Christoph Renné

Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis

Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis), but may also contribute to vascular thrombosis. Hereditary deficiency of factor XII (FXII), the protease that triggers the intrinsic pathway of coagulation in vitro, is not associated with spontaneous or excessive injury-related bleeding, indicating FXII is not required for hemostasis. We demonstrate that deficiency or inhibition of FXII protects mice from ischemic brain injury. After transient middle cerebral artery occlusion, the volume of infarcted brain in FXII-deficient and FXII inhibitor–treated mice was substantially less than in wild-type controls, without an increase in infarct-associated hemorrhage. Targeting FXII reduced fibrin formation in ischemic vessels, and reconstitution of FXII-deficient mice with human FXII restored fibrin deposition. Mice deficient in the FXII substrate factor XI were similarly protected from vessel-occluding fibrin formation, suggesting that FXII contributes to pathologic clotting through the intrinsic pathway. These data demonstrate that some processes involved in pathologic thrombus formation are distinct from those required for normal hemostasis. As FXII appears to be instrumental in pathologic fibrin formation but dispensable for hemostasis, FXII inhibition may offer a selective and safe strategy for preventing stroke and other thromboembolic diseases.

Molecular biology of Hodgkin's and Reed/Sternberg cells in Hodgkin's lymphoma

Hodgkins and Reed/Sternberg (HRS) cells, the tumour cells in classical Hodgkins lymphoma (HL), represent transformed B cells in nearly all cases. The detection of destructive somatic mutations in the rearranged immunoglobulin (Ig) genes of HRS cells in classical HL indicated that they originate from preapoptotic germinal centre (GC) B cells that lost the capacity to express a high‐affinity B‐cell receptor (BCR). Several aberrantly activated signalling pathways and transcription factors have been identified that contribute to the rescue of HRS cells from apoptosis. Among the deregulated signalling pathways, activation of multiple receptor tyrosine kinases in HRS cells appears to be a specific feature of HL. In about 40% of cases of classical HL the HRS cells are infected by Epstein‐Barr virus (EBV), indicating an important role of EBV in HL pathogenesis. Interestingly, nearly all cases of HL with destructive Ig gene mutations eliminating BCR expression (e.g. nonsense mutations) are EBV‐positive, suggesting that EBV‐encoded genes have a particular function to prevent apoptosis of HRS‐cell precursors that acquired such crippling mutations. This idea is further supported by the recent demonstration that isolated human GC B cells harbouring crippled Ig genes can be rescued by EBV from cell death, giving rise to lymphoblastoid cell lines. The molecular analysis of composite Hodgkins and non‐Hodgkins lymphomas indicated that many cases develop from a common GC B‐cell precursor in a multistep transformation process with both shared and distinct oncogenic events.

Inactivating SOCS1 mutations are caused by aberrant somatic hypermutation and restricted to a subset of B cell lymphoma entities

STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell-specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non-B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.

Molecular Cytogenetic Analyses of Immunoglobulin Loci in Nodular Lymphocyte Predominant Hodgkin’s Lymphoma Reveal a Recurrent IGH-BCL6 Juxtaposition

Chromosomal translocations juxtaposing different oncogenes to the immunoglobulin (IG) loci are the hallmark of various B-cell lymphomas. Because the tumor cells in nodular lymphocyte predominant Hodgkins lymphoma (NLPHL) are also derived from B cells, we examined whether NLPHL harbors chromosomal translocations that affect IG loci. Fluorescence in situ hybridization was applied to 24 NLPHL cases using probes flanking the IGH, IGK, and IGL loci as well as the BCL6 gene. Fourteen of these cases were additionally analyzed by combined immunofluorescence and fluorescence in situ hybridization. Chromosomal breakpoints in the IGH locus were detected in five NLPHL. All these cases also contained a BCL6 breakpoint. Triple-color interphase cytogenetics demonstrated the presence of an IGH-BCL6 juxtaposition, indicating a t(3;14)(q27;q32) in all five cases. There was no evidence for breakpoints affecting the IGK or IGL loci. Our results show that translocations juxtaposing the BCL6 oncogene next to the IGH locus are recurrent in NLPHL.

Host DNases prevent vascular occlusion by neutrophil extracellular traps

Blood DNases hack the NET Neutrophil extracellular traps (NETs) are lattices of processed chromatin decorated with select secreted and cytoplasmic proteins that trap and neutralize microbes. However, their inappropriate release may do more harm than good by promoting inflammation and thrombosis. Jiménez-Alcázar et al. report that two deoxyribonucleases (DNases), DNASE1 and DNASE1L3, have partially redundant roles in degrading NETs in the circulation (see the Perspective by Gunzer). Knockout mice lacking these enzymes were unable to tolerate chronic neutrophilia, quickly dying after blood vessels were occluded by NET clots. Furthermore, the damage unleashed by clots during septicemia was enhanced when these DNases were absent. Science, this issue p. 1202; see also p. 1126 Deoxyribonucleases work together to control vascular occlusion by neutrophil-induced blood clots. Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

The aberrant coexpression of several receptor tyrosine kinases is largely restricted to EBV‐negative cases of classical Hodgkin's lymphoma

