“Limiting Anthracycline-Induced Cardiotoxicity Using Amrubicin in Leukemia Systems”.
Authors: Fulbright Joy Marie , Chandra Joya 
Published: October 2019

Abstract
Abstract 3760
Poster Board III-696
The overall survival of pediatric cancer patients has greatly improved over the last forty years in part due to the introduction of anthracyclines into therapeutic regimens. However, a significant limitation of anthracyline use is cardiotoxicity by this class of drugs. The mechanism most often cited as contributing to anthracycline cardiotoxicity is the production of reactive oxygen species (ROS) and subsequent damage to mitochondria, resulting in activation of the apoptotic cascade. Because heart tissue contains more mitochondria than other organ sites, this mechanism is particularly damaging to this organ site. Newer, less toxic anthracyclines are being developed and amrubicin, a completely synthetic anthracycline with potent topoisomerase II inhibition, is one such compound and is approved in Japan for the treatment of small cell and non-small cell lung cancer. In animal studies amrubicin has demonstrated decreased cardiotoxicity compared to doxorubicin, and in Phase I/II clinical trials in lymphomas and solid tumors has demonstrated single agent activity and an improved early cardiotoxicity profile. Our goal was to elucidate the role of ROS in the decreased cardiotoxicity observed with amrubicin exposure and to test the ability of amrubicin to promote cell death in leukemia cells.
We used a rat cardiomyocyte cell line model (H9c2 cells) to quantify oxidative stress after exposure to amrubicin or other anthracyclines. Flow cytometry was used to evaluate superoxide levels after the cells were treated for 24 hours and stained with hydroethidium (HE). Interestingly, at equimolar doses (500 nM, 1 μM, 2 μM and 3 μM), cardiomyocytes treated with amrubicin produced less superoxide than cells treated with an equivalent dose of daunorubicin. These results suggest that ROS-mediated damage to cardiomyocytes is less after amrubicin exposure as compared to daunorubicin. The anti-leukemia efficacy of amrubicin was also evaluated as compared to other anthracyclines. Apoptosis induction in three different acute lymphocytic and myelocytic leukemia cell lines; Jurkat, ML-1 and KG-1 was measured using propidium iodide staining followed by flow cytometric analysis to yield cell cycle distributions. The percentage of the population with subdiploid amounts of DNA were representative of cells undergoing apoptotic DNA fragmentation. Using this method, we found that all three anthracyclines caused cell death after 24 hours of exposure. In order to assess the role of oxidative stress in anthracycline mediated cell death in leukemia cells, we measured ROS levels using equipotent doses of the drugs that elicited a 50% increase in the subdiploid population at 24 hours. In Jurkat and ML-1 cells both daunorubicin and doxorubicin significantly increased superoxide levels compared to control. Amrubicin also increased superoxide levels in Jurkat cells compared to cells treated with diluent alone. To establish a relationship between the observed oxidative stress and DNA fragmentation, we treated Jurkat cells with daunorubicin and doxorubicin with and without pretreatment with NAC (N-acetyl-cysteine), an antioxidant. NAC pretreatment significantly decreased superoxide levels, but did not abrogate the anthracyclines' promotion of apoptotic DNA fragmentation.
These results suggest that ROS may not be essential for anthracycline cytotoxicity in leukemia cells. Since ROS are promoting cardiotoxic effects, yet appear dispensible for anti-leukemia effects, anthracyclines that cause less ROS, such as amrubicin, may prove to be better treatment options in pediatric cancer patients for whom anthracycline exposure is indicated. Furthermore, our data raises the possibility that amrubicin's decreased cardiotoxicity could be enhanced by administering antioxidants to patients without compromising the drug's anti-tumor effects.

Disclosures:
Chandra: Pharmion Corporation: Research Funding.