FIELD OF THE INVENTION:

The present invention relates to an inhibitor of DHODH and an inhibitor of Chkl for use in the treatment of a cancer in a subject in need thereof.

BACKGROUND OF THE INVENTION:

Dihydroorotate dehydrogenase (EC 1.3.5.2; DHODH) is the one mitochondrial enzyme that is located on the outer surface of the inner membrane and takes part in the fourth and rate-limiting step of de novo pyrimidine biosynthesis [1]. It converts dihydroorotic acid to orotic acid whilst reducing ubiquinone to ubiquinol which makes DHODH a link between pyrimidine synthesis and respiratory electron transport chain.

DHODH has emerged as a new therapeutic target in a wide spectrum of pathologies as de novo pyrimidine synthesis is extensively used in rapidly proliferating human or parasitic cells. Much effort has been devoted to designing new inhibitors in order to overcome widespread resistance to current antimalarial drugs [2-5] inasmuch as Plasmodium proliferation relies exclusively on this pathway [6]. Series of original compounds were also synthesised as part of a program aiming at identifying new antivirals [7-11] and a new compound is currently in clinical development for the treatment of fungal infection [12]. The immunosuppressant leflunomide has been prescribed for the treatment of inflammatory response associated with rheumatoid arthritis [13-16] and the immunomodulatory properties of its active metabolite teriflunomide (TFN) led to its recent approval for the treatment of relapsing remitting multiple sclerosis [17-19]. DHODH inhibition also effectively slowed down cancer cell and tumour growth of diverse tissue origins [20-24].

These inhibitors reduce dNTP pools available for DNA replication. Limiting precursors of DNA synthesis has been reported as a source of genetic instability [25-27] and reduced processivity of enzymes at replication forks or replication fork stalling [28,29]. In order to prevent genetic instability, cells trigger a signalling pathway in which Chkl effector kinase plays a crucial role through the activation of checkpoints in response to replication or genotoxic stress [30-32]. A wide array of chemotherapeutic drugs have been combined with Chkl inhibitors in order to optimise treatment through the abrogation of checkpoints controlled by this kinase and allow accumulation of DNA damage that would jeopardize genome stability or induce cell death in a p53 -compromised background [33]. Interestingly, our recent data [34] showed that upon knockout of E4F1 transcription factor transformed cells elicited major mitochondrial dysfunctions including a drastic reduction in levels of orotic acid and downstream pyrimidine intermediates. Furthermore E4F1 also controls the expression of Chkl gene, which results in a strong down-regulation of Chkl protein expression and kinase activity in E4F1KO cells. We also observed that this combined down-regulation of mitochondrial and checkpoint activities strongly impacts on transformed cell survival, highlighting the potential interest of mimicking the deadly environment of E4F1KO cells by combining mitochondrial and checkpoint inhibitors. SUMMARY OF THE INVENTION:

The current study sought to investigate whether this antiproliferative effect of DHODH inhibitors could be enhanced by combining Chkl kinase inhibition. The pharmacological activity of DHODH inhibitor teriflunomide was more selective towards transformed mouse embryonic fibroblasts than their primary or immortalised counterparts, and this effect was amplified when cells were subsequently exposed to PF477736 Chkl inhibitor. Flow cytometry analyses revealed substantial accumulations of cells in S and G2/M phases, followed by increased cytotoxicity which was characterised by caspase 3-dependent induction of cell death. Associating PF477736 with teriflunomide significantly sensitised SUM 159 and HCC1937 human triple negative breast cancer cell lines to dihydroorotate dehydrogenase inhibition. The main characteristic of this effect was the sustained accumulation of teriflunomide-induced DNA damage as cells displayed increased phospho serine 139 H2AX (γΗ2ΑΧ) levels and concentration-dependent phosphorylation of Chkl on serine 345 upon exposure to the combination as compared with either inhibitor alone. More, the combination of the DHODH inhibitor with the Chkl inhibitor in a significant lower dose allow to minimize the off-target effects of the Chkl inhibitor. Altogether these results suggest that combining DHODH and Chkl inhibitions may be a strategy worth considering as a potential alternative to conventional chemotherapies.

Thus, the present invention relates to an inhibitor of DHODH and an inhibitor of Chkl for use in the treatment of a cancer in a subject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention relates to the combination of an inhibitor of DHODH and an inhibitor of Chkl for use in the treatment of a cancer in a subject in need thereof. In another embodiment, the invention relates to i) an inhibitor of DHODH and ii) an inhibitor of Chkl, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject in need thereof.

In another particular embodiment, the invention relates to an inhibitor of DHODH protein and ii) an inhibitor of Chkl protein as a combined preparation for simultaneous use in the treatment of cancer.

