title: Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing.
abstract: The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.
authors.affiliation: Department of Oncology, Division of Drug Discovery, Southern Research, Birmingham, AL, USA.
authors.forename: Ruowen
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
authors.forename: Jiahui
authors.initials: J
authors.lastname: Wu
authors.affiliation: Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Ferrandon
authors.affiliation: Department of Oncology, Division of Drug Discovery, Southern Research, Birmingham, AL, USA.
authors.forename: Katie J
authors.initials: KJ
authors.lastname: Glowacki
authors.affiliation: Department of Oncology, Division of Drug Discovery, Southern Research, Birmingham, AL, USA.
authors.forename: Janet A
authors.initials: JA
authors.lastname: Houghton
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D004261:DNA Replication / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D000072278:Forkhead Box Protein M1 / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D019073:HT29 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2 / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011725:Pyridines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000071676:Zinc Finger Protein GLI1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; C107585:FOXM1 protein, human; D000072278:Forkhead Box Protein M1; C551027:GANT 61; C400551:GLI1 protein, human; C468783:H2AX protein, human; D006657:Histones; C411985:NSMF protein, human; D009687:Nuclear Proteins; C109152:ORC4 protein, human; D051738:Origin Recognition Complex; D011725:Pyridines; D011743:Pyrimidines; C112224:SUPT5H protein, human; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D000071676:Zinc Finger Protein GLI1; D012319:RNA Polymerase II; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2
keywords: DNA licensing; GANT61; GLI; transcription