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Proteomics and Phosphoproteomics Profiling of Drug-Addicted BRAFi-Resistant Melanoma Cells
Bohui Li1,2, Xiangjun Kong3, Harm Post1,2
1Biomolecular Mass Spectrometry and Proteomics Group, Utrecht Institute for Pharmaceutical Science, Utrecht University, Utrecht 3584 CH, The Netherlands.
Abstract:
Acquired resistance to MAPK inhibitors limits the clinical efficacy in melanoma treatment. We and others have recently shown that BRAF inhibitor (BRAFi)-resistant melanoma cells can develop a dependency on the therapeutic drugs to which they have acquired resistance, creating a vulnerability for these cells that can potentially be exploited in cancer treatment. In drug-addicted melanoma cells, it was shown that this induction of cell death was preceded by a specific ERK2-dependent phenotype switch; however, the underlying molecular mechanisms are largely lacking. To increase the molecular understanding of this drug dependency, we applied a mass spectrometry-based proteomic approach on BRAFi-resistant BRAFMUT 451Lu cells, in which ERK1, ERK2, and JUNB were silenced separately using CRISPR-Cas9. Inactivation of ERK2 and, to a lesser extent, JUNB prevents drug addiction in these melanoma cells, while, conversely, knockout of ERK1 fails to reverse this phenotype, showing a response similar to that of control cells. Our analysis reveals that ERK2 and JUNB share comparable proteome responses dominated by reactivation of cell division. Importantly, we find that EMT activation in drug-addicted melanoma cells upon drug withdrawal is affected by silencing ERK2 but not ERK1. Moreover, transcription factor (regulator) enrichment shows that PIR acts as an effector of ERK2 and phosphoproteome analysis reveals that silencing of ERK2 but not ERK1 leads to amplification of GSK3 kinase activity. Our results depict possible mechanisms of drug addiction in melanoma, which may provide a guide for therapeutic strategies in drug-resistant melanoma.
Insights
Melanoma cells resistant to BRAF inhibitors can become dependent on the drug, creating a vulnerability. Silencing ERK2, but not ERK1, prevents this drug addiction and reactivates cell division, offering new therapeutic strategies.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- Acquired resistance to MAPK inhibitors poses a significant challenge in melanoma treatment.
- BRAF inhibitor (BRAFi)-resistant melanoma cells can develop drug dependency, a vulnerability exploitable for therapy.
- The molecular mechanisms underlying this drug addiction, particularly the role of ERK signaling, require further elucidation.
Purpose of the Study:
- To investigate the molecular mechanisms of drug addiction in BRAFi-resistant melanoma cells.
- To identify key molecular players, specifically ERK1, ERK2, and JUNB, in mediating drug dependency.
- To explore potential therapeutic strategies targeting drug-addicted melanoma cells.
Main Methods:
- Utilized a mass spectrometry-based proteomic approach on BRAFi-resistant BRAFMUT 451Lu melanoma cells.
- Employed CRISPR-Cas9 gene editing to selectively silence ERK1, ERK2, and JUNB.
- Conducted phosphoproteome analysis and transcription factor enrichment analysis.
Main Results:
- Inactivation of ERK2 and, to a lesser extent, JUNB prevented drug addiction, while ERK1 knockout did not reverse this phenotype.
- ERK2 and JUNB silencing led to comparable proteome responses, primarily characterized by the reactivation of cell division.
- Silencing ERK2, but not ERK1, affected epithelial-mesenchymal transition (EMT) activation upon drug withdrawal and amplified GSK3 kinase activity.
Conclusions:
- ERK2 and JUNB play critical roles in establishing and maintaining drug addiction in BRAFi-resistant melanoma.
- Targeting ERK2 presents a promising strategy to overcome drug resistance and exploit drug addiction in melanoma treatment.
- These findings provide insights into the molecular mechanisms of drug addiction, guiding future therapeutic interventions for drug-resistant melanoma.
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