PMCID
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Thus, the pathway must be carefully controlled to ensure appropriate responses to environmental cues.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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In normal cells, outcomes include survival, proliferation, senescence, and differentiation, but in cancer the constitutive pathway activation favors proliferation and survival.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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RAS–ERK signaling is particularly important in melanoma.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Somatic mutations occur in BRAF, NRAS, and KRAS in 43%, 20%, and 2% of melanomas respectively (www.sanger.ac.uk/genetics/CGP/cosmic/).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The mutations in RAS trap it in a GTP-bound, active conformation and mostly involve glycine 12 (G12), glycine 13 (G13), and glutamine 61 (Q61).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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A glutamic acid substitution for the valine at position 600 (BRAF) accounts for over 90% of the mutations in BRAF in cancer.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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However, over 100 other rare mutations have been described, most of which cluster to the glycine-rich loop and activation segment in the kinase domain.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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These regions normally trap BRAF in an inactive conformation by forming an atypical intramolecular interaction, and it is thought that the mutations disrupt this interaction, thereby allowing the active conformation to prevail (Wan et al., 2004).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Functional studies have shown that most of the mutations in BRAF are activating and enhance its ability to directly phosphorylate MEK (Wan et al., 2004; Garnett and Marais, 2004).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Curiously however, some mutants have impaired activity and although they cannot directly phosphorylate MEK, they appear to retain sufficient activity to bind to and transphosphorylate and activate CRAF in a RAS-independent manner (Garnett et al., 2005), allowing these mutants to activate the pathway indirectly through CRAF.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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More puzzling are mutations that occur at aspartic acid 594 (D594).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The carboxy oxygen of this highly conserved residue (the “D” of the DFG motif) plays a critical role in chelating Mg and stabilizing ATP binding in the catalytic site (Johnson et al., 1998).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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As in other kinases, mutation of this residue causes inactivation and thus cancer mutants such as BRAF cannot phosphorylate MEK, activate CRAF, or stimulate cell signaling (Ikenoue et al., 2003; Wan et al., 2004).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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These mutants therefore appear catalytically and biologically inactive and yet 34 have been found in human cancer (www.sanger.ac.uk/genetics/CGP/cosmic/).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Furthermore, while BRAF mutations (over 10,000 described) occur in a mutually exclusive manner with RAS mutations, four of the 34 kinase-dead mutants are coincident with RAS mutations, a highly significant enrichment (p < 10; Fisher's Exact Test) that suggests functional interaction.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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It has been shown that BRAF is 500-fold activated, can stimulates constitutive MEK–ERK signaling in cells (Gray-Schopfer et al., 2007) and induce melanoma in mice (Dankort et al., 2009; Dhomen et al., 2009), showing that it can be a founder mutation in melanoma.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Importantly, BRAF inhibition blocks melanoma cell proliferation and induces apoptosis in vitro and blocks melanoma xenograft growth in vivo (see Gray-Schopfer et al., 2007).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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These data validate BRAF as a driver of melanomagenesis and as a therapeutic target in melanoma, so drugs to target this pathway have been developed.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The first to be tested clinically were the multi-kinase inhibitor sorafenib and the MEK inhibitor PD184352 (CI1040).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Disappointingly, both failed to produce objective responses in patients, either because they were not sufficiently potent, or because they caused unacceptable toxicity (Halilovic and Solit, 2008).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Recently, more potent and selective BRAF inhibitors have been described.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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For example, the triarylimidazole SB590885 and the difluorophenylsulfonamine PLX4720 display excellent selectivity for BRAF in vitro and preferentially inhibit BRAF mutant cancer cell proliferation (King et al., 2006; Tsai et al., 2008).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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More importantly, BRAF-selective drugs have recently entered the clinic and are producing excellent responses in patients with BRAF mutant melanoma (Flaherty et al., 2009; Schwartz et al., 2009).