Synergistic Anti-Neuroblastoma Efficacy Using Combinatorial Cytoskeletal Inhibitors

Children with high risk neuroblastoma still have a poor response rate to existing chemotherapeutics making it imperative that new classes of compounds are developed to treat this disease. The actin cytoskeleton is an ideal chemotherapeutic target due to its role in numerous biological processes essential for tumor cell growth and survival. Targeting actin however has been problematic due to unacceptable levels of toxicity associated with impacting actin containing structures essential for normal cell function. We have developed a novel class of compounds which target tropomyosin, the second core component of an actin microfilament. By targeting the cancer associated tropomyosin, Tm5NM1, we are able to discriminate between the actin filament populations in normal and transformed cells. We have demonstrated that our first in class anti-tropomyosin compound, TR100, impacts actin filament integrity leading to tumor cell death in vitro and in vivo in neuroblastoma models. In this study we elucidate the molecular mechanisms by which disruption of the actin cytoskeleton by TR100 induces tumor cell death. We also investigate the efficacy of this novel class of compound in combination with existing chemotherapeutics. Preliminary studies have demonstrated that low exposure of neuroblastoma cells to TR100 results in a G0G1 arrest. Increased exposure leads to the activation of the mitochondrial apoptotic pathway as measured by increases in both caspase activity and mitochondrial permeability. To delineate the signaling pathways involved in anti-tropomyosin compound induced apoptosis, we have conducted a kinexus phospho-array. Using this approach we have identified the activation of key stress response pathways. Treatment of the SH-EP neuroblastoma cell line with anti-tropomyosin compounds for 8h results in reduced phospho-activity of key intermediates of the MEK/ERK pathway, in particular a significant decrease in the phosphorylation of the RSK1/2 family of kinases. Preliminary data suggest that the compounds mediate their effect through the downregulation of the MEK/ERK and p38/JNK cell survival pathways. We have now extended this study to investigate the impact of the drugs in combination with other established chemotherapeutic agents. In particular compounds which target the microtubules, another key cytoskeletal structure within the cells. In CHLA20 neuroblastoma cells, TR100 showed significant synergy when used in combination with both paclitaxel (microtubule stabilizing) and vincristine (microtubule destabilizing) with a combinatorial index Citation Format: Justine R. Stehn, Duo Chen, Mark A. Currier, David Eaves, Melissa Desouza, Matthew Moosavian, Timothy P. Cripe, Peter W. Gunning. Synergistic anti-neuroblastoma efficacy using combinatorial cytoskeletal inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B50.