WO2019145964A1 - Combination of a mek inhibitor and a cdk4/6 inhibitor for the treatment of sarcoma - Google Patents

Abstract

Methods of treating sarcoma are provided. Accordingly, there is provided a method of treating sarcoma in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Mitogen-activated protein kinase kinase (MEK) inhibitor and a CDK4/6 inhibitor.

Description

TREATMENT OF SARCOMA

RELATED APPLICATION

This application claims the benefit of priority under 35 USC § 119(e) of IL Patent Application No. 257225 filed January 29, 2018, the contents of which are incorporated herein by reference in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to treatment of sarcoma.

Sarcoma is a cancer that arises from transformed cells of mesenchymal origin. Thus, malignant tumors made of cancellous bone, cartilage, fat, muscle, vascular, or hematopoietic tissues are considered sarcomas. This is in contrast to a malignant tumor originating from epithelial cells, which are termed carcinoma.

Hyper activation of the RAS pathway is a common driver in many cancers. As can be seen in the simplified diagram of Figure 1, there are multiple ways by which oncogenic RAS can stimulate cancer cell growth and survival. Although much is known about this pathway, strategies to target RAS and downstream effectors have been only partially successful mostly due to the development of resistance and drug toxicity. In addition, compensatory signaling of parallel signaling cascades overcomes inhibition by specific agents. Combination therapies that target multiple proteins within the same pathway could be more effective and reduce the ability of cancer cells to find compensatory mechanisms.

CDK4/6 inhibition dephosphorylates Rb resulting in a block in the progression of the cell cycle from Gl-S which can lead to cell cycle arrest and even cell death (Figure 1) (1). MEK inhibitors have been used most often in tumors with RAS or BRAF activating mutations. Despite the biological rational for such a treatment, single drug therapy has been largely disappointing. A synthetic lethal interaction between KRAS and CDK4 was reported in a genetic mouse model of NSCLC.(2) Since then a small number of Phase I and II clinical trials testing a combination of MEK and CDK4/6 inhibitors have been performed for NSCLC (3,4) (results pending), and melanoma (5).

Ras mutations have been found in leiomyosarcoma, and activation of a downstream effector of the Ras protein, MEK, have been described in unclassified pleomorphic sarcoma/malignant fibrous histiocytoma as well as in osteosarcoma. Sorafenib which blocks RAF upstream of MEK, has antitumor effects in vitro on various sub-types of sarcoma (chondrosarcoma, MPNST, synovial sarcoma and desmoid tumours). In addition, Selubetinib (MEK inhibitor), had some effect in patients with advanced soft tissue sarcoma in a Phase Eli clinical trial. (6) These results are in line with studies in mouse models of NRAS mutant melanoma, where MEK inhibition primarily caused apoptosis but not cell cycle arrest (7).

Cell cycle check points perturbations, including inactivation of Rb or loss of the pl6INK4a required to maintain Rb activation, have been seen in unclassified pleomorphic sarcoma/malignant fibrous histiocytoma; and loss of the RBI tumour suppressor gene have been reported in leiomyosarcoma, malignant peripheral nerve sheath tumour and osteosarcoma.(6) A Phase II trial of CDK4 inhibitor was associated with a favorable progression-free rate in patients with CDK4-amplified liposarcoma.(8) In a second trial, Palbociclib was associated with favorable progression-free survival (PFS) in liposarcoma without patient selection for CDK4 amplification. (9).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of a Mitogen-activated protein kinase kinase (MEK) inhibitor and a CDK4/6 inhibitor for use in the treatment of sarcoma.

According to an aspect of some embodiments of the present invention there is provided a method of treating sarcoma in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Mitogen-activated protein kinase kinase (MEK) inhibitor and a CDK4/6 inhibitor, thereby treating the sarcoma in the subject.

According to some embodiments of the invention, the sarcoma is metastatic sarcoma.

According to some embodiments of the invention, the metastatic sarcoma is bone metastasis.

According to some embodiments of the invention, the metastatic sarcoma is soft tissue metastasis.

According to some embodiments of the invention, the sarcoma is essentially unresponsive or poorly responsive to individual treatment with the MEK inhibitor or the CDK4/6 inhibitor, as determined by an Ex- vivo organ culture (EVOC) system.

