SETDB1 antagonist, preferably mithramycin or a SETDB1 -specific R Ai, for use in the treatment of lung cancer in a subject having gene amplification of SETDB1. The invention also relates to a method for predicting the clinical response of a subject suffering from lung cancer to a treatment with a SETDB1 antagonist based on the amplification of the SETDB1 gene in a sample from said subject; and to a method for selecting a lung cancer patient for being treated with a SETDB1 antagonist.

BACKGROUND OF THE INVENTION Routine cancer management using chemotherapy, whether as definitive or adjuvant therapy, has improved patient's absolute survival when compared with non- chemotherapy control. However, not all the chemotherapeutic treatments available are suitable for all patients. The efficacy of chemotherapeutic drugs in patients suffering from cancer is influenced by the presence of certain genetic markers. Patients whose tumours have low probability to respond to a chemotherapeutic treatment may omit chemotherapy altogether or may be candidates for alternative treatments, avoiding unnecessary therapeutic side effects.

Therefore, there is a necessity for a personalized approach for the treatment of the disease, particularly in cancers such as lung cancer.

Lung cancer is one of the leading causes of worldwide death, and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, with 1.2 million new cases worldwide each year. NSCLC resulted in more than one million deaths worldwide in 2001 and is the leading cause of cancer-related mortality in both men and women (31% and 25%, respectively). The other main type of lung cancer, namely small-cell lung cancer (SCLC), is most often more rapidly and widely metastatic than non- small cell lung carcinoma and involves early hilar and mediastinal lymph nodes. Without treatment, SCLC has the most aggressive clinical course of any type of pulmonary tumor, with median survival from diagnosis of only 2 to 4 months.

Advanced lung cancers are treated with chemotherapy. There are a number of possible therapies for drug cancer but there are still many patients who receive chemotherapy from which they do not benefit, typically experiencing unnecessary toxicity and a negative impact on their quality of life. Therefore, the advent of the predictive value of any new biomarker is essential in order to improve the patient's outcome by supporting the fitting of lung cancer patient with the most effective personalized anticancer treatment available.

Gene amplification is a common mechanism in deregulated oncogene expression, mainly in solid tumours. The identification of amplified target genes is of great importance for cancer diagnosis and prognosis, and ultimately for designing targeted therapies. An example is the oncogene HER2 in breast cancer where the copy number amplification is associated to poor outcome and is a predictive factor of response to anti-HER2 therapy (Susini T et al. 2010. Gynecol. Oncol, 116(2): 234- 239).

Mithramcycin A was described to be capable of inhibiting the growth of human lung cancer cell lines CLl-5 and H647 and inhibiting the invasion phenotypes of CLl-5 cells. However, said effects are due to the ability of mithramyicin of reactivating SLIT2 expression by decreasing methylation of SLIT2 promoter, and to the inhibition of DNMT1 (Ruo-Kai Lin and Yi-Ching Wang. 2007. Bioformosa, 42, 55-62).

Watanabe H. et al. (Watanabe H. et al. 2008. Cancer Cell Int, 8: 15) discloses that expression of SETDB1 is increased in non-small cell lung cancer cell lines. However, SETDB1 silencing of immortalized and transformed human bronchial epithelial cells by siRNA treatment did not alter the cell growth rate or the DNA synthesis level and did not induce apoptosis, therefore suggesting that the inhibition of SETDB1 is not a suitable treatment for lung cancer.

However, there is still a need for further markers useful for predicting the response of lung cancer patients to a chemotherapeutic treatment.

BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the invention relates to a SETDBl antagonist for use in the treatment of lung cancer in a subject having SETDBl gene amplification.

In another aspect, the invention relates to an in vitro method for predicting the clinical response of a subject suffering from lung cancer to a treatment with a SETDBl antagonist comprising detecting whether the SETDBl gene is amplified in a sample from said subject, wherein amplification of the SETDBl gene is indicative of a good clinical response of the subject to the treatment with said antagonist.

In yet another aspect, the invention relates to an in vitro method for selecting a lung cancer patient for being treated with a SETDBl antagonist comprising detecting whether the SETDBl gene is amplified in a sample from said patient, wherein amplification of the SETDBl gene is indicative that the subject is a candidate for a treatment with a SETDBl antagonist.

In yet another aspect, the invention relates to the use of a reagent capable of detecting the amplification of SETDBl gene in a sample from a subject suffering from lung cancer for predicting the clinical response of said subject to a treatment with a SETDBl antagonist or for selecting a lung cancer patient for being treated with a SETDBl antagonist.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Determination of SETDBl gene amplification and its association with R A and protein overexpression in lung cancer cell lines. (A) Assessment of SETDBl copy number by quantitative genomic PCR. Amplification frequency of SETDBl (evaluated with SYBR Green) was calculated by the standard curve method using the 7900HT SDS program. To define an internal control gene chromosome lp36.23 was chosen because it is the least aneuploid region among our cell lines (PEX19 gene). DNA from normal lung was used as the reference standard. Results are reported as n-fold copy number increase relative to the PEX19 gene. Quantitative RT-PCR (B) and western blot (C) demonstrate higher levels of SETDBl mRNA and protein (abl2317, Abeam, Cambridge, UK), respectively, in amplified cancer cell lines (H1437, NCI- H1395 and DMS-273) than in unamplified cells. The internal control gene was ACTB (beta-actin). Figure 2. Growth-promoting effects of SETDB1 in lung cancer. (A) Stable down-regulation of the SETDB1 gene by short hairpins using two different target sequences for DMS-273 (clones A30/A31 and clone B32-63) and NCI-H1437 (clones A56-B and B46-9). (B) The short-hairpin SETDB1 -depleted cells were less viable in the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay than in the untransfected or scrambled-shRNA transfected cells (p-values obtained by the ANOVA test), h, hours. (C) The colony formation assay showed that DMS-273 and NCI-H1437 cells stably transfected with the shRNA against SETDB1 formed significantly fewer colonies than scrambled shRNA-transfected cells (p-values obtained by the ANOVA test). Data shown are means ± SD, n =3. (D) MTT and colony formation assays performed with pools of scrambled-shRNA and SETDB1 shRNA clones of DSM-273 and NCI-H1437 lung cancer cell lines. These experiments further verify the results obtained with individual clones. All p-values were obtained by the ANOVA test. For colony formation assays, data shown are means ± SD, n = 3. h, hours. (E) In the non-amplified lung cancer cell line NCI-H1299, stable depletion of SETDB1 by means of shRNA has no effect on cell growth in the MTT assay. Below, colony formation assays show that NCI-H1299 cells stably transfected with the shRNA against SETDB1 formed a similar number of colonies than scrambled shRNA-transfected cells. All p-values were obtained by the ANOVA test. For colony formation assays data shown are means ± SD, n = 3. h, hours; n.s, nonsignificance. (F) Effect of SETDB1 shRNA-mediated depletion on the growth of DMS-273 and NCI-H1437 xenografts in nude mice. Tumor volume was monitored over time and the tumor was excised and weighed at 30 days. There was a significant decrease in tumor weight in the SETDB1 shRNA stably transfected cells (p-values obtained by the ANOVA test). Data shown are means ± SD, n =10. n.s, nonsignificance; g, grams.

Figure 3. Impact of SETDB1 in invasiveness and chemo sensitivity. (A) Effect of SETDB1 on the invasion potential of A549 cells determined by the matrigel invasion assay. Cells were transfected with 3 μg of Flag-SETDBl or empty vector in 60 mm dishes. After 24 hours cells were stimulated or not with PMA plus Io (PMA/Io) for 30 minutes. Then cells were trypsinized, and 5xl0 ⁴ cells were resuspended in free-serum media and added to the upper compartment of a transwell coated with 1 mg/mL Matrigel. Media with 10% FBS was added in the lower compartment and cells were incubated at 37°C for 42 hours. Invasive cells were fixed with PBS 4% paraformaldehide, stained with 0.5% violet crystal and visualized and photographied under a 10X magnification objective with a microscope. Invasive cells were counted using ImageJ 1.45s (Wayne Rasband, National Institutes of Health, USA) and the percentage of invasive cells was represented. Results are the mean of at least three experiments by duplicate and the significance was determined using ANOVA-test. *p<0.05. PMA, phorbol 12-myristate 13-acetate; Io, ionophore A23187. (B) Cancer cells harboring the SETDB1 gene amplification are sensitive to the decrease in cell viability caused by mithramycin. MTT assays in control scrambled shRNA DMS-273- transfected cells in comparison with three shRNA stable downregulated SETDB1 clones (A21, A30 and A31) show enhanced inhibition of viability in cells with SETDB1 gene amplification-mediated overexpression. EC50: half maximal effective concentration.

Figure 4. SETDB1 target genes in DMS-273 and NCI-H1437 lung cancer cell lines. (A) As a control for the microarray experiments, qRT-PCR experiments were performed to confirm the down-regulation of SETDB1 by means of shRNA in DMS- 273 (mix of A30 A31 and B32-63 clones) and NCI-H1437 (mix of A56-B and B49-6 clones) in comparison to their scrambled- shRNA control cells. (B) Venn diagram illustrates the most upregulated genes in both cell lines upon SETDB1 depletion (Fold Change > 3). (C) Ten transcripts upregulated in both cell lines upon SETDB1 depletion (according to the microarrays) were further selected for validation using qRT-PCR. (D) For selected candidate genes quantitative chromatin immunoprecipitation assays (qChIP) using anti-H3K9me3, anti-H3 and anti-rabbit IgG antibodies were performed. ChIP levels are represented as percentage of input chromatin precipitated for each gene promoter region. The experiments depict a reduction of H3K9me3 in DMS-273 and NCI-H1437 cells stably transfected with shRNAs against SETDB1.

