WO2015009851A1 - Methods of treating cancer in subjects afflicted with metabolic dysfunction - Google Patents

Methods of treating cancer in subjects afflicted with metabolic dysfunction Download PDF

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WO2015009851A1
WO2015009851A1 PCT/US2014/046887 US2014046887W WO2015009851A1 WO 2015009851 A1 WO2015009851 A1 WO 2015009851A1 US 2014046887 W US2014046887 W US 2014046887W WO 2015009851 A1 WO2015009851 A1 WO 2015009851A1
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compound
inhibiting
ras
signaling
oncogenic
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PCT/US2014/046887
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French (fr)
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Susumu HIRABAYASHI
Thomas J. Baranski
Ross L. Cagan
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Icahn School Of Medicine At Mount Sinai
Washington University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/06—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems

Definitions

  • Metabolic disease impacts body homeostasis, leading to a constella ion of symptoms including cardiovascular disease, blindness, neuropathy, and nephropathy.
  • Epidemiological studies have provided evidence for an association between cancer and metabolic diseases such as diabetes and obesity (Barone et al . , 2008; Calle et al , , 2003; Coughlin et al . 2004; Inoue et al . , 2006).
  • the present invention provides methods of treating a subject afflicted with a cancer and a metabolic dysfunction, which comprises administering to the subject
  • compositions comprising
  • PI3K insulin receptor
  • PI3K phosphatidylinositide 3-kinase
  • the present invention provides combinations for treating a subject afflicted with a cancer and a metabolic dysfunction comprising (a) (I) a compound which is capable of inhibiting a glycos ide hydrol se,
  • the present invention provides pharmaceutical compositions for treating a subject afflicted with a cancer and a metabolic dysfunction comprising
  • PI3K phosphatidylinositide 3 -kinase
  • A-I Developmental stage matched third instar larvae fed control diet or HDS,
  • a and B lacZ control in control diet (day 7 AEL) and HDS ⁇ day 11 AEL)
  • C and D csk ⁇ -' ⁇ in control diet (day 7 AEL) and HDS
  • A-C Cleaved Caspase-3 staining (red) of rasl 012'2 ; csk '2 ' (A and B) or inR CA , rasl al2v ; csk- ' (C! eye discs raised on indicated diets.
  • D-F TUHEL assay (red) was used to label apoptotic cell death of rasl al2v ; csk-''- (D and E) or inR c ", rasl al2v ; csk " '” eye discs raised on indicated diets.
  • HDS diverts Ras/Src-activated cells into aggressive tumors leading to larval lethality. A majority (77.5%) of rasl al2v ; esk ⁇ f ⁇ animals fed HDS spent extra days as wandering larvae with multiple secondary tumors and failed to develop into pupal stage.
  • B Developmental time course to pupariation of animals with all five genotypes fed control diet or HDS. Results are shown as mean ⁇ SEM.
  • C Quantitation of total eye disc area, GFP-positive area and GFP- negative area. Results are shown as mean ⁇ SEM of individual eye discs .
  • A-C High dietary sugar but not high fat enhanced tumor growth.
  • rasl G12v csk ⁇ -' ⁇ animals raised on (A) 2 M glucose diet, (B) Banana and (C) calorie-matched high fat diet.
  • D Laminin A staining (red) of rasl al2v ; csk-''- eye discs raised on HDS at day 9 AEL.
  • E and F Src activity levels play an important role in secondary Ras/Src tumor formation.
  • rasl al2v csk hyBa/hyBO animals fed control diet or HDS. Arrowhead indicates secondary tumor.
  • G Quantitation of the observed phenotypes.
  • the present invention provides methods of treating a subject afflicted with a cancer and a metabolic dysfunction, which comprises adminis ering to the subject
  • PI3K phosphatidylinositide 3-kinase
  • each compound administered to the subject is, independently, an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer, an RNA aptamer, a polypeptide, an antibody, an antisense oligonucleotide, an interfering RNA (RNAi ⁇ molecule, or a ribozyme.
  • a compound that is capable of inhibiting a glycoside hydrolase is administered to the subject.
  • the compound which is capable of inhibiting a glycoside hydrolase is an organic compound having a molecular weight less than 1000 Daltons .
  • the compound which is capable of inhibiting a glycoside hydrolase inhibits the catalytic activity of the glycoside hydrolase .
  • the compound which is capable of inhibiting a glycoside hydrolase inhibits the ability of the glycoside hydrolase to bind a carbohydrate.
  • the glycoside hydrolase is a glucosidase.
  • the glucosidase is a-glucosidase and the compound which is capable of inhibiting the glycoside hydrolase is an ⁇ -glucosidase inhibitor.
  • the a-glucosidase inhibitor is acarbose, or a pharmaceutically acceptable salt or ester thereof.
  • the compound which is capable of inhibiting a glycoside hydrolase is an antisense oligonucleotide, an R Ai molecule, or a ribozyme that targets ⁇ -glucosidase-encoding mR A and reduces -glucosidase expression.
  • the compound which is capable of inhibiting a glycoside hydrolase is a compound approved for use in treating type 2 diabetes mellitus.
  • a compound that is capable of inhibiting the canonical Wg/Wnt pathway signaling is administered to the subject.
  • the canonical Wg/Wnt pathway signaling is canonical Wg/Wnt undead pathway signaling.
  • the compound that is capable of inhibiting canonical Wg/Wnt pathway signaling is a Wg/Wnt inhibitor.
  • the compound that is capable of inhibiting the canonical Wg/Wnt pathway signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets Wg/Wnt-encoding mRNA and reduces Wg/Wnt expression.
  • the compound that is capable of inhibiting the canonical Wg W pathway signaling is pyrvinium, or a pharmaceutically acceptable salt or ester thereof.
  • a compound that is capable of inhibiting Ras oncogenic pathway signaling is administered to the subject.
  • the compound that is capable of inhibiting Ras oncogenic pathway signaling is a Ras inhibitor.
  • the compound that is capable of inhibiting Ras oncogenic pathway signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets Ras-encoding mRNA and reduces Ras expression.
  • a compound that is capable of inhibiting Src oncogenic pathway signaling is administered to the subject.
  • the compound that is capable of inhibiting Src oncogenic pathway signaling is a Src inhibitor.
  • the compound that is capable of inhibiting Src oncogenic pathway signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets Src-encoding mRNA and reduces Src expression.
  • a compound that is capable of inhibiting Ras/Src oncogenic pathway signaling is administered to the subject.
  • the compound that is capable of inhibiting Ras/Src oncogenic pathway signaling is AD81, or a pharmaceutically acceptable salt or ester thereof.
  • a compound that inhibits Tor oncogenic pathway signaling is administered to the subject.
  • the compound that is capable of inhibiting Tor oncogenic pathway signaling is a Tor inhibitor.
  • the inhibitor of Tor oncogenic pathway signaling is rapamycin.
  • the inhibitor of Tor oncogenic pathway signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets Tor-encoding mRNA and reduces Tor expression.
  • a compound that is capable of inhibiting PI3K metabolic signaling is administered to the subject.
  • the compound that inhibits PI3K metabolic signaling is a PI3K inhibitor.
  • the PI3K inhibitor is wortmannin, or a pharmaceutically acceptable salt or ester thereof.
  • the compound that inhibits PI3K metabolic signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets PI3K-encoding mRNA and reduces PI3K expression .
  • a compound that is capable of inhibiting InR metabolic signaling is administered to the subject.
  • the compound that is capable of inhibiting InR metabolic signaling is an InR inhibitor.
  • the compound that is capable of inhibiting InR metabolic signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets InR-encoding mRNA and reduces InR expression.
  • a compound that is capable of inhibiting InR PI3K metabolic signaling is administered to the subject.
  • a compound that is capable of inhibiting Jnk metabolic signaling is administered to the subject.
  • the compound that is capable of inhibiting Jnk metabolic signaling is a Jnk inhibitor.
  • the compound that is capable of inhibiting Jnk metabolic signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets Jnk-encoding mRNA and reduces Jnk expression.
  • Some embodiments comprise administering to the subject an a- glucosidase inhibitor and a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling.
  • the a-glucosidase inhibitor is acarbose and the compound which is capable of inhibiting canonical Wg/Wnt pathway signaling is pyrviniuin.
  • Some embodiments comprise administering to the subject an a- glucosidase inhibitor and a compound which is capable of inhibiting Ras/Src oncogenic pathway signaling.
  • the ⁇ -glucosidase inhibitor is acarbose and the compound which is capable of inhibiting Ras/Src oncogenic pathway signaling is AD81.
  • Some embodiments comprise administering to the subject a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling and a compound which is capable of inhibiting Ras/Src oncogenic pathway signaling.
  • the compound which is capable of inhibiting canonical Wg/Wnt pathway signaling is pyrvinium and the compound which is capable of inhibiting Ras/Src oncogenic pathway signaling is AD81.
  • Some embodiments comprise administering to the subject an a- glucosidase inhibitor, a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, and a compound which is capable of inhibiting Ras/Src oncogenic pathway signaling.
  • the ⁇ -glucosidase inhibitor is acarbose
  • the compound which is capable of inhibiting canonical Wg/Wnt pathway signaling is pyrvinium
  • the compound which is capable of inhibiting Ras/Src oncogenic pathway signaling is AD81.
  • the subject has an increased level of circulating insulin compared to a subject not afflicted with the metabolic dysfunction.
  • the metabolic dysfunction is a metabolism-related disease.
  • the metabolism-related disease is obesity. In some embodiments, the metabolism-related disease is diabetes. In some embodiments, the diabetes is type-2 diabetes.
  • the metabolic dysfunction comprises hyperglycemia, hyperinsulinemia, or insulin resistance.
  • the cancer is breast cancer, hepatocellular carcinoma, colorectal cancer, or pancreatic cancer.
  • the cancer is breast cancer.
  • the breast cancer is progesterone-receptor- negative breast cancer.
  • the cancer is pancreatic cancer.
  • the pancreatic cancer is pancreatic duc al adenocarcinoma .
  • the subject is a mammalian subject.
  • the mammalian subject is a human subject.
  • treating the subjec comprises reducing the progression of cancer in the subject.
  • treating the subject comprises reducing the progression of a tumor in the subject.
  • treating the subjec comprises prolonging survival of the subject.
  • cells of the cancer expre s a higher amount of I R than cells from normal tissue of the same type.
  • cells of the cancer have a higher amount of InR signaling than cells from normal tissue of the same type.
  • cells of the cancer express a higher amount of fibroblast growth factor ( FGF) than cells from normal tissue of the same type .
  • FGF fibroblast growth factor
  • cells of the cancer comprise a loss-of -function mutation in an adenomatous polyposis coli (APC) gene.
  • APC adenomatous polyposis coli
  • cells o the cancer express an oncogenic fi- catenin mutant .
  • cells of the cancer express an oncogenic Ras mutant .
  • the oncogenic Ras mutant is an oncogenic K-Ras mutant .
  • the oncogenic K-Ras mutant has substitution, wherein the numbering of the K-Ras amino acid sequence is relative to the sequence set forth in SEQ ID MO: 1 or 2.
  • the oncogenic K-Ras mutant has a G12V subs ution.
  • the oncogenic K-Ras mutant protein is a R ⁇ Ras GI v mutant protein.
  • the oncogenic K-Ras mutant has a GI2D substitution .
  • the oncogenic K-Ras mutant is a K ⁇ Ras GI2D mutant.
  • the oncogenic K-Ras mutant has a G13X substitution, wherein the numbering of the K-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 1 or 2.
  • the oncogenic K-Ras mutant has a Q61X substitution, wherein the numbering of the K-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 1 or 2.
  • the oncogenic Ras mutant is an oncogenic KF-Ras mutant .
  • the oncogenic N-Ras mutant has a G12X substitution, wherein the numbering of the N-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 3.
  • the oncogenic N-Ras mutant is a N-Ras G12 or a N- Ras G ;;D mutant.
  • the oncogenic N-Ras mutant has a G13X substitution, wherein the numbering of the N-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 3.
  • the oncogenic N-Ras mutant has a Q61X substitution, wherein the numbering of the N-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 3.
  • the oncogenic Ras mutant is an oncogenic H-Ras mutant .
  • the oncogenic H-Ras mutant has a G12X substitution, wherein the numbering of the H-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 4 or 5.
  • the oncogenic H-Ras mutant is a H-Ras Gi2 or a H-Ras G12D mutant .
  • the oncogenic H-Ras mutant has a G13X substitution, wherein the numbering of the H-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO : 4 or 5. In some embodiments , the oncogenic H-Ras mutant has a Q61X substitution , wherein the numbering of the H-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 4 or 5. In some embodiments , cells of the cancer express an oncogenic Src mutant .
  • cells of the cancer express an oncogenic Csk mutant .
  • the oncogenic Csk mutant has reduced activity compared to wild-type Csk.
  • the oncogenic Csk mutant is a Csk ⁇ 108 mutant.
  • the oncogenic Csk mutant is truncated.
  • the oncogenic Csk mutant is a Csk cl56stop mutant.
  • the cells of the cancer express a lower amount of Csk than cells from normal tissue of the same type.
  • the cells of the cancer express a higher amount of Csk than cells from normal tissue of the same type.
  • the cancer comprises or is in the form of at leas one tumor .
  • the cancer is metastatic.
  • compositions comprising
  • PI3K phosphatidylinositide 3-kinase
  • PI3K phosphatidylinositide 3-kinase
  • the present invention provides combinations for treating a subj ect afflicted with a cancer and a metabolic dysfunction comprising
  • the present invention provides pharmaceutical composi ions for treating a subject afflicted with a cancer and a metabolic dysfunction comprising
  • K-Ras Amino acid sequences of K-Ras are accessible in public databases by the accession numbers P01116 (Isoform 2A (identifier: P01116-1S; Isoforrti 2B (identifier: P01116-2 ) ) and NP_004976.2, and CCDS number CCDS8702.1, and are set forth herein as SEQ ID NOs : 1 and 2.
  • Nucleotide sequences for K-Ras cDNA and the sequence of the K-Ras gene are accessible in public databases, e.g. from the Gene ID for K-Ras, which is Gene ID 3845.
  • N-Ras The amino acid sequence of N-Ras is accessible in public databases by the accession numbers P01111 and NP consult002515.1, and is set forth herein as SEQ ID NO: 3.
  • Nucleotide sequences for N-Ras cDNA and the sequence of the N-Ras gene are accessible in public databases, e.g. from the Gene ID for N-Ras , which is Gene ID 4893.
  • H-Ras Amino acid sequences of H-Ras are accessible in public databases by the accession numbers P01112 and NP_00112391 .1 (Isoform 1), and P01112-2 and (Isoform 2), and are set forth herein as SEQ ID NOS: 4 and 5.
  • Nucleotide sequences for H-Ras cDNA and the sequence of the H-Ras gene are accessible in public databases, e.g. from the Gene ID for H-Ras, which is Gene ID 3265.
  • the amino acid sequence of Csk is accessible in public databases by the accession numbers P41240 and NP_004374, and is set forth Herein as SEQ ID NO: 6.
  • Nucleotide sequences for Csk cDNA and the sequence of the Csk gene are accessible in public databases, e.g. from the Gene ID for Csk, which is Gene ID 1445.
  • Wortmannin is availaix >0 LC Laboratories
  • wortmannin The CAS Registry number for wortmannin is 19545-26-7.
  • the structure of wortmannin is:
  • Raparaycin is available from LC Laboratories (R-5000; LC Laboratories, Woburn, MA, USA) (www.lclabs.com/PRODFILE/P-R/R-5000.php4) .
  • the CAS Registry number for rapamycin is 53i23-88-9.
  • Embodiments of the subject invention relate to compounds which are capable of inhibiting a glucoside hydrolase.
  • the glucoside hydrolase is an alpha-glucos idase .
  • Acarbose is available from Sigma (A8980; Sigma, St. Louis, MO,
  • the CAS registry number for acarbose is 56180-94-0.
  • the structure of acarbose is:
  • Sucrose is a disaccharide that is broken down (converted) into two mono - saccharides glucose and fructose by an enzyme called alpha- glucosidase.
  • Acarbose inhibits enzymatic activity of alpha- glucosidase. Therefore, acarbose inhibits the conversion of sucrose into monosaccharides.
  • AD81 was provided by A. Dar and K. Shokat UCSF. AD81, and methods of synthesizing AD81 are described in: Dar, A.C., Das, T.K., Shokat, K.M., and Cagan, R.L. (2012). Chemical genetic discovery of targets and anti-targets for cancer polypharmacology. Nature 486, 80-84, the entire contents of which are incorporated herein by reference.
  • the structure of AD81 is:
  • a compound of the invention is an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer , an RNA aptamer, a polypeptide, an antibody, an antisense oligonucleotide, an interfering RNA (RNAi ) molecule, or a ribozyme.
  • RNAi interfering RNA
  • Ester derivatives of compounds may be generated from a carboxylic acid group in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Ester derivatives may serve as pro-drugs that can be converted into compounds of the invention by serum esterases .
  • Compounds used in the methods of the present invention may be prepared by techniques well know in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.
  • a compound may be in a salt form.
  • a “salt” is a salt of the instant compound which has been modified by making acid or base salts of the compounds.
  • the salt is pharmaceutically acceptable.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines.
  • pharmaceutically acceptable salt refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the invention. These salts can be prepared in si tu during the final isolation and purification of a compound, or by separately reacting a purified compound in its free acid form with a suitable organic or inorganic base, and isolating the salt thus formed .
  • Antibodies may be employed in embodiments of the invention .
  • the antibody is a monoclonal antibody.
  • an "antibody” as used herein is defined broadly as a protein that characteristically immunoreacts with an epitope (antigenic determinant) of an antigen.
  • the basic structural unit of an antibody is composed of two identical heavy chains and two identical light chains, in which each heavy and light chain consists of amino terminal variable regions and carboxy terminal constant regions.
  • Antibodies of the invention include polyclonal antibodies , monoclonal antibodies (mAbs ⁇ , chimeric antibodies, CDR-grafted antibodies, humanized antibodies, human antibodies , catalytic antibodies , multispecific antibodies , as well as fragments, regions or derivatives thereof provided by known techniques, including, for example, enzymatic cleavage, peptide synthesis or recombinant techniques.
  • monoclonal antibody means an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants , each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies , and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by 5 Kohler and Milstein, Mature 256:495-97 (1975! , or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • the monoclonal antibodies may also be isolated from phage display libraries using the techniques described, for example, in Clackson et al . , N ture 352:624-28 (1991) and Marks et al . , J. Mol , Biol. 10 222 (3) :581-97 (1991) .