According to some embodiments of the invention, the MEK inhibitor is selected from the group consisting of Trametinib (GSK1120212), Cobimetinib, Selumetinib, Binimetinib (MEK162), PD-325901, Cl- 1040, PD035901, Refametinib, Pimasertib and TAK-733.

According to some embodiments of the invention, the CDK4/6 inhibitor is selected from the group consisting of palbociclib, ribociclib and abemaciclib. According to some embodiments of the invention, the MEK inhibitor is Trametinib and the CDK4/6 inhibitor is Palbociclib.

According to some embodiments of the invention, the MEK inhibitor and the CDK4/6 inhibitor are co-formulated.

According to some embodiments of the invention, the MEK inhibitor and the CDK4/6 inhibitor are in separate formulations.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings and images. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a schematic diagram showing the two major signaling pathways present within many cancer cells showing mutations that promote cancer and the sites of action of CDK4/6 inhibitors and MEK inhibitors.

FIGs. 2A-B show representative photomicrographs of sarcoma biopsies from two patients cut with a vibrotome to 250 mM slices, placed upon titanium grid inserts with media and incubated at 37 °C in 80 % 0₂ and 5 % C0₂ for a total of 5 days. The tissues were treated with the indicated drugs for 4 days following 1 day acclimatization. Each condition including non- treated control was tested in duplicates or triplicates. Following treatment, the tissue was fixed in paraformaldehyde, embedded in paraffin blocks and processed for hematoxylin & eosin staining. The slides were scanned in a Pannoramic Scanner and evaluated by an independent expert cancer pathologist. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to treatment of sarcoma.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Sarcoma is a cancer that arises from transformed cells of mesenchymal origin. Mutations or amplifications of proteins within RAS signaling pathway promote cell cycle progression and cell proliferation in many cancers. Although much is known about this pathway, strategies to target RAS and downstream effectors have been only partially successful.

Whilst reducing the present invention to practice, the present inventors have now uncovered that a combination of Mitogen-activated protein kinase kinase (MEK) and CDK4/6 inhibitors had a synergistic effect on treatment of sarcoma.

As is illustrated hereinunder and in the examples section, which follows, the present inventors show that EVOCs from biopsies of two patients with metastatic sarcoma responded only weakly or not at all to treatment with a MEK inhibitor or a CDK4/6 inhibitor alone but there was a dramatic response when both drugs were used together (Example 1, Figures 2A-B).

Consequently, the present teachings suggest the use of a combined treatment with a MEK inhibitor and a CDK4/6 inhibitors in the treatment of sarcoma.

Hence, according to an aspect of the invention there is provided a therapeutically effective amount of a Mitogen-activated protein kinase kinase (MEK) inhibitor and a CDK4/6 inhibitor for use in the treatment of sarcoma.

According to an additional or an alternative aspect of the invention there is provided a method of treating sarcoma in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Mitogen-activated protein kinase kinase (MEK) inhibitor and a CDK4/6 inhibitor, thereby treating the sarcoma in the subject.

According to a specific embodiment the sarcoma is essentially unresponsive or poorly responsive to individual treatment with said MEK inhibitor or said CDK4/6 inhibitor, as determined by an Ex- vivo organ culture (EVOC) system, see the Examples section which follows.

Ex-vivo organ culture (EVOC) systems used in cancer biology are cultures of precision- cut slices of the patient’s tumor. EVOC has been used for diverse applications including the study of drug toxicity, viral uptake, susceptibility of tumors to radiation or specific anti-cancer drugs [see e.g. Vaira et al. (2010) Proc. Natl. Acad. Sci. U. S. A. 107, 8352-8356; Vickers et al. (2004) Chem. Biol. Interact. 150, 87-96; de Kanter et al. (2002) Curr. Drug Metab. 3, 39-59; Stoff-Khalili et al. (2005) Breast Cancer Res. BCR 7, Rl 141-1152; Merz et al. (2013) Neuro- Oncol. 15, 670-681; Gerlach et al. (20l4)Rr. J. Cancer 110, 479-488; Meijer et al. (2013) Br. J. Cancer 109, 2685-2695; Grosso et al. (2013) Cell Tissue Res. 352, 671-684; Vaira et al. (2010) PNAS 107, 8352-8356; Roife et al. (2016) Clin. Cancer Res. June 3, 1-10; Maund et al. (2014) Lab. Invest. 94, 208-221; Vickers et al. (2004) Toxicol Sci. 82(2):534-44; Zimmermann et al. (2009) Cytotechnology 61(3): 145-152); Parajuli et al. (2009) In Vitro Cell.Dev.Biol.— Animal 45:442-450; Koch et al. (2014) Cell Communication and Signaling 12:73; Graaf et al. Nature Protocols (2010) 5: 1540-1551; Majumder et al. Nat. Commun. 6, 6169 (2015); US Patent Application Publication Nos: US2014/0228246, US 2010/0203575 and US2014/0302491; and International Patent Application Publication Nos: W02002/044344 and WO2018/185760; the contents of which are fully incorporated herein by reference.

