The role of transcription factor BATF3 in CD30+ lymphomas and genetic characterization of composite lymphomas

Eine veränderte Aktivität von Transkriptionsfaktoren ist Kennzeichen vieler maligner Lymphome, trotzdem sind die Schlüsseldefekte dieser veränderten Aktivität in der Lymphom-Pathogenese bisher nicht vollkommen aufgeklärt. Die Tumorzellen des klassischen Hodgkin-Lymphoms (engl. classical Hodgkin Lymphoma, cHL), des primären mediastinalen B-Zell-Lymphoms (PMBL) und des anaplastisch großzelligem Lymphoms (ALCL) entstehen zwar aus unterschiedlichen Vorläuferzellen (prä-apoptotischen B-Zellen, thymischen B-Zellen und T-Zellen), sind jedoch durch Gemeinsamkeiten wie deregulierte JAK/STAT-Aktivität und Ausprägung von CD30 und AP-1 (engl. Activator protein-1)-Faktoren gekennzeichnet. AP-1-Faktoren regulieren eine Vielzahl physiologischer Prozesse, allerdings sind sie auch in der Pathogenese von Lymphomen beteiligt. Im PMBL, cHL und ALCL sind hohe Transkriptionslevel von dem AP-1-Faktor BATF3 zu detektieren und die bis dahin unbekannte Funktion von BATF3 in der Lymphompathogenese sollte im Rahmen dieser Arbeit untersucht werden. BATF3-Protein wurde in den Tumorzellen des cHLs, PMBLs und ALCLs sowie in den Tumorzellen des CD30+ diffus großzelligem B-Zell-Lymphoms detektiert, das heißt BATF3 ist bevorzugt in CD30+-Lymphomen exprimert. Als Interaktionspartner von BATF3 sind die AP-1-Faktoren JUN und JUNB in cHL- und ALCL-Zelllinien charakterisiert worden. Die Herunterregulation von BATF3 ist toxisch für CD30+-Lymphom-Zelllinien und führt zur erhöhten Apoptose und verminderten Zellproliferation in cHL- und ALCL-Zelllinien. Das Onkogen MYC konnte unter den Zielgenen von BATF3 identifiziert werden. So führt die Herunterregulation von BATF3 auch zu einer Reduktion von MYC in cHL-, ALCL- und einer von zwei PMBL-Zelllinien. MYC-Ausprägung wird durch die Bindung von BATF3 und JUN an den MYC-Promotor reguliert. Direkt an den BATF3-Promotor binden phosphorylierte STAT-Faktoren in cHL- und ALCL-Zelllinien und so führt konstitutive JAK/STAT-Signalaktivität zur Expression von BATF3 in cHL-, ALCL- und vermutlich auch PMBL-Tumorzellen. Folglich konnte im Rahmen dieser Arbeit BATF3 als essentieller Faktor für das Überleben von cHL- und ALCL-Zelllinien charakterisiert werden und die onkogene Achse der STAT-vermittelten Induktion von BATF3 identifiziert werden, welche die Ausprägung von MYC reguliert. In dem zweiten Teil dieser Arbeit wurden Kombinationslymphome bestehend aus einem cHL und Non-Hodgkin-Lymphom auf genetische Läsionen untersucht, um Einblicke in die Mehrschritt-Pathogenese von Lymphomen zu erhalten. Analysen der variablen (V) Region von Immunglobulin (Ig)-Genen konnten eine klonale Verwandtschaft durch gleiche IgV-Genumlagerungen in zwei von drei untersuchten Kombinationslymphomen nachweisen. Die Exom-Sequenzierung der drei Kombinationslymphome konnte erfolgreich durchgeführt werden. Die Validierung und funktionelle Charakterisierung der gefundenen genetischen Läsionen wird helfen die Krankheitsentstehung von Lymphomen besser zu verstehen.Malignant lymphomas are characterized by deregulated activity of transcription factors. However, key defects of their aberrant activity in lymphoma pathogenesis have not been fully elucidated yet. The malignant cells of classical Hodgkin Lymphoma (cHL), primary mediastinal B-cell lymphoma (PMBL) and anaplastic large cell lymphoma (ALCL) are derived from different progenitor cells (pre-apoptotic germinal center B lymphocytes, thymic B lymphocytes and T lymphocytes), nevertheless they share similarities such as deregulated JAK/STAT activity and CD30 and AP-1 (activator protein-1) expression. AP-1 factors regulate different physiological processes but are also involved in lymphoma pathogenesis. In PMBL, cHL and ALCL high transcriptional levels of the AP-1 factor BATF3 are detected and in this project the previously unknown function of BATF3 in lymphoma pathogenesis should be investigated. BATF3 protein is predominantly expressed in tumor cells of CD30+ lymphomas, namely cHL, PMBL, ALCL and CD30+ diffuse large B cell lymphoma. BATF3 heterodimerizes with the AP-1 factors JUN and JUNB in cHL and ALCL cells. Downregulation of BATF3 is toxic for CD30+ lymphoma cell lines and causes increased apoptosis and decreased cell proliferation in cHL and ALCL cell lines. The oncogene MYC could be identified as a BATF3 target gene and downregulation of BATF3 leads to reduction of MYC in cHL, ALCL and in one of two PMBL cell lines. BATF3 and JUN bind to the MYC promoter region and regulate its expression. Phosphorylated STAT factors are localized at the BATF3 promoter in cHL and ALCL cell lines, thus constitutive JAK/STAT signaling activity leads to BATF3 expression in cHL, ALCL and presumably also in PMBL cells. Hence, BATF3 could be characterized as an essential factor for the survival of cHL and ALCL cell lines, and for the first time an oncogenic axis of STAT-mediated BATF3 upregulation, which regulates MYC expression, could be identified. In the second part of this thesis composite lymphomas consisting of a cHL and a Non Hodgkin Lymphoma have been analyzed for genetic lesions to gain insights into the multistep process of lymphoma pathogenesis. The analysis of variable immunoglobulin (IgV) regions could identify a clonal relationship in two of three analyzed composite lymphomas by identical IgV gene rearrangements. Whole exome sequencing of three analyzed composite lymphomas has been successfully performed. Validation studies and functional characterization of identified genetic lesions will help to improve our understanding of lymphoma pathogenesis

