Project 10: Role of RAC2 and CYBA in the development of anthracycline-induced cardiotoxicity using patient-specific iPSCs

PI Göttingen: K. Streckfuß-Bömeke; PI London: A.M. Shah; PhD: A. Maus - finished PhD, Double degree

Scientific background and preliminary results

Doxorubicin (DOX), an effective chemotherapeutic drug, is limited in its clinical applications due to cumulative dose-dependent cardiotoxicity. Even at relatively low doses of DOX, the risk of cardiotoxicity (ACT) is estimated at 7.8% to 8.8%. At present it is not possible to predict which patients will be affected or to protect patients who will likely suffer this devastating side effect. Oxidative stress due to anthracycline “redox cycling” is currently the most widely accepted cause of chronic ACT, although the condition cannot be prevented with antioxidants. This suggests a more complex pathophysiology, which may involve poisoning of topoisomerase II beta in cardiomyocytes. Recently, it was shown, that survivors of aggressive B-cell lymphomas or haematopoietic cell transplantation have a genetic susceptibility to doxorubicin-induced heart failure. They show an accumulation of genetic polymorphisms in oxidative stress involved genes, e.g. in the subunits RAC2 and CYBA (p22phox) of the NADPH-oxidase (NOX), or the doxorubicin efflux transporter ABCC1 and 2. The involvement of the NOXs in ACT is supported by the cardiac resistance to doxorubicin observed by two different laboratories in mice deficient for a subunit of this enzyme. The family of NOXes with its seven members plays an important role in the production of reactive oxygen species (ROS) in many cells. From all NOXes, especially NOX2 and NOX4 are mainly expressed in the heart and are involved in the development of heart failure. Nox2 has been further implicated in various aspects of (cardio)vascular diseases including vascular smooth muscle and endothelial cell hypertrophy and proliferation, inflammation, and atherosclerosis. The expression levels of the Rac isoforms 1 and 2, together with specificity of signaling, control their relative importance in NOX2 and NOX4 activation. Until now it is not known (1) in which way genetic variations in different NOX-subunits lead to the cardiac disease, (2) which mechanisms are involved, and (3) which cardiac cell types including cardiomyocytes (CMs) and smooth muscle cells (SMCs) play which role in the development of ACT. The ability to generate human induced pluripotent stem cells (hiPSCs) provides a unique opportunity for modeling heart disease and for investigating the underlying mechanisms of ACT on a patient-specific level.

Hypotheses of the PhD project

We hypothesize that based on the ps-iPSC-CMs data RAC2 (and CYBA, NCF4) play a major role in the development of ACT. In the project, the doctoral researcher will test the hypotheses that

1. the Nox-subunits, especially RAC2 (and CYBA, NCF4) are differentially expressed on protein level and/or active in fibroblasts, iPSC-CMs and iPSC-SMCs of the same ACT-patients/controls after DOX-treatment.

2. an overexpression and/or downregulation of RAC2 (and CYBA, NCF4) in genetically modified isogenic iPSC lines will result in an alteration of the redox state and cardiac function after differentiation.

3. SNPs in RAC2 and CYBA (RAC2-T7508A and CYBA-T214C) will result in an altered pattern of interaction partners in ps-iPSC-CMs in comparison to control-iPSC-CMs.

4. ps-iPSC-CMs +/- SNPs can be used as a platform to screen cardiotoxicity of novel anthracycline analogous and cardioprotective agents to develop novel therapeutic modalities for the prediction and prevention of ACT.

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