Unveiling the role of tyrosine kinases in doxorubicin-induced cardiotoxicity and beyond

This editorial refers to ‘TAOK1 suppression improves doxorubicin-induced cardiomyopathy by preventing cardiomyocyte death and dysfunction’, by T. Suga et al., https://doi.org/10.1093/cvr/cvaf022. Extensive research has examined numerous key protein kinases in the heart and cardiomyocytes (CMs), emphasizing their potential as therapeutic targets.1 Beyond cardiovascular diseases, these enzymes, which possess substrate binding sites and catalytic active sites, are also considered excellent targets for small molecule therapies and are extensively studied in cancer treatment. Due to their promiscuity, specifically targeting kinases involved in CM survival, while avoiding those linked to cancer biology, remains a significant challenge. In the era of single-cell transcriptomics and proteomics, where the focus often lies on evaluating the expression levels of specific genes and proteins while neglecting their function or modulation, a functional screening approach that directly assesses the activity of kinases and their role within specific disease contexts is highly commendable. Exploiting a wide range of CRISPR/Cas9 kinase silencing approaches in human pluripotent stem cell-derived CMs (hPSC-CMs), Suga et al.2 identified TAOK1 [thousand and one (TAO) amino acid kinase 1], a STE20 kinase family member, as a key regulator of Doxorubicin (DOX)-induced CM injury. Authors demonstrated that TAOK1 suppression mitigates DOX-induced cardiomyocyte death, sarcomere disarray, contractile dysfunction, DNA damage, and mitochondrial dysfunction. Moreover, TAOK1 inhibition did not induce DOX resistance in cancer cells, preserving its anti-cancer efficacy. This has significant implications in the field of cardio-oncology. It positions TAOK1 inhibition as a promising cardioprotective strategy for patients undergoing DOX treatment, providing a pathway to safeguard cardiac health without compromising the anti-cancer effects of DOX. Among the highest-scoring kinases, aside from TAOK1, protein kinase A regulatory subunit 1-α (PRKAR1A) and uridine-cytidine kinase-like-1 (UCKL-1) are both implicated in tumorigenesis, making them less viable therapeutic targets despite their potential relevance to DOX-induced cardiotoxicity. PRKAR1A encodes the R1α subunit of PKA, and its downregulation has been linked to cancer development, including cardiac myxoma.3 Similarly, UCKL-1, involved in the pyrimidine salvage pathway, is associated with cancer cell proliferation.4 The specific ranking obtained from the screening provides an alternative perspective. It allows for the identification of molecules at one extreme of the ranking (those with the lowest RRA scores) that are relevant for CM survival following Dox treatment (Figure 1). Interestingly, among others, kinases targeted for anticancer therapies, such as ERBB1-4, which is inhibited by trastuzumab (a drug frequently combined with doxorubicin in cancer treatment, a cocktail known for its cardiotoxic effect), are effectively positioned within that part of the graph. One aspect that has not been thoroughly analysed in this paper is the impact of inhibiting kinases on electrical activity and Ca²⁺ handling in CMs, which may significantly influence cardiotoxicity and arrhythmogenic outcomes.5 An intriguing aspect of Suga’s work is the in vivo validation of a screening conducted on hPSC-CMs, where AAV-mediated TAOK1 silencing improved cardiac function in a mouse model of DOX-induced cardiomyopathy. While often criticized for not being fully matured and for expressing embryonic markers, hPSC-CMs remain the only viable option for long-term human CM culture. As shown in this paper, they excel as a platform for functional screening, which requires subsequent validation, and are valuable not only for reproducing disease-specific phenotypes (e.g. long QT syndrome) but also for addressing broader patient populations. Finally, a hidden meaning that can be read between the lines lies on the use of two doses of Dox for some experiments. High-dose 1.0 μM was used to induce cell death in hPSC-CMs due to its clear cytotoxic effects while a lower concentration of 0.5 μM was employed in specific assays. It is worth noting that such a low-dose concentration aligns with those used in other