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"Interestingly, treatment with the BRAF inhibitor Vemurafenib decreased EphA2 levels in BRAF MT cells, but not in KRAS MT cells ( xref ); the sustained MAPK signaling following Vemurafenib or siBRAF in the KRAS MT CRC cells is consistent with previous studies showing that inhibition of BRAF in the presence of oncogenic RAS induces BRAF binding to CRAF, leading to CRAF hyper-activation and sustained MEK1/2-ERK1/2 activation ( xref )."
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"However, upon CRAF overexpression, the RAS–RAF–MEK–ERK pathway was activated [154], which is not surprising, because the molecule is BRAF‐specific, unable to efficiently prevent CRAF dimerization [152], and it has been reported that the paradox can be driven by CRAF bound to BRAF‐specific inhibitor in BRAF−/− cells [108, 147]."
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"By the use of probes based on the principle of fluorescence resonance energy transfer, we found that this amino-terminal B-Raf-specific region is essential for homo-dimerization of B-Raf and hetero-dimerization of B-Raf and c-Raf at the plasma membrane, followed by phosphorylation of Thr118 in the amino-terminal B-Raf-specific region."
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"One possible explanation for this phenotype is that PLX8394, originally selected for its ability to prevent BRAF and CRAF heterodimer formation in the context of mutant or GTP bound RAS, is not able to disrupt homo-dimerization of BRAF fusion kinases that are stabilized by an additional dimerization domain encoded by the 5 ' partner."
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"LY3009120 suppresses BRAF-CRAF heterodimer activity and downstream signaling despite it strengthening formation of BRAF-CRAF heterodimers; by contrast, cobimetinib promotes kinase activity of RAF heterodimer in KRAS-dependent tumors by increasing the BRAF-CRAF heterodimers.We observed erianin inhibited phosphorylation of CRAF S338 in SK-MEL-2 (Fig. 2j)."
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"Previous studies have shown higher KRAS binding affinities when the CRD is added to the RBD , and we believe this directly correlates with the accelerated nucleotide turnover observed, as CRAF-RBDCRD induced faster hydrolysis than CRAF-RBD.Most notably, oncogenic KRAS mutants exhibited higher affinity for truncated forms of CRAF and BRAF complexes compared to the wild-type protein (Fig. 2)."
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"Selective inhibition of B-Raf drives oncogenic RAS dependent BRAF binding to C-Raf, CRAF activation and mitogen activated protein kinase kinase (MEK)-extracellular signal regulated kinase (ERK) signalling, revealing another paradigm of BRAF mediated signalling that promotes tumour progression [XREF_BIBR]."
sparser
"Previously it was reported that BRAF V600E kinase activity was reduced when it heterodimerized with CRAF ( xref ), so we hypothesized that KIT could drive formation of BRAF V600E :CRAF complexes, which would have less activity than uncomplexed BRAF V600E . To test this, we used a bioluminescence resonance energy transfer (BRET) assay to measure the interaction between Renilla Luciferase-tagged CRAF (RLuc-CRAF) and Venus-tagged BRAF V600E (V-BRAF V600E )."
sparser
"Oncogenic activation of this pathway can arise from mutations in any of the pathway components or promoters. xref Typically, the important downstream mediators in the MAPK cell signaling pathway are simulated by the activation of RAS which then signals downstream via the interaction between the serine–threonine kinases, BRAF and CRAF. xref , xref In BRAF wild-type cells, the BRAF kinase is activated by homo- or heterodimer formation with other RAF isoforms such as ARAF or CRAF. xref , xref In BRAF -mutated cells, the BRAF kinase remains constitutively activated in a monomeric state, with a >400-fold increase in kinase activity. xref – xref Upon activation of RAF, there is an interaction with downstream MEK kinases (MEK1 and MEK2) which initiates MEK phosphorylation that in turn facilitates an activating phosphorylation of ERK which promotes oncogenesis. xref , xref , xref Ultimately, the activation of ERK results in cellular proliferation along with migration and angiogenesis, accompanied by a deterrence of apoptosis, thereby promoting malignant cell growth. xref In the pathogenesis of melanoma, there is staunch reliance upon this signaling cascade."
sparser
"It is noteworthy that mutation of BRAF Ser365, another known 14-3-3 binding site, to Ala in the context of Ser729 being still present dramatically reduces both basal and AMPK stimulated 14-3-3 binding ( xref ) and allows BRAF-CRAF dimerization and ERK activation to be maintained in the presence of AICAR ( xref and xref )."
