ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells
Background Most melanoma patients with $ BRAF^{V600E} $ positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently...
Ausführliche Beschreibung
Autor*in: |
Cesi, Giulia [verfasserIn] |
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Sprache: |
Englisch |
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2017 |
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Anmerkung: |
© The Author(s). 2017 |
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Übergeordnetes Werk: |
Enthalten in: Molecular cancer - London : Biomed Central, 2002, 16(2017), 1 vom: 08. Juni |
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Übergeordnetes Werk: |
volume:16 ; year:2017 ; number:1 ; day:08 ; month:06 |
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DOI / URN: |
10.1186/s12943-017-0667-y |
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SPR029064538 |
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245 | 1 | 0 | |a ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells |
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520 | |a Background Most melanoma patients with $ BRAF^{V600E} $ positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant $ BRAF^{V600E} $ has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in $ BRAF^{V600E} $ and in $ BRAF^{WT} $/$ NRAS^{mut} $ harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions In $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% $ NRAS^{mut} $, 11.9% $ NF1^{mut} $). | ||
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650 | 4 | |a BRAF inhibitors |7 (dpeaa)DE-He213 | |
650 | 4 | |a MEK inhibitors |7 (dpeaa)DE-He213 | |
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700 | 1 | |a Walbrecq, Geoffroy |4 aut | |
700 | 1 | |a Zimmer, Andreas |4 aut | |
700 | 1 | |a Kreis, Stephanie |4 aut | |
700 | 1 | |a Haan, Claude |4 aut | |
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10.1186/s12943-017-0667-y doi (DE-627)SPR029064538 (SPR)s12943-017-0667-y-e DE-627 ger DE-627 rakwb eng Cesi, Giulia verfasserin aut ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background Most melanoma patients with $ BRAF^{V600E} $ positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant $ BRAF^{V600E} $ has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in $ BRAF^{V600E} $ and in $ BRAF^{WT} $/$ NRAS^{mut} $ harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions In $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% $ NRAS^{mut} $, 11.9% $ NF1^{mut} $). Melanoma (dpeaa)DE-He213 Metabolism (dpeaa)DE-He213 RAS/RAF/MEK/ERK pathway (dpeaa)DE-He213 NRAS (dpeaa)DE-He213 BRAF inhibitors (dpeaa)DE-He213 MEK inhibitors (dpeaa)DE-He213 Pyruvate dehydrogenase (dpeaa)DE-He213 Pyruvate dehydrogenase kinases (dpeaa)DE-He213 Reactive oxygen species (dpeaa)DE-He213 PDK inhibitors (dpeaa)DE-He213 Walbrecq, Geoffroy aut Zimmer, Andreas aut Kreis, Stephanie aut Haan, Claude aut Enthalten in Molecular cancer London : Biomed Central, 2002 16(2017), 1 vom: 08. Juni (DE-627)355987619 (DE-600)2091373-4 1476-4598 nnns volume:16 year:2017 number:1 day:08 month:06 https://dx.doi.org/10.1186/s12943-017-0667-y kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 16 2017 1 08 06 |
spelling |
10.1186/s12943-017-0667-y doi (DE-627)SPR029064538 (SPR)s12943-017-0667-y-e DE-627 ger DE-627 rakwb eng Cesi, Giulia verfasserin aut ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background Most melanoma patients with $ BRAF^{V600E} $ positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant $ BRAF^{V600E} $ has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in $ BRAF^{V600E} $ and in $ BRAF^{WT} $/$ NRAS^{mut} $ harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions In $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% $ NRAS^{mut} $, 11.9% $ NF1^{mut} $). Melanoma (dpeaa)DE-He213 Metabolism (dpeaa)DE-He213 RAS/RAF/MEK/ERK pathway (dpeaa)DE-He213 NRAS (dpeaa)DE-He213 BRAF inhibitors (dpeaa)DE-He213 MEK inhibitors (dpeaa)DE-He213 Pyruvate dehydrogenase (dpeaa)DE-He213 Pyruvate dehydrogenase kinases (dpeaa)DE-He213 Reactive oxygen species (dpeaa)DE-He213 PDK inhibitors (dpeaa)DE-He213 Walbrecq, Geoffroy aut Zimmer, Andreas aut Kreis, Stephanie aut Haan, Claude aut Enthalten in Molecular cancer London : Biomed Central, 2002 16(2017), 1 vom: 08. Juni (DE-627)355987619 (DE-600)2091373-4 1476-4598 nnns volume:16 year:2017 number:1 day:08 month:06 https://dx.doi.org/10.1186/s12943-017-0667-y kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 16 2017 1 08 06 |
allfields_unstemmed |
