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Similar effects of various Erk inhibitors: interception of negative feedback, activation of p38, imposing cell cycle arrest, and maintaining cell viability

ABSTRACT: The MAP kinases Erk1/2 are the major mediators of the oncogenicity of the Receptor tyrosine kinase (RTK)-Ras-Raf-MEK cascade. As components of this pathway are often mutated and constantly active in cancer, Erks are regarded as promising targets for therapy for many types of malignancies. Yet, while active Erks are promoting abnormal cell proliferation, they simultaneously also inhibit upstream components of the pathway. Erks’ inhibition causes, therefore, reactivation of proto-oncogenic proteins hence Erks’ inhibitors must be applied causiously. Here we studied the effects of Erks’ inhibitors on cells transformed by oncogenic variants of Erk1, Erk1R84H and Erk1R84S, in which the negative feedback effect is so powerful that Erks’ phosphorylation could not be monitored. We show that provision of either BVD523, SCH772984, ASN007, Temuterkib or GCD0994, led to re-appearance of high phosphorylation levels of Erks, suggesting that all inhibitors intercepted Erks-mediated negative feedback activity. We found that inhibitors-mediated Erks’ re-phosphorylation is dependent entirely on MEK activation and that the highly phosphorylated Erk molecules are catalytically inactive. All inhibitors caused cell cycle arrest at the G1 phase, by inducing elevation of p21Cip1/Waf1 and Klf4, but did not affect cell viability for at least 72 hours. They also caused activation of the MAP/stress kinase p38. Proteome and phosphoproteome analysis of BVD523-treated cells revealed effect of the drug on the Rho GTPase pathway and on MAPK targets. The 5 inhibitors were further tested on a battery of 9 cancer-derived cell lines and in many of them most of the effects were similar to those observed with the Erk1R84H- and Erk1R84S-transformed NIH3T3 cells

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Spleen, Liver, Cell Culture, Muscle, Kidney

SUBMITTER: Alon Savidor

LAB HEAD: Prof. David Engelberg

PROVIDER: PXD069633 | Pride | 2026-05-18

REPOSITORIES: Pride

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			Action	DRS
	HFX_DE_17337_RH1_DAY1_08122022.raw	Raw
	HFX_DE_17337_RH1_DAY3_08122022.raw	Raw
	HFX_DE_17337_RH1_DAY3_BVD_08122022.raw	Raw
	HFX_DE_17337_RH2_DAY1_07122022.raw	Raw
	HFX_DE_17337_RH2_DAY3_07122022.raw	Raw

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Project description:The extracellular signal-regulated kinases (Erks), Erk1 and Erk2, are crucial components of the receptor tyrosine kinase (RTK)-Ras-Raf-MEK pathway, which is frequently mutated and constitutively activated in various cancers. Despite Erk activity being commonly observed in cancer, the role of direct Erk activation in driving tumorigenesis remains unproven. This study explores the oncogenic potential of intrinsically active Erk1 mutants, particularly Erk1R84H, in both in vivo and in vitro models.We established a transgenic mouse model enabling tissue-specific and temporally controlled expression of Erk1R84H. Upon inducing Erk1R84H expression in the liver, we observed the development of hepatocellular carcinoma (HCC) characterized by increased liver weight, necrosis, and significant loss of body weight. Intriguingly, the phosphorylated/active form of Erk1R84H was dramatically downregulated during HCC development, becoming almost undetectable in mature tumors. This phenomenon was mirrored in NIH3T3 cells transformed by Erk1R84H, where TEY phosphorylation was significantly reduced as transformed foci appeared.Our proteomic and phosphoproteomic analyses of stable clones expressing Erk1R84H or Erk1R84S revealed substantial changes in protein expression and kinase activity. Key findings included the upregulation of CDK1, AURKB, and CDK2, and the downregulation of DNA repair and circadian rhythm-related kinases. Notably, Upp1, a crucial enzyme in nucleotide metabolism, was highly upregulated in clones transformed by Erk1R84S or Erk1R84H, suggesting its role in supporting the transformed phenotype.Furthermore, we demonstrated that the feedback inhibition mechanism suppressing Erk phosphorylation is dependent on Erk activity itself. Inhibition of Erk1R84H or Erk1R84S with Erk inhibitors restored TEY phosphorylation, highlighting the importance of precise rather very low Erk activity in maintaining the transformed state.In summary, Erk1R84H is confirmed as an efficient oncoprotein capable of inducing HCC in vivo and transforming cells in culture. The mechanism it uses for transformation, involving significant down regulation of its own activity underscores the notion that too high oncogenic activity is not favored by the tumor and proposes a modified therapeutic approach of targeting Erk for its activation rather than inhibition.

Project description:The extracellular signal-regulated kinases (Erks), Erk1 and Erk2, are crucial components of the receptor tyrosine kinase (RTK)-Ras-Raf-MEK pathway, which is frequently mutated and constitutively activated in various cancers. Despite Erk activity being commonly observed in cancer, the role of direct Erk activation in driving tumorigenesis remains unproven. This study explores the oncogenic potential of intrinsically active Erk1 mutants, particularly Erk1R84H, in both in vivo and in vitro models. We established a transgenic mouse model enabling tissue-specific and temporally controlled expression of Erk1R84H. Upon inducing Erk1R84H expression in the liver, we observed the development of hepatocellular carcinoma (HCC) characterized by increased liver weight, necrosis, and significant loss of body weight. Intriguingly, the phosphorylated/active form of Erk1R84H was dramatically downregulated during HCC development, becoming almost undetectable in mature tumors. This phenomenon was mirrored in NIH3T3 cells transformed by Erk1R84H, where TEY phosphorylation was significantly reduced as transformed foci appeared. Our proteomic and phosphoproteomic analyses of stable clones expressing Erk1R84H or Erk1R84S revealed substantial changes in protein expression and kinase activity. Key findings included the upregulation of CDK1, AURKB, and CDK2, and the downregulation of DNA repair and circadian rhythm-related kinases. Notably, Upp1, a crucial enzyme in nucleotide metabolism, was highly upregulated in clones transformed by Erk1R84S or Erk1R84H, suggesting its role in supporting the transformed phenotype. Furthermore, we demonstrated that the feedback inhibition mechanism suppressing Erk phosphorylation is dependent on Erk activity itself. Inhibition of Erk1R84H or Erk1R84S with Erk inhibitors restored TEY phosphorylation, highlighting the importance of precise rather very low Erk activity in maintaining the transformed state. In summary, Erk1R84H is confirmed as an efficient oncoprotein capable of inducing HCC in vivo and transforming cells in culture. The mechanism it uses for transformation, involving significant down regulation of its own activity underscores the notion that too high oncogenic activity is not favored by the tumor and proposes a modified therapeutic approach of targeting Erk for its activation rather than inhibition.

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Similar effects of various Erk inhibitors: interception of negative feedback, activation of p38, imposing cell cycle arrest, and maintaining cell viability

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