BRAF inhibitor resistance in melanoma: from resistance mechanisms to therapeutic innovations

Abstract

BRAF inhibitors (BRAFi) have transformed the treatment of BRAF mutant melanoma, but inherent and acquired resistance remains a major barrier to curative outcomes. Resistance arises from interconnected mechanisms: genetic alterations reactivating the MAPK pathway or bypass cascades (e.g., PI3K/AKT/RTK), epigenetic modulation, metabolic reprogramming, and the tumor microenvironment (TME) remodeling. Despite extensive research into these mechanisms, a cohesive framework linking each resistance module to targeted therapeutic strategies is lacking. This review systematically categorizes resistance into intrinsic and acquired subtypes: intrinsic resistance is driven by constitutive molecular traits of BRAF mutant melanoma (e.g., persistent MAPK activation, baseline PI3K/AKT hyperactivity), while acquired resistance emerges via therapeutic pressure-induced genetic mutations, epigenetic shifts, metabolic reprogramming, or TME modifications. For each identified resistance mechanism, we provide a detailed examination of corresponding therapeutic advancements. These encompass the development of next-generation BRAFi, strategically designed combination therapies, epigenetic modulators, immunotherapeutic approaches, and RNA-based therapeutic agents. Furthermore, we underscore the pivotal role of state-of-the-art technologies, such as liquid biopsies, single-cell multi-omics analyses, and artificial intelligence, in facilitating precise resistance monitoring and personalized therapy selection. By integrating these insights, we present a structured, translationally focused framework to guide basic research and clinical decision-making, ultimately advancing precision salvage therapy and trials aimed at preventing or overcoming BRAFi resistance.

Introduction

Metastatic melanoma has historically represented a formidable therapeutic challenge, characterized by its aggressive biology and refractoriness to conventional chemotherapy, which resulted in a dismal prognosis for affected individuals [1, 2]. The discovery that activating BRAF mutations are harbored by approximately 50% of cutaneous melanomas, alongside their prevalence in 40% of papillary thyroid carcinomas and 10% of colorectal cancers, established BRAF-mutant malignancies as the inaugural paradigm for genotype-directed therapy in solid tumors [3–5]. This foundational insight catalyzed the development and widespread clinical deployment of selective BRAF inhibitors (BRAFi) for the management of BRAF-mutated melanoma [6, 7], thyroid carcinoma [8, 9], and colorectal carcinoma [10, 11]. The regulatory approval of vemurafenib, followed by dabrafenib and encorafenib, has cemented the combination of BRAF and MEK inhibition (BRAFi ± MEKi) as the standard-of-care for metastatic disease, yielding objective response rates exceeding 60% and conferring the first substantial extension in overall survival [12, 13]. Beyond their canonical on-target effects, agents such as vemurafenib, dabrafenib, and binimetinib have been shown to exert ancillary immunomodulatory functions that contribute to prolonged progression-free survival in melanoma [14–16]. Furthermore, structural elucidation of the mechanisms by which RAS-driven paradoxical MAPK activation can compromise first-generation inhibitors has propelled the rational design of next-generation allosteric inhibitors and quinazoline-based analogues with enhanced potency and selectivity [17].

Notwithstanding these transformative therapeutic advances, the attainment of durable clinical benefit remains an unmet need for a significant proportion of patients [18]. Disease progression, driven by the emergence of therapeutic resistance, typically ensues within 6–12 months of treatment initiation [19, 20]. This pervasive limitation arises from a complex interplay of intrinsic tumor properties and adaptive, acquired resistance mechanisms forged under the selective pressure of therapy, encompassing genomic instability, epigenetic plasticity, metabolic reprogramming, and dynamic crosstalk with the tumor microenvironment. A bibliometric analysis spanning the period from 2003 To 2024 underscores the exponential expansion of research in this domain, with leading contributions from the United States, China, and Italy, and reveals a strategic pivot in scientific inquiry from the initial characterization of BRAF mutations and MAPK pathway reactivation toward the exploration of synergistic immunotherapy combinations, novel therapeutic regimens, and the roles of non-apoptotic cell death modalities [21]. To synthesize this rapidly evolving and multifaceted body of knowledge, we present this comprehensive review.

This review aims to develop a unified, translationally oriented framework that not only maps the molecular architecture of BRAFi resistance but also directly couples each resistance mechanism to its rationally derived therapeutic strategy (Table 1). We commence by delineating the molecular underpinnings of intrinsic resistance, which is predicated upon constitutive features of the tumor that confer baseline insensitivity. Subsequently, we deconstruct acquired resistance into four interconnected and non-mutually exclusive dimensions: genetic alterations such as secondary mutations and bypass track activation; non-genetic reprogramming mediated through epigenetic modifications and transcriptional network rewiring; adaptive metabolic shifts that support tumor survival under drug pressure; and reciprocal remodeling of the tumor microenvironment that fosters a protective niche. For each of these mechanistic categories, we provide a detailed appraisal of emerging salvage strategies, ranging from advanced targeted combinations and novel small-molecule inhibitors to epigenetic modulators, metabolic interventions, and innovative immunotherapeutic approaches. Finally, we highlight the pivotal role of cutting-edge technologies including liquid biopsy-based circulating free DNA (cfDNA) analysis, single-cell multi-omics, and artificial intelligence in enabling real-time, dynamic monitoring of resistance evolution to inform precision-guided therapeutic decision-making. Collectively, these insights underscore that overcoming the profound heterogeneity and dynamic adaptability of BRAFi resistance requires a therapeutic paradigm that is simultaneously multi-targeted, mechanism-guided, and responsive to real-time tumor evolution.

Table 1.

BRAFi resistance in melanoma: genetic, epigenetic, metabolic, and microenvironmental mechanisms with targeted and emerging therapies

Category	Core Mechanisms	Ref
Intrinsic Resistance	• BRAF-V600E keeps MAPK “on” • ↑PI3K/AKT & RTKs (IGF-1R, EGFR)	[22–25]
Acquired – genetic	• ↑Secondary BRAF/MEK/NRAS • ↑YAP/TAZ • ↓STAG2/3 • ↑TERT-promoter	[24, 26–28]
Acquired – non-genetic	• ↑DNA methylation (PDE4D • ↑cGAS-STING) • ↑SAMMSON • ↑miR-211 • ↑Glutaminolysis • ↑Polyamine	[29–33]
Tumor micro-environment	• ↑CAF IL-6/TNF-α/M-CSF • ↑Lipid-loaded fibroblasts • ↑Soluble CD73	[34–37]
Targeted combinations	• BRAFi + MEKi ± PI3Ki • PROTACs • PARPi • FAKi • Hsp90i	[38–41]
Epigenetic/metabolic	• DNMTi, EZH2i • PDE4Di • Glutaminase • CSE • SREBP blockade	[42–47]
Immuno-combinations	• BRAF/MEK + PD-1 blockade • HDACi or STAT3i to restore T-cell influx	[48–52]
Emerging technologies	• ↑cfDNA tracks BRAF-V600E • scRNA-seq maps resistant clones • AI predicts combos; early imaging guides switch	[53–59]

Intrinsic resistance to BRAF inhibitors in melanoma: molecular basis and clinical implications

MAPK signaling pathway dysregulation in BRAF mutant melanoma

BRAF encodes a serine/threonine kinase, the core activator of the MAPK/ERK pathway [1, 60]. It is central to the regulation of cell proliferation, differentiation, migration and survival [61, 62]. Mutations in the BRAF gene (predominantly in exons 11 and 15) occur in ~ 7% of all cancers, with the V600E variant (exon 15) being the most common [63]. Mutations in the BRAF gene lead to the constitutive activation of the BRAF protein, driving normal cells to undergo continuous division and potentially culminating in oncogenesis [64]. BRAF mutations are in ~ 7% of cancers, including 100% hairy cell leukemia, 50–60% melanoma, 30–50% papillary thyroid cancer, 10–20% colorectal cancer, and 3–5% NSCLC [65]. BRAF V600E renders the kinase RAS-independent, constitutively activating downstream RAS/RAF/MEK/ERK signaling and promoting tumor proliferation, invasion, and metastasis [66].

Monomeric BRAF V600E, the canonical class I mutant, operates independently of RAS and drives high-level ERK signaling [67]. Consequently, in BRAF V600E-mutant tumors, BRAF V600E exists predominantly as a drug-sensitive monomer. Current RAF inhibitors selectively target BRAF monomers, exhibiting substantially weaker inhibition of RAF dimers [22]. BRAF V600E alone remains inhibitor-sensitive, but resistance arises when elevated RAS-GTP drives mutant kinase dimerization with wild-type BRAF or CRAF [68]. The formation of BRAF mutant dimers represents another significant mechanism underlying aberrant signaling pathway transduction in the BRAF pathway. Elevated BRAF dimerization not only confers increased drug resistance but also operates independently of upstream RAS activation, perpetuating downstream RAF/MEK/ERK signaling [69]. Apart from BRAF dimerization, RAS gene mutations can likewise induce aberrant activation of the BRAF signaling pathway. Activated RAS mutations can coexist with hypoactive or kinase-inactivated BRAF mutants. These BRAF mutants, whether hypoactive or kinase-inactivated, activate the MEK/ERK signaling pathway in a RAS-dependent manner, promoting a malignant phenotype in tumor cells [23]. CEP55 drives acral melanoma progression via MAPK activation, causing BRAFi resistance, yet its overexpression predicts favorable immunotherapy responses, making it a therapeutic target [70]. In summary, BRAF gene mutations drive tumor progression via two mechanisms: independent activation (single mutations, mutant dimers) and RAS co-mutation, both mechanisms promote a malignant tumor phenotype. Consequently, the clinical investigation and application of BRAFi can attenuate the malignant progression of tumors by inhibiting the aberrant activation of BRAF-mediated signaling pathways.

Clinical efficacy, heterogeneity, and limitations of BRAF inhibitors in BRAF mutant melanoma

BRAFi rechallenge after progression yielded significant survival benefit in a BRAF V600 melanoma patient, suggesting that loss of evolutionary advantage in resistant clones restores cancer cell sensitivity to therapy [71]. The current clinical use of BRAFi is predominantly in combination with MEK inhibitors (MEKi) [72–74]. Dabrafenib and trametinib became the first FDA-approved targeted therapy on March 16, 2023, for systemic treatment of pediatric BRAF V600E low-grade glioma [75]. In clinical practice, BRAFi have demonstrated strong efficacy and a tolerable safety profile for BRAF V600E–mutant non-small cell lung cancer and glioma, with side effects that remain manageable [76–78]. These results have led the FDA to grant dabrafenib breakthrough therapy designation [79, 80].

Treating BRAF-mutant tumors effectively calls for either BRAFi alone or combined with MEKi, as demonstrated by SECOMBIT's strategy of using sequential combinations followed by immunotherapy maintenance [81, 82]. Long-term survival is possible for some BRAF-mutant patients. Encorafenib plus binimetinib, approved by the FDA in 2023 for BRAF V600E metastatic NSCLC in adults, illustrates this benefit [83, 84]. Transcriptome analysis reveals FAK activation mediates BRAF/MEKi resistance in melanoma, and FAKi plus avutometinib overcomes this by disrupting MAPK-RhoA-FAK-AKT feedback [38]. However, resistance to BRAFi (monotherapy or combination with MEK inhibitors) remains a significant clinical challenge despite their substantial therapeutic efficacy [85, 86]. For instance, vemurafenib and PLX8394 show off-target effects causing paradoxical endothelial MAPK activation and barrier impairment, unlike dabrafenib or encorafenib. Understanding these off-target effects is crucial for optimizing therapeutic regimens and minimizing adverse events in BRAF mutant melanoma treatment [87]. Vemurafenib and dabrafenib for V600E melanoma downregulate oncogenic RIPK4, a BRAF homolog, via direct binding and ERK1/2 modulation, enhancing efficacy and circumventing resistance via off-target effects [88]. BRAFi resistance leaves melanoma cells with heightened replication stress and an S-phase dependency that can be exploited to overcome MAPK pathway resistance [89]. In summary, BRAFi have made significant clinical headway against BRAF V600E solid tumors. Recent molecular insights from Asian melanoma populations also point toward the necessity of tailored treatments, especially for acral and mucosal forms [90]. However, the growing problem of resistance alongside ongoing drug development efforts has pushed BRAFi resistance reversal to the forefront, and scientists are now working hard to find answers.