  • hybrida or "hybridoma cell line” refers to a cell line derived by cell fusion, or somatic cell hybridization, between a normal lymphocyte and an immortalized lymphocyte tumor line.
  • B ceil hybridomas are created by fusion of normal B cells of defined antigen specificity with a myeloma cell line, to yield immortal cell lines that produce monoclonal antibodies.
  • techniques for producing human B cell hybridomas are well known in the art [Kozbor et al . , Immunol. Today 4:72 (1983); Cole et al., in
  • epitope refers to a portion of a molecule (the antigen) that is capable of being bound by a binding agent, e.g., an antibody, 25 at one or more of the binding agent's antigen binding regions.
  • Epitopes usually consist of specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • an antibody is a humanized antibody.
  • Humanized 30 antibodies means antibodies that contain minimal sequence derived from non-human immunoglobulin sequences.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hyper variable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor 35 antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205, each herein incorporated by reference.
  • framework residues of the human immunoglobulin are replaced by corresponding non-human residues (see, for example, U.S. Pat. NOB . 5,585,089; 5,693,761; 5,693,762, each herein incorporated by reference).
  • humanised antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain desired af inity ⁇ .
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or subst.antia.lly all of the framework regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • antibodies are xenogeneic or modified antibodies produced in a non-human mammalian host, more particularly a transgenic mouse, characterised by inactivated endogenous immunoglobulin (Ig) loci.
  • Ig immunoglobulin loci
  • transgenic animals competent endogenous genes for the expression of light and heavy subunits of host immunoglobulins are rendered non-functional and substituted with the analogous human immunoglobulin loci.
  • transgenic animals produce human antibodies in the substantial absence of light or heavy host immunoglobulin subunits. See, for example, U.S. Pat. No. 5,939,598, the entire contents of which are incorporated herein by ref rence .
  • polyclonal antisera or monoclonal antibodies can be made using standard methods.
  • a mammal e.g., a mouse, hamster, or rabbit
  • An immunogenic form of a molecule or protein which elicits an antibody response in the mammal.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies.
  • antisera can be obtained, and, if desired IgG molecules corresponding to the polyclonal antibodies may be isolated from the sera.
  • antibody producing cells can be harvested from an immunized animal and fused wi h myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art.
  • Hybridoma cells can be screened immunochemically for production of antibodies which are specifically reactive with the oligopeptide, and monoclonal antibodies isolated.
  • Antisense oligonucleotides are nucleotide sequences which are com lementary to a specific DWA or R A sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of target gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates , phosphorothioates , phosphorodithioates, alkylphosphonothioates , alkylphosphonates, phosphoramidates , phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (Nicholls et al . , 1993, J Immunol Meth 165:81-91). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 1, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a target polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent nucleotides, can provide sufficient targeting specificity for a target mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides in length.
  • Noncomplementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular target polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a target polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal rifaose.
  • Modified bases and/or sugars such as arabinose instead of ribose , or a 3', 5 1 -substituted oligonucleotide in which the 3 ' hydroxy1 group or the 5 ' phospha e group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art.
  • Ribozymes are RNA molecules with catalytic activity (Uhlmann et al . , 1987, Tetrahedron. Lett. 215, 3539-3542). Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences .
  • the coding sequence of a polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art.
  • the cleavage activity of ribozymes can be targeted to specific R As by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.
  • Specific ribozyme cleavage sites within an RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC . Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection , electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in. which it is desired to decrease target gene expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or VAS element, and a transcriptional teminator signal, for controlling transcription of ribozymes in the cells (U.S. 5,641,673). Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • RNAi interfering RNA
  • RKAi involves mRNA degradation.
  • the use of RNAi has been described in Fire et al . , 1998, Carthew et al . , 2001, and Elbashir et al . , 2001, the contents of which are incorporated herein by reference.
  • Interfering RNA or small inhibitory RNA (RNAi) molecules include short interfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), and micro-RNAs (miRNAs) in all stages of processing, including shRNAs, pri-miRNAs, and pre-miRNAs . These molecules have different origins: siRNAs are processed from double-stranded precursors (dsRNAs) with two distinct strands of base-paired RNA; siRNAs that are derived from repetitive sequences in the genome are called rasiRNAs; miRNAs are derived from a single transcript that forms base-paired hairpins. Base pairing of siRNAs and miRNAs can be perfect (i.e., fully complementary! or imperfect, including bulges in the duplex region.
  • Interfering RNA molecules encoded by recombinase-dependent transgenes of the invention can be based on existing shRNA, siRNA, piwi-interacting RNA (piRIJA) , micro RNA (miRNA) , double-stranded RNA (dsRNA) , antisense RNA, or any other RNA species that can be cleaved inside a cell to form interfering R As, with compatible modifications described herein.
  • an "shRNA molecule” includes a conventional stem- loop shRNA, which forms a precursor miRNA (pre-miRNA) .
  • shRNA also includes mioro-RNA embedded shRNAs (miRNA-based shRNAs ! , ' wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA.
  • a shRNA may form a primary miRNA (pri-miRNA) or a structure very similar to a natural pri-miRNA.
  • the pri-miRNA is subsequently processed by Drosha and its cofactors into pre-miRNA. Therefore, the term "shRNA” includes pri-miRNA (shRNA-mir) molecules and pre-miRNA molecules.
  • a “stem- loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion).
  • the terms “hairpin” and “fold-back” structures are also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art.
  • the secondary structure does not require exact base-pairing.
  • the stem can include one or more base mismatches or bulges.
  • the base-pairing can be exact, i.e. not include any mismatches.
  • RNAi-expressing construct or "RNAi construct” is a generic term that includes nucleic acid preparations designed to achieve an RNA interference effect.
  • An RNAi-expressing construct comprises an RNAi molecule that can be cleaved in vivo to form an siRNA or a mature shRNA.
  • an RNAi construct is an expression vector capable of giving rise to a siR A or a mature shRNA in vivo.
  • Non- 1imiting examples of vectors that may be used in accordance with the present invention are described herein and will be well known to a person having ordinary skill in the art. Exemplary methods of mak g and deli ering long or shor RNAi constructs can be found, for example, in O01/68836 and WO01/75164.
  • RNAi is a powerful tool for in vi tro and in vivo studies of gene function in mammalian cells and for therapy in both human and veterinary contexts. Inhibition of a target gene is sequence- specific in that gene sequences corresponding to a portion of the RNAi sequence, and the target gene itself, are specifically targeted for genetic inhibition. Multiple mechanisms of utilizing RNAi in mammalian cells have been described. The first is cytoplasmic delivery of siRNA molecules, which are either chemically synthesized or generated by DICER-digestion of dsRNA. These siRNAs are introduced into cells using standard transfection method . The siRNAs enter the RISC to silence target mRNA expression.
  • shRNA short hairpin RNA
  • shR A is modeled on micro interf ring RNA (miRNA) , an endogenous trigger of the RNAi pathway ⁇ Lu et al . , 2005, Advances in Genetics 54: 117-142, Fewell et al 2006, Drug Discovery Today 11: 975-982).
  • miRNA micro interf ring RNA
  • Conventional shRNAs which mimic pre-miRNA, are transcribed by RNA Polymerase II or III as single-stranded molecules that form stem- loop structures . Once produced , they exit the nucleus , are cleaved by DICER, and enter the RISC as siRNAs.
  • shRNA is modeled on primary miRNA ishRNAmir) , rather than pre- miRNA transcripts (Fewell et al . , 2006 ) .
  • An example is the rniR-30 miRNA construct .
  • the use of this transcript produces a more physiological shRNA that reduces toxic effects.
  • the shRNAmir is first cleaved to produce shRNA, and then cleaved again by DICER to produce siRNA.
  • the siRNA is then incorporated into the RISC for target mRNA degradation.
  • aspects of the present invention relate to RNAi molecules that do not require DICER cleavage. See, e.g., U.S. Patent No. 8,273,871, the entire contents of which are incorporated herein by reference.
  • RISC RNA Induced Silencing Complex
  • RLC RISC-loading complex
  • the guide strand leads the RISC to cognate target KIRNAB in a sequence- specific manner and the Slicer component of RISC hydrolyses the phosphodiester bound coupling the target mRNA nucleotides paired to nucleotide 10 and 11 of the RNA guide strand.
  • Slicer forms together with distinct classes of small RNAs the RNAi effector complex, which is the core of RISC. Therefore, the "guide strand" is that portion of the double-stranded RNA that associates with RISC, as opposed to the "passenger strand,” which is not associated with RISC.
  • the number of nucleotides which is complementary to a target sequence is 16 to 29, 18 to 23, or 21-23, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.
  • Isolated RNA molecules can mediate RN i . That is, the isolated RNA molecules of the present invention mediate degradation or block expression of mRNA that is the transcriptional product of the gene. For convenience, such mRNA may also be referred to herein as mRNA to be degraded.
  • RNA, RNA molecule(s), RNA segment(s) and RNA fragment (s) may be used interchangeably to refer to RNA that mediates RNA interference.
  • RNA double-stranded RNA
  • small interfering RNA hairpin RNA
  • single-stranded RNA isolated RNA (partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA)
  • altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA) .
  • Nucleotides in the RNA molecules of the present invention can also comprise nonstandard nucleotides, including non- naturaliy occurring nucleotides or deoxyribonucleotides . Collectively, all such altered RNAi molecules are referred to as analogs or analogs of naturally-occurring RNA. RNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi.
  • RNA molecules that mediates RNAi interacts with the RNAi machinery such that it directs the machinery to degrade particular mR As or to otherwise reduce the expression of the target protein.
  • the present invention relates to RNA molecules that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi inhibition by cleavage or blocking expression of the target mRNA.
  • an RNAi molecule of the invention is introduced into a mammalian cell in an amount sufficient to attenuate target gene expression in a sequence specific manner.
  • the RNAi molecules of the invention can be introduced into the cell directly, or can be compiexed with cationic lipids, packaged within liposomes, or otherwise delivered to the cell.
  • the RNAi molecule can be a synthetic RNAi molecule, including RNAi molecules incorporating modified nucleotides, such as those with chemical modifications to the 2 ' -OH group in the ribose sugar backbone, such as 2 ' -O-methyl (2'OMe), 2'-fluoro (2'P) substitutions, and those containing 2'0Me, or 2'F, or 2 ' -deoxy, or "locked nucleic acid" CLNA) modifications.
  • an RNAi molecule of the invention contains modified nucleotides that increase the stability or half-life of the RNAi molecule in vivo and/or in vitro.
  • the RNAi molecule can comprise one or more aptamers, which interact (s) with a target of interest to form an a tamer : target complex.
  • the aptamer can be at the 5' or the 3' end of the RNAi molecule.
  • Aptamers can be developed through the SELEX screening process and chemically synthesi ed .
  • An ap amer is general ly chosen to preferentially bind to a target .
  • Suitable targets include small organic molecules, polynucleotides , polypep ides , and proteins . Proteins can be cell sur ace proteins , extracellular proteins, membrane proteins, or serum proteins, such as albumin .
  • Such target molecules may be in ernalized by a cell, thus effecting cellular uptake of the shR A .
  • Other potential targets include organelles, viruses, and cells.
  • the R A molecules of the present invention in general comprise an RNA portion and some additional, portion, for example a deoxyribonnc1 otide portion.
  • the total number of nucleotides in the RNA molecule is suitably less than in order to be effective mediators of RMAi .
  • the number of nucleotides is 16 to 29, more preferably 18 to 23, and most preferably 21-23.
  • administering compounds in embodiments of the invention can be effected or performed using any of the various methods and delivery systems known to those skilled in the ar .
  • the administering can be, for example, intravenous , oral , intramuscular , intravascula , intraarterial , intracoronary, intramyocardial , intraperitoneal, and subcutaneous .
  • Other non-limiting examples include topical adminis ration, or coating of a device to be placed within the subjec .
  • administration is effected by injection or via a cathete .
  • Injectable drug delivery systems may be employed in the methods described he ein include solutions , suspensions , gels .
  • Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g. , hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch) , diluents (e.g. , lactose and other sugars, starch, dicalcium phosphate and cellulosic materials) , disintegrating agents (e.g. , starch polymers and cellulosic materials) and lubricating agents (e.g. , stearates and talc ) .
  • binders e.g. , hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch
  • diluents e.g. , lactose and other sugars, starch, dicalcium phosphate
  • Solutions , suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans , cellulosics and sugars), humectants (e.g., sorbitol), soiubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens , and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents ⁇ e.g., EDTA) ,
  • suspending agents e.g., gums, zanthans , cellulosics and sugars
  • humectants e.g., sorbitol
  • soiubilizers e.g., ethanol, water, PEG and prop
  • the term "effective amount" refers to the quantity of a component that is sufficient to treat a subject without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention, i.e. a therapeutically effective amount.
  • the specific effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.
  • a pharmaceutically acceptable carrier is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the compounds to the subject.
  • the carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier.
  • the compounds used in the methods of the present invention can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • the unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration.
  • the compounds can be administered alone or mixed with a pharmaceutically acceptable carrier.
  • This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used.
  • the active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form.
  • suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders.
  • Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
  • suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and ef rvescent preparations reconstituted from effervescent granules.
  • suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and ef rvescent preparations reconstituted from effervescent granules.
  • Such iiguid Such iigui
  • dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Oral dosage forms optionally contain flavorants and coloring agents.
  • Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen .
  • the dosage of a compound of the invention administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of the compound and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.
  • a dosage unit of the compounds of the invention may comprise a compound alone, or mixtures of a compound with additional compounds used to treat cancer.
  • the compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions.
  • the compounds may also be administered in intravenous (bolus or infusion) , intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection or other methods, into the eye, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
  • a compound of the invention can be administered in a mixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • a pharmaceutically acceptable carrier suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • the unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration.
  • the compounds can be administered alone but are generally mixed with a pharmaceutically acceptable carrier.
  • This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. In one embodiment the carrier can be a monoclonal antibody.
  • the active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form.
  • suitable solid carriers include lactose, sucrose, gelatin and agar.
  • Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow- inducing agents, and melting agents.
  • liquid dosage forms examples include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid dosage forms may contain, for exam le , suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Oral dosage forms optionally contain flavorants and coloring agents .
  • Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
  • Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
  • the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.
  • Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth , or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
  • a compound of the invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
  • the compounds may be administered as components of tissue- targeted emulsions.
  • a compound of the invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug.
  • Such polymers ⁇ include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol , polyhydroxyethylasparta- midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues.
  • a compound may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters , polyacetals , polydihydropyrans , polycyanoacylates , and crosslinked or amphipathic block copolymers of hydrogels .
  • biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters , polyacetals , polydihydropyrans , polycyanoacylates , and crosslinked or amphipathic block copolymers of hydrogels .
  • Gelatin capsules may contain a compound of the Invention and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets . Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets . Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the
  • a compound may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol , glycerol , water, and the like .
  • suitable 1 iquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solven s/ including es ers , emulsions , syrups or elixirs , suspensions , solutions and/or suspensions reconsti uted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules .
  • Such 1 iquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents .
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • water a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient , suitable stabilizing agents , and if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilising agents.
  • citric acid and its salts and sodium EDTA are also used.
  • parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol .
  • preservatives such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol .
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.
  • a compound may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art .
  • the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.
  • Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
  • the compounds and compositions thereof of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.
  • Insulin resistance evasion is due to Wingless-mediated Insulin Receptor upregulation
  • Drosophila a standard diet supplemented to 1.0 M sucrose high dietary sucrose or "HDS” led to metabolic defects reminiscent of specific aspects of type 2 diabetes including insulin resistance, hyperglycemia, elevated Drosophila Insulin-like peptide (DILP) levels, and accumulation of body fat (Musselman et al . , 2011).
  • DILP Drosophila Insulin-like peptide
  • body fat body fat
  • Drosophila cancer models fed HDS were compared to those fed a control (0.15 M sucrose) diet.
  • Combined elevation of Ras and Src pathway activities is a common motif in multiple cancer types including breast, colorectal, and pancreatic (Ishizawar and Parsons, 2004; Morton et al . , 2010).
  • oncogenic K- as plus Src isoforms cooperate to accelerate the onset of pancreatic ductal adenocarcinoma in mice models, and Ras plus Src act together to promote tumorigenesis in Drosophila (Shields et al . , 2011; Vidal et al . , 2007) .
  • This model was further developed by pairing transgenes that express oncogenic rasl al2v in the eye with the genotypically null cskfi 156staB allele of Csk, a primary negative regulator of Src kinase. Clones were labeled with GFP to visualize tumor progression. In control eyes, GFP expression was comparable in control diet vs. HDS, indicating that high sucrose feeding did not alter transgene expression levels ( Figures 1A and IB) ,
  • control clones decreased from 35.2% (day 7 AEL) to 13.5% (day 9 AEL) of total area while HDS clones progressively increased, reaching 58.9% at day 11 AEL and 93.7% at day 13 AEL ( Figures 1K-1S) .
  • These analyses further support a role for HDS in enhancing growth of rasl at2v ;csk '! ' ' tumors.
  • Example 2 HDS but not high f t induced cancer phenotypea
  • Example 3 HDS promoted aspects of invasive migration and distant secondary tumors
  • Insulin PI3 signaling did not mediate all effects of HDS. While the triple combination inR CA , rasl elw ; csk' ⁇ fed a control diet exhibited strongly enhanced expansion of tumors within the eye field, overgrowth was not accompanied by metastasis-like behavior or secondary tumor formation. Further, though elevated MP1 expression was observed the basement membrane was still retained ( Figures 2C and 2G) . This result again emphasizes that tumor overgrowth and metastasis are separable processes.
  • Example 6 Tumors exhibit aspects of an 'undead' phenot pe
  • a central hallmark of mammalian cancer is the ability of transformed cells to evade apoptosis.
  • Drosophila developing eye and wing epithelia keeping otherwise apoptotic cells alive ('undead') by blocking ef ector caspase activity leads to aberrant tissue overgrowth due to compensatory proliferation of surrounding cells, a process designed to maintain tissue size and integrity (reviewed in (Bergmann and Stellar, 2010; Fan and Bergmann, 2008; Martin et al . , 2009) , An antibody that recognizes the cleaved, active isoform of human caspase-3 was used, a validated readout for activity of the Drosophila initiator caspase Drone (Fan and Bergmann, 2010) .
