EFFECTS OF DOXORUBICIN ON HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES OBTAINED FROM PATIENTS SENSITIVE AND RESISTANT TO ANTHRACYCLINE-INDUCED CARDIOTOXICITY

Abstract

<h3>Background</h3> Anthracyclines (AC) are chemotherapeutic drugs used for treatment of various types of cancer. However, up to 26% of patients develop anthracycline-induced cardiotoxicity (AIC), ranging from mild arrhythmias to severe ventricular dysfunction. This study investigates the ability of human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) to recapitulate in vitro the susceptibility that certain patients have to develop AIC. <h3>Methods</h3> Patients treated with cumulative dose ≥200 mg/m2 of AC were recruited, as well as age- and gender-matched healthy volunteers (HV). iPSCs were derived from erythroblasts, evaluated for pluripotency and genomic stability, and differentiated into CM. At day 30, CMs were treated for 72h with 0.01-100µM Doxorubicin (DOX) alone or in presence of 1-100µM cardioprotectant Dexrazoxane (DRZ), 1-100µM of its metabolite ADR925 or 1mM N-acetylcysteine (NAC). Treated CMs were evaluated for viability and sarcomeric organization. Next-Generation Sequencing (NGS) was performed for cardiac targets in iPSC (5 AC-sensitive, 5 AC-resistant). <h3>Results</h3> iPSCs presented normal karyotype (G-banding assay) and pluripotency (RT-PCR: Oct4, Sox2, Nanog, Klf4; flow cytometry: OCT4, SOX2, NANOG; spontaneous differentiation into three germ layers RT-PCR: Tubulinβ3, Bmp4, Afp). CMs expressed cardiac Troponin T (cTnT) (AC-S: 82.45±14.09%; HV: 90.45±4.99%; n=6). Increasing doses of DOX led to higher mortality rates (AC-S: 0.1µM–15.09±6.33%; 1µM–46.62±13.27%; 10µM–64.46±21.99%; 100µM–96.1±5.55%; n=10; HV: 0.1µM–3.65±16.37%; 1µM–27.35±25.56%; 10µM–56.2±25.82%; 100µM–89.75±11.63%; n=6). Cotreatment with cardioprotectants showed no significant difference in cell viability (IC50 in µM AC-S: DOX=3.18±2.95, n=10; DOX+100µM DRZ=2.84±2.24, n=5, p=0.82; DOX+100µM ADR925=5.56±2.91, n=4, p=0.19; DOX+1mM NAC=2.45±2.51, n=6, p=0.62; HV: DOX=10.23±10.07, n=6; DOX+100µM DRZ=1.39, n=1; DOX+100µM ADR925=6.56±7.02, n=3, <i>p</i>=0.59; DOX+1mM NAC=3.64±4.72, n=4, <i>p</i>=0.26). cTnT staining revealed sarcomeric disorganization for DOX≥10µM. NGS sequencing revealed 49 nonsynonymous variants, none of which associated with AIC. Only one of those variants, located at desmoplakin gene, was present in all 4 AC-S patients and absent in AC-R ones. <h3>Conclusion</h3> iPSC from AC-S and AC-R patients were successfully generated and differentiated into CM. Viability tests recapitulated the patient's clinical cardiotoxicity and confirmed iPSC-CM as a platform for drug screening.