Whole-exome sequencing of genomic DNA extracted from patient-derived melanoma cell lines resistant to vemurafenib (PLX) or trametinib (TRA)
ABSTRACT: Whole-exome sequencing was performed on DNA samples extracted from seven melanoma cell lines resistant to either vemurafenib (BRAF V600E inhibitor) or trametinib (MEK1/2 inhibitor). The aim of the experiment was to search for genetic alterations responsible for phenotypic diversity of melanoma cell lines reported at the level of cell morphology, activity of signaling pathways essential for melanoma development and progression, and resistance to targeted therapeutics.
Project description:Whole-exome sequencing was performed on DNA samples extracted from seven melanoma cell lines resistant to either vemurafenib (BRAF V600E inhibitor) or trametinib (MEK1/2 inhibitor). The aim of the experiment was to search for genetic alterations responsible for phenotypic diversity of melanoma cell lines reported at the level of cell morphology, activity of signaling pathways essential for melanoma development and progression, and resistance to targeted therapeutics.
Project description:The first clinical trial testing the combination of targeted therapy with a BRAF inhibitor vemurafenib and immunotherapy with a CTLA-4 antibody ipilimumab was terminated early due to significant liver toxicities, possibly due to paradoxical activation of the MAPK pathway by BRAF inhibitors in tumors with wild type BRAF. MEK inhibitors can potentiate the MAPK inhibition in tumor, while potentially alleviating the unwanted paradoxical MAPK activation. With a mouse model of syngeneic BRAFV600E driven melanoma (SM1), we tested whether the addition of the MEK inhibitor trametinib would enhance the immunosensitization effects of the BRAF inhibitor dabrafenib. Combination of dabrafenib and trametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression. Bioluminescent imaging and tumor infiltrating lymphocyte (TIL) phenotyping showed increased effector infiltration to tumors with dabrafenib, trametinib or dabrafenib plus trametinib with pmel-1 ACT combination. Intracellular IFN gamma staining of the TILs and in vivo cytotoxicity studies showed trametinib was not detrimental to the effector functions in vivo. Dabrafenib increased tumor associated macrophages and T regulatory cells (Tregs) in the tumors, which can be overcome by addition of trametinib. Microarray analysis revealed increased melanoma antigen, MHC expression, and global immune-related gene upregulation with the triple combination therapy. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen specific ACT, we tested the triple combination of dabrafenib, trametinib with anti-PD1 therapy, and observed superior anti-tumor effect to SM1 tumors. Our findings support the testing of these combinations in patients with BRAFV600E mutant metastatic melanoma. SM1 tumors were implanted into C57BL/6 mice. Mice were treated by ACT of pmel-1 splenocytes or C57BL/6 splenocytes as control. Pmel-1 treated mice were additionally treated with either vehicle, dabrafenib, trametinib, or combination of both drugs and control mice were treated with vehicle or combination of both drugs.
Project description:Rapid resistance to BRAF inhibitors in BRAFV600-mutant metastatic melanoma has produced an urgent need for new treatment options. BRAF inhibitor resistance commonly involves reactivation of mitogen-activated protein kinase (MAPK) signaling and yet inhibition of downstream kinases has not circumvented resistance, partly because MAPK is regulated via a complex network of feedback mechanisms that influence pathway rebound. To examine the transcriptome responses of melanoma cells to MAPK inhibition, a panel of 11 BRAFV600-mutant melanoma cell lines were treated with control (DMSO), 100nM dabrafenib alone (i.e BRAF inhibitor monotherapy) or 100nM dabrafenib + 10nM trametinib (i.e combination BRAF + MEK inhibition) for 24h. Overall design: Eleven melanoma cell lines were grown in DMEM/10%FBS and treated with either 100nM dabrafenib and/or 10nM trametinib or DMSO for 24h.
Project description:aCGH of human melanoma cell lines comparing parental (drug sensitve) vs isogenic drug resistant-derived subline Two condition experiment: two BRAF-V600E mutant cell lines (drug sensitive - parental baseline) vs two derived sublines after chronic exposure to the MEK inhibitor trametinib (drug resistant) are compared
Project description:Vemurafenib is a BRAF inhibitor with specificity for the most common BRAF mutant encountered in melanomas (BRAFV600E). Vemurafenib suppresses the proliferation of BRAF mutant human melanoma cells by suppressing downstream activation of the MEK/ERK mitogen activated protein kinases. We used microarrays to examine the transcriptional response of a vemurafenib-sensitive BRAFV600E human melanoma cell line (A375) to vemurafenib in order to further delineate the mechanisms by which BRAFV600E drives cell proliferation and energy metabolism in human melanoma. BRAFV600E A375 human melanoma cells were treated with vehicle (0.1% DMSO) or 10 uM vemurafenib for 24 h after which total RNA was extracted. Cells were prepared and RNA was extracted in 3 separate batches (three different cell stocks on three separate days) providing three independent replicates (n=3). Paired replicates (prepared from the same stock of cells on the same day) are denoted by A, B and C.