As used herein, the term "DHODH" for "dihydroorotate dehydrogenase" denotes an enzyme which catalyzes the fourth enzymatic step, the ubiquinone-mediated oxidation of dihydroorotate to orotate, in de novo pyrimidine biosynthesis. This protein is a mitochondrial protein located on the outer surface of the inner mitochondrial membrane (IMM). Inhibitors of this enzyme are used to treat autoimmune diseases such as rheumatoid arthritis. (Entrez Gene ID number: 1723; mR A sequences references RefSeq: NM 001361.4; protein sequence reference RefSeq: NP 001352.2; Uniprot: Q02127).

As used herein and according to all aspects of the invention, the term "CHK1" for "replication checkpoint 1" also known as CHEK1 refers to the human gene encoding a DNA replication checkpoint kinase that signals the DNA replication fork stalling and phosphorylates cdc25, an important phosphatase in cell cycle control, particularly for entry into mitosis (Entrez Gene ID number: 1111; mRNA sequences references RefSeq: NM_001114121, NM_001114122.2, NM_001244846.1, NM_001274.5 , NM_001330427.1, NM 001330428.1; protein sequence reference RefSeq: NP 001107593, NP 001107594.1, NP_001231775.1, NP_001265.2, NP_001317356.1, NP_001317357.1).

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 a p53 -deficient cancer. In a particular embodiment, the cancer is a breast cancer. More particularly, triple negative breast cancer and high-grade serous ovarian carcinoma.

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.]).

The terms "DHODH inhibitor" or "Chkl inhibitor" denotes molecules or compound which can inhibit the activity of the proteins (e.g. inhibit the kinase, polymerase, reductase activity of the proteins) or a molecule or compound which destabilizes the proteins. In particular, an inhibitor of DHODH can inhibit the pyrimidine synthesis and particularly inhibit the reduction of ubiquinone to ubiquinol. In particular, an inhibitor of Chkl can inhibit the phosphorylation of cdc25.

The term "DHODH inhibitor" or "Chkl inhibitor" also denotes inhibitors of the expression of the gene coding for the proteins.

In one embodiment, the inhibitors according to the invention 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.

In one embodiment, the inhibitor of DHODH according to the invention may be the leflunomide or its active metabolite, the teriflunomide (TFN) of formula (I):

Formula (I)

In one embodiment, the inhibitor of DHODH according to the invention may be the Leflunomide or its active metabolite, the IPP- AO 17- A04 (TFN) of formula (II) :

Fomula (II)

In one embodiments, the DHODH inhibitor is an inhibitor described in the articles: Davies M et al 2009; Booker ML et al 2010; Coteron JM et al 2011; Skerlj RT et al 2011 ; Reyes P et al 1982; Bonavia A et al 2011; Hoffmann H-H et al 2011; Munier-Lehmann H et al 2015; Lucas-Hourani M et al 2015; Munier-Lehmann H et al 2013.

In one embodiments, the DHODH inhibitor is the compounds 2-(3' -ethoxy-3,5- difluorobiphenyl-4-ylamino)nicotinic acid; 2-(3,5-difluoro-3 -methoxybiphenyl-4- ylamino)nicotinic acid; 2-(3' -cyclopropoxy-3,5-difluorobiphenyl-4-ylamino)nicotinic acid; 2-(3,5-difluoro-3 -methoxybiphenyl-4-ylamino)-5-methylnicotinic acid; or a pharmaceutically acceptable salt or N-oxide thereof as described in the patent application WO2009153043.

In one embodiments, the DHODH inhibitor is the ASLAN003 compound from the company ASLAN Pharma as described in aslanpharma.com/drug/aslan003/.

In one embodiments, the DHODH inhibitor is a compound as described in the patent application WO2017037292.

In one embodiment, the inhibitor of Chkl according to the invention may be the PF477736 compound of formula (III):

Fomula (III)

In one embodiment, the inhibitor of Chkl according to the invention may be the Chkl inhibitor 7-hydroxystaurosporine as described in D Sampath, et al, 2005.

In one embodiments, the Chkl inhibitor is an inhibitor described in Prudhomme, Recent Patents on Anti-Cancer Drug Discovery, 2006, 1, 55-68; Expert Opin. Ther. Patents (2011) 21(8): 1191-1210; and Cell Cycle (201 1) 10: 13, 2121-2128, each of which is incorporated herein by reference.

In another embodiment, the Chkl inhibitor is selected from the group consisting of

AZD7762, LY2603618, LY2606368 (prexasertib); PF-00477736, and SCH 900776 (See Cancer Res. (2010) 70(12): 4972 et seq.; Clin. Cancer Res. (2010) 16(7): 2076-2084; and Shibata et al, Cancer Sci. (2011), each of which is incorporated herein by reference).

