Global view of the RAF-MEK-ERK module and its immediate downstream effectors

Abstract

Small molecule inhibitors of BRAF and MEK have proven effective at inhibiting tumor growth in melanoma patients, however this efficacy is limited due to the almost universal development of drug resistance. To provide advanced insight into the signaling responses that occur following kinase inhibition we have performed quantitative (phospho)-proteomics of human melanoma cells treated with either dabrafenib, a BRAF inhibitor; trametinib, a MEK inhibitor or SCH772984, an ERK inhibitor. Over nine experiments we identified 7827 class I phosphorylation sites on 4960 proteins. This included 54 phosphorylation sites that were significantly down-modulated after exposure to all three inhibitors, 34 of which have not been previously reported. Functional analysis of these novel ERK targets identified roles for them in GTPase activity and regulation, apoptosis and cell-cell adhesion. Comparison of the results presented here with previously reported phosphorylation sites downstream of ERK showed a limited degree of overlap suggesting that ERK signaling responses may be highly cell line and cue specific. In addition we identified 26 phosphorylation sites that were only responsive to dabrafenib. We provide further orthogonal experimental evidence for 3 of these sites in human embryonic kidney cells over-expressing BRAF as well as further computational insights using KinomeXplorer. The validated phosphorylation sites were found to be involved in actin regulation, which has been proposed as a novel mechanism for inhibiting resistance development. These results would suggest that the linearity of the BRAF-MEK-ERK module is at least context dependent.

In-silico modelling of the mitogen-activated kinase (MAPK) pathway in colorectal cancer: mutations and targeted therapy

“…This makes the MAPK path a very natural target of drugs which contrast the negative effects of the mutation of KRAS. In particular, the response of active ERK to the delivery of drugs has received special attention (Hamis et al, 2021;Lavoie et al, 2020;Pappalardo et al, 2016;Santini et al, 2019). Finally, we observe that the high value of the relative difference δ i of the protein p-p-ERK is due to the smallness of the related physiological equilibrium value.…”

“…Here we have examined the response of the mutated network to the effects of combination of the two drugs at variable doses have also been simulated. In fact, it is well known that drug combinations may counterbalance, e.g., the onset of drug-resistant tumour subclones (Hamis et al, 2021;Morkel et al, 2015;Santini et al, 2019).…”

“…Here we present a further set of complementary results on the MAPK pathway. with a number of remarks on the physiology of the MAPK cascade that are scattered over the literature (Morkel et al, 2015;Santini et al, 2019;Guo et al, 2020;Lavoie et al, 2020) For example, the first two columns of each panel compare physiological equilibria (purple) with those mutated by the GoF of KRAS (blue). We observe that the mutated value of k-Ras remains rather small, although increased as a consequence of the mutation.…”

Functional proteomics of patient derived head and neck squamous cell carcinoma cells reveal novel applications of trametinib

“…S3A). 19 , 20 Basal proteomic levels of cell lysate showed no significant correlations with drug response.…”

Cutaneous Melanoma Classification: The Importance of High-Throughput Genomic Technologies

“…Other mechanisms responsible for MAPK reactivation and sustained ERK signaling include alterations in MEK and NF1 genes. Additionally, the overexpression of the RAF isoform, Raf-1 Proto-Oncogene, Serine/Threonine Kinase (CRAF), can induce resistance to BRAF inhibitors by MEK activation or by paradoxical transactivation of RAF dimers, promoting ERK signaling (224,225). Similarly, poor response to BRAF inhibitors in patients with BRAF-mutant melanoma has been correlated to concurrent loss-of-function mutations in the PTEN gene, which can lead to the reactivation of the PI3K/AKT pathway (226).…”