Activities of multiple cancer-related pathways are associated with BRAF mutation and predict the resistance to BRAF/MEK inhibitors in melanoma cells

Drug resistance is a major obstacle in the targeted therapy of melanoma using BRAF/MEK inhibitors. This study was to identify BRAF V600E-associated oncogenic pathways that predict resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors. We took in silico approaches to analyze the activities of 24 cancer-related pathways in melanoma cells and identify those whose activation was associated with BRAF V600E and used the support vector machine (SVM) algorithm to predict the resistance of BRAF-mutated melanoma cells to BRAF/MEK inhibitors. We then experimentally confirmed the in silico findings. In a microarray gene expression dataset of 63 melanoma cell lines, we found that activation of multiple oncogenic pathways preferentially occurred in BRAF-mutated melanoma cells. This finding was reproduced in 5 additional independent melanoma datasets. Further analysis of 46 melanoma cell lines that harbored BRAF mutation showed that 7 pathways, including TNFα, EGFR, IFNα, hypoxia, IFNγ, STAT3, and MYC, were significantly differently expressed in AZD6244-resistant compared with responsive melanoma cells. A SVM classifier built on this 7-pathway activation pattern correctly predicted the response of 10 BRAF-mutated melanoma cell lines to the MEK inhibitor AZD6244 in our experiments. We experimentally showed that TNFα, EGFR, IFNα, and IFNγ pathway activities were also upregulated in melanoma cell A375 compared with its sub-line DRO, while DRO was much more sensitive to AZD6244 than A375. In conclusion, we have identified specific oncogenic pathways preferentially activated in BRAF-mutated melanoma cells and a pathway pattern that predicts resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors, providing novel clinical implications for melanoma therapy.     

Conjunctival melanoma copy number alterations and correlation with mutation status,tumor features,and clinical outcome

Relatively little is known about the genetic aberrations of conjunctival melanomas (CoM) and their correlation with clinical and histomorphological features as well as prognosis. The aim of this large collaborative multicenter study was to determine potential key biomarkers for metastatic risk and any druggable targets for high metastatic risk CoM. Using Affymetrix single nucleotide polymorphism genotyping arrays on 59 CoM, we detected frequent amplifications on chromosome (chr) 6p and deletions on 7q, and characterized mutation‐specific copy number alterations. Deletions on chr 10q11.21‐26.2, a region harboring the tumor suppressor genes, PDCD4, SUFU, NEURL1, PTEN, RASSF4, DMBT1, and C10orf90 and C10orf99, significantly correlated with metastasis (Fisher's exact, p ≤ 0.04), lymphatic invasion (Fisher's exact, p ≤ 0.02), increasing tumor thickness (Mann–Whitney, p ≤ 0.02), and BRAF mutation (Fisher's exact, p ≤ 0.05). This enhanced insight into CoM biology is a step toward identifying patients at risk of metastasis and potential therapeutic targets for systemic disease.     

Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma

The effect of NRAS mutations on the pathological features and clinical outcomes in patients with cutaneous melanoma was compared with that of tumors containing BRAF(V600E) mutations and tumors wild type for both (WT). Clinical outcome data were obtained from a prospective cohort of 249 patients. Mutations involving NRAS and BRAF(V600E) were detected by PCR and were sequence verified. Cox proportional hazards regression was performed to relate NRAS and BRAF mutations to clinical outcome. Seventy-five percentage of NRAS mutations occurred in tumors >1 mm thick (BRAF(V600E) 40%, WT 34%); 75% of NRAS mutations had >1 mitosis/mm(2) (BRAF(V600E) 40%, WT 55%). When compared to WT, multivariate analysis of melanoma-specific survival (MSS) identified NRAS mutations as an adverse prognostic factor [hazard ratio (HR) 2.96; P = 0.04] but not BRAF(V600E) mutations (HR 1.73; P = 0.23). NRAS mutations were associated with thicker tumors and higher rates of mitosis when compared to BRAF(V600E) and WT melanoma and independently of this, with shorter MSS.     

