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Treatment of BRAF-mutant metastatic melanoma with mitogen-activated protein kinase (MAPK) pathway targeted therapies (BRAF/MEK inhibitors) and immune checkpoint inhibitors has revolutionised management and improved outcomes for patients with advanced stage disease. However, acquired resistance to MAPK inhibitor therapy develops in the majority of patients at approximately 12 months and multiple mechanisms lead to resistance. Understanding the mechanisms of resistance is therefore critical for the development of more effective therapeutic strategies in BRAF-mutant melanoma. Recently, several distinct mechanisms of resistance to BRAF-inhibition have been proposed based on data obtained in experimental melanoma cell models and small series of human tumour samples. These include reactivation of the MAPK pathway resulting in continued extracellular signal-regulated kinase activation and activation of parallel signalling pathways including the PI3K-mTOR (phosphoinositide 3-kinase–mammalian target of rapamycin) pathway. Alterations in how the cells of the immune system respond to melanoma cells treated with targeted therapy may also influence response and progression. In this review, we discuss these mechanisms and identify potential therapeutic strategies to overcome resistance which, in turn, will lead to improved outcomes for patients with metastatic melanoma. [ABSTRACT FROM AUTHOR]

Alterations in the p14ARF tumor suppressor are frequent in many human cancers and are associated with susceptibility to melanoma, pancreatic cancer, and nervous system tumors. In addition to its p53-regulatory functions, p14ARF has been shown to influence ribosome biogenesis and to regulate the endoribo-nuclease B23, but there remains considerable controversy about its nucleolar role. We sought to clarify the activities of p14ARF by studying its interaction with ribosomes. We show that p14ARF and B23 interact within the nucleolar 60 S preribosomal particle and that this interaction does not require rRNA. In contrast to previous reports, we found that expression of p14ARF does not significantly alter ribosome biogenesis but inhibits polysome formation and protein translation in vivo. These results suggest a ribosome-dependent p14ARF pathway that regulates cell growth and thus complements p53-dependent p14ARF functions. [ABSTRACT FROM AUTHOR]

Demonstrates that the tumor suppressor factor p14 forms a complex with p120 to modulate cell cycle progression. Formation of the complex; Experimental procedures; Regulation of transcription factor activity.

Abstract: The ARF tumour suppressor is a product of the INK4a/ARF locus; a sequence that is frequently altered in human cancer. ARF is upregulated by oncogenic stimuli and is a critical regulator of p53 stability through interactions with the mdm2 and ARF-BP1/Mule ubiquitin ligases. Cellular stress signals liberate ARF from the nucleolus where it is bound to B23/nucleophosmin. This nucleolar location of ARF may serve as a reservoir for the rapid induction of p53, but may also serve to co-ordinate effects on cell cycle, survival and growth. The biological functions of ARF interactions with other binding partners remain uncertain, but ARF-mediated sumoylation may represent a unifying effector pathway. [Copyright &y& Elsevier]

