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 PTCH1^R135X/+ 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 PTCH1^R135X/+ 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.

牙源性角化囊肿（OKCs）是牙源性上皮起源的常见囊性病变，可偶发或与无回生性基底细胞癌综合征（NBCCS）联合发生。OKC具有局部侵略性，会引起颌骨的明显破坏，并且容易复发。PTCH1突变（〜80％的人经常在NBCCS相关的和散发的OKC的上皮中检测到，提示PTCH1失活可能会组成性激活声波刺猬（SHH）信号传导，并在疾病发病机理中起主要作用。因此，SHH信号的小分子抑制剂可能代表了OKC的新治疗策略。然而，在OKC外植体培养过程中，由于OKC的分子机制研究受到了有限的上皮细胞产量的限制。在这里，我们构建的同基因PTCH1 ^{R135X / +}细胞模型PTCH1通过使用簇状规则间隔的短回文重复序列（CRISPR）/ CRISPR相关的杂合突变（即OKC患者中已经鉴定出的c.403C> T（p.R135X））进入人类胚胎干细胞系而失活9（Cas9）系统。这之后是上皮分化的诱导。使用此体外等基因细胞模型，我们验证了PTCH1 ^{R135X / +}杂合突变会由于PTCH1导致SHH信号的配体独立激活单位不足。发现该激活被SHH途径抑制剂GDC-0449以剂量依赖性方式下调。此外，通过抑制激活的SHH信号传导，在这些诱导的细胞中观察到的增强的增殖受到抑制，这表明GDC-0449可能代表了OKC治疗期间使用的SHH途径的有效抑制剂。