Intrinsic resistance stems from the inherent molecular makeup of BRAF mutant melanoma, where constant MAPK activation and baseline bypass signaling create initial drug insensitivity. Most patients who initially respond to BRAFi still progress within 6 to 12 months as acquired resistance develops, an evolutionary adaptation driven by treatment pressure. Tumors remodel their genetic circuits, epigenetic states, metabolic pathways, or microenvironment to escape BRAF blockade, layering these adaptations atop intrinsic traits to form a complex defense that demands thorough mechanistic investigation.

Acquired resistance to BRAF inhibitors: adaptive evolution via genetic, epigenetic, metabolic and the tumor microenvironment remodeling

Acquired resistance develops when tumors initially sensitive to BRAFi lose response during treatment, driven by genetic mutations, epigenetic shifts, metabolic rewiring, or changes in the tumor microenvironment (TME). This differs from intrinsic resistance, which exists prior to therapy. Instead, acquired resistance emerges under the selective pressure of BRAFi itself, allowing drug-tolerant cell populations to survive and expand. We organize these adaptive processes into four interconnected categories: genetic alterations, epigenetic dysregulation, metabolic reprogramming, and tumor microenvironment remodeling.

Genetic mechanisms of adaptive escape

BRAF mutation specific genetic escape routes

MAPK re-activation, achieved either by secondary BRAF alterations, RTK up-regulation or MEK/ERK mutations, remains the dominant escape route in BRAF mutant melanoma (Figs. 1 and 2). Down-stream rewiring via PI3K/AKT or JNK/mTOR is discussed only when genotype-specific [91–93]. Furthermore, a piggyBac screen identified BRAFi resistance genes in melanoma, revealing convergent activation of MAPK, PI3K-AKT, and notably Hippo pathways. Hippo effector TAZ (WWTR1) mediates BRAFi resistance through receptor tyrosine kinase regulation and NEDD4L interactions. This reveals potential targets to overcome resistance [94]. In BRAF V600E melanoma, acquired mutations in COP1 or DET1 stabilize ETV1, ETV4, and ETV5, driving resistance to MAPK inhibitors. This finding underscores the critical role of this E3 ligase in ERK-driven cancers [26]. MAP3K3 phosphorylates YAP at Ser405 to block FBXW7 degradation, sustaining YAP activity that drives BRAF/CDK4/6 inhibitor resistance, and targeting MAP3K3 restores sensitivity by lowering YAP [95]. Persistent BRAF V600E activity drives melanoma as a therapeutic target, causing oncogenic MAPK hyper-activation and intrinsic BRAFi resistance [27, 96]. BRAF mutations dictate metastatic potential and clinical outcome; therefore, precise molecular identification of BRAF alterations is indispensable for risk stratification and targeted therapeutic decision-making [97–100]. Heightened ABL1/2 phosphorylates MAP3K1 and 14–3-3-ε, recruiting MAP4K1 to reactivate MAP3K1-MEK-ERK and re-induce MYC. This MAPK pathway mechanism transcriptionally upregulates MYC to sustain melanoma resistance [101]. ERK5 signaling is amplified in dabrafenib-resistant BRAF V600E melanoma, sustaining proliferation and survival after chronic exposure, offering a target to dismantle acquired resistance [102]. Although WM9 and Hs294T survived BRAF/MEKi, they showed raised ERK/AKT/p38/JNK and altered receptors (EGFR/ErbB2/MET/PDGFRβ up, ErbB3 down). They also displayed elevated drug metabolism and EMT/CSC traits [103]. Of note, androgen receptor is elevated in BRAFi resistant melanoma and drives EGFR and SERPINE1 upregulation to maintain resistance [104]. MAPK alterations (KRAS/BRAF amplification, MEK1 mutations) confer RAF/EGFR or RAF/MEK resistance by sustaining pathway activity, promoting tumor drug resistance [105, 106]. TERT promoter mutations can act synergistically with TPP1 promoter mutations to enhance telomere maintenance and permanence in melanoma, allowing continued melanoma proliferation despite therapeutic intervention [107–110]. This mechanism operates independent of BRAF genotype and has been observed across all melanoma subtypes. Its overexpression reduces the sensitivity of BRAF mutant melanomas to BRAFi and MEKi, promoting the development of drug resistance in these tumors [111, 112]. Upregulation of IGF1R and INSR expression has been observed in BRAF mutant melanoma cell lines that are resistant to BRAFi and MEKi [24, 113]. Patients with BRAF mutations and elevated IGF1R and INSR expression exhibit poorer overall survival [24]. Because comparable IGF1R/INSR overexpression has also been documented in BRAF wild-type melanomas, this represents a pan-genotype bypass pathway. Genomic deletions producing BRAF splice variants conferring RAF-inhibitor resistance are actionable vulnerabilities, while cfDNA detection of BRAF/TERT alterations serves as a prognostic biomarker in melanoma [53, 114–116].

Fig. 1.

MAPK Reactivation and Parallel Bypass Pathways Confer BRAF Inhibitor Resistance in Melanoma

Fig. 2.

Gain-of-function BRAF mutations sustain oncogenic MAPK signaling, attenuating BRAF-inhibitor efficacy and driving intrinsic resistance; concomitant PI3K/AKT-pathway hyper-activation, together with amplified or ligand-stimulated IGF1R, INSR and EGFR, further potentiates resistance in BRAF mutant tumors

Furthermore, loss-of-function mutations in STAG2 or STAG3, adhesion protein complex subunits, occur in BRAF mutant melanoma [117, 118]. The loss of function, particularly of the STAG2 protein, impacts the expression of dual-specificity phosphatase 6 (DUSP6), resulting in resistance to BRAFi [118]. This leads to heightened resistance of melanoma to BRAFi and that is restricted to the BRAF mutant context [118]. BRAF mutant drug-resistant melanoma, the activation of p21-activated kinase (PAK) in cells that have acquired drug resistance reactivates the JNK and mTOR pathways. This bypasses the ERK pathway and inhibits apoptosis, promoting the proliferation of melanoma cells [119]. The convergence of MAPK (via ERK reactivation) and mTORC1 signaling pathways is crucial for driving cyclin D1 protein production, enabling cell cycle reentry and drug escape [120]. Because this PAK-driven bypass has also been observed in NRAS- and NF1-mutant melanomas, it qualifies as a genotype-independent resistance mechanism. The stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) is overexpressed in BRAF mutant melanoma cells [121, 122]. This overexpression confers drug resistance and reprograms signaling from MEK/ERK to PI3K/AKT in BRAF mutant melanoma, promoting malignant progression [123]. Moreover, ALDH1A3-ACSS2 enzymatic coupling in melanoma links acetaldehyde metabolism to histone H3 acetylation of neural crest lineage genes, driving transcriptional heterogeneity and BRAFi resistance via chromatin remodeling [124]. ALDH2 acts as a detoxifying enzyme, and its reduced expression in metastatic melanoma predicts poorer survival. This downregulation also drives resistance to BRAF/MEKi, positioning ALDH2 as a potential therapeutic target [28]. EGFR pathway activation confers BRAFi resistance in vitro and in vivo and activates RTK and AKT in EGFR-activated melanoma cells [125, 126]. This phenotype occurs in both BRAF mutant and wild-type melanomas, indicating a pan-genotype bypass mechanism causing uninhibited proliferation and increased malignant progression [25, 127, 128]. PTRF has been identified as a key contributor to acquired drug resistance in melanoma by upregulating EGFR expression, leading to reduced sensitivity to BRAFi and their combinations with MEKi [129]. Building on recent evidence, in vemurafenib-resistant melanoma cells the EGF/EGFR-YAP1/TEAD2 axis drives nuclear accumulation of YAP1, which transcriptionally up-regulates the endoplasmic reticulum Ca²⁺ sensor STIM1, amplifies store-operated calcium entry and fuels both tumor progression and continued drug resistance [130].

Furthermore, the ATP-Binding Cassette (ABC) transporter family is a major factor mediating drug resistance in tumors [131–133]. It facilitates drug efflux, preventing efficient intracellular drug accumulation and promoting resistance in BRAF mutant melanoma cells. This mechanism is observed in all melanoma subtypes and therefore constitutes a genotype-independent contributor to resistance in BRAF mutant melanoma cells [134, 135]. In the presence of functional ABCG2, the inhibitory effect of vemurafenib on BRAF V600E-mutant melanoma cells is attenuated. The tolerance of tumor cells to the drug is enhanced. These findings suggest that ABCG2 confers resistance to BRAFi in melanoma cells [136, 137]. Vemurafenib enhances ABCB1-mediated drug efflux, increasing resistance and decreasing sensitivity to vemurafenib in BRAF mutant melanomas, because ABCB1 up-regulation has also been reported in BRAF wild-type melanomas treated with chemotherapy or immunotherapy, this represents a pan-genotype efflux-based resistance mechanism [138]. In BRAF V600-mutant melanoma brain metastases, intrinsic vemurafenib resistance is driven by blood–brain barrier P-gp and BCRP limiting drug entry. As shown by superior responses in ABCB1a/b, ABCG2 knockout mice, whereas acquired resistance emerges rapidly and independently of pharmacokinetics or target blockade [139].UBE3C, identified as an E3 ubiquitin ligase for mutant BRAF V600E, facilitates its ubiquitination and degradation, suggesting a novel strategy to overcome secondary resistance to BRAFi like Vemurafenib by targeting UBE3C [140]. Therefore, studies targeting the ABC transporter family represent a potential approach to elucidate the mechanisms of acquired resistance to vemurafenib in BRAF V600E-mutant melanoma cells.

Pan-genotype genetic drivers of cross-resistance

Although our focus is on resistance in BRAF mutant tumors, melanoma cells carrying NRAS, NF1, KIT mutations or triple-WT also exhibit intrinsic or acquired insensitivity to BRAF-targeted therapy [141–145]. We outline these pan-genotypic mechanisms and identify the relationship between these genes and the MAPK signaling pathway, thereby justifying alternative treatment strategies. Patients with NRAS mutant tumors lack a druggable BRAF mutation and show high intrinsic and acquired resistance to MEKi, so they do not benefit from these therapies [146]. In NRAS-mutant melanoma, the MAPK signaling pathway plays a negative feedback role in which ERK is hyperactivated and EGR1 is overexpressed, and this mechanism is regulated by eIF4F [96, 146, 147]. Meanwhile, using a BRAF V600E melanoma patient derived tumor xenograft model, researchers uncovered new acquired resistance drivers GPR39, CD27, SLC15A3, IFI27, PDGFA, and ABCB1, which act via immune modulation, microenvironment shifts or drug efflux and represent fresh targets to combat BRAFi resistance [148]. Furthermore, the emergence of melanoma resistance is associated with KIT mutations and also occurs independently of secondary KIT mutations through reactivation of the MAPK and PI3K/AKT pathways [149]. Updates on BRAFi from 2018–2023 show that RAC1 mutations, PTEN loss, and NF1/CCND1 alterations fuel acquired resistance across cancers, driving the need for next-generation compounds with tighter structure–activity profiles and fewer off-target effects [150]. NRAS, NF1, KIT mutations and triple wild type tumors can mediate resistance through shared mechanisms such as reactivation of the MAPK or PI3K/AKT pathways, providing a theoretical basis for subsequent combination targeted strategies.

BRAF-mutant melanoma typically evades therapy through several mutation-restricted mechanisms, including MAPK pathway reactivation, PI3K/AKT signaling rewiring, upregulation of ABC efflux pumps, and exploitation of ALDH1A1 or STAG2 loss. Concurrently, alterations in RAS, KIT, or triple wild-type tumors provide ancillary reinforcement of MAPK and PI3K signaling across diverse genetic contexts. Therefore, effective strategies should prioritize concurrent blockade of MAPK and PI3K axes, pharmacologic inhibition of ABC transporters, and targeted modulation of epigenetic and microenvironmental cues to surmount resistance and prolong clinical benefit. Beyond genetic alterations, non-genetic mechanisms marked by reversible epigenetic modifications and transcriptional reprogramming are equally crucial in mediating acquired resistance. These processes frequently collaborate with genetic drivers to enhance tumor cell survival under therapeutic pressure from BRAFi.