Background: Adjuvant dabrafenib plus trametinib therapy improves relapse-free survival in patients with resected stage III melanoma. We aimed to ascertain the proportion of patients who would have a pathological response and a response according to Response Evaluation Criteria in Solid Tumors (RECIST) after neoadjuvant dabrafenib plus trametinib therapy for resectable clinical stage III melanoma.Methods: NeoCombi was a single-arm, open-label, single-centre, phase 2 study done at Melanoma Institute Australia (Sydney, NSW, Australia). Eligible patients were adults (aged ≥18 years) with histologically confirmed, resectable, RECIST-measurable, clinical stage IIIB-C (American Joint Committee on Cancer [AJCC] 7th edition), BRAFV600-mutant melanoma, and had an Eastern Cooperative Oncology Group performance status of 1 or lower. Patients received 150 mg dabrafenib orally, twice daily, plus 2 mg trametinib orally, once daily, for 52 weeks (12 weeks of neoadjuvant therapy before complete resection of the pre-therapy tumour bed, and 40 weeks of adjuvant therapy thereafter). CT and PET scans were done at baseline and before resection. The primary outcomes were the proportion of patients achieving a complete pathological response and the proportion of patients achieving a response according to RECIST at week 12, analysed as per protocol. This trial is registered with ClinicalTrials.gov, NCT01972347, and follow-up of patients is ongoing.Findings: Between Aug 20, 2014, and April 19, 2017, 40 patients were screened, of whom 35 eligible patients were enrolled, received neoadjuvant dabrafenib plus trametinib, and underwent resection. At the data cutoff (Sept 24, 2018), median follow-up was 27 months (IQR 21-36). At resection, 30 (86%) patients achieved a RECIST response; 16 (46%; 95% CI 29-63) had a complete response and 14 (40%; 24-58) had a partial response. Five patients (14%; 95% CI 5-30) had stable disease, and no patients progressed. After resection and pathological evaluation, all 35 patients achieved a pathological response, of whom 17 (49%; 95% CI 31-66) patients had a complete pathological response and 18 (51%; 95% CI 34-69) had a non-complete pathological response. Treatment-related serious adverse events occurred in six (17%) of 35 patients and grade 3-4 adverse events occurred in ten (29%) patients. No treatment-related deaths were reported.Interpretation: Neoadjuvant dabrafenib plus trametinib therapy could be considered in the management of RECIST-measurable resectable stage III melanoma as it led to a high proportion of patients achieving a complete response according to RECIST and a high proportion of patients achieving a complete pathological response, with no progression during neoadjuvant therapy.Funding: GlaxoSmithKline; Novartis; National Health and Medical Research Council, Australia; and Melanoma Institute Australia. [ABSTRACT FROM AUTHOR]

Highlights • Spin column-based kits outperform magnetic bead-based commercial cfDNA kits. • The Qiagen spin column-based cfDNA kit remains the gold standard. • The Qiagen magnetic bead-based cfDNA kit is an alternative when factoring in price. Abstract Analysis of liquid biopsies and the identification of non-invasive biomarkers for the diagnosis and prognosis of solid tumors has grown exponentially over the last few years. This has led to an increasing number of commercial kits optimised for the purification of circulating free (cf) DNA and RNA/miRNA from biofluids such as plasma, serum and urine. To optimise and standardise current practices we sought to evaluate the performance of spin column-based and magnetic bead-based commercial kits. The following commercial cfDNA purification kits were analysed in this study: QIAamp circulating nucleic acid kit (Qiagen, Germany); Plasma/serum cell-free circulating DNA Purification midi kit (Norgen Biotek, Canada); QIAamp minElute ccfDNA mini kit (Qiagen); Maxwell RSC ccfDNA plasma kit (Promega, USA); MagMAX cell-free DNA isolation kit (Applied Biosystems, USA); and NextPrep-Mag cfDNA isolation kit (Bioo Scientific, USA). Extracted DNA from the plasma of healthy individuals, either nonspiked or spiked with DNA fragments or cfDNA, was evaluated for recovery using either a BioRad Experion or ddPCR analysis. This study represents the first to use a comprehensive size distribution of spiked-in DNA fragments to evaluate commercial cfDNA kits. The commonly used spin column-based Qiagen QIAamp circulating nucleic acid kit was found to be the most consistent performing kit across the two evaluation assays employed. The Qiagen QIAamp minElute ccfDNA mini kit represented the best performing magnetic bead-based kit and provides an alternative based on lower cost/sample with a simpler workflow than spin column-based kits. [ABSTRACT FROM AUTHOR]

