c 1799t a p v600e mutations  (Pyrosequencing Inc)

Journal: The Journal of Experimental Medicine

Article Title: KRAS or BRAF mutations cause hepatic vascular cavernomas treatable with MAP2K–MAPK1 inhibition

doi: 10.1084/jem.20192205

Figure Lengend Snippet: Endothelial Kras G12D or Braf V600E gain-of-function mutations cause hepatic vascular cavernomas in mice. (A and B) Dissected livers and H&E-stained liver frontal sections show vascular cavernomas (black arrows) in tamoxifen-treated Kras G12D F/+ ; Cdh5 CreERT2 (A) and Braf V600E F/+ ; Cdh5 CreERT2 (B) mice ( n = 3). Pdpn (green) and Flt4 (red) coimmunofluorescent staining with Hoechst nuclear counterstain (blue) on Kras G12D F/+ ; Cdh5 CreERT2 (A) and Braf V600E F/+ ; Cdh5 CreERT2 (B) liver sections shows contribution of Flt4 + and Pdpn-negative endothelial cells to hepatic vascular cavernomas (white arrows). n = 3. Scale bars: 500 µm (top row), 100 µm (second and third row from top), and 10 µm (bottom row). Two independent experiments. (C and D) Dissected Kras G12D F/+ ; Lyve1 Cre (C) and Braf V600E F/+ ; Lyve1 Cre (D) embryonic day 14.5 (E14.5) embryos show reduced liver size (red arrow; n = 3). H&E-stained Kras G12D F/+ ; Lyve1 Cre (C) and Braf V600E F/+ ; Lyve1 Cre (D) E14.5 liver frontal sections show vascular cavernomas (black arrows; n = 3). Coimmunofluorescent staining with Hoechst nuclear counterstain (blue) shows contribution of Lyve1 + (green) and Pecam1 + (red) endothelial cells (white arrows) to hepatic vascular cavernomas. n = 3. Scale bars: 500 µm (top row), 100 µm (second and third row from top), and 10 µm (bottom row). Two independent experiments. (E) Dissected Braf V600E F/+ ; Lyve1 Cre livers show reduction in liver size at E13.5 and progressive development of hepatic vascular cavernomas (black arrows). n = 3. Scale bars: 500 µm. (F) Lyve1 (green), Hif1α (red), and Cleaved caspase 3 (CC3, white) coimmunofluorescent staining with Hoechst nuclear counterstain (blue) on Braf V600E F/+ ; Lyve1 Cre (F) and Kras G12D F/+ ; Lyve1 Cre (G) E13.5 liver frontal sections shows hypoxia and apoptosis in clusters of nonendothelial hepatic cells near vascular cavernomas (white arrows). n = 3. Scale bars: 100 µm (top row) and 10 µm (bottom row). Two independent experiments. Littermates were used as controls for all experiments. All experimental data verified in at least two independent experiments. BD, bild duct; LL, left lobe; LML, left medial lobe; LS, liver sinusoid; LV, lymphatic vessel; PV, portal vein.

Article Snippet: The RKO human colon cell line, with c. 1799T>A (p.V600E) BRAF mutation, was used as positive control (ATCC).

Techniques: Staining

Journal: The Journal of Experimental Medicine

Article Title: KRAS or BRAF mutations cause hepatic vascular cavernomas treatable with MAP2K–MAPK1 inhibition