Non-genetic resistance: epigenetic and transcriptional reprogramming

DNA methylation-mediated resistance

Epigenetic profiling has revealed distinct BRAFi resistance mechanisms, highlighting the critical role of epigenetic plasticity during acquired resistance [29]. Epigenetic alterations contributing to intrinsic resistance mechanisms to BRAFi in BRAF mutant melanoma cells include DNA methylation, noncoding RNA regulation and protein modification [30, 74, 151, 152]. Overexpression of DNA methyltransferase is closely associated with the malignant progression of BRAF mutant melanoma cells [153–155]. It is not only overexpressed in BRAFi-resistant BRAF mutant melanomas but also highly expressed in metastatic melanomas regardless of BRAF status, representing a pan-genotype epigenetic driver [31, 156]. Moreover, demethylation of the CpG island upstream of the phosphodiesterase 4D (PDE4D) gene promoter can lead to increased PDE4D expression in drug-resistant melanoma cell lines. This occurs preferentially in BRAF mutant tumors and enhances RAF1 activation, contributing to increased resistance to BRAFi in that sp-ecific context [42]. DNA methylation remodeling is a key driver of melanoma immune escape [157, 158]. Promoter hypermethylation of cGAS and STING impairs STING signaling, downregulates MHC class I expression, and blunts cytotoxic T cell recognition and killing, thereby fostering immune evasion and resistance [159]. Hypermethylation of circulating cell free SHOX2 DNA in plasma correlates with anti PD-1 response and can serve as an early companion biomarker for melanoma patients receiving anti PD-1 therapy [54]. Elevated DNA methylation of the T cell costimulatory receptor TNFRSF9 is associated with shorter progression free survival, whereas high TNFRSF9 mRNA expression together with low TNFRSF9 methylation predicts longer overall survival, providing a rationale for targeting TNFRSF9 DNA methylation in immunotherapy [160]. Therefore, DNA methylation may represent a promising target for epigenetic and targeted combination therapies (Fig. 3a).

Fig. 3.

Epigenetic Drivers of BRAF-Inhibitor Resistance in Melanoma: DNA Methylation, Non-coding RNAs, and Histone/Ubiquitin Modifications. a DNA Methylation Rewiring in BRAF Mutant Melanoma: from PDE4D RAF1 Reactivation and cGAS STING Immune Escape to TNFRSF9 SHOX2 Liquid Biopsy Biomarkers for Epigenetic Combination Therapy. b Noncoding RNAs comprising microRNAs, circular RNAs and long non-coding RNAs exhibit crosstalk and independently enhance PI3K/AKT and MAPK signaling, thereby driving BRAF-inhibitor resistance in melanoma cells. c Post-translational histone and ubiquitin modifications that confer BRAF inhibitor resistance in melanoma

Non-coding RNA networks

Various non-coding RNAs, from miRNAs to lncRNAs and circRNAs, influence how BRAF mutant melanoma develops and resists BRAFi by regulating Wnt and MAPK pathways, marking them as valuable for both detection and treatment [161]. Resistance to BRAFi mediated by ncRNAs operates through distinct mechanisms that align with three functional themes, each associated with a particular class of ncRNA.

Altered miRNA expression shows a tight association with BRAFi resistance emergence in melanomas carrying BRAF mutations. These miRNAs tune essential signaling pathways and gene programs that keep tumor cells alive and dividing when faced with BRAFi treatment pressure [162–166]. For instance, pigmented melanoma cells resist BRAFi by upregulating miR-192-5p, miR-211-5p, GPR143, and OCA2, which drive melanosome maturation and trafficking, suggesting that pigmentation-based resistance mechanisms represent potential combination targets [167]. Furthermore, certain miRNAs facilitate therapeutic resistance by reactivating the MAPK/PI3K signaling axis. Notably, miR-19a, highly expressed in BRAF mutant melanoma cells, promotes resistance to BRAFi by targeting immunoglobulin-like domain-containing protein 1 (LRIG1), a mechanism specific to the BRAF mutant context [168]. Additionally, miR-19a is abundantly present in exosomes secreted by BRAFi-resistant melanoma cells, which in turn propagate drug resistance to other BRAFi-sensitive melanoma cells [168]. Conversely, let-7a miRNA enhances the synergistic apoptotic effects of dabrafenib and trametinib combination therapy in melanoma cells by targeting caspase-3 [169]. The expression of miR-211 augments melanoma cell resistance to vemurafenib and cobimetinib, a resistance associated with elevated ERK5 phosphorylation, as miR-211 directly inhibits DUSP6, thereby sustaining ERK5 activation to promote proliferation and counteract BRAFi [170, 171]. Moreover, ectopic expression of miR-204-5p and miR-211-5p in untreated human melanoma cells confers resistance to vemurafenib and enhances in vivo tumor growth, with their combined overexpression persistently activating Ras and inducing MAPK upregulation upon vemurafenib exposure, specifically within BRAF mutant melanomas [162]. It is noteworthy that vemurafenib efficacy in melanoma is constrained, and drug resistance arises only when the basal level of miR-211 surpasses that of miR-204, an effect mediated by MITF-dependent pro-pigmentation activity [172]. Recent studies have also highlighted the upregulated expression of specific miRNAs, such as miR-4443 and miR-4488, in drug-resistant melanomas, which promote migratory and invasive phenotypes by downregulating the intermediate filament protein nestin, further exacerbating drug resistance [173]. Conversely, miR-224-5p acts as a tumor suppressor in melanoma by directly targeting PAK4 to inhibit the CRAF/MEK/ERK pathway, thereby reversing acquired resistance to BRAFi [32]. Thus, identifying miRNAs that confer resistance to BRAFi in drug-resistant melanoma could pave the way for mechanism-based strategies to mitigate resistance and enhance clinical outcomes.

The regulation of signal transduction pathways is intricately linked to the development of adaptive drug resistance in BRAF mutant melanomas, with lncRNAs playing a central role. Therapeutic pressure exerted by BRAFi induces alterations in signaling cascades, leading to the activation of compensatory mechanisms that enable tumor cells to evade the inhibitory effects of the drug. These adaptive changes are crucial for tumor cell survival and contribute significantly to the emergence of resistance. LncRNAs are essential for the growth and survival of human BRAF mutant melanoma cells. However, overexpression of certain lncRNAs can specifically mediate resistance of BRAF mutant melanoma to BRAFi within the mutant setting. For instance, overexpression of the lncRNA SAMMSON enhances resistance to RAF inhibitors in BRAF mutant melanoma by modulating CARF-p53 signaling, thereby contributing to adaptive resistance [174, 175]. This highlights the importance of lncRNAs in fine-tuning cellular responses to BRAFi therapy and their potential as therapeutic targets.

Circular RNAs (circRNAs) exhibit significant upregulation in drug-resistant melanoma cells, where they serve as critical signaling modulators that attenuate the sensitivity of melanoma to BRAFi. Mechanistically, circRNAs function as molecular sponges, sequestering multiple microRNAs (miRNAs) and thereby activating various canonical signaling pathways implicated in drug resistance [176–179]. Notably, circFCHO2, an elevated circRNA, directly interacts with DND1 to activate PI3K/AKT signaling, thereby enhancing melanoma aggressiveness in vitro [180]. This underscores the multifaceted role of circRNAs in driving tumor malignancy and drug resistance. In a broader context of ncRNA involvement, the overexpressed lncRNA U73166 has been implicated in mediating RNA processing, cell invasion, and the induction of a more aggressive tumor phenotype, as well as conferring resistance to vemurafenib in BRAF-mutant melanoma cells. However, to maintain a strict focus on circRNAs, it is pertinent to highlight that other, yet-to-be-fully-characterized circRNAs likely play analogous roles in melanoma resistance [181]. The identification and targeted inhibition of circRNAs that confer resistance to BRAFi in drug-resistant melanoma cells represent a novel and promising therapeutic strategy to overcome resistance and restore melanoma sensitivity to BRAFi (Fig. 3b).

Histone and ubiquitin modifications

Protein modification plays a crucial regulatory role in tumor cells, with altered protein conformations participating in cell signaling, metabolic regulation and the manifestation of malignant cellular phenotypes [182–184] (Fig. 3c). Conversion of histone H3 lysine 27 methylation to acetylation opens chromatin and drives BRAFi resistance in mutant melanoma cells [185]. In these resistant cells overexpression of the E3 ligases Skp2 and UBE3C leads to ubiquitination of MST2 pathway proteins while CDKN2A loss or mutation disrupts MDM2 regulation causing p53 degradation [140, 186, 187]. The resulting impairment of ubiquitin signaling and loss of p53 activity heightens tumor aggressiveness and increases the refractory nature of resistant melanoma. Deubiquitination of cyclic GMP-AMP synthase (cGAS) by ubiquitin-specific peptidase 18 (USP18) alters the protein conformation, rendering it structurally stable. This subsequently increases cGAS expression in BRAF V600E-mutant melanomas, promoting resistance to BRAFi specifically within the BRAF mutant context [188]. Grigore et al. showed that chronic exposure of BRAF V600E-mutant melanoma cells to the selective BRAFi vemurafenib significantly increases H3K36me2/3. Because elevated H3K36 methylation rewires transcription toward pro-survival programs, these findings implicate H3K36 hypermethylation as an epigenetic driver of vemurafenib resistance [189]. Sirtuin 5, a member of the sirtuin deacylase family, maintains histone acetylation and methylation levels in melanoma cells and thereby sustains oncogenic transcriptional programs [190]. Inactivation of the neddylation signaling pathway by tetracaine hydrochloride effectively suppresses melanoma cell proliferation and alleviates vemurafenib resistance, highlighting a novel non-genetic mechanism underlying drug tolerance [191].

In summary, varying degrees of epigenetic alterations can confer resistance to BRAFi in BRAF mutant melanomas. Meanwhile, elucidating the diverse mechanisms underlying these epigenetic changes may provide a potential therapeutic strategy to enhance the sensitivity of resistant melanomas to BRAFi. Collectively documented hypermethylation of key gene promoters together with aberrant expression of miR19a, miR211 and the lncRNA SAMMSON, orchestrates transcriptional reprogramming in melanoma. Pharmacologic deployment of DNA methyltransferase inhibitors sequence-specific silencing of relevant non coding RNAs, and modulation of protein modifications may resensitize melanoma cells to BRAFi by reversing these epigenetic constraints.

Metabolic reprogramming: adaptive metabolic liabilities in acquired resistance

Metabolic reprogramming serves as a critical adaptive strategy during acquired resistance, where melanoma cells remodel their metabolic routes, such as ramping up glutaminolysis and polyamine production, to survive BRAFi-induced growth suppression and oxidative damage. Bioinformatics analysis identifies EGFR as a ferroptosis-related driver of BRAF V600E melanoma resistance. Meanwhile, lactate promotes LSD1 lactylation, which suppresses ferroptosis via the LSD1-FosL1-TFRC axis. Conversely, MitoCur-1 blocks USP14 to inactivate GPX4, deplete GSH, and accumulate ferrous iron, thereby reactivating ferroptosis. Together these findings position ferroptosis modulation as a promising unified approach to resensitize resistant tumors and enhance immunotherapy efficacy [192–194]. Adding to this resistance landscape, reprogramming of the transsulfuration pathway, especially upregulation of cystathionine gamma lyase (CSE), drives acquired resistance to BRAF V600E inhibitors in melanoma. The enzyme boosts cystine and cysteine metabolism to produce persulfides and hydrogen sulfide, which shield cells from drug induced oxidative stress and meet increased energy demands. Preclinical studies show that combining BRAF V600E inhibitors with CSE inhibitors curbs proliferative relapse and extends progression free survival [43]. Polyamine biosynthesis and EIF5A hypusination downstream of c-Myc now explain a new vemurafenib resistance route in melanoma. CRISPR screens pointed to AMD1 as a druggable node and metabolomic plus proteomic data showed that resistant tumors boost polyamines which heightens EIF5A hypusination mitochondrial translation and oxidative phosphorylation. Sustained c-Myc keeps the polyamine flux high and blocking this pathway re-sensitizes tumors in vitro and in vivo spotlighting polyamine synthesis as a clinically actionable target to improve BRAFi efficacy [195].

