III inhibition, or protein synthesis inhibition (Figure 22; Haaf et al. (1996), Exp Cell Res. 224(1):163).

Example 2

The inhibition of RNA polymerase II transcription by alpha-amanitin during 5 hours resulted in the loss of nucleolar structure as depicted by the re-distribution of UBF (upstream binding factor for RNA polymerase I) and in the reduction of rRNA production (500 μΜ of ethynyl uridine were added in the cell culture medium during the last 2 hours of drug treatment (Figure 2). In addition, a reduction of rRNA production after 5 hours RNA polymerase II inhibition by alpha-amanitin was also quantitatively measured by northern blot and quantitative RT-PCR (Figure 17).

Example 1 and 2 together illustrate that specific inhibition of RNA polymerase II induces both loss of nucleolar structure and function. This suggests that RNA polymerase II transcripts are essential for both nucleolar structure and function.

Example 3 Nucleoli can be isolated from HeLa S3 cells and their RNA content analyzed by high- throughput RNA sequencing. As depicted on Figure 3, it is observed that RNA transcripts are produced from regions, which are found both within the rDNA gene and somewhere else on the genome (basically, those regions have the same or similar RNA sequences over 50 to 100 nucleotides). Those regions are the peaks (dashed line) seen on Figure 3. These are mainly simple repeats or Alu repeat elements.

Example 4

The RNA polymerase II transcripts, which are supposed to have an essential role in nucleolar structure and function, should be sensitive to RNA polymerase II inhibition treatment. A reduction of their expression is expected. In Figure 4, the normalized RNA-Seq read distribution in the IGS after various drug treatments shows a selected peak that is clearly sensitive to inhibition of RNA polymerase II and not to inhibition of RNA polymerase I. The corresponding sequence was selected as target for RNA knockdown.

Example 5

Anti-sense oligonucleotides (ASO) were designed following the description depicted on Figure 5. These targeted the selected peak from Figure 4 (ASO-IGS-32777) and with small variations in the sequence other peaks were as well targeted (for example ASO-IGS-37019) (See Table 1 for a summary of the used ASO). Another sequence of the IGS was selected (ASO-IGS-13848) as control. It appeared that the selected peak in the IGS is transcribed from an Alu repeat element. As depicted on Figure 19, Alu repeat-containing RNA transcripts (aluRNAs) can be produced from intronic regions of primary transcripts (Wang & Huang (2014), Nucleus. 25(2): PMID: 24637839). Depending on the respective orientation of the Alu repeat and the gene, the aluRNA can be produced in the sense or in the antisense direction (Figure 19A). Our RNA-Seq analysis shows that aluRNAs can be found in both directions (sense and antisense) within isolated nucleoli (Figure 19B). Other sequences found in Alu repeat elements were therefore tested (for example ASO-IGS-32933). See as well example 8 below. For each ASO, a corresponding reverse-complement ASO was tested separately. The efficient transfection of each cell was assessed by visualizing the distribution of the ASO (Cy3 fluorescence signal). It is observed that ASO are distributed in the various compartments of the cell (cytoplasm, nucleoplasm, nucleoli).

Table 1: List of ASOs and their respective variants. Differences are indicated in bold upper case. The effect on nucleolar structure 14 hours after transfection is indicated and defined as "normal", "abnormal" (clear morphological aberrations) or "dispersed" (dispersion of the structure within the nucleoplasm). The effect on nucleolar structure was evaluated via fluorescence microscopy analysis.

rev

Figure 6A shows some examples of ASO sequences where nucleolar structure and function were not affected (ASO-IGS-13848) or presenting small aberrations (ASO-IGS-36587 or 32745). Figure 6B presents some examples of conditions where treatment with ASO (or reverse-complement ASO) resulted in large aberrations or dispersion of the nucleolar structure (the distribution of the protein B23 is used as readout). Note that the presence of structural nucleolar aberrations is associated with a nucleolar dysfunction as inferred from the reduction of rRNA production (RNA staining). Northern blot and quantitative RT-PCR measurements confirmed the reduction of rRNA production after ASO treatment against aluRNAs (Figure 18).

Examples 3, 4 and 5 together illustrate that RNA polymerase II transcripts containing Alu repeat sequences are alpha-amanitin sensitive and can be targeted by various ASO treatments. The resulting loss of nucleoli structure by dispersion and loss of nucleoli function by reduction of rRNA production are very similar to the effects observed after treatment with alpha-amanitin. This suggests that the phenotype observed after alpha-amanitin is due to a reduction of aluRNA production.