According to a specific embodiment the sarcoma is essentially unresponsive or poorly responsive to individual treatment with said MEK inhibitor or said CDK4/6 inhibitor, as determined by the EVOC system described in the Examples section which follows.

As used herein“sarcoma” refers to a malignant tumor that develops from transformed cells of mesenchymal origin. These include, malignant tumors made of, without limitation, cancellous bone, cartilage, fat, muscle, vascular, or hematopoietic tissues. Methods of diagnosing and monitoring sarcoma are well known in the art and include, but not limited to MRI, CT, PET, bone scan, biopsy.

According to an embodiment of the invention, the sarcoma is metastatic.

According to an embodiment of the invention, the metastatic sarcoma is bone metastasis.

According to an embodiment of the invention, the metastatic sarcoma is soft tissue metastasis.

Non-limiting Examples of sarcomas which can be treated according to some embodiments of the invention include Askin's tumor, Sarcoma botryoides, Chondrosarcoma, Ewing's sarcoma, Malignant hemangioendothelioma, Malignant schwannoma, Osteosarcoma and Soft tissue sarcomas including but not limited to Alveolar soft part sarcoma, Angiosarcoma, Cystosarcoma phyllodes, Dermatofibro sarcoma protuberans, Desmoid tumor, Desmoplastic small round cell tumor, Epithelioid sarcoma, Extraskeletal chondrosarcoma, Extraskeletal osteosarcoma, Fibrosarcoma, Gastrointestinal stromal tumor, Hemangiopericytoma, Hemangio sarcoma, Kaposi's sarcoma, Leiomyosarcoma, Liposarcoma, Lymphangiosarcoma, undifferentiated pleomorphic sarcoma, Malignant peripheral nerve sheath tumor, Neurofibrosarcoma, Plexiform fibrohistiocytic tumor, Rhabdomyosarcoma and Synovial sarcoma. Table 1 hereinbelow provides exemplary non-limiting list of sarcomas which can be treated according to some embodiments of the invention.

Table 1

As used herein“Mitogen-activated protein kinase kinase” (also known as MAP2K,

MEK, MAPKK) is a kinase enzyme which phosphorylates mitogen-activated protein kinase (MAPK). MEK is classified as EC 2.7.12.2.

According to specific embodiments, MEK is MEK1.

As used herein,“MEK1” is the polypeptide expression product of the MAP2K1 gene (Gene ID: 5604). According to a specific embodiment, the MEK1 refers to the human MEK1, such as provided in Accession Number NP_002746.

According to specific embodiments, MEK is MEK2.

As used herein,“MEK2” is the polypeptide expression product of the MAP2K2 gene (Gene ID: 5605). According to a specific embodiment, the MEK2 refers to the human MEK2, such as provided in Accession Number NP_l09587.

A MEK inhibitor refers typically to a molecule that selectively inhibits the mitogen- activated protein kinase kinase enzymes MEK1 and/or MEK2 either at the level of protein activity or the level of expression, as further described hereinbelow. They can be used to affect the MAPK/ERK pathway which is often overactive in some cancers. The selective inhibition of CDK4 and CDK6 is referred to herein as CDK4/6 inhibition or CDK4/6 inhibitors or any grammatical deviation of same.