Figure 5. Effect of mithramycin on SETDB1 -amplified lung cancer cell lines. (A) Western blot experiments show that lung cancer cell lines harboring the SETDB1 amplification (DMS-273, NCI-H1437 and NCI-H1395) undergo a decrease of the protein upon mithramycin treatment. (B) Mithramycin diminishes the viability of SETDB1 -amplified DMS-273 cells. This experiment, an MTT assay performed with another scrambled-shRNA DMS-273 clone, further demonstrates a higher sensitivity of the scrambled-shRNA DMS-273-transfected cells compared with the 3 different shRNA SETDB 1 clones (A21, A30 and A31). (C) Similar results were obtained comparing a pool of control scrambled-shRNA DMS-273-transfected cells with a pool of SETDB 1- depleted clones (A21, A30, A31 and B32-63). EC ₅₀ : half maximal effective concentration.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have discovered that, surprisingly, the histone methyltransferase SETDB 1 undergoes gene amplification in non-small and small cell lung cancer cell lines and primary tumors and that the increased gene dosage of SETDB 1 is associated with enhanced sensitivity to the growth-inhibitory effect mediated by the drug mithramycin. The inventors have also found that the reduction of SETDB 1 expression in gene-amplified cells by using shRNA against SETDB 1 has cancer-growth inhibitory features. In this sense, SETDB 1 gene amplification has potential value as a genetic marker for predicting a good response to a SETDB 1 antagonist, preferably mithramycin or a SETDB 1 -specific RNAi, in lung cancer patients. Based on these findings, the inventors have developed the methods of the present invention in their different embodiments that will be described now in detail.

The results provided in the example of the present invention clearly show a significant association of SETDB 1 gene amplification with a good response to mithramycin or a SETDB 1 -specific RNAi. Thus, these results suggest that subjects suffering from lung cancer with SETDB 1 gene amplification are candidates for a treatment with a SETDB 1 antagonist, preferably with mithromycin or a SETDB 1- specific RNAi.

THERAPEUTIC METHODS OF THE INVENTION

The results obtained in the present invention show that SETDB 1 gene amplification in lung tumours indicates a high probability of a successful therapeutic treatment with a SETDB 1 antagonist, an indication that this may be the best treatment available for such patients. SETDBl antagonists would be the choice as first line treatment for patients with SETDBl gene amplification.

In another aspect, the invention relates to a SETDBl antagonist for use in the treatment of lung cancer in a subject having SETDBl gene amplification.

Alternatively, the invention relates to the use of a SETDBl antagonist for the manufacture of a medicament for the treatment of a subject suffering from lung cancer wherein said subject has SETDBl gene amplification.

Alternatively, the invention relates to a method for the treatment of lung cancer in a subject comprising administering to said subject of a SETDBl antagonist wherein said subject has SETDBl gene amplification.

The term "SETDBl gene", as used herein, refers to the gene which codes for the enzyme histone H3 lysine 9-specific methyltransferase (EC 2.1.1.43) which is involved in the transcriptional silencing of euchromatic genes and retro elements. The gene that encodes said histone methyltransferase (Gene ID: 9869, human) resides in a recurrently amplified chromosome lq21 interval. Said gene is also known as ESET, KMT1E, H3- K9-HMTase4, KG IT, TDRD21 or KIAA0067. The term "SETDBl gene" encompasses the SETDBl gene of any mammalian species, including but not being limited to domestic and farm animals (cows, horses, pigs, sheep, goats, dogs, cats or rodents), primates and humans. Preferably, the SETDBl gene is human. In the present invention "human SETDBl gene" is understood as the gene defined in the NCBI database, version October 30, 2012 (positions 150898815 to 150937220 in the genomic region shown in the NCBI database under accession number NC OOOOOl .10).

As used herein, the term "SETDBl antagonist" refers to any agent capable of inhibiting or decreasing the histone methyltransferase activity of SETDBl, including those agents which prevent the expression of the SETDBl gene, causing reduced levels of mRNA or SETDBl protein, as well as agents which inhibit SETDBl causing a decrease in the activity of the enzyme. Said agent can be any organic or inorganic molecule, including modified and unmodified nucleic acids such as antisense nucleic acids, RNA interference (RNAi) agents such as siRNA, shRNA or miRNA, peptides, proteins, peptidomimetics, receptors, ligands, antibodies and small organic molecules that are useful in treating lung cancer. The SETDBl antagonist inhibits the histone methyltransferase activity of SETDBl by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or even 100% in a subject compared to the SETDBl activity prior to, or in the absence of, administration of the agent.

The person skilled in the art knows how to determine if an agent is a SETDB 1 antagonist using standard assays for determining the mR A expression levels such as RT-PCR, RNA protection analysis, Northern procedure, in situ hybridization, microarray technology and the like.

The compounds which cause reduced levels of SETDBl protein can be identified using standard assays for determining the protein expression levels such as immunoblot or Western blot, ELISA (adsorption enzyme immunoanalysis), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS- ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of protein microarrays or biochip which include specific antibodies or assays based on colloidal precipitation in formats such as reagent strips.

The determination of the inhibiting capacity of the biological activity of

SETDBl is detected using standard assays to measure the activity of SETDBl histone methyltransferase. An assay to assess if a compound is a SETDBl antagonist suitable for the present invention consists of comparing the catalytic ability of SETDBl to methylate H3K9 before and after the addition of the compound. In one assay, wells of a multi-well plate are coated with an appropriate immobilized substrate, such as an assembled recombinant nucleosomes or a histone peptide (preferably including an H3K9-comprising target peptide for SETDBl) immobilised via biotin-streptavidin linkage. To each well is added a reaction solution comprising SETDBl, S-adenosyl- methionine co-factor and the compound to be tested. If the candidate compound acts as an inhibitor of SETDBl then methylation of amino acid residues on histone H3 (comprised within the nucleosome substrate) or on the H3 peptide can be reduced or prevented. The determination of methylation status of the lysine residues in the histone H3/peptide can be determined by use of an antibody that specifically binds to the methylated target lysine residue in histone H3 (i.e. H3K9me3). The antibody can be linked to a colour generating reaction, so as to form an ELISA-type assay. In this way, wells of the multi-well plate that show a colour reaction correspond to reactions where inhibition of SETDB1 has not occurred, whereas candidate compounds present in the unco loured wells are identified as candidate inhibitors of SETDB1 activity.

In a preferred embodiment the SETDB1 antagonist is an agent that silences said enzyme, preferably a SETDB1 -specific RNAi. The term "SETDB1 -specific RNAi" refers to an RNA interference (RNAi) agent such as siRNA, shRNA or miRNA capable of decreasing the expression of SETDB1 protein. An enzyme is silenced when its expression is decreased with respect to the expression basal levels by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%.

The RNAi has to be specific for the mRNA of SETDB1 ("SETDB1 -specific RNAi"). This refers to the capacity of the nucleic acids for binding specifically to their target sequence and not to other sequence. The specificity is validated by performing BLAST analysis, by the use of negative controls, and by determining changes in mRNA levels of unrelated genes.

The expression "basal levels" refers to the level of expression of a gene in a normal cell in conditions in which no manipulation of said cell has taken place.

The expression "expression levels" or its grammatical equivalent, as used herein, means a measurement of the amount of nucleic acid, e.g. RNA or mRNA, or protein of a gene in a cell, or alternatively, the level of activity of a gene or protein in said cell.

As the person skilled in the art understands, the expression levels of SETDB1 gene can be measured by determining the mRNA expression levels of said gene or by determining the protein levels encoded by said gene, i.e. the SETDB1 protein.

In order to measure the mRNA levels of the SETDB1 gene, the cell may be treated to physically, mechanically or chemically disrupt cell structures, to release intracellular components into an aqueous or organic solution to prepare nucleic acids for further analysis. The nucleic acids are extracted from the sample by procedures known to the skilled person using commercially available reagents. RNA is then extracted from frozen or fresh samples by any of the methods typical in the art, for example, Sambrook, J., et al., 2001. Molecular cloning: A Laboratory Manual, 3 ^rd ed., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3. Preferably, care is taken to avoid degradation of the RNA during the extraction process.

Determination of the levels of SETDB1 mRNA can be carried out by any method known in the art such as qPCR, northern blot, RNA dot blot, TaqMan®, tag based methods such as serial analysis of gene expression (SAGE) including variants such as LongSAGE and SuperSAGE, microarrays, nucleic acid sequence based amplification (NASBA), Fluorescence In Situ Hybridization, including variants such as Flow-FISH, qFiSH and double fusion fish (D-FISH) as described in WO2010030818, Femino et al. (Science, 1998, 280:585-590), Levsky et al. (Science, 2002, 297:836-840) or Raj et al. (PLoS Biology, 2006, 4:e309).

In order to normalize the values of mRNA expression among the different samples, it is possible to compare the expression levels of the mRNA of interest in the test samples with the expression of a control RNA. A "Control RNA" as used herein, relates to a RNA whose expression levels do not change or change only in limited amounts cells. Preferably, the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions. Examples of housekeeping genes for use in the present invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH, PSMB4, tubulin and β-actin.

The expression levels of the SETDB1 gene may also be measured by determining the protein levels encoded by said gene, i.e. the SETDB1 protein or variants thereof. Practically any conventional method can be used within the context of the present invention to quantify the levels of SETDB1 protein such as Western blot or immunohistochemistry.

The value obtained for the expression of SETDB1 nucleic acid or protein levels in a modified cell is then compared with basal levels of SETDB1 in an unmodified cell. This allows detecting a decrease in the expression of SETDB1 under basal levels. A decrease in the expression under basal levels, as used herein, refers to a change of expression of a given gene with respect to the expression basal levels of at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%.

The SETDB1 -specific RNAi can be a siRNA (small interfering RNA), which is a class of double-stranded RNA molecule, 20-25 nucleotides in length with phosphorylated 5 ' ends and hydroxylated 3 ' ends with two overhanging nucleotides that interferes with the expression of specific genes with complementary nucleotide sequence. siRNAs can be used in a naked form and incorporated in a vector as shRNA.

The SETDB1 -specific RNAi can be a shRNA (short hairpin RNA), which is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral vectors using polymerase II and polymerase III promoters.

siRNAs have been described in Brummelkamp et al, Science 296; 550-

553,2002, Jaque et al ., Nature 418; 435-438, 2002, Elbashir S. M. et al. (2001) Nature, 411 : 494-498, McCaffrey et al. (2002), Nature, 418: 38-39; Xia H. et al. (2002), Nat. Biotech. 20: 1006-1010, Novina et al. (2002), Nat. Med. 8: 681-686, and U.S. Application No. 20030198627.

In a preferred embodiment the SETDB1 -specific RNAi is a nucleic acid selected from the group consisting of a small interfering RNA (siRNA) and a short hairpin RNA (shRNA) specific for the mRNA of said enzyme.

