BH3 mimetics potentiate pro-apoptotic activity of encorafenib in BRAFV600E melanoma cells.

BRAFV600- and MEK1/2-targeting therapies rarely produce durable response in melanoma patients. We investigated five BRAFV600E melanoma cell lines derived from drug-naïve tumor specimens to assess cell death response to encorafenib (Braftovi), a recently FDA-approved BRAFV600 inhibitor. Drug-naïve cell lines (i) did not harbor damaging alterations in genes encoding core apoptotic machinery, but they differed in (ii) mitochondrial priming as demonstrated by whole-cell BH3 profiling, and (iii) levels of selected anti-apoptotic proteins. Encorafenib modulated the balance between apoptosis-regulating proteins as it upregulated BIM and BMF, and attenuated NOXA, but did not affect the levels of pro-survival proteins except for MCL-1 and BCL-XL in selected cell lines. Induction of apoptosis could be predicted using Dynamic BH3 profiling. The extent of apoptosis was dependent on both (i) cell-intrinsic proximity to the apoptotic threshold (initial mitochondrial priming) and (ii) the abundance of encorafenib-induced BIM (iBIM; drug-induced change in priming). While co-inhibition of MCL-1 and BCL-XL/BCL-2 was indispensable for apoptosis in drug-naïve cells, encorafenib altered cell dependence to MCL-1, and reliance on BCL-XL/BCL-2 was additionally found in cell lines that were highly primed to apoptosis by encorafenib. This translated into robust apoptosis when encorafenib was combined with selective BH3 mimetics. Our study provides a mechanistic insight into the role of proteins from the BCL-2 family in melanoma cell response to targeted therapy, and presents preclinical evidence that (i) MCL-1 is a druggable target to potentiate encorafenib activity, whereas (ii) pharmacological inhibition of BCL-XL/BCL-2 might be relevant but only for a narrow group of encorafenib-treated patients.

17-Aminogeldanamycin Inhibits Constitutive Nuclear Factor-Kappa B (NF-κB) Activity in Patient-Derived Melanoma Cell Lines.

Melanoma remains incurable skin cancer, and targeting heat shock protein 90 (HSP90) is a promising therapeutic approach. In this study, we investigate the effect of 17-aminogeldanamycin, a potent HSP90 inhibitor, on nuclear factor-kappa B (NF-κB) activity in BRAFV600E and NRASQ61R patient-derived melanoma cell lines. We performed time-lapse microscopy and flow cytometry to monitor changes in cell confluence and viability. The NF-κB activity was determined by immunodetection of phospho-p65 and assessment of expression of NF-κB-dependent genes by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Constitutive activity of p65/NF-κB was evident in all melanoma cell lines. Differences in its level might be associated with genetic alterations in CHUK, IL1B, MAP3K14, NFKBIE, RIPK1, and TLR4, while differences in transcript levels of NF-κB-inducible genes revealed by PCR array might result from the contribution of other regulatory mechanisms. 17-Aminogeldanamycin markedly diminished the level of phospho-p65, but the total p65 protein level was unaltered, indicating that 17-aminogeldanamycin inhibited activation of p65/NF-κB. This conclusion was supported by significantly reduced expression of selected NF-κB-dependent genes: cyclin D1 (CCND1), C-X-C motif chemokine ligand 8 (CXCL8), and vascular endothelial growth factor (VEGF), as shown at transcript and protein levels, as well as secretion of IL-8 and VEGF. Our study indicates that 17-aminogeldanamycin can be used for efficient inhibition of NF-κB activity and the simultaneous diminution of IL-8 and VEGF levels in the extracellular milieu of melanoma.

Inhibitors of HSP90 in melanoma.

HSP90 (heat shock protein 90) is an ATP-dependent molecular chaperone involved in a proper folding and maturation of hundreds of proteins. HSP90 is abundantly expressed in cancer, including melanoma. HSP90 client proteins are the key oncoproteins of several signaling pathways controlling melanoma development, progression and response to therapy. A number of natural and synthetic compounds of different chemical structures and binding sites within HSP90 have been identified as selective HSP90 inhibitors. The majority of HSP90-targeting agents affect N-terminal ATPase activity of HSP90. In contrast to N-terminal inhibitors, agents interacting with the middle and C-terminal domains of HSP90 do not induce HSP70-dependent cytoprotective response. Several inhibitors of HSP90 were tested against melanoma in pre-clinical studies and clinical trials, providing evidence that these agents can be considered either as single or complementary therapeutic strategy. This review summarizes current knowledge on the role of HSP90 protein in cancer with focus on melanoma, and provides an overview of structurally different HSP90 inhibitors that are considered as potential therapeutics for melanoma treatment.

17-Aminogeldanamycin selectively diminishes IRE1α-XBP1s pathway activity and cooperatively induces apoptosis with MEK1/2 and BRAFV600E inhibitors in melanoma cells of different genetic subtypes.

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.