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"Nonetheless, it has been suggested that selective BRAF inhibition induces RAS dependent dimerization of CRAF with WT BRAF, but not BRAF V600E, as well as the formation of CRAF homodimers, leading to subsequent activation of CRAF and downstream MEK-ERK signaling (XREF_FIG) [XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR]."
sparser
"To test this hypothesis, we treated macrophages with the highly selective B-Raf inhibitor GDC-0879 ( xref ), because B-Raf inhibition with GDC-0879 in the presence of activated Ras induces B-Raf binding to c-Raf, leading to c-Raf hyperactivation and thereby elevated MEK/ERK signaling ( xref ; xref ; xref )."
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"We investigated the relationship between cRaf and BRaf and found that active Ras induced heterodimerization of cRaf and BRaf, an effect that was dependent on the serine residue at position 621 of cRAF : Moreover, we also found that cRaf COOH-terminus constitutively associated with BRaf, whereas the NH (2) terminus did not, even in the presence of active RAS : These data suggest that Ras induces the cRaf and BRaf complex formation through the exposure of 14-3-3 binding sites in the COOH-terminus of cRAF : Thus, Ras induced cRaf-Braf heterodimerization may explain the observed cooperativity of cRaf and BRaf in cells responding to growth factor signals."
sparser
"In those cells without BRAF mutations, BRAF inhibitors have been shown to promote BRAF-CRAF heterodimer or CRAF-CRAF homodimer formation, in which one BRAF inhibitor bound RAF protein transactivates the adjacent CRAF subunit of the dimer, leading to downstream activation of MEK-ERK ( xref ; xref ; xref )."
sparser
"One of the best characterized mechanisms of resistance to both MEK inhibitors and type I RAF inhibitors is CRAF-mediated bypass due to loss of negative feedback on the RAF node or activation of RTKs upstream leading to the formation of BRAF–CRAF heterodimers to maintain signaling through the RAS–MAPK pathway ( xref , xref )."
sparser
"Type II RAF inhibitors, which selectively target BRAF–CRAF heterodimers, and the “paradox breaker” plixorafenib, which disrupts the formation of BRAF dimers, both were developed in an attempt to target CRAF-mediated bypass to other classes of RAF and MEK inhibitors, whereas sparing ARAF activity ( xref – xref )."
sparser
"This was the first study to show RAF dimerisation without using artificial dimerisation constructs and also showed that MEK1/2 activation by BRAF:CRAF heterodimers was caused by the RAS-mediated recruitment of RAF dimers to the plasma membrane, as a KRAS G12V variant (aa 1–166) lacking C-terminal membrane targeting residues failed to increase MEK1/2 phosphorylation despite co-immunoprecipitating with, and increasing the formation of, BRAF:CRAF heterodimers."
sparser
"Packer et al. xref reported that the binding of weak RAF inhibitors (imatinib, nilotinib and dasatinib) to BRAF and CRAF drives BRAF:CRAF heterodimer as well as BRCF and CRAF homodimer formation in the presence of oncogenetic RAS, thus leading to paradoxical activation of both BRAF and CRAF, followed by the activation of MEK and ERK."
sparser
"This review will focus on recent structural and biochemical studies that have provided ‘snapshots’ into the RAF regulatory cycle, revealing structures of the autoinhibited BRAF monomer, active BRAF and CRAF homodimers, as well as HSP90/CDC37 chaperone complexes containing CRAF or BRAF V600E . In addition, we will describe the insights obtained regarding how BRAF transitions between its regulatory states and examine the roles that various BRAF domains and 14-3-3 dimers play in both maintaining BRAF as an autoinhibited monomer and in facilitating its transition to an active dimer."
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"Hence, these results indicated that PID1 could accelerate Sorafenib-induced Raf-1 activation and facilitate the formation of Raf-1/BRAF heterodimerization, thereby blocking AKT activation via Raf-1-dependent pathway and leading to increased sensitivity of hepatoma cells to Sorafenib."
sparser
"Successful strategies targeting this tunable-combinatorial signaling complex may include those inhibiting CRAF function (e.g., omni- or pan-RAF inhibitors), V600E BRAF-CRAF interaction, V600E BRAF- MUT MEK interaction/scaffolding, and MEK activation (e.g., phosphorylation by RAF)."
sparser
"To determine whether the NtA motif was required on both the “activator” and the “receiver”, as is implied by current models, we tested whether a CRAF “receiver” mutant that cannot be phosphorylated on residues 338–341 (AAFF substituted for SSYY), could be activated by activator forms of BRAF and CRAF ( xref )."
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"In normal physiology, the MAPK pathway is well regulated: upstream activation signal → RAS activation (from inactive GDP-bound to active GTP bound form) → dimerization of autoinhibited RAF to an active dimer (e.g., heterodimerization of BRAF and CRAF) → MEK phosphorylation and activation → ERK phosphorylation and activation."
