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[Preprint]. 2025 Nov 18:2025.11.18.688995. [Version 1] doi: 10.1101/2025.11.18.688995

Conserved Gαq-PLCβ-PKC signaling mediates trametinib resistance in BRAFV600E melanoma

Jingyi Ke 1,2,3,4,5, Zi Wang 1,2,3,4,5, Zhengyang Guo 1,2,3,4,5, Guangshuo Ou 1,2,3,4,5,*, Wei Li 6,*
PMCID: PMC12667813  PMID: 41332585

Abstract

Oncogenic mutations in BRAF, most commonly V600E, drive constitutive activation of the MAPK pathway in ~50% of melanomas. While MEK inhibitors such as trametinib, alone or in combination with BRAF inhibitors, are clinically effective, their therapeutic effects are restricted due to the development of drug resistance. Here we establish a Caenorhabditis elegans model carrying the oncogenic lin-45(V627E) allele, functionally equivalent to human BRAFV600E, to investigate mechanisms of trametinib resistance. Genetic suppressor screening and transcriptomic profiling reveal that the EGL-30/Gαq-PLCβ-PKC axis promotes resistance by sustaining MAPK activity under MEK blockade. Pharmacological inhibition of PLCβ or PKC synergizes with trametinib to abolish MAPK signaling and restore drug sensitivity in worms. Importantly, this resistance mechanism is conserved in human melanoma: combined MEK and Gαq-PLCβ-PKC inhibition markedly enhances trametinib efficacy in BRAFV600E melanoma cells and xenografts, and reverses acquired resistance in trametinib-resistant melanoma sublines. Together, our results identify a conserved Gαq-PLCβ-PKC pathway as a driver of trametinib resistance and provide preclinical evidence for its co-targeting with MEK inhibition as a therapeutic strategy in BRAFV600E melanoma.

Introduction

Melanoma is the most aggressive form of skin cancer and accounts for the majority of skin cancer-related deaths worldwide (Schadendorf et al., 2018). Approximately half of melanomas harbor activating BRAF mutations, most frequently V600E, which constitutively activates the RAS-RAF-MEK-ERK (MAPK) pathway and drives tumor growth and survival (Davies et al., 2002; Halle and Johnson, 2021; Wan et al., 2004). Targeted therapies with BRAF inhibitors (BRAFi) such as vemurafenib or dabrafenib, alone or in combination with MEK inhibitors (MEKi) such as trametinib or cobimetinib, have substantially improved clinical outcomes (Adashek et al., 2022). Combined BRAFi/MEKi therapy is now the standard of care for patients with BRAFV600E melanoma (Dummer et al., 2018). However, responses are transient and most patients ultimately relapse due to the development of resistance, underscoring the urgent need for novel therapeutic strategies that can extend durability of MAPK pathway inhibition (Cohen and Sullivan, 2019).

Resistance to BRAFi/MEKi arises through diverse mechanisms, including MAPK pathway reactivation and engagement of compensatory signaling circuits such as MET and PI3K-AKT-mTOR (Bahar et al., 2023; Braicu et al., 2019; Kakadia et al., 2018). Despite intensive investigation, the heterogeneity and complexity of resistance networks remain incompletely understood, limiting the rational design of effective combination therapies. Evolutionarily conserved model systems that faithfully recapitulate acquired drug resistance can accelerate discovery of fundamental resistance mechanisms and uncover new therapeutic vulnerabilities.

The nematode Caenorhabditis elegans offers a powerful genetic model for studying conserved signaling networks. The Ras/MAPK cascade is highly conserved between C. elegans and humans: LIN-45, the sole worm RAF ortholog, shares 53% identity with human BRAF, while MEK-2 and MPK-1 share 52% and 81% identity with human MEK1 and ERK, respectively (Altschul et al., 1997). Importantly, the LIN-45(V627E) substitution is functionally equivalent to the oncogenic human BRAFV600E mutation (de la Cova and Greenwald, 2012; Han et al., 1993; Kim et al., 2018).

In C. elegans, the Ras-MAPK cascade affects multiple developmental and physiological processes including germline development and vulval precursor cell (VPC) fate specification (Sternberg and Han, 1998; Sundaram, 2013), offering sensitive readouts for genetic and pharmacologic perturbation. Complete loss of MAPK signaling causes sterility (Church et al., 1995; Hajnal and Berset, 2002; Hsu et al., 2002; Lackner and Kim, 1998; Lee et al., 2007; Robinson-Thiewes et al., 2021), whereas reduced signaling produces a vulvaless (Vul) phenotype and hyperactivation induces ectopic VPC induction and a multivulva (Muv) phenotype (Han and Sternberg, 1990; Lackner et al., 1994; Saffer et al., 2011; Wu et al., 1995). Misregulated MAPK signaling can also yield a protruding vulva (Pvl) due to defects in vulval lineages or the vulval–uterine connection (Cordeddu et al., 2009; Eisenmann and Kim, 2000; Flex et al., 2014). Importantly, Muv phenotypes caused by MAPK pathway hyperactivation can be suppressed by targeted inhibitors, while pharmacologic inhibition of MAPK activity in wild-type animals produces a Vul phenotype, and together these tractable and quantifiable responses have established C. elegans as a powerful in vivo platform for high-throughput screening of MAPK pathway inhibitors (Bae et al., 2012; Gorgoń et al., 2024; Hara and Han, 1995; Ji et al., 2019).