CDK4 and CDK6 are cyclin-dependent kinases that control the transition between the Gl and S phases of the cell cycle. The S phase is the period during which the cell synthesizes new DNA and prepares itself to divide during the process of mitosis. CDK4/6 activity is typically deregulated and overactive in cancer cells. There can be amplification or overexpression of the genes encoding cyclins or of the genes encoding the CDKs themselves. Additionally, loss of endogenous INK4 inhibitors, by gene deletion, mutation, or promoter hypermethylation, can also lead to overactivity of CDK4 and CDK6. CDK4 and CDK6 are classified as EC 2.7.11.22. As use herein,“CDK4” is the polypeptide expression product of the CDK4 gene (Gene ID: 1019). According to a specific embodiment, the CDK4 refers to the human CDK4, such as provided in Accession Number NP_000066.

As use herein,“CDK6” is the polypeptide expression product of the CDK6 gene (Gene ID: 1021). According to a specific embodiment, the CDK6 refers to the human CDK6, such as provided in Accession Number: NP_00l 138778,

NP_00l250.

A CDK4/6 inhibitor refers typically to a molecule (e.g. chemical or drug) that inhibits both CDK4 and CDK6. However, a combination of drugs for affecting inhibition of CDK4 and CDK6 individually is also contemplated.

As used herein, the term“inhibitor” or“inhibit” refers to a decrease of at least 5 % in activity and/or expression in the presence of the molecule in comparison to same in the absence of the molecule, as determined by e.g. PCR, ELISA, Western blot analysis, activity assay (e.g. enzymatic, kinase, binding), cell cycle arrest (as determined by e.g. flow cytometry), increased cell death (as determined by e.g. TUNEL assay, Annexin V).

According to a specific embodiment, the decrease is in at least 10 %, 20 %, 30 %, 40 % or even higher say, 50 %, 60 %, 70 %, 80 %, 90 %, 95 % or 100 %.

Decreasing activity and/or expression of the protein (MEK, CDK4 and/or CDK6) can be effected at the genomic (e.g. homologous recombination and site specific endonucleases) and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents) or on the protein level (e.g., small molecules, aptamers, inhibitory peptides, antagonists, enzymes that cleave the polypeptide, antibodies and the like).

The inhibition may be either transient or permanent.

According to specific embodiments, the inhibitor affects the activity of the protein (MEK, CDK4 and/or CDK6). Such an inhibitor is typically a small molecule chemical or a peptide, as further described hereinbelow. Measures should be taken to use molecules that penetrate the cell membrane or modified to enter through the cell membrane.

According to specific embodiments, the inhibitor affects the expression of the protein (MEK, CDK4 and/or CDK6). Such inhibitors are well known in the art and typically include nucleic acid molecules that mediate their function through genome editing or RNA silencing.

Thus, inhibiting expression of MEK, CDK4 and/or CDK6 can be achieved by RNA silencing. Exemplary RNA silencing agents include dsRNAs, siRNAs, miRNAs, shRNAs and antisense.

Inhibiting expression of MEK, CDK4 and/or CDK6 can also be achieved by inactivating the gene via introducing targeted mutations involving loss-of function alterations (e.g. point mutations, deletions and insertions) in the gene structure.

Methods of introducing nucleic acid alterations to a gene of interest are well known in the art [see for example Menke D. Genesis (2013) 51: - 618; Capecchi, Science (1989) 244:1288-1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; US Patent Nos. 8771945, 8586526, 6774279 and UP Patent Application Publication Nos. 20030232410, 20050026157, US20060014264; the contents of which are incorporated by reference in their entireties] and include targeted homologous recombination, site specific recombinases (e.g. Cre recombinase and Flp recombinase), PB transposases and genome editing by engineered nucleases (e.g. meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR/Cas system). Agents for introducing nucleic acid alterations to a gene of interest can be designed publically available sources or obtained commercially from Transposagen, Addgene and Sangamo Biosciences.

Alternatively or additionally, inhibiting MEK, CDK4 and/or CDK6 can be achieved at the protein level.

According to specific embodiments, the inhibitor is a small molecule or a peptide which binds and/or interferes with MEK, CDK4 and/or CDK6 protein activity.

According to specific embodiments, the inhibitor is a molecule which binds to and/or cleaves MEK, CDK4 and/or CDK6. Such molecules can be a small molecule, an inhibitory peptide. Examples of CDK4/6 inhibitors that can be used according to some embodiments of the invention include, but are not limited to palbociclib, ribociclib and/or abemaciclib.

According to specific embodiments, the CDK4/6 inhibitor is Palbociclib.