  • Insulin pathway activation alone was sufficient to mimic this block: inR CA , rasl G12v ; csk ⁇ ' animals fed a control diet retained Drone caspase activity but, again, were resistant to apoptotic cell death as assessed by TUNEL ( Figures 4C, 4F and 10A) ,
  • One candidate to block apoptosis in transformed cells is the caspase inhibitor Drosophila Inhibitor of apoptosis protein 1 (Diapl), which inhibits caspases downstream of Drone (Ditzel et al., 2008) .
  • Example 8 Canonical Wingless/Wnt pathway signaling promotes Insulin sensitivity by regulating InR expression
  • inR mRHA levels were significantly upregulated in rasl 0 ""'; csk ⁇ ' ⁇ eye clones in animals fed HDS ( Figure 51! .
  • the result was an increase in phosphorylated, activated InR that was strongly enhanced when the tissue was challenged with exogenous Insulin (Figure 5J) .
  • the diet-dependent increase in inR m NA levels was suppressed by reducing activity of the Wg target Tcf (rasl G12v ; csk ' ' r tcf m ; Figure 51) .
  • Acarbose is an inhibitor of alpha-glucosidase that is used to treat type 2 diabetes mellitus (Anderson, 2005) . Feeding acarbose in the presence of HDS led to reduced tumor growth and improved rasl allv ; csk ⁇ animal survival ( Figures 6B and 6J) , Pyrvinium, a small-molecule inhibitor of canonical Wht-signaling pathway (Thorne et al . , 2010), suppressed HDS-induced tumor growth at a concentration that did not affect growth of wild type clones ( Figures 6C and 12B) : 41.9% of HDS- and pyrvinium-fed animals achieved pupariation (Figure 6J) .
  • AD81 also suppressed tumor growth and larval lethality of inR CA , rasl el2v ; csk ⁇ / ⁇ animals fed a control diet, demonstrating AD81 can act tumor- autonomously (Figure 12C! .
  • Targeting sugar or the Ras , Src , Pi3K , or Wg pa hways showed significant but limited efficacy on rasl al!v ; csk ⁇ ' ⁇ tumors in animals fed HDS.
  • Combining acarbose with pyrvinium, acarbose with AD81, or pyrvinium with AD81 enhanced survival to permit 64.
  • UAS ⁇ rasl G12v , UAS ⁇ inR A1325D (inR CA ), a*t «»* , csk' lm , UAS-tcP*, UAS-bsk ⁇ , UAS ⁇ HA ⁇ wg flies were obtained from the Bloomington Drosophila Stock Center.
  • UAS-wg mAi flies were obtained from Vienna Drosophila R Ai Center. The following stocks were kindly provided to us: FRT82B, cskP L56SCOB by A. O'Reilly and M. Simon; scrib 1 by D. Bilder; ey(3.5)- FLP1 by G. Haider.
  • GFP green fluorescent protein
  • Larval eyes were dissected in PBS, fixed in 4% paraformaldehyde (PFA) in PBS or PLP-fixative (2.5% paraformaldehyde, 0.075 M lysine. 0.25% (w/v) Ma-periodate in phosphate buffer) , washed in PBT ( PBS containing 0.1% Triton X-100) , and incubated with primary antibodies in PAXDG (PBS containing 1% BSA, 0.3% Triton X-100, 0.3% deoxycholate , and 5% goat serum) , followed by washing and incubation with secondary antibodies in PAXDG.
  • PFA paraformaldehyde
  • PLP-fixative 2.5% paraformaldehyde, 0.075 M lysine.
  • 0.25% (w/v) Ma-periodate in phosphate buffer) washed in PBT ( PBS containing 0.1% Triton X-100) , and incubated with primary antibodies in PAX
  • Tissues were counter-stained with TO-PRO-3 (Invitrogen) and mounted in Vectashield mounting media (Vector Laboratories).
  • Antibodies used were: rabbit anti-Cleaved Caspase-3 (Aspl75; Cell Signaling), mouse anti-dMMPl (DSHB: Developmental Studies Hybridoma Bank), mouse anti-Wingless (DSHB), mouse anti-foeta-galactosidase (DSHB) , rabbit anti-Laminin beta 1 (Abeam), rabbit anti-phospho-Histone H3 (SerlO; Millipore) , rabbit anti-Diapl (gift from H.
  • Drugs solubilized in DMSO were diluted directly into the fly medium and vortexed extensively to obtain homogeneous culture.
  • Drugs used were: wortmannin and rapamycin (LC Laboratories); acarbose and pyrvinium pamoate (Sigma) .
  • AD compounds were provided by A. Dar and K. Shokat COCSF) .
  • flies with the genotype ey ⁇ 3.5) FLP1; act>y+>gal4; FRT82B, tub-gal80 were crossed with flies with the following genotypes: (a) UAS-lacZ; FRT82B; (b) UAS-rasl el2v ; FRT82B, cskfi 15esc °v/TM6b (c) UAS inR A132S , UAS-rasl al2v ; FRT82B, cskP 1Msca "/TM6b; (d) UAS-rasl G12v ; FRT82B, csk , akt M226 /TM6b.
  • Total R A was extracted from the eye discs using QIAGEN R easy Mini Kit. Oligo-dT primers and Superscript RT-II reverse transcriptase (Invitrogen) was used to synthesize first strand DNA. PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) and analyzed on Applied Biosystems StepOnePlus real-time PCR system.
  • Results were normalized to Kinesin fflRNA, and RpL32 was used as a control.
  • the following primers were used : Kinesin-f , 5'-
  • GCTGGAC TCGGTCGTAGAG-3 ' (SEQ ID NO: 7) ; inesin- , 5'- CTTTTCATAGCGTCGCTTCC-3 ' (SEQ ID NO: 8) ; RpL32-f , 5'-
  • GCAGGAAGCCCTCGATGA-3 ' (SEQ ID NO: 12); chico-f , 5 ' -ATCAGGCGATGCGGTC 3' (SEQ ID NO: 13); chico-r, 5 ' -ACATAGCGCTCAGTATCG-3 ' (SEQ ID NO 14); dLnk-f , 5 ' -GTGTTCGCCTAAAGACAAATGAAAT-3 ⁇ (SEQ ID NO: 15); dLnk- 5 ' -TGTTAGCGTTGTGGGATCCAA-3 ' (SEQ ID NO: 16).
  • Drosophila as a model to explore the effects of diet on tumor progression. Feeding Drosophila a diet high in carbohydrates was previously demonstrated to direct metabolic dysfunction including hyperglycemia, hyperinsulinemia and insulin-resistance. The examples described herein demonstrate that high dietary sugar also converts Ras/Src transformed tissue from localized growths to aggressive tumors with emergent metastases. While most tissues displayed insulin resistance, Ras/Src tumors retained insulin pathway sensitivity, increased the ability to import glucose, and resisted apoptosis. High dietary sugar increased canonical Wingless Wht pathway activity, which upregulated Insulin Receptor gene expression to promote insulin sensitivity.
  • the result is a feed-forward circuit that amplified diet-mediated malignant phenotypes within Ras/Src transformed tumors.
  • the examples described herein demonstrate the potential of combinatorial drug intervention to treat diet-enhanced malignant tumors.
  • Obesity and type 2 diabetes are typically associated with chronic hyperinsulinemia : levels of insulin in the blood rise to compensate for insulin resistance. Increased circulating insulin levels are in turn a risk factor tor the development of hepatocellular carcinoma and colorectal cancer (Donadon et al . , 2009; Kaaks et al . , 2000). Together with the well-documented mitogenic effects of insulin ⁇ Ish- Shaiom et al . , 1997), this evidence suggests a role for hyperinsulinemia as a promoter of enhanced tumorigenesis in obese and diabetic patients. However, the development of insulin resistance in metabolism-related diseases raises a key question: how do tumors overcome insulin resistance to take advantage of increasing insulin levels? The examples herein utilise Dros phila to explore the effects of high dietary sugar on tumor progression in vivo .
  • Ras/Src-activated tumors remained sensitive to insulin in dietary conditions that promoted insulin resistance in surrounding tissues.
  • the examples herein demonstrate that increased insulin/PI3K signaling in turn prevents apoptosis and promotes canonical Wingless /Wnt mitogenic signaling in Ras/Src tumors.
  • Our results provide functional data that, in the face of high dietary sugar, Ras/Src-activated cells escape insulin resistance to exhibit enhanced growth that is dependent on Wingless nt mitogenic signaling.
  • the examples herein provide evidence that Ras/Src- activated cells increase insulin pathway-sensitivity in an otherwise insulin-resistant environment, allowing them to take advantage of high circulating glucose.
  • the result is a Wg- and Jnk- dependent enhancement of tumor progression.
  • the results herein support a model in which Ras/Src cells become hypersensitive to circu.la.ting insulin, leading to efficient activation of insulin/PI3K signaling (Figure 5K) .
  • a key consequence of increased insulin/PI3K signaling was prevention of apoptosis and promotion of elevated levels of Wg expression and activity; these two aspects promoted an 'undead' -like cell phenotype that further enhanced proliferation.
  • the Drosophila nt ortholog Wg promotes tumors' ability to evade insulin-resistance through its regulation of inR expression in the presence of HDS feeding, which otherwise promotes diabetes-like phenotypes (Figure 5H; (Musselman et al . , 2011).
  • High Wg expression requires both Ras/Src-activation and HDS, promoting inR expression through canonical Wg-dTcf signaling ( Figure 51).
  • Tumors with Wat-activation may represent tumor types that respond and synergize with metabolic defects. Wnt-acti ation is frequently observed in many tumor types including those with strong association with diabetes such as hepatocellular carcinomas and colorectal cancers; activating mutations in ⁇ -catenin and loss of function mutation in APC is frequently found in human hepatocellular carcinoma and colorectal cancers (Fodde et al . , 2001; Laurent-Puig and Zucman-Rossi , 2006). Incidence of pancreatic cancer has a particularly strong association with diabetes, suggesting its constituent mutation load may interface with metabolic dysfunction.
  • Activating mutations in K-Ras plus up-regulation of Src activity are commonly found in pancreatic ductal adenocarcinoma (PDA) (Almoguera et al . , 1988; Morton et al . , 2010), and a mouse K-Ras/Src pancreatic model developed PDA with shortened latency (Shields et al . , 2011).
  • Wnt ligands and transcriptional target genes of S-catenin are highly expressed in PDA (Pasca di Magliano et al., 2007), a phenomenon reminiscent of observations in the examples herein linking diet, Ras/Src, and Wnt signaling.
  • the data herein indicate that limiting dietary sucrose directly or through blocking its intermediates through compounds such as Acarbose may reduce important aspects of tumor risk and progression in individuals with metabolism-related diseases.
  • Combining acarbose with drugs that target the triggering Ras/Src pathways plus components of the amplification circuit provided the most robust therapeutic impact on diet-enhanced tumors in our model.
  • the cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila. Cell Death Differ 17, 534-539.
  • LRP6 enhances glucose metabolism by promoting TCF7L2- dependent insulin receptor expression arid IGF receptor stabilization in humans. Cell Me ab 17, 197-209.

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Abstract

Methods for treating a subject afflicted with a cancer and a metabolic dysfunction which comprises administering (i) a compound that inhibits a glycoside hydrolase or (ii) a compound that inhibits Wg/Wnt pathway signaling and (iii) a compound that inhibits Ras/Src oncogenic pathway signaling.

Description

METHODS OF TREATING CANCER IM SUBJECTS AFFLICTED WITH
METABOLIC DYSrtJHCTION
This application claims priority of U.S. Provisional Patent Application No. 61/937,239, filed February V, 201 nd U.S. Pr ovis iona ] Patent App L ication Mo. 61 /847,016, filed July 16, 2013, the entire contents f each of which are hereby incorporated herein by roccr'incG.
This hiver.t ion was made with government support under grant: numbers 0iCA170 9b and P.O 1 -CAl 09730 awarded by the National Cane«r Institute, and ki! 1 DK069940 awarded by the National Institutes of Health. The U.S. Government has certain i ig!its in the invention.
This application ineorpor t ¾s-by- ref erence nucleotide and/or amino acid sequences which are present in the file named " ! 101 ; ί;..η02Β_*ί5-Ίΰ7„Α.._Γ(;τ Jiequencebi t ing_REB . txt" , w ich is 14.1 kilobytes in size, and which was created July 16, 2014 in the IBM-PC machine format, having an operating system compatibility with MS- Windows , which is contained in the text file filed July 16, 2014 as part of this applioat-i on _
Throughout this application, various publications are referenced, including referenced in parenthesis, full citations for publications referenced in parenthesis may be found list.ed at the end of the specification immediately preceding the claims. The disclosures of all referenced publications in their entireties are hereby incorporated by reference into this application in order to more fully do;scribe the state of the art to which this invention pertains.
Background of Invention
The prevalence of metabolism-related diseases including type 2 diabetes and obesity has been increasing worldwide. Metabolic disease impacts body homeostasis, leading to a constella ion of symptoms including cardiovascular disease, blindness, neuropathy, and nephropathy. Epidemiological studies have provided evidence for an association between cancer and metabolic diseases such as diabetes and obesity (Barone et al . , 2008; Calle et al , , 2003; Coughlin et al . 2004; Inoue et al . , 2006).
New methods and compositions for treating cancer are needed.
S¾smiary of the Invention
The present invention provides methods of treating a subject afflicted with a cancer and a metabolic dysfunction, which comprises administering to the subject
(a) ( i ) a compound which is capable of inhibiting a glycoside hydrolase,
( ii ) a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, or
{ iii ) both a compound of (a) ( i) and a compound of (a) { ii ) ; and
(b) at least one other compound which is capable of inhibiting
( i ) insulin receptor ( InR) metabolic signaling,
( ii } phosphatidylinositide 3-kinase { PI3K) metabolic signaling,
(iii) InR/PI3K metabolic signaling,
( iv) c-Jun N-terminal kinase (Jnk) metabolic signaling,
(v) target of rapamycin (Tor ) oncogenic p thway signaling,
(vi ) Ras oncogenic pathway signaling,
( ii ) Src oncogenic pa hway signaling, or
(viii) Ras/Src oncogenic pathway signaling, each such compound being administered in an amount such that, when administered in combination , adminis ration of the compounds is effective to treat the sub ect .
Aspects of the present invention relate to the use of a composition comprising
(a) (i) a compound which is capable of inhibiting a glycoside hydrolase,
(ii) a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, or
(iii) both a compound of (a) (i) and a compound of (a) (ii); and
(b) at least one other compound which is capable of inhibiting
{ i ) insulin receptor ( InR) metabolic signaling, { ίί ) phosphatidylinositide 3 -kinase (PI3K) metabolic signaling,
(iii) InR/PI3K metabolic signaling,
tiv) c-Jun N-terminal kinase (Jnk) metabolic signaling, (v) target of rapamycin (Tor) oncogenic pathway signaling,
(vi ) Ras oncogenic pathway signaling,
(vi i. ] Src oncogenic pathway signaling, or
(viii) Ras/Src oncogenic pathway signaling, tor treating a subj ct afflicted wi h a cancer and a metabolic dysfunction.
Aspects of the present invention relate to the use of
(a) ( i ) a compound which is capable of inhibi ing a glycoside hydrolase,
(ii) a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, or
( iii ) both a compound of (a) ( i ) and a compound of (a) (ii) ; and
(b) at least one other compound which is capable of inhibiting
( i) insulin receptor ( InR) metabolic signaling,
(ii) phosphatidylinositide 3-kinase (PI3K) metabolic signaling,
(iii) InR/PI3K metabolic signaling,
(iv) c-Jun N-terminal kinase (Jnk) metabolic signaling,
( ) target of rapamycin (Tor) oncogenic pathway signaling,
(vi) Ras oncogenic pathway signaling,
(vii) Src oncogenic pathway signaling, or
(viii) Ras/Src oncogenic pathway signaling, in the manufacture of a medicament for the treatment of a subj ect afflicted with a cancer and a metabolic dysfunction. The present invention provides combinations for treating a subject afflicted with a cancer and a metabolic dysfunction comprising (a) (I) a compound which is capable of inhibiting a glycos ide hydrol se,
(ii) a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, or
( iii ) both a compound of (a) (i ) and a compound of (a) (ii); and
(b) at least one other compound which is capable of inhibiting
£ i ) insulin receptor ( InR) metabolic signaling , ( ii ) phosphatidylinosi i.de -kinase ( PI3K) me abolic signaling,
( iii ) lnR/Pl3 metabolic signaling,
( iv) c-Jun N-terminal kinase (Jnk) metabolic signaling,
(v) target of rapamycin (Tor) oncogenic pathway signaling,
(vi) Ras oncogenic pathway signaling,
(vii) Src oncogenic pathway signaling, or
Eviii) Ras/Src oncogenic pathway signaling. The present invention provides pharmaceutical compositions for treating a subject afflicted with a cancer and a metabolic dysfunction comprising
(a) (i) a compound which is capable of inhibiting a glycoside hydrolase,
(ii) a compound which is capable of inhibiting canonical
Wg/Wnt pathway signaling, or
(iii) both a compound of (a) (i) and a compound of (a) (ii) ; and
(b) at least one other compound which is capable of inhibiting
£ i) insulin receptor ( InR) metabolic signaling,
{ ii) phosphatidylinositide 3 -kinase (PI3K) metabolic signaling,
(iii) InR/PI3 metabolic signaling,
( iv) c-Jun N-terminal kinase (Jnk) metabolic signaling,
£v) target of rapamycin (Tor) oncogenic pathway signaling, ( i ) Ras oncogenic pathway signaling,
(vii ) Src oncogenic pa thway signaling, or
(viii) Ras/Src oncogenic pathway signaling.
