WO2024056659A1 - Method for treating prostate cancer and other epithelial cancers - Google Patents

Abstract

The present invention relates to the treatment of cancer. Here, the inventors identified a subset of prostate cancer patients showing an up-regulation of the epigenetic enzymes SUV4-20H1 and SUV4-20H2, two methyltransferases responsible for the di- and tri- methylation of histone H4 at lysine 20 (H4K20me2/3). Consistent with this, the inventors demonstrate that the pharmacological inhibition of both SUV4-20H1 and SUV4-20H2 enzymes by the chemical compound A196 (14) leads to the complete loss of H4K20me2/3 states in prostate cancer cells. Although displaying epigenetic reprograming at genome-wide levels, cancer cells display any significant impairment in their survival or proliferation, thereby demonstrating that the inhibition of SUV4-20H1 and SUV4-20H2 is not toxic per se. Yet, the inventors showed that the pharmacological inhibition of SUV4-2H1 and SUV4-20H2 subtly affects DNA repair mechanisms and the levels of trapped topoisomerase II (TOPO2) complex in silent chromatin regions upon TOPO2 poisons. This creates in vitro as well as in vivo a lethal synergy between A196 and the TOPO2-poison etoposide in prostate cancer cells. Altogether, the results of the inventors showed that the simultaneous inhibition of SUV4-20H and TOPO2 enzymatic activity constitutes indeed a new therapeutic approach for the treatment of advanced or metastatic prostate cancers, which are particularly addicted to SUV4-20H2 and TOPO2 activities. Other cancers could also benefit of this drug combination, since the co-treatment of A196 and etoposide induces similar lethal synergy in other epithelial cancer cells such as breast cancer cell lines. Thus, the present invention relates to a combination of a SUV4-20H inhibitor and a TOPO2 inhibitor for use in the treatment of a cancer in a subject in need thereof.

Description

METHOD FOR TREATING PROSTATE CANCER AND OTHER EPITHELIAL

CANCERS

FIELD OF THE INVENTION:

The present invention relates to a combination of a SUV4-20H inhibitor and a TOPO2 inhibitor for use in the treatment of a cancer in a subject in need thereof.

BACKGROUND OF THE INVENTION:

Prostate Cancer (PCa) is one of the most common malignancies in men and still remains a major cause of death by cancer (1). Because androgen receptor (AR) mediated signaling pathway is a key driver of PCa growth and survival, androgen deprivation is the mainstay treatment for patients with advanced PCa (1,2). However, despite initial response, up to 30% of patients evolve toward a more aggressive stage in which tumor cells become resistant to castration (2,3). Although second generation of AR-directed therapy, such as abiraterone and enzalutamide, and chemotherapeutic agents can further improve the overall survival and quality of life, the efficiency of these treatments remains limited and most patients develop therapy resistances (2,3). To date, castration-resistant PCas are still incurable with a median survival that does not exceed three years, underscoring the need for new therapeutic approaches. Mechanisms underlying castration-resistance in PCa usually involve the androgen receptor (AR) itself, including gene amplification, activating mutations, and alternative splicing (2,4,5). A large subset of metastatic PCa also shows lineage reprogramming (5), global chromatin structural changes (6) and silencing of AR expression (2), suggesting that epigenetic modifications also contribute to therapy resistance in PCa. Indeed, epigenetic alterations appear highly recurrent in advanced PCa (5), a facet that could be exploited for development of new therapeutic strategies.

Epigenetics refer to chromatin-associated mechanisms by which DNA methylation, non-coding RNA processing, nucleosome remodeling and histone modifications contribute to regulate DNA-templated processes, including transcription, recombination, DNA replication and repair (5). In the context of prostate cancer, pathologists revealed that the progression of the disease and its aggressiveness often coincide with the formation of hyperchromatic nuclei with dispersed but highly compact chromatin foci (6). At molecular levels, these chromatin structure alterations could result from the excessive formation of stable and heterochromatin silencing structures enriched in specific epigenetic marks, such as the methylation of histone H3 at lysine 9 and of histone H4 at lysine 20 (5,7). Chemically, lysine methylation entails the addition of one, two or three methyl groups. This creates unique functional biological outcomes for each degree of methylation according to the binding of specific proteins at each lysine and its methylation state (8). In this regard, the regulation of histone H4K20 methylation in PCa is particularly intriguing (7). Whereas AR seems to interact with the lysine methyltransferase SET8 and contributes to promote SET8-mediated H4K20 monomethylation (H4K20mel) enrichment at AR-target genes (9), castration-resistant prostate tissues are characterized by a decrease in H4K20mel and an up-regulation of the higher H4K20 di -methyl (H4K20me2) and tri -methyl (H4K20me3) states catalyzed by SUV4-20H1 and SUV4-20H2 enzymes (5,7,10). The inventors therefore hypothesized that the regulation of H4K20 methylation states and the corresponding epigenetic enzymes may represent valuable markers in PCa progression and might play a critical role in the progression of the disease and in the response to therapy.

SUMMARY OF THE INVENTION:

