Fabienne Meggetto

Acquisition of viral receptor by NK cells through immunological synapse.

Occasional EBV infection of human NK cells may lead to malignant diseases such as naso-pharyngeal NK lymphoma although NK cells do not express CD21, the primary receptor for EBV. Here we show that during early EBV infection in patients, NK cells attacked EBV-infected autologous B cells. In vitro, NK cells activated by conjugation to CD21+ B-EBV cell targets transiently acquired a weak CD21+ phenotype by synaptic transfer of few receptor molecules onto their own membrane. In the presence of viral particles, these ectopic receptors allowed EBV binding to the novel NK cell host. Hence, trans-synaptic acquisition of viral receptor from target cells might constitute an unsuspected mode of infection for otherwise unreachable lymphoid hosts.

Sporadic Early-Onset Colorectal Cancer Is a Specific Sub-Type of Cancer: A Morphological, Molecular and Genetics Study

Sporadic early onset colorectal carcinoma (EOCRC) which has by definition no identified hereditary predisposition is a growing problem that remains poorly understood. Molecular analysis could improve identification of distinct sub-types of colorectal cancers (CRC) with therapeutic implications and thus can help establish that sporadic EOCRC is a distinct entity. From 954 patients resected for CRC at our institution, 98 patients were selected. Patients aged 45–60 years were excluded to help define “young” and “old” groups. Thirty-nine cases of sporadic EOCRC (patients≤45 years with microsatellite stable tumors) were compared to both microsatellite stable tumors from older patients (36 cases, patients>60 years) and to groups of patients with microsatellite instability. Each group was tested for TP53, KRAS, BRAF, PIK3CA mutations and the presence of a methylator phenotype. Gene expression profiles were also used for pathway analysis. Compared to microsatellite stable CRC from old patients, sporadic EOCRC were characterized by distal location, frequent synchronous metastases and infrequent synchronous adenomas but did not have specific morphological characteristics. A familial history of CRC was more common in sporadic EOCRC patients despite a lack of identified hereditary conditions (p = 0.013). Genetic studies also showed the absence of BRAF mutations (p = 0.022) and the methylator phenotype (p = 0.005) in sporadic EOCRC compared to older patients. Gene expression analysis implicated key pathways such as Wnt/beta catenin, MAP Kinase, growth factor signaling (EGFR, HGF, PDGF) and the TNFR1 pathway in sporadic EOCRC. Wnt/beta catenin signaling activation was confirmed by aberrant nuclear beta catenin immunostaining (p = 0.01). This study strongly suggests that sporadic EOCRC is a distinct clinico-molecular entity presenting as a distal and aggressive disease associated with chromosome instability. Furthermore, several signaling pathways including the TNFR1 pathway have been identified as potential biomarkers for both the diagnosis and treatment of this disease.

A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor

A monoclonal anti-CD160 antibody inhibits the growth of new vessels in pathological ocular and tumor neoangiogenesis but not in healthy tissues.

Evidence for early infection of nonneoplastic natural killer cells by Epstein-Barr virus.

ABSTRACT We examined lymph nodes and tonsils from patients with infectious mononucleosis by combined detection of EBV-encoded RNA and a specific marker of natural killer (NK) cells, PEN5. A small number of Epstein-Barr virus (EBV) latently infected nonneoplastic NK cells were detected. Our data demonstrate that NK cells are natural targets of EBV and that infection of these cells is an early event observed during primary EBV infection.

ALK+ALCLs induce cutaneous, HMGB-1–dependent IL-8/CXCL8 production by keratinocytes through NF-κB activation

Anaplastic large-cell lymphomas (ALCLs) bearing the t(2;5) translocation (ALK(+)ALCLs) are frequently characterized by skin colonization and associated with a poor prognosis. Using conditional transgenic models of anaplastic lymphoma kinase-positive (ALK(+)) lymphomas and human ALK(+)ALCL cell lines, in the present study, we show that high-mobility-group box-1 (HMGB-1), a proinflammatory cytokine, is released by ALK(+) cells, and demonstrate extracellular HMGB-1-stimulated secretion of the IL-8 chemokine by HaCaT keratinocytes through the involvement of MMP-9, PAR-2, and the NF-κB pathway. Furthermore, we demonstrate that, in vitro, IL-8 is able to induce the invasiveness of ALK(+) cells, which express the IL-8 receptors CXCR1 and CXCR2. In vitro and in vivo, HMGB-1 inhibition achieved by glycyrrhizin treatment led to a drastic reduction in ALK(+) cell invasiveness. The pathophysiological relevance of our observations was confirmed by demonstrating that the HMGB-1 and IL-8 receptors are expressed in ALK(+)ALCL biopsies. We have also shown that IL-8 secretion is correlated with leukemic dissemination of ALK(+) cells in a significant number of patients. The results of the present study demonstrate for the first time a relationship among the pro-inflammatory mediators HMGB-1, MMP-9, PAR-2, and IL-8. We propose that these mediators create a premetastatic niche within the skin, thereby participating in ALK(+) lymphoma epidermotropism.