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20
25
30
35 Brief Description of tha Drawings
Figure 1, HDS diverts Rms/Src-activated cells into aggressive tumors
(A-I) Developmental stage matched third instar larvae fed control diet or HDS, (A and B) lacZ control in control diet (day 7 AEL) and HDS {day 11 AEL) (C and D) csk~-'~ in control diet (day 7 AEL) and HDS
(day 11 AEL) , (E and F) raslGIi, in control diet (day 8 AEL) and HDS
(day 12 AEL), (G, H and I) rasWs cetr'- in control diet (day 9 AEL) and HDS (day 13 AEL) . The latter demonstrated secondary tumors in a subset of animals (arrowhead in I). (A' -H ' ) , Matching dissected eye epithelial tissue stained with DAPI (red) . (J) Quantitation of the observed pheno ypes . Blue bar: eye discs without overgrowth (e.g.; Fig. 1G) . Red bar: eye discs with tumor overgrowth and overall enlarged tissue size (e.g.; Fig. 11). Green bar: animals with secondary tumors (e.g.; Fig. II). Results are shown as mean ± SEM. ( -R) Chronological age matched larvae fed control diet or HDS. rasLc'12v csk~<~ animals shown at day 7 AEL (K and L) , day 9 AEL (M and M) , day 11 AEL (O and P) , and day 13 AEL (Q and R) . (Κ', M' , ' , P' and R' ) , Matching dissected eye epithelial tissue stained with DAPI
(red) . (S) Quantitation of the percentage of GFP-positive clone area percentage relative to total eye tissue area. Results are shown as mean ± SEM of individual eye discs. * Not assessed due to early
(small discs, Day 7) or late (everted pupal discs) stages. See also Figure 7. Figure 2. Ras/Src-tumors in HDS spread into hemolymph and colonized near trachea to form secondary tumors
(A-D) Laminin A staining (red) of developmental stage matched raslI v; cskr^ or inJ?CA, rasl 12v; csk'" eye discs raised on indicated diets. (E-H) MP1 staining (red) of developmental stage matched raslG12v; csk~'~ or inRCA, raslal2,/;csk-/- eye discs raised on indicated diets. (Ϊ-Κ) Ventral view of raslal3 ; csk' animals raised on HDS (I). Zoom up of the inset is shown in (J) , and the extracted hemolymph on mineral oil is shown in (K) . (L-O) ra.slG13v; csk' ' animals raised on HDS with growing secondary tumor (arrow) . GFP (M) , bright field (BF) (N) and merged image (0) of the dissected secondary tumor is shown. (P) phospho-Histone H3 staining (p-HH3; red) of raslel2v; csk~'- secondary tumo . (Q) C itin-binding probe staining (red) of raslal2v; csk~'~ secondary tumor attached to trachea. Zoom up of the inset is shown in the middle panel. (Ft) Branchless staining (bnl red) of a raslQ12v; csk'2' secondary tumor attached to trachea. (S) Phenotype quantitation. Blue bar; % animals with loss of Laminin staining in the eye discs. Red bar: % animals with GFP-positive cells in the heitiolymph . Green bar: % animals with secondary tumors. Results are shown as mean ± SEM. See also Figure 8.
Figure 3, Ras/Src-activated cells evade insulin resistance
(A and B) Developmental stage matched animals raised on HDS with the genotype, (A) raslG12v; cskr'~ , (B) raslsi2' ; csk'2', akthyBo2hrpa , (C and D) Developmental stage matched animals raised on control diet with the genotype, (C) raslal2v; csk"2' , (D) inRci, raslel2v; csk' ' . Mote that inR°A; raslG12v; csk2" animals fed control diet led to overgrowth but not secondary tumor formation. (E) inRCA, raslG12v; csk' " animals fed HDS. (Α'-Ε') Matching dissected eye epithelial tissue stained with DAPI (red) . (F) Quantitation of the observed phenotypes . The three color bars are explained in the legend of Fig. lj. Results are shown as mean ± SEM. (G and H) Dissected eye tissue of la.cZ (G) or raslal2v; csk'2" (H) animals fed control diet or HDS were treated with or without Insulin, and total Akt , phospho-Akt (p-Akt) , and Syntaxin (Syt) levels were examined by immunoblotting . The results of immunoblots were quantitated using Image J software and p-Akt/Syt values relative to subject without Insulin stimulation were determined. Results are shown as mean ± SEM. (I) Dissected eye tissue from raslal v; csk-''- animals fed HDS were treated with Insulin, and immunostained with anti-phospho-Akt (p-Akt) antibody. (J-0) Glucose uptake was examined by uptake of 2-NBDG of the dissected eye tissue from inRCA, raslal2v; csk'2' (J), raslcl v; csk"2" (K-M) , lacZ (N) , or raslal2v;csk- -,akt {0) animals fed oontrol diet or HDS, with or without Insulin stimulation. See also Figure 9.
Figure 4. Ras/Src-tumors are resistant to apoptosis in high sucrose diet
(A-C) Cleaved Caspase-3 staining (red) of rasl012'2; csk'2 ' (A and B) or inRCA, raslal2v; csk- ' (C! eye discs raised on indicated diets. (D-F) TUHEL assay (red) was used to label apoptotic cell death of raslal2v; csk-''- (D and E) or inRc", raslal2v; csk"'" eye discs raised on indicated diets. !G-JS β-gaiactosidase (S-gal) staining (red) of eye discs from retslG12v; csk" " , diapl-lacZ animals in a control diet (G) , HDS at day 13 AEL (H) , HDS at day 11 AEL (1), and inRC*, rasl012v; csk''" , diapl -lacZ animals in a control diet (J) . (K-N) Wingless (Wg) staining (red) of eye discs from raslG12v; csk~ ~ animals in a control diet ( ) , HDS at day 13 AEL (L) HDS at day 11 AEL (M) , and inRcl, raslG1 v; csk'2' , diapl-lacZ in a control diet (N) . See also Figure 10.
Figure 5. Wg mediates Ras/Src-tumorigenesis in HDS
(A-D) Animals raised on HDS with the genotype, (A) raslG1 v;csk~ " , (B) raslG12v;csk" ",wg M, <C) raslG12v; csk"2" , tc£m, (D) rasl012'2; csk"2', hskm. (A'-D'j Matching dissected eye epithelial tissue stained with DAPI (red) , (E and P) Wingless (Wg) staining (red) of _
eye discs raised on HDS (E) and iriRCA, raslG12v; csk"2" , bsk™ eye discs raised on a control diet (F) . (G) Dissected eye tissue of LacZ control or HA-Wg animals fed HDS were treated with or without Insulin, and total Akt , phospho-Akt (p-Akt ) , and Syntaxin (Syt) levels were examined by immunoblotting. The results of immunoblots were quantitated using Image J software. Results are shown as mean ± SEM. (H and I) Histogram showing the levels of rpL32, inR, chico, and InJt mRNAs measured by quantitative RT-PCR. Total RNA was isolated from LacZ-expressing control eye discs (H) or raslG12v; csk"2" eye discs (I) raised on a control diet (Black bar) or HDS (White bar) or raslel2v; csk"2", tcfD" eye discs raised on HDS (Grey bar) . Results are shown as mean ± SEM. Asterisks indicate statistically significant difference (*, p<0.01; **, p<0.05) . (J) Dissected eye tissue of raslG12'2; csk"2' animals fed control diet or HDS were treated with or without Insulin, and phospho-Insulin Receptor (p-InR) and Syntaxin (Syt) levels were examined by immunoblotting. (K) Model of diet-mediated tumorigenesis of Ras/Src-activated cells. See also Figure 11. Figure 6» Combinatorial multi-node drug treatment for Ras/Src-tumors la HDS
(Ά-Η) raslai2v f < csk~f'~ animals fed HDS containing (A) 0.05% DMSO, (B) 20 uM acarbose, iC) 25 μΜ pyrvinium, (D) 50 μΜ AD81, (E) 20 μΜ acarbose plus 25 μΜ pyrvinium, (Fj 20 uM acarbose plus 50 μΜ AD81, (G! 25 μΜ pyrvinium plus 50 μΜ AD81, (H) 20 μΜ acarbose plus 25 μΜ pyrvinium lus 50 μΗ AD81. All phenotypes were assessed at day 17 AEL , (I) Cell extracts from dissected eye tissue of raslal2v; csk"'' animals fed HDS supplemented with DMSO, AD80 or AD81 were examined by imrmmoblotting . (J) Percent pupariation of DMSO- or drug-treated rasla, V; csk-''~ animals was determined at day 17 AEL, Results are shown as mean ± SEM. See also Figure 12.
Figure 7. HDS diverts Ras/Sro-activated cells into aggressive tumors, Related to Figure 1
(A) HDS diverts Ras/Src-activated cells into aggressive tumors leading to larval lethality. A majority (77.5%) of raslal2v; esk~f~ animals fed HDS spent extra days as wandering larvae with multiple secondary tumors and failed to develop into pupal stage. (B) Developmental time course to pupariation of animals with all five genotypes fed control diet or HDS. Results are shown as mean ± SEM. (C) Quantitation of total eye disc area, GFP-positive area and GFP- negative area. Results are shown as mean ± SEM of individual eye discs .
Figure 8. HDS but not high fat induced cancer phenotypes. Related to Figure 2
(A-C) High dietary sugar but not high fat enhanced tumor growth. raslG12v; csk~-'~ animals raised on (A) 2 M glucose diet, (B) Banana and (C) calorie-matched high fat diet. (D) Laminin A staining (red) of raslal2v; csk-''- eye discs raised on HDS at day 9 AEL. (E and F) Src activity levels play an important role in secondary Ras/Src tumor formation. raslal2v;cskhyBa/hyBO animals fed control diet or HDS. Arrowhead indicates secondary tumor. (G) Quantitation of the observed phenotypes. The three color bars are explained in the legend of Fig. 1J. Results are shown as mean ± SEM. (H and I) raslG12v; scrih^/' animals fed control diet or HDS. (J) Quantitation of the observed phenotypes. The three color bars are explained in the legend of Fig. IJ. Results are shown as mean ± SEM. figure 9. The PI3K pathway mediates diet-induced ttamorieenesis, Related to Figure 3
raslal2v; csk~''~ animals fed HDS containing 0,1% DMSO, 50 uM wortmannin and 10 μΜ rapamycin.
Figure 10. Ras/Src-tumors are resistant to apoptosis in high sucrose diet. Related to Figure 4
{A) Quantitation of Cleaved Caspase-3 and TI EL positive clones. Results are shown as mean + SEM. (B-D) Diapl staining (red) of raslG1 v; csk'^ (B and C ) , ίη^°Λ, raslG12v; csk" " (D) eye discs raised on indicated diets. (E and P) Diapl and Wingless (Wg) staining (red) of rasloi2v; csk"'1^, aktl,^a h>'"a eye discs raised on HDS. (G) Cleaved Caspase- 3 staining (red) of secondary tumors from raslel ,/; csk~'~ animals raised on KDS . (H) 13~galactosidase staining (red) of secondary tumors from raslG12v;csk~ diapl -lacZ animals raised on HDS. Figure 11. Wg mediates Ras/Src-tumorigenesis in HDS, Related to Figure 5
(A) Over-expression of InR alone failed to elevate Wg levels. Wg staining (red) of inRCA eye discs raised on a control diet. (B-D) Reducing Wg levels rescue larval lethality of Ras/Src-activated cells in HDS. raslel2v; csk-'■ ,wgmAi (B) , raslG12v;csk'-'-, tcf™ (C) , raslGl2v; csk~ hskDM (D) animals fed HDS developed into pupal stage. Percentage of animals developed into pupal stage is indicated.
Figure 12. Combinatorial multi-node drug treatment for Ras/Src- tumors in HDS, Related to Figure 6
(A) Developmental time course to pupariation of raslG12v; csk~J~ animals fed HDS with (i) DMSO or (ii) 20 μΜ aoarbose plus 25 μΜ pyrvinium plus 50 μΜ AD81. (B) Pyrvinium did not affect normal growth of the LacZ-expressing control clones. LacZ animals fed HDS containing DMSO or 25 μΜ pyrvinium. Dissected eye epithelial tissue stained with DAPI (red) is also shown. (C) AD81 suppressed tumor growth and larval lethality of inRCA, raslG1 v; csk"~ animals. inRc"-,rasla3v;csk"''" animals were fed a control diet containing DMSO or ADS i .
Pgtailad Description of the Invention,
The present invention provides methods of treating a subject afflicted with a cancer and a metabolic dysfunction, which comprises adminis ering to the subject
(a) { i 3 a compound which is capable of inhibiting a glycoside hydrolase,
(ii) a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, or
(iii) both a compound of (a) {i} and a compound of {a) (ii); and
(b) at least one other compound which is capable of inhibiting
(i) insulin receptor (InR) metabolic signaling,
(ii) phosphatidylinositide 3-kinase ( PI3K) metabolic signaling,
(iii) InR/PI3 metabolic signaling,
(iv) c-Jun N-terminal kinase (Jnk) metabolic signaling,
( ) target of rapamycin (Tor) oncogenic pathway signaling,
(vi ) Ras oncogenic pathway signaling,
(vii) Src oncogenic pathway signaling, or
(viii) Ras/Src oncogenic pathway signaling, each such compound being administered in an amount such that, when administered in combination, administration of the compounds is effective to treat the subject.
In some embodiments, each compound administered to the subject is, independently, an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer, an RNA aptamer, a polypeptide, an antibody, an antisense oligonucleotide, an interfering RNA (RNAi } molecule, or a ribozyme.
In some embodiments , a compound that is capable of inhibiting a glycoside hydrolase is administered to the subject. In some embodiment*, the compound which is capable of inhibiting a glycoside hydrolase is an organic compound having a molecular weight less than 1000 Daltons . In some embodiments, the compound which is capable of inhibiting a glycoside hydrolase inhibits the catalytic activity of the glycoside hydrolase .
In some embodiments, the compound which is capable of inhibiting a glycoside hydrolase inhibits the ability of the glycoside hydrolase to bind a carbohydrate.
In some embodiments, the glycoside hydrolase is a glucosidase. In some embodiments, the glucosidase is a-glucosidase and the compound which is capable of inhibiting the glycoside hydrolase is an α-glucosidase inhibitor.
In some embodiments, the a-glucosidase inhibitor is acarbose, or a pharmaceutically acceptable salt or ester thereof.
In some embodiments, the compound which is capable of inhibiting a glycoside hydrolase is an antisense oligonucleotide, an R Ai molecule, or a ribozyme that targets α-glucosidase-encoding mR A and reduces -glucosidase expression.
In some embodiments, the compound which is capable of inhibiting a glycoside hydrolase is a compound approved for use in treating type 2 diabetes mellitus.
In some embodiments, a compound that is capable of inhibiting the canonical Wg/Wnt pathway signaling is administered to the subject.
In some embodiments, the canonical Wg/Wnt pathway signaling is canonical Wg/Wnt undead pathway signaling.
In some embodiments, the compound that is capable of inhibiting canonical Wg/Wnt pathway signaling is a Wg/Wnt inhibitor.
In some embodiments, the compound that is capable of inhibiting the canonical Wg/Wnt pathway signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets Wg/Wnt-encoding mRNA and reduces Wg/Wnt expression.
In some embodiments, the compound that is capable of inhibiting the canonical Wg W pathway signaling is pyrvinium, or a pharmaceutically acceptable salt or ester thereof.
In some embodiments, a compound that is capable of inhibiting Ras oncogenic pathway signaling is administered to the subject.
In some embodiments, the compound that is capable of inhibiting Ras oncogenic pathway signaling is a Ras inhibitor.
In some embodiments, the compound that is capable of inhibiting Ras oncogenic pathway signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets Ras-encoding mRNA and reduces Ras expression.
In some embodiments, a compound that is capable of inhibiting Src oncogenic pathway signaling is administered to the subject.
In some embodiments, the compound that is capable of inhibiting Src oncogenic pathway signaling is a Src inhibitor.
In some embodiments, the compound that is capable of inhibiting Src oncogenic pathway signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets Src-encoding mRNA and reduces Src expression.
In some embodiments, a compound that is capable of inhibiting Ras/Src oncogenic pathway signaling is administered to the subject.
In some embodiments, the compound that is capable of inhibiting Ras/Src oncogenic pathway signaling is AD81, or a pharmaceutically acceptable salt or ester thereof.
In some embodiments, a compound that inhibits Tor oncogenic pathway signaling is administered to the subject.
In some embodiments, the compound that is capable of inhibiting Tor oncogenic pathway signaling is a Tor inhibitor.
In some embodiments, the inhibitor of Tor oncogenic pathway signaling is rapamycin.
In some embodiments, the inhibitor of Tor oncogenic pathway signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets Tor-encoding mRNA and reduces Tor expression. In some embodiments, a compound that is capable of inhibiting PI3K metabolic signaling is administered to the subject.
In some embodiments, the compound that inhibits PI3K metabolic signaling is a PI3K inhibitor.
In some embodiments, the PI3K inhibitor is wortmannin, or a pharmaceutically acceptable salt or ester thereof.
In some embodiments, the compound that inhibits PI3K metabolic signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets PI3K-encoding mRNA and reduces PI3K expression .
In some embodiments, a compound that is capable of inhibiting InR metabolic signaling is administered to the subject.
In some embodiments, the compound that is capable of inhibiting InR metabolic signaling is an InR inhibitor.
In some embodiments, the compound that is capable of inhibiting InR metabolic signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets InR-encoding mRNA and reduces InR expression. In some embodiments, a compound that is capable of inhibiting InR PI3K metabolic signaling is administered to the subject.
In some embodiments, a compound that is capable of inhibiting Jnk metabolic signaling is administered to the subject.
In some embodiments, the compound that is capable of inhibiting Jnk metabolic signaling is a Jnk inhibitor. In some embodiments, the compound that is capable of inhibiting Jnk metabolic signaling is an antisense oligonucleotide, an RNAi molecule, or a ribozyme that targets Jnk-encoding mRNA and reduces Jnk expression. Some embodiments comprise administering to the subject an a- glucosidase inhibitor and a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling.
In some embodiments, the a-glucosidase inhibitor is acarbose and the compound which is capable of inhibiting canonical Wg/Wnt pathway signaling is pyrviniuin.
Some embodiments comprise administering to the subject an a- glucosidase inhibitor and a compound which is capable of inhibiting Ras/Src oncogenic pathway signaling.
In some embodiments, the οί-glucosidase inhibitor is acarbose and the compound which is capable of inhibiting Ras/Src oncogenic pathway signaling is AD81.
Some embodiments comprise administering to the subject a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling and a compound which is capable of inhibiting Ras/Src oncogenic pathway signaling.
In some embodiments, the compound which is capable of inhibiting canonical Wg/Wnt pathway signaling is pyrvinium and the compound which is capable of inhibiting Ras/Src oncogenic pathway signaling is AD81.
Some embodiments comprise administering to the subject an a- glucosidase inhibitor, a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, and a compound which is capable of inhibiting Ras/Src oncogenic pathway signaling.
In some embodiments, the α-glucosidase inhibitor is acarbose, the compound which is capable of inhibiting canonical Wg/Wnt pathway signaling is pyrvinium, and the compound which is capable of inhibiting Ras/Src oncogenic pathway signaling is AD81.
In some embodiments, the subject has an increased level of circulating insulin compared to a subject not afflicted with the metabolic dysfunction.
