WO2015165975A1 - Methods and compositions for treating myeloid neoplasias - Google Patents

Abstract

The invention relates to a P2Y6 receptor agonist for use in a method for treating myeloid neoplasias such as chronic myelomonocytic leukemia (CMML).

Description

METHODS AND COMPOSITIONS FOR TREATING MYELOID NEOPLASIAS

FIELD OF THE INVENTION:

The invention relates to the field of medicine, in particular, to oncology. The invention thus relates to the treatment of myeloid neoplasias, for instance Myelodysplastic /Myeloproliferative disorders such as chronic myelomonocytic leukemia (CMML).

BACKGROUND OF THE INVENTION:

Hematopoiesis is maintained by a hierarchical system where hematopoietic stem cells

(HSCs) give rise to multipotent progenitors, which in turn differentiate into all types of mature blood cells. Clonal disorders in this system lead to Acute Myeloid Leukemia (AML), Myeloproliferative Neoplasms (MPNs), Myelodysplastic Syndromes (MDS) and Myelodysplastic/Myeloproliferative disorders .

Among these disorders, myelodysplastic/myeloproliferative neoplasms include four myeloid diseases grouped in 1999 by the WHO: chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), atypical chronic myeloid leukemia (aCML) and unclassified myelodysplastic/myeloproliferative syndromes (U-MDS/MPS). Monocytes have the unique property among peripheral blood cells to migrate into tissues and to further differentiate into morphologically and functionally heterogenous cells that include macrophages, myeloid dendritic cells and osteoclasts^1, . In the absence of differentiation, circulating monocytes are believed to undergo apoptotic cell death. Monocytes once stimulated by an inflammatory response, activate pro-survival pathways, migrate to tissues and differentiate into macrophages³. The differentiation of human peripheral blood monocytes into macrophages can be recapitulated ex vivo by macrophage colony-stimulating factor, also known as colony-stimulating factor- 1 (CSF-1)⁴. Previous studies have established that physiological monocyte differentiation triggered by CSF-IR engagement is critically dependent on the formation of a multimolecular complex consisting of Fas Associated Protein with Death Domain (FADD), Flice-inhibitory protein (FLIP), Caspase-8, Receptor Interacting Serine Threonine kinase 1 (RIPK1) and other protein partners, caspase-8 acting as the initiatory caspase in this process⁵' ⁶. More recently, it was established that autophagy also plays a crucial role during CSF-l-induced differentiation of human monocytes⁷' ⁸. Autophagy (macroautophagy) is an evolutionary conserved catabolic pathway that delivers cytoplasmic substrates, such as damaged organelles and cytoplasmic proteins, to lysosomes for degradation⁹' ¹⁰. There is growing evidence that supports a role of autophagy in the rapid cellular changes necessary for proper differentiation of different cell types^11"13. However, the molecular mechanisms that regulate autophagy in the context of cell differentiation have remained largely unexplored. Induction of autophagy critically requires unc-51-like autophagy activating kinase 1 (ULK1) that acts in a multi-molecular complex to initiate autophagy¹⁴. ULK1 phosphorylation on Ser 555 by AMP-dependent protein kinase (AMPK) is one of the main mechanisms leading to induction of autophagy via the phosphorylation and activation of the VPS34 lipid kinase¹⁵.

A pathological situation in which the differentiation of monocytes is clearly altered is chronic myelomonocytic leukemia (CMML)¹⁶. CMML is associated with monocytosis and characterised by defects in monocyte to macrophage differentiation¹⁷. It previously was shown that recombinant alpha-defensins (HNPs) inhibit macrophage colony-stimulating factor (M-CSF)-driven differentiation of human peripheral blood monocytes of a CD14⁺/CD24^" phenotype from healthy donors or CMML patients into macrophages.

It thus was proposed that a population of immature dysplastic granulocytes with a CD147CD24⁺ phenotype contributes to the CMML phenotype through production of alpha- defensins 1-3 (HNPl-3) that suppress the differentiation capabilities of monocytes with a CD14⁺/CD24^" phenotype and it was also suggested a role for purinergic receptor in monocyte survival and differentiation. Indeed, P2Y2, P2Y4 or P2Y6 down-regulation using antisense oligonucleotides inhibited M-CSF-induced differentiation of monocytes. It was also shown that UTP and UDP inhibit the inhibitory effect of HNPl-3 on human monocytes from healthy donors in competitive experiments.

Accordingly, it results that until now the therapeutic effect of a P2Y6 receptor agonist on M-CSF-driven differentiation of human monocytes of a CD14⁺/CD24^" phenotype from CMML patients into macrophages has never been demonstrated nor suggested.

SUMMARY OF THE INVENTION:

In a first aspect, the invention relates to a P2Y6 receptor agonist for use in a method for treating myeloid neoplasias. In a second aspect, the invention relates to P2Y6 receptor agonist for use in a method for promoting monocyte to macrophage differentiation in a patient in need thereof.

In a third aspect, the invention relates to a pharmaceutical composition for use in a method for treating myeloid neoplasias comprising a P2Y6 receptor agonist and a pharmaceutically acceptable carrier.

In a fourth aspect, the invention relates to a pharmaceutical composition for use in a method for promoting monocyte to macrophage differentiation in a patient in need thereof comprising a P2Y6 receptor agonist and a pharmaceutically acceptable carrier.

In a fifth aspect, the invention relates to a pharmaceutical composition or a kit-of-part comprising a P2Y6 receptor agonist and an additional therapeutic agent.

DETAILED DESCRIPTION OF THE INVENTION:

The invention is based on the discovery by the inventors that triggering P2Y6 with its physiological ligand (UDP) or using P2Y6 agonist (MRS2693) restores autophagy and normal differentiation in CMML patients. Collectively these findings highlight an essential role for P2Y6-mediated autophagy during differentiation of human monocytes and pave the way for future therapeutic intervention in CMML and more generally in myeloid neoplasias.

Accordingly, in a first aspect, the invention relates to a P2Y6 receptor agonist for use in a method for treating myeloid neoplasias.

The term "myeloid neoplasias" (MNs) encompasses several classes of clonal hematopoietic disorders characterized by defects in myeloid maturation and/or proliferation. MNs comprise Acute Myeloid Leukemia (AML), Myelodysplastic Syndromes (MDS) Myeloproliferative Neoplasms (MPNs) and Myelodysplastic/Myeloproliferative disorders.