Example 6

RNA FISH (Figure 7) shows the presence of endogenous aluRNAs (RNA containing an Alu repeat element) in the nucleoli. This confirms the findings of our RNA-Seq analysis (Figure 19B) and supports an essential structural and functional role for aluRNAs in the nucleoli.

Example 7

Bax is a downstream effector of Bid in the caspase activation pathways via mitochondria, and can be activated also through other pathways (Wolter et al., "Movement of Bax from the cytosol to mitochondria during apoptosis". J Cell Biol 1997; 139: 1281-92). The cellular localization of Bax is a marker of induction of apoptosis. A punctuated distribution of Bax- eGFP indicates the induction of cellular events leading to apoptosis (Bax translocation to mitochondria, which became highly fragmented in the early stages of apoptosis). As observed in Figure 8, in control Mock (Lipofectamin only) cells, most of the cells are not adversely affected (cytoplasmic/nuclear distribution of Bax). A quantification of the number of cells being apoptotic after 14 hours in this experiment is shown is Figure 8B.

In the ASO-treated cells, many cells are apoptotic (punctuated distribution of Bax). Noteworthy, some cells show fragmented nucleoli (white arrows) without any sign of apoptosis induction. This means that the fragmentation of the nucleoli occurs prior to apoptosis induction. Further analysis via FACS (fluorescence-activated cell sorting) demonstrates a clear increase of the proportion of apoptotic cells 14 hours after ASO-treatment against aluRNAs as compared to the controls (untreated, only Lipofectamin and control ASO) in the two cancer cell lines we tested (Figure 20). In addition, the FACS analysis shows a clear increase of the proportion of dead cells at 14 hours post-transfection and a further increase at 24 hours post- transfection (Figure 21).

Since many cells became apoptotic or died after specific ASO treatment, aluRNA knockdown results in loss of nucleolar structure and function, and subsequently cell death.

Example 8

When trying to define more precisely a possible genomic origin (other than in the IGS of the rDNA) for the aluRNA peak (Figure 4), it was observed that the same sequence and similar close variant of this sequence can be found in Alu repeat elements of the different families: AluS, AluY and AluJ. Such Alu repeats are embedded within introns of primary transcripts produced by RNA polymerease II. A closer investigation of 20 sequences of each family (Figure 9 for AluS, Figure 10 for AluY and Figure 11 for AluJ) shows that specific positions, refer to as position 1 to 5 in the Figures 9 to 11, have an increased homology between the various Alu repeats of each family. Moreover, an alignment of the respective regions 1 to 5 shows that the homology between those regions is in fact verified through all the Alu repeat families (Figure 12).

Example 9

Nucleoli from a cancer sample are isolated according to standard methods. The transcriptome of said nucleoli is sequenced e.g. by a random primed high-throughput sequencing method; from the sequence data obtained, it is derived which Alu sequences are comprised in the nucleoli of the cancer cells and the inhibitor of nucleolar organisation is chosen accordingly. Due to sequence homologies between the sequences of Alu repeats, it is expected that an inhibitor can hit several aluRNAs at once. Depending on the sequences, the pool of aluRNA targets will vary. It is expected that the inhibitors which are going to be the most efficient (leading to nucleoli dispersion, reduction of rRNA production and cell death) will be the ones which will manage to induce the degradation of a critical number of aluRNAs. Critical means that enough aluRNAs will be degraded so that disruption of nucleolar structure and function will lead to p53 activation and eventually cell death (Burger et al. loc. cit). Example 10

An array of potentially effective inhibitor of nucleolar organisation polynucleotides is produced; after isolating and labelling RNA from cancer cell nucleoli, said labelled RNA is hybridized against the array of potentially effective inhibitor of nucleolar organisation polynucleotides, thereby identifying inhibitor of nucleolar organisation polynucleotides binding to the nucleolar aluRNAs. Inhibitor of nucleolar organisation polynucleotides are chosen accordingly.

Example 11

Tumor cells are contacted with at least one candidate inhibitor of nucleolar organisation polynucleotide. After incubation, in-situ staining for nucleophosmin (B23/NPM protein) is performed. Fractionated nucleoli indicate that the cell is sensitive to the respective candidate inhibitor of nucleolar organisation polynucleotide.

Example 12

Tumor cells are contacted with at least one candidate inhibitor of nucleolar organisation polynucleotide. After incubation, the amount of nascent rRNA is determined; one possible method therefor is presented in example 5, but other methods can be based on radioactive labeling of nascent RNAs or Real Time PCR -related methods. A reduced amount of nascent rRNA indicates that the cell is sensitive to the respective candidate inhibitor of nucleolar organisation polynucleotide.

