WO2017160797A1

WO2017160797A1 - Combination therapy with c-myc nucleic acid inhibitors and selective cdk7 inhibitors

Info

Publication number: WO2017160797A1
Application number: PCT/US2017/022246
Authority: WO
Inventors: Marc Abrams; Edmond CHIPUMURO
Original assignee: Dicerna Pharmaceuticals, Inc.
Priority date: 2016-03-16
Filing date: 2017-03-14
Publication date: 2017-09-21

Abstract

Disclosed herein are methods for the treatment of cancer, comprising administering to a subject a c-Myc nucleic acid inhibitor molecule and a therapeutically effective amount of a selective CDK7 inhibitor. Also disclosed herein is a pharmaceutical composition comprising a therapeutically effective amount of a c- Myc nucleic acid inhibitor molecule; a therapeutically effective amount of a selective CDK7 inhibitor; and at least one pharmaceutical carrier.

Description

COMBINATION THERAPY WITH C-MYC NUCLEIC ACID INHIBITORS AND

SELECTIVE CDK7 INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[001 ] This application claims the benefit of, and relies on the filing date of, U.S. provisional patent application number 62/390,059, filed 16 March 2016 the entire disclosure of which is incorporated herein by reference.

SEQUENCE LISTING

[002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on 10 March 2017, is named 0243_0004-PCT_SL.txt and is 1 ,338 bytes in size.

FIELD

[003] The present disclosure relates generally to combination therapy using a nucleic acid inhibitor molecule that reduces expression of the c-Myc gene and a selective inhibitor of CDK7.

BACKGROUND

[004] The c-Myc gene is a key molecular regulator of cellular growth and differentiation. The c-Myc protein is a transcription factor that activates expression of many genes via binding of consensus sequences (Enhancer Box sequences (E-boxes)) and recruitment of histone acetyltransferases (HATs). The c-Myc protein can also act as a transcriptional repressor. By binding Miz-1 transcription factor and displacing the p300 co- activator, Myc inhibits expression of Miz-1 target genes. In addition, Myc has a direct role in the control of DNA replication (Dominguez-Sola et al. Nature 448 (7152): 445-51 ).

[005] Various mitogenic signaling pathways, including Wnt, Shh and EGF (via the RAS/RAF/MEK/ERK pathway), have been demonstrated to activate c-Myc. The role of c- Myc in modifying the expression of its target genes has been shown to cause numerous biological effects. The first to be discovered was its capability to drive cell proliferation (upregulates cyclins, downregulates p21 ), but c-Myc also plays an important role in regulating cell growth (upregulates ribosomal RNA and proteins), apoptosis (downregulates Bcl-2), differentiation and stem cell self-renewal. Deregulated MYC expression is found in many commonly occurring human cancers including liver, colon, breast, prostate, lung, and bladder cancer. It is estimated that increased MYC expression contributes to the cause of at least 40% of all human cancers. Dang et al., Clin. Cancer Res., 15 (21 ):6479-83 (2009).

[006] Despite advances in understanding the multi-faceted role that c-Myc plays in gene expression and cell growth and how mutations and/or altered expression of c-Myc can play a role in tumorigenesis, there remains a need for compositions that can treat disease associated with c-Myc expression, such as cancer.

SUMMARY

[007] The inventors herein have discovered that combining certain therapeutic approaches with nucleic acid inhibition of the c-Myc gene can result in synergistic inhibition of tumor growth. In particular, combining c-Myc inhibition with selective CDK7 inhibition yields effective tumor inhibition (see, e.g., Examples 2-3 and Figures 2-3).

[008] Disclosed herein are methods of treating a c-Myc-associated disease or disorder, comprising administering to a subject a therapeutically effective amount of a c- Myc nucleic acid inhibitor molecule and a therapeutically effective amount of a selective CDK7 inhibitor. In certain embodiments, the subject is a human. In certain embodiments, the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule.

[009] Also provided is a pharmaceutical composition comprising a c-Myc nucleic acid inhibitor molecule for use in treating a c-Myc-associated disease or disorder, wherein the composition is administered in combination with a selective CDK7 inhibitor.

[010] In certain embodiments of these methods and compositions for use in treating a c-Myc-catenin-associated disease or disorder, the c-Myc-catenin-associated disease or disorder is a c-Myc-associated cancer, such as liver, colon, breast, prostate, lung, and bladder cancer.

[01 1 ] In certain embodiments of these methods and compositions for use in treating a c-Myc-catenin-associated disease or disorder, the selective CDK7 inhibitor is THZ1 (see, e.g. , Kwiatkowski et al. (2014); Nature 51 1 (751 1 ): 616-620), BS-181 (see, e.g. , AN et al. (2009); Cancer Res 69(15): 6208-6215), and SY-351 (Syros Pharmaceuticals). In other embodiments, the selective CDK7 inhibitor is selected from those disclosed in WO 2015/058163, WO 2015/154022, WO 2015/154038, WO 2015/154039, WO 2015/058140, and WO 2014/063068. [012] In certain embodiments of these methods and compositions for use in treating a c-Myc-catenin-associated disease or disorder, the c-Myc nucleic acid inhibitor molecule is formulated with a lipid nanoparticle.

[013] Further disclosed herein are pharmaceutical compositions comprising a therapeutically effective amount of a c-Myc nucleic acid inhibitor molecule; a therapeutically effective amount of a selective CDK7 inhibitor; and at least one pharmaceutical excipient.

[014] Both the foregoing general summary and the following detailed description are exemplary only and are not restrictive of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written

description, serve to explain certain principles of the compositions and methods disclosed herein.

