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A novel RAF kinase inhibitor with DFG-out binding mode: high efficacy in BRAF-mutant tumor xenograft models in the absence of normal tissue hyperproliferation

ABSTRACT: Purpose: Seek for differential gene expression in vemurafenib-resistant A375 tumors vs. untreated controls to provide a rationale for resistance mechanism Methods: mRNA profiles of vemurafenib-resistant A375 tumors and untreated control tumors were generated by transcriptome sequencing of A375 melanoma bearing mice. Since our xenograft samples contain a mixture of human and mouse RNAs we mapped RNASeq reads against a hybrid human/mouse genome. We than removed reads of potential mouse origin by taking only reads that map uniquely to human chromosomes. On average 23% of reads were removed as potential mouse reads. We than took the remaining reads (on average 77% per sample) to determine the gene expression levels for each sample. Normalized expression levels of 5 resistant samples were compared to 4 untreated control samples to detect differnetially regulated genes which may contribute to vemurfenib resistance Results: Expression levels of several genes were consistently altered in all resistant samples. Expression of e.g. genes encoding SPRY2, SPRY4, DUSP6, CCND1, PIK3R3, FGFR1, EPHA4, MCL1, and IGF1R was down-regulated, whereas expression of PDGFC, VEGFC, ABCB9 and KITLG was increased. Conclusions: Our study reports several differentially expressed genes which may contribute to vemurafenib resistance in A375 tumor bearing mice RNA sequencing of genes expressed in A375 tumors bearing mice treated with vemurafenib until in vivo resistance appeared vs. untreated A375 tumors

ORGANISM(S): Homo sapiens

SUBMITTER: Andreas Schlattl

PROVIDER: E-GEOD-74729 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A novel RAF kinase inhibitor with DFG-out binding mode: high efficacy in BRAF-mutant tumor xenograft models in the absence of normal tissue hyperproliferation

Project description:Purpose: Seek for differential gene expression in vemurafenib-resistant A375 tumors vs. untreated controls to provide a rationale for resistance mechanism Methods: mRNA profiles of vemurafenib-resistant A375 tumors and untreated control tumors were generated by transcriptome sequencing of A375 melanoma bearing mice. Since our xenograft samples contain a mixture of human and mouse RNAs we mapped RNASeq reads against a hybrid human/mouse genome. We than removed reads of potential mouse origin by taking only reads that map uniquely to human chromosomes. On average 23% of reads were removed as potential mouse reads. We than took the remaining reads (on average 77% per sample) to determine the gene expression levels for each sample. Normalized expression levels of 5 resistant samples were compared to 4 untreated control samples to detect differnetially regulated genes which may contribute to vemurfenib resistance Results: Expression levels of several genes were consistently altered in all resistant samples. Expression of e.g. genes encoding SPRY2, SPRY4, DUSP6, CCND1, PIK3R3, FGFR1, EPHA4, MCL1, and IGF1R was down-regulated, whereas expression of PDGFC, VEGFC, ABCB9 and KITLG was increased. Conclusions: Our study reports several differentially expressed genes which may contribute to vemurafenib resistance in A375 tumor bearing mice

2016-01-01 | GSE74729 | GEO

A four-arm study of A375 melanoma cell line-derived xenograft tumors in NSG mice treated with Vemurafenib and CB839

Project description:Melanoma is a rare but deadly form of skin cancer, which is often treated with BRAF inhibitors such as Vemurafenib (referred to as PLX4032). Whilst Vemurafenib prolongs the survival of patients, BRAF inhibitor resistance inevitably occurs in most cases. Previous studies demonstrated that metabolic rewiring occurs in BRAF inhibitor resistance and causes dependence on glutamine. To investigate whether this vulnerability could be exploited with clinically relevant drugs, we used the BRAF inhibitor, Vemurafenib, and the glutaminase imhibitor, CB839 to treat A375-derived melanoma xenografted tumors. We showed that whilst CB839 did not significantly affect the growth of A375-derived tumors compared to those given a vehicle, the addition of CB839 to Vemurafenib treatment had a significant anti-tumor effect. Tumors were taken at the endpoint (Max tumor length 15mm) from the 6 mice in each treatment group and cut into fragments and stored in RNAlater for RNAseq analysis. RNA extraction was performed on 1-3 fragments per tumor to make up 200-300mg of tissue.

2023-04-07 | E-MTAB-12811 | biostudies-arrayexpress

Analysis of Amino Acid Variants in Malignant Melanoma Cells Resistant to BRAF inhibition

Project description:Analysis of patient-specific nucleotide variants is a cornerstone of personalised medicine. Although only 2% of the genomic sequence is protein-coding, mutations occurring in these regions have the potential to influence protein structure and may have severe impact on disease aetiology. Of special importance are variants that affect modifiable amino acid residues, as protein modifications involved in signal transduction networks cannot be analysed by genomics. Proteogenomics enables analysis of proteomes in context of patient- or tissue-specific non-synonymous nucleotide variants. Here, we developed a proteogenomics workflow and applied it to study resistance to serine/threonine-protein kinase B-raf (BRAF) inhibitor (BRAFi) vemurafenib in malignant melanoma cell line A375. This approach resulted in high identification and quantification of non-synonymous nucleotide variants and (phospho)proteins. We integrated multi-omic datasets to reconstruct the perturbed signalling networks associated with BRAFi resistance and to predict drug therapies with the potential to disrupt BRAFi resistance mechanism in A375 cells. Notably, we showed that aurora kinase A (AURKA) inhibition is effective and specific against BRAFi resistant A375 cells. Furthermore, we investigated nucleotide variants that interfere with protein post-translational modification (PTM) status and potentially influence cell signalling. Mass spectrometry (MS) measurements confirmed variant-driven PTM changes in 12 proteins; among them was the runt-related transcription factor 1 (RUNX1) displaying a variant on a known phosphorylation site S(Ph)276L. We confirmed the loss of phosphorylation site by MS and demonstrated the impact of this variant on RUNX1 interactome.

