TREATMENT OF NEUROBLASTOMA WITH HISTONE DEACETYLASE INHIBITORS

BACKGROUND

Neuroblastoma is an extra-cranial solid cancer arising from the neural crest and is among the most common cancers in infants less than 1 year of age. Approximately one child per 100,000 is diagnosed with neuroblastoma, resulting in 650 new cases each year in the United States. Current treatment for this high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy, and retinoic acid. Half of the children with neuroblastoma have high risk disease and 20%-50% of those children will fail to respond adequately to current therapies, illustrating a clear unmet medical need.

SUMMARY

Provided herein are methods of treating neuroblastoma comprising administering to a subject in need thereof a histone deacetylase inhibitor. Also provided herein is a pharmaceutical combination for treating neuroblastoma, comprising a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, and retinoic acid or a pharmaceutically acceptable salt thereof. In one embodiment, the retinoic acid is all-trans-retinoic acid (ATRA). In another embodiment, the HDAC inhibitor is an HDAC1/2 inhibitor. In another embodiment, the HDAC inhibitor is an HDAC1/2-specific inhibitor.

In an aspect, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof. In yet another aspect, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical combination comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof, and retinoic acid or a pharmaceutically acceptable salt thereof.

In embodiments of these aspects, the retinoic acid is ATRA. In another embodiment of these aspects, the HDAC inhibitor is an HDAC1/2 inhibitor. In another embodiment, the HDAC inhibitor is an HDAC1/2-specific inhibitor. In another embodiment, the subject was previously refractory to ATRA.

In another embodiment of these aspects, the HDAC inhibitor is a compound of Formula I:

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or a pharmaceutically acceptable salt thereof.

In another embodiment of these aspects, the compound of Formula I is:

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or a pharmaceutically acceptable salt thereof.

In another embodiment of these aspects, the HDAC1/2-specific inhibitor is a compound of Formula II, or a pharmaceutically acceptable salt thereof.

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In yet another embodiment, the compound of Formula II is:

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or a pharmaceutically acceptable salt thereof.

In another embodiment of these aspects, the HDAC inhibitor is:

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or a pharmaceutically acceptable salt thereof.

In still another embodiment, the combination further comprises a pharmaceutically acceptable carrier.

In another aspect, provided herein is a method for predicting whether a neuroblastoma patient will respond to treatment with a pharmaceutical combination comprising an HDAC inhibitor and retinoic acid comprising the steps of:

a) administering to the patient a pharmaceutical combination comprising an HDAC inhibitor and retinoic acid;

b) taking a biological sample from the patient;

c) measuring the expression level of one or more genes selected from the group consisting of BMP4, RGS16, IER3, RGL1, SGK, CTSH, ETS1, ETS1, DUSP6, SIPA1L2, EGR1, FOS, HSPA5, NPC2, PQLC3, CFD, DHRS2, POU4F1, MYLIP, AIF1L, HMMR, SCPEP1, MERTK, LOC338758, CIB1, COL5A1, CTSL2, IFI6, CGN, CPVL, PPP2R2B, CCDC99, CYP2J2, BAMBI, HSPA1A, RN7SK, ITPR1, SPA17, ESRRG, CLDN11, ST6GALNAC3, STAT1, PPP1R3C, CRY1, RYBP, FSTL5, PRSS35, SERPINE2, HMMR, GLRX, LMO4, IL13RA2, IGSF3, NEK1, CAST, PAG1, STK3, NPTX2, CAP1, HSPA2, SDF2L1, ACO1, MAP4K2, CRYZ, DNCL1, CREG1, RHBDF2, PYGL, LRRC1, LOC730432, SERPINI1, CBR4, RAB23, VCL, ETV5, TIPARP, ALS2, SDCBP, FERMT2, TJP1, POP5, LCMT2, CEP55, PLCB1, KIAA1618, BCL2L12, PDGFD, CDC14B, CRELD2, FLJ35767, SCN9A, LOC441089, PLS1, CYP26B1, RET, RET, CRABP2, CYP26A1, ATP7A, TSPAN1, NFKBIZ, DHRS3, RARB, PLAT, VGF, PTGER2, PCDH18, ENPP2, NAV2, RARB, PLS3, CYP1B1, LOC387763, PCDH18, PDZRN3, ENPP2, RET, MMP11, TRAF3IP2, LOC375295, PRKCH, TMX4, CYP26A1, EFNB2, TMX4, PDZRN3, FNDC5, NCOA3, THBS1, LOXL4, CHRNA3, NAV2, IRF9, REPS2, FRMD6, NEDD4L, FOXC1, RARA, REPS2, ABCA1, GNG2, PDZRN3, CHRNA3, SMOC1, AKR1C3, PRMT6, ALX3, NEDD9, RND3, C10orf33, CDKN1A, ACSL3, PLS3, CRISPLD1, CRISPLD1, PCDH20, RPL26, LOC729236, JARID2, RNU6-1, HOXD1, ATP6AP2, SPRY4, REC8, FZD7, TMEM50B, RDH10, RN5S9, NPTN, G3BP2, ITGA1, NPTN, UBLCP1, IL10RB, ARMET, SH2B3, ADD3, ACSL3, RNU6-15, LOC653158, SGK1, ZFAND6, BCHE, HSD17B12, SNORA79, LIPA, G3BP1, LAMC1, CNN2, ABCB1, GLCE, FLOT1, SPRED1, VASN, XPR1, CYB5R4, FAM69A, XPR1, SC5DL, TMEM19, DNAJB11, HSP90B1, PAPSS1, FGFR1OP2, WDR1, HSD17B12, WDR44, OSTF1, SGK1, S100A10, SIPA1, SCGN, PLS1, RALB, TMC6, EXTL2, PNPLA8, YIPF1, GPR177, TRAM2, CXorf57, MYCNOS, COQ10B, PIGM, ELMOD1, DNAJB6, LOC653156, REC8, TMBIM4, TJP1, USP8, OSBPL3, CPVL, DUSP5, CADM1, SEC24D, MYADM, LOC285359, MYL12A, C3orf59, BCL6, EPB41L5, CXorf45, ZSWIM6, DCBLD2, LAMP2, HLA-B, LOC401076, TXNDC9, PCDH17, YIPF1, LOC729646, PTGR1, IGF2R, EPB41L5, LOC100129685, PAQR8, RPGR, FBLN2, GCA, GPR126, PI15, GNS, ALG13, TP53INP1, NPPA, USP38, PSMA4, C5orf32, PRKCA, SEC22B, DNAJC10, UTP14C, TULP4, HIF1A, DYNC1I1 ANKRD57, PON2, BMPR2, SLC4A8, ATP2B1, DAD1, RAB3IP, RPPH1, PRG2, PRKAR1A, ZMYM1, CLINT1, TMCO1, PDGFD, USP9X, AADACL4, BCL2L12, ALPL, LOC653079, CCDC128, HDAC1, HLA-E, INTS6, TMEM166, NDFIP2, EDEM3, FER1L4, CHUK, C10orf75, LOC389342, RNASEL, LOC100131205, TMEM205, RRBP1, ALCAM, ATG4C, MEGF9, C1orf97, STRADB, SREBF1, SUOX, RAB8B, SPRY1, ARL6IP1, C12orf34, RPAP3, LOC728782, PLEKHA6, KLF10, CD44, SNORA8, CDH24, DLK1, PTCHD1, SLC6A15, STMN4, MIAT, C16orf53, PCOLCE, TYMS, ASAM, FLJ25404, ICA1, SLC6A15, DUSP26, SH2D3C, LRFN4, CENPV, DDX17, C16orf53, CLASP2, ARMCX1, ICA1, LAMB1, CLK1, TH, P4HTM, D4S234E, MTA1, TUB, PHF17, TAGLN3, SYTL4, ARHGDIG, ABR, SNORA18, H2AFY2, ST6GAL1, DUSP8, TFAP2B, RCN1, ZNF536, F12, SCRG1, LRRTM2, GRIN1, SEZ6L2, GRM8, CENTA1, HDGF, JAM2, DDR2, MYT1, PCGF2, CNTNAP1, EML5, C1orf43, BRSK1, N4BP2L1, TCEAL7, TAGLN3, NME4, DLK1, SNHG7, MEG3, ATP1A1, LOC100131866, LOC728452, LOC441763, LOC651816, CALML4, CD320, TRAP1, ST3GAL4, LOC647251, VIM, DCN, TRERF1, SLC29A1, C2orf48, INSM2, CACNA1H, ILVBL, NELL1, LOC648210, TUBA1A, ACTG1, LOC100008588, LOC100133565, TUBB, LOC92755, LOC100133372, TUBA1C, ACTB, RTN1, LOC642817, FLJ39632, LOC91561, LOC645691, LOC100131609, PHOX2B, LOC388654, RPLP0, PHOX2B, IRF2BP2, TMEM132A, CCT7, SIX3, LOC645436, LOC648210, HMGA1, LOC148430, RPS2, LOC645385, ALDOA, LOC728698, EEF1G, LOC728643, RPLP0, SORBS2, MYCN, GUSBL1, SORBS2, RPS9, LOC729926, C1orf43, LOC100008589, GTF2IP1, ATP1A1, LOC646294, LOC391075, LOC402112, ALDOA, LOC728565, LOC646785, RPS9, TPI1, TCP1, LOC644063, APP, LOC440589, LOC284821, LOC100129553, PGAM1, LOC643357, PRMT1, PLD6, LOC647000, PRDX2, HAND2, LOC100131609, GTF2IP1, MATR3, ATF4, LOC100132528, LOC347544, LOC440589, PLCXD3, LOC728658, LOC651149, PRDX2, SNHG7, LOC729779, NCL, LOC285053, MTHFD2, SMA4, LOC441775, CAPRIN1, LOC648695, LOC648249, HIST3H2A, LOC644774, ZIC2, NPIP, SSR2, LGALS3BP, TSPO, LOC387867, NDUFA4L2, GREM1, LOC728732, SPAG9, TH, MPST, NPDC1, ACP1, ATP2C1, CASC3, LOC441506, LOC646531, PQBP1, LOC100008589, LOC100128771, B3GNT6, RNF5P1, LOC153561, NUMA1, NXPH1, RELN, SNORA67, TTC8, NFKBIA, SPTBN1, LOC100132394, GAB2, LOC652900, GLCCI1, CKAP5, LOC388707, SNRPN, SMA5, CNBP, MYT1L, LOC100128266, CD276, PHB2, HDGF2, FLJ22184, SCARB1, RBMX, MBTPS1, TMOD1, LOC441013, LOC643531, MIR1978, ATN1, FBLN1, GUSBL1, BIN1, CAMKV, LOC728658, LOC440349, HDAC9, SMA4, UNC5A, LOC390354, UNG, PRMT1, FTL, 3-Sep, ATCAY, PYCR1, RANBP1, GNG4, TAGLN2, LOC440157, CUEDC2, NFIX, TH1L, SUMO2, SORL1, DEAF1, LOC92755, CKAP4, C12orf24, TUBB4Q, LOC728139, PRRT2, LOC100130561, TACC2, MAP1B, PKMYT1, UCK2, LOC652489, IRF2BP2, EEF1D, RALY, PFKP, CCDC136, RNF165, NOMO1, TCF3, LOC401537, TNPO1, ST8SIA2, STMN2, APIP, ATP1A1, LOC649150, PKD1, LOC643300, PLOD3, SDHA, GPX7, THOC4, PRRX2, SGPP2, APEX1, PHF2, CABC1, LOC100134241, LOC732007, CCT6A, FTL, THOC3, PRR7, MCM2, C9orf86, CSNK1E, MGAT3, FEZ1, PODXL2, ENO2, LMO3, WDR5, LOC399804, PKM2, PLEKHG3, PLD6, B4GALNT4, GUSBL1, PCBP4, C12orf57, LOC651198, GAPDH, LOC402251, PALM, PCK2, ACO2, TIAL1, PTPRD, MARCKSL1, 3-Sep, PISD, PTK7, FAF1, SLC35F3, H2AFX, GNL3, FAM57B, CDK5R1, TNIP1, EEF1D, TRPC4AP, RAD51AP1, PSCD1, RELN, SIGMAR1, STXBP1, LOC643873, SKP2, HNRPK, FEZ1, HNRNPL, ADM, DBNDD2, LOC643668, NGFRAP1, FOXK1, CENTG3, NME3, EIF4A1, LOC100131735, SAC3D1, LOC100134364, TMSB10, IDH2, DPM3, PRKCZ, EIF4H, GAS6, NHP2, CNTFR, LOC440927, LOC286444, LOC100133840, TSC22D3, KIAA0195, LOC728873, BIN1, RSL1D1, N4BP2L1, NIPSNAP1, GPSM1, COLEC11, TNC, LOC100129585, NDUFV1, TPT1, ZNF423, UCKL1, MDK, TIGA1, LOC727761, FAM125B, LOC157627, SDC1, SLC10A4, SCAMP5, DAPK1, LOC389141, HRK, LOC100132060, PNMA3, DYRK2, MRPS24, LOC648927, FRZB, KLF11, LOC644237, LOC648024, TNRC4, HNRNPK, CALD1, PWWP2B, WDR45L, LOC440595, HDAC9, TRIM28, ADAR, TMEM101, PEG10, HNRNPA3, LOC100134648, LOC728411, GAPDH, GRIA4, CACNA1H, SNHG3-RCC1, EEF1A1, SLC4A2, TUBB3, PIM1, ZNRD1, ZNF536, RPL13A, DBNDD1, TXNDC5, PDZD4, SLC27A3, and RPL12 in the biological sample from the patient;

d) determining whether there is a greater than 2-fold increase in expression of one or more genes selected from the group consisting of BMP4, RGS16, IER3, RGL1, SGK, CTSH, ETS1, ETS1, DUSP6, SIPA1L2, EGR1, FOS, HSPA5, NPC2, PQLC3, CFD, DHRS2, POU4F1, MYLIP, AIF1L, HMMR, SCPEP1, MERTK, LOC338758, CIB1, COL5A1, CTSL2, IFI6, CGN, CPVL, PPP2R2B, CCDC99, CYP2J2, BAMBI, HSPA1A, RN7SK, ITPR1, SPA17, ESRRG, CLDN11, ST6GALNAC3, STAT1, PPP1R3C, CRY1, RYBP, FSTL5, PRSS35, SERPINE2, HMMR, GLRX, LMO4, IL13RA2, IGSF3, NEK1, CAST, PAG1, STK3, NPTX2, CAP1, HSPA2, SDF2L1, ACO1, MAP4K2, CRYZ, DNCL1, CREG1, RHBDF2, PYGL, LRRC1, LOC730432, SERPINI1, CBR4, RAB23, VCL, ETV5, TIPARP, ALS2, SDCBP, FERMT2, TJP1, POP5, LCMT2, CEP55, PLCB1, KIAA1618, BCL2L12, PDGFD, CDC14B, CRELD2, FLJ35767, SCN9A, LOC441089, PLS1, CYP26B1, RET, RET, CRABP2, CYP26A1, ATP7A, TSPAN1, NFKBIZ, DHRS3, RARB, PLAT, VGF, PTGER2, PCDH18, ENPP2, NAV2, RARB, PLS3, CYP1B1, LOC387763, PCDH18, PDZRN3, ENPP2, RET, MMP11, TRAF3IP2, LOC375295, PRKCH, TMX4, CYP26A1, EFNB2, TMX4, PDZRN3, FNDC5, NCOA3, THBS1, LOXL4, CHRNA3, NAV2, IRF9, REPS2, FRMD6, NEDD4L, FOXC1, RARA, REPS2, ABCA1, GNG2, PDZRN3, CHRNA3, SMOC1, AKR1C3, PRMT6, ALX3, NEDD9, RND3, C10orf33, CDKN1A, ACSL3, PLS3, CRISPLD1, CRISPLD1, PCDH20, RPL26, LOC729236, JARID2, RNU6-1, HOXD1, ATP6AP2, SPRY4, REC8, FZD7, TMEM50B, RDH10, RN5S9, NPTN, G3BP2, ITGA1, NPTN, UBLCP1, IL10RB, ARMET, SH2B3, ADD3, ACSL3, RNU6-15, LOC653158, SGK1, ZFAND6, BCHE, HSD17B12, SNORA79, LIPA, G3BP1, LAMC1, CNN2, ABCB1, GLCE, FLOT1, SPRED1, VASN, XPR1, CYB5R4, FAM69A, XPR1, SC5DL, TMEM19, DNAJB11, HSP90B1, PAPSS1, FGFR1OP2, WDR1, HSD17B12, WDR44, OSTF1, SGK1, S100A10, SIPA1, SCGN, PLS1, RALB, TMC6, EXTL2, PNPLA8, YIPF1, GPR177, TRAM2, CXorf57, MYCNOS, COQ10B, PIGM, ELMOD1, DNAJB6, LOC653156, REC8, TMBIM4, TJP1, USP8, OSBPL3, CPVL, DUSP5, CADM1, SEC24D, MYADM, LOC285359, MYL12A, C3orf59, BCL6, EPB41L5, CXorf45, ZSWIM6, DCBLD2, LAMP2, HLA-B, LOC401076, TXNDC9, PCDH17, YIPF1, LOC729646, PTGR1, IGF2R, EPB41L5, LOC100129685, PAQR8, RPGR, FBLN2, GCA, GPR126, PI15, GNS, ALG13, TP53INP1, NPPA, USP38, PSMA4, C5orf32, PRKCA, SEC22B, DNAJC10, UTP14C, TULP4, HIF1A, DYNC1I1, ANKRD57, PON2, BMPR2, SLC4A8, ATP2B1, DAD1, RAB3IP, RPPH1, PRG2, PRKAR1A, ZMYM1, CLINT1, TMCO1, PDGFD, USP9X, AADACL4, BCL2L12, ALPL, LOC653079, CCDC128, HDAC1, HLA-E, INTS6, TMEM166, NDFIP2, EDEM3, FER1L4, CHUK, C10orf75, LOC389342, RNASEL, LOC100131205, TMEM205, RRBP1, ALCAM, ATG4C, MEGF9, C1orf97, STRADB, SREBF1, SUOX, RAB8B, SPRY1, ARL6IP1, C12orf34, RPAP3, LOC728782, PLEKHA6, and KLF10, or a greater than 2-fold decrease in expression of one or more genes selected from the group consisting of CD44, SNORA8, CDH24, DLK1, PTCHD1, SLC6A15, STMN4, MIAT, C16orf53, PCOLCE, TYMS, ASAM, FLJ25404, ICA1, SLC6A15, DUSP26, SH2D3C, LRFN4, CENPV, DDX17, C16orf53, CLASP2, ARMCX1, ICA1, LAMB1, CLK1, TH, P4HTM, D4S234E, MTA1, TUB, PHF17, TAGLN3, SYTL4, ARHGDIG, ABR, SNORA18, H2AFY2, ST6GAL1, DUSP8, TFAP2B, RCN1, ZNF536, F12, SCRG1, LRRTM2, GRIN1, SEZ6L2, GRM8, CENTA1, HDGF, JAM2, DDR2, MYT1, PCGF2, CNTNAP1, EML5, C1orf43, BRSK1, N4BP2L1, TCEAL7, TAGLN3, NME4, DLK1, SNHG7, MEG3, ATP1A1, LOC100131866, LOC728452, LOC441763, LOC651816, CALML4, CD320, TRAP1, ST3GAL4, LOC647251, VIM, DCN, TRERF1, SLC29A1, C2orf48, INSM2, CACNA1H, ILVBL, NELL1, LOC648210, TUBA1A, ACTG1, LOC100008588, LOC100133565, TUBB, LOC92755, LOC100133372, TUBA1C, ACTB, RTN1, LOC642817, FLJ39632, LOC91561, LOC645691, LOC100131609, PHOX2B, LOC388654, RPLP0, PHOX2B, IRF2BP2, TMEM132A, CCT7, SIX3, LOC645436, LOC648210, HMGA1, LOC148430, RPS2, LOC645385, ALDOA, LOC728698, EEF1G, LOC728643, RPLP0, SORBS2, MYCN, GUSBL1, SORBS2, RPS9, LOC729926, C1orf43, LOC100008589, GTF2IP1, ATP1A1, LOC646294, LOC391075, LOC402112, ALDOA, LOC728565, LOC646785, RPS9, TPI1, TCP1, LOC644063, APP, LOC440589, LOC284821, LOC100129553, PGAM1, LOC643357, PRMT1, PLD6, LOC647000, PRDX2, HAND2, LOC100131609, GTF2IP1, MATR3, ATF4, LOC100132528, LOC347544, LOC440589, PLCXD3, LOC728658, LOC651149, PRDX2, SNHG7, LOC729779, NCL, LOC285053, MTHFD2, SMA4, LOC441775, CAPRIN1, LOC648695, LOC648249, HIST3H2A, LOC644774, ZIC2, NPIP, SSR2, LGALS3BP, TSPO, LOC387867, NDUFA4L2, GREM1, LOC728732, SPAG9, TH, MPST, NPDC1, ACP1, ATP2C1, CASC3, LOC441506, LOC646531, PQBP1, LOC100008589, LOC100128771, B3GNT6, RNF5P1, LOC153561, NUMA1, NXPH1, RELN, SNORA67, TTC8, NFKBIA, SPTBN1, LOC100132394, GAB2, LOC652900, GLCCI1, CKAP5, LOC388707, SNRPN, SMA5, CNBP, MYT1L, LOC100128266, CD276, PHB2, HDGF2, FLJ22184, SCARB1, RBMX, MBTPS1, TMOD1, LOC441013, LOC643531, MIR1978, ATN1, FBLN1, GUSBL1, BIN1, CAMKV, LOC728658, LOC440349, HDAC9, SMA4, UNC5A, LOC390354, UNG, PRMT1, FTL, 3-Sep, ATCAY, PYCR1, RANBP1, GNG4, TAGLN2, LOC440157, CUEDC2, NFIX, TH1L, SUMO2, SORL1, DEAF1, LOC92755, CKAP4, C12orf24, TUBB4Q, LOC728139, PRRT2, LOC100130561, TACC2, MAP1B, PKMYT1, UCK2, LOC652489, IRF2BP2, EEF1D, RALY, PFKP, CCDC136, RNF165, NOMO1, TCF3, LOC401537, TNPO1, ST8SIA2, STMN2, APIP, ATP1A1, LOC649150, PKD1, LOC643300, PLOD3, SDHA, GPX7, THOC4, PRRX2, SGPP2, APEX1, PHF2, CABC1, LOC100134241, LOC732007, CCT6A, FTL, THOC3, PRR7, MCM2, C9orf86, CSNK1E, MGAT3, FEZ1, PODXL2, ENO2, LMO3, WDR5, LOC399804, PKM2, PLEKHG3, PLD6, B4GALNT4, GUSBL1, PCBP4, C12orf57, LOC651198, GAPDH, LOC402251, PALM, PCK2, ACO2, TIAL1, PTPRD, MARCKSL1, 3-Sep, PISD, PTK7, FAF1, SLC35F3, H2AFX, GNL3, FAM57B, CDK5R1, TNIP1, EEF1D, TRPC4AP, RAD51AP1, PSCD1, RELN, SIGMAR1, STXBP1, LOC643873, SKP2, HNRPK, FEZ1, HNRNPL, ADM, DBNDD2, LOC643668, NGFRAP1, FOXK1, CENTG3, NME3, EIF4A1, LOC100131735, SAC3D1, LOC100134364, TMSB10, IDH2, DPM3, PRKCZ, EIF4H, GAS6, NHP2, CNTFR, LOC440927, LOC286444, LOC100133840, TSC22D3, KIAA0195, LOC728873, BIN1, RSL1D1, N4BP2L1, NIPSNAP1, GPSM1, COLEC11, TNC, LOC100129585, NDUFV1, TPT1, ZNF423, UCKL1, MDK, TIGA1, LOC727761, FAM125B, LOC157627, SDC1, SLC10A4, SCAMP5, DAPK1, LOC389141, HRK, LOC100132060, PNMA3, DYRK2, MRPS24, LOC648927, FRZB, KLF11, LOC644237, LOC648024, TNRC4, HNRNPK, CALD1, PWWP2B, WDR45L, LOC440595, HDAC9, TRIM28, ADAR, TMEM101, PEG10, HNRNPA3, LOC100134648, LOC728411, GAPDH, GRIA4, CACNA1H, SNHG3-RCC1, EEF1A1, SLC4A2, TUBB3, PIM1, ZNRD1, ZNF536, RPL13A, DBNDD1, TXNDC5, PDZD4, SLC27A3, and RPL12, as compared to normalized gene expression level of the gene(s), indicating that the patient will respond to the pharmaceutical combination.

In one embodiment of the method for predicting whether a neuroblastome patient will respond to treatment with an HDAC inhibitor and retinoic acid, the gene having a greater than 2-fold increase in expression has a greater than 4-fold increase in expression, and the gene is selected from the group consisting of CYP26A1, CYP26B1, DHRS3, CRABP2, RARB, PTGER2, ETS1, IER3, RET, NFKBIZ, DUSP6, CDKN1A, PCDH18, CTSH, ATP7A, HSPA5, and ACSL3.