In one embodiments, the DHODH inhibitor is the GDC-0575 compound from the company genentech as described in gene.com/medical-professionals/pipeline/.

In still another embodiment, an inhibitor of the Chkl according to the invention may be a compound as described in the patent application WO2008132500.

In one embodiment, the inhibitor according to the invention (inhibitors of DHODH or Chkl) is an antibody. Antibodies or directed against DHODH or Chkl can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against DHODH or Chkl can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Ko filer and Milstein (1975); the human B-cell hybridoma technique (Cote et al, 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti- DHODH or Chkl single chain antibodies. Coumpounds useful in practicing the present invention also include anti- DHODH or Chkl antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to DHODH or Chkl .

Humanized anti- DHODH or anti- Chkl antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

Then, for this invention, neutralizing antibodies of DHODH or Chkl are selected. In a particular embodiment, the anti-Chkl antibody according to the invention may be the sc-8408 antibody as send by Santa Cruz biotechnology. In another embodiment, the antibody according to the invention is a single domain antibody against DHODH or Chkl . The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody®". According to the invention, sdAb can particularly be llama sdAb. The term "VHH" refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term "complementarity determining region" or "CDR" refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in- vitro maturation.

VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen- specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the "Hamers patents" describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The "Hamers patents" more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

Then, for this invention, neutralizing aptamers of DHODH or Chkl are selected.

In one embodiment, the compound according to the invention is a polypeptide.

In a particular embodiment the polypeptide is an antagonist of DHODH or Chkl and is capable to prevent the function of DHODH or Chkl . Particularly, the polypeptide can be a mutated DHODH or Chkl protein or a similar protein without the function of DHODH or Chkl .

In one embodiment, the polypeptide of the invention may be linked to a cell- penetrating 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 bio materials 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 half- life, 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 DHODH or Chkl inhibitor according to the invention is an inhibitor of DHODH or Chkl gene expression.

Small inhibitory RNAs (siRNAs) can also function as inhibitors of DHODH or Chkl expression for use in the present invention. DHODH or Chkl gene expression 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 DHODH or CHkl 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 DHODH or CHkl 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 endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of DHODH or CHkl 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 DHODH or CHkl 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'-0-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 DHODH or CHkl . 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 siR A 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 R A 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 nonessential 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 microencapsulation.

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 another embodiment, the invention relates to a method for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of DHODH and an inhibitor of Chk 1.

In order to test the functionality of a putative DHODH or Chkl inhibitor a test is necessary. For that purpose, to identify DHODH inhibitors, the pyrimidine synthesis or the reduction of ubiquinone to ubiquinol can be tested. The impact of the inhibitors on DHODH enzymatic activity can also be tested using enzymatic assays as described in Knecht and Loffler, 1998; Miller et al, 1968 or in Yin S et al. Sci Rep. 2017 Jan 13;7:40670. To identify Chkl inhibitors, the impact of these inhibitors on the phosphorylation of cdc25 and the auto- phosphorylation of Chkl can be tested. Inhibition of Chkl kinase activity can also be tested by an in vitro Kinase assay performed with purified Chkl (immuno-precipitated from Human cells or recombinant protein) and a purified protein substrate of Chkl, in presence of labelled ATP.

Therapeutic composition

Another object of the invention relates to a therapeutic composition comprising an inhibitor of DHODH and an inhibitor of Chkl according to the invention for use in the treatment of 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), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) 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, procarbazine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, 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 (PDCDl, 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-IDOl 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. The combination of teriflunomide and PF477736 results in increased antiproliferative effect in transformed mouse embryonic fibroblasts. Primary, immortalised or transformed mouse embryonic fibroblasts were exposed for 24 hours to increasing concentrations of teriflunomide ± transformed MEF ICIO PF477736 (0.7 μΜ) (which was added 30 minutes after the beginning of exposure to TFN) (A) or IPP-A017-A04 ± transformed MEF ICIO PF477736 (B), and grown in drug-free medium for three doubling times. Mean ± SD, n=3 independent experiments. * p < 0.05, ** p < 0.01 as determined by two-tailed unpaired t-test.

Figure 2. Pharmacological activity of PF477736 as a single agent or in combination with teriflunomide in triple negative breast cancer cell lines. (A) SUM 159, HCC1937, BT549 and HCC38 cells were exposed to increasing concentrations of PF477736 for 24 hours and grown in drug-free medium for three doubling times. Mean ± SD, n=3 independent experiments. (B) BT549 and (C) HCC38 cells were exposed for 24 hours to increasing concentrations of teriflunomide ± ICIO PF477736 (0.05 μΜ and 0.02 μΜ respectively, added 30 minutes after the beginning of exposure to TFN) and grown in drug- free medium for three doubling times. Mean ± SD, n=3 independent experiments.

