WO2013153139A1

WO2013153139A1 - Methods for the treatment and diagnosis of acute leukemia

Info

Publication number: WO2013153139A1
Application number: PCT/EP2013/057530
Authority: WO
Inventors: Pierre BROUSSET; Wilfried VALLERON; Laure BERQUET
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université Paul Sabatier (Toulouse III)
Priority date: 2012-04-11
Filing date: 2013-04-11
Publication date: 2013-10-17

Abstract

The present invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising measuring in a sample obtained from said patient, at least one snoRNAs of the DLK1-DI03 locus. The invention relates also to a compound which inhibits the expression of a snoRNA of the DLK1-DI03 locus for use in the treatment of acute leukemia in a patient in need thereof.

Description

METHODS FOR THE TREATMENT AND DIAGNOSIS OF ACUTE LEUKEMIA

FIELD OF THE INVENTION:

BACKGROUND OF THE INVENTION:

In cancer cells, much effort has been devoted to the understanding of microRNA expression. Other classes of non-coding RNAs (ncRNAs) were originally considered products of constitutively-expressed housekeeping genes or representing transcriptional noise so until now have been largely neglected. Small nucleolar RNAs (snoRNAs) are 60 to 300 nucleo tide- long ncRNAs that are excised from the intron regions of pre-mRNAs, usually those encoding fundamental housekeeping proteins (Kiss T., 2002). There are two structurally distinct classes of snoRNAs, called the box C/D and H/ACA snoRNAs, which function mainly as guide RNAs in the site-specific 2 '-O-methylation and pseudouridylation of rRNAs, respectively (Kiss T., 2002). However, a subgroup of so-called "orphan" snoRNAs exists that lacks any apparent complementarity to cellular RNAs and therefore has unknown function. A few orphan snoRNAs, such as SNORD1 12 to SNORD1 16, are encoded by multiple, tandemly- arranged intronic genes that display high sequence similarity (Cavaille J. et al, 2000).

Recently, new unexpected regulatory functions have been described for human snoRNAs. In terms of pathologies, the human HBII-52 cluster that expresses several variants of the SNORD115 snoRNA was implicated in a neurodevelopmental disorder, the Prader- Willi syndrome (Gallagher RC. et al, 2002). Moreover, recent reports suggest that particular snoRNAs could be relevant to oncogenesis and solid cancer prognosis (Gee HE. et al, 2011). Despite, these studies, snoRNAS involvement in cancers, especially in leukemia has not been clearly demonstrated. SUMMARY OF THE INVENTION:

Thus, the invention relates to The present invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising measuring in a sample obtained from said patient, at least one snoR As of the DLK1-DI03 locus.

The invention relates also to a compound which inhibits the expression of a snoR A of the DLK1-DI03 locus for use in the treatment of acute leukemia in a patient in need thereof.

DETAILED DESCRIPTION OF THE INVENTION:

Definitions: Throughout the specification, several terms are employed and are defined in the following paragraphs.

As used herein, the term, snoRNAs for "Small nucleolar RNAs" denotes a class of small RNA molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs. There are two main classes of snoRNA, the C/D box snoRNAs which are associated with methylation, and the H/ACA box snoRNAs which are associated with pseudouridylation. snoRNAs are commonly referred to as guide RNAs but should not be confused with the guide RNAs that direct RNA editing in trypanosomes. All the snoRNAs pertaining to the invention are known per se and sequences of them are publicly available from the data bases http ://www-snorna.biotoul. fr/ index.php and http://www.ensembl.org. The snoRNAs of the invention are listed in Table A: snoRNAs snoRNAs Accession

number

SNORD112 (14q(0)) NR 003080

SNORD113-4 (14q(I-4)) NR_003232.1

SNORD113-6 (14q(I-6)) NR_003234.1

SNORD113-7 (14q(I-7)) NR_003235.1 SNORD113-8 (14q(I-8)) NR 003236.1

SNORD113-9 (14q(I-9)) NR_003237.1

SNORD114-1 (14q(II-l)) NR 003193.1

SNORD 114-3 (14q(II-3)) NR 003195.1

SNORD114-9 (14q(II-9)) NR 003201.1

SNORD 114-12 (14q(II- 12)) NR 003205.1

SNORD 114-14 (14q(II- 14)) NR 003207.1

SNORD114-17 (14q(II-17)) NR 003210.1

SNORD 114-22 (14q(II-22)) NR 003215.1

SNORD 114-26 (14q(II-26)) NR 003219.1

Table A: list of the snoRNAs according to the invention

As used herein, the term "patient" denotes a mammal. In a preferred embodiment of the invention, a patient according to the invention refers to any patient (preferably human) afflicted with or susceptible to be afflicted with acute leukemia.

Diagnostic method

A first aspect of the invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising measuring in a sample obtained from said patient, at least one snoRNAs of the DLK1-DI03 locus. In one embodiment, the invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising a step of measuring in a sample obtained from said patient the expression level of at least one snoRNAs selected from the groups consisting of SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113- 8, SNORD113-9, SNORD114-1, SNORD114-3, SNORD114-9, SNORD114-12, SNORD114-14, SNORD114-17, SNORD114-22 or SNORD114-26.

Typically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 snoRNAs are measured. In another embodiment, the invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising a step of measuring in a sample obtained from said patient the expression level of all snoR As of the group consisting of SNORD112, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9, SNORD 114-1.

In another embodiment, the invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising a step of measuring in a sample obtained from said patient the expression level of all the snoRNAs SNORD114-1.

In one embodiment, the acute leukemia may be an acute myeloblastic, an acute lymphoblastic or acute promyelocytic leukemia.

The method of the invention may further comprise a step consisting of comparing the expression level of at least one snoRNAs in the sample with a control, wherein detecting differential in the expression level of the snoRNAs between the sample and the control is indicative of having or a risk of having or developing an acute leukemia. The control may consist in sample associated with a healthy patient not afflicted with acute leukemia or in a sample associated with a patient afflicted with acute leukemia.

In one embodiment, hight expression level of at least one snoRNAs of the DLK1- DI03 locus is indicative of patient having or at risk of having or developing an acute leukemia. According to the invention, measuring the expression level of the snoRNAs of the invention in the sample obtained from the patient can be performed by a variety of techniques.

For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted snoRNAs is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR). Preferably quantitative or semiquantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

In a particular embodiment, the determination comprises contacting the sample with selective reagents such as probes or primers and thereby detecting the presence, or measuring the amount of snoRNAs originally in the sample. Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a snoRNAs array. The substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a detectable complex, such as a snoRNAs hybrid, to be formed between the reagent and the snoRNAs of the sample.

