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WO2014092572A1 - Méthodes améliorées de traitement du cancer à l'aide d'un agent génotoxique - Google Patents

Méthodes améliorées de traitement du cancer à l'aide d'un agent génotoxique Download PDF

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Publication number
WO2014092572A1
WO2014092572A1 PCT/NL2013/050892 NL2013050892W WO2014092572A1 WO 2014092572 A1 WO2014092572 A1 WO 2014092572A1 NL 2013050892 W NL2013050892 W NL 2013050892W WO 2014092572 A1 WO2014092572 A1 WO 2014092572A1
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Prior art keywords
agent
cancer
cells
treatment
susceptibility
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Inventor
Ramakrishnaiah SIDDAPPA
Jordi Carreras PUIGVERT
Dimitrios TYPAS
Louise Marie-Agnes Maximiliane Freiin VON STECHOW
Erik Hendrik Julius DANEN
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Universiteit Leiden
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Universiteit Leiden
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • 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
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of cancer therapy, more specifically to methods and means for treating an individual suffering from cancer with a genotoxic agent.
  • the methods and means of the invention will improve a response to treatment with a ge no toxic agent.
  • a damage provides the risk of toxicity and the accumulation of mutations and chromosomal instability, potentially resulting in malignant transformation (Ciccia and Elledge, 2010. Mol Cell 40: 179-2042010;
  • DDR DNA damage response
  • Regulators of genome stability have been identified, including infrared- induced double strand brakes repair foci, and genotoxic stress-induced apoptosis (Arora et al 2010, Gynecol Oncol 118:220-227; Kolas et al. 2007, Science 318: 1837-1640; M&cKeigan et al. 2005, Nat Cell Biol 7: 591 -600; Paulsen et al, 2009. Mol Cell 35: 228-239).
  • the DDE is a highl conserved process but somatic cells, ste cells, and cancer cells show variations on the theme through, developmental-, tissue specific-, or oncogenic alterations in expression of components of the DDR.
  • p5B plays a major role in the DDR in somatic cells while its role in embryonic stem cells (ESC) is debated and p53 signaling is inactivated in the majority of cancer cells (Harper et al. 2007. MoL Cell 28: 739-745; Aladjem et aL 1998. Current Biology 8: 145 - 155).
  • the large variety of genotoxieily-induced defects reflects the pleiotropic nature of DNA damage normally occurring through ongoing cellular metabolism and exposure to environmental mutagens,
  • DDR DNA damage response
  • the present invention provides a susceptibility agent, for use in a method for treatment of an individual suffering from cancer, whereby said
  • susceptibility agent is combined with a genotoxic agent.
  • a susceptibility agent according to the invention improves treatment of the individual by the genotoxic agent, compared to treatment with the genotoxic agent in the absence of a susceptibility agent.
  • the invention also provides a method for treatment of an individual suffering from cancer, comprising (a) providing said individual with a genotoxic agent; and (b) providing said individual with a susceptibility agent, whereby treatment is improved compared to treatment in the absence of said susceptibility agent.
  • the invention also provides use of a susceptibility agent, in the preparation of a medicament for treatment of an individual suffering from cancer, whereby said medicament is combined with, a genotoxic agent, whereby treatment of the individual is improved compared to treatment in the absence of said susceptibility agent.
  • gene refers to an agent that induces damage in the genomic DNA of a cell that arrests the cell cycle, activates repair
  • genomic DNA damage includes base modifications, single strand breaks, and crosslinks, such as intrastrand and interstrand cross-links.
  • a preferred genotoxic agent is selected from an alkylating agent such as nitrogen mustard, e.g.
  • cyclophosphamide mechlorethamine or in us tine, uramustine and/or uracil mustard, melphalan, chlorambucil, ifosfamide; nitrosourea, including carnmstine, lomustine, stre tozocin; an alkyl sulfonate such as bnsulfan, an ethylenime such as thiotepa and analogues thereof, a hydrazine/triazine such as dacarba ⁇ ine, altretamine, niito3 ⁇ 4olomide, temoKolomide, altretamine, procarbazine, dacarba3 ⁇ 4ine and temozolomide; an intercalating agent such as a platinum-based compound like cisplatin, carb platin, nedapiatin, oxaHplatin and sa raplatin; antbraeyelines such as doxorubicin,
  • a further preferred genotoxic agent is provided by radiation, including ultraviolet radiation and gamma radiation.
  • a more preferred genotoxic agent is selected from gamma radiation, a platinum-based compound and or an anthracyclin,
  • a most preferred genotoxic agent is cisplatin and/or doxorubicin.
  • Cisplatin is administered at 2 to 8 mg kg every 3 to 4 weeks or at 20 mg m2/day for 5 days every 3 to 4 weeks; at 40 mg-120 mg/m2 every 8 to 4 weeks.
  • Cisplatin is preferably administered for treatment of advanced bladder cancer, malignant melanoma and osteogenic sarcomas.
  • Cisplatin is preferably administered by injection or infusion, preferably by intravenous, intraarterial or intraperitoneal injection or infusion.
  • antbracyclms sneh as doxorubicin, daunorabicin, epirnbicin and idarubiein are routinely administered at 40-75 mg m2, every 3 weeks for treatment of, for example, breast cancer, uterine cancer, ovarian cancer, and lung cancer.
  • gamma radiation is administered in a dose that depends on the tumour type, whether radiation is given alone or with chemotherapy, before or after snrge y, the success of surgery as is known to the skilled person.
  • a dose that depends on the tumour type, whether radiation is given alone or with chemotherapy, before or after snrge y, the success of surgery as is known to the skilled person.
  • curative treatment of a solid epithelial tumor radiation dose raging from 50-70 Gy is administered, In case of lymphomas 20-40 Gy is given.
  • This dose is given in daily fraction called the fraction schedule.
  • adnlts receive 1,8-2 Gy per fraction.
  • the typical treatment schedule is 5 days per week. These small frequent doses allow healthy cells time to grow back, repairing damage inflicted by the radiation.
  • Daily fractions of radiation are given using an external source or an internal source such as implants.
  • Said individnal may snffer from breast cancer, ovarian cancer, lung cancer, liver cancer, head and neck cancer, squamous cell carcinoma, bladder cancer, colorectal cancer, cervical cancer, renal cell carcinoma, stomach cancer, prostate cancer, melanoma, brain cancer, and/or esophageal cancer.
  • a susceptibility agent as herein defined, is an agent that improves treatment of au individual suffering from cancer by a genotosde agent.
  • a susceptibility agent makes cancer cells more sensible to the DNA damaging effects of a genotoxie agent
  • a susceptibility agent might hamper or inhibit the DNA damage response (DDR) in cancer cells, or parts of the DDR, and thereby improves the response of cancer cells towards a genotoxie agent.
  • DDR DNA damage response
  • a preferred susce ibility agent inhibits expression and/or activity of a product of a gene selected from the group consisting of FRKDl, BITSP15, ARIH1, UBE1X, RPL7L1, USPS and EHP.
  • Protein kinase Bl encodes serine/tkreonine-protem kinase Dl (EC 2.7.11,13).
  • Alternative names for the serine/threonine -protein kinase are PKC-mu, PKB1, PRKCM1, PKC-mu, PKCM1, and PKC-MU.