Here, we introduce lin-45(V627E) mutant worms as a tractable genetic platform for modeling trametinib resistance. Using unbiased transcriptomic profiling and genetic suppressor screens, we identify the EGL-30/Gαq-PLCβ-PKC axis as a conserved mediator of MEK inhibitor resistance. We further demonstrate this pathway promotes resistance not only in worms but also in BRAFV600E melanoma cells and xenografts, and that pharmacological blockade of Gαq-PLCβ-PKC signaling restores trametinib sensitivity. Our findings reveal a conserved resistance mechanism and provide a rationale for combined MEK and PLCβ/PKC inhibition as a therapeutic strategy for BRAFV600E melanoma.

Results

A lin-45(V627E) C. elegans Model Reveals Acquired Trametinib Resistance.

To establish a C. elegans model that recapitulates the acquired trametinib resistance observed in BRAFV600E-driven tumors, we exposed day 1 adult wild-type (WT) and lin-45(V627E) mutant hermaphrodites (P0 generation) to the FDA-approved MEK inhibitor trametinib at concentrations ranging from 1 to 100 μM (Fig. 1A). As previously reported, all lin-45(V627E) hermaphrodites (n = 300) display Pvl phenotype (Fig. 1C) (Wang et al., 2024). In WT animals, 1 μM trametinib induced a vulvaless (Vul) phenotype in 20% of F1 progeny, and higher doses (10 μM and 100 μM) completely abolished vulval development, reflecting efficient suppression of MAPK signaling. In contrast, lin-45(V627E) mutants responded in a dose-dependent manner: 1 μM and 10 μM trametinib suppressed the Pvl phenotype in 100% of F1 progeny, whereas 100 μM exposure unexpectedly restored protruding vulval morphology in all mutants, a sharp contrast to the complete Vul phenotype observed in WT worms under the same condition (Fig. 1D).

Figure 1: A lin-45(V627E) C. elegans model reveals acquired trametinib resistance under high concentration treatment.

Figure 1:

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(A) Experimental workflow of brood size quantification and vulva phenotype analysis in wild-type (WT) or lin-45(V627E) worms treated with DMSO or different concentrations of trametinib.

(B) Dose-dependent effects of trametinib (Tram) on brood size of WT and lin-45(V627E) F1 animals. Mann-Whitney U test and Dunn’s multiple comparisons test were used to determine statistical significance. n >35. **** P < 0.0001, ns: not significant.

(C) Representative bright-field images of vulva from lin-45(V627E) mutants and wild-type, treated with DMSO and 10 μM trametinib (Tram). n > 35. Scale bar, 10 μm and 100 μm.

(D) Quantification of vulva phenotypes in lin-45(V627E) mutants and wild-type (WT) treated with DMSO or different concentrations of trametinib. n >100.

To further evaluate MAPK pathway inhibition, we quantified brood sizes of F1 animals. Compared to DMSO-treated controls, WT F1 animals exposed to 1 μM trametinib showed severe brood size reduction (250 ± 49 vs. 23 ± 20, p < 0.0001; Fig. 1B). Complete sterility occurred in WT F1 animals treated with 10 μM or 100 μM trametinib (Fig. 1B), suggesting a complete blockade of the BRAF/MEK/ERK pathway . These observations are in line with the sterile phenotypes observed in null alleles of lin-45/Raf, mek-2/MEK, or mpk-1/ERK (Hsu et al., 2002; Sundaram, 2013).

DMSO-treated lin-45(V627E) F1 animals maintained normal brood sizes comparable to those of DMSO-treated WT animals (241 ± 59 vs. 250 ± 49, p = 0.54). Notably, 1 μM trametinib had no significant effect on the brood size of lin-45(V627E) F1 animals compared to DMSO-treated mutants (236 ± 53 vs. 241 ± 59, p > 0.99, Fig. 1B), suggesting that this concentration is insufficient to inhibit the constitutively active BRAF/MEK/ERK signaling in the mutant background. In contrast, treatment with 10 μM trametinib nearly abolished fertility in lin-45(V627E) F1 animals (4 ± 7 vs. 241 ± 59, p < 0.0001; Fig. 1B), indicating effective pathway suppression at this concentration. Strikingly, increasing the dose to 100 μM paradoxically restored brood size to ~63% of DMSO-treated mutant levels (153 ± 61 vs. 241 ± 59, p = 0.0001; Fig. 1B), implying that lin-45(V627E) worms had acquired resistance to high-dose MEK inhibition.

This dose-dependent resistance phenotype likely arises through reactivation of MAPK signaling or engagement of alternative signaling pathways. This phenomenon mirrors observations in BRAF-mutant melanoma cells, which acquire trametinib resistance following prolonged exposure to escalating drug concentrations (Hartman et al., 2020; Tripathi et al., 2020). Collectively, these findings establish lin-45(V627E) worms as a genetically tractable in vivo model for dissecting the molecular mechanisms underlying acquired trametinib resistance.