The term "myeloid" refers to bone marrow-derived cells, including granulocytes and monocytes. The term "monocytes" refers to a type of leukocytes (representing about 3 to 8% of total leukocytes) produced by the bonne marrow from haematopoietic stem cell precursors called monoblasts. Classical monocytes are characterized by a high expression level of the CD14 cell surface receptor (CD14⁺ cells). The term "granulocytes" refers to a type of leukocytes characterized by the presence of granules in their cytoplasm. Granulocytes are also characterized by a high expression level of the CD24 cell surface receptor (CD24⁺ cells). Said monocytes and granulocytes may be selected by well known methods in the art (such as flow cytometry) using the specific surface markers expressed on the cells (i.e. CD14 and CD24).

In one embodiment of the invention, said myeloid neoplasias are associated with monocytosis (which refers to an increase in the number of monocytes circulating in the peripheral blood) and/or granulocytosis (which refers to an increase in the number of granulocytes circulating in the peripheral blood).

In one embodiment of the invention, said myeloid neoplasias are Myelodysplastic/Myeloproliferative disorders which are clonal myeloid disorders that possess both dysplastic and proliferative features comprising chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), atypical chronic myeloid leukemia (aCML) and unclassified myelodysplastic/myeloproliferative syndromes (U-MDS/MPS). In a particular embodiment of the invention, the Myelodysplastic/Myeloproliferative disorder is chronic myelomonocytic leukemia (CMML). CMML is be characterized pathologically by persistent monocytosis is greater than 1>< 10⁹/L in the peripheral blood; no Philadelphia chromosome or BCR/ABL fusion gene; fewer than 20% blasts in the blood or bone marrow; dysplasia involving one or more myeloid lineages.

CMML is associated with monocytosis and is characterized by defects in monocyte to macrophage differentiation. Defects in monocyte differentiation are likely provoked by granulocytes, more particularly immature granulocytes which are CD147CD24⁺ cells. Said CD147CD24⁺ immature CMML granulocytes inhibit macrophage differentiation of monocytes through alpha-defensin secretion.

In one embodiment, CMML is associated with an increased level of immunosuppressive CD147CD24⁺ cells.

A CMML associated with an increased level of immunosuppressive CD147CD24⁺ cells corresponds to a patient having a PBMC fraction with more than 1%, preferably more than 10%, and more preferably more than 20%, of CD147CD24⁺ cells.

In another embodiment, CMML is not associated with an increased level of immunosuppressive CD147CD24⁺ cells. The term "P2Y receptors" refer to 7TM (containing seven transmembrane domains) receptors that couple to G protein-dependent and -independent signaling pathways, including ion channels. Eight human subtypes of the P2Y receptor family have been defined. According to a dendrogram relating sequence homology, the P2Y1 , P2Y2, P2Y4, P2Y6, and P2Y1 1 receptors form a cluster of preferentially Gq-coupled receptors, and the P2Y12, P2Y13, and P2Y14 receptors form a cluster of preferentially Gi-coupled receptors.

P2Y6 agonists include monucleoside 5 '-diphosphates and dinucleoside mono-, di-, and triphosphates (polyphosphates).

Monucleoside 5 '-diphosphates useful in this application include uridine 5 '-diphosphate

(UDP) and its analogues of general Formula I.

UDP and its analo ues are depicted by general Formula I:

wherein:

Xi , and X₂ are each independently either O^" or S^";

Y is H or OH;

Pvi is selected from the group consisting of O, imido, methylene, and dihalomethylene (e. g., dichloromethylene, difluoromethylene);

P 2 is selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkoxyl, nitro and azido;

Ri is selected from the group consisting of nothing, H, alkyl, acyl (including arylacyl), and arylalkyl; and R4 is selected from the group consisting of-OR',-SR', NR', and NR'R", wherein R' and R" are independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, alkoxyl, and aryloxyl, and with the proviso that R' is absent when R4 is double bonded from an oxygen or sulfur atom to the carbon at the 4-position of the pyrimidine ring.

As used herein, the term "alkyl" refers to Ci-10 inclusive, linear, branched, or cyclic, saturated or unsaturated (i. e., alkenyl and alkynyl) hydrocarbon chains, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, allenyl and optionally substituted arylalkenyl and arylalkyny groups. As used herein, the term "acyl" refers to an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent (i.e., as represented by RCO-, wherein R is an alkyl or an aryl group). As such, the term "acyl" specifically includes arylacyl groups. Specific examples of acyl groups include acetyl and benzoyl. As used herein, the term "aryl" refers to 5 and 6membered hydrocarbon and heterocyclic aromatic rings. Examples of aryl groups include cyclop entadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, isothiazole, isoxazole, pyrazole, pyrazine, pyrimidine, and the like. The term "alkoxyl" as used herein refers to Ci-10 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including for example methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, and pentoxy. The term "aryloxyl" as used herein refers to aryloxy such as phenyloxyl, and alkyl, halo, or alkoxyl substituted aryloxyl. As used herein, the terms "substituted alkyl" and "substituted aryl" include alkyl and aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl or alkyl group are replaced with another atom or functional group, for example, halogen, aryl, alkyl, alkoxy, hydroxy, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto. The terms "halo," "halide," or "halogen" as used herein refer to fluoro, chloro, bromo, and iodo groups.

Compounds illustrative of the compounds of Formula I include those disclosed in WO 99/09998; the reference is incorporated herein by reference. Formula I compounds, for example, include : uridine 5'-diphosphate (UDP); uridine 5'-0- (2-thio diphosphate) (UDPPS); 5-bromouridine 5'-diphosphate (5-BrUDP); 5-(l-phenylethynyl)-uridine 5 '-diphosphate (5- (l-phenylethynyl)UDP); 5-methyluridine '-diphosphate (5-methylUDP) ; 4-hexylthiouridine 5'diphosphate (4-hexylthioUDP); 4-mercaptouridine 5 '-diphosphate (4-mercaptoUDP); 4methoxyuridine 5 '-diphosphate (4-methoxyUDP); 4-(N-morpholino)uridine 5 '-diphosphate (4-(N-morpholino)UDP; 4-hexyloxyuridine 5 '-diphosphate (4-hexyloxyUDP); N,N- dimethylcytidine 5 '-diphosphate (Ν,Ν-dimethylCDP); N-hexylcytidine 5 '-diphosphate (NhexylCDP); and N-cyclopentylcytidine 5 '-diphosphate (N-cyclopentylCDP).

Preferred compounds of Formula I include UDP

Certain compounds of Formula I (e.g., UDP and UDP S) are known and may be made in accordance with known procedures or variations thereof, which will be apparent to those skilled in the art. Alternatively, UDP, and other analogs thereof are also commercially available from vendors such as Sigma (St. Louis, MO) and Pharmacia (Uppsala, Sweden).