  • Dual specificity phosphatase 15 encodes dual specificity phosphatase 15 (EC 8.1.3.483).
  • Alternative names for the phosphatase are vaccinia virus VII 1 -related dual-specific protein phosphatase (VR ' Yl), dual specificity protein phosphatase 15, dual specificity phosphatase-like 15, and
  • ligase H7 AP2, HHARI, monocyte protein 6 (MOP-6), UbeH7 -binding protein, IJbcM4 nteracting protein, ubiquitin-eonjugating enzyme E2 ⁇ binding protein 1,
  • Alternative gene names are ARI, ⁇ 6, and UBCH7BP.
  • Eukaryotic niRNAs are mostly recruited to the ribosome through their 5' 7-methylguanosine cap (Kong and Lasko 2012. Nat Rev Genet 13; 383-394,).
  • the rate -limiting step of eukaryotic cap- dependent translation initiation is the binding of the translation initiation factor eIF4F to the mRNA S'eap structure.
  • EIF4F is composed of the eap5 binding protein eIF4E, the R A helicase eiF4A and the scaffold protein eiF4G (G ngras et al. 1999, Armu Rev Bioehera 68, 913-963: Gross et al. 2003. Cell 115, 739-750).
  • ARIHl is a mediator of a protective DNA damage response in cancer ceils that prevents aberrant translation, after genotoxic stress.
  • a preferred susceptibility agent inhibits the expression and/or activity of ARIHl, and thereby improves the response of cancer cells towards a genotoxic agent.
  • Eukaryotic translation initiation factor 4E family member 2 (4EHP or EIF4E2) encodes eukaryotic translation initiation factor 4E family member 2,
  • Alternative names for the protein are eukaryotic translation initiation factor 4E ike 3 (EIF4EL3), mRNA cap-binding protein 4E11P, mRNA cap- ? binding protein type 3.
  • EIF4EL3 eukaryotic translation initiation factor 4E ike 3
  • 4E11P mRNA cap-binding protein
  • 4E11P mRNA cap- ? binding protein type 3.
  • 4SHP is a competitive inhibitor of the translation initiation factor eIF4E.
  • 4EHP translocates to the 5 s rnRNA-cap and arrests nsRNA translation upon DNA damage, in an ARIHl-dependent manner.
  • downmodulation of 4EHF hampers or prevents DNA damage-induced translation arrest.
  • Downmodulation of 4EHP improves the response of cancer cells to genotoxie stress.
  • a preferred susceptibility agent inhibits the expression and/or activity of 4EHP and thereby improves the response of cancer cells towards a genotoxic agent,
  • Ubiqui tin-activating enzyme El encodes a protein that catalyzes the first step in iihiquitm conjugation to mark cellular proteins for degradation.
  • Alternative names for the phosphatase are ubiquitin-like modifier activating enzyme 1 (IJBAI A1S9: A1ST; GXPL UBEl. A1S9T, AMCX1, POC2G, SMAX2, and UBA1A.
  • Ribosomal protein L? ⁇ like 1 (RPL7L1) encodes ribosomal protein L7 ⁇ like 1.
  • An alternative name for the protein is 80S ribosomal protein L7 fke.
  • UBPY1 Uluqxii tin-specific-processing protease 8
  • Methods for do wnmodulatin g and/or inhibiting expression and or activity of a product of a gene selected from the group consisting of AEIHl, DUSP15, UBEIX, RPL7L1, USpS, 4EHP and PRKD1 are known in the art. and include RNA antisense expression, RNA interference and introduction, of small inhibitor molecules.
  • a preferred susceptibility agent according to the invention is an antisense RNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIH1, DUSF15, UBEXX, RPL7L1, USp8 s 4E.HP and PRKD1
  • a further preferred susceptibility agent according to the invention is an antisense RNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIHI, DUSP15, RPL7L1, USp8 s 4EHP and PRKD ' L
  • Yet a further preferred susce tibility agent according to the invention is an anti sense RNA compound,, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of
  • a further preferred susceptibility agent according to the invention is an antisense RNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIHI, DUSP15, RPL7L1 and PRKD1.
  • a farther preferred susceptibility agent according to the invention is an antisense RNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIH I, DUSP15 and PKKD L
  • a further preferred susceptibility agent according to the invention is an antisense SNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIHI and DUSPX5
  • Yet a further preferred susceptibility agent according to the invention is an antisense RNA compound, a targeting RNA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of ARIHI.
  • a susceptibility agent according to the invention preferably an antisense RNA compound, a targeting ENA molecule, and/or a small inhibitor molecule that downmodulates and/or inhibits expression and/or activity of a product of a gene selected from the group consisting of ARIH ' l, DUSP15, UBS IX, EPL7L1, USp8, 4 ⁇ and PRKD1, is preferably used in a method for treatment of a individual suffering from cancer, whereby said
  • susceptibilit agent is combined with a genotoxic agent
  • Antisense RNA compounds a e commonly used as researcli reagents and diagnostics.
  • antisense oligonucleotides which are able to inhibit gene expression with extraordinar specificity, are often used by those of ordinary skill to elucidate the function of particular genes.
  • the specificity and sensitivity of antisense compounds is harnessed by those of skill in the art for therapeutic uses, Antisense oligonucleotides and antisense
  • oligonucleotides and polynucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides and antisense polynucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides and polynucleotides can be useful therapeutic modalities that can be configured to he useful in treatment regimes for treatment of cells, tissues and animals, especially humane.
  • polynucleotide refers to an polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DMA) or mimetics thereof.
  • RNA ribonucleic acid
  • DMA deoxyribonucleic acid
  • This term includes oligonucleotides composed of naturally- occurring nucleobases, sugars and cova!ent iutemucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occnrring portions which function similarly.
  • modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
  • a preferred polymer is or corresponds to more than 30 nucleotides, more preferred more than 50 nucleotides, more preferred more than 100 nucleotides, more preferred more than 200 nucleotides, more preferred more than 500 nucle
  • oligonucleotide refers to an oligomer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • oligonucleotides composed of naturally- occurring nucleobases, sugars and covalent intemucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.
  • Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
  • antisense oligonucleotides and antisense polynucleotides are a preferred form of antisense compound
  • the present invention comprehends other oligomeric or polymeric antisense compounds, including but not limited to oligonucleotide mimetics and polynucleotide mimetics such as are described below.
  • Preferrd oHgonucleotic antisense compounds in accordance with this invention preferably comprise from about 8 to about 30
  • nucleobases Particularly preferred are antisense oligonucleotides
  • nucleoside is a base-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines .
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2 3' or 5 ' hydroxyl moiety of the sugar.
  • the phosphate groups eovalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • oligonucleotides containing modified backbones or non-natural internucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • phosphorus atom in their internncleoside backbone can also he considered to be oligonucleosides.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioat.es, chiral phosphorothioates,
  • phosphorodithioates phosphotriesters, aminoalkyl-phosphotriesters, methyl and other a Iky I phosphonates including 3 ! -alkyIene phosphonat.es and chiral phosphonates, phosphinates, phosphoramidates including 3 '-ammo phosphoramidate and aminoalkylphosphoramidates ⁇
  • thionoalkylphosphotriesters and boranophosphates having normal 3' -5' linkages, 2' -5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are Hnked 3' ⁇ 5 ! to 5' -3 f or 2" - 5 ! to 5 ' ⁇ 2'.
