Abstract 5495: GANT61-induced cell death involves inhibition of transcription and DNA licensing at GLI sites in the promoters of GLI-dependent target genes in human colon carcinoma cells

Abstract

Abstract The GLI genes, GLI1 and GLI2, are transcriptional regulators of the Hedgehog signaling response, binding at promoters to GACCACCCA-like consensus sequences. From genetic and biochemical studies, GLI2 is the primary mediator of HH signaling, which activates GLI1 to transcriptionally regulate target genes. In cancers both GLI1 and GLI2 are oncogenes, aberrantly and constitutively activated by oncogene-driven signaling pathways, in particular KRAS/BRAF in colon cancer. GANT61, a specific GLI inhibitor, induced extensive cytotoxicity in human models of colon cancer, indicating GLI to be a critical target in cancer cell survival. We have determined FOXM1 to be a transcriptional target of GLI1 at a GCCCACCCA consensus sequence. In HT29 cells, inhibition of GLI binding to the FOXM1 promoter by GANT61 led to inhibition of binding of the pause-release factors DSIF, NELF, and p-TEFb in the region of the TSS, with inhibition of binding of Pol II at both GLI and TSS sites. In R-loop regions, RNA:DNA hybrid formation was reduced at both the GLI and TSS sites in the FOXM1 promoter by GANT61, sensitive to RNaseH. Bisulfite conversion/PCR also determined >50% reduction in ssDNA (C->T conversions) in the GLI binding region in GANT61-treated cells. Data support GANT61-induced inhibition of GLI-dependent transcription at the PIC before R-loops are formed. Pretreatment of HT29 cells with α-amanitin (Pol II inhibitor) reduced GANT61-induced γH2AX foci, indicating the importance of transcription in GANT61-induced DNA damage. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication were linked. By co-immunoprecipitation, GLI1 bound the DNA replication licensing factors ORC4, CDT1, and MCM2, decreased in the presence of GANT61. By confocal microscopy, significant co-localization of GLI1 and ORC4 foci was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. In addition to non-transcriptional mechanisms of interaction between GLI1 and DNA licensing factors, CDT1 was found to be a transcription target of GLI1 at a putative GACCACCCG consensus sequence in the promoter. ChIP analysis determined significant enrichment of γH2AX at this site in GANT61-treated HT29 cells. Overexpression of CDT1 in HT29 cells reduced GANT61-induced cell death and cleavage of caspase-3, indicating a significant role of DNA replication licensing in the mechanism of induction of GANT61-induced cell death. In summary, we have demonstrated inhibition of GLI-dependent transcription by GANT61 at the PIC during the initiation of RNA synthesis, and the importance of inhibiting DNA replication licensing at sites in promoter regions that bind the transcription factor GLI1 by both non-transcriptional and transcriptional mechanisms. Supported by NCI Award 1 RO1 CA183921-01A1 and by Southern Research Institute Citation Format: Ruowen Zhang, Jiahui Wu, Sylvain Ferrandon, Katie J. Glowacki, Janet A. Houghton. GANT61-induced cell death involves inhibition of transcription and DNA licensing at GLI sites in the promoters of GLI-dependent target genes in human colon carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5495. doi:10.1158/1538-7445.AM2017-5495