The SETDB1 antagonist can be a small organic molecule. In a preferred embodiment the SETDB1 antagonist is mithramycin or a pharmaceutically acceptable salt, solvate, polymorph or cocrystal thereof. In a more preferred embodiment the SETDB1 antagonist is mithramycin or a pharmaceutically acceptable salt thereof.

"Mithramycin", as used herein, is also referred to as plicamycin or mithramycin A. It is an antineoplastic antibiotic produced by Streptomyces plicatus which acts as RNA synthesis inhibitor.

The chemical name of mithramycin is (lS)-5-deoxy-l-C-((2S,3S)-7-{[2,6- dideoxy-3-0-(2,6-dideoxy-P-D-arabino-hexopyranosyl)-P-D-arab ino- hexopyranosyl]oxy}-3-{[2,6-dideoxy-3-C-methyl-P-D-ribo-hexop yranosyl-(l→3)-2,6- dideoxy-P-D-arabino-hexopyranosyl-(l→3)-2,6-dideoxy-P-D-ar abino- hexopyranosyl]oxy } -5 , 10-dihydroxy-6-methyl-4-oxo- 1 ,2,3 ,4-tetrahydroanthracen-2-yl)- 1-O-methyl-D-xylulose and its chemical structure is represented by the formula depicted in Figure 3B.

The term "pharmaceutically acceptable" refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability.

The term "pharmaceutically acceptable salt" embraces salts with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids, for example and without limitation hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic, hydroiodic and nitric acid and organic acids, for example and without limitation citric, fumaric, maleic, malic, mandelic, ascorbic, oxalic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic, cyclohexylsulfamic (cyclamic) or p-toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases, for example and without limitation alkyl amines, arylalkyl amines and heterocyclic amines.

The term "solvate" according to this invention is to be understood as meaning any solid form of mithramycin which has another molecule attached to it via non- covalent bonding. Examples of solvates include hydrates and alcoholates, preferably C1-C6 alcoholates, e.g. methanolate.

The term "polymorph" according to this invention is to be understood as a specific crystalline form of a compound that can crystallize in different forms.

The term "cocrystal", as used herein, is to be understood as a crystalline structure composed of mithramycin and at least another component.

The terms "lung cancer" or "lung tumour" refer to the physiological condition in mammals characterized by unregulated cell growth in tissues of the lung. The term lung cancer is meant to refer to any cancer of the lung and includes non- small cell lung carcinomas and small cell lung carcinomas. In an embodiment, the lung cancer is non- small cell lung cancer (NSCLC). In another embodiment the lung cancer is small cell lung cancer (SCLC).

The term non-small cell lung cancer (NSCLC), as used herein, refers to a group of heterogeneous diseases grouped together because their prognosis and management is roughly identical and includes, according to the histologic classification of the World Health Organization/International Association for the Study of Lung Cancer (Travis WD et al. Histological typing of lung and pleural tumours. 3 ^rd ed. Berlin: Springer- Verlag, 1999):

(i) squamous cell carcinoma (SCC), accounting for 30% to 40% of NSCLC, starts in the larger breathing tubes but grows slower meaning that the size of these tumours varies on diagnosis.

(ii) adenocarcinoma is the most common subtype of NSCLC, accounting for 50%) to 60%o of NSCLC, which starts near the gas-exchanging surface of the lung and which includes a subtype, the bronchio alveolar carcinoma, which may have different responses to treatment.

(iii) large cell carcinoma is a fast-growing form that grows near the surface of the lung. It is primarily a diagnosis of exclusion, and when more investigation is done, it is usually reclassified to squamous cell carcinoma or adenocarcinoma.

(iv) adenosquamous carcinoma is a type of cancer that contains two types of cells: squamous cells (thin, flat cells that line certain organs) and gland-like cells.

(v) carcinomas with pleomorphic, sarcomatoid or sarcomatous elements. This is a group of rare tumours reflecting a continuum in histologic heterogeneity as well as epithelial and mesenchymal differentiation.

(vi) carcinoid tumour is a slow-growing neuroendocrine lung tumour and begins in cells that are capable of releasing a hormone in response to a stimulus provided by the nervous system.

(vii) carcinomas of salivary gland type begin in salivary gland cells located inside the large airways of the lung.

(viii) unclassified carcinomas include cancers that do not fit into any of the aforementioned lung cancer categories. In a particular embodiment the NSCLC is selected from squamous cell carcinoma of the lung, large cell carcinoma of the lung and adenocarcinoma of the lung.

The term small cell lung cancer (SCLC), as used herein, refers to a proliferation of small cells with unique and strict morphological features, containing dense neurosecretory granules which give this tumor an endocrine/paraneoplastic syndrome associated. Most cases arise in the larger airways (primary and secondary bronchi). These cancers grow quickly and spread early in the course of the disease.

The term "treat" or "treatment" refers to a therapeutic treatment, as well as a prophylactic or prevention method, wherein the goal is to prevent or reduce an unwanted physiological change or disease, such as lung cancer. Beneficial or desired clinical results include, but not limiting, release of symptoms, reduction of the length of the disease, stabilized pathological state (specifically not deteriorated), retardation in the disease's progression, improve of the pathological state and remission (both partial and total), both detectable and not detectable. "Treatment" can mean also to prolong survival, compared to the expected survival if the treatment is not applied. Those who need the treatment include those who are suffering from lung cancer and have SETDB1 gene amplification, as well as those with tendency to suffer from lung cancer.

The term "subject", as used herein, refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, e.g., human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats or rodents. Preferably, the subject is a male or female human of any age or race. In the context of the present invention, the subject is a subject suffering from lung cancer or previously diagnosed with lung cancer and having SETDB1 gene amplification. In a preferred embodiment the subject is a mammal, preferably a human being.

The term "gene amplification" (and variants such as "amplification of a gene") refer to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. The copies of the gene are not necessarily located in the same chromosome. The duplicated region (a stretch of amplified DNA) is often referred to as an "amplicon". The amount of the messenger R A (mR A) produced, i.e. the level of gene expression, also increases in proportion to the number of copies made of the particular gene. The gene amplification can be detected in any kind of sample. The term "sample", as used herein, relates to any sample which can be obtained from the subject and which contains genetic material. The present method can be applied to any kind of biological sample from a subject, such as a biopsy sample, tissue, cell or fluid (serum, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat, milk), brain extracts, samples obtained by bronchial lavage, bronchoscopy, fine needle aspiration biopsy (FNAB) and the like. In a particular embodiment, the subject has SETDB1 amplification in a tissue sample, preferably a lung tumour tissue sample. Said sample can be obtained by conventional methods, e.g., biopsy, by using methods well known to those of ordinary skill in the related medical arts. Methods for obtaining the sample from the biopsy include gross apportioning of a mass, or microdissection or other art- known cell-separation methods. Tumour cells can additionally be obtained from fine needle aspiration cytology. In a preferred embodiment samples are obtained by bronchial lavage. In another preferred embodiment samples are obtained by fine needle aspiration biopsy (FNAB) .

Generally, tissue samples are prepared by fixing and embedding the tissue in a medium (such as paraffin). In other examples, samples include a cell suspension which is prepared as a monolayer on a solid support (such as a glass slide) for example by smearing or centrifuging cells onto the solid support. In further examples, fresh frozen (for example, unfixed) tissue sections may be used in the methods disclosed herein.

In some examples an embedding medium is used. An embedding medium is an inert material in which tissues and/or cells are embedded to help preserve them for future analysis. Embedding also enables tissue samples to be sliced into thin sections. Embedding media include paraffin, celloidin, OCT™ compound, agar, plastics, or acrylics.

Many embedding media are hydrophobic; therefore, the inert material may need to be removed prior to histological or cytological analysis, which utilizes primarily hydrophilic reagents. The term deparaffinization or dewaxing is broadly used herein to refer to the partial or complete removal of any type of embedding medium from a biological sample. For example, paraffin-embedded tissue sections are dewaxed by passage through organic solvents, such as toluene, xylene, limonene, or other suitable solvents. The process of fixing a sample can vary. Fixing a tissue sample preserves cells and tissue constituents in as close to a life-like state as possible and allows them to undergo preparative procedures without significant change. Fixation arrests the autolysis and bacterial decomposition processes that begin upon cell death, and stabilizes the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing, such as for in situ hybridisation (ISH).

Tissues can be fixed by any suitable process, including perfusion or by submersion in a fixative. Fixatives can be classified as cross-linking agents (such as aldehydes, e.g., formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non- aldehyde cross-linking agents), oxidizing agents (e.g., metallic ions and complexes, such as osmium tetroxide and chromic acid), protein-denaturing agents (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy's fixative, methacarn, Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid), microwaves, and miscellaneous fixatives (e.g., excluded volume fixation and vapor fixation). Additives may also be included in the fixative, such as buffers, detergents, tannic acid, phenol, metal salts (such as zinc chloride, zinc sulfate, and lithium salts), and lanthanum.

The most commonly used fixative in preparing samples for immunohistochemistry (IHC) is formaldehyde, generally in the form of a formalin solution (4 percent formaldehyde in a buffer solution, referred to as 10 percent buffered formalin). In one example, the fixative is 10 percent neutral buffered formalin.

PREDICTIVE METHODS OF THE INVENTION

The authors of the present invention have observed that amplification of the SETDBl gene is indicative of a good response to a SETDBl -specific shRNA and to mithramycin in SETDBl -amplified lung cancer cell lines. The example of the present invention describes that mithramycin diminishes the viability of SETDBl -amplified lung cancer cell lines.

Thus, in another aspect, the invention relates to an in vitro method for predicting the clinical response of a subject suffering from lung cancer to a treatment with a SETDBl antagonist comprising detecting whether the SETDBl gene is amplified in a sample from said subject, wherein amplification of the SETDB1 gene is indicative of a good clinical response of the subject to the treatment with said antagonist.

The term "predicting", as used herein, refers to the determination of the likelihood that the subject suffering from lung cancer will respond favorably to a therapy with a SETDB1 antagonist. Especially, the term "prediction", as used herein, relates to an individual assessment of the expected response of a subject suffering from lung cancer if the tumour is treated with a SETDB 1 antagonist.

The term "clinical response", as used herein, refers to the response of the subject suffering from lung cancer to a SETDB 1 antagonist. Standard criteria (Miller, et al. Cancer, 1981; 47:207-14) that can be used herewith to evaluate the response to chemotherapy include response, stabilization and progression.

