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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.

Cathepsin D (CTSD) is a lysosomal protease and a marker of poor prognosis in breast cancer. However, the cells responsible for this association and the function of CTSD in cancer are still incompletely understood. By using a conditional CTSD knockout mouse crossed to the transgenic MMTV-PyMT breast cancer model we demonstrate that CTSD deficiency in the mammary epithelium, but not in myeloid cells, blocked tumor development in a cell-autonomous manner. We show that lack of CTSD impaired mechanistic Target of Rapamycin Complex 1 (mTORC1) signaling and induced reversible cellular quiescence. In line, CTSD-deficient tumors started to grow with a two-month delay and quiescent Ctsd-/- tumor cells re-started proliferation upon long-term culture. This was accompanied by rewiring of oncogenic gene expression and signaling pathways, while mTORC1 signaling remained permanently disabled in CTSD-deficient cells. Together, these studies reveal a tumor cell-autonomous effect of CTSD deficiency, and establish a pivotal role of this protease in the cellular response to oncogenic stimuli., The lysosomal aspartic protease Cathepsin D (CTSD) is associated with breast cancer progression. Here the authors show that selective inactivation of CTSD in mammary epithelium delays tumor onset due to impaired mTORC1 signaling, but resumes malignant growth due to compensatory oncogenic pathways

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.

This corrects the article DOI: 10.1038/nm.4484.

As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC‐PCP‐SILAC, we analyzed protein–protein interactions of hyperactive BRAFV600E and wild‐type BRAF (BRAFWT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAFV600E resides in large complexes of higher molecular mass and activity, while BRAFWT is confined to smaller, slightly less active complexes. However, expression of oncogenic K‐RasG12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAFWT into large, active complexes, whereas pharmacological inhibition of BRAFV600E has the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors.

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)

Background The Gab2 docking protein acts as an important signal amplifier downstream of various growth factor receptors and Bcr-Abl, the driver of chronic myeloid leukaemia (CML). Despite the success of Bcr-Abl tyrosine kinase inhibitors (TKI) in the therapy of CML, TKI-resistance remains an unsolved problem in the clinic. We have recently shown that Gab2 signalling counteracts the efficacy of four distinct Bcr-Abl inhibitors. In the course of that project, we noticed that two clinically relevant drugs, imatinib and dasatinib, provoke distinct alterations in the electrophoretic mobility of Gab2, its signalling output and protein interactions. As the signalling potential of the docking protein is highly modulated by its phosphorylation status, we set out to obtain more insights into the impact of TKIs on Gab2 phosphorylation. Findings Using stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS), we show now that imatinib and dasatinib provoke distinct effects on the phosphorylation status and interactome of Gab2. This study identifies several new phosphorylation sites on Gab2 and confirms many sites previously known from other experimental systems. At equimolar concentrations, dasatinib is more effective in preventing Gab2 tyrosine and serine/threonine phosphorylation than imatinib. It also affects the phosphorylation status of more residues than imatinib. In addition, we also identify novel components of the Gab2 signalling complex, such as casein kinases, stathmins and PIP1 as well as known interaction partners whose association with Gab2 is disrupted by imatinib and/or dasatinib. Conclusions By using MS-based proteomics, we have identified new and confirmed known phosphorylation sites and interaction partners of Gab2, which may play an important role in the regulation of this docking protein. Given the growing importance of Gab2 in several tumour entities we expect that our results will help to understand the complex regulation of Gab2 and how this docking protein can contribute to malignancy.

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 CD8CD107aIFN-γ 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.