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"It is unclear why PLX4720 only induces weak binding of BRAF to CRAF, but this may stem from its unique property of displacing the alpha-C helix of BRAF when it binds and suggests that this helix is important for BRAF binding to CRAF, something that will only be resolved when the BRAF : CRAF crystal structure is solved."
sparser
"In addition, we reported that DGKη interacted with C-Raf and B-Raf (mitogen-activated kinase (MAPK) kinase kinase (MAPKKK)) in response to epidermal growth factor (EGF) stimulation and regulated the Raf–MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)–ERK pathway [ xref ]."
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"We have also used RNAi to examine the potential role of other proteins implicated in BRAF and CRAF complex formation or pathway activation, including the scaffold proteins KSR, Sprouty2 and RKTG and the small G protein RHEB, but our preliminary results have not revealed obvious roles for these proteins."
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"Thus, BRAF appears to possess a higher intrinsic dimerization propensity than RAF1.The Western blot analysis in Fig. 4B demonstrates that all fusion proteins were efficiently produced and that cells co-expressing RAF1-LgBiT/BRAF-SmBiT and BRAF-LgBiT/BRAF-SmBiT dimer displayed enhanced MEK phosphorylation compared to the other combinations.Although the considerably weaker MEK/ERK phosphorylation displayed by cells expressing RAF1-LgBiT/RAF1-SmBiT homodimers fits well to the data in Fig. 4A and the notion that these homo-dimers are less active than BRAF/RAF1 heterodimers and BRAF/BRAF homodimers [21], it is impossible to directly compare the Nluc activities of BRAF-LgBiT/BRAF-SmBiT and RAF1-LgBiT/RAF1-SmBiT homodimers, as the distinct epitope-tags cannot be used to confirm equal expression between HA- and Myc-tagged RAFs."
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"Our inability to demonstrate an obvious role for KSR in mediating BRAF binding to CRAF or CRAF activation by BRAF suggests that the mechanism underlying dimerization here may be different from those described in flies, but clearly additional studies are required to investigate further the role of scaffold proteins in mediating the phenomena we report."
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"Although technical difficulties precluded analysis of B-Raf immunoprecipitates by Western blotting, the data collectively indicate that PMA/PKCα induces the formation of A-Raf/B-Raf, A-Raf/C-Raf, and B-Raf/C-Raf heterodimers and that there is redundancy in the ability of Raf isoforms and dimers to mediate PKCα-induced growth-suppressive ERK signaling."
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"Activation of RAF (or RAF priming) is triggered by its binding to active RAS-GTP, which induces RAF conformational changes (a shift of the DFG motif and αC helix to IN positions), followed by the homo- and heterodimerization of BRAF and CRAF, which further enhances their catalytic activity."
sparser
"While mutant BRAF V600E is constitutively active and has a limited role in dimerization, xref the BRAF-CRAF heterodimer is believed to be
the primary species in both native signaling and paradoxical activation. xref , xref , xref Genetic and biochemical results
have repeatedly implicated CRAF as the primary species responsible
for phosphorylating MEK in paradoxical activation and native signaling. xref − xref , xref − xref Specifically, inhibitor-bound
BRAF is implicated in promoting heterodimerization with unbound CRAF,
causing transactivation of CRAF through an allosteric mechanism at
the protein–protein interface between protomer kinase domains. xref , xref "
sparser
"Recent research has indicated
the potential for selective inhibition of CRAF-driven signaling to
be a more effective and tolerated therapeutic approach for mutant
RAS tumors. xref , xref Identifying selective CRAF kinase
inhibitors that would block BRAF-CRAF heterodimer signaling could
thus be a valuable drug for the treatment of mutant RAS-driven tumors,
but would present a major challenge for medicinal chemistry."
sparser
"Studies have shown that A- and B-Raf kinase mutants can interact with endogenous kinase-competent C-Raf monomers to drive aberrant signaling in cancer. xref , xref , xref We hypothesize that the C-Raf D486A mutation ablates kinase activity but does not detrimentally affect B-Raf binding."
sparser
"Among the mechanisms promoting BRAFi resistance that affect the MAPK signalling pathway, several lead to the dysregulation of RAF1 activity: hyperactivation of RTKs, loss of NF1, mutations in NRAS, increased expression of RAF1, RAF1-BRAF heterodimers, and loss of ERK negative feed-back loops [ xref , xref , xref ]."
