Comparative analysis of RAF depletion vs. MAPK inhibition
Ontology highlight
ABSTRACT:
PROVIDER: EGAS00001005743 | EGA |
REPOSITORIES: EGA
Similar Datasets
Project description:KRAS, mutated in ~30% of all cancers, activates the RAF-MEK-ERK signaling cascade. It has previously been shown that CRAF is required for growth of KRAS mutant lung tumors but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescued growth inhibition suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrated enrichment of CRAF:ARAF dimers in KRAS mutant cells and depletion of both CRAF and ARAF rescued the CRAF loss phenotype. Mechanistically, CRAF depletion caused sustained ERK activation and induction of cell cycle arrest. Treatment with low dose MEK or ERK inhibitor rescued the CRAF loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting dimerization or degrading CRAF may have therapeutic benefit.
2021-10-29 | MSV000088285 | MassIVE
Project description:Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines, and then applied Multiplexed Inhibitor Bead/Mass Spectrometry (MIB/MS) chemical proteomics to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transforming activities, and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGFBR1, ILK), and pharmacologic inhibition of the upregulated kinase, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacologic inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death than WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.
2022-02-17 | PXD025819 | Pride
Project description:Mutations activating the KRAS GTPase or the BRAF kinase are frequent in colorectal cancer and are thought to constitutively activate the terminal mitogen-activated protein kinase, ERK. Using mass cytometry, we found graded phosphorylation of ERK anti-correlated with cell differentiation in patient-derived colorectal cancer organoids, and unexpectedly this gradient was observed independently of KRAS mutational status. We therefore investigated differentiation-dependent signal transduction elicited by oncogenic KRAS or BRAF in transgenic mouse organoid models. Reporter, single cell transcriptome and mass cytometry analyses showed that transgenic expression of KRASG12V activated ERK in a cell type-specific pattern. Furthermore, expression of oncogenic KRAS induced the formation of RAS-ERK-responsive cells. In contrast, transgenic expression of BRAFV600E triggered high ERK activity and downstream gene expression in all intestinal cell types, followed by epithelial disorganisation. We analysed signal transduction to ERK using single cell-resolved network perturbation data in transgenic organoids. Network reconstruction followed by quantitative modelling revealed that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identify dual-specificity phosphatases as candidate modulators of MEK to ERK signal transduction. Our experiments highlight key differences between ERK activity elicited by the BRAF or KRAS oncogenes in colorectal cancer and find unexpected functional heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.
2019-03-31 | GSE115242 | GEO
Project description:The oncogenic proteins expressed in human cancer cells are exceedingly difficult targets for drug discovery due to intrinsic properties of the Ras GTPase switch. As a result, recent efforts have largely focused on inhibiting Ras-regulated kinase effector cascades, particularly the Raf/MEK/ERK and PI3 kinase/Akt/mTOR pathways. We constructed murine stem cell leukemia virus (MSCV) vectors encoding oncogenic K-RasD12 with additional “second site” amino acid substitutions that that impair PI3 kinase/Akt or Raf/MEK/ERK activation and performed bone marrow transduction/transplantation experiments in mice. In spite of attenuated signaling properties, defective K-Ras oncoproteins induced aggressive clonal T lineage acute lymphoblastic leukemia (T-ALL). These leukemias exhibited a high frequency of somatic Notch1 mutations, which is also true of human T-ALL. Multiple independent T-ALLs restored full oncogenic Ras activity by acquiring “third site” mutations within the viral KrasD12 transgenes. Other leukemias with undetectable PTEN and elevated phosphoryated Akt levels showed a similar gene expression profile to human early T progenitor (ETP) T-ALL. Expressing oncoproteins that are defective for specific functions is a general strategy for assessing requirements for tumor maintenance and uncovering potential mechanisms of drug resistance in vivo. In addition, our observation that defective Kras oncogenes regain potent cancer initiating activity strongly supports simultaneously targeting distinct components of Ras signaling networks in the substantial fraction of cancers with RAS mutations. WT Balb/c mice were lethally irradiated and transplanted with WT Balb/c bone marrow cells transduced with MSCV-IRES-Kras mutant-GFP vectors. Mice developed T-cell lymphoproliferative disease.