Nucleic acids exhibiting sequence complementarity or homology to the snoRNAs of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).

The probes and primers are "specific" to the snoRNAs they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

Accordingly, the present invention concerns the preparation and use of snoRNAs arrays or snoRNAs probe arrays, which are macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of snoRNAs molecules positioned on a support or support material in a spatially separated organization. Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted. Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters. Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non-identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter. Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of snoR As-complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample. A variety of different array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art. Useful substrates for arrays include nylon, glass, metal, plastic, latex, and silicon. Such arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like.

After an array or a set of snoR As probes is prepared and/or the snoRNAs in the sample or snoRNAs probe is labeled, the population of target nucleic acids is contacted with the array or probes under hybridization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity in view of the particular assay being performed. Suitable hybridization conditions are well known to those of skill in the art and reviewed in Sambrook et al. (2001). Of particular interest in many embodiments is the use of stringent conditions during hybridization. Stringent conditions are known to those of skill in the art.

Alternatively, snoRNAs quantification method may be performed by using stem-loop primers for reverse transcription (RT) followed by a real-time TaqMan® probe. Typically, said method comprises a first step wherein the stem-loop primers are annealed to snoRNAs targets and extended in the presence of reverse transcriptase. Then snoRNAs-specific forward primer, TaqMan® probe, and reverse primer are used for PCR reactions. Quantitation of snoRNAs is estimated based on measured CT values.

Many snoRNAs quantification assays are commercially available from Qiagen (S. A.

Courtaboeuf, France) or Applied Biosystems (Foster City, USA).

Expression level of a snoRNA may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a snoRNA by comparing its expression to the expression of a mRNA that is not a relevant for determining patient having or at risk of having or developing an acute leukemia, e.g., a housekeeping mRNA that is constitutively expressed. Suitable mRNA for normalization includes housekeeping mRNAs such as the 5S rRNA. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources. A further aspect of the invention relates to a method for monitoring the efficacy of a treatment for acute leukemia.

Methods of the invention can be applied for monitoring the treatment (e.g., drug compounds) of the patient. For example, the effectiveness of an agent to affect the expression level of the snoR As (as herein after described) according to the invention can be monitored during treatments of patients receiving acute leukemia treatments.

As used herein, "acute leukemia treatment" relates to any type of acute leukemia therapy undergone by the acute leukemia patients previously to collecting the cancerous tissue samples, including anticancer treatment.

Accordingly, the present invention relates to a method for monitoring the treatment of patient affected with an acute leukemia, said method comprising the steps consisting of:

i) diagnosis of acute leukemia before said treatment by performing the method of the invention

ii) diagnosis of acute leukemia after said treatment by performing the method of the invention

iii) and comparing the results determined a step i) with the results determined at step ii) wherein a difference between said results is indicative of the effectiveness of the treatment. In another aspect, the invention relates to a method for diagnosis acute leukemia in a patient comprising a step consisting of determining the expression level of at least one snoRNAs of the DLK1-DI03 locus.

In still another aspect, the invention relates to a method for diagnosis acute leukemia in a patient comprising a step consisting of determining the expression level of at least one snoRNAs selected from the group consisting of SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9, SNORD 114-1, SNORD114-3, SNORD114-9, SNORD114-12, SNORD114-14, SNORD114-17, SNORD114-22 or SNORD 114-26.in a sample obtained from said patient.

In a preferred embodiment, the acute leukemia may be an acute myeloblastic, an acute lymphoblastic or acute promyelocytic leukemia.

Typically, the sample according to the invention may be a blood, plasma, serum, lymph or bone marrow. In a preferred embodiment, said sample is blood or bone marrow.

Typically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 snoRNAS are measured. The term "detecting" as used above includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control. Typically snoR As expression level may be measured for example by RT-PCR performed on the sample.

Kits:

A further object of the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of the snoRNAs clusters of the invention in the sample obtained from the patient. The kits may include probes, primers macroarrays or microarrays as above described.

For example, the kit may comprise a set of snoRNAs probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers. The kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.

Alternatively the kit of the invention may comprise amplification primers (e.g. stem- loop primers) that may be pre-labelled or may contain an affinity purification or attachment moiety. The kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.

In a particular embodiment, the kit of the invention relates to a kit for identifying whether a patient has or is at risk of having or developing an acute leukemia, comprising means for measuring, in a sample obtained from said patient, at least one snoRNAs of the DLK1-DI03 locus.

In another particular embodiment, the kit of the invention relates to a kit for identifying whether a patient has or is at risk of having or developing an acute leukemia, comprising means for measuring, in a sample obtained from said patient, at least one snoRNAs selected from the group consisting of SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9, SNORD114-1, SNORD114-3, SNORD114-9, SNORD114-12, SNORD114-14, SNORD114-17, SNORD114-22 or SNORD114-26. In a particular embodiment, the kit of the invention relates to a kit which further comprise means for comparing the expression level of the snoRNAs in the sample with a control, wherein detecting differential in the expression level of the snoRNAs between the sample and the control is indicative of having, or a risk of having or developing an acute leukemia. The control may consist in sample associated with a healthy patient not afflicted with acute leukemia or in a sample associated with a patient afflicted with acute leukemia.

Therapeutic method:

A second aspect of the invention relates to a compound which inhibits the expression of a snoRNA of the DLK1-DI03 locus for use in the treatment of acute leukemia in a patient in need thereof. In one embodiment, the snoRNA to inhibit is selected from the group consisting of

SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9, SNORD 114-1, SNORD114-3, SNORD114-9, SNORD114-12, SNORD114-14, SNORD114- 17, SNORD 114-22 or SNORD 114-26.

In another embodiment, the snoRNA to inhibit is the SNORD 114-1.

As used herein, a compound which inhibits the expression of a snoRNA may be identifying by high throughput screening.

As used herein, the term "compound which inhibits the expression of a snoRNA" refers to any nucleotidic compound able to prevent the action of the selected snoRNA. The compound of the present invention is a compound that inhibits or reduces the activity of the selected snoRNA for example by inhibiting the production of the snoRNA selected. In this case, the production of selected the snoRNA in the organism after treatment is less than the amount produced prior to treatment. One skilled in the art can readily determine whether the selected snoRNA expression has been inhibited in the organism, using for example the techniques for determining snoRNA transcript level.

