MM, once thought to be a single entity, is now considered a molecular and developmentally diverse disease. This observation allows for the generation and application of novel therapies specifically targeted to the molecular pathway lesions of a patient's disease. Such an approach affords the opportunity to positively affect the historically low survival rates in melanoma and serves as a molecular tool to help dissect the oncogenic events that drive this disease.
The effect of aberrant ERK signaling in MM is well described [7
]. Consequently, the clinical application of selective MAPK inhibitors is currently underway. PLX4032 is a potent inhibitor of the mutated form of BRAF. An early phase 1 trial of this compound in MM has been promising [14
]. However, concurrent preclinical molecular research is required to supplement clinical findings and to help direct the proper clinical application of PLX4032, including how to predict and overcome the development of drug resistance.
We tested the efficacy of PLX4032 in a large panel of well-characterized MM cell lines. The presence of BRAFm predicted for, but did not guarantee, a response to therapy. In addition to a BRAFm, samples that had a concurrent MC1Rv and a more differentiated melanocytic genotype had a preferential response. This provides valuable information that could be used to screen and select future clinical trial participants and to retrospectively analyze tissue and outcome results from the completed phase 1 trial.
Phosphoprofiling of our samples before and after PLX4032 treatment confirmed the inhibition of p-ERK signaling in all cell lines with a BRAFm
regardless of response. This confirms the specificity of PLX4032 and the dependency of most BRAFm
MM cells on p-ERK signaling. SKMEL31, sensitive, was an exception to this. After exposure to PLX4032, SKMEL31 had an increase in p-ERK signaling, much like NRAS mutated or BRAFwt
samples. It is possible that SKMEL31 has a reverse mutant allele-specific imbalance in which the wild-type allele is increased compared to the mutant [24
]. The presence of the mutated allele must be sufficient to drive its malignant phenotype and confer sensitivity to PLX4032. However, the imbalance in favor of the wild-type allele could explain why its post-PLX4032 p-ERK signaling pattern is more consistent with a BRAFwt
cell line. Of the 23 MM cell lines with a BRAFm
, 15 are heterozygous. Evaluation of the chromatogram of these cell lines revealed SKMEL31 and SKMEL3 as the only two lines that have a higher peak of the wild-type allele compared with the mutant. Interestingly, SKMEL3 is the most resistant cell line in the panel. It is possible that this reverse allelic imbalance will explain the phosphosignaling pattern of SKMEL31 and provide a way to predict resistance in patients who are heterozygous for the BRAFm
Abrogation of MAPK signaling in the PLX4032-resistant BRAFm
cell lines suggests that a small population of MM samples exist that have aberrant ERK signaling but can survive in the absence of this pathway's functional input. These samples (BRAFm
PLX4032-resistant) were heterozygous for the BRAFm
, tended to be in the NPG, and were without a MC1Rv
. Further validation of the unique qualities of this population may identify alternate signaling pathways, treatment options, and those patients with a BRAFm
who would not benefit from single-agent RAF inhibition. Also, our analysis clearly suggests that patients with NRASm
tumors would not benefit from PLX4032. As expected, the application of PLX4032 in these cells did not abrogate p-ERK signaling. Instead, selective BRAF inhibition seemed to activate the MAPK pathway as previously described [46,47
]. A correlation in response to PLX4032 was noted in the BRAF mutated samples that have a concurrent MC1R polymorphism. BRAFm
/MC1R normal cell lines were resistant to the effects of PLX4032. This suggests that melanoma cells that have BRAFm
are more dependent on aberrant p-ERK signaling as driven by the BRAFm
. The dominant effect of both pathways may converge on MITF signaling. This may explain why cell lines in the DMG that are BRAFm
seem to be more sensitive to PLX4032 than cell lines in the NPG that are BRAFm
The application of PLX4032 in the MM cell lines shows the downstream effect of ERK signaling in BRAF mutated samples. We performed cell cycle and apoptotic analyses before/after exposure to the drug. Interruption of ERK signaling decreased cellular proliferation and the ability to escape apoptosis. Our gene expression data suggest that BMF and PUMA mediate the restoration of apoptosis. Our expression analysis also confirmed that MAPK activity in BRAF mutated MM is resistant to negative feedback inhibition of DUSP and SPRY and that MYC, FOSL1, EGR1, and ETV 1, 4, and 5 are the main transcriptional activators of ERK as driven by a BRAFm.