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"A recent study reported that a subset of MEK inhibitors that are inactive in RAS-mutant cancers (AZD6244, GDC-0973) promotes BRAF and CRAF heterodimer formation allowing feedback activation of MEK and ERK, whereas RAS active MEK inhibitors (GDC-0623, G-573) stabilize a nonproductive RAF and MEK complex preventing MEK feedback activation."
sparser
"Invitro studies showed that these compounds (PLX7904 and PLX8394) overcame several mechanisms of resistance to BRAF inhibitors and did not promote RAS-mutant cell growth.[58] Crystalography analyses demonstrated that in contrast to vemurafenib, CRAF-BRAF dimer formation was not promoted, likely accounting for their lack of paradoxical MAPK promotion."
sparser
"Phosphorylated Ksr can also function as a transactivator; however, since Raf binding to Ksr induces limited kinase activity xref , in quiescent cells the constitutive association of Ksr with B-Raf may serve to prevent C-Raf binding to B-Raf, safeguarding against undue activation of the pathway xref ."
sparser
"Alternatively, but not excluding the first model, it might be possible that BRAF ΔLNVTAP>F and BRAF ΔLNVTAP>Y , which are less likely occupied by type I 1/2 compounds, are further activated by drug-bound WT BRAF or RAF1, as both isoforms take up dabrafenib and encorafenib in the single-digit nanomolar range ( xref – xref )."
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"Of the three classes, only compounds that target the first type of mutations have demonstrated clear clinical benefit.The use of first-generation RAF inhibitors, such as dabrafenib or vemurafenib, led to better understanding the mechanisms of resistance and shed light on the importance of homo- or heterodimerization of B-Raf and C-Raf as critical in intrinsic or drug-induced resistance [92]."
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"Plasmids for the induced dimerization of RAF1 and BRAF were generated in two steps.Using the iDimerize plasmids pC -RHE and pC EN-F1 (former ARIAD Pharmaceuticals, now Takara Bio) as template, the ORFs encoding FRB and FKBP respectively were amplified by PCR and cloned into pcDNA3.1(+) (Invitrogen), giving the pFRB and pFKBP vectors."
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"5
,
20
It remains undetermined whether all RAF isoforms have a similar autoinhibited conformation, whether the functional dimerization of KDs in the cytosol impacts the dimerization mode of KRAS in complex with the RBD–CRD on the membrane, and why the heterodimeric RAF1‐BRAF complex predominates in the RAS‐driven signaling pathway.3
Conclusion."
sparser
"Furthermore, disrupting CRAF-mediated MEK activation is proved essential to effectively inhibit MAPK signaling pathway in KRAS mutant tumors. xref Unlike the type I½ BRAF inhibitor vemurafenib, erianin inhibited BRAF-CRAF heterodimer and it was also reflected in the suppression of MAPK signaling pathway in SK-MEL-2 (Fig. xref )."
sparser
"Several studies indicated erianin impacted ERK and AKT/mTOR pathways, xref – xref and the cross-talk between the RAF-MAPK and AKT/mTOR pathways in the proliferation was noticed through the interaction of CRAF and AKT. xref Mechanism analysis indicated that CRAF protomer activation and enhancement of phospho-AKT partly revealed the resistant mechanism of BRAF inhibitors. xref Usually, BRAF inhibitors induce paradoxical activation of MAPK pathway by reinforcing dimerization of BRAF-CRAF in RAS-driven cancers."
sparser
"Erianin targets CRAF isoform, decreasing the formation of BRAF-CRAF heterodimers in SK-MEL-2 cell line (Fig. xref ), which was different from pan-RAF inhibitor LY3009120 and MEK inhibitor cobimetinib. xref , xref Although LY3009120 and cobimetinib both elevate BRAF-CRAF heterodimers, their mechanisms are different."
sparser
"Underlying the mechanism for the autocrine growth promotion, there are three indispensable molecular events disclosed in this study: enrichment of CRAF through inhibition of proteasomal degradation, CRAF stabilization effects of GSTP1 facilitation of the RAF dimer (CRAF-CRAF and CRAF-BRAF) formation, and activation of catalytic activity of CRAF."
reach
"The analysis of the BRAF V600E / NRAS melanoma mouse model showed an unexpected finding related to the effect of BRAF inhibitors in these cells : in fact, BRAF inhibitors induce RAS dependent binding of BRAF to CRAF, consequent activation first of CRAF and then of MEK-ERK signaling [XREF_BIBR]."
sparser
"G12C KRAS -mutant, xref , xref , xref so an alternative is to target downstream effectors in the RAF/MEK/ERK pathway, which has led to the development of RAF, MEK and ERK drugs. xref However, in KRAS -mutant cells, BRAF-selective drugs such as vemurafenib (PLX4032) and dabrafenib xref cause paradoxical hyperactivation of the RAF-ERK pathway through formation of BRAF-CRAF homo- and hetero-dimers. xref Unfortunately, targeting MEK downstream of RAF with drugs such as trametinib xref is ineffective in KRAS -mutant cancers because of feedback mechanisms xref and adverse side-effects, xref and therefore these drugs have been unsuccessful in KRAS -mutant PDAC, CRC and NSCLC. xref , xref "
sparser
"However, the presence of constitutively activated BRAF dimers or activated RAS does not preclude a mutation from being classified as class I. When there is high-level upstream RTK signaling, V600-mutated BRAF can form a dimer with a partner BRAF or CRAF molecule, resulting in a dimer resistant to anti-EGFR antibodies [ xref , xref , xref ]."
