Abstract A299: Select microtubule inhibitors increase lysosome acidity and promote lysosomal disruption in acute myeloid leukemia (AML) cells.

Abstract AML is a hematological malignancy for which the standard of care therapy has remained unchanged for almost 30 years. Novel therapeutic approaches are therefore urgently needed for the treatment of this heterogeneous disease. To identify new strategies for the treatment of AML, we screened a natural product library for compounds cytotoxic to AML cells and identified Deoxysappanone B 7,4’-dimethyl ether. Deoxysappanone B is a homoisoflavanoid compound extracted primarily from the dried heartwood of Caesalpinia sappan, a medicinal plant native to South-East Asia. However, anticancer activity of this compound has not been previously described and its molecular targets are largely unknown. In subsequent validation studies, Deoxysappanone B possessed anti-leukemic activity in 6 tested AML cell lines with nanomolar IC50s and was preferentially cytotoxic to primary AML cells and stem/progenitor cells over normal hematopoietic cells. To understand its mechanism of action, we performed chemo-genomic profiling of Deoxysappanone B in S. cerevisiae and identified enrichment of genes related to mitotic cell cycle as well as vacuolar acidification, therefore pointing to microtubules and lysosomes’ proton-pumping vacuolar (V)-ATPase as potential targets. We confirmed Deoxysappanone B's action as a microtubule inhibitor and localized its binding site near to that of colchicine via in-vitro tubulin polymerization and competitive binding assays. We also showed that Deoxysappanone B reversibly induces cell cycle arrest and cell death in a panel of AML cell lines as well as overcomes some mechanisms of resistance to vinca alkaloids. Validating the functional importance of tubulin as a target for Deoxysappanone B-mediated cell death, epidermoid carcinoma cells with a tubulin mutation were more resistant to Deoxysappanone B compared to their parental counterpart. In addition to inhibiting tubulin polymerization, Deoxysappanone B also increased lysosome acidity as measured by a V-ATPase enzymatic assay as well as staining with LysoSensor™ Yellow/Blue DND-160 and confocal microscopy. The sustained increase in lysosome acidity ultimately led to lysosomal disruption as evidenced by acridine orange staining. Supporting a tubulin-mediated effect on lysosomes, nocodazole, although not vinblastine, vincristine, paclitaxel or colchicine, produced a similar increase in lysosome acidity and lysosomal disruption. The effects on lysosomes were functionally relevant as pre-treatment with bafilomycin A1, a lysosomal V-ATPase inhibitor, partially abrogated the cytotoxic effect of Deoxysappanone B. Thus, our data provide insight into a novel mechanism of action of select microtubule inhibitors in the context of AML. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A299. Citation Format: Dannie Bernard, Marinella Gebbia, Swayam Prabha, Marcela Gronda, Neil MacLean, Xiaoming Wang, Rose Hurren, Mahadeo A. Sukhai, Eunice E. Cho, Morris F. Manolson, Alessandro Datti, Jeffrey Wrana, Rima Al-Awar, Ahmed Aman, Corey Nislow, Guri Giaever, Aaron D. Schimmer. Select microtubule inhibitors increase lysosome acidity and promote lysosomal disruption in acute myeloid leukemia (AML) cells. [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 A299.