In another aspect, provided herein is a method for treating a neuroblastoma patient with a pharmaceutical combination comprising an HDAC inhibitor and retinoic acid, wherein the neuroblastoma patient is screened for response to the pharmaceutical combination, and if it is determined that the neuroblastoma patient will respond to the pharmaceutical combination, administering a therapeutically effective amount of the pharmaceutical combination to thereby treat the patient,

wherein the screening for response to the pharmaceutical combination comprises:

a) administering to the patient a pharmaceutical combination comprising an HDAC inhibitor and retinoic acid;

b) taking a biological sample from the patient;

c) measuring the expression level of one or more genes selected from the group consisting of BMP4, RGS16, IER3, RGL1, SGK, CTSH, ETS1, ETS1, DUSP6, SIPA1L2, EGR1, FOS, HSPA5, NPC2, PQLC3, CFD, DHRS2, POU4F1, MYLIP, AIF1L, HMMR, SCPEP1, MERTK, LOC338758, CIB1, COL5A1, CTSL2, IFI6, CGN, CPVL, PPP2R2B, CCDC99, CYP2J2, BAMBI, HSPA1A, RN7SK, ITPR1, SPA17, ESRRG, CLDN11, ST6GALNAC3, STAT1, PPP1R3C, CRY1, RYBP, FSTL5, PRSS35, SERPINE2, HMMR, GLRX, LMO4, IL13RA2, IGSF3, NEK1, CAST, PAG1, STK3, NPTX2, CAP1, HSPA2, SDF2L1, ACO1, MAP4K2, CRYZ, DNCL1, CREG1, RHBDF2, PYGL, LRRC1, LOC730432, SERPINI1, CBR4, RAB23, VCL, ETV5, TIPARP, ALS2, SDCBP, FERMT2, TJP1, POP5, LCMT2, CEP55, PLCB1, KIAA1618, BCL2L12, PDGFD, CDC14B, CRELD2, FLJ35767, SCN9A, LOC441089, PLS1, CYP26B1, RET, RET, CRABP2, CYP26A1, ATP7A, TSPAN1, NFKBIZ, DHRS3, RARB, PLAT, VGF, PTGER2, PCDH18, ENPP2, NAV2, RARB, PLS3, CYP1B1, LOC387763, PCDH18, PDZRN3, ENPP2, RET, MMP11, TRAF3IP2, LOC375295, PRKCH, TMX4, CYP26A1, EFNB2, TMX4, PDZRN3, FNDC5, NCOA3, THBS1, LOXL4, CHRNA3, NAV2, IRF9, REPS2, FRMD6, NEDD4L, FOXC1, RARA, REPS2, ABCA1, GNG2, PDZRN3, CHRNA3, SMOC1, AKR1C3, PRMT6, ALX3, NEDD9, RND3, C10orf33, CDKN1A, ACSL3, PLS3, CRISPLD1, CRISPLD1, PCDH20, RPL26, LOC729236, JARID2, RNU6-1, HOXD1, ATP6AP2, SPRY4, REC8, FZD7, TMEM50B, RDH10, RN5S9, NPTN, G3BP2, ITGA1, NPTN, UBLCP1, IL10RB, ARMET, SH2B3, ADD3, ACSL3, RNU6-15, LOC653158, SGK1, ZFAND6, BCHE, HSD17B12, SNORA79, LIPA, G3BP1, LAMC1, CNN2, ABCB1, GLCE, FLOT1, SPRED1, VASN, XPR1, CYB5R4, FAM69A, XPR1, SC5DL, TMEM19, DNAJB11, HSP90B1, PAPSS1, FGFR1OP2, WDR1, HSD17B12, WDR44, OSTF1, SGK1, S100A10, SIPA1, SCGN, PLS1, RALB, TMC6, EXTL2, PNPLA8, YIPF1, GPR177, TRAM2, CXorf57, MYCNOS, COQ10B, PIGM, ELMOD1, DNAJB6, LOC653156, REC8, TMBIM4, TJP1, USP8, OSBPL3, CPVL, DUSP5, CADM1, SEC24D, MYADM, LOC285359, MYL12A, C3orf59, BCL6, EPB41L5, CXorf45, ZSWIM6, DCBLD2, LAMP2, HLA-B, LOC401076, TXNDC9, PCDH17, YIPF1, LOC729646, PTGR1, IGF2R, EPB41L5, LOC100129685, PAQR8, RPGR, FBLN2, GCA, GPR126, PI15, GNS, ALG13, TP53INP1, NPPA, USP38, PSMA4, C5orf32, PRKCA, SEC22B, DNAJC10, UTP14C, TULP4, HIF1A, DYNC1I1, ANKRD57, PON2, BMPR2, SLC4A8, ATP2B1, DAD1, RAB3IP, RPPH1, PRG2, PRKAR1A, ZMYM1, CLINT1, TMCO1, PDGFD, USP9X, AADACL4, BCL2L12, ALPL, LOC653079, CCDC128, HDAC1, HLA-E, INTS6, TMEM166, NDFIP2, EDEM3, FER1L4, CHUK, C10orf75, LOC389342, RNASEL, LOC100131205, TMEM205, RRBP1, ALCAM, ATG4C, MEGF9, C1orf97, STRADB, SREBF1, SUOX, RAB8B, SPRY1, ARL6IP1, C12orf34, RPAP3, LOC728782, PLEKHA6, KLF10, CD44, SNORA8, CDH24, DLK1, PTCHD1, SLC6A15, STMN4, MIAT, C16orf53, PCOLCE, TYMS, ASAM, FLJ25404, ICA1, SLC6A15, DUSP26, SH2D3C, LRFN4, CENPV, DDX17, C16orf53, CLASP2, ARMCX1, ICA1, LAMB1, CLK1, TH, P4HTM, D4S234E, MTA1, TUB, PHF17, TAGLN3, SYTL4, ARHGDIG, ABR, SNORA18, H2AFY2, ST6GAL1, DUSP8, TFAP2B, RCN1, ZNF536, F12, SCRG1, LRRTM2, GRIN1, SEZ6L2, GRM8, CENTA1, HDGF, JAM2, DDR2, MYT1, PCGF2, CNTNAP1, EML5, C1orf43, BRSK1, N4BP2L1, TCEAL7, TAGLN3, NME4, DLK1, SNHG7, MEG3, ATP1A1, LOC100131866, LOC728452, LOC441763, LOC651816, CALML4, CD320, TRAP1, ST3GAL4, LOC647251, VIM, DCN, TRERF1, SLC29A1, C2orf48, INSM2, CACNA1H, ILVBL, NELL1, LOC648210, TUBA1A, ACTG1, LOC100008588, LOC100133565, TUBB, LOC92755, LOC100133372, TUBA1C, ACTB, RTN1, LOC642817, FLJ39632, LOC91561, LOC645691, LOC100131609, PHOX2B, LOC388654, RPLP0, PHOX2B, IRF2BP2, TMEM132A, CCT7, SIX3, LOC645436, LOC648210, HMGA1, LOC148430, RPS2, LOC645385, ALDOA, LOC728698, EEF1G, LOC728643, RPLP0, SORBS2, MYCN, GUSBL1, SORBS2, RPS9, LOC729926, C1orf43, LOC100008589, GTF2IP1, ATP1A1, LOC646294, LOC391075, LOC402112, ALDOA, LOC728565, LOC646785, RPS9, TPI1, TCP1, LOC644063, APP, LOC440589, LOC284821, LOC100129553, PGAM1, LOC643357, PRMT1, PLD6, LOC647000, PRDX2, HAND2, LOC100131609, GTF2IP1, MATR3, ATF4, LOC100132528, LOC347544, LOC440589, PLCXD3, LOC728658, LOC651149, PRDX2, SNHG7, LOC729779, NCL, LOC285053, MTHFD2, SMA4, LOC441775, CAPRIN1, LOC648695, LOC648249, HIST3H2A, LOC644774, ZIC2, NPIP, SSR2, LGALS3BP, TSPO, LOC387867, NDUFA4L2, GREM1, LOC728732, SPAG9, TH, MPST, NPDC1, ACP1, ATP2C1, CASC3, LOC441506, LOC646531, PQBP1, LOC100008589, LOC100128771, B3GNT6, RNF5P1, LOC153561, NUMA1, NXPH1, RELN, SNORA67, TTC8, NFKBIA, SPTBN1, LOC100132394, GAB2, LOC652900, GLCCI1, CKAP5, LOC388707, SNRPN, SMA5, CNBP, MYT1L, LOC100128266, CD276, PHB2, HDGF2, FLJ22184, SCARB1, RBMX, MBTPS1, TMOD1, LOC441013, LOC643531, MIR1978, ATN1, FBLN1, GUSBL1, BIN1, CAMKV, LOC728658, LOC440349, HDAC9, SMA4, UNC5A, LOC390354, UNG, PRMT1, FTL, 3-Sep, ATCAY, PYCR1, RANBP1, GNG4, TAGLN2, LOC440157, CUEDC2, NFIX, TH1L, SUMO2, SORL1, DEAF1, LOC92755, CKAP4, C12orf24, TUBB4Q, LOC728139, PRRT2, LOC100130561, TACC2, MAP1B, PKMYT1, UCK2, LOC652489, IRF2BP2, EEF1D, RALY, PFKP, CCDC136, RNF165, NOMO1, TCF3, LOC401537, TNPO1, ST8SIA2, STMN2, APIP, ATP1A1, LOC649150, PKD1, LOC643300, PLOD3, SDHA, GPX7, THOC4, PRRX2, SGPP2, APEX1, PHF2, CABC1, LOC100134241, LOC732007, CCT6A, FTL, THOC3, PRR7, MCM2, C9orf86, CSNK1E, MGAT3, FEZ1, PODXL2, ENO2, LMO3, WDR5, LOC399804, PKM2, PLEKHG3, PLD6, B4GALNT4, GUSBL1, PCBP4, C12orf57, LOC651198, GAPDH, LOC402251, PALM, PCK2, ACO2, TIAL1, PTPRD, MARCKSL1, 3-Sep, PISD, PTK7, FAF1, SLC35F3, H2AFX, GNL3, FAM57B, CDK5R1, TNIP1, EEF1D, TRPC4AP, RAD51AP1, PSCD1, RELN, SIGMAR1, STXBP1, LOC643873, SKP2, HNRPK, FEZ1, HNRNPL, ADM, DBNDD2, LOC643668, NGFRAP1, FOXK1, CENTG3, NME3, EIF4A1, LOC100131735, SAC3D1, LOC100134364, TMSB10, IDH2, DPM3, PRKCZ, EIF4H, GAS6, NHP2, CNTFR, LOC440927, LOC286444, LOC100133840, TSC22D3, KIAA0195, LOC728873, BIN1, RSL1D1, N4BP2L1, NIPSNAP1, GPSM1, COLEC11, TNC, LOC100129585, NDUFV1, TPT1, ZNF423, UCKL1, MDK, TIGA1, LOC727761, FAM125B, LOC157627, SDC1, SLC10A4, SCAMP5, DAPK1, LOC389141, HRK, LOC100132060, PNMA3, DYRK2, MRPS24, LOC648927, FRZB, KLF11, LOC644237, LOC648024, TNRC4, HNRNPK, CALD1, PWWP2B, WDR45L, LOC440595, HDAC9, TRIM28, ADAR, TMEM101, PEG10, HNRNPA3, LOC100134648, LOC728411, GAPDH, GRIA4, CACNA1H, SNHG3-RCC1, EEF1A1, SLC4A2, TUBB3, PIM1, ZNRD1, ZNF536, RPL13A, DBNDD1, TXNDC5, PDZD4, SLC27A3, and RPL12 in the biological sample from the patient;

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows that HDAC1/2i induce gene expression consistent with differentiation and that in combination with ATRA, the differentiation effect is enhanced; data for Compound A and ATRA on SK-N-BE(2) cells shown.

FIG. 1B shows that HDAC1/2i induce gene expression consistent with differentiation and that in combination with ATRA, the differentiation effect is enhanced;

data for Compound B and ATRA on SK-N-BE(2) cells shown.

FIG. 1C shows that HDAC1/2i induce gene expression consistent with differentiation and that in combination with ATRA, the differentiation effect is enhanced; inhibition of HDAC2 activity in live cells with potency in the 0.5-3 μM range shown.

FIG. 1D shows that Compound B induces histone acetylation in neuroblastoma cells as shown by Western blot at 24 hours.

FIG. 2A shows that the addition of 1 μM or 3 μM ATRA, which has potent differentiation activity, has little effect on HDAC1/2i mediated toxicity, indicating that HDAC1/2i toxicity is independent of ATRA, and occurs at concentrations greater than those needed for differentiation.

FIG. 2B shows that Caspase 3/7 activity is increased at concentrations ≧2 μM Compound B, and that the increased activity is independent of the presence of ATRA, indicating that HDAC1/2i toxicity is independent of ATRA, and occurs at concentrations greater than those needed for differentiation.

FIG. 3A shows that differentiation is induced by non-cytotoxic concentrations of Compound B; data for Compound B on SK-N-BE(2) cells shown.

FIG. 3B shows that differentiation is induced by non-cytotoxic concentrations of Compound A; data for Compound A on SK-N-BE(2) cells shown.

FIG. 4A shows that Compound B combines with ATRA to enhance suppression of neuroblastoma proliferation; data for 1 μM Compound B shown.

FIG. 4B shows that Compound B combines with ATRA to enhance suppression of neuroblastoma proliferation; data for 3 μM Compound B shown.

FIG. 5A shows that Compound A combines with ATRA to enhance suppression of neuroblastoma proliferation; data for 0.75 μM Compound A shown.

FIG. 5B shows that Compound A combines with ATRA to enhance suppression of neuroblastoma proliferation; data for 2 μM Compound A shown.

FIG. 6A shows that Compound B induced increased expression of the master cell cycle regulator p21 as a single agent and in a dose-dependent manner; Compound B enhances ATRA-mediated cell cycle arrest.

FIG. 6B shows that single-agent ATRA reduces s-phase frequency and increases the sub-G1 population, and that this effect is enhanced by Compound B in a dose dependent manner; Compound B enhances ATRA-mediated cell cycle arrest.

FIG. 6C shows the population of each stage of the cell cycle at various concentrations of Compound B and ATRA alone, or in combination; Compound B enhances ATRA-mediated cell cycle arrest.

FIG. 7 shows that Compound B combines with ATRA to enhance morphology changes consistent with differentiation.

FIG. 8 shows that Compound B combines with ATRA to enhance NF-M staining.

FIG. 9 shows that Compound B combines with ATRA to reduce neuroblastoma outgrowth.

FIG. 10 shows that Compound B (HDAC1/2i) shows stronger combination effect with ATRA than Compound C (HDAC3i; N-(2-amino-4-fluorophenyl)-8-cyclopropyl-7-(piperazin-1-yl)quinoline-3-carboxamide) on cell proliferation and dendrite outgrowth.

FIG. 11 shows that the combination of Compound B with ATRA completely blocks cluster formation of NB at concentrations lower than Compound C (HDAC3i).

FIG. 12A shows gene expression changes and overlaps of SK-N-BE(2) cells treated with 3 μM Compound B, 1 μM ATRA, or a combination of both at 37° C. over 2 hours when compared to a solvent (DMSO) control.

FIG. 12B shows gene expression changes and overlaps of SK-N-BE(2) cells treated with 3 μM Compound B, 1 μM ATRA, or a combination of both at 37° C. over 48 hours when compared to a solvent (DMSO) control.

FIG. 13 shows that ATRA alters binding positions of retinoic acid receptor (RAR) to chromatin, and this altered binding is enhanced by Compound B.

FIG. 14A shows a model for HDACi enhancement of ATRA-mediated differentiation.

FIG. 14B shows a simplified model for HDACi enhancement of ATRA-mediated differentiation.

FIG. 15 shows that HDAC1/2 inhibitors in combination with ATRA disrupt the Wnt signaling pathway.

FIG. 16 shows that Retinoic Acid-activated AKT is reduced by HDAC1/2i.

FIG. 17 shows the modulation of proteins involved in cell-cycle progression by HDAC1/2i.

FIG. 18 shows a diagram of pathways that were consistently enriched or suppressed by HDAC1/2 inhibition.

FIG. 19 shows that the combination of an HDAC1/2i (compound with retinoic acid enhanced cleavage of caspase 3 and 9 and PARP, consistent with apoptotic death.

FIG. 20 shows that pro-E2F signaling proteins CDK4 and 6 were decreased in the combination while the inhibitory protein p21 was increased.

FIG. 21 shows that treatment with HDAC1/2i+RA enhanced RA-induced expression of the RARβ gene.

FIG. 22 shows that treatment with HDAC1/2i+RA increased RARβ protein levels.

FIG. 23 shows that RAR binding to the RARβ gene promoter was enhanced in a combination setting (Compound B+ATRA) relative to either single agent alone.

FIG. 24 shows that Cyp26b1, a protein induced by RA and negative regulator of RAR signaling, was decreased in a combination setting (Compound B+ATRA) as measured by gene expression and protein levels.

FIG. 25 shows that DHRS3, a protein induced by RA and negative regulator of RAR signaling, was decreased in a combination (Compound B+ATRA) setting as measured by gene expression and protein levels.

FIG. 26 shows the HDAC1/2 inhibitor Compound E combines with RA to slow neuroblastoma tumor growth in vivo. (Error bars illustrate the standard error of the mean).

FIG. 27A shows that ATRA has a cytotoxic effect on neuroblastoma cells as a single agent.

FIG. 27B shows that Compound B has a cytotoxic effect on neuroblastoma cells as a single agent.

FIG. 27C shows the combination index (CI) values plotted against the fraction affected (Fa), demonstrating synergistic combination (CI values less than 1) across a wide range of Fa values.

FIG. 27D shows the combination matrix of ATRA with Compound B and illustrates the fraction of cells affected (dead cells, Fa value) and combinations where the CI value was less than 0.7 (underlined), suggesting potent synergy.

FIG. 28A shows that ATRA has a cytotoxic effect on neuroblastoma cells as a single agent.

FIG. 28B shows that Compound A has a cytotoxic effect on neuroblastoma cells as a single agent.

FIG. 28C shows the combination index (CI) values plotted against the fraction affected (Fa), demonstrating synergistic combination (CI values less than 1) across a wide range of Fa values.

FIG. 28D shows the combination matrix of ATRA with Compound A and illustrates the fraction of cells affected (dead cells, Fa value) and combinations where the CI value was less than 0.7 (underlined), suggesting potent synergy.

DETAILED DESCRIPTION

Provided herein are methods of treating neuroblastoma in a subject in need thereof, comprising administering to the subject an HDAC inhibitor. Also provided herein are combinations comprising an HDAC inhibitor and retinoic acid, for the treatment of neuroblastoma in a subject in need thereof. Also provided herein are methods for treating neuroblastoma in a subject in need thereof, comprising administering to the subject an effective amount of the above combination comprising an HDAC inhibitor and retinoic acid. Also provided herein is a method for predicting whether a neuroblastoma patient will respond to treatment with a combination comprising an HDAC inhibitor and retinoic acid.

DEFINITIONS

Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

The term “about” generally indicates a possible variation of no more than 10%, 5%, or 1% of a value. For example, “about 25 mg/kg” will generally indicate, in its broadest sense, a value of 22.5-27.5 mg/kg, i.e., 25±2.5 mg/kg.

The term “alkyl” refers to saturated, straight- or branched-chain hydrocarbon moieties containing, in certain embodiments, between one and six, or one and eight carbon atoms, respectively. Examples of C_1-6-alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl moieties; and examples of C_1-8-alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.

The number of carbon atoms in an alkyl substituent can be indicated by the prefix “C_x-y,” where x is the minimum and y is the maximum number of carbon atoms in the substituent. Likewise, a C_Xchain means an alkyl chain containing x carbon atoms.

The term “cycloalkyl” denotes a monovalent group derived from a monocyclic or polycyclic saturated carbocyclic ring compound. Examples of C_3-8-cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; and examples of C_3-12-cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl, and bicyclo [2.2.2] octyl.

The term “heterocycloalkyl” refers to a cycloalkyl ring system, as described herein, of which one ring atom is selected from S, O, N and Si; zero, one or two ring atoms are additional heteroatoms independently selected from S, O, N and Si; and the remaining ring atoms are carbon.

The term “aryl” refers to a mono- or poly-cyclic carbocyclic ring system having one or more aromatic rings, fused or non-fused, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.

The term “heteroaryl” refers to a mono- or poly-cyclic (e.g., bi-, or tri-cyclic or more) fused or non-fused, moieties or ring system having at least one aromatic ring, having from five to ten ring atoms of which one ring atom is selected from S, O, N and Si; zero, one or two ring atoms are additional heteroatoms independently selected from S, O, N and Si; and the remaining ring atoms are carbon. Heteroaryl includes, but is not limited to pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

The term “halo” refers to a halogen, such as fluorine, chlorine, bromine, and iodine.

The term “HDAC” refers to histone deacetylases, which are enzymes that remove the acetyl groups from the lysine residues in core histones, thus leading to the formation of a condensed and transcriptionally silenced chromatin. There are currently 18 known histone deacetylases, which are classified into four groups. Class I HDACs, which include HDAC1, HDAC2, HDAC3, and HDAC8, are related to the yeast RPD3 gene. Class II HDACs, which include HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, are related to the yeast Hda1 gene. Class III HDACs, which are also known as the sirtuins are related to the Sir2 gene and include SIRT1-7. Class IV HDACs, which contains only HDAC11, has features of both Class I and II HDACs. The term “HDAC” refers to any one or more of the 18 known histone deacetylases, unless otherwise specified.

The term “HDAC1/2i” or “HDAC1/2 inhibitor” means that the compound binds to HDAC1 and HDAC2.

The term “HDAC1/2-specific” means that the compound binds to HDAC1 and DAC2 to a substantially greater extent, such as 5×, 10×, 15×, 20× greater or more, than to any other type of HDAC enzyme, such as HDAC3 or HDAC6. That is, the compound is selective for HDAC1 and HDAC2 over any other type of HDAC enzyme. For example, a compound that binds to HDAC1 and HDAC2 with an IC₅₀of 10 nM and to HDAC3 with an IC₅₀of 50 nM is HDAC1/2-specific. On the other hand, a compound that binds to HDAC1 and HDAC2 with an IC₅₀of 50 nM and to HDAC3 with an IC₅₀of 60 nM is not HDAC1/2-specific.

The term “combination” refers to two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such combination of therapeutic agents can be in the form of a single pill, capsule, or intravenous solution. However, the term “combination” also encompasses the situation when the two or more therapeutic agents are in separate pills, capsules, or intravenous solutions. Likewise, the term “combination therapy” refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, or in separate containers (e.g., capsules) for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein. Further, in an embodiment, the two or more therapeutic agents may be administered independently, at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic, effect.

The term “neuroblastoma” encompasses all kinds of neuroblastomas and refers to a sarcoma of nervous system origin, composed chiefly of neuroblasts and affecting mostly infants and children up to 10 years of age. Most neuroblastomas arise in the autonomic nervous system (sympathico-blasroma) or in the adrenal medulla. Specifically, all stages of a neuroblastoma are comprised by the term. Staging is carried out, preferably, according to the International Neuroblastoma Staging System (INS S) (Brodeur 1993, J Clin Oncol 11: 1466-1477). In principle, this surgical-based staging distinguishes five basic stages of neuroblastoma: Stage I: Localized tumour confined to the area of origin. Complete gross resection with or without microscopic residual disease; identifiable ipsilateral and contralateral lymph node negative for tumour. Stage II: Unilateral tumour with incomplete gross resection; identifiable ipsilateral and contralateral lymph node negative for tumour (stage II a), with ipsilateral lymph node positive for tumour, identifiable contralateral lymph node negative for tumour (stage II b). Stage III: Tumour infiltrating across the midline with or without regional lymph node involvement; or unilateral tumour with contralateral lymph node involvement or midline tumour with bilateral lymph node involvement. Stage IV: Dissemination of tumour to distant lymph nodes, bone marrow, liver, or other organs except as defined in stage IVS. Stage IVS: Localized primary tumour as defined for stage 1 or 2 with dissemination limited to liver, skin, and bone marrow (<10% of nucleated marrow cells are tumor cells).

The term “inhibitor” is synonymous with the term antagonist.

Histone Deacetylase (HDAC) Inhibitors

Provided herein are methods for treating neuroblastoma in a subject in need thereof. Also provided herein are pharmaceutical combinations for the treatment of neuroblastoma in a subject in need thereof.

The methods and combinations of the invention comprise an HDAC inhibitor. The HDAC inhibitor can be any HDAC inhibitor. Thus, the HDAC inhibitor can be specific or non-specific to a particular type of histone deacetylase enzyme. Preferably, the HDAC inhibitor is an HDAC1/2 inhibitor. More preferably, the HDAC inhibitor is an HDAC1/2-specific inhibitor.

In some embodiments, the HDAC inhibitor is a compound of Formula I:

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or a pharmaceutically acceptable salt thereof,

wherein,

ring B is aryl or heteroaryl;

R¹is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or C_1-6-alkyl; and

R is H or C_1-6-alkyl.

In one embodiment, R¹is an aryl or heteroaryl, each of which is substituted by halo.

Representative compounds of Formula I include, but are not limited to:

embedded image

or pharmaceutically acceptable salts thereof.

The preparation and properties of HDAC inhibitors according to Formula I are provided in International Patent Application No. PCT/US2011/021982, the entire contents of which are incorporated herein by reference. The HDAC inhibitory profile of Compound A is found in Example 3, Table 1.

In another embodiment, the HDAC1/2-specific inhibitor is a compound of Formula II:

embedded image

or a pharmaceutically acceptable salt thereof,

wherein,

R¹is aryl or heteroaryl;

R²and R³are each independently selected from C_3-6-cycloalkyl, C_1-6-alkyl-OR⁶, C_1-6-alkyl-C_3-6-cycloalkyl, C_1-6-alkyl-heterocycloalkyl, C_2-6-alkenyl;

R⁶is H or C_1-6-alkyl; and

R⁷is H or C_3-6-cycloalkyl.

Compounds of Formula II are represented by, but not limited to, Compound B, or pharmaceutically acceptable salts thereof.

embedded image

The preparation and properties of HDAC1/2-specific inhibitors according to Formula II are provided in US Patent Publication No. 2014-0128391, the entire contents of which are incorporated herein by reference. The HDAC inhibitory profile of Compound B is found in Example 3, Table 1.

In another embodiment, the HDAC inhibitor is Compound E, or a pharmaceutically acceptable salt thereof.

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The preparation and properties of the HDAC inhibitor Compound E are provided in US Patent Publication No. 2014-0128391, the entire contents of which are incorporated herein by reference. The HDAC inhibitory profile of Compound E is found in Example 3, Table 1.

In some embodiments, the compounds described herein are unsolvated. In other embodiments, one or more of the compounds are in solvated form. As known in the art, the solvate can be any of pharmaceutically acceptable solvent, such as water, ethanol, and the like.

Combinations/Pharmaceutical Combinations

Provided herein are combinations for the treatment of neuroblastoma in a subject in need thereof. Provided in some embodiments are combinations, comprising an HDAC inhibitor and retinoic acid for the treatment of neuroblastoma in a subject in need thereof. In some embodiments of the combinations, the retinoic acid is ATRA, or a pharmaceutically acceptable salt thereof. In other embodiments of the combinations, the retinoic acid is 13-cis-retinoic acid, or a pharmaceutically acceptable salt thereof.

In some embodiments, the HDAC inhibitor is a compound of Formula I:

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or a pharmaceutically acceptable salt thereof.

In an embodiment, the compound of Formula I is:

embedded image

or a pharmaceutically acceptable salt thereof.

In an embodiment, the compound of Formula I is:

embedded image

or a pharmaceutically acceptable salt thereof.

In other specific embodiments, the HDAC1/2-specific inhibitor is a compound of Formula II:

embedded image

or a pharmaceutically acceptable salt thereof.

In preferred embodiments, the compound of Formula II is:

embedded image

or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the HDAC inhibitor is:

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or a pharmaceutically acceptable salt thereof.

In certain embodiment, the retinoic acid is all-trans-retinoic acid, which is also known as tretinoin and has the following structure:

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In another embodiment, the retinoic acid is 13-cis-retinoic acid, or isotretinoin, having the following structure:

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In yet another embodiment, the retinoic acid is 9-cis-retinoic acid, or alitretinoin, having the following structure:

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In some embodiments of the combinations, retinoic acid can be the free acid or a pharmaceutically acceptable salt thereof.

Although the compounds of Formulas I and II, and Compound E, are depicted in their neutral forms, in some embodiments, these compounds are used in a pharmaceutically acceptable salt form. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

Administration/Dose

In some embodiments, the HDAC inhibitor (a compound of Formula I or II, or Compound E) is administered alone. In some embodiments, the HDAC inhibitor (a compound of Formula I or II, or Compound E) is administered simultaneously with retinoic acid. Simultaneous administration typically means that both compounds enter the patient at precisely the same time. However, simultaneous administration also includes the possibility that the HDAC inhibitor and retinoic acid enter the patient at different times, but the difference in time is sufficiently miniscule that the first administered compound is not provided the time to take effect on the patient before entry of the second administered compound. Such delayed times typically correspond to less than 1 minute, and more typically, less than 30 seconds. In one example, wherein the compounds are in solution, simultaneous administration can be achieved by administering a solution containing the combination of compounds. In another example, simultaneous administration of separate solutions, one of which contains the HDAC inhibitor and the other of which contains retinoic acid, can be employed. In one example wherein the compounds are in solid form, simultaneous administration can be achieved by administering a composition containing the combination of compounds. Alternatively, simultaneous administration can be achieved by administering two separate compositions, one comprising the HDAC inhibitor and the other comprising retinoic acid.