Activation of inflammatory responses in human U937 macrophages by particulate matter collected from dairy farms: an in vitro expression analysis of pro-inflammatory markers

Abstract Background The purpose of the present study was to investigate activation of inflammatory markers in human macrophages derived from the U937 cell line after exposure to particulate matter (PM) collected on dairy farms in California and to identify the most potent components of the PM. Methods PM from different dairies were collected and tested to induce an inflammatory response determined by the expression of various pro-inflammatory genes, such as Interleukin (IL)-8, in U937 derived macrophages. Gel shift and luciferase reporter assays were performed to examine the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Toll-like-receptor 4 (TLR4). Results Macrophage exposure to PM derived from dairy farms significantly activated expression of pro-inflammatory genes, including IL-8, cyclooxygenase 2 and Tumor necrosis factor-alpha, which are hallmarks of inflammation. Acute phase proteins, such as serum amyloid A and IL-6, were also significantly upregulated in macrophages treated with PM from dairies. Coarse PM fractions demonstrated more pro-inflammatory activity on an equal-dose basis than fine PM. Urban PM collected from the same region as the dairy farms was associated with a lower concentration of endotoxin and produced significantly less IL-8 expression compared to PM collected on the dairy farms. Conclusion The present study provides evidence that the endotoxin components of the particles collected on dairies play a major role in mediating an inflammatory response through activation of TLR4 and NF-κB signaling

