Abstract
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 BRAFV600E 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 PERKL21del variant revealed by whole-exome sequencing in melanoma cell lines. 17-aminogeldanamycin exhibited similar activity in NRASQ61R melanoma cells that harbored a heterozygous inactivating variant of NAD(P)H:quinone oxidoreductase 1 (NQO1P187S). In addition, 17-aminogeldanamycin acted cooperatively with trametinib (an inhibitor of MEK1/2) and vemurafenib (an inhibitor of BRAFV600E) 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/BRAFV600E 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.
Highlights
Genomic classification has been a gauge for clinical management of melanoma patients by using immunotherapy or targeted inhibitors of BRAFV600 and MEK1/2 [1]
We have demonstrated that 17-aminogeldanamycin perturbates endoplasmic reticulum (ER) homeostasis, predominantly by interfering with activity of IRE1α-dependent pathway, and induces apoptosis in melanoma cells of the BRAFV600E and NRASQ61R subtypes. 17-aminogeldanamycin takes
Quantification of cleaved PARP (cPARP) level after 24 h of cell incubation with drugs is shown in the Online Resource 7a several advantages over geldanamycin and other geldanamycin derivatives as supported by previously published and present results showing that 17-aminogeldanamycin (i) is more potent than geldanamycin against melanoma cells; (ii) exerts anti-melanoma activity at lower concentrations compared with IC50 values for other geldanamycin analogues assessed in different human cancer cell lines [26], and (iii) attenuates self-triggered increase in HSP70 transcript level
Summary
Genomic classification has been a gauge for clinical management of melanoma patients by using immunotherapy or targeted inhibitors of BRAFV600 and MEK1/2 [1]. Lack of hot-spot BRAF, RAS, or NF1 driver mutations in the triple wild-type subtype accounting for 6–20% of melanomas [2, 3], and variability of phenotype of patientderived melanoma cell lines representing the same genetic subtype [4] enforce combining both genetic and phenotypic traits to achieve more accurately stratification of melanoma patients. Phenotype-based approaches can limit the number of potential therapeutic targets by pointing to master regulators of cell identity as demonstrated by selection of either MEK or HSP90, whose inhibition substantially affected 75% of melanoma cell lines [5]. Expression of HSP90 substantially increases from nevi to melanoma resulting in high HSP90 level in more than 50% of melanoma tumors, and augments with advanced melanoma stage [9, 10]. It has been demonstrated that HSP90 isoform present in melanoma-derived exosomes contributes to creation of a pre-metastatic niche by ‘educating’ bone marrow progenitors [12]
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