Therapy-exposed BRAF mutant melanoma contains persister cells that spatial transcriptomics identifies as upregulating oxidative phosphorylation and invasion while downregulating proliferation. Their dependence on DUSPs, reticulon 4, and CDK2 for survival exposes vulnerable intervals where intervention could prevent resistance emergence [196]. In BRAFi-resistant melanoma cells, amplified glutaminolysis fuels survival, making glutaminase a metabolic vulnerability for overcoming resistance [33]. Metabolic reprogramming in resistant melanoma cells is not an isolated adaptive process, it is tightly intertwined with remodeling of the tumor microenvironment, where stromal cells and soluble factors further shape metabolic vulnerabilities and reinforce drug tolerance.

The tumor microenvironment remodeling: stromal-immune synergistic resistance

TME comprising stromal cells, extracellular matrix, and soluble molecules, plays a pivotal role in melanoma progression and treatment resistance, highlighting its potential as a therapeutic target [34]. The progression of malignant tumors from early to advanced stages is significantly influenced by TME [197–199]. Mechanical and architectural signals within the tumor microenvironment significantly influence acral melanoma pathogenesis, inducing DNA damage and promoting a malignant phenotype, as revealed by a novel 3D in vitro platform [35]. Alterations in TME can promote resistance to BRAFi in BRAF mutant melanoma cells [200–203]. Fibroblasts in older men selectively drive melanoma cell invasiveness and therapeutic resistance to BRAFi, this phenomenon has been documented across both BRAF mutant and BRAF wild-type melanomas and therefore represents a pan-genotype microenvironmental driver [204]. Likewise aging fibroblasts in elderly melanoma patients secrete more lipids and co cultured young melanoma cells increase lipid uptake via fatty acid transporter FATP2 which reduces their sensitivity to targeted inhibitors [205]. BRAFi induce the activation of cancer-associated fibroblasts (CAFs), which in turn drive stromal remodeling and immune-evasion TME in melanoma [206–208]. Activation of β-catenin in CAFs stimulates the secretion of periostin, promoting resistance of specifically in BRAF mutant melanoma cells [209]. At the same time, Rho/MRTF pathway activation drives aggressive behavior in vemurafenib-resistant murine melanomas through enhanced stress fiber formation, MRTF-A nuclear entry and immune checkpoint upregulation provide a new target to overcome resistance and improve outcomes [210]. Drug-resistant cells display elevated IL-6 secretion, the IL-6–202 and IL-6–205 transcript variants confer BRAFi resistance in BRAF mutant melanoma [211]. Moreover, BRAFi resistant melanoma cells shed elevated soluble CD73, an ectonucleotidase that produces immunosuppressive adenosine and fuels resistance. Nutrient stress drives MMP-9 mediated CD73 release, which is blocked by CD73 inhibitors. While BRAFi treatment lowers CD73 in sensitive cells, resistant cells upregulate both membrane and soluble CD73, linking CD73 to acquired BRAF-targeted therapy resistance [212]. Vemurafenib-resistant melanoma cells not only modulate dendritic cell (DC) activation and cytokine production within TME, influencing DC maturation, but also exhibit elevated expression levels of IFN-γ, IL-8, VEGF, CD147/basigin and MMP-2 compared to their parental cell counterparts, these alterations have been observed in both BRAF mutant and BRAF wild-type melanomas, qualifying as pan-genotype mechanisms [213]. TNF-α derived from macrophages within TME can drive melanoma cells to develop resistance to MAPK inhibitors through the lineage-specific transcription factor MITF (microphthalmia-associated transcription factor), an event that occurs preferentially in BRAF mutant melanoma cells [36]. Elevated secretion of macrophage colony-stimulating factor (M-CSF) in drug-resistant melanoma cells and higher levels of M-CSF induce a drug-resistant phenotype in melanoma cells [214, 215]. This promotes BRAFi resistance in BRAF V600E mutant melanoma while increasing tumor migration and growth, and correlates with poor prognosis in patients with BRAF V600E mutant metastatic melanoma [214]. Cancer stem cells (CSCs) within the melanoma TME, regulated by the paracrine renin-angiotensin system, contribute to treatment resistance, highlighting the potential of targeting this system to enhance the efficacy of targeted therapy and immunotherapy [216]. Furthermore, BRAFi-resistant melanoma cells silence RICTOR/mTORC2 to boost mitochondrial respiration via the NAMPT-ETC axis and simultaneously overexpress and secrete NAMPT and NNMT free or in extracellular vesicles, sculpting a microenvironment that reinforces both intrinsic and extrinsic resistance to BRAF/MEK therapy [37, 217]. Therefore, the aforementioned findings indicate that alterations in TME can contribute to resistance in tumor cells to BRAFi. Some of these alterations are pan-genotype, whereas others are restricted to the BRAF mutant context. Investigation of TME-associated factors may represent a promising therapeutic target for overcoming BRAFi resistance. In addition to the paracrine signals and stromal remodeling mediated by CAFs and immune cells, autophagy emerges as a central hub that integrates TME cues with intracellular survival pathways, further amplifying BRAFi resistance in a bidirectional crosstalk between tumor cells and their microenvironment (Fig. 4a).

Fig. 4.

Tumor Microenvironment and Autophagy Networks in BRAF-Inhibitor Resistance: Myofibroblasts, Immune Cytokines, and Autophagy-Driven Feedback Circuits. a Tumor associated myofibroblasts and infiltrating immune cells including dendritic cells and macrophages orchestrate a cytokine milieu comprising TNF α, M CSF and IL 6 that confers BRAF inhibitor resistance in melanoma. b Autophagy-Driven Resistance Circuitry in BRAF-Mutant Melanoma: from Ambra1 Loss and BMAL1 Repression to USP18-cGAS Deubiquitination, LaminB1 Modulation, and ATP-LC3-ERK Feedback

Autophagy functions as a central metabolic and signaling hub that dynamically sculpts the tumor microenvironment by regulating immune cell infiltration, cytokine release, and extracellular matrix remodeling [218–222] (Fig. 4b). This bidirectional interaction promotes immune evasion and therapy resistance while simultaneously providing actionable targets for combination cancer therapies [223–225]. As an example, loss of Ambra1 (autophagy and Beclin-1 regulator 1) upregulates multiple cytokines and chemokines, which in turn suppresses regulatory T cell infiltration and thereby promotes melanoma immune escape and drug resistance [220]. BMAL1 (aryl hydrocarbon receptor nuclear translocator-like) is markedly down-regulated in melanoma, impairing autophagy and increasing tumor-associated neutrophil infiltration to reshape the tumor microenvironment and drive malignant progression [226]. Autophagy plays a cytoprotective role in melanoma cells treated with BRAFi such as vemurafenib and dabrafenib, and its induction promotes tumor cell survival, contributing to drug resistance. Therefore, targeting autophagy represents a promising adjuvant strategy to enhance the efficacy of BRAF-targeted therapies and overcome resistance [227].

BRAFi-resistant melanoma cells exhibit increased basal levels of autophagosome markers and decreased autophagic flux [228, 229]. Conditioned media derived from these drug-resistant cells enhance the resistance of BRAFi-sensitive melanoma cells [230, 231]. Cellular autophagy has been associated with various mechanisms, such as the deubiquitination of cyclic GMP-AMP synthase (cGAS) by USP18. This process specifically triggers resistance-protective autophagy in BRAF mutant melanoma cells, conferring resistance to vemurafenib and other BRAFi [188]. Interfering with nuclear protein laminB1 enhances vemurafenib sensitivity in BRAF-mutated melanoma cells by promoting DNA damage and inhibiting protective autophagy, revealing laminB1 as a potential diagnostic marker and therapeutic target [232]. Vemurafenib-resistant cells exhibit heightened autophagic flux and ATP efflux, sustaining ERK autocrine signaling [233]. As drug resistance increases, autophagic flux increases and the co-localization of intracellular ATP with the pro-autophagic marker LC3 is enhanced [233].

Collectively, CAF-dependent β-catenin activation and macrophage-secreted M-CSF or IL-6 isoforms sustain proliferative and survival signaling within the resistant TME. Immune cell derived M -CSF IL 6 and TNF alpha drive stromal support and enhance BRAF mutant melanoma resistance to BRAFi. We conclude that BRAFi escape is not a single-pathway revival but a multi-layer rewiring of genetics, epigenetics and the tumor micro-environment. Looking forward, real-time integration of cfDNA methylation scores with single-cell enhancer maps should allow the first pre-emptive switch before imaging escape is even visible. Unsupervised analysis identifies key immune response genes and matrisome components as shared contributors to heterogeneous resistance mechanisms against BRAFi and MEKi in melanoma, highlighting their potential as therapeutic targets [234].

Mechanism-driven strategies to overcome resistance: targeted, epigenetic, immune and novel therapies

Resistance arises from intertwined genetic, epigenetic, metabolic, and microenvironmental processes, requiring multi-pronged therapeutic designs. Such integrated approaches bridge the gap between laboratory findings and clinical efficacy.

Targeted combination therapies: genomics-guided precision regimens

The goal of individualized precision therapy is to achieve the optimal treatment match for each patient, enhancing therapeutic efficacy and minimizing the risks and costs associated with ineffective treatments [235–239]. This treatment paradigm, which incorporates patient genomics and molecular profiling, is particularly crucial in cancer treatment, as tumors from different patients may exhibit distinct genetic and molecular profiles, necessitating personalized treatment strategies (Table 2). In clinical settings, individualized treatment plans can be developed based on patients' genomic characteristics and tumor molecular features [72, 240–243]. These plans may encompass palliative care, the selection of appropriate targeted agents and immunotherapies, and combination therapies [244–247]. They are all aimed at improving patients' quality of life while minimizing treatment-related adverse effects [248, 249].

Table 2.

Genomics-guided and targeted combination strategies to overcome tumor drug resistance via inhibition, degradation, and pathway modulation

Strategy Class	Key Combination/Agent	Mechanism Trampled	Ref
PARP add-on	• PARP inhibitor after BRAF/MEK failure	• Exploits increased replication stress • Deepens response	[39]
T-type calcium block	• Cav3.1 blocker + vemurafenib	• Inhibits autophagy-mediated survival • Restores apoptosis	[40]
PI3K rescue	• Neratinib (pan-ERBB/HERi) + BRAF/MEK in PTEN-null tumors	• Reverses PTEN-loss–driven PI3K/AKT hyper-activation & restores sensitivity	[41]
ABL1/2 block	• Nilotinib + BRAF ± MEKi	• Silences ABL-driven MAPK re-activation & prevents emergence of resistance	[101]
Genomics-guided matching	•Tailor combo to each patient’s mutation profile	• Cuts futile therapy & cost; boosts response	[235–239]
PROTAC degradation	•PROTAC that degrades BRAF-V600E	• Catalytic removal of mutant BRAF → deeper MAPK shut-off, slower resistance	[250]
Genome-wide scan	•Whole-genome sequencing of resistant tumor	• Finds actionable second-site mutations for next combo	[166, 251–256]
FANCD2 degradation	•Protocatechuic aldehyde + temozolomide	• Degrades FANCD2, boosts cytotoxicity regardless of MGMT status	[257]
Proteasome + ion channel	•Proteasomei + Kv1.3 blocker	• Synergistic apoptosis in sensitive & resistant cells	[258]

The strategies outlined, which encompass approaches from ABL1/2 inhibition to PROTAC-mediated protein degradation, are precisely tailored to counteract the genetic escape mechanisms. Such as MAPK pathway reactivation and PI3K/AKT bypass signaling, described earlier. This alignment underscores the meticulous design of therapeutic interventions to specifically target the underlying mechanisms of resistance.

A comprehensive genome-wide analysis of drug-resistant melanoma patients, leveraging advanced genomic techniques, holds significant potential to elucidate the underlying genetic mechanisms of resistance [166, 251–253]. This can provide crucial insights to inform the development of highly personalized treatment regimens and optimized clinical strategies tailored to the specific genetic profiles of individual patients. Utilizing whole-genome sequencing to analyze a patient's tumor sample enables the identification of specific gene mutations, copy number variations and gene expression profiles [254–256]. This comprehensive approach elucidates the mechanisms underlying drug resistance and informs the development of targeted therapies, enhancing the precision and efficacy of treatment strategies [39, 250].

