Small molecule inhibition of RAS/MAPK signaling ameliorates developmental pathologies of Kabuki Syndrome

Abstract

Kabuki Syndrome (KS) is a rare disorder characterized by distinctive facial features, short stature, skeletal abnormalities, and neurodevelopmental deficits. Previously, we showed that loss of function of RAP1A, a RAF1 regulator, can activate the RAS/MAPK pathway and cause KS, an observation recapitulated in other genetic models of the disorder. These data suggested that suppression of this signaling cascade might be of therapeutic benefit for some features of KS. To pursue this possibility, we performed a focused small molecule screen of a series of RAS/MAPK pathway inhibitors, where we tested their ability to rescue disease-relevant phenotypes in a zebrafish model of the most common KS locus, kmt2d. Consistent with a pathway-driven screening paradigm, two of 27 compounds showed reproducible rescue of early developmental pathologies. Further analyses showed that one compound, desmethyl-Dabrafenib (dmDf), induced no overt pathologies in zebrafish embryos but could rescue MEK hyperactivation in vivo and, concomitantly, structural KS-relevant phenotypes in all KS zebrafish models (kmt2d, kmd6a and rap1). Mass spectrometry quantitation suggested that a 100 nM dose resulted in sub-nanomolar exposure of this inhibitor and was sufficient to rescue both mandibular and neurodevelopmental defects. Crucially, germline kmt2d mutants recapitulated the gastrulation movement defects, micrognathia and neurogenesis phenotypes of transient models; treatment with dmDf ameliorated all of them significantly. Taken together, our data reinforce a causal link between MEK hyperactivation and KS and suggest that chemical suppression of BRAF might be of potential clinical utility for some features of this disorder.

The Nuclear Translocation of Mitogen-Activated Protein Kinases: Molecular Mechanisms and Use as Novel Therapeutic Target

“…Among them are RASopathies, which include the Noonan, Costello and cardio-facio-cutaneous (CFC) syndromes [103, 104], extracranial arteriovenous malformation [105], and Kabuki syndrome [106]. These last disorders can be induced by activating the mutation of MEK (CFC) or upstream components of the ERK1/2 cascade [107].…”

Inhibition of Notch signaling rescues cardiovascular development in Kabuki Syndrome

“…A recent study revealed a link between RAS/MAPK pathway hyperactivation and the neurological and craniofacial defects in the context of KS knockdown in zebrafish (28). In line with this, chemical inhibition of a downstream target of this pathway (BRAF inhibitor), partially rescued the craniofacial and neuroanatomical phenotype of kmt2ddepleted zebrafish larvae in transient knockdown and kmt2d +/heterozygous crosses (29). These findings suggest a pathway involved in some aspects of KS-neurological defects and establishes the utility of zebrafish for drugs screening in KS.…”

Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

“…We focused on three different genes ( anln, kmt2d , and smchd1 ) for which (a) we have substantial experience in this model organism and (b) which give reproducible, quantitative defects in kidney morphogenesis (Hall et al, 2018), mandibular and neuronal development (Tsai et al, 2018), and craniofacial morphogenesis (Shaw et al, 2017). For each locus, we used sgRNAs that had the following three characteristics.…”

“…For each locus, we used sgRNAs that had the following three characteristics. First, for each of the three genes, we selected an sgRNA with demonstrated high efficiency (100%) and an sgRNA with low efficiency (∼30%), as determined by heteroduplex analysis and Sanger sequencing of cloned PCR products (Hall et al, 2018; Shaw et al, 2017; Tsai et al, 2018) (Suppl. Figure 1).…”

“…We used CHOPCHOP (Labun et al, 2016) to identify sgRNAs targeting a sequence within the coding regions of the target genes and sgRNAs were in vitro transcribed using the GeneArt precision gRNA synthesis kit (Thermo Fisher, Waltham, MA) according to the manufacturer’s instructions. See Supplemental Figure S1, Table S1, and references (Hall et al, 2018; Shaw et al, 2017; Tsai et al, 2018) for details on targeting sequences/locations and sgRNA efficiency. Zebrafish embryos from a single clutch from a natural mating of a ZDR background founder pair were either uninjected or injected into the cell at the 1-cell stage with a 1 nl cocktail of 100 pg/nl sgRNA, 200 pg/nl Cas9 protein (PNA Bio, Newbury Park, CA), or a combination of both reagents.…”