WO2023078906A1 - Method for treating acute myeloid leukemia - Google Patents

Abstract

The present invention relates the treatment of acute myeloid leukemia (AML). The inventors and others have shown that CDK6 is a vulnerability to exploit in AML. Because of the limits of CDK6 inhibition (reversible effects and limited efficacy in vivo as monotherapy), the inventors started a project aiming at combining CDK6 inhibitors with another molecule to induce irreversible differentiation of blast cells and target leukemic stem cells (CFU-L). They tested 20 molecules/drugs alone and in combination with Palbociclib with the goal of identifying a synergic combination. Surprisingly, they showed that LSD1 inhibitor (like TCP) can be used in synergy with CDK6 inhibitor. Thus, the invention relates to a combination ofan inhibitor of CDK6 and an inhibitor of LSD1 for use in the treatment of acute myeloid leukemia (AML) in a subject in need thereof.

Description

METHOD FOR TREATING ACUTE MYELOID LEUKEMIA

FIELD OF THE INVENTION:

The present invention relates to a combination of an inhibitor of CDK6 and an inhibitor of LSD 1 for use in the treatment of acute myeloid leukemia (AML) in a subject in need thereof.

BACKGROUND OF THE INVENTION:

Acute myeloid leukemia (AML) is characterized by the accumulation of immature blood cells (blasts) that are blocked in differentiation, in the bone marrow. Leukemic cells are heterogeneous: the leukemic bulk is made of multiple clones with variable reconstitution properties (very few progenitors with stem cell potential and a majority of cells with limited reconstitution potential).

The most critical issues in AML are treatment resistance and relapse. Therefore, it is necessary to target the entire population of leukemic cells, including the progenitors endowed with reconstitution properties (LSC, leukemic stem cell activity).

CDK6 is a serine/threonine kinase involved in progression of the G1 phase of cell cycle. In addition, independently of its role in cell cycle, CDK6 regulates gene transcription through association with transcriptions factors, and modifies epigenetic marks.

CDK6 is now an established therapeutic target in AML. CDK6 targeting offers several advantages. First, since CDK6 is overexpressed in all AML samples, it is likely a common vulnerability shared by all/most AML clones. Second, CDK6 plays a role in the leukemic stem cell compartment. Third, AML cells at relapse, resistant to chemotherapy and/or to targeted FLT3 therapy, remain sensitive to CDK4/6 inhibitors.

LSD1 is a lysine demethylase which regulates directly and indirectly epigenetic marks at the level of histones. The direct activity of LSD 1 is to remove the methyl groups of histone H3K4me and H3K9me. LSD1 is assembled in large multiprotein complexes which regulate chromatin, such as CoREST (corepressor for element- 1 -silencing transcription factor complex) in hematopoietic cells. Such complexes incorporate other epigenetic factors including HDACs (histone de-acetylases) and LSD1 is the protein responsible for the recruitment to specific regions through interaction with GFI1.

Hematopoietic stem cells have a specific epigenetic landscape. LSD1 controls part of this cell identity molecular signature. Therefore, loss of expression or inhibition of LSD1 favors differentiation and loss of stem cell properties. In vitro, AML and small cell lung cancer cell lines have been shown to be the most sensitive cells to LSD1 inhibition.

SUMMARY OF THE INVENTION:

The inventors and others have shown that CDK6 is a vulnerability to exploit in AML. Because of the limits of CDK6 inhibition (reversible effects and limited efficacy in vivo as monotherapy), the inventors started a project aiming at combining CDK6 inhibitors with another molecule to induce irreversible differentiation of blast cells and target leukemic stem cells (LSCs or CFU-L). They tested 20 molecules/drugs alone and in combination with CDK6 inhibitor Palbociclib with the goal of identifying a synergic combination. Surprisingly, they showed that LSD1 inhibitor (like TCP) can be used in synergy with CDK6 inhibitor.

Thus, the present invention relates to a combination of an inhibitor of CDK6 and an inhibitor of LSD 1 for use in the treatment of acute myeloid leukemia (AML) in a subject in need thereof. Particularly, the invention is defined by its claims.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention relates to a combination of an inhibitor of CDK6 and an inhibitor of LSD 1 for use in the treatment of acute myeloid leukemia (AML) in a subject in need thereof.