The term "response", as used herein, can be a complete response (or complete remission) which is the disappearance of all detectable malignant disease or a partial response which is defined as approximately >50% decrease in the sum of products of the largest perpendicular diameters of one or more lesions (tumour lesions), no new lesions and no progression of any lesion. Subjects achieving complete or partial response were considered "responders", and all other subjects were considered "non- responders". As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for all (i.e. 100 percent) of the subjects to be identified. The term, however, requires that a statistically significant portion of subjects can be identified (e.g. a cohort in a cohort study). 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. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley and Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%), at least 90 percent, at least 95 percent, at least 97 percent, at least 98 percent or at least 99 percent. The p- values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60 percent, at least 70 percent, at least 80 percent or at least 90 percent of the subjects of a population can be properly identified by the method of the present invention. The term "stabilization", as used herein, is defined as a <50% decrease or a <25% increase in tumour size.

The term "progression", as used herein, is defined as an increase in the size of tumour lesions by >25% or appearance of new lesions.

Any other parameter which is widely accepted for comparing the efficacy of alternative treatments can be used for determining a response to treatment and include, without limitation:

• disease-free progression which, as used herein, describes the proportion of subjects in complete remission who have had no recurrence of disease during the time period under study.

• disease-free survival (DFS), as used herewith, is understood as the length of time after treatment for a disease during which a subject survives with no sign of the disease.

• objective response which, as used in the present invention, describes the proportion of treated subjects in whom a complete or partial response is observed.

• tumour control which, as used in the present invention, relates to the proportion of treated subjects in whom complete response, partial response, minor response or stable disease > 6 months is observed.

· progression free survival which, as used herein, is defined as the time from start of treatment to the first measurement of cancer growth.

• Time to progression (TTP), as used herein, relates to the time after a disease is treated until the disease starts to get worse. The term "progression" has been previously defined.

· six-month progression free survival or "PFS6" rate which, as used herein, relates to the percentage of subjects wherein free of progression in the first six months after the initiation of the therapy and

• median survival which, as used herein, relates to the time at which half of the subjects enrolled in the study are still alive.

The method of the second aspect of the invention comprises detecting whether the SETDB1 gene is amplified in a sample from said subject. In a preferred embodiment detecting the amplification of the SETDB1 gene comprises determining the copy number of the SETDB1 gene.

The term "copy number", as used herein, refers to the number of copies of a nucleic acid molecule in a cell. The copy number includes the number of copies of one or more genes or portions thereof in genomic (chromosomal) DNA of a cell. In a normal cell (such as a non- tumour cell), the copy number of a gene (or any genomic DNA) is usually about two (one copy on each member of a chromosome pair). In some examples, the copy number of a gene or nucleic acid molecule includes an average copy number taken from a population of cells.

Methods of determining the copy number of a gene or chromosomal region are well known to those of skill in the art. Said methods include, without limitation, in situ hybridisation (such as fluorescent, chromogenic, or silver in situ hybridisation), comparative genomic hybridisation, or polymerase chain reaction (such as real-time quantitative PCR). In a particular embodiment, the copy number of the SETDB1 gene is determined by in situ hybridisation (ISH), such as fluorescence in situ hybridisation (FISH), chromogenic in situ hybridisation (CISH), or silver in situ hybridisation (SISH). In a preferred embodiment, the copy number of the SETDB1 gene is determined by FISH.

The term "fluorescent in situ hybridisation" or FISH, as used in the present invention, refers to a cytogenetic technique that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. As the probe for FISH, a DNA fragment, a PCR product, a cDNA, a PAC clone or a BAC clone (each of which has a sequence of interest) may be used. In a typical method using FISH, a DNA probe (a CHKA probe) is labeled with a fluorescent dye or a hapten, typically in the form of fluor-dUTP, digoxigenin-dUTP, biotin-dUTP or hapten-dUTP that is incorporated into the DNA using enzymatic reactions, such as nick translation or PCR. The sample containing the genetic material (chromosome samples) may be smears on slide glass prepared from cultured cells of an isolated cancer. Alternatively, chromosome samples may be slide samples sliced from formalin- fixed, paraffin-embedded cancer-containing tissue blocks. After hardening for prevention of falling off from the slide glass during hybridisation process, chromosome sample slides are denatured by formamide treatment. The labeled probe is then hybridized to the sample containing genetic material on slides under appropriate conditions, which will be determined by the skilled person in the art. After hybridisation, the labeled sample is visualized either directly (in the case of a fluor- labeled probe) or indirectly (using fluorescently labeled antibodies to detect a hapten- labeled probe).

In the case of CISH, the probe is labeled with digoxigenin, biotin, or fluorescein and hybridised to the sample containing genetic material (chromosome or nuclear preparations) under appropriate conditions. The probe is detected with an anti- digoxigenin, -biotin or -fluorescein antibody, which is either conjugated to an enzyme (such as horseradish peroxidase or alkaline phosphatase) that produces a coloured product at the site of the hybridized probe in the presence of an appropriate substrate (such as DAB, NBT/BCIP, etc.), or with a secondary antibody conjugated to the enzyme. SISH is similar to CISH, except that the enzyme (such as horseradish peroxidase) conjugated to the antibody (either anti- hapten antibody or a secondary antibody) catalyzes deposition of metal nanoparticles (such as silver or gold) at the site of the hybridized probe. For any ISH method, SETDB1 gene copy number may be determined by counting the number of fluorescent, coloured, or silver spots on the chromosome or nucleus.

Any label that can be attached to a nucleic acid molecule (such as SETDB1 nucleic acid) can be used to label the SETDB1 probes, thereby permitting detection of the nucleic acid molecule. Examples of labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labelling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1998).

In a preferred embodiment the determination of the copy number of the SETDB1 gene is carried out by in situ hybridisation.

In situ hybridisation comprises contacting a sample containing genetic material with a polynucleotide under conditions adequate for the hybridisation of the polynucleotide with the SETDB 1 gene or genes present in the sample and determining the copy number of the SETDB 1 gene based on the hybridisation of the polynucleotide with the sample containing genetic material.

The "conditions adequate for the hybridisation" will be adjusted by a skilled person in the art and will vary depending upon the nature of the hybridisation method and the composition and length of the hybridizing nucleic acid sequences of the composition of the invention. Generally, the temperature of hybridisation and the ionic strength (such as the sodium concentration) of the hybridisation buffer will determine the stringency of hybridisation. Calculations regarding hybridisation conditions for attaining particular degrees of stringency are discussed in Sambrook et αί, (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY (chapters 9 and 11) and in Ausubel et ah, Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley and Sons, 1999.

The authors have identified a genomic fragment that allows the detection of the amplification of the SETDB 1 gene. Thus, in a more preferred embodiment the in situ hybridisation is carried out with a polynucleotide comprising the human genomic fragment present in the RP11-42A12 BAC (SEQ ID NO: 1) or a variant thereof which hybridizes under stringent conditions to said polynucleotide.

The BAC is hereinafter referred using the NCBI-recommended clone nomenclature. For instance, the term "RP11-42A12" indicates a clone in BAC library "RPH". More precisely, it is a clone found in microtiter dish "42" at the intersection of row A and column "12".

The term "RP11-42A12" corresponds to a BAC which comprises a region of chromosome 1 having a length of 156757 bp and starting at position 150878829 and ending at position 151035585 according to the sequence of chromosome 1 in the USC Human Genome Browser (GRCh37/hgl9 Assembly of Feb. 2009). Its nucleotide sequence is shown in SEQ ID NO: 1.

The term "polynucleotide", as used herein, shall mean any single or double stranded nucleotide polymer of genomic origin or some combination thereof, which by virtue of its origin the isolated polynucleotide (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

The term "human genomic fragment", as used in the present invention refers to a specific fragment comprising a gene or a fragment of a gene of human origin or a variant thereof. A person skilled in the art will understand that the present invention relates not only to the genomic fragments mentioned in the present invention, but also to variants thereof. A person skilled in the art will also understand that the variant of the human genomic fragment can be of any mammal, such as simians, mice, rats, pigs, dogs, rabbits, cats, cows, horses and goats.

The term "variant of the human genomic fragment" according to the present invention relates to any polynucleotide in which one or more nucleotides of the polynucleotides according to the invention are deleted, added or substituted. As is known in the state of the art, the identity between two nucleotide sequences is determined by comparing the sequence of a first and a second nucleic acid. Variants according to the present invention include nucleic acid sequences presenting at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90% or 95% similarity or identity with the original nucleotide sequence. The degree of identity between two nucleic acids is determined by using computational algorithms and methods which are widely known by the persons skilled in the art. The identity between two nucleic acid sequences will preferably be determined by using the BLAST algorithm [BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990)].

The variant of the human genomic fragments include fragments which preserve the capacity to hybridize under stringent conditions to the polynucleotides comprising the human genomic fragments present in the BAC as mentioned above.

"Stringent hybridisation" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of a hybrid is a function of ionic strength, temperature, G/C content, and the presence of chaotropic agents. The Tm values for polynucleotides can be calculated using known methods for predicting melting temperatures (see, e.g., Baldino et al, Methods Enzymology 168:761-777; Bolton et al, 1962, Proc. Natl. Acad. Sci. USA 48: 1390; Bresslauer et al, 1986, Proc. Natl. Acad. Sci USA 83:8893-8897; Freier et al, 1986, Proc. Natl. Acad. Sci USA 83:9373-9377; Kierzek et al, Biochemistry 25:7840- 7846; Rychlik et al, 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, Nucleic Acids Res 19:698; Sambmok et al, ad supra); Suggs et al, 1981, In Developmental Biology Using Purified Genes (Brown et al, eds.), pp. 683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol 26:227-259. All publications are incorporated herein by reference). In some embodiments, the polynucleotide encodes the polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence comprising the genomic fragment present in the BAC as defined above.

The term "moderately stringent conditions" as used herein refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60 percent identity, preferably about 75 percent identity, about 85 percent identity to the target DNA, with greater than about 90 percent identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridisation in 50% formamide, 5X Denhardfs solution, 5X SSPE, 0.2 % SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 42°C.

