Effect of the sonic hedgehog inhibitor GDC-0449 on an in vitro isogenic cellular model simulating odontogenic keratocysts

Abstract

Odontogenic keratocysts (OKCs) are common cystic lesions of odontogenic epithelial origin that can occur sporadically or in association with naevoid basal cell carcinoma syndrome (NBCCS). OKCs are locally aggressive, cause marked destruction of the jaw bones and have a propensity to recur. PTCH1 mutations (at ∼80%) are frequently detected in the epithelia of both NBCCS-related and sporadic OKCs, suggesting that PTCH1 inactivation might constitutively activate sonic hedgehog (SHH) signalling and play a major role in disease pathogenesis. Thus, small molecule inhibitors of SHH signalling might represent a new treatment strategy for OKCs. However, studies on the molecular mechanisms associated with OKCs have been hampered by limited epithelial cell yields during OKC explant culture. Here, we constructed an isogenic PTCH1R135X/+ cellular model of PTCH1 inactivation by introducing a heterozygous mutation, namely, c.403C>T (p.R135X), which has been identified in OKC patients, into a human embryonic stem cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. This was followed by the induction of epithelial differentiation. Using this in vitro isogenic cellular model, we verified that the PTCH1R135X/+ heterozygous mutation causes ligand-independent activation of SHH signalling due to PTCH1 haploinsufficiency. This activation was found to be downregulated in a dose-dependent manner by the SHH pathway inhibitor GDC-0449. In addition, through inhibition of activated SHH signalling, the enhanced proliferation observed in these induced cells was suppressed, suggesting that GDC-0449 might represent an effective inhibitor of the SHH pathway for use during OKC treatment. Using gene-edited cell cultures, Chinese researchers have gathered useful insights into the genetic origins and treatment of benign oral tumours called odontogenic keratocysts (OKCs). A total of 80% of OKCs are associated with mutations in the gene PTCH1, which are thought to activate a signalling pathway that drives OKC tumour growth. However, research is hindered by OKC cell culturing limitations. Tiejun Li, from the Peking University School and Hospital of Stomatology, and colleagues created an OKC cell culture model that uses CRISPR gene editing to introduce a PTCH1 gene mutation into human stem cells before differentiating them into epithelial cells. Using their model, the team confirm that PTCH1 mutation activates tumour-associated signalling, which the basal cell carcinoma drug vismodegib greatly reduces. Vismodegib also reduces the overproliferation of mutant cells and offers a potential treatment option for OKC patients.