Noonan syndrome-associated Raf1 mutants with increased or decreased kinase activity differentially activate Erk and cause distinct syndromic phenotypes

¹Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada
²Campbell Family Cancer Research Insitute, Ontario Cancer Institute and Princess Margaret Hospital,  University Health Network, Toronto, ON, Canada
³Department of Physiology and Medicine, Heart and Stroke/Richard Lewar Centre, University of Toronto, Toronto, ON, Canada
⁴Department of Human Health & Nutritional Sciences, University of Guelph, Guelph, ON, Canada

Noonan syndrome (NS) is one of several  “RASopathies” caused by mutations in different components of the RAS-RAF-MEK-ERK MAPK pathway. Germ line mutations in RAF1 (encoding the serine-threonine kinase RAF1) account for ~3-5% of NS. Unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with HCM.  Surprisingly, other NS-associated RAF1 mutations show normal or decreased kinase activity. To explore the pathogenesis of NS-associated RAF1 mutations, we generated  “knock-in” mice that express kinase-activating (L613V) and impaired (D486N) RAF1 mutants, respectively. Like NS patients, L613V/+ mice had short stature, craniofacial dysmorphia and hematologic abnormalities. Valvuloseptal development was normal, but L613V/+ mice exhibited eccentric cardiac hypertrophy and aberrant cardiac fetal gene expression, and decompensated following pressure overload. D486N heterozygotes and, surprisingly, homozygotes, were obtained at the expected Mendelian ratio. Male D486N/+ mice did not show NS phenotypes, whereas D486N/+ females had mildly decreased body size. D486N/D486N mice showed cardiac hypertrophy, but not dilatation, and one third had severe growth defects. Agonist-evoked MEK/ERK activation was enhanced in multiple cell types from either Raf1^L613V or Raf1^D486N mutants, although L613V had a much stronger effect. Moreover,  in response to agonist stimulation, both types of  Raf1 mutant formed more heterodimers with Braf than did wild-type Raf1, and shRNA and mutational analysis indicate that heterodimers are required for mutant action. Hence, NS-associated kinase-active and impaired  RAF1 alleles act as gain-of-function mutants on the RAS-RAF-MEK-ERK pathway by promoting increased heterodimerization with BRAF. Finally, post-natal MEK inhibition normalized all NS defects in L613V/+ mice. We conclude that different NS genes have intrinsically distinct pathological effects, that enhanced MEK-ERK activity is critical for HCM and other RAF1-mutant NS phenotypes, and suggest a mutation-specific approach to  RASopathy therapy.