AEBP1 upregulation confers acquired resistance to BRAF (V600E) inhibition in melanoma

An activating BRAF (V600E) kinase mutation occurs in approximately half of melanomas. Recent clinical studies have demonstrated that vemurafenib (PLX4032) and dabrafenib, potent and selective inhibitors of mutant v-raf murine sarcoma viral oncogene homolog B1 (BRAF), exhibit remarkable activities in patients with V600 BRAF mutant melanomas. However, acquired drug resistance invariably develops after the initial treatment. Identification of acquired resistance mechanisms may inform the development of new therapies that elicit long-term responses of melanomas to BRAF inhibitors. Here we report that increased expression of AEBP1 (adipocyte enhancer-binding protein 1) confers acquired resistance to BRAF inhibition in melanoma. AEBP1 is shown to be highly upregulated in PLX4032-resistant melanoma cells because of the hyperactivation of the PI3K/Akt-cAMP response element-binding protein (CREB) signaling pathway. This upregulates AEBP1 expression and thus leads to the activation of NF-κB via accelerating IκBa degradation. In addition, inhibition of the PI3K/Akt-CREB-AEBP1-NF-κB pathway greatly reverses the PLX4032-resistant phenotype of melanoma cells. Furthermore, increased expression of AEBP1 is validated in post-treatment tumors in patients with acquired resistance to BRAF inhibitor. Therefore, these results reveal a novel PI3K/Akt-CREB-AEBP1-NF-κB pathway whose activation contributes to acquired resistance to BRAF inhibition, and suggest that this pathway, particularly AEBP1, may represent a novel therapeutic target for treating BRAF inhibitor-resistant melanoma.     

Clinicopathological characteristics associated with BRAFK601E and BRAFL597 mutations in melanoma

BRAF mutations at codons L597 and K601 occur uncommonly in melanoma. Clinical and pathological associations of these mutations were investigated in a cohort of 1119 patients with known BRAF mutation status. A BRAF mutation was identified in 435 patients; Mutations at L597 and the K601E mutation were seen in 3.4 and 3.2% of these, respectively. K601E melanomas tended to occur in male patients, a median age of 58 yr, were generally found on the trunk (64%) and uncommonly associated with chronically sun‐damaged (CSD) skin. BRAF L597 melanomas occurred in older patients (median 66 yr), but were associated with CSD skin (extremities or head and neck location – 73.3%, P = 0.001). Twenty‐three percent of patients with V600E‐ and 43% of patients with K601E‐mutant melanomas presented with nodal disease at diagnosis compared to just 14% of patients with BRAF wild‐type tumors (P = 0.001 and 0.006, respectively). Overall, these mutations represent a significant minority of BRAF mutations, but have distinct clinicopathological phenotypes and clinical behaviors.     

Genetic and morphologic features for melanoma classification

Melanoma is comprised of biologically distinct subtypes. The defining clinical, histomorphologic, and molecular features are not fully established. This study sought to validate the association between genetic and histomorphologic features previously described and to determine their reproducibility and association with important clinical variables. Detailed clinical and histomorphologic features of 365 primary cutaneous melanomas were assessed by 11 pathologists and correlated with mutation status of BRAF and NRAS. There was substantial agreement in the quantitative assessment of histomorphologic features showing similar or better interobserver reproducibility than the established World Health Organization classification scheme. We confirmed that melanomas with BRAF mutations showed characteristic morphologic features (P < 0.0001) and metastasized more frequently to regional lymph nodes (P = 0.046). Importantly, melanomas without mutations were a heterogeneous group, with a subset having very similar clinical and morphological features as those with BRAF mutation raising the possibility that they are biologically related. Our study confirms an association between histomorphologic features, mutation status, and pattern of metastasis, providing criteria for a refined melanoma classification aimed at defining biologically homogeneous disease subgroups.     

