WO2021067914A1

WO2021067914A1 - Combination therapy for acute myeloid leukemia, myelodysplastic syndromes and other blood diseases and disorders

Info

Publication number: WO2021067914A1
Application number: PCT/US2020/054213
Authority: WO
Inventors: Stavroula Kousteni; Azra Raza; Abdullah ALI; Ioanna MOSIALOU
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2019-10-03
Filing date: 2020-10-05
Publication date: 2021-04-08

Abstract

The present disclosure provides for methods and compositions for preventing and treating myelodysplastic syndromes, acute myeloid leukemia, and other diseases and disorders of the blood, using a combination therapy including all-trans retinoic acid (ATRA) which inhibits or decreases β-catenin, and a ΤΟΡβ inhibitor.

Description

COMBINATION THERAPY FOR ACUTE MYELOID LEUKEMIA, MYELODYSPLASTIC SYNDROMES AND OTHER BLOOD DISEASES AND

DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the priority to U.S. provisional patent application serial no. 62/910,057 filed October 3, 2019, which is hereby incorporated by reference in its entirety.

FIELD

This disclosure is in the field of preventing and treating myelodysplastic syndromes, acute myeloid leukemia, and other diseases and disorders of the blood, using a combination therapy including all-trans retinoic acid (ATRA) which inhibits or decreases β-catenin, and a TGFβ inhibitor.

BACKGROUND

Myelodysplastic syndromes (MDS) are a heterogenous group of clonal stem-cell disorders characterized by the failure of hematopoietic stem cells to fully differentiate into mature blood cells, genetic instability, and peripheral blood cytopenias (reduced number of blood cells), such as low red blood cell count (anemia), or platelet count (thrombocytopenia). MDS often transforms into leukemia. Gangat et al., 2016.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The five-year survival rate is about 26% for patients with AML.

Activated β-catenin signaling in osteoblasts has been found in patients with MDS and MDL. This activated β-catenin alters the differentiation of blood cell progenitors and can promote leukemic transformation. See c-owned U.S. Patent No. 10,350,216.

Current management of MDS relies on frequent blood transfusions to improve low blood cell counts caused by the differentiation defects; however, repeated transfusions carry significant clinical and economic drawbacks, highlighting a need for better therapeutic interventions. Chemotherapy is used to treat AML.

Low doses of all-trans retinoic acid (ATRA) are used in chemotherapy regimens to induce maturation and differentiation of blood cell progenitors. Ferrero et al., 2014. ATRA has been shown to inhibit β-catenin signaling, a reduction in β-catenin protein levels, and regulates differentiation of osteoblasts (bone-forming cells). Green et al. 2017. While ATRA has been used as a chemotherapeutic agent, there exists a need for improved results using ATRA for the use in treating or preventing blood disorders.

SUMMARY

The present disclosure is based upon the discovery that the constitutive activation of β-catenin in osteoblasts induces myelodysplasia (MDS) and acute myeloid leukemia (AML) and that treatment with both all-trans retinoic acid (ATRA), which reduces β-catenin in osteoblasts and an agent which inhibits TGFβ has a synergistic effect on the treatment of MDS and MDL.

Thus, one embodiment of the present disclosure is a method of treating and/or preventing a blood disorder including myelodysplastic syndrome, comprising administering to a subject in need thereof, a therapeutically effective amount of all-trans retinoic acid (ATRA) and an agent that inhibits TGFβ.

A further embodiment of the present disclosure is a composition for treating and/or preventing a blood disorder including myelodysplastic syndrome comprising all-trans retinoic acid (ATRA) and an agent that inhibits TGFβ.

A further embodiment of the present disclosure is a kit for treating and/or preventing a blood disorder including myelodysplastic syndrome including a therapeutically effective amount of all-trans retinoic acid (ATRA) and an agent that inhibits TGFβ.

Disorders of the blood other than MDS that can be treated and/or prevented by the methods and compositions disclosed herein include but are not limited to myelodysplastic syndrome (MDS), aplastic anemia, and anemia associated with kidney disease.

A further embodiment of the present disclosure is a method of treating and/or preventing acute myeloid leukemia, comprising administering to a subject in need thereof, a therapeutically effective amount of all-trans retinoic acid (ATRA) and an agent that inhibits TGFβ.

