Search

Additional file 2: Figure S2. Side-by-side comparison of growth factor versus oncogenic KRASG12V induced BRAF-LgBiT/RAF1-SmBIT heterodimerization. HEK293T cells were transfected with either empty pMIGor pMIG/KRASG12V and the empty control vectors pLgBiT-N/pSmBit-N or the BRAF-LgBiT and RAF1-SmBiT expression vectors. Cells were either left untreated or stimulated with 100 ng/ml EGF for the indicated timepoints. Note the EGF induced increase in cells expressing BRAF-LgBiT/RAF1-SmBiT that is absent in cells transfected with the empty control vectors. Also note the consistent and highly significant increase in Nluc activity in unstimulated KRASG12V expressing cells compared to EGF stimulated cells transfected with pMIG e.V.

Additional file 3: Figure S3. Effects of more complex DIF mutations in BRAF on its heterodimerization with RAF1. HEK293T cells were transfected with either empty pLgBiT-N/pSmBiT-N control plasmidsor expression vectors encoding RAF1WT-SmBiT and the indicated BRAF-LgBiT proteins, either in combination with pMIG e.V.or pMIG KRASG12V. Cells were either treated with 10 µM Sorafenib or DMSO for 4 h prior to measurement. Shown is the mean of three to four biological replicates. HEK293T cells were transfected with either empty pLgBiT-N/pSmBiT-N control plasmids or expression vectors encoding RAF1WT-SmBiT and the indicated BRAF-LgBiT proteins, either in combination with pMIG e.V. or pMIG KRASG12V. Cells were either treated with 10 µM Sorafenib or DMSO) for 4 h prior to measurement. RAF1WT-SmBiT fusion proteins were immunoprecipitated using anti-Myc antibody. Following Western blotting, immunoprecipitatesand TCLs were probed with anti-HA and anti-Myc antibodies. Shown is a representative experiment from two biological replicates.

Additional file 1: Figure S1. Co-immunoprecipitation using anti-c-Myc antibodies confirms the enhanced heterodimerization between BRAF-LgBiT and RAF1-SmBiT as well as between RAF1-LgBiT and BRAF-SmBiT proteins in the presence of either KRASG12V or Sorafenib. HEK293T cells were co-transfected with the indicated plasmids and either pMIG empty vectoror pMIG/KRASG12V. Four hours prior to lysis, cells were treated with either 10 μM Sorafenib or the equivalent volume of DMSO. Myc-tagged RAF1-SmBiT or RAF1-LgBiT proteins were immuno-purified using anti-Myc antibody. Top: Immunoprecipitates were loaded on two gels. Purified RAF1 proteins were either detected using anti-Myc or RAF1 antibodies, while co-purified HA-tagged BRAF proteins were detected with anti-HA or anti-BRAF F7 antibodies. Note the expected increased BRAF/RAF1 heterodimerization by KRASG12V or sorafenib. Bottom: Analysis of total cellular lysates demonstrates expression of all Nluc components and confirms activation of the RAS/RAF/MEK/ERK-pathway by KRASG12V.

Additional file 4.

Additional file 12: Figs. S1���12. Figure S1: Differentially expressed genes in HDF after vemurafenib treatment. Figure S2: Down-regulated processes in HDF by vemurafenib treatment. Figure S3: Effect of vemurafenib on HDF motility. Figure S4: Enrichment of tumoral stroma signatures in HDF after vemurafenib treatment. Figure S5: Effect of vemurafenib on MaMel viability. Figure S6: Comparison of transcriptional profiles in BRAFWT and BRAFV600E mutant cell lines after vemurafenib treatment. Figure S7: Effect of vemurafenib on BRAFWT melanoma cell lines. Figure S8: Accessibility within promoter regions and transcription start sites (TSS) in HDF. Figure S9: Pearson correlation of chromatin accessibility and gene expression changes in HDF after vemurafenib treatment. Figure S10: Receiver operating characteristic (ROC) and precision-recall (PR) curves. Figure S11: Most significant gene-sets (KEGG pathways) showing a relation between promoter accessibility and gene expression changes after vemurafenib treatment in HDF. Figure S12: Effect of trametinib on MAPK/ERK pathway activation.

Correlation of biological replicates. GAB2 protein complexes were enriched by IP. Contaminating proteins were removed and ratios normalized to GAB2. Proteins changing significantly interactions with GAB2 by indicated inhibitor treatments are highlighted red. Signigicant affeceted proteins are determined using Significance A (MaxQuant, p

(A/B) Western Blot quantification of Fig. 1d and 1G, n = 3, using FusionCapt 7.06 (Vilber Lourmat, Germany). (PDF 184 kb)

(A/B) Ba/F3 vector cells and cells transformed with pBABE Bcr-Abl were exposed to the indicated inhibitors or DMSO for 48 h. Cells were stained with 7-AAD and assessed for viability (A) or metabolic activity (MTT assay) (B). (C) KBM5 and KBM5-T315I cells were exposed to the indicated inhibitors or DMSO for 48 h. Cells were assessed for metabolic activity (MTT assay) (D/E) K562 cells overexpressing Lyn or hyperactive Lyn Y508F were exposed to the indicated inhibitors or DMSO for 48 h. Cells were stained with 7-AAD and assessed for viability (D) or metabolic activity (MTT assay) (E). Relevant statistically significant effects are indicated by asterisks, all statistical data can be found above. (PDF 176 kb)

Correlation of biological replicates. GAB2 protein complexes were enriched by IP. Contaminating proteins were removed and ratios normalized to GAB2. Proteins changing significantly interactions with GAB2 by indicated inhibitor treatments are highlighted red. Signigicant affeceted proteins are determined using Significance A (MaxQuant, p

Supplementary Methods and Supplementary Statistics. (PDF 297 kb)

(A/B) Western Blot quantification of Fig. 1d and 1G, n = 3, using FusionCapt 7.06 (Vilber Lourmat, Germany). (PDF 184 kb)

showing supplementary materials and methods. (DOCX 31Â kb)

showing supplementary materials and methods. (DOCX 31Â kb)

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F–myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient–derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1–mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Contains fulltext : 190745.pdf (Publisher’s version ) (Closed access) Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD(+) leukemia cells. This synergized with the allogeneic CD8(+) T cell response, leading to long-term survival in six mouse models of FLT3-ITD(+) AML. Sorafenib-related IL-15 production caused an increase in CD8(+)CD107a(+)IFN-gamma(+) T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD(+) AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8(+) T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.