  • Preferred modified oligonucleotide backbones that do not include a
  • phosphorus atom therein have backbones that are formed by short chain alkyl or cyeloa!kyl internncleoside linkages, mixed heteroatom and alky! or cycioalkyl internncleoside linkages, or one or more short chain heteroatomic or heterocyclic internncleoside linkages.
  • These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; form acetyl and thiofonnaeetyl backbones; methylene form acetyl and thioforxnacetyl
  • backbones a!kene containing backbones; sulfamate backbones;
  • both the sugar and the internncleoside linkage, i.e.. the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • a peptide nucleic acid (PNA) an oligonucleotide mimetic that lias been shown to have excellent hybridisation properties.
  • PNA peptide nucleic acid
  • nucieobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Modified oligonucleotides may also contain one or more substituted sugar moieties, Preferred oligonucleotides comprise one of the following at the 2' position ; OH; F; 0-, S-, or N-alkyl: Q- , S-, or N-alkenyl; 0 » , S- OF N-alkynyl; or O- alkyl-O alkyl, wherein the alkyl, alkenyl and alkyny).
  • CIO alkyl may he substituted or unsuhstituted C to CIO alkyl or 02 to CIO alkenyl and alkynyl. Particularly preferred are 0[(CH2)nQ]mCH3, 0(CH2)nOCH3, 0(CH2)nNH2,
  • oligonucleotides comprise one of the following at the 2* position: CI to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkvL G-alkaryl or 0-araikyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3 f NH2, heterocycloalkyl heterocyeloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, a intercalator, a group for
  • a preferred modification includes 2 ' -methoxyethoxy (2 ' -0-CH2CH2OCH3, also known as 2 ' -0- (2- methoxyethyl) or 2 ' -MOE) (Martin et al. 1995, Helv. CM . Acta 78: 488-504) i.e., an alkoxyalkoxy group.
  • a further preferred modification includes 2 ' ⁇ dimethylaminooxyethoxy, i.e., a O (CH2) 20 (CH3) 2 group, also known as 2 f -DMAOE, as described in examples herein below.
  • Oligonucleotides may also have sugar mimetics such as eyclob tyl moieties in place of the pentofuranosyl sugar.
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein,
  • nue!eobases include the purine bases adenine (A) and guanine (G) , and the pyrimidine bases thymine (T) , eytosine (C) and uracil (U) .
  • Modified nucleohases include other synthetic and natural
  • mieleobases such as 5 ⁇ met yl eytosine (5-me-C) , 5-l.tydroxymetbyl cytosine, xanthine, hypoxanthine , 2-aminoadenme, 6 methyl and other alkyl derivatives of adenine and guanine, 2-propy). and other alky!
  • 8-substituted adenines and guanines 5 halo particularly 5-faromo, 5-trii3uoromethyl and other 5- substituted uracils and eytosin.es, 7-methylguanine and 7-niethyladenine, 8- aza guanine and 8-azaadenine, 7 ⁇ dea3 ⁇ 4aguanine and 7-dea3 ⁇ 4aademne and 3- deazagua ne and 3 ⁇ deazaadenine.
  • oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance or modify pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance of the oligonucleotide.
  • Conjugate groups are routinely used in the chemical arts and are linked directl or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an
  • oligomerie compound such as an oligonucleotide.
  • Conjugate groups include without limitation, mtercaiators, reporter molecules, polyamines,
  • polyamides polyethylene glycols, thioetbers, polye there, eholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenantbridine, anthraquinone, adaniantane, aeridine,
  • conjugate group or groups may be directly attached to oligonucleotides in oligomerie
  • conjugate group or groups are attached to oligonucleotides by a conjugate linking group.
  • conjugate linking group In certain sucn
  • conjugate linking groups including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein.
  • Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilising groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound.
  • a bifunctional linking moiety comprises a. hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group.
  • the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units
  • functional groups that are routinely used in a bifunctional Unking moiety include, but are not limited to, electrophiles for reacting with nucleophilie groups and nueleophiles for reacting with
  • bifunctional linking moieties include amino, hydroxy!, earboxyiic acid, thiol, unsaiurations (e.g., double or triple bonds), and the like.
  • the present invention also includes antisense compounds which are chimeric compounds.
  • the term "chimeric" antisense compound”, as used herein refers to an antisense compound, particularly an oligonucleotide, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA: DMA or ENA: RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the RNA strand 18 of an RNA: DNA duplex.
  • Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified
  • oligonucleotides oligemic! eosides and/or oligonucleotide niimetics as described, above.
  • Such compounds have also been referred to in the art as hybrids or gapmers.
  • the antisense compounds need in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
  • the compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule
  • liposomes structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • the antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other
  • prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions, in particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [ (S-acetyl-2- thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 in WO 94/28764.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • Pharmaceutically acceptable base addition salts arc formed with metals or amines, such as alkali and alkaline earth metals or organic amines , Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are ⁇ , ⁇ ' - dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methyiglucamine. and procaine.
  • the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form may he regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
  • the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar IS solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.
  • a "pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines.
  • Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates.
  • Suitable pharmaceutically acceptable salts include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic earboxylic, sulfonic, sulfo or phospho acids or N ⁇ substituted s ilfamie acids, for example acetic acid, propionic acid, glyeolic acid, succinic acid, maleie acid, hydroxyrnaleic acid, methylmaleic acid, fu marie acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, henmic acid, cinnamie acid, mandelic acid, salicylic acid, 4 ⁇ aminosalicylic acid, 2-phenoxyben3 ⁇ 4oic acid, 2-acetoxybengoic acid,
  • naphthalene-l 5-disulfonic acid, 2- or S-phosphoglycerate, gIncose ⁇ 8- phosphate, N-cyclohe ⁇ ylsnJfamic acid (with the formation of cyc!amates) , or with other acid organic compounds, such as ascorbic acid, Pharmaceutically acceptable salts of compounds may also he prepared with a
  • Suitable pharmaceutically acceptable cation include but are sot limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyandries such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromie acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,
  • a therapeutic amount or dose of an antisense RNA compound of the present invention may range from about 0.1 to 500 mg/kg/day, such, as 0.1, 0.5, 1.0, 1.5, 2.0, 3.0, 5.0, 10, 25, 50, 100, 200, or 500 mg/kg/day.
  • a targeting may range from about 0.1 to 500 mg/kg/day, such, as 0.1, 0.5, 1.0, 1.5, 2.0, 3.0, 5.0, 10, 25, 50, 100, 200, or 500 mg/kg/day.
  • the resulting solution can be administered, for example by infusion with a portable volumetric infusion pump.
  • An antisense RNA compound is preferably provided as an expression
  • Said antisense RNA compound, or a DNA sequence coding for an antisense RNA compound is preferably linked to one or more control sequences, i.e. regulatory DNA sequences, in such a manner that said antisense RNA compound is highly expressed in a cancer cell.
  • Control sequences that are operably linked to the sequences encoding the targeting RNA molecule include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in.
  • promoter is well-known in the art and encompasses nucleic acid regions ranging in s1 ⁇ 2e and complexity from minimal promoters to promoters including upstream elements and enhancers.