Detrimental effects of melanocortin‐1 receptor (MC1R) variants on the clinical outcomes of BRAF V600 metastatic melanoma patients treated with BRAF inhibitors

Melanocortin‐1 receptor (MC1R) plays a key role in skin pigmentation, and its variants are linked with a higher melanoma risk. The influence of MC1R variants on the outcomes of patients with metastatic melanoma (MM) treated with BRAF inhibitors (BRAFi) is unknown. We studied the MC1R status in a cohort of 53 consecutive BRAF‐mutated patients with MM treated with BRAFi. We also evaluated the effect of vemurafenib in four ^V600BRAF melanoma cell lines with/without MC1R variants. We found a significant correlation between the presence of MC1R variants and worse outcomes in terms of both overall response rate (ORR; 59% versus 95%, P = 0.011 univariate, P = 0.028 multivariate analysis) and progression‐free survival (PFS) shorter than 6 months (72% versus 33%, P = 0.012 univariate, P = 0.027 multivariate analysis). No difference in overall survival (OS) was reported, probably due to subsequent treatments. Data in vitro showed a significant different phosphorylation of Erk1/2 and p38 MAPK during treatment, associated with a greater increase in vemurafenib IC50 in MC1R variant cell lines.     

Role and therapeutic potential of PI3K-mTOR signaling in de novo resistance to BRAF inhibition

BRAF inhibition is highly active in BRAF-mutant melanoma, but the degree and duration of responses is quite variable. Improved understanding of the mechanisms of de novo resistance may lead to rational therapeutic strategies with improved efficacy. Proteomic analysis of BRAF-mutant, PTEN-wild-type human melanoma cell lines treated with PLX4720 demonstrated that sensitive and de novo resistant lines exhibit similar RAS-RAF-MEK-ERK pathway inhibition, but the resistant cells exhibited durable activation of S6 and P70S6K. Treatment with the mTOR inhibitor rapamycin blocked activation of P70S6K and S6, but it also increased activation of AKT and failed to induce cell death. Combined treatment with rapamycin and PX-866, a PI3K inhibitor, blocked the activation of S6 and AKT and resulted in marked cell death when combined with PLX4720. The results support the rationale for combined targeting of BRAF and the PI3K-AKT pathways and illustrate how target selection will be critical to such strategies.     

Genomic analysis and clinical management of adolescent cutaneous melanoma

Melanoma in young children is rare; however, its incidence in adolescents and young adults is rising. We describe the clinical course of a 15‐year‐old female diagnosed with AJCC stage IB non‐ulcerated primary melanoma, who died from metastatic disease 4 years after diagnosis despite three lines of modern systemic therapy. We also present the complete genomic profile of her tumour and compare this to a further series of 13 adolescent melanomas and 275 adult cutaneous melanomas. A somatic BRAF^V600E mutation and a high mutational load equivalent to that found in adult melanoma and composed primarily of C>T mutations were observed. A germline genomic analysis alongside a series of 23 children and adolescents with melanoma revealed no mutations in known germline melanoma‐predisposing genes. Adolescent melanomas appear to have genomes that are as complex as those arising in adulthood and their clinical course can, as with adults, be unpredictable.     

BRAF mutations in cutaneous melanoma are independently associated with age, anatomic site of the primary tumor, and the degree of solar elastosis at the primary tumor site

Oncogenic BRAF mutations are more frequent in cutaneous melanoma occurring at sites with little or moderate sun-induced damage than at sites with severe cumulative solar ultraviolet (UV) damage. We studied cutaneous melanomas from geographic regions with different levels of ambient UV radiation to delineate the relative effects of cumulative UV damage, age, and anatomic site on the frequency of BRAF mutations. We show that BRAF-mutated melanomas occur in a younger age group on skin without marked solar elastosis and less frequently affect the head and neck area, compared to melanomas without BRAF mutations. The findings indicate that BRAF-mutated melanomas arise early in life at low cumulative UV doses, whereas melanomas without BRAF mutations require accumulation of high UV doses over time. The effect of anatomic site on the mutation spectrum further suggests regional differences among cutaneous melanocytes.     

