Acyl protein thioesterase 1 and 2 (APT-1, APT-2) inhibitors palmostatin B, ML348 and ML349 have different effects on NRAS mutant melanoma cells

Abstract

Oncogenic NRAS mutations are frequent in melanoma and lead to increased downstream signaling and uncontrolled cell proliferation. Since the direct inhibition of NRAS is not possible yet, modulators of NRAS posttranslational modifications have become an area of interest. Specifically, interfering with NRAS posttranslational palmitoylation/depalmitoylation cycle could disturb proper NRAS localization, and therefore decrease cell proliferation and downstream signaling. Here, we investigate the expression and function of NRAS depalmitoylating acyl protein thioesterases 1 and 2 (APT-1, APT-2) in a panel of NRAS mutant melanoma cells. First, we show that all melanoma cell lines examined express APT-1 and APT-2. Next, we show that siRNA mediated APT-1 and APT-2 knock down and that the specific APT-1 and -2 inhibitors ML348 and ML349 have no biologically significant effects in NRAS mutant melanoma cells. Finally, we test the dual APT-1 and APT-2 inhibitor palmostatin B and conclude that palmostatin B has effects on NRAS downstream signaling and cell viability in NRAS mutant melanoma cells, offering an interesting starting point for future studies.

Lysophospholipases cooperate to mediate lipid homeostasis and lysophospholipid signaling

“…However, when both enzymes are knocked out, the cells are unable to regulate levels of LysoPLs, leading to aberrant LysoPLdependent signaling, an unregulated Lands cycle, and associated phenotypic and morphological changes to the cells. This work is one of the first documented studies of LYPLA activity in a cellular setting and gives a clear answer to why inhibiting only LYPLA1 or LYPLA2 seems to have little to no effect (52,86). We have focused here on the role of these proteins in maintaining LysoPL homeostasis, but their redundancy in substrate specificity may also apply to their other major function, namely, the thioesterase activity that results in the depalmitoylation of protein substrates (87).…”

“…This suggests a role for LYPLA2 in endocannabinoid metabolism. Furthermore, LYPLA1 and LYPLA2 are among a small pool of enzymes known to exhibit thioesterase activity on palmitoylated proteins such as the G 5 subunit of heterotrimeric G proteins and Ras (50)(51)(52). This posttranslational modification serves to alter protein conformation and/or tether a cytosolic protein to a lipid membrane with the addition of a lipophilic acyl moiety covalently bound to cysteine residues via a thioester linkage (53)(54)(55)(56).…”

“…This posttranslational modification serves to alter protein conformation and/or tether a cytosolic protein to a lipid membrane with the addition of a lipophilic acyl moiety covalently bound to cysteine residues via a thioester linkage (53)(54)(55)(56). LYPLA1 and LYPLA2 act to remove these acyl modifications as part of a dynamic palmitoylation process, regulating the subcellular location and conformation of a variety of cellular proteins (50,52,54,57).…”

APT2 Inhibition Restores Scribble Localization and S -Palmitoylation in Snail-Transformed Cells

“…Importantly, this effect is cell-line dependent, since ML349 attenuation of MEK activation is more robust in MDCK cells than MCF10A cells. Indeed, ML348 and ML349 have little effect on NRAS or BRAF mutant melanoma cell lines (Vujic et al, 2016), highlighting the context-dependent role for APT enzymes in cell polarity and growth signaling. Looking forward, ongoing advances in metabolic labeling and pulse-chase strategies open new opportunities to profile APT2 substrates by chemical proteomics, which will likely benefit from analysis in defined models of cell polarity.…”

Searching for the Chokehold of NRAS Mutant Melanoma

“…This was later discovered to be due to alternative lipid modifications mediated by the related enzyme geranylgeranyltransferase that functionally substitutes for farnesylation (Whyte et al, 1997). Similarly, inhibitors of posttranslational palmitoylation, such as palmostatin B, which also aim to disrupt the membrane localization of RAS, were effective in preclinical models of NRAS mutant cells but have not (yet) translated into clinical applications (Vujic et al, 2016; Xu et al, 2012). The clinical value of targeting other post-translational modifications of RAS such as phosphorylation, nitrosylation, monoubiquitination, and acetylation, which mainly affect the subcellular localization of the protein, is currently unknown.…”