Suitable compounds according to the invention include double-stranded RNA (such as short- or small-interfering RNA or "siRNA"), antagosnoRNAs, antisense nucleic acids, and enzymatic RNA molecules such as ribozymes. Each of these compounds can be targeted to a given snoRNA and destroy or induce the destruction of the target snoRNA. For example, expression of a given snoRNA can be inhibited by inducing RNA interference of the snoRNA with an isolated double-stranded RNA ("dsRNA") molecule which has at least 90%, for example 95%, 98%, 99% or 100%, sequence homology with at least a portion of the snoRNA. In one embodiment, the dsRNA molecule is a "short or small interfering RNA" or "siRNA". siRNA useful in the present methods comprise short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length. The siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter "base-paired"). The sense strand comprises a nucleic acid sequence which is substantially identical to a nucleic acid sequence contained within the target snoRNA.

As used herein, a nucleic acid sequence in a siRNA which is "substantially identical" to a target sequence contained within the target mRNA is a nucleic acid sequence that is identical to the target sequence, or that differs from the target sequence by one or two nucleotides. The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area. The siRNA can also be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides.

One or both strands of the siRNA can also comprise a 3' overhang. As used herein, a

"3" overhang" refers to at least one unpaired nucleotide extending from the 3 '-end of a duplexed RNA strand. Thus, in one embodiment, the siRNA comprises at least one 3' overhang of 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length. In a preferred embodiment, the 3' overhang is present on both strands of the siRNA, and is 2 nucleotides in length. For example, each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu"). The siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in U.S. published patent application 2002/0173478 to Gewirtz and in U.S. published patent application 2004/0018176 to Reich et al, the entire disclosures of which are herein incorporated by reference.

Expression of a given snoRNA can also be inhibited by an antisense nucleic acid. As used herein, an "antisense nucleic acid" refers to a nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-peptide nucleic acid interactions, which alters the activity of the target RNA. Antisense nucleic acids suitable for use in the present methods are single-stranded nucleic acids (e.g., RNA, DNA, RNA-DNA chimeras, PNA) that generally comprise a nucleic acid sequence complementary to a contiguous nucleic acid sequence in a snoRNA. Preferably, the antisense nucleic acid comprises a nucleic acid sequence that is 50-100% complementary, more preferably 75-100% complementary, and most preferably 95-100% complementary to a contiguous nucleic acid sequence in an snoRNA. Nucleic acid sequences for the snoRNAs are provided in Table A. Without wishing to be bound by any theory, it is believed that the antisense nucleic acids activate RNase H or some other cellular nuclease that digests the snoRNA/antisense nucleic acid duplex.

In one embodiment the inhibitor is an antagosnoRNA and/or an antisense oligonucleotide.

The term "antagosnoRNA" as used herein refers to a chemically engineered small

RNA that is used to silence the selected snoRNA. The antagosnoRNA is complementary to the specific snoRNA target with either mis-pairing or some sort of base modification. AntagosnoRNAs may also include some sort of modification to make them more resistant to degradation. In one embodiment the antagosnoRNA is a chemically engineered cholesterol- conjugated single-stranded RNA analogue.

Inhibition of snoRNAs can also be achieved with antisense 2'-0-methyl (2'-0-Me) oligoribonucleotides, 2'-0-methoxyethyl (2'-0-MOE), phosphorothioates, locked nucleic acid (LNA), morpholino oligomers or by use of lentivirally or adenovirally expressed antagomirs (Stenvang and Kauppinen (2008), Expert Opin. Biol. Ther. 8(1):59-81). Furthermore, MOE (2'-0-methoxyethyl phosphorothioate) or LNA (locked nucleic acid (LNA) phosphorothioate chemistry)-modification of single-stranded RNA analogous can be used to inhibit snoRNA activity.

Antisense nucleic acids can also contain modifications of the nucleic acid backbone or of the sugar and base moieties (or their equivalent) to enhance target specificity, nuclease resistance, delivery or other properties related to efficacy of the molecule. Such modifications include cholesterol moieties, duplex intercalators such as acridine or the inclusion of one or more nuclease-resistant groups.

Antisense nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described below. Exemplary methods for producing and testing are within the skill in the art; see, e.g., Stein and Cheng (1993), Science 261 : 1004 and U.S. Pat. No. 5,849,902 to Woolf et al, the entire disclosures of which are herein incorporated by reference.

Expression of a given snoRNA can also be inhibited by an enzymatic nucleic acid. As used herein, an "enzymatic nucleic acid" refers to a nucleic acid comprising a substrate binding region that has complementarity to a contiguous nucleic acid sequence of a snoRNA, and which is able to specifically cleave the snoRNA. Preferably, the enzymatic nucleic acid substrate binding region is 50-100% complementary, more preferably 75-100% complementary, and most preferably 95-100% complementary to a contiguous nucleic acid sequence in a snoRNA. The enzymatic nucleic acids can also comprise modifications at the base, sugar, and/or phosphate groups. An exemplary enzymatic nucleic acid for use in the present methods is a ribozyme.

The enzymatic nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described below. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in Werner and Uhlenbeck (1995), Nucl. Acids Res. 23:2092-96; Hammann et al. (1999), Antisense and Nucleic Acid Drug Dev. 9:25-31; and U.S. Pat. No. 4,987,071 to Cech et al, the entire disclosures of which are herein incorporated by reference. The compound of the invention can be obtained using a number of standard techniques. For example the compound of the invention can be chemically synthesized or recombinantly produced using methods known in the art. Typically, the compound of the invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, 111., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK). In some embodiments, of the invention, a synthetic compound of the invention contains one or more design elements. These design elements include, but are not limited to: (i) a replacement group for the phosphate or hydroxyl of the nucleotide at the 5' terminus of the complementary region; (ii) one or more sugar modifications. In certain embodiments, a synthetic compound of the invention has a nucleotide at its 5' end of the complementary region in which the phosphate and/or hydroxyl group has been replaced with another chemical group (referred to as the "replacement design"). In some cases, the phosphate group is replaced, while in others, the hydroxyl group has been replaced. In particular embodiments, the replacement group is biotin, an amine group, a lower alkylamine group, an acetyl group, 2'0-Me (2'oxygen-methyl), DMTO (4,4'-dimethoxytrityl with oxygen), fluorescein, a thiol, or acridine, though other replacement groups are well known to those of skill in the art and can be used as well. In particular embodiments, the sugar modification is a 2'0-Me modification. In further embodiments, there is one or more sugar modifications in the first or last 2 to 4 residues of the complementary region or the first or last 4 to 6 residues of the complementary region.

In a particular embodiment, the compound of the invention is resistant to degradation by nucleases. One skilled in the art can readily synthesize nucleic acids which are nuclease resistant, for example by incorporating one or more ribonucleotides that are modified at the 2'-position into the snoRNAs. Suitable 2'-modified ribonucleotides include those modified at the 2'-position with fluoro, amino, alkyl, alkoxy, and O-allyl.

The present invention also relates to a vector comprising a compound according to the invention for use in the treatment of acute leukemia.