Targeting autophagy enhances the anti-tumoral action of crizotinib in ALK-positive anaplastic large cell lymphoma

Anaplastic Lymphoma Kinase-positive Anaplastic Large Cell Lymphomas (ALK+ ALCL) occur predominantly in children and young adults. Their treatment, based on aggressive chemotherapy, is not optimal since ALCL patients can still expect a 30% 2-year relapse rate. Tumor relapses are very aggressive and their underlying mechanisms are unknown. Crizotinib is the most advanced ALK tyrosine kinase inhibitor and is already used in clinics to treat ALK-associated cancers. However, crizotinib escape mechanisms have emerged, thus preventing its use in frontline ALCL therapy. The process of autophagy has been proposed as the next target for elimination of the resistance to tyrosine kinase inhibitors. In this study, we investigated whether autophagy is activated in ALCL cells submitted to ALK inactivation (using crizotinib or ALK-targeting siRNA). Classical autophagy read-outs such as autophagosome visualization/quantification by electron microscopy and LC3-B marker turn-over assays were used to demonstrate autophagy induction and flux activation upon ALK inactivation. This was demonstrated to have a cytoprotective role on cell viability and clonogenic assays following combined ALK and autophagy inhibition. Altogether, our results suggest that co-treatment with crizotinib and chloroquine (two drugs already used in clinics) could be beneficial for ALK-positive ALCL patients.

Conditional TPM3-ALK and NPM-ALK transgenic mice develop reversible ALK-positive early B-cell lymphoma/leukemia

NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) and TPM3-ALK (nonmuscular tropomyosin 3-anaplastic lymphoma kinase) are oncogenic tyrosine kinases implicated in the pathogenesis of human ALK-positive lymphoma. We report here the development of novel conditional mouse models for ALK-induced lymphomagenesis, with the use of the tetracycline regulatory system under the control of the EmuSRalpha enhancer/promoter. The expression of either oncogene resulted in the arrest of the differentiation of early B cells and lymphomagenesis. We also observed the development of skin keratoacanthoma lesions, probably because of aberrant ALK expression in keratinocytes. The inactivation of the ALK oncogene on doxycycline treatment was sufficient to induce sustained regression of both hematopoietic tumors and skin disease. Importantly, treatment with the specific ALK inhibitor (PF-2341066) also reversed the pathologic states, showing the value of these mouse models for the validation of ALK tyrosine kinase inhibitors. Thus, our results show (1) that NPM-ALK and TPM3-ALK oncogenes are sufficient for lymphoma/leukemia development and required for tumor maintenance, hence validating ALK as potentially effective therapeutic target; and (2) for the first time, in vivo, the equal tumorigenic potential of the NPM-ALK and TPM3-ALK oncogenic tyrosine kinases. Our models offer a new tool to investigate in vivo the molecular mechanisms associated with ALK-induced lymphoproliferative disorders.

Development of a conditional bioluminescent transplant model for TPM3-ALK-induced tumorigenesis as a tool to validate ALK-dependent cancer targeted therapy.