Dinucleoside polyphosphates are depicted by general Formula II:

wherein:

X is O, methylene, difluoromethylene, imido;

n = 0, 1 or 2;

m = 0, 1 or 2;

n + m = 0, 1, 2, 3 or 4; B and B' are each independently a purine residue or a pyrimidine residue linked through the 9- or 1- position, respectively;

Z = OH or ₃;

Z' = OH or N₃;

Y = H or OH; and

Y '= H or OH.

The ribosyl moieties are in the D-configuration, as shown, but may be L-, or D-and L. The D-configuration is preferred.

The selective P2Y6 receptor agonist compounds useful for this invention have selectivity of P2Y6 receptor over P2Y2 receptor or P2Y4 receptor.

Specific examples of selective P2Y6 receptor agonists of the invention, include, but are not limited to, mononucleoside 5 '-diphosphates, dinucleoside monophosphate, dinucleoside diphosphates, or dinucleoside triphosphates such as those disclosed in the PCT application No. WO2007002945 and Jacobson KA, Ivanov AA, de Castro S, Harden TK, Ko H. Development of selective agonists and antagonists of P2Y receptors Purinergic Signal. Mar 2009; 5(1): 75-89; uridine di- and tri-phosphate derivatives such as those disclosed in the PCT application No. WO2012073237; uridine '-diphosphate analogues such as those disclosed in Besada P, Shin DH, Costanzi S, Ko H, Mathe C, Gagneron J, Gosselin G, Maddileti S, Harden TK, Jacobson KA; Structure-activity relationships of uridine 5'- diphosphate analogues at the human P2Y6 receptor. J Med Chem. 2006 Sep 7;49(18):5532-43 and in Ginsburg-Shmuel Tl, Haas M, Schumann M, Reiser G, Kalid O, Stern N, Fischer B; 5- OMe-UDP is a potent and selective P2Y(6)-receptor agonist; J Med Chem. 2010 Feb 25;53(4):1673-85.

In one embodiment, the selective P2Y6 receptor agonist is an uridine 5 '-diphosphate analogue.

In a particular embodiment, the selective P2Y6 receptor agonist is MRS2693 (5- Iodouridine-5'-0-diphosphate or 5-Iodo-UDP) as described in Besada et al. 2006 (as well as its salts) having the following formula:

In another particular embodiment, the selective P2Y6 receptor agonist is Methoxyuridine 5 '-diphosphate (OMe-UDP), as described in Ginsburg-Shmuel et al. 2010, well as its salts such as the 5-OMe-UDP trisodium salt.

In another aspect, the invention relates to a P2Y6 receptor agonist for use in a method for promoting monocyte to macrophage differentiation in a patient in need thereof.

The term "differentiation" refers to the process by which cells matures from one cell type to another cell type.

Preferably, promoting monocyte to macrophage differentiation as disclosed herein will result in greater than about 5% or about 10% of differentiation of monocytes into macrophages in response to CSF-1 in a patient in need thereof. Even more preferably, greater than about 25%, 30% or 35% of the monocytes will be differentiated into macrophages.

In one embodiment, the patient in need thereof is suffering from a myeloid neoplasia (e.g. suffering from CMML).

Pharmaceutical compositions:

Another aspect of the invention relates to a pharmaceutical composition for use in a method for treating myeloid neoplasias comprising a P2Y6 receptor agonist as described above and a pharmaceutically acceptable carrier. Another aspect of the invention relates to a pharmaceutical composition for use in a method for promoting monocyte to macrophage differentiation in a patient in need thereof comprising a P2Y6 receptor agonist and a pharmaceutically acceptable carrier. A P2Y6 receptor agonist of the invention as above described may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical 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, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

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. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. To prepare pharmaceutical compositions, an effective amount of a P2Y6 receptor agonist according to the invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the compositions can be brought about by the use in the compositions of agents delaying absorption (e.g. aluminium monostearate and gelatine). The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The P2Y6 receptor agonist according to the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Another aspect of the invention relates to a method for treating myeloid neoplasias in a patient in need thereof comprising a step of administering a therapeutically effective amount of a P2Y6 receptor agonist the invention to said patient.

In a particular embodiment, the myeloid neoplasia is CMML

In a particular embodiment, the P2Y6 receptor agonist is MRS2693.

In another particular embodiment, the P2Y6 receptor agonist is 5-OMe-UDP.

As used herein, the term "therapeutically effective amount" is intended for a minimal amount of active agent, which is necessary to impart therapeutic benefit to a patient. For example, a "therapeutically effective amount of a compound" to a subject is an amount of the compound that induces, ameliorates or causes an improvement in the pathological symptoms, disease progression, or physical conditions associated with the disease affecting the patient.

According to the invention, a "therapeutically effective amount of a P2Y6 receptor agonist" is one which is sufficient to achieve a desired biological effect, in this case

i) inducing the differentiation of monocytes and/or

ii) inhibiting the suppressor activity of granulocytes.

As used herein, the term "treating" a disorder or a condition refers to reversing, alleviating or inhibiting the process of one or more symptoms of such disorder or condition.

As used herein, the term "patient" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient according to the invention is a human.

In one embodiment, the patient in need thereof is suffering from a myeloid neoplasia (e.g. suffering from CMML).

Pharmaceutical compositions of the invention may comprise an additional therapeutic agent.

Combination therapies of the invention:

In other aspects, the P2Y6 receptor agonist may be administered to a patient with an appropriate additional therapeutic agent useful in the treatment of the condition from which the patient suffers or is susceptible to; examples of such agents include a chemotherapeutic agent such as DNA methyltransferase inhibitors.

The administration of the P2Y6 receptor agonist and the other therapeutic agent, (e.g., a chemotherapeutic agent) can be carried out simultaneously, e.g., as a single composition or as two or more distinct compositions using the same or different administration routes. Alternatively, or additionally, the administration can be done sequentially, in any order. Alternatively, or additionally, the steps can be performed as a combination of both sequentially and simultaneously, in any order. Accordingly, in one aspect, the invention relates to a pharmaceutical composition comprising a P2Y6 receptor agonist according to the invention and an additional therapeutic agent. In another aspect, the invention relates to a pharmaceutical composition comprising a P2Y6 receptor agonist according to the invention and an additional therapeutic agent for use in a method for treating myeloid neoplasias. In still another aspect, the invention relates to a pharmaceutical composition comprising a P2Y6 receptor agonist according to the invention and an additional therapeutic agent for use in a method for promoting monocyte to macrophage differentiation in a patient in need thereof.

In another aspect, the invention relates to a kit-of-part comprising a P2Y6 receptor agonist according to the invention and an additional therapeutic agent.

