Helen Rizos

BRAF Inhibitor Resistance Mechanisms in Metastatic Melanoma: Spectrum and Clinical Impact

Purpose: Multiple BRAF inhibitor resistance mechanisms have been described, however, their relative frequency, clinical correlates, and effect on subsequent therapy have not been assessed in patients with metastatic melanoma. Experimental Design: Fifty-nine BRAFV600-mutant melanoma metastases from patients treated with dabrafenib or vemurafenib were analyzed. The genetic profile of resistance mechanisms and tumor signaling pathway activity was correlated with clinicopathologic features and therapeutic outcomes. Results: Resistance mechanisms were identified in 58% progressing tumors and BRAF alterations were common. Gene expression analysis revealed that mitogen-activated protein kinase (MAPK) activity remained inhibited in 21% of resistant tumors, and the outcomes of patients with these tumors were poor. Resistance mechanisms also occurred in pretreatment biopsies and heterogeneity of resistance mechanisms occurred within patients and within tumors. There were no responses to subsequent targeted therapy, even when a progressing tumor had a resistance mechanism predicted to be responsive. Conclusions: Selecting sequential drugs based on the molecular characteristics of a single progressing biopsy is unlikely to provide improved responses, and first-line therapies targeting multiple pathways will be required. Clin Cancer Res; 20(7); 1965–77. ©2014 AACR.

A melanoma-associated germline mutation in exon 1β inactivates p14ARF

The INK4a/ARF locus encodes the cyclin dependent kinase inhibitor, p16INK4a and the p53 activator, p14ARF. These two proteins have an independent first exon (exon 1α and exon 1β, respectively) but share exons 2 and 3 and are translated in different reading frames. Germline mutations in this locus are associated with melanoma susceptibility in 20–40% of multiple case melanoma families. Although most of these mutations specifically inactivate p16INK4a, more than 40% of the INK4a/ARF alterations located in exon 2, affect both p16INK4a and p14ARF. We now report a 16 base pair exon 1β germline insertion specifically altering p14ARF, but not p16INK4a, in an individual with multiple primary melanomas. This mutant p14ARF, 60ins16, was restricted to the cytoplasm, did not stabilize p53 and was unable to arrest the growth of a p53 expressing melanoma cell line. This is the first example of an exon 1β mutation that inactivates p14ARF, and thus implicates a role for this tumour suppressor in melanoma predisposition.

Increased MAPK reactivation in early resistance to dabrafenib/trametinib combination therapy of BRAF-mutant metastatic melanoma

One-third of BRAF-mutant metastatic melanoma patients treated with combined BRAF and MEK inhibition progress within 6 months. Treatment options for these patients remain limited. Here we analyse 20 BRAF(V600)-mutant melanoma metastases derived from 10 patients treated with the combination of dabrafenib and trametinib for resistance mechanisms and genetic correlates of response. Resistance mechanisms are identified in 9/11 progressing tumours and MAPK reactivation occurred in 9/10 tumours, commonly via BRAF amplification and mutations activating NRAS and MEK2. Our data confirming that MEK2(C125S), but not the synonymous MEK1(C121S) protein, confers resistance to combination therapy highlight the functional differences between these kinases and the preponderance of MEK2 mutations in combination therapy-resistant melanomas. Exome sequencing did not identify additional progression-specific resistance candidates. Nevertheless, most melanomas carried additional oncogenic mutations at baseline (for example, RAC1 and AKT3) that activate the MAPK and PI3K pathways and are thus predicted to diminish response to MAPK inhibitors.

Response of BRAF mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis

UNLABELLED Deregulated glucose metabolism fulfills the energetic and biosynthetic requirements for tumor growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF-mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF-mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including hypoxia-inducible factor-1α, MYC, and MONDOA (MLXIP), drives glycolysis downstream of BRAF(V600), is critical for responses to BRAF inhibition, and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor (BRAFi)-resistant melanoma cells. Thus, we provide a proof-of-principle for treatment of melanoma with combinations of BRAFis and glycolysis inhibitors. SIGNIFICANCE BRAF is suppress glycolysis and provide strong clinical benefi t in BRAF V600 melanoma. We show that BRAF inhibition suppresses glycolysis via a network of transcription factors that are critical for complete BRAFi responses. Furthermore, we provide evidence for the clinical potential of therapies that combine BRAFis with glycolysis inhibitors.