Here, the inventors identified a subset of prostate cancer patients showing an upregulation of the epigenetic enzymes SUV4-20H1 and SUV4-20H2, two methyltransferases responsible for the di- and tri-methylation of histone H4 at lysine 20 (H4K20me2/3) (11,12). They also found that the up-regulation of SUV4-20H2, but not of SUV4-20H1, coincides with a poor prognosis and the appearance of metastases in patients (Figurel). These results suggest an important role of these enzymes in prostate cancer progression. Consistent with this, the inventors demonstrate that the pharmacological inhibition of both SUV4-20H1 and SUV4- 20H2 enzymes by the chemical compound Al 96 (14), leads to the complete loss of H4K20me2/3 states (Figure 2). These epigenetic alterations upon Al 96 impairs replication fork progression and chromatin compaction (Figure 2). However, prostate cancer cell survival and proliferation remained unaffected, indicating that the inhibition of SUV4-20H1 and SUV4-20H2 is tolerated by prostate cancerous cells (Figure 2). However, the inventors showed that the pharmacological inhibition of SUV4-2H1 and SUV4-20H2 also affects DNA repair mechanisms (Figure 5), which creates a specific synthetic lethality with innocuous concentrations of topoisomerase II (TOPO2) inhibitors (Figure 3). A similar enhanced sensitivity for TOPO2 inhibitors is observed with cancer cells where SUV4-20H1 and SUV4- 20H2 are genetically inactivated by crips-Cas9 approaches (Figure 4). Altogether, the results of the inventors showed that the simultaneous inhibition of SUV4-20H and TOPO2 enzymatic activity constitutes a new therapeutic approach for the treatment of the metastatic stages of prostate cancer, which are particularly addicted to SUV4-20H2 and TOPO2 activity. Other cancers could also benefit of this strategy (Figure 6). The inhibition of SUV4-20H catalytic activity also strongly improves the efficacy of TOPO2 inhibitors allowing to reduce the effective dose of these compounds. This is a critical issue in clinic due to the cardio-toxicity (16,17) of these FDA-approved drugs notably in elderly, who make up the majority of prostate cancer patients.

Thus, the present invention relates to a combination of a SUV4-20H inhibitor and a TOPO2 inhibitor for use in the treatment of a cancer in a subject in need thereof. Particularly, the invention is defined by its claims.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors showed that the synergic use of SUV4-20H inhibitors with a TOPO2 inhibitor enhances the sensitivity of the cancerous cells to the impairment of TOPO2 activities and allows the use of less quantity of TOPO2 inhibitor.

Thus, a first aspect of the present invention relates to a combination of a SUV4-20H inhibitor and a TOPO2 inhibitor for use in the treatment of a cancer in a subject in need thereof.

In a particular aspect, the present invention relates to i) a SUV4-20H inhibitor and ii) a TOPO2 inhibitor, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject in need thereof.

In a particular aspect, the present invention relates to i) a SUV4-20H inhibitor and ii) a TOPO2 inhibitor for simultaneous, separate or sequential use in the treatment of cancer in a subject in need thereof.

In another particular aspect, the invention also relates to a SUV4-20H inhibitor to improve the efficiency of a TOPO2 inhibitor in the treatment of cancer.

In a particular embodiment, the SUV4-20H inhibitor targets both SUV4-20H1 and SUV4-20H2) enzymes.

In another embodiment, the invention relates to i) a specific SUV4-20H1 protein inhibitor and ii) a TOPO2 inhibitor, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject in need thereof. In still another embodiment, the invention also relates to i) a specific SUV4-20H2 protein inhibitor and ii) a TOPO2 inhibitor as a combined preparation for simultaneous use in the treatment of cancer.

In still another embodiment, the invention also relates to i) a specific SUV-20H1 inhibitor, ii) a specific SUV4-20H2 inhibitor and iii) a TOPO2 inhibitor as a combined preparation for simultaneous use in the treatment of cancer.

As used herein, the terms “SUV4-20H1” (also known as KMT5B) and “SUV4-20H2” (also known as KMTB5C) have their general meaning in the art and refer to epigenetic enzymes responsible for the di-methylation and tri-methylation of histone H4 at lysine 20 (H4K20me2 and H4K20me3 respectively), which have been linked to chromatin compaction, gene regulation, DNA repair and proper cell-cycle progression.

For human SUV4-20H1, the Entrez Gene ID number is 51111 and the Uniprot number is Q4FZB7. For human SUV4-20H2, the Entrez gene ID number is 84787 and the Uniprot number is Q86Y97. According to the invention, the general term “SUV4-20H” is used for the enzymes “SUV4-20H1” and “SUV4-20H2”.

As used herein, the term “topoisomerase II” or “TOPO2” denotes topoisomerases that cut both strands of the DNA helix simultaneously in order to manage DNA tangles and supercoils. They use the hydrolysis of ATP, unlike Type I topoisomerase. In this process, these enzymes change the linking number of circular DNA by ±2. Topoisomerases are ubiquitous enzymes, found in all living organisms.

The term “TOPO2 inhibitor” denotes molecule or compound that impair the action of the type II topoisomerases (named TOPO2). TOPO2 inhibitors influence the essential cellular processes of TOPO2. Some topoisomerase inhibitors prevent topoisomerases from performing DNA strand breaks. In contrast, other TOPO2 inhibitors, deemed TOPO2 poisons, associate with topoisomerase-DNA complexes and prevent the re-ligation step of the topoisomerase mechanisms, thereby trapping TOPO2 into chromatin and transforming this enzyme into a DNA damaging agent.

The term “TOPO2 inhibitor” also denotes inhibitors of the expression of the gene coding for the TOPO2 proteins.

In a particular embodiment, the TOPO2 inhibitor according to the invention is a TOPO2 poison. The terms “SUV4-20H inhibitor” denotes molecule or compound that is able to inhibit simultaneously the activity or expression of both SUV4-20H1 and SUV4-20H2. In particular, the SUV4-20H inhibitor of the present invention is a compound that is able to inhibit the catalytic activity of both SUV4-20H1 and SUV4-20H2. The term “SUV4-20H inhibitor” can also denote inhibitors of the expression of the genes coding for the proteins SUV4-20H1 and SUV4-20H2.

The term “SUV4-20H1 inhibitor” denotes molecule or compound that inhibit specifically SUV4-20H1 but not SUV4-20H2. It also denotes inhibitor of the expression of the gene coding for SUV4-20H1.

The term “SUV4-20H2 inhibitor” denotes molecule or compound that inhibit specifically SUV4-20H2 but not SUV4-20H1. It also denotes inhibitor of the expression of the gene coding for SUV4-20H2.