The terms "kit", "product" or "combined preparation", as used herein, define especially a "kit-of-parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e. simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners to be administered in the combined preparation can be varied. The combination partners can be administered by the same route or by different routes. When the administration is sequential, the first partner may be for instance administered 1, 2, 3, 4, 5, 6, 12, 18 or 24 h before the second partner.

Another aspect of the invention is a kit-of-part comprising a P2Y6 receptor agonist according to the invention and an additional therapeutic agent and an additional therapeutic active agent for simultaneous, separate or sequential use in a method for treating myeloid neoplasias.

Another aspect of the invention is a kit-of-part comprising a P2Y6 receptor agonist according to the invention and an additional therapeutic agent and an additional therapeutic active agent for simultaneous, separate or sequential use in a method for promoting monocyte to macrophage differentiation in a patient in need thereof.

Combination P2Y6 receptor agonist with an additional therapeutic agent:

It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.

In one embodiment, the P2Y6 receptor agonist may be used in combination with a chemotherapeutic agent useful for myeloid neoplasias.

Non-limiting examples of chemotherapeutic agents which can be used in combination treatments of the present invention include, for example, hydroxyurea, DNA methyltransferase inhibitors, retinoic acid (RA), arsenic derivatives, nucleoside analogues and monoclonal antibodies.

DNA methyltransferase inhibitors are well known from the skilled person.

In a preferred embodiment, said DNA methyltransferase inhibitor is a cytosine analogue and is preferably selected among azacitidine, decitabine and zebularine. Most preferably, said DNA methyltransferase inhibitor is azacitidine or decitabine. Decitabine or 5-aza-2'-deoxycytidine (trade name DACOGEN) is the compound 4- amino- 1 -(2-deoxy-b-D-erythro-pentofuranosyl)- 1 ,3 ,5-triazin-2( 1 H)-one. Decitabine is indicated for the treatment of myelodysplastic syndromes (MDS) including previously treated and untreated, de novo and secondary MDS of all French-American-British subtypes (refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and chronic myelomonocytic leukemia - CMML) and Intermediate- 1, Intermediate-2, and High-Risk International Prognostic Scoring System groups.

Azacitidine (trade name VIDAZA) is the compound 4-amino-l-[beta]-D- ribofuranosyl-s-triazin-2(lH)-one. Azacitidine is an anti-neoplastic pyrimidine nucleoside analog used to treat several subtypes of myelodysplastic syndrome. Azacitidine is specifically indicated for the treatment of the following myelodysplastic syndrome subtypes: refractory anemia, refractory anemia with ringed sideroblasts (if accompanied by neutropenia or thrombocytopenia or requiring transfusions), refractory anemia with excess blasts, refractory anemia with excess blasts in transformation and chronic myelomonocytic leukemia (CMML).

By "retinoic acid" is meant all-trans retinoic acid (ATRA) or cis derivatives of ATRA such as 9-cis retinoic acid or 13-cis retinoic acid, vitamin A (retinol), carotene or rexinoids or pharmacologically acceptable salts thereof.

In a preferred embodiment, said retinoic acid is ATRA.

By "arsenic derivative" is meant a common naturally occurring substance that can exist under three inorganic forms: red (arsenic disulfide or As4S4 referred to as realgar or pararealgar), yellow (arsenic trisulfide or As2S3 referred to as arsenikon, aurum pigmentum or orpiment) and white (arsenic trioxide or As203).

In a preferred embodiment, said arsenic trioxide (As203).

By "nucleoside analogue" is meant a molecule that act like nucleosides in DNA synthesis used as chemotherapy agents, in particular those used in myeloid neoplasias.

In one embodiment, said nucleoside analogue is a purine nucleoside analogue such as acadesine, acadesine precursors or acadesine derivatives.

Examples of such purine nucleoside analogues are disclosed in the US patent application US2013109648 and in the international patent application WO2012143624.

In a preferred embodiment, said purine nucleoside analogue is acadesine (AICA- riboside) or acadesine 5'-monophosphate.

By "monoclonal antibody" is meant an antibody used as chemotherapy agents, in particular those used in myeloid neoplasias. In one embodiment, said monoclonal antibody is selected form the group consisting of and anti-TNFa (e.g. Adalimumab or Infliximab), an anti-CD25 monoclonal antibody (e.g. Basiliximab), an anti-CD33 monoclonal antibody (e.g. Lintuzumab), an anti-CD44 monoclonal antibody, an anti-CD47 monoclonal antibody

In one embodiment, the P2Y6 receptor agonist may be used in combination with a P2Y2 receptor agonist and/or a P2Y4 receptor agonist.

P2Y2/P2Y4 receptor agonists include uridine 5 '-triphosphate (UTP) and its analogues of general Formula II.

UTP and its analogues are depicted by general Formula II:

wherein:

Xi , X2 and X3 are each independently either O^" or S^",

Y is H or OH;

Pvi , P 2, P3 and R4 are defined as in Formula I above.

Preferably, X2 and X3 are O^", Ri is oxygen or imido, and R2 is H.

Particularly preferred compounds of Formula II include uridine 5 '-triphosphate (UTP), 2-ThioUTP, uridine 5'-0-(3-thiotriphosphate) (UTPyS).

Other specific examples of P2Y2 receptor agonists include MRS2768 (Uridine-5'- tetraphosphate δ-phenyl ester and its salts including MRS2768 tetrasodium salt), MRS2498 and P'-diridine 5')-P⁴-(2'-deoxycytidine 5 ')tetraphosphate or a salt thereof, such as a tetrasodium salt (INS37217 - Denufosol) as disclosed in the US patent 8,609,066. Other specific example of P2Y4 receptor agonist is 2'-azido-dUTP and MRS4062 (N⁴- phenylpropoxycytidine-5'-0-triphosphate and its salts including MRS4062 triethylammonium salt).

In another aspect, the invention relates to a P2Y2 or a P2Y4 receptor agonist for use in a method for treating myeloid neoplasias.

In a particular embodiment of the invention, the myeloid neoplasia is CMML.

In another aspect, the invention relates to a P2Y2 or a P2Y4 receptor agonist for use in a method for promoting monocyte to macrophage differentiation in a patient in need thereof.

In another aspect, the invention relates to a pharmaceutical composition for use in a method for treating myeloid neoplasias comprising a P2Y2 or a P2Y4 receptor agonist and a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a pharmaceutical composition for use in a method for promoting monocyte to macrophage differentiation in a patient in need thereof comprising a P2Y2 or a P2Y4 receptor agonist and a pharmaceutically acceptable carrier.

P2Y2 or P2Y4 receptor agonists useful in these aspects have been described above and includes UTP, UTPyS, MRS2768, MRS2498 and INS37217 - Denufosol. The invention will be further illustrated by the following examples. However, these examples should not be interpreted in any way as limiting the scope of the present invention.