The term "highly stringent conditions", as used herein refers generally to conditions that are about 10 degrees centigrade or less from the thermal melting temperature Tm as determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein). Exemplary high stringency conditions can be provided, for example, by hybridisation in conditions equivalent to 50% formamide, 5X Denhardfs solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.1X SSPE, and 0.1% SDS at 65 °C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1%) (w:v) SDS at 65°C degrees centigrade and washing in 0.1X SSC containing 0.1 %> SDS at 65°C.

Other high stringency hybridisation conditions, as well as moderately stringent conditions, are described in the references cited above. In a preferred embodiment, the human genomic fragments are found within cloning vehicles in order to allow their propagation. The skilled person will understand that the cloning vehicles are those which can accommodate human genomic fragments having the size of the genomic fragments according to the invention. Thus, preferably, the cloning vehicle is an artificial chromosome. The cloning vehicle can comprise an artificial chromosome comprising a bacterial artificial chromosome (BAC), a bacteriophage PI -derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC). In a preferred embodiment, the human genomic fragments are provided in BACs.

The term "bacterial artificial chromosome" (BAC), as used herein, refers to a

DNA construct, based on a functional fertility plasmid (or F-plasmid), which has a low copy number because of the strict control of replication, used for transforming and cloning in bacteria, usually E. coli. The BAC's usual insert size is 150-350 kbp in which medium-sized segments of DNA (100,000 to 300,000 bases in length) that come from another species are cloned into bacteria. Once the foreign DNA has been cloned into the bacteria's chromosome, many copies of it can be made (amplified) and sequenced. In addition, BAC libraries of human genomic DNA have more complete and accurate representation of the human genome than libraries in cosmids or yeast artificial chromosomes. BACs are described in further detail in U.S. Application Nos. 10/659,034 and 61/012,701, which are hereby incorporated by reference in their entireties.

The BACs are commercially available, for example, the RP11 BAC library is available from the BAC Resources PAC (Children's Hospital Oakland Research Institute), from Empire Genomics, imaGenes or Invitrogen.

In another example, SETDB 1 gene copy number is determined by comparative genomic hybridisation (CGH). See, e.g., Kallioniemi et al, Science 258:818-821, 1992; U.S. Pat. Nos. 5,665,549 and 5,721,098. In one example, CGH includes the following steps. DNA from tumour tissue (such as a lung cancer sample) and from normal control tissue (reference, such as a non-tumour sample) is labeled with different detectable labels, such as two different fluorophores. After mixing tumour and reference DNA along with unlabeled human Cot-1 DNA (placental DNA that is enriched for repetitive DNA sequences such as the Alu and Kpn family) to suppress repetitive DNA sequences, the mix is hybridized to normal metaphase chromosomes. The fluorescence intensity ratio along the chromosomes is used to evaluate regions of DNA gain or loss in the tumour sample. In a further example, SETDBl gene copy number is determined by array CGH (aCGH). See, e.g., Pinkel and Albertson, Nat. Genet. 37:S11-S17, 2005; Pinkel et al, Nat. Genet. 20:207-211, 1998; Pollack et al, Nat. Genet. 23:41-46, 1999. Similar to standard CGH, tumour and reference DNA are differentially labeled and mixed. However, for aCGH, the DNA mixture is hybridized to a slide containing hundreds or thousands of defined DNA probes (such as probes that are homologous to portions of the SETDBl gene). The fluorescence intensity ratio at each probe in the array is used to evaluate regions of DNA gain or loss in the tumour sample, which can be mapped in finer detail than CGH, based on the particular probes which exhibit altered fluorescence intensity. In one example, the array is an Agilent Human Genome CGH 44B Oligo Microarray (Agilent Technologies, Santa Clara, CA). In another example, the CGH array is a Whole Genome Tiling, Custom, or Chromosome specific Tiling Array (for example, a Chromosome 15 Tiling Array) as provided by Roche NimbleGen, Inc. (Madison, WI).

In general, CGH (and aCGH) does not provide information as to the exact number of copies of a particular genomic DNA or chromosomal region. Instead, CGH provides information on the relative copy number of one sample (such as a tumour sample, for example a lung cancer sample) compared to another (such as a control sample, for example a non-tumour cell or tissue sample). Thus, CGH is most useful to determine whether SETDBl gene copy number of a sample is increased or decreased as compared to a control sample (such as a non-tumour cell or tissue sample or a reference value).

In another preferred embodiment the determination of the copy number of the

SETDBl gene is carried out by quantitative genomic PCR.

Additional methods that may be used to determine copy number of the SETDBl gene are known to those of skill in the art. Such methods include, but are not limited to

Southern blotting, multiplex ligation-dependent probe amplification (MLPA; see, e.g., Schouten et al., Nucl. Acids Res. 30:e57, 2002), and high-density SNP genotyping arrays (see, e.g. WO 98/030883). Also disclosed herein is a method of scoring (for example, enumerating) copy number of a gene in a sample from a subject (such as a subject with neoplastic disease), wherein the sample is stained by ISH (such as FISH, SISH, CISH, or a combination of two or more thereof) for the gene of interest and wherein individual copies of the gene are distinguishable in cells in the sample. Typically, the method includes identifying individual cells in a sample with the highest number of signals per nucleus for the gene (such as the strongest signal in the sample), counting the number of signals for the gene in the identified cells, and determining an average number of signals per cell, thereby scoring the gene copy number in the sample. In additional embodiments, the method further includes counting the number of signals for a reference (such as a chromosomal locus known not to be abnormal, for example, centromeric DNA) and determining an average ratio of the number of signals for the gene to the number of signals for the reference per cell.

The scoring method includes identifying individual cells in the sample (such as a tissue section or tumour core) having the highest number of signals (such as the highest number of spots per cell or the brightest intensity of staining) for the gene of interest in the cells in the sample. Thus, the disclosed method does not determine gene copy number in a random sampling of cells in the sample. Rather, the method includes specifically counting gene copy number in those cells that have the highest gene copy number in the sample. In some examples, identifying the individual cells having the highest number of signals for the gene includes examining a sample stained by ISH for the gene under low power microscopy (such as about 20X magnification). Cells with the strongest signal (for example, highest amplification signal under higher power) are identified for counting by eye or by an automated imaging system. In some examples, such as when the sample is a tissue section, the sample is examined (for example, visually scanned) to identify a region that has a concentration of tumour cells that has amplification of the gene. Gene copy number in the cells with highest amplification in the selected region is then counted. In other examples, such as when the sample is a tumour core (such as a tumour microarray), most of the sample is visible in the field of view under low power magnification and the individual cells (such as tumour cells) with the strongest signal (for example, highest amplification signal under high power) are separately identified for counting. In particular examples, the cells chosen for counting the gene copy number may be non-consecutive cells, such as cells that are not adjacent to or in contact with one another. In other examples, at least some of the cells chosen for counting the gene copy number may be consecutive cells, such as cells that are adjacent to or in contact with one another.

The disclosed methods include counting the number of ISH signals (such as fluorescent, coloured, or silver spots) for the gene in the identified cells. The methods may also include counting the number of ISH signals (such as fluorescent, coloured or silver spots) for a reference (such as a chromosome-specific probe) in the identified cells. In some examples, the number of spots per cells is distinguishable in the identified cells and the number of spots are counted (or enumerated) and recorded. In other examples, one or more of the identified cells may include a cluster, which is the presence of multiple overlapping signals in a nucleus that cannot be counted (or enumerated). In particular examples, the number of copies of the gene (or chromosome) may be estimated by the person (or computer, in the case of an automated method) scoring the slide. For example, one of skill in the art of pathology may estimate that a cluster contains a particular number of copies of a gene (such as 10, 20, or more copies) based on experience in enumerating gene copy number in a sample. In other examples, the presence of a cluster may be noted as a cluster, without estimating the number of copies present in the cluster.

The number of cells identified for counting is a sufficient number of cells that provides for detecting a change (such as an increase or decrease) in gene copy number. In some examples, the number of cells identified for counting is at least about 20, for example, at least 25, 30, 40, 50, 75, 100, 200, 500, 1000 cells, or more. In a particular example, about 50 cells are counted. In other examples, every cell in the sample or every cell in a microscope field of vision, or in a number of microscope fields (such as at least 2 microscope fields, at least 3, at least 4, at least 5, at least 6 microscope fields, and the like) which contains 3 or more copies of the gene of interest (such as 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) is counted. In some examples, the biological sample is a tumour sample, such as a tumour which potentially includes a gene amplification. Exemplary biological samples include neoplastic cells or tissues, which may be isolated from solid tumours, including lung cancer (e.g., non-small cell lung cancer, such as lung squamous cell carcinoma). The inventors have found that lung cancer cells having SETDB1 gene amplification have eighteen genes repressed. The inventors have validated the genes that appear in Figures 4C and 4D. Therefore, in another preferred embodiment, detecting the amplification of the SETDB1 gene comprises detecting decreased expression levels of one or more genes selected from the group consisting of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGK1 and TFAP2A with respect to a reference value.

The expression "expression levels" or its grammatical equivalent, as used herein, means a measurement of the amount of nucleic acid, e.g. RNA or mRNA, or protein of a gene in a cell, or alternatively, the level of activity of a gene or protein in said cell.

As the person skilled in the art understands, the expression levels of said genes can be measured by determining the mRNA expression levels of said genes or by determining the protein levels encoded by said genes.

In order to measure the mRNA levels of said genes, the cell may be treated to physically, mechanically or chemically disrupt cell structures, to release intracellular components into an aqueous or organic solution to prepare nucleic acids for further analysis. The nucleic acids are extracted from the sample by procedures known to the skilled person using commercially available reagents. RNA is then extracted from frozen or fresh samples by any of the methods typical in the art, for example, Sambrook, J., et al., 2001. Molecular cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3. Preferably, care is taken to avoid degradation of the RNA during the extraction process.

Determination of the levels of mRNA of said genes can be carried out by any method known in the art such as qPCR, northern blot, RNA dot blot, TaqMan®, tag based methods such as serial analysis of gene expression (SAGE) including variants such as LongSAGE and SuperSAGE, microarrays, nucleic acid sequence based amplification (NASBA), Fluorescence In Situ Hybridization, including variants such as Flow-FISH, qFiSH and double fusion fish (D-FISH) as described in WO2010030818, Femino et al. (Science, 1998, 280:585-590), Levsky et al. (Science, 2002, 297:836-840) or Raj et al. (PLoS Biology, 2006, 4:e309). In a particular embodiment de expression levels of said genes are determined by a microarray. In another particular embodiment the expression levels of said genes are determined by quantitative PCR, preferably Real-Time PCR.