doi: 10.1084/jem.20192205

Figure Lengend Snippet: Endothelial Kras G12D or Braf V600E gain-of-function mutations cause hepatic vascular cavernomas and embryonic lethality in mice. (A and B) Lyve1 (green) and Pecam1 (red) coimmunofluorescent staining with Hoechst nuclear counterstain (blue) on tamoxifen-treated adult Kras G12D F/+ ; Cdh5 CreERT2 (A) and Braf V600E F/+ ; Cdh5 CreERT2 (B) liver sections shows contribution of Lyve1 + and Pecam1 + endothelial cells to hepatic vascular cavernomas (white arrows). n = 3. Scale bars: 100 μm (top row) and 10 µm (bottom row). Two independent experiments. (C and D) Dissected livers and H&E-stained liver frontal sections show vascular cavernomas (black arrows) in tamoxifen-treated Kras G12D F/+ ; Cdh5 CreERT2 (C) and Braf V600E F/+ ; Cdh5 CreERT2 (D) neonatal mice. Lyve1 (green) and Pecam1 (red) coimmunofluorescent staining with Hoechst nuclear counterstain (blue) on Kras G12D F/+ ; Cdh5 CreERT2 (C) and Braf V600E F/+ ; Cdh5 CreERT2 (D) liver sections shows contribution of Lyve1 + and Pecam1 + endothelial cells to hepatic vascular cavernomas (white arrows). n = 3. P, postnatal day. Scale bars: 500 μm (top row), 100 µm (second and third rows from top), and 10 µm (bottom row). Two independent experiments. (E and F) Genotyping tables of embryos or pups derived from Kras G12D F/+ mice crossed with either Lyve1 Cre/+ (E) or Lyve1 Cre/Cre (F) mice. (G and H) Genotyping tables of embryos derived from Braf V600E F/F (G) or Braf V600E F/+ (H) mice crossed with either Lyve1 Cre/+ (G) or Lyve1 Cre/Cre (H) mice. χ 2 P value 0.94 (E, E13.5), 0.09 (E, P0), 0.0001 (F), 0.857 (G, E11.5), 0.285 (G, E14.5), 1.0 (H). LS, liver sinusoid; PV, portal vein.

Article Snippet: The RKO human colon cell line, with c. 1799T>A (p.V600E) BRAF mutation, was used as positive control (ATCC).

Techniques: Staining, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: KRAS or BRAF mutations cause hepatic vascular cavernomas treatable with MAP2K–MAPK1 inhibition

doi: 10.1084/jem.20192205

Figure Lengend Snippet: Mice with endothelial Kras G12D or Braf V600E gain-of-function mutations exhibit normal proliferation of hepatic endothelial cells. (A and B) Ki67 (red) and Cdh5 (green) coimmunofluorescent staining with Hoechst nuclear counterstain (blue) on tamoxifen-treated Kras G12D F/+ ; Cdh5 CreERT2 (A) and Braf V600E F/+ ; Cdh5 CreERT2 (B) adult murine liver sections. White arrows show Ki67 + cells. n = 3. Scale bars: 100 µm (top row) and 10 µm (bottom row). Two independent experiments. FDR, false discovery rate. (C and D) Quantitation of proliferating Cdh5 + cells in tamoxifen-treated control, Kras G12D F/+ ; Cdh5 CreERT2 (C), and Braf V600E F/+ ; Cdh5 CreERT2 (D) embryonic murine livers. n = 3. Unpaired t test, P > 0.8 (C) and P > 0.4 (D). Two independent experiments. Data represent the mean ± SEM. (E and F) Ki67 (red) and Cdh5 (green) coimmunofluorescent staining with Hoechst nuclear counterstain (blue) on Kras G12D F/+ ; Lyve1 Cre/+ (C) and Braf V600E F/+ ; Lyve1 Cre/+ (D) embryonic murine liver sections. White arrows show Ki67 + cells. n = 3. E, embryonic day. Scale bars: 100 µm (top row) and 10 µm (bottom row). Two independent experiments. (G and H) Quantitation of proliferating Cdh5 + cells in control, Kras G12D F/+ ; Lyve1 Cre/+ (G), and Braf V600E F/+ ; Lyve1 Cre/+ (H) mutant murine livers. n = 3. Unpaired t test, P > 0.8. Two independent experiments. Data represent the mean ± SEM. N.S., not significant. BD, bile duct; LS, liver sinusoid; PV, portal vein.