In another embodiment, the invention relates to i) an inhibitor of CDK6 and ii) an inhibitor of LSD 1, as a combined preparation for simultaneous, separate or sequential use in the treatment of an AML in a subject in need thereof.

In a particular embodiment the invention relates to the combination of an inhibitor of CDK6 and an inhibitor of LSD 1 for use in the treatment of acute myeloid leukemia (AML) in a subject in need thereof wherein the combination allows the differentiation of the blasts and reduce the number of leukemic progenitors with clonogenic potential.

In another particular embodiment, the invention relates to i) an inhibitor of CDK6 protein and ii) an inhibitor of LSD 1 protein as a combined preparation for simultaneous use in the treatment of cancer.

As used herein, the term “CDK6” for “cell division protein kinase 6” denotes an enzyme encoded by the CDK6 gene. It is regulated by cyclins, more specifically by Cyclin D proteins and Cyclin-dependent kinase inhibitor proteins. The protein encoded by this gene is a member of the cyclin-dependent kinase, (CDK) family, which includes CDK4. This kinase is a catalytic subunit of the protein kinase complex, important for the G1 phase progression and Gl/S transition of the cell cycle and the complex is composed also by an activating sub-unit; the cyclin D. The activity of this kinase first appears in mid-Gl phase, which is controlled by the regulatory subunits including D-type cyclins and members of INK4 family of CDK inhibitors (Entrez Gene ID number: 1021; Uniprot number: Q00534).

As used herein and according to all aspects of the invention, the term “LSD1” for “Lysine-specific histone demethylase 1A” also known as lysine (K)-specific demethylase 1A (KDM1A) refers to a protein in humans that is encoded by the KDM1A gene. LSD1 is a flavin-dependent monoamine oxidase, which can demethylate mono- and di-methylated lysines, specifically histone 3, lysines 4 and 9 (H3K4 and H3K9). This enzyme can have roles critical in embryogenesis and tissue-specific differentiation, as well as oocyte growth. KDM1A was the first histone demethylase to be discovered though more than 30 have been described ((Entrez Gene ID number: 23028; Uniprot number: 060341).

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. Particularly, the subject according to the invention is a human suffering from an AML. In some embodiments, the subject according to the invention is a human suffering from a pediatric AML. In some embodiments, the subject is an adult, a child or an infant.

As used herein, the terms "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 terms "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 “CDK6 inhibitor” or “LSD1 inhibitor” denotes molecules or compound which can inhibit the activity of the proteins (e.g. inhibit the kinase or demethylase activity of the protein (enzyme)) or a molecule or compound which destabilizes the proteins. In particular, an inhibitor of CDK6 can inhibit the serine/threonine kinase activity of the enzyme. In particular, an inhibitor of LSD 1 can inhibit the phosphorylation of cdc25.

The term “CDK6 inhibitor” or “LSD1 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 CDK6 according to the invention may be the palbociclib, the ribociclib or the abemaciclib (see for example Uras Iris Z et al, Int J Mol Sci. 2020).

In one embodiment, the inhibitor of LSD1 according to the invention may be the tranylcypromine (or Trans-2-phenylcyclopropylamine HC1) (or TCP), the ladademstat dihydrochloride (or ORY-1001), the GSK-2879552, the IMG-7289, the INCB059872, the CC-90011, the GSK-LSD1 or the ORY-2001 (see for example Fang Yuan et al, Journal of Hematology & Oncology 2019 or Kyung Soo Hong et al, Signal Transduct Target Ther.

2020).

In one embodiment, the inhibitors according to the invention (inhibitor of CDK6 or LSD1) is an antibody. Antibodies directed against CDK6 or LSD1 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 CDK6 or LSD1 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 Kohler 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- CDK6 or LSD1 single chain antibodies. Compounds useful in practicing the present invention also include anti- CDK6 or LSD1 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 CDK6 or LSD1.

Humanized anti- CDK6 or anti- LSD1 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 CDK6 or LSD1 are selected.

In a particular embodiment, the anti-CDK6 antibody according to the invention may be the ZRB1490 antibody as send by Merck.

In a particular embodiment, the anti -LSD 1 antibody according to the invention may be the AB37165 antibody as send by Bioz.

In another embodiment, the antibody according to the invention is a single domain antibody against CDK6 or LSD1. 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 consist 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 CDK6 or LSD1 are selected.