10.1186/s12943-017-0667-y doi (DE-627)SPR029064538 (SPR)s12943-017-0667-y-e DE-627 ger DE-627 rakwb eng Cesi, Giulia verfasserin aut ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background Most melanoma patients with $ BRAF^{V600E} $ positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant $ BRAF^{V600E} $ has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in $ BRAF^{V600E} $ and in $ BRAF^{WT} $/$ NRAS^{mut} $ harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions In $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% $ NRAS^{mut} $, 11.9% $ NF1^{mut} $). Melanoma (dpeaa)DE-He213 Metabolism (dpeaa)DE-He213 RAS/RAF/MEK/ERK pathway (dpeaa)DE-He213 NRAS (dpeaa)DE-He213 BRAF inhibitors (dpeaa)DE-He213 MEK inhibitors (dpeaa)DE-He213 Pyruvate dehydrogenase (dpeaa)DE-He213 Pyruvate dehydrogenase kinases (dpeaa)DE-He213 Reactive oxygen species (dpeaa)DE-He213 PDK inhibitors (dpeaa)DE-He213 Walbrecq, Geoffroy aut Zimmer, Andreas aut Kreis, Stephanie aut Haan, Claude aut Enthalten in Molecular cancer London : Biomed Central, 2002 16(2017), 1 vom: 08. Juni (DE-627)355987619 (DE-600)2091373-4 1476-4598 nnns volume:16 year:2017 number:1 day:08 month:06 https://dx.doi.org/10.1186/s12943-017-0667-y kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 16 2017 1 08 06 |
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10.1186/s12943-017-0667-y doi (DE-627)SPR029064538 (SPR)s12943-017-0667-y-e DE-627 ger DE-627 rakwb eng Cesi, Giulia verfasserin aut ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background Most melanoma patients with $ BRAF^{V600E} $ positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant $ BRAF^{V600E} $ has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in $ BRAF^{V600E} $ and in $ BRAF^{WT} $/$ NRAS^{mut} $ harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions In $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% $ NRAS^{mut} $, 11.9% $ NF1^{mut} $). Melanoma (dpeaa)DE-He213 Metabolism (dpeaa)DE-He213 RAS/RAF/MEK/ERK pathway (dpeaa)DE-He213 NRAS (dpeaa)DE-He213 BRAF inhibitors (dpeaa)DE-He213 MEK inhibitors (dpeaa)DE-He213 Pyruvate dehydrogenase (dpeaa)DE-He213 Pyruvate dehydrogenase kinases (dpeaa)DE-He213 Reactive oxygen species (dpeaa)DE-He213 PDK inhibitors (dpeaa)DE-He213 Walbrecq, Geoffroy aut Zimmer, Andreas aut Kreis, Stephanie aut Haan, Claude aut Enthalten in Molecular cancer London : Biomed Central, 2002 16(2017), 1 vom: 08. Juni (DE-627)355987619 (DE-600)2091373-4 1476-4598 nnns volume:16 year:2017 number:1 day:08 month:06 https://dx.doi.org/10.1186/s12943-017-0667-y kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 16 2017 1 08 06 |
allfieldsSound |
10.1186/s12943-017-0667-y doi (DE-627)SPR029064538 (SPR)s12943-017-0667-y-e DE-627 ger DE-627 rakwb eng Cesi, Giulia verfasserin aut ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background Most melanoma patients with $ BRAF^{V600E} $ positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant $ BRAF^{V600E} $ has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in $ BRAF^{V600E} $ and in $ BRAF^{WT} $/$ NRAS^{mut} $ harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions In $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% $ NRAS^{mut} $, 11.9% $ NF1^{mut} $). Melanoma (dpeaa)DE-He213 Metabolism (dpeaa)DE-He213 RAS/RAF/MEK/ERK pathway (dpeaa)DE-He213 NRAS (dpeaa)DE-He213 BRAF inhibitors (dpeaa)DE-He213 MEK inhibitors (dpeaa)DE-He213 Pyruvate dehydrogenase (dpeaa)DE-He213 Pyruvate dehydrogenase kinases (dpeaa)DE-He213 Reactive oxygen species (dpeaa)DE-He213 PDK inhibitors (dpeaa)DE-He213 Walbrecq, Geoffroy aut Zimmer, Andreas aut Kreis, Stephanie aut Haan, Claude aut Enthalten in Molecular cancer London : Biomed Central, 2002 16(2017), 1 vom: 08. Juni (DE-627)355987619 (DE-600)2091373-4 1476-4598 nnns volume:16 year:2017 number:1 day:08 month:06 https://dx.doi.org/10.1186/s12943-017-0667-y kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 16 2017 1 08 06 |
language |
English |
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Enthalten in Molecular cancer 16(2017), 1 vom: 08. Juni volume:16 year:2017 number:1 day:08 month:06 |
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Enthalten in Molecular cancer 16(2017), 1 vom: 08. Juni volume:16 year:2017 number:1 day:08 month:06 |
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findex.gbv.de |
topic_facet |
Melanoma Metabolism RAS/RAF/MEK/ERK pathway NRAS BRAF inhibitors MEK inhibitors Pyruvate dehydrogenase Pyruvate dehydrogenase kinases Reactive oxygen species PDK inhibitors |
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container_title |
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Cesi, Giulia @@aut@@ Walbrecq, Geoffroy @@aut@@ Zimmer, Andreas @@aut@@ Kreis, Stephanie @@aut@@ Haan, Claude @@aut@@ |
publishDateDaySort_date |
2017-06-08T00:00:00Z |
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However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant $ BRAF^{V600E} $ has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. 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Cesi, Giulia |