sparser
"CR1 contains two key regions, the RAS‐binding domain (RBD) and the cysteine‐rich domain (CRD), which combine with RAS‐GTP and are indispensable for cell membrane recruitment. xref , xref , xref An important function of CR1 is to inhibit CR3 and hence keep BRAF inactive. xref , xref When CR1 and RAS‐GTP are combined, this inhibition is relieved, and BRAF is activated (phosphorylation of the activation segment (AS) is also required). xref CR3 contains several key regions, namely, the P‐loop (also known as the glycine‐rich loop, located in the N‐region), an αC helix (important for the formation of BRAF‐CRAF dimers), a dimerization interface (DIF), a catalytic loop, a DFG motif, and the AS."
sparser
"This mutation can increase RAF dimer formation and promote ERK pathway signal expression. xref In the BRAF gene, a kind of splice variant that deletes a part of the exon that removes the RBD participates in the resistance to BRAF inhibitors by increasing the tendency to form homologous dimers and enhancing the MAPK signaling pathway. xref , xref , xref A study supported that the AGAP3‐BRAF fusion protein, which lacks the RBD, may be a novel resistance mechanism. xref Some researchers also demonstrated that BRAF, when inhibited, can be transformed into CRAF or ARAF through a kinase transformation that continues to activate MAPK, leading to resistance to BRAF inhibitors. xref In addition, in RAS mutations, BRAF can bind to CRAF when BRAF is inhibited, thereby promoting the MAPK pathway."
sparser
"► BRAF activates ERK but in some circumstances BRAF inhibitors can induce tumor growth ► BRAF inhibitors drive BRAF-CRAF binding, activating ERK in cells with oncogenic RAS ► Kinase-dead mutants of BRAF have the same effect as BRAF inhibitors ► Oncogenic RAS and kinase-dead BRAF cooperate to induce melanoma in mice"
sparser
"It is unclear why PLX4720 only induces weak binding of BRAF to CRAF, but this may stem from its unique property of displacing the α-C helix of BRAF when it binds ( xref ) and suggests that this helix is important for BRAF binding to CRAF, something that will only be resolved when the BRAF:CRAF crystal structure is solved."
sparser
"Furthermore, the assembly process for a signaling‐competent hetero‐tetrameric complex comprising KRAS and RAF dimers may be affected by the relative stoichiometry between RAS, RAF, and other RAS effectors and regulators, which varies in many types of normal or KRAS‐driven cancer cells. [ xref ] It has been argued that KRAS nanoclusters represent proximal monomers simply because of their recruitment by KRAS‐lipid interactions with specific size‐limited domains on the membrane. [ xref ] However, PRE and mutagenesis studies have demonstrated that KRAS self‐assembly involves physical interaction between the catalytic GTPase domains of KRAS. [ xref , xref ] It remains undetermined whether all RAF isoforms have a similar autoinhibited conformation, whether the functional dimerization of KDs in the cytosol impacts the dimerization mode of KRAS in complex with the RBD–CRD on the membrane, and why the heterodimeric RAF1‐BRAF complex predominates in the RAS‐driven signaling pathway."
sparser
"We have also used RNAi to examine the potential role of other proteins implicated in BRAF-CRAF complex formation or pathway activation, including the scaffold proteins KSR, Sprouty2 and RKTG and the small G protein RHEB, but our preliminary results have not revealed obvious roles for these proteins."
sparser
"Our inability to demonstrate an obvious role for KSR in mediating BRAF binding to CRAF or CRAF activation by BRAF suggests that the mechanism underlying dimerization here may be different from those described in flies, but clearly additional studies are required to investigate further the role of scaffold proteins in mediating the phenomena we report."
sparser
"LY3009120 inhibits MEK1/2 phosphorylation by inhibiting kinase activity in BRAF–CRAF heterodimers and retards the development of tumors carrying KRAS mutations while inducing a more significant dimerization. xref However, when used as a single agent, the required dose of RAF inhibitor being effective in KRAS‐mutated models is significantly higher than that in the BRAF‐V600E model."
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"L779450 induced B-Raf and C-Raf dimerization was not reduced in MEFs expressing the R615H- and C809Y KSR1 mutants unable to bind B-Raf; whereas B-Raf and C-Raf dimerization was abolished in cells expressing S297A and S392A-KSR 1 that exhibits enhanced inhibitor induced B-Raf binding (XREF_FIG)."
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"In contrast, equivalent C-Raf and B-Raf dimerization was observed in KSR -/- and WT-KSR1 MEFs treated with PLX4720, which does not promote KSR1 and B-Raf binding, and PLX4720 induced C-Raf and B-Raf dimerization was significantly increased in both cells lines when Ras V12 was expressed (XREF_FIG)."