As used herein, a vector may be a plasmid or a viral vector which express a compound like a siRNA. Thus, as used herein, the vector denotes a DNA structure in which a specific sequence; like a sequence encoding a siRNA, is contained.

Alternatively, the compound of the invention can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing RNA from a plasmid include, e.g., the U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art. The recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the compound of the invention in the organism. The compound of the invention that is expressed from recombinant plasmids can be isolated from cultured cell expression systems by standard techniques. The compound of the invention which is expressed from recombinant plasmids can also be delivered to, and expressed directly in, the organism. The use of recombinant plasmids to deliver the compound of the invention to organism is discussed in more detail below.

The compound of the invention can be expressed from a separate recombinant plasmid, or can be expressed from a unique recombinant plasmid. Preferably, the compound of the invention is expressed as the nucleic acid precursor molecules from a single plasmid, and the precursor molecules are processed into the functional compound by a suitable processing system.. Other suitable processing systems include, e.g., the in vitro Drosophila cell lysate system as described in U.S. published application 2002/0086356 to Tuschl et al. and the E. coli RNAse III system described in U. S . published patent application 2004/0014113 to Yang et al, the entire disclosures of which are herein incorporated by reference.

Selection of plasmids suitable for expressing the compound of the invention, methods for inserting nucleic acid sequences into the plasmid to express the gene products, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art. See, for example, Zeng et al. (2002), Molecular Cell 9: 1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are herein incorporated by reference.

In one embodiment, a plasmid expressing the compound of the invention comprises a sequence encoding a compound precursor under the control of the CMV intermediate early promoter. As used herein, "under the control" of a promoter means that the nucleic acid sequences are located 3' of the promoter, so that the promoter can initiate transcription of the compound coding sequences.

The compound of the invention can also be expressed from recombinant viral vectors. It is contemplated that the compound of the invention can be expressed from separate recombinant viral vectors, or from a unique viral vector. The compound expressed from the recombinant viral vectors can either be isolated from cultured cell expression systems by standard techniques, or can be expressed directly in organism. The use of recombinant viral vectors to deliver the compound to organism is discussed in more detail below. The recombinant viral vectors of the invention comprise sequences encoding the compound of the invention and any suitable promoter for expressing the compound sequences. Suitable promoters include, for example, the U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the compound in organism.

Any viral vector capable of accepting the coding sequences for the compound of the invention can be used; for example, vectors derived from adenovirus (AV); adeno associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of the viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J. E. et al. (2002), J Virol 76:791801, the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing said compound of the invention into the vector, methods of delivering the viral vector to the cells of interest, and recovery of the expressed compound products are within the skill in the art. See, for example, Dornburg (1995), Gene Therap. 2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum. Gene Therap. 1 :5-14; and Anderson (1998), Nature 392:25-30, the entire disclosures of which are herein incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. A suitable AV vector for expressing the compound of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia et al. (2002), Nat. Biotech. 20: 1006-1010, the entire disclosure of which is herein incorporated by reference. Suitable AAV vectors for expressing the compound of the invention, methods for constructing the recombinant AAV vector, and methods for delivering the vectors into target cells are described in Samulski et al. (1987), J. Virol. 61 :3096-3101 ; Fisher et al. (1996), J. Virol, 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference. Preferably, the compound of the invention is expressed from a single recombinant AAV vector comprising the CMV intermediate early promoter.

In one embodiment, a recombinant AAV viral vector of the invention comprises a nucleic acid sequence encoding a compound precursor in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter. As used herein, "in operable connection with a polyT termination sequence" means that the nucleic acid sequences encoding the sense or antisense strands are immediately adjacent to the polyT termination signal in the 5' direction. During transcription of the compound sequences from the vector, the polyT termination signals act to terminate transcription. The compound according to the invention can be administered to a patient by any means suitable for delivering these compounds to the patient. For example, the compound can be administered by methods suitable to transfect cells of the patient with these compounds, or with nucleic acids comprising sequences encoding these compounds. Preferably, the cells are transfected with a plasmid or viral vector comprising sequences encoding at least one compound.

The compound can be administered to a patient by any suitable enteral or parenteral administration route. Suitable enteral administration routes for the present methods include, e.g., oral, rectal, or intranasal delivery. Suitable parenteral administration routes include, e.g., intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., intra-retinal injection, or subretinal injection); subcutaneous injection or deposition, including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation.

In the present methods, a compound can be administered to the patient either as naked RNA, in combination with a delivery reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising sequences that express the compound. Suitable delivery reagents include, e.g, the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), and liposomes.

Recombinant plasmids and viral vectors comprising sequences that express the compound, and techniques for delivering such plasmids and vectors to organism, are discussed above.

In a preferred embodiment, liposomes are used to deliver a compound (or nucleic acids comprising sequences encoding them) to a patient. Liposomes can also increase the blood half-life of the gene products or nucleic acids. Liposomes suitable for use in the invention can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half- life of the liposomes in the blood stream.

A variety of methods are known for preparing liposomes, for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which are herein incorporated by reference. The liposomes for use in the present methods can comprise a ligand molecule that targets the liposome to organism. Ligands which bind to receptors prevalent in organism, such as monoclonal antibodies that bind to organism antigens, are preferred. The liposomes for use in the present methods can also be modified so as to avoid clearance by the mononuclear macrophage system ("MMS") and reticuloendothelial system ("RES"). Such modified liposomes have opsonization-inhibition moieties on the surface or incorporated into the liposome structure. In a particularly preferred embodiment, a liposome of the invention can comprise both opsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer that significantly decreases the uptake of the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference. Opsonization inhibiting moieties suitable for modifying liposomes are preferably water- soluble polymers with a number-average molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N- vinyl pyrrolidone; linear, branched, or dendrimeric polyamido amines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; animated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups. Preferably, the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".

The opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl- ethanolamine lipid-soluble anchor, and then bound to a membrane. Similarly, a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive animation using Na(CN)BH3 and a solvent mixture, such as tetrahydrofuran and water in a 30: 12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes. Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" micro vasculature. Thus, tissue characterized by such microvasculature defects will efficiently accumulate these liposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., USA, 18:6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation of the liposomes in the liver and spleen. Thus, liposomes that are modified with opsonization-inhibition moieties are particularly suited to deliver the compound (or nucleic acids comprising sequences encoding them) to the organism. One skilled in the art can readily determine a therapeutically effective amount of said compound to be administered to a given patient, by taking into account factors such as the size and weight of the patient; the extent of disease penetration; the age, health and sex of the patient; the route of administration; and whether the administration is regional or systemic. An effective amount of said compound can be based on the approximate or estimated body weight of a patient to be treated. Preferably, such effective amounts are administered parenterally or enterally, as described herein. For example, an effective amount of the compound is administered to a patient can range from about 5-10000 micrograms/kg of body weight, and is preferably between about 5-3000 micrograms/kg of body weight, and is preferably between about 700-1000 micrograms/kg of body weight, and is more preferably greater than about 1000 micrograms/kg of body weight. One skilled in the art can also readily determine an appropriate dosage regimen for the administration of the compound to a given patient. For example, the compound can be administered to the patient once (e.g., as a single injection or deposition).