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The aim of this study was to better understand the responses that melanoma cells make to BRAF-selective inhibitors and thereby to provide a molecular basis for the design of clinical trials using BRAF drugs.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We also wished to examine if kinase-dead BRAF and oncogenic RAS functionally interact in vivo.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We selected four drugs for our studies (Figures S1A–S1D).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Sorafenib is a class II (inactive conformation binder) drug (Wan et al., 2004) that inhibits BRAF at 40 nM, CRAF at 13 nM, and several other kinases in the low nM range (Wilhelm et al., 2004).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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It is the least-selective drug that we used.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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PLX4720 is a class I (active conformation binder) inhibitor that is highly selective and inhibits BRAF at 13 nM (Tsai et al., 2008).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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885-A (Figure S1C) is a close analog of the class I inhibitor SB590885 (King et al., 2006) that is also highly selective for BRAF.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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It inhibits BRAF at 2 nM (Figure S1E), is ineffective against a panel of 64 other protein kinases (Table S1), and preferentially blocks BRAF mutant cancer cell proliferation (Figure S1F).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Finally, we also used the potent and selective MEK inhibitor PD184352 (Sebolt-Leopold et al., 1999).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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As expected, all four drugs blocked ERK activity in BRAF mutant A375 melanoma cells (Figure 1A; see Table S2).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Similarly, all four drugs inhibited ERK in SkMel24, SkMel28, D25, and WM266.4 cells, another four lines that express mutant BRAF (Figure S1G).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We also tested the drugs in D04, MM415, MM485, and WM852 NRAS mutant cells (Table S2).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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As expected, PD184352 and sorafenib inhibited ERK in all of these lines (Figure 1A).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Surprisingly, however, PLX4720 and 885-A caused an unexpected increase in ERK activity in the NRAS mutant cells (Figure 1A).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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NRAS or CRAF depletion by RNA interference (RNAi) blocked MEK/ERK activation by PLX4720 and 885-A in NRAS mutant cells (Figure 1B and 1C) and we show that 885-A activated CRAF in these cells (Figure 1D).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We previously reported that oncogenic RAS requires CRAF but not BRAF to activate MEK (Dumaz et al., 2006) and consistent with this, BRAF is inactive in NRAS mutant cells (Figure 1E).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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These data therefore present an intriguing paradox.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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BRAF is not active and is not required for MEK/ERK activation in RAS mutant cells.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Nevertheless, BRAF inhibitors hyperactivate CRAF and MEK in these cells, so we studied the underlying mechanism(s).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Wild-type BRAF binds to CRAF in a RAS-dependent manner and although this binding is weak, it leads to CRAF activation (Garnett et al., 2005).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Since RAS and CRAF are required for ERK activation by PLX4720 and 885-A, we investigated if these drugs induce BRAF binding to CRAF.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Endogenous BRAF was immunoprecipitated from melanoma cells and western blotted for endogenous CRAF.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We show that CRAF did not bind to BRAF in untreated or PD184352 treated WM852, D04, MM415, or MM485 cells (Figure 2A), demonstrating that MEK inhibition does not induce binding.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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In contrast, sorafenib and 885-A induced strong binding of BRAF to CRAF in all four lines (Figure 2A).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We also performed the experiment in the inverse manner, immunoprecipitating CRAF and showing that BRAF binding was strongly induced by sorafenib and 885-A (Figure 2A).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Curiously, PLX4720 did not appear to induce BRAF binding to CRAF, but previous studies have shown that ERK phosphorylates BRAF in a negative-feedback loop that destabilizes its binding to CRAF (Rushworth et al., 2006).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We show that PD184352 stabilizes BRAF binding to CRAF in the presence of PLX4720 (Figure 2B), demonstrating that PLX4720 does induce binding, albeit less strongly than the other drugs.