According to the invention, the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

In a particular embodiment, the cancer is an epithelial cancer. As used herein, the term “Epithelial cancer” or “Carcinoma” refers to cancer with epithelial cells origin. This includes skin cancer, mucous cancer or gland cancer. Most epithelial cancers comprise prostate cancer, lung cancer, pancreas cancer, colon cancer, bladder cancer, ovary cancer and breast cancer (18).

In a particular embodiment, the cancer is a prostate cancer, particularly an aggressive metastatic prostate cancer or a resistant or prostate cancer. In a particular embodiment, the aggressive or resistant prostate cancer is a metastatic prostate cancer. As used herein, the term “resistant prostate cancer” denotes a prostate cancer resistant to castration or any current therapies against this type of cancer (hormonotherapy and chemotherapy).

According to the invention, a prostate cancer used herein refers to a disorder characterized by proliferation and scattering of malignant prostate gland cells derived from a single clone. It is diagnosed using standard diagnostic criteria. Typically, elevated levels of prostate-specific antigen (PSA) in blood test, a digital rectal exam followed or not by a prostate biopsy to measure the Gleason score.

In a particular embodiment, the cancer is a breast cancer. As used herein, the term “Breast cancer” refers to cancers affecting breast cells, in particular, duct cells, lobule cells or glandular tissue cells.

As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

In one embodiment, the inhibitors according to the invention (inhibitors of TOPO2 and SUV4-20H (SUV4-20H1 and SUV4-20H2) may be a low molecular weight compound, e. g. a small organic molecule (natural or not).

The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

Inhibitors of SUV4-20H are well known in the art and typically include the compounds and its derivatives described in Bromberg, et al. 2017 and notably the compound Al 96 of formula I:

Formula I

Inhibitors of TOPO2 are well known in the art and can be divided in two groups according to their mode of action: toposisom erases catalytic inhibitors and toposiomerase poisons. The TOPO2 catalytic inhibitors typically include but are not limited to MST-16; ICFR-187, dexrazoxane, novobiocin, Merbarone and Anthracylcine aclarubicin. The TOPO2 poisons typically include but are not limited to the etoposide, the mitoxantrone, the doxorubicin and the teniposide. In one embodiment, the compound according to the invention can be a polypeptide. Accordingly, the polypeptide is an antagonist of SUV4-20H1, SUV4-20H2 or TOPO2 and is capable to prevent the enzymatic activities of SUV4-20H1, SUV4-20H2 or TOPO2. Particularly, the polypeptide can be a mutated SUV4-20H1, SUV4-20H2 or TOPO2 protein or a similar protein without the function of SUV4-20H1, SUV4-20H2 or TOPO2.

In one embodiment, the polypeptide of the invention may be linked to a cellpenetrating peptide” to allow the penetration of the polypeptide in the cell.

The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).

The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e- amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa). In addition, to the polymer backbone being important in maintaining circulatory halflife, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

In another embodiment, the SUV4-20H1, SUV4-20H2 or TOPO2 inhibitor according to the invention can be an inhibitor of the expression of the genes encoding for SUV4-20H1, SUV4-20H2 or TOPO2 respectively. Small inhibitory RNAs (siRNAs) can also function as inhibitors of SUV4-20H1, SUV4-20H2 or TOPO2 expression for use in the present invention. The expression of SUV4-20H1, SUV4-20H2 or TOPO2 genes can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that SUV4-20H1, SUV4-20H2 or TOPO2 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of SUV4-20H1, SUV4-20H2 or TOPO2 gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of SUV4-20H1, SUV4-20H2 or TOPO2 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of SUV4-20H1, SUV4-20H2 or TOPO2 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing SUV4-20H1, SUV4-20H2 or TOPO2. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno- associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sulati on .

In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes For example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

In a particular embodiment, an endonuclease can be used to abolish the expression of the gene, transcript or protein variants of SUV4-20H1, SUV4-20H2 or TOPO2.

Indeed, as an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, new technologies provide the means to manipulate the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone non homologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707- 714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534/genetics.H3.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836-843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA- directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

In order to test the functionality of a putative SUV4-20H or TOPO2 inhibitor a test is necessary. For that purpose, to identify a SUV4-20H inhibitor the measure of the steady-state levels of histone H4K20 methylation states by immunoblot with specific antibodies for each H4K20 methylation degree is the most appropriate test. To identify a TOPO2 inhibitor, DNA supercoiled plasmid assay is the most appropriate test, in addition to measure the putative presence of DNA damage signals by immunofluorescence and immunoblot with antibodies against conventional DNA damage markers.

In another embodiment, the invention relates to a method for treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of SUV4-20H inhibitor and a TOPO2 inhibitor.

Therapeutic composition A second aspect of the invention relates to a therapeutic composition comprising a SUV4-20H inhibitor and a TOPO2 inhibitor for use in the treatment of a cancer in a subject in need thereof.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising an agonist, antagonist or inhibitor of the expression according to the invention and a further therapeutic active agent. For example, anti-cancer agents may be added to the pharmaceutical composition as described below.

Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), and Bendamustine (Treanda).

Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, campathecins, bleomycin, dactinomycin, plicamycin, L-asparaginase, epimbicm, 5 -fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.

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

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

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

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

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

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

In yet another embodiment, the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.

Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).

Typically, the checkpoint blockade cancer immunotherapy agent is an antibody.

In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti -IDO 1 antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: SUV4-20H1 and SUV4-20H2 expression is up-regulated in prostate cancer and SUV4-20H2 expression is associated with poor patient survival and decrease of disease-free interval. (A) comparison of SET8, SUV4-20H1 and SUV4-20H2 mRNA level between benign-normal (52 patient samples) prostate samples and local (354 patient samples) or metastatic (79 patient samples) tumor samples from TCGA cohort. (B) Kaplan- Meier analysis of overall survival (OS) probability of SUV4-20H2 expression in prostate TCGA cohort. (C) Kaplan-Meier analysis of disease-free interval (DFI) probability of SUV4- 20H2 expression in prostate TCGA cohort.