studies to mimic cellular senescence in CMs. In CMs, DOX has been shown to induce cell death at high concentrations, whereas lower concentrations result in a milder phenotype consistent with senescence.6–8 Notably, these low-dose models are more clinically relevant, as they mirror the conditions experienced by patients who receive multiple low doses of DOX, leading to plasma levels ranging from 12.54 to 620.01 ng/mL (0.0231–1.14 μM), with tissue concentrations likely being even lower.9 Suga et al.2 utilized a low dose of Dox, thereby preserving library diversity and enabling evaluation under less toxic conditions. This approach allowed for the assessment of a certain degree of contractile dysfunction while maintaining spontaneous beating. At such low doses, mitogen-activated protein kinase (MAPK) p38 (MAPK14) phosphorylation was significantly elevated, accompanied by evidence of mitochondrial dysfunction but with minimal, if any, cellular death. Senescent CMs display increased oxidative stress, abnormal mitochondrial morphology, reduced adenosine triphosphate (ATP) production, and elevated levels of phosphorylated p38MAPK, without significant cytotoxicity.7,8 Although a more comprehensive assessment of senescence hallmarks—such as the upregulation of senescence-associated beta-galactosidase (SA-β-gal) and cyclin-dependent kinase inhibitors like p16INK4a (CDKN2A) and p21CIP1,6,7 (CDKN1A)—would be necessary to fully substantiate this hypothesis, it can be postulated that at 0.5 μM of DOX, a relevant percentage of CMs entered a state of senescence. Evidence of senescence has also been observed in human heart samples from patients with DOX-induced cardiomyopathy, obtained during left ventricular assist device implantation, when compared with control hearts.8 This state likely resulted in mildly dysfunctional yet partially contractile cells, which could serve as a compensatory mechanism. However, when the dose exceeded the capacity of this mechanism (primarily observed in in vitro or in vivo models), the cells transitioned to apoptosis. These suggest that senescence may play a role in preserving cardiac function in patients receiving DOX. This assumes that the removal of senescent CMs using ‘senolytic’ strategies to target antiapoptotic pathways specifically upregulated in senescent cells, thus promoting cell death in those cells, which have recently become quite popular, could make heart tissue more susceptible to other stressors.8 Interestingly, studies in different cell types have demonstrated that miR-24 can attenuate cellular senescence by directly targeting TAOK1.10 In Suga et al. p38 MAPK phosphorylation was significantly increased at low doses of DOX in the presence of TAOK1 overexpression. Therefore, targeting of kinases may be conceived for reducing the senescence markers along with apoptosis reduction and eventually enabling senescent cells to re-enter the cell cycle. Therefore, caution should be taken, as the senescence process limits tumorigenesis by promoting the spread of senescence and inducing secondary cell death in tumours. A schematic depict of study design by Suga et al.2 CRISPR/Cas9-based tyrosine kinase screening was conducted in induced Pluripotent Stem Cells (iPSC)-CMs exposed to low-dose doxorubicin to identify genes critical for cell survival. Surviving cells were analysed, and sgRNAs enriched in these cells were ranked by RRA (Robust Rank Aggregation) scores. TAOK1 emerged as a top-ranked candidate. Functional validation of TAOK1 inhibition demonstrated improved iPSC-CM survival, reduced p38 phosphorylation, reactive oxygen species (ROS), and enhanced ATP levels and contractility in-vitro. In-vivo, inhibition of TAOK1 in a doxorubicin-induced cardiotoxicity (DIC) mouse model reduced apoptosis, fibrosis, and hypertrophy while improving heart functionality. Notably, TAOK1 inhibition did not affect tumor cell viability. These results suggest TAOK1 as an anti-apoptotic kinase and potential target for cardiotoxicity prevention in CMs without compromising anti-cancer efficacy. Other kinases identified may be relevant for cardiomyocyte survival but could act as potential inducers of cardiotoxicity. L.B. is the recipients of a research grant by Cecilia-Augusta Foundation, Lugano, Switzerland. No new data were generated or analysed in support of this research.