SAMHD1 is recurrently mutated in T-cell prolymphocytic leukemia

T-cell prolymphocytic leukemia (T-PLL) is an aggressive malignancy with a median survival of the patients of less than two years. Besides characteristic chromosomal translocations, frequent mutations affect the ATM gene, JAK/STAT pathway members, and epigenetic regulators. We here performed a targeted mutation analysis for 40 genes selected from a RNA sequencing of 10 T-PLL in a collection of 28 T-PLL, and an exome analysis of five further cases. Nonsynonymous mutations were identified in 30 of the 40 genes, 18 being recurrently mutated. We identified recurrently mutated genes previously unknown to be mutated in T-PLL, which are SAMHD1, HERC1, HERC2, PRDM2, PARP10, PTPRC, and FOXP1. SAMHD1 regulates cellular deoxynucleotide levels and acts as a potential tumor suppressor in other leukemias. We observed destructive mutations in 18% of cases as well as deletions in two further cases. Taken together, we identified additional genes involved in JAK/STAT signaling (PTPRC), epigenetic regulation (PRDM2), or DNA damage repair (SAMHD1, PARP10, HERC1, and HERC2) as being recurrently mutated in T-PLL. Thus, our study considerably extends the picture of pathways involved in molecular pathogenesis of T-PLL and identifies the tumor suppressor gene SAMHD1 with ~20% of T-PLL affected by destructive lesions likely as major player in T-PLL pathogenesis

Aryl hydrocarbon receptor signaling regulates NF-κB RelB activation during dendritic-cell differentiation.

How the aryl hydrocarbon receptor (AhR) regulates dendritic-cell (DC) differentiation is unknown. We show that activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused enhanced differentiation from immature DCs (IDCs) to mature DCs (MDCs) in the bone-marrow-derived DCs (BMDC) from B6 wild-type mice but not in the BMDCs from AhR-null mice as indicated by the expression of CD11c and class II major histocompatibility complex (MHC). Enhanced maturation of BMDCs was associated with elevated levels of CD86 and an increased AhR-dependent nuclear accumulation of nuclear factor-kappa-light-chain enhancer of activated B cell (NF-κB) member RelB in BMDCs. The expression of interleukin (IL) 10 and chemokine DC-CK1 was suppressed, whereas that of CXCL2, CXCL3 and IL-22 was significantly increased in AhR-activated BMDCs. Furthermore, TCDD induced expression of the regulatory enzymes indoleamine 2,3-dioxygenase (IDO1) and indoleamine 2,3-dioxygenase-like 1 (IDO2). Increased expression of IDO2 was associated with coexpression of the cell-surface marker CCR6. Interestingly, mRNA expression of the chemokine receptor CCR6 was drastically decreased in AhR-null IDCs and MDCs. Overall, these data demonstrate that AhR modifies the maturation of BMDCs associated with the induction of the regulatory enzyme IDO and altered expression of cytokine, chemokines and DC-specific surface markers and receptors

Aryl hydrocarbon receptor signaling regulates NF‐κB RelB activation during dendritic‐cell differentiation

How the aryl hydrocarbon receptor (AhR) regulates dendritic-cell (DC) differentiation is unknown. We show that activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused enhanced differentiation from immature DCs (IDCs) to mature DCs (MDCs) in the bone-marrow-derived DCs (BMDC) from B6 wild-type mice but not in the BMDCs from AhR-null mice as indicated by the expression of CD11c and class II major histocompatibility complex (MHC). Enhanced maturation of BMDCs was associated with elevated levels of CD86 and an increased AhR-dependent nuclear accumulation of nuclear factor-kappa-light-chain enhancer of activated B cell (NF-κB) member RelB in BMDCs. The expression of interleukin (IL) 10 and chemokine DC-CK1 was suppressed, whereas that of CXCL2, CXCL3 and IL-22 was significantly increased in AhR-activated BMDCs. Furthermore, TCDD induced expression of the regulatory enzymes indoleamine 2,3-dioxygenase (IDO1) and indoleamine 2,3-dioxygenase-like 1 (IDO2). Increased expression of IDO2 was associated with coexpression of the cell-surface marker CCR6. Interestingly, mRNA expression of the chemokine receptor CCR6 was drastically decreased in AhR-null IDCs and MDCs. Overall, these data demonstrate that AhR modifies the maturation of BMDCs associated with the induction of the regulatory enzyme IDO and altered expression of cytokine, chemokines and DC-specific surface markers and receptors