Figure 2: the chemical compound A196 lead to SUV4-20H inhibition and H4K20 methylation alterations without apparent toxicity in prostate cancer cell lines. (A) Immunoblot analysis of the methylation levels of H4K20 in osteosarcoma cell line U2OS and 2 metastatic prostate cancer cell lines, LNCaP and DU145, treated or not with different concentrations of A-96 for 72h. (B) Representative images and quantitative FRET/FLIM analysis of the compaction levels of the genome in cells treated with 4 pM of Al 96 for three days, the least compacted zones are in black and the most compacted in grey.

Figure 3: Synthetic lethality of A196 and TOPO2 inhibitors in metastatic prostate cancer cell lines. (A) Viability matrix and synergy scores (synergy > 20, additivity when values are between O and 20) after 3 days of treatment with increasing doses of Al 96 and etoposide (core and intensity of gray depending on the degree of synergy. B) Viability matrix and synergy scores (synergy > 20, additivity between O and 20) after 3 days of treatment with increasing doses of A196 and mitoxantrone in DU145 cells. Score and intensity of gray depending on the degree of synergy. (C) Clonogenicity tests and survival of prostate cancer cell lines treated as indicated for 72h. (D) Clonogenicity tests and survival of prostate cancer cell lines treated as indicated for 72h. (E) Survival of DU145 metastatic prostate cancer cell lines following exposure to the indicated doses of A- 196 and etoposide for 72h. The percentage of dead cells was estimated using PI/DAPI staining.

Figure 4: (A) Immunoblot analysis for SUV4-20H1, SUV4-20H2 and the methylation levels of H4K20 in DU145 WT, KOSUV4-20H1 (clone F10-C7), KOSUV4-20H2 (clone CO4-C4) and Double KO (clone CIO) clones. (B) Cell proliferation of DU145 WT, KO SUV4-20H1 (F10-C7), KO SUV4-20H2 (C04-C4) and Double KO (CIO) clones. Cells were counted as phase objective count using the Incucyte. Noted that the loss of one enzyme increase cell proliferation compared to control DU145 cells, whereas the loss of both enzymes have the opposite effect. (C) Cell proliferation of DU145 WT, KO SUV4-20H1, KO SUV4- 20H2 and Double KO clones treated with different concentrations of etoposide for 4 days. Cells were counted as phase objective count using the Incucyte and statistical significance was performed using GraphPad Prism 8. ***P<0.0001 multiple t-test. Noted of, the higher sensitivity of double KO clones compared to single KO clones and the control parental DU145 cell line.

Figure 5 : A196 enhances the formation of TOPO2-DNA complexes trapped by etoposide. (A) immunoblots showing the levels of stableTopo2-DNA complexes in response to drug treatments as indicated. To this end, DU145 cells were treated or not with A196 for 3 days. Cells were then collected 3 hours after or not etoposide treatment and total proteins extracted were subjected to immunoblots with anti -topoisomerase 2. Anti -histone H3 immunoblot serves as loading control for DNA-bound proteins. (B) quantification of immunoblots showed above. Experiments n=3. * p<0.001; ** p<000.1

Figure 6: The enhanced response to TOPO2 inhibitors upon A196 is due to defects in DNA repair signaling pathways mediated by DNA-PK kinase. (A) Number of foci formation for y-H2AX and 53BP1 in DU145 cells treated or not with A-196 for 72h, then pulsed with 0.45pM of etoposide for 24h. y-H2AX and 53BP1 foci were detected with Zeiss microscopy and counted using Cellprofiler 4.2.1 and statistical significance was performed using GraphPad Prism 8. (Bl et B2) DU145 cells were refreshed with a new medium after etoposide elimination with maintenance of A-196 or DMSO all the time of treatment. Then, cells were collected at the time points indicated in the diagram. y-H2AX and 53BP1 foci were counted and cells with >5 foci are considered as positive cells. Statistical significance was performed using GraphPad Prism 8. (C) Immunoblot analysis of the methylation levels of H4K20 in DU145 cells treated with A-196 for 72h followed with etoposide pulse for 24h. (D) Immunoblot analysis of DNA-PKcs phosphorylation S2056 and ATM phosphorylation SI 981 in DU145 cells collected at the time points indicated in the diagram. ***P<0.0001 Mann- Whitney runk sum test.

Figure 7: In vivo combination of etoposide and A196 increases therapeutic efficacy without no significant toxicity. Intact athymic nude male received 10⁶ DU145 cells by subcutaneous injection. All mice were divided in four cohorts: Vehicle (DMSO+cornoil), etoposide, Al 96 and combination. (A) Body weight measurement of treated mice after three weeks of treatment as indicated. (B) tumor growth volume of treated mice as indicated and after three weeks of treatment. ** p<0.0001.

Figure 8: A196 inhibitor enhances etoposide-induced lethality in breast cancer cell line MCF7. (A) Survival of MCF-7 breast cancer cell line following exposure to the indicated doses of A-196 for 18, 24, 48 and 72h. The percentage of dead cells was estimated using proliferation assay. (Bl and B2) Survival of MCF-7 breast cancer cell line treated with A-196 and etoposide as indicated for 48h. The percentage of dead cells was estimated using proliferation assay. (C) Immunoblot analysis of the methylation levels of H4K20 in MCF-7 cells treated with A-196 and/or etoposide for 48h. (D) Immunoblot analysis of y-H2AX in MCF-7 cells collected after 48h of treatment. Statistical significance was performed using GraphPad Prism 8. ***P<0.0001 multiple t-test.

EXAMPLE:

Material & Methods

Prostate cancer cell culture and reagents.