According to a specific embodiment, an inhibitor is a small molecule. Examples of MEK inhibitors that can be used according to some embodiments of the invention include, but are not limited to, Trametinib (GSK1120212), Cobimetinib and/or XL518. Other examples include, but are not limited to, Selumetinib, Binimetinib (MEK162), PD- 325901, CI-1040, PD035901, Refametinib, Pimasertib and TAK-733.

According to specific embodiments, the MEK inhibitor is a Trametinib.

Another agent which can be used along with some embodiments of the invention to inhibit MEK, CDK4 and/or CDK6 is an aptamer. As used herein, the term“aptamer” refers to double stranded or single stranded RNA molecule that binds to specific molecular target, such as a protein. Various methods are known in the art which can be used to design protein specific aptamers. The skilled artisan can employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for efficient selection as described in Stoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering (2007) 24(4):381-403).

According to specific embodiments the inhibitor is an antibody or antibody fragment capable of specifically binding MEK, CDK4 and/or CDK6 S. As MEK, CDK4 and CDK6 are localized intracellularly, an antibody or antibody fragment capable of specifically binding MEK, CDK4 and/or CDK6 is typically an intracellular antibody. Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

It will be appreciated that a non-functional analogue of at least a catalytic or binding portion of MEK, CDK4 and/or CDK6 can be also used as an inhibitor.

According to a specific embodiment, a MEK inhibitor also encompasses an upstream activator inhibitor e.g., RAF inhibitor or a downstream effector inhibitor e.g., ERK inhibitor (see e.g., Figure 1).

According to a specific embodiment, a MEK inhibitor also encompasses an upstream activator inhibitor e.g., RAF inhibitor.

According to a specific embodiment the MEK inhibitor specifically inhibits MEK and not an activator or effector thereof.

According to a specific embodiment the CDK4/6 inhibitor specifically inhibits CDK4/6 and not an activator or effector thereof.

According to specific embodiments, the MEK and the CDK4/6 inhibitors of the present invention can be administered to a subject in combination with other established (e.g. gold standard) or experimental therapeutic regimen to treat sarcoma including, but not limited to analgesics, chemotherapeutic agents, radiotherapeutic agents, cytotoxic therapies (conditioning), hormonal therapy, antibodies and other treatment regimens (e.g., surgery) which are well known in the art.

Each of the active ingredients of some embodiments of the invention can be administered to a subject (e.g., human diagnosed with sarcoma) per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the active ingredient i.e., CDK4/6 inhibitor and/or MEK inhibitor accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a subop timal delivery method.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

The term“tissue” refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.

Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (i.e., MEK, CDK4/6 inhibitors) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., sarcoma) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1

P-1)·

Dosage amount and interval may be adjusted individually to provide effective (e.g., tissue) levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

For instance, the doses used can be those which are used in the clinic and in trials as long as efficacy is achieved. One of ordinary skill in the art would know how to calculate an effective dose, such as by using EVOC.

An exemplary dose of Trametinib include, without limitation, 2 mg/orally/day.

An exemplary dose of Cobimetinib include, without limitation, 60 mg/orally/day, 21 days on 7 days off.

An exemplary dose of Palbociclib include, without limitation, 125 mg/orally/day, 21 days on 7 days off.

An exemplary dose of Ribociclib include, without limitation, 600 mg/orally/day, 21 days on 7 days off.

An exemplary dose of Ademaciclib include, without limitation, 150 mg/orally/day.

It will be appreciated that the synergistic effect obtained between the MEK inhibitor and the CDK4/6 inhibitor can reduce the doses used individually.

Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

According to specific embodiments, the MEK inhibitor and the CDK4/6 inhibitor are packaged in separate containers (i.e. in separate formulations).

According to specific embodiments, the MEK inhibitor and the CDK4/6 inhibitor are in a co-formulation.

The term“treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition, e.g. sarcoma) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

As used herein, the term“preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

As used herein, the term“subject” includes mammals, preferably human beings at any age which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology.