In other embodiments, the HDAC inhibitor and retinoic acid are not administered simultaneously. In some embodiments, the HDAC inhibitor is administered before retinoic acid. In other embodiments, retinoic acid is administered before the HDAC inhibitor. In other embodiments, the first administered compound is provided time to take effect on the patient before the second administered compound is administered. Generally, the difference in time does not extend beyond the time for the first administered compound to complete its effect in the patient, or beyond the time the first administered compound is completely or substantially eliminated or deactivated in the patient.

In some embodiments, one or both of the HDAC inhibitor and retinoic acid are administered in a therapeutically effective amount or dosage. A “therapeutically effective amount” is an amount of HDAC inhibitor (a compound of Formula I or II, or Compound E) or retinoic acid that, when administered to a patient by itself, effectively treats neuroblastoma. An amount that proves to be a “therapeutically effective amount” in a given instance, for a particular subject, may not be effective for 100% of subjects similarly treated for the disease or condition under consideration, even though such dosage is deemed a “therapeutically effective amount” by skilled practitioners. The amount of the compound that corresponds to a therapeutically effective amount is strongly dependent on the type of cancer, stage of the cancer, the age of the patient being treated, and other facts. In general, therapeutically effective amounts, e.g., retinoic acid, are known in the art.

In other embodiments, one or both of the HDAC inhibitor and retinoic acid are administered in a sub-therapeutically effective amount or dosage. A sub-therapeutically effective amount is an amount of HDAC inhibitor (a compound of Formula I or II, or Compound E) or retinoic acid that, when administered to a patient by itself, does not completely inhibit over time the biological activity of the intended target.

Whether administered in therapeutic or sub-therapeutic amounts, the combination of the HDAC inhibitor and retinoic acid should be effective in treating a neuroblastoma. For example, a sub-therapeutic amount of a compound of retinoic acid can be an effective amount if, when combined with an HDAC inhibitor (a compound of Formula I or II, or Compound E), the combination is effective in the treatment of neuroblastoma. For example, a sub-therapeutic amount of a compound of retinoic acid can be an effective amount if, when combined with an HDAC inhibitor (a compound of Formula I or II, or Compound E), the combination is effective in the treatment of neuroblastoma, wherein the combination is administered at dosages that would not be effective when one or both of the compounds are administered alone, but which amounts are effective in combination.

In some embodiments, the combination of compounds exhibits a synergistic effect (i.e., greater than additive effect) in the treatment of neuroblastoma. The term “synergistic effect” refers to the action of two agents, such as, for example, an HDAC inhibitor and retinoic acid, producing an effect, for example, slowing the symptomatic progression of neuroblastoma or symptoms thereof, which is greater than the simple addition of the effects of each drug administered alone. A synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

In preferred embodiments, the combinations and methods provided herein include an HDAC inhibitor of Formula I and retinoic acid. Thus, in one embodiment, the combinations and methods include Compound A and retinoic acid. In another embodiment, the combination and methods include Compound F and retinoic acid. In other preferred embodiments, the combinations and methods provided herein include an HDAC inhibitor of Formula II and retinoic acid. Thus, in one embodiment, the combinations and methods include Compound B and retinoic acid. In still other preferred embodiments, the combinations and methods provided herein include Compound E and retinoic acid.

In different embodiments, depending on the combination and the effective amounts used, the combination of compounds can inhibit neuroblastoma growth, achieve neuroblastoma stasis, or even achieve substantial or complete neuroblastoma regression.

While the amounts of an HDAC inhibitor and retinoic acid should result in the effective treatment of neuroblastoma, the amounts, when combined, are preferably not excessively toxic to the patient (i.e., the amounts are preferably within toxicity limits as established by medical guidelines). In some embodiments, either to prevent excessive toxicity or provide a more efficacious treatment, or both, of neuroblastoma, a limitation on the total administered dosage is provided. Typically, the amounts considered herein are per day; however, half-day and two-day or three-day cycles also are considered herein.

Different dosage regimens can be used to treat neuroblastoma. In some embodiments, a daily dosage, such as any of the exemplary dosages described above, is administered once, twice, three times, or four times a day for three, four, five, six, seven, eight, nine, or ten days. Depending on the stage and severity of the cancer, a shorter treatment time (e.g., up to five days) can be employed along with a high dosage, or a longer treatment time (e.g., ten or more days, or weeks, or a month, or longer) can be employed along with a low dosage. In some embodiments, a once- or twice-daily dosage is administered every other day. In some embodiments, each dosage contains both an HDAC inhibitor and retinoic acid to be delivered as a single dosage, while in other embodiments each dosage contains an HDAC inhibitor or retinoic acid to be delivered as separate dosages.

Compounds of Formula I or II, or Compound E, or their pharmaceutically acceptable salts or solvate forms, in pure form or in an appropriate pharmaceutical composition, can be administered via any of the accepted modes of administration or agents known in the art. The compounds can be administered, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally. The dosage form can be, for example, a solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages. A particular route of administration is oral, particularly one in which a convenient daily dosage regimen can be adjusted according to the degree of severity of the disease to be treated.

As discussed above, the HDAC inhibitor and retinoic acid pharmaceutical combination can be administered in a single unit dose or separate dosage forms. Accordingly, the phrase “pharmaceutical combination” includes a combination of two drugs in either a single dosage form or separate dosage forms, i.e., the pharmaceutically acceptable carriers and excipients described throughout the application can be combined with an HDAC inhibitor and retinoic acid in a single unit dose, as well as individually combined with an HDAC inhibitor and retinoic acid when these compounds are administered separately.

Auxiliary and adjuvant agents can include, for example, preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms is generally provided by various antibacterial and antifungal agents, such as, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, can also be included. Prolonged absorption of an injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. The auxiliary agents also can include wetting agents, emulsifying agents, pH buffering agents, and antioxidants, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, and the like.

Solid dosage forms can be prepared with coatings and shells, such as enteric coatings and others well-known in the art. They can contain pacifying agents and can be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds also can be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., the HDAC inhibitors or retinoic acid described herein, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethyl formamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a solution or suspension.

Generally, depending on the intended mode of administration, the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of the compounds described herein, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of a pharmaceutically acceptable excipient. In one example, the composition will be between about 5% and about 75% by weight of a compound described herein, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.

Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. Reference is made, for example, to Remington's Pharmaceutical Sciences, 18th Ed. (Mack Publishing Company, Easton, Pa., 1990).

Methods of the Invention

Provided herein are methods for treating neuroblastoma in a subject in need thereof comprising administering to the subject an HDAC inhibitor. Also provided herein are methods for treating neuroblastoma in a subject in need thereof comprising administering to the subject a pharmaceutical combination of the invention. Thus, provided herein are methods for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an HDAC inhibitor. Also provided herein are methods for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a combination comprising an HDAC inhibitor and retinoic acid. In one embodiment, the subject was previously refractory to retinoic acid (e.g., ATRA or 13-cis-retinoic acid).

In an embodiment, the HDAC inhibitor and retinoic acid are administered at dosages and/or over time intervals producing a synergistic effect.

The subject considered herein is typically a human. However, the subject can be any mammal for which treatment is desired. Thus, the methods described herein can be applied to both human and veterinary applications.

The terms “treating” or “treatment” indicates that the method has, at the least, mitigated abnormal cellular proliferation. For example, the method can reduce the rate of neuroblastoma growth in a patient, or prevent the continued growth or spread of the neuroblastoma, or even reduce the overall reach of neuroblastoma.

As such, in one embodiment, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Compound A and retinoic acid, or pharmaceutically acceptable salts thereof.

In another embodiment, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Compound F and retinoic acid, or pharmaceutically acceptable salts thereof.

In another embodiment, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Compound B and retinoic acid, or pharmaceutically acceptable salts thereof.

In another embodiment, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Compound E and retinoic acid, or pharmaceutically acceptable salts thereof.

In another embodiment, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Formula I and retinoic acid, or pharmaceutically acceptable salts thereof.

In another embodiment, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Formula II and retinoic acid, or pharmaceutically acceptable salts thereof.

In yet another embodiment, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Formula I, or a pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.

In still another embodiment, provided herein is a method for treating neuroblastoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt thereof.

Also provided herein are methods for inhibiting migration or invasion, or both, of neuroblastoma cells. In particular, provided herein are methods for inhibiting migration or invasion, or both, of neuroblastoma cells, in a subject in need thereof. Specifically, provided herein are methods for inhibiting migration or invasion, or both, of neuroblastoma cells in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an HDAC inhibitor of Formula I or II, or Compound E. Also provided herein are methods for inhibiting migration or invasion, or both, of neuroblastoma cells in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a combination comprising an HDAC inhibitor of Formula I or II, or Compound E, and retinoic acid.

Provided herein are methods for decreasing cell viability of cancer cells by administering a combination comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for inducing differentiation of cancer cells by administering a combination comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for inducing apoptosis of cancer cells by administering a combination comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for decreasing cell cycle progression comprising administering a combination comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for increasing cellular apoptosis comprising administering a combination comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for suppressing transcriptional regulators in cancer comprising administering a combination therapy comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for enhancing neuroblastoma differentiation comprising administering a combination therapy comprising an HDAC inhibitor and retinoic acid. In one embodiment, differentiation is induced by a non-cytotoxic amount of the HDAC inhibitor.

Provided herein are methods for suppressing neuroblastoma proliferation comprising administering a combination therapy comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for enhancing alteration of the binding position of retinoic acid receptor to chromatin comprising administering a combination comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for reducing Wnt signaling comprising administering a combination comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for enhancing one or more of a signaling pathway selected from the group consisting of KRAS signaling (up), protein secretion, pancreas beta cells, TNFA signaling via NFKB, bile acid metabolism, complement, coagulation, adipogenesis, estrogen response (early), fatty acid metabolism, estrogen response (late), epithelial mesenchymal transition, IL2-STATS signaling, interferon gamma response, apoptosis, interferon alpha response, and IL6-JAK-STAT3, or suppressing one or more of a signaling pathway selected from the group consisting of MYC target (V2), E2F target, MYC target (V1), and DNA repair comprising administering a combination therapy comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for enhancing one or more of a signaling pathway selected from the group consisting of KRAS signaling (up), protein secretion, pancreas beta cells, TNFA signaling via NFKB, bile acid metabolism, complement, coagulation, adipogenesis, estrogen response (early), fatty acid metabolism, estrogen response (late), epithelial mesenchymal transition, IL2-STATS signaling, interferon gamma response, apoptosis, interferon alpha response, and IL6-JAK-STAT3, and suppressing one or more of a signaling pathway selected from the group consisting of MYC target (V2), E2F target, MYC target (V1), and DNA repair comprising administering a combination therapy comprising an HDAC inhibitor and retinoic acid.

Provided herein are methods for enhancing one or more of a signaling pathway selected from the group consisting of KRAS signaling (up), protein secretion, pancreas beta cells, TNFA signaling via NFKB, bile acid metabolism, complement, coagulation, adipogenesis, estrogen response (early), fatty acid metabolism, estrogen response (late), epithelial mesenchymal transition, IL2-STATS signaling, interferon gamma response, apoptosis, interferon alpha response, and IL6-JAK-STAT3, and suppressing one or more of a signaling pathway selected from the group consisting of MYC target (V2), E2F target, MYC target (V1), and DNA repair comprising administering an HDAC inhibitor.

Provided herein are methods for enhancing one or more of a signaling pathway selected from the group consisting of KRAS signaling (up), protein secretion, pancreas beta cells, TNFA signaling via NFKB, bile acid metabolism, complement, coagulation, adipogenesis, estrogen response (early), fatty acid metabolism, estrogen response (late), epithelial mesenchymal transition, IL2-STATS signaling, interferon gamma response, apoptosis, interferon alpha response, and IL6-JAK-STAT3, and suppressing one or more of a signaling pathway selected from the group consisting of MYC target (V2), E2F target, MYC target (V1), and DNA repair comprising administering retinoic acid.

Kits

In other embodiments, kits are provided. Kits according to the invention include package(s) comprising compounds or compositions of the invention. In some embodiments, kits comprise an HDAC inhibitor, or a pharmaceutically acceptable salt thereof, and retinoic acid, or a pharmaceutically acceptable salt thereof.

The phrase “package” means any vessel containing compounds or compositions presented herein. In some embodiments, the package can be a box or wrapping. Packaging materials for use in packaging pharmaceutical products are well-known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

The kit can also contain items that are not contained within the package, but are attached to the outside of the package, for example, pipettes.

Kits can further contain instructions for administering compounds or compositions of the invention to a patient. Kits also can comprise instructions for approved uses of compounds herein by regulatory agencies, such as the United States Food and Drug Administration. Kits can also contain labeling or product inserts for the compounds. The package(s) or any product insert(s), or both, can themselves be approved by regulatory agencies. The kits can include compounds in the solid phase or in a liquid phase (such as buffers provided) in a package. The kits can also include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.

EXAMPLES

Examples have been set forth below for the purpose of illustration and to describe certain specific embodiments of the invention. However, the scope of the claims is not to be in any way limited by the examples set forth herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, derivatives, formulations or methods of the invention can be made without departing from the spirit of the invention and the scope of the appended claims. Definitions of the variables in the structures in the schemes herein are commensurate with those of corresponding positions in the formulae presented herein.

It is demonstrated herein that next generation selective and orally bioavailable HDAC1/2 inhibitors can induce gene expression changes in neuroblastoma cells consistent with differentiation. The action of HDAC1/2 inhibitors potently enhances the retinoic acid differentiation effect at sub-optimal concentrations of retinoic acid or HDAC inhibitor, as well as with intermittent (pulse) HDAC1/2 inhibition. Retinoic acid alone and in combination with HDAC1/2 inhibitors is able to slow cell proliferation in long term growth assays and alter morphology in a manner consistent with differentiation. A gene expression pattern associated with retinoic acid-induced neuroblastoma differentiation is similarly induced by inhibition of HDAC1/2. The observed enhancement of differentiation by selective HDAC1/2 inhibitors occurs at concentrations below that required for cell death as evidenced by viability assays and caspase 3/7 activation. Acute toxicity is induced by elevated concentrations of HDAC1/2 inhibitors, and synergy is observed in combination with retinoic acid. Ongoing studies exploring global gene expression changes, ChIP-seq examining retinoic acid receptor and HDAC1/2 chromatin binding, and activity of the selective HDAC1/2 inhibitor in combination with retinoic acid in animal models of neuroblastoma is discussed. Taken together, these findings support a role for selective HDAC1/2 inhibitors in combination with retinoic acid for the treatment of patients with high risk neuroblastoma.

The syntheses of the compounds of Formula I (Compound A) are provided in PCT/US2011/021982; this application is incorporated herein by reference in its entirety. The syntheses of compounds of Formula II (Compound B) are provided in U.S. Patent Publication No. 2014-0128391; this application is incorporated herein by reference in its entirety.

Example 1
Synthesis of 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide (Compound A)

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Reaction Scheme:

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Experimental Procedure

Synthesis of Intermediate 2: A mixture of aniline (3.7 g, 40 mmol), compound 1 (7.5 g, 40 mmol), and K₂CO₃(11 g, 80 mmol) in DMF (100 ml) was degassed and stirred at 120° C. under N₂overnight. The reaction mixture was cooled to r.t. and diluted with EtOAc (200 ml), then washed with saturated brine (200 ml×3). The organic layers were separated and dried over Na₂SO₄, evaporated to dryness and purified by silica gel chromatography (petroleum ethers/EtOAc=10/1) to give the desired product as a white solid (6.2 g, 64%).

Synthesis of Intermediate 3: A mixture of compound 2 (69.2 g, 1 equiv.), 1-chloro-2-iodobenzene (135.7 g, 2 equiv.), Li₂CO₃(42.04 g, 2 equiv.), K₂CO₃(39.32 g, 1 equiv.), Cu (1 equiv. 45 μm) in DMSO (690 ml) was degassed and purged with nitrogen. The resulting mixture was stirred at 140° C. Work-up of the reaction gave compound 3 at 93% yield.

Synthesis of Intermediate 4: 2N NaOH (200 ml) was added to a solution of compound 3 (3.0 g, 9.4 mmol) in EtOH (200 ml). The mixture was stirred at 60° C. for 30 min After evaporation of the solvent, the solution was neutralized with 2N HCl to give a white precipitate. The suspension was extracted with EtOAc (2×200 ml), and the organic layers were separated, washed with water (2×100 ml), brine (2×100 ml), and dried over Na₂SO₄. Removal of the solvent gave a brown solid (2.5 g, 92%).

Synthesis of Intermediate 6: A mixture of compound 4 (2.5 g, 8.58 mmol), compound 5 (2.52 g, 12.87 mmol), HATU (3.91 g, 10.30 mmol), and DIPEA (4.43 g, 34.32 mmol) was stirred at r.t. overnight. After the reaction mixture was filtered, the filtrate was evaporated to dryness and the residue was purified by silica gel chromatography (petroleum ethers/EtOAc=2/1) to give a brown solid (2 g, 54%).

Synthesis of 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide (Compound A): A mixture of the compound 6 (2.0 g, 4.6 mmol), sodium hydroxide (2N, 20 mL) in MeOH (50 ml) and DCM (25 ml) was stirred at 0° C. for 10 min Hydroxylamine (50%) (10 ml) was cooled to 0° C. and added to the mixture. The resulting mixture was stirred at r.t. for 20 min After removal of the solvent, the mixture was neutralized with 1M HCl to give a white precipitate. The crude product was filtered and purified by pre-HPLC to give a white solid (950 mg, 48%).

Example 2
Synthesis of N-(2-amino-5-(thiophen-2-yl)phenyl)-2-cyclopropyl-1-(2-morpholinoethyl)-1H-indole-5-carboxamide (Compound B)

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Reaction Scheme

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Experimental Procedure

Step 1: To a solution of compound 1 in DCE was added POBr₃and imidazole. The reaction was stirred at 80° C. overnight. Water and DCM were added to the reaction, and the organic layer was separated, washed with brine, and dried under reduced pressure to give compound 2.

Step 2: To a solution of compound 2 in DMSO was added compound a and KOH. The resulting reaction mixture was stirred at 45° C. for 4 h, quenched with H₂O, and extracted with EA. The combined organic layers were purified by gel chromatography to yield the desired product, compound 3.

Step 3: A mixture of compound 3, cyclopropyl boronic acid, Pd(OAc)₂, tricyclohexylphosphine, and K₃PO₄in toluene and water was stirred at 100° C. under N₂atmosphere overnight. The mixture was cooled, filtered, and concentrated to obtain a residue, which was purified by Prep-TLC to get compound 4.

Step 4: A mixture of compound 4 and NaOH in EtOH and THF was stirred at 60° C. for 5 h. The mixture was concentrated to obtain a residue, to which was added aq. sat. citric acid and extracted with EA. The organic layers were separated, dried, filtered and concentrated to obtain compound 5.

Step 5: A mixture of compound 5, tert-butyl 2-amino-4-(thiophen-2-yl)phenylcarbamate, HOAT, EDCI, and DIPEA in DMF was stirred at 55° C. for overnight. Water was added to the mixture, and extracted with EA. The organic layers were separated, dried, filtered, and concentrated to get a residue, which was purified by Prep-TLC to afford compound 6.

Step 6: To a solution of compound 6 in DCM was added TFA and stirred at r.t. for 1 h. The mixture was concentrated to obtain a residue, which was purified by Prep-HPLC to afford compound 7. ¹H NMR (500 MHz, DMSO) δ 9.63 (s, 1H), 8.16 (s, 1H), 7.79-7.73 (m, 1H), 7.51 (d, J=2.1 Hz, 2H), 7.36 (d, J=5.1 Hz, 1H), 7.29 (dd, J=8.3, 2.1 Hz, 1H), 7.25 (d, J=3.5 Hz, 1H), 7.05 (dd, J=5.0, 3.6 Hz, 1H), 6.82 (d, J=8.3 Hz, 1H), 6.24 (s, 1H), 5.12 (s, 2H), 4.43 (s, 2H), 3.57 (s, 5H), 2.77-2.58 (m, 2H), 2.09 (s, 1H), 1.02 (d, J=8.0 Hz, 2H), 0.76 (d, J=4.4 Hz, 2H). LCMS: m/z=487.2 (M+H)+.

Example 3
Targeted HDAC1/2 Inhibitors Combine with ATRA to Enhance Genetic Differentiation Markers (48 Hours) (FIG. 1)

A subset of the fingerprint genes are assessed for each treatment group, and a score is assigned to each gene based on the degree of expression and weighted with a (+) or (−) sign based on if it increased or decreased as predicted. The scores are summed to generate a single Index value for each treatment group, set to ATRA signal and compared. It is observed that HDAC1/2i induce gene expression consistent with differentiation and that in combination with ATRA, the differentiation effect is enhanced.

Compound B inhibited HDAC isoforms 1 & 2 in a biochemical assay (Table 1) as well as HDAC2 activity in live cells with potency in the 0.5-3 μM range (FIG. 1C). Further, Compound B induces histone acetylation in neuroblastoma cells as studied by western blot at 24 hours (FIG. 1D). A differentiation Index Score based on a gene signature indicates that Compound B induces gene expression changes consistent with differentiation (FIG. 1B), and that the effect is enhanced in combination with retinoic acid.

TABLE 1

Biochemical Activity of Isoform-Selective

HDAC Inhibitors (IC₅₀in nM)

HDAC1
HDAC2
HDAC3
HDAC6

Compound A
33
54
61
5

Compound B
6
36
445
—

Compound C
2000
619
57
—

Compound D*
2123
2570
11223
7

Compound E
4
15
114
—

Comparator A (MS-275)
115
86
26

*Compound D: 2-((1-(3-fluorophenyl)cyclohexyl)amino)-N-hydroxypyrimidine-5-carboxamide

Neuroblastoma cells were treated with the indicated compounds and a basket of genes associated with differenation were monitored (FIG. 1A). The Differenation Index is a metric that describes the global movement of genes associated with differenation and an increased score suggests expression patterns associated with retinoic acid induced differentation. The combination of the listed compounds with retinoic acid results in a greater differentation index score, illustrating enhanced differentiation in the combination setting.

Example 4
HDAC1/2i Toxicity is Independent of Retinoic Acid and Occurs at Concentrations Greater than Those Needed for Differentiation (FIG. 2)

Compound B caused tumor cell death at concentrations ≧2 μM, which is greater than what is needed to induce differentiation (FIG. 2A). The addition of 1 μM or 3 μM of ATRA, which has potent differentiation activity, has little effect on the HDAC1/2i mediated toxicity. Similar observations were made when assessing caspase activation, with an increase in activity at concentrations ≧2 μM and little to no enhancement by retinoic acid (FIG. 2B). These results suggest that acute toxicity is driven by HDAC1/2i and is independent of retinoic acid activity.

Example 5
Differentiation is Induced by Non-Cytotoxic Concentrations of Compound A and Compound B (48 Hours) (FIG. 3)

Cells were cultured for 48 hours at the indicated concentration of drug, and then cell viability was measured by Promega CellTiter Glo assay and caspase activity was measured by Promega Caspase3/7 Glo assay. Minimal cell death and caspase 3/7 induction is observed at 1 μM of Compound B or Compound A, which are concentrations that can induce differentiation (FIGS. 3A and 3B). Anti-tumor activity is evident at 3 μM of treatment.

Example 6
Compound B Combines with ATRA to Enhance Suppression of Neuroblastoma Proliferation (FIG. 4)

Cells were cultured over a 7 day period and manually counted at days 3, 5, and 7. Both 1 μM and 3 μM of Compound B alone can suppress proliferation similar to ATRA, and the combination enhances suppression (FIGS. 4A and 4B). Combination effect of Compound B/ATRA on proliferation is evident at all of the time points assessed.

HDAC1/2i Enhances ATRA-mediated Suppression of Proliferation. Compound B caused a decrease in proliferation over time at concentrations that induce differentiation (FIG. 4A) Enhanced effects are observed with the Compound B/ATRA combination, particularly after extended time. The effects were enhanced by higher concentrations of HDAC1/2i (FIG. 4B).

Example 7
Compound a Combines with ATRA to Enhance Suppression of Neuroblastoma Proliferation (FIG. 5)

Cells were cultured over a 7 day period and manually counted at days 3, 5, and 7. Both 0.75 μM and 2 μM of Compound A alone can suppress proliferation similar to ATRA, and the combination enhances suppression (FIGS. 5A and 5B). Combination effect of Compound A/ATRA on proliferation is evident at all of the time points assessed.

Example 8
Compound B Enhances ATRA-Mediated Suppression of Proliferation and Cell Cycle Arrest (FIG. 6)

Cells were cultured for 7 days with the indicated treatments and then stained with the Invitrogen Click-iT EdU kit and the Invitrogen FxCycle PR/Rnase kits to assess frequency of cells in each stage of the cell cycle. Single-agent ATRA reduces s-phase frequency and increases the sub-G1 population. The effect is enhanced by Compound B in a dose dependent manner.

Compound B induced increased expression of the master cell cycle regulator p21 as a single agent and in a dose-dependent manner (FIG. 6A). In combination with ATRA, the effect on p21 is enhanced at concentrations that induce gene expression changes consistent with differentiation. At 7 days of treatment, combination effects are observed on the cell cycle, with a decreasing frequency of s-phase cells and an increasing sub-G1 population (FIG. 6B). Neuroblastoma cells were treated with the indicated compounds and assessed for their cell cycle characteristics (FIG. 6C). EDU is increased in cells in s-phase while PI intensity indicates relative levels of DNA. Using these metrics, we illustrate the frequency of cells cells in the indicated states of the cell cycle. These data indicated that the combination of the indicated compounds with ATRA cause a reduction in the frequency of s-phase cells, suggesting decreased growth, while increasing the sub-G1 population, suggesting cell death.

Example 9
Compound B Combines with ATRA to Enhance Morphology Changes Consistent with Differentiation (FIG. 7)

Cells were cultured for 3, 5, and 7 days with the indicated treatments. Images were captured on an inverted microscope. Dendrite outgrowth induced by ATRA is enhanced by the Compound B/ATRA combination, particularly at the 3 day time point. Retinoic acid caused the outgrowth of dendrites over time, with the strongest effects at 5 and 7 days of treatment. Compound B as a single agent does not alter morphology, but surprisingly, in combination with ATRA, Compound B enhances the ability of ATRA to induce morphology changes consistent with differentiation. The HDAC1/2i enhancement effect on retinoic acid is particularly noticeable at earlier time points.

Example 10
Compound B Combines with ATRA to Enhance NF-M Staining (FIG. 8)

Cells were cultured for 7 days with the indicated treatments on glass slides. Cells were fixed and stained with antibodies for NF-M and nucleus. Images were captured on a fluorescent microscope. Compound B caused a general increase in NF-M staining. The combination of ATRA and Compound B cause both increased neurite formation and increased NF-M staining.

Example 11
Compound B Combines with ATRA to Reduce Neuroblastoma Outgrowth (FIG. 9)

Cells were cultured for 15, 20 or 25 days with the indicated treatments. Cells were imaged on an inverted microscope at days 15 and 20. On day 25, the cells were stained with Crystal Violet dye and imaged by cell phone camera Importantly, the combination of ATRA and Compound B suppressed the growth of ATRA-resistant colonies.

Example 12
Compound B (HDAC1/2i) Shows Stronger Combination Effect with ATRA than Compound C (HDAC3i) on Cell Proliferation and Dendrite Outgrowth (FIG. 10)

Example 13
Compound B/ATRA Combination Completely Blocks Cluster Formation of Neuroblastoma at Concentrations Lower than Compound C (FIG. 11)

Cells were cultured for 15, 20 or 25 days with the indicated treatments. Cells were imaged on an inverted microscope at days 15 and 20. On day 25, the cells were stained with Crystal Violet dye and imaged. It was observed that the combination activity is mediated by HDAC1/2i.