2011-09-30 | E-GEOD-28687 | biostudies-arrayexpress
Project description:Mutational activation of the KRAS oncogene is a major genetic driver of pancreatic ductal adenocarcinoma (PDAC) growth. KRAS-dependent PDAC growth is mediated primarily through persistent activation of the RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade, one of the most extensively studied cancer signaling networks. While substrates of RAF and MEK kinases are highly restricted, ERK1/2 has been attributed to over 1,000 substrates. In this study, we used the highly selective ERK1/2 inhibitor, SCH772984, and proteomic and phosphoproteomic analyses to extend the repertoire of ERK-dependent phosphosites and phosphoproteins in PDAC. We validated the specificity of SCH772984 in our cell lines using multiplexed inhibitor beads coupled with mass spectrometry (MIB/MS). We then performed phosphoproteomics and global proteomics in a panel of PDAC cell lines and identified 5,117 ERK-dependent phosphosites on 2,252 proteins, of which 88% and 67%, respectively, were not previously associated with ERK. We then utilized our recently annotated serine/threonine kinome motif database to dissect the phosphoproteome and reveal an expansive ERK-regulated kinase network. We found that ERK- and immediate downstream kinase RSK-substrate motifs predominated after one hour of ERK inhibition, whereas cell cycle regulatory cyclin-dependent kinase motifs predominated by 24 h, reflecting a highly dynamic ERK-dependent phosphoproteome. We find compensatory activation of HIPK, CLK, PKN, PAK, and DYRK family kinases. Finally, using the genome-wide CRISPR-Cas9 dataset in the Cancer Dependency Map portal (DepMap), we determined that approximately 18% of ERK dependent phosphoproteins are essential for pancreatic cancer growth, and these are enriched in nuclear proteins. Together, our findings provide a system-wide profile of the mechanistic basis for ERK-driven pancreatic cancer growth.
2024-05-29 | PXD043585 | Pride
Project description:Mutational activation of the KRAS oncogene is a major genetic driver of pancreatic ductal adenocarcinoma (PDAC) growth. KRAS-dependent PDAC growth is mediated primarily through persistent activation of the RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade, one of the most extensively studied cancer signaling networks. While substrates of RAF and MEK kinases are highly restricted, ERK1/2 has been attributed to over 1,000 substrates. In this study, we used the highly selective ERK1/2 inhibitor, SCH772984, and proteomic and phosphoproteomic analyses to extend the repertoire of ERK-dependent phosphosites and phosphoproteins in PDAC. We validated the specificity of SCH772984 in our cell lines using multiplexed inhibitor beads coupled with mass spectrometry (MIB/MS). We then performed phosphoproteomics and global proteomics in a panel of PDAC cell lines and identified 5,117 ERK-dependent phosphosites on 2,252 proteins, of which 88% and 67%, respectively, were not previously associated with ERK. We then utilized our recently annotated serine/threonine kinome motif database to dissect the phosphoproteome and reveal an expansive ERK-regulated kinase network. We found that ERK- and immediate downstream kinase RSK-substrate motifs predominated after one hour of ERK inhibition, whereas cell cycle regulatory cyclin-dependent kinase motifs predominated by 24 h, reflecting a highly dynamic ERK-dependent phosphoproteome. We find compensatory activation of HIPK, CLK, PKN, PAK, and DYRK family kinases. Finally, using the genome-wide CRISPR-Cas9 dataset in the Cancer Dependency Map portal (DepMap), we determined that approximately 18% of ERK dependent phosphoproteins are essential for pancreatic cancer growth, and these are enriched in nuclear proteins. Together, our findings provide a system-wide profile of the mechanistic basis for ERK-driven pancreatic cancer growth.
2024-05-29 | PXD048534 | Pride
Project description:Mutational activation of the KRAS oncogene is a major genetic driver of pancreatic ductal adenocarcinoma (PDAC) growth. KRAS-dependent PDAC growth is mediated primarily through persistent activation of the RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade, one of the most extensively studied cancer signaling networks. While substrates of RAF and MEK kinases are highly restricted, ERK1/2 has been attributed to over 1,000 substrates. In this study, we used the highly selective ERK1/2 inhibitor, SCH772984, and proteomic and phosphoproteomic analyses to extend the repertoire of ERK-dependent phosphosites and phosphoproteins in PDAC. We validated the specificity of SCH772984 in our cell lines using multiplexed inhibitor beads coupled with mass spectrometry (MIB/MS). We then performed phosphoproteomics and global proteomics in a panel of PDAC cell lines and identified 5,117 ERK-dependent phosphosites on 2,252 proteins, of which 88% and 67%, respectively, were not previously associated with ERK. We then utilized our recently annotated serine/threonine kinome motif database to dissect the phosphoproteome and reveal an expansive ERK-regulated kinase network. We found that ERK- and immediate downstream kinase RSK-substrate motifs predominated after one hour of ERK inhibition, whereas cell cycle regulatory cyclin-dependent kinase motifs predominated by 24 h, reflecting a highly dynamic ERK-dependent phosphoproteome. We find compensatory activation of HIPK, CLK, PKN, PAK, and DYRK family kinases. Finally, using the genome-wide CRISPR-Cas9 dataset in the Cancer Dependency Map portal (DepMap), we determined that approximately 18% of ERK dependent phosphoproteins are essential for pancreatic cancer growth, and these are enriched in nuclear proteins. Together, our findings provide a system-wide profile of the mechanistic basis for ERK-driven pancreatic cancer growth.
