WO 2011/124669
`
`PCT/EP2011/055482
`
`15
`
`ligands
`
`
`
`
`
`5
`
`10
`
`15
`
`20
`
`25
`
`label, for detecting hybridization. A wide variety of appropriate indicators are known in the art
`including, fluorescent,
`radioactive,
`enzymatic
`or
`other
`Probes typically comprise single-stranded nucleic acids of between 10 to 1000
`nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and
`700, typically of
`between
`20
`and
`500.
`Primers
`typically
`of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a
`nucleic acid of interest, to be amplified. The probes and primers are "specific"
`to the nucleic
`acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization
`conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or
`6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate). For instance,
`the probes and primers can
`be selected
`from
`the
`Taqman
`Applied
`ones
`cited
`in
`The nucleic acid primers or probes used herein may be assembled as a kit. Such a kit
`includes consensus primers and molecular probes. A preferred kit also includes the components
`necessary to determine if amplification has occurred. The kit may also include, for example,
`PCR buffers and enzymes; positive control sequences, reaction control
`primers; and instructions
`for amplifying
`and
`detecting
`the
`specific
`sequences.
`In another preferred embodiment, the expression level is determined by DNA chip
`analysis. Such DNA
`chip
`or
`nucleic
`acid
`
`microarray consists probes that
`are chemically
`
`
`to attached a substrate, which
`
`
`can be a microchip, a glass slide or a microsphere-
`sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica
`or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise
`nucleic acids
`such
`as
`cDNAs
`or
`
`oligonucleotides be about 10
`to
`
`that about
`
`60 pairs. To may base
`determine the expression level, a sample from a test subject, optionally first subjected to a
`reverse transcription, is labelled and contacted with the microarray in hybridization conditions,
`leading to the formation of complexes between target nucleic acids that are complementary to
`probe sequences attached to
`
`the microarray surface. The labelled hybridized complexes are then
`detected and can be quantified or semi-quantified. Labelling may be achieved by various
`methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray
`hybridization technology are available to the man skilled in the art (see e.g. the review by
`30 Hoheisel, et 2006)
`Other methods for determining the expression level of said genes include the
`determination of
`the
`quantity
`
`
`
`of encoded by said proteins genes.
`
`01727
`
`MYLAN - EXHIBIT 1004 (Part 10 of 13)
`
`

`
`WO 2011/124669
`
`PCT/EP2011/055482
`
`16
`
`(e.
`
`added.
`
`Such methods comprise contacting
`a biological sample with binding partner capable of a
`
`
`selectively interacting with a marker protein present in the sample. The binding partner is
`generally an antibody
`that
`may
`be
`polyclonal
`or
`monoclonal,
`The presence of the protein can be detected using standard electrophoretic and
`immunodiagnostic techniques, including immunoassays
`such as competition, direct reaction, or
`sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination
`tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays;
`radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally
`include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or
`dye molecules, or other methods for detecting the formation of a complex between the antigen
`and the antibody
`or
`antibodies
`reacted
`therewith.
`The aforementioned assays generally involve separation of unbound protein in a liquid
`phase from a
`solid
`phase
`support
`to
`which
`antigen-antibody
`g.,
`in the practice of
`the
`
`
`include invention substrates such
`as
`nitrocellulose
`which can be used
`in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells);
`polystyrene latex {e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper;
`nylon membranes;
`activated beads, magnetically
`responsive
`
`beads, the like.
`and
`a microtiter plate
`More particularly,
`an ELISA method can be used, wherein
`the wells of
`
`are coated with an antibody against the protein to be tested. A biological sample containing or
`suspected of containing
`the marker protein is then added to the coated wells. After a period of
`incubation sufficient to allow the formation of
`
`antibody-antigen complexes,
`the plate(s) can be
`washed to remove
`unbound
`moieties
`
`and labeled secondary a
`
`detectably binding molecule
`
`
`The secondary binding molecule
`is allowed to react with any captured sample marker protein,
`the plate washed and the presence of the secondary binding molecule detected using methods
`25 well known in the art.
`The invention further provides a tool for implementing said methods, e.g. a DNA chip
`acids to at least 5,6,1, 8, 9, 10, that
`
`c o m p r i sing a
`solid
`support which
`carries
`nucleic
`
`15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 500 or 1000 genes selected from the
`group consisting of the genes listed in Tables 1 to 6, optionally Tables 1 and 2. Optionally, the
`DNA chip further carries nucleic acids that are specific to at least one gene selected from the
`the genes listed in Tables 3 to 6, optionally Tables 3 and 4. In a preferred
`
`group consisting of
`embodiment, the DNA chip carries nucleic acids that are specific to genes of Table 6, and
`optionally of one,
`
`
`
`
`several genes of Table or all 5. Optionally,
`the DNA chip may further
`include
`nucleic acids specific of additional genes from Tables 1-4.The DNA chip can further comprise
`
`5
`
`10
`
`15
`
`20
`
`30
`
`01728
`
`

`
`WO 2011/124669
`
`PCT/EP2011/055482
`
`17
`
`5
`
`10
`
`Tables
`
`detection
`
`nucleic acids for control gene, for instance a positive and negative control or a nucleic acid for
`an ubiquitous gene in order to normalize the results. In addition, the present invention also
`provides a kit for implementing
`said methods comprising detection means
`that are specific
`to at
`least 5, 6, 7, 8,
`9,
`10, 15,
`20,
`25,
`30, 150, 200, 300, 500 or 1000 genes 40,
`
`
`50,
`
`of
`selected from the group consisting
`the listed in Tables genes
`
`1
`6,
`to
`optionally
`Optionally, the kit further comprises detection means that are specific to at least one gene
`selected from the group consisting
`of
`
`the listed in Tables genes
`
`3
`to
`6,
`optionally
`In a preferred embodiment, the kit carries detection means that are specific to genes of Table 6,
`and optionally of one, several or all genes of Table 5. Optionally, the kit may further include
`detection means
`for
`
`additional genes from Tables
`1-4.