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

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

Die EGFR-Signalübertragung steuert viele verschiedene zelluläre Prozesse in allen Arten von Epithelzellen, einschließlich des Darmepithels. Diese Prozesse reichen von Proliferation und Wachstum über Differenzierung bis hin zu Autophagie und Apoptose. Die vorliegende Studie zielt darauf ab, die Signalveränderungen zu charakterisieren, die im Darmepithel als Reaktion auf EGF-induzierten Hungerstress stattfinden. Kontraintuitiv führte eine 24-stündige EGF-Starre zu einer deutlichen Phosphorylierung von EGFR, MEK1/2 und ERK1/2, was auf eine Aktivierung dieser Signalachse in Darmzellen hindeutet. Diese Veränderungen waren am signifikantesten in den undifferenzierten CD44-reichen Krypta-Basiszellen. Interessanterweise war die EGF-Starvation-induzierte ERK1/2-Phosphorylierung mit der Hochregulierung einer Untergruppe von ERK-Zielgenen verbunden, bei denen es sich zumeist um primäre Zielgene handelt. Die Überexpression des EGFR-Liganden HBEGF und des FGFR-Liganden FGF1 in ausgehungerten Zellen könnte für die hungerbedingte Zunahme der MAPK-Aktivität verantwortlich sein, obwohl eine erhöhte Sekretion dieser Liganden durch ausgehungerte Organoide nicht bestätigt werden konnte. Dennoch wird die kompensatorische Ligandensekretion durch die Beobachtung gestützt, dass die erneute Zugabe von EGF zu ausgehungerten Organoiden die pERK1/2-Spiegel auf den Ausgangswert zurücksetzt, was bedeutet, dass EGF mit einem anderen von ausgehungerten Zellen sezernierten Liganden um den EGFR konkurriert. Zusätzlich zu HBEGF wurde festgestellt, dass andere Gene, die für den Schutz, das Überleben und die Regeneration des Darmepithels bekannt sind, in ausgehungerten Organoiden überexprimiert werden, wie z. B. Reg3b. Insgesamt können die in dieser Studie berichteten EGF-induzierten Veränderungen der MAPK-Signalübertragung und der globalen Genexpression als ein überlebensförderndes Programm interpretiert werden, das bevorzugt in Darmstammzellen und frühen Vorläuferzellen aktiviert wird. EGFR signaling drives many different cellular processes in all kinds of epithelial cells including the intestinal epithelium. Such processes range from proliferation and growth to differentiation to autophagy and apoptosis. The present study aims to characterize signaling changes that take place in the intestinal epithelium in response to EGF starvation-induced stress using epithelial organoids derived from the mouse duodenum and human colorectal tumor tissue. Counterintuitively, 24 h EGF starvation induced a prominent phosphorylation of EGFR, MEK1/2 and ERK1/2 indicating an activation of this signaling axis in intestinal cells. These changes were most significant in the undifferentiated CD44-high crypt base cells. Interestingly, EGF starvation-induced ERK1/2 phosphorylation was associated with upregulation of a subset of ERK target genes that were mostly primary-response targets. Overexpression of the EGFR ligand HBEGF and the FGFR ligand FGF1 in starved cells may account for starvation-driven increase in MAPK activity, although an increased secretion of these ligands by starved organoids was not confirmed. Nevertheless, compensatory ligand secretion is still supported by the observation that EGF re-addition to starved organoids restores pERK1/2 levels to baseline which implies that EGF competes for EGFR with some other ligand secreted by starved cells. In addition to HBEGF, other genes known to promote protection, survival and regeneration of the intestinal epithelium were found to be overexpressed in starved organoids such as Reg3b. Collectively, EGF starvation-induced changes in MAPK signaling and global gene expression reported in this study can be interpreted as a pro-survival program that gets activated preferentially in intestinal stem cells and early progenitors.

CLL is a frequent lymphoproliferative disorder of B cells, representing the most common type of leukemia in Western countries. The remarkable clinical success of several BCR signaling inhibitors improving CLL patient’s survival indicates an essential role of BCR signaling and BCR-associated kinases in the pathogenesis of CLL. However, acquired drug resistance is still a crucial issue that leads to relapse of the disease and unfavorable outcome. By generating mouse models allowing the inducible inactivation of BCR signaling componentsin CLL cells,we established a tool for the investigation of CLL signal transduction and the discovery of potential new targets in order to provide therapeutic alternatives for CLL patients. Our results identified the surface expression of the BCR signaling complex as a uniquely important regulator of CLL survival. Similarly, the small GTPase RHOA was found to be an important effector of BCR signaling that is critical to maintain CLL cell viability. Deletion of the SYK-encoding gene in murine Eµ-TCL1 CLL B cells also resulted in CLL cell remission, however, the persistence of a considerable fraction of SYK-deficient CLL cells questions the importance of SYK in CLL cell survival. Interestingly, we observed that SYK-deficiency in early developmental stages of B cells in combination with TCL1 overexpression caused malignant pre-B cell transformation possibly resulting in pre-B ALL. We also revealed that Pten-deletion accelerated the onset of CLL leukemogenesis and thereby confirmed that an increased PI3K- signaling pathway supports CLL progression. Additionally, we revealed that PTEN expression is downregulated in approximately two-third of human CLL patients, likely caused by increased expression levels of the miRNA family miR-29 and the protein PAX5. Moreover, deletion of Btk in CLL B cells resulted in an egress of CLL cells from lymphatic organs into the periphery of Eµ-TCL1 transgenic mice. This phenomenon was also observed in CLL patients treated with the BTK inhibitor ibrutinib. Thus, themb1-CreERT2;BTKfl/fl;Eµ-TCL1 mouse model possibly can be used to further study the role of lymphocytosis in the treatment of CLL. Altogether, our findings contributed to a better understanding of SYK and BTK inhibitor effectiveness in CLL therapy and describe the BCR, RHOA and the PTEN-targeting miRNA-29 family as potential new therapeutic targets for CLL treatment.

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.