[016] Figs. 1A-B show the results of an in vitro study with Hep3B (A) and Hep G2 (B) cells, where the combination of a c-Myc nucleic acid inhibitor molecule (MYC2) with a CDK7 inhibitor (THZ1 ) achieved a potent reduction in c-Myc mRNA.

[017] Fig. 2 show a titration curve from an in vitro study with HepG2 cells, where the combination of a c-Myc nucleic acid inhibitor molecule (MYC2) with a CDK7 inhibitor (THZ1 ) at lower doses induced significantly higher cellular cytotoxicity and showed synergy as compared to the individual agents.

[018] Fig. 3 shows that combination therapy in mice harboring Hep3B tumor cells with a c-Myc nucleic acid inhibitor molecule (MYC2) with a CDK7 inhibitor (THZ1 ) enhances anti-tumor efficacy as compared to treatment with either of MYC2 or THZ1 individually.

[019] Fig. 4 shows that combination therapy in mice harboring Hep3B tumor cells with a c-Myc nucleic acid inhibitor molecule (MYC2) with a CDK7 inhibitor (THZ1 ) resulted in downregulation of c-Myc mRNA (58% downregulation with combination versus 25% downregulation with each individual agent). The c-Myc mRNA was measured 24 hours after administration of the last dose.

[020] Fig. 5 shows one non-limiting embodiment of a double-stranded c-Myc nucleic acid inhibitor molecule, having of a sense (or passenger) strand (SEQ ID NO: 1 ) and an antisense (guide) strand (SEQ ID NO: 2). This c-Myc nucleic acid inhibitor molecule is referred to herein as MYC2.

[021 ] Fig. 6 shows one non-limiting embodiment of a double-stranded c-Myc nucleic acid inhibitor molecule, having of a sense (or passenger) strand (SEQ ID NO: 3) and an antisense (guide) strand (SEQ ID NO: 4). This c-Myc nucleic acid inhibitor molecule is referred to herein as MYC1 .

[022] Fig. 7 shows one non-limiting embodiment of a lipid nanoparticle that can used to formulate the c-Myc nucleic acid inhibitor molecule. The LNP includes the following core lipids: DL-048 (cationic lipid) and DSG-MPEG (pegylated lipid), and the following envelope lipids: DL-103 (cationic lipid), DSPC, cholesterol, and DSPE-MPEG (pegylated lipid).

DETAILED DESCRIPTION

Definitions

[023] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[024] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus for example, a reference to "a method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[025] Administer: As used herein, "administering" a composition to a subject means to give, apply or bring the composition into contact with the subject. Administration can be accomplished by any of a number of routes, including, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal and intradermal.

[026] Approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[027] C-Myc: As used herein, "c-Myc" refers to either to a polypeptide or a nucleic acid sequence encoding such a c-Myc protein (such as Genbank Accession Nos. NP_002458.2 and NP_034979.3). c-Myc transcripts include the sequences of Genbank Accession Nos. NM_002467.4 and NM_010849.4.

[028] c-Myc-associated disease or disorder. As used herein, a "c-Myc-associated" disease or disorder refers to a disease or disorder that is associated with altered c-Myc expression, level and/or activity. A "c-Myc-associated" disease or disorder includes cancer and/or proliferative diseases, conditions, or disorders, including liver, colon, breast, prostate, lung, and bladder cancer.

[029] CDK7 Inhibitor. As used herein, the term "CDK7 inhibitor" refers to a compound or agent that reduces the activity of Cyclin-Dependent Kinase 7 ("CDK7").

[030] Selective CDK7 Inhibitor. As used herein, the term "selective CDK7 inhibitor" refers to a CDK7 inhibitor that reduces the activity of CDK7 more than it reduces the activity of any other cyclin-dependent kinase ("CDK"). For the purposes of this application, the CDK inhibitors flavopiridol, BMS-387032, PHA-793887, Roscovitine are not selective CDK7 inhibitors as each has been shown to have a lower inhibitory activity toward CDK7 than toward at least one other CDK (see Table 1 , Kwiatkowski et al. (2014); Nature 51 1 (751 1 )).

[031 ] Excipient As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a composition, for example to provide or contribute to a desired consistency or stabilizing effect.

[032] Nucleic acid inhibitor molecule: As used herein, the term "nucleic acid inhibitor molecule" refers to an oligonucleotide molecule that reduces or eliminates the expression of a target gene wherein the oligonucleotide molecule contains a region that specifically targets a sequence in the target gene mRNA. Typically, the targeting region of the nucleic acid inhibitor molecule comprises a sequence that is sufficiently complementary to a sequence on the target gene mRNA to direct the effect of the nucleic acid inhibitor molecule to the specified target gene. For example, a "c-Myc nucleic acid inhibitor molecule" reduces or eliminates the expression of a c-Myc gene. The nucleic acid inhibitor molecule may include natural ribonucleotides, natural deoxyribonucleotides, and/or modified nucleotides. The modified nucleotides include modifications such as substitution on positions on the sugar ring, modifications of the phosphoester linkages between nucleotides, non-natural bases, and non-natural alternative carbon structures such as locked nucleic acids ("LNA") (see below) and unlocked nucleic acids ("UNA") (see below).

[033] MYC1: As used herein "MYC1 " refers to a nucleic acid inhibitor molecule that targets the c-Myc gene and has a sense strand with a nucleic acid sequence consisting of SEQ ID NO: 3 and an antisense strand with a nucleic acid sequence consisting of SEQ ID NO: 4.