Example 13:

Figure 15 shows the evolution in time of the changes in the nucleolar structure (as revealed by nucleophosmin staining, NPM) after alpha- amanitin treatment (inhibition of RNA polymerase II) and ASO-treatment targeting aluRNAs. First, the nucleoli are swelling and becoming clearly bigger. Finally the structure dispersed into numerous smaller particles throughout the nucleoplasm. In fact, structural aberrations are appearing already after 1 hour alpha- amanitin treatment and 6 hours ASO treatment.

Tumor cells were contacted with candidate inhibitor of nucleolar organisation polynucleotides as specified herein below, and, after incubation for 14 hours, the state of nucleolar structure was evaluated. Exemplary tumor cells used were DU145 cells (prostate cancer cells), HeLa cells (cervix cancer cells), MCF7 cells (breast cancer cells), U20S cells (bone cancer cells), SF188 cells (pediatric glioblastoma cells) and JOPACA cells (pancreatic cancer cells). All tumor cells tested showed increased numbers of abnormal and/or dispersed nucleoli after transfection of any of the five ASO-oligonucleotides (selected within the consensus regions as defined in Figure 12) as compared to transfection of a control oligonucleotide (Figure 16B- G). HUVEC cells (human umbilical vein cells) were used as control cells and showed only a minor increase in the number of abnormal nucleoli; dispersed nucleoli were not observed (Figure 16 H).

Example 14:

Using genomic integration of lacO arrays in U20S cells, it possible to recruit GFP-tagged proteins using a Lacl protein fused to a GFP-binder (GBP) as illustrated in Figure 13A (Chung et al. (2011), J Cell Sci. 124(Pt 21):3603-18). By tethering nucleolin (NCL) or nucleophosmin (NPM) to lacO arrays, an accumulation of endogenous aluRNAs at those loci is achieved (Figure 13B). This demonstrates the interaction of those two major nucleolar proteins with aluRNAs. As a control, another nucleolar proteins, known to interact with RNA (Mayer et al. (2006), Mol. Cell 22, 351), namely TIP5, does not interact with aluRNAs. This suggests that the interaction of aluRNAs with NCL and NPM is important for the structure and function of the nucleoli. Indeed, disruption of the nucleolus structure is achieved via NCL siRNAs (Ugrinova et al. (2007), BMC Mol Biol. 8:66) and NPM (Amin et al. (2008) Biochem J. 415(3):345) and a similar phenotype as the one observed after RNA polymerase II inhibition or aluRNA ASO treatment is obtained after simultaneous knockdown of NCL and NPM (Figure 14). Example 14 strongly supports the hypothesis that aluRNAs are involved in the maintenance of nucleolar structure and functions by forming complexes with NCL and NPM. The reduction of a critical proportion of either or several of the partners (aluRNAs, or NCL and NPM) results in strong and similar nucleolar structural and functional defects. According to the model of p53-dependent ribosomal-stress checkpoint, this will activate the p53-dependent apoptotic signaling pathway (Bywater et al. (2012), Cancer Cell 22:51; Boulon et al. (2010), Mol Cell 40:216).

Example 15

Alu-containing RNAs (aluRNAs) have a similar structure, as depicted in Figure 19 A, although the underlying sequences are homologous but different. On short portions, aluRNAs are very similar, but on their full length of about 300 nucleoltides, each Alu sequence is unique. The fact that most of the transcribed Alu repeats are transcribed by RNA polymerase II results in a strong link between the gene expression program of a cell and its aluRNA content (Wang & Huang (2014), loc. cit). It is therefore expected that each cell will present some specific aluRNA content. In consequence, a certain degree of liberty exists concerning the design of ASO to target the aluRNAs of various cell types. As observed on Figure 16, ASOs have variable effects from cells to cells (see for example position 3 in the various graphs in Figure 16).

Moreover, a second degree of liberty exists in the design of the ASO sequence. As illustrated in Table 1, for a same position, alternative sequences can be similarly efficient. As already discuss in the example 9, this certainly illustrates some variability in the pool of aluRNAs, which are targeted by each ASO, the important point to succeed with the ASO treatment being to reach a critical reduction of the aluRNA population within the cell nucleus. Thus, any ASO resulting in a critical decrease of the aluRNAs population will be a good ASO to kill a defined cell type by provoking nucleolar disruption and subsequent apoptosis induction (Bywater et al. (2012), Cancer Cell 22:51; Boulon et al. (2010), Mol Cell 40:216).

