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BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS

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Science Advances  14 Aug 2019:
Vol. 5, no. 8, eaav8463
DOI: 10.1126/sciadv.aav8463
  • Fig. 1 Dynamics of the BRAF KinCon reporters.

    (A) Schematic depiction of the intramolecular Rluc-PCA–based BRAF kinase conformation reporter (KinCon reporter). Upstream RAS activation (EGF), RAS mutations, RAF mutations, or mutation-specific cancer drugs modulate opened, intermediate, or closed full-length RAF kinase conformations, resulting in an increase or decrease of Rluc-PCA–emitted cellular bioluminescence. (B) BRAF conformations (alterations of Rluc-PCA bioluminescence) were measured using transiently transfected HEK293 cells. Immunoblotting shows BRAF, F[1]-BRAF-F[2], and F[1]-BRAFV600E-F[2] expression levels and P-ERK1/2 levels (representative experiment; ±SD). RLU, relative light units. (C) Time-dependent effects of EGF (200 ng/ml) on BRAF conformations in the presence of hemagglutinin (HA)–tagged HRAS variants (±SEM; n = 4 independent experiments). (D) Impact of indicated BRAFi and MEKi on shown BRAF KinCon reporters (±SEM; n = 9 independent experiments; 3-hour treatments, 1 μM, HEK293 cells). (E) Dose-dependent recordings of BRAF conformations upon indicated BRAFi exposure for 3 hours. RLU signals have been normalized on the twofold elevated BRAFV600E KinCon reporter expressions. n = 8, 6, and 6 independent experiments are shown for vemurafenib, dabrafenib, and encorafenib, respectively (±SEM; amalgamated data from 24- and 48-hour reporter expressions). Student’s t test was used to evaluate statistical significance. Confidence levels: *P < 0.05, **P < 0.01, and ***P < 0.001. wt, wild-type; n.s., not significant.

  • Fig. 2 Correlations of BRAF conformations and activities.

    (A) Dose-dependent recordings of BRAF conformations upon PLX8394 treatment (3 hours; n = 4 independent experiments are shown; ±SEM). (B) Dose-dependent determination of P-ERK1/2 levels immediately after PLX8394 treatment (1-hour treatments; quantification from n = 4 independent experiments; ±SEM). (C) Dose-dependent correlations of BRAF-V600E KinCon reporter–dependent P-ERK/P-MEK activities and BRAF-V600E conformations upon PLX8394 exposure. (D) Time-dependent effect of PLX8394 on BRAF KinCon conformations (HEK293 cells; ±SEM from n = 4 independent experiments). (E) Schematic depiction of the modular structure of BRAF; patient mutations in the A-loop and P-loop are indicated (RBD, RAS-binding domain; CRD, cysteine-rich domain). The expression normalized values for BRAF KinCon reporter conformations in % of RLU and the impact of PLX8394 on indicated wild-type and mutant BRAF conformations (±SEM from n = 4 independent samples; representative of at least n = 3 independent experiments) are shown. (F) The cis-regulatory prediction (Cis-regPred) profile of BRAF is indicated. Pink lines at the top show the coordinates of both cis-regulatory elements (CREs). Student’s t test was used to evaluate statistical significance. Confidence levels: *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 Quantification of GTP-controlled and RAFi-affected RAS:RAF complexes.

    (A) IPs of transiently overexpressed and BRAFV600E using flag-tagged antibodies from SW480 cells following 1-hour RAFi exposure (quantification is shown from n = 6 independent experiments; ±SEM). (B) Illustration of the Rluc-PCA biosensor strategy to quantify PPIs of RAS:BRAF in vivo; fragments 1 and 2 of Rluc-PCA (-F[1] and -F[2]). Elevated PPIs induce complementation of Rluc-PCA fragments (PPI reporter). (C) Structure illustration indicates the localization of R188 and R166 in the RAS:RBD(BRAF) binding interface [Protein Data Bank (PDB): 4G0N and 2L05]. Following coexpression of Rluc-PCA fragment tagged pairs of full-length RAS isoforms/mutants and full-length BRAF, we determined GTP-dependent PPIs (±SEM; n = 4 independent experiments). Following coexpression of Rluc-PCA fragment tagged pairs of RAS isoforms/mutants and full-length BRAF, we determined GTP-dependent PPIs (±SD; representative experiment of n = 3 independent experiments). (D) Following coexpression of indicated luciferase and PPI reporter constructs, we treated cells for 3 or 16 hours with 10 μM vemurafenib. The PPI analyses of n = 4 independent experiments (HEK293; ±SEM) are shown. We have normalized the PPI values on the untreated PPI signals of each tested protein pair. Student’s t test was used to evaluate statistical significance. Confidence levels: *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 RAFi elevate mutated RAS:BRAF complexes.