[034] MYC2: As used herein "MYC2" refers to a nucleic acid inhibitor molecule that targets the c-Myc gene and has a sense strand with a nucleic acid sequence consisting of SEQ ID NO: 1 and an antisense strand with a nucleic acid sequence consisting of SEQ ID NO: 2.

[035] Reduce(s): The term "reduce" or "reduces" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid inhibitor molecules (e.g., c-Myc siNA molecules) or exemplary inhibitors (e.g., selective CDK7 inhibitors), the term generally refers to the reduction in the expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, below that observed in the absence of the nucleic acid inhibitor molecules.

[036] RNAi inhibitor molecule: The term "RNAi inhibitor molecule" as used herein refers to either (a) a double stranded nucleic acid inhibitor molecule ("dsRNAi inhibitor molecule") having a sense strand (passenger) and antisense strand (guide), where the antisense strand is used by the Argonaute 2 (Ago 2) endonuclease in the cleavage of the target mRNA or (b) a single stranded nucleic acid inhibitor molecule ("ssRNAi inhibitor molecule") having a single antisense strand that is used by Ago2; wherein the RNAi inhibitor molecule makes use of at least part of the cell's RNA interference ("RNAi") machinery to reduce or eliminate expression of the target gene.

[037] Subject As used herein, the term "subject" means any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human. The terms "individual" or "patient" are intended to be interchangeable with "subject." Nucleic Acid Inhibitor Molecules

[038] Various structures have been used for nucleic acid inhibitor molecules. For example, early work focused on double-stranded nucleic acid molecules with each strand having sizes of 19-25 nucleotides with at least one 3' overhang of 1 to 5 nucleotides (see, e.g. , U S Patent No. 8,372,968). Subsequently, longer double-stranded RNA molecules that get processed in vivo by the Dicer enzyme to active siRNA molecules were developed (see, e.g. , U.S. Patent No. 8,883,996). Later work developed extended double-stranded nucleic acid inhibitor molecules where at least one end of at least one strand is extended beyond the double-stranded targeting region of the molecule, including structures where one of the strands includes a thermodynamically-stabilizing tetraloop structure (see, e.g., U.S. Patent No. 8,513,207, U.S. Patent NO. 8,927,705, and WO 2010/033225, which are incorporated by reference for their disclosure of these double-stranded nucleic acid inhibitor molecules). Those structures include single-stranded extensions (on one or both sides of the molecule) and double-stranded extensions. Furthermore, recent efforts have demonstrated activity of single-stranded RNAi molecules (see, e.g.,Matsui et al. (2016), Molecular Therapy, accepted article preview online February 23 2016; doi: 10.1038/mt.2016.39). And, antisense molecules have been used for decades to reduce expression of specific target genes. A number of variations on the common themes of these structures have been developed for a range of targets. The c-Myc nucleic acid inhibitor molecules of the present invention can be based on any of the above structures and their variations described in the literature. c-Myc nucleic acid inhibitor molecules also include micro-RNA (miRNA) and short hairpin RNA (shRNA) molecules, such as those described in U.S. Published Application No. 2009/00991 15.

[039] Typically, many of the nucleotide subunits of the nucleic acid inhibitor molecules are modified to improve various characteristics of the molecule such as resistance to nucleases and lowered immunogenicity (see, e.g., Bramsen et al. (2009), Nucleic Acids Res., 37, 2867-2881 ). In certain embodiments, every nucleotide of a nucleic acid inhibitor molecule is modified. In certain embodiments, substantially all of the nucleotides of a nucleic acid inhibitor molecule are modified. In certain embodiments, more than half of the nucleotides of a nucleic acid inhibitor molecule are modified. In certain embodiments, less than half of the nucleotides of a nucleic acid inhibitor molecule are modified. In certain embodiments, none of the nucleotides of a nucleic acid inhibitor molecule are modified. Modifications can occur in groups on the oligonucleotide chain or different modified nucleotides can be interspersed.

[040] Many nucleotide modifications have been used in the oligonucleotide field. Modifications can be made on any part of the nucleotide, including the sugar moiety, the phosphoester linkage, and the nucleobase. Typical examples of nucleotide modification include, but are not limited to, 2'-fluoro-, 2'-OMethyl-, 5-methylcytosine. In certain embodiments, the nucleic acid inhibitor molecules of the invention include one or more deoxyribonucleotides. Typically, the nucleic acid inhibitor molecules contain 5 or fewer deoxyribonucleotides. In certain embodiments, the nucleic acid inhibitor molecules of the invention include one or more ribonucleotides.

[041 ] In certain embodiments, the ring structure of the sugar moiety is modified, including, but not limited to, Locked Nucleic Acids ("LNA") (see, e.g., Koshkin et al. (1998), Tetrahedron 54, 3607-3630) and Unlocked Nucleic Acids ("UNA") (see, e.g., Snead et al. (2013), Molecular Therapy - Nucleic Acids, 2, e103 (doi: 10.1038/mtna.2013.36)).

[042] The 5' end of the oligonucleotide is an oft modified position. In certain embodiments, a hydroxyl group is attached to the 5' end of the oligonucleotide of a nucleic acid inhibitor molecule of the invention. In certain embodiments, a phosphate group is attached to the 5' end of the oligonucleotide of a nucleic acid inhibitor molecule of the invention. In certain embodiments, the 5' end is attached to chemical moiety that mimics the electrostatic and steric properties of a phosphate group ("phosphate mimic") (see, e.g., Prakash et al. (2015), Nucleic Acids Res., advance access published March 9, 2015 (doi: 10.1093/narlgkv162). Many phosphate mimics have been developed that can be attached to the 5' end (see, e.g., U.S. Patent No. 8,927,513). Other modifications have been developed for the 5' end of oligonucleotides (see, e.g., WO 201 1/133871 ).

c-Myc nucleic acid inhibitor molecules

[043] As disclosed herein, a c-Myc nucleic acid inhibitor molecule can be combined with selective CDK7 inhibitor for treating c-Myc-associated disease or disorders, such as cancer. We have shown that these combinations can produce synergetic effects as compared to the administration of each agent individually.