Example 16: Methods used in the work underlying the present invention

Cell culture

Wild-type HeLa, HeLa S3 and MCF7 cell lines were cultured as described (Caudron-Herger et al. (2011), "Coding RNAs with a non-coding function: maintenance of an open chromatin structure". Nucleus; 2, 410-424). For microscopy purposes, cells were grown on glass coverslips.

DU145 cells were cultured in RPMI like wild- type HeLa. U20S and U20S F6B2 cells were cultured in DMEM like MCF7 cells. U20S F6B2 cell line is described in (Jegou et al. (2009), Mol. Biol. Cell; 20, 2070-2082). SF188 pedriatric glioblastoma cells were cultured like MCF7 cells but using DMEM high glucose medium. JOPACA cells were cultured in IMDM medium. HUVEC cells were cultures as described (Melnik et al. (2011), Nature Methods; 8, 963-968).

FACS (fluorescence-activated cell sorting) Living cells were collected, counted and about 10e5 cells were labeled for 15 min with FITC Annexin V (BioLegend, #640905) and with TO-PR03 (Life Technologies, #T3605) in FACS buffer (10 mM Hepes pH 7.4, 2.5 mM CaCl₂ and 140 mM NaCl). Annexin V is used to target and specifically identify apoptotic cells. TO-PR03 is used to target and identify dead cells as it will enter and label the DNA within dead cells but not within living or apoptotic cells. A number of 20000 cells were analyzed on a flow cytometer for each sample in two or three replicates.

Plasmids

GFP-tagged proteins were generated by cloning the corresponding cDNAs into pEGFP-Cl (Life Technologies). The pEGFP-NCL vector was obtained from Addgene (#28176). pEGFP- NPM was kindly provided by Mitsuru Okuwaki (University of Tsukuba, Japan). The GFP- TAM-ATl+2wt (labeled as TIP5) and GBP-LacI-mRFP plasmids have been described (ZiUner et al. (2013), Nucleic Acids Res; 41, 5251-5262) and (Chung et al. (2011), Journal of cell science; 124, 3603-3618).

Drug treatment

For specific inhibition of RNA polymerase II activity, cells were treated with alpha- amanitin as described (ibd). For specific inhibition of RNA polymerase I activity, cells were similarly treated with actinomycin D (AMD) at a concentration of 50 ng/ml. For specific inhibition of RNA polymerase III, cells were treated with the inhibitor ML-60218 at a concentration of 54 μΜ. For inhibition of protein translation, cells were treated with cycloheximide (CHX) at a concentration of 50 μg/ml.

Immunofluorescence

For immunofluorescence staining, antibodies against C23 (#sc-56622, Santa Cruz biotechnology, inc), B23 (#sc-13057, Santa Cruz biotechnology, inc), UBF (#sc-13125, Santa Cruz biotechnology, inc) and RNA polymerase I (the serum was a gift from the Ingrid Grummt laboratory, DKFZ, Heidelberg, Germany) were used and it was continued as described (ibd).

Click chemistry

Cells were incubated as indicated with ethynyl uridine (EU) and further processed following the protocol of the manufacturer (Life Technologies, Germany, #C 10329). Microscopy

Laser scanning microscopy images were acquired as described (ibd). RNA isolation

Nucleoli from HeLa S3 cells were isolated following the procedure as described in (Sullivan et al. (2001), "Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli". Embo J 20: 2867-2874). RNA from nucleoli or cell pellets was purified as described (Caudron-Herger et al. (2011), "Coding RNAs with a non-coding function: maintenance of an open chromatin structure." Nucleus; 2, 410-424).

RNA sequencing

For high-throughput sequencing of the RNA samples, the libraries preparation and sequencing reactions were the same as described (ibd). For the nucleoli RNA fraction, the rRNA depletion step was omitted. For the total RNA preparation of non-treated and drug-treated HeLa S3 cells, the RNA samples were rRNA depleted as described (ibd).

RNA sequence analysis (RNA-Seq)

Reads were aligned with Bowtie (Langmead et al., "Ultrafast and memory-efficient alignment of short DNA sequences to the human genome". Genome biology, 10(3), R25) as indicated either on the GRCh37/hgl9 (2009) assembly version of the human genome or on the human rDNA gene (GenBank U13369) reporting all hits without mismatches, with and without trimming of the 3' and 5' ends. The respective position of each read was assessed from the resulting alignment and used for calculating the distribution of the reads within the intergenic spacer (IGS) of the rDNA gene. When several RNA samples were compared, the read distributions were normalized respectively to the total number of mapped reads for each sample.