Background Acquired resistance to BRAF inhibitors (BRAFi) is a near-universal phenomenon caused by numerous genetic and non-genetic alterations. In this study, we evaluated the spectrum, onset, pattern of progression, and subsequent clinical outcomes associated with specific mechanisms of resistance. Methods We compiled clinical and genetic data from 100 patients with 132 tissue samples obtained at progression on BRAFi therapy from 3 large, previously published studies of BRAFi resistance. These samples were subjected to whole-exome sequencing and/or polymerase chain reaction-based genetic testing. Results Among 132 samples, putative resistance mechanisms were identified in 58%, including NRAS or KRAS mutations (20%), BRAF splice variants (16%), BRAF V600E/K amplifications (13%), MEK1/2 mutations (7%), and non-mitogen-activated protein kinase pathway alterations (11%). Marked heterogeneity was observed within tumors and patients; 18 of 19 patients (95%) with more than one progression biopsy had distinct/unknown drivers of resistance between samples. NRAS mutations were associated with vemurafenib use (p = 0.045) and intracranial metastases (p = 0.036), and MEK1/2 mutations correlated with hepatic progression (p = 0.011). Progression-free survival and overall survival were similar across resistance mechanisms. The median survival after disease progression was 6.9 months, and responses to subsequent BRAF and MEK inhibition were uncommon (2 of 15; 13%). Post-progression outcomes did not correlate with specific acquired BRAFi-resistance mechanisms. Conclusions This is the first study to systematically characterise the clinical implications of particular acquired BRAFi-resistance mechanisms in patients with BRAF -mutant melanoma largest study to compile the landscape of resistance. Despite marked heterogeneity of resistance mechanisms within patients, NRAS mutations correlated with vemurafenib use and intracranial disease involvement. [ABSTRACT FROM AUTHOR]

BRAF and MEK inhibitors, alone or in combination, are highly active in the 40% of patients with BRAF mutant metastatic melanoma. Despite this activity resistance often develops in patients treated with these agents. This review summarises the biology of the mitogen activated protein kinase (MAPK) pathway, with particular reference to the effects of BRAF and MEK inhibitors in BRAF mutant melanoma. The clinical and molecular predictors of response and mechanisms of resistance are discussed in detail along with the biological rationale and evidence for future treatment strategies in both MAPK inhibitor naïve and resistant BRAF mutant melanoma. [ABSTRACT FROM AUTHOR]

BARD1 is a breast cancer tumor suppressor with multiple domains and functions. BARD1 comprises a tandem BRCT domain at the C-terminus, and this sequence has been reported to target BARD1 to distinct subcellular locations such as nuclear DNA breakage sites and the centrosome through binding to regulatory proteins such as HP1 and OLA1, respectively. We now identify the BRCT domain as a binding site for p53. We first confirmed previous reports that endogenous BARD1 binds to p53 by immunoprecipitation assay, and further show that BARD1/p53 complexes locate at mitochondria suggesting a cellular location for p53 regulation of BARD1 apoptotic activity. We used a proximity ligation assay to map three distinct p53 binding sequences in human BARD1, ranging from weak (425–525) and modest (525–567) to strong (551–777 comprising BRCT domains). Deletion of the BRCT sequence caused major defects in the ability of BARD1 to (1) bind p53, (2) localize to the cytoplasm and mitochondria, and (3) induce Bax oligomerization and apoptosis. Our data suggest that BARD1 can move to mitochondria independent of p53, but subsequently associates with p53 to induce Bax clustering in part by decreasing mitochondrial Bcl-2 levels. We therefore identify a role for the BRCT domain in stimulating BARD1 nuclear export and mitochondrial localization, and in assembling mitochondrial BARD1/p53 complexes to regulate specific activities such as apoptotic function. [ABSTRACT FROM AUTHOR]

