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.
Highlights
Mutations in lysine (K)-specific methyltransferase 2D (KMT2D, known as MLL2)[8,9,10,11] and lysine (K)-specific demethylase 6 A (KDM6A)[11,12,13] are mutated in ~75% and 5% of Kabuki Syndrome (KS) cases, respectively
In KS, we have shown that loss of the RAF1-inhibitory activity of RAP1A or RAP1B is the likely driver of developmental pathologies, not least because we were able to rescue convergence and extension (CE)-driven phenotypes by suppressing RAF1 genetically in rap[1] mutants
We found that 22/26 small molecules induced no appreciable pathology at a concentration of 100 nM (Fig. 1b); we selected that dose for subsequent experiments
Summary
Mutations in lysine (K)-specific methyltransferase 2D (KMT2D, known as MLL2)[8,9,10,11] and lysine (K)-specific demethylase 6 A (KDM6A)[11,12,13] are mutated in ~75% and 5% of KS cases, respectively Subsequent to these discoveries, we reported that mutations in the genes coding for two RAS-related proteins, RAP1A and RAP1B, can cause KS and the phenotypically-overlapping Hadziselimovic syndrome. We were able to phenocopy this rescue by downregulating MEK signaling by exposing morphant KS embryos to the small molecule tool compound PD184161, a MEK inhibitor[15] Together, these results suggested that some of the features found in KS patients overlap mechanistically with the “RASopathies,” a group of disorders caused by germline mutations in genes that encode components or regulators of the RAS/MAPK pathway[20,21]. Drug discovery efforts have led to the development of clinically approved inhibitors that are prescribed for these cancers[24]
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