scholarly journals Combined BRAF and MEK Inhibition With Dabrafenib and Trametinib in BRAF V600–Mutant Colorectal Cancer

2015 ◽  
Vol 33 (34) ◽  
pp. 4023-4031 ◽  
Author(s):  
Ryan B. Corcoran ◽  
Chloe E. Atreya ◽  
Gerald S. Falchook ◽  
Eunice L. Kwak ◽  
David P. Ryan ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Protein Kinase ◽  
Partial Response ◽  
Microsatellite Instability ◽  
Mutational Analysis ◽  
Braf Inhibitor ◽  
Complete Response ◽  
Mitogen Activated Protein Kinase ◽  
Pik3ca Mutations ◽  
Mitogen Activated Protein

Purpose To evaluate dabrafenib, a selective BRAF inhibitor, combined with trametinib, a selective MEK inhibitor, in patients with BRAF V600–mutant metastatic colorectal cancer (mCRC). Patients and Methods A total of 43 patients with BRAF V600–mutant mCRC were treated with dabrafenib (150 mg twice daily) plus trametinib (2 mg daily), 17 of whom were enrolled onto a pharmacodynamic cohort undergoing mandatory biopsies before and during treatment. Archival tissues were analyzed for microsatellite instability, PTEN status, and 487-gene sequencing. Patient-derived xenografts were established from core biopsy samples. Results Of 43 patients, five (12%) achieved a partial response or better, including one (2%) complete response, with duration of response > 36 months; 24 patients (56%) achieved stable disease as best confirmed response. Ten patients (23%) remained in the study > 6 months. All nine evaluable during-treatment biopsies had reduced levels of phosphorylated ERK relative to pretreatment biopsies (average decrease ± standard deviation, 47% ± 24%). Mutational analysis revealed that the patient achieving a complete response and two of three evaluable patients achieving a partial response had PIK3CA mutations. Neither PTEN loss nor microsatellite instability correlated with efficacy. Responses to dabrafenib plus trametinib were comparable in patient-derived xenograft–bearing mice and the biopsied lesions from each corresponding patient. Conclusion The combination of dabrafenib plus trametinib has activity in a subset of patients with BRAF V600–mutant mCRC. Mitogen-activated protein kinase signaling was inhibited in all patients evaluated, but to a lesser degree than observed in BRAF-mutant melanoma with dabrafenib alone. PIK3CA mutations were identified in responding patients and thus do not preclude response to this regimen. Additional studies targeting the mitogen-activated protein kinase pathway in this disease are warranted.

scholarly journals Nuclear Export and Plasma Membrane Recruitment of the Ste5 Scaffold Are Coordinated with Oligomerization and Association with Signal Transduction Components

2003 ◽  
Vol 14 (6) ◽  
pp. 2543-2558 ◽  
Author(s):  
Yunmei Wang ◽  
Elaine A. Elion
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Binding Site ◽  
Nuclear Export ◽  
Mutational Analysis ◽  
Active Form ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade

The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a Gβγ dimer (Ste4/Ste18) at the plasma membrane in a recruitment event that requires prior nuclear shuttling of Ste5. Genetic evidence suggests that Ste5 must oligomerize to function, but its impact on Ste5 function and localization is unknown. Herein, we show that oligomerization affects Ste5 activity and localization. The majority of Ste5 is monomeric, suggesting that oligomerization is tightly regulated. Increasing the pool of Ste5 oligomers increases association with Ste11. Remarkably, Ste5 oligomers are also more efficiently exported from the nucleus, retained in the cytoplasm by Ste11 and better recruited to the plasma membrane, resulting in constitutive activation of the mating mitogen-activated protein kinase cascade. Coprecipitation tests show that the RING-H2 domain is the key determinant of oligomerization. Mutational analysis suggests that the leucine-rich domain limits the accessibility of the RING-H2 domain and inhibits export and recruitment in addition to promoting Ste11 association and activation. Our results suggest that the major form of Ste5 is an inactive monomer with an inaccessible RING-H2 domain and Ste11 binding site, whereas the active form is an oligomer that is more efficiently exported and recruited and has a more accessible RING-H2 domain and Ste11 binding site.

scholarly journals Mechanism of Mpk1 Mitogen-Activated Protein Kinase Binding to the Swi4 Transcription Factor and Its Regulation by a Novel Caffeine-Induced Phosphorylation

2009 ◽  
Vol 29 (24) ◽  
pp. 6449-6461 ◽  
Author(s):  
Andrew W. Truman ◽  
Ki-Young Kim ◽  
David E. Levin
Keyword(s):  
Cell Wall ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Dna Binding ◽  
Binding Site ◽  
Mutational Analysis ◽  
Cell Wall Integrity ◽  
Mitogen Activated Protein Kinase ◽  
Activation Mechanism ◽  
Mitogen Activated Protein