Epigenetic changes are widespread in melanoma and contribute to the pathogenic biology of this disease. In the present study, we show that I-BET151, which belongs to a new class of drugs that target the BET family of epigenetic 'reader' proteins, inhibits melanoma growth in vivo and induced variable degrees of apoptosis in a panel of melanoma cells. Apoptosis was caspase dependent and associated with G1 cell cycle arrest. All melanoma cells tested had increased levels of the BH3 proapoptotic protein BIM, which appeared to be regulated by the BRD2 BET protein and to some extent by BRD3. In contrast, knockdown experiments indicated that inhibition of BRD4 was associated with decreased levels of BIM. Apoptosis was dependent on BIM in some but not all cell lines, indicating that other factors were determinants of apoptosis, such as downregulation of antiapoptotic proteins revealed in gene expression arrays. G1 cell cycle arrest appeared to be mediated by p21 and resulted from inhibition of the BRD4 protein. The activity of BET protein inhibitors appears independent of the BRAF and NRAS mutational status of melanoma, and further studies to assess their therapeutic role in melanoma are warranted. [ABSTRACT FROM AUTHOR]

Cancer cells commonly undergo chronic endoplasmic reticulum (ER) stress, to which the cells have to adapt for survival and proliferation. We report here that in melanoma cells intrinsic activation of the ER stress response/unfolded protein response (UPR) is, at least in part, caused by increased outputs of protein synthesis driven by oncogenic activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and promotes proliferation and protects against apoptosis induced by acute ER stress. Inhibition of oncogenic BRAFV600E or MEK-attenuated activation of inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) signaling of the UPR in melanoma cells. This was associated with decreased phosphorylation of eukaryotic initiation factor 4E (eIF4E) and nascent protein synthesis and was recapitulated by knockdown of eIF4E. In line with this, introduction of BRAFV600E into melanocytes led to increases in eIF4E phosphorylation and protein production and triggered activation of the UPR. Similar to knockdown of glucose-regulated protein 78 (GRP78), inhibition of XBP1 decelerated melanoma cell proliferation and enhanced apoptosis induced by the pharmacological ER stress inducers tunicamycin and thapasigargin. Collectively, these results reveal that potentiation of adaptation to chronic ER stress is another mechanism by which oncogenic activation of the MEK/ERK pathway promotes the pathogenesis of melanoma. [ABSTRACT FROM AUTHOR]

Approximately 50% of melanomas require oncogenic B-RAFV600E signaling for proliferation, survival, and metastasis, and the use of highly selective B-RAF inhibitors has yielded remarkable, although short-term, clinical responses. Reactivation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may therefore provide new strategies for managing melanoma. In this report, we applied whole-genome expression analyses to reveal that oncogenic B-RAFV600E regulates genes associated with epithelial-mesenchymal transition in normal cutaneous human melanocytes. Most prominent was the B-RAF-mediated transcriptional repression of E-cadherin, a keratinocyte-melanoma adhesion molecule whose loss is intimately associated with melanoma invasion and metastasis. Here we identify a link between oncogenic B-RAF, the transcriptional repressor Tbx3, and E-cadherin. We show that B-RAFV600E induces the expression of Tbx3, which potently represses E-cadherin expression in melanocytes and melanoma cells. Tbx3 expression is normally restricted to developmental embryonic tissues and promoting cell motility, but it is also aberrantly increased in various cancers and has been linked to tumor cell invasion and metastasis. We propose that this B-RAF/Tbx3/E-cadherin pathway has a critical role in promoting the metastasis of B-RAF-mutant melanomas. [ABSTRACT FROM AUTHOR]

Cellular senescence permanently restricts the replicative capacity of cells in response to various stress signals, including aberrant activation of oncogenes. The presence of predictive senescence markers in human premalignant lesions suggests that senescence may function as a genuine tumor suppressor. These markers are not exclusive to the senescence program, however, and it is possible that their expression in vivo does not discriminate irreversible from reversible forms of proliferative arrest. In this study, we aimed to clarify whether human nevus cells can be distinguished from primary and transformed melanocytes by examining the expression of eight senescence markers, including those previously purported to define nevi as senescent tumors. Specifically, we analyzed effectors of senescence, including p16INK4a, p53, and DNA damage (γ-H2AX), as well as predictive markers of senescence including Ki67, PML, senescence-associated β-galactosidase, heterochromatic foci (H3K9Me, 4′-6-diamidino-2-phenylindole), and nuclear size. We found that these commonly accepted senescence markers do not in fact distinguish nevi from precursor/normal and transformed/malignant melanocytes. We conclude that on the basis of current evidence it cannot be reasonably inferred that nevi are permanently growth arrested via senescence. [ABSTRACT FROM AUTHOR]

