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.     

Inhibition of autophagy enhances the effects of the AKT inhibitor MK‐2206 when combined with paclitaxel and carboplatin in BRAF wild‐type melanoma

This study investigates the mechanism of action behind the long‐term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK‐2206 in combination with paclitaxel and carboplatin. Although single agent MK‐2206 inhibited phospho‐AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK‐2206 with paclitaxel and carboplatin was cytotoxic in long‐term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase‐dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti‐oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.     

Clinical and pathological associations of the activating RAC1 P29S mutation in primary cutaneous melanoma

Activating mutations in the GTPase RAC1 are a recurrent event in cutaneous melanoma. We investigated the clinical and pathological associations of RAC1^P29S in a cohort of 814 primary cutaneous melanomas with known BRAF and NRAS mutation status. The RAC1^P29S mutation had a prevalence of 3.3% and was associated with increased thickness (OR=1.6 P = 0.001), increased mitotic rate (OR=1.3 P = 0.03), ulceration (OR=2.4 P = 0.04), nodular subtype (OR=3.4 P = 0.004), and nodal disease at diagnosis (OR=3.3 P = 0.006). BRAF mutant tumors were also associated with nodal metastases (OR=1.9 P = 0.004), despite being thinner at diagnosis than BRAF WT (median 1.2 mm versus 1.6 mm, P < 0.001). Immunohistochemical analysis of 51 melanomas revealed that 47% were immunoreactive for RAC1. Melanomas were more likely to show RAC1 immunoreactivity if they were BRAF mutant (OR=5.2 P = 0.01). RAC1 may therefore be important in regulating the early migration of BRAF mutant tumors. RAC1 mutations are infrequent in primary melanomas but may accelerate disease progression.     

Recurrent BRAF kinase fusions in melanocytic tumors offer an opportunity for targeted therapy

BRAF is the most prevalent oncogene and an important therapeutic target in melanoma. In some cancers, BRAF is activated by rearrangements that fuse its kinase domain to 5′ partner genes. We examined 848 comparative genomic hybridization profiles of melanocytic tumors and found copy number transitions within BRAF in 10 tumors, of which six could be further characterized by sequencing. In all, the BRAF kinase domain was fused in‐frame to six N‐terminal partners. No other mutations were identified in melanoma oncogenes. One of the seven melanoma cell lines without known oncogenic mutations harbored a similar BRAF fusion, which constitutively activated the MAP kinase pathway. Sorafenib, but not vemurafenib, could block MAP kinase pathway activation and proliferation of the cell line at clinically relevant concentrations, whereas BRAF^V600E mutant melanoma cell lines were significantly more sensitive to vemurafenib. The patient from whom the cell line was derived showed a durable clinical response to sorafenib.     

Clinical and therapeutic implications of BRAF mutation heterogeneity in metastatic melanoma

Heterogeneity of BRAF mutation in melanoma has been a controversial subject. Quantitative data on BRAF allele frequency (AF) are sparse, and the potential relationship with response to BRAF inhibitors (BRAFi) in patients with metastatic melanoma is unknown. We quantitatively measured BRAF AF in a cohort of treatment naïve metastatic melanoma samples by pyrosequencing and correlated with survival data in patients treated with BRAFi as part of their clinical care. Fifty‐two samples from 50 patients were analysed. BRAF V600E mutations were detected in 71.1% of samples followed by V600K (25%) and V600R (3.9%). There was a wide range of AF from 3.9% to 80.3% (median 41.3%). In 33 patients treated with BRAFi, there was no difference in overall or progression‐free survival when the patients were categorized into high or low AF groups. There was no correlation between AF and degree of response, and no difference in survival based on genotype.     

Near‐genomewide RNAi screening for regulators of BRAFV600E‐induced senescence identifies RASEF,a gene epigenetically silenced in melanoma

The activation of oncogenes in primary cells blocks proliferation by inducing oncogene‐induced senescence (OIS), a highly potent in vivo tumor‐suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near‐genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF^V600E‐induced arrest. With genome‐wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence‐associated (SA)‐β‐galactosidase activity, interleukins, and tumor suppressor p15^INK4B. Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.     