  • the promoter is typically selected from promoters that are functional in mammalian cells, although promoters functional in other e karyotic cells may he used.
  • the type of promoter is chosen to accomplish a useful expression profile for said targeting RNA molecule in the context of said replication competent
  • promoters are ENA polymerase II promoters, such as a viral promoter, for example SV40 early gene promoter and GM ' V promoter and RNA polymerase III promoters, including but not limited to the U8, Hi and tRNA (Val) promoters are especially suitable for the invention, but other promoters are not excluded.
  • ENA polymerase II promoters such as a viral promoter, for example SV40 early gene promoter and GM ' V promoter and RNA polymerase III promoters, including but not limited to the U8, Hi and tRNA (Val) promoters are especially suitable for the invention, but other promoters are not excluded.
  • a preferred promoter/enhancer drives specific expression of the antisense RNA compound in target cancer cells.
  • specific expression refers to a high level of expression from the promoter/enhancer in target cells, when compared to other cells or other cell types.
  • TiGER Tissue-specific Gene Expression and Regulation
  • CRM eis-re gulatory module
  • promoter/enhancer for expression in hepatocellular carcinoma cells is the alpha -fetoprotein promoter/enhancer; for expression in breast cancer cells is the alp a-lactalhumin (ALA) promoter/enhancer, for expression in prostate cancer cells is the PSA/FMSA and/or the kallikrein-2 promoter/enhancer, for expression in melanoma cells is the tyrosinase promoter/enhancer
  • the antisen.se RNA compound and control sequences are preferably incorporated in a vector.
  • said vector is a plasmid.
  • a plasmid into cells preferably mammalian cells
  • Preferred vector are viral vectors, including but not limited to an adenoviral vector, an adeno-associated viral vector, and a retroviral vector
  • a preferred retroviral vector is provided by a replication non-competent murine leukemia virus (MLV) and a replication non-competent human immunodeficiency virus 1 and 2.
  • a therapeutic amount or dose of an expression vector comprising an antisense ENA compound of the present invention may range from about 1*10E9 to 1*1E13 copies/kg, or from about 1*I0E10 to i*lE12 copies/kg.
  • treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about l*i0E10 to 1* IE.1.2 copies/kg of an expression vector comprising an antisense RNA compound of this invention in single or multiple doses.
  • copies refers to plasmid numbers for p!asmid vectors, and to particle numbers for viral vectors, as is known to the skilled person.
  • RNA interference is a conserved cellular surveillance system that recognises double-stranded RNA (dsENA) and activates a sequence-specific degradation of ENA species homologous to the dsR A (Hannon (2002). Nature 418: 244-251). in addition, RNAi can cause transcriptional gene silencing by RNA- directed promoter DNA methyl&tion and or histone methylation (Kawasaki and Taira 2004, Nature 431: 211-217; Morris et al 2004. Science 305: 1239-1292).
  • dsENA double-stranded RNA
  • RNAi can cause transcriptional gene silencing by RNA- directed promoter DNA methyl&tion and or histone methylation (Kawasaki and Taira 2004, Nature 431: 211-217; Morris et al 2004. Science 305: 1239-1292).
  • RNAi -related processes have been described in almost all e karyo ic organisms, including protozoa, flies, nematodes, insects, parasites, and mouse and human cell lines (reviewed in: Zamore 2001. Nat, Struct, Biol. 8: 746-750; Hannon 2002, Nature 418: 244-251; Agrawal et al 2003. Microbiol. MoL Biol, Rev. 67: 657-885), By now, RNA interference is the most widely used method to specifically downregulate genes for functional studies.
  • RNAi is mediated by a targeting RNA molecule capable of decreasing expression of a target gene through the process of RNA interference.
  • Said targeting RNA molecule preferably comprises a double stranded portion, preferably having a length of at least 19 nucleotides (per strand).
  • the double stranded portion preferably has a len gt of less than 30 nucleotides, and said RNA molecule preferably comprises a short hairpin SNA (shRNA), or comprises a double-stranded structure consisting of two different RNA molecules having complementary regions of preferably the complete length of the RNA molecules. In the latter case, the said RNA molecules can be transcribed from different promoters.
  • Said RNA molecules are preferably susceptible to the action (i.e.
  • target gene is understood to indicate that the process of decreasing expression of said gene occurs with specificity towards ARIHl, DUSP15, UBE1X, RPL7L1, USp8, 4EH.P and/or PRK 1.
  • the targeting RNA molecule can be provided to cancer ceils by any method known in the art.
  • the nncleic acids can be coupled to a nanoparticle of an inert solid (commonly gold) which is then shot directly into a target cell's nucleus.
  • magnet-assisted transfeetion uses magnetic force to deliver nucleic acids into target cells after associating said nucleic acids with magnetic nanopartieles.
  • inipalefection is carried out by impaling cells by elongated anostruetures and arrays of such
  • nanostrueture s such as carbon nanofibers or silicon nanowires which ha e been inaction aliped with nucleic acid molecules.
  • a therapeutic amount or dose of a targeting RNA molecule of the present invention may range from about 0.1 to 500 mg kg day, such as 0.1, (1,5, 1.0, 1,5, 2.0, 3.0, 5.0, 10, 25, 50, 100, 200, or 500 nig/kg/day.
  • a targeting compound is preferably provided in 0,9% NaCL
  • the resulting solution can he administered, for example by infusion with a portable volumetric infusion pump.
  • Said targeting ENA molecule, or a DNA sequence codi g for a targeting RNA molecule is preferably linked to one or more control sequences, i.e. regulatory DNA sequences, in such a manner that said targeting RNA molecule is highly expressed in a cancer cell.
  • Control sequences that are operahly linked to the sequences encoding the targeting RNA molecule include promoters/enhancers and other expression regulation signals. These control sequences may be selected to he compatible with the host cell for which the expression vector is designed to be used in.
  • promoter is well-known in the art and encompasses nucleic acid regions ranging in sise and complexity from minimal promoters to promoters including upstream elements and enhancers.
  • the promoter is typically selected from promoters that are functional in mammalian cells, although promoters functional in other eukaryotic cells may be used.
  • the type of promoter is chosen to accomplish a useful expression profile for said targeting ENA molecule in the context of said replication competent adenovirus.
  • RNA polymerase III promoters including but not limited to the U6, HI and tENA( ⁇ al)
  • said DNA sequence coding for a targeting RNA molecule is functionally linked to one or more control sequences, i.e. regulatory DNA sequences, in such a manner that said targeting RNA molecule is only expressed or is expressed at a higher level in a cell into which the targeting RNA molecule is introduced tinder certain conditions that can be modulated by an external signal, where the term "external" means having its origin outside of the DNA fragment
  • Preferred targeting ENA molecules are directed to the target sequences depicted in Table 1. It is further preferred that the targeting RNA molecules is or comprises one or more targeting siRNA/shENA molecules that are depicted in Table 1. These targeting siRNA/shRNA molecules are
  • the targeting ENA molecule and control sequences are preferably
  • said vector is a plasmid.
  • a preferred vector is a viral vector, including but not limited to n adenoviral vector, an adeno-assoeiated viral vector, and a retroviral vector.