Neuroblastoma cells were treated with Compound B or the HDAC3-selective inhibitor Compound C in a long term growth assay. Compound B at 1 μM of exposure reduced neuroblastoma cell growth as a single agent and strongly suppressed retinoic acid resistant colonies in a combination setting (FIG. 11). Compound C, in contrast, required higher concentrations to mediate a similar effect (FIG. 11). These data suggest that at equimolar treatments, HDAC1/2i more potently enhances retinoic acid than HDAC3i.

Example 14
Gene Expression Changes are Enhanced by the Combination of HDAC1/2i and ATRA Relative to Single Agent Treatment (FIG. 12)

Gene expression from cells treated with ATRA single agent and combination of ATRA and HDAC1/2i were assessed at 2 hrs and 48 hrs of treatment (FIG. 12A). The 48 hr results were assessed by gene set enrichment analysis against the Broad Institute C6 Msig database, which revealed several differentially regulated pathways involved in development, survival and differentiation (FIG. 12B). A function analysis of the data using the Ingenuity IPA platform was performed by Qiagen; overlap is observed in the top hits between treatment groups.

Genes that statistically increase over 2-fold relative to the DMSO control were mapped on a Venn diagram (FIG. 12B). Genes in each section of the Venn diagram of FIG. 12B, are listed in Tables 3-9.

Gene set enrichment analysis using gene expression profiles from HDAC1/2i-treated cells and the Hallmark gene sets of the Molecular Signatures Database (MSigDB, Broad Institute) revealed a list of pathways that were consistently enriched by HDAC1/2i (FIG. 18). Pathways of KRAS signaling, protein secretion, pancreas beta cells, TNFA signaling via NFKB, bile acid metabolism, complement, coagulation, adipogenesis, estrogen response (early), fatty acid metabolism, estrogen response (late), epithelial mesenchymal transition, IL2-STATS signaling, interferon gamma response, apoptosis, interferon alpha response, and IL6-JAK-STAT3 signaling were enriched in genes up-regulated by HDAC1/2i either alone or in combination with RA, whereas pathways of MYC target (V2), E2F target (a proxy for cell cycle progression), MYC target (V1), and DNA repair were enriched in genes down-regulated by HDAC1/2i alone or in combination with RA. Caspase activation was examined and it was found that HDAC1/2i+RA enhanced cleavage of caspase 3 & 9 and PARP, consistent with apoptotic death (FIG. 19). When proteins involved in the E2F signaling pathway were examined, it was found that the pro-E2F signaling proteins CDK4 & 6 were decreased in the combination while the inhibitory protein p21 was increased (FIG. 20). Further, retinoblastoma (Rb) protein, a key regulator of the E2F transcription factor, was hypo-phosphorylated, suggesting E2F is in a repressed state.

Microarray Experimental Design

Neuroblastoma cell line SK-N-BE(2) cells were treated with 3 μM Compound B, 1 μM ATRA, or a combination of both at 37° C. over 2 hours (FIG. 12A) or 48 hours (FIG. 12B) and compared to the solvent (DMSO) control.

TABLE 2

Pathways enriched in the 48 hr ATRA treated group relative to the combination setting

NAME
Description
Set Size
NES
FDR q-val

PDGF_ERK_DN.V1_DN
ERK inactivation by inhibitors
145
1.66
0.07

WNT_UP.V1_UP
WNT1 overexpression
176
1.48
0.11

MYC_UP.V1_UP
MYC overexpression
170
1.40
0.21

HOXA9_DN.V1_DN
Genes decreased after HOXA9 knockdown
184
1.39
0.18

GCNP_SHH_UP_LATE.V1_UP
SHH stimulation in neuron precursors
170
1.36
0.17

GCNP_SHH_UP_EARLY.V1_UP
SHH stimulation in neuron precursors
170
1.29
0.17

AKT_UP_MTOR_DN.V1_DN
Genes decreased after AKT1 overexpression
180
1.28
0.16

CYCLIN_D1_UP.V1_UP
Overexpression of Cyclin D1
184
1.26
0.19

TABLE 3

FC (abs)
Regulation

(Moderated
(Moderated

T-Test
T-Test

[Compound B]
[Compound B]

Vs [DMSO]
Vs [DMSO]

P <= 0.05
P <= 0.05

FC >= 2.0)
FC >= 2.0)
Symbol
Definition

2.5698965
up
TLE4

Homo sapiens transducin-like

enhancer of split 4 (E(sp1) homolog,

Drosophila) (TLE4), mRNA.

2.5064785
up
STC1

Homo sapiens stanniocalcin 1

(STC1), mRNA.

2.3753035
up
RALGPS1

Homo sapiens Ral GEF with PH

domain and SH3 binding motif 1

(RALGPS1), mRNA.

2.2640777
up
ARC

Homo sapiens activity-regulated

cytoskeleton-associated protein

(ARC), mRNA.

2.2220922
up
FAM83D

Homo sapiens family with sequence

similarity 83, member D (FAM83D),

mRNA.

2.1907196
up
RASD2

Homo sapiens RASD family,

member 2 (RASD2), mRNA.

2.1847532
up
RHOB

Homo sapiens ras homolog gene

family, member B (RHOB), mRNA.

2.1649334
up
C5orf37

Homo sapiens chromosome 5 open

reading frame 37 (C5orf37), mRNA.

2.143248
up
NOTCH3

Homo sapiens Notch homolog 3

(Drosophila) (NOTCH3), mRNA.

2.1351867
up
SRBD1

Homo sapiens S1 RNA binding

domain 1 (SRBD1), mRNA.

2.1036422
up
OLFML2A

Homo sapiens olfactomedin-like 2A

(OLFML2A), mRNA.

2.0986614
up
CKS2

Homo sapiens CDC28 protein kinase

regulatory subunit 2 (CKS2), mRNA.

2.0979898
up
NEK2

Homo sapiens NIMA (never in

mitosis gene a)-related kinase 2

(NEK2), mRNA.

2.0797677
up
SULF2

Homo sapiens sulfatase 2 (SULF2),

transcript variant 1, mRNA.

2.071442
up
DPM1

Homo sapiens dolichyl-phosphate

mannosyltransferase polypeptide 1,

catalytic subunit (DPM1), mRNA.

2.065949
up

BX088936 Soares_testis_NHT

Homo sapiens cDNA clone

IMAGp998G123255;

IMAGE: 1292195, mRNA sequence

2.0578866
up
FGFR3

Homo sapiens fibroblast growth

factor receptor 3 (achondroplasia,

thanatophoric dwarfism) (FGFR3),

transcript variant 2, mRNA.

2.0553508
up
CPNE4

Homo sapiens copine IV (CPNE4),

mRNA.

2.0535789
up
RGS5

Homo sapiens regulator of G-protein

signaling 5 (RGS5), mRNA.

2.0502908
up
PPM1E

Homo sapiens protein phosphatase

1E (PP2C domain containing)

(PPM1E), mRNA.

2.0453603
up
FAM149B1

Homo sapiens family with sequence

similarity 149, member B1

(FAM149B1), mRNA.

2.0264914
up
ARL6IP1

Homo sapiens ADP-ribosylation

factor-like 6 interacting protein 1

(ARL6IP1), mRNA.

2.0142202
up
FGFR4

Homo sapiens fibroblast growth

factor receptor 4 (FGFR4), transcript

variant 3, mRNA.

2.0096514
up
RPS6KA2

Homo sapiens ribosomal protein S6

kinase, 90 kDa, polypeptide 2

(RPS6KA2), transcript variant 2,

mRNA.

2.2990463
down
GAL

Homo sapiens galanin prepropeptide

(GAL), mRNA.

2.163846
down
SHISA5

Homo sapiens shisa homolog 5

(Xenopus laevis) (SHISA5), mRNA.

2.104913
down
TXNDC5

Homo sapiens thioredoxin domain

containing 5 (endoplasmic reticulum)

(TXNDC5), transcript variant 1,

mRNA.

2.0841973
down
SHD

Homo sapiens Src homology 2

domain containing transforming

protein D (SHD), mRNA.

2.0804224
down
PRNP

Homo sapiens prion protein (PRNP),

transcript variant 2, mRNA.

2.0670848
down
CPXM1

Homo sapiens carboxypeptidase X

(M14 family), member 1 (CPXM1),

mRNA.

2.0438597
down
ROBO2

Homo sapiens roundabout, axon

guidance receptor, homolog 2

(Drosophila) (ROBO2), mRNA.

2.0305886
down
PCGF1

Homo sapiens polycomb group ring

finger 1 (PCGF1), mRNA.

2.0155308
down
LDLR

Homo sapiens low density

lipoprotein receptor (familial

hypercholesterolemia) (LDLR),

mRNA.

2.0127265
down
CD151

Homo sapiens CD151 molecule

(Raph blood group) (CD151),

transcript variant 2, mRNA.

TABLE 4

FC (abs)
Regulation

(Moderated
(Moderated

T-Test
T-Test

[Compound B]
[Compound B]

Vs [DMSO]
Vs [DMSO]

P <= 0.05
P <= 0.05
Gene

FC >= 2.0)
FC >= 2.0)
Symbol
Definition

2.286589
up
BNIP3

Homo sapiens

BCL2/adenovirus E1B

19 kDa interacting protein 3

(BNIP3), nuclear gene

encoding mitochondrial

protein, mRNA.

2.023285
up
RGS4

Homo sapiens

regulator of G-protein

signalling 4 (RGS4),

mRNA.

2.220195
down
FAM163A

Homo sapiens

family with sequence

similarity 163, member A

(FAM163A), mRNA.

2.08247
down
FAM163A

Homo sapiens

family with sequence

similarity 163, member A

(FAM163A), mRNA.

TABLE 5

FC (abs)
Regulation

(Moderated
(Moderated

T-Test
T-Test

[ATRA]
[ATRA]

Vs [DMSO]
Vs [DMSO]

P <= 0.05
P <= 0.05

FC >= 2.0)
FC >= 2.0)
Symbol
Definition

8.72177
up
GDF10

Homo sapiens growth differentiation

factor 10 (GDF10), mRNA.

4.283083
up
DKK2

Homo sapiens dickkopf homolog 2

(Xenopus laevis) (DKK2), mRNA.

3.677953
up
TNS3

Homo sapiens tensin 3 (TNS3),

mRNA.

3.677291
up
TLX2

Homo sapiens T-cell leukemia

homeobox 2 (TLX2), mRNA.

3.319324
up
NTRK2

Homo sapiens neurotrophic tyrosine

kinase, receptor, type 2 (NTRK2),

transcript variant b, mRNA.

3.18521
up
ADAMTS9

Homo sapiens ADAM

metallopeptidase with

thrombospondin type 1 motif, 9

(ADAMTS9), mRNA.

2.99289
up
TSHZ3

Homo sapiens teashirt zinc finger

homeobox 3 (TSHZ3), mRNA.

2.986171
up
RBP1

Homo sapiens retinol binding protein

1, cellular (RBP1), mRNA.

2.937687
up
DYSF

Homo sapiens dysferlin, limb girdle

muscular dystrophy 2B (autosomal

recessive) (DYSF), mRNA.

2.80848
up
DICER1

Homo sapiens Dicer1, Dcr-1 homolog

(Drosophila) (DICER1), transcript

variant 2, mRNA.

2.800005
up
C20orf103

Homo sapiens chromosome 20 open

reading frame 103 (C20orf103),

mRNA.

2.795654
up
SLCO2A1

Homo sapiens solute carrier organic

anion transporter family, member 2A1

(SLCO2A1), mRNA.

2.722439
up
DKK2

Homo sapiens dickkopf homolog 2

(Xenopus laevis) (DKK2), mRNA.

2.714301
up
SCHIP1

Homo sapiens schwannomin

interacting protein 1 (SCHIP1),

mRNA.

2.66298
up
ARHGAP28

Homo sapiens Rho GTPase activating

protein 28 (ARHGAP28), transcript

variant 1, mRNA.

2.651171
up
YPEL2

Homo sapiens yippee-like 2

(Drosophila) (YPEL2), mRNA.

2.644729
up
SNORA59A

Homo sapiens small nucleolar RNA,

H/ACA box 59A (SNORA59A), small

nucleolar RNA.

2.596732
up
DICER1

Homo sapiens Dicer1, Dcr-1 homolog

(Drosophila) (DICER1), transcript

variant 2, mRNA.

2.565
up
VPS13D

Homo sapiens vacuolar protein sorting

13 homolog D (S. cerevisiae)

(VPS13D), transcript variant 1,

mRNA.

2.547531
up
NR0B1

Homo sapiens nuclear receptor

subfamily 0, group B, member 1

(NR0B1), mRNA.

2.490577
up
C15orf52

Homo sapiens chromosome 15 open

reading frame 52 (C15orf52), mRNA.

2.455256
up
HOXD8

Homo sapiens homeobox D8

(HOXD8), mRNA.

2.452022
up
TBX3

Homo sapiens T-box 3 (TBX3),

transcript variant 1, mRNA.

2.399388
up
ARHGAP28

Homo sapiens Rho GTPase activating

protein 28 (ARHGAP28), transcript

variant 1, mRNA.

2.388347
up
CCND1

Homo sapiens cyclin D1 (CCND1),

mRNA.

2.382721
up
KIAA1641
PREDICTED: Homo sapiens

KIAA1641, transcript variant 7

(KIAA1641), mRNA.

2.380837
up
TEX101

Homo sapiens testis expressed 101

(TEX101), mRNA.

2.364498
up
HERC2P2

Homo sapiens hect domain and RLD 2

pseudogene 2 (HERC2P2) on

chromosome 15.

2.329918
up
ZBED5

Homo sapiens zinc finger, BED-type

containing 5 (ZBED5), mRNA.

2.324694
up
MPPED2

Homo sapiens metallophosphoesterase

domain containing 2 (MPPED2),

mRNA.

2.311865
up
LOC642530
PREDICTED: Homo sapiens

hypothetical protein LOC642530

(LOC642530), mRNA.

2.293098
up
HOXD3

Homo sapiens homeobox D3

(HOXD3), mRNA.

2.275352
up
LHFPL2

Homo sapiens lipoma HMGIC fusion

partner-like 2 (LHFPL2), mRNA.

2.255434
up

PREDICTED: Homo sapiens

hypothetical LOC400043

(LOC400043), mRNA

2.207207
up

Homo sapiens cDNA: FLJ21199 fis,

clone COL00235

2.203424
up
ZBED5

Homo sapiens zinc finger, BED-type

containing 5 (ZBED5), mRNA.

2.197959
up
SGIP1

Homo sapiens SH3-domain GRB2-

like (endophilin) interacting protein 1

(SGIP1), mRNA.

2.196478
up
ANTXR1

Homo sapiens anthrax toxin receptor 1

(ANTXR1), transcript variant 1,

mRNA.

2.184386
up
BHLHB2

Homo sapiens basic helix-loop-helix

domain containing, class B, 2

(BHLHB2), mRNA.

2.181258
up
LBX2

Homo sapiens ladybird homeobox 2

(LBX2), mRNA.

2.174702
up
HNRPM

Homo sapiens heterogeneous nuclear

ribonucleoprotein M (HNRPM),

transcript variant 1, mRNA.

2.171866
up
PPP1R10

Homo sapiens protein phosphatase 1,

regulatory (inhibitor) subunit 10

(PPP1R10), mRNA.

2.157885
up
HNRNPM

Homo sapiens heterogeneous nuclear

ribonucleoprotein M (HNRNPM),

transcript variant 1, mRNA.

2.133766
up
LIMA1

Homo sapiens LIM domain and actin

binding 1 (LIMA1), mRNA.

2.12906
up
C4orf6

Homo sapiens chromosome 4 open

reading frame 6 (C4orf6), mRNA.

2.116109
up
HEBP2

Homo sapiens heme binding protein 2

(HEBP2), mRNA.

2.108607
up
SEMA5A

Homo sapiens sema domain, seven

thrombospondin repeats (type 1 and

type 1-like), transmembrane domain

(TM) and short cytoplasmic domain,

(semaphorin) 5A (SEMA5A), mRNA.

2.107324
up
AJAP1

Homo sapiens adherens junctions

associated protein 1 (AJAP1),

transcript variant 2, mRNA.

2.106345
up
DCX

Homo sapiens doublecortin (DCX),

transcript variant 4, mRNA.

2.094997
up
POPDC2

Homo sapiens popeye domain

containing 2 (POPDC2), mRNA.

2.092158
up
TTC30B

Homo sapiens tetratricopeptide repeat

domain 30B (TTC30B), mRNA.

2.087778
up
CYTL1

Homo sapiens cytokine-like 1

(CYTL1), mRNA.

2.087406
up
DLGAP5

Homo sapiens discs, large

(Drosophila) homolog-associated

protein 5 (DLGAP5), mRNA.

2.057107
up
AKAP12

Homo sapiens A kinase (PRKA)

anchor protein (gravin) 12 (AKAP12),

transcript variant 1, mRNA.

2.052444
up
EIF4G2

Homo sapiens eukaryotic translation

initiation factor 4 gamma, 2 (EIF4G2),

transcript variant 1, mRNA.

2.045954
up
FAM40A

Homo sapiens family with sequence

similarity 40, member A (FAM40A),

mRNA.

2.042676
up
HNRNPM

Homo sapiens heterogeneous nuclear

ribonucleoprotein M (HNRNPM),

transcript variant 2, mRNA.

2.038353
up
PPARG

Homo sapiens peroxisome

proliferator-activated receptor gamma

(PPARG), transcript variant 2, mRNA.

2.032579
up
SNAI2

Homo sapiens snail homolog 2

(Drosophila) (SNAI2), mRNA.

2.015555
up
GMPPA

Homo sapiens GDP-mannose

pyrophosphorylase A (GMPPA),

transcript variant 2, mRNA.

2.014441
up
TM4SF4

Homo sapiens transmembrane 4 L six

family member 4 (TM4SF4), mRNA.

2.378933
down
KAL1

Homo sapiens Kallmann syndrome 1

sequence (KAL1), mRNA.

2.103935
down
SC4MOL

Homo sapiens sterol-C4-methyl

oxidase-like (SC4MOL), transcript

variant 1, mRNA.

2.073776
down
VIM

Homo sapiens vimentin (VIM),

mRNA.

2.070505
down
RPL9

Homo sapiens ribosomal protein L9

(RPL9), transcript variant 2, mRNA.

2.048986
down
RPL37

Homo sapiens ribosomal protein L37

(RPL37), mRNA.

2.036535
down
LOC399491
PREDICTED: Homo sapiens

misc_RNA (LOC399491), miscRNA.

2.033366
down
PRSS12

Homo sapiens protease, serine, 12

(neurotrypsin, motopsin) (PRSS12),

mRNA.

2.029949
down
ADAM15

Homo sapiens ADAM

metallopeptidase domain 15

(ADAM15), transcript variant 2,

mRNA.

2.029488
down
LAMA5

Homo sapiens laminin, alpha 5

(LAMA5), mRNA.

2.016979
down
PDXP

Homo sapiens pyridoxal (pyridoxine,

vitamin B6) phosphatase (PDXP),

mRNA.

TABLE 6

FC (abs)
Regulation

(Moderated
(Moderated

T-Test
T-Test

[Compound B]
[Compound B]

Vs [DMSO]
Vs [DMSO]

P <= 0.05
P <= 0.05

FC >= 2.0)
FC >= 2.0)
Symbol
Definition

2.120727
down
CD44

Homo sapiens CD44 molecule (Indian

blood group) (CD44), transcript

variant 4, mRNA.

2.416998
down
SNORA8

Homo sapiens small nucleolar RNA,

H/ACA box 8 (SNORA8), small

nucleolar RNA.

2.51326
down
CDH24

Homo sapiens cadherin-like 24

(CDH24), transcript variant 1, mRNA.

2.641853
down
DLK1

Homo sapiens delta-like 1 homolog

(Drosophila) (DLK1), mRNA.

2.737174
down
PTCHD1

Homo sapiens patched domain

containing 1 (PTCHD1), mRNA.

3.346026
down
SLC6A15

Homo sapiens solute carrier family 6

(neutral amino acid transporter),

member 15 (SLC6A15), transcript

variant 2, mRNA.

4.589464
down
STMN4

Homo sapiens stathmin-like 4

(STMN4), mRNA.

2.028951
up
BMP4

Homo sapiens bone morphogenetic

protein 4 (BMP4), transcript variant 3,

mRNA.

2.07585
up
RGS16

Homo sapiens regulator of G-protein

signalling 16 (RGS16), mRNA.

2.293513
up
IER3

Homo sapiens immediate early

response 3 (IER3), mRNA.

2.303625
up
RGL1

Homo sapiens ral guanine nucleotide

dissociation stimulator-like 1 (RGL1),

mRNA.

2.430483
up
SGK

Homo sapiens serum/glucocorticoid

regulated kinase (SGK), mRNA.

2.749795
up

Homo sapiens mRNA full length

insert cDNA clone EUROIMAGE

151432

3.030697
up
CTSH

Homo sapiens cathepsin H (CTSH),

transcript variant 1, mRNA.

3.177024
up
ETS1

Homo sapiens v-ets erythroblastosis

virus E26 oncogene homolog 1 (avian)

(ETS1), mRNA.

3.274463
up
ETS1

Homo sapiens v-ets erythroblastosis

virus E26 oncogene homolog 1 (avian)

(ETS1), mRNA.

3.331427
up
DUSP6

Homo sapiens dual specificity

phosphatase 6 (DUSP6), transcript

variant 2, mRNA.

3.41655
up
SIPA1L2

Homo sapiens signal-induced

proliferation-associated 1 like 2

(SIPA1L2), mRNA.

4.000809
up
EGR1

Homo sapiens early growth response 1

(EGR1), mRNA.

7.405237
up
FOS

Homo sapiens v-fos FBJ murine

osteosarcoma viral oncogene homolog

(FOS), mRNA.

TABLE 7

FC (abs)
Regulation

(Moderated
(Moderated

T-Test
T-Test

[Compound B]
[Compound B]

Vs [DMSO]
Vs [DMSO]

P <= 0.05
P <= 0.05
Gene

FC >= 2.0)
FC >= 2.0)
Symbol
Definition

3.7700295
up
HSPA5

Homo sapiens heat shock 70 kDa

protein 5 (glucose-regulated protein,

78 kDa) (HSPA5), mRNA.

3.463821
up
NPC2

Homo sapiens Niemann-Pick

disease, type C2 (NPC2), mRNA.

3.382726
up
PQLC3

Homo sapiens PQ loop repeat

containing 3 (PQLC3), mRNA.

3.2470052
up
CFD

Homo sapiens complement factor D

(adipsin) (CFD), mRNA.

3.2319236
up
DHRS2

Homo sapiens

dehydrogenase/reductase (SDR

family) member 2 (DHRS2),

transcript variant 1, mRNA.

3.1507273
up
POU4F1

Homo sapiens POU class 4

homeobox 1 (POU4F1), mRNA.

3.0529962
up
MYLIP

Homo sapiens myosin regulatory

light chain interacting protein

(MYLIP), mRNA.

3.006488
up
AIF1L

Homo sapiens allograft inflammatory

factor 1-like (AIF1L), transcript

variant 1, mRNA.

2.889204
up

AGENCOURT_10229596

NIH_MGC_141 Homo sapiens

cDNA clone IMAGE: 6563923 5,

mRNA sequence

2.848912
up
HMMR

Homo sapiens hyaluronan-mediated

motility receptor (RHAMM)

(HMMR), transcript variant 2,

mRNA.

2.7428732
up
SCPEP1

Homo sapiens serine

carboxypeptidase 1 (SCPEP1),

mRNA.

2.741772
up
MERTK

Homo sapiens c-mer proto-oncogene

tyrosine kinase (MERTK), mRNA.

2.7384179
up
LOC338758
PREDICTED: Homo sapiens

hypothetical protein LOC338758

(LOC338758), mRNA.

2.7313623
up
CIB1

Homo sapiens calcium and integrin

binding 1 (calmyrin) (CIB1), mRNA.

2.7183042
up
COL5A1

Homo sapiens collagen, type V,

alpha 1 (COL5A1), mRNA.

2.6359165
up
CTSL2

Homo sapiens cathepsin L2

(CTSL2), mRNA.

2.6211162
up
IFI6

Homo sapiens interferon, alpha-

inducible protein 6 (IFI6), transcript

variant 3, mRNA.

2.6199353
up
CGN

Homo sapiens cingulin (CGN),

mRNA.

2.6197765
up
CPVL

Homo sapiens carboxypeptidase,

vitellogenic-like (CPVL), transcript

variant 1, mRNA.

2.6172824
up
PPP2R2B

Homo sapiens protein phosphatase 2

(formerly 2A), regulatory subunit B,

beta isoform (PPP2R2B), transcript

variant 4, mRNA.

2.613672
up
CCDC99

Homo sapiens coiled-coil domain

containing 99 (CCDC99), mRNA.

2.5982802
up
CYP2J2

Homo sapiens cytochrome P450,

family 2, subfamily J, polypeptide 2

(CYP2J2), mRNA.

2.5876853
up
BAMBI

Homo sapiens BMP and activin

membrane-bound inhibitor homolog

(Xenopus laevis) (BAMBI), mRNA.

2.5142672
up
HSPA1A

Homo sapiens heat shock 70 kDa

protein 1A (HSPA1A), mRNA.

2.5097656
up
RN7SK

Homo sapiens RNA, 7SK small

nuclear (RN7SK), non-coding RNA.

2.4892557
up
ITPR1

Homo sapiens inositol 1,4,5-

triphosphate receptor, type 1

(ITPR1), transcript variant 2, mRNA.

2.4447675
up
SPA17

Homo sapiens sperm autoantigenic

protein 17 (SPA17), mRNA.

2.4395416
up
ESRRG

Homo sapiens estrogen-related

receptor gamma (ESRRG), transcript

variant 2, mRNA.

2.4309964
up
CLDN11

Homo sapiens claudin 11

(oligodendrocyte transmembrane

protein) (CLDN11), mRNA.

2.4051182
up
ST6GALNAC3

Homo sapiens ST6 (alpha-N-acetyl-

neuraminyl-2,3-beta-galactosyl-1,3)-

N-acetylgalactosaminide alpha-2,6-

sialyltransferase 3 (ST6GALNAC3),

mRNA.

2.3992615
up
STAT1

Homo sapiens signal transducer and

activator of transcription 1, 91 kDa

(STAT1), transcript variant alpha,

mRNA.

2.382162
up
PPP1R3C

Homo sapiens protein phosphatase 1,

regulatory (inhibitor) subunit 3C

(PPP1R3C), mRNA.

2.3813944
up
CRY1

Homo sapiens cryptochrome 1

(photolyase-like) (CRY1), mRNA.

2.3754704
up
RYBP

Homo sapiens RING1 and YY1

binding protein (RYBP), mRNA.

2.365169
up
FSTL5

Homo sapiens follistatin-like 5

(FSTL5), mRNA.

2.316434
up
PRSS35

Homo sapiens protease, serine, 35

(PRSS35), mRNA.

2.3060548
up
SERPINE2

Homo sapiens serpin peptidase

inhibitor, clade E (nexin,

plasminogen activator inhibitor type

1), member 2 (SERPINE2), mRNA.