In order to normalize the values of mRNA expression among the different samples, it is possible to compare the expression levels of the mRNA of interest in the test samples with the expression of a control RNA. A "Control RNA" as used herein, relates to a RNA whose expression levels do not change or change only in limited amounts cells. Preferably, the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions. Examples of housekeeping genes for use in the present invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH, PSMB4, tubulin and β-actin.

In one embodiment relative gene expression quantification is calculated according to the comparative Ct method using GAPDH, β-actin or PSMB4 as an endogenous control and commercial RNA controls as calibrators. Final results are determined according to the formula 2-(ACt sample-ACt calibrator), where ACT values of the calibrator and sample are determined by substracting the CT value of the target gene from the value of the control gene.

The expression levels of said genes may also be measured by determining the protein levels or variants thereof encoded by said genes. Practically any conventional method can be used within the context of the present invention to quantify the levels of said proteins such as Western blot or immunohistochemistry.

As previously mentioned, variants of said proteins can be used for measuring the expression levels of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGK1 or TFAP2A genes in order to put into practice the method of the invention.

Thus, variants of the ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGK1 or TFAP2A proteins may be: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code; (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups; (iii) one in which the protein is an alternative splice variant of the proteins of the present invention and/or; (iv) fragments of the proteins. The fragments include proteins generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants are deemed to be within the scope of those skilled in the art from the teaching herein.

Variants according to the present invention include amino acid sequences that are at least 60%, 70%, 80%>, 90%>, 95% or 96% similar or identical to the original amino acid sequence. As known in the art the "similarity" between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein to a sequence of a second protein. The degree of identity between two proteins is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990)].

The proteins can be post-translationally modified. For example, post- translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Additionally, the proteins may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.

In a particular embodiment said variant is a mammal variant, preferably a human variant, more preferably with at least 60%, 70%, 80%>, 90%>, 95%> or 96%> similarity or identity to the original amino acid sequence.

The ADRB2 gene, as used herein, refers to the gene which codes for the protein adrenergic, beta-2-, receptor surface; also named adrenoceptor beta 2, surface. The human gene (UniGene Hs. 2551) is defined in the NCBI database under accession number NC 000005.9 (positions 148206156 to 148208197 in the genomic region), version October 30, 2012.

The ANXA3 gene, as used herein, refers to the gene which codes for the protein annexin A3. The human gene (UniGene Hs.480042) is defined in the NCBI database under accession number NC_000004.11 (positions 79472673 to 79531605 in the genomic region), version October 30, 2012. The DNER gene, as used herein, refers to the gene which codes for the protein delta/notch-like EGF repeat containing. The human gene (UniGene Hs.234074) is defined in the NCBI database under accession number NC_000002.11 (positions 230222345 to 230579286 in the genomic region), version October 30, 2012.

The FGFR2 gene, as used herein, refers to the gene which codes for the protein fibroblast growth factor receptor 2. The human gene (UniGene Hs.533683) is defined in the NCBI database under accession number NC OOOOl 0.10 (positions 123237844 to 123357972 in the genomic region), version October 30, 2012.

The GPR64 gene, as used herein, refers to the gene which codes for the protein G protein-coupled receptor 64. The human gene (UniGene Hs.146978) is defined in the NCBI database under accession number NC 000023.10 (positions 19007425 to 19140755 in the genomic region), version October 30, 2012.

The IGFBP7 gene, as used herein, refers to the gene which codes for the protein insulin-like growth factor binding protein 7. The human gene (UniGene Hs.479808) is defined in the NCBI database under accession number NC_000004.11 (positions 57896939 to 57976551 in the genomic region), version October 30, 2012.

The IL6 gene, as used herein, refers to the gene which codes for the protein interleukin 6. The human gene (UniGene Hs.654458) is defined in the NCBI database under accession number NC 000007.13 (positions 22765503 to 22771621 in the genomic region), version October 30, 2012.

The NAV3 gene, as used herein, refers to the gene which codes for the protein neuron navigator 3. The human gene (UniGene Hs.655301) is defined in the NCBI database under accession number NC_000012.11 (positions 78224685 to 78606790 in the genomic region), version October 30, 2012.

The SGK1 gene, as used herein, refers to the gene which codes for the protein serum/glucocorticoid regulated kinase 1. The human gene (UniGene Hs.510078) is defined in the NCBI database under accession number NC_000006.11 (positions 134490384 to 134639250 in the genomic region), version October 30, 2012.

The TFAP2A gene, as used herein, refers to the gene which codes for the protein transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha). The human gene (UniGene Hs.519880) is defined in the NCBI database under accession number NC_000006.11 (positions 10393419 to 10419892 in the genomic region), version October 30, 2012.

The value obtained for the expression of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGKl and/or TFAP2A nucleic acid or protein levels in a sample from the subject suffering from lung cancer is then compared with a reference value.

This comparison allows detecting a decrease in the expression of one or more of said genes with respect to the reference value. The expression "decreased expression levels" of one or more genes selected from the group consisting of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGKl and TFAP2A means a decrease in the expression of a given gene with respect to the reference value of at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%.

The reference value can be determined by techniques well known in the state of the art, for example, determining the median value of expression levels of each of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGKl or TFAP2A genes measured in a collection of tumour tissue in biopsy samples from subjects suffering from lung cancer who do not have amplification of SETDB1 gene or from normal tissue. In a preferred embodiment the expression levels of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGKl or TFAP2A genes are measured in a collection of lung tumour tissue in biopsy samples from subjects suffering from lung cancer who do not have amplification of the SETDB1 gene or from normal lung tissue. Once this median value is established, the level of this marker expressed in tumour tissues from the subject can be compared with this median value, and thus be assigned a level of "decreased" (low) or "increased" (high) expression level. The collection of samples from which the reference level is derived will preferably be constituted from subjects suffering from the same type of lung cancer or a mixture of lung tissues from normal individuals not affected of lung cancer. Alternatively, the use of a reference value used for determining whether the expression level of a gene is "increased" or "decreased" could correspond to the median value of expression levels of each gene measured in a RNA sample obtained by pooling equal amounts of RNA from each of the tumour samples obtained by biopsy from subjects suffering from lung cancer who do not have amplification of the SETDB1 gene, preferably from subjects suffering from the same type of lung cancer. Once this median value is established, the level of this marker expressed in tumour tissues from subjects can be compared with this median value, and thus be assigned a level of "increased" or "decreased". For example, a decrease in expression levels below the reference value of at least 0.9-fold, 0.75-fold, 0.2-fold, 0.1 -fold, 0.05-fold, 0.025-fold, 0.02-fold, 0.01 -fold, 0.005-fold or even less compared with reference value is considered as "decreased" expression level.

In the present invention, the "reference value" may be an arbitrary cut-off point, established according to ROC methodology. Once this cut-off point is established, the level of this marker expressed in tumour tissues from the subject can be compared with this cut-off point, and thus be assigned a level of "low" expression if it is under this cutoff.

Once the level of the amplification of the SETDB1 gene has been determined

(for example, when the SETDB1 gene copy number has been determined or when a comparison between the expression levels of one or more of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGKl or TFAP2A genes and the reference value has been made), the method of the invention allows making a prediction as to whether the subject will show a good clinical response to a treatment with a SETDB1 antagonist, preferably to a SETDB1 -specific RNAi or to mithramycin. Thus, patients having amplification of the SETDB1 gene will have a good clinical response to the treatment with said antagonist.

The expression "good clinical response", as used herein, means that the subject will show a favourable response to the treatment with a SETDB 1 antagonist. As will be understood by those skilled in the art, such the assessment of the probability, although preferred to be, may usually not be correct for 100% of the subjects to be analyzed. The term, however, requires that a statistically significant portion of subjects can be identified as having a predisposition of responding to the chemotherapeutic treatment. 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. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%), at least 70%>, at least 80%>, at least 90%> at least 95%>. . The p- values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60 percent, at least 70 percent, at least 80 percent or at least 90 percent of the subjects of a population can be properly identified by the method of the present invention.

In some examples, an increased SETDB1 gene copy number includes SETDB1 gene copy number per nucleus (such as average SETDB1 gene copy per nucleus) in the sample of greater than about two copies of the gene per nucleus (such as greater than 2, 3, 4, 5, 10 or 20 copies). In other examples, an increased SETDB1 gene copy number includes a ratio of SETDB1 gene copy number to Chromosome 1 copy number (such as an average SETDB1 gene: Chromosome 1 ratio) in the sample of greater than about 2 (such as a ratio of greater than 2, 3, 4, 5, 10, or 20). In further examples, an increased SETDB 1 copy number includes an increase in SETDB 1 gene copy number relative to a control (such as an increase of about 1.5-fold, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 20-fold, or more).

In other examples, both the SETDB 1 gene and Chromosome 1 DNA (such as Chromosome 1 centromeric DNA) are detected in a sample from the subject, for example by ISH. Chromosome 1 -specific probes are well known in the art and include commercially available probes. The SETDB 1 gene and Chromosome 1 DNA may be detected on the same sample (for example, on a single slide or tissue section, such as a dual colour assay) or in different samples from the same subject (for example, SETDB 1 gene is detected on one slide and Chromosome 1 DNA is detected on a matched slide from the same subject, such as a single colour assay). The SETDB 1 gene and Chromosome 1 DNA are detected with two different detectable labels for dual colour assay (such as two different fluorophores, two different chromogens, or a chromogen and metal nanoparticles). The SETDB 1 gene and Chromosome 1 DNA may be detected with the same label for single colour assay. The SETDB 1 gene and Chromosome 1 DNA copy number may be determined by counting the number of fluorescent, coloured, or silver spots on the chromosome or nucleus. A ratio of SETDB 1 gene copy number and Chromosome 1 DNA number is then determined. Thus, in a particular embodiment, if the sample of the subject which has been amplified has more than 2 copies of the SETDBl gene, it is indicative of a good clinical response of the subject to the treatment with a SETDBl antagonist. In a particular embodiment, if the sample of the subject which has been amplified has more than 6 copies of the SETDBl gene, it is indicative of a good clinical response of the subject to the treatment with a SETDBl antagonist.