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"In further support that KSR1 can compete with C-Raf for inhibitor induced binding to B-Raf, C-Raf and B-Raf dimerization was consistently reduced in WT-KSR1 MEFs versus KSR -/- MEFs when cells were treated with any of the Raf inhibitors capable of promoting KSR1 and B-Raf binding (XREF_FIG), and C-Raf and B-Raf dimerization was still reduced in L779450 treated WT-KSR1 MEFs even when Ras V12 was expressed (XREF_FIG)."
sparser
"One intriguing phenomenon was that even a catalytically compromised B-Raf was capable of inducing kinase activity of Raf-1 in trans in a manner dependent on a physical interaction between B-Raf and Raf-1, suggesting that the underlying mechanism is independent of a simple transautophosphorylation route [ xref , xref - xref ]."
reach
"Using these live-cell assays for Ras–Raf interaction, coupled with similarly designed reporters for monitoring Raf-dimerization, the authors proposed a model in which these first-generation inhibitors induce Raf–Ras binding through disruption of the Raf auto-inhibitory state, coupled with BRaf/CRaf-heterodimerization facilitated by Ras dimers/nanoclusters, thereby increasing Ras activation."
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"Interestingly, it was shown that in the TERTp mutant breast cancer cell line MDA-MB-231, small molecule mediated dimerization between BRAF and CRAF (a proposed mechanism of this paradoxical activation) was insufficient to recapitulate the activation These studies, and our analysis of CTRP data, suggest that paradoxical activation mechanisms may be less relevant in a subset of tumors, including TERTp mutants."
sparser
"In a series of subsequent experiments, we showed that GSTP1 in mKRAS cells interacted with CRAF at its N-terminal domain to protect it from degradation by the ubiquitin-dependent proteasome pathway and to facilitate an essential step for signaling in the MAPK pathway involving the formation of RAF dimers; homodimer of CRAF and heterodimer of CRAF with BRAF (Fig. xref ). xref )"
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"RAS mutations reportedly conferred resistance to osimertinib.4 Activation of RAS signaling in patients with melanoma also conferred resistance to BRAF inhibitors.30 On activation of RAS signaling, RAS-driven heterodimerization of BRAF and CRAF increases, enhancing drug resistance.31 Together, the data suggest that RAS mutations may mediate resistance to EGFR plus BRAF co-inhibition.Our work had several limitations."
sparser
"A recent study reported that a subset of MEK inhibitors that are inactive in RAS-mutant cancers (AZD6244, GDC-0973) promotes BRAF-CRAF heterodimer formation allowing feedback activation of MEK and ERK, whereas RAS active MEK inhibitors (GDC-0623, G-573) stabilize a nonproductive RAF-MEK complex preventing MEK feedback activation ( xref )."
sparser
"Targeting the MEK5-ERK5 pathway using MEK5 inhibitor BIX02189 or treatment with RAF inhibitor LY3009120 blocked cell growth to a greater extent in A1847-T cells than parental, demonstrating the dependency of trametinib–resistant cells for MEK5 and CRAF-BRAF signaling ( xref – xref )."
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"Targeting MEK downstream of BRAF and CRAF heterodimers using PD0325901 subsequent to RAF inhibitor treatment reduced ERK1/2 rebound and simultaneous treatment of tumor xenografts with both RAF and MEK inhibitors led to more pronounced tumor growth inhibition than either treatment alone."
sparser
"Indeed, clinically approved BRAF V600E inhibitors, such as vemurafenib, dabrafenib, and encorafenib, are only effective with RAF monomers like BRAF V600 , and not BRAF and CRAF dimers, and can lead to paradoxical activation of the EGFR/MAPK pathway through ERK-mediated regulatory feedback. xref Conversely, MEKi such as trametinib, binimetinib, and cobimetinib, prevent MEK phosphorylating ERK1/2, thus avoiding its dimerization and nuclear translocation. xref However, trametinib, cobimetinib, and also RO5126766 (a potent RAF/MEKi;still recruiting) alone were not proven active in this subset of patients. xref , xref , xref Again, this can be explained by redundant signaling through upstream RTK and activation of parallel signal transduction cascades bypassing MEK inhibition and reactivating ERK signaling. xref Likewise, preliminary results of ERK inhibitor monotherapy with LY3214996 or CC-90003 did not show marked activity (,). xref , xref Given the poor outcome with monotherapies, combinations exploiting vertical and/or horizontal (on parallel pathways) blockade have been assessed."
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"Moreover, BRAF inhibitors drive RAS dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling in oncogenic RAS [XREF_BIBR] revealing a paradigm of BRAF mediated signaling that promotes tumour progression with clinical implications [XREF_BIBR] and highlighting the importance of understanding pathway signaling in clinical practice and of genotyping tumours prior to administering BRAF selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects such as increased invasion [XREF_BIBR]."