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

In a particular embodiment the polypeptide is an antagonist of CDK6 or LSD1 and is capable to prevent the function of CDK6 or LSD1. Particularly, the polypeptide can be a mutated CDK6 or LSD1 protein or a similar protein without the function of CDK6 or LSD1.

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 CDK6 or LSD1 inhibitor according to the invention is an inhibitor of CDK6 or LSD1 gene expression.

Small inhibitory RNAs (siRNAs) can also function as inhibitors of CDK6 or LSD1 expression for use in the present invention. CDK6 or LSD1 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 CDK6 or LSD1 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 CDK6 or LSD1 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 CDK6 or LSD1 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 CDK6 or LSD1 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 CDK6 or LSD1. 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 sul ati 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 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 CDK6 and an inhibitor of LSD 1.

In order to test the functionality of a putative CDK6 or LSD1 inhibitor a test is necessary. For that purpose, to identify CDK6 inhibitors, the assay of reference is the phosphorylation status of RB protein visualized by western-blot (Wang Lisheng et al., Blood 2007). To identify LSD1 inhibitors, several assays are available, the most common being: (i) the histone H3 methylation status by western-blot (detection of H3K4 me2 using specific antibodies) and (ii) an in vitro assay based on histone 3 peptides containing monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity, with antibody- mediated quantitation of the demethylated product by fluorescence (dissociation-enhanced lanthanide fluorescence immunoassay) (J Biomol Screen. 2014 Jul; 19(6):973-8).

Therapeutic composition

Another object of the invention relates to a therapeutic composition comprising an inhibitor of CDK6 and an inhibitor of LSD1 according to the invention for use in the treatment of an AML 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 (daunorubicine or idarubicine), fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, geni stein, 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. Effect of single and combination treatments on differentiation of AML cell lines. The indicated AML cell lines, cultivated on a layer of HS5 stromal cells, were treated for 4 days either with single molecules or the combination of Palbociblib + TCP inhibitors. (A) Expression of myeloid markers CDl lb and CD86 were quantified by FACS analysis after treatment. (B) The cell morphology is shown following treatment. Cells were centrifuged on a slide and stained with MGG. (C) Expression of CD86 on treated M0LM14 and PL21 cell lines.

Figure 2. Reduction of CFU-L activity following double treatment (combination). MV4-11 (A), M0LM14 (B) and PL21 (C) cells were treated with TCP, Palbociclib or both TCP+Palbociclib for 4 days. Following treatment, cells were washed and the number of CFU- L was determined using methy cellulose cultures. Colonies were counted 10-12 days later.

Figure 3. Efficiency of the combination treatment on primary AML samples. As for cell lines, primary AML cultures were treated for 4 days with Palbociclib and/or TCP. (A) Expression of CDl lb and CD86 markers following treatment. (B) Examples of cell morphology following treatment from two patient samples. (C) CFU-L activity following treatment. (D) Secondary cultures to evaluate the persistence of CFU-L activity. (E) Efficiency of the combination treatment on pediatric AML samples. Eight primary pediatric AML samples were treated for 4 days with Palbociclib and/or TCP. Following treatments, cells were seeded to assess CFU-L activity a colony formation assay on methylcellulose. The figure shows that all samples have significant reduced number of colonies.

Figure 4. Other CDK6i or LSDli show the same activity as Palbociclib or TCP. (A) Effects of CDK6 inhibitor Riboci clib on the apparition of CDl lb marker and CFU-L activity. (B) Effects of LSD1 inhibitor ORY-lOOlon the apparition of CDl lb marker and CFU-L activity.

Figure 5. Reduced expression of CDK6 or LSD1 recapitulate CDK6 or LSD1 inhibition. Specific siRNAs were transfected to reduce the expression of either LSD1 or CDK6. CDK6 siRNAs were combined with LSD1 inhibitor TCP (A) while LSD1 siRNAs were combined or not with CDK6 inhibitor Palbociclib (B).

Figure 6. Synergism between CDK6 and LSD1 inhibitors. A dose response of CDK6 and LSD1 inhibitors was done using 5 concentrations of each drug, on MV4-11 cells. The read out was the % of cells showing CDl lb expression. The results were analyzed using SynergyFinder software. (A) Illustration of the results obtained with Palbociclib and TCP. A ZIP score >10 indicates synergy. (B) ZIP synergy scores of all tested combinations (CDK6i left column, LSDli first line).

