CRISPRi/a screens in human iPSC-cardiomyocytes identify glycolytic activation as a druggable target for doxorubicin-induced cardiotoxicity.

Doxorubicin is limited in its therapeutic utility due to its life-threatening cardiovascular side effects. Here, we present an integrated drug discovery pipeline combining human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs), CRISPR interference and activation (CRISPRi/a) bidirectional pooled screens, and a small-molecule screening to identify therapeutic targets mitigating doxorubicin-induced cardiotoxicity (DIC) without compromising its oncological effects. The screens revealed several previously unreported candidate genes contributing to DIC, including carbonic anhydrase 12 (CA12). Genetic inhibition of CA12 protected iCMs against DIC by improving cell survival, sarcomere structural integrity, contractile function, and calcium handling. Indisulam, a CA12 antagonist, can effectively attenuate DIC in iCMs, engineered heart tissue, and animal models. Mechanistically, doxorubicin-induced CA12 potentiated a glycolytic activation in cardiomyocytes, contributing to DIC by interfering with cellular metabolism and functions. Collectively, our study provides a roadmap for future drug discovery efforts, potentially leading to more targeted therapies with minimal off-target toxicity.