Differentiation of cucumber mosaic virus isolates using the polymerase chain reaction

A procedure based on the polymerase chain reaction (PCR) has been developed to classify cucumber mosaic cucumovirus (CMV) isolates accurately into two subgroups. Two CMV-specific primers that flank the CMV capsid protein gene were used to amplify a DNA fragment of approximately 870 bp. Restriction enzyme analysis of this fragment produces distinct restriction patterns that assign the CMV isolate into one of two subgroups. These two restriction groups correlate with the previously established CMV subgroupings; this PCR-based method may provide a simple alternative to the serological assays used for typing CMV isolates.

Apoptosis of human melanoma cells induced by inhibition of B-RAFV600E involves preferential splicing of bimS

Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, BimS, remains largely unappreciated. Here, we show that inhibition of the mutant B-RAFV600E triggers preferential splicing to produce BimS, which is particularly important in induction of apoptosis in B-RAFV600E melanoma cells. Although the specific B-RAFV600E inhibitor PLX4720 upregulates all three major isoforms of Bim, BimEL, BimL, and BimS, at the protein and mRNA levels in B-RAFV600E melanoma cells, the increase in the ratios of BimS mRNA to BimEL and BimL mRNA indicates that it favours BimS splicing. Consistently, enforced expression of B-RAFV600E in wild-type B-RAF melanoma cells and melanocytes inhibits BimS expression. The splicing factor SRp55 appears necessary for the increase in BimS splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of BimS and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in BimS splicing is also mirrored in freshly isolated B-RAFV600E melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAFV600E inhibitors.

Acquired BRAF inhibitor resistance: A multicenter meta-analysis of the spectrum and frequencies, clinical behaviour, and phenotypic associations of resistance mechanisms

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.

WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors

About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAF(V600E/K)) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance.

Circulating tumor DNA to monitor treatment response and detect acquired resistance in patients with metastatic melanoma

Repeat tumor biopsies to study genomic changes during therapy are difficult, invasive and data are confounded by tumoral heterogeneity. The analysis of circulating tumor DNA (ctDNA) can provide a non-invasive approach to assess prognosis and the genetic evolution of tumors in response to therapy. Mutation-specific droplet digital PCR was used to measure plasma concentrations of oncogenic BRAF and NRAS variants in 48 patients with advanced metastatic melanoma prior to treatment with targeted therapies (vemurafenib, dabrafenib or dabrafenib/trametinib combination) or immunotherapies (ipilimumab, nivolumab or pembrolizumab). Baseline ctDNA levels were evaluated relative to treatment response and progression-free survival (PFS). Tumor-associated ctDNA was detected in the plasma of 35/48 (73%) patients prior to treatment and lower ctDNA levels at this time point were significantly associated with response to treatment and prolonged PFS, irrespective of therapy type. Levels of ctDNA decreased significantly in patients treated with MAPK inhibitors (p < 0.001) in accordance with response to therapy, but this was not apparent in patients receiving immunotherapies. We show that circulating NRAS mutations, known to confer resistance to BRAF inhibitors, were detected in 3 of 7 (43%) patients progressing on kinase inhibitor therapy. Significantly, ctDNA rebound and circulating mutant NRAS preceded radiological detection of progressive disease. Our data demonstrate that ctDNA is a useful biomarker of response to kinase inhibitor therapy and can be used to monitor tumor evolution and detect the early appearance of resistance effectors.

Two arginine rich domains in the p14ARF tumour suppressor mediate nucleolar localization.

The INK4a/ARF locus encodes two distinct tumour suppressors, p16INK4a and p14ARF, that regulate cell cycle progression via the pRB and p53 pathways, respectively. The ARF protein inhibits hdm2 activity, leading to the stabilization of the p53 tumour suppressor and cell cycle inhibition. The amino-terminal domain of human p14ARF and of the mouse homologue, p19ARF, is sufficient for these effects. This domain is also sufficient for the nucleolar localization of the mouse ARF protein. In contrast, we show that the human ARF protein requires two arginine rich domains, one in the amino- and the other in the carboxy-terminus, for nucleolar targeting. The amino-terminal nucleolar-targeting domain of p14ARF is also important for ARF-hdm2 binding and cell cycle inhibition. The carboxy-terminal p14ARF nucleolar localization domain lies within the shared INK4a/ARF exon 2, and is mutated in a small number of melanoma-prone kindreds. The INK4a/ARF exon2-mutations could affect the function of both the p16INK4a and p14ARF tumour suppressors.