`In
`particular,
`the
`pair of primers, a probe or an antibody. The kit can further comprise control reagents and other
`necessary reagents.
`In a particular embodiment, the genes, preferably additional genes are selected for the
`tool or kit as above detailed for the methods of the invention. Preferably,
`the at least 5 genes,
`preferably additional genes, are selected from the group consisting of ENST00000399723,
`BI836406, C10orf79, AK022962, TMTC1, LOC728295, SUSD5, WNT6, BC044624, AY358241,
`ZNF251, ST6GAL2, LOC643401, NOV, CLGN, PROM1, SPEF2, FLRT2, RGS2, FOXP2, TRIM55,
`
`
`THAP5, PKD2L1, RP4-692D3.1, CB985069, ARL14, AY831680, XRN1, ZNF248, BC016022, PLAG1,
`THC2724353, THC2488083, C5orf41, BMS1P5, BMS1, THC2627008, PLA2G4A, DPY19L2, VCX2,
`PPP1R1C, GLT25D2, KIAA1841, IFIT2, ZNF596, TSPAN19, BC029907, C10orfl07, ZNF594,
`AMPD1, C21orf88, THC2694827, HSPC105, IFI44, THC2662262, FAM84A, DNAH7, KHDRBS2,
`NANP, AK091357, N4BP2L1, FAM105A, CA941346, CCDC68, CASC1, FAM90A12, PBX1,
`THC2739159, KCNQ2, ANXA1, AL122040, THC2655194, ENST00000342608, DSC2, ENOX1, IL13,
`BG571904, BX455216, LOC729085, BG188151, LOC729409, Clorfl03, PPP1R14C, NAIP, C13or01,
`25 GOLGA8E, AK022848, CXorf22, KIF5C, LRRCC1, FAM81B, ID2, CMYA5, Clorfl94, TTC18,
`tcag7.1314, ZNF385B, ADAMTS6, RHOU, ENST00000378850, C2orf55, GPR83, LRRIQ1, WDR31,
`DEFB126, ARMETL1, LOC642826, LOC129881, C2orfl3, THC2553512, ACVR1C, ZNF207,
`ANTXR1, CHD9, THC2526838, ABCA12, TncRNA, FKTN, PTPRG, ZNF233, ENST00000370378,
`FANK1, PCM1, SERPINI1, ARID4B, KIAA1377, FGF7, CY339166, LINCR, DA834198, CFH, SCG2,
`30 ARHGEF10, DA093175, GOLGA8A, AK021467, LOC283666, FLJ35767, THC2725553, ZNF430,
`CCDC141, MAP3K13, CCDC66, THC2727226, THC2528990, THC2718728, THC2507829,
`AK123972, EDEM3, DB304731, TPD52L1, MFAP5, EHF, NCF2, TRIM6, PERLD1, ATXN1, INHBB,
`CR627122, JAM3, CXCL14, CR594735, FLJ11235, C15orf52, LIMCH1, LOH11CR2A, BX281122,
`GPR110, ARNT2, ATP6V0A4, PDGFRB, ELA3B, NEDD9, MYH6, SLC35F2, HAS3, COLEC12,
`SLC3A2, AW993939, RUNX2, SUSD3, PLAU, SLC22A3, FCRL4, DOCK2, SOX3, THC2616558,
`RNASET2, LOC100130360, IL1R2, MGAT5B, TCF7L1, AF222857, AHNAK, HOXB8, S100A16,
`
`15
`
`20
`
`35
`
`01729
`
`

`
`WO 2011/124669
`
`PCT/EP2011/055482
`
`18
`
`5
`
`10
`
`15
`
`INSIG1 and DCDC2. More preferably, the genes are selected from the group consisting of
`ENST00000399723, BI836406, C10orf79, AK022962, TMTC1, LOC728295, SUSD5, WNT6,
`BC044624, AY358241, ZNF251, ST6GAL2, LOC643401, NOV, CLGN, PROM1, SPEF2, FLRT2,
`RGS2, FOXP2, TRIMS5, PKD2L1, RP4-692D3.1, CB985069, ARL14, AY831680, XRN1, THAP5,
`ZNF248, BC016022, PLAG1, THC2724353, THC2488083, C5orf41, BMS1P5, BMS1, THC2627008,
`PLA2G4A, DPY19L2, VCX2, PPP1R1C, GLT25D2, KIAA1841, IFIT2, ZNF596, TSPAN19,
`BC029907, C10orfl07, ZNF594, AMPD1, C21orf88, THC2694827, HSPC105, IFI44, THC2662262,
`FAM84A, DNAH7, KHDRBS2, NANP, AK091357, N4BP2L1, FAM105A, CA941346, CCDC68,
`CASC1, FAM90A12, PBX1, THC2739159, KCNQ2, ANXA1, AL122040, THC2655194,
`ENST00000342608, DSC2, ENOX1,
`IL13, BG571904, BX455216, LOC729085, BG188151,
`LOC729409, Clorfl03, PPP1R14C, NAIP, C13orf31, GOLGA8E, AK022848, CXorf22, K1F5C,
`TPD52L1, MFAP5, EHF, NCF2, TRIM6, PERLD1, ATXN1, ESTHBS, CR627122, JAMS, CXCL14,
`CR594735, FLJ11235, C15orf52, LIMCH1, LOH11CR2A, BX281122, GPR110, ARNT2, ATP6Y0A4,
`PDGFRB, ELA3B, NEDD9, MYH6, SLC35F2, HAS3, COLEC12, SLC3A2, AW993939, RUNX2 and
`SUSD3. Even more preferably, the genes are selected from
`the group consisting of
`ENST00000399723, BI836406, C10orf79, AK022962, TMTC1, LOC728295, SUSD5, WNT6,
`BC044624, AY358241, ZNF251, ST6GAL2, LOC643401, NOV, CLGN, PROM1, SPEF2, FLRT2,
`RGS2, FOXP2, TRIM55, PKD2L1, RP4-692D3.1, TPD52L1, MFAP5, EHF, NCF2, TRIM6, PERLD1,
`ATXN1, INHBB, CR627122, JAMS, CXCL14 and CR594735. In the most preferred embodiment,
