Transcriptional responses of melanoma cells to BRAF inhibition
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA432707 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6666200.fastq.gz | Fastqsanger.gz | |||
| SRR6666201.fastq.gz | Fastqsanger.gz | |||
| SRR6666202.fastq.gz | Fastqsanger.gz | |||
| SRR6666203.fastq.gz | Fastqsanger.gz | |||
| SRR6666204.fastq.gz | Fastqsanger.gz |
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Project description:Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to MAPK pathway inhibition we identified the combination PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ERBB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and RTK mutational status. Layered over base-line resistance was substantial upregulation of many ERBB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ERBB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes. 12 BRAF mutant melanomas and 4 melanomas with WT BRAF were exposed plx4720 treatment to evaluate their responses after 8 hours of treatment. 5 of the 12 BRAF mutant melanomas responses were also evaluated in response to the treatment of lapatinib alone, masitinib alone, the combination of lapatinib with plx4720, or the combination of masitinib with plx4720. All samples were run in at least triplicate.
2015-09-16 | E-GEOD-68453 | biostudies-arrayexpress
Project description:A major contributor to poor sensitivity to anti-cancer kinase inhibitor therapy is drug-induced cellular adaptation, whereby remodeling of signaling and gene regulatory networks permits a drug-tolerant phenotype. Here, we resolve the scale and kinetics of critical subcellular events following oncogenic kinase inhibition and preceding cell cycle re-entry, using mass spectrometry-based phosphoproteomics and RNA sequencing to capture molecular snapshots within the first minutes, hours, and days of BRAF kinase inhibitor exposure in a human BRAF-mutant melanoma model of adaptive therapy resistance. By enriching specific phospho-motifs associated with mitogenic kinase activity, we monitored the dynamics of thousands of growth- and survival-related protein phosphorylation events under oncogenic BRAF inhibition and drug removal. We observed early and sustained inhibition of the BRAF-ERK axis, gradual downregulation of canonical cell cycle-dependent signals, and three distinct and reversible phase transitions toward quiescence. Statistical inference of kinetically-defined regulatory modules revealed a concerted response to oncogenic BRAF inhibition and a dominant compensatory induction of SRC family kinase (SFK) signaling, which we found to be at least partially driven by accumulation of reactive oxygen species via impaired redox homeostasis. This induction sensitized cells to co-treatment with an SFK inhibitor across a panel of patient-derived melanoma cell lines and in an orthotopic mouse xenograft model, underscoring the translational potential for measuring the temporal dynamics of signaling and transcriptional networks under therapeutic challenge.
2024-02-27 | PXD048479 | Pride
Project description:Nearly 50% of cutaneous melanomas carry activating mutations on the BRAF oncogene, and the combination of BRAF- and MEK-inhibitors (BRAF/MEKi) is frequently used for their clinical management. One major drawback of BRAF/MEKi targeted therapy is the rapid development of therapeutic resistance, which can occur via multiple mechanisms, including the metabolic rewiring of cancer cells that often involves the upregulation of mitochondrial bionergetics and NAD+ biosynthetic pathways. mTORC2 is a signaling complex that requires the presence of its essential RICTOR subunit to play its regulatory functions in cell growth and metabolism. mTORC2 is believed to play mostly pro-oncogenic roles in several tumor types, including melanoma. However, bioinformatics analysis of TCGA melanoma patients’ database revealed that low RICTOR levels in tumors correlate with an overall worse clinical outcome. GSEA analysis of low-RICTOR tumors evidenced also a gene expression signature suggestive of activation of mitochondrial energy producing pathways. On these bases, we have hypothesized that inhibition of mTORC2 activity may render BRAFV600E melanoma cells resistant to BRAFi/MEKi. We show here that RICTOR/mTORC2-deficient cells are intrinsically tolerant to BRAFi/MEKi, and anticipate the onset of resistance to BRAFi after sustained drug exposure both in vitro and in vivo, indicating that mTORC2 activity normally opposes the acquisition of targeted therapy resistance in BRAFV600E melanomas. Mechanistically, RICTOR-deficient cells show an enhanced mitochondrial respiratory potential and increased expression of nicotinamide phosphoribosyltransferase (NAMPT) protein, the rate-limiting enzyme of NAD+ salvage pathway, and pharmacological inhibition of these processes in RICTOR-deficient cells is sufficient to restore sensitivity to BRAFi. Thus, our work identifies a novel role for mTORC2 in favoring the responses of BRAF-mutated melanoma cells to targeted therapy, and suggest that the evaluation of the intratumor level of RICTOR may help to predict the responses of melanoma patients to these treatments.