In some embodiments, the metabolic dysfunction is a metabolism- related disease.
In some embodiments, the metabolism-related disease is obesity. In some embodiments, the metabolism-related disease is diabetes. In some embodiments, the diabetes is type-2 diabetes.
In some embodiments, the metabolic dysfunction comprises hyperglycemia, hyperinsulinemia, or insulin resistance.
In some embodiments, the cancer is breast cancer, hepatocellular carcinoma, colorectal cancer, or pancreatic cancer.
In some embodiments, the cancer is breast cancer.
In some embodiments, the breast cancer is progesterone-receptor- negative breast cancer.
In some embodiments, the cancer is pancreatic cancer. In some embodiment , the pancreatic cancer is pancreatic duc al adenocarcinoma .
In some embodiments, the subject is a mammalian subject.
In some embodiments, the mammalian subject is a human subject.
In some embodiments , treating the subjec comprises reducing the progression of cancer in the subject.
In some embodiments, treating the subject comprises reducing the progression of a tumor in the subject.
In some embodiments , treating the subjec comprises prolonging survival of the subject.
In some embodiments , cells of the cancer expre s a higher amount of I R than cells from normal tissue of the same type.
In some embodiments, cells of the cancer have a higher amount of InR signaling than cells from normal tissue of the same type.
In some embodiments , cells of the cancer express a higher amount of fibroblast growth factor ( FGF) than cells from normal tissue of the same type .
In some embodiments, cells of the cancer comprise a loss-of -function mutation in an adenomatous polyposis coli (APC) gene.
In some embodiments , cells o the cancer express an oncogenic fi- catenin mutant .
In some embodiments, cells of the cancer express an oncogenic Ras mutant .
In some embodiments , the oncogenic Ras mutant is an oncogenic K-Ras mutant . In some embodiments, the oncogenic K-Ras mutant has substitution, wherein the numbering of the K-Ras amino acid sequence is relative to the sequence set forth in SEQ ID MO: 1 or 2. In some embodiments, the oncogenic K-Ras mutant has a G12V subs ution.
In some embodiments, the oncogenic K-Ras mutant protein is a R~ RasGI v mutant protein.
In some embodiments, the oncogenic K-Ras mutant has a GI2D substitution .
In some embodiments, the oncogenic K-Ras mutant is a K~RasGI2D mutant.
In some embodiments, the oncogenic K-Ras mutant has a G13X substitution, wherein the numbering of the K-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 1 or 2.
In some embodiments, the oncogenic K-Ras mutant has a Q61X substitution, wherein the numbering of the K-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 1 or 2. In some embodiments, the oncogenic Ras mutant is an oncogenic KF-Ras mutant .
In some embodiments, the oncogenic N-Ras mutant has a G12X substitution, wherein the numbering of the N-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 3.
In some embodiments, the oncogenic N-Ras mutant is a N-RasG12 or a N- RasG ;;D mutant.
In some embodiments, the oncogenic N-Ras mutant has a G13X substitution, wherein the numbering of the N-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 3.
In some embodiments, the oncogenic N-Ras mutant has a Q61X substitution, wherein the numbering of the N-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 3.
In some embodiments , the oncogenic Ras mutant is an oncogenic H-Ras mutant . in some embodiments, the oncogenic H-Ras mutant has a G12X substitution, wherein the numbering of the H-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 4 or 5.
In some embodiments, the oncogenic H-Ras mutant is a H-RasGi2 or a H-RasG12D mutant .
In some embodiments , the oncogenic H-Ras mutant has a G13X substitution, wherein the numbering of the H-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO : 4 or 5. In some embodiments , the oncogenic H-Ras mutant has a Q61X substitution , wherein the numbering of the H-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 4 or 5. In some embodiments , cells of the cancer express an oncogenic Src mutant .
In some embodiments , cells of the cancer express an oncogenic Csk mutant .
In some embodiments , the oncogenic Csk mutant has reduced activity compared to wild-type Csk.
In some embodiments, the oncogenic Csk mutant is a Csk^108 mutant.
In some embodiments, the oncogenic Csk mutant is truncated.
In some embodiments, the oncogenic Csk mutant is a Cskcl56stop mutant.
In some embodiments , the cells of the cancer express a lower amount of Csk than cells from normal tissue of the same type.
In some embodiments, the cells of the cancer express a higher amount of Csk than cells from normal tissue of the same type.
In some embodiments, the cancer comprises or is in the form of at leas one tumor .
In some embodiments, the cancer is metastatic.
Aspects of the present invention relate to the use of a composition comprising
(a) (i) a compound which is capable of inhibiting a glycoside hydrolas ,
( ii) a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, or
(iii) both a compound of (a) (i) and a compound of {a) (ii) ; and
(b) at least one other compound which is capable of inhibiting
{ i ) insulin receptor ( InR) metabolic signaling,
(ii) phosphatidylinositide 3-kinase (PI3K) metabolic signaling,
(iii) InR/Pl3K metabolic signaling,
(iv) c-Jun N-terminal kinase (Jnk) metabolic signaling,
(v) target of rapamycin (Tor) oncogenic pathway signaling,
(vi) Ras oncogenic pathway signaling,
(vii ) Src oncogenic pathway signaling , or
(viii) Ras/Src oncogenic pathway signaling, for treating a subject afflicted with a cancer and a metabolic dysfunction.
Aspects of the present invention relate to the use of
(a) (i ) a compound which is capable of inhibiting a glycoside hydrolase, ( ii ) a compound which is capable of inhibiting canonical Wg/ nt pathway signaling, or
(iii) both a compound of {a} { i ) and a compound of (a) (ii); and
(b) at least one other compound which is capable of inhibiting
{ i ) insulin receptor (InR) metabolic signaling,
(ii) phosphatidylinositide 3-kinase (PI3K) metabolic signaling,
(iii) InR/Pl3K metabolic signaling,
( iv) c-Jun N-terminal kinase (Jnk) metabolic signaling,
{v) target of rapamycin (Tor) oncogenic pathway signaling,
(vi) Ras oncogenic pathway signaling,
(vii) Src oncogenic pathway signaling, or
(viii ) Ras/Src oncogenic pathway signaling, the manufacture of a medicament f r the treatment of a subj ect flicted with a cancer and a metabolic dysfunction. The present invention provides combinations for treating a subj ect afflicted with a cancer and a metabolic dysfunction comprising
(a} ( i ) a compound which is capable of inhibiting a glycoside hydrolase,
( ii ) a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, or
{iii} both a compound of (a) (i) and a compound of (a) (ii) ; and
(b) at least one other compound which is capable of inhibiting
( i } insulin receptor ( InR) metabolic signaling,
(ii ) phosphatidylinositide 3-kinase ( PI3K) metabolic signaling,
(iii) InR/Pl3K metabolic signaling,
(iv) c-Jun H-terminal kinase (Jnk) metabolic signaling, (v) target of rapamycin (Tor) oncogenic pathway signaling,
(vi) Ras oncogenic pathway signaling, (vii) Src oncogenic pathway signaling, or
(viii) Ras/Src oncogenic pathway signaling.
The present invention provides pharmaceutical composi ions for treating a subject afflicted with a cancer and a metabolic dysfunction comprising
{a) ( i } a compound which is capable of inhibiting a glycoside hydrolase,
{ ii ) a compound which is capable of inhibiting canonical
Wg/Wnt pathway signaling, or
fiii) both a compound of (a) (i) and a compound o (a) (ii) ; and
(b) at least one other compound which is capable of inhibiting
(i ) insulin receptor ( InR) metabolic signaling,
(ii) phosphatidylinositide 3-kinase { PI3K) metabolic signaling,
(iii} InR/P13 metabolic signaling,
(iv) c-Jun N-terminal kinase (Jnk) metabolic signaling,
(v) target of rapamycin (Tor} oncogenic pathway signaling,
(vi) Ras oncogenic pathway signaling,
(vii) Src oncogenic pathway signaling, or
(viii) Ras/Src oncogenic pathway signaling.
Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.
It is understood that where a parameter range is provided, all integers within that range, and tenths thereof , are also provided by the invention . For example, "0.2-5 mg/kg/day" is a disclosure of 0.2 mg/kg/day, 0.3 mg/kg/day, 0.4 mg/kg/dayf 0.5 mg/kg/day, 0.6 mg/kg/day etc. up to 5.0 mg/kg/day. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs . As used herein, "about" in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.
Amino acid sequences of K-Ras are accessible in public databases by the accession numbers P01116 (Isoform 2A (identifier: P01116-1S; Isoforrti 2B (identifier: P01116-2 ) ) and NP_004976.2, and CCDS number CCDS8702.1, and are set forth herein as SEQ ID NOs : 1 and 2. Nucleotide sequences for K-Ras cDNA and the sequence of the K-Ras gene are accessible in public databases, e.g. from the Gene ID for K-Ras, which is Gene ID 3845.
The amino acid sequence of N-Ras is accessible in public databases by the accession numbers P01111 and NP„002515.1, and is set forth herein as SEQ ID NO: 3. Nucleotide sequences for N-Ras cDNA and the sequence of the N-Ras gene are accessible in public databases, e.g. from the Gene ID for N-Ras , which is Gene ID 4893.
Amino acid sequences of H-Ras are accessible in public databases by the accession numbers P01112 and NP_00112391 .1 (Isoform 1), and P01112-2 and (Isoform 2), and are set forth herein as SEQ ID NOS: 4 and 5. Nucleotide sequences for H-Ras cDNA and the sequence of the H-Ras gene are accessible in public databases, e.g. from the Gene ID for H-Ras, which is Gene ID 3265. The amino acid sequence of Csk is accessible in public databases by the accession numbers P41240 and NP_004374, and is set forth Herein as SEQ ID NO: 6. Nucleotide sequences for Csk cDNA and the sequence of the Csk gene are accessible in public databases, e.g. from the Gene ID for Csk, which is Gene ID 1445. Compounds of the Inver
Wortmannin is availaix >0 LC Laboratories,
Woburn, MA, USA) {www.lclab3.com/PRODFILE/S-Z/W-2990.php4) . The CAS Registry number for wortmannin is 19545-26-7. The structure of wortmannin is:
Figure imgf000027_0001
Raparaycin is available from LC Laboratories (R-5000; LC Laboratories, Woburn, MA, USA) (www.lclabs.com/PRODFILE/P-R/R-5000.php4) . The CAS Registry number for rapamycin is 53i23-88-9. The structure of
Figure imgf000027_0002
Embodiments of the subject invention relate to compounds which are capable of inhibiting a glucoside hydrolase. In some embodiments, the glucoside hydrolase is an alpha-glucos idase . Acarbose is available from Sigma (A8980; Sigma, St. Louis, MO,
OSA) (ww . sigmaaldrich . com/catalog/product/sigma/a8980?lang=enSregion= US). The CAS registry number for acarbose is 56180-94-0. The structure of acarbose is:
Figure imgf000028_0001
Sucrose is a disaccharide that is broken down (converted) into two mono - saccharides glucose and fructose by an enzyme called alpha- glucosidase. Acarbose inhibits enzymatic activity of alpha- glucosidase. Therefore, acarbose inhibits the conversion of sucrose into monosaccharides.
As used herein, "pyrvinium" means pyrvinium pamoate. Pyrvinium pamoate is available from Sigma (P0027 Sigma, St. Louis, MO, USA) (www . sigmaaldrich. com/catalog/product/sigma/p0027?lang=ensregion«US) . The CAS registry number for pyrvinium pamoate is 3546-41-6 alt hydrate is:
Figure imgf000028_0002
AD81 was provided by A. Dar and K. Shokat UCSF. AD81, and methods of synthesizing AD81 are described in: Dar, A.C., Das, T.K., Shokat, K.M., and Cagan, R.L. (2012). Chemical genetic discovery of targets and anti-targets for cancer polypharmacology. Nature 486, 80-84, the entire contents of which are incorporated herein by reference. The structure of AD81 is:
Figure imgf000028_0003
In some embodiments, a compound of the invention is an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer , an RNA aptamer, a polypeptide, an antibody, an antisense oligonucleotide, an interfering RNA (RNAi ) molecule, or a ribozyme.
Ester derivatives of compounds may be generated from a carboxylic acid group in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Ester derivatives may serve as pro-drugs that can be converted into compounds of the invention by serum esterases . Compounds used in the methods of the present invention may be prepared by techniques well know in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.
Compounds used in the methods of the present invention may be prepared by techniques described in Vogel ' s Textbook of Practical Organic Chemistry, A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, P.W.G. Smith, (Prentice Hall) 5th Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5th Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.
In some embodiments, a compound may be in a salt form. As used herein, a "salt" is a salt of the instant compound which has been modified by making acid or base salts of the compounds. In the case of the use of compounds of the invention for treatment of cancer, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines. The term "pharmaceutically acceptable salt" In this respect , refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the invention. These salts can be prepared in si tu during the final isolation and purification of a compound, or by separately reacting a purified compound in its free acid form with a suitable organic or inorganic base, and isolating the salt thus formed .
Antibodies
Antibodies may be employed in embodiments of the invention . In some embodiments the antibody is a monoclonal antibody.
An " antibody" as used herein is defined broadly as a protein that characteristically immunoreacts with an epitope (antigenic determinant) of an antigen. As is known in the art, the basic structural unit of an antibody is composed of two identical heavy chains and two identical light chains, in which each heavy and light chain consists of amino terminal variable regions and carboxy terminal constant regions. Antibodies of the invention include polyclonal antibodies , monoclonal antibodies (mAbs } , chimeric antibodies, CDR-grafted antibodies, humanized antibodies, human antibodies , catalytic antibodies , multispecific antibodies , as well as fragments, regions or derivatives thereof provided by known techniques, including, for example, enzymatic cleavage, peptide synthesis or recombinant techniques.
As used herein, "monoclonal antibody" means an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants , each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies , and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by 5 Kohler and Milstein, Mature 256:495-97 (1975! , or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolated from phage display libraries using the techniques described, for example, in Clackson et al . , N ture 352:624-28 (1991) and Marks et al . , J. Mol , Biol. 10 222 (3) :581-97 (1991) .
The term "hybridoma" or "hybridoma cell line" refers to a cell line derived by cell fusion, or somatic cell hybridization, between a normal lymphocyte and an immortalized lymphocyte tumor line. In
15 particular, B ceil hybridomas are created by fusion of normal B cells of defined antigen specificity with a myeloma cell line, to yield immortal cell lines that produce monoclonal antibodies. In general, techniques for producing human B cell hybridomas, are well known in the art [Kozbor et al . , Immunol. Today 4:72 (1983); Cole et al., in
20 Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. 77-96 (1985) ] .
The term "epitope" refers to a portion of a molecule (the antigen) that is capable of being bound by a binding agent, e.g., an antibody, 25 at one or more of the binding agent's antigen binding regions.
Epitopes usually consist of specific three-dimensional structural characteristics, as well as specific charge characteristics.
In some embodiments, an antibody is a humanized antibody. "Humanized 30 antibodies" means antibodies that contain minimal sequence derived from non-human immunoglobulin sequences. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hyper variable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor 35 antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205, each herein incorporated by reference. In some instances, framework residues of the human immunoglobulin are replaced by corresponding non-human residues (see, for example, U.S. Pat. NOB . 5,585,089; 5,693,761; 5,693,762, each herein incorporated by reference). Furthermore, humanised antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain desired af inity}. Tn general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or subst.antia.lly all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. For further details see Jones et al . , Nature 331:522-25 (1986); Riechmann et al., Mature 332:323-27 (1988); and Presta, Curro Opin. Struct. Biol. 2:593-96 (1992), each of which is incorporated herein by referenc .
In some embodiments, antibodies are xenogeneic or modified antibodies produced in a non-human mammalian host, more particularly a transgenic mouse, characterised by inactivated endogenous immunoglobulin (Ig) loci. In such transgenic animals, competent endogenous genes for the expression of light and heavy subunits of host immunoglobulins are rendered non-functional and substituted with the analogous human immunoglobulin loci. These transgenic animals produce human antibodies in the substantial absence of light or heavy host immunoglobulin subunits. See, for example, U.S. Pat. No. 5,939,598, the entire contents of which are incorporated herein by ref rence .
Those skilled in the art will be aware of how to produce antibody molecules of the present invention. For example, polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenic form of a molecule or protein which elicits an antibody response in the mammal. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained, and, if desired IgG molecules corresponding to the polyclonal antibodies may be isolated from the sera.
To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused wi h myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. Hybridoma cells can be screened immunochemically for production of antibodies which are specifically reactive with the oligopeptide, and monoclonal antibodies isolated.
Oligonucleotides
Antisense oligonucleotides are nucleotide sequences which are com lementary to a specific DWA or R A sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of target gene products in the cell.
Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates , phosphorothioates , phosphorodithioates, alkylphosphonothioates , alkylphosphonates, phosphoramidates , phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
Modifications of gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (Nicholls et al . , 1993, J Immunol Meth 165:81-91). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a target polynucleotide. Antisense oligonucleotides which comprise, for example, 1, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a target polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent nucleotides, can provide sufficient targeting specificity for a target mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides in length. Noncomplementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular target polynucleotide sequence. Antisense oligonucleotides can be modified without affecting their ability to hybridize to a target polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal rifaose. Modified bases and/or sugars, such as arabinose instead of ribose , or a 3', 51 -substituted oligonucleotide in which the 3 ' hydroxy1 group or the 5 ' phospha e group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art.
Ribozymes
Ribozymes are RNA molecules with catalytic activity (Uhlmann et al . , 1987, Tetrahedron. Lett. 215, 3539-3542). Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences . The coding sequence of a polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art. For example, the cleavage activity of ribozymes can be targeted to specific R As by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.
Specific ribozyme cleavage sites within an RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC . Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection , electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in. which it is desired to decrease target gene expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or VAS element, and a transcriptional teminator signal, for controlling transcription of ribozymes in the cells (U.S. 5,641,673). Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
RNA Interference
Some embodiments the invention relate to an interfering RNA (RNAi) molecule. RKAi involves mRNA degradation. The use of RNAi has been described in Fire et al . , 1998, Carthew et al . , 2001, and Elbashir et al . , 2001, the contents of which are incorporated herein by reference.
Interfering RNA or small inhibitory RNA (RNAi) molecules include short interfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), and micro-RNAs (miRNAs) in all stages of processing, including shRNAs, pri-miRNAs, and pre-miRNAs . These molecules have different origins: siRNAs are processed from double-stranded precursors (dsRNAs) with two distinct strands of base-paired RNA; siRNAs that are derived from repetitive sequences in the genome are called rasiRNAs; miRNAs are derived from a single transcript that forms base-paired hairpins. Base pairing of siRNAs and miRNAs can be perfect (i.e., fully complementary! or imperfect, including bulges in the duplex region.