`the genes are selected from the group consisting of ENST00000399723, BI836406, C10orf79,
`AK022962, TMTC1, LOC728295, SUSD5, WNT6, BC044624, TPD52L1, MFAP5, EHF, NCF2,
`TRIM6, PERLD1, ATXN1,
`INHBB and CR627122. Optionally, at least one further gene is selected
`for the tool or kit, said gene being selected from the group consisting of the genes listed in
`Tables 3 and 4, preferably TFPI2, PCDH7, SMAD9, AK090762, RAB39B, BF831953, AL050204,
`25 VCX, ITGA2, CXCR4, SLC16A10, PDE1A, MAL, KRT80, FXYD2 and AK3L1, more preferably
`TFPI2, PCDH7, SMAD9, AK090762, RAB39B, BF831953, AL050204, VCX, CXCR4, SLC16A10,
`PDE1A, MAL, and even more preferably
`
`TFPI2, PCDH7, SMAD9, CXCR4 and SLC16A10.
`The present invention also relates to the use of a DNA chip or a kit of the invention for
`preparing a kit for predicting or monitoring whether a patient affected by a cancer is responsive
`to a treatment with a molecule of the taxoid family. Preferably,
`the cancer is selected from the
`group consisting of the breast cancer, the
`lung cancer, the prostate
`
`cancer, gastric cancer the
`
`and
`the head and neck cancer. More preferably the cancer is the prostate cancer. In a preferred
`embodiment, the molecule of the taxoid family is selected from the group consisting of
`docetaxel, larotaxel, cabazitaxel (XRP6258), BMS-184476, BMS-188797, BMS-275183,
`ortataxel, RPR 109881A, RPR 116258, NBT-287, PG-paclitaxel, ABRAXANE®, Tesetaxel,
`
`20
`
`30
`
`35
`
`01730
`
`

`
`WO 2011/124669
`
`PCT/EP2011/055482
`
`19
`
`5,
`
`6,
`
`
`of
`
`cell-line
`
`IDN 5390, Taxoprexin, DHA-paclitaxel, and MAC-321. More preferably, the molecule of the
`taxoid family
`is
`docetaxel.
`screening or identifying a compound
`concerns methods for
`The present invention further
`suitable for improving the treatment of a cancer with a molecule of the taxoid family or for
`reducing the resistance development during the treatment of a cancer with a molecule of the
`taxoid family.
`least
`at
`a method comprises: 1) providing cell-line with
`
`
`
`the
`In a first embodiment,
`8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 500 or 1000 genes over-
`expressed and/or under-expressed respectively selected from the group
`of over-expressed genes
`
`of Tables 1, 3 and 5, optionally of Table 1, and under-expressed genes of Tables 2, 4 and 5,
`optionally of Table 2; 2) contacting said cell-line with a test compound; 3) determining the
`expression
`level of said at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150,
`200, 300, 500 or 1000 genes; and, 4) selecting the compound which decreases the expression
`level of over-expressed
`genes
`and
`increases
`
`the expression under-expressed genes. More
`level
`preferably,
`the genes
`are
`selected
`from
`genes
`the
`one,
`and
`are
`the genes of
`Table
`6
`selected,
`optionally
`or all genes of
`Table
`5.
`In a second embodiment, the method comprises: 1) providing a cell-line sensitive to the
`molecule of
`the
`taxoid
`family;
`2)
`contacting
`said
`
`of the taxoid family; 3) determining the expression level of said at least 5, 6, 7, 8, 9, 10, 15, 20,
`25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 500 or 1000 genes selected from the genes
`listed in Tables 1 to 6, optionally of Tables 1 and 2; and, 4) selecting the compound which
`
`
`of inhibits the appearance an over-expression
`
`
`an and/or under-expression of
`
`least at 5, 6, 7, 8, 9,
`
`10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 500 or 1000 genes respectively
`selected from the group of genes of over-expressed genes of Tables 1, 3 and 5, optionally of
`Table 1, and under-expressed
`genes
`of
`Tables
`2,
`4
`the genes are selected from the genes
`of
`
`Tables 5 and 6. Still more preferably,
`
`at least the genes
`of Table 6 are
`selected,
`and
`optionally
`
`one, of Table 5.