2.3006706
up
HMMR

Homo sapiens hyaluronan-mediated

motility receptor (RHAMM)

(HMMR), transcript variant 1,

mRNA.

2.2822168
up
GLRX

Homo sapiens glutaredoxin

(thioltransferase) (GLRX), mRNA.

2.271626
up
LMO4

Homo sapiens LIM domain only 4

(LMO4), mRNA.

2.2702909
up
IL13RA2

Homo sapiens interleukin 13

receptor, alpha 2 (IL13RA2),

mRNA.

2.269596
up
IGSF3

Homo sapiens immunoglobulin

superfamily, member 3 (IGSF3),

transcript variant 1, mRNA.

2.257454
up
NEK1

Homo sapiens NIMA (never in

mitosis gene a)-related kinase 1

(NEK1), mRNA.

2.243685
up
CAST

Homo sapiens calpastatin (CAST),

transcript variant 8, mRNA.

2.223723
up
PAG1

Homo sapiens phosphoprotein

associated with glycosphingolipid

microdomains 1 (PAG1), mRNA.

2.2132776
up
STK3

Homo sapiens serine/threonine

kinase 3 (STE20 homolog, yeast)

(STK3), mRNA.

2.211524
up
NPTX2

Homo sapiens neuronal pentraxin II

(NPTX2), mRNA.

2.2004776
up
CAP1

Homo sapiens CAP, adenylate

cyclase-associated protein 1 (yeast)

(CAP1), mRNA.

2.1915953
up
HSPA2

Homo sapiens heat shock 70 kDa

protein 2 (HSPA2), mRNA.

2.1893888
up
SDF2L1

Homo sapiens stromal cell-derived

factor 2-like 1 (SDF2L1), mRNA.

2.1769004
up
ACO1

Homo sapiens aconitase 1, soluble

(ACO1), mRNA.

2.166546
up
MAP4K2

Homo sapiens mitogen-activated

protein kinase kinase kinase kinase 2

(MAP4K2), mRNA.

2.1538491
up
CRYZ

Homo sapiens crystallin, zeta

(quinone reductase) (CRYZ),

mRNA.

2.1500034
up
DNCL1

Homo sapiens dynein, cytoplasmic,

light polypeptide 1 (DNCL1),

mRNA.

2.1493442
up
CREG1

Homo sapiens cellular repressor of

E1A-stimulated genes 1 (CREG1),

mRNA.

2.1460152
up
RHBDF2

Homo sapiens rhomboid 5 homolog

2 (Drosophila) (RHBDF2), transcript

variant 2, mRNA.

2.145611
up

Homo sapiens cDNA FLJ43160 fis,

clone FCBBF2000199

2.1441228
up
PYGL

Homo sapiens phosphorylase,

glycogen, liver (PYGL), mRNA.

2.1436455
up
LRRC1

Homo sapiens leucine rich repeat

containing 1 (LRRC1), mRNA.

2.1423635
up
LOC730432
PREDICTED: Homo sapiens similar

to serine/threonine/tyrosine

interacting protein, transcript variant

1 (LOC730432), mRNA.

2.132023
up
SERPINI1

Homo sapiens serpin peptidase

inhibitor, clade I (neuroserpin),

member 1 (SERPINI1), mRNA.

2.1283884
up
CBR4

Homo sapiens carbonyl reductase 4

(CBR4), mRNA.

2.1136534
up
RAB23

Homo sapiens RAB23, member RAS

oncogene family (RAB23), transcript

variant 1, mRNA.

2.1128619
up
VCL

Homo sapiens vinculin (VCL),

transcript variant 1, mRNA.

2.112098
up
ETV5

Homo sapiens ets variant gene 5 (ets-

related molecule) (ETV5), mRNA.

2.109667
up
TIPARP

Homo sapiens TCDD-inducible

poly(ADP-ribose) polymerase

(TIPARP), mRNA.

2.109442
up
ALS2

Homo sapiens amyotrophic lateral

sclerosis 2 (juvenile) (ALS2),

mRNA.

2.0956025
up
SDCBP

Homo sapiens syndecan binding

protein (syntenin) (SDCBP),

transcript variant 2, mRNA.

2.0934463
up
FERMT2

Homo sapiens fermitin family

homolog 2 (Drosophila) (FERMT2),

mRNA.

2.0914667
up
TJP1

Homo sapiens tight junction protein

1 (zona occludens 1) (TJP1),

transcript variant 1, mRNA.

2.088695
up
POP5

Homo sapiens processing of

precursor 5, ribonuclease P/MRP

subunit (S. cerevisiae) (POP5),

transcript variant 3, mRNA.

2.081764
up
LCMT2

Homo sapiens leucine carboxyl

methyltransferase 2 (LCMT2),

mRNA.

2.05315
up
CEP55

Homo sapiens centrosomal protein

55 kDa (CEP55), mRNA.

2.044371
up
PLCB1

Homo sapiens phospholipase C, beta

1 (phosphoinositide-specific)

(PLCB1), transcript variant 1,

mRNA.

2.0366564
up
KIAA1618

Homo sapiens KIAA1618

(KIAA1618), mRNA.

2.034572
up
BCL2L12

Homo sapiens BCL2-like 12 (proline

rich) (BCL2L12), transcript variant

3, mRNA.

2.0263453
up
PDGFD

Homo sapiens platelet derived

growth factor D (PDGFD), transcript

variant 2, mRNA.

2.0171542
up
CDC14B

Homo sapiens CDC 14 cell division

cycle 14 homolog B (S. cerevisiae)

(CDC14B), transcript variant 2,

mRNA.

2.0131624
up
CRELD2

Homo sapiens cysteine-rich with

EGF-like domains 2 (CRELD2),

mRNA.

2.0129623
up
FLJ35767

Homo sapiens FLJ35767 protein

(FLJ35767), mRNA.

2.0035446
up
SCN9A

Homo sapiens sodium channel,

voltage-gated, type IX, alpha subunit

(SCN9A), mRNA.

2.0030117
up
LOC441089

Homo sapiens CRSP8 pseudogene

(LOC441089), non-coding RNA.

2.0013416
up
PLS1

Homo sapiens plastin 1 (I isoform)

(PLS1), mRNA.

3.2073772
down
MIAT

Homo sapiens myocardial infarction

associated transcript (non-protein

coding) (MIAT), non-coding RNA.

3.134669
down
C16orf53

Homo sapiens chromosome 16 open

reading frame 53 (C16orf53),

mRNA.

3.0669296
down
PCOLCE

Homo sapiens procollagen C-

endopeptidase enhancer (PCOLCE),

mRNA.

2.9874208
down
TYMS

Homo sapiens thymidylate

synthetase (TYMS), mRNA.

2.9546156
down
ASAM

Homo sapiens adipocyte-specific

adhesion molecule (ASAM), mRNA.

2.881155
down
FLJ25404
PREDICTED: Homo sapiens

hypothetical protein FLJ25404,

transcript variant 2 (FLJ25404),

mRNA.

2.7996004
down
ICA1

Homo sapiens islet cell autoantigen

1, 69 kDa (ICA1), transcript variant

2, mRNA.

2.7547011
down
SLC6A15

Homo sapiens solute carrier family

6, member 15 (SLC6A15), transcript

variant 1, mRNA.

2.6473744
down
DUSP26

Homo sapiens dual specificity

phosphatase 26 (putative) (DUSP26),

mRNA.

2.6301363
down
SH2D3C

Homo sapiens SH2 domain

containing 3C (SH2D3C), transcript

variant 2, mRNA.

2.6298933
down
LRFN4

Homo sapiens leucine rich repeat and

fibronectin type III domain

containing 4 (LRFN4), mRNA.

2.5819318
down
CENPV

Homo sapiens centromere protein V

(CENPV), mRNA.

2.569341
down
DDX17

Homo sapiens DEAD (Asp-Glu-Ala-

Asp) box polypeptide 17 (DDX17),

transcript variant 1, mRNA.

2.5645072
down
C16orf53

Homo sapiens chromosome 16 open

reading frame 53 (C16orf53),

mRNA.

2.5115252
down
CLASP2

Homo sapiens cytoplasmic linker

associated protein 2 (CLASP2),

mRNA.

2.4085312
down
ARMCX1

Homo sapiens armadillo repeat

containing, X-linked 1 (ARMCX1),

mRNA.

2.395616
down
ICA1

Homo sapiens islet cell autoantigen

1, 69 kDa (ICA1), transcript variant

2, mRNA.

2.3802598
down
LAMB1

Homo sapiens laminin, beta 1

(LAMB1), mRNA.

2.3713975
down
CLK1

Homo sapiens CDC-like kinase 1

(CLK1), mRNA.

2.3289754
down
TH

Homo sapiens tyrosine hydroxylase

(TH), transcript variant 3, mRNA.

2.3156798
down
P4HTM

Homo sapiens prolyl 4-hydroxylase,

transmembrane (endoplasmic

reticulum) (P4HTM), transcript

variant 3, mRNA.

2.3148339
down
D4S234E

Homo sapiens DNA segment on

chromosome 4 (unique) 234

expressed sequence (D4S234E),

transcript variant 2, mRNA.

2.2813866
down
MTA1

Homo sapiens metastasis associated

1 (MTA1), mRNA.

2.2809587
down
TUB

Homo sapiens tubby homolog

(mouse) (TUB), transcript variant 2,

mRNA.

2.2759655
down
PHF17

Homo sapiens PHD finger protein 17

(PHF17), transcript variant S,

mRNA.

2.2701757
down
TAGLN3

Homo sapiens transgelin 3

(TAGLN3), transcript variant 3,

mRNA.

2.2348192
down
SYTL4

Homo sapiens synaptotagmin-like 4

(granuphilin-a) (SYTL4), mRNA.

2.230719
down
ARHGDIG

Homo sapiens Rho GDP dissociation

inhibitor (GDI) gamma

(ARHGDIG), mRNA.

2.218432
down
ABR

Homo sapiens active BCR-related

gene (ABR), transcript variant 2,

mRNA.

2.200697
down
SNORA18

Homo sapiens small nucleolar RNA,

H/ACA box 18 (SNORA18), small

nucleolar RNA.

2.1834235
down
H2AFY2

Homo sapiens H2A histone family,

member Y2 (H2AFY2), mRNA.

2.1802266
down
ST6GAL1

Homo sapiens ST6 beta-

galactosamide alpha-2,6-

sialyltranferase 1 (ST6GAL1),

transcript variant 2, mRNA.

2.1800709
down
DUSP8

Homo sapiens dual specificity

phosphatase 8 (DUSP8), mRNA.

2.1764905
down
TFAP2B

Homo sapiens transcription factor

AP-2 beta (activating enhancer

binding protein 2 beta) (TFAP2B),

mRNA.

2.1661515
down
RCN1

Homo sapiens reticulocalbin 1, EF-

hand calcium binding domain

(RCN1), mRNA.

2.1537797
down
ZNF536

Homo sapiens zinc finger protein

536 (ZNF536), mRNA.

2.147822
down
F12

Homo sapiens coagulation factor XII

(Hageman factor) (F12), mRNA.

2.1474955
down
SCRG1

Homo sapiens scrapie responsive

protein 1 (SCRG1), mRNA.

2.1445231
down
LRRTM2

Homo sapiens leucine rich repeat

transmembrane neuronal 2

(LRRTM2), mRNA.

2.141449
down
GRIN1

Homo sapiens glutamate receptor,

ionotropic, N-methyl D-aspartate 1

(GRIN1), transcript variant NR1-2,

mRNA.

2.1393347
down
SEZ6L2

Homo sapiens seizure related 6

homolog (mouse)-like 2 (SEZ6L2),

transcript variant 2, mRNA.

2.1239994
down
GRM8

Homo sapiens glutamate receptor,

metabotropic 8 (GRM8), mRNA.

2.1140378
down
CENTA1

Homo sapiens centaurin, alpha 1

(CENTA1), mRNA.

2.1079326
down
HDGF

Homo sapiens hepatoma-derived

growth factor (high-mobility group

protein 1-like) (HDGF), mRNA.

2.1008873
down
JAM2

Homo sapiens junctional adhesion

molecule 2 (JAM2), mRNA.

2.0997539
down
DDR2

Homo sapiens discoidin domain

receptor tyrosine kinase 2 (DDR2),

transcript variant 2, mRNA.

2.0729117
down
MYT1

Homo sapiens myelin transcription

factor 1 (MYT1), mRNA.

2.0711784
down
PCGF2

Homo sapiens polycomb group ring

finger 2 (PCGF2), mRNA.

2.0665834
down

AGENCOURT_15463101 Human

Anterior Horn Homo sapiens cDNA

clone IMAGE: 30516556 5, mRNA

sequence

2.0662923
down
CNTNAP1

Homo sapiens contactin associated

protein 1 (CNTNAP1), mRNA.

2.0568583
down
EML5

Homo sapiens echinoderm

microtubule associated protein like 5

(EML5), mRNA.

2.0407145
down
C1orf43

Homo sapiens chromosome 1 open

reading frame 43 (C1orf43),

transcript variant 1, mRNA.

2.0184758
down
BRSK1

Homo sapiens BR serine/threonine

kinase 1 (BRSK1), mRNA.

2.0163672
down
N4BP2L1

Homo sapiens NEDD4 binding

protein 2-like 1 (N4BP2L1),

transcript variant 2, mRNA.

2.0149431
down
TCEAL7

Homo sapiens transcription

elongation factor A (SII)-like 7

(TCEAL7), mRNA.

2.0054402
down
TAGLN3

Homo sapiens transgelin 3

(TAGLN3), transcript variant 3,

mRNA.

2.0032253
down
NME4

Homo sapiens non-metastatic cells 4,

protein expressed in (NME4),

nuclear gene encoding mitochondrial

protein, mRNA.

TABLE 8

FC (abs)
Regulation

(Moderated
(Moderated

T-Test
T-Test

[ATRA +
[ATRA +

Compound B]
Compound B]

Vs [DMSO]
Vs [DMSO]

P <= 0.05
P <= 0.05
Gene

FC >= 2.0)
FC >= 2.0)
Symbol
Definition

74.06221
up
CYP26B1

Homo sapiens cytochrome P450,

family 26, subfamily B, polypeptide 1

(CYP26B1), mRNA.

11.48934
up
RET

Homo sapiens ret proto-oncogene

(RET), transcript variant 4, mRNA.

11.17362
up
RET

Homo sapiens ret proto-oncogene

(RET), transcript variant 2, mRNA.

9.271276
up
CRABP2

Homo sapiens cellular retinoic acid

binding protein 2 (CRABP2), mRNA.

6.779234
up
CYP26A1

Homo sapiens cytochrome P450,

family 26, subfamily A, polypeptide 1

(CYP26A1), transcript variant 2,

mRNA.

6.748227
up
ATP7A

Homo sapiens ATPase, Cu++

transporting, alpha polypeptide

(Menkes syndrome) (ATP7A),

mRNA.

6.434992
up
TSPAN1

Homo sapiens tetraspanin 1

(TSPAN1), mRNA.

5.608586
up
NFKBIZ

Homo sapiens nuclear factor of kappa

light polypeptide gene enhancer in B-

cells inhibitor, zeta (NFKBIZ),

transcript variant 2, mRNA.

5.604396
up
DHRS3

Homo sapiens

dehydrogenase/reductase (SDR

family) member 3 (DHRS3), mRNA.

5.54892
up
RARB

Homo sapiens retinoic acid receptor,

beta (RARB), transcript variant 1,

mRNA.

5.007533
up
PLAT

Homo sapiens plasminogen activator,

tissue (PLAT), transcript variant 1,

mRNA.

4.24417
up
VGF

Homo sapiens VGF nerve growth

factor inducible (VGF), mRNA.

4.239555
up
PTGER2

Homo sapiens prostaglandin E

receptor 2 (subtype EP2), 53 kDa

(PTGER2), mRNA.

4.087792
up
PCDH18

Homo sapiens protocadherin 18

(PCDH18), mRNA.

3.82108
up
ENPP2

Homo sapiens ectonucleotide

pyrophosphatase/phosphodiesterase 2

(ENPP2), transcript variant 2, mRNA.

3.81664
up
NAV2

Homo sapiens neuron navigator 2

(NAV2), transcript variant 2, mRNA.

3.78091
up
RARB

Homo sapiens retinoic acid receptor,

beta (RARB), transcript variant 2,

mRNA.

3.767821
up
PLS3

Homo sapiens plastin 3 (T isoform)

(PLS3), mRNA.

3.743108
up
CYP1B1

Homo sapiens cytochrome P450,

family 1, subfamily B, polypeptide 1

(CYP1B1), mRNA.

3.659243
up
LOC387763
PREDICTED: Homo sapiens

hypothetical LOC387763

(LOC387763), mRNA.

3.580283
up
PCDH18

Homo sapiens protocadherin 18

(PCDH18), mRNA.

3.53702
up
PDZRN3
PREDICTED: Homo sapiens PDZ

domain containing RING finger 3

(PDZRN3), mRNA.

3.466291
up
ENPP2

Homo sapiens ectonucleotide

pyrophosphatase/phosphodiesterase 2

(ENPP2), transcript variant 2, mRNA.

3.462251
up
RET

Homo sapiens ret proto-oncogene

(RET), transcript variant 2, mRNA.

3.351228
up
MMP11

Homo sapiens matrix metallopeptidase

11 (stromelysin 3) (MMP11), mRNA.

3.276531
up
TRAF3IP2

Homo sapiens TRAF3 interacting

protein 2 (TRAF3IP2), transcript

variant 2, mRNA.

3.15566
up
LOC375295
PREDICTED: Homo sapiens

hypothetical gene supported by

BC013438 (LOC375295), mRNA.

3.148894
up
PRKCH

Homo sapiens protein kinase C, eta

(PRKCH), mRNA.

3.136123
up
TMX4

Homo sapiens thioredoxin-related

transmembrane protein 4 (TMX4),

mRNA.

3.130793
up
CYP26A1

Homo sapiens cytochrome P450,

family 26, subfamily A, polypeptide 1

(CYP26A1), transcript variant 2,

mRNA.

3.128071
up
EFNB2

Homo sapiens ephrin-B2 (EFNB2),

mRNA.

3.121308
up
TMX4

Homo sapiens thioredoxin-related

transmembrane protein 4 (TMX4),

mRNA.

3.114901
up
PDZRN3

Homo sapiens PDZ domain containing

ring finger 3 (PDZRN3), mRNA.

3.075643
up
FNDC5

Homo sapiens fibronectin type III

domain containing 5 (FNDC5),

mRNA.

3.050035
up
NCOA3

Homo sapiens nuclear receptor

coactivator 3 (NCOA3), transcript

variant 1, mRNA.

2.929809
up
THBS1

Homo sapiens thrombospondin 1

(THBS1), mRNA.

2.929415
up
LOXL4

Homo sapiens lysyl oxidase-like 4

(LOXL4), mRNA.

2.869473
up
CHRNA3

Homo sapiens cholinergic receptor,

nicotinic, alpha 3 (CHRNA3), mRNA.

2.818317
up
NAV2

Homo sapiens neuron navigator 2

(NAV2), transcript variant 2, mRNA.

2.737314
up
IRF9

Homo sapiens interferon regulatory

factor 9 (IRF9), mRNA.

2.682665
up
REPS2

Homo sapiens RALBP1 associated

Eps domain containing 2 (REPS2),

transcript variant 1, mRNA.

2.665766
up
FRMD6

Homo sapiens FERM domain

containing 6 (FRMD6), mRNA.

2.638139
up
NEDD4L

Homo sapiens neural precursor cell

expressed, developmentally down-

regulated 4-like (NEDD4L), mRNA.

2.627141
up
FOXC1

Homo sapiens forkhead box C1

(FOXC1), mRNA.

2.519936
up
RARA

Homo sapiens retinoic acid receptor,

alpha (RARA), transcript variant 1,

mRNA.

2.379357
up
REPS2

Homo sapiens RALBP1 associated

Eps domain containing 2 (REPS2),

transcript variant 1, mRNA.

2.351435
up
ABCA1

Homo sapiens ATP-binding cassette,

sub-family A (ABC1), member 1

(ABCA1), mRNA.

2.325886
up
GNG2

Homo sapiens guanine nucleotide

binding protein (G protein), gamma 2

(GNG2), mRNA.

2.30321
up
PDZRN3
PREDICTED: Homo sapiens PDZ

domain containing RING finger 3

(PDZRN3), mRNA.

2.293642
up
CHRNA3

Homo sapiens cholinergic receptor,

nicotinic, alpha 3 (CHRNA3), mRNA.

2.217881
up
SMOC1

Homo sapiens SPARC related

modular calcium binding 1 (SMOC1),

transcript variant 1, mRNA.

2.062672
up
AKR1C3

Homo sapiens aldo-keto reductase

family 1, member C3 (3-alpha

hydroxysteroid dehydrogenase, type

II) (AKR1C3), mRNA.

2.040579
up
PRMT6

Homo sapiens protein arginine

methyltransferase 6 (PRMT6),

mRNA.

2.027333
up
ALX3

Homo sapiens aristaless-like

homeobox 3 (ALX3), mRNA.

2.025153
up
NEDD9

Homo sapiens neural precursor cell

expressed, developmentally down-

regulated 9 (NEDD9), transcript

variant 1, mRNA.

2.022927
up
RND3

Homo sapiens Rho family GTPase 3

(RND3), mRNA.

2.008617
up
C10orf33

Homo sapiens chromosome 10 open

reading frame 33 (C10orf33), mRNA.

15.87051
down
DLK1

Homo sapiens delta-like 1 homolog

(Drosophila) (DLK1), mRNA.

5.487316
down
SNHG7

Homo sapiens small nucleolar RNA

host gene 7 (non-protein coding)

(SNHG7), transcript variant 1, non-

coding RNA.

4.849351
down
MEG3

Homo sapiens maternally expressed 3

(non-protein coding) (MEG3),

transcript variant 1, non-coding RNA.

XR_001346-XR_001372

4.700442
down
ATP1A1

Homo sapiens ATPase, Na+/K+

transporting, alpha 1 polypeptide

(ATP1A1), transcript variant 1,

mRNA.

3.83497
down
LOC100131866
PREDICTED: Homo sapiens

misc_RNA (LOC100131866),

miscRNA.

3.625778
down
LOC728452
PREDICTED: Homo sapiens similar

to nuclear pore membrane protein 121

(LOC728452), mRNA.

3.402146
down
LOC441763
PREDICTED: Homo sapiens

hypothetical LOC441763

(LOC441763), mRNA.

3.160639
down
LOC651816
PREDICTED: Homo sapiens similar

to Ubiquitin-conjugating enzyme E2S

(Ubiquitin-conjugating enzyme E2-24

kDa) (Ubiquitin-protein ligase)

(Ubiquitin carrier protein) (E2-EPF5)

(LOC651816), mRNA.

3.153724
down
CALML4

Homo sapiens calmodulin-like 4

(CALML4), transcript variant 1,

mRNA.

2.967706
down
CD320

Homo sapiens CD320 molecule

(CD320), mRNA.

2.859124
down
TRAP1

Homo sapiens TNF receptor-

associated protein 1 (TRAP1), mRNA.

2.724363
down
ST3GAL4

Homo sapiens ST3 beta-galactoside

alpha-2,3-sialyltransferase 4

(ST3GAL4), mRNA.

2.705246
down
LOC647251
PREDICTED: Homo sapiens similar

to maternally expressed 3

(LOC647251), mRNA.

2.618742
down
VIM

Homo sapiens vimentin (VIM),

mRNA.

2.558125
down
DCN

Homo sapiens decorin (DCN),

transcript variant C, mRNA.

2.26428
down
TRERF1

Homo sapiens transcriptional

regulating factor 1 (TRERF1), mRNA.

2.220506
down
SLC29A1

Homo sapiens solute carrier family 29

(nucleoside transporters), member 1

(SLC29A1), nuclear gene encoding

mitochondrial protein, transcript

variant 4, mRNA.

2.172436
down
C2orf48

Homo sapiens chromosome 2 open

reading frame 48 (C2orf48), mRNA.

2.163782
down
INSM2

Homo sapiens insulinoma-associated 2

(INSM2), mRNA.

2.131659
down
CACNA1H

Homo sapiens calcium channel,

voltage-dependent, T type, alpha 1H

subunit (CACNA1H), transcript

variant 1, mRNA.

2.097367
down
ILVBL

Homo sapiens ilvB (bacterial

acetolactate synthase)-like (ILVBL),

mRNA.

2.053179
down
NELL1

Homo sapiens NEL-like 1 (chicken)

(NELL1), mRNA.

TABLE 9

FC (abs)
Regulation

(Moderated
(Moderated

T-Test
T-Test

[ATRA +
[ATRA +

Compound B]
Compound B]

Vs [DMSO]
Vs [DMSO]

P <= 0.05
P <= 0.05

FC >= 2.0)
FC >= 2.0)
Symbol
Definition

5.310678
up
CDKN1A

Homo sapiens cyclin-dependent

kinase inhibitor 1A (p21, Cip1)

(CDKN1A), transcript variant 1,

mRNA.

4.607131
up
ACSL3

Homo sapiens acyl-CoA synthetase

long-chain family member 3

(ACSL3), transcript variant 2, mRNA.

3.865349
up
PLS3

Homo sapiens plastin 3 (T isoform)

(PLS3), mRNA.

3.724138
up
CRISPLD1

Homo sapiens cysteine-rich secretory

protein LCCL domain containing 1

(CRISPLD1), mRNA.

3.32357
up
CRISPLD1

Homo sapiens cysteine-rich secretory

protein LCCL domain containing 1

(CRISPLD1), mRNA.

3.273512
up
PCDH20

Homo sapiens protocadherin 20

(PCDH20), mRNA.

3.241042
up
RPL26

Homo sapiens ribosomal protein L26

(RPL26), mRNA.

3.095257
up
LOC729236
PREDICTED: Homo sapiens

misc_RNA (LOC729236), miscRNA.

3.081601
up
JARID2

Homo sapiens jumonji, AT rich

interactive domain 2 (JARID2),

mRNA.

2.992321
up
RNU6-1

Homo sapiens RNA, U6 small nuclear

1 (RNU6-1), small nuclear RNA.

2.854101
up
HOXD1

Homo sapiens homeobox D1

(HOXD1), mRNA.

2.770691
up
ATP6AP2

Homo sapiens ATPase, H+

transporting, lysosomal accessory

protein 2 (ATP6AP2), mRNA.

2.743215
up
SPRY4

Homo sapiens sprouty homolog 4

(Drosophila) (SPRY4), mRNA.

2.741689
up
REC8

Homo sapiens REC8 homolog (yeast)

(REC8), transcript variant 1, mRNA.

2.721933
up
FZD7

Homo sapiens frizzled homolog 7

(Drosophila) (FZD7), mRNA.

2.706069
up
TMEM50B

Homo sapiens transmembrane protein

50B (TMEM50B), mRNA.

2.705729
up
RDH10

Homo sapiens retinol dehydrogenase

10 (all-trans) (RDH10), mRNA.

2.689977
up
RN5S9

Homo sapiens RNA, 5S ribosomal 9

(RN5S9), ribosomal RNA.

2.68436
up
NPTN

Homo sapiens neuroplastin (NPTN),

transcript variant beta, mRNA.

2.676171
up
G3BP2

Homo sapiens GTPase activating

protein (SH3 domain) binding protein

2 (G3BP2), transcript variant 3,

mRNA.

2.675857
up
ITGA1

Homo sapiens integrin, alpha 1

(ITGA1), mRNA.

