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ERK signaling plays an essential role in the IFNg-mediated apoptosis of melanoma cells. [CRISPR screen]

ABSTRACT: Interferon-gamma (IFNg) exerts potent growth inhibitory (antiproliferative/pro-apoptotic) effects on a wide range of tumors, including melanoma. While several terminal effectors of cell cycle arrest and apoptosis have been identified, a definitive account of the signaling pathway leading to these events is lacking. We set up a chemical genomics screen and a whole genome targeting CRISPR/Cas9 screen in a growth inhibition (GI)-sensitive patient-derived melanoma (PDM) cell line. Drug screening revealed that treatment with RAF and ERK inhibitors disrupted IFNg GI in PDM cells. For the CRISPR screen, the gene-level analysis showed that the magnitude of enrichment for guide RNAs targeting ERK2 in IFNg-treated cells was comparable to core IFNg signaling proteins like IFNGR2, JAK1, JAK2, and STAT1. We validated the involvement of ERK by detecting a sustained increase in phospho-ERK1/2 (p-ERK) levels following IFNg treatment. In live imaging experiments, we found that an ERK inhibitor (ERKi) blocks the induction of cell death in 17 of 23 (~74%) IFNg-sensitive PDM lines covering all the MAPK mutant and triple wildtype molecular subtypes. Through gene expression analysis, we found that IFNg-ERK signaling triggered the expression of stress response genes and subsequent upregulation of DR5 and NOXA, which were both critical for the induction of cell death. In summary, the IFNg-ERK signaling axis mediates cell death downstream of IFNg treatment in melanoma cells. Our results provide a new understanding of the IFNg-mediated GI pathway that will also be crucial to define mechanisms of GI resistance in tumor cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE235237 | GEO | 2023/10/06

REPOSITORIES: GEO

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ERK signaling plays an essential role in the IFNg-mediated apoptosis of melanoma cells. [RNAseq]

Project description:Interferon-gamma (IFNg) exerts potent growth inhibitory (antiproliferative/pro-apoptotic) effects on a wide range of tumors, including melanoma. While several terminal effectors of cell cycle arrest and apoptosis have been identified, a definitive account of the signaling pathway leading to these events is lacking. We set up a chemical genomics screen and a whole genome targeting CRISPR/Cas9 screen in a growth inhibition (GI)-sensitive patient-derived melanoma (PDM) cell line. Drug screening revealed that treatment with RAF and ERK inhibitors disrupted IFNg GI in PDM cells. For the CRISPR screen, the gene-level analysis showed that the magnitude of enrichment for guide RNAs targeting ERK2 in IFNg-treated cells was comparable to core IFNg signaling proteins like IFNGR2, JAK1, JAK2, and STAT1. We validated the involvement of ERK by detecting a sustained increase in phospho-ERK1/2 (p-ERK) levels following IFNg treatment. In live imaging experiments, we found that an ERK inhibitor (ERKi) blocks the induction of cell death in 17 of 23 (~74%) IFNg-sensitive PDM lines covering all the MAPK mutant and triple wildtype molecular subtypes. Through gene expression analysis, we found that IFNg-ERK signaling triggered the expression of stress response genes and subsequent upregulation of DR5 and NOXA, which were both critical for the induction of cell death. In summary, the IFNg-ERK signaling axis mediates cell death downstream of IFNg treatment in melanoma cells. Our results provide a new understanding of the IFNg-mediated GI pathway that will also be crucial to define mechanisms of GI resistance in tumor cells.

2023-10-06 | GSE235235 | GEO

ULK1 as a novel therapeutic target to enhance immune checkpoint therapies against melanoma

Project description:Immune checkpoint inhibitors (ICIs) have transformed the treatment of melanoma. However, the majority of patients have primary or acquired resistance to ICIs, limiting durable responses and patient survival. Interferon-gamma (IFNg) signaling and the expression of IFNg-stimulated genes correlate with either response or resistance to ICIs, in a context-dependent manner. While IFNg-inducible immunostimulatory genes are required for response to ICIs, chronic IFNg signaling induces the expression of immunosuppressive genes, promoting resistance to these therapies. Here, we show that high levels of ULK1 correlate with poor survival in melanoma patients and overexpression of ULK1 in melanoma cells enhances IFNg-induced expression of immunosuppressive genes, with minimal effects on the expression of immunostimulatory genes. In contrast, genetic or pharmacological inhibition of ULK1 reduces expression of IFNg-induced immunosuppressive genes. ULK1 binds IRF1 in the nuclear compartment of melanoma cells, controlling its binding to the PD-L1 promoter region. Additionally, pharmacological inhibition of ULK1 in combination with anti-PD-1 therapy further reduces melanoma tumor growth and enhances anti-tumor immune responses in vivo. Our data suggest that targeting ULK1 represses IFNg-dependent immunosuppression. These findings support the combination of ULK1 drug-targeted inhibition with ICIs for the treatment of melanoma patients to improve response rates and patient outcomes.

2023-02-02 | PXD035347 | Pride

GNPS - Combining HPLC Activity Profiling with Advanced UPLC-HRMS/MS Annotation to Accelerate the Discovery of Bioactive Natural Products

Project description:Dataset containing multiple Hyptis and Artemisia spp. used for the discovery of natural products inhibiting aberrant signaling, namely MAPK/ERK and PI3K/AKT, in melanoma

2021-12-22 | MSV000088606 | GNPS

Conserved interferon-gamma signaling and decreased immune exclusion programs in responses to immune checkpoint blockade therapy

Project description:Purpose: We treated melanoma cell lines with IFNG to assess which ones would be resistant or responsive to T-cell attack. Methods: We treated the melanoma cell lines with IFNG and then sequenced them at 6 hours to assess changes the transcriptome. Results: Unless the cell lines had JAK1 or JAK2 KO, they responded very consistently with human melanocytes (normals), revealing a consistent signature for IFNG response in melanoma. Conclusions: IFNG signaling is conserved in melanoma cell lines, and there are genes that go down in IFNG that allow immune infiltration to make immune blockade response possible.