The Hodgkin‐Reed/Sternberg (HRS) cells of classical Hodgkins lymphoma (HL) aberrantly express up to 7 different receptor tyrosine kinases (RTK) with extensive heterogeneity regarding the number and combinations of expressed RTKs in individual cases and a more prominent coexpression in nodular‐sclerosis (ns) than mixed‐cellularity (mc) HL. To investigate whether RTK expression patterns are related to other pathogenetic mechanisms and clinical behaviour, we analysed a large collection of EBV+ and EBV− cases of ns and mc subtype and cases with relapses for expression of the 7 RTKs. No specific relation of any RTK to a specific group of cases was observed. The analysis of average numbers of expressed RTKs per case as a measure for strength of overall RTK signalling revealed a relation with the histological subtype and the EBV‐status. RTK coexpression was significantly higher in EBV− nsHL cases compared to both EBV− and EBV+ mcHL cases. Among mcHL cases RTK coexpression was significantly higher in EBV− compared to EBV+ cases. Coexpression of 3 and more RTKs was largely restricted to EBV− cases. The inverse correlation between strong RTK signalling and presence of EBV may indicate that RTK signalling can at least partially replace the role of EBV in HRS cell pathogenesis. For cases with aberrant coexpression of several RTKs inclusion of RTK inhibitors in therapy regimens may be a novel option.

In angioimmunoblastic T-cell lymphoma, neoplastic T cells may be a minor cell population. A molecular single-cell and immunohistochemical study

The significance of T-cell proliferations in angioimmunoblastic lymphoma (AILD) is still enigmatic. Although classified as a malignant T-cell lymphoma in the World Health Organisation lymphoma classification, some cases of AILD lack dominant T-cell clones. In a previous study, based on single-cell polymerase chain reaction (PCR), we obtained similar results as studies of AILD using Southern blot or conventional PCR: some cases of AILD contained large T-cell clones, and, in other cases, T-cell clones were undetectable. As in single-cell studies, only a limited number of cells could be investigated; thus, we wanted to gain more insight into the amount and distribution of tumour cells. By applying triple immunofluorescent staining with antibodies directed against T-cell receptor Vβ-family-specific epitopes, we investigated T-cell populations in AILD and their localisation in the tissue in relation to B cells (CD20) and follicular dendritic cells (CD21). In two of five cases investigated, only a minority of the T-cells compartment belonged to the tumour clone. Neoplastic T cells were found throughout the tissue, including areas dominated by B cells.

Pathogenesis of Hodgkin's lymphoma.

Abstract:  In Hodgkins lymphoma (HL), the B cell origin of the tumour cells, the Hodgkin and Reed‐Sternberg (HRS) cells, has been disclosed by molecular single cell analysis about 10 yr ago. This finding formed the basis for various studies aimed to better understand the pathogenesis of this peculiar malignancy and the pathophysiology of the HRS cells. Work of our groups in this regard was focussed recently on two main topics, namely the study of differential gene expression in HRS cells and the pathogenesis of composite lymphomas. Composite lymphomas are combinations of HL and B cell non‐Hodgkin lymphomas, that turned out to be often clonally related. By molecular analysis of several composite lymphomas for potential transforming events, we identified examples of both shared as well as distinct transforming events. Comparing gene expression profiles of HL‐derived cell lines with the corresponding profiles from other B cell lymphomas and normal B cell subsets revealed a global down‐regulation of the B cell‐specific gene expression signature in HRS cells. Moreover, we identifed aberrant expression and activity of multiple receptor tyrosine kinases in HRS cells of classical and to a lesser extend lymphocyte predominant HL, which appears to be a unique feature of HL, and may offer novel strategies for treatment.

Expression and functional relevance of cannabinoid receptor 1 in Hodgkin lymphoma.

Background Cannabinoid receptor 1 (CB1) is expressed in certain types of malignancies. An analysis of CB1 expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date. Design and Methods We examined the distribution of CB1 protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB1 signaling on cell survival was investigated. Results A predominant expression of CB1 was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB1-abundance and displayed a dose-dependent decline of viability under CB1 inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells. Conclusions The present study identifies CB1 as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.

Induction of Endoplasmic Reticulum Stress by Sorafenib and Activation of NF-κB by Lestaurtinib as a Novel Resistance Mechanism in Hodgkin Lymphoma Cell Lines

Hodgkin-Reed/Sternberg (HRS) cells of classical Hodgkin lymphoma show aberrant expression and activation of several receptor tyrosine kinases (RTK) in the majority of cases. Therefore, we tested whether tyrosine kinase inhibitors (TKI) already in clinical use or late stages of clinical trials have antiproliferative effects on HRS cell lines and evaluated the targets, affected signaling pathways, and mechanisms of cell death and resistance. Sorafenib and lestaurtinib had antiproliferative effects on HRS cell lines at concentrations achievable in patients. Sorafenib inhibited platelet-derived growth factor receptor (PDGFR) α, TRKA and RON, caused decreases in total and phosphorylated amounts of several signaling molecules, and provoked caspase-3–independent cell death, most likely due to endoplasmic reticulum stress as indicated by upregulation of GADD34 and GADD153 and phosphorylation of PERK. Lestaurtinib inhibited TRKA, PDGFRα, RON, and JAK2 and had only a cytostatic effect. Besides deactivation, lestaurtinib also caused activation of signaling pathways. It caused increases in CD30L and TRAIL expression, and CD30L/CD30 signaling likely led to the observed concomitant activation of extracellular signal–regulated kinase 1/2 and the alternative NF-κB pathway. These data disclose the possible use of sorafenib for the treatment of Hodgkin lymphoma and highlight NF-κB activation as a potential novel mechanism of resistance toward TKIs. Mol Cancer Ther; 12(2); 173–83. ©2012 AACR.