2.665102
up
NPTN

Homo sapiens neuroplastin (NPTN),

transcript variant alpha, mRNA.

2.656279
up
UBLCP1

Homo sapiens ubiquitin-like domain

containing CTD phosphatase 1

(UBLCP1), mRNA.

2.65175
up
IL10RB

Homo sapiens interleukin 10 receptor,

beta (IL10RB), mRNA.

2.626543
up
ARMET

Homo sapiens arginine-rich, mutated

in early stage tumors (ARMET),

mRNA.

2.614088
up
SH2B3

Homo sapiens SH2B adaptor protein 3

(SH2B3), mRNA.

2.60153
up
ADD3

Homo sapiens adducin 3 (gamma)

(ADD3), transcript variant 3, mRNA.

2.59546
up
ACSL3

Homo sapiens acyl-CoA synthetase

long-chain family member 3

(ACSL3), transcript variant 1, mRNA.

2.591278
up
RNU6-15

Homo sapiens RNA, U6 small nuclear

15 (RNU6-15), small nuclear RNA.

2.589584
up
LOC653158
PREDICTED: Homo sapiens similar

to hypothetical protein MGC40405,

transcript variant 1 (LOC653158),

mRNA.

2.577032
up
SGK1

Homo sapiens serum/glucocorticoid

regulated kinase 1 (SGK1), transcript

variant 1, mRNA.

2.574045
up
ZFAND6

Homo sapiens zinc finger, AN1-type

domain 6 (ZFAND6), mRNA.

2.567282
up
BCHE

Homo sapiens butyrylcholinesterase

(BCHE), mRNA.

2.558458
up
HSD17B12

Homo sapiens hydroxysteroid (17-

beta) dehydrogenase 12 (HSD17B12),

mRNA.

2.549309
up
SNORA79

Homo sapiens small nucleolar RNA,

H/ACA box 79 (SNORA79), small

nucleolar RNA.

2.54709
up
LIPA

Homo sapiens lipase A, lysosomal

acid, cholesterol esterase (LIPA),

transcript variant 2, mRNA.

2.536986
up
G3BP1

Homo sapiens GTPase activating

protein (SH3 domain) binding protein

1 (G3BP1), transcript variant 1,

mRNA.

2.509722
up
LAMC1

Homo sapiens laminin, gamma 1

(formerly LAMB2) (LAMC1),

mRNA.

2.505713
up
CNN2

Homo sapiens calponin 2 (CNN2),

transcript variant 2, mRNA.

2.488804
up
ABCB1

Homo sapiens ATP-binding cassette,

sub-family B (MDR/TAP), member 1

(ABCB1), mRNA.

2.481133
up
GLCE

Homo sapiens glucuronic acid

epimerase (GLCE), mRNA.

2.477101
up
FLOT1

Homo sapiens flotillin 1 (FLOT1),

mRNA.

2.465013
up
SPRED1

Homo sapiens sprouty-related, EVH1

domain containing 1 (SPRED1),

mRNA.

2.463788
up
VASN

Homo sapiens vasorin (VASN),

mRNA.

2.456818
up
XPR1

Homo sapiens xenotropic and

polytropic retrovirus receptor (XPR1),

mRNA.

2.450285
up
CYB5R4

Homo sapiens cytochrome b5

reductase 4 (CYB5R4), mRNA.

2.449843
up
FAM69A

Homo sapiens family with sequence

similarity 69, member A (FAM69A),

mRNA.

2.435929
up
XPR1

Homo sapiens xenotropic and

polytropic retrovirus receptor (XPR1),

mRNA.

2.426759
up
SC5DL

Homo sapiens sterol-C5-desaturase

(ERG3 delta-5-desaturase homolog,

S. cerevisiae)-like (SC5DL), transcript

variant 1, mRNA.

2.410317
up
TMEM19

Homo sapiens transmembrane protein

19 (TMEM19), mRNA.

2.406851
up
DNAJB11

Homo sapiens DnaJ (Hsp40) homolog,

subfamily B, member 11 (DNAJB11),

mRNA.

2.399452
up
HSP90B1

Homo sapiens heat shock protein

90 kDa beta (Grp94), member 1

(HSP90B1), mRNA.

2.389103
up
PAPSS1

Homo sapiens 3′-phosphoadenosine

5′-phosphosulfate synthase 1

(PAPSS1), mRNA.

2.387213
up

Homo sapiens primary neuroblastoma

cDNA, clone: Nbla10111, full insert

sequence

2.376022
up
FGFR1OP2

Homo sapiens FGFR1 oncogene

partner 2 (FGFR1OP2), mRNA.

2.368677
up
WDR1

Homo sapiens WD repeat domain 1

(WDR1), transcript variant 2, mRNA.

2.362372
up
HSD17B12

Homo sapiens hydroxysteroid (17-

beta) dehydrogenase 12 (HSD17B12),

mRNA.

2.358175
up
WDR44

Homo sapiens WD repeat domain 44

(WDR44), mRNA.

2.354716
up
OSTF1

Homo sapiens osteoclast stimulating

factor 1 (OSTF1), mRNA.

2.338338
up
SGK1

Homo sapiens serum/glucocorticoid

regulated kinase 1 (SGK1), transcript

variant 1, mRNA.

2.338305
up
S100A10

Homo sapiens S100 calcium binding

protein A10 (S100A10), mRNA.

2.333673
up
SIPA1

Homo sapiens signal-induced

proliferation-associated gene 1

(SIPA1), transcript variant 2, mRNA.

2.317173
up
SCGN

Homo sapiens secretagogin, EF-hand

calcium binding protein (SCGN),

mRNA.

2.315298
up
PLS1

Homo sapiens plastin 1 (I isoform)

(PLS1), mRNA.

2.314937
up
RALB

Homo sapiens v-ral simian leukemia

viral oncogene homolog B (ras

related; GTP binding protein)

(RALB), mRNA.

2.308636
up
TMC6

Homo sapiens transmembrane

channel-like 6 (TMC6), mRNA.

2.302612
up
EXTL2

Homo sapiens exostoses (multiple)-

like 2 (EXTL2), transcript variant 1,

mRNA.

2.2959
up
PNPLA8

Homo sapiens patatin-like

phospholipase domain containing 8

(PNPLA8), mRNA.

2.274158
up
YIPF1

Homo sapiens Yip1 domain family,

member 1 (YIPF1), mRNA.

2.256347
up
GPR177

Homo sapiens G protein-coupled

receptor 177 (GPR177), transcript

variant 1, mRNA.

2.251912
up
TRAM2

Homo sapiens translocation associated

membrane protein 2 (TRAM2),

mRNA.

2.246115
up
CXorf57

Homo sapiens chromosome X open

reading frame 57 (CXorf57), mRNA.

2.239622
up
MYCNOS
PREDICTED: Homo sapiens

misc_RNA (MYCNOS), miscRNA.

2.233039
up
COQ10B

Homo sapiens coenzyme Q10

homolog B (S. cerevisiae) (COQ10B),

mRNA.

2.232365
up
PIGM

Homo sapiens phosphatidylinositol

glycan anchor biosynthesis, class M

(PIGM), mRNA.

2.231697
up
ELMOD1

Homo sapiens ELMO/CED-12

domain containing 1 (ELMOD1),

mRNA.

2.228658
up
DNAJB6

Homo sapiens DnaJ (Hsp40) homolog,

subfamily B, member 6 (DNAJB6),

transcript variant 1, mRNA.

2.225392
up
LOC653156
PREDICTED: Homo sapiens similar

to hCG1782414 (LOC653156),

mRNA.

2.224361
up
REC8

Homo sapiens REC8 homolog (yeast)

(REC8), transcript variant 1, mRNA.

2.223486
up
TMBIM4

Homo sapiens transmembrane BAX

inhibitor motif containing 4

(TMBIM4), mRNA.

2.22101
up
TJP1

Homo sapiens tight junction protein 1

(zona occludens 1) (TJP1), transcript

variant 2, mRNA.

2.212364
up
USP8

Homo sapiens ubiquitin specific

peptidase 8 (USP8), mRNA.

2.208929
up
OSBPL3

Homo sapiens oxysterol binding

protein-like 3 (OSBPL3), transcript

variant 4, mRNA.

2.203524
up
CPVL

Homo sapiens carboxypeptidase,

vitellogenic-like (CPVL), transcript

variant 2, mRNA.

2.197295
up
DUSP5

Homo sapiens dual specificity

phosphatase 5 (DUSP5), mRNA.

2.194288
up
CADM1

Homo sapiens cell adhesion molecule

1 (CADM1), transcript variant 1,

mRNA.

2.193162
up
SEC24D

Homo sapiens SEC24 related gene

family, member D (S. cerevisiae)

(SEC24D), mRNA.

2.189749
up
MY ADM

Homo sapiens myeloid-associated

differentiation marker (MYADM),

transcript variant 4, mRNA.

2.185441
up
LOC285359

Homo sapiens phosducin-like 3

pseudogene (LOC285359) on

chromosome 3.

2.177089
up
MYL12A

Homo sapiens myosin, light chain

12A, regulatory, non-sarcomeric

(MYL12A), mRNA.

2.175736
up
C3orf59

Homo sapiens chromosome 3 open

reading frame 59 (C3orf59), mRNA.

2.169516
up
BCL6

Homo sapiens B-cell CLL/lymphoma

6 (zinc finger protein 51) (BCL6),

transcript variant 1, mRNA.

2.168062
up
EPB41L5

Homo sapiens erythrocyte membrane

protein band 4.1 like 5 (EPB41L5),

mRNA.

2.167211
up
CXorf45

Homo sapiens chromosome X open

reading frame 45 (CXorf45), transcript

variant 1, mRNA.

2.161228
up
ZSWIM6
PREDICTED: Homo sapiens zinc

finger, SWIM-type containing 6

(ZSWIM6), mRNA.

2.158413
up
DCBLD2

Homo sapiens discoidin, CUB and

LCCL domain containing 2

(DCBLD2), mRNA.

2.157928
up
LAMP2

Homo sapiens lysosomal-associated

membrane protein 2 (LAMP2),

transcript variant LAMP2B, mRNA.

2.157778
up
HLA-B

Homo sapiens major

histocompatibility complex, class I, B

(HLA-B), mRNA.

2.155381
up

PREDICTED: Homo sapiens

hypothetical LOC389089

(LOC389089), mRNA

2.154519
up
LOC401076
PREDICTED: Homo sapiens

misc_RNA (LOC401076), miscRNA.

2.153577
up
TXNDC9

Homo sapiens thioredoxin domain

containing 9 (TXNDC9), mRNA.

2.149624
up
PCDH17

Homo sapiens protocadherin 17

(PCDH17), mRNA.

2.148683
up

Homo sapiens cDNA FLJ26539 fis,

clone KDN09310

2.147121
up
YIPF1

Homo sapiens Yip1 domain family,

member 1 (YIPF1), mRNA.

2.135235
up
LOC729646
PREDICTED: Homo sapiens

misc_RNA (LOC729646), miscRNA.

2.133704
up
PTGR1

Homo sapiens prostaglandin reductase

1 (PTGR1), mRNA.

2.133145
up
IGF2R

Homo sapiens insulin-like growth

factor 2 receptor (IGF2R), mRNA.

2.133046
up
EPB41L5

Homo sapiens erythrocyte membrane

protein band 4.1 like 5 (EPB41L5),

mRNA.

2.132543
up
LOC100129685
PREDICTED: Homo sapiens

hypothetical protein LOC100129685

(LOC100129685), mRNA.

2.130848
up
PAQR8

Homo sapiens progestin and adipoQ

receptor family member VIII

(PAQR8), mRNA.

2.124304
up
RPGR

Homo sapiens retinitis pigmentosa

GTPase regulator (RPGR), transcript

variant B, mRNA.

2.122092
up
FBLN2

Homo sapiens fibulin 2 (FBLN2),

transcript variant 2, mRNA.

2.121826
up
GCA

Homo sapiens grancalcin, EF-hand

calcium binding protein (GCA),

mRNA.

2.120949
up
GPR126

Homo sapiens G protein-coupled

receptor 126 (GPR126), transcript

variant a2, mRNA.

2.118829
up

Homo sapiens mRNA; cDNA

DKFZp564C152 (from clone

DKFZp564C152)

2.118362
up
PI15

Homo sapiens peptidase inhibitor 15

(PI15), mRNA.

2.116085
up
GNS

Homo sapiens glucosamine (N-

acetyl)-6-sulfatase (Sanfilippo disease

IIID) (GNS), mRNA.

2.110086
up
ALG13

Homo sapiens asparagine-linked

glycosylation 13 homolog

(S. cerevisiae) (ALG13), mRNA.

2.109918
up
TP53INP1

Homo sapiens tumor protein p53

inducible nuclear protein 1

(TP53INP1), mRNA.

2.109562
up
NPPA

Homo sapiens natriuretic peptide

precursor A (NPPA), mRNA.

2.108881
up
USP38

Homo sapiens ubiquitin specific

peptidase 38 (USP38), mRNA.

2.108092
up
PSMA4

Homo sapiens proteasome (prosome,

macropain) subunit, alpha type, 4

(PSMA4), mRNA.

2.10809
up
C5orf32

Homo sapiens chromosome 5 open

reading frame 32 (C5orf32), mRNA.

2.095516
up
PRKCA

Homo sapiens protein kinase C, alpha

(PRKCA), mRNA.

2.094473
up

Homo sapiens cDNA clone

IMAGE: 5268658

2.090239
up
SEC22B

Homo sapiens SEC22 vesicle

trafficking protein homolog B

(S. cerevisiae) (SEC22B), mRNA.

2.084241
up
DNAJC10

Homo sapiens DnaJ (Hsp40) homolog,

subfamily C, member 10 (DNAJC10),

mRNA.

2.083668
up
UTP14C

Homo sapiens UTP14, U3 small

nucleolar ribonucleoprotein, homolog

C (yeast) (UTP14C), mRNA.

2.082504
up
TULP4

Homo sapiens tubby like protein 4

(TULP4), transcript variant 2, mRNA.

2.080901
up
HIF1A

Homo sapiens hypoxia-inducible

factor 1, alpha subunit (basic helix-

loop-helix transcription factor)

(HIF1A), transcript variant 2, mRNA.

2.079186
up
DYNC1I1

Homo sapiens dynein, cytoplasmic 1,

intermediate chain 1 (DYNC1I1),

mRNA.

2.077066
up
ANKRD57

Homo sapiens ankyrin repeat domain

57 (ANKRD57), mRNA.

2.073996
up
PON2

Homo sapiens paraoxonase 2 (PON2),

transcript variant 1, mRNA.

2.071366
up
BMPR2

Homo sapiens bone morphogenetic

protein receptor, type II

(serine/threonine kinase) (BMPR2),

mRNA.

2.069789
up
SLC4A8

Homo sapiens solute carrier family 4,

sodium bicarbonate cotransporter,

member 8 (SLC4A8), transcript

variant 2, mRNA.

2.067038
up
ATP2B1

Homo sapiens ATPase, Ca++

transporting, plasma membrane 1

(ATP2B1), transcript variant 1,

mRNA.

2.066323
up
DAD1

Homo sapiens defender against cell

death 1 (DAD1), mRNA.

2.063078
up
RAB3IP

Homo sapiens RAB3A interacting

protein (rabin3) (RAB3IP), transcript

variant beta 1, mRNA.

2.062607
up
RPPH1

Homo sapiens ribonuclease P RNA

component H1 (RPPH1), RNase P

RNA.

2.059798
up
PRG2

Homo sapiens proteoglycan 2, bone

marrow (natural killer cell activator,

eosinophil granule major basic

protein) (PRG2), mRNA.

2.056469
up
PRKAR1A

Homo sapiens protein kinase, cAMP-

dependent, regulatory, type I, alpha

(tissue specific extinguisher 1)

(PRKAR1A), transcript variant 1,

mRNA.

2.056032
up
ZMYM1

Homo sapiens zinc finger, MYM-type

1 (ZMYM1), mRNA.

2.050947
up
CLINT 1

Homo sapiens clathrin interactor 1

(CLINT1), mRNA.

2.050481
up
TMCO1

Homo sapiens transmembrane and

coiled-coil domains 1 (TMCO1),

mRNA.

2.048413
up
PDGFD

Homo sapiens platelet derived growth

factor D (PDGFD), transcript variant

1, mRNA.

2.047608
up
USP9X

Homo sapiens ubiquitin specific

peptidase 9, X-linked (USP9X),

transcript variant 4, mRNA.

2.046355
up
AADACL4

Homo sapiens arylacetamide

deacetylase-like 4 (AADACL4),

mRNA.

2.046026
up
BCL2L12

Homo sapiens BCL2-like 12 (proline

rich) (BCL2L12), transcript variant 3,

mRNA.

2.045556
up
ALPL

Homo sapiens alkaline phosphatase,

liver/bone/kidney (ALPL), transcript

variant 1, mRNA.

2.040306
up
LOC653079
PREDICTED: Homo sapiens

misc_RNA (LOC653079), miscRNA.

2.037734
up
CCDC128

Homo sapiens coiled-coil domain

containing 128 (CCDC128), mRNA.

2.034251
up
HDAC1

Homo sapiens histone deacetylase 1

(HDAC1), mRNA.

2.032832
up
HLA-E

Homo sapiens major

histocompatibility complex, class I, E

(HLA-E), mRNA.

2.032317
up
INTS6

Homo sapiens integrator complex

subunit 6 (INTS6), transcript variant

2, mRNA.

2.032247
up
TMEM166

Homo sapiens transmembrane protein

166 (TMEM166), mRNA.

2.031952
up
NDFIP2

Homo sapiens Nedd4 family

interacting protein 2 (NDFIP2),

mRNA.

2.031459
up
EDEM3

Homo sapiens ER degradation

enhancer, mannosidase alpha-like 3

(EDEM3), mRNA.

2.030964
up
FER1L4

Homo sapiens fer-1-like 4 (C. elegans)

(FER1L4) on chromosome 20.

2.029642
up
CHUK

Homo sapiens conserved helix-loop-

helix ubiquitous kinase (CHUK),

mRNA.

2.028674
up
C10orf75
PREDICTED: Homo sapiens

misc_RNA (C10orf75), miscRNA.

2.025385
up
LOC389342
PREDICTED: Homo sapiens similar

to QM protein, transcript variant 11

(LOC389342), mRNA.

2.025148
up
RNASEL

Homo sapiens ribonuclease L (2′,5-

oligoisoadenylate synthetase-

dependent) (RNASEL), mRNA.

2.024918
up
LOC100131205
PREDICTED: Homo sapiens

hypothetical protein LOC100131205

(LOC100131205), mRNA.

2.021271
up
TMEM205

Homo sapiens transmembrane protein

205 (TMEM205), mRNA.

2.019676
up
RRBP1

Homo sapiens ribosome binding

protein 1 homolog 180 kDa (dog)

(RRBP1), transcript variant 1, mRNA.

2.017824
up
ALCAM

Homo sapiens activated leukocyte cell

adhesion molecule (ALCAM),

mRNA.

2.014654
up
ATG4C

Homo sapiens ATG4 autophagy

related 4 homolog C (S. cerevisiae)

(ATG4C), transcript variant 7, mRNA.

2.014039
up
MEGF9

Homo sapiens multiple EGF-like-

domains 9 (MEGF9), mRNA.

2.013871
up
C1orf97

Homo sapiens chromosome 1 open

reading frame 97 (C1orf97), mRNA.

2.013175
up
STRADB

Homo sapiens STE20-related kinase

adaptor beta (STRADB), mRNA.

2.01128
up
SREBF1

Homo sapiens sterol regulatory

element binding transcription factor 1

(SREBF1), transcript variant 1,

mRNA.

2.010292
up
SUOX

Homo sapiens sulfite oxidase

(SUOX), nuclear gene encoding

mitochondrial protein, transcript

variant 1, mRNA.

2.007771
up
RAB8B

Homo sapiens RAB8B, member RAS

oncogene family (RAB8B), mRNA.

2.006673
up
SPRY1

Homo sapiens sprouty homolog 1,

antagonist of FGF signaling

(Drosophila) (SPRY1), transcript

variant 1, mRNA.

2.006646
up
ARL6IP1

Homo sapiens ADP-ribosylation

factor-like 6 interacting protein 1

(ARL6IP1), mRNA.

2.005585
up
C12orf34

Homo sapiens chromosome 12 open

reading frame 34 (C12orf34), mRNA.

2.003665
up
RPAP3

Homo sapiens RNA polymerase II

associated protein 3 (RPAP3), mRNA.

2.00341
up
LOC728782
PREDICTED: Homo sapiens similar

to ribosomal protein L21

(LOC728782), mRNA.

2.002626
up
PLEKHA6

Homo sapiens pleckstrin homology

domain containing, family A member

6 (PLEKHA6), mRNA.

2.000571
up
KLF10

Homo sapiens Kruppel-like factor 10

(KLF10), transcript variant 2, mRNA.

8.136414
down
LOC648210
PREDICTED: Homo sapiens similar

to Heterogeneous nuclear

ribonucleoprotein A1 (Helix-

destabilizing protein) (Single-strand

RNA-binding protein) (hnRNP core

protein A1) (HDP) (LOC648210),

mRNA.

6.259928
down
TUBA1A

Homo sapiens tubulin, alpha 1a

(TUBA1A), mRNA.

6.153608
down
ACTG1

Homo sapiens actin, gamma 1

(ACTG1), mRNA.

5.798153
down
LOC100008588

Homo sapiens 18S ribosomal RNA

(LOC100008588), non-coding RNA.

5.754853
down
LOC100133565
PREDICTED: Homo sapiens similar

to hCG23738 (LOC100133565),

mRNA.

5.722868
down
TUBB

Homo sapiens tubulin, beta (TUBB),

mRNA.

5.397586
down
LOC92755
PREDICTED: Homo sapiens

misc_RNA (LOC92755), miscRNA.

5.187223
down
LOC100133372
PREDICTED: Homo sapiens

misc_RNA (LOC100133372),

miscRNA.

4.373462
down
TUBA1C

Homo sapiens tubulin, alpha 1c

(TUBA1C), mRNA.

4.353879
down
ACTB

Homo sapiens actin, beta (ACTB),

mRNA.

4.222068
down
RTN1

Homo sapiens reticulon 1 (RTN1),

transcript variant 3, mRNA.

4.119943
down
LOC642817
PREDICTED: Homo sapiens

hypothetical LOC642817

(LOC642817), mRNA.

4.083703
down
FLJ39632
PREDICTED: Homo sapiens

misc_RNA (FLJ39632), miscRNA.

4.034211
down
LOC91561
PREDICTED: Homo sapiens similar

to ribosomal protein S2, transcript

variant 3 (LOC91561), mRNA.

3.980733
down
LOC645691
PREDICTED: Homo sapiens similar

to heterogeneous nuclear

ribonucleoprotein A1 (LOC645691),

mRNA.

3.887888
down
LOC100131609
PREDICTED: Homo sapiens

misc_RNA (LOC100131609),

miscRNA.

3.809681
down
PHOX2B

Homo sapiens paired-like homeobox

2b (PHOX2B), mRNA.

3.807991
down
LOC388654
PREDICTED: Homo sapiens similar

to laminin receptor 1 (ribosomal

protein SA) (LOC388654), mRNA.

3.683417
down
RPLP0

Homo sapiens ribosomal protein,

large, P0 (RPLP0), transcript variant

1, mRNA.

3.642376
down
PHOX2B

Homo sapiens paired-like homeobox

2b (PHOX2B), mRNA.

3.548049
down
IRF2BP2

Homo sapiens interferon regulatory

factor 2 binding protein 2 (IRF2BP2),

transcript variant 1, mRNA.

3.533466
down
TMEM132A

Homo sapiens transmembrane protein

132A (TMEM132A), transcript

variant 2, mRNA.

3.521768
down
CCT7

Homo sapiens chaperonin containing

TCP1, subunit 7 (eta) (CCT7),

transcript variant 1, mRNA.

3.502324
down
SIX3

Homo sapiens sine oculis homeobox

homolog 3 (Drosophila) (SIX3),

mRNA.

3.466076
down
LOC645436
PREDICTED: Homo sapiens similar

to Heterogeneous nuclear

ribonucleoprotein A1 (Helix-

destabilizing protein) (Single-strand

binding protein) (hnRNP core protein

A1) (HDP-1) (Topoisomerase-

inhibitor suppressed) (LOC645436),

mRNA.

3.374453
down
LOC648210
PREDICTED: Homo sapiens similar

to Heterogeneous nuclear

ribonucleoprotein A1 (Helix-

destabilizing protein) (Single-strand

RNA-binding protein) (hnRNP core

protein A1) (HDP) (LOC648210),

mRNA.

3.339326
down
HMGA1

Homo sapiens high mobility group

AT-hook 1 (HMGA1), transcript

variant 1, mRNA.

3.33026
down
LOC148430
PREDICTED: Homo sapiens

misc_RNA (LOC148430), miscRNA.

3.297835
down
RPS2

Homo sapiens ribosomal protein S2

(RPS2), mRNA.

3.2824
down
LOC645385
PREDICTED: Homo sapiens similar

to heterogeneous nuclear

ribonucleoprotein A1 (LOC645385),

mRNA.

3.280514
down
ALDOA

Homo sapiens aldolase A, fructose-

bisphosphate (ALDOA), transcript

variant 2, mRNA.

3.279588
down
LOC728698
PREDICTED: Homo sapiens

misc_RNA (LOC728698), miscRNA.

3.266189
down
EEF1G

Homo sapiens eukaryotic translation

elongation factor 1 gamma (EEF1G),

mRNA. XM_935976 XM_935977

XM_935978 XM_935979

3.255224
down
LOC728643

Homo sapiens heterogeneous nuclear

ribonucleoprotein A1 pseudogene

(LOC728643), non-coding RNA.

3.249131
down
RPLP0

Homo sapiens ribosomal protein,

large, P0 (RPLP0), transcript variant

2, mRNA.

3.246931
down
SORBS2

Homo sapiens sorbin and SH3 domain

containing 2 (SORBS2), transcript

variant 1, mRNA.

3.222426
down
MYCN

Homo sapiens v-myc

myelocytomatosis viral related

oncogene, neuroblastoma derived

(avian) (MYCN), mRNA.

3.216845
down
GUSBL1

Homo sapiens glucuronidase, beta-like

1 (GUSBL1), non-coding RNA.

3.189626
down
SORBS2

Homo sapiens sorbin and SH3 domain

containing 2 (SORBS2), transcript

variant 1, mRNA.

3.171891
down
RPS9

Homo sapiens ribosomal protein S9

(RPS9), mRNA.

3.160895
down
LOC729926
PREDICTED: Homo sapiens

misc_RNA (LOC729926), miscRNA.

3.14376
down
C1orf43

Homo sapiens chromosome 1 open

reading frame 43 (C1orf43), transcript

variant 1, mRNA.

3.11659
down
LOC100008589

Homo sapiens 28S ribosomal RNA

(LOC100008589), non-coding RNA.

3.103844
down
GTF2IP1

Homo sapiens general transcription

factor II, i, pseudogene 1 (GTF2IP1)

on chromosome 7.

3.101386
down
ATP1A1

Homo sapiens ATPase, Na+/K+

transporting, alpha 1 polypeptide

(ATP1A1), transcript variant 1,

mRNA.

3.069661
down
LOC646294
PREDICTED: Homo sapiens

misc_RNA (LOC646294), miscRNA.