[044] Specific c-Myc nucleic acid inhibitor molecules are known, as disclosed for example in U.S. Published Application Nos. 2014/0107178 and 2009/00991 15, which are incorporated by reference for their disclosure of these c-Myc nucleic acid inhibitor molecules.

[045] In certain embodiments, the c-Myc nucleic acid inhibitor molecule is a molecule disclosed in U.S. Published Application No. 2014/0107178.

[046] In certain embodiments, the c-Myc nucleic acid inhibitor molecules of the invention are dsRNAi inhibitor molecules where the double-stranded region of the molecule is between 15 and 40 nucleotides in length. In certain of those embodiments, the double- stranded region is between 20 and 30 nucleotides in length. In certain of those embodiments, the double-stranded region is 21 , 22, 23, 24, 25, or 26 nucleotides in length.

[047] In certain embodiments, the c-Myc nucleic acid inhibitor molecules of the invention are dsRNAi inhibitor molecules where the sense strand is between 18 and 66 nucleotides in length. In certain of those embodiments, the sense strand is between 25 and 45 nucleotides in length. In certain embodiments, the sense strand is between 30 and 40 nucleotides in length. In certain embodiments, the sense strand is 37, 38, 39, or 40 nucleotides in length. In certain embodiments, the sense strand is between 25 and 30 nucleotides in length. In certain of those embodiments, the sense strand is 25, 26, or 27 nucleotides in length.

[048] In certain embodiments, the c-Myc nucleic acid inhibitor molecules of the invention are dsRNAi inhibitor molecules where the antisense strand is between 18 and 66 nucleotides in length. Typically, the antisense strand comprises a sequence that is sufficiently complementary to a sequence in the target gene mRNA to direct the effect of the nucleic acid inhibitor molecule to the target gene. In certain embodiments, the antisense strand comprises a sequence that is fully complementary with a sequence contained in the target gene mRNA where the fully complementary sequence is between 18 and 40 nucleotides long. In certain of those embodiments, the antisense strand is between 20 and 50 nucleotides in length. In certain embodiments, the antisense strand is between 20 and 30 nucleotides in length. In certain embodiments, the antisense strand is 21 , 22, 23, 24, 25, 26, 27, or 28 nucleotides in length. In certain embodiments, the antisense strand is between 35 and 40 nucleotides in length. In certain of those embodiments, the antisense strand is 36, 37, 38, or 39 nucleotides in length.

[049] In certain embodiments, the c-Myc nucleic acid inhibitor molecules of the invention are single-stranded nucleic acid molecules. [050] In certain embodiments, the antisense strand of the c-Myc nucleic acid inhibitor molecule comprises the sequence of SEQ ID NO: 2. In certain embodiments, the antisense strand of the c-Myc nucleic acid inhibitor molecule consists of the sequence of SEQ ID NO: 2. In certain embodiments, the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule and the sense strand comprises the sequence of SEQ ID NO: 1. In certain embodiments, the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule and the sense strand consists of the sequence of SEQ ID NO: 1 . In certain embodiments, the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecules and the sense strand comprises the sequence of SEQ ID NO: 1 and the antisense strand comprises the sequence of SEQ ID NO: 2. In certain embodiments, the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule where the sense strand consists of the sequence of SEQ ID NO: 2 and the antisense strand consists of the sequence of SEQ ID NO: 2. See Figure 5.

[051 ] In other embodiments, the antisense strand of the c-Myc nucleic acid inhibitor molecule comprises the sequence of SEQ ID NO: 4. In certain embodiments, the antisense strand of the c-Myc nucleic acid inhibitor molecule consists of the sequence of SEQ ID NO: 4. In certain embodiments, the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule and the sense strand comprises the sequence of SEQ ID NO: 3. In certain embodiments, the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule and the sense strand consists of the sequence of SEQ ID NO: 3. In certain embodiments, the c- Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule and the sense strand comprises the sequence of SEQ ID NO: 3 and the antisense strand comprises the sequence of SEQ ID NO: 4. In certain embodiments, the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule and the sense strand consists of the sequence of SEQ ID NO: 3 and the antisense strand consists of the sequence of SEQ ID NO: 4. See Figure 6.

[052] The level or activity of a c-Myc RNA can be determined by a suitable method now known in the art or that is later developed. It can be appreciated that the method used to measure a target RNA and/or the "expression" of a target gene can depend upon the nature of the target gene and its encoded RNA. For example, where the target c-Myc RNA sequence encodes a protein, the term "expression" can refer to a protein or the c-Myc RNA/transcript derived from the c-Myc gene (either genomic or of exogenous origin). In such instances the expression of the target c-Myc RNA can be determined by measuring the amount of c-Myc RNA/transcript directly or by measuring the amount of c-Myc protein. Protein can be measured in protein assays such as by staining or immunoblotting or, if the protein catalyzes a reaction that can be measured, by measuring reaction rates. All such methods are known in the art and can be used. Where target c-Myc RNA levels are to be measured, art-recognized methods for detecting RNA levels can be used (e.g., RT-PCR, Northern Blotting, etc.). In targeting c-Myc RNAs, it is also anticipated that measurement of the efficacy of the nucleic acid inhibitor molecule in reducing levels of c-Myc RNA or protein in a subject, tissue, in cells, either in vitro or in vivo, or in cell extracts can also be used to determine the extent of reduction of c-Myc -associated phenotypes (e.g., disease or disorders, e.g., cancer or tumor formation, growth, metastasis, spread, etc.). The above measurements can be made on cells, cell extracts, tissues, tissue extracts or other suitable source material.

