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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

RNAi screens identify short hairpins sensitizing to BRAFi or ERKi treatment04.01 melanoma cells were transduced with the kinome shRNA library (which was divided in four pools each containing ˜1,000 hairpins) with a multiplicity of infection (MOI) of 0.5. After selection with puromycin, cell pools were divided into four samples: one as a reference control, one was treated with DMSO as a control, one was treated with 0.15 μM PLX4720 and one was treated with 0.015 μM SCH772984. After 4 and 7 days of treatment, cells were harvested. Genomic DNA was isolated and deep sequenced. shRNAs that dropped out in the treated sample are highlighted by dashed empty circles and indicated by red arrows compared to the control. Hairpins that dropped out in the untreated sample compared to the reference sample are highlighted by dashed empty circles and indicated by green arrows.Venn diagram depicting the overlap of identified hairpins in the PLX4720 and SCH772984 sensitizer screens. On the right, the BRAF pathway and the two inhibitors used are depicted.
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fig04: RNAi screens identify short hairpins sensitizing to BRAFi or ERKi treatment04.01 melanoma cells were transduced with the kinome shRNA library (which was divided in four pools each containing ˜1,000 hairpins) with a multiplicity of infection (MOI) of 0.5. After selection with puromycin, cell pools were divided into four samples: one as a reference control, one was treated with DMSO as a control, one was treated with 0.15 μM PLX4720 and one was treated with 0.015 μM SCH772984. After 4 and 7 days of treatment, cells were harvested. Genomic DNA was isolated and deep sequenced. shRNAs that dropped out in the treated sample are highlighted by dashed empty circles and indicated by red arrows compared to the control. Hairpins that dropped out in the untreated sample compared to the reference sample are highlighted by dashed empty circles and indicated by green arrows.Venn diagram depicting the overlap of identified hairpins in the PLX4720 and SCH772984 sensitizer screens. On the right, the BRAF pathway and the two inhibitors used are depicted.

Mentions: Parallel to the proteomic analysis, we set out to perform an unbiased and function-based genomic screen to identify potential drug target(s) that can be used in combination with BRAF inhibitors. Specifically, we performed a sensitizer screen for PLX4720 with a lentiviral shRNA library containing ∼4,000 shRNAs targeting ∼500 different kinases. We transduced the human melanoma cell line 04.01 with a multiplicity of infection (MOI) of 0.5 to limit the likelihood of multiple integrations per cell, avoiding competition for the RNAi silencing machinery. After pharmacologic selection, cells were treated for 1 week with either DMSO (control) or low-concentration PLX4720 (IC20). Next-generation sequencing was used to identify hairpins selectively depleted in the treated sample compared to the control, with the aim to identify drug enhancers (FigA). As already described in the introduction, many resistance mechanisms to BRAF inhibition involve re-activation of ERK. Also ERK inhibitors are currently being developed to treat melanoma (Morris et al, 2013). Therefore, we performed an ERK inhibitor (SCH772984) sensitization screen in parallel. For both screens, we selected the hairpins that were significantly depleted (P < 0.05) in the treated sample by at least 1/3 after 1 week of treatment. We excluded hairpins that were enriched after 4 days of treatment, to minimize the number of false-positive hits. We compared the identified hairpins from both screens (PLX4720 and SCH772984) and found an overlap of 59 (Fig4B, Supplementary Table S4). Selecting the genes that were silenced by at least two hairpins in both screens (to exclude potential off-target effects and to increase the likelihood that the hits functionally interacted with the BRAF/MEK/ERK pathway) yielded five potential hits: AAK1, PLK4, IGF1R, MET and ROCK1.


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)

RNAi screens identify short hairpins sensitizing to BRAFi or ERKi treatment04.01 melanoma cells were transduced with the kinome shRNA library (which was divided in four pools each containing ˜1,000 hairpins) with a multiplicity of infection (MOI) of 0.5. After selection with puromycin, cell pools were divided into four samples: one as a reference control, one was treated with DMSO as a control, one was treated with 0.15 μM PLX4720 and one was treated with 0.015 μM SCH772984. After 4 and 7 days of treatment, cells were harvested. Genomic DNA was isolated and deep sequenced. shRNAs that dropped out in the treated sample are highlighted by dashed empty circles and indicated by red arrows compared to the control. Hairpins that dropped out in the untreated sample compared to the reference sample are highlighted by dashed empty circles and indicated by green arrows.Venn diagram depicting the overlap of identified hairpins in the PLX4720 and SCH772984 sensitizer screens. On the right, the BRAF pathway and the two inhibitors used are depicted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4300494&req=5

fig04: RNAi screens identify short hairpins sensitizing to BRAFi or ERKi treatment04.01 melanoma cells were transduced with the kinome shRNA library (which was divided in four pools each containing ˜1,000 hairpins) with a multiplicity of infection (MOI) of 0.5. After selection with puromycin, cell pools were divided into four samples: one as a reference control, one was treated with DMSO as a control, one was treated with 0.15 μM PLX4720 and one was treated with 0.015 μM SCH772984. After 4 and 7 days of treatment, cells were harvested. Genomic DNA was isolated and deep sequenced. shRNAs that dropped out in the treated sample are highlighted by dashed empty circles and indicated by red arrows compared to the control. Hairpins that dropped out in the untreated sample compared to the reference sample are highlighted by dashed empty circles and indicated by green arrows.Venn diagram depicting the overlap of identified hairpins in the PLX4720 and SCH772984 sensitizer screens. On the right, the BRAF pathway and the two inhibitors used are depicted.
Mentions: Parallel to the proteomic analysis, we set out to perform an unbiased and function-based genomic screen to identify potential drug target(s) that can be used in combination with BRAF inhibitors. Specifically, we performed a sensitizer screen for PLX4720 with a lentiviral shRNA library containing ∼4,000 shRNAs targeting ∼500 different kinases. We transduced the human melanoma cell line 04.01 with a multiplicity of infection (MOI) of 0.5 to limit the likelihood of multiple integrations per cell, avoiding competition for the RNAi silencing machinery. After pharmacologic selection, cells were treated for 1 week with either DMSO (control) or low-concentration PLX4720 (IC20). Next-generation sequencing was used to identify hairpins selectively depleted in the treated sample compared to the control, with the aim to identify drug enhancers (FigA). As already described in the introduction, many resistance mechanisms to BRAF inhibition involve re-activation of ERK. Also ERK inhibitors are currently being developed to treat melanoma (Morris et al, 2013). Therefore, we performed an ERK inhibitor (SCH772984) sensitization screen in parallel. For both screens, we selected the hairpins that were significantly depleted (P < 0.05) in the treated sample by at least 1/3 after 1 week of treatment. We excluded hairpins that were enriched after 4 days of treatment, to minimize the number of false-positive hits. We compared the identified hairpins from both screens (PLX4720 and SCH772984) and found an overlap of 59 (Fig4B, Supplementary Table S4). Selecting the genes that were silenced by at least two hairpins in both screens (to exclude potential off-target effects and to increase the likelihood that the hits functionally interacted with the BRAF/MEK/ERK pathway) yielded five potential hits: AAK1, PLK4, IGF1R, MET and ROCK1.

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