2024-05-29 | PXD048532 | Pride
Project description:Mutational activation of the KRAS oncogene is a major genetic driver of pancreatic ductal adenocarcinoma (PDAC) growth. KRAS-dependent PDAC growth is mediated primarily through persistent activation of the RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade, one of the most extensively studied cancer signaling networks. While substrates of RAF and MEK kinases are highly restricted, ERK1/2 has been attributed to over 1,000 substrates. In this study, we used the highly selective ERK1/2 inhibitor, SCH772984, and proteomic and phosphoproteomic analyses to extend the repertoire of ERK-dependent phosphosites and phosphoproteins in PDAC. We validated the specificity of SCH772984 in our cell lines using multiplexed inhibitor beads coupled with mass spectrometry (MIB/MS). We then performed phosphoproteomics and global proteomics in a panel of PDAC cell lines and identified 5,117 ERK-dependent phosphosites on 2,252 proteins, of which 88% and 67%, respectively, were not previously associated with ERK. We then utilized our recently annotated serine/threonine kinome motif database to dissect the phosphoproteome and reveal an expansive ERK-regulated kinase network. We found that ERK- and immediate downstream kinase RSK-substrate motifs predominated after one hour of ERK inhibition, whereas cell cycle regulatory cyclin-dependent kinase motifs predominated by 24 h, reflecting a highly dynamic ERK-dependent phosphoproteome. We find compensatory activation of HIPK, CLK, PKN, PAK, and DYRK family kinases. Finally, using the genome-wide CRISPR-Cas9 dataset in the Cancer Dependency Map portal (DepMap), we determined that approximately 18% of ERK dependent phosphoproteins are essential for pancreatic cancer growth, and these are enriched in nuclear proteins. Together, our findings provide a system-wide profile of the mechanistic basis for ERK-driven pancreatic cancer growth.
2024-05-29 | PXD048531 | Pride
Project description:Mutational activation of the KRAS oncogene is a major genetic driver of pancreatic ductal adenocarcinoma (PDAC) growth. KRAS-dependent PDAC growth is mediated primarily through persistent activation of the RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade, one of the most extensively studied cancer signaling networks. While substrates of RAF and MEK kinases are highly restricted, ERK1/2 has been attributed to over 1,000 substrates. In this study, we used the highly selective ERK1/2 inhibitor, SCH772984, and proteomic and phosphoproteomic analyses to extend the repertoire of ERK-dependent phosphosites and phosphoproteins in PDAC. We validated the specificity of SCH772984 in our cell lines using multiplexed inhibitor beads coupled with mass spectrometry (MIB/MS). We then performed phosphoproteomics and global proteomics in a panel of PDAC cell lines and identified 5,117 ERK-dependent phosphosites on 2,252 proteins, of which 88% and 67%, respectively, were not previously associated with ERK. We then utilized our recently annotated serine/threonine kinome motif database to dissect the phosphoproteome and reveal an expansive ERK-regulated kinase network. We found that ERK- and immediate downstream kinase RSK-substrate motifs predominated after one hour of ERK inhibition, whereas cell cycle regulatory cyclin-dependent kinase motifs predominated by 24 h, reflecting a highly dynamic ERK-dependent phosphoproteome. We find compensatory activation of HIPK, CLK, PKN, PAK, and DYRK family kinases. Finally, using the genome-wide CRISPR-Cas9 dataset in the Cancer Dependency Map portal (DepMap), we determined that approximately 18% of ERK dependent phosphoproteins are essential for pancreatic cancer growth, and these are enriched in nuclear proteins. Together, our findings provide a system-wide profile of the mechanistic basis for ERK-driven pancreatic cancer growth.
2024-05-29 | PXD048529 | Pride
Project description:Objective Oncogenic Kras-activated robust Mek/Erk signals phosphorylate to the tuberous sclerosis complex (Tsc) and deactivates mammalian target of rapamycin (mTOR) suppression in pancreatic ductal adenocarcinoma (PDAC); however, Mek and mTOR inhibitors alone have demonstrated minimal clinical antitumor activity. Design We generated transgenic mouse models in which mTOR was hyperactivated either through the Kras/Mek/Erk cascade, by loss of Pten or through Tsc1 haploinsufficiency. Primary cancer cells were isolated from mouse tumours. Oncogenic signalling was assessed in vitro and in vivo, with and without single or multiple targeted molecule inhibition. Transcriptional profiling was used to identify biomarkers predictive of the underlying pathway alterations and of therapeutic response. Results from the preclinical models were confirmed on human material. Results Reduction of Tsc1 function facilitated activation of Kras/Mek/Erk-mediated mTOR signalling, which promoted the development of metastatic PDACs. Single inhibition of mTOR or Mek elicited strong feedback activation of Erk or Akt, respectively. Only dual inhibition of Mek and PI3K reduced mTOR activity and effectively induced cancer cell apoptosis. Analysis of downstream targets demonstrated that oncogenic activity of the Mek/Erk/Tsc/mTOR axis relied on Aldh1a3 function. Moreover, in clinical PDAC samples, ALDH1A3 specifically labelled an aggressive subtype. Conclusions These results advance our understanding of Mek/Erk-driven mTOR activation and its downstream targets in PDAC, and provide a mechanistic rationale for effective therapeutic matching for Aldh1a3-positive PDACs.
2015-10-05 | GSE48643 | GEO