Another object of the invention relates to a method for treating acute leukemia comprising administering to a subject in need thereof a therapeutically effective amount of a compound as described above.

As used herein, the term "treating" or "treatment", denotes reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

In another embodiment, the acute leukemia may be an acute myeloblastic, an acute lymphoblastic or acute promyelocytic leukemia.

Pharmaceutical compositions:

The compound of the invention may be used or prepared in a pharmaceutical composition.

In one embodiment, the invention relates to a pharmaceutical composition comprising a compound as described above and a pharmaceutical acceptable carrier for use in the treatment of acute leukemia in a patient in need thereof. The compounds of the invention are preferably formulated as pharmaceutical compositions, prior to administering to a patient, according to techniques known in the art. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.

The present pharmaceutical formulations comprise compound (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically- acceptable carrier. The pharmaceutical formulations of the invention can also comprise compound which are encapsulated by liposomes and a pharmaceutically-acceptable carrier. Preferred pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3%> glycine, hyaluronic acid and the like.

Pharmaceutical compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.

For solid pharmaceutical compositions of the invention, conventional nontoxic solid pharmaceutically acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%), preferably 25%>-75%>, of the compound. A pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20%) by weight, preferably 1%>-10%> by weight, of the compound encapsulated in a liposome as described above, and a propellant. A carrier can also be included as desired; e.g., lecithin for intranasal delivery. Pharmaceutical compositions of the invention may include any further agent which is used in the prevention or treatment of acute leukemia. For example, the anti- acute leukemia may include an anticancer agent. For example, said anticancer agents include but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photo sensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, anthracyclines, MDR inhibitors and Ca2+ ATPase inhibitors.

Additional anticancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.

Additional anticancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.

In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron.

In another embodiment, the further therapeutic active agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.

In still another embodiment, the other therapeutic active agent can be an opioid or non-opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.

In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.

In one embodiment, said additional active agents may be contained in the same composition or administrated separately.

In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of acute leukemia.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES: Figure 1: The DLK1-DI03 locus is deregulated in APL samples. Inside the DLK1- DI03 locus (a), three classes of ncRNA (long ncRNA, microRNA and clustered snoRNA) are represented. Due to imprinting, the genes are either paternally (Pat) or maternally (Mat) expressed under the control of an intergenic differentially-methylated region (IG-DMR). The microRNA and snoRNA loci are represented by diagonally and vertically hatched boxes, (b) The signal intensities of DLK1-DI03 snoRNAs in AML samples assayed on GeneChip microRNA arrays demonstrated that only a small set of snoRNA was expressed. Shaded boxes represent signal noise from the chip and _st and x st designed two different probes for one snoRNA. Only the snoRNAs whose expression was significantly different from noise signal were represented on the graph, (c) The snoRNA signature obtained using microarrays was confirmed by qPCR in 8 APL (PR), 18 AML PML-RARalpha-negative (nPR) and 7 CD33+ myeloid controls, (d) We confirmed a specific DLK1-DI03 microRNA profile of APL samples by GeneChip microRNA array. Expression of microRNAs in 4 APL and 8 AML PML-RARa-negative samples was normalized to those of 4 CD33+ myeloid controls. Only significant changes in microRNA expression were presented on the graphs. The unpaired t-test was used to determine the statistical significance of the differences (p<0.05: *; p<0.01 : ** and p<0.001 : ***). (Each graph represents means ± SEM)

Figure 2: DLK1-DI03 snoRNA expression was lost by ATRA treatment and increased by PML-RARalpha. (a) SNORD112 [14q(0)], SNORD113-6 SNORD113-7, SNORD113-8 and SNORD113-9) [14q(I-6,-7,-8,-9)] and SNORD114-1 [14q(II-l)] expressions were monitored at days 3 and 5 by qPCR. Expression of snoRNAs in ATRA- treated cells was normalized to those under control conditions and expression fold changes were represented on the graph, (b) KG1 cells were transfected with a plasmid expressing PML-RARalpha. Expression of PML-RARalpha was checked by RT-PCR 48 and 72h after transfection. (c) Expression of 14q(0), 14q(I-6,-7,-8,-9), and [14q(II-l)] were monitored by qPCR until 48h after transfection. A paired t-test was used as the statistical test (p<0.05 : *; p<0.01 : **). (Each graph represents means ± SEM) Figure 3: The 14q(II-l) snoRNA variant impacts on cell growth and cell cycle, (a)

14q(II-l) [SNORD 114-1] was cloned and transfected into a K562 cell line. Its over- expression was verified by qPCR 48h after transfection. (b) To over-express the 14q(II-l) snoRNA, sequences containing the intron (in which the snoRNA sequence was present) and its flanking exon were cloned. We ensured that only the mature snoRNA sequence was over- expressed by RT-PCR for the snoRNA and intronic lariat sequences. "Blank" corresponds to RT-PCR on the mature snoRNA sequence using cDNA template from DLK1-DI03 negative cells. Cell growth of 14q(II-l) over-expressing cells was estimated using an MTS proliferation assay (c) and Malassez counting (d) and compared to cells transfected with the empty vector. Conversely, 14q(II-l) silencing by siRNA was realized and the effective targeting was checked by qPCR (e). (f) Cell growth was estimated 24 and 48h after transfection by Malassez counting. Paired t-tests were used as a statistical test (p<0.05 : *; p<0.01 : ** and p<0.001 : ***). (Each graph represents means ± SEM) Figure 4: 14q(II-l) snoRNA variant silencing induces a decrease in cell growth via the RB pathways, (a) 14q(II-l) snoRNA silencing by siRNA was realized in a K562 cell line. We first verified the decrease in cell growth 48h after transfection, then evaluated cell cycle distribution of transfected cells by IP staining and flow cytometry analysis. Graph represented means ± SEM of cell frequency in each phase of the cell cycle. Cell doublets were excluded by the PE-A/PE-W method. A paired t-test was used as the statistical test (p<0.05: *; p<0.01 : ** and p<0.001 : ***). (Each graph represents means ± SEM)

Figure 5: 14q(II-l) snoRNA effect on cell growth and Rb/pl6 pathways is confirmed in APL blasts, (a) Blasts from APL patients were transfected with siRNA- 14q(II-l) or siRNA negative control (siRNA-Neg). Effective targeting of the 14q(II-l) snoRNA was evaluated by qPCR and means ± SEM of expression fold change were represented on the graph, (b) Cell growth of APL blasts after 48h of siRNA treatment was estimated using Mallassez cell counting. Paired t-tests were used as the statistical test (p<0.05 : *; p<0.01 : ** and p<0.001 :

Table 1: Clinical and biological characteristics EXAMPLE:

Material & Methods Patients and Healthy donors

Fresh and thawed samples from acute myeloblasts leukemia (AML) or acute lymphoblastic leukemia (ALL) patients (Table 1) and from healthy donors (bone marrow, CD3+, CD 19+ and CD33+ cells) were obtained.