Figure 7. Effect of the combination therapy on normal CD34+ human cells. CD34+ cells purified from human cord bloods (N=3) were treated with the same protocol as AML samples, for 4 days with Palbociclib and/or TCP. (A) Following treatment, the changes in CD34 and CDl lb markers were evaluated by FACS. (B) The effect of the treatments on progenitors was evaluated by colony quantification on methylcellulose media (CFU assay). (C) Blood counts in normal mice treated with the combination. C57BL/6 mice were treated with the combination for 4 weeks and blood counts were compared to 5 control animals.

Figure 8. Combination of CDK6 and LSD1 inhibitors reduces leukemia in a mouse model of AML. (A) Detection of luciferase positive human leukemic cells by imaging after 4 weeks of treatment. (B) Relative number of leukemic cells in bone marrow and spleen of mice treated with single molecules or the combination.

Figure 9. Combination of CDK6 and LSD1 inhibitors reduces leukemia of PDX mice. Mice were transplanted with a primary AML sample. Ten mice per group were then given intraperitoneal injections of either with PBS, or ORY-1001, or Palbociclib or the combination of ORY + Palbociclib. After 4 weeks of treatments, the presence of human leukemic cells were evaluated in the bone marrow (A) and in the spleen (B).

EXAMPLE:

Material & Methods

Cell line culture

Human AML cell lines MV4-11 and MOLM-14 were provided by Cell Biology Institute (Okayama, Japan). Human AML cell line PL21 was purchased from DSMZ cell collection. The human stroma cell lines HS5 was obtained from the ATCC. All cells were cultured at 5% CO2 and 37°C, in Gibco RPMI 1640 media, supplemented with 10% fetal bovine serum (FBS) and 1% Glutamine.

Chemical compounds

For in vitro studies, CDK6 inhibitors (Palbociclib, Ribociclib, and Abemaciclib) were purchased from Carbosynth (FA65120 and FR106415) and MedChem Express (HY-16297A), respectively. LSD1 inhibitors (Trans-2 -phenylcyclopropylamine HC1 (=TCP), ladademstat dihydrochloride (=ORY-1001), GSK2879552 2HC1 were purchased from Sigma Aldrich (P8511), MedChem Express (HY-12782T) and Selleckchem (S7796) respectively. Drugs were dissolved in DMSO. The final concentration of DMSO was always below 0.1% for cellular assays. For in vitro studies, the four-days treatments were performed as 1-day CDK6i alone, followed by 3-days CDK6i+LSDli.

For in vivo studies, Palbociclib HC1 was purchased from MedChem Express (HY- 50767A) and dissolved in PBS at 5 mg/mL and used at 25 mg/kg. ORY-1001 (ladademstat dihydrochloride) was purchased from MedChem Express (HY-12782T) and used at 0.0125 mg/kg.

Primary samples

Human primary AML cells were obtained from the biobanks of Institut Paoli Calmettes (Marseille, France) and Centre de Recherche en Cancerologie de Toulouse (Toulouse, France) after approval of the project by the respective local review boards. Patients were informed and gave writing consent in compliance with French and European regulations. Cells were cultured in 5% CO2 at 37°C, at a concentration of 5.105 to 1.106 cells/mL in IMDM media, supplemented with 20% fetal bovine serum (FBS), ImM Glutamine and the following cytokines: lOOng/ml SCF, lOOng/ml FLT3-L, 5ng/ml IL-3 (all three from PeproTech) + lOng/ml GM-CSF(Berlex).

For normal CD34+ hematopoietic cells, three independent cord blood samples were grown in the same media as AML primary samples, and immature cells were purified using the CD34 Microbead Kit UltraPure Human (Miltenyi 130-100-453).

Flow cytometry assays

Anti -human CD 11b was purchased from Beckman Coulter (AO7796), anti -human CD14 was from BD Horizon (367124), anti-human CD86 from BD Biosciences (560357), and anti-human CD163 was purchased from BD Pharmigen (563697). Live/Dead stain kit purchased from Invitrogen (L10120) was used in all FACS analyses.

For the development of Patient-Derived Xenografts, and engraftment monitoring, antihuman CD45 was purchased from Biolegend (304029), anti-mouse CD45 was purchased from Life Technologies (47-0451-82), anti-human CD33 was purchased from BD Pharmingen (555450).