Figure 3. The combination of teriflunomide and low dose of PF477736 (ICIO) reduces proliferation of SUM159 and HCC1937 triple negative breast cancer cell lines. (A) SUM 159 and (B) HCC1937 cells were exposed for 24 hours to increasing concentrations of teriflunomide ± ICIO PF477736 (2.5 μΜ and 0.29 μΜ respectively, added 30 minutes after the beginning of exposure to TFN) and grown in drug-free medium for three doubling times. Mean ± SD, n=3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by two-tailed unpaired t-test.

Figure 4: The combination of teriflunomide and PF477736 reduces proliferation of HCC1937 triple negative breast cancer cell line. HCC1937 cells were exposed for 24 hours to increasing concentrations of teriflunomide ± ICIO PF477736 (0.40 μΜ), added 30 minutes after the beginning of exposure to TFN) and grown in drug-free medium for three doubling times. Mean ± SD, n=3 independent experiments.

Figure 5: The combination of teriflunomide and PF477736 reduces proliferation of MDA-MB-231 triple negative breast cancer cell lines. MDA-MB-231 cells were exposed for 24 hours to increasing concentrations of teriflunomide ± ICIO PF477736 (0.25 μΜ, added 30 minutes after the beginning of exposure to TFN) and grown in drug-free medium for three doubling times. Mean ± SD, n=3 independent experiments.

EXAMPLE:

Material & Methods

Chemicals

The active metabolite of leflunomide, teriflunomide (A771726, 2-cyano-3-hydroxy-N- (4-(trifluoromethyl)phenyl)but-2-enamide, see Suppl. Fig. 1), was purchased from Selleck Chemicals LLC (Houston, TX). DHODH inhibitor IPP-A017-A04 (5-cyclopropyl-2-(4-(2,6- difluorophenoxy)-3-isopropoxy-5-methyl-lH-pyrazol-l-yl)-3-fl uoropyridine, see Suppl. Fig. 1) was synthesised according to the published procedure [10] and Chkl inhibitor PF477736 ((2R)-2-Amino-2-cyclohexyl-N-[2-( 1 -methyl- 1 H-pyrazol-4-yl)-6-oxo-5 ,6-dihydro- 1 H- [l,2]diazepino[4,5,6-cd]indol-8-yl]-acetamide, see Suppl. Fig.l) was purchased from Axon Medchem (Groningen, Netherlands). Drugs were kept at -20°C as 10 mM stock solutions in DMSO. Paclitaxel was supplied by Fresenius-Kabi. Camptothecin, gemcitabine, hydroxyurea, sulforhodamine B, 7 AminoActinomycin D, propidium iodide and RNase A were purchased from Sigma Aldrich.

Cell culture

Primary, p53KO and p53KO mouse embryonic fibroblasts transformed by HaRasV12

[34] were grown in DMEM-GlutaMax supplemented with 10 % fetal bovine serum, 100 μg/ml streptomycin and 100 units/ml penicillin. SUM 159 triple negative breast cancer cells were obtained from Asterand Bioscience, UK, and grown in Ham's F 12 medium supplemented with 5 % fetal bovine serum, 10 μg/ml insulin, 1 μg/ml hydrocortisone, 100 μ^πιΐ streptomycin and 100 units/ml penicillin. HCC1937 (ATCC-CRL-2336), HCC38 (ATCC-CRL-2314) and BT 549 (ATCC HTB-122) triple negative breast cancer cells were obtained from American Type Culture Collection. HCC1937 and HCC38 cells were grown in RPMI supplemented with 10 % fetal bovine serum, 100 μg/ml streptomycin and 100 units/ml penicillin. BT-549 cells were grown in Dulbecco Modified Eagle Medium supplemented with 10 % fetal bovine serum, 100 μg/ml streptomycin and 100 units/ml penicillin. All cell types were grown at 37°C in a humidified atmosphere containing 5% C02 and were regularly checked for the absence of mycoplasma contamination.

Growth inhibition assay

Inhibition of cell growth for each individual drug or combinations was assessed using the sulforhodamine B technique [35]. Two thousand SUM 159 or BT-549 breast cancer cells, HaRasV12 transformed p53KO or p53KO mouse embryonic fibroblasts, fifteen hundred HCC38 cells, one thousand HCC1937 cells or four thousand primary mouse embryonic fibroblasts were seeded onto 96-well plates on day 1. On day 2, cells were exposed for 24 hours to increasing concentrations of terifiunomide, IPP-A017-A04 or PF477736. Medium was then replaced and cells grown for three doubling times, fixed with 12.5% trichloroacetic acid in PBS, washed with water and stained with 0.4 % sulforhodamine B in 1% acetic acid. Each well was washed 5 times with acetic acid to remove unbound dye and protein bound SRB was extracted with 10 mM Tris base pH 10.5. Optical density (540 nm) in each well was measured using a Tecan® infinite 200 PRO multimode reader. Average +/- SD values from three independent experiments were plotted using GraphPad Prism software.

