Gli1/ DNA interaction is a druggable target for Hedgehog‐dependent tumors

Abstract

Hedgehog signaling is essential for tissue development and stemness, and its deregulation has been observed in many tumors. Aberrant activation of Hedgehog signaling is the result of genetic mutations of pathway components or other Smo-dependent or independent mechanisms, all triggering the downstream effector Gli1. For this reason, understanding the poorly elucidated mechanism of Gli1-mediated transcription allows to identify novel molecules blocking the pathway at a downstream level, representing a critical goal in tumor biology. Here, we clarify the structural requirements of the pathway effector Gli1 for binding to DNA and identify Glabrescione B as the first small molecule binding to Gli1 zinc finger and impairing Gli1 activity by interfering with its interaction with DNA. Remarkably, as a consequence of its robust inhibitory effect on Gli1 activity, Glabrescione B inhibited the growth of Hedgehog-dependent tumor cells in vitro and in vivo as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. The identification of the structural requirements of Gli1/DNA interaction highlights their relevance for pharmacologic interference of Gli signaling.

Targeting GLI factors to inhibit the Hedgehog pathway

“…By virtual screening of a natural compounds library against these hot spots and monitoring Hh inhibition at GLI1 level, we have identified GlaB (Figures 3 and 5B (Figures 4 and 5). GlaB has proved to inhibit Hh signaling in multiple cancer cells and CSCs, as well as the in vivo MB and BCC tumor growth in orthotopic xenograft mice and in allograft mice models, respectively (Table 1) [106]. Our work has raised the importance to investigate the mechanism of GLI1-mediated transcription, and the identification of the structural requirements of GLI1/DNA interaction have highlighted their relevance for pharmacological interference of GLI1 signaling by direct GLI1 antagonists.…”

“…For this reason, the challenge in this field is the understanding of the molecular mechanisms that regulate GLI-mediated transcription. In this regard, the recently reported identification of the structural requirements of GLI1/DNA interaction, stands as a promising tool for discovering small molecules capable of inhibiting Hh pathway by directly targeting GLI [106]. The discovery of the natural compound, GlaB, able to impair Hh oncogenic activity by inhibiting GLI1/DNA interaction, provides a proof-of-principle for the therapeutic relevance of such an approach, focused on the unique downstream GLI1 transcriptional effector rather than on multiple upstream oncogenic deregulated signals.…”

“…NMR and (EMSA) Electrophoretic Mobility Shift Assay experiments have confirmed that GlaB binds to GLI1 in correspondence of ZF4 and ZF5 affecting GLI1/DNA interaction and also emphasizing the role of K350 and K340 in this event[106] …”

“…Recently, our own research group has established a multidisciplinary drug discovery program focusing on the identification of natural products as direct GLI1 antagonists. Starting from the crystallographic structure of the GLI1-ZF domain in a complex with DNA [105], we have performed a mixed computational and experimental structure-based study to identify K350, R354, R380, and K381 residues as the strongest hot spots for GLI1/DNA interaction and transcriptional functions ( Figure 5) [106]. This information was subsequently used to discover pharmacological agents able to interfere with this basic process.…”

Mitogen-activated protein kinases and Hedgehog-GLI signaling in cancer: A crosstalk providing therapeutic opportunities?

“…Agents such as GANT58/GANT61 [151] and HPI 1-4 [152] act by blocking GLI processing, activation and/or transcriptional activity. Recently, other GLI inhibitors have been reported, including ATO (arsenic trioxide) [153], an already approved therapeutic for acute promyelocytic leukemia, the anti-pinworm pyrvinium [154] and glabrescione B, a small molecule that interferes with the binding of GLI to the DNA [155]. HH pathway can be blocked also by disrupting the interaction between HH proteins and PTCH, using anti-HH monoclonal antibody 5E1 [156] or the macrocyclic small-molecule robotnikinin [157], both exhibiting antitumor activity.…”