CDK7 Inhibitors

[053] As herein described, the term "CDK7" refers to the cyclin dependent kinase 7 enzyme. Cyclin dependent kinases are a family of protein kinases integrally involved in regulating the cell cycle. CDK1 plays an important role in determining mitotic progression. In higher eukaryotes, CDK2 is important for DNA replication, while CDK4 and CDK6 help control cell cycle entry. Other CDKs, such as CDK7, CDK9, and CDK9 regulate transcription. CDK5 has post-mitotic function in specialized tissue.

[054] Given their role in regulating the cell cycle, aberrant expression of CDKs has been linked to numerous cancers, and agents that target CDK activity have been an attractive target for development of anti-tumor therapies.

[055] Certain CDK inhibitors target more than one CDK, such as CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK8, CDK9, and CDK1 1 . In certain instances, these nonspecific CDK inhibitors may also target CDK7, but to a lesser extent than at least one of the CDKs targeted by the non-specific CDK inhibitor. Recently, however, selective CDK7 inhibitors have been identified. These selective CDK7 inhibitors target only CDK7 or target CDK7 with a higher inhibitory activity than any other CDK.

[056] In certain embodiments, the selective CDK7 inhibitor is selected from those disclosed in WO 2015/058163, WO 2015/154022, WO 2015/154038, WO 2015/154039, WO 2015/058140, and WO 2014/063068. [057] Selective CDK7 inhibitors include, but are not limited to, THZ1 (see, e.g., Kwiatkowski et al. (2014); Nature 51 1 (751 1 ): 616-620), BS-181 (see, e.g., AN et al. (2009); Cancer Res 69(15): 6208-6215), and SY-351 (Syros Pharmaceuticals). In one embodiment, the CDK7 inhibitor is THZ1 .

[058] In one embodiment, the selective CDK7 inhibitor is a covalent inhibitor, such as THZ1 .

Pharmaceutical Compositions

[059] The present disclosure provides pharmaceutical compositions comprising a c- Myc nucleic acid inhibitor molecule and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprising the c-Myc nucleic acid inhibitor molecule and the pharmaceutically acceptable excipient further comprises a selective CDK7 inhibitor.

[060] The pharmaceutically acceptable excipients useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15^th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, including vaccines, and additional pharmaceutical agents. Suitable pharmaceutical excipients include, for example, 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. In general, the nature of the excipient will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, buffers, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid excipients can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, a surface active agent, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In certain embodiments, the pharmaceutically acceptable excipient is non-naturally occurring. [061 ] The pharmaceutical composition according to certain embodiments disclosed herein, may comprise at least one ingredient, which may belong to the same or different categories of excipients, including at least one disintegrant, at least one diluent, and/or at least one binder.

[062] Typical non-limiting examples of the at least one disintegrant that may be added to the pharmaceutical composition according to embodiments disclosed herein, include, but are not limited to, povidone, crospovidone, carboxymethylcellulose, methylcellulose, alginic acid, croscarmellose sodium, sodium starch glycolate, starch, formaldehyde-casein, and combinations thereof.

[063] Typical non-limiting examples of the at least one diluents that may be added to the pharmaceutical composition according to embodiments disclosed herein, include, but are not limited to, maltose, maltodextrin, lactose, fructose, dextrin, microcrystalline cellulose, pregelatinized starch, sorbitol, sucrose, silicified microcrystalline cellulose, powdered cellulose, dextrates, mannitol, calcium phosphate, and combinations thereof.

[064] Typical non-limiting examples of the at least one binder that may be added to the pharmaceutical composition according to embodiments disclosed herein, include, but are not limited to, acacia, dextrin, starch, povidone, carboxymethylcellulose, guar gum, glucose, hydroxypropyl methylcellulose, methylcellulose, polymethacrylates, maltodextrin, hydroxyethyl cellulose, and combinations thereof.

[065] Suitable preparation forms for the pharmaceutical compositions disclosed herein include, for example, tablets, capsules, soft capsules, granules, powders, suspensions, aerosols, emulsions, microemulsions, nanoemulsions, unit dosage forms, rings, films, suppositories, solutions, creams, syrups, transdermal patches, ointments, or gels.

[066] The c-Myc nucleic acid inhibitor molecule may be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, including, for example, liposomes and lipids such as those disclosed in U.S. Patent Nos. 6,815,432, 6,586,410, 6,858,225, 7,81 1 ,602, 7,244,448 and 8, 158,601 ; polymeric materials such as those disclosed in U.S. Patent Nos. 6,835,393, 7,374,778, 7,737, 108, 7,718, 193, 8, 137,695 and U.S. Published Patent Application Nos. 201 1/0143434, 201 1/0129921 , 201 1/0123636, 201 1/0143435, 201 1/0142951 , 2012/0021514, 201 1/0281934, 201 1/0286957 and 2008/0152661 ; capsids, capsoids, or receptor targeted molecules for assisting in uptake, distribution or absorption.