A further embodiment of the present disclosure is a composition for treating and/or preventing acute myeloid leukemia comprising all-trans retinoic acid (ATRA) and an agent that inhibits TGFβ.

A further embodiment of the present disclosure is a kit for treating and/or preventing acute myeloid leukemia including a therapeutically effective amount of all-trans retinoic acid (ATRA) and an agent that inhibits TGFβ.

In some embodiments, the subject has activated or nuclear β-catenin in their osteoblasts or MDS cells. In some embodiments, the agent that inhibits TGFβ is A83-01.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1 is a bar graph showing Lef1 mRNA levels in cells pretreated with Wnt3A (Wnt3A) and/or TGFβ (TGFb) alone, treated with ATRA alone (ATRA), TGFβ inhibitor A83-01 alone, or in the following combinations of pretreatment and treatment: ATRA + Wnt3A, TGFb + A83-01, Wt3a + TGFb, Wnt3a + TGFb + ATRA, Wnt3a + TGFb + A83-01, Wnt3a + TGFb + ATRA + A83-01, and Wnt3a + A83-01.

Figure 2 is a bar graph showing Jagged1 mRNA levels in cells pretreated with Wnt3A (Wnt3A) and/or TGFβ (TGFb) alone, treated with ATRA alone (ATRA), TGFβ inhibitor A83-01 alone, or in the following combinations of pretreatment and treatment: ATRA + Wnt3A, TGFb + A83-01, Wnt3a + TGFb, Wnt3a + TGFb + ATRA, Wnt3a + TGFb + A83-01, Wnt3a + TGFb + ATRA + A83-01, and Wnt3a + A83-01.

DETAILED DESCRIPTION

Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

The term “subject” as used in this application means an animal with an immune system such as avians and mammals. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates. Avians include, but are not limited to, fowls, songbirds, and raptors. Thus, the invention can be used in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications

The term “patient” as used in this application means a human subject. In some embodiments of the present invention, the “patient” is known or suspected of having or being at risk of developing leukemia, another blood cancer, a blood disorder, or other disease related to abnormal hematopoiesis.

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or delays or minimizes or mitigates one or more symptoms associated with the disease or disorder, or results in a desired beneficial change of physiology in the subject.

The terms “treat”, “treatment”, and the like refer to a means to slow down, relieve, ameliorate or alleviate at least one of the symptoms of the disease or disorder, or reverse the disease or disorder after its onset.

The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or minimize the extent of the disease or disorder or slow its course of development.

The term “in need thereof’ would be a subject known or suspected of having or being at risk of developing MDS, AML, another blood disease or blood disorder or other disease related to abnormal hematopoiesis or known to have activated or nuclear β-catenin in their osteoblasts.

A subject in need of treatment would be one that has already developed the disease or disorder. A subject in need of prevention would be one with risk factors of MDS, AML or another blood cancer, disease or disorder, and/or symptoms of abnormal hematopoiesis or known to have activated or nuclear β-catenin in their osteoblasts.

The term “agent” as used herein means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, biologies, small organic molecules, antibodies, nucleic acids, peptides, and proteins.

As used herein “an adverse effect” is an unwanted reaction caused by the administration of a drug.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. , the limitations of the measurement system, i.e. , the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

Treatment with Both ATRA and a TGFβ Inhibitor has a Synergistic Effect on MDS

The inventors have previously shown that both a mouse model of MDS and AML,, and human patients with MDS and AML., have β-catenin in the nucleus of their osteoblasts as opposed to the localization of β-catenin in the membrane in healthy controls. It was also shown that certain genes are overexpressed in subjects with MDS and AML. These genes included JAGGED-1 and DLL-1, and β-catenin target genes, including but not limited to, Axin-2, Tcf-1, Tcf-3, and Lef-1. See co-owned US Patent No. 10,350,216, herein incorporated by reference in its entirety.

All-trans retinoic acid or ATRA is a retinoid which are drugs that are relatives of Vitamin A. Retinoids control normal cell growth, cell differentiation, and cell death during embryonic development and in certain tissues later in life. Retinoids effects on the cells are controlled by receptors on the nucleus of each cell.