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"The dimerization between C-Raf and B-Raf promotes ERK signaling; however, complex formation of KSR and B-Raf actually limits ERK activation [XREF_BIBR], which suggests that KSR in cells being treated with Raf kinase inhibitors may actually limit paradoxical, rebound ERK activation in response to inhibitor induced Raf dimerization and activation."
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"Phosphorylated Ksr can also function as a transactivator; however, since Raf binding to Ksr induces limited kinase activity XREF_BIBR, in quiescent cells the constitutive association of Ksr with B-Raf may serve to prevent C-Raf binding to B-Raf, safeguarding against undue activation of the pathway XREF_BIBR."
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"As Dixon et al. (2016) demonstrated in their technical report that the LgBiT/SmBiT complementation system can be in principle applied to BRAF/RAF1 heterodimers [48], we decided to explore this system in detail for its suitability to monitor the dimerisation dynamics of RAF and KSR proteins in various settings."
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"To test the suitability of the Nluc system for studying RAF homo- and heterodimerization and to confirm the biological activity of these tagged RAF proteins, we transiently transfected HEK293T cells with constructs allowing the analysis of BRAF/RAF1 heterodimerization and the appropriate LgBiT-N/SmBiT-N control vectors encoding either LgBiT or SmBiT proper."
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"The additive effect between Raf and Mek inhibitors on CRaf PM translocation was observed when measuring CRaf and BRaf hetero-dimerization and CRaf kinase activation induced by GDC0879 in the KRasG12D and CRaf cells (XREF_FIG) as well as in H226 NSCLC cells where only endogenous proteins are expressed (XREF_SUPPLEMENTARY)."
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"As our analysis revealed that mutations at MLK3 are most probably functionally relevant, occurring almost exclusively in MSI carcinomas, and that MLK3 is described as a component of the multiprotein BRAF and RAF1 complex, we decided to analyse how mutations in MLK3 (all types) correlated with mutations in KRAS and/or BRAF in our series."
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"Therefore and because structural RAF mutants have not been investigated in detail with split luciferase reporters, we studied the contribution of these three events for BRAF/RAF1 heterodimerization by using BRAF in the LgBiT and RAF1 in the SmBiT context.First, we investigated the role of the RAS/RAF interaction by introducing the well-known R89L and R188L substitutions [58, 59] into the RBDs of RAF1 and BRAF, respectively."
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"Nevertheless, Sorafenib was still able to slightly increase Nluc activity in cells expressing RBD mutants of BRAF and RAF1, suggesting that drug induced heterodimerization is not entirely RAS dependent.Although the DIF plays an essential role for allosteric transactivation and a critical role for the formation of BRAF homo- and to a lesser extent BRAF/RAF1 heterodimers that are stable enough to survive co-immunoprecipitation procedures [29, 38], little is known as to how DIF mutations impair RAF dimerization in living cells."
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"In contrast, introduction of a DIF mutation in only one of the protomers significantly impaired dimerization induced by the combination of KRAS with Sorafenib, which was further reduced upon introduction of DIF mutations into both protomers.Although R509 plays a very critical role in allosteric transactivation, its histidine substitution is often insufficient to abrogate BRAF dimerization in co-immunoprecipitation experiments, in particular in the context of BRAF/RAF1 heterodimers [29]."
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"This data demonstrates that KRAS induced BRAF/RAF1 heterodimers are potently stabilized by this dimer targeting RAFi.Using Endurazine, a substrate that, according to the manufacturer, provides an initially weaker but more long-lasting Nluc signal than Furimazine and Vivazine, we were able to show that Naporafenib continues to drive BRAF-LgBiT/RAF1SmBiT dimers for up to 24 h (Fig. 3C)."
reach
"Dixon et al. have used the BRAF-LgBiT/RAF1-SmBiT combination for their initial demonstration of the split Nluc system [48] and while our manuscript was in an advanced stage of preparation, Murphy et al. also applied this pair to demonstrate the potent induction of BRAF/RAF1 heterodimers in MEFs harboring oncogenic Nras knock-in alleles [73]."
sparser
"ERBB stimulation efficiently induces BRAF-RAF1 heterodimers, xref which confer resistance to RAFi. xref , xref , xref We have previously shown that this type of resistance can be overcome by combining two structurally different RAFi, but that the exact combinations depend on cell-type-specific traits, such as genetic mutations and feedback loops, and drug properties, such as allosteric effects and ON/OFF binding rates. xref , xref Therefore, we utilized our cell-line-specific models to predict if a combination of two conformation-specific RAFi can synergize to inhibit ERK signaling in PSN1, PANC1, and Capan2 cells."