To identify the RNA transcripts containing Alu element sequences, a list of human Alu repeats was produced using the RepeatMasker track in the Table Browser (www.genome.ucsc.edu). The function "intersect" of the genome arithmetic suite bedtools⁴³ was applied to list the transcripts overlapping with Alu repeats. Heatmaps were produced using the programs seqMINER (Ye, T. et al., Nucleic Acids Res 39, e35 (2011)) and ngs.plot (Shen, L., Shao, N., Liu, X. & Nestler, E. ngs.plot: An easy-to-use global visualization tool for next-generation sequencing data. Icahn School of Medicine at Mount Sinai, New York (2013)).

Anti-sense oligo (ASO) treatment

ASO were design as described (Ideue et al., "Efficient oligonucleotide-mediated degradation of nuclear noncoding RNAs in mammalian cultured cells". R A, 15(8), 1578-1587). They bear a Cy3-dye at their 5' end.

We used Lipofectamin (Life Technologies, Germany, #11668-019) to transfect the ASO at a final concentration of 40 nM and following the instruction of the manufacturer. After 14 hours or otherwise indicated (see Figure 15), the cells were fixed for 10 min in PBS containing 4% paraformaldehyde and further processed for immunofluorescence. If needed, the RNA transcription level was monitored, by click chemistry using 500 μΜ ethynyl uridine during the last 2 hours incubation. HUVEC cells were transfected with ASO via electroporation as those cells cannot be transfected efficiently using usual transfection reagents. siRNA

For siRNA-mediated knockdown of NCL and NPM, 1.5 x 10⁵ cells were transfected with Lipofectamin2000 and siRNAs against NCL (Dharmacon L-003854-00-0005) or NPM (Dharmacon L-015737-00-0005). On-TARGETplus Non-targeting Pool (Dharmacon, D- 001810-10-50) was used as control siRNA. 48 hours after transfection, cells were processed for immunofluorescence microscopy or for western blot analysis using antibodies against NCL (sc-13057), NPM (sc53175), or histone H3 (abl791, Abeam).

Northern blot

RNA was purified from unfractionated cells (Caudron-Herger, M. et al., Nucleus 2, 410-424 (2011)). Pre-rRNA was assayed by Northern blotting using a radiolabeled 5'-ETS antisense riboprobe (from +150 to +1) or by RT-qPCR as reported before (Hoppe, S. et al., Proc. Natl. Acad. Sci. USA 106, 17781-17786 (2009)).

RNA FISH (fluorescence in situ hybridization)

Cells grown on glass coverslips were incubated with CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgC12, 10 mM PIPES, 0.5% triton) containing either Vanadyl Ribonucleoside Complex (VRC) or RNase A for 5min. After a short wash in PBS, cells were fixed for 12 min in PBS containing 4% PFA. After a short PBS wash and an ethanol series (70%, 85% and 100%) of 3 min each, coverslips were air-dried. A drop of 2 to 5 μΐ of digoxigenin-labeled FISH probe (final concentration 50 ng/ml in 50% formamide, 10% Dextran and 2x SSC (3M NaCl, 300 mM Na-citrate pH 7.0)) was put on a slide and the coverslips up side down on top of it. Seal and incubate overnight at 37 °C in a wet chamber. On the next day, proceed to the following washing steps: (i) 2x 15 min in 2x SSC buffer containing 50% formamide, (ii) 10 min at 40°C in 0.2x SSC containing 0.1% Tween, (iii) 5 min in 2x SSC and (iv) 5 min PBS. The cells can then be further processed for immunofluorescence.

FISH probes used on Figure 7:

Control RNA: AAC CCT AAC CCT AAC CCT AAC CCT AAC CCT AAC CCT AAC CCT AAC CCT AA (SEQ ID NO: 23)

Alul: CTG TAG TCC CAG CTA CTC GGG AGG CTG AGG CAG GAG AAT CGC TTG AAC CC (SEQ ID NO: 24)

Alu2: TCA GTG GCT CAC GCC TGT CAT CCC AGC ACT TTG GGA GGC CGA GGC GGG CG (SEQ ID NO: 25).