Transcriptome Profiling and Genetic Screening Identify EGL-30/Gαq Signaling as a Mediator of Trametinib Resistance in C. elegans.

To elucidate the molecular mechanisms underlying acquired trametinib resistance in lin-45(V627E) mutants, we performed transcriptome profiling of trametinib-sensitive and -resistant lin-45(V627E) mutants. Specifically, we compared F1 animals derived from lin-45(V627E) hermaphrodites treated with 10 μM trametinib (sensitive) and 100 μM trametinib (resistant). Principal component analysis (PCA) of RNA-seq revealed a clear separation between these two populations, indicating distinct gene expression patterns (Fig. S1A). Differential expression analysis identified 443 genes that were significantly upregulated (fold change > 2 and adjusted P-value (Padj) < 0.05) in trametinib-resistant animals (Fig. 2C). Gene Ontology (GO) enrichment analysis on these genes revealed significant upregulation of genes associated with G-protein signaling pathway, including the egl-30 gene, which encodes the C. elegans Gαq subunit orthologous to human GNAQ/GNA11, within the heterotrimeric G-protein complex (fold change = 2.3, Padj = 0.008; Fig. 2C–D and S1B).

Figure 2: EGL-30/Gαq-PLCβ-PKC signaling is required for acquired trametinib resistance in lin-45(V627E) mutants.

Figure 2:

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(A) Schematic representation of the genetic screening process using EMS mutagenesis on lin-45(V627E) worms to identify suppressors exhibiting restored vulval phenotype.

(B) Quantification of vulva phenotypes in lin-45(V627E), lin-45(V627E); egl-30(R25C), lin-45(V627E); egl-30(S6F) and lin-45(V627E); egl-30(c.594–1G>A) mutants. n >50.

(C) Volcano plot of up- (red) and down-regulated (blue) genes in lin-45(V627E) F1 animals treated with 100 μM trametinib compared with those treated with 10 μM trametinib. Fold change > 2 and adjusted P-value (Padj) < 0.05. n = 3 biological replicates per treatment.

(D) Dot plot of gene ontology (GO) enrichment of the up-regulated genes in the lin-45(V627E) F1 animals treated with 100 μM trametinib.

(E) Dose-dependent effects of trametinib (Tram) on brood size of lin-45(V627E); egl-30(R25C) F1 animals. Dunn’s multiple comparisons test were used to determine statistical significance. n >35. **** P < 0.0001, ns: not significant.

(F) Brood sizes of WT and lin-45(V627E) F1 animals treated with DMSO, 50 μM U73122, 50 μM sotrastaurin (Sotra), 100 μM trametinib (Tram), or the combination. Dunn’s multiple comparisons test was used to determine statistical significance. n >35. **** P < 0.0001.

Supporting this finding, a forward genetic suppressor screen uncovered a recessive allele, egl-30(R25C), which suppressed the Pvl phenotype in lin-45(V627E) mutants (Fig. 2A). Similar phenotypes were ovserved when we introduced other egl-30 loss-of-function mutations into the lin-45(V627E) background (Fig. 2B).

To evaluate the functional role of egl-30 in trametinib resistance, we analyzed brood sizes in lin-45(V627E); egl-30(R25C) double mutants. Under DMSO treatment, the double mutants exhibited a severe fertility defect (Fig. 2E), producing significantly fewer progeny than either WT (13 ± 7 vs. 250 ± 49, p < 0.0001) or lin-45(V627E) single mutants (13 ± 7 vs. 241 ± 59, p < 0.0001). Similar to lin-45(V627E) single mutants, the double mutants remained fertile at 1 μM trametinib but became sterile at 10 μM (Fig. 2E). Notably, while 100 μM trametinib partially restored fertility in lin-45(V627E) single mutants (63% of DMSO controls), this rescue was substantially impaired in the double mutants, with brood size reduced to 12% of DMSO controls (2 ± 2 vs.13 ± 7; Fig. 2E). These findings indicate that reduced EGL-30/Gαq activity compromises the resistance phenotype, supporting a critical role for Gαq signaling in mediating high-dose trametinib resistance.

EGL-30/Gαq Signaling Promotes Trametinib Resistance in C. elegans through Its Canonical Downstream Pathway.

To further explore how EGL-30/Gαq signaling contributes to trametinib resistance, we examined its canonical downstream signaling cascade. Gαq typically activates phospholipase Cβ (PLCβ), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). DAG subsequently activates protein kinase C (PKC), which phosphorylates a range of downstream targets, including C-Raf, thereby initiating MAPK pathway activation (Kolch et al., 1993). Based on this well-established signaling cascade, we hypothesized that the EGL-30/Gαq-PLCβ-PKC axis enables lin-45(V627E) mutants to bypass MEK inhibition and maintain MAPK activity under high-dose trametinib treatment, thereby enabling lin-45(V627E) worms to retain fertility.