`several
`In a third embodiment, the method comprises: 1) providing a cell-line with at least one
`
`or
`
`gene over-expressed and/or under-expressed respectively selected
`
`from the group consisting
`
`of
`
`ENST00000399723, BI836406, C10orf79, AK022962, TMTC1, LOC728295, SUSD5, WNT6,
`BC044624, AY358241, ZNF251, ST6GAL2, LOC643401, NOV, CLGN, PROM1, SPEF2, FLRT2,
`RGS2, FOXP2, TRIMS5, PKD2L1, RP4-692D3.1, CB985069, ARL14, AY831680, XRN1, THAP5,
`ZNF248, BC016022, PLAG1, THC2724353, THC2488083, C5orf41, BMS1P5, BMS1, THC2627008,
`PLA2G4A, DPY19L2, VCX2, PPP1R1C, GLT25D2, KIAA1841, IFIT2, ZNF596, TSPAN19,
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`01731
`
`

`
`WO 2011/124669
`
`PCT/EP2011/055482
`
`20
`
`BC029907, ClOorflO?, ZNF594, AMPD1, C21orf88, THC2694827, HSPC105, IFI44, THC2662262,
`FAM84A, DNAH7, KHDRBS2, NANP, AK091357, N4BP2L1, FAM105A, CA941346, CCDC68,
`CASC1, FAM90A12, PBX1, THC2739159, KCNQ2, ANXA1, AL122040, THC2655194,
`ENST00000342608, DSC2, ENOX1,
`IL13, BG571904, BX455216, LOC729085, BG188151,
`LOC729409, Clorfl03, PPP1R14C, NAIP, C13orDl, GOLGA8E, AK022848, CXorf22, KIF5C,
`LRRCC1, FAM81B, ID2, CMYA5, Clorfl94, TTC18, tcag7.1314, ZNF385B, ADAMTS6, RHOU,
`ENST00000378850, C2orf55, GPRS 3, LRRIQ1, WDR31, DEFB126, ARMETL1, LOC642826,
`LOC129881, C2orfl3, THC2553512, ACVR1C, ZNF207, ANTXR1, CHD9, THC2526838, ABCA12,
`TncRNA, FKTN, PTPRG, ZNF233, ENST00000370378, FANK1, PCM1, SERPINI1, ARID4B,
`K1AA1377, FGF7, CV339166, LINCR, DA834198, CFH, SCG2, ARHGEF10,
`DA093175, GOLGA8A,
`AK021467, LOC283666, FLJ35767, THC2725553, ZNF430, CCDC141, MAP3K13, CCDC66,
`THC2727226, THC2528990, THC2718728, THC2507829, AK123972, EDEM3, DB304731, preferably
`ENST00000399723, BI836406, C10orf79, AK022962, TMTC1, LOC728295, SUSD5, WNT6,
`BC044624, AY358241, ZNF251, ST6GAL2, LOC643401, NOV, CLGN, PROM1, SPEF2, FLRT2,
`RGS2, FOXP2, TRIMS5, PKD2L1, RP4-692D3.1, CB985069, ARL14, AY831680, XRN1, THAP5,
`ZNF248, BC016022, PLAG1, THC2724353, THC2488083, C5orf41, BMS1P5, BMS1, THC2627008,
`PLA2G4A, DPY19L2, YCX2, PPP1R1C, GLT25D2, KIAA1841, IFIT2, ZNF596, TSPAN19,
`BC029907, C10orfl07, ZNF594, AMPD1, C21orf88, THC2694827, HSPC105, IFI44, THC2662262,
`FAM84A, DNAH7, KHDRBS2, NANP, AK091357, N4BP2L1, FAM105A, CA941346, CCDC68,
`CASC1, FAM90A12, PBX1, THC2739159, KCNQ2, ANXA1, AL122040, THC2655194,
`ENST00000342608, DSC2, ENOX1,
`IL13, BG571904, BX455216, LOC729085, BG188151,
`LOC729409, Clorfl03, PPP1R14C, NAIP, C13orI31, GOLGA8E, AK022848, CXorf22 and KIF5C,
`more preferably ENST00000399723, BI836406, C10orf79, AK022962, TMTC1, LOC728295, SUSD5,
`WNT6, BC044624, AY358241, ZNF251, ST6GAL2, LOC643401, NOV, CLGN, PROM1, SPEF2,
`FLRT2, RGS2, FOXP2, TRIM55, PKD2L1 and RP4-692D3.1, even more preferably
`
`ENST00000399723, BI836406, C10orf79, AK022962, TMTC1, LOC728295, SUSD5, WNT6 and
`
`BC044624 for the over-expressed genes, and TPD52L1, MFAP5, EHF, NCF2, TRIM6, PERLD1,
`
`INHBB, CR627122, JAM3, CXCL14, CR594735, FLJ11235, C15orf52, LIMCH1,
`ATXN1,
`LOH11CR2A, BX281122, GPR110, ARNT2, ATP6V0A4, PDGFRB, ELA3B, NEDD9, MYH6,
`SLC35F2, HAS3, COLEC12, SLC3A2, AW993939, RUNX2, SUSD3, PLAU, SLC22A3, FCRL4,
`DOCK2, SOX3, THC2616558, RNASET2, LOC100130360, IL1R2, MGAT5B, TCF7L1, AF222857,
`AHNAK, HOXB8, S100A16, INSIG1 and DCDC2, preferably TPD52L1, MFAP5, EHF, NCF2,
`TRIM6, PERLD1, ATXN1, INHBB, CR627122, JAM3, CXCL14, CR594735, FLJ11235, C15orf52,
`LIMCH1, LOH11CR2A, BX281122, GPR110, ARNT2, ATP6V0A4, PDGFRB, ELA3B, NEDD9,
`MYH6, SLC35F2, HAS3, COLEC12, SLC3A2, AW993939, RUNX2 and SUSD3, more preferably
`
`TPD52L1, MFAP5, EHF, NCF2, TRIM6, PERLD1, ATXN1,
`
`INHBB, CR627122,
`
`JAM3, CXCL14 and
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`01732