Circulating cell‐free microRNAs in cutaneous melanoma staging and recurrence or survival prognosis

Cutaneous melanoma is a skin cancer with increasing incidence. Identification of novel clinical biomarkers able to detect the stage of disease and suggest prognosis could improve treatment and outcome for melanoma patients. Cell‐free microRNAs (cf‐miRNAs) are the circulating copies of short non‐coding RNAs involved in gene expression regulation. They are released into the interstitial fluid, are detectable in blood and other body fluids and have interesting features of ideal biomarker candidates. They are stable outside the cell, tissue specific, vary along with cancer development and are sensitive to change in the disease course such as progression or therapeutic response. Moreover, they are accessible by non‐invasive methods or venipuncture. Some articles have reported different cf‐miRNAs with the potential of diagnostic tools for melanoma staging, recurrence and survival prediction. Although some concordance of results is already emerging, differences in analytical methods, normalization strategies and tumour staging still will require further research and standardization prior to clinical usage of cf‐miRNA analysis. This article reviews this literature with the aim of contributing to a shared focusing on these new promising tools for melanoma treatment and care.     

Selective RAF inhibitor impairs ERK1/2 phosphorylation and growth in mutant NRAS,vemurafenib‐resistant melanoma cells

The RAF inhibitor vemurafenib achieves remarkable clinical responses in mutant BRAF melanoma patients. However, vemurafenib is burdened by acquired drug resistance and by the side effects associated with its paradoxical activation of the ERK1/2 pathway in wild‐type BRAF cells. This paradoxical effect has driven the development of a new class of RAF inhibitors. Here, we tested one of these selective, non‐paradox‐inducing RAF inhibitors termed paradox‐breaker‐04 (PB04) or PLX7904. Consistent with its design, PB04 is able to efficiently inhibit activation of ERK1/2 in mutant BRAF melanoma cells but does not hyperactivate ERK1/2 in mutant RAS‐expressing cells. Importantly, PB04 inhibited ERK1/2 phosphorylation in mutant BRAF melanoma cells with acquired resistance to vemurafenib/PLX4720 that is mediated by a secondary mutation in NRAS. Consistent with ERK1/2 reactivation driving the re‐acquisition of malignant properties, PB04 promoted apoptosis and inhibited entry into S phase and anchorage‐independent growth in mutant N‐RAS‐mediated vemurafenib‐resistant cells. These data indicate that paradox‐breaker RAF inhibitors may be clinically effective as a second‐line option in a cohort of acquired vemurafenib‐resistant patients.     

Dual MEK/AKT inhibition with trametinib and GSK2141795 does not yield clinical benefit in metastatic NRAS‐mutant and wild‐type melanoma

Aberrant MAPK and PI3K pathway signaling may drive the malignant phenotype in NRAS‐mutant and BRAF^WT NRAS^WT metastatic melanoma. To target these pathways, NRAS‐mutant and BRAF^WT NRAS^WT patients received oral trametinib at 1.5 mg daily and GSK2141795 at 50 mg daily in a two‐cohort Simon two‐stage design. Participants had adequate end‐organ function and no more than two prior treatment regimens. Imaging assessments were performed at 8‐week intervals. A total of 10 NRAS‐mutant and 10 BRAF^WT NRAS^WT patients were enrolled. No objective responses were noted in either cohort. The median PFS and OS were 2.3 and 4.0 months in the NRAS‐mutant cohort and 2.8 and 3.5 months in the wild‐type cohort. Grade 3 and grade 4 adverse events, primarily rash, were observed in 25% of patients. We conclude that the combination of trametinib and GSK2141795 does not have significant clinical activity in NRAS‐mutant or BRAF^WT NRAS^WT melanoma.     

Cooperative induction of apoptosis in NRAS mutant melanoma by inhibition of MEK and ROCK

No effective targeted therapy is currently available for NRAS mutant melanoma. Experimental MEK inhibition is rather toxic and has only limited efficacy in clinical trials. At least in part, this is caused by the emergence of drug resistance, which is commonly seen for single agent treatment and shortens clinical responses. Therefore, there is a dire need to identify effective companion drug targets for NRAS mutant melanoma. Here, we show that at concentrations where single drugs had little effect, ROCK inhibitors GSK269962A or Fasudil, in combination with either MEK inhibitor GSK1120212 (Trametinib) or ERK inhibitor SCH772984 cooperatively caused proliferation inhibition and cell death in vitro. Simultaneous inhibition of MEK and ROCK caused induction of Bim_EL, PARP, and Puma, and hence apoptosis. In vivo, MEK and ROCK inhibition suppressed growth of established tumors. Our findings warrant clinical investigation of the effectiveness of combinatorial targeting of MAPK/ERK and ROCK in NRAS mutant melanoma.     

A novel ATM‐dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM‐dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM‐dependent checkpoint arrest, and over‐expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM‐dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over‐express PLK1, and a significant proportion of melanomas have high levels of PLK1 over‐expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM‐dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected.