Abstract A147: Pixantrone induces cell death through mitotic perturbations and subsequent aberrant cell divisions in solid tumor cell lines.

Abstract

Background and Aim: Pixantrone (Pix) is an aza-anthracenedione which has demonstrable activity in patients with non-Hodgkin's lymphoma. Pix has both alkylating and intercalating activity, but the mechanism of cell killing is unclear. Here we sought to elucidate the mechanisms by examining the effects of Pix on a number of cancer cells. Specifically, we assessed the impact Pix has on the cell cycle, DNA damage response and their relationships to cell killing. Methods: Cell lines derived from ovarian, colorectal, pancreatic and breast cancers were tested for response to Pix. We used a combination of FACs and microscopy to determine whether Pix altered cell cycle kinetics. To determine effects on cell death, short term (MTS) and long term (clonogenic) assays were performed. Apoptosis was determined by Annexin V staining and by biochemical markers as ascertained by western blotting. Moreover, live cell video-microscopy was used to assesses mechanisms of cell death. Results: We found that concentrations of Pix that were sufficient to induce cell death in clonogenic assays did not perturb cell cycle dynamics. We show that sensitivity to Pix did not correlate with levels of topoisomerase II protein, arguing that cell death was not due to inhibition of topoisomerase II. Immunofluorescence staining showed that cells treated with killing doses of Pix lacked detectable phospho-H2AX foci. This contrasted with doxorubicin which induced robust phospho-H2AX foci that was accompanied by a G2 cell cycle arrest. However, we did find a number of DNA damage and repair proteins formed discrete foci after treatment including pATM, pChk1, and 53BP1. How this relates to the standard DNA damage response is currently unclear. In addition, treatment of cells with Pix interfered with chromosome segregation during mitosis that were detected by the presence of chromosome bridges during mitosis and formation of micro/multi-nuclei after cell division. Long-term studies showed that cells do not immediately die after aberrant division, but only die after 3-4 abnormal rounds of mitoses, consistent with our observations that cell viability was reduced only after 3-4 days of Pix treatment. We addressed whether p53 was a determinant of Pix sensitivity by testing a pair of isogenic cell lines which differed in p53 status. We found that cells lacking p53 were less sensitive to Pix and took longer to die (>3 days) as compared to cells with wildtype p53 cells. Given that pChk1 foci were detected in Pix-treated cells, we tested the effects of Chk1 inhibitors in combination with Pix. We found that the combination of treatments killed more efficiently than either drug alone. Conclusion: Our studies show that Pix does not elicit a DNA damage response that leads to cell cycle arrest. The mechanism of cell killing appears to be by impairing chromosome segregation that generates severely aneuploid cells. Interestingly, the aberrant progeny cells do not die immediately but continued to divide for approximately 4 divisions before dying. The molecular mechanisms by which Pix impairs mitosis remains to be elucidated. We propose that the generation of micro nuclei as a result of aberrant division may serve as a biomarker of Pix efficacy and is a concept we are testing further. Finally, the finding that Pix appears to synergize with Chk1 inhibitors opens a window of opportunity to enhancement of tumor cell killing. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A147. Citation Format: Neil Beeharry, Xijun Zhu, Vignish Murali, Mitchell R. Smith, Timothy Yen. Pixantrone induces cell death through mitotic perturbations and subsequent aberrant cell divisions in solid tumor cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A147.

Rethinking Drugs from Chemistry to Therapeutic Opportunities: Pixantrone beyond Anthracyclines

“…γH2AX foci, markers of DNA damage, were observed in some micro­nuclei but not in main nuclei. Moreover, apoptosis occurred only after three or four waves of aberrant mitoses and showed a limited dependence on p53. , Other elegant studies maintained that PIX was a good inhibitor of topoisomerase IIα and caused formation of DSB in K562 cells from human chronic myeloid leukemia . Interestingly, however, such effects surfaced when PIX was used at ≥5 μM, which was appreciably higher than the transient plasma C max of PIX in clinical studies. , We believe this is an important issue that needs to be put into context.…”

“…The first round of formation of chromosomal bridges does not commit cells to death. As it was said earlier, cell death occurred only after multiple waves of aberrant mitoses, presumably because mitotic checkpoints do not adequately intercept cells carrying dysfunctional centromeres and kinetochores . Repeat rounds of chromosome non-disjunction and micronuclei formation eventually lead to the appearance of highly multi­nucleated cells, a widely anticipated outcome of mis-segregation events. , …”

“…The anti­tumor activity of anthra­cyclines and congeners correlates much better with plasma exposure over time rather than C max . In the case of PIX, topo­isomerase IIα-independent aberrant mitoses were observed in cells exposed to as low as 100 nM PIX, , a concentration level that reproduced steady-state plasma levels of PIX after C max and throughout primary and terminal half-lives . It is safe to conclude that higher than pharmacologically relevant concentrations of PIX do inhibit topo­isomerase IIα, while lower and clinically more relevant concentrations spare topo­isomerase IIα and/or act primarily by altering mitotic fidelity.…”

“…Possible cause-and-effect relations between such unique mechanisms of DNA alkylation and mitotic aberrations remain unknown at this point in time. Pharmacologic inhibition of pChk1 enhanced growth inhibition effects of PIX, but we do not know whether this was caused by a unopposed mitotic entry of cells that carried centromere–kinetochore abnormalities or durable DNA adducts or both kinds of lesions …”

Pixantrone–Rituximab Versus Gemcitabine–Rituximab In Relapsed/Refractory Aggressive Non-Hodgkin Lymphoma

“…Moreover, pixantrone does not generate oxygenfree radicals since it cannot bind iron, which is the putative mechanism for the cardiac toxicity of anthracycline and anthracenediones. Recent findings suggest that pixantrone induces a latent type of DNA damage that impairs the fidelity of mitosis, without triggering DNA damage response or mitotic checkpoint activation, but is lethal after successive rounds of aberrant division [15]. This mechanism of cell killing appears to be by impairing chromosome segregation that generates severely aneuploid cells.…”