Project description:Inhibitors of the MAPKs, BRAF and 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 6-7 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 demonstrate 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 signalling, decrease in BrdU incorporation and S-phase inhibition. This cytostatic effect was also associated diminished levels of cyclin D1 and p-pRb.. Two short-term melanoma cultures generated from resistant tumour biopsies also responded to MAPK inhibition with comparable inhibitory changes in proliferation and MAPK signalling. 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. Total RNA obtained from melanooma cell lines treated for 24h with dabrafenib, trametinib or combination of dabrafenib and trametinib
Project description:V600E being the most common mutation in BRAF, leads to constitutive activation of the MAPK signaling pathway. The majority of V600E BRAF positive melanoma patients treated with the BRAF inhibitor vemurafenib showed initial good clinical responses but relapsed due to acquired resistance to the drug. The aim of the present study was to identify possible biomarkers associated with the emergence of drug resistant melanoma cells. To this end we analyzed the differential gene expression of vemurafenib-sensitive and vemurafenib resistant brain and lung metastasizing melanoma cells. The major finding of this study is that the in vitro induction of vemurafenib resistance in melanoma cells is associated with an increased malignancy phenotype of these cells. Resistant cells expressed higher levels of genes coding for cancer stem cell markers (JARID1B, CD271 and Fibronectin) as well as genes involved in drug resistance (ABCG2), cell invasion and promotion of metastasis (MMP-1 and MMP-2). We also showed that drug-resistant melanoma cells adhere better to and transmigrate more efficiently through lung endothelial cells than drug-sensitive cells. The former cells also alter their microenvironment in a different manner from that of drug-sensitive cells. Biomarkers and molecular mechanisms associated with drug resistance may serve as targets for therapy of drug-resistant cancer. Overall design: 6 Samples (arrays) were analyzed. We generated pairwise comparisons between Vemurafenib and control DMSO treatments of various metastatic variants, using Partek Genomics Suite. Genes with fold-change difference of ≥1.25 or <-1.25 were selected.
Project description:We used phosphoproteomics to compare the responses of the ERK1/2 inhibitors, SCH772984 and GDC0994, and the MKK1/2 inhibitor, trametinib. These are compared with responses to the MKK1/2 inhibitor, selumetinib (AZD6244), previously measured by our lab in the same metastatic melanoma cell line. In three replicate experiments, we quantified a total of 12,805 class I phosphosites on 3,819 proteins in the trametinib-SCH772984-DMSO experiment, and 7,074 class I phosphosites on 2,453 in the GDC0994-SCH772984-DMSO experiment. This included 466 phosphosites that reproducibly decreased in response to at least one inhibitor in the trametinib-SCH772984-DMSO experiment and 414 phosphosites in the GDC0994-SCH772984-DMSO experiment. The results demonstrate linearity in signaling through the MAP kinase pathway. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates. SILAC Experimental Design Experiment 1 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – Trametinib Replicate 2: Heavy – SCH772984, Medium – Trametinib, Light – DMSO Replicate 3: Heavy – Trametinib, Medium – DMSO, Light – SCH772984 SILAC Experimental Design Experiment 2 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – GDC0994 Replicate 2: Heavy – SCH772984, Medium – GDC0994, Light – DMSO Replicate 3: Heavy – GDC0994, Medium – DMSO, Light – SCH772984 File List 1. Zipped MaxQuant search results folder containing index and output folders for each raw file, ‘combined’ output folder, and mqpar.xml MaxQuant search parameters file 2. Individual raw files of phosphopeptide-enriched ERLIC fractions 3. Zipped MaxQuant version used for analysis 4. FASTA file containing Uniprot human identifications 5. Instructions for viewing annotated spectra
Project description:Despite the successful use of drugs targeting the MAPK signaling pathway and immunotherapy in melanoma, the majority of patients with metastatic disease still undergo disease progression indicating a gradual development of therapy resistance. In the present study, microarray analyses were performed on BRAF inhibitor sensitive melanoma cells A375 and corresponding vemurafenib (PLX4032) - resistant cells A375_X1 to describe changes in the transcriptome that might play a role in drug resistance. For each cell line the microarray experiment was performed in duplicates.
Project description:Mutations in the NRAS oncogene are present in up to 20% of melanoma. Here, we show that interferon alpha-inducible protein 6 (IFI6) is necessary for NRASQ61K-induced transformation and melanoma growth. IFI6 was transcriptionally upregulated by NRASQ61K, and knockdown of IFI6 resulted in DNA replication stress due to dysregulated DNA replication via E2F2. This stress consequentially inhibited cellular transformation and melanoma growth via senescence or apoptosis induction depending on the RB and p53 pathway status of the cells. NRAS-mutant melanoma were significantly more resistant to the cytotoxic effects of DNA replication stress-inducing drugs, and knockdown of IFI6 increased sensitivity to these drugs. Pharmacological inhibition of IFI6 expression by the MEK inhibitor trametinib, when combined with DNA replication stress-inducing drugs, blocked NRAS-mutant melanoma growth. Collectively, we demonstrate that IFI6, via E2F2 regulates DNA replication and melanoma development and growth, and this pathway can be pharmacologically targeted to inhibit NRAS-mutant melanoma. YUGASP cells stably expressing a non-silencing shRNA or two individual shRNAs against IFI6 were used to prepare the total RNA, which was then used to analyze for gene expression using Illumina expression array.