As used herein the phrase“treatment regimen” refers to a treatment plan that specifies the type of treatment, dosage, schedule and/or duration of a treatment provided to a subject in need thereof (e.g., a subject diagnosed with a pathology). The selected treatment regimen can be an aggressive one which is expected to result in the best clinical outcome (e.g., complete cure of the pathology) or a more moderate one which may relief symptoms of the pathology yet results in incomplete cure of the pathology. It will be appreciated that in certain cases the more aggressive treatment regimen may be associated with some discomfort to the subject or adverse side effects (e.g., a damage to healthy cells or tissue). The type of treatment can include a surgical intervention (e.g., removal of lesion, diseased cells, tissue, or organ), a cell replacement therapy, an administration of a therapeutic drug (e.g., receptor agonists, antagonists, hormones, chemotherapy agents) in a local or a systemic mode, an exposure to radiation therapy using an external source (e.g., external beam) and/or an internal source (e.g., brachytherapy) and/or any combination thereof. The dosage, schedule and duration of treatment can vary, depending on the severity of pathology and the selected type of treatment, and those of skills in the art are capable of adjusting the type of treatment with the dosage, schedule and duration of treatment.

As used herein the term“about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term“consisting of’ means“including and limited to”.

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases“ranging/ranges between” a first indicate number and a second indicate number and“ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1- 4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);“Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

MATERIALS AND METHODS

Tissue slices - Cancerous tissues were obtained from human biopsies of two human patients with metastatic sarcoma. From one patient a tissue from an open biopsy of a bone metastasis was received. From the second a core needle biopsy from a peripheral soft tissue metastasis was received. Following harvesting, the fresh tissue was immediately placed in ice- cold Phosphate-buffered saline (PBS, Biological Industries, Kibbutz Beit Haemek, Israel) or ice- cold culture medium (RPMI Biological Industries). The tissue was sliced to ~ 250 mM slices using a sterile vibrotome (Compresstome TM VF-300, Precisionary Instruments, Greenville, NC, USA).

Culturing procedure - Tissue slices were placed individually in 6-wells plates (Corning CC-3516 Getter, Petach-Tikva, Israel) on top of titanium grid inserts (MA0036 Well Inserts, Alabama R&D, Alabama, USA). Each well contained 4 ml of DMEM-F12 media supplemented with Gentamycin 50ug/mL, Amphotericin B 2.5 pg / mL, Fetal Calf Serum 5 - 10 %, Penicillin 100 I.U./ mL / Streptomycin 100 pg / ml, L-Glutamine 1 % 200 mM. The plates were incubated in a humidified incubator at 37 ^°C with 80 % 0₂ and 5 % C0₂ for a total of 5 days. Following 1 day acclimatization, the medium was replaced with fresh medium containing: DMSO, Palbociclib 10 pM, Trametinib 1 pM, Doxorubicin 40 pM, AZD4745 50 pM, Taxol 30 pM, Gemcitibine 20 pM, Olaparib 40 pM, Pazopanib 40 pM (all from AdooQ Bioscience, Irvine, CA, USA), or a combination of same, as indicated. The medium was replaced once every 48 hours with fresh drug/DMSO for 4 days. Each condition (including non-treated control) was tested in duplicates or triplicates. Histological evaluation - At the end of culturing, the tissues were fixed overnight in 4 % Paraformalydehyde (PFA) and embedded in paraffin blocks. Following the slices were paraffinized, blocked and cut to 4-5 mM slices for Hematoxylin & Eosin (H&E) staining. Following, the slides were scanned in a Pannoramic Scanner (Pannoramic SCAN II, 3DHISTECH, Budapest, Hungary) and evaluated by an independent expert cancer pathologist.

EXAMPLE 1

Ex Vivo Organotypic Culture (EVOC) in Two Sarcoma Patients

The present inventors performed EVOC from biopsies of two patients with metastatic sarcoma.

From one patient a tissue from an open biopsy of a bone metastasis was received. From the second a core needle biopsy from a peripheral soft tissue metastasis was received. EVOCs from both patients received similar drugs and drug combinations at identical doses. The assay is described in the legend to Figure 2 and in the Materials and Methods section hereinabove. Results can be seen in Figure 2. The cancerous tissues from both patients responded only weakly or not at all to treatment with a MEK inhibitor or a CDK4/6 inhibitor alone but there was a dramatic response when both drugs were used together. Interestingly, the cancerous tissues from the two patients responded differently to doxorubicin (one responded almost completely while the other did not respond at all, Figure 2A). All experiments were performed in duplicates or triplicates and were reviewed by an independent pathologist.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. REFERENCES

( other references are cited in the application)

1. Sherr CJ, Beach D, Shapiro GI. Targeting CDK4 and CDK6: From Discovery to Therapy. Cancer Discov 2016 6(4) 353-67.

2. Puyol M, Martin A, Dubus P, et.al. A Synthetic Lethal Interaction between K-Ras Oncogenes and Cdk4 Unveils a Therapeutic Strategy for Non-small Cell Lung Carcinoma. Cancer Cell 2010 18(1) 63-73.