Prostate cancer cell lines were purchased from ATCC. DU145 cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C, 5% CO2. LnCaP and PC3 cell lines were cultured in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C, 5% CO2. Etoposide, mitoxantrone and Al 96 were purchased from Sigma and MecdChemexpress and resuspended at 10 mM in DMSO.

Immunoblot.

For immunoblot analysis, cells washed with phosphate-buffered saline (PBS) were lysed in Laemmli buffer. After measuring protein quantity by Bradford, equal amounts of protein were resolved by SDS-PAGE, transferred to a nitrocellulose membrane and probed with one of the following antibodies: anti-H4K20mel (CS, ref 9724) anti-H4K20me2(CS, #9759), anti-H4K20me3 (CS, #5737), anti-SUV4-20Hl (HPA063648), anti-SUV4-20H2 (HPA052294), anti -histone H4 (CS, #2935), anti -histone H3 (CS, #4499), anti -tubulin (Sigma), anti DNA-PK (CS, #4602), Anti-phosphoDNA-PK S2056 (CS, #6876), anti-ATM (CS, #2873), anti-phospho-ATM S1981 (CS, #13050).

DNA repair foci - immunofluorescence assays.

DU145 cells were seeded and treated after 24hr with A196 4pM for 72hr. Then, cells were transferred on coverslips in 6-wells plate at 200,000 cells/well. After 24hr, cells were treated with etoposide for 24hr. Following this treatment, cells were washed with medium and a fresh medium was added with or without the maintenance of Al 96 and then fixed with paraformaldehyde 4% in PBS at room temperature after 0, 6, 24 and 48hr of etoposide removal. After fixation, cells were washed with PBS, and permeabilized for 10 min at room temperature with PBS 0.25% Triton-X 100. Then, cells were washed with PBS and incubated for about 1 hr at room temperature in 5%BSA PBS 0.1% Tween20 prior to incubation overnight at 4°C with 5-H2AX and 53BPlprimary antibodies diluted in blocking buffer (Dilution 1/500). Cells were then washed with PBS 0.5% Tween-20 and incubated with appropriate secondary mouse and rabbit antibodies coupled to Alexa Fluor 568 or 488 respectively diluted in blocking buffer (Dilution: 1/1000) for 2hr at room temperature. At last, cells were washed with PBS 0.5% Tween-20 and stained with DAPI for 10 min at room temperature, and coverslips were mounted with Prolong Gold antifade reagent. For imaging, images were acquired with a Zeiss apotome microscope fitted with a 63 objective. Zen Blue 3.3 was used to adjust brightness and contrast of corresponding images. Nuclear gH2AX and 53BP1 foci were quantified with Cellprofiler software.

Crispr/Cas9-mediated SUV4-20H1 and SUV4-20H2 KO models.

Knock-out of SUV4-20H1 and SUV4-20H2 were generated by CRISPR-Cas9 technology in prostate DU145 cell line. Briefly, DU145 cells were transfected using Jet-PEI with the plasmid pSpCAS9-2A-GFP and the lentiviral vector U6-gRNA2A-TagBFP expressing BFP and guide RNAs targeting SUV4-20H1 (HS5000028803) or SUV4-20H2 (HS5000023102). The guide RNAs were purchased from the Sanger Whole genome CRISPR arrayed library (Merck). Two days after transfection, cells were sorted by flow cytometry for GFP and BFP expression and positive clones were grown for three weeks without selection for Cas9 and RNA guides expression. The cellular clones inactivated for SUV4-20H1 or SUV4-20H2 were then identified by western blotting using antibodies against H4K20 methylation, SUV4-20H1 and SUV4-20H2. The inactivation of SUV4-20H1 or SUV4-20H2 encoding genes were then confirmed by DNA sequencing. The double SUV4-20H1 and SUV4-20H2 KO cells were generated by transfection of SUV4-20H2-KO DU145 cell line with the guide RNA targeting SUV4-20H1.

Measure of cell proliferation and sensitivity to drug treatments.

Cell viability upon drug treatments was evaluated using resazurin viability assays and cell proliferation and growth rate was measured with the incucyte cell-by-cell analysis module following the manufacturer recommendations (Sartorius).

Drug combination screening For drug combination screening, prostate cancer cell lines were treated with increasing concentration of each drug alone or in combination for three days. The percentage of viable and dead cells were then evaluated using the Celigo flow cytometer after DAPI and propidium iodide (PI) staining and following the manufacture recommendations (Nexcelom). The interaction between the drugs tested in vitro was quantified with a concentration matrix test, in which increasing concentration of each single drug were assessed with all possible combinations of the other drugs. For each combination, the percentage of expected growing cells in the case of effect independence was calculated according to the Bliss equation: fuc=fuAfuB, where fuc is the expected fraction of cells unaffected by the drug combination in the case of effect independence, and fuA and fuB are the fractions of cells unaffected by treatment A and B, respectively. The difference between the fuc value and the fraction of living cells in the cytotoxicity test was considered as an estimation of the interaction effect, with positive values indicating synergism and negative values antagonism.

Flow cytometry analysis.

Cells were incubated with 300pM BrdU (Sigma) for Ih, fixed with a 70% ethanol solution, then permeabilized with 0,2% Triton X-100 for lOmin. Then, cells were treated with 0,2N HC1 before staining with mouse antibody to BrdU (1:30 diluted in PBS with 0,2% Tween20, 1% Bovine Serum Albumine, Becton Dickinson) for Ih at room temperature, followed by 1 hour incubation with an FITC-conjugated antibody (BD; 1 :300). DNA was then counterstained by overnight incubation with 7- Amino- Actinomycin D (7AAD, 1 :50, Sigma) in the presence of RNAse. Data acquisition was obtained with Gallios flow cytometer (BD) and analysis was performed with Flow Jo software.

Colony formation assays.

