Abstract 669: A unique acquired resistance mechanism to agents that target GLI (GANT61) in the Hedgehog (HH) signaling pathway in human colon cancer cells.
Authors: Agyeman Akwasi , Mazumder Tapati , Houghton Janet 
Published: August 2014

In normal cellular processes, the HH signaling pathway has critical roles in embryonic development, tissue patterning, stem cell function, and differentiation. Canonical HH signaling engages the transmembrane receptor PTCH, the intermediary signaling molecule SMO, and the transcriptional regulators of target genes and the HH signaling response, GLI1 and GLI2. In colon cancers, GLI is aberrantly regulated by oncogenic signaling pathways, driving cell survival and genomic instability involved in oncogenesis, progression and metastasis. Using the small molecule inhibitor of GLI1/GLI2 transcription, GANT61, we have demonstrated that inhibition of HH signaling at the level of GLI induces extensive cell death in human colon carcinoma cell line models in contrast to targeting SMO. Cells with acquired resistance to SMO inhibitors also remain highly sensitive to GANT61, underscoring GLI as a critical target in colon cancer. We have now identified a previously undescribed, novel mechanism by which cancer cells evade cell death following protracted, targeted exposure of GLI by GANT61. The classic pharmacologic approach of stepwise increasing drug selection did not produce a proliferative colon cancer cell line resistant to GANT61, confirming the critical importance of GLI genes to cell survival. In contrast, prolonged exposure (1-3 weeks) of HT29 cells to semi-lethal concentrations of GANT61 (10 μM; HT29-G10), induced GLI1/GLI2 inhibition, reduced proliferation, decreased appearance of γH2AX nuclear foci, and increased methylation of histone H3K9 (H3K9me1-3) within the GLI1 and GLI2 promoters. Methylation of H3K9 lysine residues is correlated with either an inactive or silent state of gene expression, and can exist in mono-, di-, or tri- methylated states. It has been demonstrated by others that methylation at H3K9 in the promoters of p53, p21Waf1 or DHFR genes, can regulate cell proliferation or function at the G1/S transition. In addition to GLI1 and GLI2, H3K9 methylation was also observed in HT29-G10 cells in the promoter regions of FOXM1 and CDC6, G1/S regulatory genes. These are GLI target genes, which regulate the G1/S transition, or PreReplication Complex assembly at initiation of DNA replication, respectively. Data suggest that the GLI2-&gt;GLI1-&gt;FOXM1-&gt;CDC6 promoters are coordinately regulated by H3K9 methylation in a pathway to promote gene silencing, reduce gene transcription, and reduce cell cycle transition at G1/S, constituting a unique mechanism of acquired resistance to drugs that target GLI. It is anticipated that these approaches will provide new insight into critical targets that determine HH-dependent survival, and lead to the translation of agents that target or regulate GLI function.

Citation Format: Akwasi Agyeman, Tapati Mazumder, Janet Houghton. A unique acquired resistance mechanism to agents that target GLI (GANT61) in the Hedgehog (HH) signaling pathway in human colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 669. doi:10.1158/1538-7445.AM2013-669