Wongchenko MJ et al. meticulously analyzed and evaluated the gene expression profiles of BRAF mutant melanoma samples, including the expression of cell-cycle-related genes, in a bid to precisely target these profiles for the treatment of BRAF mutant melanoma [257]. In BRAF mutant melanoma that has acquired resistance to BRAFi, secondary gene mutations fuel the survival of resistant cells, and inhibitors targeting these mutations can halt their progression. For instance, nilotinib, an FDA-approved ABL1/2 tyrosine-kinase inhibitor, restores sensitivity to BRAFi ± MEKi in resistant melanoma. In vitro it abrogates cell viability and triggers apoptotic commitment. In vivo it elicits sustained regression of BRAFi/MEKi-refractory xenografts. And, when implemented as upfront therapy, effectively precludes the evolution of resistance [101]. PARP inhibitors produce partial or near-complete responses in advanced BRAF V600-mutant melanoma after BRAF/MEK progression, pointing to a synergistic PARPi plus BRAF/MEK regimen for refractory disease [40]. PROTAC-directed degradation of BRAF V600E demonstrates superior MAPK pathway inhibition and apoptotic induction compared to standard BRAFi in transcriptomic studies. This enhanced efficacy and diminished resistance risk highlight PROTACs as a compelling therapeutic avenue for resistant melanoma [258]. Protocatechuic aldehyde (PA) demonstrates a synergistic effect with temozolomide (TMZ) in enhancing cytotoxicity against both BRAFi-sensitive and -resistant melanoma cells by promoting FANCD2 degradation, offering a novel combinatorial chemotherapy strategy regardless of MGMT status [41]. The Cav3.1 gene isoform of the T-type calcium channel (TTCC) is highly expressed in vemurafenib-resistant BRAF mutant melanomas. TTCC blockers have been shown to induce apoptosis and impair tumor cell migration and invasion by inhibiting autophagy-mediated resistance in drug-resistant melanoma cells and in mouse xenograft models [259]. Thus, Cav3.1 may serve as a marker for the sensitivity of drug-resistant melanoma cells to combination therapy. TTCC blockers may provide a potential targeted therapy for vemurafenib-resistant BRAF V600E-mutant melanoma. A novel therapeutic approach combining proteasome inhibition (Proteasomei) with Kv1.3 potassium channel blockade demonstrates synergistic effects in inducing apoptosis in both drug-sensitive and BRAFi-resistant melanoma cells, offering a promising strategy to overcome therapy resistance and potentially extending to other BRAF mutant tumors [260]. In BRAF V600E melanoma, PTEN loss-of-function mutations occur in approximately 40 percent of tumors, leading to hyperactivated PI3K/AKT signalling that compromises the efficacy of combined BRAF/MEK blockade. Treatment with the pan-ERBB/HER inhibitor (pan-ERBB/HERi) neratinib restores drug sensitivity in these PTEN-null cells and effectively reverses the resistant phenotype [261].

In summary, integrative genomic profiling via whole-exome and long-read sequencing deciphers clonal architecture and bypass lesions driving BRAF/MEKi resistance in melanoma. Precision combination protocols that co-target ABL1/2, PARP, PROTAC-directed BRAF V600E degradation, FANCD2 proteolysis, TTCC or Kv1.3 blockade, and PI3K/AKT re-sensitization with pan-ERBBi achieve synchronized blockade of adaptive signaling, collateral survival circuits, and immune-evasive reprogramming, yielding augmented objective responses with attenuated off-target toxicity. While genomics-guided combination therapies effectively block known resistance pathways, novel small-molecule inhibitors expand the therapeutic arsenal by targeting previously undruggable nodes or non-oncogene addictions that emerge during BRAFi resistance.

Novel small-molecule inhibitors: targeting resistance drivers beyond MAPK

Phosphoenolpyruvate carboxykinase 1 (PCK1) is upregulated in vemurafenib-resistant melanomas, activating PI3K/Akt and reducing ROS. Targeted inhibition of PCK1 with 3-mercaptopropionic acid restores vemurafenib sensitivity [262]. Equally important, RIDR-PI-103, a ROS-activated PI3K prodrug, blocks the PI3K/Akt pathway under high ROS and halts proliferation in BRAFi and MEKi -resistant melanoma, while diclofenac increases intracellular ROS and activates the p38/p53 pathway to augment cytotoxicity and apoptosis in BRAFi-resistant cells, offering new ways to beat resistance [263, 264]. Disulfiram (DSF), a copper ionophore, overcomes BRAFi resistance in melanoma by inducing mitochondrial dysfunction via copper-dependent oxidative stress and TXNIP upregulation, independent of MAPK inhibition, offering a novel therapeutic avenue [265]. Likewise, acyclic retinoid overcomes vemurafenib resistance in BRAF V600E melanoma by simultaneously blocking MAPK and PI3K/AKT/mTOR signaling, highlighting its promise as a new therapeutic agent [266]. VERU-11, a microtubule inhibitor, disrupts microtubule polymerization and induces microtubule depolymerization in drug-resistant melanoma cells. When combined with vemurafenib, it blocks the cell cycle, induces apoptosis and inhibits cell migration. This combination also increases p53 expression levels and suppresses the activation of the ERK/Akt signaling pathway [267]. The quinolinol small molecules MMRi62 and its derivative SC-62–1 induce p53-independent apoptosis in melanoma cells, including those resistant to BRAFi, by covalently binding to over 1500 cellular proteins and altering multiple metabolic and stress response pathways, pointing to their potential as multi-targeting agents to overcome BRAFi resistance [268]. C-terminal Hsp90 inhibitors (KU757, KU758) overcome MEK/BRAFi resistance in melanoma: they had similar IC50 in A375 and resistant A375 MEKi. KU757 downregulated resistance pathways (oxidative phosphorylation, AKT/PI3K/MTOR) and cell cycle, upregulated apoptosis, with hub genes (e.g., NDUFA7, CDC20) suggesting clinical benefit [269]. 3-bromopyruvate beats vemurafenib alone against resistant melanoma by blocking glycolysis and EMT, halting proliferation and invasion while killing resistant cells [270]. A novel antibody therapeutic, such as an Indolium1, has been shown to inhibit the proliferation, migration and invasion of BRAF mutant, vemurafenib-resistant melanoma cells. This effect is achieved by inhibiting the expression of the oncogenic factor pRB and inducing the expression of the tumor suppressor EPHA3, restoring the drug sensitivity of resistant cells [271].

ENOX2, the cell-surface disulfide-thiol exchanger that powers melanoma redox cycling, has emerged as a double-duty asset. High tumor levels track with shorter overall, disease-specific and metastasis-free survival, and the small-molecule inhibitor phenoxodiol shuts down ENOX2 activity, suppresses cell viability and can prevent acquired vemurafenib resistance [272]. Marine fungal extracts are providing new anticancer candidates, particularly mortiamide-D. This Mortierella-derived cyclic peptide achieves selective toxicity, eradicating both drug-sensitive and resistant melanoma cells at micromolar levels while remaining harmless to normal keratinocytes. Structure–activity work shows that swapping D-Ile for D-Arg at position 7 boosts membrane penetration and cytotoxic potency, underscoring mortiamides as optimizable scaffolds for future melanoma drugs that could be deployed alongside ENOX2 blockers to forestall resistance and improve patient outcomes [273]. Researchers have designed new benzimidazole derivatives containing oxindole and non-oxindole structures that simultaneously block BRAF V600E and ABL2 kinase activity. These dual inhibitors kill resistant melanoma cells, shut down key survival signals including p-CrkL and p-ERK1/2, halt cell cycle progression in G1 phase, and offer a viable strategy to tackle treatment-resistant disease [274].

Lipid metabolism-dependent processes and genes regulating lipid metabolism, such as SREBP-1, promote adipogenesis in BRAF mutant melanoma cells. This results in enhanced therapeutic resistance of tumor cells to targeted agents, conferring acquired resistance to these therapies [275–277]. However, targeting lipid metabolism can attenuate the resistance of BRAF mutant melanomas to BRAFi. Avasimibe, an SREBP inhibitor, can act synergistically with vemurafenib, increasing the occurrence of ferroptosis in tumor cells. This enhances the sensitivity of drug-resistant cells to the drug [278]. BRAF-mutated melanoma cells (A375, FO-1) developed distinct resistance signatures after long-term RAF inhibitor (dabrafenib/AZ628) exposure (dabrafenib-resistant: high proliferation, AZ628-resistant: slow-cycling, ferroptosis-susceptible). Antiretrovirals doravirine/cabotegravir reduced resistant cell viability, with doravirine specifically activating apoptosis and inhibiting growth in proliferative resistant cells via upregulating p16Ink4a/p27Kip1 [279]. In BRAF-mutated malignant melanoma, resistance to the second-generation BRAFi Encorafenib is mediated by NCOA4-regulated iron trafficking, leading to altered iron metabolism and ferritinophagy, as evidenced by increased levels of NCOA4, FTH1, and intracellular iron in resistant A375 cells [280]. Desmoplakin, a potential anticancer agent, can reverse the upregulation of the pentose phosphate pathway and lipogenesis in vemurafenib-resistant BRAF mutant melanoma cells. This drug not only upregulates the expression of the lactophilin family of protein-coding genes but also enhances T-cell activity, exerting an antitumor effect [281]. Dihydrotanshinone I (DHT), a lipophilic compound from Salvia miltiorrhiza, enhances BRAF/MEKi efficacy in BRAF mutant melanoma by directly inhibiting STAT3/SOX2 signaling, thereby overcoming primary and secondary resistance via dual MAPK-STAT3 pathway blockade [282]. The Rho/MRTF axis and its effector pirin drive melanoma drug resistance, and blocking Rho/MRTF with CCG-257081 restores vemurafenib sensitivity while preventing emergence of resistance by amplifying apoptosis, positioning pirin or Rho/MRTF inhibition as a promising tactic to overcome therapy failure [283]. Xanthohumol (XN), a natural compound derived from hops, enhances the sensitivity of melanoma cells to vemurafenib by reducing membrane cholesterol and increasing fluidity, thereby facilitating greater drug uptake and improving therapeutic efficacy with minimal toxicity to non-tumor cells [44]. A novel small molecule, EPS496, has been identified as a selective inhibitor of p300, a histone acetyltransferase implicated in transcriptional regulation and resistance mechanisms in melanoma. EPS496 demonstrated high binding stability and potent anti-proliferative activity in both normal and vemurafenib-resistant BRAF V600E mutated melanoma cells, offering a promising therapeutic strategy to overcome vemurafenib resistance [284]. In summary, studies targeting lipid metabolism may provide a viable strategy for the clinical management of vemurafenib-resistant BRAF mutant melanoma and for improving patient outcomes.

In summary, post-genomic metabolic liabilities PCK1, SREBP1, NCOA4 and ENOX2 propagate BRAFi/MEKi refractoriness; targeted small-molecule, ionophore and degradative interventions enforce ferroptotic oxidative stress and G1 arrest MAPK-independently. Single-cell PDO lipidomics will prioritize these non-oncogene addictions for adaptive, metabolism-driven phase I trials.

Epigenetic and metabolic interventions: reversing adaptive reprogramming

Although epigenetic modifications are known to contribute to resistance to BRAFi in melanoma, the interplay between these alterations and associated signaling pathways remains a component of the resistance mechanism that is not yet fully elucidated. Thus, an in-depth investigation of epigenetics could provide a theoretical basis for the development of novel small-molecule inhibitors. Integrated multi-omics analysis of proteins and RNAs can elucidate the epigenetic regulation of proteins and RNAs in tumor cells [285].