As shown below, treatment of osteoblastic cells with ATRA inhibited the induction of β-catenin target genes and abrogated nuclear localization of β-catenin. Treatment with ATRA also inhibited β-catenin signaling and improved anemia, thrombocytopenia, and survival of leukemic mice (Example 1). Treatment of a human subject with MDS by administering ATRA at 120 mg/ m² per day resulted in an alleviation of symptoms as well as an inhibition of the expression of β-catenin in his osteoblasts (Example 2).

More remarkably, the treatment of osteoblasts with both ATRA and a TOEb inhibitor resulted in a synergistic effect on the inhibition of expression of both Lef1 and Jagged1, genes shown to be overexpressed in MDS and AML (Example 3).

The combination therapy disclosed herein of ATRA and an agent that inhibits TGFβ is a more effective treatment of MDS and AML than either ATRA or the agent that inhibits TGFβ used alone. The combination therapy using the ATRA and the agent that inhibits TGFβ has a more potent therapeutic effect on MDS and AML than the ATRA or the agent that inhibits TGFβ alone allowing the decrease in dosage and/or increase in administration interval, thereby decreasing adverse effects.

Methods of Treatment and/or Prevention of MDS

A method of combination therapy for the treatment and/or prevention of MDS and AML is provided by the present disclosure. The method includes the co-administration of ATRA and an agent that inhibits TGFβ to a subject in need thereof. The ATRA and the agent that inhibits TGFβ can be used in amounts that are therapeutically effective when combined which amount may be determined by the skilled medical practitioner. The method may further include prior to the co-administration of ATRA and an agent that inhibits TGFβ, a step of identifying a subject in need of treatment or prevention of MDS and AML. For example, the step of identifying may include diagnosing a subject as having MDS and AML. One such test for identification of a subject in need of treatment would be a subject with increased activated or nuclear β-catenin in their osteoblasts. In one embodiment of the present disclosure, the subject to which the co-administration of the ATRA and agent that inhibits TGFβ is administered has been shown to have or known to have activated or nuclear β- catenin in their osteoblasts

Co-administration may be conducted by administering a mixed formulation (e.g., a single composition of the ATRA and the agent that inhibits TGFβ), as described herein. Alternatively, the ATRA and the agent that inhibits TGFβ can be administered separately. The co-administration can be conducted by a first step of administering the ATRA and a second step of administering the agent that inhibits TGFβ, wherein the first and second steps may be conducted simultaneously or sequentially. In the case of the sequential administration, any order of administration can be used and separated by any suitable time interval. The ATRA and the agent that inhibits TGFβ can be used in amounts that are therapeutically effective when combined which amount may be determined by the skilled medical practitioner.

By the co-administration of the ATRA and the agent that inhibits TGFβ, synergistic effects can be obtained as compared to the use of either single active agent alone, i.e. , without the other. Pharmaceutical Compositions and Methods of Administration

The present disclosure encompasses the administration of agents. Preferred methods of administration of the agents include oral; mucosal, such as nasal, sublingual, vaginal, buccal, or rectal; parenteral, such as subcutaneous, intravenous, bolus injection, intramuscular, or intraarterial; or transdermal administration to a subject. Thus, the agent must be in the appropriate form for administration of choice.

Such compositions for administration may comprise a therapeutically effective amount of the agents and a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human, and approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. “Carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, cachets, troches, lozenges, dispersions, suppositories, ointments, cataplasms (poultices), pastes, powders, dressings, creams, plasters, patches, aerosols, gels, liquid dosage forms suitable for parenteral administration to a patient, and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable form of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration· Pharmaceutical compositions adapted for oral administration may be capsules, tablets, powders, granules, solutions, syrups, suspensions (in non-aqueous or aqueous liquids), or emulsions. Tablets or hard gelatin capsules may comprise lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid polyols. Solutions and syrups may comprise water, polyols, and sugars. An active agent intended for oral administration may be coated with or admixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract. Thus, the sustained release may be achieved over many hours and if necessary, the active agent can be protected from degradation within the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of an active agent at a particular gastrointestinal location due to specific pH or enzymatic conditions.