2020-07-24 | GSE154996 | GEO

RNA-sequencing of murine B16F10 melanoma cells with IFNg and FK866, a Nampt inhibitor

Project description:NAMPT is expressed in all cells during normal cell homeostasis. However, melanoma cells have a higher energetic demand and increase NAMPT expression. Our data show that IFNg signaling upregulates NAMPT in both human and mouse melanoma cells leading to increased cell growth and expression kinetics. The purpose of this experiment was to identify how IFNg inducible Nampt alters cell kinetics and we did this by treating melanoma cells with the combination of IFNg and FK866.

2023-07-01 | GSE207060 | GEO

RNA sequencing of rhabdomyosarcoma RD cells transfected with NRAS siRNAs for 48 h or treated with 300nM ERKi SCH772984 for 24 h

Project description:To compare gene transcription changes induced by NRAS knockdown or ERK inhibition, we performed RNA-seq analyses in the NRAS Q61H RD cells after 48 h NRAS suppression by 2 independent NRAS siRNAs, or 24 hours after ERK inhibitor treatment (ERKi).

2021-12-15 | GSE180127 | GEO

DNA methylation analysis of melanoma cell lines SK28 and MEL501

Project description:Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints. As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed. We reasoned that the base excision repair enzyme Thymine DNA Glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation. Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence and death by mitotic alterations, alters the transcriptome and methylome, and impairs xenograft tumor formation. Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity. These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.

2018-12-18 | GSE123928 | GEO

BRAF siRNA NHGRI_Liang_BRAF

Project description:BRAF, one of three RAF serine/threonine kinases (ARAF, BRAF and CRAF), plays a major role in the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway, which mediates cellular responses to growth signals. Recently a high frequency (~60%-70%) of activating BRAF mutations (predominantly V600E) has been reported in malignant melanoma. In order to identify the downstream effects of BRAF signaling on melanoma cell growth and gene expression, cDNA microarray analysis was carried out following BRAF siRNA or MEK1/2 inhibitor (U0126) treatment. Keywords: time series, siRNA time series, siRNA, drug treatment

2013-07-07 | E-GEOD-5481 | biostudies-arrayexpress

MKK2 is sufficient but not necessary for proliferation and anchorage-independent growth of SK-MEL-28 cells.

Project description:Though mitogen activated protein kinase kinases (MKK or MEK) 1 and 2 are widely assumed to be functionally redundant some reports indicate they possess distinct biologic activities. To test the hypothesis that MEK1 and MEK2 signaling pathways are interchangeable we used two complementary approaches to determine the necessity and sufficiency of individual MEK1 and MEK2 signaling pathways for human melanoma SK-MEL-28 cell proliferation. To test the necessity we targeted MEK1 and/or MEK2 using specific siRNAs. An effect on proliferation was observed only when both MEK1 and MEK2 were knocked down indicating that neither of the individual MEK isoforms is necessary for SK-MEL-28 cell proliferation. To test the sufficiency we inhibited multiple MEK and MKK signaling pathways in SK-MEL-28 cells with anthrax lethal toxin (LeTx) a MEK/MKK-specific protease and rescued individual MEK signaling pathways by expressing a cleavage-resistant form of MEK (MEKcr). In this fashion ERK activation was retained only in MEK2cr-expressing cells but not in MEK1cr-expressing cells following LeTx treatment. Microarray analysis revealed groups of non-overlapping downstream transcriptional targets of MEK1 and MEK2 and indicated a substantial rescue effect of MEK2cr on proliferation pathways. Furthermore LeTx efficiently inhibited the cell proliferation and anchorage-independent growth of SK-MEL-28 cells expressing MKK1cr but not MEK2cr. These results not only indicate that in this cellular context MEK2 signaling pathway alone is sufficient for ERK activation melanoma cell proliferation and anchorage-independent growth but MEK1 is not but also demonstrate that MEK1 and MEK2 signaling pathways are not redundant and interchangeable for melanoma cell proliferation. We conclude that while MEK2 alone is sufficient for SK-MEL-28 cell proliferation MEK1 can conditionally compensate for loss of MEK2. SK-MEL-28 melanoma cells +/- cleavage resistant MKK1/MKK2

2011-06-24 | E-GEOD-23930 | biostudies-arrayexpress

MAGE-A1 Promotes Proliferation, Migration and Invasion of Human Melanoma Cells through RNA-SEQ Analysis via Affecting C-JUN-IL8-ARHGAP29 Network

Project description:To define the role of MAGE-A1 in melanoma growth and metastasis, we performed RNA-seq analysis on MAGE-A1 overexpression (OE) and knockdown (KD) models in A375 human melanoma cell line. Our results revealed that overexpression of MAGE-A1 dramatically promoted proliferation, migration, and invasion of human melanoma cells in vitro and down-regulated of MAGE-A1 inhibited tumor cell proliferation and invasion. Furthermore, MAGE-A1 exerts its tumor promoting activity via activating including ERK-MAPK signaling pathway by RNA-seq analysis. mRNA profiles of MAGE-A1 over expression (OE), knockdown (KD), pcDNA-vector control, and pRNAT-scramble control in A375 cell line were generated using Ion torrent

2016-01-04 | E-GEOD-66859 | biostudies-arrayexpress

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