RNA preparation and cDNA synthesis

Total RNA was prepared by extraction with TRIzol® (Invitrogen) from mononuclear- sorted cells (CD33+, CD3+ and CD 19+) and fresh or thawed samples (from AML and ALL patients). RNA integrity was evaluated using the RNA 6000 Nano Chip kit (Agilent Technologies). Only RNAs with a RIN > 7.5 were used. RNAs were reverse-transcribed into cDNA using 3μΜ of random primer mix (Biolabs) and Superscript III reverse transcriptase (Invitrogen). For high throughput quantitative PCR, RNAs were polyadenylated by Poly(A) Polymerase Tailing Kit (EPICENTRE® Biotechnologies) before reverse transcription. GeneChip microRNA arrays

Four hundred nanograms of total RNA were hybridized on GeneChip microRNA array (Affymetrix). Unlogged data were treated with dChip software with a specific cut-off (filter) to generate dendrograms. The cut-off value of 0.5 applied to microRNAs could not be used for snoRNAs because their expression was lower, thus, we used a cut-off value of 0.25. Rows (corresponding to RNAs of interest) were standardized by respective RNA means of expression and distant metric system applied is correlation. Sample clustering was carried out by the centroid method. Significance analysis of microarrays (SAM) was used to analyze statistical significance of the results. High-throughput quantitative PCR

The Fluidigm high-throughput quantitative PCR method (Biomark) was used as previously described (Narsinh KH. et al, 2011). Data were analyzed as for qPCR using 5S rRNA as the housekeeping gene (GAPDH and ACTIN were also tested). Non- supervised clustering was realized using dChip software as for microarray analysis with a cut-off value of 0.85. The unpaired t-test was used for statistical analyses.

Quantitative PCR (qPCR) and data analysis

For real-time PCR, the 25 μΐ reaction contained lxSYBR Green PCR Master Mix (Sigma Aldrich), with each primer at 0.3μΜ, and 5μ1 of cDNA (see above) diluted at 1 :5. The primer efficiency (>85%) was checked prior to the experiments. 2-ACT (threshold cycle) values were used for the analysis (5S rRNA was selected as the housekeeping gene).

In vitro differentiation of Acute Promyelocytic Leukemia (APL) blast cells

APL blasts were cultured in the presence of ΙμΜ ATRA (Sigma) diluted in ethanol, or in and 5 days, the percentage of differentiated cells was assessed by cell morphology with May-Grunwald-Giemsa (MGG) stained cyto centrifuge slides and by expression of CD l ib as determined by flow cytometry. The monoclonal antibodies used for staining were: CDl lb- PE-Cy7 (BD Biosciences) and CD33-PE-Cy5 (BD Biosciences) and isotype-matched control conjugates. Flow cytometry was performed using a BD-LSRII flow cytometer (BD Biosciences).

PML-RARalpha transient transfection into a KG-1 cell line

A cDNA encoding PML-RARalpha (kindly provided by Valerie Lallemand- Breitenbach, Hospital St Louis, Paris) was cloned into an expression vector pCDNA3 (Invitrogen). KG-1 cells (ATCC CCL-246) were transfected with pCDNA3 or pCDNA3-P/R using nucleofection Amaxa Technology (Lonza) and Ingenio universal solution (Minis Bio LLC). SnoRNA expression was monitored by RT-qPCR after 48 and 72h of transfection. PML-RARalpha expression was monitored by RT-PCR using primers.

14q(II-l) over-expression and silencing

Sequences containing snoRNA (Figure 3b) were amplified by a Pfu-Ultra enzyme, sequenced and then cloned. The latter construct or empty vector were transiently transfected by AMAXA technology (Solution V, T16-program) in K562 cell line (ATCC CCL-243). Cell growth was evaluated by Malassez cell counting or MTS-Proliferation assay (Promega). Over-expression of 14q(II-l) snoRNA was checked by qPCR using primers designed.

14q(II-l) silencing in K562 cell line was realized using siRNA (siRNA-14q(II-l), Sigma; siRNA negative control, Eurogentec) transfection (25nM as a final siRNA concentration) using Lipofectamine RNAiMax (Invitrogen). Effective targeting after 48h was analysed by qPCR as described above. Total Rb and pi 6 protein expressions were evaluated by western blotting using a mouse anti-human total Rb monoclonal antibody (Sc-102), mouse anti-human pl6 (CDKN2A) antibody (Sc-9968) and anti-P-actin (MAB 150L). Rb phosphorylation on Ser780 was analysed using rabbit anti-human phosphor-ser780-Rb (Sc- 9307). Cell Cycle analysis

Cells were washed with PBS and fixed in cold 70% ethanol for 20min. Cells were then washed twice with PBS-0.1% BSA and once with PBS then labeled using propidium iodide staining for 30min (Invitrogen). Cell cycle distribution was evaluated by fluorescence analysis on a FACScan cytometer. Cell doublets were excluded and 20,000 events per condition were analyzed.

Results

Global down-regulation of snoRNAs in ALL and AML

To characterize specific snoRNA expression patterns in AML, we first used GeneChip microRNA arrays that allow the simultaneous screening of almost all snoRNAs and microRNAs that are listed in the ENSEMBL and snoRNABase databases (http://www.ensembl.org and http://www-snorna.biotoul.fr/). SnoRNA expression levels were screened in a cohort of twelve AML samples and compared to four CD33+ myeloid-sorted cells from healthy donors. By using non-supervised clustering, we observed a global down- regulation of snoRNAs compared to their non-neoplastic counterparts, thus distinguishing leukemic cells from normal cells (data not shown). To verify that these observations were robust, we used the same method to analyze acute lymphoblastic leukemia (ALL) samples matched with CD3/CD19-sorted cells. This confirmed a global snoRNA down-regulation which was the main characteristic differentiating healthy donors from cancer samples (data not shown). SnoRNA profiles from CD3+ and CD 19+ control samples were also analyzed and demonstrated that the latter can be regarded as a single control group (data not shown). To evaluate the accuracy of the snoRNA signatures, we also analyzed microRNA expression profiles and compared them to those reported in the literature. We successfully reproduced the microRNA signatures previously described for AML and ALL, confirming that our experimental design was robust (data not shown) (Marcucci G. et al, 2008).

