Single-Nucleus Transcriptomics Reveal Mechanisms of Cardiovascular Protection Against Ponatinib-Induced Cardiotoxicity Through ROCKs Deletion in Cardiomyocytes

Ponatinib is a potent tyrosine kinase inhibitor and primarily used in the treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL). Its broad anti-cancer capability is restricted to CML or Ph + ALL harboring T315I mutant breakpoint cluster region gene fused with the Abelson murine leukemia gene 1 due to its potential cardiotoxicity (CTX) side effect. It is crucial to better understand the mechanisms of ponatinib's CTX to maximize its beneficial therapeutic effects while reducing CTX. We established a ponatinib-induced CTX mouse model, which was used in this study for evaluating long-term effects of ponatinib on cardiac structure and function. We first assessed the role of Rho-associated coiled-coil containing kinases 1 and 2 (ROCK1 and ROCK2) in mediating CTX on mouse models characterized by the inducible cardiomyocyte-specific double ROCK knockout (DKO). Next, we expanded the study with single nucleus RNA sequencing (snRNA-seq) to reveal critical differentially expressed genes and interrelated biological pathways. Finally, we hypothesized the most probable underlying cellular and molecular mechanisms by synthesizing the mouse model and transcriptomic findings. Important findings include that double ROCK deletion in cardiomyocytes prevented ponatinib-induced cardiac dilation and dysfunction and suppressed serum levels of several vascular toxicity biomarkers. Analysis of snRNA-seq revealed cell-type-specific transcriptomic signatures attributable to ponatinib treatment; however, they were attenuated in DKO mouse hearts. The associated genes include extracellular matrix- and thrombogenesis-related genes in cardiomyocytes, myofibroblast activation-related genes in fibroblasts, and pathological activation-related genes in endothelial cells (EC). The hypothesized mechanism is to integrate transcriptomic signatures of these three functional associated cell types with prior established roles of ROCK1 and ROCK2 in regulating actin cytoskeleton rearrangement. Hence, the contributory roles of cardiomyocyte ROCK in CTX and vascular toxicity have opened a new field for future experimental validation: drug-activated ROCK activities and cytoskeleton reorganization occurred in cardiomyocytes, fibroblasts and EC form the driving forces responsible for CTX induction.