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Introduction: Noonan Syndrome (NS) is caused by mutations that affect the RAS/ERK1/2-MAPK pathway. More than 90% of NS individuals with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated-HCM. Methods: Here, we used patient-derived RAF1S257L/+ and CRISPR-Cas9-generated isogenic control iPSC-derived cardiomyocytes (iCMs) to delineate the molecular mechanisms underlying HCM in NS-associated RAF1 mutations. Results: We found that mutant iCMs phenocopy the pathology seen in NS by exhibiting hypertrophy and structural defects (n=5). At the molecular level, while RAF1S257L/+ iCMs had elevated RAF1 and MEK1/2 activities, ERK1/2 itself was modestly enhanced. To test whether ERK1/2 activation was responsible for increased size of RAF1S257L/+ iCMs, we inhibited MEK1/2 activity with three different inhibitors (U0126, PD or Tram) and found that, while neither PD nor Tram reduced mutant iCMs area, both rescued myofibrillar disarray (n=3), suggesting that MEK1/2 and/or ERK1/2 regulate myofilament organization but not cell size. Since, PD and Tram block MEK1/2 activation by RAF, whereas U0126 prevents activation of ERK1/2 by active MEK1/2, we hypothesized that MEK1/2 rather than ERK1/2 regulates myofilament organization. Accordingly, we found that specific inhibition or ERK1/2 activity by ectopic expression of MKP3 did not rescue myofibrillar disarray in mutant RAF1 iCMs. Strikingly, U0126 was able to reduce iCMs surface area, suggesting that an ERK1/2-independent pathway was responsible for hypertrophy. Since U0126 also targets MEK5, we treated RAF1S257L/+ iCMs with a specific MEK5/ERK5 inhibitor and found that hypertrophy was drastically reduced (n=3), indicating that the ERK5 pathway triggered hypertrophy in RAF1S257L/+ iCMs. Finally, RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iCMs and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways and known as critical regulators of cardiac development, as well as extracellular matrix, cytoskeletal and actin assembly homeostasis that could contribute to the NS-associated HCM. Conclusion: Taken together, our study identifies the molecular mechanisms by which NS RAF1 mutations cause HCM and reveals downstream effectors that could serve as therapeutic targets for treatment of HCM in NS patients. [ABSTRACT FROM AUTHOR]