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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In addition to inducing BRAF binding to CRAF in NRAS mutant cells, 885-A and sorafenib also induce this binding in WM1791c melanoma cells and in SW620 and HCT116 colorectal carcinoma cells (Figure 2C), all of which express mutant KRAS (Table S2).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Importantly, no strong binding of BRAF to CRAF was seen in A375 cells even in the presence of PD184352 and the drugs did not induce strong BRAF binding to CRAF in two other BRAF mutant melanoma cell lines (Figure 2D and Figure S2).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Thus, sorafenib, 885-A and PLX4720 all induced BRAF binding to CRAF in NRAS or KRAS mutant cells, but not in BRAF mutant cells, showing that BRAF inhibition per se did not induce this binding; it only occurred when BRAF was inhibited in the presence of oncogenic RAS.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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To confirm the essential role of RAS, we show that a CRAF mutant (CRAF) that cannot bind to RAS (Fabian et al., 1994) did not bind to BRAF (Figure 3A and Figure S3A) and the corresponding mutant of BRAF (BRAF) did not bind to CRAF (Figure 3B and see Figure S3B).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We also prepared membrane/cytosol fractionations of RAS mutant cells and show that under normal conditions over 40% of CRAF is in the membrane, whereas BRAF is largely cytosolic (Figure 3C).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Notably, 885-A treatment leads to strong recruitment of BRAF to the membrane fraction, whereas CRAF is only weakly affected (Figure 3C).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We also show that under normal conditions, EGF did not induce BRAF binding to CRAF in PMWK cells, a line that is wild-type for BRAF and RAS (Table S2).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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However, in the presence of 885-A, EGF induced robust binding of BRAF to CRAF in PMWK cells and this resulted in sustained pathway activation (Figure 3D).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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This shows that BRAF binding to CRAF is induced in the presence of both oncogenic RAS and activated wild-type RAS.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We note that sorafenib and 885-A induce a mobility shift in BRAF in SDS-gels (Figure 2A).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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BRAF also undergoes a mobility shift in PLX4720 treated cells in the presence of PD184352 (Figure 2B).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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This mobility shift is reduced when immunoprecipitated BRAF is treated with calf intestinal alkaline phosphatase (CIP; Figure 3E) and PD184352 pretreatment reduced, but did not ablate the magnitude of the shift induced by 885-A (Figure 3F).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Importantly, in vitro CIP treatment and cell pretreatment with PD184352 did not prevent BRAF binding to CRAF (Figures 3E and 3F).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Together, these data suggest that the BRAF bound to CRAF is hyperphosphorylated through MEK–ERK-dependent and MEK–ERK-independent mechanisms, but that this phosphorylation is not required for BRAF binding to CRAF.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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To test directly if BRAF binding to CRAF is driven by 885-A binding to BRAF, we mutated the so-called “gatekeeper threonine” (T529) of BRAF to asparagine (T529N).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Since BRAF is not active in RAS mutant melanoma cells (Figure 1E), we measured BRAF activity using transient expression in COS cells (Wan et al., 2004).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The results show that BRAF is still activated by HRAS, NRAS and KRAS (Figure 4A and Figure S4A).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Importantly, BRAF is ∼170-fold less sensitive to 885-A than wild-type BRAF (17 nM versus 2869 nM; Figure 4B) and 885-A did not stimulate its binding to CRAF (Figure 4C), proving that drug binding to BRAF drives BRAF binding to CRAF.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Next, we expressed a kinase-dead version of BRAF (BRAF) in D04 cells and show that it forms a constitutive complex with CRAF (Figure 4D) and that it activates MEK constitutively (Figure 4E, compare lanes 1, 4, and 7).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Notably, 885-A does not further enhance MEK activation driven by BRAF (Figure 4E, compare lanes 4, 6 to 7, 9), presumably because it cannot further inhibit this already inactive kinase.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Two other kinase-dead BRAF mutants, the classical catalytic lysine mutant (BRAF), and BRAF, a mutant found in human cancer (Wan et al., 2004), also activate MEK in D04 cells (Figure 4F).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Thus, it is BRAF inhibition and not drug binding that drives BRAF binding to CRAF.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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This experiment also shows that MEK activation driven by kinase-dead BRAF is inhibited by sorafenib (Figures 4E and 4F).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Indeed, cell responses to sorafenib appear to be paradoxical.