TY - JOUR A1 - Lodde, Georg A1 - Forschner, Andrea A1 - Hassel, Jessica A1 - Wulfken, Lena M. A1 - Meier, Friedegund A1 - Mohr, Peter A1 - Kähler, Katharina A1 - Schilling, Bastian A1 - Loquai, Carmen A1 - Berking, Carola A1 - Hüning, Svea A1 - Schatton, Kerstin A1 - Gebhardt, Christoffer A1 - Eckardt, Julia A1 - Gutzmer, Ralf A1 - Reinhardt, Lydia A1 - Glutsch, Valerie A1 - Nikfarjam, Ulrike A1 - Erdmann, Michael A1 - Stang, Andreas A1 - Kowall, Bernd A1 - Roesch, Alexander A1 - Ugurel, Selma A1 - Zimmer, Lisa A1 - Schadendorf, Dirk A1 - Livingstone, Elisabeth T1 - Factors influencing the adjuvant therapy decision: results of a real-world multicenter data analysis of 904 melanoma patients JF - Cancers N2 - Adjuvant treatment of melanoma patients with immune-checkpoint inhibition (ICI) and targeted therapy (TT) significantly improved recurrence-free survival. This study investigates the real-world situation of 904 patients from 13 German skin cancer centers with an indication for adjuvant treatment since the approval of adjuvant ICI and TT. From adjusted log-binomial regression models, we estimated relative risks for associations between various influence factors and treatment decisions (adjuvant therapy yes/no, TT vs. ICI in BRAF mutant patients). Of these patients, 76.9% (95% CI 74–80) opted for a systemic adjuvant treatment. The probability of starting an adjuvant treatment was 26% lower in patients >65 years (RR 0.74, 95% CI 68–80). The most common reasons against adjuvant treatment given by patients were age (29.4%, 95% CI 24–38), and fear of adverse events (21.1%, 95% CI 16–28) and impaired quality of life (11.9%, 95% CI 7–16). Of all BRAF-mutated patients who opted for adjuvant treatment, 52.9% (95% CI 47–59) decided for ICI. Treatment decision for TT or ICI was barely associated with age, gender and tumor stage, but with comorbidities and affiliated center. Shortly after their approval, adjuvant treatments have been well accepted by physicians and patients. Age plays a decisive role in the decision for adjuvant treatment, while pre-existing autoimmune disease and regional differences influence the choice between TT or ICI. KW - melanoma KW - adjuvant treatment KW - checkpoint blocker KW - targeted therapy KW - BRAF KW - PD-1 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239583 SN - 2072-6694 VL - 13 IS - 10 ER - TY - JOUR A1 - Glutsch, Valerie A1 - Amaral, Teresa A1 - Garbe, Claus A1 - Thoms, Kai-Martin A1 - Mohr, Peter A1 - Hauschild, Axel A1 - Schilling, Bastian T1 - Indirect Comparison of Combined BRAF and MEK Inhibition in Melanoma Patients with Elevated Baseline Lactate Dehydrogenase JF - Acta Dermato-Venereologica N2 - The approval of BRAF and MEK inhibitors has signifi-cantly improved treatment outcomes for patients with BRAF-mutated metastatic melanoma. The 3 first-line targeted therapy trials have provided similar results, and thus the identification of predictive biomarkers may generate a more precise basis for clinical deci-sion-making. Elevated baseline lactate dehydrogenase (LDH) has already been determined as a strong prog-nostic factor. Therefore, this indirect analysis compa-red subgroups with elevated baseline LDH across the pivotal targeted therapy trials co-BRIM, COMBI-v and COLUMBUS part 1. The Bucher method was used to compare progression-free survival, objective response rate and overall survival indirectly. The results show a non-significant risk reduction for progression in the subgroup with elevated baseline LDH receiving vemu-rafenib plus cobimetinib compared with dabrafenib plus trametinib and encorafenib plus binimetinib. Al-though an indirect comparison, these data might pro-vide some guidance for treatment recommendations in melanoma patients with elevated LDH. KW - melanoma KW - BRAF KW - lactate dehydrogenase Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230190 VL - 100 ER - TY - JOUR A1 - Eisenhardt, Anja E. A1 - Sprenger, Adrian A1 - Röring, Michael A1 - Herr, Ricarda A1 - Weinberg, Florian A1 - Köhler, Martin A1 - Braun, Sandra A1 - Orth, Joachim A1 - Diedrich, Britta A1 - Lanner, Ulrike A1 - Tscherwinski, Natalja A1 - Schuster, Simon A1 - Dumaz, Nicolas A1 - Schmidt, Enrico A1 - Baumeister, Ralf A1 - Schlosser, Andreas A1 - Dengjel, Jörn A1 - Brummer, Tilman T1 - Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles JF - Oncotarget N2 - B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase. KW - BRAF KW - proteomics KW - phosphorylation KW - sorafenib KW - protein-protein interaction Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166529 VL - 7 IS - 18 ER - TY - JOUR A1 - Grimm, Johannes A1 - Hufnagel, Anita A1 - Wobser, Marion A1 - Borst, Andreas A1 - Haferkamp, Sebastian A1 - Houben, Roland A1 - Meierjohann, Svenja T1 - BRAF inhibition causes resilience of melanoma cell lines by inducing the secretion of FGF1 JF - Oncogenesis N2 - Approximately half of all melanoma patients harbour activating mutations in the serine/threonine kinase BRAF. This is the basis for one of the main treatment strategies for this tumor type, the targeted therapy with BRAF and MEK inhibitors. While the initial responsiveness to these drugs is high, resistance develops after several months, frequently at sites of the previously responding tumor. This indicates that tumor response is incomplete and that a certain tumor fraction survives even in drug-sensitive patients, e.g., in a therapy-induced senescence-like state. Here, we show in several melanoma cell lines that BRAF inhibition induces a secretome with stimulating effect on fibroblasts and naive melanoma cells. Several senescence-associated factors were found to be transcribed and secreted in response to BRAF or MEK inhibition, among them members of the fibroblast growth factor family. We identified the growth factor FGF1 as mediator of resilience towards BRAF inhibition, which limits the pro-apoptotic effects of the drug and activates fibroblasts to secrete HGF. FGF1 regulation was mediated by the PI3K pathway and by FRA1, a direct target gene of the MAPK pathway. When FGFR inhibitors were applied in parallel to BRAF inhibitors, resilience was broken, thus providing a rationale for combined therapeutical application. KW - melanoma KW - senescence KW - BRAF KW - tumor Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177261 VL - 7 IS - 71 ER - TY - THES A1 - Alcantarino Menescal, Luciana T1 - In vivo characterization of genetic factors involved in Xmrk driven melanoma formation in Medaka (Oryzias latipes): a closer look at braf, Stat5 and c-myc T1 - In vivo Charakterisierung genetischer Faktoren mit Einfluss auf Xmrk induzierte Melanome in Medaka (Oryzias latipes): Untersuchung von braf, Stat5 und c-myc. N2 - Melanoma arises from the malignant transformation of melanocytes and is one of the most aggressive forms of human cancer. In fish of the genus Xiphophorus, melanoma development, although very rarely, happens spontaneously in nature and can be induced by interspecific crossing. The oncogenic receptor tyrosine kinase, Xmrk, is responsible for melanoma formation in these fishes. Since Xiphophorus are live-bearing fishes and therefore not compatible with embryonic manipulation and transgenesis, the Xmrk melanoma model was brought to the medaka (Oryzias latipes) system. Xmrk expression under the control of the pigment cell specific mitf promoter leads to melanoma formation with 100% penetrance in medaka. Xmrk is an orthologue of the human epidermal growth factor receptor (EGFR) and activates several downstream signaling pathways. Examples of these pathways are the direct phosphorylation of BRAF and Stat5, as well as the enhanced transcription of C-myc. BRAF is a serine-threonine kinase which is found mutated at high frequencies in malignant melanomas. Stat5 is a transcription factor known to be constitutively activated in fish melanoma. C-myc is a transcription factor that is thought to regulate the expression of approximately 15% of all human genes and is involved in cancer progression of a large number of different tumors. To gain new in vivo information on candidate factors known to be involved in melanoma progression, I identified and analysed BRAF, Stat5 and C-myc in the laboratory fish model system medaka. BRAF protein motifs are highly conserved among vertebrates and the results of this work indicate that its function in the MAPK signaling is maintained in medaka. Transgenic medaka lines carrying a constitutive active version of BRAF (V614E) showed more pigmented skin when compared to wild type. Also, some transiently expressing BRAF V614E fishes showed a disrupted eye phenotype. In addition, I was able to identify two Stat5 copies in medaka, named Stat5ab/a and Stat5ab/b. Sequence analysis revealed a higher similarity between both Stat5 sequences when compared to either human Stat5a or Stat5b. This suggests that the two Stat5 copies in medaka arose by an independent duplication processes. I cloned these two Stat5 present in medaka, produced constitutive active and dominant negative gene versions and successfully established transgenic lines carrying each version under the control of the MITF promoter. These lines will help to elucidate questions that are still remaining in Stat5 biology and its function in melanoma progression, like the role of Stat5 phosphorylation on tumor invasiveness. In a third project during my PhD work, I analysed medaka C-myc function and indentified two copies of this gene in medaka, named c-myc17 and c-myc20, according to the chromosome where they are located. I produced conditional transgenic medaka lines carrying the c-myc17 gene coupled to the hormone binding domain of the estrogen receptor to enable specific transgene activation at a given time point. Comparable to human C-myc, medaka C-myc17 is able to induce proliferation and apoptosis in vivo after induction. Besides that, C-myc17 long-term activation led to liver hyperplasia. In summary, the medaka models generated in this work will be important to bring new in vivo information on genes involved in cancer development. Also, the generated transgenic lines can be easily crossed to the melanoma developing Xmrk medaka lines, thereby opening up the possibility to investigate their function in melanoma progression. Besides that, the generated medaka fishes make it possible to follow the whole development of melanocytes, since the embryos are transparent and can be used for high throughput chemical screens. N2 - Melanome entstehen durch die krankhafte Transformation von Melanozyten und sind eine der aggressivsten Krebsarten beim Menschen. In Fischen der Gattung Xiphophorus können, wenn auch sehr selten, spontan Melanome entstehen oder durch spezielle Artenkreuzungen induziert werden. Grundlage für das Entstehen der Melanome in diesen Fischen ist die Rezeptortyrosinkinase Xmrk. Da alle Xiphophorus-Arten lebendgebärend sind und keine Manipulationen an Embryonen vorgenommen werden können, wurde ein Xmrk Melanommodel für Medaka (Oryzias latipes) etabliert. Die Expression von Xmrk in Pigmentzellen dieser Fischart resultiert mit 100%iger Penetranz in Melanomen. Das Xmrk ist ein Ortholog des menschlichen „epidermal growth factor“ (EGFR) und aktiviert verschiedene nachgeschaltete Signalwege. Beispiele für diese Aktivierungen sind die Phosphorylierung von BRAF, Stat5 und die erhöhte Expression von c-myc. BRAF ist eine Serin-Threoninkinase, welche oft in malignen Melanomen mutiert ist. Stat5 ist ein Transkriptionsfaktor, welcher dauerhaft in Fischtumoren aktiviert ist. C-myc ist ein Transkriptionsfaktor, welcher etwa 15% aller menschlichen Gene sowie die Entstehung vieler menschlicher Tumore reguliert. Um neue Einsichten in die Funktion der Kanidatengene im Prozess der Melanomentstehung in vivo zu erlangen, habe ich Orthologe von BRAF, Stat5 und C-myc bei Medaka identifiziert und analysiert. Die Domänen des BRAF Proteins sind hoch konserviert in allen Vertebraten. Weiterhin deuten die Ergebnisse meiner Arbeit auf eine Beibehaltung der Funktionen im MAPK Signalweg hin. Transgene Medakalinien, welche eine dauerhaft aktive Version des BRAF Gens (V614E) exprimieren, weisen einerseits eine stärkere Hautpigmentierung auf. Weiterhin treten in diesen Fischen Veränderungen der Augen auf. In einem weiteren Projekt meiner Arbeit gelang es mir, zwei Kopien des Stat5 Gens im Medaka zu identifizieren, Stat5ab/a und Stat5ab/b. Sequenzanalysen zeigten eine höhere Übereinstimmung zwischen den beiden Genkopien, als zwischen denen von Medaka und Menschen. Dieses Ergebnis deutet darauf hin, dass die beiden Medaka Gene durch eine unabhängige Duplikation entstanden. In meiner Arbeit habe ich beide Gene des Medakas kloniert und jeweils eine konstitutiv aktive und eine dominant negative Version der Gene hergestellt. Weiterhin konnte ich erfolgreich für jede Genversion eine transgene Medakalinie etablieren, welche die verschiedenen Genvarianten unter der Kontrolle des pigmentzellspezifischen Promoters des mitf Gens exprimieren. Diese Linien werden in Zukunft helfen, den Einfluss von Stat5 Signalen auf den Prozess der Melanomverbreitung und dessen Invasivität zu erklären. In einem dritten Projekt meiner Doktorarbeit untersuchte ich das Vorkommen und die Funktion der C-myc Gene des Medakas. Ich konnte zwei Genkopien identifizieren, c-myc17 und c-myc20, welche auf unterschiedlichen Chromosomen lokalisiert sind. Ich konnte induzierbare, stabil transgene Linien herstellen, welche ein Fusionsprotein aus C-myc17 und der Hormonbindungsdomäne des Östrogenrezeptors von Maus exprimiert. Diese Linie ermöglichte eine induzierbare Aktivität des Transgens. Vergleichbar zum menschlichen MYC ist C-myc17 fähig, nach Aktivierung Proliferation und Apoptose in vivo auszulösen. Dauerhafte Aktivierung über einen längeren Zeitraum führt in diesen Linien zu Hyperplasie in Leber. Die verschiedenen Fischmodelle, die während dieser Arbeit generiert wurden, werden essentiell sein, um neue Einsichten in die Rolle diese Faktoren während der Krebsentwicklung in vivo zu erlangen. Weiterhin ermöglichen diese transgenen Linien durch einfaches Auskreuzen auf Xmrk Linien, deren Einfluss auf die Verbreitung von Melanomen zu untersuchen. Letztendlich sind mit diesen Linien auch Untersuchungen der Entwicklung von Pigmentzellen über Zeit möglich, da die Embryonen transparent sind und sich für chemisches Hochdurchsatz-Screening eignen. KW - Japankärpfling KW - Melanom KW - Myc KW - Molekulargenetik KW - melanoma KW - medaka KW - BRAF KW - Stat5 KW - c-myc KW - melanoma KW - medaka KW - BRAF KW - Stat5 KW - c-myc Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70762 ER -