Abstract 1759: Targeting GLI-dependent transcription by GANT61 in human colon carcinoma cells (CC); a new therapeutic approach
Authors: Agyeman Akwasi , Jha Babal K. , Mazumder Tapati , Houghton Janet A. 
Published: May 2015

Colorectal cancer constitutes the second leading cause of cancer mortality in the United States with limited therapeutic options and poor survival for advanced stage disease. The GLI genes, GLI1 and GLI2, are transcription factors, which act at the distal end of the Hedgehog pathway (SHH-&gt;PTCH-&gt;SMO-&gt;GLI) to regulate organogenesis and differentiation in the colon. In cancer GLI1 and GLI2 are activated, are oncogenes, and we have shown are critical to the survival of colon carcinoma cells. Inhibition of GLI by the small molecule inhibitor, GANT61, induces extensive cell death downstream of γH2AX foci formation in CC. GANT61 rapidly reduces GLI binding to consensus sequences in target gene promoters (1 hr) and inhibits GLI1- and GLI2- dependent transcription. Using the known crystal structure of the five zinc finger GLI1-DNA complex we demonstrated, by computational docking, that GANT61 binds to the GLI1 protein, but not to the DNA binding site, via H bonds at amino acids E119 and E167. These sites lie within the groove between zinc fingers 2 and 3, on the opposite surface from, but in close proximity to, the GLI1-DNA binding regions. GANT61 is not observed to dock with DNA. The predicted binding constant for GANT61 (Kd = 2.6 μM) is consistent with GANT61 concentrations required to induce cell death in CC. Surface plasmon resonance (Biacore) confirmed the model predictions using immobilized GLI1 or DNA and GANT61 or GANT61/DNA as anylate, respectively (KD= 7.5 µM; IC50= 3.2μM). Following GANT61 treatment of CC we demonstrated transcriptional stalling of RNA Polymerase II (Pol II) at sites of early elongation adjacent to GLI binding regions at target gene promoters. This is associated with modification of promoter-bound pause (DSIF, NELF) or pause-release (P-TEFb) factors. Single-stranded DNA (R-loops) is detected at sites of stalled Pol II; γH2AX foci are induced. Consistent with inhibition of transcription, GANT61 induces γH2AX foci in S-phase and non-S-phase cells. A transient intra-S-phase checkpoint is induced and cells accumulate in early S without further progression prior to the onset of cell death. The GLI1 target gene FOXM1, and its transcriptional target CDC6, regulate the G1/S transition and initiation of DNA replication, and are decreased in GANT61-treated cells. Data suggest that: 1) GANT61 inhibits GLI-mediated transcription by direct binding to GLI; 2) GLI-dependent Pol II transcription is stalled at early elongation in GANT61-treated cells, with formation of R-loops and DNA DSBs; 3) GLI1, FOXM1 and CDC6 regulate events at G1/S and initiation of DNA replication that controls outcome of the GANT61 response. These studies open the door to exploring the role of GLI as a therapeutic target. Since dysregulated HH signaling occurs in a wide variety of human cancers, the proposed studies are of broad impact.

Citation Format: Akwasi Agyeman, Babal K. Jha, Tapati Mazumder, Janet A. Houghton. Targeting GLI-dependent transcription by GANT61 in human colon carcinoma cells (CC); a new therapeutic approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1759. doi:10.1158/1538-7445.AM2014-1759