Extended Data Fig. 5: GNAQ/11-mutant cells produce high levels of IP3.
From: INPP5A phosphatase is a synthetic lethal target in GNAQ and GNA11-mutant melanomas
a, b, Quantitative analysis of IP3 (a) and IP4 (b) levels of 92.1-doxycycline-inducible shRNA cells expressing empty vector or the indicated INPP5A cDNA 72 h after doxycycline treatment. Data are presented as relative fold change to no dox, IP3, n = 3 and IP4, n = 2 independent experiments. FC, fold change. c, Schematic depiction of the metabolic flux of IP3 upon activating the Gα proteins, GNAQ and GNA11. LiCl inhibits the enzymatic activity of inositol monophosphatases (IMPases), leading to the stability of IP1, a downstream metabolite of IP3. IP1 accumulation is used as a surrogate for IP3 synthesis by activated GNAQ/11. d, Bar graph showing homologous time-resolved fluorescence (HTRF)-based IP1 accumulation in the indicated cell lines. Ratio of HTRF signal in cells treated with DMSO or GNAQ/11 inhibitor (FR900359 10 nM) for 16 h is shown. HTRF is a competitive immunoassay that measures cellular levels of IP1 which is used as a surrogate for the levels of IP3 produced as described in (c). HTRF signal is inversely proportional to the endogenous levels of IP1. GNAQ/11Mut UM cells exhibit enhanced IP3 synthesis driven by their oncogenic mutations compared to GNAQ/11WT cells. Upon inhibition of GNAQ/11 activity by FR, IP3 synthesis is reduced leading to lower accumulation of endogenous IP1 and thus increased HTRF signal, n = 4 independent experiments for DMSO and n = 2 for FR data for all cell lines except MP41 and 92.1 DMSO n = 6, Mel202 DMSO n = 3, MP46, OMM2.5, Mel285, Mel290 FR n = 4. Mut, mutant, WT, wild-type. e, Bar graph showing HTRF-based IP1 accumulation in HEK293A-Cas9 parental cells or cells expressing the indicated HA-GNAQ cDNAs. Cells were treated with DMSO or increasing concentrations of the GNAQ/11 inhibitor (FR900359) for 16 h. An increase in HTRF signal, which is indicative of reduced IP3 synthesis, was achieved at lower FR concentrations in cells expressing GNAQ-R183Q compared to GNAQ-Q209L or GNAQ-Q209P -expressing cells, suggesting that R183Q mutation results in intrinsically less active mutant GNAQ/11 proteins. DMSO, n = 2, FR, n = 3 biologically independent samples. WT, wild-type. All data are presented as mean ± s.e.m.; P values were determined by two-way analysis of variance (ANOVA) with multiple comparisons (a, e) or two-tailed, unpaired Student’s t-test (d).
