Therese M. Becker

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.

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.

The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a

BackgroundCDKN2A/p16INK4ais frequently altered in human cancers and it is the most important melanoma susceptibility gene identified to date. p16INK4a inhibits pRb phosphorylation and induces cell cycle arrest, which is considered its main tumour suppressor function. Nevertheless, additional activities may contribute to the tumour suppressor role of p16INK4a and could help explain its specific association with melanoma predisposition. To identify such functions we conducted a yeast-two-hybrid screen for novel p16INK4a binding partners.ResultsWe now report that p16INK4a interacts with the chromatin remodelling factor BRG1. We investigated the cooperative roles of p16INK4a and BRG1 using a panel of cell lines and a melanoma cell model with inducible p16INK4a expression and BRG1 silencing. We found evidence that BRG1 is not required for p16INK4a-induced cell cycle inhibition and propose that the p16INK4a-BRG1 complex regulates BRG1 chromatin remodelling activity. Importantly, we found frequent loss of BRG1 expression in primary and metastatic melanomas, implicating this novel p16INK4a binding partner as an important tumour suppressor in melanoma.ConclusionThis data adds to the increasing evidence implicating the SWI/SNF chromatin remodelling complex in tumour development and the association of p16INK4a with chromatin remodelling highlights potentially new functions that may be important in melanoma predisposition and chemoresistance.

Oncogene-Induced Senescence Does Not Require the p16INK4a or p14ARF Melanoma Tumor Suppressors

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 p16(INK4a) 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 p16(INK4a) and/or p14ARF is not sufficient to execute the oncogene-induced senescence program. In this study, we examined the impact of melanoma-associated N-RAS(Q61K) on melanocyte senescence and utilized RNA-interference vectors to directly assess the individual contribution of human p14ARF and p16(INK4a) 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 p16(INK4a), and highlight the need to thoroughly explore INK4a/ARF-independent molecular pathways of senescence in human melanocytes.

IGFBP7 is not required for B-RAF-induced melanocyte senescence.

Induction of senescence permanently restricts cellular proliferation after oncogenic stimulation thereby acting as a potent barrier to tumor development. The relevant effector proteins may therefore be fundamental to cancer development. A recent study identified IGFBP7 as a secreted factor mediating melanocyte senescence induced by oncogenic B-RAF, which is found commonly in cutaneous nevi. In contrast to the previous report, we demonstrate that B-RAF signaling does not induce IGFBP7 expression, nor the expression of the IGFBP7 targets, BNIP3L, SMARCB1, or PEA15, in human melanocytes or fibroblasts. We also found no correlation between B-RAF mutational status and IGFBP7 protein expression levels in 22 melanoma cell lines, 90 melanomas, and 46 benign nevi. Furthermore, using a lentiviral silencing strategy we show that B-RAF induces senescence in melanocytes and fibroblasts, irrespective of the presence of IGFBP7. Therefore, we conclude that the secreted protein IGFBP7 is dispensable for B-RAF(V600E)-induced senescence in human melanocytes.

Acquired resistance to BRAF inhibition can confer cross-resistance to combined BRAF/MEK inhibition

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.

Antiproliferative Effects of Continued Mitogen-Activated Protein Kinase Pathway Inhibition following Acquired Resistance to BRAF and/or MEK Inhibition in Melanoma