To test this, we treated lin-45(V627E) adults with either the PLCβ inhibitor U73122 or the PKC inhibitor Sotrastaurin, alone or in combination with trametinib. Sotrastaurin, currently in Phase II clinical trials, demonstrates minimal off-target activity even at elevated concentrations (Klaeger et al., 2017). Treatment with 50 μM U73122 or 50 μM Sotrastaurin alone had minimal effects on fertility compared to DMSO controls (U73122: 240 ± 32 vs. 241 ± 59, p = 0.33; Sotrastaurin: 222 ± 40 vs. 241 ± 59, p = 0.01; Fig. 2F). In contrast, co-treatment with 100 μM trametinib and either inhibitor resulted in complete sterility (Fig. 2F), phenocopying the genetic suppression of egl-30(R25C). Together, these findings establish the EGL-30/Gαq-PLCβ-PKC signaling axis as a critical mediator of adaptive resistance to MEK inhibition, and highlight it as a potential therapeutic target in MAPK-driven malignancies.

Combined Inhibition of Gαq-PLCβ-PKC Signaling and MEK Synergistically Suppresses Proliferation of BRAFV600E Melanoma Cell Lines.

To assess whether the resistance mechanism identified in C. elegans is conserved in human cancer cells, we investigated whether inhibition of the Gαq-PLCβ-PKC pathway enhances the sensitivity of BRAFV600E melanoma cells to MEK inhibition. We first performed shRNA-mediated knockdown of two Gαq paralogs, GNAQ and GNA11, in A375 melanoma cells harboring the BRAFV600E mutation (Fig. 3C). While knockdown of GNAQ/GNA11 had minimal effect on basal proliferation (Fig. 3A), it markedly reduced the IC50 of trametinib in A375 cells (0.14 nM vs. 0.04 nM and 0.04 nM, p < 0.01; Fig. 3B). Knockdown of GNAQ/11 markedly reduced p-ERK levels, indicating that Gαq is required for ERK phosphorylation (Fig. 3D). Thus, loss of Gαq signaling potentiated trametinib sensitivity, indicating a synergistic interaction between MEK inhibition and Gαq blockade.

Figure 3. Synergistic inhibition of A375 cell proliferation by Gαq/11–PLCβ–PKC blockade and MEK inhibition.

Figure 3.

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(A) A375 cells transduced with empty vector control (Ctrl) or shRNA targeting both GNAQ and GNA11 (shGαq/11) were treated with vehicle or trametinib for 72 h. Cell viability was measured by CellTiter-Glo, and dose-response curves are shown (mean ± SD, n = 3 independent experiments).

(B) IC50 values of trametinib in A375 cells transduced with Ctrl or shGαq/11. n = 3, ** P < 0.01.

(C) Knockdown efficiency of A375 cells transduced with shGαq/11 was verified by western blot.

(D) Western blot analysis of indicated proteins in A375 cells transduced with Ctrl or shGαq/11 treated with DMSO or trametinib at the indicated concentrations for 24 h. A representative blot (left) ( n = 3 independent experiments ) and quantification of pERK (right) are shown, ** P < 0.01, **** P < 0.0001, ns: not significant.

(E) Dose-response curves of viability (left) and Chou-Talalay combination index (CI) diagram (right) of A375 cells treated with U73122 in combination with trametinib at the indicated concentrations for 72 h (mean ± SD, n = 3). CI < 0.3, strong synergy (red); 0.3–0.9, synergy; 0.90–1.1, additive; ≥ 1.1, antagonism (blue).

(F) Dose-response curves of viability (left) and CI diagram (right) of A375 cells treated with sotrastaurin (Sotra) in combination with trametinib at the indicated concentrations for 72 h (mean ± SD, n = 3).

(G) Western blot analysis of indicated proteins in A375 cells treated with DMSO, or 10 μM sotrastaurin (Sotra) or 5 μM U73122, alone or with trametinib at the indicated concentrations for 24 h. A representative blot (left) ( n = 3 independent experiments ) and quantification of pERK (right) are shown, * P < 0.1, ** P < 0.01.

We next assessed pharmacological inhibition of the Gαq-PLCβ-PKC pathway in combination with trametinib. Co-treatment with trametinib and the PLCβ inhibitor U73122 or the PKC inhibitor sotrastaurin produced robust synergistic effects on A375 cell proliferation across a broad range of concentrations (Fig. 3E–F). Drug interactions were quantified using the Chou-Talalay combination index (CI) (Chou and Talalay, 1984). Notably, in most dose combinations, CI values for trametinib-U73122 and trametinib-sotrastaurin were <0.3 (Fig. 3E–F), further supporting strong synergy between MEK inhibition and suppression of the Gαq-PLCβ-PKC pathway. Consistent with these effects on proliferation, trametinib alone reduced ERK phosphorylation, while combined treatment produced a more pronounced and synergistic suppression of ERK activation (Fig. 3G). These findings in human melanoma cells parallel the genetic interactions observed in lin-45(V627E) nematodes, highlighting the evolutionary conservation of this resistance mechanism.

Combining Trametinib with PLCβ/PKC Inhibitors Suppresses Tumor Growth in Vivo.