To further confirm these results we used the BioMark digital PCR system (Fluidigm) for high-throughput expression studies on 62 selected snoRNAs, amongst which 46 significantly varied on microarrays. The remaining 16 snoRNAs, 5 guide and 11 orphan RNAs, did not vary on the microarrays. In addition, we also tested 4 spliceosomal small nuclear RNAs (U2, U4, U6 and U12) and two snoRNAs (U8 and U3) which are synthesized from independent genes. The 5S ribosomal RNA was used to normalize snoRNA expression due to its stability and length. We extended our RT-qPCR experiments to 26 AML cases. Non-supervised clustering indicated that the snoRNA expression profiles of the AML samples were different from those of CD33+ myeloid cells (data not shown), CD34+ progenitors or total bone marrow (data not shown). It is worth mentioning, however, that Flt3-ITD-mutated AML co-segregated with controls, suggesting that this group of leukemia is distinct with regard to snoRNA profiles (data not shown). To confirm the identity of snoRNA expression patterns obtained in ALL cases by microarray analysis, we also checked the delineation of ALL samples compared to "lymphoid controls" in a larger cohort (25 ALL cases). In leukemic samples, sixty-one percent of snoRNAs tested were significantly under-expressed compared to control cells (data not shown). This is in-keeping with the global under- expression reported for microRNAs in several types of cancers. Thus, RT-qPCR experiments validated the global down-regulation of snoRNA expression previously noticed on micro arrays (data not shown). Specific snoRNA expression profiles identify APL cases

Although snoRNAs appeared to be globally down-regulated in leukemia cells, few snoRNAs located into clusters are over-expressed in some AML cases. Their expression was associated or not to specific chromosomal translocations (data not shown). Thus, through microarray analysis we noticed ectopic expression of a set of snoRNAs from the DLK1-DI03 locus in APL samples carrying identical PML-RARalpha bcrl translocations (data not shown). DLK1-DI03 snoRNAs are located within the introns of the maternally expressed gene 8 (Meg8), which carries 1 , 9 and 31 highly related copies of SNORD 1 12 [14q(0)], SNORD113 [14q(I>] and SNORD114 [14q(II)] snoRNA genes respectively (Figure la). As expected (due to their restricted expression pattern), they were not detected in controls (healthy CD33+ myeloid cells) or in PML/RARa-negative AML samples (Figures lb and lc). Using microarrays, we observed that only eight sequence variants of SNORD114, five variants of SNORD113 and one SNORD112 were expressed in APL patients (Figure lb). Of note, the level of expression of these particular snoRNAs varied significantly within the same cluster.

DLK1-DI03 snoRNA expression profiles were confirmed by RT-qPCR (Figure lc).

Due to their high sequence homology, we selected only the snoRNAs for which we could design specific primers: SNORD112 [14q(0)], SNORD113-6 [14q(I-6)], SNORD113-7 [14q(I-7)], SNORD113-8 [14q(I-8)], SNORD113-9 [14q(I-9)], and SNORD114-1 [14q(II-l)]. We first tested SNORDl 13-3 whose expression did not vary when assayed by microarrays. RT-qPCR confirmed the lack of expression indicating that, despite high sequence homology of SNORD113 snoRNAs, the PCR-based quantification was specific.

Several micro RNA genes are located around the SNOR112-114 gene cluster in the DLK1-DI03 locus and seem to have the same expression pattern as their neighboring snoRNAs. To validate snoRNA results from microarrays in APL, we examined the expression of the micro RNAs from the DLK1-DI03 locus in APL and PML-RARalpha-negative cells using GeneChip microRNA arrays. When compared to AML samples, we confirmed the significant over-expression of hsa-miR-127, miR-370 and miR-154, as already reported in APL, as well as eleven other micro RNAs (Figure Id) (Dixon-Mclver A. et al, 2008).

PML-RARalpha impacts on DLK1-DI03 snoRNA expression.

To test whether snoRNA expression from the DLK1-DI03 locus is controlled by PML-RARa we treated leukemic blast cells from three APL patients in vitro with ATRA which directly targets PML-RARalpha, leading t o granulocytic differentiation. Blast differentiation was monitored by MGG staining and CD l ib over-expression (data not shown). RT-qPCR revealed that expression of the SNORD112, SNORD113 and SNORD114 gene cluster in the DLK1-DI03 locus decreased during differentiation (Figure 2a). Interestingly, levels of the six snoRNAs did not decrease at the same time. After three days of treatment, only SNORD113-8 [14q(I-8)] was significantly under-expressed. After five days, all snoRNAs were significantly decreased. These findings demonstrate that expression of orphan snoRNAs encoded within the DLK1-DI03 locus is linked to PML-RARalpha. Moreover, these data corroborate the notion that within the 14q polycistronic snoRNA clusters expression of individual snoRNAs is regulated in a surprisingly independent fashion. To further investigate whether the PML-RARa protein chimera influenced snoRNA expression in the DLK1-DI03 locus, we transfected the human AML Ml (non-APL) cell line KG-1 with a plasmid that expressed PML-RARalpha bcrl . At both two and three days after transfection, we verified expression of PML-RARalpha mRNA (Figure 2b) and measured the levels of SNORD112, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9 and SNORD114-1 snoRNAs by RT-qPCR. In non-transfected control KG-1 cells, there was a low level of basal expression of all these snoRNAs (<2-12 relative expression compared to 5S rRNA). Transient expression of PML-RARalpha resulted in an up-regulation of all the snoRNAs tested at 48h post-transfection (Figure 2c). Similar data were obtained when we expressed PML-RARalpha in KGl-a cells, an AML M0 (non-APL) cell line (data not shown). The SNORD114-1 [14q(II-l)] variant is implicated in cell growth through cell cycle regulation.