Interfering RNA molecules encoded by recombinase-dependent transgenes of the invention can be based on existing shRNA, siRNA, piwi-interacting RNA (piRIJA) , micro RNA (miRNA) , double-stranded RNA (dsRNA) , antisense RNA, or any other RNA species that can be cleaved inside a cell to form interfering R As, with compatible modifications described herein.
As used herein, an "shRNA molecule" includes a conventional stem- loop shRNA, which forms a precursor miRNA (pre-miRNA) . *shRNA" also includes mioro-RNA embedded shRNAs (miRNA-based shRNAs ! ,' wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. When transcribed, a shRNA may form a primary miRNA (pri-miRNA) or a structure very similar to a natural pri-miRNA. The pri-miRNA is subsequently processed by Drosha and its cofactors into pre-miRNA. Therefore, the term "shRNA" includes pri-miRNA (shRNA-mir) molecules and pre-miRNA molecules.
A "stem- loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion). The terms "hairpin" and "fold-back" structures are also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem can include one or more base mismatches or bulges. Alternatively, the base-pairing can be exact, i.e. not include any mismatches.
"RNAi-expressing construct" or "RNAi construct" is a generic term that includes nucleic acid preparations designed to achieve an RNA interference effect. An RNAi-expressing construct comprises an RNAi molecule that can be cleaved in vivo to form an siRNA or a mature shRNA. For example, an RNAi construct is an expression vector capable of giving rise to a siR A or a mature shRNA in vivo. Non- 1imiting examples of vectors that may be used in accordance with the present invention are described herein and will be well known to a person having ordinary skill in the art. Exemplary methods of mak g and deli ering long or shor RNAi constructs can be found, for example, in O01/68836 and WO01/75164.
Use of RNAi
RNAi is a powerful tool for in vi tro and in vivo studies of gene function in mammalian cells and for therapy in both human and veterinary contexts. Inhibition of a target gene is sequence- specific in that gene sequences corresponding to a portion of the RNAi sequence, and the target gene itself, are specifically targeted for genetic inhibition. Multiple mechanisms of utilizing RNAi in mammalian cells have been described. The first is cytoplasmic delivery of siRNA molecules, which are either chemically synthesized or generated by DICER-digestion of dsRNA. These siRNAs are introduced into cells using standard transfection method . The siRNAs enter the RISC to silence target mRNA expression. Another mechanism is nuclear delivery, via viral vectors, of gene expression cassettes expressing a short hairpin RNA (shRNA) . The shR A is modeled on micro interf ring RNA (miRNA) , an endogenous trigger of the RNAi pathway {Lu et al . , 2005, Advances in Genetics 54: 117-142, Fewell et al 2006, Drug Discovery Today 11: 975-982). Conventional shRNAs , which mimic pre-miRNA, are transcribed by RNA Polymerase II or III as single-stranded molecules that form stem- loop structures . Once produced , they exit the nucleus , are cleaved by DICER, and enter the RISC as siRNAs.
Another mechanism is identical to the second mechanism, except that the shRNA is modeled on primary miRNA ishRNAmir) , rather than pre- miRNA transcripts (Fewell et al . , 2006 ) . An example is the rniR-30 miRNA construct . The use of this transcript produces a more physiological shRNA that reduces toxic effects. The shRNAmir is first cleaved to produce shRNA, and then cleaved again by DICER to produce siRNA. The siRNA is then incorporated into the RISC for target mRNA degradation. However, aspects of the present invention relate to RNAi molecules that do not require DICER cleavage. See, e.g., U.S. Patent No. 8,273,871, the entire contents of which are incorporated herein by reference.
For mRNA degradation, translational repression, or deadeny1ation, mature miRNAs or siRMAs are loaded into the RNA Induced Silencing Complex (RISC) by the RISC-loading complex (RLC) . Subsequently, the guide strand leads the RISC to cognate target KIRNAB in a sequence- specific manner and the Slicer component of RISC hydrolyses the phosphodiester bound coupling the target mRNA nucleotides paired to nucleotide 10 and 11 of the RNA guide strand. Slicer forms together with distinct classes of small RNAs the RNAi effector complex, which is the core of RISC. Therefore, the "guide strand" is that portion of the double-stranded RNA that associates with RISC, as opposed to the "passenger strand," which is not associated with RISC.
It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi inhibition by cleavage or blocking expression of the target mRNA. In preferred RNA molecules, the number of nucleotides which is complementary to a target sequence is 16 to 29, 18 to 23, or 21-23, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.
Isolated RNA molecules can mediate RN i . That is, the isolated RNA molecules of the present invention mediate degradation or block expression of mRNA that is the transcriptional product of the gene. For convenience, such mRNA may also be referred to herein as mRNA to be degraded. The terms RNA, RNA molecule(s), RNA segment(s) and RNA fragment (s) may be used interchangeably to refer to RNA that mediates RNA interference. These terms include double-stranded RNA, small interfering RNA (siRNA), hairpin RNA, single-stranded RNA, isolated RNA (partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA) , as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA) . Nucleotides in the RNA molecules of the present invention can also comprise nonstandard nucleotides, including non- naturaliy occurring nucleotides or deoxyribonucleotides . Collectively, all such altered RNAi molecules are referred to as analogs or analogs of naturally-occurring RNA. RNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi.
As used herein the phrase "mediate RNAi" refers to and indicates the ability to distinguish which mRNA molecules are to be afflicted with the RNAi machinery or process. RNA that mediates RNAi interacts with the RNAi machinery such that it directs the machinery to degrade particular mR As or to otherwise reduce the expression of the target protein. In one embodiment, the present invention relates to RNA molecules that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi inhibition by cleavage or blocking expression of the target mRNA.
In some embodiments, an RNAi molecule of the invention is introduced into a mammalian cell in an amount sufficient to attenuate target gene expression in a sequence specific manner. The RNAi molecules of the invention can be introduced into the cell directly, or can be compiexed with cationic lipids, packaged within liposomes, or otherwise delivered to the cell. In certain embodiments the RNAi molecule can be a synthetic RNAi molecule, including RNAi molecules incorporating modified nucleotides, such as those with chemical modifications to the 2 ' -OH group in the ribose sugar backbone, such as 2 ' -O-methyl (2'OMe), 2'-fluoro (2'P) substitutions, and those containing 2'0Me, or 2'F, or 2 ' -deoxy, or "locked nucleic acid" CLNA) modifications. In some embodiments, an RNAi molecule of the invention contains modified nucleotides that increase the stability or half-life of the RNAi molecule in vivo and/or in vitro. Alternatively, the RNAi molecule can comprise one or more aptamers, which interact (s) with a target of interest to form an a tamer : target complex. The aptamer can be at the 5' or the 3' end of the RNAi molecule. Aptamers can be developed through the SELEX screening process and chemically synthesi ed . An ap amer is general ly chosen to preferentially bind to a target . Suitable targets include small organic molecules, polynucleotides , polypep ides , and proteins . Proteins can be cell sur ace proteins , extracellular proteins, membrane proteins, or serum proteins, such as albumin . Such target molecules may be in ernalized by a cell, thus effecting cellular uptake of the shR A . Other potential targets include organelles, viruses, and cells.
As noted above, the R A molecules of the present invention in general comprise an RNA portion and some additional, portion, for example a deoxyribonnc1 otide portion. The total number of nucleotides in the RNA molecule is suitably less than in order to be effective mediators of RMAi . In preferred RNA molecules , the number of nucleotides is 16 to 29, more preferably 18 to 23, and most preferably 21-23.
Administration
"Administering" compounds in embodiments of the invention can be effected or performed using any of the various methods and delivery systems known to those skilled in the ar . The administering can be, for example, intravenous , oral , intramuscular , intravascula , intraarterial , intracoronary, intramyocardial , intraperitoneal, and subcutaneous . Other non-limiting examples include topical adminis ration, or coating of a device to be placed within the subjec . In embodiments , administration is effected by injection or via a cathete .
Injectable drug delivery systems may be employed in the methods described he ein include solutions , suspensions , gels . Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g. , hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch) , diluents (e.g. , lactose and other sugars, starch, dicalcium phosphate and cellulosic materials) , disintegrating agents (e.g. , starch polymers and cellulosic materials) and lubricating agents (e.g. , stearates and talc ) . Solutions , suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans , cellulosics and sugars), humectants (e.g., sorbitol), soiubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens , and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents {e.g., EDTA) ,
As used herein, the term "effective amount" refers to the quantity of a component that is sufficient to treat a subject without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention, i.e. a therapeutically effective amount. The specific effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.
The compounds used in embodiments the present invention can be administered in a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the compounds to the subject. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier. The compounds used in the methods of the present invention can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and ef rvescent preparations reconstituted from effervescent granules. Such iiguid. dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen .
Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al . , 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S, Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.
The dosage of a compound of the invention administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of the compound and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.
A dosage unit of the compounds of the invention may comprise a compound alone, or mixtures of a compound with additional compounds used to treat cancer. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion) , intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection or other methods, into the eye, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
A compound of the invention can be administered in a mixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone but are generally mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. In one embodiment the carrier can be a monoclonal antibody. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow- inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for exam le , suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents . Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
Specific examples of pharmaceutical acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U. S. Pat. No. 3,903,297, issued Sept. 2, 1975.
Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth , or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
A compound of the invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue- targeted emulsions. A compound of the invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers■ include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol , polyhydroxyethylasparta- midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, a compound may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters , polyacetals , polydihydropyrans , polycyanoacylates , and crosslinked or amphipathic block copolymers of hydrogels .
Gelatin capsules may contain a compound of the Invention and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets . Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
For oral administration in liquid dosage form, a compound may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol , glycerol , water, and the like . Examples of suitable 1 iquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solven s/ including es ers , emulsions , syrups or elixirs , suspensions , solutions and/or suspensions reconsti uted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules . Such 1 iquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents .
Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient , suitable stabilizing agents , and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilising agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol . Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.
A compound may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art . To be adminis ered in the orm of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
The compounds and compositions thereof of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.
Aspects of the present invention are also described in: Hirabayashi et al , , {2013) Transformed Drosophila Cells Evade Diet-Mediated Insulin Resistance through Wingless Signaling, Cell {In Press} , dx . doi . org/10.1016/j . cell .2013.06.030, the entire contents of which are incorporated herein by reference.
All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.
This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as defined in the claims which follow thereafter .
Experimental D tails
Exam les are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only .
Highlights :
· High dietary sugar enhances Ras/Src-mediated transformation in Drosophila
• Ras /Src-activated tumors evade diet-mediated insulin resistance
• Insulin resistance evasion is due to Wingless-mediated Insulin Receptor upregulation
· Rational targeting of multiple pathways can reduce diet-enhanced tumors
Example 1. High dietary sucrose enhanced tumor growth
Feeding Drosophila a standard diet supplemented to 1.0 M sucrose (high dietary sucrose or "HDS") led to metabolic defects reminiscent of specific aspects of type 2 diabetes including insulin resistance, hyperglycemia, elevated Drosophila Insulin-like peptide (DILP) levels, and accumulation of body fat (Musselman et al . , 2011). To explore the link between metabolic dysfunction and tumor progression Drosophila cancer models fed HDS were compared to those fed a control (0.15 M sucrose) diet. Combined elevation of Ras and Src pathway activities is a common motif in multiple cancer types including breast, colorectal, and pancreatic (Ishizawar and Parsons, 2004; Morton et al . , 2010). Experimentally, oncogenic K- as plus Src isoforms cooperate to accelerate the onset of pancreatic ductal adenocarcinoma in mice models, and Ras plus Src act together to promote tumorigenesis in Drosophila (Shields et al . , 2011; Vidal et al . , 2007) . This model was further developed by pairing transgenes that express oncogenic raslal2v in the eye with the genotypically null cskfi156staB allele of Csk, a primary negative regulator of Src kinase. Clones were labeled with GFP to visualize tumor progression. In control eyes, GFP expression was comparable in control diet vs. HDS, indicating that high sucrose feeding did not alter transgene expression levels (Figures 1A and IB) ,
Genotypically csk null clones alone were rarely obtained when fed either control diet or HDS (Figures 1C and ID) due to Src-mediated apoptosis ( idal et al . , 2006). Also as previously reported (Pagliarini and Xu, 2003), targeting rasl""" to the eye resulted in isolated benign tumors that in turn led to lethality as pupae (Figure IE) . raslG12v animals fed HDS exhibited at most a mild increase in growth within the eye field (Figure IF) . Eyes with elevated Ras plus Src activity iraslal2v; csk~''~) raised on a control diet displayed fewer and smaller GFP-positive clones compared to slGi '~expressing clones alone (Figures IE and 1G) , indicating that raslai-v expression cannot overcome elimination of csk mutant cells.
By striking contrast, clones displayed strongly increased proliferation in 96.5% of raslc,1:v; csk'- ' larvae fed HDS. The result was striking overgrowth and a significantly enlarged GFP-positive eye field that dominated the head region (Figures 1H and II) . This overgrowth had consequences on animal viability: a majority (77.5%) of raslG12v; csk"'' ' animals fed HDS failed to initiate pupariation, instead dying as larvae with multiple secondary tumors (Figure 7A) . Together, these results indicate that the metabolic status of the animals—as influenced by diet—strongly affects the outcome of Ras /Src-mediated tumorigenesis in the eye epithelium.
HDS feeding led to an approximately four day delay in completion of larval development (Musselman et al . , 2011) (Figure 7B); therefore, phenotypes were compared between developmental stage matched animals fed HDS vs. control diet (late third instar larvae) . To rule out the possibility that enhanced tumor growth in HDS fed animals was due to their older chronological age, progressive clone sizes in chronological age matched animals were also analyzed (Figures 1K-1S) At day 9 after egg laying (AEL) , HDS fed larval eye tissue was significantly smaller than age-matched controls (12.9%; Figures 1M' , IN' and 7C) but the percentage area occupied by GFP-positive clones was already significantly larger: 13.5% in control diet, 42.8% in HDS {Figures 1M' , IN' and IS) . This difference widened over time: control clones decreased from 35.2% (day 7 AEL) to 13.5% (day 9 AEL) of total area while HDS clones progressively increased, reaching 58.9% at day 11 AEL and 93.7% at day 13 AEL (Figures 1K-1S) . These analyses further support a role for HDS in enhancing growth of raslat2v;csk'!'' tumors.
Example 2 , HDS but not high f t induced cancer phenotypea
Diets containing 2.0 M glucose enhanced raslG12v;csk" " growth at a level similar to diets containing 1.0 M sucrose (Figure 1H and 8A) . Other diets including pure banana that have similar carbohydrate content yielded similar tumor enhancement (Figure 8B) , further emphasizing the specific role of high dietary carbohydrates. By contrast, rasl 2v; csk'^ tumors failed to show enhanced tumor growth in a calorie-matched high fat diet (Figure 8C) , indicating that high levels of dietary carbohydrates—but not high total caloric levels- are responsible for the enhanced cancer phenotype. This is consistent with observations that a high carbohydrate diet in general leads to metabolic defects in Drosophila not observed with calorie-matched high fat or high protein diets (Musselman et al . , 2011) .
Example 3. HDS promoted aspects of invasive migration and distant secondary tumors
Basement membrane degradation by Matrix Metalloproteases (MMPs) is a key early step in the initiation of cell spreading and metastasis (Deryugina and Quigley, 2006) . Eye epithelia from raslG12v; csJc~''~ animals fed a control diet displayed an intact basement membrane as assessed by Laminin A (Figure 2A) . HDS feeding led to broad loss of Laminin A; loss was already evident at day 9 AEL (Figure 2B and 8D) . However, raslG12v; csAr^-mediated up-regulation of MMPl expression was independent of dietary sugar (Figures 2E and 2F) . The data herein suggests that HDS enhances breakdown of the basement membrane to facilitate spreading and metastasis-like behavior of transformed cells, at least in part by mechanisms independent of MMPl expression levels . Larvae fed HDS consistently {93.0%) displayed GFP-labeled raslal2v; csk~ - cells scat ered throughout their hemolymph, a blood an.aj.ocj (Figures 21-2K and 2S ) - 19.1% of these larvae also developed secondary raBG12v;csk~ " tumors distant from the eye field that continued to proliferate (Figures 2L, 2P and 2S) secondary tumors were rarely (0.8%) observed in animals fed control food (Figure 23} . Whereas the percentage of animals with loss of Laminin in the eye tissue correlated with the percentage of animals with GFP-positive cells in the hemolymph, readily scorable secondary tumors were found in only a subset (19.1%) of the animals fed HDS (Figure 2S) .
Combining rasV312v with a weaker csk allele, csk :'we <rasl0""; ci**""*"") led to a reduced penetrance of secondary tumors (Figures 8E, 8F, 8F ' and 8G) , suggesting that levels of Src activity play a primary role in distant Ras/Src tumor formation. scribbled, which encodes a protein required to maintain apical/basal cell polarity was also examined (Bilder and Perriaon, 2000). As previously reported (Pagliarini and Xu, 2003), targeting raslG12v; scrib~'~ to the eye led to substantial overgrowth; however, secondary tumors in these animals were not observed when fed either a control diet or HDS (Figures 8H, 81 and 8J) . Without wishing to be bound by any scientific theory, these results indicate a specific relationship between Ras and Csk/Src activities that, when combined with HDS, synergize to produce secondary tumors. The data also indicate that tumor overgrowth and secondary tumor formation are separable processes and, similar to mammals (Mehlen and Puisieux, 2006), only a small subset of circulating cells successfully seed a secondary tumor . Larger secondary raslal2v; csJc * tumors were typically tightly associated with the tracheal system, a tubular network that transports oxygen (Figures 2L-20) . A Chitin-binding probe (Devine et al . , 2005) indicated that secondary tumors enwrapped tracheal branches (Figure 2Q) . Interestingly, Chitin was abnormally deposited within these tumor-imbedded trachea (Figure 2Q) , suggesting either tracheal alteration or de novo branching. Consistent but not proving the latter view, secondary raslG12v;csk~'~ tumors expressed Branchless (Figure 2R) , a fly FGF ortholog capable of promoting de novo trachea branching {Sutherland et al . , 1996). Together, these results indicate that HDS enhances or establishes multiple aspects of tumorigenesis in the presence of activated as/Src including proliferation, a block in apoptosis, invasive migration, and establishment of secondary tumors distant from the primary tumor.