Cells were seeded at 50000 cells/well in 12-well plate. After 24hr, cells were treated with combinational concentrations of A196 and etoposide for 72hr. Following treatment, drugs with media were removed and cells were harvested and resuspended in fresh medium at 200 cells/ml. Cells were seeded in new 12-well plates and incubated at 37°c for 14 days to allow the formation of colonies. After 14 days, medium was removed, and cells were fixed with methanol for 20 min then washed and incubated with crystal violet staining solution for 5min in order to stain the colonies.

DNA fiber assays.

DU145 cells were plated at 75000 cells/well in 6-well plates and allowed to adhere for 24 hr. Cells were then treated with A196 for 72hr. Subsequently, DNA was labeled for 30 minutes with 25 pM 5-iodo-20-deoxyuridine (IdU) and washed with medium, then treated with 50pM 5-chloro-20-deoxyuridine (CldU) for 30 minutes. After labeling, cells were collected with trypsin, washed with PBS, and resuspended in PBS at 500 cells/pL. Cell solution was placed on a slide for 3 minutes, followed by lysis for 3 minutes with 7pL of spreading buffer (0.5% SDS, 200 mM Tris-HCl, pH 7.5, and 50 mM EDTA). Slides were tilted to a 45° angle to allow fibers to spread, allowed to dry for 20 minutes, fixed in 3: 1 methanol: acetic acid for 10 minutes. After fixation, slides were rehydrated in water for 5 minutes, denatured with 2.5 M HC1 for Ihr 30 minutes, blocked with 6%BSA PBS 0.1% Tween-20 for 1 hr. Then, slides were incubated with primary anti-IdU and anti-CldU (45 minutes, 1 : 100) followed with secondary antibodies Alexa 488 anti -mouse and anti-rat-Cy3 respectively (30 minutes, 1 : 100) in PBS 0.1% TritonX-100 at 37°. Slides were washed with PBS and mounted with ProLong Gold antifade. Track lengths were measured using ImageJ software.

Chromatin compaction assays.

Chromatin compaction was evaluated by FLIM-FRET measurements in cells stably expressing H2B-~GFP alone or both H2B-GFP and mCherry-H2B. Fluorescence Lifetime Imaging Microscopy (FLIM) was performed using an inverted laser scanning multiphoton microscope LSM780 (Zeiss) equipped with temperature and CO2 controlled environmental black walls chamber. Measurements were acquired in live cells at 37°C, 5% CO2 and with a 40*oil immersion lens NA 1.3 Plan Apochromat objective from Zeiss. Two photon excitation was achieved using a Chameleon Ultra II tunable (680-1080 nm) laser (Coherent) to pump a mode locked frequency doubled Ti: Sapphire laser that provided sub 150 femtosecond pulses at a 80 Mhz repetition rate. Enhanced detection of the emitted photons was afforded by the use of the HPM 100 module (Hamamatsu R10467 40 GaAsP hybrid PMT tube). The fluorescence lifetime imaging capability was provided by TCSPC electronics (SPC 830; Becker & Hickl GmbH). TCSPC measures the time elapsed between laser pulses and the fluorescence photons. EGFP and mCherry fluorophores were used as a FRET pair. Fluorescence lifetime measurements were acquired over 60 sec and fluorescence lifetimes were calculated for all pixels in the field of view (256^256 pixels).

FLIM analysis was performed by using the SPCImage software (Becker & Hickl, GmbH). The FRET efficiency was calculated by comparing the FLIM values obtained for the EGFP donor fluorophores in presence and absence of the mCherry acceptor fluorophores. FRET efficiency (E% FRET) was derived by applying the following equation: E FRET = 1 - (TDA/TD), where TDA is the mean fluorescence lifetime of the donor (H2B-eGFP) and TD is the mean fluorescence lifetime of H2B-eGFP (in absence of acceptor) that are present in the same field of view. FRET efficiency values from 20 to 30 cells was then normalized and graphically represented using excel or GraphPad Prism softwares.

Purification and analysis of etoposide-trapped topoisomerase II complex

DU145 cells were plated and treated after 24h with A-196 4pM for 72h. Then, cells were transferred into a 6-well plate at 300,000 cells/well with maintenance of A-196. After 24h, cells were treated with different concentrations of etoposide for 3h. Following this treatment, cells were washed with PBS, harvested with trypsin, and washed with cold PBS. After gentle centrifugation, pellets were resuspended in lysis buffer (150 mM NaCl, 1 mM EDTA, 0.5% IGEPAL CA-630, 2X HALT Protease and Phosphatase Inhibitor Cocktail [Thermo Fisher Scientific] 20 mM Tris-HCl, pH 8.0) complemented with 100 U/mL Heparin (H3393, Sigma- Aldrich) for 15 minutes on ice. After the incubation, lysates were centrifuged at 15000 rpm for 5 minutes at 4°C and pellets were resuspended again with lysis buffer. To facilitate the migration of the protein in immunoblotting, a short chromatin sonication was performed with Bioruptor PICO (Diagenode) with 2 cycles - 30 sec ON and 30 sec Off each. Protein concentration was then determined with Nanodrop by measuring the absorbance at 280 nm. Before loading, heparin-based extracts were diluted with Laemmli buffer and Immunoblotting was performed with the following antibodies: anti-Topoisomerase Ila (sc- 365916) and anti-histone H3 (CS, #4499).

In vivo Animal studies

The Institute Animal Care and French APAFIS committee approved all mouse protocols used in this study (French national agreement #28412). One million (IxlO⁶) DU145 cells were subcutaneously injected into intact athymic nude male mice. Treatment was initiated when tumor size reached -100 mm3, and mice were randomized into four treatment groups: (1) Vehicle (20% DMSO/corn oil, 2x week, intraperitoneal (i.p.) injection), (2) Etoposide (0.1 mg/mouse, 2x week, i.p.), (3) A196 (0.3mg/mouse, 2x week, i.p.), (4) combination. Mice were weighed weekly to monitor for toxicity and tumor growth was assessed by serial caliper measurements once a week.

