The Anti-Proliferative Effects of Tricyclic Coumarin GUT-70 as An Hsp90 Inhibitor In Mantle Cell Lymphoma

Abstract 3977

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, requiring novel anticancer strategies. Since p53 inactivating mutations occur primarily in the aggressive and refractory MCL variants, targeting p53-independent signaling pathways is of considerable interest. We previously reported the cytotoxic efficacy of a newly discovered tricyclic coumarin GUT-70 (5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl) -10-propyl-2H,8H-benzo[1,2-b;3,4-b ]dipyran-8-one (C₂₃H₂₆O₅) (synthesized at Nippon Shinyaku, Kyoto, Japan), originally derived from Calophyllum brasiliense, with more pronounced apoptotic effects in mutant-p53 MCL cells than in wild type-p53 cells (Jin et al., ASH abstract 2009). Some of the coumarin antibiotics are known to bind to chaperone molecule 90-kDa heat shock proteins (Hsp90) and induce degradation of Hsp90 client proteins including key components of multiple signaling pathways for cell proliferation and/or survival. In this study, the mechanisms of action of GUT-70 were investigated in MCL cell lines with known p53 mutation status (wt-p53: JVM-2, Granta-519, mt-p53: Jeko-1, MINO). GUT-70 demonstrated a dose-dependent binding affinity to Hsp90 (competitive binding assay using fluorescently labeled geldanamycin), increased ubiquitinated proteins accumulation, and further induced degradation of Hsp90 client proteins, including cyclin D1, Raf-1, Akt, and mt-p53, or increased Hsp70, a marker of Hsp90 inhibition. The downregulation of constitutively overexpressed cyclin D1 in MCL by GUT-70 was accompanied by p27 accumulation, decreased Rb phosphorylation, and impeded cell cycle progression in the wt-p53 JVM2 and Granta 519. However, GUT-70 induced apoptosis in mt-p53-bearing MINO and Jeko cells which was accompanied by only minimal cell cycle arrest. These findings suggest that apoptosis induction by GUT-70 in mt-p53 cells is in part independent from cell cycle arrest is known to protect cells from apoptosis. Moreover, the mt-p53 depletion by GUT-70 will further diminish its “gain of functions” for cell proliferation and anti-apoptosis.

To determine if GUT-70 might potentiate the apoptotic effects of commonly used chemotherapeutic agents, we assessed the combination effects of GUT-70 with bortezomib (BTZ), a selective inhibitor of the 26S proteasome, and with doxoubicin (DOX), a conventional chemotherapeutic agent drug for MCL. Synergistic anti-proliferative effects of GUT-70 and BTZ or GUT-70 and DOX combinations were observed in both wt-p53 and mt-p53 MCL cells at 48 h post-exposure (combination index; GUT-70/Bortezomib; 0.59 for JVM2, 0.73 forMINO, GUT-70/Doxorubicin; 0.37 for JVM2, 0.35 for MINO). In conclusion, our results demonstrate that the novel anticancer agent tricyclic coumarin GUT-70, an Hsp90 inhibitor, has potential utility for mt-p53 bearing MCL cells, and in the combination therapy of MCL.