Karabanovich, Galina

ABSTRACT The role of DNA topoisomerase II beta (TOP2B) in cardiomyocyte differentiation is poorly understood. To address this, human induced pluripotent stem cells (hiPSC) were differentiated into cardiomyocytes (CM) that were wild type (WT) or contained a genomic deletion of Topoisomerase 2B (BKO). Both WT and BKO hiPSC could be induced to differentiate into sheets of beating cardiomyocytes. BKO hiPSC take slightly longer to differentiate into sheets of beating CM than WT iPSC. RNA was prepared from both undifferentiated and differentiated WT and BKO hiPSC. RNA-seq was used to examine gene expression changes when the WT and BKO hiPSC were differentiated into CM. Gene expression changes following differentiation of BKO cells were largely similar to those in WT cells. In addition, the differentiated WT CM were treated with dexrazoxane (ICRF-187), a TOP2 catalytic inhibitor that targets both TOP2A and TOP2B, or topobexin, a new TOP2B selective catalytic inhibitor. Topobexin inhibition partially phenocopied a TOP2B deletion and thereby providing an alternative to TOP2B gene knockout in many cell lines. In future, hiPSC derived CM with and without TOP2B and inhibition by topobexin ex vivo CM could be used to study anthracycline-induced cardiotoxicity and to screen for cardioprotectants.

Abstract The role of DNA topoisomerase II beta (TOP2B) in cardiomyocyte differentiation is poorly understood. To address this, Human induced pluripotent stem cells (hiPSC) were differentiated into cardiomyocytes (CM) that are wildtype or contain a genomic deletion of Topoisomerase 2B (BKO). Both WT and BKO hiPSC could be induced to differentiate into sheets of beating cardiomyocytes. BKO hiPSC take slightly longer to differentiate into sheets of beating CM than WT iPSC. RNA was prepared from both undifferentiated and differentiated WT and BKO hiPSC. RNA seq was used to examine gene expression changes when the WT and BKO hiPSC were differentiated into CM. Gene expression changes following differentiation of BKO cells were largely similar to those in WT cells. In addition, the differentiated WT CM were treated with dexrazoxane (ICRF-187), a TOP2 catalytic inhibitor that targets both TOP2A and TOP2B, or topobexin, a new TOP2B selective catalytic inhibitor. Topobexin inhibition partially phenocopied a TOP2B deletion and thus providing an alternative to TOP2B gene knockout in many cell lines. In future, hiPSC derived CM with and without TOP2B and inhibition by topobexin ex vivo CM could be used to study anthracycline-induced cardiotoxicity and to screen for cardioprotectants. Highlights Used CRISPR-Cas9 to delete TOP2B from hiPSC Produced beating cardiomyocytes from both WT and TOP2B null hiPSC Transcriptome analysis of WT and TOP2B null hiPSC and derived cardiomyocytes RNA seq showed he specific TOP2B inhibitor topobexin largely phenocopies TOP2B gene inactivation in iPSC derived cardiomyocytes. Topobexin inhibition could be used as an alternative to a TOP2B gene knockout in many different cell types, speeding up the analysis of the function of TOP2B.

Anthracyclines are potent chemotherapeutic agents, yet their clinical use is hampered by their cardiotoxicity. Anthracyclines achieve potent anti- cancer effects primarily via TOP2A, but at the same time they induce a dose limiting cardiotoxicity through TOP2B. The use of a catalytic TOP2 inhibitors such as dexrazoxane (ICRF 187) significantly reduces the incidence of anthracycline-induced cardiotoxicity. However, dexrazoxane targets the highly conserved TOP2 dimer interface that is identical between TOP2A and TOP2B, fostering persisting concerns that its inhibition of TOP2A isoenzyme may interfere with anthracycline anticancer effects and augment anthracycline-induced myelosuppression. Thus, isoform- selective TOP2 inhibitors that can control the DNA damage caused by TOP2A vs TOP2B are a significant unmet clinical need. We have developed a new class of highly potent and TOP2B-selective catalytic inhibitors through rational design by building upon the general pharmacophore present in isolates from Calophyllum Brasiliense. Using X-ray crystallography, we demonstrate that our newly developed compounds occupy a previously unidentified druggable binding-site in the TOP2 ATPase domain and function as a physical barrier to block the conformational change triggered by ATP hydrolysis. We have named the class of inhibitors that target this site “obex”, the Latin word for obstacle. The obex binding site contains residues that differ between the TOP2A and TOP2B isoenzymes, based on which we rationally designed, prepared, and systematically evaluated a targeted panel of obex inhibitors. We have identified the lead molecule, namely, topobexin, as the first- in-class TOP2B-specific obex inhibitor that can prevent anthracycline-induced DNA damage. This first TOP2 isoform-specific inhibition underscores the broader potential to improve drug specificity and minimize adverse effects in various medical treatments.