EXAMPLE: Material & Methods

Reagents and antibodies - Human CSF-1 was purchased from Miltenyi. Cycloheximide was purchased from Sigma- Aldrich. Dorsomorphin, STO-609, MRS2578, UDP, MRS2179, MRS2693 were from Tocris and U73122 from ENZO life Sciences. Anti- AMPK, Phospho-AMPK (Thrl72), AMP a2, Phospho-(Ser/Thr)-AMPK Substrate (P-S/T2- 102), PLCy2, Phospho-PLCy2 (Y759), PLCy3, Phospho-UL l (Ser555), LKB1, LC3-B antibodies were purchased from Cell signaling Technology. Actin, CSF-1R, P2Y6 (SC- 20127) and HSP90 antibodies were from Santa Cruz Biotechnology. HRP-conjugated rabbit anti-goat or mouse was purchased from Dako and HRP-conjugated goat anti-rabbit from Cell Signaling. Patient's samples - Patients and volunteers signed an informed consent according to the Declaration of Helsinki and to recommendations of an independent scientific review board. Chronic-phase CMML diagnosis was based on WHO criteria. Patients were newly diagnosed or had previously diagnosed hematopoietic disease and were followed every 3 months. They were either untreated or received supportive care or cytotoxic treatment, in most cases hydroxyurea.

Cell sorting and differentiation - Blood samples were collected using ethylene diamine tetraacetic acid-containing tubes. Mononucleated cells were first isolated by Ficoll Hypaque. Then, we used the autoMACS™ Separator (Miltenyi) to perform cell enrichment. A first negative selection that included antibodies targeting CD3, CD7, CD16, CD19, CD56, CD123, and glycophorin A was used for monocyte enrichment. In CMML samples, CD14⁺ and CD 14^" populations were further enriched using an anti-CD 14 antibody. Human purified monocytes were grown in RPMI 1640 medium with glutamax-I (Gibco) supplemented with 10% (vol/vol) fetal bovine serum (Gibco). Macrophage differentiation (adhesion to culture flasks and fibroblast-like shape) was visualized using standard optics (Leica) equipped with a Moticam 2500 camera (Motic). Phase images of culture were recorded with a 20 0.30 PHI objective with Motic Image Plus software (Motic).

Flow cytometry - Differentiation, cell surface expression of P2Y6 and autophagy were studied by flow cytometry. To analyse macrophagic differentiation of monocytes, cells were washed with ice-cold phosphate buffered saline (PBS), incubated at 4°C for 10 min in PBS/bovine serum albumin (BSA 0.5%) with anti-CD71 and anti-CD163 or an isotype control (Miltenyi). Finally, cells were washed and fixed in 2% paraformaldehyde. To analyse cell surface expression of P2Y6, cells were washed with ice-cold phosphate buffered saline (PBS), incubated at 4°C for 1 h in PBS/bovine serum albumin (BSA 0.5%) with P2Y6 (1 : 100) or an isotype control (Santa Cruz Biotechnology), washed and incubated with secondary antibody (1 : 1500) during 30 min. Finally, cells were washed and fixed in 2% paraformaldehyde. Cyto-ID^® Autophagy Detection Kit was used for monitoring autophagic activity at the cellular level according to the manufacturer's instructions (Enzo Life Sciences). Briefly, the 488 nm excitable green fluorescent detection reagent supplied in the Cyto-ID^® Autophagy Detection Kit becomes brightly fluorescent in vesicles produced during autophagy. Fluorescence was measured by the use of an autoMACS® Pro Separator (Miltenyi).

Immunoblot assays - Cells were lysed for 30 min at 4°C in lysis buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 20 mM EDTA, 100 mM NaF, 10 mM Na3V04, complete protease inhibitor mixture (CPIM, Roche, Indianapolis, IN), 1% Triton X-100]. Lysates were centrifuged at 20,000 g (15 min, 4°C) and supernatants were supplemented with concentrated sodium dodecyl sulfate (SDS). 50 μg of proteins were separated and transferred following standard protocols before analysis with chemi- luminescence detection kit (GE Healthcare).

Reverse- Transcription and real-time polymerase chain reaction. RNA was prepared from 5.10⁶ cells and with the RNeasy Mini Kit according to manufacturer's protocol (Qiagen). Each cDNA was prepared using superscript II RT and random primers (Invitrogen). Real-time polymerase chain reaction (PCR) was performed with SyBR Green detection protocol (Applied Biosystems). Briefly, 5 ng of total cDNA, 125 nM (each) primers, and 10 μί SyBR Green mixture were used in a total volume of 20 μί. Detection of multiple endogenous control (actin, L32 and ubiquitin) were used to normalize the results. Specific forward and reverse primers are accessible upon request. siRNA knockdown - Small interfering (si) RNAs were introduced into monocytes by nucleoporation (Amaxa) of 5 x 10⁶ monocytes in 100 μΐ_^ of nucleofector solution with 15 nmol of siRNA. Cells were incubated for 24 h with 5mL of prewarmed complete medium, and CSF-1 was subsequently added. We used siRNAs (Invitrogen) targeting AMPK (HSS108454), PLCy2 (HSS108098), CaMKK (HSS173805), P2Y6 (HSS143211), PLCy3 (HSS108082), CSF-1R (HSS102358), LKB1 (VHS50411) and, luciferase as a negative control.

Functional assay - (Gentamicin protection assay) - Monocytes (10⁶) were infected for 20 min in RPMI 1640 medium supplemented with 10% FCS with ampicillin resistant E.coli K12 (MOI=50). Cells were washed 3 times before incubation for 20 min with RPMI 1640 medium supplemented with 10% FCS and gentamicin (50 μg/mL) for internalized bacteria measurement. Cells were lysed in PBS 0.1 % Triton X-100 and bacteria counted on LB plates containing ampicillin (100 μg/mL). Mean value of triplicates representative of four independent experiments. Calcium flux assay - Monocytes were loaded with Fluo-4 Direct Calcium Assay Kit according to the manufacturer's instructions (Molecular Probes). Calcium release was measured by the use of an autoMACS® Pro Separator (Miltenyi).

Immunofluorescence - Transfected cells were washed with ice-cold phosphate buffered saline (PBS), incubated at 4°C for 1 h in PBS/bovine serum albumin (BSA 0.5%) with P2Y6 (1 : 100) or an isotype control (Santa Cruz Biotechnology), washed and incubated with secondary antibody (1 :1500) during 30 min. Finally, cells were washed and fixed in 2% paraformaldehyde and spun on to a microscope slide for 4 min at 800 g in a Cytospin 3 apparatus (Shandon Thermo Electron Corp). Cells were then mounted on coverslips and analyzed by confocal microscopy (Carl Zeiss).