Sequence alignment

Human Alu-repeat sequences were retrieved from the RepeatMasker (UCSC table browser). For each of the 3 Alu-families (Alus, AluY and AluJ), 20 sequences were selected and aligned using the free accessible alignment tool MultAlin (Corpet F et al., "Multiple sequence alignment with hierarchical clustering". Nucleic Acids Res. 1988 Nov 25;16(22): 10881-90.).

Claims

1. An inhibitor of nucleolus organisation, wherein said inhibitor of nucleolus organisation is a polynucleotide.

2. The inhibitor of nucleolus organisation of claim 1, wherein the inhibitor of nucleolus organisation inhibits expression or causes degradation of polynucleotides comprising at least 12 contiguous nucleotides of at least one of SEQ ID NO: l to 10 or of at least one of SEQ ID NO: 103 to 105; or wherein the inhibitor of nucleolus organisation inhibits expression or causes degradation of polynucleotides comprising at least 12 contiguous nucleotides of a reverse complement of at least one of SEQ ID NO: l to 10 or of at least one of SEQ ID NO: 103 to 105.

3. The inhibitor of nucleolus organisation of claim 1 or 2, wherein the inhibitor of nucleolus organisation comprises

i) a nucleotide sequence corresponding to the reverse complement of a stretch of 10 contiguous nucleotides at least 70% conserved between at least 90% of Alu sequences of the AluS family of sequences, of a stretch of 10 contiguous nucleotides at least 70% conserved between at least 90% of Alu sequences of the AluY family of sequences, or of a stretch of 10 contiguous nucleotides at least 70% conserved between at least 90% of Alu sequences of the AluJ family of sequences,

ii) a reverse complement of a nucleotide sequence according to i), and/or

iii) a nucleotide sequence being at least 70% identical to a nucleotide sequence of i) and/or ii).

4. The inhibitor of nucleolus organisation of claim 3, wherein the inhibitor of nucleolus organisation comprises a reverse complement of a nucleotide sequence corresponding to a stretch of 10 nucleotides at least 70% conserved between at least 90% of Alu sequences of the AluS family of sequences and/or a nucleotide sequence being at least 70% identical thereto, wherein the members of the AluS family are located on the human Chromosome 1 at nucleotide positions 39624-39924, 169374-169679, 101056- 101352, 247373-247669, 144606-144899, 174518-174820, 101822-102122, 167495- 167792, 76893-77201, 129999-130313, 102976-103280, 249293-249604, 175915- 176246, 111081-111386, 149396-149703, 163848-164153, 80805-81096, 165007- 165310, 164359-164695, and 168485-168786.

5. The inhibitor of nucleolus organisation of any one of claim 1 to 4, wherein the inhibitor of nucleolus organisation comprises

i) a nucleotide sequence corresponding to at least 12 contiguous nucleotides of at least one of SEQ ID NO: 1 to 10 or of at least one of SEQ ID NO: 103 to 105, ii) a reverse complement of a nucleotide sequence of i), and/or

6. The inhibitor of nucleolus organisation of any one of claims 1 to 5, wherein the inhibitor of nucleolus organisation comprises

i) a nucleotide sequence having at least one of SEQ ID NO: 11 to 22, or SEQ ID NO: 106, or of SEQ ID 229-237, and/or

ii) a nucleotide sequence being at least 70% identical to a nucleotide sequence of i).

7. The inhibitor of nucleolus organisation of any one of claims 1 to 6, wherein the inhibitor is an anti- sense oligo.

8. A vector comprising the inhibitor of nucleolus organisation of any one of claims 1 to 6.

9. An inhibitor of nucleolus organisation according to any one of claims 1 to 7 for use in medicine.

10. An inhibitor of nucleolus organisation according to any one of claims 1 to 7 for use in the treatment of cancer.

11. A host cell comprising the inhibitor of nucleolus organisation according to any one of claims 1 to 7 and/or the vector according to claim 8.

12. Use of an inhibitor of nucleolus organisation according to any one of claims 1 to 7 for the inhibition of cancer cell proliferation.

13. A device comprising the inhibitor of nucleolus organisation according to any one of claims 1 to 7, a vector according to claim 8, and/or a host cell according to 11.

14. A kit comprising the inhibitor of nucleolus organisation according to any one of claims 1 to 7, a vector according to claim 8, or a host cell according to claim 11 and/or an instruction manual.

15. A method of inhibiting cancer cell proliferation, comprising

a) contacting a cancer cell with an effective amount of inhibitor of nucleolus organisation according to any one of claims 1 to 7, and/or a vector according to claim 8, and

b) thereby inhibiting cancer cell proliferation.