[067] In certain embodiments, the nucleic acid inhibitor molecules are formulated in a lipid nanoparticle. Lipid-nucleic acid nanoparticles typically form spontaneously upon mixing lipids with nucleic acid to form a complex. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be optionally extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). To prepare a lipid nanoparticle for therapeutic use, it may desirable to remove solvent (e.g., ethanol) used to form the nanoparticle and/or exchange buffer, which can be accomplished by, for example, dialysis or tangential flow filtration. Methods of making lipid nanoparticles containing nucleic acid interference molecules are known in the art, as disclosed, for example in U.S. Published Patent Application Nos. 2015/0374842 and 2014/0107178.

[068] In certain embodiments, the LNP comprises a liposome comprising a cationic liposome and a pegylated lipid. The LNP can further comprise one or more envelope lipids, such as a cationic lipid, a structural lipid, a sterol, a pegylated lipid, or mixtures thereof.

[069] Cationic lipids for use in LNPs are known in the art, as discussed for example in U.S. Published Patent Application Nos. 2015/0374842 and 2014/0107178. Typically, the cationic lipid is a lipid having a net positive charge at physiological pH. In certain embodiments, the cationic liposome is DODMA, DOTMA, DL-048, or DL-103. In certain embodiments the structural lipid is DSPC, DPPC or DOPC. In certain embodiments, the sterol is cholesterol. In certain embodiments, the pegylated lipid is DMPE-PEG, DSPE- PEG, DSG-PEG, DMPE-PEG2K, DSPE-PEG2K, DSG-PEG2K, DSPE-MPEG, or DSG- MPEG. In one embodiment, the cationic lipid is DL-048, the pegylated lipid is DSG-MPEG and the one or more envelope lipids are DL-103, DSPC, cholesterol, and DSPE-MPEG.

[070] These pharmaceutical compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 1 1 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

[071 ] In certain embodiments, the pharmaceutical compositions described herein are for use in treating a c-Myc-catenin-associated disease or disorder. In certain embodiments, the pharmaceutical composition for use in treating a c-Myc-catenin- associated disease or disorder comprises a c-Myc nucleic acid inhibitor molecule, wherein the composition is administered in combination with a selective CDK7 inhibitor (e.g., THZ1 ).

Methods of Administration/Treatment

[072] Typically, the c-Myc nucleic acid inhibitor molecules of the invention are administered intravenously or subcutaneously. However, the pharmaceutical compositions disclosed herein may also be administered by any method known in the art, including, for example, oral, buccal, sublingual, rectal, vaginal, intraurethral, topical, intraocular, intranasal, and/or intraauricular, which administration may include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salves, ointments, or the like. Administration may also be via injection, for example, intraperitoneally, intramuscularly, intradermally, intraorbitally, intracapsularly, intraspinally, intrasternally, or the like.

[073] The therapeutically effective amount of the compounds disclosed herein may depend on the route of administration and the physical characteristics of the patient, such as general state, weight, diet, and other medications. As used herein, a therapeutically effective amount means an amount of compound or compounds effective to prevent, alleviate or ameliorate disease or condition symptoms of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art and generally range from about 0.5 mg to about 3000 mg of the small molecule agent or agents per dose per patient.

[074] In one aspect, the pharmaceutical compositions disclosed herein may be useful for the treatment or prevention of symptoms related to a c-Myc-catenin-associated disease or disorder. One embodiment is directed to a method of treating a c-Myc-catenin- associated disease or disorder, comprising administering to a subject a therapeutically effective amount of a c-Myc nucleic acid inhibitor molecule and a therapeutically effective amount of a selective CDK7 inhibitor, such as THZ1 . [075] Typically, the c-Myc nucleic acid inhibitor molecule is administered separately from, and on a different schedule than, a small molecule therapeutic that is in combination with the nucleic acid inhibitor molecule. For example, when used as a single agent, THZ1 is currently prescribed as a daily oral dose (typically about 1 -2 mg/day). The c-Myc nucleic acid inhibitor molecule, on the other hand, is likely to be administered through an intravenous or subcutaneous route with doses given once a week, once each two weeks, once a month, once every three months, twice a year, etc. The subject may already be taking the small molecule therapeutic at the initiation of the administration of the c-Myc nucleic acid inhibitor molecule. In other embodiments, the subject may begin administration of both the small molecule therapeutic and the c-Myc nucleic acid inhibitor molecule at about the same time. In other embodiments, the subject may begin taking the small molecule therapeutic after the initiation of administration of the c-Myc nucleic acid inhibitor molecule.

[076] In certain embodiments for the methods of treatment disclosed herein, one pharmaceutical composition may comprise the c-Myc nucleic acid inhibitor molecule and a separate pharmaceutical composition may comprise the selective CDK7 inhibitor, such as THZ1 .

[077] In other embodiments, the c-Myc nucleic acid inhibitor molecule may be administered simultaneously with the selective CDK7 inhibitor, such as THZ1 .

[078] Accordingly, in certain embodiments for the methods of treatment disclosed herein, a single pharmaceutical composition may comprise both the c-Myc nucleic acid inhibitor molecule and the selective CDK7 inhibitor, such as THZ1 . Thus, in one embodiment of the treatment methods disclosed herein, a single pharmaceutical composition is administered to the subject, wherein the single pharmaceutical composition comprises both the c-Myc nucleic acid inhibitor molecule and the selective CDK7 inhibitor, such as THZ1 .