It is worth noting that pharmacological modulation of epigenetic machinery and associated metabolism offers a promising therapeutic strategy for patients with resistant melanoma. In advanced disease upregulation of PDE4D methylation is observed and selective PDE4 inhibition effectively counters resistant melanoma cells [42]. Inhibition of EZH2, an epigenetic regulator linked to melanoma progression and BRAF therapy resistance, enhances vemurafenib activity in BRAFi-resistant cells by downregulating PLK1, leading to decreased viability, cell-cycle arrest, and increased apoptosis, indicating that targeting EZH2 or PLK1 together with BRAFi offers a potential new therapeutic strategy for BRAF mutant melanomas [286]. DNA methylation also drives the exhaustion of cytotoxic T cells during tumor progression and histone methyltransferase EZH2 cooperates with DNA methylation to promote resistance. Consequently, combined targeting of DNA methylation and EZH2 may be essential to overcome immunotherapy resistance in melanoma [45, 287]. LncRNAs play a critical regulatory role in melanoma biology [288, 289]. A combined CRISPR activation and small-molecule screen mapped lncRNAs that drive BRAFi resistance in melanoma, uncovering druggable lncRNAs and combination treatment options [290]. Suppression of lncRNA ROSALIND elevates reactive oxygen species and protein oxidation thereby inducing severe mitochondrial respiratory dysfunction and compromising melanoma cell viability [291]. Knockdown of lncRNA U73166 and LINC01291 markedly inhibits melanoma cell migration and invasion. Consequently lncRNA-directed therapies may serve as novel biomarkers and effective therapeutic targets for melanoma [46, 181]. Metabolic regulatory nodes such as autophagy play a pivotal role in melanoma [292, 293]. Pharmacological blockade of autophagy represents a potential strategy to circumvent therapeutic resistance [294]. The small molecule inhibitor SPP86 suppresses autophagy and exerts antineoplastic activity through attenuation of PI3K/AKT signaling [295]. Moreover, the antibiotic eravacycline induces autophagy while promoting M1 macrophage polarization thereby enhancing the efficacy of anti PD-1 therapy in melanoma [296]. Similarly, elevated expression of the histone lysine methyltransferase Suv39h1 promotes tumor immune escape, and inhibiting this gene enhances CD8 + T-cell infiltration [297].

Moreover, recent insights into oxidative stress in melanoma reveal its paradoxical role in tumor progression, where it initially promotes tumorigenesis but later hinders metastasis, and highlight redox metabolic reprogramming as a key factor in acquired resistance to BRAFi/MEKi, suggesting that modulating the antioxidant system could enhance therapeutic efficacy [47]. Mechanistically, the involvement of Nrf2, a key regulator of antioxidant responses, in the acquired resistance to BRAFi and MEKi in melanoma has been illuminated, where its upregulation and stabilization by DUB3 contribute to therapy resistance, and targeting Nrf2 or DUB3 presents a promising approach to reverse this resistance [298]. In patient-derived melanoma cells, the rewiring of oxidative phosphorylation, preservation of pyruvate dehydrogenase activity, and maintenance of high glutathione levels have been identified as crucial contributors to the onset of PLX4032 resistance, suggesting that targeting glutathione biosynthesis and/or pyruvate dehydrogenase activity in combination with PLX4032 may offer a viable strategy to overcome drug resistance in BRAF-mutated melanoma patients [299]. Cholesterol and its metabolite 27-hydroxycholesterol (27HC) have been implicated in promoting resistance to vemurafenib in melanoma by activating Rap1-PI3K/AKT signaling, suggesting that targeting 27HC synthesis could be a potential strategy to overcome treatment resistance [300]. In summary, combinatorial blockade of EZH2–DNMT–lncRNA axes, glutathione-dependent OXPHOS, 27HC-Rap1–PI3K signaling and autophagic flux reactivates cytotoxic T cells, exhausts antioxidant capacity and enforces ferroptosis, thereby ablating BRAFi/MEKi refractoriness. These convergent epigenetic, metabolic and redox interventions constitute a resistance-agnostic backbone for forthcoming adaptive basket trials in advanced melanoma.

Immunotherapy combinations: the tumor microenvironment-targeted immuno-oncology strategies

TME shapes immune escape and drug resistance, thereby dictating therapeutic response in melanoma [301, 302]. Targeting this niche with targeted and immune therapies may achieve durable disease control [48, 303, 304]. The combination of the BRAFi and the MEKi has been widely employed in clinical practice for the treatment of BRAF mutant melanoma and has demonstrated significant efficacy in improving overall survival among patients (ClinicalTrials.gov identifier NCT01902173) [49, 305, 306]. However, as resistance develops, immune cells within the tumor also undergo corresponding changes, and novel therapies such as immunotherapy have therefore been introduced. For instance, vemurafenib resistance concurrently up-regulates MICA/ULBP2 and TRAIL-RII, markedly enhancing NK-cell degranulation and IFN-γ secretion. Serum levels of sMICA, sB7H6, PD-L1 and CEACAM1 correlate with clinical response, providing mechanistic and biomarker rationale for combining BRAFi/MEKi with NK-directed immunotherapy to overcome acquired resistance [307]. Moreover, RNA-seq and bioinformatics pinpoint a CD20 + MM subset that is intrinsically resistant to BRAFi, and pairing BRAFi with anti-CD20 antibodies markedly boosts killing of these cells, offering a promising way to prevent recurrence in melanoma patients carrying this CD20 + subpopulation [308]. The combination of atezolizumab, vemurafenib and cobimetinib increases the expression of lactate dehydrogenase (LDH) and programmed death ligand 1 (PD-L1) in TME and enhances the inhibitory effects of BRAFi on tumor cells. This combination significantly improves progression-free survival (PFS) in patients with advanced-stage BRAF V600E-mutant melanoma [309, 310]. Over the past two decades, melanoma treatment has moved from low-efficacy cytokine-based immunotherapy to PD-1 antibodies as the standard for advanced disease, alongside FDA-approved BRAFi and BRAF/MEK combinations for BRAF mutant tumors, changes that have produced the largest survival improvements seen in any cancer during the 2010s [311]. As an illustration, a LASSO model singled out high platelet-to-lymphocyte ratio, acral or mucosal subtype, BRAF mutation, low globulin, and multiple metastatic sites as key predictors of primary PD-1 inhibitor resistance in metastatic melanoma, giving clinicians early warning flags and hints for countermeasures [312]. In BRAFi/MEKi-refractory melanoma, EGFR overexpression was identified as a potential biomarker for responsiveness to second-line immune checkpoint inhibitors (ICIs), as demonstrated by increased CD8 + T effector cells and activation of the EGFR-STAT signaling pathway in resistant tumors, which retained sensitivity to ICIs, contrasting with NRAS-driven resistance showing cross-resistance to ICIs [313]. Moreover, the combination of atezolizumab, vemurafenib and cobimetinib can also serve as a first-line therapeutic option for patients with unresectable, advanced BRAF V600E-mutation-positive melanoma. This combination has demonstrated safety and tolerability in targeted therapy [6]. Subsequently, sequential administration of PD-1 checkpoint inhibitors (nivolumab or pembrolizumab) after first-line BRAFi/MEKi failure provides the greatest survival benefit to patients with advanced BRAF mutant melanoma [314].

The use of immunotherapy, either alone or in combination with targeted therapies, can help determine a patient’s eligibility for treatment with immune checkpoint inhibitors [315]. Liu S et al. loaded CpG or BMS-202 into liposomes and conjugated them to OT-1 CD8 + T cells (CD8-T-LP-CpG/CD8-T-LP-BMS-202), markedly reducing PMN-MDSCs, M2 macrophages, and Tregs while increasing the infiltration of cytotoxic and effector-memory CD8 + T cells within mouse tumors. This platform combines an immunomodulator with adoptive T cells, offering a readily translatable strategy to disrupt the immunosuppressive microenvironment of solid tumors and enhance adoptive T-cell therapy [316–318]. Likewise, inhibiting the Rho/MRTF pathway with CCG-257081 lowers PDL1 in BRAFi-resistant melanoma, boosts CD8 + T and B cell infiltration while reducing tumor-associated macrophages, and thereby restores anti-PD1 efficacy to overcome resistance and enhance immunotherapy response [319]. Nivolumab, an anti-PD-1 antibody, synergizes with CD26-high CD4⁺ T cells to produce clinical benefit in patients with PD-1-resistant melanoma, and their overall survival is longer than 12 months [50]. Yet BRAF blockade heightens tumor immunogenicity by upregulating immune activation genes, drawing activated cDC1 and cDC2 into the tumor and draining nodes, and this myeloid remodeling is essential for the CD4 + and CD8 + T cell response that drives therapeutic efficacy [320]. Combination therapy involving BRAFi, MEKi and PD-1 checkpoint inhibitors in patients with BRAF V600E-mutant melanoma not only enhances antitumor efficacy but also improves outcomes for a subset of metastatic patients by increasing the frequency of durable responses [321]. In melanoma resistant to vemurafenib, the cell-permeable STAT3 inhibitory peptide APTSTAT3 9R increases cytotoxic T lymphocyte (CTL) infiltration into TME. Combined with anti-PD-1 antibodies, this therapy reduces myeloid suppressor cell and tumor-associated macrophage infiltration, while enhancing CD8 + T cell infiltration and cytotoxicity, significantly inhibiting tumor growth [322]. Likewise, within TME, Tim-3 and STAT3 are upregulated in anti-PD-1-resistant melanoma, which limits Treg-mediated immunosuppression, and targeting STAT3 significantly enhances PD-1 immunotherapy in mouse melanoma models [323]. Furthermore, recent research has identified Diosmetin (DIOS), a naturally occurring flavonoid compound, which significantly augments the antitumor activity of BRAFi in melanoma by concurrently suppressing the MAPK and JAK2/STAT3 signaling pathways. DIOS not only exhibits potent antiproliferative effects but also downregulates PD-L1 expression in melanoma cells, thereby enhancing intratumoral T cell infiltration and activating antitumor immune responses, providing a novel adjuvant strategy for BRAF mutant melanoma therapy [324].

The contribution of integrin and TGF-β signaling to vemurafenib resistance in metastatic melanoma is well established. Strategic co-targeting of ITGA5, ITGB3, PAI1, or p21 alongside vemurafenib generates synergistic suppression of proliferation, invasion, and clonogenicity in resistant populations, presenting a rational approach to circumvent resistance and boost clinical benefit [51]. The triple combination of vemurafenib, a Toll-like receptor 7 (TLR7) agonist, and a PD-1 antibody synergistically enhances the induction of melanoma antigen gp100-specific T cells in a BRAF mutant melanoma mouse model [325]. This augments T-cell immunoreactivity and promotes the efficacy of tumor-specific T cells, tumor-specific T cells can be used to treat patients with metastatic melanoma and have demonstrated feasibility and safety(NCT02424916) [325, 326]. Romidepsin plus IFN-α2b reverses vemurafenib resistance in primary melanoma cells by blocking tumorigenic signals, restoring immune responses silenced via histone deacetylation, and boosting immunogenicity, offering a promising approach for BRAFi-refractory metastatic disease [52]. In TME, HDAC inhibitors transcriptionally up-regulate PD-1/PD-L1 on both tumor and immune cells, sensitizing BRAF mutant-bearing mice to PD-1/PD-L1 blockade [327]. Concurrent HDAC and MAPK/MEKi synergistically enhances PD-1 blockade efficacy, increases intratumoral CD8 + T-cell infiltration, and yields significant antitumor activity [328, 329]. Thus, research has increasingly focused on combination therapies as potential effective treatment strategies for overcoming resistance to BRAFi in BRAF mutant melanoma within clinical settings (Table 3).

Table 3.