`
`

`
`WO 2011/124669
`
`PCT/EP2011/055482
`
`21
`
`CR594735, even more preferably TPD52L1, MFAP5, EHF, NCF2, TRIM6, PERLD1, ATXN1,
`
`INHBB and CR627122 for the under-expressed genes; 2) contacting said cell-line with a test
`
`compound; 3) determining the expression level of said at least one gene; and, 4) selecting the
`
`compound which decreases the expression level of over-expressed genes and increases the
`
`5
`
`expression
`
`level
`
`of
`
`under-expressed
`
`genes.
`
`In a fourth embodiment, the method comprises 1) providing a cell-line with the genes
`PCDH7, KHDRBS2, AUTS2, and C2orf55 being over-expressed and the genes JAM3, DCDC2,
`MFAP5, SLC3A1, AKAP12, ZNF649,
`RNASET2, NCF2, DLC1, CXCR4, CR594735,
`TRIM6,
`and MBNL3 being under-expressed; 2) contacting said cell-line with a test compound; 3)
`determining
`the
`
`expression level of said genes; and, 4) selecting
`the compound which decreases
`the expression level of one
`or several of the over-expressed genes and increases
`
`the expression
`level of one or
`several
`of
`the
`
`under-expressed genes.
`In a fifth embodiment, the method comprises 1) providing a cell-line sensitive to the
`molecule of
`cell-line
`the
`taxoid
`family;
`2)
`contacting
`said
`of the taxoid family; 3) determining
`the expression level of the genes JAM3, PCDH7, DCDC2,
`KHDRBS2, MFAP5, AUTS2, C2orf55, SLC3A1, AKAP12, ZNF649, RNASET2, NCF2,
`DLC1, CXCR4, CR594735, TRIM6, and MBNL3; and, 4) selecting the compound which
`inhibits the appearance of an over-expression of the genes PCDH7, KHDRBS2, AUTS2, and
`C2orf55 and/or an under-expression
`
`of the genes JAM3, DCDC2, MFAP5, SLC3A1, AKAP12,
`ZNF649, RNASET2, NCF2,
`DLC1,
`CXCR4,
`
`CR594735, MBNL3.
`TRIM6,
`Preferably,
`the cell-line is a cancer cell-line. In particular,
`the cancer cell-line is specific
`of the targeted cancer. For instance, if the prostate cancer is
`to be treated, then the cell-line is a
`prostate cancer
`cell-line.
`In a preferred embodiment, the molecule of the taxoid family is selected from the group
`consisting of docetaxel, larotaxel, cabazitaxel (XRP6258), BMS-184476, BMS-188797, BMS-
`275183, ortataxel, RPR 109881A, RPR 116258, NBT-287, PG-paclitaxel, ABRAXANE®,
`Tesetaxel, IDN 5390, Taxoprexin, DHA-paclitaxel, and MAC-321. More preferably, the
`molecule of the taxoid family is docetaxel. Preferably, the cancer is selected from the group
`consisting of the breast cancer, the lung cancer, the prostate cancer, the gastric cancer and the
`head and neck
`cancer. More
`preferably
`the
`cancer
`is
`
`10
`
`15
`
`20
`
`25
`
`30
`
`The example illustrates
`
`the
`
`invention
`
`without
`
`limiting
`
`its
`
`EXAMPLE
`
`01733
`
`

`
`WO 2011/124669
`
`PCT/EP2011/055482
`
`22
`
`Methods
`Cell culture and
`clones
`of selection docetaxel-resistant
`
`
`The human androgen-independent IGR-CaPl cell line recently obtained for a localized
`prostate cancer was maintained in RPMI medium complemented
`
`with 10% FBS and antibiotics.
`Docetaxel-resistant clones were selected by culturing the cells in docetaxel in a dose-escalation
`manner. Initial culture was done in 5nM docetaxel. Cellular clones surviving in the presence of
`5nM docetaxel were maintained in culture during four passages, and then the concentration of
`docetaxel in the medium was increased
`to 12nM, 25nM, 50nM, lOOnM and 200nM. The same
`selection methodology was followed with each increase in docetaxel concentration. Once cells
`were freely dividing in each dose of docetaxel mediums, they were considered as resistant and
`labelled IGR-CaPl-R. Cell cultures were maintained at 70% confluency and medium was
`changed every 48 h.
