Doxorubicin, although one of the oldest anti-cancer agents, is highly effective in treating a wide range of cancers and is still utilized in 70% of allchildhood cancer treatments. However, its utility is limited by its cardiac toxicity, occurring in p to 65% of long-term survivors of childhood cancer. Children are more susceptible to this life-threatening side effect than adults. We have found two genetic variants associated with dramatically altered risk of doxorubicin cardiotoxicity. One protective variant is in the gene SLC28A3, an anti-cancer drug transporter and one risk variant is in RARG, a nuclear receptor and transcription factor that alters expression of other genes. Although these studies represent an advance in using a patient's genetics to guide doxorubicin usage (pharmacogenomics), the true effect of these gene variants is far from proven. Additional criteria that must be met include (1) Confirmation in other patient cohorts; (2) Validation, using a model system, that the gene variant alters cardiotoxicity; (3) Validation of a mechanism for its effects (e.g. does a loss-of-function change in a drug transporter lead to decreased intracellular drug levels and decreased toxicity); and (4) Demonstration that reversion of the variant to the normal (wild-type) gene rescues the altered toxicity effect. Patient-derived hiPSC-CMs (human induced pluripotent stem cell-derived cardiomyocytes) represent a novel technology which has been applied to understanding disease mechanisms and to screening drugs for toxicity. Although hiPSC-CMs do not replicate all aspects of mature cardiomyocytes, we show that hiPSC-CMs from patients who have had doxorubicin cardiotoxicity show increased doxorubicin damage compared to cells from patients without cardiotoxicity. We hypothesize that hiPSC-CMs represent a model platform for studying the validity and mechanisms of gene variants in regulating doxorubicin cardiotoxicity. Aim 1: To develop hiPSC lines with the gene variant in SLC28A3 and examine for decreased susceptibility to doxorubicin cardiotoxicity. Cells will be derived (a) directly from patients with the gene variant; and (b) by genetically inducing the same gene alterations in a control hiPSC line. Doxorubicin toxicity will be quantified by assays of cell function and viabilit. Aim 2: To develop hiPSC lines with the candidate gene variant in RARG and examine for increased susceptibility to in vitro doxorubicin cardiotoxicity. Aim 3: To explore the mechanism(s) by which each variant alters doxorubicin cardiotoxicity. (a) Expression of each candidate gene will be increased or decreased in a control hiPSC-CM line; (b) The variant will be reverted to normal (wild-type) in hiPSC-CMs from patients with each variant (c) We will then explore the specific mechanisms by which each variant affects doxorubicin cardiotoxicity. Aim 4: To utilize our platform to validate additional high risk hits. We will duplicate the above studies or other variants, chosen by meeting a strict definition of high probability and replicability.