Abstract 4689: Targeting constitutive GLI activation in colon carcinomas and cancers with oncogenic KRAS signaling
Authors: Tapati Mazumdar, Babal K. Jha, Akwasi Agyeman, Janet A. Houghton
Journal: Cancer Research (2015)

Abstract The GLI genes, GLI1 and GLI2, encode transcription factors that regulate target genes at the distal end of the canonical Hedgehog signaling (HH) pathway (SHH-&amp;gt;PTCH-&amp;gt;SMO-&amp;gt;GLI), tightly regulated in embryonic development, tissue patterning and differentiation. In cancers, both GLI1 and GLI2 are oncogenes, aberrantly and constitutively activated. Oncogene-driven signaling pathways, in particular KRAS/BRAF in colon cancer, circumvent the HH-GLI axis to further drive GLI activation, which serves as a nodal channel for oncogenic signals, and provides a pivotal role for GLI in cell survival. GANT61, a small molecule inhibitor of GLI-dependent transcription, terminates oncogenic HH-GLI and KRAS/BRAF-GLI signaling, inducing DNA damage and extensive cell death in seven human colon carcinoma cell line models examined. The overall goal is to understand how GANT61 induces cell death by inhibiting GLI-dependent transcription mechanisms. We have demonstrated that GANT61 is: a) specific for binding to GLI, and does not bind to DNA or to other transcription factors; b) selective for cancer cells vs normal cells that lack constitutive GLI activation. Inhibition of GLI1 and GLI2 by GANT61 rapidly reduces GLI binding to consensus sequences in target gene promoters and inhibits GLI-dependent transcription. Stalling of Pol II occurs adjacent to GLI binding regions at target gene promoters (FOXM1, BCL-2), associated with inhibition of GLI binding, enrichment of Pol II, and modification of promoter-bound pause (DSIF, NELF) or pause-release (P-TEFb) factors within a 300bp region of the FOXM1 promoter. Single-stranded DNA and RNA:DNA hybrids determining R-loop formation (distributed throughout the nucleoplasm) occur at sites of stalled Pol II, inhibited by RNAseH. Consistent with transcriptional inhibition, GANT61 induced DNA damage (γH2AX foci) in S-phase and non-S-phase cells, recognized at the initiation of S-phase (G1/S), prior to the onset of cell death. This may be due to one of two mechanisms: 1) The GLI1 target gene FOXM1, and its transcriptional target CDC6, downregulated in GANT61-treated cells, regulate Pre-Replication Complex assembly at G1/S thereby inhibiting the initiation of DNA replication; this controls outcome of the GANT61 response, dependent on GLI; 2) Transcriptional R-loop formation can conflict with DNA replication in GANT61-treated cells. These events inhibit initiation of DNA replication from unfired origins at early replicons and inhibit further progression through S-phase. GLI1 and/or GLI2 are constitutively activated in epithelial cancers of the GI tract, brain tumors, melanoma, pediatric solid tumors, liver, lung, breast, pancreatic and prostate cancers. Further, KRAS is mutated in one third of all human cancers and in 50% of colon carcinomas. Targeting GLI therefore has the potential for high impact in therapeutics, and is anticipated to lead to new approaches and therapeutic strategies for treating cancer. Citation Format: Tapati Mazumdar, Babal K. Jha, Akwasi Agyeman, Janet A. Houghton. Targeting constitutive GLI activation in colon carcinomas and cancers with oncogenic KRAS signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4689. doi:10.1158/1538-7445.AM2015-4689