Article Snippet: The RKO human colon cell line, with c. 1799T>A (p.V600E) BRAF mutation, was used as positive control (ATCC).

Techniques: Staining, Quantitation Assay, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: KRAS or BRAF mutations cause hepatic vascular cavernomas treatable with MAP2K–MAPK1 inhibition

doi: 10.1084/jem.20192205

Figure Lengend Snippet: Mice with Kras G12D or Braf V600E gain-of-function mutations within macrophages exhibit normal normal sinusoidal and hepatic development. (A and B) Dissected Kras G12D F/+ ; LysM Cre (A) and Braf V600E F/+ ; LysM Cre (B) embryos show normal liver size (black arrows). H&E-stained Kras G12D F/+ ; LysM Cre (A) and Braf V600E F/+ ; LysM Cre (B) liver frontal sections show normal sinusoidal capillaries (black arrows). n = 3. E, embryonic day. Scale bars: 500 µm (top row), 1,000 µm (middle row), and 100 µm (bottom row). Two independent experiments. (C and D) Mice with Kras G12D or Braf V600E gain-of-function mutations within endothelial cells exhibit normal normal spleen morphology and sinusoids. Spleen dissected from adult tamoxifen-treated Kras G12D F/+ ; Cdh5 CreERT2 (C) and Braf V600E F/+ ; Cdh5 CreERT2 (D) mice. H&E-stained Kras G12D F/+ ; Cdh5 CreERT2 (C) and Braf V600E F/+ ; Cdh5 CreERT2 (D) spleen sagittal sections show normal morphology. Lyve1 (green) and Pecam1 (red) coimmunofluorescent staining with Hoechst nuclear counterstain (blue) on Kras G12D F/+ ; Cdh5 CreERT2 (C) and Braf V600E F/+ ; Cdh5 CreERT2 (D) spleen sagittal sections. n = 3. Scale bars: 500 µm (first and second rows), 100 µm (third row), and 50 µm (fourth row). Two independent experiments. White arrows show normal Lyve1 + vessels. A, artery; CrRL, cranial right lobe; LK, left kidney; LL, left lobe; LML, left medial lobe; LV, left ventricle; RK, right kidney; RML, right medial lobe; RV, right ventricle; S, stomach.

Article Snippet: The RKO human colon cell line, with c. 1799T>A (p.V600E) BRAF mutation, was used as positive control (ATCC).

Techniques: Staining

Journal: The Journal of Experimental Medicine

Article Title: KRAS or BRAF mutations cause hepatic vascular cavernomas treatable with MAP2K–MAPK1 inhibition

doi: 10.1084/jem.20192205

Figure Lengend Snippet: Endothelial Kras G12D or Braf V600E gain-of-function mutations aberrantly enhance zipper-like contiguous expression of adherens junctional proteins at sinusoidal endothelial cell–cell contacts. (A) Distribution of differentially up-regulated transcripts (red), down-regulated transcripts (blue), and unchanged transcripts (gray) in E11.5 Braf V600E F/+ ; Lyve1 Cre livers compared with littermate Control E11.5 livers ( n = 3). FDR, false discovery rate. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway gene set enrichment analysis (GSEA) of differentially regulated, identified transcripts in E11.5 Braf V600E F/+ ; Lyve1 Cre livers ( n = 3). (C) Heatmap of top differentially regulated transcripts within KEGG pathway categories of focal adhesion and cell adhesion ( n = 3). (D and E) Flt4 and Cdh5 immunofluorescent staining with Hoechst nuclear counterstain (blue) on sections of human KRAS c.35 G>A (p.G12D) and BRAF c.1798G>A (p.V600M) liver (D) and Cdh5 immunofluorescent staining with Hoechst nuclear counterstain (blue) on murine Kras G12D F/+ ; Lyve1 Cre liver (E) and murine Braf V600E F/+ ; Lyve1 Cre livers (E). n = 3. White arrows show normal button-like discontiguous expression of junctional proteins between sinusoidal endothelial cells. Red arrows show abnormal zipper-like contiguous expression of adherens junctional proteins at sinusoidal endothelial cell–cell contacts. Scale bars in D: 100 µm (first column from left) and 10 µm (second and third columns from left); scale bars in E: 10 µm (first column from left) and 5 µm (second column from left). All experimental data verified in at least two independent experiments.