Immuno-oncology tactics to overcome resistance: PD-1/PD-L1 boost, NK activation, multi-pathway blockade, metabolic reprogramming, biomarkers, and target degradation

Intervention Focus	Tested Agent/Regimen	Immune Pathway Modulated	Ref
CD26-high CD4 help	Nivolumab + CD26-high CD4⁺ T cells	• Prolongs OS > 12 months in PD-1-refractory patients	[50]
Integrin/TGF-β axis block	ITGA5/ITGB3/PAI1/p21 inhibitors + vemurafenib	• ↓Proliferation • ↓Invasion • Colony formation of resistant cells	[51]
HDAC + IFN boost	Romidepsin + IFN-α2b with vemurafenib	• Re-awakens silenced immune genes • Immunogenicity	[52]
NK-cell ligand up-regulation	Add NK-directed Rx to vemurafenib	• ICA/ULBP2 & TRAIL-RII up-regulated → ↑ NK degranulation & IFN-γ • Serum sMICA/sB7H6/PD-L1/CEACAM1 correlate with response	[307]
CD20⁺ resistant subset elimination	Anti-CD20 mAb + BRAFi	• Edicates CD20⁺ resistant sub-pop • Pevents recurrence	[308]
PD-L1-boost triplet (IMspire150 regimen)	Atezolizumab + vemurafenib + cobimetinib	• ↑LDH & PD-L1 in TME • Improves PFS vs targeted alone	[309, 310]
LASSO predictive signature	High PTL, acral/mucosal, BRAF mutation, low globulin, multiple mets	• Early warning of primary PD-1 resistance • Guides counter-measures	[312]
EGFR biomarker for ICI response	EGFR over-expression in BRAF/MEK-refractory tumors	• Identifies ICI-sensitive subset • EGFR-STAT activation linked to retained ICI benefit vs NRAS-driven cross-resistance	[313]
Sequential PD-1 blockade	Nivolumab/pembrolizumab after BRAF/MEK	•Greatest OS benefit when PD-1 given second-line	[314]
metabolic T-cell help (liposome-conjugated)	CpG or BMS-202-liposome conjugated to OT-1 CD8⁺ T cells	• ↓PMN-MDSC/M2/Treg • ↑Cytotoxic & memory CD8⁺ T cells	[316–318]
Rho/MRTF pathway inhibition	CCG-257081 + anti-PD-1	• ↓PDL1 • ↑CD8⁺/B cells • ↓TAM • Restores anti-PD-1 efficacy	[319]
cDC1/2 recruitment (BRAFi alone effect)	BRAFi alone	• ↑Immune-activation gene signature • Recruits cDC1/2 • Fuels CD4⁺/CD8⁺ T-cell response	[320]
STAT3 peptide inhibition	APTSTAT3 9R peptide + anti-PD-1	• ↑CTL infiltration •↓ MDSC & TAM reverses vemurafenib resistance	[322]
Tim-3/STAT3 dual hit	STAT3 inhibition + anti-PD-1	• Reverses Tim-3–mediated Treg suppression • Boosts PD-1 efficacy	[323]
Diosmetin adjuvant	Diosmetin flavonoid + BRAFi	• Dual MAPK & JAK2/STAT3 suppression • ↓PD-L1 • ↑T-cell influx	[324]
TLR7 agonist triplet	Vemurafenib + TLR7 agonist + PD-1 Ab	• ↑Gp100-specific T cells • Augments T-cell immunoreactivity	[325]

Emerging therapeutic approaches: CRISPR, nanomedicine, and herbal-derived agents

The development of novel targeted therapies can enhance the efficacy of BRAFi and reduce adverse effects. Their combination with BRAFi can overcome resistance in BRAF mutant melanoma cells. This approach also provides a viable strategy for improving patient outcomes, representing a significant direction in contemporary cancer therapy. CRISPR/Cas9 enables precise in vivo gene editing and is emerging as a central platform for cancer therapy. Using targeted CRISPR-Cas systems in relevant HER2-positive ovarian cancer models results in powerful permanent gene correction, induces considerable tumor regression, and expands the landscape of druggable targets to enable durable anti-cancer efficacy [39]. Goh CJH et al. performed a genome-wide CRISPR/Cas9 screen to identify genes that regulate resistance to Vemurafenib in melanomas harboring the BRAF V600E mutation. Their study pinpointed several key genes, including NF1/2, CUL3, MED10/12, FOXD3, ANAPC11. Alterations in these genes have been shown to confer resistance to Vemurafenib in drug-resistant melanoma cases [330]. Genome-scale CRISPR-Cas9 transcriptional activation screening identified three BRAFi resistance-associated lncRNA genes (SNHG16, NDUFV2-AS1, and LINC01502) in melanoma, revealing a lncRNA-miRNA-mRNA regulatory network that provides new insights into the mechanisms underlying BRAFi resistance [331]. Equally significant, CD133-positive melanoma stem cells have been shown to acquire resistance to trametinib. CRISPR–Cas9-mediated, precise knockdown of CD133 effectively triggers melanoma-cell apoptosis, providing compelling evidence that CD133 is a high-value target for combination therapeutic strategies [332]. The evolution of BRAF-targeted therapies in melanoma underscores the necessity for innovative strategies to overcome therapeutic resistance, highlighting emerging targets such as ERK5 and CD73, and the potential of advanced tools like mRNA vaccines and CRISPR-Cas9 in personalized oncology [333]. In BRAF-mutated melanoma, increased expression of the cancer stem cell marker CD271, driven by Nox4-derived reactive oxygen species (ROS), mediates resistance to BRAFi like vemurafenib. Inhibition of Nox with DPI reduces CD271 expression, ERK and Akt signaling, and epithelial-mesenchymal transition (EMT), thereby suppressing drug resistance and metastatic potential [334].

The development of drug resistance in melanoma is closely associated with mutations in the BRAF gene, making it a prime target for gene-editing therapies utilizing CRISPR/Cas9 technology. However, the delivery of CRISPR/Cas9 remains a significant challenge. Nanoparticles based on nanotechnology, such as multifunctional lipid nanoparticles, have been shown to effectively deliver CRISPR/Cas9-sgRNA ribonucleoprotein complexes to target the BRAF gene in murine models. This delivery system efficiently edits melanoma cells and reduces BRAF expression. It inhibits melanoma progression [335]. Red-blood-cell-membrane nanocages that co-deliver CRISPR–Cas9 and ACC inhibitors achieve potent, dual metabolic–genetic suppression of tumor in vivo [336]. Complementing this, intratumoral PLNPs containing Cas9/sgPLK-1 plasmid reduce A375 tumor burden by > 67%, highlighting the platform’s strong translational promise [337]. Two distinct lipid-based delivery systems show promise against BRAFi-resistant melanoma. An oral nanocomplex combining BRD4 PROTAC ARV-825 with vemurafenib, and a gene therapy approach co-delivering PTEN plasmid with BRD4 PROTAC, both suppress c-Myc, demonstrate synergistic cytotoxicity in vitro, and achieve significant tumor suppression and apoptosis in vivo [338, 339]. Consequently, the development of targeted therapies aimed at these specific genes holds promise as a viable strategy for the treatment of melanoma patients. Nanotechnology-based delivery systems hold significant potential for effectively targeting tumors. Furthermore, the tumor suppressor protein p53 is often inactivated in BRAF mutant melanomas. However, its activator SLMP53-2 targets p53 to restore transcriptional activity, curbing melanoma aggressiveness and synergizing with vemurafenib to resensitize resistant tumor cells and exert anti-tumor effects [340]. The p53 family members TP53, TP63 and TP73 participate in acquired MAPK inhibitor resistance in melanoma, with specific isoforms showing elevated expression that directly fuels tumor cell proliferation, survival and therapy evasion, pointing to these proteins as potential therapeutic targets to overcome resistance [341]. Furthermore, encorafenib, a novel BRAFi, has been approved for the treatment of melanoma and colorectal cancer [342–344]. Encorafenib was found to inhibit drug efflux mediated by the ABC transporter protein ABCB1, reducing drug resistance in BRAF mutant melanoma cells and exerting antitumor effects [345]. Thus, encorafenib may serve as a therapeutic agent for tumors characterized by BRAF mutations and overexpression of the ABCC1 transporter protein.

Ezrin, a cytoskeletal protein involved in cell junctions in tumor cells, is significantly associated with the development of resistance to vemurafenib in BRAF V600E-mutant melanoma. Targeting this protein not only enhances the sensitivity of drug-resistant cells to vemurafenib but also synergistically enhances the antitumor effects of vemurafenib when combined with its inhibitor in drug-resistant melanoma cells [346]. The expression of cysteine sulfinyl reductase Sestrin2 is elevated in vemurafenib-resistant BRAF mutant melanomas. Knockdown of Sestrin2 enhances the sensitivity of these drug-resistant cells to vemurafenib. Guo et al. demonstrated that combining an mTOR inhibitor with Sestrin2 knockdown and vemurafenib synergistically enhances the antiproliferative and pro-apoptotic effects of vemurafenib in drug-resistant melanoma [347]. Furthermore, combining statins, which are inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), with disopyramide, an inhibitor of sterol regulatory element-binding protein 2 (SREBP2), may enhance the efficacy of vemurafenib in patients with BRAF V600E-mutant, drug-resistant melanoma. This combination may represent a significant advancement in the treatment of resistant melanoma [348]. Mechanistically, Kisspeptin-54, a KiSS1-derived peptide, enhances vemurafenib-induced apoptosis in BRAF mutant melanoma cells according to their PLX4032 sensitivity while modulating apoptotic regulators, revealing a new route to defeat BRAFi resistance [349]. Connexin43 (Cx43) has been identified to enhance the response to BRAFi/MEKi by reducing DNA repair capacity, promoting persistent DNA damage, and inducing cellular senescence, thereby offering a novel therapeutic strategy to overcome drug resistance in advanced BRAF mutant tumors [350]. PF-07799933, a brain-penetrant pan-mutant BRAFi, shows preclinical and clinical activity against V600 and non-V600 mutants and beats current RAF resistance through dose escalation guided by pharmacokinetics, offering a promising new option [351]. Thus, the development of novel targeted agents and their integration with BRAFi holds promise for the treatment of BRAF mutant, drug-resistant melanoma.

Herbal-derived small molecules and standardized botanical extracts have emerged as a complementary strategy to overcome BRAFi resistance in melanoma [352–354]. Emerging studies show that violacein, a natural pigment derived from Chromobacterium violaceum, significantly enhances the efficacy of vemurafenib in BRAF-mutated melanoma spheroids by downregulating key mediators such as fatty acid synthase (FASN), thereby inducing apoptosis and improving treatment response [355]. In pre-clinical models, cumingianoside A, a triterpenoid saponin isolated from the leaves and fine twigs of cumingia latifolia, has been shown to block the cell cycle and and supress autophagy-mediated resistance in vemurafenib-resistant BRAF mutant melanoma cells [356]. Cumingianoside A demonstrated significant inhibitory effects on tumor growth in xenograft mouse models, whether used alone or in combination with vemurafenib [356]. Vitexin compound 1 from Zingiber officinale inhibits BRAFi-resistant melanoma cells through ROS elevation and consequent DNA damage. This monomer does not adversely affect normal cells [357]. Furthermore, curcumin, as a single therapeutic agent, can enhance ROS production and disrupt mitochondrial membrane potential. It can induce apoptosis in drug-resistant BRAF mutant melanoma cells. Combining curcumin with gefitinib enhances the ability to inhibit the proliferation of drug-resistant cells [358]. Ailanthone, a novel c-Jun inhibitor, enhances the efficacy of anti-PD-1 therapy in melanoma and attenuates the immunosuppressive function of Tregs within the tumor microenvironment [359]. Cyclic membrane-active peptides, such as cyclic tachyplesin I (cTI), demonstrate potent efficacy in killing proliferative, non-proliferative, and drug-resistant melanoma cells, including those with the BRAF V600E mutation, without inducing resistance, offering a promising alternative therapeutic strategy to combat acquired drug resistance in melanoma [360]. Although these compounds exhibit low toxicity in vitro and in vivo, their clinical value remains to be established by well-designed randomised controlled trials. At present, no herbal products have received FDA approval for melanoma, and some plant-derived compounds such as aristolochic acid are recognized carcinogens. Therefore, rigorous pharmacological standardisation and Phase I/II safety studies are essential before translating these agents into clinical practice. Based on the resistance patterns outlined in this review, monotherapy or combined targeted agents only delay the onset of resistant disease and do not achieve lasting control. These findings indicate that ERK and PI3K inhibitors plus immunotherapy should be moved to the first line. Initiating randomized triple or quadruple regimens at cycle one represents the logical next phase, shifting from reactive rescue to a proactive first strike with emerging targeted agents and technologies.

Emerging technologies shaping resistance management and future directions

To maximize the efficacy of the mechanism-driven therapies discussed in the aforementioned section, emerging technologies offer transformative tools for real-time resistance monitoring, precise patient stratification, and optimization of treatment regimens for closing the gap between preclinical discoveries and clinical application.

