IPI-493

Outcomes of melanoma patient treatment remain unsatisfactory despite accessibility of oncoprotein-targeting drugs and immunotherapy. Here, we reported that 17-aminogeldanamycin more potently activated caspase-3/7 in BRAF^V600E melanoma cells than geldanamycin, another inhibitor of heat shock protein 90 (HSP90). 17-aminogeldanamycin alleviated self-triggered compensatory increase in HSP70 mRNA level and induced endoplasmic reticulum (ER) stress, which was followed by selective diminution of cytoprotective IRE1α-XBP1s pathway activity of unfolded protein response (UPR), inhibition of ERK1/2 activity and induction of apoptosis. Concomitantly, ATF6/p50 level and expression of PERK-dependent genes, CHOP and BIM, remained unaltered. This might result from an inframe deletion in EIF2AK3 leading to a PERK^L21del variant revealed by whole-exome sequencing in melanoma cell lines. 17-aminogeldanamycin exhibited similar activity in NRAS^Q61R melanoma cells that harbored a heterozygous inactivating variant of NAD(P)H:quinone oxidoreductase 1 (NQO1^P187S). In addition, 17-aminogeldanamycin acted cooperatively with trametinib (an inhibitor of MEK1/2) and vemurafenib (an inhibitor of BRAF^V600E) in induction of apoptosis in melanoma cell lines as evidenced by in-cell caspase-3/7 activation and PARP cleavage that occurred earlier compared with either drug used alone. As trametinib and vemurafenib did not significantly affect HSP70 and GRP78 transcript levels, cooperation of MEK/BRAF^V600E inhibitors and 17-aminogeldanamycin might result from a concurrent inhibition of the RAS/RAF/MEK/ERK cascade and IRE1α-dependent signaling, and cell-intrinsic ER homeostasis can determine the extent of the drug cooperation. Our study indicates that 17-aminogeldanamycin takes several advantages compared with other HSP90-targeting compounds, and can complement activity of BRAF/MEK inhibitors in melanoma cells of different genetic subtypes.

Geldanamycin (GM) is an inhibitor of heat shock protein 90 (Hsp90) with potent and broad anti‐cancer properties, but with unacceptable levels of hepatotoxicity. These characteristics have led to the devise of a range of less toxic GM structural analogues. Redox properties and thiol reactivity are central to the therapeutic and toxicologic effects of quinones, and the question arises as to whether the toxicity of GM and its analogues relates to the reduction potential of the quinone. Using pulse radiolysis, we have previously determined the one‐electron reduction potentials (vs. the normal hydrogen electrode [NHE]) at pH 7.0 of GM, 17‐(dimethylaminoethylamino)‐17‐demethoxygeldanamycin (17‐DMAG), and 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG) to be −62±7 mV, −194±6 mV, and −273±8 mV, respectively. These experimental results are now used to establish a predictive relationship for the reduction potential of GM analogues based on the Hammett para substituent constants. These values correlate well with the drugs effects in vivo. We show that cytotoxicity of the benzoquinone ansamycin increases as its respective reduction potential increases, even after blocking of the C19‐position from nucleophilic addition. We conclude that the cytotoxicity is directly related to the reduction potential of the quinone/semiquinone couple.