Article Snippet: The RKO human colon cell line, with c. 1799T>A (p.V600E) BRAF mutation, was used as positive control (ATCC).

Techniques: Expressing, Staining

Journal: The Journal of Experimental Medicine

Article Title: KRAS or BRAF mutations cause hepatic vascular cavernomas treatable with MAP2K–MAPK1 inhibition

doi: 10.1084/jem.20192205

Figure Lengend Snippet: Endothelial Kras G12D or Braf V600E gain-of-function mutations aberrantly enhance expression of adherens junctional proteins. (A) Analysis of differentially regulated transcripts show expression changes in E11.5 Braf V600E F/+ ; Lyve1 Cre livers ( n = 3). (B) KEGG pathway gene set enrichment analysis (GSEA) of differentially regulated, identified transcripts in E11.5 Braf V600E F/+ ; Lyve1 Cre livers ( n = 3). NES, normalized enriched score. (C and D) Flt4 (green) and Pecam1 (red) coimmunofluorescent staining with Hoechst nuclear counterstain (blue) on sections of murine Braf V600E F/+ ; Lyve1 Cre embryonic liver (C) and murine Kras G12D F/+ ; Lyve1 Cre liver (D). Red arrows show increased Flt4 expression. E, embryonic day. Scale bars: 100 µm (top row) and 10 µm (bottom row). n = 3. Two independent experiments. (E and F) Genotyping tables of embryos derived from Mapk1 F/+ ; Kras G12D F/+ mice (E) or Mapk1 F/+ ; Braf V600E F/+ (F) mice crossed with Mapk1 F/+ ; Lyve1 Cre/+ mice. χ 2 P values 0.262 (E) and 0.991 (F).

Article Snippet: The RKO human colon cell line, with c. 1799T>A (p.V600E) BRAF mutation, was used as positive control (ATCC).

Techniques: Expressing, Staining, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: KRAS or BRAF mutations cause hepatic vascular cavernomas treatable with MAP2K–MAPK1 inhibition