Results

In order to identify epigenetic targets for prostate cancer treatments, the inventors explored the transcriptomic data from the Cancer Genome Atlas (TCGA) and asked whether the cancer-related H4K20 methyltransferases, which are responsible for the methylation of histone H4 tail at lysine (K) 20, are differentially expressed between benign, local and metastatic primary prostate tumors. Whereas the expression of the H4K20 monomethyltransferase SET8 remained unchanged between benign and local prostate tumors or decreased in metastatic patient samples, a significant increase in SUV4-20H1 and SUV4- 20H2 expression is positively correlated with the appearance of prostate tumors (Figure 1A). Moreover, SUV4-20H2 expression, but not of SUV4-20H1, was even higher in metastatic tumor samples (Figure 1). These tumor samples mainly corresponds to aggressive prostate tumors and those resistance to AR therapy (2,3). Consistent with this, Kaplan-Meyer analysis of TCGA data revealed that higher SUV4-20H2 expression in patient prostate tumor samples was markedly associated with decrease overall survival (OS) (Figure IB) and disease-free interval (DFI) (Figure 1C).

According to the higher expression of SUV4-20H1 and SUV4-20H2 in primary prostate tumors, the inventors provide evidence that the pharmacological inhibition of SUV4- 20H1/2 enzymes by the chemical compound A196 (13,14) leads to the complete loss of H4K20me2/3 states in metastatic PCa cell lines independently to their AR status (LNCAP are AR positive cells while DU145 are AR negative cells) (Figure 2A). Same results are observed with other cancer cell lines such as US2OS cells, which derived from osteosarcoma (Figure 2A).

As shown in figure 2B, FRET-FLIM approaches with histone-fluorescent proteins reveal that the treatment of cancerous cells with Al 96 reduces the compaction of chromatin. This is accompanied by a significant acceleration of replication fork progression as measured by DNA fiber assays (data not shown). However, FACS analysis show similar cell-cycle distribution between untreated and A196-treated cells, indicating that changes in DNA replication upon Al 96 does not affect cell cycle progression (data not shown). Moreover, costaining with propidium iodide and DAPI show the absence of death cells (data not shown) and the proliferation rate between untreated and treated cells are similar (data not shown). In conclusion, although DNA replication and chromatin structure are modified upon Al 96, prostate cancer cell survival and proliferation are not affected by this chemical compound.

The specific epigenetic alterations induced by Al 96 without apparent cellular toxicity let us to determine whether Al 96 could induce specific synthetic lethality with FDA- approved drugs for cancer treatment. If such drug synergy may exist, this may lead to potential new chemotherapy for prostate cancer treatment. To this end, the inventors screen for FDA-approved compounds and search for specific toxicity in combination with Al 96 treatment in metastatic prostate cancer cell lines. For this screen, the inventors use the celigo imaging cytometer to measure at different concentrations of each drug, alone or in combination, cancer cell viability by imaging and quantifying DAPI and propidium iodide (PI) cell staining. By this approach, the inventors discover that the chemical compound Al 96 leads to a specific lethal synergy with innocuous concentrations of etoposide (15), a well characterized topoisomerase II (TOPO2) poison used for several cancer treatment (Figure 3 A). An additive lethality is also observed with mitoxantrone (15), another TOPO2 inhibitor already approved for prostate cancer treatment (Figure 3B). Consistent with these results, colony formation experiments show that the combination of Al 96 with etoposide or mitoxantrone lead to a strong synergy in AR-positive as well as in AR-negative prostate cancer cells (Figure 3C). At the molecular levels, the drug combination of the present invention causes the accumulation of cancerous cells in G2/M phase of the cell cycle followed by the death of most of these cells in a drug concentration dependent manner (Figure 3D and 3E). Using Crispr/Cas9-induced knock out of SUV4-20H1 and SUV4-20H2 enzymes in prostate cancer cell lines, the inventors demonstrate that the enhanced sensitivity of prostate cancer cells to TOPO2 poisons is indeed related to the enzymatic inhibition of both SUV4- 20H1 and SUV4-20H2 proteins (Figures 4A and 4B). Loss of one or the other SUV4-20H enzyme is not sufficient to induce a strong lethal synergy with etoposide (see Figure 4C). This demonstrates that the synergy effect of the compound Al 96 with TOPO2 inhibitors necessarily require the inhibition of SUV4-20H1 as well as SUV4-20H2 enzymes. From a mechanistic point of view, the inventors demonstrate that the Al 96 treatment strongly increased the levels of TOPO2-DNA complexes trapped by etoposide (Figure 5), thereby leading to perturb chromatin structure and to enhance DNA breaks. Moreover, the effectiveness of the drug combination of the present invention is related to the defects of proliferating cancerous cells to repair DNA breaks induced by TOPO2-DNA complexes when the enzymatic activity of SUV4-20H1 and SUV4-20H2 enzymes are inhibited (Figures 6A- 5B). These defects to repair TOPO2-induced DNA breaks in A196-treated cells are related to alterations in H4K20 methylation states, notably the loss of H4K20m3 and H4K20me states (Figure 6C). Accordingly, cancerous cells treated with the combination of Al 96 and etoposide displayed a specific impairment of DNA-PK-mediated DNA damage signaling pathways, as observed by the maintenance of high levels of DNA-PK phosphorylation, but not of ATM phosphorylation, after drug treatment (Figure 6D). The anti -tumor activity of Al 96 alone or in combination with etoposide was also evaluated in vivo by the inventors. Xenografted prostate tumor bearing mice (athymic nude mouse models) were treated with vehicle (20% DMSO + com oil, 2x week), etoposide (0.1 mg per mouse, 2x week), A 196 (0.3 mg per mouse, 2x week) or combination. No significant toxicity was observed in all therapies studies as shown by body weight measurement (Figure 7 A). However, while Al 96 or etoposide treatments had no significant impact by their own in these experiments, the inventors showed that the combination treatment significantly delayed xenografted prostate tumor growth (Figure 7B). These results demonstrate the potential superior anti -tumor activity and therapeutic efficacy of the A196-etoposide drug combination with minimal acute toxicity in vivo. Finally, the inventors show that the synthetic lethality induced by the combination Al 96 and etoposide is not specific to metastatic prostate cancer cell lines, but can also occur in other cancer cell types such as the breast cancer cell line MCF7 (see Figure 8).