All flow cytometry data have been acquired on a BD LSRFortessa cell analyser, and analysed with Diva software.

Colony forming cell (CFC) assays

1x103 to 3x103 treated-AML cell lines were seeded in 1 mL Methocult H4230 medium (StemCell Technologies) and plated into 35mm plates. The number of colonies was evaluated 10 days later. Colonies were stained with MTT tetrazolium dye. Colony number was calculated using ImageJ and Colony-counter plugin.

For primary AML patient samples, 1x105 cells were seeded in 1 mL Methocult H4535 without EPO medium (StemCell Technologies) and plated into 35mm plates. The number of colonies was then evaluated ten days later.

Cell morphology assays

5x104 AML cells were deposited on a SuperFrost microscope slide (Thermo Scientific), with a Cytospin centrifuge (Thermo Scientific). The slide was then stained with May-Griinwald-Giemsa solutions and imaged with an Apotome microscope.

Biostatistical analyses

All treatment conditions were compared using One-way analysis of variance (ANOVA-1) followed by Tukey’s multiple comparison test, or Bonferroni post-test. A p- value < 0.05 is considered as significant. Results are represented as bars and plots with mean +/- SD, or as boxes with Min to Max whiskers. All conditions are compared with One-way analysis of variance (ANOVA-1) followed by Tukey’s multiple comparison test, or Bonferroni post-test. A p-value < 0.05 is considered as significant.

Synergy studies

Synergy assays were assessed by measuring the appearance of a differentiation marker by flow cytometry (anti -human CD1 lb). Each drug was used at 5 concentrations, based on 2- fold dilutions. Synergy scores were finally assessed with SynergyFinder web-application; and synergy distribution was represented with ZIP based-model. SynergyFinder analyses full dose response curves for single agents and mixtures and allows quantitative synergy scoring with Chou-Talalay method (Dose-based) and Bliss scoring (Effect-based). A synergy score <10 considers an antagonistic interaction, from -10 to 10 the interaction is likely to be additive, whereas a synergistic interaction is detected for a score >10.

In vivo experiments with bioluminescent AML cell line

All animal experiments were performed according to French Guidelines for Animal Handling in the Cancerology Research Center of Marseille Animal Facility (agreement D13 055 04). Experimental protocols were approved by the Ethical Committee no. 14 under the declaration number 02294.01.

MOLM14-GFP+Luciferase+ cells suspended in PBS IX were implanted intravenously (2x 105 cells/lOOpL/mouse) in 6-8 weeks male NOD scid gamma (NSG) mice (Charles River). Post implantation, bioluminescence was measured to homogenize assign four mice groups: vehicle control, ORY-1001 (LSDli), Palbociclib (CDK6i) and combination group. Treatments were initiated following implantation, with a kinetic of 4-days ON / 3-days OFF, during 4 weeks. All mice were treated intraperitoneally with PBS IX for vehicle control group, ORY-1001 0.0125 mg/kg alone, Palbociclib 25 mg/kg alone, or the combination of ORY-1001 + Palbociclib.

Bioluminescence imaging was performed once weekly after treatment initiation, and body weights were measured daily, as well as mouse behaviour. At the end of experiment, mice were sacrificed to quantify hCD45+hCD33+ cells in bone marrow and spleen.

In vivo experiments with Patient-Derived Xenograft (PDX) samples

All animal studies were performed according to protocols approved by the French Authorities: Adaptive Therapeutics Animal Care and Use Committee (APAFiS n° 6743).

NSG mice were engrafted intravenously with 1x105 primary AML cells. Detection of blasts in the peripheral blood was periodically monitored to allocate mice into homogeneous groups. Treatments were initiated once leukemic blasts were detected. All mice were treated intraperitoneally with PBS1X as vehicle control, ORY-1001 0.0125 mg/kg alone, Palbociclib 25 mg/kg alone, or the combination of ORY-1001 + Palbociclib; with 4-days ON / 3-days OFF, during 4 weeks. At the end of the 4 weeks, peripheral blood, bone marrow from the legs and spleen were isolated to monitor hCD45+hCD33+ cells and murine CD45 positive cells.

Results

1- Association of CDK6 and LSD1 inhibitors promote differentiation of AML cell lines.

Treatment of AML cell line MV4-11 for 4 days with the combination of CDK6i and LSDli induce differentiation while single treatments show very modest effects.