Example 4. The P13K pathway mediates diet-induced tumorigonesis
Genetically reducing PI3K activity through a hypomorphic allele of akt ( raslsl2v; csk-''", akthwt">a; Figure 3B) or through feeding of the ΡΪ3Κ inhibitor wortmannin (Figure 9) led to strongly reduced tumor cell survival in the presence of HDS. The Tor-class inhibitor raparaycin displayed a similar reduction in tumor tissue (Figure 9); in addition, animals were significantly smaller with an extended third instar larval stage as previously reported (Zhang et al . , 2000). Conversely, co-expressing the constitutively active Insulin receptor isoform inRA ( inRA, raslf;)' ; csk'' ') in animals fed a control diet prevented elimination of clones from the epithelia. The result was tumor overgrowth in which 91.0% of larvae displayed an enlarged eye disc phenotype (Figures 3D and 3F) . inRCA, raslal v; csk' ~ animals did not exhibit any further increase in primary tumor size when raised on HDS (Figure 3E) , nor did they exhibit enhanced tumors when compared to raslal2v; csk~-'~ in HDS (compare Figure 1J and 3F) . Without wishing to be bound by any scientific theory, the conclusion herein is that components of the Insul in/PI3K pathway are required autonomously within the raslGl v; csJc~/_-transformed cells for enhancement by HDS.
Of note, Insulin PI3 signaling did not mediate all effects of HDS. While the triple combination inRCA, raslelw; csk'^ fed a control diet exhibited strongly enhanced expansion of tumors within the eye field, overgrowth was not accompanied by metastasis-like behavior or secondary tumor formation. Further, though elevated MP1 expression was observed the basement membrane was still retained (Figures 2C and 2G) . This result again emphasizes that tumor overgrowth and metastasis are separable processes.
Example 5. Rae/Src-ac ivated cells remain sensitive to Insulin and glucose
The results herein suggest that insulin signaling is required to mediate HDS-enhanced growth. However, eye epithelial tissue in control animals raised on HDS exhibited reduced phosphorylated, activated Akt upon Insulin stimulation, an indication of insulin resistance as previously reported {Figure 3G (Musselman et al . , 2011). This led to a central question in tumor metabolism studies: How does a diet that promotes insulin resistance also enhance tumors in an Insulin/PI3K-dependent manner?
In contrast to controls, eye tissue within raslal2v; csi"'" animals fed HDS exhibited an increase in baseline Akt when compared to control diet-fed animals (Figure 3H) , suggesting these tissues were in fact not insulin resistant. Consistent with this view, challenging eye tissue from HDS animals with exogenous Insulin led to a strong increase in phospho-Akt (Figure 3Hi . Insulin-stimulated increase in phospho-Akt was observed autonomously within the raslGli"; csk~/- clones (Figure 31). These data indicate that activating the as and Src pathways reversed HDS-induced insulin resistance, leading to hypersensitive responsiveness to Insulin. The result was increased sugar flux: in the presence or absence of exogenous Insulin, rasli;12v; csk~'- animals raised on HDS displayed a significant increase in glucose uptake to a level comparable to the triple combination inRCf-, raslG1~v;csk'/- (Figures 3J-3N) . Reducing akt activity (rasl01 v;csk-'-, aicfw*"") blocked glucose uptake (Figure 30). Without wishing to be bound by any scientific theory, the conclusion herein is that raslm2v;csk~'~ cells escape diet-induced insulin resistance to utilize both increased insulin responsiveness and insulin pathway-dependent glucose uptake. The mechanisms by which transformed cells evade insulin resistance were explored next.
Example 6. Tumors exhibit aspects of an 'undead' phenot pe
A central hallmark of mammalian cancer is the ability of transformed cells to evade apoptosis. In Drosophila developing eye and wing epithelia, keeping otherwise apoptotic cells alive ('undead') by blocking ef ector caspase activity leads to aberrant tissue overgrowth due to compensatory proliferation of surrounding cells, a process designed to maintain tissue size and integrity (reviewed in (Bergmann and Stellar, 2010; Fan and Bergmann, 2008; Martin et al . , 2009) , An antibody that recognizes the cleaved, active isoform of human caspase-3 was used, a validated readout for activity of the Drosophila initiator caspase Drone (Fan and Bergmann, 2010) . Broad Drone activity was observed in rasl012v; csk~ " clones of animals raised on a control diet (Figure 4A and 10A) . A significant proportion of these cells were undergoing apoptotic cell death as assessed by TI EL staining (Figure 4D and 10A} .
In the presence of HDS, ra.slal2v; csk~'~ clones also exhibited broad cytosolic Drone activity (Figure 4B and 10A) . However, these clones were almost completely negative for apoptotic cell death as assessed by TUNEL and DAPI analysis (Figure 4E and 10A; data not shown) ; a few cells outside of the clone region were TUNEL positive. Insulin pathway activation alone was sufficient to mimic this block: inRCA, raslG12v; csk~ ' animals fed a control diet retained Drone caspase activity but, again, were resistant to apoptotic cell death as assessed by TUNEL (Figures 4C, 4F and 10A) , One candidate to block apoptosis in transformed cells is the caspase inhibitor Drosophila Inhibitor of apoptosis protein 1 (Diapl), which inhibits caspases downstream of Drone (Ditzel et al., 2008) . diapl gene expression and Diapl protein levels were strongly increased in the raslG12v; csk'/' clones of animals raised in HDS compared to animals fed a control diet (Figures 4G-4I, 10B and IOC) . The triple combination inRCA, raslal2v; csk''- was sufficient to elevate diapl expression and Diapl protein levels in animals fed a control diet, whereas reduced aJ t activity (rasleuvicskr'-, aj tiw failed to elevate Diapl in HDS animals (Figures 4J, 10D and 10E) . These results indicate that elevation of insulin pathway signaling through HDS leads to elevated Diapl and a block in apoptotic cell death downstream of cleaved, activated Drone. Interestingly, secondary tumors retained diapl expression but lost Drone activity (Figure 10G and 10H) .
Ex mple 7. Increased Wingless/Wat-signaling mediates diet-induced tujBorigenesis .
Previous work in Drosophila compensatory proli eration found that 'undead' cells also elevate expression of the Writ ortholog Wingless (Wg) ; this mitogenic signal in turn directs local hyperplasia (Perez-Garijo et al . , 2009). In raslG1 v; csk^2' eye clones, low levels of Wg protein were detected within the clones in animal fed a control diet (Figure 4K) . Notably, Wg protein levels were strongly unregulated in animals fed HDS (Figures 4L and 4M) . Reducing ake activity (raslG12v; csk~r'~, akthY"'/hyrx') prevented this elevation (Figure iOF) , while the triple combination inRCA f rasl 2v; csk"2' displayed increased Wg levels in animals fed a control diet (Figure 4N) . Activation of InR alone was not sufficient to elevate Wg levels (Figure 11A) . Without wishing to be bound by any scientific theory, the conclusion herein is that combining elevated InR, Ras, and Src activities leads to elevated Wg expression in discrete tumors.
Elevation of Wg contributed to tumor progression. Reducing Wg activity (raslG12'"'; csk"2", wg"1"'1) in animals fed HDS led to strong suppression of tumor growth (Figure SB) . Expression of a dominant- negative isoform of the downstream pathway effector Tcf tcf™ {raslG1 v;csk~'~, tcfDS) also suppressed tumor growth (Figure 5C) , indicating that canonical Wg signaling is required for high sucrose- induced enhancement of tumo igenesis . As a result, while only 22.5% of raslG12v; csk'2' animals successfully initiated pupariation when fed HDS, 100% of ra.slel2v;csk-'-, wg™" or raslel2v; csr'-, tcf™ animals successfully pupariated when fed HDS (Figures 11B and 11C) . In 'undead' cells, Jnk pathway signaling is required for ectopic Wg expression (Ryoo et al . , 2004). Expressing a dominant-negative isoform of the Drosophila Jnk ortholog Basket bskm {raslal v; csk~'~ ,bskD") suppressed tumor growth in HDS (Figures 5D and 11D) and blocked the elevation in Wg protein levels (Figure 5E) , indicating that Wg upregulation is downstream of Bsk. Wg was also blocked in inRci, raslal2v; csk~'-, bskBN eye clones of animals fed a control diet, indicating Bsk is in turn downstream of InR with regard to elevated Wg (Figure 5F) . Together, these results indicate that raslal2v; cak' cells achieve an 'undead' status through Diapl, which then activates a inR-Jnk-Wg pathway that promotes cell autonomous proliferation. It was next asked whether this ability of Wg to mediate key aspects of HDS insulin pathway activity within Ras/Src-activated tumors extended to evasion of insulin resistance.
Example 8. Canonical Wingless/Wnt pathway signaling promotes Insulin sensitivity by regulating InR expression
In organ culture experiments from animals fed HDS, challenging control tissue with exogenous Insulin led to little or no increase in pathway activity as assessed by phosphorylated Akt (Figure 5G) ; significant downregulation of inR mRHA was also observed (Figure 5H) , which presumably at least partially accounts for the tissues' insulin resistance. Two other members of the Insulin pathway, Chico/IRS and Lnk SH2B, showed no change in transcript levels (Figure 5H) , suggesting that diet-directed downregulation is specific to InR. In contrast, tissue expressing Wg responded to exogenous Insulin by strongly activating pathway signaling (Figure 5G) . This suggests that—in addition to Insulin regulating Wg—Wg regulates Insulin signaling, potentially through regulation of inR expression .
Consistent with this model, inR mRHA levels were significantly upregulated in rasl0""'; csk~'~ eye clones in animals fed HDS (Figure 51! . The result was an increase in phosphorylated, activated InR that was strongly enhanced when the tissue was challenged with exogenous Insulin (Figure 5J) . Importantly, the diet-dependent increase in inR m NA levels was suppressed by reducing activity of the Wg target Tcf (raslG12v; csk' ' r tcfm; Figure 51) . These results indicate that canonical Wg signaling promotes insulin sensitivity by upregulating inR transcripts. That is, transformed tissue evaded insulin resistance through a feed forward mechanism by which elevated Wg directs elevated InR (Figure 5K) .
Example 9, Combinatorial multi-node drug treatments for diet-induced Ras/Src -tumoriqenesia
Identifying an HDS-dependent ' undead' cell phenotype in raslal2v; csk~ tissue suggested specific points of therapeutic intervention: (i) conversion of dietary sucrose, iii) InR/PI3 metabolic signaling, !iii) canonical Wg/ nt pathway 'undead' cell signaling, and (iv) Ras plus Src oncogenic pathways (Fig. 5K) . Nearly three-quarters of raslG12v; csk~:'- animals raised on HDS failed to pupariate due to aggressive eye tumors; pupariation rate was utilized as a quantitative viability assay to take a stepwise approach towards identifying an optimized whole animal therapeutic cocktail. Time to pupariation was not affected by drug treatments when compared to vehicle (DMSO) treatment alone; pupariation rates were assessed at day 17 AEL , after pupariation curves reached a plateau (Figure 12A) .
Acarbose is an inhibitor of alpha-glucosidase that is used to treat type 2 diabetes mellitus (Anderson, 2005) . Feeding acarbose in the presence of HDS led to reduced tumor growth and improved raslallv; csk~ animal survival (Figures 6B and 6J) , Pyrvinium, a small-molecule inhibitor of canonical Wht-signaling pathway (Thorne et al . , 2010), suppressed HDS-induced tumor growth at a concentration that did not affect growth of wild type clones (Figures 6C and 12B) : 41.9% of HDS- and pyrvinium-fed animals achieved pupariation (Figure 6J) . Polypharmacological anti-cancer compounds were recently developed (Dar et al., 2012) that target the Ras, Src, and Tor pathways as single agents. These represent the central cellular pathways that mediate HDS-induced tumor growth in raslG12v; csk' ' animals and, indeed, the compound AD81 suppressed Ras/Src-tumorigenesis and larval lethality of animals fed HDS (Figures 6D and 6J) . Biochemical analysis of dissected eye tissues supported inhibition of Ras, Src and Tor pathways by AD81 feeding, though not by the close analog AD80 (Figure 61; (Dar et al . , 2012). Furthermore, AD81 also suppressed tumor growth and larval lethality of inRCA, raslel2v; csk~/~ animals fed a control diet, demonstrating AD81 can act tumor- autonomously (Figure 12C! . Targeting sugar or the Ras , Src , Pi3K , or Wg pa hways showed significant but limited efficacy on raslal!v; csk~'~ tumors in animals fed HDS. Combining acarbose with pyrvinium, acarbose with AD81, or pyrvinium with AD81 enhanced survival to permit 64. % f 62.9%, and 68.5%, survival of raslal v; csk~ animals to pupariation in the presence of HDS, respectively (Figures 6E-6G, and 6J) . Strikingly, the triple combination acarbose-pyrvinium~AD81 achieved a 91.2% rescue level of raslal3v; csk~J~ animals fed HDS {Figures 6H and 6J) , far higher than any single or double drug combination we have tested. Without wishing to be bound by any scientific theory, the conclusion herein is that targeting multiple nodes in a rational manner may be required to achieve an optimal therapeutic index, balancing efficacy with minimal whole animal toxicity. Example 10. Experimental Procedures for Examples 1-9
Fly Stocks
UAS~raslG12v, UAS~inRA1325D (inRCA), a*t«»* , csk'lm, UAS-tcP*, UAS-bsk∞, UAS~HA~wg flies were obtained from the Bloomington Drosophila Stock Center. UAS-wgmAi flies were obtained from Vienna Drosophila R Ai Center. The following stocks were kindly provided to us: FRT82B, cskPL56SCOB by A. O'Reilly and M. Simon; scrib1 by D. Bilder; ey(3.5)- FLP1 by G. Haider.
To create eyeless-driven green fluorescent protein (GFP) -labeled clones, flies with the genotype ey(3.5) -FLP1 ; act>y+>ga!4, UAS-GFP; FRT82B, tub-gal80 were crossed with flies with the following genotypes: (a) UAS-lacZ; FRT82B; (b) UAS-lacZ; FRT82B, csk?l5ssc°'/TM6b; (c) UAS- aslG1 v; FRT82B; (d) UAS-raslel v; FRT82B, Csk°156sc°e/TMSb; (e) UAS-raslG12v; FRT82B, csk2lmVTM6b; (f) UAS-raslG12v; FRT82B, scrib1/TM6b; (g) UAS-inRA1325D, UAS-raslG12v; FRT82B, csk >/TM6b; (h) UAS-raslG12v; FRT82B, cskP1"^™, akt0422i;/TM6b; (i) OAS-raslal2v; FRT82B, cskPls6scef, UAS-wgmA1/TM6b; (j) UAS-raslG12v; FRT82B, csk^15^"", UAS~tcfm/TM6b; (k) UAS-raslel2v; FRT82B, csk9156st<">, UAS-bskm/TM6b. (1) UAS-inRA1325D, UAS- raslcl2v; FRT82B, cskP1Ms o", UAS-bskm/TM6b. (m) UAS-inRA13 5n; FRT82B. (n) FRT82B, UAS-HA-wg. Cultures
Cultures were carried out on Bloomington semi-defined medium (described by the Bloomington Drosophil stock center) with modifications. Detailed recipes for control diet, HDS , and high fat diet is previously described (Musselman et al . , 2011). The following final concentrations of carbohydrates were included; 0.15 Ϊ sucrose (control diet), 1.0 M sucrose (HDS), 2.0 M glucose (high glucose diet) . Cultures were performed at 25°C unless otherwise noted. Immunofluorescence and Tt EL Labeling
Larval eyes were dissected in PBS, fixed in 4% paraformaldehyde (PFA) in PBS or PLP-fixative (2.5% paraformaldehyde, 0.075 M lysine. 0.25% (w/v) Ma-periodate in phosphate buffer) , washed in PBT ( PBS containing 0.1% Triton X-100) , and incubated with primary antibodies in PAXDG (PBS containing 1% BSA, 0.3% Triton X-100, 0.3% deoxycholate , and 5% goat serum) , followed by washing and incubation with secondary antibodies in PAXDG. Tissues were counter-stained with TO-PRO-3 (Invitrogen) and mounted in Vectashield mounting media (Vector Laboratories). Antibodies used were: rabbit anti-Cleaved Caspase-3 (Aspl75; Cell Signaling), mouse anti-dMMPl (DSHB: Developmental Studies Hybridoma Bank), mouse anti-Wingless (DSHB), mouse anti-foeta-galactosidase (DSHB) , rabbit anti-Laminin beta 1 (Abeam), rabbit anti-phospho-Histone H3 (SerlO; Millipore) , rabbit anti-Diapl (gift from H. Steller) , rabbit anti-phospho-Akt (Ser473; Cell Signaling) , rat anti-Branchless (gift from M. Krasnow) . Secondary Alexa 568-conjugated anti-mouse and anti-rabbit antibodies were used (Molecular Probes). To label tracheal lumen, Rhodamine- conjugated Chitin-binding Probe was used (New England BioLabs). TUNEL (terminal deoxynucleotidyl transferase-rnediated deoxyuridine- triphosphate nick end-labeling) was performed using the TMR-Red In Situ Cell Detection Kit (Roche Diagnostics). Samples were fixed for 15 minutes in 4% PFA in PBT, washed in PBT and permeabilized in 100 mM sodium citrate/0.1% TritonX-100 at 65°C for 30 minutes, washed in PBT, and free DNA ends were fluorescein-labeled . Fluorescent and confocal images were taken with Leica DM5500 microscope equipped with DFC340 FX Monochrome Digital Camera and TSC SPE confocal microscope. Drugs
Compounds solubilized in DMSO were diluted directly into the fly medium and vortexed extensively to obtain homogeneous culture. Drugs used were: wortmannin and rapamycin (LC Laboratories); acarbose and pyrvinium pamoate (Sigma) . AD compounds were provided by A. Dar and K. Shokat COCSF) .
Western Blotting
Wandering third instar larvae were rinsed in PBS, the eye-discs were dissected out, and placed in Schneider's Drosophila Medium on ice. The eye-discs were incubated with or without 5 μΜ recombinant human Insulin (Sigma) for 15 minutes at room temperature. Eye discs were transferred to a micro centrifuge tube, spun down briefly. The supernatant were discarded and the remaining precipitates were suspended in SDS sample buffer at a concentration of 10 μΐ mg tissue and used to generate Western blots. Antibodies used targeted: total Akt (Cell Signaling), phospho-Drosopiiila Akt (Ser505; Cell Signaling), phospho-IGF-1 Receptor β (Tyrll31 ) /Insulin Receptor β (Tyrll46 ; Cell Signaling), and Syntaxin (DSHB) . Secondary antibodies were from Cell Signaling.
