Deep Myocardial Phenotyping of the Noonan Syndrome-causing Mutation RIT1F82L/+ In Vitro and RAS/MAPK Signalling as a Therapeutic Target
Doctoral thesis
Date of Examination:2023-12-11
Date of issue:2024-10-23
Advisor:Dr. George Kensah
Referee:Dr. George Kensah
Referee:Prof. Dr. Thomas Meyer
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Abstract
English
Noonan syndrome (NS) belongs to the RASopathies, which describe a group of rare developmental disorders caused by heterozygous germline mutations leading to a dysregulation of the Ras/mitogen-activated protein kinase (RAS/MAPK) pathway. Some of the most severe symptomatic burdens for infants diagnosed with NS are cardiac anomalies often manifesting in early-onset hypertrophic cardiomyopathy (HCM). Due to a lack of knowledge about underlying pathomechanisms, no approved targeted therapeutics are available for NS-associated HCM yet. Therefore, they can only be treated symptomatically and often heart transplantation is the last resort. Recently, hopes for a novel treatment option were raised, when two infant NS patients with mutations in RIT1 were treated off-label with the cancer drug trametinib (TB). TB is a highly selective reversible allosteric MEK1/2 inhibitor modulating the activity of the RAS/MAPK pathway. In both patients, TB treatment had a remarkably beneficial effect on the cardiac function by even regressing HCM and improving extra-cardiac symptoms such as somatic growth. In the project at hand, an in vitro model for the RIT1-associated myocardial phenotype was established by using induced pluripotent stem cells (iPSCs) generated from one of the aforementioned patients. The heterozygous mutation p.F82L in RIT1 was corrected by CRISPR/Cas9 technology to provide an isogenic control. iPSC-derived cardiomyocytes were used to generate miniaturised 3D NS myocardium in order to study its function over a course of nine weeks in vitro. A distinct phenotype was observed that manifested in severe myocardial remodelling, including structural, metabolic, and electrophysiological changes. This resulted in the development of a fibrotic sheath and impaired myocardial function. Moreover, this study suggests that the progression of this phenotype is further enhanced by a feedback loop. In a reverse translational manner, application of different TB treatment regimens showed that the emergence of the many phenotypical features can be suppressed. Late-onset treatment even rescued a severely manifested phenotype to a large extent, reflecting the clinical response of the iPSC donor to TB. In summary, this project showed that the presented human in vitro disease model was able to mimic a clinical phenotype and, thereby, helps to identify and understand novel pathomechanisms of inherited cardiomyopathies. Furthermore, it was shown by the reverse translational use of TB that this model system has the potential to predict the efficacy of a drug as well as potential side effects in vitro.
Keywords: RASopathy; Noonan syndrome; Myocardial tissue engineering; Disease modelling; MEK inhibition; Reverse Translation; Hypertrophic cardiomyopathy