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We show that although sorafenib inhibits ERK (Figure 1A), it induces BRAF binding to CRAF (Figure 2A), CRAF activation (Figure 4G) and CRAF phosphorylation on S338 (Figure 4G, inset), a critical event in CRAF activation (Mason et al., 1999).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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To test directly the role of CRAF in cells when BRAF is inhibited, we mutated its gatekeeper threonine to asparagine (CRAF).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Notably, CRAF still binds to BRAF in sorafenib and 885-A treated cells (Figure 4H), demonstrating that drug binding to CRAF is not required for BRAF binding to CRAF.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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More importantly, in the presence of CRAF, sorafenib activates rather than inhibits the pathway (Figure 4H, compare lanes 3 and 7).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We therefore posit that sorafenib induces paradoxical activation of CRAF because it inhibits BRAF and drives CRAF activation, but simultaneously binds to and inhibits CRAF.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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In agreement with this model, we show that two other pan-RAF inhibitors, ZM336372 and RAF265 also induce BRAF binding to CRAF, but without activating ERK (see Figure S4B).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Our data establish that inhibition of BRAF in the presence of oncogenic RAS hyperactivates CRAF, MEK, and ERK.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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To investigate the consequences of this in vivo, we used conditionally targeted alleles of oncogenic Kras (Kras) and kinase-dead Braf (Braf) in transgenic mice.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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These alleles use Cre-recombinase/LoxP-Stop-LoxP (LSL) technology to regulate inducible expression of mutant proteins from the endogenous mouse genes to ensure normal levels of protein expression.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The Kras allele has been described (Jackson et al., 2001), and we recently developed the Braf allele.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Briefly, exon 15 of endogenous Braf was targeted to mutate D594 to alanine (D594A; see Figure 5A).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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To prevent expression of Braf in all cells, an LSL cassette was inserted between exon 14 and the mutated exon 15.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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This contains a minigene for exons 15–18 of Braf, a transcription terminator and a Neo selection marker to ensure that only Braf is expressed.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Removal of the LSL cassette by Cre-recombinase reveals the mutated exon 15 and Braf is expressed.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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These mice were crossed to Tyr::CreERT2 mice (Yajima et al., 2006), in which the tyrosinase promoter is used to express tamoxifen-activated Cre-recombinase (CreERT2) in the melanocytes.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Since CreERT2 is activated by tamoxifen, this approach provides exquisite spatial and temporal control over Kras and Braf expression.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Kras, Braf, and Tyr::CreERT2 mice were crossed to generate Kras;Tyr::CreERT2, Braf;Tyr::CreERT2, or Kras;Braf;Tyr::CreERT2 mice.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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In all cases, the conditionally targeted alleles were balanced over a corresponding wild-type allele.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Mice were treated with tamoxifen at 2–3 months of age to induce mutant protein expression.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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We have recently shown that in this model, Braf induces skin hyperpigmentation, nevus formation, and melanoma (Dhomen et al., 2009).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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In contrast, Braf did not induce skin hyperpigmentation, nevi (data not shown) or tumors (Figure 5C).
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PMC2872605
|
Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Kras induced weak tail darkening after 5–6 months (Figure 5B) but did not induce either nevi (data not shown) or tumors (Figure 5C).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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However, when Braf and Kras were combined, they induced a conspicuous skin phenotype.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Within 2–3 months the ears (data not shown), tails (Figure 5B), and paws (Figure 5D) darkened visibly.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The mice did not develop nevi, but within 6 months, they all developed large, rapidly growing oligo-pigmented tumors (Figures 5C and 5E).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The tumors displayed evidence of ulceration (Figure 5F) and were composed largely of spindle cells that exhibit features of malignancy, including cellular atypia, nuclear pleomorphism, and conspicuous nucleoli (Figure 5G).
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