Amrubicin Targets Nuclear Matrix-Bound Topoisomerase II In Human Myeloid Leukemia Cells

Abstract 1830

Amrubicin, a synthetic anthracycline which is approved in Japan for the treatment of lung cancer, is currently being evaluated in a global phase III trial for second line treatment of SCLC. Anthracyclines can exert cytotoxicity through inhibiting topoiosomerase II (Topo II), resulting in the blockade of DNA transcription and replication. Similar to etoposide, Amrubicin also is thought to induce covalent DNA protein complexes (stabilized DNA-topo II complexes) and double strand DNA breaks. In this study, we compared the cytotoxic and DNA damage-inducing effects of amrubicin to two other Topo II inhibitors and evaluated their effects on purified human Topo II as well as on the MV4-11 leukemia cell line. Cellular cytotoxicity assays demonstrated that amrubicin, etoposide and daunorubicin induced MV4-11 leukemia cell death at EC₅₀'s of 0.7 μM, 1.8 μM and 0.2 μM, respectively. In addition, DNA damage as represented by H2AX phosphorylation, was observed following drug treatment but was less extensive with amrubicin than with daunorubicin. One level of DNA organization consists of the folding of chromatin into 50–100 kb loops, which are attached at their bases to the nuclear matrix. The matrix attachment regions (MARS) of DNA contain strong Topo II binding sites and mediate the attachment of the chromatin loops to the nuclear matrix. We examined the effects of amrubicin on purified human Topo II, nuclear matrix-bound Topo II, and Topo II activity in MV4-11 leukemia cells in order to gain insights into the mechanism of amrubicin-induced apoptosis. The results were compared to those obtained with the “classical” Topo II-active drugs, etoposide and daunorubicin. Amrubicin, etoposide and daunorubicin inhibited the DNA strand passage reaction of purified human Topo II by stabilizing the formation of Topo II-DNA cleavable complexes (at IC₅₀ values of 20 μM, 750 μM and 4 mM, respectively). In MV4-11 leukemia cells, cleavable complex stabilization by IC50 concentrations of etoposide and daunorubicin resulted in extensive DNA fragmentation from 1–50 kb. In contrast, DNA cleavage by amrubicin was limited to the formation of >50 kb fragments even after 24 hrs of continuous drug exposure. The >50 kb fragments were similar in size to the 50–100 kb chromatin loops that are attached to the nuclear matrix. The hypothesis that amrubicin inhibits selectively nuclear matrix-bound Topo II resulting in detachment of the chromatin loops from the matrix was examined directly by using isolated nuclear matrix preparations from MV4-11 cells. Amrubicin was much more potent in inducing chromatin loop release from isolated nuclear matrix preparations than either etoposide or daunorubicin despite the fact that all three drugs stabilized Topo II-DNA cleavable complex formation. Chromatin loop release by amrubicin was Mg⁺⁺ dependent and reversible by 4 mM ATP indicating that it was Topo II mediated. Taken together, our results suggest that in MV4-11 cells amrubicin selectively targets nuclear matrix-bound Topo II, while etoposide and daunorubicin are less selective and induce generalized and extensive DNA damage. Exposure to etoposide and daunorubicin is associated with risk of myelodysplastic syndrome and secondary acute myeloid leukemia, which may be related to their ability to induce generalized DNA damage. As a more selective DNA damaging agent, further clinical development of amrubicin is warranted.