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 all childhood 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.

Public Health Relevance

If we are successful in demonstrating the validity of our platform for any of the candidate variants in doxorubicin cardiotoxicity, we will have provided physicians with a valuable tool to devise patient-specific chemotherapy protocols. Ultimately, for patients with high risk variants, protocols could reduce exposure to doxorubicin. In contrast, for patients with low risk variants, higher dose protocols could be used safely, leading to enhanced cancer cure rates without increased risk of cardiotoxicity. Finally, we can prepare a panel of hiPSC-CMs with each validated variant facilitate the development of new less toxic anthracycline analogues as well as cardioprotective agents. (End of Abstract)

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HL123655-01
Application #
8755003
Study Section
Special Emphasis Panel (ZHL1-CSR-H (M1))
Program Officer
Adhikari, Bishow B
Project Start
2014-08-08
Project End
2016-06-30
Budget Start
2014-08-08
Budget End
2015-06-30
Support Year
1
Fiscal Year
2014
Total Cost
$201,442
Indirect Cost
$75,933
Name
Stanford University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Jung, Gwanghyun; Fajardo, Giovanni; Ribeiro, Alexandre J S et al. (2016) Time-dependent evolution of functional vs. remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation. FASEB J 30:1464-79
Gottlieb, Roberta A; Bernstein, Daniel (2016) Mitochondrial remodeling: Rearranging, recycling, and reprogramming. Cell Calcium 60:88-101
Aminkeng, Folefac; Bhavsar, Amit P; Visscher, Henk et al. (2015) A coding variant in RARG confers susceptibility to anthracycline-induced cardiotoxicity in childhood cancer. Nat Genet 47:1079-84
Bernstein, Daniel; Burridge, Paul (2014) Patient-Specific Pluripotent Stem Cells in Doxorubicin Cardiotoxicity: A New Window Into Personalized Medicine. Prog Pediatr Cardiol 37:23-27