Statistical analysis - The statistical analysis was perfomed using paired t test and significance was considered when /?_values were lower than 0.05. Results are expressed as mean ± SEM.

Results

CSF-l-induced differentiation of human monocytes is associated with induction of AMPKal expression and activation. When stimulated with CSF-1, human monocytes differentiate into macrophages as shown by both an increase in cell adherence and acquisition of specific markers such as CD71 and CD 163. After 4 days more than 80% of myeloid cells were found to be positive for CD71 and CD 163 expression. Increased cell surface expression correlated with a rise in CD71 and CD 163 mRNA expression. During differentiation, monocytes accumulated the AMPK protein. Increased expression of AMPK was detected 1 day after CSF-1 stimulation and was maximal 3-4 days later. The CSF-l-induced augmentation in AMPK expression tightly correlated with an increased phosphorylation of AMPK on Thrl72. Indeed, there was a strict correlation between CSF-l-induced expression of AMPK and its phosphorylation on Thrl72 during human monocyte differentiation. We next sought to identify the AMPK alpha catalytic subunits ( l and/or a2) expressed during human monocyte differentiation. Of note, unstimulated monocytes exhibited undetectable levels of AMPKa2 mRNA and of the corresponding protein and no induction of AMPKa2 mRNA was detected during CSF-1 -mediated monocyte differentiation, indicating that only AMPKal was expressed in differentiating human monocytes. Importantly, the increased expression of AMPKal was not accompanied by a concomitant rise in AMPKal mRNA level, as shown by real-time qPCR analysis. These findings suggest that increased expression of AMPKal is regulated at the post-transcriptional level during CSF-1 -mediated physiological differentiation of human monocytes and accordingly, cycloheximide treatment of monocytes inhibited CSF-1 -mediated accumulation of AMPKal expression).

The CaMKKp-AMPKal axis is required for human monocyte differentiation.

The increased expression of AMPKal during monocyte differentiation prompted us to investigate whether it could play a role in this process. Pharmacological inhibition of AMPK by dorsomorphin (DRS, 2 μΜ) dampened CSF-1 -induced AMPK phosphorylation on Thrl72 and inhibited CSF-1 -mediated monocyte differentiation, as illustrated by the drastic decrease in the double-positive CD71⁺/CD163⁺ cell population at day 2. Accordingly, inhibition of AMPKal expression by a specific siRNA resulted in a concomitant decrease of AMPK phosphorylation on Thrl72 and a reduction of AMPK substrates phosphorylation. In light with the effect of DRS on monocyte differentiation, AMPK knockdown also led to a robust decrease of the double-positive CD71⁺/CD163⁺ cell population. It has been reported previously that LKB1 and CaMKKp can both phosphorylate AMPK on Thrl72 resulting in its activation¹⁸' ¹⁹. Of note, we found that human monocytes failed to express the LKB1 mRNA. in order to investigate if very low or technically undetectable levels of LKBl were nevertheless expressed by human monocytes, we analysed whether LKBl silencing could affect CSF-1 -induced monocyte differentiation. LKBl silencing failed to affect CSF-1 - induced monocyte differentiation strongly suggesting that LKBl is neither involved in AMPKal phosphorylation nor in differentiation of human monocytes induced by CSF-1. The efficiency of the siRNA targeting LKBl was verified on K562 cell line.

Consequently, we hypothesized that CaMKKp might act as the upstream kinase responsible for AMPKal phosphorylation and activation in human monocytes. Accordingly, the specific CaMKKP inhibitor STO-609 was as efficient as DRS to inhibit phosphorylation of AMPK on Thrl72 and AMPK substrates and CSF- 1 -induced differentiation of human monocytes. The importance of CaMKK in this process was further highlighted using a siRNA that targets specifically CaMKKP □. CaMKK silencing resulted in decreased phosphorylation of AMPKal on Thrl72 and inhibition of monocyte differentiation as did knockdown of AMPKal . Pharmacological inhibition of CaMKK by STO-609 or AMPK by DRS or knockdown of CaMKKP or AMPKal furthermore induced a significant decrease in the phagocytic function of macrophages. Taken together our data clearly illustrate the key role of the CaMKKP-AMPKal axis in the differentiation of human monocytes into macrophages and the acquisition of phagocytic function. PLCy2 downstream of the CSF-1 receptor is required for monocyte differentiation but not AMPK activation. To decipher further the signalling events that link the CSF-1 receptor to the induction of monocyte differentiation, we next studied the implication of calcium release that is a prerequisite for CaMKKp activation²⁰. It is well established that PLCy2 interacts with the CSF-1 receptor to induce release of Ca²⁺²¹. Accordingly, CSF-1 triggered a rapid release of Ca ⁺ in human monocytes that was abrogated by the pan-PLC inhibitor U73122. STO-609 that inhibits CaMKKp Ddownstream of Ca²⁺ release was used as a negative control and failed to dampen CSF-1 -induced Ca ⁺ release but induced for an unknown reason a delayed response. U73122, a pan-PLC inhibitor also inhibited CSFl-mediated PLCy2 and AMPK phosphorylation on Thrl72, suggesting that the effect of CSF-1 on AMPK phosphorylation is PLC dependent. Of note, knock down of the CSF-1R decreased AMPK expression and phosphorylation in identical conditions. Finally, knock down of PLCy2 failed to affect CSF-1 -induced AMPK phosphorylation at day 1 but efficiently inhibited CSF- 1 -induced monocyte differentiation at day 2. Collectively, these findings show that PLCy2 downstream of CSF-1 receptor activation is required for CSF1- induced differentiation of human monocytes, but dispensable for AMPK phosphorylation and activation.

