Investigations on the Mechanisms of Resistance to Pixantrone in Tumor Cells.

Abstract

Pixantrone is an aza-anthracenedione currently undergoing Phase 3 clinical trials and has shown significant activity in non-Hodgkin's lymphoma. Preclinical studies with the aza-anthracenediones demonstrated a remarkable structure activity requirement, i.e., the ring nitrogen must be in the 2 position for anti-tumor activity. More importantly, Pixantrone which lacks hydroxyl groups at either the 1 or 4 positions of the chromophore, lacked any cardiotoxicity in pre-clinical trials. These pre-clinical findings have been substantiated during clinical development of Pixantrone. Given that there is a very real possibility that Pixantrone will progress to broader clinical use, we reasoned that understanding potential mechanisms by which tumor cells may become resistant to the drug were essential. By continuous in vitro exposure of MCF-7 cells to increasing concentrations of Pixantrone, we have developed a cell line (MCF7/aza) 20 fold resistant to the drug. These cells are cross resistant with mitoxantrone, surprisingly to a much great degree of resistance to mitoxantrone than to Pixantrone. Two compounds BBR 3438 and BBR 3576, represent a new class of anticancer drugs- the aza-anthrapyrazoles- have entered clinical trials and both were cross resistant in the MCF 7/aza cells. Using western blotting techniques we have demonstrated that the MCF 7/aza cells express elevated levels of BCRP but not Pgp. Further, resistance to Pixantrone was reversed with fumitremorgin C, a classic BCRP inhibitor that reverses mitoxantrone resistance in BCRP expressing cells. Resistance to Pixantrone was not reversed by verapamil, the classic Pgp inhibitor that reverses mitoxantrone resistance in MDR cells. Microscope evaluation of cells treated with Pixantrone demonstrated a unique intracellular distribution of the drug in MCF 7/aza cells in that the drug was sequestered in cytoplasmic vesicles rather than in the nucleus. More recently wehave completed a microarray analysis of MCF 7/Aza compared to MCF 7 cells and not surprisingly found a large number of genetic alterations in the resistant phenotype. One of the most striking alterations was a greater than 30 fold increase in BCRP. The results of our studies indicate that Pixantrone, a non-cardiotoxic mitoxantrone analogue, induced a resistant phenotype more closely related to that seen with mitoxantrone than the multi-drug resistance seen with many other anti-cancer drugs. It is of interest that an aza-anthracenedione, BBR 2378, was not cross resistant in MCF 7/aza cells. The unique structural feature of BBR 2378 is the presence of tertiary amines at the terminus of both sides arms of the compound in contrast to Pixantrone, BBR 3438, BBR 3576 or mitoxantrone all of which lack tertiary amine side arms. BBR 2378 has been previously been shown to be active in MDR cell linesas well. These results suggest that side arms play a pivotal role in determining activity or resistance in a variety of drug resistance tumor phenotypes.