Small Interfering RNA Transfection

Transformed mouse embryonic fibroblasts were transfected with 700 pmoles of either a mix of 4 target-specific 19-25 nt small interfering RNA (siRNA) for mouse Chkl (Santa Cruz Biotechnology sc 29270), or SASI_Mm01_00087610 and SASI_Mm01_00087610 MISSION® siRNA for mouse DHODH (Sigma Aldrich), or 1400 pmoles of the combination of Chkl and DHODH siRNA. SUM159 cells were transfected with 700 pmoles of either SASI_Hs02_00326304 and SASI_Hs02_00326304_AS MISSION® siRNA for Human Kinase Chkl (Sigma Aldrich), or SASI_Hs01_00246561 and SASI HsOl 00246561 AS MISSION® siRNA for human DHODH (Sigma Aldrich) and 1400 pmoles of the combination of Chkl and DHODH siRNA. A 20-25 nt siRNA (Santa Cruz Biotechnology sc- 37007) was used as a control. Transfection of siRNA duplexes was performed for 5 hours using lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's instructions. Samples were collected 48 hours post-transfection and probed for Chkl and DHODH protein levels.

Flow cytometric analysis of cell cycle phase distribution

Exponentially growing cells were exposed to teriflunomide, IPP-A017-A04, PF4777736, 0.1 μΜ camptothecin or 5 mM hydroxyurea (used as positive controls) or the combination of a DHODH inhibitor and PF477736 for 24 hours then grown in drug-free medium. At each time point (8, 24 and 48 hours after the beginning of the exposure), cells were washed once with ice-cold PBS, trypsinised and counted. One million cells per sample were fixed in ice-cold 70% ethanol and stored at -20°C until analysis. They were then washed in PBS before being suspended in 0.5 ml staining solution (5 μg/ml 7-AminoActinomycin D or 25 μg/ml propidium iodide, 200 μg/ml ribonuclease A in PBS) and incubated at 37°C for 30 min. Analysis of 10000 events was performed on a FACSCalibur flow cytometer (Becton Dickinson). DNA fluorescence was collected in linear mode using a doublet discrimination gate and cell cycle distribution was analysed using FlowJo software (Tree Star). Each experiment was performed three times.

Quantification of cell death using multiparametric flow cytometry

Exponentially growing cells were exposed to teriflunomide, IPP-A017-A04, PF4777736, 0.1 μΜ camptothecin, 5 mM hydroxyurea or the combination of a DHODH inhibitor and PF477736 for 24 hours then grown in drug-free medium. At each time point, cells were washed with PBS, trypsinised, pooled with floating cells and counted. The annexin V and 7-AAD (or propidium iodide) dual labelling of apoptotic cells was conducted using the annexin V-FLUOS Staining Kit from Roche Applied Science according to the manufacturer's instructions. Two million cells were washed twice with PBS and once with binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM CaC12, pH 7.4). They were then incubated at room temperature in the dark for 30 min in 2 μΐ annexin- V-Fluos reagent and 5 μg/ml 7-AAD (or 25 μg/ml propidium iodide) in binding buffer. Analysis of 20000 events was performed on a FACSCalibur flow cytometer. DNA fluorescence was collected in logarithmic mode and cell viability was quantitated using FlowJo software. The population of dead cells was calculated as the sum of events in upper (left + right) and lower right quadrants in "annexin- V-FITC" vs "7-AAD" dot plots. Each experiment was performed three times.

Immunoblotting

Exponentially growing cells were exposed to teriflunomide, IPP-A017-A04, PF4777736, 0.1 μΜ camptothecin, 40 μΜ gemcitabine, or the combination of a DHODH inhibitor and PF477736 for up to 24 hours. At each mentioned time point, cells were collected, washed with ice-cold PBS and pellets were resuspended in lysis buffer (50 mM Tris HC1 pH 7.4 containing 100 mM NaCl, 50 mM NaF, 40 mM □ -glycerophosphate, 5 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 100 μΜ PMSF, 1 μΜ leupeptin, 1 μΜ pepstatin A and 1 μΜ aprotinin) and incubated on ice for 20 minutes. Lysates were then centrifuged for 10 min at 13000 rpm and 4°C and supernatant protein concentrations were determined using the bicinchoninic acid assay.

