Resistance to vemurafenib resulting from a novel mutation in the <scp>BRAFV</scp>600<scp>E</scp> kinase domain

Wagenaar, Timothy R.; Ma, Leyuan; Roscoe, Benjamin P.; Park, Sung Mi; Bolon, Daniel N.; Green, Michael R.

doi:10.1111/pcmr.12171

Cited by 55 publications

(49 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Mirroring the clinical paradigm of acquired Vemurafenib (from now on indicated as PLX4032) resistance [42,43], we used the well characterized BRAF-mutated A375 metastatic cell line, and two independent isogenic metastatic melanoma cell models of acquired resistance (PLX4032-sensitive: 451-LU or M1617 and PLX4032-resistant: 451-LU/RES or M1617/RES [10]) to investigate modalities for overcoming or bypassing drug resistance. The resistance mechanism of 451-LU/RES and M1617/RES has been previously described and found to largely rely on a switch to the CRAF or ARAF isoforms resulting in MEK1 hyper-activation after PLX4032 treatment [10].…”

Section: Mek Inhibitor U0126 Sensitizes Plx4032-resistant Melanoma mentioning

confidence: 99%

Concurrent MEK and autophagy inhibition is required to restore cell death associated danger-signalling in Vemurafenib-resistant melanoma cells

Martin

Dudek-Perić

Maes

et al. 2015

Biochemical Pharmacology

View full text Add to dashboard Cite

Vemurafenib (PLX4032), an inhibitor of BRAF(V600E), has demonstrated significant clinical anti-melanoma effects. However, the majority of treated patients develop resistance, due to a variety of molecular mechanisms including MAPK reactivation through MEK. The induction of a cancer cell death modality associated with danger-signalling resulting in surface mobilization of crucial damage-associated-molecular-patterns (DAMPs), e.g. calreticulin (CRT) and heat shock protein-90 (HSP90), from dying cells, is emerging to be crucial for therapeutic success. Both cell death and danger-signalling are modulated by autophagy, a key adaptation mechanism stimulated during melanoma progression. However, whether melanoma cell death induced by MAPK inhibition is associated with danger-signalling, and the reliance of these mechanisms on autophagy, has not yet been scrutinized. Using a panel of isogenic PLX4032-sensitive and resistant melanoma cell lines we show that PLX4032-induced caspase-dependent cell death and DAMPs exposure in the drug-sensitive cells, but failed to do so in the drug-resistant cells, displaying heightened MEK activation. MEK inhibitor, U0126, treatment sensitized PLX4032-resistant cells to death and re-established their danger-signalling capacity. Only melanoma cells exposing death-induced danger-signals were phagocytosed and induced DC maturation. Although the PLX4032-resistant melanoma cells displayed higher basal and drug-induced autophagy, compromising autophagy, pharmacologically or by ATG5 knockdown, was insufficient to re-establish their PLX4032 sensitivity. Interestingly, autophagy abrogation was particularly efficacious in boosting cell death and ecto-CRT/ecto-HSP90 in PLX4032-resistant cells upon blockage of MEK hyper-activation by U0126. Thus combination of MEK inhibitors with autophagy blockers may represent a novel treatment regime to increase both cell death and danger-signalling in Vemurafenib-resistant metastatic melanoma.

show abstract

Section: Mek Inhibitor U0126 Sensitizes Plx4032-resistant Melanoma mentioning

confidence: 99%

Concurrent MEK and autophagy inhibition is required to restore cell death associated danger-signalling in Vemurafenib-resistant melanoma cells

Martin

Dudek-Perić

Maes

et al. 2015

Biochemical Pharmacology

View full text Add to dashboard Cite

show abstract

“…Deep mutational scanning is a powerful tool for exploring the molecular basis of protein function (7,15,25,26). However, restrictions on functional assays have limited its general applicability, particularly for enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…Previous work in enzyme sequence-function mapping has used in vivo assays that couple an enzyme's function to cellular growth (7,(24)(25)(26). These in vivo selections are limited not only in the types of enzyme functions that can be analyzed, but also by the range of experimental conditions compatible with the intracellular environment.…”

Section: Romero Et Almentioning

confidence: 99%

Dissecting enzyme function with microfluidic-based deep mutational scanning

Romero

Tran

Abate

2015

Proc. Natl. Acad. Sci. U.S.A.