[079] In certain embodiments, the c-Myc nucleic acid inhibitor molecule is administered at a dosage of 20 micrograms to 10 milligrams per kilogram body weight of the recipient per day, 100 micrograms to 5 milligrams per kilogram, 0.25 milligrams to 2.0 milligrams per kilogram, or 0.5 to 2.0 milligrams per kilogram.

[080] In certain embodiments, the c-Myc nucleic acid inhibitor molecule is administered once daily, once weekly, once monthly, once every two months, once a quarter, twice a year, or once yearly. In certain embodiments, the c-Myc nucleic acid inhibitor molecule is administered once or twice every 2, 3, 4, 5, 6, or 7 days. The compositions (containing both agents or a single, individual agent) can be administered once monthly, once weekly, once daily (QD), once every other day, or divided into multiple monthly, weekly, or daily doses, such as twice daily, three times a day or once every two weeks. In certain embodiments, the compositions can be administered once a day for two, three, four, five, six, or at least seven days. Although the agents can be administered simultaneously, typically each agent will be administered on a different schedule, particularly if the agents are administered via different routes.

[081 ] Alternatively, continuous intravenous infusion sufficient to maintain therapeutically effective concentrations in the blood are contemplated. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age or weight of the subject, and other diseases present.

[082] Treatment of a subject with a therapeutically effective amount of an agent can include a single treatment or, preferably, can include a series of treatments. In certain embodiments, the treatment schedule includes a first loading dosage or phase, which is typically a higher dosage or frequency, followed by a maintenance dosage or phase, which is typically a lower dosage or frequency than the loading dosage/phase. Typically, the treatment continues until disease progression or unacceptable toxicity occurs.

[083] In certain embodiments, the c-Myc nucleic acid inhibitor molecules can be inserted into expression constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art. Expression constructs can be delivered to a subject by, for example, inhalation, orally, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91 , 3054-3057).

[084] The expression constructs may be constructs suitable for use in the appropriate expression system and include, but are not limited to retroviral vectors, linear expression cassettes, plasm ids and viral or virally-derived vectors, as known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or H1 RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct, e.g., Tuschl (2002, Nature Biotechnol 20: 500-505).

[085] One aspect is directed to methods of treating a c-Myc-catenin-associated disease or disorder, comprising administering to a subject (preferably a human) a therapeutically effective amount of a c-Myc nucleic acid inhibitor molecule and a therapeutically effective amount of a selective CDK7 inhibitor, such as THZ1 .

[086] In one embodiment, the c-Myc nucleic acid inhibitor molecule is a double stranded nucleic acid. In certain of those embodiments, the sense strand comprises or consists of the sequence of SEQ ID NO: 1 and the antisense strand comprises of consists of the sequence of SEQ ID NO: 2. In certain of those embodiments, the sense strand comprises or consists of the sequence of SEQ ID NO: 3 and the antisense strand comprises of consists of the sequence of SEQ ID NO: 4. In one embodiment the c-Myc nucleic acid inhibitor molecule is formulated with a lipid nanoparticle. In one embodiment, the c-Myc nucleic acid inhibitor molecule is administered intravenously.

[087] In one embodiment, the method of treatment comprises administering to a subject (preferably a human) a therapeutically effective amount of a c-Myc nucleic acid inhibitor molecule and a therapeutically effective amount of a selective CDK7 inhibitor. In one embodiment, the selective CDK7 inhibitor is THZ1 . In one embodiment, THZ1 is administered orally. In one embodiment, trametinib is administered at a dosage of about 0.5 to 5 mg daily or every other day.

[088] In one embodiment, the selective CDK7 inhibitor is THZ1 , which is administered orally, and the c-Myc nucleic acid inhibitor molecule is a double stranded nucleic acid, wherein the double-stranded region of the molecule is between 18 and 40 nucleotides in length, including, for example, a double stranded nucleic acid having a sense strand and an antisense strand, wherein the sense strand comprises or consists of the sequence of SEQ ID NO: 3 and the antisense strand comprises of consists of the sequence of SEQ ID NO: 4. The c-Myc nucleic acid inhibitor molecule can be formulated with a lipid nanoparticle and administered intravenously. EXAMPLES

[089] EXAMPLE 1 : MYC2 Construct

[090] A nucleic acid inhibitor molecule that targets the c-Myc gene was constructed ("MYC2"). MYC2 has a 26 base pair passenger strand and a 38 base pair guide strand that form a duplex region consisting of 26 base pairs. Figure 5. The 5' end of the passenger strand consists of a 10-base pair, single stranded overhang, and the 3' end of the guide strand consists of a two-base pair single-stranded, overhang. Figure 5. The MYC2 construct was formulated in EnCore LNP.

[091] EXAMPLE 2: In Vitro Potency and Cell Viability Assays

[092] Human cell lines Hep3B and HepG2 were obtained from ATCC (Manassas, VA) and grown in RPMI/DMEM medium supplemented with 10% FBS.

[093] Hep3B and HepG2 cells were transfected using Lipofectamine RNAiMAX (Life Technologies, Grand Island, NY). Before the transfections, MYC2 was incubated at room temperature for 20 minutes with RNAiMAX in OptiMEM (Life Technologies). Reverse transfections at various concentrations (ranging from 0.064 to 1000pM) were done in a 96- well plates containing 20 000 cells/well. 18-hrs after transfections, THZ1 or DMSO was added and cells were harvested 24hr post-transfections. Total RNA was isolated (SV96 Total RNA Isolation System, Promega, Madison, Wl) and cDNA was reverse transcribed (Superscript II, Life Technologies). RNA knockdown was quantified by real-time qPCR on an Applied Biosystems 7900 HT (Carlsbad, CA). In addition to qPCR for MYC, qPCR was performed for two housekeeping genes (HPRT1 and SRSF9) for normalization. We observed that Hep3B or HepG2 cells at low doses of THZ1 or MYC2 alone show marginal MYC mRNA. However, when two agents were combined, a more robust MYC mRNA knockdown was achieved (Fig. 1).