Twenty-five microgram proteins were resolved in 7.5 % polyacrylamide gels and transferred onto nitrocellulose membranes (Whatman). Membranes were incubated overnight with either mouse monoclonal anti-Chkl (G4, Santa Cruz Biotechnology), rabbit anti- Phospho-Chkl (Ser345 or Ser296, Cell Signalling Technology), mouse monoclonal anti-Chk2 (clone 7, Upstate), mouse monoclonal anti-DHODH (E-8, Santa Cruz Biotechnology) or mouse monoclonal anti-D-actin (clone AC-15, Sigma) antibodies. For cleaved caspase-3 and gamma-H2AX assays, 15 microgram proteins were resolved in 15 % polyacrylamide gels. Membranes were incubated overnight with either anti-phospho-histone (Serl39) H2AX (1/500, Merck Millipore), cleaved caspase-3 (Aspl75) (Cell Signalling Technology) or anti- tubulin (Sigma) rabbit antibodies. Signals were visualised using horseradish-conjugated antibodies and Luminata Chemiluminescent detection substrate (Millipore).

Immuno fluorescence

Exponentially growing cells were seeded in chamber slides, exposed to teriflunomide, IPP A017-A04, PF4777736, 0.1 μΜ camptothecin or the combination of a DHODH inhibitor and PF477736 for up to 24 hours. Cells were washed twice with PBS, incubated in 4% paraformaldehyde in PBS for 10 minutes, washed in PBS for 5 minutes, blocked with PBS containing 2% bovine serum albumin and 0.5% triton X-100, and incubated overnight at 4°C with anti-phospho-histone (Serl39) H2AX (1/500, Merck Millipore) antibody. Slides were washed, incubated with a FITC AlexaFluor 488 conjugated goat anti-rabbit IgG antibody for 1 hour at room temperature, and washed in PBS. Slides were mounted and image acquisition was performed on a LSM780 confocal microscope (Zeiss, Germany) at the Montpellier RIO Imaging facility (Campus CNRS route de Mende). Images were processed using ImageJ software.

Immunohistochemistry analysis

Five tumour-bearing mice were monitored in each group on days 2 and 3 of the protocol for pharmacodynamic assessment of the aforementioned regimens by immunohistochemistry. Tumours were harvested 24 or 48 hours after the beginning of each treatment and were fixed with formalin, all sections were counterstained with hematoxylin and eosin. Samples were also probed for Ki67 and cleaved caspase-3 levels. Quantitative analysis of Ki67 section staining was performed using ImageJ software and caspase-3 cleavage was quantitated using Aperio ImageScope software.

Statistical analyses

In vitro data expressed as mean values +/- SD of three independent experiments were analysed using Statview 5.0 software (Informer Technologies) using a two-tailed unpaired Student t test. In vivo data were analysed according to a non-linear mixed-effect model using Stata ver.13 (Stata Corporation, College Station, TX, USA). Kaplan-Meier survival curves were compared using the Log-rank test.

Results

Pharmacological activity of DHODH inhibitors in transformed mouse embryonic fibroblasts

The antiproliferative effect of DHODH inhibitor teriflunomide (TFN) was determined in primary, p53KO and p53KO mouse embryonic fibroblasts transformed by HaRasV12 derived from the same embryo [34] (data not shown). While a 24-hour exposure to TFN had a limited effect on primary and immortalised cells, it strongly reduced proliferation (monitored three doubling times after the end of this exposure) of transformed cells in a concentration- dependent manner (p < 0.01). This differential effect was also observed when these cell populations were exposed to another DHODH inhibitor, IPP-A017-A04 [10], and the antiproliferative effect of both compounds was partly reversed by concomitant exposure to 50 μg/ml uridine (data not shown). To characterise this antiproliferative effect transformed MEFs were then exposed to TFN and cell cycle distribution was assessed for up to 48 hours (data not shown). Exposure to a high TFN concentration (IC90) induced a significant S phase accumulation at 24 hours followed with the appearance of a sub-Gl population at 48 hours. At lower concentrations such as IC50 and IC70 these effects were barely detectable at these time points.