Figure 1A illustrates the acquisition of early (CDl lb) and late (CD86) differentiation markers.

Figure IB illustrates cell morphology changes characteristics of myeloid differentiation, that includes increased cell size, higher cytoplasmic/nucleus ratio, apparition of rough cytoplasmic membrane, and changes in cytoplasm composition illustrated by color lightening.

The same results were obtained using other AML cell lines including MOLM-14 and PL21 (Figure 1C).

2- Clonogenic leukemic progenitors are impaired by the combination treatment.

Leukemic progenitors able to restore the leukemia are rare in the leukemic cell population. Their numbers correlate with the capacity to engraft in mice. In vitro, the number of leukemic progenitors (CFU-L) can be estimated with the colony forming unit (CFU) assay based on colony formation in methylcellulose media.

MV4-11 AML cells treated for 4 days either with CDK6i or LSDli show a slight decrease in CFU-L, while combination treatment results in robust reduction of leukemic progenitors (Figure 2A).

The same result was obtained using M0LM14 and PL21 AML cell lines (Figure 2B and 2C).

3- The combination treatment is efficient on primary AML patient samples and results in exhaustion of leukemic progenitors.

Adult AML samples We have used frozen samples obtained from the CRCM/IPC biobank to test our combination on primary AML samples. The samples were treated as above for cell lines except for the composition of culture media (see methods).

As for the cell lines, the combination significantly increased leukemic blasts differentiation monitored using FACS analysis of differentiation markers (Figure 3A) and morphology of the cells (Figure 3B).

The number of CFU-L cells was also greatly reduced (Figure 3C).

To evaluate whether the remaining CFU-L cells were equivalent to the control or are being exhausted by the treatment, similar number of CFU-L from the primary methylcellulose were replated on a secondary methylcellulose. Figure 3D shows that the remaining CFU-L obtained following Palbociclib treatment are equivalent to control non-treated cells. By contrast, cells treated with the combination do not maintain their clonogenic potential (Figure 3D), indicating that the treatment also induces differentiation of the leukemic progenitors.

Pediatric AML samples

The combination treatment is also efficient on pediatric AML samples. AML is clinically and cytogenetically different in children. We evaluated whether pediatric AML cells responded to CDK6 and LSD1 inhibitors.

Pediatric AML samples were treated for 4 days as above. Then the effects of the treatments on the immature leukemic progenitor cells were estimated using the CFU assay as depicted in Figure 3C. The number of CFU-L cells was slightly reduced when treated with single inhibitors and greatly reduced by the combination in all samples tested (Figure 3E).

4- Other CDK6 and LSD1 inhibitors show the same features.

CDK6 inhibitor Ribociclib (Figure 4A) and LSD1 inhibitor ORY-1001 (Figure 4B) induce the same effects as Palbociclib and TCP, on differentiation markers and CFU assay (Figure 4).

We also confirmed using various combinations of 3 CDK6 inhibitors and 3 LSD1 inhibitors that all combinations induced differentiation marks (Figure 6).

5- RNA interference confirms that CDK6 and LSD1 are the targets of the respective specific drugs.

To demonstrate that CDK6 is the actual target of Palbociclib or other CDK6 inhibitors, we repeated the experiments but this time CDK6 was targeted using RNA interference in combination with TCP (Figure 5A). Similarly, we have LSD1 inhibitors can be replaced by RNA interference using siRNAS targeting LSD1 (Figure 5B). These results confirmed that CDK6 and LSD1 are the actual targets of CDK6 and LSD1 inhibition.

6- Synergy between CDK6 and LSD1 inhibitors.

On several experiments, it seemed that the effects obtained with the combination were greatly superior to the addition of the single treatments (see for instance Figure 1A or Figure 2B). To characterize the combination, we used the SynergyFinder tool which allows to define a ZI synergy score between two drugs. A ZIP score between -10 and 10 indicated additivity of the two compounds, while a score >10 indicated synergy.

MV4-11 cells were treated with various concentrations of Palbociclib and TCP and we quantified CDl lb expression by FACS analysis. The ZIP score was 29, indicating strong synergy between the two compounds (Figure 6A).

Similarly, all other combinations using either Palbociclib, Ribociclib or Abemaciclib as CDK6 inhibitor, and TCP, ORY-1001, or GSK2879552 showed synergy (Figure 6B).