3.067323
down
LOC391075
PREDICTED: Homo sapiens

misc_RNA (LOC391075), miscRNA.

3.065105
down
LOC402112
PREDICTED: Homo sapiens

misc_RNA (LOC402112), miscRNA.

3.040335
down
ALDOA

Homo sapiens aldolase A, fructose-

bisphosphate (ALDOA), transcript

variant 3, mRNA.

3.033488
down
LOC728565
PREDICTED: Homo sapiens similar

to Beta-glucuronidase precursor

(LOC728565), mRNA.

3.02633
down
LOC646785
PREDICTED: Homo sapiens

misc_RNA (LOC646785), miscRNA.

3.002853
down
RPS9

Homo sapiens ribosomal protein S9

(RPS9), mRNA.

2.996558
down
TPI1

Homo sapiens triosephosphate

isomerase 1 (TPI1), mRNA.

2.988225
down
TCP1

Homo sapiens t-complex 1 (TCP1),

transcript variant 1, mRNA.

2.981205
down
LOC644063
PREDICTED: Homo sapiens similar

to heterogeneous nuclear

ribonucleoprotein K (LOC644063),

mRNA.

2.977413
down
APP

Homo sapiens amyloid beta (A4)

precursor protein (APP), transcript

variant 3, mRNA.

2.972969
down
LOC440589
PREDICTED: Homo sapiens similar

to ribosomal protein S2, transcript

variant 3 (LOC440589), mRNA.

2.94257
down
LOC284821
PREDICTED: Homo sapiens similar

to ribosomal protein L13a

(LOC284821), mRNA.

2.913472
down
LOC100129553
PREDICTED: Homo sapiens

misc_RNA (LOC100129553),

miscRNA.

2.911284
down
PGAM1

Homo sapiens phosphoglycerate

mutase 1 (brain) (PGAM1), mRNA.

2.884164
down
LOC643357
PREDICTED: Homo sapiens similar

to SMT3 suppressor of mif two 3

homolog 2 (LOC643357), mRNA.

2.862033
down
PRMT1

Homo sapiens protein arginine

methyltransferase 1 (PRMT1),

transcript variant 2, mRNA.

2.849308
down
PLD6

Homo sapiens phospholipase D

family, member 6 (PLD6), mRNA.

2.848781
down
LOC647000
PREDICTED: Homo sapiens similar

to tubulin, beta 5 (LOC647000),

mRNA.

2.848405
down
PRDX2

Homo sapiens peroxiredoxin 2

(PRDX2), nuclear gene encoding

mitochondrial protein, transcript

variant 3, mRNA.

2.834647
down
HAND2

Homo sapiens heart and neural crest

derivatives expressed 2 (HAND2),

mRNA.

2.828951
down
LOC100131609
PREDICTED: Homo sapiens

misc_RNA (LOC100131609),

miscRNA.

2.828043
down
GTF2IP1

Homo sapiens general transcription

factor II, i, pseudogene 1 (GTF2IP1)

on chromosome 7.

2.821679
down
MATR3

Homo sapiens matrin 3 (MATR3),

transcript variant 1, mRNA.

2.819402
down
ATF4

Homo sapiens activating transcription

factor 4 (tax-responsive enhancer

element B67) (ATF4), transcript

variant 2, mRNA.

2.81394
down
LOC100132528
PREDICTED: Homo sapiens

misc_RNA (LOC100132528),

miscRNA.

2.810629
down
LOC347544
PREDICTED: Homo sapiens similar

to ribosomal protein L18a

(LOC347544), mRNA.

2.803635
down
LOC440589
PREDICTED: Homo sapiens similar

to ribosomal protein S2, transcript

variant 3 (LOC440589), mRNA.

2.795735
down
PLCXD3

Homo sapiens phosphatidylinositol-

specific phospholipase C, X domain

containing 3 (PLCXD3), mRNA.

2.794639
down
LOC728658
PREDICTED: Homo sapiens similar

to 23 kD highly basic protein,

transcript variant 1 (LOC728658),

mRNA.

2.784671
down
LOC651149
PREDICTED: Homo sapiens similar

to 60S ribosomal protein L3 (L4)

(LOC651149), mRNA.

2.761892
down
PRDX2

Homo sapiens peroxiredoxin 2

(PRDX2), nuclear gene encoding

mitochondrial protein, transcript

variant 3, mRNA.

2.755405
down
SNHG7

Homo sapiens small nucleolar RNA

host gene 7 (non-protein coding)

(SNHG7), transcript variant 2, non-

coding RNA.

2.755216
down
LOC729779
PREDICTED: Homo sapiens

misc_RNA (LOC729779), miscRNA.

2.739008
down
NCL

Homo sapiens nucleolin (NCL),

mRNA.

2.734261
down
LOC285053
PREDICTED: Homo sapiens similar

to ribosomal protein L18a, transcript

variant 1 (LOC285053), mRNA.

2.723704
down
MTHFD2

Homo sapiens

methylenetetrahydrofolate

dehydrogenase (NADP+ dependent) 2,

methenyltetrahydrofolate

cyclohydrolase (MTHFD2), nuclear

gene encoding mitochondrial protein,

transcript variant 2, mRNA.

2.723382
down
SMA4

Homo sapiens SMA4 (SMA4),

mRNA.

2.709106
down
LOC441775
PREDICTED: Homo sapiens similar

to 60S ribosomal protein L18

(LOC441775), mRNA.

2.7038
down
CAPRIN1

Homo sapiens cell cycle associated

protein 1 (CAPRIN1), transcript

variant 1, mRNA.

2.69812
down
LOC648695
PREDICTED: Homo sapiens similar

to retinoblastoma binding protein 4,

transcript variant 5 (LOC648695),

mRNA.

2.695318
down
LOC648249
PREDICTED: Homo sapiens similar

to 40S ribosomal protein SA (p40)

(34/67 kDa laminin receptor) (Colon

carcinoma laminin-binding protein)

(NEM/1CHD4) (Multidrug resistance-

associated protein MGr1-Ag),

transcript variant 3 (LOC648249),

mRNA.

2.689333
down
HIST3H2A

Homo sapiens histone cluster 3, H2a

(HIST3H2A), mRNA.

2.68172
down
LOC644774
PREDICTED: Homo sapiens similar

to Phosphoglycerate kinase 1

(LOC644774), mRNA.

2.675767
down
ZIC2

Homo sapiens Zic family member 2

(odd-paired homolog, Drosophila)

(ZIC2), mRNA.

2.6746
down
NPIP

Homo sapiens nuclear pore complex

interacting protein (NPIP), mRNA.

2.671463
down
SSR2

Homo sapiens signal sequence

receptor, beta (translocon-associated

protein beta) (SSR2), mRNA.

2.663006
down
LGALS3BP

Homo sapiens lectin, galactoside-

binding, soluble, 3 binding protein

(LGALS3BP), mRNA.

2.642011
down
TSPO

Homo sapiens translocator protein

(18 kDa) (TSPO), transcript variant

PBR-S, mRNA.

2.637904
down
LOC3 87867
PREDICTED: Homo sapiens similar

to 40S ribosomal protein SA (p40)

(34/67 kDa laminin receptor) (Colon

carcinoma laminin-binding protein)

(NEM/1CHD4) (Multidrug resistance-

associated protein MGr1-Ag)

(LOC387867), mRNA.

2.63311
down
NDUFA4L2

Homo sapiens NADH dehydrogenase

(ubiquinone) 1 alpha subcomplex, 4-

like 2 (NDUFA4L2), mRNA.

2.631414
down
GREM1

Homo sapiens gremlin 1, cysteine

knot superfamily, homolog

(Xenopus laevis) (GREM1), mRNA.

2.62583
down
LOC728732
PREDICTED: Homo sapiens

misc_RNA (LOC728732), miscRNA.

2.623359
down
SPAG9

Homo sapiens sperm associated

antigen 9 (SPAG9), mRNA.

2.606464
down
TH

Homo sapiens tyrosine hydroxylase

(TH), transcript variant 2, mRNA.

2.604067
down
MPST

Homo sapiens mercaptopyruvate

sulfurtransferase (MPST), nuclear

gene encoding mitochondrial protein,

transcript variant 2, mRNA.

2.579552
down
NPDC1

Homo sapiens neural proliferation,

differentiation and control, 1

(NPDC1), mRNA.

2.572909
down
ACP1

Homo sapiens acid phosphatase 1,

soluble (ACP1), transcript variant 4,

mRNA.

2.565894
down
ATP2C1

Homo sapiens ATPase, Ca++

transporting, type 2C, member 1

(ATP2C1), transcript variant 4,

mRNA.

2.562114
down
CASC3

Homo sapiens cancer susceptibility

candidate 3 (CASC3), mRNA.

2.555915
down
LOC441506
PREDICTED: Homo sapiens

misc_RNA (LOC441506), miscRNA.

2.553831
down
LOC646531
PREDICTED: Homo sapiens similar

to nuclease sensitive element binding

protein 1 (LOC646531), mRNA.

2.541955
down
PQBP1

Homo sapiens polyglutamine binding

protein 1 (PQBP1), transcript variant

3, mRNA.

2.536753
down
LOC100008589

Homo sapiens 28S ribosomal RNA

(LOC100008589), non-coding RNA.

2.533471
down
LOC100128771
PREDICTED: Homo sapiens

misc_RNA (LOC100128771),

miscRNA.

2.524265
down
B3GNT6

Homo sapiens UDP-GlcNAc:betaGal

beta-1,3-N-

acetylglucosaminyltransferase 6

(B3GNT6), mRNA.

2.515392
down
RNF5P1
PREDICTED: Homo sapiens ring

finger protein 5 pseudogene 1

(RNF5P1), misc RNA.

2.512245
down
LOC153561
PREDICTED: Homo sapiens

hypothetical protein LOC153561

(LOC153561), mRNA.

2.511474
down
NUMA1

Homo sapiens nuclear mitotic

apparatus protein 1 (NUMA1),

mRNA.

2.506295
down
NXPH1

Homo sapiens neurexophilin 1

(NXPH1), mRNA.

2.499662
down
RELN

Homo sapiens reelin (RELN),

transcript variant 1, mRNA.

2.495256
down
SNORA67

Homo sapiens small nucleolar RNA,

H/ACA box 67 (SNORA67), small

nucleolar RNA.

2.49162
down
TTC8

Homo sapiens tetratricopeptide repeat

domain 8 (TTC8), transcript variant 1,

mRNA.

2.489073
down
NFKBIA

Homo sapiens nuclear factor of kappa

light polypeptide gene enhancer in B-

cells inhibitor, alpha (NFKBIA),

mRNA.

2.478279
down
SPTBN1

Homo sapiens spectrin, beta, non-

erythrocytic 1 (SPTBN1), transcript

variant 1, mRNA.

2.476282
down

Homo sapiens cDNA FLJ45619 fis,

clone BRTHA3027318

2.466702
down
LOC100132394
PREDICTED: Homo sapiens

hypothetical protein LOC100132394

(LOC100132394), mRNA.

2.465765
down
GAB2

Homo sapiens GRB2-associated

binding protein 2 (GAB2), transcript

variant 1, mRNA.

2.463514
down
LOC652900
PREDICTED: Homo sapiens similar

to SEZ6L2 protein (LOC652900),

mRNA.

2.459292
down
GLCCI1

Homo sapiens glucocorticoid induced

transcript 1 (GLCCI1), mRNA.

2.455777
down
CKAP5

Homo sapiens cytoskeleton associated

protein 5 (CKAP5), transcript variant

1, mRNA.

2.450269
down
LOC388707
PREDICTED: Homo sapiens

misc_RNA (LOC3 88707), miscRNA.

2.446437
down
SNRPN

Homo sapiens small nuclear

ribonucleoprotein polypeptide N

(SNRPN), transcript variant 3, mRNA.

2.446257
down
SMA5

Homo sapiens SMA5 (SMA5),

mRNA.

2.439987
down
CNBP

Homo sapiens CCHC-type zinc finger,

nucleic acid binding protein (CNBP),

mRNA.

2.437716
down
MYT1L

Homo sapiens myelin transcription

factor 1-like (MYT1L), mRNA.

2.435687
down
LOC100128266
PREDICTED: Homo sapiens

misc_RNA (LOC100128266),

miscRNA.

2.434372
down
CD276

Homo sapiens CD276 molecule

(CD276), transcript variant 1, mRNA.

XM_945872 XM_945874

2.429116
down
PHB2

Homo sapiens prohibitin 2 (PHB2),

transcript variant 2, mRNA.

2.421669
down
HDGF2

Homo sapiens hepatoma-derived

growth factor-related protein 2

(HDGF2), transcript variant 1, mRNA.

2.410991
down
FLJ22184

Homo sapiens hypothetical protein

FLJ22184 (FLJ22184), mRNA.

2.407718
down
SCARB1

Homo sapiens scavenger receptor

class B, member 1 (SCARB1),

mRNA.

2.405512
down
RBMX

Homo sapiens RNA binding motif

protein, X-linked (RBMX), mRNA.

2.404951
down
MBTPS1

Homo sapiens membrane-bound

transcription factor peptidase, site 1

(MBTPS1), mRNA.

2.395497
down
TMOD1

Homo sapiens tropomodulin 1

(TMOD1), mRNA.

2.394954
down
LOC441013
PREDICTED: Homo sapiens

misc_RNA (LOC441013), miscRNA.

2.394569
down
LOC643531
PREDICTED: Homo sapiens

misc_RNA (LOC643531), miscRNA.

2.385242
down
MIR1978

Homo sapiens microRNA 1978

(MIR1978), microRNA.

2.384901
down
ATN1

Homo sapiens atrophin 1 (ATN1),

transcript variant 1, mRNA.

2.383114
down
FBLN1

Homo sapiens fibulin 1 (FBLN1),

transcript variant A, mRNA.

2.373436
down
GUSBL1

Homo sapiens glucuronidase, beta-like

1 (GUSBL1), non-coding RNA.

2.36863
down
BIN1

Homo sapiens bridging integrator 1

(BIN1), transcript variant 1, mRNA.

2.366588
down
CAMKV

Homo sapiens CaM kinase-like

vesicle-associated (CAMKV), mRNA.

2.366183
down
LOC728658
PREDICTED: Homo sapiens similar

to 23 kD highly basic protein,

transcript variant 1 (LOC728658),

mRNA.

2.363276
down
LOC440349
PREDICTED: Homo sapiens similar

to nuclear pore complex interacting

protein, transcript variant 1

(LOC440349), mRNA.

2.360442
down
HDAC9

Homo sapiens histone deacetylase 9

(HDAC9), transcript variant 3,

mRNA.

2.356692
down
SMA4

Homo sapiens SMA4 (SMA4),

mRNA.

2.346119
down
UNC5A

Homo sapiens unc-5 homolog A

(C. elegans) (UNC5A), mRNA.

2.345477
down
LOC390354
PREDICTED: Homo sapiens similar

to ribosomal protein L18a; 60S

ribosomal protein L18a, transcript

variant 36 (LOC390354), misc RNA.

2.336991
down
UNG

Homo sapiens uracil-DNA

glycosylase (UNG), nuclear gene

encoding mitochondrial protein,

transcript variant 1, mRNA.

2.332026
down
PRMT1

Homo sapiens protein arginine

methyltransferase 1 (PRMT1),

transcript variant 3, mRNA.

2.330827
down
FTL

Homo sapiens ferritin, light

polypeptide (FTL), mRNA.

2.329446
down
3-Sep

Homo sapiens septin 3 (SEPT3),

transcript variant B, mRNA.

2.325651
down
ATCAY

Homo sapiens ataxia, cerebellar,

Cayman type (caytaxin) (ATCAY),

mRNA.

2.322724
down
PYCR1

Homo sapiens pyrroline-5-carboxylate

reductase 1 (PYCR1), transcript

variant 2, mRNA.

2.322228
down
RANBP1

Homo sapiens RAN binding protein 1

(RANBP1), mRNA.

2.320466
down
GNG4

Homo sapiens guanine nucleotide

binding protein (G protein), gamma 4

(GNG4), transcript variant 2, mRNA.

2.317229
down
TAGLN2

Homo sapiens transgelin 2

(TAGLN2), mRNA.

2.313841
down
LOC440157

Homo sapiens hypothetical gene

supported by AK096951; BC066547

(LOC440157), mRNA.

2.30987
down
CUEDC2

Homo sapiens CUE domain

containing 2 (CUEDC2), mRNA.

2.307863
down
NFIX

Homo sapiens nuclear factor I/X

(CCAAT-binding transcription factor)

(NFIX), mRNA.

2.307834
down
TH1L

Homo sapiens TH1-like (Drosophila)

(TH1L), transcript variant 2, mRNA.

2.306791
down

AGENCOURT_14354957

NIH_MGC_191 Homo sapiens cDNA

clone IMAGE: 30413554 5, mRNA

sequence

2.305834
down
SUMO2

Homo sapiens SMT3 suppressor of

mif two 3 homolog 2 (S. cerevisiae)

(SUMO2), transcript variant 2,

mRNA.

2.305034
down
SORL1

Homo sapiens sortilin-related

receptor, L(DLR class) A repeats-

containing (SORL1), mRNA.

2.304166
down
DEAF1
PREDICTED: Homo sapiens

deformed epidermal autoregulatory

factor 1 (Drosophila) (DEAF1),

mRNA.

2.298846
down
LOC92755
PREDICTED: Homo sapiens

misc_RNA (LOC92755), miscRNA.

2.294239
down
CKAP4

Homo sapiens cytoskeleton-associated

protein 4 (CKAP4), mRNA.

2.292942
down
C12orf24

Homo sapiens chromosome 12 open

reading frame 24 (C12orf24), mRNA.

2.28912
down
TUBB4Q

Homo sapiens tubulin, beta

polypeptide 4, member Q (TUBB4Q),

mRNA.

2.286832
down
LOC728139
PREDICTED: Homo sapiens

misc_RNA (LOC728139), miscRNA.

2.286763
down
PRRT2

Homo sapiens pro line-rich

transmembrane protein 2 (PRRT2),

mRNA.

2.28539
down
LOC100130561
PREDICTED: Homo sapiens similar

to high-mobility group (nonhistone

chromosomal) protein 1-like 10,

transcript variant 2 (LOC100130561),

mRNA.

2.284874
down

Homo sapiens mRNA; cDNA

DKFZp686J0156 (from clone

DKFZp686J0156)

2.281905
down
TACC2

Homo sapiens transforming, acidic

coiled-coil containing protein 2

(TACC2), transcript variant 2, mRNA.

2.27488
down
MAP1B

Homo sapiens microtubule-associated

protein 1B (MAP1B), mRNA.

2.273095
down
PKMYT1

Homo sapiens protein kinase,

membrane associated

tyrosine/threonine 1 (PKMYT1),

transcript variant 2, mRNA.

2.272674
down
UCK2

Homo sapiens uridine-cytidine kinase

2 (UCK2), mRNA.

2.272521
down
LOC652489
PREDICTED: Homo sapiens similar

to SMT3 suppressor of mif two 3

homolog 2 (LOC652489), mRNA.

2.271163
down
IRF2BP2

Homo sapiens interferon regulatory

factor 2 binding protein 2 (IRF2BP2),

transcript variant 1, mRNA.

2.270466
down
EEF1D

Homo sapiens eukaryotic translation

elongation factor 1 delta (guanine

nucleotide exchange protein)

(EEF1D), transcript variant 1, mRNA.

2.269964
down
RALY

Homo sapiens RNA binding protein,

autoantigenic (hnRNP-associated with

lethal yellow homolog (mouse))

(RALY), transcript variant 2, mRNA.

2.269945
down
PFKP

Homo sapiens phosphofructokinase,

platelet (PFKP), mRNA.

2.265796
down
CCDC136

Homo sapiens coiled-coil domain

containing 136 (CCDC136), mRNA.

2.262251
down
RNF165

Homo sapiens ring finger protein 165

(RNF165), mRNA.

2.259489
down
NOMO1

Homo sapiens NODAL modulator 1

(NOMO1), mRNA.

2.259268
down
TCF3

Homo sapiens transcription factor 3

(E2A immunoglobulin enhancer

binding factors E12/E47) (TCF3),

mRNA.

2.257809
down
LOC401537
PREDICTED: Homo sapiens

misc_RNA (LOC401537), miscRNA.

2.257748
down
TNPO1

Homo sapiens transportin 1 (TNPO1),

transcript variant 2, mRNA.

2.254448
down
ST8SIA2

Homo sapiens ST8 alpha-N-acetyl-

neuraminide alpha-2,8-

sialyltransferase 2 (ST8SIA2), mRNA.

2.254105
down
STMN2

Homo sapiens stathmin-like 2

(STMN2), mRNA.

2.250269
down
APIP

Homo sapiens APAF1 interacting

protein (APIP), mRNA.

2.243515
down
ATP1A1

Homo sapiens ATPase, Na+/K+

transporting, alpha 1 polypeptide

(ATP1A1), transcript variant 2,

mRNA.

2.241412
down
LOC649150
PREDICTED: Homo sapiens similar

to eukaryotic translation elongation

factor 1 alpha 2 (LOC649150),

mRNA.

2.240263
down
PKD1

Homo sapiens polycystic kidney

disease 1 (autosomal dominant)

(PKD1), transcript variant 1, mRNA.

2.236215
down
LOC643300
PREDICTED: Homo sapiens similar

to 60 kDa heat shock protein,

mitochondrial precursor (Hsp60) (60

kDa chaperonin) (CPN60) (Heat

shock protein 60) (HSP-60)

(Mitochondrial matrix protein P1)

(P60 lymphocyte protein) (HuCHA60)

(LOC643300), mRNA.

2.234636
down
PLOD3

Homo sapiens procollagen-lysine, 2-

oxoglutarate 5-dioxygenase 3

(PLOD3), mRNA.

2.233062
down
SDHA

Homo sapiens succinate

dehydrogenase complex, subunit A,

flavoprotein (Fp) (SDHA), nuclear

gene encoding mitochondrial protein,

mRNA.

2.230375
down
GPX7

Homo sapiens glutathione peroxidase

7 (GPX7), mRNA.

2.226073
down
THOC4
PREDICTED: Homo sapiens THO

complex 4 (THOC4), mRNA.

2.224401
down
PRRX2

Homo sapiens paired related

homeobox 2 (PRRX2), mRNA.

2.224325
down
SGPP2
PREDICTED: Homo sapiens

sphingosine-1-phosphate phosphotase

2 (SGPP2), mRNA.

2.223073
down
APEX1

Homo sapiens APEX nuclease

(multifunctional DNA repair enzyme)

1 (APEX1), transcript variant 1,

mRNA.

2.222308
down
PHF2

Homo sapiens PHD finger protein 2

(PHF2), mRNA.

2.221914
down
CABC1

Homo sapiens chaperone, ABC1

activity of bc1 complex homolog

(S. pombe) (CABC1), nuclear gene

encoding mitochondrial protein,

mRNA.

2.220262
down
LOC100134241
PREDICTED: Homo sapiens

hypothetical protein LOC100134241

(LOC100134241), mRNA.

2.218515
down
LOC732007
PREDICTED: Homo sapiens similar

to Phosphoglycerate mutase 1

(Phosphoglycerate mutase isozyme B)

(PGAM-B) (BPG-dependent PGAM

1) (LOC732007), mRNA.

2.211864
down
CCT6A

Homo sapiens chaperonin containing

TCP1, subunit 6A (zeta 1) (CCT6A),

transcript variant 1, mRNA.

2.208987
down
FTL

Homo sapiens ferritin, light

polypeptide (FTL), mRNA.

2.208881
down
THOC3

Homo sapiens THO complex 3

(THOC3), mRNA.

2.207744
down
PRR7

Homo sapiens proline rich 7 (synaptic)

(PRR7), mRNA.

2.207275
down
MCM2

Homo sapiens minichromosome

maintenance complex component 2

(MCM2), mRNA.

2.203152
down
C9orf86

Homo sapiens chromosome 9 open

reading frame 86 (C9orf86), mRNA.

2.201281
down
CSNK1E

Homo sapiens casein kinase 1, epsilon

(CSNK1E), transcript variant 1,

mRNA.

2.199816
down
MGAT3

Homo sapiens mannosyl (beta-1,4-)-

glycoprotein beta-1,4-N-

acetylglucosaminyltransferase

(MGAT3), transcript variant 1,

mRNA.

2.19858
down
FEZ1

Homo sapiens fasciculation and

elongation protein zeta 1 (zygin I)

(FEZ1), transcript variant 2, mRNA.

2.198326
down
PODXL2

Homo sapiens podocalyxin-like 2

(PODXL2), mRNA.

2.194836
down
ENO2

Homo sapiens enolase 2 (gamma,

neuronal) (ENO2), mRNA.

2.193517
down
LMO3

Homo sapiens LIM domain only 3

(rhombotin-like 2) (LMO3), transcript

variant 1, mRNA.

2.191548
down
WDR5

Homo sapiens WD repeat domain 5

(WDR5), transcript variant 1, mRNA.

2.190587
down
LOC399804
PREDICTED: Homo sapiens

misc_RNA (LOC399804), miscRNA.

2.190034
down
PKM2

Homo sapiens pyruvate kinase, muscle

(PKM2), transcript variant 2, mRNA.

2.18845
down
PLEKHG3

Homo sapiens pleckstrin homology

domain containing, family G (with

RhoGef domain) member 3

(PLEKHG3), mRNA.

2.187572
down
PLD6

Homo sapiens phospholipase D

family, member 6 (PLD6), mRNA.

2.187389
down
B4GALNT4

Homo sapiens beta-1,4-N-acetyl-

galactosaminyl transferase 4

(B4GALNT4), mRNA.

2.185219
down
GUSBL1

Homo sapiens glucuronidase, beta-like

1 (GUSBL1), mRNA.

2.18114
down
PCBP4

Homo sapiens poly(rC) binding

protein 4 (PCBP4), transcript variant

1, mRNA.

2.177992
down
C12orf57

Homo sapiens chromosome 12 open

reading frame 57 (C12orf57), mRNA.

2.17742
down
LOC651198
PREDICTED: Homo sapiens similar

to hCG2036706 (LOC651198),

mRNA.

2.173998
down
GAPDH

Homo sapiens glyceraldehyde-3-

phosphate dehydrogenase (GAPDH),

mRNA.

2.173798
down
LOC402251
PREDICTED: Homo sapiens similar

to eukaryotic translation elongation

factor 1 alpha 2 (LOC402251),

mRNA.

2.169406
down
PALM

Homo sapiens paralemmin (PALM),

transcript variant 1, mRNA.

2.168301
down
PCK2

Homo sapiens phosphoenolpyruvate

carboxykinase 2 (mitochondrial)

(PCK2), nuclear gene encoding

mitochondrial protein, transcript

variant 1, mRNA.

2.167464
down
ACO2

Homo sapiens aconitase 2,

mitochondrial (ACO2), nuclear gene

encoding mitochondrial protein,

mRNA.

2.166496
down
TIAL1

Homo sapiens TIA1 cytotoxic

granule-associated RNA binding

protein-like 1 (TIAL1), transcript

variant 2, mRNA.

2.165684
down
PTPRD

Homo sapiens protein tyrosine

phosphatase, receptor type, D

(PTPRD), transcript variant 2, mRNA.

2.165212
down
MARCKSL1

Homo sapiens MARCKS-like 1

(MARCKSL1), mRNA.

2.163887
down
3-Sep

Homo sapiens septin 3 (SEPT3),

transcript variant B, mRNA.