    (A) Dose-dependent treatments of HEK293 cells with increasing concentrations of dabrafenib, encorafenib, and vemurafenib (3 hours) after transient coexpression of indicated PPI reporters. The obtained bioluminescence signals have been normalized on the interaction of RBD:HRASG12V and on the signals obtained with 0.1 nM drug treatment. The PPI analyses of n = 7 independent experiments (HEK293; ±SEM) are shown. (B) Following coexpression of indicated PPI reporters, we treated HEK293 cells with increasing concentrations of PLX8394 (3 hours). The obtained bioluminescence signals were normalized on the interaction of RBD:HRASG12V and on the signals obtained with 0.1 nM drug treatment. The PPI analyses of n = 4 independent experiments (HEK293; ±SEM) are shown. Co-IP of flag-tagged BRAFV600E following exposure to 1 μM PLX8394 (HRASG12V coexpression). Quantification from n = 3 independent experiments is shown (±SEM). (C) Following coexpression of indicated PPI reporters (HEK293), we analyzed the impact of indicated RAFi on cellular PPI using the Rluc-PCA as readout. The PPI analyses of at least n = 7 independent experiments (HEK293; ±SEM) are shown. Student’s t test was used to evaluate statistical significance. Confidence levels: *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 Impact of mutations and BRAFi on RAS:RAF complex formation.

    (A) Schematic depiction of analyzed PPIs. (B) Used reporter constructs are indicated. Impact of vemurafenib or PLX8394 exposure (3 hours, 1 μM) on indicated PPIs measured using the Rluc-PCA as readout (n = 4 independent experiments; ±SD). (C) Impact of vemurafenib exposure (3 hours, 1 μM) on indicated CRAF:RAS and BRAF:HRAS interactions in the presence or absence of HA-tagged BRAF or BRAFV600E (±SEM; n = 3 independent experiments). (D) Impact of vemurafenib and PLX8394 exposure (3 hours, 1 μM) on indicated RAF:RAS and RBD:HRAS interactions in the presence or absence of BRAFV600E and BRAFR509H/V600E. The PPI analyses of n = 4 independent experiments (HEK293, ±SEM) are shown. We have normalized the PPI RLUs on the untreated PPI signals. (E) Following coexpression of indicated Rluc-PCA fragment-tagged protein pairs and HRAS variants, we determined PPIs (±SEM from n = 4 independent experiments). The RLU values have been normalized on the expression of F[2]-tagged hybrid proteins. Student’s t test was used to evaluate statistical significance. Confidence levels: *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Perturbation of tetrameric RAS:RAF complexes in intact cells.

    (A) Illustration of strategies on how to interfere with tetrameric RAS:RAF complexes. (B) Structure illustration indicates the localization of W75 and W77 in the RAS:NS1 binding interface (PDB: 5E95). (C) Localization study of YFP-tagged NS1 and NS1neg in the absence or presence of HA-HRAS in HEK293 cells. Scale bars, 25 μm. RAF dimerization analyses using indicated PPI reporters, NS1 variants, and upon coexpression of HRAS variants (n = 3; ±SEM). (D) RAF dimerization analyses using indicated PPI reporter constructs and coexpression of HRAS variants (n = 3; ±SEM). (E) PPI reporter analyses of transiently expressed reporter protein pairs with and without indicated HRAS variants and following 1-hour treatment with PLX8394. Quantifications from n = 5 independent experiments are shown (±SEM). (F) IP of flag-tagged HRASG12V in the presence of wild-type and RAS-binding deficient (R88A) CRAF-NanoLuc (NL) variants from BRAF-V600E–positive melanoma cells A375. The PKA RI subunit serves as unrelated prey. Quantifications from n = 4 independent experiments are shown (±SEM). Student’s t test was used to evaluate statistical significance. Confidence levels: *P < 0.05, **P < 0.01, and ***P < 0.001. (G) Systematic analyses using the full-length BRAF reporter platform unveiled the sequence of reorganizations of macromolecular kinase complexes upon αC-OUT BRAFi binding to the catalytic kinase pocket of mutated BRAF (left protomer, e.g., V600E mutation = *). Dose-dependent αC-OUT BRAFi binding to mutant BRAF promotes intermediate RAF conformations (1), thereby elevating BRAF*:RASGTP interactions (2) and endorsing the formation of RAS nanocluster/dimer–organized RASGTP:BRAF*:RAF:RASGTP tetramers (3), which may lead to the activation of a complexed second RAF protomer (4). RAS-specific monobodies and RBD:RAS interface mutations counteract BRAFi-promoted RAS:RAF complexes.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaav8463/DC1

    Table S1. Used expression constructs, materials, and cell lines.

    Fig. S1. BRAF KinCon reporter activities and BRAF profiling.

    Fig. S2. Impact of defined kinase mutations on BRAF KinCon reporter dynamics.

    Fig. S3. BRAF KinCon reporter dynamics.

    Fig. S4. Complex formation of RAS:BRAF and PPI reporter dynamics.

    Fig. S5. Impact of vemurafenib on indicated PPIs.

    Fig. S6. Impact of BRAFi on binary RAS:RAF interactions.

    Fig. S7. PPI reporter dynamics and NS1 monobody IPs.

    Fig. S8. Validation of PPI dynamics and impact of RAFi on RAF conformations.

  • Supplementary Materials

    This PDF file includes:

    • Table S1. Used expression constructs, materials, and cell lines.
    • Fig. S1. BRAF KinCon reporter activities and BRAF profiling.
    • Fig. S2. Impact of defined kinase mutations on BRAF KinCon reporter dynamics.
    • Fig. S3. BRAF KinCon reporter dynamics.
    • Fig. S4. Complex formation of RAS:BRAF and PPI reporter dynamics.
    • Fig. S5. Impact of vemurafenib on indicated PPIs.
    • Fig. S6. Impact of BRAFi on binary RAS:RAF interactions.
    • Fig. S7. PPI reporter dynamics and NS1 monobody IPs.
    • Fig. S8. Validation of PPI dynamics and impact of RAFi on RAF conformations.

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