To test whether DLK1-DI03 snoRNAs played a role in APL pathogenesis, we selected the SNORD114-1 [14q(II-l)] variant to analyze its impact on cell growth. This variant was chosen because among the snoRNAs it displayed the highest expression of the locus and because expression of this variant was sustained the longest following treatment with ATRA. The 14q(II-l) variant was cloned and transiently trans fected into the K562 cell line which represents the best model described for studying the DLK1-DI03 locus (i.e., this locus is transcriptionally active in these cells). To up-regulate 14q(II-l) snoRNA, a vector was constructed that contained the intron hosting the snoRNA sequence and its flanking exons. RT-qPCR quantification of expression of the 14q(II-l) variant revealed a mean fold change of 57 in transfected cells (Figure 3a). We then sought to ensure that snoRNA processing from this construct was effective. RT-PCR was carried out with primers specific to the intronic lariat (the flanking sequences of snoRNA) or primers specific to the mature snoRNA sequence. Thus, we noticed a strong over-expression of the mature snoRNA sequence compared to that of the intron lariat (Figure 3b). Over-expression of the 14q(II-l) variant in the K562 cell line induced a significant increase in cell proliferation as detected with both an MTS cell proliferation assay (Figure 3c) and Malassez counting (Figure 3d). Conversely, snoRNA silencing carried out with 25nM of siRNA 14q(II-l) in K562 cells caused a 50% reduction in snoRNA expression (Figure 3e) and a significant decrease in cell growth at 24 and 48h (Figure 4f). We then looked at the cell cycle distribution in siRNA- treated cells and demonstrated that after 48h of treatment 14q(II-l) snoRNA targeting induced a significant increase of cells in G0/G1 phase and, conversely, a significant decrease in S phase (Figure 4a).

The Rb/ l6 pathways are involved in snoRNA-mediated cell growth.

The effect of 14q(II-l) snoRNA on cell growth and the cell cycle prompted us to focus on what signaling pathways were potentially activated. Indeed, in K562 cells important cell cycle regulators such as pl6(CDKN2A) and p53 are known to be inactivated. Therefore, we investigated the impact of 14q(II-l) snoRNA silencing on the Rb pathway using western blot analysis and noticed a clear-cut over-expression of the corresponding protein (data not shown). This result is in-keeping with those described above for cell cycle distribution. To validate these observations, we analyzed the effect of 14q(II-l) over-expression on Rb. As expected, we observed an under-expression of total Rb protein in 14q(II-l) over-expressing cells (data not shown). In addition, we noticed that the remaining Rb protein was hyper- phosphorylated on serine-780 compared to that under control conditions (data not shown), suggesting that cell cycle activity was increased. To test whether this effect on Rb was also observed in APL patients, 14q(II-l) silencing by siRNA was performed using APL blasts from five patients. We confirmed an effective targeting of the 14q(II-l) variant at the RNA level (Figure 5a). Interestingly, a cell growth decrease of 30% was also observed after 48h (Figure 5b). We therefore analyzed the expression of total Rb protein in APL blasts transfected with siRNA-14q(II-l) or an siRNA-negative control. In three of these patients we confirmed an increase in Rb expression after 14q(II-l) silencing (data not shown). In the two remaining patients, Rb expression was almost undetectable whereas p l 6 expression was detected. It has been previously reported that in cancer cells pl6 is often deleted or mutated and Rb/pl6 expressions are inversely regulated (27-29). In APL cells with a basal level of pl6, siRNA-14q(II-l) fosters the expression of the protein (data not shown). Similar to the K562 cell line, we could not find p53 expression (using western blot analysis with two different antibodies) in APL patients' cells. Overall, we demonstrated that 14q(II-l) snoRNA modulated cell growth through the Rb pathway in both a K562 cell line and in APL cells ex vivo. Interestingly, in some cases we observed that the pl6 pathway could be an alternative regulator of the cell cycle. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Cavaille J, Buiting K, Kiefmann M, Lalande M, Brannan CI, Horsthemke B, et al. Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization. Proc Natl Acad Sci U S A 2000 Dec 19; 97(26): 14311-14316.

Dixon-Mclver A, East P, Mein CA, Cazier JB, Molloy G, Chaplin T, et al. Distinctive patterns of microRNA expression associated with karyotype in acute myeloid leukaemia. PLoS One 2008; 3(5): e2141.

Gallagher RC, Pils B, Albalwi M, Francke U. Evidence for the role of PWCR1/HBII- 85 C/D box small nucleolar RNAs in Prader-Willi syndrome. Am J Hum Genet 2002 Sep; 71(3): 669-678. Gee HE, Buffa FM, Camps C, Ramachandran A, Leek R, Taylor M, et al. The small- nucleolar RNAs commonly used for micro RNA normalisation correlate with tumour pathology and prognosis. Br J Cancer 2011 Mar 15; 104(7): 1168-77

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Claims

CLAIMS:

1. A method of identifying a patient having or at risk of having or developing an acute leukemia, comprising measuring in a sample obtained from said patient, at least one snoRNAs of the DLK1-DI03 locus.

2. The method according to claim 1 wherein the snoRNASs are selected from the group consisting of SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113- 8, SNORD113-9, SNORD 114-1, SNORD114-3, SNORD114-9, SNORD114-12, SNORD114-14, SNORD114-17, SNORD114-22 or SNORD114-26.

3. The method according to claim 1 or 2 which further comprise a step consisting of comparing the expression level of the snoRNAs in the sample with a control, wherein detecting differential in the expression level of the snoRNAs between the sample and the control is indicative of having or a risk of having or developing an acute leukemia.

4. The method according to any claims 1 to 3 wherein the acute leukemia is an acute myeloblasts, an acute lymphoblastic leukemia or an acute promyelocytic leukemia.

5. The method according to any claims 1 to 4, for monitoring the efficacy of a treatment for acute leukemia.

6. A compound which inhibits the expression of a snoRNA of the DLK1-DI03 locus for use in the treatment of acute leukemia in a patient in need thereof.

7. The compound for use according to the claim 6 wherein the snoRNA to inhibit is selected from the group consisting of SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9, SNORD 114-1, SNORD114-3, SNORD 114-9, SNORD 114-12, SNORD 114-14, SNORD 114-17, SNORD 114-22 or SNORD114-26.

8. The compound for use according to the claim 7 wherein the snoRNA to inhibit is the SNORD 114-1.

9. The compound for use according to claims 6 to 8 wherein said compound is selected from the group consisting of double-stranded RNA, antagosnoR As, antisense nucleic acids and enzymatic RNA molecules.

10. The compound for use according to claims 6 to 9 wherein the acute leukemia is an acute myeloblastic or an acute lymphoblastic leukemia.

11. A vector comprising a compound according to any claims 6 to 10 for use in the treatment of acute leukemia in a patient in need thereof.

12. A pharmaceutical composition for use in the treatment of acute leukemia comprising a compound according to any one of claims 6 to 11 and a pharmaceutically acceptable carrier.

13. A method for treating acute leukemia comprising administering to a subject in need thereof a therapeutically effective amount of a compound which inhibits the expression of a snoRNA of the DLK1-DI03 locus.