`Transcription
`Reverse
`and
`Total RNA Preparation
`Total RNA from parental and docetaxel-resistant IGR-CaPl cells was isolated using
`TriReagent (Sigma-Aldrich) and purified with RNeasy Micro Kit (Qiagen) according to
`manufacturer's protocols. Quality of RNA preparation, based on the RNA Integrity Number
`(RIN), was assessed using the Agilent RNA 6000 Nano Kit as developed on the Agilent 2100
`Bioanalyzer device (Agilent Technologies,
`Palo Alto, CA). All specimens included in
`this
`displayed a RIN of 10. RNA
`
`
`frozen samples were in nuclease-free water
`(Qiagen).
`Oligo Microarray Technology
`Parental and resistant-cell line total RNAs were directly compared by using Agilent
`oligonucleotide dual-color
`
`technology, running dye-swap and duplicate experiments. Total RNA
`from the parental IGR-CaPl cell line without treatment was used as the RNA reference. Total
`RNA from IGR-CaPl cells resistant to treatment with 5nM, 12nM, 25nM, 50nM, lOOnM and
`200nM of docetaxel respectively, were used as samples. Probe synthesis and labeling were
`performed by Agilent's
`Low
`
`Low Fluorescent Linear Amplification
`
`
`input Kit.
`Hybridization
`performed on
`the Agilent
`4x44K Human 1A (G4112F) long (60-bp) oligonucleotide
`
`microarrays
`(Agilent Technologies) by using reagents and protocols provided by the manufacturer. Feature
`extraction software
`the
`provided
`by
`
`
`
`Agilent A.9.5.3.1) was (Version used
`to
`quantify
`fluorescent
`images
`and
`to
`normalize
`
`results and lowess subtraction using
`
`
`the linear method.
`The methodology described below is based on a dose-dependent gene expression
`changes:
`
`study
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`was
`
`01734
`
`

`
`WO 2011/124669
`
`PCT/EP2011/055482
`
`23
`
`5-parameters
`
`logistic
`
`regression
`
`a
`
`profiles
`
`Under the hypothesis of a clone enrichment, and/or a biological effect due to drug
`increasing, monotonically
`increasing
`or
`decreasing
`expression identified by using
`
`(T-B)
`y g = B +
`model:
`[i + io^-'^f
`where y% is the log.ratio of treatment vs. reference for the gene g, x is the drug-dose in
`Logio[nM], and B,
`T,
`p
`are,
`
`respectively, value, the estimated maximal the
`
`xc,
`estimated
`value, the slope
`at
`the
`inflexion
`point,
`and
`the
`asymmetric
`the
`For each probe,
`parameters
`
`were initialized with first
`
`observed
`values,
`and
`an iterative method of gradient (the Newton-Raphson method). The aim of this iterative
`algorithm
`is
`to minimize
`the
`
`quadratic weighted of residuals:
`
`sum
`1
`\y i.fit-y i.obs\
`
`S = £. w. (y. .fit - y. .obs) 2 where w.
`
`robust
`a
`values.
`
`linear
`
`regression
`
`for each probe, by
`of the fitting was measured,
`
`The performance
`observed
`of the fitted values
`against
`the
`increasing
`drug
`the
`Probes potentially
`associated
`with
`RLRp-value < le-5, and (fold change| > 2 between the first and the last dose (resp: 5 and
`200nM), considering
`the
`fold
`
`change estimated by
`the
`5-parameters
`logistic
`regression
`Calculations and graphic visualizations were performed in R (free software version 2.6.2), by
`using the package
`"MASS"
`
`(version and supplemental scripts, 7.2-40),
`
`in R language, written in
`the lab (F. Commo).
`
`were
`
`5
`
`10
`
`15
`
`20
`
`RESULTS
`
`Generation of acquired resistance to Docetaxel in vitro. Prostate cancer IGR-CaPl
`cells were used to generate successive docetaxel-resistant
`cell lines. The addition of docetaxel
`
`induced a selection process, whereby large majority a
`
`of
`cells
`
`initially underwent cell
`death
`the ability
`to proliferate was
`regained.
`The
`
`IGR-CaPl inventors obtained resistant (IGR-CaPl-R)
`
`clones which
`survived
`in
`medium
`containing
`
`respectively 5nM, 25nM, 50nM, lOOnM and
`200nM of docetaxel. Cell cycle analysis was done to show acquired resistance to drug. The
`resistant cell lines showed cell cycle similar to the parental IGR-CaPl cells, suggesting that
`acquired resistance
`had
`been
`gained
`(not
`shown).
`
`25
`
`until
`
`12nM,
`
`Genome-wide analysis of IGR-CaPl docetaxel-resistant lines using microarray.
`30 Human genome-wide analysis of gene expression changes was realized in order to stringently
`
`01735
`
`

`
`WO 2011/124669
`
`PCT/EP2011/055482
`
`24
`
`identify human genes that might represent the molecular signature of
`to
`resistance or sensitivity
`used
`lines
`were
`docetaxel in prostate
`
`cancer. Untreated IGR-CaPl parental
`cell
`Such genes were those for which expression changes (at least one probe in case of multiple
`probe sets per gene)
`
`appeared as drug-dependent,
`in
`
`the of criterion described sense
`
`above.