P2Y6 engagement activates the PLCyS-CaMKKp-AMPKal pathway that promotes human monocyte differentiation. We reported previously that P2Y6 is required for CSF-l-induced human monocyte differentiation¹⁷. In line with this observation, we thus postulated that the CaMKKp-AMPKal pathway was triggered following an engagement of P2Y6. In agreement with this hypothesis, CSF-1 significantly increased P2Y6 mR A levels, while mRNA expression of other P2Y receptors was reduced in identical conditions. Increased expression of P2Y6 mRNA by CSF-1 was associated with an increased cell surface expression of P2Y6. We next analyzed whether Ca²⁺ influx downstream of P2Y6 could initiate CaMKK and AMPKal activation upon CSF-1 treatment. Interestingly, CSF-1 - mediated increase in Ca²⁺ was abolished by MRS2578, a P2Y6 antagonist, suggesting that CaMKK was activated downstream of P2Y6. Importantly, stimulation of monocytes with UDP, a physiological ligand of P2Y6 resulted in a robust Ca²⁺ release, that was abrogated by the P2Y6 antagonist MRS2578. Inhibition of Ca²⁺ influx by MRS2578 correlated with a concomitant inhibition of AMPK accumulation and activation at day 1, as shown by a decreased expression and phosphorylation of AMPK on Thrl72. In addition, inhibition of P2Y6 by MRS2578 resulted in a significant inhibition of CSF-1 -mediated monocyte differentiation and phagocytic function. Of note, the specific P2Y1 antagonist, MRS2179 failed to affect CSF-1 -induced monocyte differentiation in identical conditions. In addition and contrarily to MRS2578, MRS2179 didn't affect AMPK phosphorylation on Thrl72. All together these data highlight the specific role of P2Y6 in the regulation of autophagy and monocyte differentiation. To gain insights into AMPK l regulation during monocyte differentiation, we took advantage of a siR A approach aiming at inhibiting individually the expression of the P2Y6 or PLCy3. PLCy3 has been reported as the specific PLC isoform activated downstream of P2Y6²². Individual silencing of both proteins induced a significant inhibition of AMPK expression and phosphorylation on Thrl72 in monocytes stimulated for 1 day with CSF-1. Since it has been recently demonstrated that the P2Y6 antibody commonly used for Western Blot detection of P2Y6 was poorly specific²³, we checked P2Y6 expression using a specific monoclonal antibody that recognizes the native form of P2Y6 by flow cytometry and immunofluorescence. P2Y6 silencing could be efficiently validated with this antibody by flow cytometry analysis and immunofluorescence. Of note, P2Y6 and PLCy3 knockdown potently inhibited CSF-1 -mediated monocyte differentiation at day 2. Taken together, these findings show that in CSF-l-treated monocytes P2Y6 signalling activates the PLCy3 -C ΑΜΚΚβ- AMPKa 1 pathway.

P2Y6-AMPK pathway mediates autophagy induction and monocyte differentiation. Autophagy induction by AMPK required ULK1 -dependent phosphorylation and activation of the Vps34 lipid kinase¹⁴' ²⁴. Therefore, we investigated whether ULK1 was involved in autophagy induction and monocyte differentiation mediated by CSF-1. To this aim, we used both pharmacological and siRNA approaches. DRS (2 μΜ) prevented an accumulation of the double positive CD71⁺/CD163⁺ cell population that corresponds to macrophages. Inhibition of differentiation by DRS also correlated with a reduction of the number of autolysosomes in cells treated with CSF-1 and DRS, as assessed by the CytoID assay. Of note, DRS also reduced CSF-1 -induced AMPK phosphorylation on Thrl72 and phosphorylation of ULK1 on Ser 555. We also confirmed that AMPK knockdown with a specific siRNA significantly reduced CSF-1 -induced monocyte differentiation and autolysosome formation. Importantly, knockdown of AMPK reproduced the effect of DRS, as shown by the inhibition of AMPK expression and phosphorylation on Thrl72, the reduction of ULK1 phosphorylation on Ser 555 and the decrease in LC3-I conversion into LC3-II. Finally, in contrast to M S2179, MRS2578 also inhibited LC3-II conversion highlighting the specific role of P2Y6 in the regulation of autophagy. All together, our findings established that the P2Y6-AMPKal-ULKl pathway is required for CSF-1 -mediated induction of autophagy and differentiation of human monocytes.

Impaired differentiation in CMML patients can be overcame by UDP and P2Y6 agonists through reactivation. We previously established the occurrence in CMML patients of an immature CD147CD24⁺ granulocytic subpopulation that represses the differentiation of the CD14⁺/CD24^" patient's blasts¹⁷. This CD147CD24⁺ population that arose from the same leukemic clone than the CD14VCD24^" population produces high levels of alpha defensins that in turn block the differentiation of CD14⁺/CD24^" monocytes through inhibition of P2Y6 signalling. Incubation of highly purified monocytes from a CMML patient with CSF-1 resulted in altered monocyte differentiation (48.1% double-positive CD71⁺/CD163⁺ monocytes versus 70.2% in cells in which the suppressive CD147CD24⁺ monocyte subpopulation has been depleted). A normal CSF-l-induced monocyte differentiation was restored by the physiological P2Y6 ligand UDP (100 μΜ) (77.4% DP cells) or the P2Y6 agonist MRS2693 (30 μΜ) (72.2% DP cells). To investigate the potential role of AMPK in the effect of P2Y6 agonists, we looked for AMPK expression and autophagy induction in monocytes from a CMML patient treated with CSF-1. After 4 days of treatment, there was a significant increase in both AMPK expression and phosphorylation on Thrl72 in monocytes from CMML patients treated with the combination of CSF-1 or UDP as compared to CMML patients treated with CSF-1 alone. This increased in AMPK activation correlated both with a higher level of differentiation and an increase in the conversion of LC3-I into LC3-II. A similar situation was observed when monocytes from CMML patients were incubated with the combination of CSF-1 and MRS2693. Increased AMPK expression was confirmed by quantification of the AMPK/Actin ratio in cells from CMML patients treated with the combination of CSF-1 plus UDP or MRS2693. Taken together, our data show that the defect in CSF-1 -mediated autophagy and monocyte differentiation found in CMML patients can be overcame by P2Y6 engagement.

DISCUSSION: Autophagy is an evolutionary conserved catabolic process for the degradation of long- live molecules and organelles that also plays a crucial role during the differentiation of a wide range of cell types^25"27. Of note, differentiation of hematopoietic cells requires intense energy consumption, membrane remodelling and/or organelles elimination, as exemplified for myeloid, megakaryocyte or erythroi^'d differentiation⁷' ^η' ²⁸' ²⁹. Physiological monocyte differentiation triggered by CSF-IR engagement requires the formation of a multimolecular complex consisting of FADD, FLIP, Caspase-8, RIP 1 and other protein partners in which caspase-8 acts as the initiatory caspase to induce the cleavage of key protein substrates (RIPKl, nucleophosmin...) that orchestrate the differentiation process^{6, 30}. Accordingly, inhibition of caspase activation with pan-caspase inhibitors or by caspase-8 silencing is sufficient to dampen differentiation of human monocytes into macrophages.