ABSTRACT The Mpk1 mitogen-activated protein kinase (MAPK) of the cell wall integrity signaling pathway uses a noncatalytic mechanism to activate the SBF (Swi4/Swi6) transcription factor. Active Mpk1 forms a complex with Swi4, the DNA-binding subunit of SBF, conferring the ability to bind DNA. Because SBF activation is independent of Mpk1 catalytic activity but requires Mpk1 to be in an active conformation, we sought to understand how Mpk1 interacts with Swi4. Mutational analysis revealed that binding and activation of Swi4 by Mpk1 requires an intact D-motif-binding site, a docking surface common to MAPKs that resides distal to the phosphorylation loop but does not require the substrate-binding site, revealing a novel mechanism for MAPK target regulation. Additionally, we found that Mpk1 binds near the autoinhibitory C terminus of Swi4, suggesting an activation mechanism in which Mpk1 substitutes for Swi6 in promoting Swi4 DNA binding. Finally, we show that caffeine is an atypical activator of cell wall integrity signaling, because it induces phosphorylation of the Mpk1 C-terminal extension at Ser423 and Ser428. These phosphorylations were dependent on the DNA damage checkpoint kinases, Mec1/Tel1 and Rad53. Phosphorylation of Ser423 specifically blocked SBF activation by preventing Mpk1 association with Swi4, revealing a novel mechanism for regulating MAPK target specificity.

scholarly journals Single cell analysis of colorectal cancer identifies mitogen-activated protein kinase as a key driver of tumor cell plasticity

2020 ◽  
Author(s):  
Florian Uhlitz ◽  
Philip Bischoff ◽  
Stefan Peidli ◽  
Anja Sieber ◽  
Benedikt Obermayer ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Protein Kinase ◽  
Single Cell ◽  
Cancer Cell ◽  
Developmental Trajectories ◽  
Mitogen Activated Protein Kinase ◽  
Colorectal Cancer Cell ◽  
Protein Kinase Activity ◽  
Normal Colon ◽  
Mitogen Activated Protein

AbstractIn colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to identify differences in cellular composition between normal colon and colorectal cancer, and to define signals controlling cancer cell development.We used single cell RNA and protein profiling to analyze tumors and matched normal tissues of twelve colorectal cancer patients. RNA metabolic labelling followed by single cell RNA sequencing in patient-derived normal colon and colorectal cancer organoids was employed to define colorectal cancer cell developmental trajectories.We find that colorectal cancer tissues exhibited consistent changes in cellular composition in the epithelial, immune and stromal compartments across patients compared to normal colon. Tumor epithelial cells displayed patient-specific gene expression often correlating with somatic copy number alterations, but mainly organized into patient-overarching clusters. These clusters were defined by cell type-specific transcriptional programs related to stem, transient-amplifying and immature goblet cells, and showed differential expression for signatures of oncogenic traits such as replication stress. Patient-derived colorectal cancer organoids exhibited developmental trajectories forming along a gradient of mitogen-activated protein kinase activity that were Wnt-independent. Likewise, colorectal cancer cell types of patient samples were organized by mitogen-activated protein kinase activity.Our single-cell analyses provide a subtyping system for colorectal cancer cells based on transcriptional readout of morphogenetic signals and oncogenic traits. We provide evidence that mitogen-activated protein kinase signaling, a key pathway for targeted therapy, is a main driver of colorectal cancer cell plasticity.

scholarly journals The Mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes.

1997 ◽  
Vol 17 (11) ◽  
pp. 6419-6426 ◽  
Author(s):  
C H de Moor ◽  
J D Richter
Keyword(s):  
Protein Kinase ◽  
Mutational Analysis ◽  
Cyclin B1 ◽  
Mitogen Activated Protein Kinase ◽  
Coding Region ◽  
Cdc2 Kinase ◽  
Cytoplasmic Polyadenylation ◽  
Response Elements ◽  
Mitogen Activated Protein ◽  
Maternal Mrnas

Cytoplasmic polyadenylation controls the translation of several maternal mRNAs during Xenopus oocyte maturation and requires two sequences in the 3' untranslated region (UTR), the U-rich cytoplasmic polyadenylation element (CPE), and the hexanucleotide AAUAAA. c-mos mRNA is polyadenylated and translated soon after the induction of maturation, and this protein kinase is necessary for a kinase cascade culminating in cdc2 kinase (MPF) activation. Other mRNAs are polyadenylated later, around the time of cdc2 kinase activation. To determine whether there is a hierarchy in the cytoplasmic polyadenylation of maternal mRNAs, we ablated c-mos mRNA with an antisense oligonucleotide. This prevented histone B4 and cyclin A1 and B1 mRNA polyadenylation, indicating that the polyadenylation of these mRNAs is Mos dependent. To investigate a possible role of cdc2 kinase in this process, cyclin B was injected into oocytes lacking c-mos mRNA. cdc2 kinase was activated, but mitogen-activated protein kinase was not. However, polyadenylation of cyclin B1 and histone B4 mRNA was still observed. This demonstrates that cdc2 kinase can induce cytoplasmic polyadenylation in the absence of Mos. Our data further indicate that although phosphorylation of the CPE binding protein may be involved in the induction of Mos-dependent polyadenylation, it is not required for Mos-independent polyadenylation. We characterized the elements conferring Mos dependence (Mos response elements) in the histone B4 and cyclin B1 mRNAs by mutational analysis. For histone B4 mRNA, the Mos response elements were in the coding region or 5' UTR. For cyclin B1 mRNA, the main Mos response element was a CPE that overlaps with the AAUAAA hexanucleotide. This indicates that the position of the CPE can have a profound influence on the timing of cytoplasmic polyadenylation.

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