  • a preferred retroviral vector is provided by a replication non- competent murine leukemia virus (MLV) and a replication non-competent human immunodeficiency virus 1 and 2.
  • the invention also provides methods and means for formulating the replication non-competent viruses according to the invention that can be used to preserve said replication competent viruses and to administer said replication non-competent viruses to cells.
  • the formulations are used to administer said replication non-competent viruses to cells in vitro, in another variation the formulations are used to administer said replication non-competent viruses to cells in vivo.
  • the invention furthermore provides methods for administering the
  • formulations according to the invention to cells, leading to infection of said cells with the replication non-competent viruses of the invention.
  • the methods are used to administer said formulations to cells in vitro, in another variation the methods are used to administer said formulations to cells in vivo.
  • Methods for providing an expression vector comprising a targeting RNA molecule to cancer cells are known in the art an include ex vivo
  • a therapeutic amount or dose of an expression vector comprising a targeting RNA molecule of the present invention may range from about 1*10E9 to 1*1E13 copies/kg, or from about 1*10E10 to 1*1E12 28 copies/kg.
  • treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 1*10E1G to 1*1E12 copies/kg of an expression vector comprising a targeting RNA molecule of this invention in single or multiple doses.
  • copies refers to plasmid numbers for plasmid vectors, arid to particle numbers for viral vectors, as is known to the skilled person.
  • a further preferred susceptibility agent according to the invention is an inhibitor molecule, such as a large or a small inhibitor molecule.
  • Known large inhibitor molecules include, for example, antibodies that are directed to a gene product of ARM 1, DUSF1.5, UBE1X, EFL7L1, USp8, 4EHF and/or PRKDl.
  • the term antibody includes conventional antibodies such as an IgG which comprises two heavy and two light chain .molecules. Further included are dimers, diabodies, triabodies, bispecific, trispeeific, tetraspecifie antibody formats, monovalent, divalent, trivalent, tetravalent or other multivalent antibody formats, or any portion or fragment thereof such as, hut not limited to, single domain antibodies, F(ab').sub.2, Fab, Fab", Facb, Fc, and scFv,
  • Lipodin ⁇ Ab® Lipodin ⁇ Ab®
  • tbe vesicles comprise an amphophilic block copolymer having a hydropbilie and a hydrophobic block.
  • nucleic acid encoding one or more antibodies against a gene product of ARIH1, BUSP15, UBE1X, RPL7L1, USp8, 4EHP and/or FRKD1 can be incorporated into a gene delivery vector which further comprises control sequences that are operah!y linked to tbe sequences encoding the antibody or antibodies.
  • control sequences include promoters/enhancers and other exp ession regulation signals. These control sequences ma be selected to be compatible with the host cell for which the expression vector is designed to be used in.
  • the term promoter is well- known in the art and encompasses nucleic acid regions ranging in :m and complexity from minimal promoters to promoters including upstream elements and enhancers.
  • the promoter is typically selected from promoters that are functional in mammalian cells, although promoters functional in other eukaryo ie cells may be used.
  • the type of promoter is chosen to accomplish a useful expression profile for said targeting RNA molecule in the context of said replication competent adenovirus.
  • Preferred promoters are ENA polymerase II promoters, as are indicated hereinabove.
  • Preferred vectors are plasmids and viral vectors, preferably adenoviral, retroviral or adeno-assoeiated viral vectors.
  • Antibodies against a gene product of AR!H!, DUSP15, UBElX, RPL7L1, USpS, 4EHP and/or PRKD1 are known in the art.
  • other companies such as Nevus Bioiogicals
  • PRKDI small inhibitor molecules of PRKDI
  • CID 2011758 (5 ⁇ (3- c orophenyl) ⁇ N 4-(morphoIin-4-ylmethyI)phenyI]foran-2"Carboxamide; Tocris Bioscience), CID 755873 (2, 3 ; 4 ; 5-tetrab dro-7-bydroxy- 1 H ⁇
  • a therapeutic amount or dose of a small inhibitor molecule of the present invention may range from about 0.1 nag/kg to about 500 mg kg, alternatively from about 1 to about 50 mg kg.
  • treatment regimens accordmg to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of a small inhibitor molecule of this invention per day in single or multiple doses.
  • Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.
  • the invention also provides compositions of the replication non-competent viruses according to the invention and cells in which the replicatio non- competent viruses according to the invention induce expression of a susceptibility agent.
  • the invention further provides a susceptibility agent for use according to the invention, whereby the susceptibility agent is administered before, during or after treatment an individual suffering from cancer with a genotoxic agent. It is preferred that a susceptibility agent for use according to the invention is provided prior or adjacent to treatment of an individual suffering from cancer with a genotoxic agent.
  • Said individual suffering from cancer is preferably a mammal, more preferably a human.
  • RNAi screen for ubiqidtination / sumoylation enzymes identifies cisplatin response modulators, (A) Hits identified in primary screens;
  • FIG. 2 Silencing ARIHl sensitises to genotoxie stress.
  • A ES cell viability after treatment with 10 microM cisplatin (CP), 10 mg/ml mitomycin C (MMC), 150nm etoposide (ETO), 250nM doxorubicin (DOX), 250 microM diethylmaleathe (DSM) S 1 microM thapsigargm (THAPS), or ⁇
  • Vincristine Vine
  • B ES cell viability in presence of control or ARIHl siENA after treatment with CP, Etc, or MMC.
  • C ES cell viability in presence of Kifll -siENA (only for PBS), GFP-siR A, p53-8_KNA, or 3 individual siENA sequences targeting AEIH1 after treatment with vehicle control, CP, DOX, DEM, THAPS or VINC (normalized to siGFF).
  • D ASIHl protein levels and H2B loading control in U20S expressing shcontrol or 3 individual sbRNAs targeting ARIHl, followed by bulk puromycin selection. Percentages indicate remaining ARIHl expression.
  • FIG. 5 ARIHl mediates eisplatin-induced mRNA translation arrest.
  • A Methionine incorporation in U20S cells after treatment with 15 microM CP for 2h, 4h or 8h or cyclohexamide (CHX) for lb. Alexa546 signal (reflecting amount of newly synthesized proteins) / number of nuclei (DAPI) is shown.
  • B Methionine incorporation in sheontrol and two different sbARIHl U20S cell lines after treatment with CP for indicated times. Effect of co-treatment with 15 microM CP and 2.5 microM salubrinal (SAL) for 2b. is shown and CHX is used as positive control. Normalized to Alexa546 fluorescence / nucleus value in PBS for each cell line, (C-E) Cell survival in cells
  • SMARTpools from indicated gene families affecting cell viability under control (PBS) condition pie diagram
  • B Graphs show -seore ranking in primary screen of SMARTpools after exclusion of those affecting general viability. Pie diagrams show number of SMARTpools reducing (left) or enhancing (right) CP-induced loss of viability [absolute Z ⁇ Score>L5; p ⁇ 0.05
  • C Confirmation of hits from primary screen by deeon volution using 4 individual siRNAs against each target gene.
  • D Number of primary hits confirmed (dark lb medium dark) and rejected (light).