212

183

View full text Add to dashboard Cite

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a hightemperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNAsequencing technologies, enables high-throughput mapping of enzyme sequence space.protein engineering | droplet-based microfluidics | high-throughput DNA sequencing E nzymes are powerful biological catalysts capable of remarkably accelerating the rates of chemical transformations (1). The molecular bases of these rate accelerations are often complex, using multiple steps, multiple catalytic mechanisms, and relying on numerous molecular interactions, in addition to those provided by the main catalytic groups. This complexity imposes a significant barrier to understanding how an enzyme's sequence impacts its function and, thus, on our ability to rationally design biocatalysts with new or enhanced functions (2-4).Comprehensive mappings of sequence-function relationships can be used to dissect the molecular basis of protein function in an unbiased manner (5). Growth selections or in vitro binding screens can be combined with next-generation DNA sequencing to generate detailed mappings between a protein's sequence and its biochemical properties, such as binding affinity, enzymatic activity, and stability (6-9). This deep mutational scanning approach has been used to study the structure of the protein fitness landscape, discover new functional sites, improve molecular energy functions, and identify beneficial combinations of mutations for protein engineering. However, these methods rely on functional assays coupled to cell growth or protein binding, severely limiting the types of proteins that can be analyzed. For example, most enzymes of biological or industrial relevance cannot be analyzed using existing methods because they do not catalyze a reaction that can be directly coupled to cell growth. Experimental advances are needed to broaden the applicability of deep mutational scanning to the diverse palette of functions performed by enzymes.In this paper, we present a general method for mapping protein sequence-func...

show abstract

“…This study also quantified mutational rates and demonstrated that for different mutations (e.g., G/A or A/C) these rates spanned more than two orders of magnitude. Systematic analyses of engineered mutations have been extended beyond microbes to investigate drug resistance in mammalian cells (Wagenaar et al 2014). This study of the V600E BRAF oncogene systematically identified mutations in the kinase active site that enable cultured cells to grow in the presence of drug therapy.…”

Section: Concluding Thoughtsmentioning

confidence: 99%

Viewing Protein Fitness Landscapes Through a Next-Gen Lens

Boucher¹,

Cote²,

Flynn³

et al. 2014

Genetics

View full text Add to dashboard Cite

High-throughput sequencing has enabled many powerful approaches in biological research. Here, we review sequencing approaches to measure frequency changes within engineered mutational libraries subject to selection. These analyses can provide direct estimates of biochemical and fitness effects for all individual mutations across entire genes (and likely compact genomes in the near future) in genetically tractable systems such as microbes, viruses, and mammalian cells. The effects of mutations on experimental fitness can be assessed using sequencing to monitor time-dependent changes in mutant frequency during bulk competitions. The impact of mutations on biochemical functions can be determined using reporters or other means of separating variants based on individual activities (e.g., binding affinity for a partner molecule can be interrogated using surface display of libraries of mutant proteins and isolation of bound and unbound populations). The comprehensive investigation of mutant effects on both biochemical function and experimental fitness provide promising new avenues to investigate the connections between biochemistry, cell physiology, and evolution. We summarize recent findings from systematic mutational analyses; describe how they relate to a field rich in both theory and experimentation; and highlight how they may contribute to ongoing and future research into protein structure-function relationships, systems-level descriptions of cell physiology, and population-genetic inferences on the relative contributions of selection and drift.

show abstract

Resistance to vemurafenib resulting from a novel mutation in the BRAFV600E kinase domain

Cited by 55 publications

References 24 publications

Concurrent MEK and autophagy inhibition is required to restore cell death associated danger-signalling in Vemurafenib-resistant melanoma cells

Concurrent MEK and autophagy inhibition is required to restore cell death associated danger-signalling in Vemurafenib-resistant melanoma cells

Dissecting enzyme function with microfluidic-based deep mutational scanning

Viewing Protein Fitness Landscapes Through a Next-Gen Lens

Contact Info

Product

Resources

About