Pharmacological activity of Chkl inhibitor PF477736 in combination with DHODH inhibition in primary, immortalised and transformed mouse embryonic fibroblasts

Primary, immortalised and transformed MEFs were also exposed to increasing concentrations of Chkl inhibitor PF477736. Consistent with previous observations in human cancer cells [33], the antiproliferative effect of this compound was also more prominent in transformed MEFs than in their immortalised or primary counterparts (data not shown). These transformed fibroblasts were then exposed to ranging (IC10, IC50 and IC90) concentrations of PF477736 and cell cycle distribution was assessed at the 48-hour time point (data not shown). Profiles were indicative of a concentration-dependent cell cycle accumulation in S and G2/M phases along with the appearance, at the highest PF477736 concentrations, of hyperploid and sub-Gl cells. Of note these effects were undetectable upon exposure to IC10 PF477736. The same PF477736 concentration was then combined with ranging TFN concentrations to assess potentiation (Fig. 1A). A moderate but significant potentiation effect was detected in immortalised and transformed cells (n = 3 independent experiments) suggesting that as little as IC10 PF477736 sensitised these cell models to DHODH inhibitors. Accordingly a similar potentiation effect of IC10 PF477736 occurred when combined with IPP A017 A04 (Fig. IB).

In transformed cells this potentiation effect was significant (p = 0.0328) when IC10 PF477736 was combined with 10 μΜ TFN (IC70) (Fig 1A). Cell cycle distribution was therefore monitored at these optimal concentrations for up to 48 hours (data not shown). Cells exposed to this combination already accumulated in S phase 8 hours after the beginning of the time course. At 24 hours they further accumulated in S as well as G2/M. At this time point a substantial hyperploid population was also detectable. Finally, at a later time point (48h), sub Gl cells were clearly detected upon exposure to this TFN + PF477736 combination. Importantly, neither cell cycle perturbation nor cell death was observed with either inhibitor alone. A similar trend was noticed in cells exposed to the IPP A017 A04 + PF477736 combination (data not shown).

DNA damage signalling in response to DHODH and Chkl inhibitors in transformed mouse embryonic fibroblasts

Nucleotide depletion is a major cause of replication stress and DNA damage. Cells cope with these insults by triggering a protective signalling pathway that involves Chkl kinase activation by phosphorylation. Thus the combination of antimetabolites such as gemcitabine and Chkl inhibitors was reported to induce DNA double strand breaks [36]. We therefore hypothesized that cells exposed to DHODH and Chkl inhibitors would accumulate DNA damage as monitored by the presence of γΗ2ΑΧ staining (H2AX phosphorylation on serine 139) and ATR-dependent phosphorylation of Chkl on serine 345. Gamma H2AX staining and Chkl phosphorylation were indeed reported as pharmacodynamic markers of chemopotentiation and Chkl inhibition in combinations of genotoxic drugs and Chkl inhibitors [37].

While the amount and intensity of γΗ2ΑΧ foci were limited in transformed cells exposed to IC70 TFN or IC10 PF477736 alone, cell exposure to the combination of these inhibitors resulted in a significant increase in γΗ2ΑΧ staining (data not shown), indicative of substantial DNA damage. Consistently the phosphorylation of Chkl on serine 345 was also strongly stimulated in these TFN + PF477736-treated cells (data not shown) whereas it was undetectable in cells exposed to either inhibitor alone. Similar results were obtained when cells were exposed to a combination involving IPP-A017-A04 DHODH inhibitor. Interestingly, the intensity of Chkl phosphorylation on serine 345 in the presence of PF477736 was dependent on TFN concentration (data not shown). This suggested that Chkl phosphorylation on serine 345, which is a hallmark of Chkl activation, could be used as a quantitative marker of DHODH inhibition in this setting.

Of note DNA damage is also known to stimulate Chk2 phosphorylation [38] as exemplified here by exposure of transformed MEFs to camptothecin (CPT) used as a positive control (data not shown). Accordingly Chk2 phosphorylation was detected in lysates prepared from cells exposed to DHODH + Chkl inhibitors.

Cell death in transformed mouse embryonic fibroblasts exposed to the combination of DHODH and Chkl inhibitors

The induction of massive DNA damage suggested by γΗ2ΑΧ staining and Chkl / Chk2 phosphorylation prompted us to assess cell fate in response to these compounds and their combination. In order to assess cell death, annexin V/7-AAD staining was performed in cell populations that were collected 48 hours after the beginning of the time course. Dot plots (data not shown) showed a larger population of annexin V positive/7-AAD positive cells upon exposure to the combination as compared with matched controls or each individual inhibitor. These annexin V/7-AAD profiles were indicative of both apoptosis and necrosis. Upon quantification (data not shown), this increase in mortality was highly significant (p = 0.0002 as compared with TFN ; p = 0.0089 as compared with PF477736 and p = 0.0245 as compared with camptothecin). Again a similar trend was noticed upon exposure to the IPP AO 17 A04 + PF477736 combination as compared with single compounds or matched controls (data not shown).

