Hedgehog (Hh) signalling is evolutionarily conserved pathway that is critical during development and maintenance of stem cells in adults. Activation of the Hedgehog pathway can occur through either ligand-dependent or ligand-independent mechanisms. While mutations in key Hh signalling components (PTCH1, SUFU, SMO, GLI1) result in ligand-dependent signalling and constitutive activation in some cancers, the vast majority of cancers in which Hh signalling is implicated do not have pathway mutations and are therefore driven upstream at the level of Hh ligand. Despite this, there are no biomarkers that can predict which tumours are dependent on Hh ligand and are likely to be responsive to Hh pathway inhibition.

Using a panel of osteosarcoma cell lines derived from either radiation-induced or conditional Trp53 and Rb1 genetic inactivation murine models of osteosarcoma, we observed a dramatic increase in Gli1 mRNA, a downstream target of the Hh pathway, in response to Hh ligand stimulation in Trp53 and Rb1 deficient cells. This was accompanied by a significant increase in primary cilia frequency, a critical organelle for Hh pathway activation. Importantly, Hh response to ligand was abolished using the SMO inhibitor LDE225. Using osteosarcoma allograft models, we showed that LDE225-mediated inhibition of the Hh pathway resulted in reduced tumour growth and increased survival in ciliated Trp53 and Rb1-deficient allografts but no response in a non-ciliated Trp53 and Rb1 wildtype-allograft.

The sustained clinical use of SMO inhibitors ultimately leads to development of resistance. We next evaluated the efficacy of Itraconazole, a SMO inhibitor previously demonstrated to be effective in Hh-therapy resistant ligand-independent tumour models, in our ligand-dependent osteosarcoma models. We show that Itraconazole inhibits Hh pathway activation in response to ligand leading to reduced Gli1 mRNA in vitro, and reduced tumour growth and increased survival in vivo.

Collectively, this data implicates Trp53, Rb1 and primary cilia as a potential biomarker of Hh ligand responsiveness in osteosarcoma and response to Hh pathway inhibition by SMO inhibitors LDE225 and Itraconazole.

Citation Format: Vijesh G. Vaghjiani, Catherine Cochrane, Neil Watkins, Stuart Mudge, Jason E. Cain. Inhibition of Hedgehog signalling in ligand dependent tumours [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 84.