To assess the in vivo efficacy of combined MEK and PLCβ/PKC inhibition, we established A375 xenografts in nude mice. Tumors in vehicle-treated animals grew rapidly, whereas monotherapies with trametinib, U73122, or sotrastaurin significantly delayed tumor progression relative to vehicle controls. Notably, combination treatments (U73122 + trametinib and sotrastaurin + trametinib) resulted in markedly enhanced tumor suppression compared to either agent alone (Fig. 4), indicating a synergistic anti-tumor effect in vivo. Importantly, treatment with trametinib, sotrastaurin, or their combination had no discernible effect on body weight, suggesting good tolerability. In contrast, mice receiving U73122 alone or in combination with trametinib exhibited a modest but measurable reduction in body weight, indicating potential systemic effects at the tested dose (Fig. S2).

Figure 4. Combined PLCβ-PKC and MEK inhibition synergistically suppresses tumor growth.

Figure 4.

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(A) Tumor volumes of mice measured at different time points after treatment. Mice bearing parental A375 xenografts were randomized into four groups (n = 12/group) and treated with vehicle, 50 mg/kg U73122, 80 mg/kg sotrastaurin (Sotra), 0.2 mg/kg trametinib (Tram), or the indicated combinations when average tumor size reached 250 mm3. Data represent mean ± SEM; some error bars are too small to be visible. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test. **** P < 0.0001.

(B) Tumor weights at the end of treatment. Mice treated with vehicle, U73122, Sotra, Tram, or the indicated combinations were euthanized on days 8, 10, or 18 as indicated, and tumors were removed and weighed. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

(C) Representative tumor photographs from each treatment group collected at the end of treatment.

Blockade of Gαq-PLCβ-PKC Reverses Acquired Trametinib Resistance in Melanoma.

To determine whether Gαq-PLCβ-PKC activation is necessary for the maintenance of acquired trametinib resistance, we established polyclonal trametinib-resistant A375 cell lines (A375-TR) through prolonged exposure to increasing concentrations of the drug (Fig. 5A). As expected, parental A375 cells were highly sensitive to trametinib (IC50 = 0.5 nM), whereas A375-TR cells displayed a significantly higher IC50 (266 nM, p = 0.01, Fig. 5B). Under high concentrations of trametinib treatment, A375-TR cell line retained higher levels of phosphorylated ERK compared to the parental cells (Fig. 5C).

Figure 5. Synergistic inhibition of trametinib-resistant A375 (A375-TR) cell proliferation by Gαq/11–PLCβ–PKC blockade and MEK inhibition.

Figure 5.

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(A) Parental A375 and A375-TR cells were treated with vehicle or trametinib for 72 h. Cell viability was measured by CellTiter-Glo, and dose-response curves are shown (mean ± SD, n = 3 independent experiments).

(B) IC50 values of trametinib in A375 and A375-TR cells. n = 3, ** P < 0.01.

(C) Western blot analysis of indicated proteins in A375 and A375-TR cells treated with DMSO or trametinib at the indicated concentrations for 24 h. A representative blot (left) (n = 3 independent experiments) and quantification of pERK (right) are shown, * P < 0.1.

(D) A375-TR cells transduced with empty vector control (Ctrl) or shRNA targeting both GNAQ and GNA11 (shGαq/11) were treated with vehicle or trametinib for 72 h. Cell viability was measured by CellTiter-Glo, and dose-response curves are shown (mean ± SD, n = 3 independent experiments).

(E) IC50 values of trametinib in A375-TR cells transduced with Ctrl or shGαq/11. n = 3, **p < 0.01, *** P < 0.001.

(F) Knockdown efficiency of A375-TR cells transduced with shGαq/11 was verified by western blot.

(G) Western blot analysis of indicated proteins in A375-TR cells transduced with Ctrl or shGαq/11 treated with DMSO or trametinib at the indicated concentrations for 24 h. A representative blot (left) (n = 3 independent experiments) and quantification of pERK (right) are shown, ** P < 0.01, **** P < 0.0001.

(H) Dose-response curves of viability (left) and Chou-Talalay combination index (CI) diagram (right) of A375-TR cells treated with U73122 in combination with trametinib at the indicated concentrations for 72 h (mean ± SD, n = 3). CI < 0.3, strong synergy (red); 0.3–0.9, synergy; 0.90–1.1, additive; ≥ 1.1, antagonism (blue).

(I) Dose-response curves of viability (left) and CI diagram (right) of A375-TR cells treated with sotrastaurin (Sotra) in combination with trametinib at the indicated concentrations for 72 h (mean ± SD, n = 3).

(J) Western blot analysis of indicated proteins in A375-TR cells treated with DMSO, 10 μM sotrastaurin (Sotra) or 5 μM U73122, alone or with trametinib at the indicated concentrations for 24 h. A representative blot (left) (n = 3 independent experiments) and quantification of pERK (right) are shown, ** P < 0.01, *** P < 0.001.

Knockdown of GNAQ/GNA11 significantly reduced the IC50 of trametinib and ERK activationin A375-TR cells (576 nM vs. 128 nM and 255 nM, p < 0.01; Fig. 5D–G), indicating that suppression of Gαq signaling restores drug sensitivity. Treatment of A375-TR cells with either the PLCβ inhibitor U73122 or the PKC inhibitor sotrastaurin substantially reduced cell viability (Fig. 5H–I). Notably, co-treatment with trametinib further potentiated the anti-proliferative effects of both inhibitors (Fig. 5H–I), consistent with a synergistic interaction. Across most dose combinations, the CI values for trametinib-U73122 and trametinib-sotrastaurin were <0.3 (Fig. 5H–I), confirming strong synergy. Combined treatment resulted in a marked and potent suppression of ERK activation in A375-TR cells (Fig. 5J). These findings indicate that sustained Gαq-PLCβ-PKC signaling is critical for maintaining the resistant phenotype and that pharmacological blockade of this axis can restore trametinib sensitivity in resistant melanoma cells.