`(Tables
`In the first analysis,
`772
`genes
`were
`over-expressed
`down-
`regulated (Tables
`
`2 and 4) in docetaxel-resistant
`cells.
`
`These genes were
`sorted out by the mean
`of the fold change observed between
`the
`
`first and the last doses of docetaxel
`
`(between 5 and 200
`nM).
`
`A second analysis was performed from
`
`data set.
`
`first
`to confirm the
`biological duplicates
`In the second analysis, only the irreversible resistance mechanisms were retained by using
`resistant cells cultured in the absence of drug during two passages before the microarray
`analysis. The second analysis generated a list of 486 genes in which 44 genes were already
`observed
`in
`the
`
`first analysis. In
`the
`
`
`list of 44 genes commons in the two analyses,
`17
`genes
`over-expressed
`and
`27 genes were down-regulated in docetaxel-resistance cells (Table 5).
`
`These
`genes were sorted out by the mean of the fold change observed between the first and the last
`doses of docetaxel
`(between
`5 200 nM).
`
`and
`selected containing 4 over-expressed genes
`
`Among these
`genes,
`subset a of 17 genes was
`
`
`and 13 under-expressed genes in docetaxel-resistance cells (Table 6). This set of genes has been
`selected by
`the
`following
`method.
`
`were
`
`5
`
`10
`
`15
`
`20
`
`01736
`
`

`
`1,032
`0,505
`0,767
`0,742
`0,499
`0,379
`1,058
`0,452
`-0,077
`1,272
`0,965
`0,523
`0,987
`0,893
`0,284
`0,644
`0,311
`0,743
`0,677
`1,118
`0,393
`0,624
`0,876
`1,334
`1,304
`0,554
`0,680
`0,820
`1,118
`0,766
`0,984
`0,773
`200nM
`
`0,696
`0,268
`0,590
`0,391
`0,387
`0,318
`1,033
`-0,009
`-0,356
`0,892
`0,461
`0,147
`0,546
`0,723
`0,113
`0,551
`0,225
`0,765
`0,590
`0,981
`0,403
`0,077
`0,827
`1,389
`1,170
`0,398
`0,354
`0,981
`1,188
`0,347
`1,070
`0,451
`lOOnM
`
`0,357
`-0,175
`0,227
`-0,037
`0,131
`0,109
`1,091
`-0,525
`-0,627
`0,649
`0,409
`0,576
`0,781
`0,368
`0,180
`0,337
`0,333
`0,241
`0,436
`0,165
`-0,067
`0,278
`0,826
`1,226
`1,045
`-0,179
`-0,459
`0,839
`1,186
`0,449
`0,857
`-0,222
`50nM
`
`0,216
`-0,074
`0,160
`-0,062
`0,117
`0,084
`0,913
`-0,408
`-0,860
`0,692
`0,071
`0,582
`0,717
`0,417
`0,040
`0,156
`0,286
`0,256
`0,568
`0,137
`-0,040
`0,042
`0,980
`1,134
`0,914
`-0,364
`-0,233
`0,643
`0,780
`0,049
`0,601
`-0,173
`25nM
`
`0,317
`-0,429
`0,398
`0,326
`0,133
`0,078
`0,386
`-0,545
`-0,365
`0,481
`0,056
`-0,104
`0,265
`0,048
`-0,382
`0,000
`-0,574
`0,064
`0,024
`0,333
`-0,541
`0,089
`0,637
`0,756
`0,593
`-0,260
`0,017
`-0,077
`0,046
`0,030
`0,130
`0,134
`12nM
`
`0,186
`-0,353
`-0,098
`-0,123
`0,103
`0,005
`0,181
`-0,437
`-0,976
`0,235
`0,353
`-0,382
`0,063
`-0,032
`-0,690
`-0,304
`-0,640
`-0,214
`-0,304
`0,111
`-0,599
`-0,365
`-0,170
`0,325
`0,165
`-0,568
`-0,490
`-0,321
`0,000
`-0,498
`-0,285
`-0,532
`5nM
`
`hs|15q11.2
`hs|8q12.1
`
`hs|10p11.21
`hs|7q31.1
`hs|3q23
`hs|3q13.12
`hs|3q26.1
`hs|4q22.1
`hs|1p34.2
`hs|Xp 11.23
`hs|8q13.1
`hs|7q31.1
`hs|1q31.2
`hs|14q31.3
`hs|5p13.2
`hs|4p15.32
`hs|4q31.1
`hs|8q24.12
`hs|5p14.1
`hs|2q12.3
`hs|8q24.3
`hs|12q23.3
`hs|1q23.3
`hs|2q35
`hs|3p22.3
`hs|1q12
`hs|12p11.22
`hs|1q23.3
`hs|10q25.1
`hs|4q22.1
`hs|1q12
`Cytoband
`
`10,428
`12,542
`13,302
`14,803
`15,296
`15,444
`18,347
`18,566
`20,197