2.161171
down
PISD

Homo sapiens phosphatidylserine

decarboxylase (PISD), mRNA.

2.160368
down
PTK7

Homo sapiens PTK7 protein tyrosine

kinase 7 (PTK7), transcript variant

PTK7-4, mRNA.

2.157614
down
FAF1

Homo sapiens Fas (TNFRSF6)

associated factor 1 (FAF1), mRNA.

2.15675
down
SLC35F3
PREDICTED: Homo sapiens solute

carrier family 35, member F3

(SLC35F3), mRNA.

2.156618
down
H2AFX

Homo sapiens H2A histone family,

member X (H2AFX), mRNA.

2.156176
down
GNL3

Homo sapiens guanine nucleotide

binding protein-like 3 (nucleolar)

(GNL3), transcript variant 3, mRNA.

2.153096
down
FAM57B

Homo sapiens family with sequence

similarity 57, member B (FAM57B),

mRNA.

2.151984
down
CDK5R1

Homo sapiens cyclin-dependent

kinase 5, regulatory subunit 1 (p35)

(CDK5R1), mRNA.

2.150896
down
TNIP1

Homo sapiens TNFAIP3 interacting

protein 1 (TNIP1), mRNA.

2.150337
down
EEF1D

Homo sapiens eukaryotic translation

elongation factor 1 delta (guanine

nucleotide exchange protein)

(EEF1D), transcript variant 1, mRNA.

2.149941
down
TRPC4AP

Homo sapiens transient receptor

potential cation channel, subfamily C,

member 4 associated protein

(TRPC4AP), transcript variant 1,

mRNA.

2.147648
down
RAD51AP1

Homo sapiens RAD51 associated

protein 1 (RAD51AP1), mRNA.

2.144922
down
PSCD1

Homo sapiens pleckstrin homology,

Sec7 and coiled-coil domains

1(cytohesin 1) (PSCD1), transcript

variant 2, mRNA.

2.141083
down
RELN

Homo sapiens reelin (RELN),

transcript variant 2, mRNA.

2.139602
down
SIGMAR1

Homo sapiens sigma non-opioid

intracellular receptor 1 (SIGMAR1),

transcript variant 1, mRNA.

2.135113
down
STXBP1

Homo sapiens syntaxin binding

protein 1 (STXBP1), transcript variant

2, mRNA.

2.134394
down
LOC643873
PREDICTED: Homo sapiens

misc_RNA (LOC643873), miscRNA.

2.134312
down
SKP2

Homo sapiens S-phase kinase-

associated protein 2 (p45) (SKP2),

transcript variant 1, mRNA.

2.131977
down
HNRPK

Homo sapiens heterogeneous nuclear

ribonucleoprotein K (HNRPK),

transcript variant 3, mRNA.

2.131822
down
FEZ1

Homo sapiens fasciculation and

elongation protein zeta 1 (zygin I)

(FEZ1), transcript variant 1, mRNA.

2.129834
down
HNRNPL

Homo sapiens heterogeneous nuclear

ribonucleoprotein L (HNRNPL),

transcript variant 2, mRNA.

2.129226
down
ADM

Homo sapiens adrenomedullin

(ADM), mRNA.

2.126514
down
DBNDD2

Homo sapiens dysbindin (dystrobrevin

binding protein 1) domain containing

2 (DBNDD2), transcript variant 3,

mRNA.

2.125708
down
LOC643668
PREDICTED: Homo sapiens similar

to peptidase (prosome, macropain)

26S subunit, ATPase 1 (LOC643668),

mRNA.

2.125221
down
NGFRAP1

Homo sapiens nerve growth factor

receptor (TNFRSF16) associated

protein 1 (NGFRAP1), transcript

variant 3, mRNA.

2.124531
down
FOXK1

Homo sapiens forkhead box K1

(FOXK1), mRNA.

2.124004
down
CENTG3

Homo sapiens centaurin, gamma 3

(CENTG3), mRNA.

2.123406
down
NME3

Homo sapiens non-metastatic cells 3,

protein expressed in (NME3), mRNA.

2.12233
down
EIF4A1

Homo sapiens eukaryotic translation

initiation factor 4A, isoform 1

(EIF4A1), mRNA.

2.121922
down
LOC100131735
PREDICTED: Homo sapiens

misc_RNA (LOC100131735),

miscRNA.

2.120917
down
SAC3D1

Homo sapiens SAC3 domain

containing 1 (SAC3D1), mRNA.

2.120508
down
LOC100134364
PREDICTED: Homo sapiens

hypothetical protein LOC100134364

(LOC100134364), mRNA.

2.119961
down
TMSB10

Homo sapiens thymosin beta 10

(TMSB10), mRNA.

2.119585
down
IDH2

Homo sapiens isocitrate

dehydrogenase 2 (NADP+),

mitochondrial (IDH2), nuclear gene

encoding mitochondrial protein,

mRNA.

2.117743
down
DPM3

Homo sapiens dolichyl-phosphate

mannosyltransferase polypeptide 3

(DPM3), transcript variant 2, mRNA.

2.117534
down
PRKCZ

Homo sapiens protein kinase C, zeta

(PRKCZ), transcript variant 1, mRNA.

2.117004
down
EIF4H

Homo sapiens eukaryotic translation

initiation factor 4H (EIF4H), transcript

variant 2, mRNA.

2.115663
down
GAS6

Homo sapiens growth arrest-specific 6

(GAS6), mRNA.

2.115582
down
NHP2

Homo sapiens NHP2

ribonucleoprotein homolog (yeast)

(NHP2), transcript variant 1, mRNA.

2.110555
down
CNTFR

Homo sapiens ciliary neurotrophic

factor receptor (CNTFR), transcript

variant 2, mRNA.

2.104631
down
LOC440927
PREDICTED: Homo sapiens similar

to 60S acidic ribosomal protein P1,

transcript variant 4 (LOC440927),

mRNA.

2.10323
down
LOC286444
PREDICTED: Homo sapiens

misc_RNA (LOC286444), miscRNA.

2.103015
down
LOC100133840
PREDICTED: Homo sapiens similar

to hCG1994151 (LOC100133840),

mRNA.

2.102011
down
TSC22D3

Homo sapiens TSC22 domain family,

member 3 (TSC22D3), transcript

variant 1, mRNA.

2.101288
down

Homo sapiens mRNA; cDNA

DKFZp686E0389 (from clone

DKFZp686E0389)

2.096581
down
KIAA0195

Homo sapiens KIAA0195

(KIAA0195), mRNA.

2.095432
down
LOC728873
PREDICTED: Homo sapiens

misc_RNA (LOC728873), miscRNA.

2.094849
down
BIN1

Homo sapiens bridging integrator 1

(BIN1), transcript variant 4, mRNA.

2.088547
down
RSL1D1

Homo sapiens ribosomal L1 domain

containing 1 (RSL1D1), mRNA.

2.08493
down
N4BP2L1

Homo sapiens NEDD4 binding

protein 2-like 1 (N4BP2L1), transcript

variant 2, mRNA.

2.079067
down
NIPSNAP1

Homo sapiens nipsnap homolog 1

(C. elegans) (NIPSNAP1), mRNA.

2.078163
down
GPSM1
PREDICTED: Homo sapiens G-

protein signalling modulator 1 (AGS3-

like, C. elegans) (GPSM1), mRNA.

2.077028
down
COLEC11

Homo sapiens collectin sub-family

member 11 (COLEC11), transcript

variant 2, mRNA.

2.074221
down
TNC

Homo sapiens tenascin C

(hexabrachion) (TNC), mRNA.

2.073396
down
LOC100129585
PREDICTED: Homo sapiens similar

to hCG2011544 (LOC100129585),

mRNA.

2.071684
down
NDUFV1

Homo sapiens NADH dehydrogenase

(ubiquinone) flavoprotein 1, 51 kDa

(NDUFV1), mRNA.

2.068496
down
TPT1

Homo sapiens tumor protein,

translationally-controlled 1 (TPT1),

mRNA.

2.067051
down
ZNF423

Homo sapiens zinc finger protein 423

(ZNF423), mRNA.

2.066952
down
UCKL1

Homo sapiens uridine-cytidine kinase

1-like 1 (UCKL1), mRNA.

2.066395
down
MDK

Homo sapiens midkine (neurite

growth-promoting factor 2) (MDK),

transcript variant 1, mRNA.

2.065846
down
TIGA1

Homo sapiens TIGA1 (TIGA1),

mRNA.

2.064929
down
LOC727761
PREDICTED: Homo sapiens similar

to Deoxythymidylate kinase

(thymidylate kinase), transcript variant

4 (LOC727761), mRNA.

2.064799
down
FAM125B

Homo sapiens family with sequence

similarity 125, member B

(FAM125B), transcript variant 1,

mRNA.

2.064142
down
LOC157627

Homo sapiens hypothetical

LOC157627 (LOC157627), non-

coding RNA.

2.063894
down
SDC1

Homo sapiens syndecan 1 (SDC1),

transcript variant 1, mRNA.

2.063298
down
SLC10A4

Homo sapiens solute carrier family 10

(sodium/bile acid cotransporter

family), member 4 (SLC10A4),

mRNA.

2.062089
down
SCAMP5

Homo sapiens secretory carrier

membrane protein 5 (SCAMP5),

mRNA.

2.061974
down
DAPK1

Homo sapiens death-associated

protein kinase 1 (DAPK1), mRNA.

2.060575
down
LOC389141
PREDICTED: Homo sapiens

misc_RNA (LOC389141), miscRNA.

2.055651
down
HRK

Homo sapiens harakiri, BCL2

interacting protein (contains only BH3

domain) (HRK), mRNA.

2.053187
down
LOC100132060
PREDICTED: Homo sapiens

hypothetical protein LOC100132060

(LOC100132060), mRNA.

2.053184
down
PNMA3

Homo sapiens paraneoplastic antigen

MA3 (PNMA3), mRNA.

2.051588
down
DYRK2

Homo sapiens dual-specificity

tyro sine-(Y)-phosphorylation

regulated kinase 2 (DYRK2),

transcript variant 1, mRNA.

2.051375
down
MRPS24

Homo sapiens mitochondrial

ribosomal protein S24 (MRPS24),

nuclear gene encoding mitochondrial

protein, mRNA.

2.051081
down
LOC648927
PREDICTED: Homo sapiens

misc_RNA (LOC648927), miscRNA.

2.050558
down
FRZB

Homo sapiens frizzled-related protein

(FRZB), mRNA.

2.049056
down
KLF11
PREDICTED: Homo sapiens Kruppel-

like factor 11 (KLF11), mRNA.

2.048945
down
LOC644237
PREDICTED: Homo sapiens

misc_RNA (LOC644237), miscRNA.

2.047743
down
LOC648024
PREDICTED: Homo sapiens similar

to eukaryotic translation initiation

factor 4A, isoform 1 (LOC648024),

mRNA.

2.04652
down
TNRC4

Homo sapiens trinucleotide repeat

containing 4 (TNRC4), mRNA.

2.045708
down
HNRNPK

Homo sapiens heterogeneous nuclear

ribonucleoprotein K (HNRNPK),

transcript variant 2, mRNA.

2.045596
down
CALD1

Homo sapiens caldesmon 1 (CALD1),

transcript variant 3, mRNA.

2.041429
down
PWWP2B

Homo sapiens PWWP domain

containing 2B (PWWP2B), transcript

variant 1, mRNA.

2.041209
down
WDR45L

Homo sapiens WDR45-like

(WDR45L), mRNA.

2.040227
down
LOC440595
PREDICTED: Homo sapiens

misc_RNA (LOC440595), miscRNA.

2.039603
down
HDAC9

Homo sapiens histone deacetylase 9

(HDAC9), transcript variant 5,

mRNA.

2.038931
down
TRIM28

Homo sapiens tripartite motif-

containing 28 (TRIM28), mRNA.

2.036902
down
ADAR

Homo sapiens adenosine deaminase,

RNA-specific (ADAR), transcript

variant 2, mRNA.

2.033741
down
TMEM101

Homo sapiens transmembrane protein

101 (TMEM101), mRNA.

2.032995
down
PEG10
PREDICTED: Homo sapiens

paternally expressed 10 (PEG10),

mRNA.

2.032149
down
HNRNPA3

Homo sapiens heterogeneous nuclear

ribonucleoprotein A3 (HNRNPA3),

mRNA.

2.031689
down
LOC100134648
PREDICTED: Homo sapiens similar

to hCG2024106, transcript variant 2

(LOC100134648), mRNA.

2.029636
down
LOC728411
PREDICTED: Homo sapiens similar

to Beta-glucuronidase precursor

(LOC728411), mRNA.

2.029015
down
GAPDH

Homo sapiens glyceraldehyde-3-

phosphate dehydrogenase (GAPDH),

mRNA.

2.028673
down
GRIA4

Homo sapiens glutamate receptor,

ionotrophic, AMPA 4 (GRIA4),

transcript variant 3, mRNA.

2.022971
down
CACNA1H

Homo sapiens calcium channel,

voltage-dependent, T type, alpha 1H

subunit (CACNA1H), transcript

variant 2, mRNA.

2.020432
down
SNHG3-RCC1

Homo sapiens SNHG3-RCC1

readthrough transcript (SNHG3-

RCC1), transcript variant 1, mRNA.

2.018636
down
EEF1A1

Homo sapiens eukaryotic translation

elongation factor 1 alpha 1 (EEF1A1),

mRNA.

2.018083
down
SLC4A2

Homo sapiens solute carrier family 4,

anion exchanger, member 2

(erythrocyte membrane protein band

3-like 1) (SLC4A2), mRNA.

2.015969
down
TUBB3

Homo sapiens tubulin, beta 3

(TUBB3), mRNA.

2.01536
down
PIM1

Homo sapiens pim-1 oncogene

(PIM1), mRNA.

2.014172
down
ZNRD1

Homo sapiens zinc ribbon domain

containing 1 (ZNRD1), transcript

variant a, mRNA.

2.013845
down
ZNF536

Homo sapiens zinc finger protein 536

(ZNF536), mRNA.

2.011526
down
RPL13A

Homo sapiens ribosomal protein L13a

(RPL13A), mRNA.

2.010711
down
DBNDD1

Homo sapiens dysbindin (dystrobrevin

binding protein 1) domain containing

1 (DBNDD1), transcript variant 1,

mRNA.

2.009193
down
TXNDC5

Homo sapiens thioredoxin domain

containing 5 (TXNDC5), transcript

variant 2, mRNA.

2.007024
down
PDZD4

Homo sapiens PDZ domain containing

4 (PDZD4), mRNA.

2.006589
down
SLC27A3

Homo sapiens solute carrier family 27

(fatty acid transporter), member 3

(SLC27A3), mRNA.

2.001083
down
RPL12

Homo sapiens ribosomal protein L12

(RPL12), mRNA.

Example 15
ATRA Modulates Binding Positions of Retinoic Acid Receptor (RAR) to the Chromatin and is Enhanced by Compound B (FIG. 13)

Retinoic acid receptor active binding sites defined in any individual treatment group by ChIP-seq at 48 hrs after treatment were stacked (y-axis) and aligned to the center of the binding peak (x-axis) (FIG. 13). ATRA treatment caused increased RAR binding (regions 1-4), which was further enhanced by HDAC1/2i across a large proportion of sites (region 1). Treatment also caused RAR binding to decrease (regions 5-7), with potent effects observed in the Compound B single agent group (region 6). Many of the genes found near region 1 binding sites are involved in regulation of retinoid signaling and are transcription factors that drive differentiation (Table 10). Pathway analysis of transcription factors near region 1 retinoic acid receptor binding sites suggest relevant pathways in neuroblastoma differentiation that might be activated (Table 11).

Regions 1-7 of FIG. 13 are defined relative to DMSO: 1, [Combo] Increased; 2, [ATRA] Increased; 3, [ATRA] [Combo] Increased; 4, [Combo] [ATRA] [Compound B] Increased; 5, [Combo] [ATRA] Decreased; 6, [Compound B] Decreased; and 7, [Compound B] [Combo] [ATRA] Decreased.

Treatment with HDAC1/2i+RA enhanced RA-induced expression of the RARβ gene (FIG. 21) and increased RARβ protein levels (FIG. 22). Further, RAR binding to the RARβ gene promoter was enhanced in the combination setting relative to either Compound B or RA alone (FIG. 23).

HDAC1/2i inhibits RA-inducible regulators of RAR signaling. Retinoic acid treatment induces a negative feedback loop that regulates RAR signaling. Cyp26b1 and DHRS3, proteins that are induced by RA and negatively regulate RAR signaling, were decreased in the combination setting as measured by gene expression and protein levels (FIG. 24 and FIG. 25).

TABLE 10

Selected Region 1 (FIG. 13) Genes Involved in RA Signaling

Gene Name
Description

HOXA,
A group of related genes that control the body plan of an

HOXB,
embryo along the anterior-posterior (head-tail) axis.

HOXC,
Coordinated activation is required for differentiation.

HOXD

NCOA2,
NCoA is a transcriptional coregulatory protein that contains

NCOA3
several nuclear receptor interacting domains and an intrinsic

histone acetyltransferase activity.

NCOA2 is recruited to DNA promotion sites by ligand-

activated nuclear receptors. NCOA2 in turn acetylates

histones, which makes downstream DNA more accessible

to transcription.

RARA,
Retinoic acid receptor isoforms that are activated by

RARB
retinoic acid

CYP26A1,
cytochrome P450 superfamily of enzymes, which catalyze

B1, C1
many reactions involved in drug metabolism and synthesis

of cholesterol, steroids and other lipids including

retinoids.

HDAC3
Histone deacetylase 3, a component of the Ncor repressive

complex known to interact with RAR, repressing RA

signaling

TABLE 11

Pathways that Overlap with Region 1 (FIG. 13) Transcription Factors

Gene Set Name

# Genes In

FDR

[# Genes (K)]
Description
Overlap (k)
k/K
p-value
q-value

HALLMARK_TNFA_ SIGNALING_VIA_NFKB [200]
Genes regulated by NF-kB in response to TNF [GeneID = 7124].
13

embedded image

1.19 e⁻⁹
5.93 e⁻⁸

HALLMARK_G2M_ CHECKPOINT [200]
Genes involved in the G2/M checkpoint, as in progression through the cell division cycle.
10

embedded image

1.12 e⁻⁶
1.87 e⁻⁵

HALLMARK_TGF_BETA_ SIGNALING [54]
Genes up-regulated in response to TGFB1 [GeneID = 7040].
6

embedded image

1.67 e⁻⁶
2.09 e⁻⁵

HALLMARK_HEDGEHOG_ SIGNALING [36]
Genes up-regulated by activation of hedgehog signaling.
5

embedded image

4.1 e⁻⁶
4.1 e⁻⁵

HALLMARK_P53_PATHWAY [200]
Genes involved in p53 pathways and networks.
7

embedded image

4.23 e⁻⁴
1.82 e⁻³

Additional RAR binding occurs after ATRA is applied. Unexpectedly, combination of ATRA and Compound B increases RAR binding sites relative to ATRA alone. Further, Compound B can reduce RAR binding as a single agent.

ChIP-Seq Experimental Design

Neuroblastoma cell line SK-N-BE(2) cells were treated with 3 μM Compound B, 1 μM ATRA, or a combination of both at 37° C. over 48 hours and compared to the solvent (DMSO) control. Antibodies to pan-retinoic acid receptor were used to pull down DNA associated with the receptor binding and sequenced. Lists of binding regions were mapped to the chromosome and “associated” with a given gene if the binding region was located within 10,000 bp up- or down-stream of the gene in question. Binding regions were represented as a heatmap where the sites were stacked on the y-axis across treatments, the “0” mark on the x-axis is the center of the binding peak and the intensity of signal indicated by color. The bracketed regions are areas of statistical significance relative to the DMSO control as indicated on the plot.

Example 16
Integration of RAR ChIP-Seq and Microarray Data Reveal Potential Drivers of HDACi Enhancement of Retinoid Activity

Table 12 lists the genes returned when the following conditions are met: 1) combo peak height >4 fold relative to the DMSO control group, and 2) combo expression >4 fold relative to the DMSO control group. RAR ChIP-seq and microarray data (48 hr) was queried to identify a list of genes near RAR binding sites that 1) showed enhanced RAR-chromatin interactions and 2) increased gene expression in the combination setting. Functional sorting suggests three key processes are activated: 1) RA metabolism, 2) RA signaling, and 3) kinase signaling.

TABLE 12

Published

RAR Binding
Gene Expression Change

RAR
Known Functions
(Peak Height)
Cmpd. B
ATRA
Combo

CYP26A1
Yes
RA Metabolism
RA
2.33
7.00
8.67
0.99
7.84
8.18

CYP26B1
Yes
RA Metabolism
Processing
1.50
11.50
10.00
1.87
116.67
77.45

DHRS3
Yes
RA Metabolism

1.00
7.50
5.50
1.03
24.14
5.60

CRABP2
Yes
RA Transport to the Nucleus
RA
0.69
3.15
4.23
1.18
12.15
9.49

RARB
Yes
RAR beta Isoform
Signaling
1.10
2.20
4.90
1.14
3.91
5.64

PTGER2
—
RA/ERK1/2 Signaling

0.75
2.13
4.50
1.41
2.48
4.73

ETS1
Yes
ERK Signaling
Potential
3.35
3.99
6.26
3.00
3.50
8.50

IER3
Yes
ERK Signaling
Non-Genomic
2.67
6.83
8.67
2.33
3.75
8.81

RET
Yes
AKT Signaling
RAR
1.71
8.86
10.57
1.34
10.78
11.62

NFKBIZ
Yes
AKT/MAPK Regulation
Signaling
3.33
4.00
14.67
1.95
2.85
6.33

DUSP6
Yes
ERK Regulation

3.00
4.00
6.00
3.33
8.59
9.96

CDKN1A
Yes
p21 master cell cycle regulator
Potential
0.50
4.75
5.00
1.93
1.25
5.86

PCDH18
Yes
Cellular Adhesion
Phenotype
4.00
3.50
7.00
2.53
3.98
4.37

CTSH
Yes
Lysosomal Function
Drivers
0.83
5.42
4.33
3.05
2.40
5.80

ATP7A
Yes
Copper Metal Homeostasis

2.20
4.60
12.40
1.98
4.30
6.80

HSPA5
—
Protein Synthesis

2.00
2.00
4.86
3.78
1.48
4.55

ACSL3
Yes
Metabolic Processes

2.00
1.00
9.00
2.23
1.68
4.63

Example 17
Model for HDACi Enhancement of RA-Mediated Differentiation (FIG. 14)

A model for HDAC1/2i contribution to retinoid-induced differentiation has emerged from analysis of RAR ChIP-seq and microarray studies (FIG. 14A). The proposed model captures key signaling routes, including the Wnt, RTK and SHH pathways.

Classical metrics of differentiation induced by ATRA are enhanced by HDAC1/2i, which include reduced proliferation, cell cycle effects and dendrite outgrowth. HDAC1/2i has direct anti-tumor effects that are retinoid independent. Gene expression changes consistent with differentiation are induced by HDAC1/2i and are enhanced in combination with retinoic acid. HDAC1/2i modulates RAR interactions with the chromatin near key genes involved in differentiation and cell growth, metabolism and survival.

FIG. 14B represents a proposed model for how HDAC1/2i impacts retinoic acid induced differentiation of neuroblastoma. RA signaling is reinforced through a positive feedback cycle due to an increase in the retinoic acid receptor. Simultaneously, enzymes that limit the intracellular pool of RA are also increased, negatively regulating RA signaling. The homeostatic balance is perturbed by HDAC1/2i+RA combination, resulting in enhanced RAR signaling, decreased levels of enzymes that reduce cellular RA, and slower proliferation by modulating cyclins and inducing p21 while also inducing caspase cleavage. The net result is enhanced differentiation, decreased proliferation, and neuroblastoma cell death

Example 18
HDAC1/2 Inhibitors in Combination with ATRA Disrupt the Wnt Signaling Pathway (FIG. 15)

The levels of Wnt-related signaling molecules were assessed following treatment (SK-N-BE2 cells; 3 days in culture) with DMSO (control), Compound B alone, ATRA alone, and Compound B in combination with ATRA. b-catenin, the key signaling molecule in the Wnt pathway, is decreased by the Compound B and ATRA combination. Activated p-LRP is decreased after Compound B single agent treatment. Disheveled (Dvl) 2 & 3 is reduced by Compound B and ATRA combination treatment. Naked2 is increased by Compound B as a single agent and in combination with ATRA. Axin1 is decreased by Compound B as a single agent and in combination with ATRA. Taken together, Compound B in combination with ATRA reduces Wnt signaling.

Example 19
Retinoic Acid-Activated AKT is Reduced by HDAC1/2i (FIG. 16)

The levels of AKT and c-RAF were assessed following treatment (SK-N-BE2 cells; 3 days in culture) with DMSO (control), Compound B alone, ATRA alone, and Compound B in combination with ATRA. Activated AKT is increased by ATRA and reduced in the combination setting with Compound B. Compound B as a single agent, and in combination with ATRA, decreases cRAF phosphorylation at residue Ser259.

Example 20
Modulation of Cell Cycle Progression Through G1 (FIG. 17)

The levels of proteins related to cell-cycle progression were assessed following treatment (SK-N-BE2 cells; 3 days in culture) with DMSO (control), Compound B alone, ATRA alone, and Compound B in combination with ATRA. p21 is increased by ATRA and enhanced by Compound B. Cyclin D1 is decreased in the combination setting relative to ATRA as a single agent. CDK2 and CDK4 are decreased in the combination setting with Compound B and ATRA.

As described herein, the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on neuroblastoma (NB) cell differentiation, proliferation and apoptosis was examined RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by microarray and retinoic acid receptor (RAR) ChIP-seq. HDAC1/2i and RA together caused increased localization of the RAR to its own RARα and RARβ promoter regions, and an increase in RAR mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26a1/b1, enzymes responsible for clearing intercellular RA, were reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i was enhancing apoptotic pathways and decreasing E2F driven cell cycle signaling.

In further experiments, enhanced apoptosis was confirmed in the combination setting by measuring caspase 3 and PARP cleavage, which is consistent with reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies. Further, the E2F-activators, CDK4 and CDK6, were reduced at the protein level in the combination setting while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB.

Example 21
HDAC1/2i Slows Neuroblastoma Tumor Growth (FIG. 26)

IMR-32 tumors were implanted in NUDE mice and Compound E and retinoic acid was administered orally once daily at the indicated doses on a 5/2 on/off schedule. Compound E treatment resulted in a dose-dependent trend toward reduced tumor growth, with an enhanced effect at the 100 mg/kg dosing group observed in combination with ATRA.

Example 22
Compound a and Compound B in Combination with ATRA Results in Synergistic Acute Toxicity Against Neuroblastoma Cells (FIGS. 27 and 28)

Neuroblastoma cells were treated with the indicated compounds, i.e., ATRA, Compound A, and Compound B, in a dose matrix. Viable cells were measured in an MTS assay and the combination index (CI) was calculated using the Chou-Talaay method. Any combinations where a CI value less than 1 is observed indicates a synergistic combination. These data illustrate the indicated compounds combine to induce synergistic neuroblastoma cell death. FIGS. 27A-D show the data obtained for Compound B plus ATRA and FIGS. 28A-D show the data obtained for Compound A plus ATRA.

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

	Number	Date	Country
	62250638	Nov 2015	US
	62148851	Apr 2015	US

TREATMENT OF NEUROBLASTOMA WITH HISTONE DEACETYLASE INHIBITORS

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