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

www.nature.com/scientificreports OPEN Received: 18 August 2017 Accepted: 12 June 2018 Published: xx xx xxxx Small molecule inhibition of RAS/ MAPK signaling ameliorates developmental pathologies of Kabuki Syndrome I-Chun Tsai1, Kelly McKnight1, Spencer U. McKinstry1, Andrew T. Maynard2, Perciliz L. Tan 1, Christelle Golzio1, C. Thomas White2, Daniel J. Price3, Erica E. Davis 1, Heather Amrine-Madsen2 & Nicholas Katsanis1 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. Kabuki syndrome (KS) is a developmental disorder characterized by a distinctive set of facial features, short stature, intellectual disability, dermatoglyphic abnormalities, and internal malformations of the cardiac, renal, gastrointestinal, and/or skeletal systems1–4. The global prevalence has been estimated at 1:32,000 births5. Current treatment options for KS do not exist, with clinical care limited to the management of individual symptoms6,7. The lack of a KS-specific treatment has motivated research into the genetic and pathomechanistic bases of the disorder, although the rarity of this syndrome continues to pose commercial and regulatory challenges in the pursuit of novel therapeutic approaches. Mutations in lysine (K)-specific methyltransferase 2D (KMT2D, also known as MLL2) 8–11 and lysine (K)-specific demethylase 6 A (KDM6A)11–13 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 also cause KS and the phenotypically-overlapping Hadziselimovic syndrome. Grounded on these observations and the known role of RAP1 in the regulation of RAS/MAPK signaling14, we showed in zebrafish embryos that dysfunction of any of kmt2d, kdm6a, rap1a, or rap1b yields anatomical developmental defects relevant to the KS phenotype through aberrant hyperactivation of MEK within the RAS pathway15. During 1 Center for Human Disease Modeling, Duke University School of Medicine, Durham, NC, 27701, USA. 2Target Sciences, GlaxoSmithKline, Research Triangle Park, NC, 27709, USA. 3Platform Technology and Science, GlaxoSmithKline, Research Triangle Park, NC, 27709, USA. I-Chun Tsai and Kelly McKnight contributed equally to this work. Correspondence and requests for materials should be addressed to N.K. (email: ) ScienTific REPOrTS | (2018) 8:10779 | DOI:10.1038/s41598-018-28709-y 1 www.nature.com/scientificreports/ gastrulation, these phenotypes manifested as convergence and extension (CE) defects; later in development, we also observed cell-cell intercalation pathologies that likely drive mandibular formation defects and, ultimately, micrognathia15. The RAS/MAPK pathway begins with RAS activation, which promotes the activation of RAF protein kinases, including ARAF, BRAF, and/or RAF1 (Supplementary Fig. 1). RAF kinases phosphorylate and activate MEK1 and/or MEK2, which in turn phosphorylate and activate ERK1 and/or ERK2. ERK1/2 is the ultimate effector; its substrates include nuclear components, transcription factors, membrane proteins, and protein kinases that control a multitude of processes such as cell cycle progression, differentiation, and growth16. In humans, the small GTPases RAP1A and RAP1B regulate RAS/MAPK signaling14. In some contexts, these proteins act to inhibit the phosphorylation of RAF1, and are thus antagonists of MAPK signal propagation17. In other contexts, RAP1A and RAP1B activate BRAF and thus agonize MAPK signaling18,19. 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 CE-driven phenotypes by suppressing RAF1 genetically in rap1 mutants. Moreover, 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 inhibitor15. 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 pathway20,21. Although each RASopathy is unique, they all share characteristics with KS, such as craniofacial dysmorphisms; musculoskeletal, cutaneous, and cardiac abnormalities; and neurocognitive impairment20. Importantly, this group of disorders has remained too rare to motivate robust ab initio drug discovery efforts. However, the RASopathies may benefit from a serendipitous advantage, in that persistent activation of the RAS/MAPK pathway has been reported in several cancers22. Most notably, activating mutations in BRAF lead to constitutive activation and phosphorylation of MEK and ERK in the RAS-RAF-MAPK signaling cascade, which are understood to contribute significantly to malignant melanoma, thyroid and colon carcinomas, as well as other cancers23. As a consequence, drug discovery efforts have led to the development of clinically approved inhibitors that are now prescribed for these cancers24. The conceptual bridge between the development of small molecule inhibitors for somatic RAS/MAPK activating mutations and their possible utility in germline disorders of this pathway has some experimental support. For example, treatment of a Raf1 mouse model of Noonan s (...truncated)