Overexpression and activation of TPM3-ALK tyrosine kinase fusion protein is a causal oncogenic event in the development of Anaplastic Large Cell Lymphoma and Inflammatory Myofibroblastic ALK-positive tumours. Thus, the development of ALK specific tyrosine kinase inhibitors is a current therapeutic challenge. Animal models are essential to assess, in vivo, the efficiency of ALK-oncogene inhibitors and to identify new and/or additional therapeutic targets in the ALK tumorigenesis pathway. Using the tetracycline system to allow conditional and concomitant TPM3-ALK and luciferase expression, we have developed a unique transplant model for bioluminescent TPM3-ALK-induced fibroblastic tumours in athymic nude mice. The reversible TPM3-ALK expression allowed us to demonstrate that this oncogene is essential for the tumour growth and its maintenance. In addition, we showed that this model could be used to precisely assess tumour growth inhibition upon ALK chemical inactivation. As proof of principle, we used the general tyrosine kinase inhibitor herbimycin A to inhibit ALK oncoprotein activity. As expected, herbimycin A treatment reduced tumour growth as assessed both by tumour volume measurement and bioluminescent imaging. We conclude that this transplant model for TPM3-ALK-induced tumours represents a valuable tool not only to accurately and rapidly evaluate in vivo ALK-targeted therapies but also to gain insight into the mechanism of ALK-positive tumour development.

Comparative analysis of oncogenic properties and nuclear factor-κB activity of latent membrane protein 1 natural variants from Hodgkin’s lymphoma’s Reed-Sternberg cells and normal B-lymphocytes

Latent membrane protein 1 (LMP1) was the first Epstein-Barr virus (EBV) latent protein found to be able to transform cell lines, and further the development of lymphomas in LMP1 transgenic mice due to its oncogenic potential. LMP1 polymorphism participates in EBV genetic diversity. In EBV-associated Hodgkin’s lymphomas, neoplastic Reed-Sternberg cells and surrounding non-tumor B-cells contain different variants of LMP1. This study shows that LMP1 variants from Reed-Sternberg cells have enhanced proliferating and clonogenic potential. Background In Epstein-Barr virus-associated Hodgkin’s lymphomas, neoplastic Reed-Sternberg cells and surrounding non-tumor B-cells contain different variants of the LMP1-BNLF1 oncogene. In this study, we raised the question of functional properties of latent membrane protein 1 (LMP1) natural variants from both Reed-Sternberg and non-tumor B-cells. Design and Methods Twelve LMP1 natural variants from Reed-Sternberg cells, non-tumor B-cells of Hodgkin’s lymphomas and from B-cells of benign reactive lymph nodes were cloned, sequenced and stably transfected in murine recombinant interleukin-3-dependent Ba/F3 cells to search for relationships between LMP1 cellular origin and oncogenic properties as well as nuclear factor-κB activation, and apoptosis protection. Results LMP1 variants of Reed-Sternberg cell origin were often associated with increased mutation rate and with recurrent genetic events, such as del15bp associated with S to N replacement at codon 309, and four substitutions I85L, F106Y, I122L, and M129I. Oncogenic potential (growth factor-independence plus clonogenicity) was consistently associated with LMP1 variants from Reed-Sternberg cells, but inconstantly for LMP1-variants from non-tumor B-cells. Analysis of LMP1 variants from both normal B-cells and Reed-Sternberg cells indicates that protection against apoptosis through activation of nuclear factor-κB - whatever the cellular origin of LMP1 - was maintained intact, regardless of the mutational pattern. Conclusions Taken together, our results demonstrate that preserved nuclear factor-κB activity and protection against apoptosis would be the minimal prerequisites for all LMP1 natural variants from both normal and tumor cells in Hodgkin’s lymphomas, and that oncogenic potential would constitute an additional feature for LMP1 natural variants in Reed-Sternberg cells.

Immunobiochemical characterization of the antigen detected by monoclonal antibody IND.64 : Evidence that IND.64 reacts with the cell proliferation associated nuclear antigen previously defined by Ki-67

SummaryThe immunohistochemical characteristics of the monoclonal antibody IND.64 are very similar to those of the monoclonal antibody Ki-67. The aim of this study was to further characterize this new antibody and to compare it with Ki-67 using immunobiochemical methods. Our results demonstrate that the similarity between the antibodies holds true even at the molecular level. Immunoblot analysis of IM-9-cell lysates with both antibodies showed a double band with apparent molecular weights of 395 kD and 345 kD, respectively. Competition ELISAs using a synthetic peptide derived from the thus far determined Ki-67 cDNA sequence as competitor, indicate that IND.64 may recognize the same epitope as Ki-67. The IND.64 epitope resides at least within a 20 amino acid sequence which also contains the Ki-67 epitope. Since IND.64 is of the IgG2b subclass, while Ki-67 is of the IgG1 subclass, the two antibodies may be useful for double immunostaining. In addition, IND.64 may help in determining the still unknown function of the anigen it recognizes.