Kumar, Rakesh

Pazopanib and paclitaxel produce cytotoxic synergy in thyroid cancer models through inhibition of aurora kinase A.

Abstract Background: We recently reported a 49% RECIST response rate in conjunction with a phase 2 clinical trial of the multi-targeted kinase inhibitor pazopanib in differentiated thyroid cancers (DTC; Lancet Oncology 2010, 11:962-72), prompting a search for synergistic pazopanib-containing doublets in thyroid cancer cell lines. Methods and Results: Among all explored pazopanib-containing combinations, paclitaxel and pazopanib produced the greatest combined cytotoxic effects (continuous exposure pazopanib, 24 h paclitaxel; Combination Index, CI, at IC90=0.3). Interestingly, analogous synergy resulted when combining pazopanib with other antimicrotubule agents including other taxanes (docetaxel), Vinca alkyloids (vincristine) and epothilones (ixabepilone) – suggesting a class effect. Further, we found this combination to be synergistic in multiple cancer cell lines including DTC, medullary and anaplastic thyroid cancer lines, and in the A549 lung cancer line. In pursuit of underlying mechanism(s), we noted that the combination produced a greater fraction of cells in G2/M phase of the cell cycle and increased apoptosis in comparison to paclitaxel alone, prompting us to examine whether pazopanib might potentiate the effects of paclitaxel on mitosis. Analysis of cell fate resulting from pazopanib and paclitaxel alone and in combination using time-lapsed video microscopy demonstrated that as low as 2.5 μM pazopanib augmented paclitaxel-induce mitotic catastrophe by 2.4 fold, while inducing no appreciable cell death alone. As pazopanib is somewhat promiscuous in inhibition of kinases, we hypothesized that observed combined effects with antimicrotubule agents might be attributed to its inhibition of cell cycle by targeting CDKs or mitosis by targeting aurora kinases. Inhibition of CDKs 1 and 2 required pazopanib concentrations 1000-times those required to inhibit its primary kinase targets, VEGF-Rs; whereas aurora kinases A and B were inhibited at much lower concentrations (IC50s, 500-1000 nM). Hence, we pursued shRNA aurora A knockdown experiments: potentiation of pazopanib effects were observed in knockdown clones, suggesting that pazopanib single-agent effects are at least in part mediated via aurora A inhibition. Moreover, augmentation of paclitaxel effects were achieved in knockdown clones that rivaled those observed for the pazopanib/paclitaxel combination. Additionally, pazopanib/paclitaxel synergy was reduced in aurora A knockdown clones (CI at IC80: vector<0.5, knockdown clones >1.0), indicating that aurora A inhibition by pazopanib is contributory to paclitaxel/pazopanib synergy. Conclusions: Pazopanib combines with antimicrotubule agents to produce cytotoxic synergy associated with augmentation of mitotic catastrophe that appears at least in part mediated by its ability to inhibit aurora kinases. Supported in part by CA125750. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3597. doi:10.1158/1538-7445.AM2011-3597

A critical role for vascular endothelial factor (VEGF) has been demonstrated in multiple myeloma (MM) pathogenesis. Here, we characterized the effect of the small-molecule VEGF receptor inhibitor pazopanib on MM cells in the bone marrow milieu. Pazopanib inhibits VEGF-triggered signaling pathways in both tumor and endothelial cells, thereby blocking in vitro MM cell growth, survival, and migration, and inhibits VEGF-induced up-regulation of adhesion molecules on both endothelial and tumor cells, thereby abrogating endothelial cell-MM cell binding and associated cell proliferation. We show that pazopanib is the first-in-class VEGF receptor inhibitor to inhibit in vivo tumor cell growth associated with increased MM cell apoptosis, decreased angiogenesis, and prolonged survival in a mouse xenograft model of human MM. Low-dose pazopanib demonstrates synergistic cytotoxicity with conventional (melphalan) and novel (bortezomib and immunomodulatory drugs) therapies. Finally, gene expression and signaling network analysis show transcriptional changes of several cancer-related genes, in particular c-Myc. Using siRNA, we confirm the role of c-Myc in VEGF production and secretion, as well as angiogenesis. These preclinical studies provide the rationale for clinical evaluation of pazopanib, alone and in combination with conventional and novel therapies, to increase efficacy, overcome drug resistance, reduce toxicity, and improve patient outcome in MM.