3. Massachusetts General Hospital Cancer Center. Clinicaltrials.gov Identifier NCT02065063.

4. NIH Clinical Trials PALBOCICLIB + PD-0325901 for NSCLC & Solid Tumors. ClinicalTrials.gov Identifier: NCT02022982

5. Sosman JA, Kittaneh M, Lolkema MPJK, et.al. A phase lb/2 study of LEE011 in combination with binimetinib (MEK162) in patients with NRAS -mutant melanoma: Early encouraging clinical activity. Jour Clin Oncol 2014 32(15) 9009.

6. Demicco EG, Maki RG Lev, DC, Lazar AJ. New Therapeutic Targets in Soft Tissue Sarcoma. Adv Anat Pathol 2012 19(3) 170-180.

7. Kwong LN, Costello JC, Liu H, et.al. Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma. Nat Med 2013 18, 1503-10.

8. Dickson MA, Tap WD, Keohan ML, et.al. Phase II trial of the CDK4 inhibitor PD0332991 in patients with advanced CDK4-amplified well-differentiated or dedifferentiated liposarcoma. J Clin Oncol 2013 31(16) 2024-8.

9. Dickson MA, Schwartz GK, Keohan ML, et.al. Progression-Free Survival Among Patients With Well-Differentiated or Dedifferentiated Liposarcoma Treated With CDK4 Inhibitor Palbociclib: A Phase 2 Clinical Trial. JAMA Oncol 2016 2(7) 937-40.

Claims

WHAT IS CLAIMED IS:

1. A therapeutically effective amount of a Mitogen-activated protein kinase kinase (MEK) inhibitor and a CDK4/6 inhibitor for use in the treatment of sarcoma.

2. A method of treating sarcoma in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Mitogen-activated protein kinase kinase (MEK) inhibitor and a CDK4/6 inhibitor, thereby treating the sarcoma in the subject.

3. The MEK inhibitor and the CDK4/6 inhibitor for use of claim 1 or the method of claim 2, wherein said sarcoma is metastatic sarcoma.

4. The MEK inhibitor and the CDK4/6 inhibitor for use or the method of claim 3, wherein said metastatic sarcoma is bone metastasis.

5. The MEK inhibitor and the CDK4/6 inhibitor for use or the method of claim 3, wherein said metastatic sarcoma is soft tissue metastasis.

6. The MEK inhibitor and the CDK4/6 inhibitor for use or the method of any one of claims 1-5, wherein said sarcoma is essentially unresponsive or poorly responsive to individual treatment with said MEK inhibitor or said CDK4/6 inhibitor, as determined by an Ex- vivo organ culture (EVOC) system.

7. The MEK inhibitor and the CDK4/6 inhibitor for use or the method of any one of claims 1-6, wherein said MEK inhibitor is selected from the group consisting of Trametinib (GSK1120212), Cobimetinib, Selumetinib, Binimetinib (MEK162), PD-325901, Cl- 1040, PD035901, Refametinib, Pimasertib and TAK-733.

8. The MEK inhibitor and the CDK4/6 inhibitor for use or the method of any one of claims 1-7, wherein said CDK4/6 inhibitor is selected from the group consisting of palbociclib, ribociclib and abemaciclib.

9. The MEK inhibitor and the CDK4/6 inhibitor for use or the method of any one of claims 1-6, wherein said MEK inhibitor is Trametinib and said CDK4/6 inhibitor is Palbociclib.

10. The MEK inhibitor and the CDK4/6 inhibitor for use of any one of claims 1 and 3- 9, being co-formulated.

11. The method of any one of claims 2-9, wherein said MEK inhibitor and said CDK4/6 inhibitor are co-formulated.

12. The MEK inhibitor and the CDK4/6 inhibitor for use of any one of claims 1 and 3- 9, being in separate formulations.

13. The method of any one of claims 2-9, wherein said MEK inhibitor and said CDK4/6 inhibitor are in separate formulations.