  • HMl mouse ES ceils derived from OLA/129 genetic background were maintained under feeder free conditions in GMEM medium containing 5x105U mouse recombinant leukemia inhibitory factor (LIF; PAA). All other cell lines were purchased from ATCC. MCF7 hurnan breast cancer cells, 4T1 mouse breast cancer cells and H1299 human non-small cell lung cancer ceils were maintained in EPMX medium. U20S human sarcoma cells were kept in DMEM. All media contained 10% FBS and 25U/ml penicillin, and 25 g/r J streptomycin.
  • LIF leukemia inhibitory factor
  • Genotoxieants included the DNA cross-linker cispiati (Cis-PtCl2(NH3)2) (provided by the Pharmacy unit of University Hospital, Leiden NL) and the inhibitors of topoisomera.se IJ-mediated DNA unwinding, doxorubicin
  • Oxidative stressor diethyl maleate (DEM), microtubule poison Vincristine, and ES stressor Tbapsigaragin were from Sigma,
  • the pan ⁇ caspase inhibitor gA ⁇ abAla-DL-Asp-fiuerometbylketone (so VAD-fink) was purchased from Bachem, the eif2alpha phosphorylation inhibitor sa!nbrina! was from Ca!biochem.
  • Z' -factors were determined for each plate, using La in A/C as a negative control and p53 as a positive control.
  • Z-seores were calculated using as a. reference i) the mean of ail test samples in the primary screen and ii) the mean of the negative control samples in the secondary deeonvolution screen (in order to prevent bias due to pre- enrichment of hits) (Birmingham et a!. 2009. Nat Methods 6: 569-575).
  • Hit determination was done using Z-seores with a cut off value of 1.5 below or above the reference and -value lower than. 0.05.
  • Enrichment of canonical pathways and formation of p53/ ubiqnitination signaling network was performed using MetaCoreTM data-mining software.
  • Apoptosis and cell cycle analysis ES cells wore exposed to vehicle or cisplatin for 8h for ceil cycle analysis or 24b. for apoptosis analysis.
  • MCF7 cells were exposed, for 24h for cell cycle analysis. Floating and attached cells were pooled and fixed in 80% ethane! overnight.
  • RNAseA were stained usi g PBS EDTA containing 7.5rnM propidium iodine and 40mg/ml RNAseA and measured by flow cytometry (FACSCanto XI; Becton Dickinson), The amount of cells in the different ceil cycle fractions or in sub G0/G1 for apoptotie cells was calculated using BD FACSDiva software. Alternatively, apoptosis was determined using live imaging of Annexin V labeling, as described
  • IJ20S cells 250 cells plate
  • shRMAs shRMAs
  • U20S cells were seeded on glass coverslips and allowed to grow for two days. Subsequently, they were irradiated with O.oGy and fixed ⁇ using 2% formaldehyde at the indicated tiniepo nts. Alter washing with PBS, post- fixation extraction took place by incubating with 0.25% Triton-X. Cells were extensively washed with PBS to remove detergent and then blocked in S% BSA, Finally eoverslips were ixnnxunostained with rabbit 53BP1 and mo se ⁇ 2 ⁇ antibodies, followed by eounterstaining with DAPI and appropriate secondary fluorescent antibodies.
  • HMI ES cells, U20S. and MCF7 breast cancer ceils were seeded in 8-well plates at a density of 0,5 million cells/ well.
  • Cells were treated with different concentrations of cisplatin for 4 h (U20S, MCF7) or 8h (ES) and proteins were harvested in lysis buffer containing liaM phenyirnethylsulfonyl fluoride (Cell Signalling), Cap binding proteins were precipitated using 7- raethyl-GTP-Sepharose 4B beads (Amersha n) as described previously (Moody et al. 2005, J Virol 79; 5499-5506). Precipitated proteins were separated on 12% SDS-PAGE gels and analyzed by immunoblotting for 4ehp (E1F4E2).
  • Cliek-iT® Metabolic Labeling Reagents for Proteins was purchased from n vitrogen and used according to manufacturers Instructions.
  • U20S cells were seeded to 80% confluence in 96 well rnicroclear plates and subsequently treated with 15 microM cisplatin for 2-8h or with 2mg/ml eydohex&mi e (CHX) for lb, or for 2b with a combination of 15 microM ciBplatin and 2.5 micreM sai brmal.
  • CHX eydohex&mi e
  • DAP! was used as coanterstain and images were acquired using a BD- pathway imaging system. Image analysis was performed using BD
  • I.I20S cells expressing different shENAs, were transiently transfeeted with FLAG-tagged ehp cD A (provided by Dong-Ex Zhang, Scripps Research Institute, La Jolia CA. - through Addgene; plasnnd 17342) (Okumura et al. 2007. Genes Dev 21: 255-260) or GFP control plasraid in OptiMEM
  • ENA was e tracted using ENeasy Plus Mini Kit from Qiagen.
  • cDNA was made from 50 ng total RNA with EevertAid H minus First strand cDNA synthesis kit (Fermentas) and real-time qPCS was subsequently performed in triplicate using SYBR green PGR (Applied Brosystems) on a 7900HT last real-time PGR system (Applied Biosystems).
  • SYBR green PGR Applied Brosystems
  • 7900HT last real-time PGR system
  • ES cells that display a robust apoptotic response to genotoxic compounds, including cispiatin, were treated with 10 microM cisplatin or vehicle and cell viability was monitored after 24h. 50
  • ubiquitination library* presumably based on the presence of predicted domains associated with ubiquitinase function, including RING, SOCS, or SPRY.
  • Ubelx Uba
  • the knockdown of the El ubiquitin enzyme Ubelx (Uba) which has recently been shown to he a crucial El enzyme in the DDR following ionizing radiation and replication stress (Moudry et aL. 2012. Cell Cycle 11: 1573-1582), resulted in a particularly strong reduction of viability (Fig 1C).
  • a large proportion of the identified hits have been previously established to regulate the transcription factor 53, which acts as a master regulator of the outcome of the DDR i various cell types including ES cells (Fig IE).
  • Three of the identified DUBs, USP7 (HAUSP), USP4, and USP5 can directly or indirectly influence p53 protein levels (Brooks and Gu 2006. Mol Cell 21: 307-315; Dayal et aL 2069, J Biol Chem 284: 5030-5041; Meuimeester et al, 2005. Mol Cell 18: 585-576; Zhang et al. 2011. EMBO J 30: 2177-2189).
  • ARIHI Parkin family ubiquitln ligase Ariadne homologue 1
  • Fig 1A,C ubiquitln ligase Ariadne homologue 1
  • ARIHI knockdown of ARIHI did not sensitize ES cells to non- enotoxic agents such as the ER stressor thapsigargin, the oxidative stressor diethyl maleate (DEM), or the microtubule poison Vincristine (Fig 2A,C).
  • non- enotoxic agents such as the ER stressor thapsigargin, the oxidative stressor diethyl maleate (DEM), or the microtubule poison Vincristine (Fig 2A,C).
  • AEIH1 is mainly implicated in maintaining viability while the cell cycle is arrested and. repair is ongoing.
  • silencing ARIHl increased the si bGl/GO fraction after treatment with cisplatin (Fig 31).
  • This effect of AEIH1 was not restricted to easpase 3- mediated apoptosis, since transient or stable silencing of ARIHl also sensitized the easpase -3 deficient hum n breast cancer ceil line MCF7 (Fig 3J-L),
  • ARIHl knockdown did not affect basal eel! cycle distribution or eisplatin-indnced cell cycle arrest in MCF7 (Fig 3M).