Pharmaceutical compositions adapted for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient over a prolonged period of time.

Pharmaceutical compositions adapted for nasal and pulmonary administration may comprise solid carriers such as powders which can be administered by rapid inhalation through the nose. Compositions for nasal administration may comprise liquid carriers, such as sprays or drops. Alternatively, inhalation directly through into the lungs may be accomplished by inhalation deeply or installation through a mouthpiece. These compositions may comprise aqueous or oil solutions of the active ingredient. Compositions for inhalation may be supplied in specially adapted devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which can be constructed so as to provide predetermined dosages of the active ingredient.

Pharmaceutical compositions adapted for rectal administration may be provided as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain anti-oxidants, buffers, baceriostats, and solutes that render the compositions substantially isotonic with the blood of the subject. Other components which may be present in such compositions include water, alcohols, polyols, glycerine, and vegetable oils. Compositions adapted for parental administration may be presented in unit-dose or multi-dose containers, such as sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile carrier, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Selection of a therapeutically effective dose or amount will be determined by the skilled artisan considering several factors which will be known to one of ordinary skill in the art. Such factors include the particular form of the inhibitor, and its pharmacokinetic parameters such as bioavailability, metabolism, and half-life, which will have been established during the usual development procedures typically employed in obtaining regulatory approval for a pharmaceutical compound. Further factors in considering the dose include the condition or disease to be treated or the benefit to be achieved in a normal individual, the body mass of the patient, the route of administration, whether the administration is acute or chronic, concomitant medications, and other factors well known to affect the efficacy of administered pharmaceutical agents. Thus, the precise dose should be decided according to the judgment of the person of skill in the art, and each patient’s circumstances, and according to standard clinical techniques.

ATRA is FDA approved of the treatment of acute promyelocytic leukemia. It is given by mouth in capsule form in 10 mg dose. Common side effects include headache, fever, nausea and vomiting. Because the present disclosure providing for a method and composition combining ATRA with an agent that inhibits TGFβ has a synergistic effect, it is contemplated that a lower dosage of ATRA can be administered and side effects reduced.

The pharmaceutical composition for the combination therapy may be a mixed formulation (e.g., a single composition comprising two or more active ingredients) of the ATRA and the agent that inhibits TGFβ. The ATRA and the agent that inhibits TGFβ can be present in any amount that is therapeutically effective when used together. The composition can be formulated to be used for simultaneous administration of the two active agents.

Alternatively, the ATRA and the agent that inhibits TGFβ can be formulated in separate compositions and the two active agents can be separately administered, concurrently, simultaneously or sequentially. In the case of the sequential administration, any order of administration can be used.

Kits

Also within the scope of the present disclosure are kits for practicing the method of the invention of preventing and/or treating MDS or AML, including a first pharmaceutical composition including ATRA and a second pharmaceutical composition including an agent that inhibits TGFβ and a package container. The ATRA and the agent that inhibits TGFβ can be used in amounts that are therapeutically effective when combined, which amounts may be determined by the skilled medical practitioner. The package container can be any container that holds or otherwise links the two compositions in individual containers together in a single unit or the package container may be a single, divided container having at least two chambers that each hold one of the compositions.

In some embodiments, the kit can comprise instructions for use in any of the methods described herein. The included instructions can comprise a description of administration of the agents to a subject to achieve the intended activity in a subject. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment.

The instructions relating to the use of the agents described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and/or alleviating a disease or disorder in a subject.

The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device, or an infusion device. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

EXAMPLES

The present invention may be better understood by reference to the following non- limiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed to limit the broad scope of the invention.

Example 1- ATRA Treatment of Cells and Leukemic Mice

Treatment of osteoblastic cells with ATRA inhibited the induction of β-catenin target genes by Wnt3a and abrogated nuclear localization of β-catenin, as shown by immunohistochemistry and western blot analysis. These effects were mediated by the retinoic acid receptor a (RARa) since silencing RARa, but not RARb or RARg, in osteoblasts abolishes the inhibitory effect of ATRA on Wnt3a-induced b-catenin signaling.

