Decrease, GLI1/2 target gene expression leads to orofacial clefting

This Adverse Outcome Pathway (AOP) describes the linkage between antagonism of the Smoothened (SMO) receptor and orofacial clefts (OFCs) (AOP 460 in the Collaborative Adverse Outcome Pathway Wiki). Sonic Hedgehog (SHH) is a major signaling pathway of intercellular signaling during embryogenesis including the morphogenesis of the face. The SHH pathway is sensitive to chemical induced disruption at multiple points including through disruption to GLI1/2 target gene expression. Activation of the SHH pathway causes a signaling cascade that culminates with the transcription of GLI transcription factors and can occur in an autocrine, paracrine, or juxtracrine manner. When GLI1/2 target gene expression is disrupted during critical windows of development, this signaling cascade is disrupted resulting in reduced outgrowth of the facial prominences and the formation of an OFC. While most of the adjacent events lack studies showing dose or time response for the relationships, there is a high biological plausibility of the proposed AOP. We performed literature searches using Pubmed with Medical Subject Headings (MeSH) terms associated with the key events (KE) of interest. Sources were initially screened for relevance through review of the title and abstract. Those selected were reviewed to determine what if any data existed to support or refute the key event relationship (KER). While this AOP is specific to mouse (mus musculus) during embryonic development, SHH and the development of the face is largely conserved between mouse and human making this AOP able to be extrapolated to risk assessment for human exposures. It is hoped that further studies will be performed to increase the weight of evidence (WoE) for this pathway. This AOP is intended to serve as a tool for risk assessment for drug and chemical exposures during embryonic development when disruption to SHH through a decrease in GLI1/2 target gene expression occurs.

Orofacial clefts (OFCs), encompassing cleft lip with or without palate (CL/P), and cleft palate only (CPO) represent the second most common birth defect in humans with a prevalence of 1-2/1,000 births (Lidral, Moreno et al. 2008). The etiology of OFCs is complex with approximately 50% of CPO and 70% of CL/P considered non-syndromic (2011). SHH signaling is required for normal facial development and plays a critical role in the growth of the facial processes that form the upper palate and lip (Bush and Jiang 2012, Kurosaka 2015). The epithelial derived SHH drives orofacial development through an induced gradient in the underlying mesenchyme  (Lan and Jiang 2009, Kurosaka 2015). This gradient of SHH induces cellular proliferation and outgrowth of the mesenchyme (Lan and Jiang 2009). The SHH pathway is sensitive to chemical disruption and can be disrupted at multiple places along the signaling cascade during critical windows for exposure and has been shown to cause OFCs (Lipinski and Bushman 2010, Heyne, Melberg et al. 2015). The targets of this disruption include ligand modification, ligand secretion, downstream sensing, and signal transduction (Jeong and McMahon 2002, Lauth, Bergström et al. 2007, Petrova, Rios-Esteves et al. 2013). Chemical modulators of the SHH pathway through antagonism of SMO have been identified including the natural alkaloid cyclopamine, both natural and synthetic pharmaceuticals, and a pesticide synergist (PBO) (Lipinski, Dengler et al. 2007, Lipinski, Song et al. 2010, Wang, Lu et al. 2012, Everson, Sun et al. 2019, Rivera-González, Beames et al. 2021).

This AOP was developed as part of a larger network of AOPs linking disruption of SHH signaling with OFCs (OECD Advisory Group on Emerging Science in Chemicals Assessment (ESCA) workplan project 1.101.). Orofacial clefts (OFCs) are one of the most common human birth defects and occur in approximately 1-2/1,000 live births (Lidral, Moreno et al. 2008). Early orofacial development involves epithelial ectoderm derived SHH ligand driving tissue outgrowth through an induced gradient of SHH dependent transcription in the underlying mesenchyme, which is thought to drive mesenchymal proliferation (Lan and Jiang 2009, Kurosaka 2015). The SHH pathway is sensitive to chemical disruption at multiple molecular targets along the signaling cascade, with exposure during critical windows in development leading to OFCs (Lipinski and Bushman 2010, Heyne, Melberg et al. 2015). The molecular targets of this disruption include SHH ligand modification with cholesterol and palmitoylate, ligand secretion, mesenchymal reception, and signal transduction (Jeong and McMahon 2002, Lauth, Bergström et al. 2007, Petrova, Rios-Esteves et al. 2013). This AOP focuses on the disruption to SHH signaling through a decrease in GLI1/2 target gene expression. To select the key events for the AOP, we used existing knowledge of the pathway along with reviews of the SHH pathway to assemble a path that was physiologically plausible. Care was taken to select events that would be of direct regulatory relevance (i.e. a method to quantify exists). To identify sources and data for each Key Event Relationship (KER), Pubmed was used. Initially results were screened for relevance off title/abstract and any of suspected relevance were reviewed in full to determine their applicability for the KER. Each KER includes a table of relevant search information (date, search terms, citations, etc). It is the hope of the authors that this AOP is used as a tool for risk assessment for drug and chemical exposures during embryonic development when disruption to SHH through decreased GLI1/2 gene expression occurs.

Table 1: Literature Search AOP 491

Overall Assessment

Annex 1 Table, Assessment of the relative level of confidence in the overall AOP based on rank ordered weight of evidence elements is attached in PDF format.

Considerations for potential applications of the AOP

The intended use of this AOP from a regulatory standpoint is to improve predictive potential of developmental hazards as they relate to the SHH pathway and OFCs. It is hoped that this AOP can be applied to data from in silico and in vitro high-throughput screening assays (HTS) to guide selection of agents for further investigation in more representative models of orofacial development. Disruption of the Sonic Hedgehog pathway has broader outcomes than just OFCs and SHH is known to play a role in many aspects of embryonic development including patterning of many systems and limb and digit development. This AOP can be used as part of an integrated assessment of toxicity and can help to guide risk assessment for potential exposures during development. 

There is a need for development of New Approach Methodologies (NAMs) to increase understanding of the relationships that exist within this AOP to provide facilitate screenings abilities. Humans are exposed to upwards of 80,000 industrial chemicals and natural products, the majority of which have not undergone any type of toxicity testing either alone or in mixtures. Even highly regulated drugs are typically not tested for safety in pregnant women for obvious reasons despite the medical need in this population (Wise 2022). To help address this, we have engineered an in vitro microphysiological model (MPM) model of orofacial development to facilitate the study of both normal and abnormal orofacial development including disruption of SHH (Johnson, Vitek et al. 2021, Reynolds, Vitek et al. 2022). Traditional high throughput screening (HTS) assays are optimized for one pathway: one readout. This oversimplifies toxicant metabolism, intercellular pathway interactions, and ultimately makes the assay not representative of real-life exposures. Problems with HTS in drug discovery have been identified including missing intercellular interactions, co-exposures, and off target safety (Macarron, Banks et al. 2011). We can learn from these identified problems and engineer in vitro systems to more accurately recapitulate the biology to give a more thorough assessment of chemical and drug exposure.

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