Discussion

Resistance to MAPK pathway inhibitors remains a major barrier to durable responses in BRAFV600E melanoma. Here, we identify a conserved Gαq-PLCβ-PKC signaling axis as a critical mediator of trametinib resistance. Using a genetically tractable C. elegans model carrying the lin-45(V627E) mutation, functionally equivalent to human BRAFV600E, we show that Gαq signaling enables adaptive MAPK reactivation under MEK inhibition. Importantly, this adaptive mechanism is conserved in human BRAFV600E melanoma, where pharmacological blockade of the Gαq-PLCβ-PKC axis restores trametinib sensitivity in cell lines and xenografts. These findings reveal a previously unrecognized resistance circuit and provide a translationally actionable strategy for improving the efficacy of MEK inhibition.

The cross-species conservation of this resistance mechanism highlights the power of C. elegans as a model for cancer drug resistance. Worms provide a rapid, whole-organism platform for genetic and pharmacologic interrogation, enabling the discovery of signaling pathways that are not easily predicted from human data alone. C. elegans has been successfully employed as an in vivo platform for screening inhibitors of the MAPK/ERK pathway (Bae et al., 2012; Gorgoń et al., 2024; Hara and Han, 1995; Ji et al., 2019). Our work advances this field by demonstrating that worms can also faithfully model acquired resistance to targeted therapies. Through whole-organism genetics, we uncovered the Gαq-PLCβ-PKC pathway as an unrecognized resistance mechanism, subsequently validated in human BRAFV600E melanoma.

Previous reports have shown that oncogenic GNAQ mutations confer intrinsic resistance to BRAF inhibition in melanoma (Maziarz et al., 2018; Turajlic et al., 2014). Our findings extend this paradigm by demonstrating that Gαq signaling also underlies acquired resistance to MEK inhibition, with the PLCβ-PKC module as the operative effector. Thus, Gαq signaling emerges as a shared mediator of both intrinsic and acquired resistance, reinforcing its clinical relevance and supporting combined MEK-PKC targeting in BRAFV600E melanoma.

Our data highlight the functional versatility of Gαq-PKC signaling across melanoma subtypes. In uveal melanoma, activating GNAQ/GNA11 mutations establish this pathway as a lineage-defining oncogenic driver, and combined Gαq-PKC and MEK inhibition synergistically induces potent antitumor effects (Chen et al., 2014; Hitchman et al., 2021; Ma et al., 2021). By contrast, in BRAFV600E cutaneous melanoma, the same pathway is co-opted as an adaptive bypass to sustain ERK signaling during MEK blockade. This convergence underscores the broad therapeutic relevance of Gαq-PKC signaling while emphasizing its context-dependent biology: driver dependence in uveal melanoma versus resistance circuitry in cutaneous melanoma.

Therapeutically, our findings provide a strong rationale for co-targeting MEK and the Gαq-PLCβ-PKC axis in BRAFV600E melanoma. Both genetic and pharmacologic inhibition of this pathway produced robust synergy with trametinib, suppressed ERK reactivation, and restored drug sensitivity in resistant cells. PKC inhibitors such as sotrastaurin, which are already in clinical evaluation, demonstrated particularly favorable combinatorial activity, supporting the feasibility of rapid translational development. Given the limited durability of current BRAFi/MEKi regimens, integrating Gαq-PKC blockade represents a promising avenue to overcome resistance and extend progression-free survival.

In conclusion, our work establishes a novel experimental pipeline that begins with mechanistic discovery in C. elegans and extends to translational validation in human melanoma. By identifying Gαq-PLCβ-PKC signaling as a conserved resistance mechanism and therapeutic target, we provide preclinical evidence for a new combination therapy strategy in BRAFV600E melanoma. Future studies should explore the broader applicability of this approach in other MAPK-driven malignancies.

Materials and methods

Worm strains and culture

C. elegans were maintained according to the standard methods (Brenner, 1974). All worms were cultivated at 20 °C on nematode growth medium (NGM) plates seeded with the Escherichia coli OP50 unless stated otherwise. Adult hermaphrodite worms were used in the live-cell imaging experiments. The wild-type strain was Bristol N2. Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

Forward genetic screens

We used forward genetic screens to isolate lin-45 (V627E) suppressors. The lin-45 (V627E) mutant animals (P0) were synchronized at the late L4 larval stage, collected in 4 mL M9 buffer, and incubated with 50 mM ethyl methanesulfonate (EMS) for 4 hours at room temperature with constant rotation. Animals were then washed with M9 three times and cultured under standard conditions. After 20 hours, adult animals were bleached. Eggs (F1) were distributed and raised on ~800 9-cm NGM plates, each containing 50 to 100 eggs. Adult F2 animals on each plate were screened. Mutant animals with normal vulva were individually cultured, and their progenies were further observed. We identified mutations using whole-genome sequencing and mapped egl-30 (R25C) on chromosome I. We confirmed gene cloning using multiple alleles and rescue experiments.