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Cesi, Giulia misc Melanoma misc Metabolism misc RAS/RAF/MEK/ERK pathway misc NRAS misc BRAF inhibitors misc MEK inhibitors misc Pyruvate dehydrogenase misc Pyruvate dehydrogenase kinases misc Reactive oxygen species misc PDK inhibitors ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells |
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ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells Melanoma (dpeaa)DE-He213 Metabolism (dpeaa)DE-He213 RAS/RAF/MEK/ERK pathway (dpeaa)DE-He213 NRAS (dpeaa)DE-He213 BRAF inhibitors (dpeaa)DE-He213 MEK inhibitors (dpeaa)DE-He213 Pyruvate dehydrogenase (dpeaa)DE-He213 Pyruvate dehydrogenase kinases (dpeaa)DE-He213 Reactive oxygen species (dpeaa)DE-He213 PDK inhibitors (dpeaa)DE-He213 |
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misc Melanoma misc Metabolism misc RAS/RAF/MEK/ERK pathway misc NRAS misc BRAF inhibitors misc MEK inhibitors misc Pyruvate dehydrogenase misc Pyruvate dehydrogenase kinases misc Reactive oxygen species misc PDK inhibitors |
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ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells |
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ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells |
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ros production induced by braf inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells |
title_auth |
ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells |
abstract |
Background Most melanoma patients with $ BRAF^{V600E} $ positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant $ BRAF^{V600E} $ has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in $ BRAF^{V600E} $ and in $ BRAF^{WT} $/$ NRAS^{mut} $ harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions In $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% $ NRAS^{mut} $, 11.9% $ NF1^{mut} $). © The Author(s). 2017 |
abstractGer |
Background Most melanoma patients with $ BRAF^{V600E} $ positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant $ BRAF^{V600E} $ has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in $ BRAF^{V600E} $ and in $ BRAF^{WT} $/$ NRAS^{mut} $ harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions In $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% $ NRAS^{mut} $, 11.9% $ NF1^{mut} $). © The Author(s). 2017 |
abstract_unstemmed |
Background Most melanoma patients with $ BRAF^{V600E} $ positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant $ BRAF^{V600E} $ has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in $ BRAF^{V600E} $ and in $ BRAF^{WT} $/$ NRAS^{mut} $ harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions In $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% $ NRAS^{mut} $, 11.9% $ NF1^{mut} $). © The Author(s). 2017 |
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ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells |
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Results We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in $ BRAF^{V600E} $ and in $ BRAF^{WT} $/$ NRAS^{mut} $ harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions In $ BRAF^{V600E} $ and $ BRAF^{WT} $/$ NRAS^{mut} $ melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% $ NRAS^{mut} $, 11.9% $ NF1^{mut} $).</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Melanoma</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Metabolism</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">RAS/RAF/MEK/ERK pathway</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">NRAS</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">BRAF inhibitors</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">MEK inhibitors</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Pyruvate dehydrogenase</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Pyruvate dehydrogenase kinases</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Reactive oxygen species</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">PDK inhibitors</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Walbrecq, Geoffroy</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Zimmer, Andreas</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Kreis, Stephanie</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Haan, Claude</subfield><subfield code="4">aut</subfield></datafield><datafield tag="773" ind1="0" ind2="8"><subfield code="i">Enthalten in</subfield><subfield code="t">Molecular cancer</subfield><subfield code="d">London : Biomed Central, 2002</subfield><subfield code="g">16(2017), 1 vom: 08. 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score |
7.400667 |