MicroRNA‐125a promotes resistance to BRAF inhibitors through suppression of the intrinsic apoptotic pathway

Melanoma patients with BRAF^V600E‐mutant tumors display striking responses to BRAF inhibitors (BRAFi); however, almost all invariably relapse with drug‐resistant disease. Here, we report that microRNA‐125a (miR‐125a) expression is upregulated in human melanoma cells and patient tissues upon acquisition of BRAFi resistance. We show that miR‐125a induction confers resistance to BRAF^V600E melanoma cells to BRAFi by directly suppressing pro‐apoptotic components of the intrinsic apoptosis pathway, including BAK1 and MLK3. Apoptotic suppression and prolonged survival favor reactivation of the MAPK and AKT pathways by drug‐resistant melanoma cells. We demonstrate that miR‐125a inhibition suppresses the emergence of resistance to BRAFi and, in a subset of resistant melanoma cell lines, leads to partial drug resensitization. Finally, we show that miR‐125a upregulation is mediated by TGFβ signaling. In conclusion, the identification of this novel role for miR‐125a in BRAFi resistance exposes clinically relevant mechanisms of melanoma cell survival that can be exploited therapeutically.     

MicroRNA and mRNA expression profiling in metastatic melanoma reveal associations with BRAF mutation and patient prognosis

The role of microRNAs (miRNAs) in melanoma is unclear. We examined global miRNA expression profiles in fresh‐frozen metastatic melanomas in relation to clinical outcome and BRAF mutation, with validation in independent cohorts of tumours and sera. We integrated miRNA and mRNA information from the same samples and elucidated networks associated with outcome and mutation. Associations with prognosis were replicated for miR‐150‐5p, miR‐142‐3p and miR‐142‐5p. Co‐analysis of miRNA and mRNA uncovered a network associated with poor prognosis (PP) that paradoxically favoured expression of miRNAs opposing tumorigenesis. These miRNAs are likely part of an autoregulatory response to oncogenic drivers, rather than drivers themselves. Robust association of miR‐150‐5p and the miR‐142 duplex with good prognosis and earlier stage metastatic melanoma supports their potential as biomarkers. miRNAs overexpressed in association with PP in an autoregulatory fashion will not be suitable therapeutic targets.     

Detailed imaging and genetic analysis reveal a secondary BRAFL505H resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three‐dimensional imaging. In all patients, only a subset of lesions progressed. Next‐generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib‐resistant patient‐derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.     

Regulation of NR4A nuclear receptor expression by oncogenic BRAF in melanoma cells

Activating mutations in the MAPK pathway effectors, NRAS or BRAF, are detected in over 70% of melanomas. Accordingly, the identification of downstream targets of constitutive MAPK signalling in melanoma represents a major goal in understanding the genesis of this disease. We report here the regulation of members of the NR4A family of nuclear receptors by the BRAF-MEK-ERK cascade in melanoma cells. Expression profiling of melanoma cells in which both the NR4A1 and NR4A2 family members have been down-regulated by siRNA revealed alterations in genes associated with proliferation, survival and invasiveness of tumour cells. Notably, the up-regulation of Wnt/β-catenin pathway antagonists, DACT1 and CITED1, following NR4A1/2 ablation suggests a possible link between NR4A and β-catenin activity in melanoma cells. Taken together, these data suggest that dysregulation of NR4A nuclear receptors expression and function by the MAPK pathway may contribute to melanoma tumourigenicity.     

Cotargeting histone deacetylases and oncogenic BRAF synergistically kills human melanoma cells by necrosis independently of RIPK1 and RIPK3

Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAF^V600E melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAF^V600E melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAF^V600E melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.