  • AR H 1 -depleted cells respond to DSBs by initiating DDE foci and arresting the cell cycle.
  • ARIHl cell survival is more severely compromised following DNA damage, which does not depend on p53 or cas ase - 3 ⁇ me di ie d apoptosis.
  • ARIHl can act as an E3 ubiquitin ligase for 4ehp (Tan et al. 2003. FEES Lett 554; 501-504.) more recently it has been established that ARIHl can ISGylate 4ehp thus enhancing its affinity for the mRNA cap structure and replacing eIF4E
  • AEIHl protein levels were enhanced following cisplatin treatment in U20S cells (Fig 4A). This conld not be explained by enhanced mRNA levels, indicating that genotoxie stress triggers enhanced synthesis or stability of the ARIH1 protein (Fig 4B).
  • Co-immuno precipitations in U20S cells showed that the increased levels of AEIHl in cisplatin treated cells led to ARIHl association with 4ehp (Fig 4C), We used 5' cap-pulldown assays to investigate whether cisplatin treatment caused 4ehp translocation to the mRNA cap.
  • RNAi screen identifies modulators of chemosensjtivity
  • B4418 mouse ESC derived from C57/B16 genetic background (provided by Dr. Monique de Waard, Erasmus Medical Center, Rotterdam ML (Kruse et ah 2007.
  • Genotoxieants included the DNA cross-linker oisplaiin (CP; Cis- PtC12(NH3)2) (provided by the Pharmacy unit of University Hospital, Leiden NL) and the inhibitors of topoisonierase Il-mediated DNA
  • Oxidative stressors included menadione (Sigma), diethyl roa!eate (DEM; Sigma), and 11202 (Merck).
  • the pan-easpase inhibitor z-Val-Ala-DL-Agpfluorometbylketone (z» VAD-fmk) was purchased from. Bacheni. Retinoie acid (RA) and LiC!2 were obtained from Sigma.
  • SB-481542 TGFbeta receptor inhibitor was obtained from Tocris Bioscience. Antibodies against p58 and pbospbo-po3 wore purchased from Novaeostra and Cell signaling, respectively.
  • Antibody against 53.BP1 was from BD Biosciences, antibody against Tubulin was obtained from Sigma, Antibody against active beta-catenin. (anti-ABC; clone 8E7) was from Millipore and antibody against p--Ser45 beta-eatenin was from Cell Signaling,
  • Example 1 As a quality control Z'-foctors were determined for each plate, as decribed in. Example 1, using Lamin A/C as a negative control and p53 as a positive control.
  • HMI ESC were treated with CP (1 microM, 5 roierofVi s or 10 ieroM or vehicle control for 8k in 3 independent experiments.
  • B4418 ESC were treated for 8h with the genotoxicants CP didoxorubicin or etoposide, or the oxidative stressors menadione, DEM or H202.
  • Total SNA was isolated using the RNAeasy kit (Qiagen) according to manufacturer's instructions. SNA quality and integrity was assessed with Agilent 21.00 Bioanalyser system (Agilent technologies). Gene expression was measured using
  • phosphatidyl serine exposure at the outer membrane leaflet was detected hy Annexin V-PITC in real-time in attached cells as described previously (Puigvert et ai 2010. Curr Protoc Cell Biol Chapter 18, Unit 18,10.1-13).
  • Extracts were prepared in SDS protein lysis sample buffer and boiled for 5 mm at 95°C. Extracts were separated by SDS-PAGE on polyacrylami.de gels, transferred to PVDF membranes, and membranes were blocked using 5% BSA.. Following incubation with primary and secondary antibodies signal was detected using a TyphoonTM 9400 from GE Healthcare.
  • G REINER pCiear 96 weii/piates coated with 1% gelatin and exposed to vehicle (PBS) or 5 ⁇ CP for indicated times.
  • RNA was extracted and real-time qPCR was subsequently performed as
  • KMAi screen identifies modulators of chemose ⁇ itivity m ESC
  • Genome Biol 7: E66 Genome Biol 7: E66 of all CP-treated plates based on si-Larnin A C and si- pod was ⁇ "0,5, indicating a strong signal to noise ratio. For hit selection, we first excluded. siRNAs that significantly reduced viability in. control conditions. This list contained expected survival genes from ail three gene libraries, such as Plkl, Oct-3/4, and Wi.pl (Fig, 6A),
  • siRNAs were ranked by Z-scores and hits were defined as [absolute Z-Seore>1.5: p ⁇ 0,06]. Using these criteria, 106 SMARTpools protected against CP and 78 sensitized (Fig, 2B), These hits entered a secondary deconvolution screen where hit confirmation was defined as at least 3 out of 4 individual siRNAs copying the effect of the SMARTpool [absolute Z-Seore>L5; p ⁇ 0,05, ranked against Lamin ⁇ C] (19). la this way, about 2.5% of all kinases, phosphatases, and. transcription factors ( ⁇ 32% of the primary screen hits) were confirmed as CP response modifiers (Fig. 6QD).
  • micro-array analyses and SILAC were employed to map global changes in mENA expression and protein phosphorylation, respectively in response to CP treatment (Fig. 1).
  • ESC were exposed to vehicle or 1, 5, or 10 mieroM CP for 8 h. followed hy NA isolation.
  • FACS analysis at 24h rom parallel plates of the same experiment confirmed dose-dependent induction of apoptosis (data not shown), A concentration-dependent inductio of differentially expressed gen.es (DEGs; p ⁇ 0.05) was observed and 2,269 DEGs were identified at 10 microM exposure. 29 of the 47 DEGs already
  • isotope-iaheled amino acids were used to distinguish hetween proteins isolated from untreated ESC and ESC treated with 5 microM CP for Ah. Isolated peptide mixtures were enriched for phosphopeptides on a titanium column and samples were analyzed by tandem mass spectrometry. Of the 8,251 identified phosphopeptides, 1,612 showed differential phosphorylation
  • siR A screens we were able to identify a panel of genes, which were shown to sensitize all four cell lines. Screening conditions were optimized based on the sensitivity of the cancer cell lines, and concentrations that did not induce complete killing were used for the screens. In 4T1 ceil, screens were carried out with a 24b treatment and a CP concentration of 5 microM and 12.5 microM. In the Mcf/ cells, incubation with CP was prolonged until 48h using a concentration of 25 microM and H1299 and Hepg2 cells were treated for 24b with 25 microM CP, Hits were ranked based on survival as well as en the observed p- value for both control and CP treated conditions.

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Abstract

La présente invention concerne un agent de prédisposition à utiliser dans une méthode de traitement d'une personne souffrant d'un cancer, moyennant quoi ledit agent de prédisposition est associé à un agent génotoxique. Ledit agent génotoxique est de préférence choisi parmi les rayons gamma, un composé à base de platine et/ou une anthracycline. Un agent de prédisposition préféré inhibe l'expression et/ou l'activité d'un produit d'un gène choisi dans le groupe constitué par AKIHL DUSP15, UBE1X, RPL7L1, USp8, 4EHP et PRKDL.