2024-05-23 | PXD045346 | Pride
Project description:Combined inhibition of BRAF, MEK and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on anti-tumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a-/-Pten-/- melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA-seq analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted pro-inflammatory macrophages and cross-priming CD103+ dendritic cells (DCs), the absence of which correlated with poor overall survival and clinical responses to ICB in melanoma patients. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo. While combined BRAF, MEK and CDK4/6 inhibition demonstrates favourable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact on anti-tumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.
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Project description:Melanoma cell lines were assessed for differences in gene expression patterns between the lines sensitive and resistant to BRAF and MEK inhibitor drugs. 22 BRAF-mutant melanoma cell lines were assessed for response to BRAF and MEK inhibitors in a 3 day drug treatment dose response assay. Based on the IC50, 18 lines were found to be responsive to BRAF or MEK inhibition and 4 were resistant. Normalised gene expression data generated from experimental replicate affymetrix arrays was assessed to identify differential patterns of inherent gene expression between the cell lines grouped as drug-responsive or drug-resistant. This were used to idenify specific candidate genes and pathways associated with inherent BRAF/MEK inhibitor drug resistance in melanoma cells.
2015-08-26 | E-GEOD-45558 | biostudies-arrayexpress
Project description:Background Main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF- mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to define in this context the specific role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit and regarded as an oncogenic driver in several tumor types, including MM. Methods After analyzing the TCGA MM patients’ database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAF V600E MM cell lines on their response to BRAF/MEKi in vitro. We performed a proteomic screening to identify proteins modulated by changes in RICTOR expression and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. Results We found that low RICTOR levels in MM correlate with an overall worse clinical outcome. GSEA of low- RICTOR tumors revealed a gene expression signature suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy producing pathway. RICTOR-deficient BRAF V600E cells are intrinsically tolerant to BRAFi/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. In RICTOR- depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, while their pharmacological inhibition restores sensitivity to BRAFi. Conclusions Our work unveils a novel tumor suppressing role for mTORC2 in the responses of BRAF V600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low- RICTOR tumors. Importantly, our findings support the concept that the evaluation of intra-tumor RICTOR levels in MM has a prognostic value, and may help predicting the response of patients to targeted therapy.
2024-06-22 | PXD050614 | Pride
Project description:NRAS and BRAF are the most commonly found genetic alterations in melanoma. It has been demonstrated that NRAS and BRAF oncogenic alterations, even affecting the same signaling pathway, represent two different clinical and biochemical entities, showing different signaling patterns and biological responses. Metabolic reprogramming is considered a novel target to control cancer; however, it is mostly unknown how the NRAS oncogene contributes to this cancer hallmark. We have showed that NRAS-mutated melanomas harbor specific metabolic alterations that render cells sensitive to RAS pathway inhibition upon metabolic stress. Gene expression analysis has confirmed different transcriptional profiles of NRAS- and BRAF- mutant cells both, under basal conditions and in response to glucose starvation. Moreover, analysis of glucose metabolism-related genes expression regulation revealed PFKFB2 as an important player linking glycolysis and RAS pathway activation.
2022-08-31 | E-MTAB-10921 | biostudies-arrayexpress
Project description:Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to MAPK pathway inhibition we identified the combination PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ERBB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and RTK mutational status. Layered over base-line resistance was substantial upregulation of many ERBB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ERBB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes.
2015-09-16 | GSE68453 | GEO