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ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma.

Smit MA, Maddalo G, Greig K, Raaijmakers LM, Possik PA, van Breukelen B, Cappadona S, Heck AJ, Altelaar AF, Peeper DS - Mol. Syst. Biol. (2014)

Bottom Line: However, most patients eventually relapse due to drug resistance.We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3.Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

Proteomic and genomic workflowsCell lysates from control samples and samples derived from 1 and 3 days after PLX4720 treatment were digested with Lys-C/Trypsin, labeled by triplex dimethyl approach and mixed in 1:1:1 ratios. For protein expression analysis, 200 μg of digested lysate was fractionated by SCX and each fraction was analyzed by LC/MS/MS to determine the relative protein expression levels for every time point compared to the control. For the phosphoproteome, 3 mg of digested lysate was fractionated by SCX and each fraction was enriched for phosphopeptides by Ti4+-IMAC prior to LC/MS/MS analysis.Melanoma cells were transduced with a lentiviral kinome library, containing ˜4,000 shRNAs targeting ˜500 kinases. Cells were treated either with DMSO (control) or with BRAFi or ERKi. Genomic DNA was isolated, and hairpins were amplified by PCR. Using deep sequencing, the hairpins that specifically dropped out in the treated sample were identified. In this case, the absence of the blue bar in deep sequencing indicates schematically a synthetic lethal effect of the shRNA and BRAFi/ERKi.
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fig01: Proteomic and genomic workflowsCell lysates from control samples and samples derived from 1 and 3 days after PLX4720 treatment were digested with Lys-C/Trypsin, labeled by triplex dimethyl approach and mixed in 1:1:1 ratios. For protein expression analysis, 200 μg of digested lysate was fractionated by SCX and each fraction was analyzed by LC/MS/MS to determine the relative protein expression levels for every time point compared to the control. For the phosphoproteome, 3 mg of digested lysate was fractionated by SCX and each fraction was enriched for phosphopeptides by Ti4+-IMAC prior to LC/MS/MS analysis.Melanoma cells were transduced with a lentiviral kinome library, containing ˜4,000 shRNAs targeting ˜500 kinases. Cells were treated either with DMSO (control) or with BRAFi or ERKi. Genomic DNA was isolated, and hairpins were amplified by PCR. Using deep sequencing, the hairpins that specifically dropped out in the treated sample were identified. In this case, the absence of the blue bar in deep sequencing indicates schematically a synthetic lethal effect of the shRNA and BRAFi/ERKi.

Mentions: To gain insight into the molecular mechanisms and signaling pathways underlying normal drug response and thereby screen for novel targets for drug sensitization, we combined (phospho)proteomic analysis and an shRNA library function-based approach. We used a low-passage human melanoma cell line, 04.01, which is sensitive to treatment with PLX4720, a preclinical vemurafenib analog. For the MS-based proteomic approach, we performed a time course experiment, monitoring the changes of the proteome and phosphoproteome after 1 and 3 days of treatment with an IC50 dose of PLX4720 (Fig1A). Alongside, we performed two shRNA screens on the same cell line to identify shRNAs that sensitize melanoma cells to targeted inhibition of either BRAF or one of its critical effectors, ERK (Fig1B; described in detail below).


ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma.

Smit MA, Maddalo G, Greig K, Raaijmakers LM, Possik PA, van Breukelen B, Cappadona S, Heck AJ, Altelaar AF, Peeper DS - Mol. Syst. Biol. (2014)

Proteomic and genomic workflowsCell lysates from control samples and samples derived from 1 and 3 days after PLX4720 treatment were digested with Lys-C/Trypsin, labeled by triplex dimethyl approach and mixed in 1:1:1 ratios. For protein expression analysis, 200 μg of digested lysate was fractionated by SCX and each fraction was analyzed by LC/MS/MS to determine the relative protein expression levels for every time point compared to the control. For the phosphoproteome, 3 mg of digested lysate was fractionated by SCX and each fraction was enriched for phosphopeptides by Ti4+-IMAC prior to LC/MS/MS analysis.Melanoma cells were transduced with a lentiviral kinome library, containing ˜4,000 shRNAs targeting ˜500 kinases. Cells were treated either with DMSO (control) or with BRAFi or ERKi. Genomic DNA was isolated, and hairpins were amplified by PCR. Using deep sequencing, the hairpins that specifically dropped out in the treated sample were identified. In this case, the absence of the blue bar in deep sequencing indicates schematically a synthetic lethal effect of the shRNA and BRAFi/ERKi.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300494&req=5

fig01: Proteomic and genomic workflowsCell lysates from control samples and samples derived from 1 and 3 days after PLX4720 treatment were digested with Lys-C/Trypsin, labeled by triplex dimethyl approach and mixed in 1:1:1 ratios. For protein expression analysis, 200 μg of digested lysate was fractionated by SCX and each fraction was analyzed by LC/MS/MS to determine the relative protein expression levels for every time point compared to the control. For the phosphoproteome, 3 mg of digested lysate was fractionated by SCX and each fraction was enriched for phosphopeptides by Ti4+-IMAC prior to LC/MS/MS analysis.Melanoma cells were transduced with a lentiviral kinome library, containing ˜4,000 shRNAs targeting ˜500 kinases. Cells were treated either with DMSO (control) or with BRAFi or ERKi. Genomic DNA was isolated, and hairpins were amplified by PCR. Using deep sequencing, the hairpins that specifically dropped out in the treated sample were identified. In this case, the absence of the blue bar in deep sequencing indicates schematically a synthetic lethal effect of the shRNA and BRAFi/ERKi.
Mentions: To gain insight into the molecular mechanisms and signaling pathways underlying normal drug response and thereby screen for novel targets for drug sensitization, we combined (phospho)proteomic analysis and an shRNA library function-based approach. We used a low-passage human melanoma cell line, 04.01, which is sensitive to treatment with PLX4720, a preclinical vemurafenib analog. For the MS-based proteomic approach, we performed a time course experiment, monitoring the changes of the proteome and phosphoproteome after 1 and 3 days of treatment with an IC50 dose of PLX4720 (Fig1A). Alongside, we performed two shRNA screens on the same cell line to identify shRNAs that sensitize melanoma cells to targeted inhibition of either BRAF or one of its critical effectors, ERK (Fig1B; described in detail below).

Bottom Line: However, most patients eventually relapse due to drug resistance.We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3.Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

No MeSH data available.


Related in: MedlinePlus