The skilled person will appreciate that the particular embodiments developed in the first aspect of the invention are also applicable to the second aspect of the invention. Therefore, in a preferred embodiment the method predicts the clinical response of a subject suffering from lung cancer to a treatment with a SETDBl antagonist, wherein the SETDBl antagonist is a SETDBl -specific RNAi. In another preferred embodiment the SETDBl antagonist is mithramycin or a pharmaceutically acceptable salt, solvate, polymorph or cocrystal thereof. In another preferred embodiment the subject is a mammal, preferably a human being. In another preferred embodiment the sample is a lung tumour tissue sample.

The terms "subject", "lung cancer", "treatment", "SETDBl antagonist", "mithramycin", "pharmaceutically acceptable salt", "solvate", "polymorph", "cocrystal", "SETDBl -specific RNAi", "SETDBl gene" and "sample" have been described in detail above in the context of the first aspect of the invention and are used with the same meaning in the context of the second aspect of the invention.

SELECTIVE METHODS OF THE INVENTION

The authors of the present invention have observed that amplification of the SETDBl gene is indicative of a good response to a SETDBl -specific shRNA and to mithramycin in SETDBl -amplified lung cancer cell lines. Therefore, the detection of the SETDBl gene amplification can be a marker useful for selecting a patient for being treated with a SETDBl antagonist. In this way, subjects can proceed directly to adequate therapies while avoiding ineffective therapies and secondary effects associated to them.

Thus, in a third aspect, the invention relates to an in vitro method for selecting a lung cancer patient for being treated with a SETDBl antagonist comprising detecting whether the SETDBl gene is amplified in a sample from said patient, wherein amplification of the SETDBl gene is indicative that the subject is a candidate for a treatment with a SETDBl antagonist.

All the embodiments of the second aspect of the invention are also applicable to the third aspect of the invention. Therefore, in a preferred embodiment the method allows selecting a lung cancer patient for being treated with a SETDBl antagonist, wherein the SETDBl antagonist is a SETDBl -specific R Ai. In another preferred embodiment the SETDBl antagonist is mithramycin or a pharmaceutically acceptable salt, solvate, polymorph or cocrystal thereof. In another preferred embodiment detecting the amplification of the SETDBl gene comprises determining the copy number of the SETDBl gene, preferably more than 2 copies, or detecting decreased expression levels of one or more genes selected from the group consisting of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGK1 and TFAP2A with respect to a reference value. In another preferred embodiment the patient is a mammal, preferably a human being. In another preferred embodiment the determination of the copy number of the SETDBl gene is carried out by in situ hybridisation, preferably with a polynucleotide comprising the human genomic fragment present in the RP11-42A12 BAC (SEQ ID NO: 1) or a variant thereof which hybridizes under stringent conditions to said polynucleotide. In another preferred embodiment the determination of the copy number of the SETDBl gene is carried out by quantitative genomic PCR. In another preferred embodiment the sample is a lung tumour tissue sample.

All the terms have been defined previously in the context of the first and second aspects of the invention and are used with the same meaning in the context of the third aspect of the invention.

KITS OF THE INVENTION AND USES THEREOF

The inventors of the present invention have found that the copy number of the SETDBl gene can be determined by FISH (fluorescent in situ hybridization) using a specific polynucleotide, namely, RPl 1-42A12.

In another aspect, the invention relates to the use of a reagent capable of detecting the amplification of SETDBl gene in a sample from a subject suffering from lung cancer for predicting the clinical response of said subject to a treatment with a SETDBl antagonist.

In another aspect, the invention relates to the use of a reagent capable of detecting the amplification of SETDBl gene in a sample from a subject suffering from lung cancer for selecting a lung cancer patient for being treated with a SETDBl antagonist.

The term "reagent", as used herein, refers to any compound or composition which can be used for detecting the amplification of SETDBl gene (for example any reagent capable of detecting the copy number of the SETDBl gene or any reagent capable of detecting decreased expression levels of one or more genes selected from the group consisting of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGK1 and TFAP2A). In a preferred embodiment the reagent is a reagent for detecting the copy number of the SETDBl gene. In another embodiment the reagent is a reagent for detecting the expression levels of one or more genes selected from the group consisting of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGK1 and TFAP2A, i.e. for detecting their genes, proteins or variants thereof. The reagent can include, optionally, reagents for detecting one or more housekeeping genes or the protein encoded by said housekeeping gene(s). This set of reagents can include, without limitation, nucleic acids capable of specifically hybridising with the SETDBl gene and/or antibodies or fragments thereof capable of specifically binding to SETDBl protein or to variants thereof (including fragments thereof containing antigenic determinants).

The reagents for use in the method of the invention may be formulated as a "kit" and thus, may be combined with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packages such as packaging intended for commercial sale, substrates to which the reagents are attached, electronic hardware components, etc.).

In a preferred embodiment, the reagents for determining the copy number of

SETDBl gene are probes for the identification of this gene, particularly a polynucleotide comprising the human genomic fragment present in the RP11-42A12

(SEQ ID NO: 1) or a variant thereof which hybridizes under stringent conditions to said polynucleotide. In another embodiment the reagents for determining the amplification of SETDB1 gene are primers and/or antibodies.

Nucleic acids capable of specifically hybridizing with the SETDB1 gene are, for example, one or more pairs of primer oligonucleotides for the specific amplification of fragments of the mRNA (or of their corresponding cDNA) of said gene.

Nucleic acids capable of specifically hybridizing with one or more genes selected from the group consisting of ADRB2, ANXA3, DNER, FGFR2, GPR64, IGFBP7, IL6, NAV3, SGK1 and TFAP2A can also be included.

As the skilled person understands, the oligonucleotide primers and probes of the kit of the invention can be used in all techniques of gene expression profiling (RT-PCR, SAGE, TaqMan, Real Time-PCR, FISH, NASBA, etc).

Antibodies, or a fragment thereof, capable of detecting an antigen, capable of specifically binding to a protein or to variants thereof are, for example, monoclonal and polyclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanised antibodies. The antibodies of the kit of the invention can be used in conventional methods for detecting protein expression levels, such as Western-blot or Western transfer, ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzymatic immunoassay), DAS- ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips, protein microarrays including specific antibodies or assays based on colloidal precipitation in formats such as dipsticks, etc.

Said reagents, specifically the probes and the antibodies, may be fixed onto a solid support, such as a membrane, a plastic or a glass, optionally treated in order to facilitate fixation of said probes or antibodies onto the support.

The kits of the invention optionally comprise additional reagents for detecting a housekeeping gene, a polypeptide encoded by a housekeeping gene or the mRNA encoded by said housekeeping gene. The availability of said additional reagent allows the normalization of measurements taken in different samples (e.g. the test sample and the control sample) to exclude that the differences in expression of the biomarker are due to a different amount of total protein in the sample rather than to real differences in relative expression levels. Housekeeping genes, as used herein, relates to genes which code for proteins which are constitutively expressed and carry out essential cellular functions. Preferred housekeeping genes for use in the present invention include β-2- microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, PSMB4, GAPDH, tubulin and β-actin.

All the particular embodiments disclosed for the methods of the present invention are applicable to the kit of the invention and uses thereof.

Particularly, in a preferred embodiment the SETDB 1 antagonist is selected from the group consisting of: (i) a SETDB 1 -specific R Ai, and (ii) mithramycin or a pharmaceutically acceptable salt, solvate, polymorph or cocrystal thereof. In another preferred embodiment the subject is a mammal, preferably a human being. In another preferred embodiment the sample is a lung tumour tissue sample.

The following example is provided as merely illustrative and is not to be construed as limiting the scope of the invention.

EXAMPLE

The inventors first screened a collection of 15 human lung cancer cell lines for SETDB 1 gene copy number alterations. These included seven non-small (A549, NCI- H1299, NCI-H1975, NCI-H1993, NCI-H2170, NCI-H1437 and NCIH1395) and eight small (HCC-33, N417, NCI-H446, NCI-H1048, NCI-H1963, NCI-H2029, DMS-114 and DMS-273) cell lung cancer types. The lung cancer cell lines were purchased from the American Type Culture Collection (ATCC) and were grown and maintained in 10% FBS in RPMI medium. Primary normal tissues such as lung epithelium and leukocytes were used as normal SETDB 1 copy number control samples. Using a quantitative genomic PCR approach, the inventors observed that two non- small (NCI-H1437 and NCI-H1395) and one small (DMS-273) cell lung cancer lines had a greater than fourfold change in SETDB 1 gene copy number (Figure 1A). This increase was particularly important in the small lung cancer cell line DMS-273 (Figure 1A). The remaining eleven lung cancer cell lines did not present any evident change in SETDB 1 gene copy number (i.e., amplification, half gene dosage or homozygous deletion). Fluorescence in situ hybridization (FISH) analyses were performed in order to confirm the presence of SETDBl gene amplification suggested to occur in the three lung cancer cell lines by the competitive genomic PCR approach. The UCSC genome browser (http://genome.ucsc.edu) was used to select the BAC clone spanning the lq21 region for the SETDBl gene: RP11-42A12. A telomeric BAC clone located in the telomeric lp36.23 region was used as a control. The BACs were obtained from the BACPAC Resource Center at the Children's Hospital Oakland Research Institute (Oakland, CA). SETDBl and telomeric probes were labeled with Spectrum Green and Red dUTP (Abbott), respectively, using a CGH Nick Translation Reagent Kit (Abbott Molecular Inc., Des Plaines, IL). The samples were counterstained with DAPI in Vectashield antifade solution. The FISH technique confirmed the presence of SETDBl gene amplification in NCI-H1437, NCI-H1395 and DMS-273 (data not shown). The applied FISH technique was validated by the observation of the normal copy number of the SETDBl gene in lymphocytes and probes were verified to give a single signal on normal commercial lymphocyte metaphase slides (CGH Reagents, Abbott, Wiesbaden, Germany). Using reported Copy Number Variation (CNV) data, the inventors have determined the size of the amplicon that contains SETDBl in the three studied lung cancer cell lines: DMS-273 (1,038,210 bp), NCI-H1437 (4,539,342 bp) and NCI-H1395 (4,623,419 bp). SETDBl is genomically located in the middle of all three amplicons, even in the case of the smallest one (DMS-273). It was calculated that NCI-H1437 and NCI-H1395 have 7 copies of SETDBl gene and DMS-273 has 17 copies of SETDBl gene.