Aberrant activation of the BRAF kinase occurs in ∼60% of melanomas, and although BRAF inhibitors have shown significant early clinical success, acquired resistance occurs in most patients. Resistance to chronic BRAF inhibition often involves reactivation of mitogen-activated protein kinase (MAPK) signaling, and the combined targeting of BRAF and its downstream target MAPK/ERK kinase (MEK) may delay or overcome resistance. To investigate the efficacy of combination BRAF and MEK inhibition, we generated melanoma cell clones resistant to the BRAF inhibitor GSK2118436. These BRAF inhibitor-resistant sublines acquired resistance through several distinct mechanisms, including the acquisition of activating N-RAS mutations and increased accumulation of COT1. These alterations uniformly promoted MAPK reactivation and most conferred resistance to MEK inhibition and to the concurrent inhibition of BRAF and MEK. These data indicate that melanoma tumors are likely to develop heterogeneous mechanisms of resistance, many of which will confer resistance to multiple MAPK inhibitory therapies. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Loss of expression of the p16INK4/CDKN2 gene in cutaneous malignant melanoma correlates with tumor cell proliferation and invasive stage," by L. Talve and colleagues in the 1997 issue.

A letter to the editor is presented regarding the effect of oncogenic B-RAFV600E on the survival of human melanocytes.

Oncogene-induced senescence is considered to act as a potent barrier to cell transformation, and has been seen in vivo during the early stages of tumor development. Human nevus cells frequently express oncogenic N-RAS or B-RAF, and are thought to be permanently growth arrested. Many studies have suggested that the p16INK4a and, to a lesser extent, the p14ARF tumor suppressor proteins act as critical triggers of oncogene-induced senescence in nevi, and thus these proteins represent major inhibitors of progression to melanoma. There have also been reports, however, showing that p16INK4a and/or p14ARF is not sufficient to execute the oncogene-induced senescence program. In this study, we examined the impact of melanoma-associated N-RASQ61K on melanocyte senescence and utilized RNA-interference vectors to directly assess the individual contribution of human p14ARF and p16INK4a genes to the N-RAS-induced senescence program. We formally show that cultured human melanocytes can initiate an effective oncogene-mediated senescence program in the absence of INK4a/ARF-encoded proteins. Our data are consistent with observations showing that senescent nevus cells do not always express p16INK4a, and highlight the need to thoroughly explore INK4a/ARF-independent molecular pathways of senescence in human melanocytes.Journal of Investigative Dermatology (2009) 129, 1983–1991; doi:10.1038/jid.2009.5; published online 12 February 2009 [ABSTRACT FROM AUTHOR]

We concurrently examine the whole genome, transcriptome, methylome, and immune cell infiltrates in baseline tumors from 77 patients with advanced cutaneous melanoma treated with anti-PD-1 with or without anti-CTLA-4. We show that high tumor mutation burden (TMB), neoantigen load, expression of IFNγ-related genes, programmed death ligand expression, low PSMB8 methylation (therefore high expression), and T cells in the tumor microenvironment are associated with response to immunotherapy. No specific mutation correlates with therapy response. A multivariable model combining the TMB and IFNγ-related gene expression robustly predicts response (89% sensitivity, 53% specificity, area under the curve [AUC], 0.84); tumors with high TMB and a high IFNγ signature show the best response to immunotherapy. This model validates in an independent cohort (80% sensitivity, 59% specificity, AUC, 0.79). Except for a JAK3 loss-of-function mutation, for patients who did not respond as predicted there is no obvious biological mechanism that clearly explained their outlier status, consistent with intratumor and intertumor heterogeneity in response to immunotherapy. [Display omitted] • Multiomic analysis predicts response but not resistance to immunotherapy • Nonresponders had no common mechanisms of resistance • Structural rearrangements and PSMB8 promoter methylation occurred in nonresponders • JAK3 mutation was a possible resistance mechanism in a patient predicted to respond Newell et al. used clinical features and multiomic analysis (WGS, RNAseq, immunohistochemistry, methylation) to show that IFNγ plus TMB most accurately predicted response to immunotherapy, but not resistance. No common mechanism of resistance was identified in keeping with tumor heterogeneity, and patients with clinical and molecular discordance were analyzed individually. [ABSTRACT FROM AUTHOR]