Brood Size Assay

Synchronized L4 worms or dauer-stage worms were transferred singly to NGM plates with E. coli OP50 bacteria cultured with Luria-Bertani broth. Worms were repeatedly transferred to a freshly seeded NGM plate every 24 h until reproduction ceased. We counted the number of progeny at their L3/L4 stages. Our measured WT brood size is consistent with the previous results. Total brood sizes were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test (GraphPad Prism 10). Data are presented as mean ± SEM, and p < 0.05 was considered statistically significant. Each data point represents an individual worm, with experiments repeated independently at least three times.

Genome editing

CRISPR-Cas9 constructs were purified with AxyPrep Plasmid Purification Miniprep Kit (Axygen, #AP-MN-P-250) and PureLink Quick PCR purification Kit (Invitrogen, #K310001) and co-injected into the gonads of young adult worms with the rol-6 (su1006) selection marker. F1 transgenic progenies were singled and screened by PCR. All variations and deletions were verified by the Sanger sequencing of the entire genes, and no other mutations were inserted on the target genes.

Microinjection and transgenic strains

For transgenic C. elegans experiments, transgenic lines were generated by injecting the DNA constructs into the gonads of the indicated worm strains. The co-injected selection marker was rol-6(su1006). At least two independent transgenic lines with the constant transmission rate (>50%) were propagated. Concentrations of DNA constructs used for generating knock-in were 50 ng/μl, and for overexpression was 20 ng/μl.

Cell culture and treatment

The human melanoma cell line A375 was a gift from Prof. Jiang Peng (Tsinghua University) and was cultured in DMEM (Gibco) containing 10 % fetal bovine serum (Gibco) and 1 % penicillin and streptomycin (Yeasen) at 37 °C with 5 % CO2. A375-TR (A375-trametinib resistant) cell lines were established from A375 cells by chronic treatment with gradually increasing concentrations of trametinib and were selected for monoclones using the limiting dilution method.

Short hairpin RNA (shRNA) constructs targeting human GNA11 and GNAQ were designed and generated via molecular cloning. Two target sequences were used: shRNA#1, 5′-CCGGAGCTCAAGCTGCTGCTGCTCGCTCGAGCGAGCAGCAGCAGCTTGAGCTTTTTTG-3′, and shRNA#2, 5′-CCGGACCCCTGGTTCCAGAACTCCTCTCGAGAGGAGTTCTGGAACCAGGGGTTTTTTG-3′. A non-targeting control shRNA was purchased from Thermo Fisher Scientific. Lentiviral particles were produced and used to infect A375 and A375-TR melanoma cells. Stable cell lines expressing shRNA were selected according to the manufacturer’s instructions using puromycin and maintained under selection for downstream experiments.

Cell viability assays and combination index calculation

The Cell-ATP Viability Detection Kit (HY-K0302; MedChemExpress) was adopted to determine the Cell viability of each group. Cells were incubated with serial dilutions of drugs alone or two-drug combinations in complete culture medium for 72 h. At least three independent experiments were performed. The curve-fitting software, GraphPad Prism version 10 (GraphPad Software, SanDiego, CA, USA), was used to calculate half-maximal inhibitory concentration values. Combination index (CI) values were calculated using the Chou-Talalay method implemented in the synergy Python package (Wooten and Albert, 2021).

Clonogenic assay

Cells were seeded in six-well plates at low density (100 cells/well) and cultured with drugs for 10 days, after which colonies were fixed with methanol and stained with 1× crystal violet. The quantification of the number of colonies and average colony area were analysed by using ImageJ.

Mouse xenografts and in vivo drug studies

All animal experiments were approved by the Animal Care and Use Committee at Tsinghua University. BALB/c-nude mice were housed in a temperature-controlled environment at around 21 °C with a 12 hr light/12 hr dark cycle and free access to food and water. 5×106 A375 BRAFV600E melanoma cells were resuspended in PBS were injected subcutaneously into the flanks of 5-week-old immunodeficient BALB/C-nude female mice (at least 6 mice per group). Tumor formation was monitored twice a week, and tumor volume based on calliper measurements was calculated by the modified ellipsoidal formula (tumor volume = 1/2(length × width2)). When tumors reached a volume of approximately 250 mm3, mice were randomized into four treatment arms: no drug, MEK inhibitor trametinib only, PLCβ inhibitor U73122/PKC inhibitor sotrastaurin only or the combination of trametinib and U73122/sotrastaurin. 100 mg/kg Sotrastaurin (#HY-10343, MCE) or 0.2 mg/kg trametinib (#C16249920, MACKLIN) were formulated in a vehicle consisting of 5% DMSO (#196055, MP Biomedicals), 40% PEG300 (#HY-Y0873, MCE), 5% Tween-80 (#HY-Y1891, MCE), and 50% sterile saline solution (v/v). Drugs were dissolved in this vehicle and administered via oral gavage at a volume of 100 μL per mouse. Treatment was performed three times per week. 50 mg/kg U73122 (#HY-13419, MCE) was ground into a fine powder and suspended in sterile saline to prepare a homogeneous suspension. Mice received 100 μL of the suspension via intraperitoneal injection. Treatment was performed three times per week. Control animals received either the vehicle solution or sterile saline alone, following the same route and dosing schedule as the corresponding treatment groups.

