Abstract 2960: Flavopiridol-mediated multiple modulatory effects synergistically increase sensitivity to TRAIL-induced cell death in human leukemia cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Previously, we described the antileukemic synergistic interactions between the cyclin-dependent kinase inhibitor flavopiridol (F) and TRAIL in multiple cell lines representing a broad spectrum of human leukemia. With the introduction of new hybrid regimens for flavopiridol-based therapies and advances in the clinical development of TRAIL/TRAIL agonists, a renewed interest in this therapeutic concept has emerged. In an attempt to identify key mechanistic signaling networks, human U937 leukemia cells were treated simultaneously with F (SelleckChem, Houston TX) and TRAIL (Alexis, Enzo, Farmingdale, NY) and analyzed for signaling events associated to cell death induction. We found a dramatic F-mediated shift from cytoprotective NF-kB, AKT and ERK pathways to pro-cell death ceramide and JNK signals. Functional studies using both pharmacological inhibitors and genetically modified cells revealed the functional relevance of these signals. Furthermore, F-mediated transcriptional modifications played key roles in determining and regulating the cross-talk between anti- and pro-cell death pathways. Co-exposure to F resulted in transcriptional down-regulation of genes involved in the regulation of pro-apoptotic JNK including XIAP, Gadd45β and phosphatases DUSP1, -10 and -11. In addition, F induced a significant reduction of multiple anti-apoptotic genes/proteins directly implicated in the pro-apoptotic cascades including (Mcl-1, XIAP, Bcl-2, Bcl-xL, c-Flip, cIAP2/3), and cell cycle modulators (cyclin D1, -E1/E2, CDKs 1,7,9,13,17, p15, p57, p21). However, although in many cases similar changes were observed in F- and TRAIL/F-treated cells, additional factors may have to exist that lead to the potent synergistic interactions observed with the drug combination. In fact, by using clonogenic assays (long term effects), we observed that while TRAIL or F-treated cells were able to recover after treatment, that was not the case in TRAIL/F-exposed cells (% clonogenic survival: T= 82, F=47, TF= 1.4). Time-course analysis by flow cytometry of cell surface localized TRAIL Death Receptors DR4 and DR5 showed that in the presence of F, either alone or in combination with TRAIL, the membrane levels of both receptors were significantly increased during the first 2-6h of treatment. Together, these findings indicate that combined exposure of human acute leukemia cells to TRAIL/FP results in an early mobilization of TRAIL death receptors (DR4, DR5) to the cell surface, effect that is temporally associated with activation of anti-apoptotic ERK, AKT and NF-kB signals; however, activation of DR4/5-mediated apoptotic pathway is later potentiated by a shift toward pro-cell death JNK/ceramide signals that, in association to F-mediated transcriptional effects, results in a dramatic increase in the sensitivity to TRAIL in otherwise TRAIL-resistant human leukemia cells. Citation Format: Swaminathan P. Iyer, Jaime Mejia, Adriana E. Rosato, Steven Grant, Roberto R. Rosato. Flavopiridol-mediated multiple modulatory effects synergistically increase sensitivity to TRAIL-induced cell death in human leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2960. doi:10.1158/1538-7445.AM2013-2960