Metastatic cutaneous melanoma is highly resistant to cytotoxic drugs, and this contributes to poor prognosis. In vivo studies on the chemosensitivity of metastatic melanoma are rare and hampered by poor response rates to systemic chemotherapeutics. Patients who undergo isolated limb infusion (ILI) with cytotoxic drugs show high response rates and are, therefore, a good cohort for studying chemosensitivity in vivo. We used tumors from patients who underwent ILI to study the role of melanoma tumor-suppressor genes and oncogenes on melanoma chemosensitivity. Prospectively acquired tumors from 30 patients who subsequently underwent ILI with melphalan and actinomycin-D for metastatic melanoma were investigated for mRNA expression levels of p14ARF, p16INK4a, and MITFm. The mutation status of B-RAF, N-RAS, and PTEN were also determined. A high percentage of tumors had activating mutations in either B-RAF (15/30) or N-RAS (10/30) and only two tumors carried altered PTEN. High expression of p16INK4a and absence of an activating B-RAF mutation independently predicted response to treatment. Further, inducible expression of p16INK4a sensitized a melanoma cell line to death induced by melphalan or actinomycin-D. This study shows that high expression of p16INK4a or the absence of activated B-RAF correlates with in vivo response of melanoma to cytotoxic drugs. [ABSTRACT FROM AUTHOR]

Therapy resistance arises from heterogeneous drug-tolerant persister cells or minimal residual disease (MRD) through genetic and nongenetic mechanisms. A key question is whether specific molecular features of the MRD ecosystem determine which of these two distinct trajectories will eventually prevail. We show that, in melanoma exposed to mitogen-activated protein kinase therapeutics, emergence of a transient neural crest stem cell (NCSC) population in MRD concurs with the development of nongenetic resistance. This increase relies on a glial cell line-derived neurotrophic factor-dependent signaling cascade, which activates the AKT survival pathway in a focal adhesion kinase (FAK)-dependent manner. Ablation of the NCSC population through FAK inhibition delays relapse in patient-derived tumor xenografts. Strikingly, all tumors that ultimately escape this treatment exhibit resistance-conferring genetic alterations and increased sensitivity to extracellular signal-regulated kinase inhibition. These findings identify an approach that abrogates the nongenetic resistance trajectory in melanoma and demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths. [Display omitted] • Tumors recurrently select either a genetic or nongenetic drug resistance trajectory • FAK signaling is activated in melanoma drug persisters with a neural crest-like state • Targeting neural crest-like cells prevents nongenetic drug resistance evolution • The cellular composition of MRD dictates the evolutionary path to resistance Marin-Bejar et al. identify focal adhesion kinase (FAK) as a vulnerability of melanoma drug persisters harboring a neural crest-like state. Targeting these cells, using a FAK inhibitor, prevents the development of nongenetic, but not genetic, resistance, indicating that the path to resistance is dictated by the cellular composition of minimal residual disease. [ABSTRACT FROM AUTHOR]

A correction to the article "Acquired Resistance to BRAF Inhibition Can Confer Cross-Resistance to Combined BRAF/MEK Inhibition" by Kavitha Gowrishankar and colleagues in the April 4, 2013 issue is presented, which discusses human melanoma–derived cell lines resistant to the BRAF gene.