More recently we established that the induction of autophagy is also a prerequisite for CSF-l-induced monocyte differentiation and acquisition of phagocytic functions⁷' ⁸. However the molecular mechanisms that link the CSF-IR to the induction of autophagy remained unknown. In the present study we deciphered the complete signalling pathway from CSF-1 receptor engagement to the induction of autophagy and monocyte differentiation. Taking advantadge of both pharmacological and siRNA approaches, we demonstrate that the physiological differentiation of human monocytes into macrophages upon CSF-IR activation requires an induction of autophagy through the purinergic receptor P2Y6. Accordingly, stimulation of human monocytes with CSF-1 increased P2Y6 mRNA accumulation and P2Y6 protein expression at the monocyte cell surface. Among all the P2Y receptors analysed in the present study, solely the expression of P2Y6 was increased by CSF-1, whereas expression of all other P2Y mRNA subtypes was reduced, reinforcing the notion of a specific role of P2Y6 during human monocyte differentiation. There are few evidence in the literature that P2Y6 receptor activation is involved in differentiation processes such as osteogenic, monocytic or dopaminergic differentiation¹⁷' ^1~33. Moreover, it has been previously reported that P2Y13 activation by ADP induced autophagy in hepatoma cell lines³⁴. But to the best of our knowledge, this is the first description showing that autophagy is induced upon the engagement of a purinergic receptor and that autophagy is essential for a differentiation process.

Understanding how physiological monocyte differentiation occurs at the molecular and cellular levels is obviously a key question in cell biology. Here we demonstrate that PLCp3 interacts with the P2Y6 receptor to induce intracellular Ca2+ production and CaMKKP activation. CaMKK in turn phosphoralyted AMPK on Thrl72 leading to its activation. Once activated, AMPK is phosphorylated and activates the Serine/Threonine Kinase ULKl on Ser 555, resulting in the induction of autophagy. Importantly, inhibition of P2Y6-mediated autophagy induction using different pharmacological approaches or specific siR A directed against AMPK reduced autophagy induction and inhibited human monocyte differentiation into macrophages. Moreover, inhibition of the P2Y6 pathway by specific inhibitors (DRS, STO-609, U73122 and MRS2578) prevented CSF-1 -induced differentiation of human monocytes without increasing the rate of cell death as assessed by annexin-V staining and PARP cleavage. The characterization of the molecular pathways that control monocyte differentiation and more precisely the discovery of the implication of the P2Y6 signalling pathway in the regulation of AMPK activation and autophagy could be of the highest interest for the treatment of myeloid malignancies, including CMML. Of note, we have identified recently a granulocytic subpopulation in the blood of CMML patients that produces high levels of alpha-defensins inhibiting the differentiation of CMML blasts. Accordingly, depletion of this granulocytic subpopulation allowed blasts of CMML patients to fully differentiate into macrophages. In light with this finding, we show here that UDP or P2Y6 agonists can restore physiological monocyte differentiation ex vivo in samples of patients suffering from CMML.

The reinduction of differentiation has been shown to be a valuable therapeutic strategy in some hematopoietic malignancies and has particularly been well exemplified in acute promyelocytic leukemia (APL). APL is caused by an arrest of leukocyte differentiation at the promyelocyte stage and All-Trans-Retinoic-Acid (ATRA) treatment that is able to reinduce differentiation has transformed APL from a highly fatal disease to a highly curable³⁵. Importantly, the antileukemic and differentiation effect of ATRA in APL has been recently linked to its abilitity to induce autophagy-dependent degradation of the PML-RARot oncoprotein^36"38. The discovery that P2Y6 ligands such as UDP or P2Y6 agonists are able to restore normal differentiation in CMML patients through reinduction of autophagy is exciting. In agreement with these findings, it has been recently reported that 5-Aminoimidazole-4- carboxamide ribonucleoside, an AMPK activator, enhanced ATRA-mediated differentiation of promyelocytic NB4 cells and also induced as a single agent the expression of cell surface markers associated with mature monocytes and macrophages in the acute myeloid leukemia cell line U93739. Decitabine and Azacitidine are two nucleoside analogues currently used as first-line treatments for high-risk MDS and CMML patients. Indeed, both agents have been previously shown to exert potent anti-leukemic effect in MDS and CMML⁴⁰' ⁴¹. Therefore, it is tempting to speculate that part of their anti-leukemic effect might also involve an induction of autophagy and myeloid differentiation. Nevertheless, the data presented herein does not only decipher the signalling pathways involved in CSF-1 -mediated induction of autophagy and monocyte differentiation, but also pave a new research avenue for future therapeutic interventions in the context of CMML.

REFERENCES:

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Claims

CLAIMS;

1. A P2Y6 receptor agonist for use in a method for treating myeloid neoplasias.

2. The P2Y6 receptor agonist for use according to claim 1, wherein said disease is associated with an increased level of CD147CD24⁺ cells.

3. The P2Y6 receptor agonist for use according to claim 1 or 2, wherein said disease is chronic myelomonocytic leukemia (CMML).

4. A P2Y6 receptor agonist for use in a method for promoting monocyte to macrophage differentiation in a patient in need thereof.

5. The P2Y6 receptor agonist for use according to any one claims 1 to 5, wherein said P2Y6 receptor agonist is selected from the group of monucleoside 5 '-diphosphates and dinucleoside mono-, di-, and triphosphates.

6. The P2Y6 receptor agonist for use according to claim 5, wherein said monucleoside 5 '-diphosphates is uridine 5 '-diphosphate (UDP) or an analogue thereof.

7. The P2Y6 receptor agonist for use according to claim 6, wherein said uridine 5'- diphosphate analogue is MRS2693 or 5-OMe-UDP.

8. A pharmaceutical composition for use in a method for treating myeloid neoplasias comprising a P2Y6 receptor agonist and a pharmaceutically acceptable carrier.

9. A pharmaceutical composition for use in a method for promoting monocyte to macrophage differentiation in a patient in need thereof comprising a P2Y6 receptor agonist and a pharmaceutically acceptable carrier.

10. A pharmaceutical composition or a kit-of-part comprising a P2Y6 receptor agonist and an additional therapeutic agent.

11. The pharmaceutical composition or a kit-of-part according to claim 10, wherein said therapeutic agent is selected from the group consisting of hydroxyurea, DNA methyltransferase inhibitors, retinoic acid, arsenic derivatives, nucleoside analogues, monoclonal antibodies, P2Y2 receptor agonists and P2Y4 receptor agonists.

12. The pharmaceutical composition or a kit-of-part according to claim 11, wherein said DNA methyltransferase inhibitor is Decitabine or Azacitidine.

13. The pharmaceutical composition or a kit-of-part according to claim 11, wherein said nucleoside analogue is Acadesine or a derivative thereof.

14. The pharmaceutical composition or a kit-of-part according to claim 11, wherein said arsenic derivative is arsenic trioxide (As203).

15. The pharmaceutical composition or a kit-of-part according to claim 11, wherein said P2Y2 receptor agonists and P2Y4 receptor agonists is uridine '-triphosphate (UTP) or an analogue thereof.