`FoldChange p.value
`
`NM 001080850 Hs.473495
`NM_033215
`Hs.433652
`Hs 85524
`NM 184086
`
`NM 002655
`BC016022
`NM 021045
`NM 182529
`NM_019001
`AY831680
`NM 025047
`
`THC2724353
`PLAG1
`BC016022
`ZNF248
`THAP5
`XRN1
`AY831680
`ARL14
`CB985069
`RP4-692D3.1
`PKD2L1
`TRIIUISS
`FOXP2
`RGS2
`NM 002923
`FLRT2
`NM_013231
`SPEF2
`NM 024867
`PROM1
`NM 006017
`CLGN
`NM 004362
`NOV
`NM 002514
`LOC643401
`BC039509
`ST6GAL2
`AB058780
`ZNF251
`BC006258
`AY358241
`AY358241
`BC044624
`BC044624
`WNT6
`NM 006522
`AB011099
`SUSD5
`LOC728295
`XR_015377
`TMTC1
`NM 175861
`AK022962
`AK022962
`C10orf79
`NM_025145
`BI836406
`BI836406
`ENST00000399723 AK090412
`Gene Symbol
`Accession#
`Genbank
`
`Hs.14968
`Hs.679496
`Hs.572001
`Hs.650237
`Hs.435103
`Hs.526752
`Hs.287702
`
`Hs.78944
`Hs.533710
`Hs.298863
`Hs.614734
`Hs.86368
`Hs.235935
`Hs.533212
`Hs.98265
`Hs.534516
`Hs.626042
`Hs.654412
`Hs.29764
`Hs.196647
`Hs.636711
`Hs.401954
`Hs.654412
`Hs.288927
`Hs.130203
`Hs.656011
`UniGenelD
`
`01737
`
`

`
`5,448
`5,522
`5,537
`5,548
`5,626
`5,704
`5,791
`5,856
`5,923
`5,940
`5,948
`5,976
`5,999
`6,007
`6,082
`6,100
`6,106
`6,131
`6,155
`6,175
`6,185
`6,191
`6,207
`6,244
`6,329
`6,543
`6,580
`6,668
`6,701
`6,850
`6,865
`6,932
`FoldChange p.value
`
`0,668
`0,110
`0,352
`0,795
`0,822
`0,253
`0,330
`0,653
`1,146
`0,130
`0,735
`0,764
`0,742
`0,642
`0,668
`0,732
`1,294
`0,811
`0,789
`0,781
`0,974
`0,445
`0,143
`0,364
`1,223
`0,787
`0,375
`0,324
`0,654
`0,824
`1,010
`0,829
`200nM
`
`0,445
`-0,126
`0,201
`0,492
`0,567
`0,237
`0,210
`0,373
`1,062
`-0,178
`0,685
`0,478
`0,440
`0,855
`0,060
`0,391
`1,232
`0,628
`0,607
`0,419
`0,583
`0,311
`0,112
`0,095
`0,877
`0,708
`0,158
`0,267
`0,801
`0,793
`0,618
`0,514
`IOO11PJI
`
`0,445
`-0,207
`-0,209
`0,144
`0,072
`0,083
`0,587
`0,356
`0,711
`-0,263
`0,248
`0,000
`0,202
`0,940
`-0,261
`-0,037
`1,312
`0,478
`0,491
`-0,051
`0,613
`-0,012
`0,284
`-0,704
`0,451
`0,659
`-0,267
`0,227
`0,656
`0,675
`0,416
`-0,005
`50nM
`
`0,248
`-0,415
`-0,078
`0,110
`0,093
`-0,185
`0,316
`0,243
`0,418
`-0,458
`0,475
`-0,421
`-0,078
`0,783
`-0,180
`-0,099
`1,258
`0,251
`0,405
`0,066
`0,031
`-0,135
`0,233
`-0,349
`0,602
`0,303
`-0,434
`-0,038
`0,457
`0,525
`0,254
`-0,011
`25nM
`
`0,000
`0,120
`-0,095
`0,204
`0,006
`-0,097
`-0,171
`0,079
`0,175
`-0,243
`0,217
`-0,008
`0,284
`0,271
`0,000
`0,212
`0,660
`0,174
`0,068
`0,000
`0,194
`0,132
`-0,383
`-0,367
`0,441
`0,309
`-0,241
`-0,162
`0,276
`0,270
`0,425
`0,000
`12nM
`
`-0,069
`-0,666
`-0,404
`0,041
`0,149
`-0,507
`-0,433
`-0,145
`0,485
`-0,646
`-0,039
`0,000
`-0,035
`0,077
`-0,338
`-0,050
`0,508
`0,022
`0,000
`-0,058
`0,181
`-0,347
`-0,557
`-0,431
`0,421
`0,157
`-0,526
`-0,500
`-0,043
`-0,013
`0,059
`-0,038
`5nM
`
`hs|18q21.2
`hs|15q11.2
`hs|5p15.2
`hs|13q13.1
`hs|6p12.3
`hs|20p11.21
`hs|6q11.1
`hs|2q32.3
`hs|2p24.3
`hs|14q32.32
`hs|1p31.1
`hs|16q23.3
`hs|Xq22.1
`hs|21q22.2
`hs|1p13.2
`hs|17p13.2
`hs|10q21.2
`hs|1p22.1
`hs|1p31.3
`hs|8p23.3
`hs|10q23.31
`hs|2p15
`hs|1q25.3
`hs|2q31.3
`hs|Xp22.31
`hs|12q14.2
`hs|1q31.1
`hs|4q24
`hs|10q11.21
`hs|10q11.22
`hs|5q35.2
`hs|17p11.2
`Cytoband
`
`Hs. 120790
`
`Hs.591751
`Hs.161220
`Hs.485528
`Hs.666255
`Hs.519794
`Hs.97403
`Hs.260855
`
`NM 025214
`CA941346
`NM