Liquid biopsies: non-invasive monitoring of resistance dynamics

cfDNA detection offers high sensitivity and specificity for clinical tunor samples harboring mutant DNA at frequencies as low as 1 to 5% [55, 361–365]. Silva S et al. employed whole-genome sequencing to create a cfDNA copy number profile for 83 melanoma patients. Their findings revealed that cfDNA is not only relatively stable in plasma but also that an increase in its copy number is strongly correlated with BRAF V600E-mutant melanoma [366]. The BRAF V600E mutation was detected in 14 (18%) of the 76 successfully amplified cfDNA samples. CfDNA concentrations were found to be significantly elevated in patients with active melanoma compared to healthy controls [366]. This finding indicates that gene copy number analysis may function as a potential prognostic indicator of survival and a biomarker for patients with active melanoma. CfDNA profiling enables comprehensive surveillance of emergent targetable and resistance-associated mutations throughout treatment [53]. This technology therefore supports precision personalization of therapy for patients with drug-refractory melanoma.

Noninvasive metabolic imaging using 1H and 1H/31P MRS captures immediate biochemical responses to dabrafenib in melanoma models. The technology tracks sensitivity through lactate and alanine accumulation and measures bioenergetic capacity via βNTP/Pi ratios, offering a promising approach to forecast patient outcomes with signaling pathway inhibitors [367]. Vemurafenib triggers a noncanonical senescence associated secretory phenotype in BRAF V600E melanoma cells, releasing CCL2, TIMP2, and NGFR that shield neighboring tumor cells from growth inhibition, so co-targeting senescent cells and these cytokines may defeat vemurafenib resistance [368]. Ranolazine, an FDA and EMA approved anti-anginal drug, rewires melanoma metabolism by blocking fatty acid oxidation and boosting the methionine salvage pathway, delaying acquired BRAFi resistance and enhancing immunogenicity to improve both targeted therapy and immunotherapy responses [56]. The new patient-derived cutaneous melanoma line MelT79 carries both BRAF V600E and the rare RET S649L mutation, giving researchers a unique tool to dissect how combined alterations fuel heterogeneity, progression, and BRAFi resistance [369].

Single-cell omics: deciphering tumor heterogeneity and resistance clones

Single-cell RNA-seq (scRNA-seq) has become the cornerstone for dissecting melanoma heterogeneity, revealing cell-state switches that underlie BRAFi resistance [370–373]. A study created and fully characterized the vemurafenib-resistant lines A375V, SK-MEL-28V, and RPMI-7951, which show altered morphology, faster growth, and greater invasiveness, providing a robust model to define resistance mechanisms and test new drugs against BRAFi resistance in melanoma [374]. Leveraging these transcriptomic maps guides the identification of targetable RNA networks and accelerates precision strategies [375, 376]. For instance, the dynamics of gene regulation can be understood through the study of protein chromatin open transitions in epigenetics using single-cell Assay for Transposase-Accessible Chromatin with sequencing scATAC-Seq technology [57]. According to scATAC-seq profiles, melanoma drug resistance is tightly linked to TME [377]. scRNA-seq analysis of tumor-specific genes reveals immune infiltration and tumor micro-environment remodeling in melanoma, improving patient prognosis prediction and the feasibility of gene-targeted therapy [378–381]. Tumor-infiltrating T cells exhibit an exhausted phenotype while cancer-associated fibroblasts accumulate, together orchestrating immune escape and resistance to immunotherapy [382–385].

AI-driven drug discovery: predicting resistance and optimizing combinations

Artificial intelligence (AI) is reshaping anti-cancer drug development by forecasting resistance before it emerges and ranking effective drug pairs within minutes [58, 386, 387]. Machine learning models that mine large-scale omics and literature data rapidly identify next-generation inhibitors and optimal combination strategies for BRAF mutant tumors, offering a powerful platform for precision oncology [388]. For instance, machine learning models can detect specific downregulation events linked to CD8 + T-cell infiltration during tumor drug resistance, offering actionable insights for immune checkpoint inhibitor development [389]. Machine learning-based prognostic signatures independently and robustly predict melanoma patient outcome, and algorithm-guided analyses further illustrate the benefit of combining kinase inhibitors with mTOR inhibitors, DNA-damaging agents or HDAC inhibitors [390]. Although artificial intelligence facilitates daily life, models trained on biased or small datasets frequently yield overfitted predictions that fail across diverse patient cohorts. Their black-box architecture hampers biological interpretation and regulatory validation, thereby restricting clinical translation [391, 392]. AI-driven identification of optimal drug combinations and resistance predictors has accelerated the development of adaptive clinical trial designs, which prioritize real-world data to rapidly translate preclinical insights into patient-centric treatments. Accumulating real-world clinical evidence is therefore essential before AI-based disease and therapeutic predictions can be reliably implemented.

Clinical trial innovations: adaptive designs for precision salvage therapy

New clinical trial designs use real-time data to quickly assign patients to the best BRAFi treatments as resistance develops. As an illustration, baseline and early [18F] FDG PET/CT-derived metabolic tumor volume and total lesion glycolysis predict progression-free survival in advanced BRAF V600-mutated melanoma on BRAFi/MEKi better than traditional SUV metrics [393]. How quickly metastases grow before treatment starts serves as a standalone predictor of survival in advanced BRAF V600 melanoma on targeted agents. Rapid baseline growth worsens prognosis regardless of other factors like LDH, where tumors have spread, overall disease load, or prior therapy lines [394]. Furthermore, a phase 1 trial exploring the combination of vemurafenib, cobimetinib, and the HSP90 inhibitor XL888 in advanced BRAF V600-mutant melanoma demonstrated significant antitumor activity, albeit with notable toxicities necessitating dose reductions. Combining HSP90 blockade with BRAFi and MEKi yielded responses in over three quarters of patients, but durability proved challenging with 7.6 months median progression-free survival and 37% five-year survival, underscoring the mixed results of this intensive regimen [59]. Re-challenging with BRAF/MEKi after a drug-free interval can still elicit objective responses in patients who previously acquired resistance, as retrospective data show measurable response rates and progression-free survival during third or fourth courses, supporting repeated cycles alongside other agents [395]. Data from SECOMBIT trial demonstrate that the combination of targeted therapies, including BRAFi and MEKi, with dual immunotherapy targeting PD-1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), significantly improves overall survival (OS) and progression-free survival (PFS) in patients with metastatic melanoma harboring the BRAF V600E mutation (NCT05732805) [81, 396]. Crucially, the combination of anti-PD-1 therapy and BRAFi has demonstrated acceptable safety in patients with advanced melanoma, and longitudinal clinical benefit can be predicted using validated biomarkers (NCT02967692) [16, 397]. Through clinical trials, including BRAFi in metastatic melanoma phase II trial (BRIM-2), BRAFi in metastatic melanoma phase III trial (BRIM-3), BRAFi in metastatic melanoma phase Ib/II combination trial (BRIM-7), cobimetinib plus vemurafenib combination phase III trial (coBRIM), researchers have demonstrated that the combination of cobimetinib and vemurafenib significantly mitigates the adverse effects associated with vemurafenib monotherapy, particularly in patients who previously exhibited poor outcomes with vemurafenib alone [257, 398, 399]. This finding not only elucidated that combination therapy more effectively suppresses the expression of cell-cycle-related genes compared to monotherapy, but also led to a significant improvement in progression-free survival (PFS) among patients. These innovative clinical trial designs not only validate the mechanism-driven therapies outlined earlier but also address unresolved questions such as optimal treatment sequencing and toxicity management, paving the way for personalized salvage therapy in BRAFi-resistant melanoma. However, the NeoTrio trial shows that sequential therapy (targeted therapy followed by neoadjuvant immunotherapy) yields higher disease-free survival than concurrent therapy, while the concurrent arm has more recurrences and the highest toxicity (NCT02858921) [400]. Thus, new clinical evidence is needed to confirm the safety of combining targeted therapy with immunotherapy.

Overall, this section emphasizes four pioneering technologies: liquid biopsy, single-cell omics, AI-driven drug discovery, and adaptive trial design. These technologies collectively have the capability to anticipate and counteract resistance to BRAFi. The crucial message is that these tools should not operate independently. Instead, an integrated framework that incorporates cfDNA monitoring, single-cell profiling, AI-guided decision-making, and adaptive randomization is vital. This integration will enable a shift from reactive rescue strategies to a genuinely proactive and precision-focused therapeutic approach.

Conclusion

Mutations in the BRAF gene, as well as other genetic and epigenetic alterations, predispose patients to recurrence and metastasis following surgery [401, 402]. For instance, patients with sigmoid colon cancer harboring both a BRAF mutation and an NTRK3 fusion gene were found to develop metastases following surgical resection [403]. The introduction of BRAFi has revolutionized the treatment landscape for BRAF mutant melanoma. However, as resistance to vemurafenib, dabrafenib in combination with trametinib and other therapies continues to emerge, the development of novel targeted agents has become a critical need, facilitating improved clinical care for patients with drug-resistant disease. Through clinical observation, periodic assessment of tumor response and the development of drug resistance allows for timely adjustment of treatment plans [404]. While BRAFi have demonstrated substantial clinical efficacy, the development of drug resistance presents a formidable challenge. Consequently, addressing the issue of tumor resistance to BRAFi is crucial for enhancing the clinical efficacy of these agents. The integration of whole-genome analysis, liquid biopsies (e.g., cell-free DNA testing), individualized therapy, new drug development and combination therapy strategies can enhance therapeutic efficacy, overcome tumor resistance and improve patient prognosis.

This article provides a detailed explanation of the theoretical underpinnings of the mechanism of action of BRAFi and explores the mechanisms of drug resistance in BRAF mutant melanoma. It outlines strategies for overcoming and reversing this resistance. It aims to promote the rational use of these agents by clinicians and mitigate tumor resistance, improve patient outcomes, and provide new research directions for investigators studying BRAF mutant melanoma. Despite the rapid accumulation of pre-clinical and clinical data, several unresolved issues temper the conclusions presented herein:(i) The causal hierarchy linking MAPK re-activation, PI3K/AKT bypass signalling and microenvironmental reprogramming remains incompletely understood, recent single-cell studies suggest that these events may occur in parallel rather than in a linear sequence, complicating the design of combination strategies [57, 325]. (ii) The prognostic value of circulating BRAF V600E cfDNA in early-stage or non-metastatic melanoma remains controversial, with discordant results across independent cohorts [115, 366]. (iii) Phase III clinical evidence is still lacking for several promising triple regimens, such as combining a BRAFi, TLR7 agonist, and PD-1 blockade, and the immune-related toxicity risks may outweigh their benefits in unselected patient populations [325]. (iv) Optimal sequencing of targeted therapy followed by immunotherapy (or vice versa) has yet to be defined, and validated predictive biomarkers for this decision are absent [81]. (v) The clinical translation of CRISPR/Cas9-based gene editing or nanoparticle delivery systems is hindered by tissue-specific targeting, off-target mutagenesis and scalable manufacturing challenges [335]. Addressing these limitations will be essential for converting mechanistic insights into durable clinical gains. This review provides new research avenues for investigators focusing on BRAF mutant melanoma to elucidate potential resistance mechanisms, identify corresponding tumor markers and develop drugs to counteract BRAFi resistance. These efforts can enhance or synergize the effects of BRAF-targeted therapies, improving therapeutic efficacy in vemurafenib-resistant melanoma and enhancing patients' quality of life.

Authors’ contributions

Yan Shang: Conceptualization, Writing – original draft, Writing –editing. Tingping Cao: Writing – original draft; Junyan Li: Writing – original draft; Juan Li: Writing – original draft. Lingnan Zhang: Writing – original draft. Qiqi Ma: Writing – original draft. Lanyan Feng: Writing – original draft. Hailong Zhao: Conceptualization, Writing – review & editing, Supervision, Funding acquisition. All authors have read and approved the final manuscript.

Funding

This study was funded by the National Natural Science Foundation of China Regional Project (Grant No.: 82060503) and the Guizhou Province Science and Technology Plan Project (Grant Nos.: QKHJC -ZK [2022] General 622, QKHJC [2019] 1334), National College Students' Innovation and Entrepreneurship Training Program (S202310661178, S202310661167, S2024106612300).

Data availability

The data referenced in this study are primarily sourced from publicly accessible databases: PubMed andweb of science. Specific literature and datasets can be accessed as follows:

PubMed: A doi has been provided after each reference.

As this study did not generate new datasets or raw data, there are no additional data to share. All necessary data and analysis results are described in detail within the article and can be accessed through the iaforementioned links.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.