Proteomic and phosphoproteomics profiling depict drug addiction mechanisms in BRAFi-resistant melanoma

ABSTRACT: Acquired resistance to MAPK inhibitors limits the clinical efficacy in melanoma treatment. We and others have recently shown that BRAF inhibitors (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 cell while, conversely, knock out of ERK1 fails to reverse this phenotype, showing a response similar to 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 strategies in drug-resistant melanoma.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Bohui Li  

LAB HEAD: Maarten Altelaar

PROVIDER: PXD026557 | Pride | 2022-02-17

REPOSITORIES: Pride