In order to confirm that exposure to Chkl and DHODH inhibitors results in similar phenotypic changes as the ones that occur upon loss of Chkl and DHODH functions, transformed mouse embryonic fibroblasts were transfected with siRNA for Chkl and DHODH and protein depletion was assessed using western blotting at 48 hours (data not shown). Flow cytometry analysis showed a significant induction of cell death upon dual siRNA knockdown as compared with single depletions or control siRNA (data not shown). These results suggested that the effects that were observed upon PF477736 and TFN exposure were representative of Chkl and DHODH inhibitions respectively.

Phenotypic effect of the combination of DHODH and Chkl inhibitors in triple negative breast cancer cell lines

The cytotoxic effect that was observed in transformed mouse embryonic fibroblasts raised the question as to whether the combination of DHODH and Chkl inhibitors would also be effective in a model of human cancer cells. Our interest was focused on triple negative breast cancer (TNBC) as this pathology is highly resistant to most conventional chemotherapies.

In order to ensure the selective effect of Chkl inhibition in this strategy, SUM 159

TNBC cells were exposed to increasing concentrations of TFN with or without a fixed concentration of PF477736. We compared the modulation of the antiproliferative effect of TFN by either 0.1 μΜ, 0.5 μΜ or 2.5 μΜ PF477736 (the latter corresponding to IC10 in this cell line, Fig. 2A). The chemopotentiation of TFN effect by PF477736 was dependent on the value of the fixed concentration of Chkl inhibitor (data not shown). The autophosphorylation of Chkl on serine 296 that occurs upon DNA damage and is considered as a relevant biomarker for Chkl kinase activity, was assessed by western blotting (data not shown). SUM159 cells were exposed to either 0.1 μΜ, 0.5 μΜ or 2.5 μΜ PF477736 alone or in combination with IC70 TFN (or 40 μΜ gemcitabine as a positive control). Phospho-Ser296 Chkl levels were reduced in a concentration-dependent manner, with the lowest level observed at 2.5 μΜ PF477736, which confirmed optimal kinase inhibition at that concentration. A concentration-dependent increase in phospho-Ser345 Chkl levels was concurrently observed when Chkl was inhibited (data not shown). Thus the increase in the antiproliferative effect of TFN was dependent on PF477736 concentration up to the IC10, and the involvement of Chkl inhibition in this phenomenon was confirmed by the concomitant decrease in the levels of Chkl phosphorylation on serine 296 within the same concentration range.

As our strategy was validated and the antiproliferative effect of PF477736 as a single agent was determined in a panel of four triple negative breast cancer cell lines including SUM159 (Fig 2A), each cell model was exposed to increasing TFN concentrations with or without IC10 PF477736. While TFN alone had almost no effect in SUM 159 cell line, its combination with IC10 Chkl inhibitor resulted in a drastic decrease in cell growth (Fig. 3 A), and the same phenomenon was observed in HCC1937 cell line (Fig. 3B) and to a lesser extent, in MDA-MB-231 cell line (Fig. 5). In contrast this combination had no significant effect in either BT549 (Fig. 2B) or HCC38 (Fig. 2C) cell lines which were however more sensitive to PF477736 than the aforementioned cell lines (Fig. 2A).

Strikingly, SUM 159 cells were strongly γΗ2ΑΧ positive as early as 4 hours upon exposure to the combination (data not shown). Furthermore western blotting analysis ((data not shown) showed that this phosphorylation event occurred to an extent that was comparable to the one induced by exposure to DNA damaging agent camptothecin at 48 hours. This indicates that the association of DHODH and Chkl inhibitors was highly effective in inducing DNA damage in these fast-proliferating TNBC cells. At 48 hours this TFN + IC10 PF477736 combination resulted in a significant cell accumulation in S and G2/M phases along with the appearance of a population of hyperploid cells ((data not shown). As described with transformed mouse embryonic fibroblasts, this was also associated with a significant increase in the percentage of dead cells ((data not shown) as compared with matched controls or each individual drug (p = 0.0046 vs DHODH inhibition ; p = 0.0089 vs Chkl inhibition). Western blotting experiments confirmed a significant induction of caspase 3 dependent apoptosis at 72 hours in cells exposed to the combination as compared with either inhibitor alone ((data not shown). The induction of a substantial caspase 3 -dependent cell death was also observed when Chkl depleted cells were exposed to teriflunomide ((data not shown).

Altogether these data confirmed that combining IC10 Chkl inhibitor with a DHODH inhibitor can convert two cytostatic effects into cytotoxicity in both mouse and human cancer cells in culture.

Of note similar combination experiments were also performed on all TNBC cell lines with chkl inhibitors at IC20 with limited gain compared to IC10 (for example see experiments on HCC1937 cell line at IC10 PF477736 + TFN (Fig. 3B) or at IC10 PF477736 + TFN (Fig. 4 or Fig. 5)).

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.

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