PCT/NL2013/050892 2012-12-12 2013-12-12 Méthodes améliorées de traitement du cancer à l'aide d'un agent génotoxique Ceased WO2014092572A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020038976A1 (fr) * 2018-08-23 2020-02-27 Roche Innovation Center Copenhagen A/S Oligonucléotides antisens ciblant l'usp8

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024510A1 (fr) 1992-05-25 1993-12-09 Centre National De La Recherche Scientifique (Cnrs) Composes biologiquement actifs de type phosphotriesters
WO1994026764A1 (fr) 1993-05-12 1994-11-24 Centre National De La Recherche Scientifique (Cnrs) Oligonucleotides phosphorothioates triesters et procede de preparation
WO2009140304A1 (fr) * 2008-05-12 2009-11-19 Genomic Health Inc. Tests pour prédire une sensibilité de patients atteints de cancer à des options de traitement de chimiothérapie
US20110111036A1 (en) 2008-05-15 2011-05-12 Biocompatibles Uk Limited Intracellular antibody delivery

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024510A1 (fr) 1992-05-25 1993-12-09 Centre National De La Recherche Scientifique (Cnrs) Composes biologiquement actifs de type phosphotriesters
WO1994026764A1 (fr) 1993-05-12 1994-11-24 Centre National De La Recherche Scientifique (Cnrs) Oligonucleotides phosphorothioates triesters et procede de preparation
WO2009140304A1 (fr) * 2008-05-12 2009-11-19 Genomic Health Inc. Tests pour prédire une sensibilité de patients atteints de cancer à des options de traitement de chimiothérapie
US20110111036A1 (en) 2008-05-15 2011-05-12 Biocompatibles Uk Limited Intracellular antibody delivery

Non-Patent Citations (55)

* Cited by examiner, † Cited by third party
Title
AGRAWAL ET AL., MICROBIOL. MOL. BIOL. REV., vol. 67, 2003, pages 657 - 685
ALADJEM ET AL., CURRENT BIOLOGY, vol. 8, 1998, pages 145 - 155
ARORA ET AL., GYNECOL. ONCOL., vol. 118, 2010, pages 220 - 227
BERGINK; JENTSCH, NATURE, vol. 458, 2009, pages 461 - 467
BIRMINGHAM ET AL., NAT METHODS, vol. 6, 2009, pages 569 - 575
BOUTROS ET AL., GENOME BIOL, vol. 7, 2006, pages R66
BRAUNSTEIN ET AL., MOL CELL BIOL, vol. 29, 2009, pages 5645 - 5656
BROOKS; GU, MOL CELL, vol. 21, 2006, pages 307 - 315
CICCIA; ELLEDGE, MOL CELL, vol. 40, 2010, pages 179 - 2042010
CONNOLLY ET AL., MOL CELL BIOL, vol. 26, 2006, pages 3955 - 3965
DAYAL ET AL., J BIOL CHEM, vol. 284, 2009, pages 5030 - 5041
DE VRIES ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 99, 2002, pages 2948 - 2953
FU ET AL., PROC NATL ACAD SCI USA, vol. 107, 2010, pages 4579 - 4584
GINGRAS ET AL., ANNU REV BIOCHEM, vol. 68, 1999, pages 913 - 963
GROSS ET AL., CELL, vol. 115, 2003, pages 739 - 750
HANEY, PHARMACOGENOMICS, vol. 8, 2007, pages 1037 - 1049
HANNON, NATURE, vol. 418, 2002, pages 244 - 251
HARPER ET AL., MOL. CELL, vol. 28, 2007, pages 739 - 745
JACKSON; BARTEK, NATURE, vol. 461, 2009, pages 1071 - 1078
JUNG ET AL., MOL CELL BIOL, vol. 31, 2011, pages 3997 - 4006
KAWASAKI; TAIRA, NATURE, vol. 431, 2004, pages 211 - 217
KOLAS ET AL., SCIENCE, vol. 318, 2007, pages 1637 - 1640
KONG; LASKO, NAT REV GENET, vol. 13, 2012, pages 383 - 394
KRUSE ET AL., MUTAT. RES., vol. 617, 2007, pages 58 - 70
KUMAR ET AL., BIOL CHEM, vol. 275, 2000, pages 10779 - 10787
LEE ET AL., BIOCHEM BIOPHYS RES COMMS, vol. 337, 2005, pages 701
LI ET AL., INT J BIOCHEM MOL BIOL, vol. 2, 2011, pages 138 - 145
LIN ET AL., MOL CELL, vol. 42, 2011, pages 237 - 249
LIU ET AL., J BIOL CHEM, vol. 286, 2011, pages 22314 - 22322
MACKEIGAN ET AL., NAT CELL BIOL, vol. 7, 2005, pages 591 - 600
MAGIN ET AL., NUCLEIC ACIDS RESEARCH, vol. 20, 1992, pages 3795 - 3796
MARTIN ET AL., HELV. CHI . ACTA, vol. 78, 1995, pages 486 - 504
MATSUOKA ET AL., SCIENCE, vol. 316, 2007, pages 1160 - 1166
MEDEMA; MACUREK, ONCOGENE, vol. 31, 2012, pages 2601 - 13
MEULMEESTER ET AL., MOL CELL, vol. 18, 2005, pages 565 - 576
MOODY ET AL., J VIROL, vol. 79, 2005, pages 5499 - 5506
MORRIS ET AL., SCIENCE, vol. 305, 2004, pages 1289 - 1292
MOUDRY ET AL., CELL CYCLE, vol. 11, 2012, pages 1573 - 1582
OKUMURA ET AL., GENES DEV, vol. 21, 2007, pages 255 - 260
PAPADAKIS ET AL., CURRENT GENE THERAPY, vol. 4, 2004, pages 89 - 113
PAULSEN ET AL., MOL. CELL, vol. 35, 2009, pages 228 - 239
PINES ET AL., MOLECULAR AND CELLULAR BIOLOGY, vol. 31, 2011, pages 4964 - 4977
PUIGVERT ET AL.: "Curr Protoc Cell Biol", 2010
REINHARDT ET AL., CELL CYCLE, vol. 10, 2011, pages 23 - 27
REINHARDT; YAFFE, CURR OPIN CELL BIOL, vol. 21, 2009, pages 245 - 255
ROY ET AL., NAT REV CANCER, vol. 12, 2012, pages 68 - 78
SABAPATHY ET AL., THE EMBO JOURNAL, vol. 16, 1997, pages 6217 - 6229
SILVERA ET AL., NAT REV CANCER, vol. 10, 2010, pages 254 - 266
SOLOZOBOVA ET AL., BMC CELL BIOL., vol. 10, 2009, pages 46
TAN ET AL., CYTOGENET CELL GENET, vol. 90, 2000, pages 242 - 245
TAN ET AL., FEBS LETT, vol. 554, 2003, pages 501 - 504
VILLEN ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 104, 2007, pages 1488 - 1493
YANG ET AL., J BIOL CHEM, vol. 284, 2009, pages 18588 - 18592
ZAMORE, NAT. STRUCT. BIOL., vol. 8, 2001, pages 746 - 750
ZHANG ET AL., EMBO J, vol. 30, 2011, pages 2177 - 2189

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020038976A1 (fr) * 2018-08-23 2020-02-27 Roche Innovation Center Copenhagen A/S Oligonucléotides antisens ciblant l'usp8

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