Inhibitors of the mitogen-activated protein kinases (MAPK), BRAF, and MAP–ERK kinase (MEK) induce tumor regression in the majority of patients with BRAF-mutant metastatic melanoma. The clinical benefit of MAPK inhibitors is restricted by the development of acquired resistance with half of those who benefit having progressed by 6 to 7 months and long-term responders uncommon. There remains no agreed treatment strategy on disease progression in these patients. Without published evidence, fears of accelerated disease progression on inhibitor withdrawal have led to the continuation of drugs beyond formal disease progression. We now show that treatment with MAPK inhibitors beyond disease progression can provide significant clinical benefit, and the withdrawal of these inhibitors led to a marked increase in the rate of disease progression in two patients. We also show that MAPK inhibitors retain partial activity in acquired resistant melanoma by examining drug-resistant clones generated to dabrafenib, trametinib, or the combination of these drugs. All resistant sublines displayed a markedly slower rate of proliferation when exposed to MAPK inhibitors, and this coincided with a reduction in MAPK signaling, decrease in bromodeoxyuridine incorporation, and S-phase inhibition. This cytostatic effect was also associated with diminished levels of cyclin D1 and p-pRb. Two short-term melanoma cultures generated from resistant tumor biopsies also responded to MAPK inhibition, with comparable inhibitory changes in proliferation and MAPK signaling. These data provide a rationale for the continuation of BRAF and MEK inhibitors after disease progression and support the development of clinical trials to examine this strategy. Mol Cancer Ther; 12(7); 1332–42. ©2013 AACR.

Vemurafenib Induces Senescence Features in Melanoma Cells

A large proportion of human melanomas harbor a mutation leading to permanent activation of the serine/threonine kinase BRAF, and as a consequence, they have developed dependence on BRAF/mitogen-activated protein kinase signaling. Accordingly, BRAF inhibitors such as Vemurafenib show a good anti-tumorigenic effect on metastases with the BRAF(V600E) mutation. Although an initial period of sustained tumor regression is usually observed after Vemurafenib treatment, tumors often relapse at the same site, and apoptosis induction of melanoma cells in vitro is incomplete. Here, we demonstrate, using a large panel of melanoma cell lines, that Vemurafenib induces features of stress-induced senescence in addition to apoptosis. This senescence phenotype is characterized by heterochromatin formation, changes in cell shape, and increased senescence-associated β-galactosidase activity. Importantly, senescence features induced by BRAF(V600E) inhibition was also detected in human melanoma cells xenografted into nude mice. Our observations provide a possible explanation for the lack of complete and durable pro-apoptotic effect of Vemurafenib in patients.

Association of p14ARF with the p120E4F transcriptional repressor enhances cell cycle inhibition

The p14ARF tumor suppressor is a key regulator of cellular proliferation and is frequently inactivated in human cancer. This tumor suppressor functions in the p53 and pRb cell cycle regulatory pathways and can effectively activate both pathways to induce growth arrest or cell death. We now report that p14ARF forms a complex with the E1A-regulated transcriptional repressor, p120E4F. p120E4F contacts p14ARF and p53 to form a ternary complex in vivo and enhances p14ARF-induced G2 cell cycle arrest in a p53-dependent manner. We suggest that the interaction of p14ARF and p120E4F forms an important link between the p14ARF and p53 tumor suppressor proteins, both of which exhibit enhanced cell cycle inhibitory activity in the presence of this transcriptional repressor.

The MAPK pathway functions as a redundant survival signal that reinforces the PI3K cascade in c-Kit mutant melanoma

Stimulation of the c-Kit receptor tyrosine kinase has a critical role in the development and migration of melanocytes, and oncogenic c-Kit mutants contribute to the progression of some melanomas. c-Kit signalling activates the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways and their relative contribution to the activities of oncogenic and ligand-dependent c-Kit remains uncertain. We show that PI3K is a major regulator of MAPK activation in response to c-Kit activity and the dominant effector of c-Kit-driven melanocyte proliferation and melanoma survival. Nevertheless, inhibition of the PI3K pathway in c-Kit mutant melanoma cells did not replicate the apoptotic efficacy of the c-Kit inhibitor, imatinib mesylate. Instead, the simultaneous suppression of the PI3K and MAPK pathways promoted a strong synergistic apoptotic effect. These data indicate that MAPK functions as a redundant survival signal that reinforces the PI3K cascade in c-Kit mutant melanoma. Thus, the concurrent inhibition of PI3K and MAPK signalling is required to suppress oncogenic c-Kit activity and may provide an effective therapeutic strategy in c-Kit mutant melanomas.