doi: 10.1084/jem.20192205

Figure Lengend Snippet: Pharmacologic inhibition of Braf V600E -Map2k or genetic ablation of Mapk1 rescue hepatic vascular cavernomas in Braf V600E F/+ ; Lyve1 Cre and Kras G12D F/+ ; Lyve1 Cre mice . (A and B) Dissected Braf V600E F/+ ; Lyve1 Cre littermate embryos treated with dabrafenib and trametinib (A) or lacking Mapk1 (B) show rescued liver size (green arrows) compared with Braf V600E F/+ ; Lyve1 Cre embryos (yellow arrow; n = 3). H&E-stained or Lyve1 (green), Pecam1 (red), and Hoechst (blue) coimmunofluorescent–stained liver sections of Braf V600E F/+ ; Lyve1 Cre littermate embryos treated with dabrafenib and trametinib (A) or lacking Mapk1 (B) show normal sinusoidal capillaries (green arrows) compared with cavernous sinusoids in Braf V600E F/+ ; Lyve1 Cre embryos (yellow arrows; n = 3). Immunofluorescent staining on liver sections of Braf V600E F/+ ; Lyve1 Cre embryos treated with dabrafenib and trametinib (A) or lacking Mapk1 (B) show normal button-like discontiguous expression of Cdh5 (brown arrows) compared with abnormal zipper-like contiguous expression of Cdh5 in Braf V600E F/+ ; Lyve1 Cre embryos (red arrows; n = 3). White arrows show normal button-like discontiguous expression of Cdh5 in control livers. Scale bars: 500 µm (top row), 1,000 µm (second row from top), 100 µm (third, fourth, and sixth rows from top), and 10 µm (fifth and seventh rows from top). Two independent experiments. (C) Dissected Kras G12D F/+ ; Lyve1 Cre littermate embryos lacking Mapk1 show rescued liver size (green arrow), normal sinusoidal capillaries (green arrows) stained with H&E or Lyve1 (green) and Pecam1 (red), and normal button-like discontiguous expression of Cdh5 (brown arrows) compared with Kras G12D F/+ ; Lyve1 Cre (yellow and red arrows). n = 3. Scale bars: 500 µm (first column from left), 1,000 µm (second column from left), 100 µm (third, fourth, and sixth columns from left), and 10 µm (fifth and seventh columns from left). Two independent experiments. (D) Endothelial activating KRAS or BRAF mutations drive hepatic vascular cavernomas via MAP2K–MAPK1 signaling pathway. Proposed model suggesting that constitutive activation of KRAS–BRAF–MAP2K–MAPK1 signaling pathway in sinusoidal endothelial cells promotes aberrant zipper-like contiguous expression of adherens junctional proteins, such as Cdh5, switching hepatic sinusoidal capillaries from branching to cavernous expansion. All experimental data verified in at least two independent experiments. CdRL, caudal right lobe; CL, caudate lobe; CrRL, cranial right lobe; LK, left kidney; LL, left lobe; LML, left medial lobe; LV, left ventricle; RK, right kidney; RML, right medial lobe; RV, right ventricle; S, stomach.

Article Snippet: The RKO human colon cell line, with c. 1799T>A (p.V600E) BRAF mutation, was used as positive control (ATCC).

Techniques: Inhibition, Staining, Expressing, Activation Assay

Journal: Blood Advances

Article Title: Vemurafenib acts as a molecular on-off switch governing systemic inflammation in Langerhans cell histiocytosis

doi: 10.1182/bloodadvances.2021005442

Figure Lengend Snippet: Clinical improvement is linked directly to the administration of vemurafenib. (A) Drug therapy of the first 420 days (60 weeks, start: day 0); vemurafenib was started on day 37 because of the worsening clinical condition during stratum I (vinblastine/prednisone), paused during initial salvage therapy of stratum III (2-CdA/Ara-C), and reduced in dose in 2 stages and discontinued during continuation therapy part 2 (6-MP/MTX/vinblastine/prednisone). Subsequently, continuation therapy part 3 (6-MP/MTX) was carried out until day 730 (supplemental Figure 1). (B) Vemurafenib resulted in immediate improvement in fever, CRP, and TP. Each cessation of vemurafenib during the chemotherapy cycles caused intermittent fever, rise in CRP, and falling levels of TP, which reversed on continuation. (C) MRT of cranial lesions, which decreased in size visibly already during therapy with vinblastine/prednisone (T1-weighted MRI). (D) CC-chemokine ligand 2 (CCL2) concentration was closely correlated with clinical signs of disease activity. Points represent the median, and error bars the top and bottom of triplicate measurements per time point. The LCH disease activity score (DAS), which stayed high during initial chemotherapy, immediately improved on start with vemurafenib. It is important to note that the DAS after 2-CdA/Ara-C is confounded by chemotherapy-induced cytopenia. (E) During vemurafenib therapy, the percentage of BRAF V600E mutant alleles increased. Following stratum III therapy, no more mutant alleles were measurable.

Article Snippet: Droplet-digital polymerase chain reaction (PCR) for BRAF V600E was performed as previously described using the Mutation Assay BRAF p.V600E c.1799T-A, Human (Bio-Rad Laboratories) and the QX200 Droplet Digital PCR System (Bio-Rad Laboratories).

Techniques: Concentration Assay, Activity Assay, Mutagenesis