[094] Next, HepG2 cells were transfected with MYC2 in 96-well. After 24 hrs, the cells were treated with various concentrations of the THZ1 (ranging from 1 nM to 10 μΜ). DMSO solvent without drug served as a control. After 48 hr of incubation, cells were analyzed for cell viability using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, Wl). As a single agent, MYC2 did not significantly affect cellular viability (ICso > 1000pM) while THZ1 was relatively potent (ICso >0.01 uM). We observed significantly higher cellular cytotoxicity when THZ1 and MYC2 were combined at very low doses (Fig.2). These results demonstrate in vitro synergistic activities when THZ1 and MYC2 were combined.

[095] EXAMPLE 3: Tumor Studies

[096] 6-8 week old Hsd:Athymic ude-Foxn1^nu mice were injected subcutaneously with Hep3B (5e6 cells+ matrigel) under the right shoulder. Tumor volume was measured twice a week to monitor tumor growth/suppression. Dosing was initiated when the tumors reached 200-250mm³. For tumor growth inhibition studies, animals were randomized and assigned to different cohorts and subjected to dosing cycles. MYC2 or LNP was given intravenously via lateral tail vein at a total volume of 10 ml/kg. THZ1 treatment was given intraperitoneal at a volume of 10 ml/kg.

[097] As a single agent, MYC2 dosed at qdX3, 2 mg/kg caused marginal tumor growth inhibition of 38% relative to vehicle-treated animals. Likewise, THZ1 alone dosed at 7.5 mg/kg twice daily inhibited the tumor growth by only 36%. Interestingly, when MYC2 and THZ1 were combined at similar doses, we observed a more robust anti-tumor activity (73%), suggesting additive/synergistic effects (Fig 3). To mechanistically understand the combination effects, we evaluated MYC expression levels in the tumors 24hr after the last dose. We observed 25% MYC mRNA knockdown in the tumors of animals treated with the single agent compare to 58% loss of MYC after the combination (Fig 4).

[098] Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure.

Claims

WHAT IS CLAIMED IS:

1 . A method of treating a c-Myc-associated cancer in a subject, comprising administering to the subject: a therapeutically effective amount of a c-Myc nucleic acid inhibitor molecule; and a therapeutically effective amount of a selective CDK7 inhibitor.

2. A pharmaceutical composition comprising a c-Myc nucleic acid inhibitor molecule for use in treating a c-Myc- associated cancer, wherein the composition is administered in combination with a selective CDK7 inhibitor.

3. The method of claim 1 or the composition of claim, wherein the selective CDK7 inhibitor is THZ1 .

4. The method of any of the preceding claims, wherein the subject is a human.

5. The method or composition of any one of the preceding claims, wherein the c-Myc nucleic acid inhibitor molecule is a short interfering RNA (siRNA).

6. The method or composition of any one of the preceding claims, wherein the c-Myc nucleic acid inhibitor molecule is a double stranded nucleic acid, wherein the double-stranded region of the molecule is between 18 and 40 nucleotides in length.

7. The method or composition of any one of the preceding claims, wherein the c-Myc nucleic acid inhibitor molecule is a double stranded nucleic acid comprising a sense and an antisense strand and a duplex region of at least 18 base pairs, wherein the sense strand is 25-34 nucleotides in length and the antisense strand is 26-38 nucleotides in length and comprises 1 -5 single-stranded nucleotides at its 3' terminus.

8. The method or composition of claim 8, wherein the sense strand comprises the sequence of SEQ ID NO: 1 .

9. The method or composition of claim 8 or 9, wherein the antisense strand comprises the sequence of SEQ ID NO: 2.

10. The method or composition of claim 8, wherein the sense strand of the c-Myc nucleic acid inhibitor molecule comprises the sequence of SEQ ID NO: 3.

1 1 . The method or composition of claim 8 or 1 1 , wherein the antisense strand of the c-Myc nucleic acid inhibitor molecule comprises the sequence of SEQ ID NO: 4.

12. The method or composition of any of the preceding claims, wherein the c-Myc nucleic acid inhibitor molecule is formulated with a lipid nanoparticle.

13. The method of claim 13, wherein the lipid nanoparticle comprises a cationic lipid and a pegylated lipid.

14. A method of treating a c-Myc-associated cancer in a subject, comprising administering to the subject: a therapeutically effective amount of a c-Myc nucleic acid inhibitor molecule; and a therapeutically effective amount of a selective CDK7 inhibitor; wherein the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule.

15. A method of treating a c-Myc-associated cancer in a subject, comprising administering to the subject: a therapeutically effective amount of a c-Myc nucleic acid inhibitor molecule; and a therapeutically effective amount of a selective CDK7 inhibitor; wherein the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule with a sense strand comprising SEQ ID NO: 3 and an antisense strand comprising SEQ ID NO: 4.

16. A method of treating a c-Myc-associated cancer in a subject, comprising administering to the subject: a therapeutically effective amount of a c-Myc nucleic acid inhibitor molecule; and a therapeutically effective amount of a selective CDK7 inhibitor; wherein the c-Myc nucleic acid inhibitor molecule is a dsRNAi inhibitor molecule with a sense strand consisting of SEQ ID NO: 3 and an antisense strand consisting of SEQ ID NO: 4.