Abstract 1667: Targeting acute myelogenous leukemia with novel combrestatin analogs and development of predictors of response
Authors: Zong Hongliang , Penthala Narsimha R. , Sonar Vijayakumar N. , Giannakakou Paraskevi , Roboz Gail J. , Crooks Peter A. , Guzman Monica L. 
Published: August 2015

Abstract
Introduction: Most adult acute myelogenous leukemia (AML) patients relapse and die of the disease. AML stem cells (AML-SCs) have been postulated to resist standard chemotherapeutic agents and thus give rise to relapse. Combretastatin A-4 (CA-4), a natural product isolated from the South African tree Combretum caffrum, has been reported to possess anti-leukemic activity in AML cell lines. To increase the repertoire of combretastatin analogs with potent anti-leukemic activity, about 100 CA-4 analogs were tested in AML cells, and studied the mechanism that AML cells resist to such microtubule (MT) inhibitors.
Methods: Cell viability/apoptosis was determined by annexin V and 7-AAD staining and flow cytometry. Cellular ROS production was measured by CellROX. Cell cycle was determined by DAPI staining and flow cytometry. Stem cell function was evaluated by colony assay and mouse xenograft assays. Tubulin depolymerization was measured by immunofluorescence or immunoblotting for microtubule fraction. Interaction of tubulin with BTAN or p38 MAPK was determined by pull down assays.
Results: One of the most potent analogs, BTAN, was further investigated in a panel of leukemic cell lines and primary AML samples. Similar to CA-4, BTAN inhibited tubulin polymerization in vitro and induced MT depolymerization and cell cycle arrest at G2/M in AML cells. BTAN induced cell death of AML blast, progenitor and stem cells via caspase activation regardless of their proliferating status and level of intracellular reactive oxygen species (ROS). In addition, BTAN impaired the physical contact of AML with stromal cells. To further identify the mechanism of resistance to BTAN, we investigated several signal transduction pathways including AKT, JAK-STAT, ERK1/2, JNK and p38. p38 MAPK inhibitor sensitized leukemia cells to BTAN treatment, and the sensitivity of AML cells to BTAN correlated with the basal level of active/phosphorylated p38 MAPK, suggesting p38 MAPK may interfere with the sensitivity to BTAN in AML. We demonstrated that tubulin interacted with p38 MAPK, especially its phosphorylated form, which was disrupted by treatment with a p38 MAPK inhibitor. In addition, in the presence of p38 inhibitor, the accumulation of BTAN was potentiated in AML cells, suggesting activated p38 may interfere with the interaction between BTAN and its tubulin targets.
Conclusion: Our data demonstrated that BTAN is a newly identified anti-AML-SC agent that works by disrupting the MT cytoskeleton and inducing caspase activation. These data suggest that the MT network is involved in the regulation of pro-survival signaling pathways even in quiescent AML-SCs, and that p38 MAPK is a key modulator for the response of AML blasts to microtubule inhibitors and that co-treatment with p38 MAPK inhibitors could be beneficial to microtubule destabilizing agents in AML.
Note: This abstract was not presented at the meeting.
Citation Format: Hongliang Zong, Narsimha R. Penthala, Vijayakumar N. Sonar, Paraskevi Giannakakou, Gail J. Roboz, Peter A. Crooks, Monica L. Guzman. Targeting acute myelogenous leukemia with novel combrestatin analogs and development of predictors of response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1667. doi:10.1158/1538-7445.AM2015-1667