Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition

Kirkpatrick, Donald S.; Bustos, Daisy; Dogan, Taner; Chan, Jocelyn; Phu, Lilian; Young, Amy; Friedman, Lori S.; Belvin, Marcia; Song, Qinghua; Bakalarski, Corey E.; Hoeflich, Klaus P.

doi:10.1073/pnas.1309473110

Cited by 53 publications

(50 citation statements)

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“…1E). It was important to see if the phosphorylation sites identified to be inhibited by the compounds used in this study were also reported to be inhibited by other MEK inhibitors (29,30). Site S135/136 in neuroblast differentiation-associated protein (AHNAK) was significantly downregulated after both BRAF and MEK inhibition and has previously been shown to be regulated in response to the MEK inhibitor I (CAS 297744-42-4) in the MEK-sensitive acute myelogenous leukemia (AML) cell line, MV4-11.…”

Section: Discussionmentioning

confidence: 99%

“…In their study, phosphorylation of S135/136 was nonresponsive to MEK inhibition in the resistant AML cell line, HEL. In a second study, melanoma cells were treated with MEKi GDC-0973 and PI3Ki GDC-0941, and immunoprecipitation of phosphopeptides was used to profile DNA damage response signaling (29). The protein SMARC4 showed initial decrease (not significant) in phosphorylation after MEKi and increased after long-term treatment with PI3Ki singularly and in combination with MEKi (29).…”

Section: Discussionmentioning

confidence: 99%

“…In a second study, melanoma cells were treated with MEKi GDC-0973 and PI3Ki GDC-0941, and immunoprecipitation of phosphopeptides was used to profile DNA damage response signaling (29). The protein SMARC4 showed initial decrease (not significant) in phosphorylation after MEKi and increased after long-term treatment with PI3Ki singularly and in combination with MEKi (29). Consistent with data presented here, S695 in SMARC4 is affected at 15 minutes with the MEKi AZD-6244 and returns to control levels by 30 minutes.…”

Section: Discussionmentioning

confidence: 99%

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Phosphoproteomics of MAPK Inhibition in BRAF-Mutated Cells and a Role for the Lethal Synergism of Dual BRAF and CK2 Inhibition

Parker

Clifton‐Bligh

Molloy

2014

Molecular Cancer Therapeutics

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Activating mutations in the MAPK pathway are prevalent drivers of several cancers. The chief consequence of these mutations is a hyperactive ERK1/2 MAPK able to promote cell proliferation, producing a critical hallmark of metastatic disease. The biochemistry of the ERK pathway is well characterized; however, how the pathway achieves different outcomes in the face of genetic aberrations of cancer and subsequent treatment with chemical inhibitors is not clear. To investigate this, we used mass spectrometry to complete a global phosphoproteomic analysis of a BRAFV600E thyroid cancer cell line (SW1736) after treatment with the mutation-selective inhibitor vemurafenib (PLX4032) and MEK1/2 inhibitor selumetinib (AZD6244). We identified thousands of phosphorylation events orchestrated in BRAFV600E cells and performed kinase landscape analysis to identify putative kinases regulated in response to MAPK blockade. The abundance of phosphopeptides containing consensus motifs for acidophilic kinases increased after short-term inhibition with these compounds. We showed that coinhibition of the pleiotropic acidophilic protein kinase CK2 (CK2) and BRAFV600E synergistically reduced proliferation in patient-derived melanomas and thyroid cancer cells harboring the BRAF lesion. We investigated this mechanism and show a role for CK2 in controlling AKT activation that was not reliant on changes to PTEN or PDK1 phosphorylation. These findings highlight a role for CK2 blockade in potentiating the antiproliferative effects of BRAF and MEK inhibition in BRAF cancers. Mol Cancer Ther; 13(7); 1894-906. Ó2014 AACR.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Phosphoproteomics of MAPK Inhibition in BRAF-Mutated Cells and a Role for the Lethal Synergism of Dual BRAF and CK2 Inhibition

Parker

Clifton‐Bligh

Molloy

2014

Molecular Cancer Therapeutics

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“…Interestingly, a proteomic study of cellular responses has identified DNA damage response signaling activation in response to dual inhibition of these pathways [206]. Among the most prominent responses were activation-related phosphorylation of ATM, and DNA-dependent protein kinases.…”

Section: Introductionmentioning

confidence: 99%

“…Among the most prominent responses were activation-related phosphorylation of ATM, and DNA-dependent protein kinases. Inhibition of these kinases in vitro enhanced cell death induced by MAPK and PI3K inhibitors [206]. …”

Section: Introductionmentioning

confidence: 99%

Pathways and therapeutic targets in melanoma

et al. 2014

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This review aims to summarize the current knowledge of molecular pathways and their clinical relevance in melanoma. Metastatic melanoma was a grim diagnosis, but in recent years tremendous advances have been made in treatments. Chemotherapy provided little benefit in these patients, but development of targeted and new immune approaches made radical changes in prognosis. This would not have happened without remarkable advances in understanding the biology of disease and tremendous progress in the genomic (and other “omics”) scale analyses of tumors. The big problems facing the field are no longer focused exclusively on the development of new treatment modalities, though this is a very busy area of clinical research. The focus shifted now to understanding and overcoming resistance to targeted therapies, and understanding the underlying causes of the heterogeneous responses to immune therapy.

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Deciphering MET‐dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics

et al. 2020

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Increasing evidence suggests that interference with growth factor receptor tyrosine kinase (RTK) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA-damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). Following an immunoaffinity-based phosphoproteomic discovery survey, we selected candidate phosphorylation sites for extensive characterization by targeted proteomics focusing on phosphorylation sites in both signaling networks. Several substrates of the DDR were confirmed to be modulated by sequential MET inhibition and IR, or MET inhibition alone. Upon combined treatment, for two substrates, NUMA1 S395 and CHEK1 S345, the gain and loss of phosphorylation, respectively, were recapitulated using invivo tumor models by immunohistochemistry, with possible utility in future translational research. Overall, we have corroborated phosphorylation sites at the intersection between MET and the DDR signaling networks, and suggest that these represent a class of proteins at the interface between oncogene-driven proliferation and genomic stability.Abbreviations ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; CDK, cell cycle-dependent kinase; CHEK1, checkpoint kinase 1; CHEK2, checkpoint kinase 2; DDA, DNA-damaging agent; DDR, DNA damage response; DNA-PKcs, catalytic subunit of the DNA-dependent protein kinase

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Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition

Abstract: Blots were performed against DDR (p53 pSer15, histone 2AX pSer139), cell survival/ cell death (AKT pThr308, cleaved PARP), and cell signaling (ERK1/2 pThr202/Tyr204) markers and controls. Actin and GAPDH served as loading controls.

Cited by 53 publications

References 42 publications

Phosphoproteomics of MAPK Inhibition in BRAF-Mutated Cells and a Role for the Lethal Synergism of Dual BRAF and CK2 Inhibition

Phosphoproteomics of MAPK Inhibition in BRAF-Mutated Cells and a Role for the Lethal Synergism of Dual BRAF and CK2 Inhibition

Pathways and therapeutic targets in melanoma

Deciphering MET‐dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics

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