7- CDK6i and LSDli combination shows little effect on normal immature (CD34+) hematopoietic cells.

Figure 7 shows that 4 days treatment using Palbociblib and TCP has little effect on normal immature hematopoietic cells: the cells remain immature as determined using the CD34 marker and do not show significant increase of CD1 lb marker (Figure 7A).

The combination did not either impair the progenitor compartment as assessed using the CFU assay (Figure 7B).

Effects of the combination treatment on physiological hematopoiesis

C57BL/6 mice were treated using the combination therapy for four weeks to evaluate putative effects on steady state normal hematopoiesis. Analyses of blood counts of treated (N=5) versus control mice (N=5) indicated: normal number of white and red blood cells, normal concentration of hemoglobin and hematocrit, and a slight increase of platelets following treatment (Figure 7C).

8- Combination treatment in vivo reduces the leukemic burden in a mouse model of

AML, To evaluate the efficiency of the treatment in vivo, a mouse model transplanted with MOLM14 AML cell line was used. The MOLM14 cells used here were modified to carry a luciferase reporter gene, allowing detection of the human leukemic cells in lived mice by non- invasive intravital imaging

Transplanted NSG mice were divided in four groups once the human blasts were detected in the blood: control mice, mice receiving CDK6 inhibitor Palbociclib, mice treated with LSD1 inhibitor ORY-1001, and mice treated with the two inhibitors (combination). Treatments were administered during 4 weeks with cycles of IP injections for 4 consecutive days followed by 3 days of rest.

After 4 weeks of treatment, we evaluated the leukemia in the mice using intravital imaging (Figure 8A) and FACS analysis to quantify the leukemic cells in bone marrow and spleen (Figure 8B). Live imaging of the mice indicated that the monotherapies slightly reduced the detection of human leukemic cells, while the combination of drugs strongly reduced the leukemia (Figure 8A). Quantification of the leukemic burden by FACS analysis of bone marrow and spleen cells confirmed that the combination was efficient to strongly decrease the number of leukemic cells in this mouse model (Figure 8B).

9- Efficiency of the combination on mice PDX models of AML,

Next, we tested the efficiency of the treatment in vivo, on a primary patient sample. For this, the same experiment as above was repeated but using this time a human AML sample. Six weeks following transplantation, control mice developed a severe AML with high invasion of the bone marrow and spleen by leukemic cells (around 80 and 90% of total cells respectively). In this model, ORY-1001 had almost no effect, while Palbociclib decreased the leukemic burden. Interestingly, the combination further decreased the number of leukemic cells (Figure 9A and 9B). In conclusion, the mouse models of AML confirmed the efficiency of the combination therapy in vivo. Furthermore, these experiments demonstrated the superiority of the combination compared to single treatments with CDK6i and LSDli. The results obtained with mice PDX models of AML were reproduced and confirmed with three other mice PDX models (data not shown).

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. Schmitt Martin L et al. Heterogeneous Antibody -Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate. J Biomol Screen. 2014 Jul; 19(6): 973 -8.

Wang Lisheng et al. Pharmacologic inhibition of CDK4/6: mechanistic evidence for selective activity or acquired resistance in acute myeloid leukemia. Blood. 2007 Sep 15;110(6):2075-83.

Claims

- 26 - CLAIMS:

1. A combination of an inhibitor of CDK6 and an inhibitor of LSD1 for use in the treatment of acute myeloid leukemia (AML) in a subject in need thereof.

2. An i) inhibitor of CDK6 and ii) an inhibitor of LSD1, as a combined preparation for simultaneous, separate or sequential use in the treatment of an AML in a subject in need thereof.

3. A combination according to the claims 1 or 2 wherein the inhibitor of CDK6 is the palbociclib, the ribociclib or the abemaciclib.

4. A combination according to the claims 1 or 2 wherein the inhibitor of LSD1 is the tranylcypromine (TCP), the ladademstat dihydrochloride (or ORY-1001), the GSK- 2879552, the IMG-7289, the INCB059872, the CC-90011, the GSK-LSD1 or the ORY- 2001.

5. A method for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of CDK6 and an inhibitor of LSD 1.

6. A therapeutic composition comprising an inhibitor of CDK6 and an inhibitor of LSD1 according to the invention for use in the treatment of an AML in a subject in need thereof.