Similarly, treatment of leukemic mice expressing constitutively active β-catenin in their osteoblasts with ATRA, inhibited β-catenin signaling as measured by expression of its target genes Axin2 and Lef1 in bone. At the same time, ATRA improved anemia and thrombocytopenia, decreased the percentage of blasts in bone marrow and blood and prolonged overall survival in these leukemic mice as compared to vehicle-treated mice.

Example 2 - ATRA Increased Platelet Stability and Decreased β-Catenin in the Osteoblasts of a Patient with MDS

A 70 year old Caucasian male with Refractory Cytopenia and Multilineage Dysplasia presented with a white blood count (WBC) of 2.1 cells/μl, hemoglobin of 15.1 g/dL, and platelets of 85,000 cells/μl. Bone marrow (BM) aspiration and biopsy showed a normocellular marrow with 4% blasts and normal cytogenetics. He was followed without any treatment and showed slowly worsening thrombocytopenia. Repeated bone marrow biopsies confirmed a diagnosis of refractory cytopenia with multilineage dysplasia (RCMD) with an International Prognostic Scoring System (IPSS) category of Intermediate-1.

When the patient’s platelets fluctuated between 25,000 to 40,000 cells/μl, he was started on ATRA, 120 mg/m² per day in 2 separate doses which was adjusted due to side effects to every other two weeks. The patient’s worsening thrombocytopenia stabilized in response to treatment with ATRA. Two somatic alterations genes recurrently mutated in AML, TET2 and SRSF2, were identified in his blood. The patient has been kept on ATRA for more than 3 years and shows a stabilized disease with almost complete remission.

In view of reports that Retinoic Acid (RA) signaling through RA-recep tor- alpha (RARA) inhibits β-catenin function, β-catenin activity on BM aspirates of this patient was analyzed pre-ATRA and post/on- ATRA. Interestingly, an increased β-catenin activity in both CD34+ cells and osteoblasts (Lin-, CD34-, RunX2+, OCN+ cells) was found in the pre- therapy BM sample but not in the post/on-therapy ones, where β-catenin activation was completely downregulated.

This data suggested that the disease stabilization was due to ATRA treatment since the platelet patient's count was stabilized in parallel with the treatment and more importantly, because the β-catenin activity was completely abrogated after it. This case report suggested the possibility of a larger trial using targeted-therapy with ATRA in patients with high β- catenin activity.

The response to ATRA treatment of was also found in a second MDS patient with active β-catenin in their osteoblasts, and not responding to other treatments.

Example 3 - Combination Treatment of ATRA and a TGFβ Inhibitor Had Synergistic Effect on the Expression of Gene Expression Markers of MDS

Materials and Methods- Primary human osteoblasts isolated from core bone biopsies were plated in 10cm² in αMEM supplemented with 10% FBS and 100 I.U/ml penicillin/ 100μg/ml streptomycin and cultured at 37°C in a 5% CO₂ atmosphere. The next day, when cells reached confluence, the medium was supplemented with 5mM β-glycerolphosphate and 100μg/ml ascorbic acid (mineralization medium), which was replaced every 2 days thereafter for 10 days. Two days before the experiment, cells were placed in αMEM supplemented with 5% CSS and one day later, cells were serum starved overnight in αMEM supplemented with 0.5% CSS. The following day, cells were pre-treated with 20ng/ml recombinant mouse Wnt3a (R&D Systems) and/or 1ng/ml recombinant mouse TGFβ (R&D) or vehicle for 3 hours to activate these signaling pathways. Subsequently, cells were treated with 1μM ATRA (Sigma) and/or 1μM TGFβ inhibitor A83-01 (Tocris) or vehicle for 3 hours. Total RNA was extracted using Trizol reagent, cDNA was prepared, and real-time PCR analyses were carried out following standard protocols.

Results- When cells were treated with both ATRA and a TGFβ inhibitor, there was a four-fold reduction in Lef1 expression as compared to treatment with ATRA or the TGFβ inhibitor alone (Figure 1). There was a five-fold reduction in Jagged1 expression when the cells were treated with both ATRA and a TGFβ inhibitor as compared to treatment with the TGFβ inhibitor alone and a fold reduction as compared to treatment with ATRA alone (Figure 2).