sparser
"While BRAF inhibitors of both classes stimulate the formation of BRAF-CRAF dimers [ xref , xref ], type-II inhibitors are considerably less efficient than type-I inhibitors in stimulating the phosphorylation of downstream targets, i.e., of MEK kinases and, henceforth, in activating the MAPK pathway."
reach
"Interestingly, Braf-null MEFs reconstituted with BRAF S729A mutant showed similar basal ERK activity to WT BRAF expressing cells, suggesting that, in these cells dimerization of BRAF with CRAF is not critical for ERK activation and that the BRAF-KSR1 interaction is more important."
reach
"Taken together, the above results strongly suggest that SHOC2 complex-mediated S259 RAF dephosphorylation is required for 14-3-3 dissociation from RAFs, MEK dissociation from BRAF, and BRAF heterodimerization with ARAF, CRAF, and KSR, but not for RAF binding to RAS (SI Appendix, Fig. S2)."
sparser
"Nevertheless, peptides generated from the dimerization interface of BRAF have been shown to prevent formation of homodimers and heterodimers of BRAF–CRAF in a BRAF-mutant or RAS-mutant cellular context xref , suggesting the possibility of an alternative strategy for developing inhibitors of RAF dimerization."
reach
"The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAF WT tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879."
sparser
"The difference between Raf inhibitor combination effects might be due to the preferred Raf binding conformation for each compound: AZD628 binds to the inactive conformation (DFG-out) of Craf, whereas both GDC0879 and PLX4720 bind to the active CRaf conformation (DFG-in) xref , with PLX4720 causing a c-helix shift that may block BRaf:CRaf heterodimer formation, as observed in our current study."
reach
"Other phosphorylation sites on CRAF and BRAF were diminished to some degree following treatment with KG5 which is not surprising considering the fact that KG5 is an allosteric inhibitor of RAF and blocks the dimerization of BRAF and CRAF XREF_BIBR thereby preventing co-activation of these molecules."
sparser
"Specifically, it would be helpful to address whether the activation of BRAF by Type II inhibitors, while strongly correlated with BBD model predictions in vitro, also depends on CRAF via BRAF-CRAF in cells and therefore overlaps with the mechanisms of paradoxical activation by Type I and aC-out inhibitors."
sparser
"Previously, we showed that Sorafenib (BAY 43–9006, Nexavar), a RAF inhibitor, blocked cAMP-dependent ERK activation, cell proliferation and in vitro cyst growth of human ADPKD cells. xref In a subsequent study, treatment with the RAF inhibitor PLX5568 caused a dose-dependent inhibition of cell proliferation and in vitro cyst growth, and attenuated PKD in the Han:SPRD Cy/+ rat; although, it also resulted in elevated renal and hepatic fibrosis. xref These studies suggest that BRAF may be a therapeutic target to slow PKD progression; however, side-effects and resistance to BRAF inhibitors remain significant clinical challenges. xref Evidence has shown that RAF inhibitors have a paradoxical effect on ERK activation due to drug-induced BRAF-RAF-1 dimerization, and this activation is not compromised if the kinase activity of one of the RAF partners is destroyed. xref , xref Another factor to consider in targeting BRAF as an anti-PKD therapy is the effect of epidermal growth factor receptor (EGFR) expression by cystic epithelial cells."
sparser
"Agent
Target
Company
Atezolizumab
PD-L1
Roche/Genentech
Mersetinib Repotrectinib Tislelizumab AMG-510 DS-6051b LOXO-195 LOXO-292 LY-3300054 PLX-8394 PLX-9486
MET, TRK, MERTK, ROS1 and others ROS1, TRK, ALK PD-1 KRAS TRK, ROS1 TRK RET PD-L1 BRAF, BRAF–CRAF KIT
Eli Lilly TP Therapeutics BeiGene/Celgene Amgen Daiichi Sankyo Loxo Oncology Loxo Oncology Eli Lilly Plexxikon Plexxikon
Development Status
Approved for several tumor type specific indications."
sparser
"Of note, BRAF inhibitors can only bind one component of each dimer, a mechanism which allows the unbound BRAF monomers to interact with CRAF monomers to form BRAF-CRAF heterodimers that ultimately trigger the re-activation of ERK signaling to reduce long-term BRAF inhibitor efficacy [ xref , xref ]."
reach
"These effects are consistent with the ability of Raf-inhibitors to transactivate Raf-1 when a PKA activated Ras promotes Raf-1 and B-Raf heterodimerization, and are inhibited by interfering with cAMP and PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib."
reach
"Meanwhile, our IP analysis showed that dasatinib induced the heterodimer of BRaf and CRaf in Ishikawa cells, but not in SKUT-2 cells (XREF_FIG), indicating that co-localization between BRaf and CAV-1 induced by dasatinib in SKUT-2 cells interferes with the formation of the BRaf and CRaf heterodimer and therefore can inactivate signaling downstream of BRaf and CRaf (MAPK pathway)."