Conclusions

Altogether, the present invention demonstrates that targeting SUV4-20H enzymes strongly improves the effectiveness of TOPO2 poisons in proliferating cancer cells in vitro and in vivo. Notably, this invention demonstrates that the combination of SUV4-20H and TOPO2 inhibitors constitutes a novel therapeutic approach for the treatment of metastatic prostate cancers, by improving the efficacy of TOPO2 inhibitors and preventing the repair of DNA breaks induced by these inhibitors in cancer cells. Another major interest of the drug combination of this present invention is the dose reduction of TOPO2 poisons, since it is an important issue in the clinic due to the cardio-toxicity (16,17) of these FDA-approved drugs notably for the elderly, who make up the majority of prostate cancer patients and have unfortunately limited until now the use of TOPO2 inhibitors for this type of patients.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. l.Wang G et al. (2018) Genetics and biology of prostate cancer. Genes & Dev

32: 1105-1140. 2. Feng, Q. & He, B. Androgen Receptor Signaling in the Development of Castration- Resistant Prostate Cancer. Front. Oncol. 9, 858 (2019).

3. Katsogiannou, M. et al. The hallmarks of castration-resistant prostate cancers. Cancer Treat. Rev. 41, 588-597 (2015).

4.Vlachostergios, P. J., Puca, L. & Beltran, H. Emerging Variants of Castration- Resistant Prostate Cancer. Curr. Oncol. Rep. 19, 32 (2017).

5. Yegnasubramanian, S., et al. Prostate Cancer Epigenetics. Cold Spring Harb. Perspect. Med. 9, (2019).

6. Ci, X. et al. Heterochromatin la Mediates Development and Aggressiveness of Prostate Cancer. Cancer Res. 78, 2691-2704 (2018).

7.Behbahani, T. E. et al. Alterations of global histone H4K20 methylation during prostate carcinogenesis. BMC Urol. 12, 5 (2012).

8. Black, J. C., et al. Histone lysine methylation dynamics: establishment, regulation, and biological impact. Mol. Cell 48, 491-507 (2012)

9. Yao, L. et al. Histone H4 Lys 20 methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer. Biochem. Biophys. Res. Commun. 450, 692-696 (2014).

10. Vieira, F. Q. et al. SMYD3 contributes to a more aggressive phenotype of prostate cancer and targets Cyclin D2 through H4K20me3. Oncotarget 6, 13644-13657 (2015).

11. Beck DB, et al. (2012) PR-Set7 and H4K20mel : at the crossroads of genome integrity, cell cycle, chromosome condensation and transcription. Genes Dev, 26, 2580-2589.

12. Jorgensen S, et al. (2013). Histone H4 lysine 20 methylation: key player in epigenetic regulation of genomic integrity Nucleic Acids Res. 41, 2797-2806.

13 Bromberg, et al. (2017) The SUV4-20 inhibitor A- 196 verifies a role for epigenetics. Nat. Chem. Biol. 13, 317-324.

14 Vilema-Enriquez G, et al. J Biol Chem. 2020 Dec 25;295(52): 17973-17985. doi: 10.1074/jbc.RA120.015533.

15 Vann KR, Oviatt AA, Osheroff N. Biochemistry. 2021 Jun l;60(21): 1630-1641. doi: 10.1021/acs. biochem. lc00240.

16 De Bono JS, et al. Lancet. 2010 Oct 2;376(9747): 1147-54. doi: 10.1016/S0140- 6736(10)61389

17 Cattrini C, Capaia M, Boccardo F, Barboro P. Cancer Treat Res Commun. 2020;25: 100221. doi: 10.1016/j.ctarc.2020.100221. Epub 2020 Oct 13. 18. Xing P, Wang S, Cao Y, Liu B, Zheng F, Guo W, Huang J, Zhao Z, Yang Z, Lin X, Sang L, Liu Z. Treatment strategies and drug resistance mechanisms in adenocarcinoma of different organs. Drug Resist Updat. 2023 Aug 22;71: 101002. doi: 10.1016/j.drup.2023.101002. Epub ahead of print. PMID: 37678078

Claims

CLAIMS:

1. A combination of a SUV4-20H inhibitor and a TOPO2 inhibitor for use in the treatment of a cancer in a subject in need thereof.

2. A i) SUV4-20H inhibitor and ii) a TOPO2 inhibitor, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject in need thereof.

3. A combination according to the claims 1 or 2 wherein the SUV4-20H is the compound Al 96 of formula I:

Formula (I)

4. A combination according to claims 1 to 3 wherein the TOPO2 inhibitor a is a TOPO2 poison.

5. A combination according to the claim 4 wherein the TOPO2 poison is the etoposide, the, mitoxantrone, the doxorubicin or the teniposide.

6. A combination according to the claims 1 to 5 wherein the cancer is an epithelial cancer prostate cancer or a breast cancer.

7. A combination according to the claim 6 wherein the cancer is a prostate cancer or a breast cancer.

8. A combination according to the claims 1 to 5 wherein the prostate cancer is an aggressive prostate cancer or a resistant prostate cancer. A therapeutic composition comprising a SUV4-20H inhibitor and a TOPO2 inhibitor for use in the treatment of a cancer in a subject in need thereof. A method for treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of SUV4-20H inhibitor and a TOPO2 inhibitor.