REFERENCES

Ferrero et al., Survival improvement of poor-prognosis AML/MDS patients by maintenance treatment with low-dose chemotherapy and differentiating agents. Ann Hematol. 2014 Aug; 93(8): pp. 1391-400. Gangat et al., Myelodysplastic syndromes: Contemporary review and how we treat.

Am J Hematol. 2016 Jan; 91(1): pp. 76-89.

Green et al., Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells. Exp Cell Res. 2017 Jan; 350(1): pp. 284-97.

Claims

1. A method of treating and/or preventing a disease or disorder of the blood comprising administering to a subject in need thereof a therapeutically effective amount of all- trans retinoic acid and an agent that inhibits TGFβ, wherein the therapeutically effective amount of all-trans retinoic acid and an agent that inhibits TGFβ is less than when either is administered alone.

2. The method of claim 1, wherein the disease of the blood is acute myeloid leukemia.

3. The method of claim 1, wherein the disorder of the blood is selected from the group consisting of myeloproliferative syndrome (MPS), myelodysplastic syndrome (MDS), aplastic anemia, and anemia associated with kidney disease.

4. The method of claim 1, wherein the agent is selected from the group consisting of chemicals, phytochemicals, pharmaceuticals, biologies, small organic molecules, antibodies, nucleic acids, peptides, and proteins.

5. The method of claim 1, wherein the agent is AR83-01.

6. The method of claim 1, wherein the subject is a mammal.

7. The method of claim 1, wherein the subject is human.

8. The method of claim 1, wherein the subject has been identified as having activated or nuclear β-catenin in its osteoblasts.

9. The method of claim 1, wherein the all-trans retinoic acid and the agent that inhibits TGFβ are administered in the same composition.

10. The method of claim 1, wherein the all-trans retinoic acid and the agent that inhibits TGFβ are administered in different compositions.

11. The method of claim 1, wherein the all-trans retinoic acid and the agent that inhibits TGFβ are administered simultaneously.

12. The method of claim 1, wherein the all-trans retinoic acid and the agent that inhibits TGFβ are administered sequentially.

13. A pharmaceutical composition for the treatment and/or prevention of a disease or disorder of the blood, comprising a therapeutically effective amount of all-trans retinoic acid and an agent that inhibits TGFβ, wherein the therapeutically effective amount of all-trans retinoic acid and an agent that inhibits TGFβ is less than when either is administered alone.

14. The composition of claim 13, wherein the disease of the blood is acute myeloid leukemia.

15. The composition of claim 13, wherein the disorder of the blood is selected from the group consisting of myeloproliferative syndrome (MPS), myelodysplastic syndrome (MDS), aplastic anemia, and anemia associated with kidney disease.

16. The composition of claim 13, wherein the agent is selected from the group consisting of chemicals, phytochemicals, pharmaceuticals, biologies, small organic molecules, antibodies, nucleic acids, peptides, and proteins.

17. The composition of claim 13, wherein the agent is AR83-01.

18. The composition of claim 13, wherein the composition further comprises a pharmaceutically acceptable carrier.

19. A kit, comprising a therapeutically effective amount of all-trans retinoic acid and a therapeutically effective amount of an agent that inhibits TGFβ and instructions as to dosage, dosing schedule, and route of administration of the agent for the treatment or prevention of a disease or disorder of the blood wherein the therapeutically effective amount of all-trans retinoic acid and an agent that inhibits TGFβ is less than when either is administered alone.

20. The kit of claim 19, wherein the disease of the blood is acute myeloid leukemia.

21. The kit of claim 19, wherein the disorder of the blood is selected from the group consisting of myeloproliferative syndrome (MPS), myelodysplastic syndrome (MDS), aplastic anemia, and anemia associated with kidney disease.

22. The kit of claim 19, wherein the agent is selected from the group consisting of chemicals, phytochemicals, pharmaceuticals, biologies, small organic molecules, antibodies, nucleic acids, peptides, and proteins.

23. The kit of claim 19, wherein the agent is AR83-01.

24. The kit of claim 19, wherein the all-trans retinoic acid and the agent that inhibits TGFβ are packaged in the same composition.

25. The kit of claim 19, wherein the all-trans retinoic acid and the agent that inhibits TGFβ are packaged in different compositions.