Glucose Dptake Assay
For the glucose uptake experiments, flies with the genotype ey{3.5) FLP1; act>y+>gal4; FRT82B, tub-gal80 were crossed with flies with the following genotypes: (a) UAS-lacZ; FRT82B; (b) UAS-raslel2v; FRT82B, cskfi15esc°v/TM6b (c) UAS inRA132S , UAS-raslal2v; FRT82B, cskP1Msca"/TM6b; (d) UAS-raslG12v; FRT82B, csk , aktM226/TM6b.
Wandering third instar larvae were rinsed in PBS, the eye-discs were dissected out, and placed in Drosophila Larval Saline (Hepes-NaOH (pH7.1), 87 mM NaCl, 40 mM KC1, 8 mM CaCl2, 8 mM MgCla, 50 mM Sucrose, 5 mM Trehalose) on ice. The eye-discs were incubated with or without 5 μΜ recombinant human Insulin (Sigma) in the presence of 2-(Af-(7- nitrobenz-2-oxa-l , 3 -diazoI-4-yl ! amino) -2-deoxyglucose (2 -NBDG :
Invitrogen) for 15 minutes at room temperature. The tissues were washed with Drosophila Larval Saline and mounted with a Vectashield (Vector Laboratories) .
Quantitative RT-PCK
Total R A was extracted from the eye discs using QIAGEN R easy Mini Kit. Oligo-dT primers and Superscript RT-II reverse transcriptase (Invitrogen) was used to synthesize first strand DNA. PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) and analyzed on Applied Biosystems StepOnePlus real-time PCR system.
Results were normalized to Kinesin fflRNA, and RpL32 was used as a control. The following primers were used : Kinesin-f , 5'-
GCTGGAC TCGGTCGTAGAG-3 ' (SEQ ID NO: 7) ; inesin- , 5'- CTTTTCATAGCGTCGCTTCC-3 ' (SEQ ID NO: 8) ; RpL32-f , 5'-
GCTAAGCTGTCGCACAAATG-3 ' (SEQ ID NO: 9) RpL32-r, 5' -
GTTCGATCCGTAACCGATGT-3 ' ί SEQ ID NO : 10); InR-f , 5' -
ACAAAATGTAAAACCTTGCAAATCC- 3 ' (SEQ ID NO: 11 ) ; InR-r, 5' -
GCAGGAAGCCCTCGATGA-3 ' (SEQ ID NO: 12); chico-f , 5 ' -ATCAGGCGATGCGGTC 3' (SEQ ID NO: 13); chico-r, 5 ' -ACATAGCGCTCAGTATCG-3 ' (SEQ ID NO 14); dLnk-f , 5 ' -GTGTTCGCCTAAAGACAAATGAAAT-3 · (SEQ ID NO: 15); dLnk- 5 ' -TGTTAGCGTTGTGGGATCCAA-3 ' (SEQ ID NO: 16).
Discussion
Risk of specific cancers increases in patients with metabolic dysfunction including obesity and diabetes. Examples described herein use Drosophila as a model to explore the effects of diet on tumor progression. Feeding Drosophila a diet high in carbohydrates was previously demonstrated to direct metabolic dysfunction including hyperglycemia, hyperinsulinemia and insulin-resistance. The examples described herein demonstrate that high dietary sugar also converts Ras/Src transformed tissue from localized growths to aggressive tumors with emergent metastases. While most tissues displayed insulin resistance, Ras/Src tumors retained insulin pathway sensitivity, increased the ability to import glucose, and resisted apoptosis. High dietary sugar increased canonical Wingless Wht pathway activity, which upregulated Insulin Receptor gene expression to promote insulin sensitivity. The result is a feed-forward circuit that amplified diet-mediated malignant phenotypes within Ras/Src transformed tumors. By targeting multiple steps in this circuit with rationally applied drug combinations, the examples described herein demonstrate the potential of combinatorial drug intervention to treat diet-enhanced malignant tumors.
Recently, the American Diabetes Association and American Cancer Society jointly published a consensus report emphasizing accumulating evidence that patients with diabetes also show elevated risks of specific cancer types (Giovannucci et al . , 2010) . The increase in metabolic diseases worldwide highlights metabolic dysfunction as an increasing issue in cancer progression. However, the mechanisms by which metabolic dysfunction contribute to cancer progression remain poorly understood.
Epidemiological studies have provided strong evidence for the association between cancer and metabolic diseases including diabetes and obesity {Barone et al . , 2008; Calle et al . , 2003; Coughlin et al . , 2004; Inoue et al . , 2006} : patients with metabolic dysfunction have both a higher incidence of specific tumors types and higher overall cancer-related mortality. For example, obese patients with progesterone receptor-negative breast cancer had a higher risk of lymph node metastasis, suggesting that metabolic dysfunction can promote tumor aggressiveness (Maehle et al . , 2004).
Obesity and type 2 diabetes are typically associated with chronic hyperinsulinemia : levels of insulin in the blood rise to compensate for insulin resistance. Increased circulating insulin levels are in turn a risk factor tor the development of hepatocellular carcinoma and colorectal cancer (Donadon et al . , 2009; Kaaks et al . , 2000). Together with the well-documented mitogenic effects of insulin { Ish- Shaiom et al . , 1997), this evidence suggests a role for hyperinsulinemia as a promoter of enhanced tumorigenesis in obese and diabetic patients. However, the development of insulin resistance in metabolism-related diseases raises a key question: how do tumors overcome insulin resistance to take advantage of increasing insulin levels? The examples herein utilise Dros phila to explore the effects of high dietary sugar on tumor progression in vivo .
Feeding Drosophila a diet supplemented to 1.0 M sucrose led to insulin resistance, hyperglycemia, increased insulin levels (hyperinsulinemia), and accumulation of fat, phenocopying important aspects of type 2 diabetes (Musselman et al . , 2011) . This model was used to explore the effects of high dietary sugar on tumor progression in vivo. On a normal diet, Csk/Src-dependent tumors were eliminated from the epithelium either alone or when paired with an oncogenic Ras isoform. In contrast, elevated dietary sugar strongly synergized with Ras/Src to generate large tumors associated with aggressive metastasis-like behavior. Using this model system, whether increased insulin levels underlie induction of malignant tumors was investigated. Importantly, Ras/Src-activated tumors remained sensitive to insulin in dietary conditions that promoted insulin resistance in surrounding tissues. The examples herein demonstrate that increased insulin/PI3K signaling in turn prevents apoptosis and promotes canonical Wingless /Wnt mitogenic signaling in Ras/Src tumors. Our results provide functional data that, in the face of high dietary sugar, Ras/Src-activated cells escape insulin resistance to exhibit enhanced growth that is dependent on Wingless nt mitogenic signaling.
Epidemiological studies provide strong evidence for an association between metabolism-related diseases and cancer but the underlying mechanisms that link these two diseases remain poorly understood. In the examples herein, it is demonstrated that dietary sucrose strongly enhances multiple aspects of Ras/Src-activated tumors in Drosophila. HDS led to stabilization of tumors within the epithelium increased proliferation, and metastasis-like behavior.
Mechanistically, the examples herein provide evidence that Ras/Src- activated cells increase insulin pathway-sensitivity in an otherwise insulin-resistant environment, allowing them to take advantage of high circulating glucose. The result is a Wg- and Jnk- dependent enhancement of tumor progression. The results herein support a model in which Ras/Src cells become hypersensitive to circu.la.ting insulin, leading to efficient activation of insulin/PI3K signaling (Figure 5K) . A key consequence of increased insulin/PI3K signaling was prevention of apoptosis and promotion of elevated levels of Wg expression and activity; these two aspects promoted an 'undead' -like cell phenotype that further enhanced proliferation. Low but detectable levels of Wg expression in raslel2v; cskr'- clones were observed in animals fed a control diet (Figure 4K) ; these clones exhibited high levels of apoptosis. In contrast, Wg accumulated at significantly higher levels in raslG12v; csk' ' tumors within animals fed HDS (Figure 4L) . High Wg levels led to elevated Diapl expression and reduced apoptosis, stabilizing raslG12v; csk'^ clones and revealing accumulation of Wg within the clone. The model described in the examples herein suggests that inhibition of cell death in 'HDS tumors' is a key aspect of Ras/Src-mediated transformation may allow cells to stably and continuously produce Wg. Wg was expressed within raslGl2v: csk~-'~ cells in the presence of HDS {Figure 4L and 4M) and phosphorylated Akt levels increased within Ras/Src clones but not detectably in nearby wild type cells (Figure 31) . Therefore, unlike the conventional undead situation—induced by Hid plus P35—a Wg mediated increase in InR expression and signaling provides a growth advantage specifically within as/Src cells rather than surrounding wild type tissue. Together, the data herein provide evidence that the 'undead' cell phenotype generates an InR-Jnk~ g-InR feed-forward mechanism that results in elevated InR expression and a reversal of insulin resistance (Figure 5K) . The result is transformed tissue that is optimized for insulin-dependent glucose uptake in an animal with hyperinsulinemia and hyperglycemia.
The 'undead' phenotype was not fully maintained in secondary tumors: Drone expression was lost in distant tumors of raelal2V;csk/~, though diapl expression was maintained (Figure 10G and 10H) . These results suggest that secondary tumors develop progressively distinct properties; understanding these changes will require additional studies. A Chitin-binding probe suggested progressive steps of tubule maturation within secondary tumor-associated trachea including potentially newly expressed Chitin scattered within immature tubules, tubules filled with solid projections of Chitin, and hollowed out mature tubules (Figure 2Q) . Additional studies will be required to confirm that this represents neo-tracheogenesis and, if so, whether this occurs through processes reminiscent of the neo- angiogenesis that is a common feature of mammalian metastasis (Bergers and Benjamin, 2003). Strikingly, secondary tumors expressed high levels of the FGF ortholog Branchless, an important step in promoting tracheogenesis during development (Figure 2R; (Sutherland et al. , 1996) ) .
The Drosophila nt ortholog Wg promotes tumors' ability to evade insulin-resistance through its regulation of inR expression in the presence of HDS feeding, which otherwise promotes diabetes-like phenotypes (Figure 5H; (Musselman et al . , 2011). High Wg expression requires both Ras/Src-activation and HDS, promoting inR expression through canonical Wg-dTcf signaling (Figure 51). These results emphasize a link between canonical Wg signaling and Insulin- signaling. Consistent with this model linking canonical Wg and Insulin signaling pathways, a recent study reports that reduced expression of skeletal muscle Insulin Receptor—linked to hyperinsuiinemia and insulin resistance—is enhanced by a mutation in the Wnt co-receptor LRP6 in humans (Singh et al . , 2013).
Tumors with Wat-activation may represent tumor types that respond and synergize with metabolic defects. Wnt-acti ation is frequently observed in many tumor types including those with strong association with diabetes such as hepatocellular carcinomas and colorectal cancers; activating mutations in β-catenin and loss of function mutation in APC is frequently found in human hepatocellular carcinoma and colorectal cancers (Fodde et al . , 2001; Laurent-Puig and Zucman-Rossi , 2006). Incidence of pancreatic cancer has a particularly strong association with diabetes, suggesting its constituent mutation load may interface with metabolic dysfunction. Activating mutations in K-Ras plus up-regulation of Src activity are commonly found in pancreatic ductal adenocarcinoma (PDA) (Almoguera et al . , 1988; Morton et al . , 2010), and a mouse K-Ras/Src pancreatic model developed PDA with shortened latency (Shields et al . , 2011). Wnt ligands and transcriptional target genes of S-catenin are highly expressed in PDA (Pasca di Magliano et al., 2007), a phenomenon reminiscent of observations in the examples herein linking diet, Ras/Src, and Wnt signaling. A clear link has yet to be established between pancreatic cancers featuring elevated Ras/Src activity and/or Wnt-pathway activation within diabetic patients. The data herein indicate that limiting dietary sucrose directly or through blocking its intermediates through compounds such as Acarbose may reduce important aspects of tumor risk and progression in individuals with metabolism-related diseases. Combining acarbose with drugs that target the triggering Ras/Src pathways plus components of the amplification circuit provided the most robust therapeutic impact on diet-enhanced tumors in our model. These results provide a potential explanation for how insulin resistant animals are at increased risk for tumorigenesis , and emphasize the importance of targeting multiple, specific nodes to achieve optimal therapeutic value. Seferegces
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Claims

A method of treating a mammalian subject afflicted with a cancer and a metabolic dysfunction, which comprises administering to the subject
(a) (i) a compound which is capable of inhibiting a glycoside hydrolase,
(ii ) a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, or
( iii ) both a compound of (a) ( i ) and a compound of (a) {ii} ; and
(b) at least one other compound which is capable of inhibiting Ras/Src oncogenic pathway signaling, each such compound being administered in an amount such that, when administered in combination, administration of the compounds is effective to treat the subj ect .
The method of claim 1, wherein each compound administered to the subject is , independently, an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer , an R A aptame , a polypeptide, an antibody, an antisense oligonucleotide, an interfering RNA (RHAi) molecule, or a ribozyme .
The method of claim 1 or 2 , wherein a compound that is capable of inhibiting a glycoside hydrolase is administered to the subject .
The method of claim 3, wherein the compound which is capable of inhibiting a glycoside hydrolase is an organic compound having a molecular weight less than 1000 Daltons.
The method of any one of claims 1-4, wherein the compound which is capable of inhibiting a glycoside hydrolase inhibits the catalytic activity of the glycoside hydrolase. The method of claim any one of claims 1-5 wherein the compound which is capable of inhibiting a glycoside hydrolase inhibits the ability of the glycoside hydrolase to bind a carbohydrate .
The method of any one of claims 1-6 , wherein the compound which is capable of inhibiting the glycoside hydrolase is an -glucosidase inhibitor .
The method of claim 7 , wherein the a-glucosidase inhibitor is acarbose, or a pharmaceutically acceptable salt or ester thereof .
The method of any one of claims 1-8 , wherein the compound which is capable of inhibiting a glycoside hydrolase is a compound approved for use in treating type 2 diabetes mellitus .
The method of any one of claims 1-9, wherein a compound that is capable of inhibiting the canonical Wg/Wnt pathway signaling is administered to the subject .
The method of claim 10, the wherein compound that is capable of inhibiting canonical Wg/Wnt pathway signaling is a Wg/Wnt inhibitor .
The method of claim 11 , wherein the compound that is capable of inhibiting the canonical Wg/Wnt pathway signaling is pyrvinium, or a pharmaceutically acceptable salt or ester thereof .
The method of any one of claims 1-12, wherein the compound that is capable of inhibiting as/Src oncogenic pathway signaling is AD81 , or a pharmaceutically acceptable salt or ester thereof .
The method of any one of claims 1-13, comprising administering to the subj ect an α-glucosidase inhibitor , a compound which is capable of inhibiting canonical g/ nt pathway signaling, and a compound which is capable of inhibiting Ras/Src oncogenic pathway signaling.
The method of claim 14, wherein the a-glucosidase inhibitor is acarbose, the compound which is capable of inhibiting canonical Wg/Wnt pathway signaling is pyrvinium, and the compound which is capable of inhibiting Ras/Src oncogenic pathway signaling is AD81.
The method of any one of claims 1-15, wherein the metabolic dysfunction is a metabolism-related disease and comprises hyperglycemia, hyperinsulinemi or insulin resistance.
The method of claim 16, wherein the metabolism-related disease is obesity or diabetes .
The method of claim 17, wherein the metabolism-related disease is diabetes, and the diabetes is type-2 diabetes.
The method of any one of claims 1-18, wherein the cancer is breast cancer, hepatocellular carcinoma, colorectal cancer, or pancreatic cancer.
The method of any one of claims 1-19, wherein the mammalian subject is a human subject.
The method of any one of claims 1-20, wherein cells of the cancer (1) express a higher amount of InR than cells from normal tissue of the same type, (2) have a higher amount of InR signaling than cells from normal tissue of the same type, (3) express a higher amount of fibroblast growth factor (FGF) than cells from normal tissue of the same type, (4) comprise a loss-of-function mutation in an adenomatous polyposis coli (APC) gene, (5) express an oncogenic E-catenin mutant, (6) express an oncogenic Src mutant or (7) express an oncogenic Csk mutant . The method of any one of claims 1-21, wherein cells of the cancer express an oncogenic K-Ras, N-Ras , or H-Ras mutant.
The method of claim 22, wherein cells of the cancer express {1} an oncogenic K-Ras mutant that has a G12X substitution, a G13X substitution or a Q61X substitution, wherein the numbering of the K-Ras amino acid sequence is relative to the sequence set forth in SEQ ID MO: 1 or 2;
(2) an oncogenic N-Ras mutant that has a G12X substitution, a G13X substitution or a Q61X substitution, wherein the numbering of the N-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO: 3; or
(3) an oncogenic H-Ras mutant that has a G12X substitution, a G13X substitution or a Q61X substitution, wherein the numbering of the N-Ras amino acid sequence is relative to the sequence set forth in SEQ ID NO : 4 or 5.
The method of any one of claims 1-23, wherein the cancer is metastatic .
A combination for treating a mammalian subject afflicted with a cancer and a metabolic dysfunction comprising
(a) (i) a compound which is capable of inhibiting a
glycoside hydrolase,
(ii) a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, or
(iii) both a compound of (a) (i) and a compound of (a) (ii) and
(b) at least one other compound which is capable of inhibiting Ras/Src oncogenic pathway signaling.
A method of treating a subject afflicted with a cancer and a metabolic dysfunction, which comprises administering to the subj ect
(a) (i) a compound which is capable of inhibiting a
glycoside hydrolase, (ii) a compound which is capable of inhibiting canonical Wg/Wnt pathway signaling, or
(iii) both a compound of (a) (i) and a compound of (a) (ii); and
(b) at least one other compound which is capable of inhibiting
(i) insulin receptor { InR) metabolic signaling,
(ii) phosphatidylinositide 3-kinase (PI3K) metabolic signaling,
(iii) InR/PI3K metabolic signaling,
(iv) c-Jun N-terminal kinase (Jnk) metabolic signaling,
(v) target of rapamycin (Tor) oncogenic pathway signaling,
(vi ) Ras oncogenic pathway signaling,
(vii) Src oncogenic pathway signaling, or
(viii) Ras/Src oncogenic pathway signaling, each such compound being administered in an amount such that, when administered in combination, administration of the compounds is effective to treat the subject.
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