Western Blot Analysis

Cells were washed three times with cold phosphate-buffered saline (PBS) to remove residual medium and debris. After washing, cells were lysed using RIPA lysis buffer (#C0121, Beyotime) supplemented with complete protease inhibitors and phosSTOP phosphatase inhibitors (Roche). Lysates were clarified by centrifugation at 14,000 × g for 15 minutes at 4 °C to remove insoluble debris. The supernatants were collected and stored at −80 °C until further analysis.

5× SDS loading buffer (#20315ES20, Yeasen) and boiled for 5 min at 95 °C for Western blotting. Proteins were separated by FastPAGE precast gel (#TSP024–15, TSINGKE) electrophoresis and transferred to a polyvinylidene fluoride membrane (#B36011, ESAYBIO). The membrane was blocked by 5% BSA and incubated overnight with the primary antibodies at 4 °C. After washing with 10× TBST buffer containing 5‰ Tween 20 (#T1081, Solarbio) three times, the membrane was incubated with the secondary antibodies for 1 h on a shaker. Protein bands were visualized by chemiluminescence using the SuperSignal West Dura (Thermo Fisher Scientific, 34076).

The following antibodies were used: alpha-Tubulin (DM1A) Mouse mAb (#3873T, CST), anti-mouse IgG HRP-linked Antibody (#7076P2, CST), anti-rabbit IgG, HRP-linked antibody (#7074P2, CST), Galpha(q) (D5V1B) rabbit mAb (#14373S, CST), p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (#4695T, CST), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb (#4370T, CST).

RNA sequencing

Synchronized worms were cultured on NGM plates with the OP50 bacteria. Day 1 adult worms were harvested 24 hours after the fourth larval (L4) stage and then lysed with TRIzol reagent (Invitrogen). For cell lines, after treatment with serial dilutions of drugs alone or two-drug combinations in complete culture medium for 24 h, A375 or A375-TR cells were lysed with TRIzol reagent (Invitrogen). Total RNA was extracted according to the manufacturer’s protocol. SUPERase•InTM RNase Inhibitor (Invitrogen) was used in each step to prevent RNA from degradation. RNA concentration was quantified using the Qubit RNA High Sensitivity Assay Kit (Invitrogen). RNA quality was assessed with the Agilent 2100 bioanalyzer system, and samples with an RNA integrity number (RIN) above 6.0 were used to construct the library, 50 ng to 500 ng of total RNA was used for library preparation using the KAPA RNA HyperPrep Kit (KAPA Biosystems). Library samples were analyzed by Agilent 2100 bioanalyzer system and Qubit for quality control and quantification. The samples were sequenced on an Illumina HiSeq platform. A total of 150-bp paired-end reads were generated.

Molecular biology

We followed the established protocols to perform the genome editing experiments in C. elegans (Dickinson et al., 2013; Friedland et al., 2013). We used the CRISPR design tool (https://zlab.bio/guide-design-resources) to select the target sequence. The sgRNA sequences were inserted to the pDD162 vector (Addgene #47549) by linearizing this vector with 15 bp overlapped primers. The resulting PCR products containing 15 bp overlapped double-strand DNA ends were treated with DpnI (NEB, #R0176V) digestion 4 hours and transformed into E. coli. The linearized PCR products were cyclized to generate plasmids by spontaneous recombination in bacteria.

Short hairpin RNAs (shRNAs) targeting GNA11 and GNAQ were designed based on published sequences. Oligonucleotides encoding the hairpin sequences were synthesized, annealed, and cloned into the vector pLKO.1 plasmid downstream of the U6 promoter via standard molecular cloning techniques following standard protocols. Ligated products were transformed into E. coli DH5α, and positive clones were confirmed by colony PCR and Sanger sequencing.

Imaging

Adult C. elegans hermaphrodites were anesthetized using 1 μM levamisole in M9 buffer, mounted on 3% agarose pads, maintained at room temperature and imaged immediately. A Zeiss LSM900 confocal microscope equipped with a 100×/1.46 oil objective and controlled by ZEN software (Carl Zeiss) was utilized. All images were acquired using a Zeiss Axio Observer (bright-field mode).

Supplementary Material

Supplement 1

Acknowledgements

This work was supported by the National Key R&D Program of China Grants 2022YFA1302700, 2024YFA1307301; and National Natural Science Foundation of China Grants 32270773 (W.L.), 32470730 (W.L.),992254306 (G.O.), 32430026 (G.O.), 32021002 (G.O.) and 31991191 (G.O.).

Footnotes

Competing interests: G.O., W.L., J.K., and Z.W. are inventors on patent application no. CN202511508363.0, submitted by Tsinghua University, which covers the combined use of MEK inhibitors and PLCβ/PKC signaling pathway inhibitors and their application in the treatment of BRAFV600E mutant tumors.

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Supplement 1
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