2021-11-04 | PXD018305 | Pride

Expression data from BRAFV600E A375 melanoma cells treated with vehicle or vemurafenib

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.

2014-05-20 | E-GEOD-42872 | biostudies-arrayexpress

Quantitative proteomics links the intermediate filament nestin to vemurafenib resistance in melanoma cell lines

Project description:Targeted inhibition of mutated kinases using selective MAP kinase inhibitors in malignant melanoma often results in temporary improvement of the metastatic disease followed by rapid development of resistance. To gain insights in molecular processes that govern resistance, we performed SILAC-based quantitative proteomics profiling of vemurafenib-resistant and -sensitive melanoma cells. Among downregulated proteins in vemurafenib-resistant cell lines we detected multiple proteins involved in cytoskeletal organization and signaling, including the intermediate filament nestin, which was one of the most down-regulated proteins. Previous studies showed that nestin is expressed in various types of solid tumors and its abundance correlates with malignant phenotype of transformed cells. However, the role of nestin in cancer cells with regard to acquired resistance is still poorly understood. We performed CRISPR/Cas9 knockout of the nestin gene (NES) in vemurafenib-sensitive cells and showed that loss of nestin leads to increased cellular proliferation and colony formation upon treatment with BRAF and MEK inhibitors. Moreover, nestin depletion led to increased invasiveness and metalloproteinase activity similar to resistant phenotype of melanoma cells. Finally, phosphoproteome analysis revealed that nestin depletion influenced signaling through integrin and PI3K-Akt-mTOR pathways and led to increased focal adhesion kinase expression and phosphorylation. Taken together, our results reveal that nestin is associated with acquired vemurafenib resistance in melanoma cell lines.

2019-05-14 | PXD010683 | Pride

scRNAseq of four syngeneic human melanoma cell lines (two replicate per cell line) to study mechanisms of resistance to the BRAF inhibitor vemurafenib

Project description:Three cell lines were derived from the A375 human melanoma (harbouring the BRAF v600e mutation; ATCC) after treatment with vemurafenib during a period of 4-5 weeks in the absence (MAPKi-resist Rep1/Rep2) or presence of the fatty acid oxidation inhibitors etomoxir (Etomoxir Rep1/Rep2) or ranolazine (Ranolazine Rep1/Rep2).

2023-06-20 | E-MTAB-12412 | biostudies-arrayexpress

miR-204-5p and miR-211-5p contribute to BRAF inhibitor resistance in melanoma

Project description:Purpose: Identification of miRNAs that enable resistance to BRAF inhibitors in melanoma suggests a mechanism-based strategy to limit resistance and improve clinical outcomes. Methods: We generated A375 melanoma cells resistant to Vemurafenib (VMF) with the goal of investigating changes in miRNA expression patterns that might contribute to resistance. Results: Increased expression of miR-204-5p and miR-211-5p occurring in VMF-resistant cells was determined to impact VMF response.

2018-01-18 | GSE107576 | GEO

Analysis of A375 and A375 clones that acquired resistance to GSK2118436 after treatment with GSK2118436 (GSK436), GSK1120212 (GSK212), or the combination of GSK2118436 and GSK1120212 for 24 hour

Project description:In an effort to understand the mechanisms of acquired resistance to BRAF inhibitors, we isolated clones that acquired resistance to the BRAF inhibitor GSK2118436 derived from the A375 BRAF V600E mutant melanoma cell line. This resistance clones acquired mutations in NRAS and MEK1. One clones, 16R6-4, acquired two mutations in NRAS – Q61K and A146T. Proliferation and western blot analyses demonstrated that these clones were insensitive to single agent GSK2118436 or GSK1120212 (an allosteric MEK inhibitor) but were sensitive to the combination of GSK2118436 and GSK1120212. To further characterize this combination, global transcriptomic analysis was performed in A375 and 16R6-4 after 24 hour treatment with GSK2118436, GSK1120212 or the combination of GSK2118436 and GSK1120212. This data set was published in Molecular Cancer Therapeutics with the title “Combined inhibition of BRAF and MEK, BRAF and PI3K/mTOR, or MEK and PI3K/mTOR overcomes acquired resistance to the BRAF inhibitor GSK2118436, mediated by NRAS or MEK mutations” by Greger, J.G., et.al. A375 and 16R6-4 (an A375 derived GSK2118436 resistance clone) were treated for 24 hours with 0.1 micromolar GSK2118436, 1 micromolar GSK2118436, 0.01 micromolar GSK1120212, 0.1 micromolar GSK2118436 + 0.01 micromolar GSK1120212, or 1 micromolar GSK2118436 + 0.01 micromolar GSK1120212.

2012-02-01 | E-GEOD-35230 | biostudies-arrayexpress

Gene expression profile in A375 melanoma cells after 45 functionally important molecules were knocked down using siRNA

Project description:Affymetrix microarray data were generated from A375 melanoma cells treated in vitro with siRNAs against 45 transcription factors and signalling molecules. 45 functionally important molecules were knocked down in A375 melanoma cells by siRNA. Then the gene expression profiles of these A375 cells, along with untreated cells and sRNA control treated cells were analysed by microarrays.

2012-03-15 | E-GEOD-31534 | biostudies-arrayexpress

A new ALK isoform transported by extracellular vesicles confers drug resistance to vemurafenib in A375 melanoma cells

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.

2019-02-28 | E-MTAB-6596 | biostudies-arrayexpress

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