The Anti-Proliferative Effects of Hsp90 Inhibitor Tricyclic Coumarin GUT-70 and Geldanamycin Analog 17-DMAG in AML Cells in Hypoxia
Authors: Jin Linhua , Tabe Yoko , Kojima Kensuke , Ruvolo Vivian , Miida Takashi , Andreeff Michael , Konopleva Marina , Kimura Shinya 
Published: October 2019

Abstract
Abstract 2480
Hypoxia plays an important role in survival and chemoresistance of AML cells in bone marrow (BM) microenvironment. In AML, constitutively activating ITD mutations of FLT3 are associated with poor prognosis by activating downstream targets involved in the PI3K/AKT, RAS/MAPK or STAT5 signaling which confers oncogenic properties such as dysregulated proliferation, resistance to apoptosis, and blocked differentiation. We and others have demonstrated that the antileukemic effects of FLT3 inhibitors was less prominent in BM-resident leukemic FLT3-ITD blasts compared to circulating blasts (Zhang et al. J Natl Cancer Inst. 2008). <>The chaperone molecule 90-kDa heat shock proteins (Hsp90) is known to be highly expressed in malignant cells, and its activity is enhanced under hypoxic conditions. We have previously reported the cytotoxic efficacy of a 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) that induced degradation of Hsp90 client proteins including key components of multiple signaling pathways involved in cell proliferation and/or survival (Jin et al. Br.J. Cacer 2011). In this study, the anti-proliferative effects and molecular mechanisms of action of GUT-70 and another HSP90 inhibitor 17-DMAG (geldanamycin analog) were investigated in AML cells with/without FLT3-ITD mutation in hypoxic condition. Cells were adapted to hypoxia by culturing under 1.0% O2 for at least 14 days prior to experiments. The anti-proliferative effects of GUT-70 or 17-DMAG treatment were examined in 3 AML cell lines with known FLT mutation status (wt-FLT3: OCI-AML3; FLT3-ITD mutated: MOLM13, MV4;11) and paired isogenic Ba/F3 cells transfected with wt-FLT3 (Ba/F3-FLT3) or FLT3-ITD (Ba/F3-ITD) (kindly provided by Dr. D. Small). GUT-70 or 17-DMAG induced cell growth inhibition in a concentration-dependent manner with stronger effects in hypoxia than in normoxia (Table 1). In normoxic condition, GUT-70 or 17-DMAG caused more pronounced increase in the apoptotic fraction and G0/G1 accumulation in FLT3/ITD harboring cells than in wt-FLT3 cells. In hypoxia, however, wt-type FLT3 expressing cells were more sensitive to GUT-70 or 17-DMAG compared to the FLT3-ITD cells. Mutation status of p53 did not affect the anti-proliferative effects of GUT-70 or 17-DMAG, as determined by comparing anti-leukemia efficacy in OCI-AML3 and MOLM13 cells infected with p53 shRNA; and in wt- vs mt-p53 bearing MV4;11 (MV4;11R) cells. <>The mechanisms of action of GUT-70 or 17-DMAG were next investigated in FLT3-ITD bearing MOLM13 and Baf3/ITD, and in wt-FLT3 expressing OCI-AML3 and Baf3/FLT cells. Both GUT-70 and 17-DMAG induced Hsp70, a marker of Hsp90 inhibition, along with the degradation of Hsp90 client proteins, c-Raf, p-ERK, and p-Akt, without a significant difference between FLT3/ITD (+/−) cells irrespective to oxygen concentrations. The downregulation of Akt downstream targets p-GSK3b and p-4EBP1 as well as FLT3 target p-STAT5 were observed.The accumulation of cell cycle inhibitors p27 and p21 or proapoptotic BH3-only protein Noxa along with caspase-9 and -3 cleavage was more pronounced in hypoxia than in normoxia.Table 1.IC50 of GUT-70 and 17-DMAG in normoxia and hypoxia (MTT assay).IC50 ofGUT-70(μM)IC50 of17-DMAG(nM)NormoxiaHypoxiaaerobic IC50/hypoxic IC50NormoxiaHypoxiaaerobic IC50/hypoxic IC50OCI-AML37.74.61.750.518.92.7MOLM133.02.11.420.610.12.0MV4;115.94.31.418.12.09.1Baf3/FLT39.24.91.933.919.81.7Baf3/ITD7.33.91.923.215.21.5
In summary, our findings indicate that HSP-90 inhibitors successfully repress the pro-survival kinase pathways and stimulate cell cycle inhibitory and pro-apoptotic factors in FLT3-ITD as well as FLT3-wt AML cells under hypoxic condition. Results suggest that HSP-90 inhibitors may be useful in the eradication of chemoresistant AML cells sequestered in hypoxic BM niches.

Disclosures:
No relevant conflicts of interest to declare.