The inventors next considered the possible existence of an association between extra copies of the SETDBl gene and overexpression of the corresponding RNA transcript and protein using quantitative reverse transcription-PCR (qRTPCR) and western blot approaches, respectively (Figure IB and 1C). They found that the expression of SETDBl for both mRNA and protein was enhanced in cancer cell lines harboring the SETDBl gene amplification event relative to non-amplified cancer cells (Figure IB and 1C). In the smallest SETDBl amplicon (present in DMS-273 cells), thirty-four genes are also co-amplified (data not shown). Using reported microarray expression data, the inventors have observed that, in addition to SETDBl, only 7 of these other 34 genes (21%) are overexpressed in DMS-273, NCI-H1437 and NCI- H1395 in comparison to unamplified lung cancer cells (NCI-H1299 and A549) (data not shown). Said seven genes are TARS2, GOLPH3L, ARNT, C10rf56, GABPB2, LYSMDl and SELENBPl . In this regard, the inventors have confirmed by qRT-PCR that six of these seven genes are overexpressed in the SETDBl amplified lung cell lines (data not shown).

Once the presence of SETDBl gene amplification and its associated overexpression in the lung cancer cell lines were demonstrated, the inventors examined its contribution to the tumorigenic phenotype in vitro and in vivo. They first analyzed the effect of SETDBl depletion in lung cancer cells harboring its gene amplification and its associated overexpression. Table 1 includes all the used short hairpin RNA (shRNA) sequences.

Table 1. Primers and shRNA target sequences

'HuSH^ System, OriGene. The kit also provides negative control sequences for the scrambled clones.

² In this case, the negative control sequence was obtained by inserting a pair of mutations: GAAGGAGCTAGCTGAGTTA (SEQ ID NO: 2).

Three SETDBl shRNA depleted clones were established for DMS-273 cells (A30, A31 and B32-63) and two clones for NCI-H1437 (A56-B and B49-6). Experiments for each clone were performed in triplicate. The inventors observed that the reduction of SETDBl expression in the gene-amplified cells had cancer growth- inhibitory features (Figure 2). Upon stable trans fection of shRNAs against SETDBl in the gene-amplified DMS-273 and NCI-H1437 lung cancer cell lines and efficient depletion of the SETDBl protein (Figure 2A), the cells proved less viable in the MTT assay (Figure 2B and 2D) and had a markedly reduced percentage colony formation density in the assay developed on plastic plate (Figures 2C and 2D). Transfection of the scramble short hairpin RNA did not reduce cell viability (Figure 2B and 2D) and had no impact on the colony formation assay (Figures 2C and 2D). Confocal microscopy experiments in the NCI-H1437 lung cancer cell line confirmed the expected nuclear staining of the SETDBl protein (DAPI co localization) and the disappearance of the nuclear signal upon SETDBl shRNA-mediated depletion (data not shown). SETDBl knock-down by the shRNA approach in a lung cancer cell line without gene amplification (NCI-H1299) did not cause a significant effect in cell growth determined by the MTT experiments or the colony formation assay (Figure 2E), suggesting the SETDBl dependence for cell growth only in the amplified cancer cells. The inventors next tested the ability of SETDBl shRNA-transfected DMS-273 and NCI-H1437 cells to form tumors in nude mice compared with scramble shRNA-transfected cells (Figure 2F). DMS-273 and NCI-H1437 scramble shRNA-transfected cells formed tumors rapidly, but cells with shRNA-mediated depletion of SETDBl had much lower tumorigenicity (Figure 2F).

The inventors also studied the oncogenic potential of SETDBl by evaluating its ability to enhance cell invasion in a lung carcinoma cell line without SETDBl gene amplification (A549) (Figure 3A). To this end, the inventors transfected the plasmids driving the expression of SETDBl and performed a matrigel invasion assay. A significant increase in the number of invasive cells upon SETDBl transfection in comparison with empty vector-transfected cells was observed (Figure 3A).

The inventors also wondered about gene targets in the amplified lung cancer cells whose expression could be regulated by SETDBl mediated H3-K9 promoter methylation and that could further explain the above observed impact in cell growth and invasiveness. To find downstream targets of SETDBl in the amplified lung cancer cell lines DMS-273 and NCI-H1437, the inventors developed expression microarray analyses (Agilent G4851B 60K) of both SETDBl shRNA depleted cell lines in comparison to their corresponding shRNA-scrambled control cell lines. The microarray expression data obtained are freely available at the Gene Expression Omnibus database: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=tdexdacy giayejy&acc=GSE4517 5. Using this approach, the inventors identified eighteen common genes repressed in both SETDBl amplified lung cancer cell lines that become upregulated at least 3-fold upon SETDBl shRNA-mediated downregulation (Table 2; Figure 4B).

Table 2. Common upregulated genes in both SETDBl-depleted lung cancer cell lines (DSM-273 and NCI-H1437)

Gene Description

ADARB2 Homo sapiens adenosine deaminase, RNA-specific, B2

ADRB2 Homo sapiens adrenergic, beta-2-, receptor, surface

ANXA3 Homo sapiens annexin A3

C8orfll Homo sapiens chromosome 8 open reading frame 31

DNER Homo sapiens delta/notch-like EGF repeat containing

FGFR2 fibroblast growth factor receptor 2

GPR64 Homo sapiens G protein-coupled receptor 64

IGFBP7 Homo sapiens insulin-like growth factor binding protein 7

IL11 Homo sapiens interleukin 11

IL32 Homo sapiens interleukin 32

IL6 Homo sapiens interleukin 6 (interferon, beta 2)

LOC100506718 PREDICTED: Homo sapiens hypothetical LOC100506718

NAV3 Homo sapiens neuron navigator 3

PLAG1 Homo sapiens pleomorphic adenoma gene 1

SGK1 Homo sapiens serum/glucocorticoid regulated kinase 1

SPANXA1 Homo sapiens sperm protein associated with the nucleus,

X-linked, family member Al

TFAP2A Homo sapiens transcription factor AP-2 alpha (activating

enhancer binding protein 2 alpha)

TFPI2 Homo sapiens tissue factor pathway inhibitor 2

The inventors also confirmed the expression changes of the candidate genes by quantitative RT-PCR (Figure 4C) and the shift in H3-K9 methylation status in their respective promoters by quantitative chromatin immunoprecitation (qChIP) using anti- H3K9me3, anti-H3 and anti-rabbit IgG antibodies (Figure 4D). The experiments depicted a reduction of H3K9me3 in DMS-273 and NCI-H1437 cells stably transfected with shRNAs against SETDBl . Related to function, gene ontology (GO) analysis of these genes determined "Regulation of cell proliferation" as the most significantly enriched biological process (FDR = 0.0006). Representative examples included the tumor suppressor roles of Delta/Notch-like epidermal growth factor-related receptor (DNER) and insulin-like growth factor binding protein 7 (IGFBP7).

The observation that the presence of the SETDBl gene amplification with associated overexpression was critical for the tumorigenesis of these lung cancer cells prompted the inventors to examine whether drugs targeting this pathway might find a therapeutic "niche" for their use in the described subset of cases with extra copies of this gene. No highly specific inhibitor for SETDBl has been described in the publicly available literature, but one agent— mithramycin— is a clinically approved anti-tumor antibiotic that binds to DNA by interacting with the minor groove and displacing transcriptional activators that bind to GC-rich binding sites. Thus, the inventors tested whether a putative growth-inhibitory effect of this drug in lung cancer cells was dependent on SETDBl expression. First, western blot analyses for the SETDBl protein in the SETDBl amplified lung cancer cell lines DMS-273, NCI-H1437 and NCI-H1395 upon the use of the drug were developed. The inventors found that mithramycin was able to inhibit SETDB1 expression in the three cell lines (Figure 5). Using the small lung cancer cell line DMS-273, harboring the previously identified SETDB1 gene amplification, in comparison with three stable short hairpin SETDB1 -depleted clones, we observed that the scramble shRNA DMS-273 cells were significantly more sensitive to the growth- inhibitory effect mediated by mithramycin than any of the depleted clones (A30, A31 and B32-63) (Figure 3B and 5). Experiments for each clone were performed in triplicate. Furthermore, we extended the cell viability experiments, using the MTT assay, to the other two SETDB1 amplified lung cancer cell lines (NCI-H1437 and NCI- H1395) and to three SETDB1 non-amplified lung cancer cell lines (DMS-114, A549 and NCI-H1299). The determination of the corresponding EC50 values further confirmed that SETDB1 amplified cell lines (EC50=13.6 nM for NCI-H1437 and EC50=14.7 nM for NCI-H1395) are more sensitive to the drug than the non-amplified cell lines (EC50=347.5 nM for DMS-114, EC50=122 nM for A549 and EC50=32 nM for NCI-H1299) (ANOVA, p<0.001). Thus, the presence of SETDB1 gene amplification could "mark" lung cancer cells that are more sensitive to the inhibition of cell viability associated with the use of mithramycin.

Finally, the inventors demonstrated that the presence of SETDB1 gene amplification was not a specific feature of in vitro grown lung cancer cell lines and that it also occurred in primary tumors of lung cancer patients. FISH analysis for the SETDB1 locus were performed using a collection of 59 primary lung tumors, corresponding to 40 non-small cell lung tumors (20 squamous cell carcinomas and 20 adenocarcinomas) and 19 small cell carcinomas (data not shown). The UCSC genome browser (http://genome.ucsc.edu) was used to select the BAC clone RPT 1-42A12 spanning the lq21 region of the SETDB1 gene. The telomeric BAC clone RPT 1-60J11 located in the telomeric lp36.23 region was used as a control. The inventors identified SETDB1 gene amplification in nine tumors corresponding to 20% (4 of 20), 20% (4 of 20) and 5%> (1 of 19) of adenocarcinoma, squamous and small cell lung cancer cases, respectively. Most importantly, the inventors also demonstrated that the presence of extra copies of SETDB1 in these nine primary tumors was associated with overexpression of the SETDB1 protein as determined by immunohistochemistry in all cases (HPA018142, Sigma-Aldrich, St. Louis, MO, USA) (data not shown). Among the remaining fifty unamplified cases, eight (14%) also showed enhanced SETDBl expression that could be associated with other upstream regulatory events. Fisher's test, two-tailed P value < 0.0001.