An interesting observation noted with the disruption of ERK signaling is the reexpression of melanocyte-specific genes and markers of melanocyte differentiation. This includes TYR, MLANA, DCT
, and MITF
. It is uncertain why these differentiation markers are decreased in MM by ERK signaling. However, it has been suggested that prolonged ERK signaling will decrease the expression of MITF with subsequent down-regulation of melanogenesis to avoid the cytotoxic effects of excess melanin production [42
]. Consistent with this is our observation that genes associated with melanosome function, RAB27A, MYO5, MLPH
, and RILP
, were reexpressed after MM cell lines with a BRAFm
were treated with PLX4032. The inhibition of aberrant ERK signaling in BRAF mutated samples, especially ones that have a MC1Rv
, may relieve the feedback inhibition of ERK and reestablish the innate drive for melanin production.
Adding to the complexity of the relationship between ERK signaling and melanocyte differentiation is the identification of a subset of melanoma samples whose genotype is more characteristic of neuronal precursors than that of typical melanocytes. These cells behave and function like melanocytes, develop into clinically indistinguishable melanomas, and are susceptible to common melanoma mutations. However, they have very distinct gene expression signatures that suggest activity of the noncanonical Wnt and TGFβ pathways at the expense of MITF, CTNNB1, and the canonical Wnt pathway. Interestingly, BRAF mutated MM samples of the NPG have an increase in melanocytic genes after PLX4032 exposure. Also, BRAF mutated MM cell lines of the DMG that develop resistance to PLX4032 have an increase in the expression of genes that are more characteristic of cells with in the NPG. These data suggest that plasticity does exist between the DMG and NPG and that cell lines of the NPG may be able to differentiate into a more melanocytic phenotype if they encounter the proper internal/external cues.
Our data show that the cells of DMG were more dependent on ERK signaling when driven by a BRAFm. Cell lines of the DMG were more sensitive to PLX4032 than were BRAFm cells in the NPG. Interestingly, those cell lines in the NPG that were clearly sensitive to PLX4032 were mostly in the NPG2, the group that was not defined by melanocytic or by neuronal genes. It is possible that this group represents a genetic bridge that links the DMG with the NPG1 and the plasticity inherent to NCC development.
We propose that cells of the NPG develop from the NC apart from the influence of SOX10 and MITF. In such, they never develop a strong melanocytic genotype and retain much of the neuronal influences of their precursors. A higher incidence of PTEN loss was noted in these cells. PTEN loss has been shown in conjunction with a BRAFm
to facilitate melanoma development [10,11
]. We noticed a predilection of the PTENm
to be in the NPG regardless of BRAF status. This suggests that the less differentiated cells of the NPG may be more dependent on the cross talk between the MAPK and PI3K pathways that is thought to potentiate melanoma formation in BRAF mutated nevi.
As seen clinically, continual exposure of melanoma to PLX4032 can promote drug resistance especially in the more differentiated samples. Western blot analysis showed reactivation of p-ERK signaling. However, a gene expression analysis did not consistently reproduce the downstream signaling patterns of a fully penetrable BRAFm. These data suggest that other mediators may contribute to the acquisition of resistance. In the samples with PLX4032 acquired resistance, we noticed increase activity of RAS and a discordant signaling pattern between MEK and ERK. This may afford some insights into points of influence on MAPK pathway apart from a BRAFm. Changes in AKT and p-AKT signaling were noted in the resistant samples compared with their sensitive parental counterparts. However, further assessment, including treatment data with selective PI3K and mTOR inhibitors, did not make a strong argument for this pathways involvement in acquired PLX4032 resistance.
A gene expression analysis implicated several other mediators that may be involved in the acquisition of resistance to PLX4032; this includes members of the platelet derived growth factor family, increased expression of angiogenic factors, and increased activity of certain MTs. Such observations afford biomarkers to screen for the development of resistance in patients who are actively being treated with PLX4032 and possible clinical avenues to pursue when resistance occurs.
PLX4032 is a selective BRAF inhibitor that seems to have significant clinical activity in BRAF mutated MM. The development of this compound provides hope in a malignancy that has been notoriously refractory to standard therapies. The preclinical use of this molecule sheds light onto its proper clinical application and affords new insight into patterns of response and resistance. Such information furthers our understanding of this terrible malignancy and opens the door to new targeted therapies and the clinical means to properly apply them in the context of well-defined pathway lesions.