Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis

CD40, a member of the tumor necrosis factor (TNF) receptor family, plays an essential role in T cell–dependent immune responses. Because CD40 is widely expressed on the surface of tumor cells in various B cell malignancies, deregulated CD40 signaling has been suggested to contribute to lymphomagenesis. In this study, we show that B cell-specific expression of a constitutively active CD40 receptor, in the form of a latent membrane protein 1 (LMP1)/CD40 chimeric protein, promoted an increase in the number of follicular and marginal zone B cells in secondary lymphoid organs in transgenic mice. The B cells displayed an activated phenotype, prolonged survival and increased proliferation, but were significantly impaired in T cell-dependent immune responses. Constitutive CD40 signaling in B cells induced selective and constitutive activation of the noncanonical NF-κB pathway and the mitogen-activated protein kinases Jnk and extracellular signal–regulated kinase. LMP1/CD40-expressing mice older than 12 mo developed B cell lymphomas of mono- or oligoclonal origin at high incidence, thus showing that the interplay of the signaling pathways induced by constitutive CD40 signaling is sufficient to initiate a tumorigenic process, ultimately leading to the development of B cell lymphomas

Rapid conditional knock-down–knock-in system for mammalian cells

RNA interference (RNAi) is a powerful tool to analyze gene function in mammalian cells. However, the interpretation of RNAi knock-down phenotypes can be hampered by off-target effects or compound phenotypes, as many proteins combine multiple functions within one molecule and coordinate the assembly of multimolecular complexes. Replacing the endogenous protein with ectopic wild-type or mutant forms can exclude off-target effects, preserve complexes and unravel specific roles of domains or modifications. Therefore, we developed a rapid-knock-down–knock-in system for mammalian cells. Stable polyclonal cell lines were generated within 2 weeks by simultaneous selection of two episomal vectors. Together these vectors mediated reconstitution and knock-down in a doxycycline-dependent manner to allow the analysis of essential genes. Depletion was achieved by an artificial miRNA-embedded siRNA targeting the untranslated region of the endogenous, but not the ectopic mRNA. To prove effectiveness, we tested 17 mutants of WDR12, a factor essential for ribosome biogenesis and cell proliferation. Loss-off function phenotypes were rescued by the wild-type and six mutant forms, but not by the remaining mutants. Thus, our system is suitable to exclude off-target effects and to functionally analyze mutants in cells depleted for the endogenous protein

Functional loss of IKBE leads to NF-KB deregulation in aggressive chronic lymphocytic leukemia

NF-?B is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-?B pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes I?B?, a negative regulator of NF-?B in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced I?B? protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that I?B? loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-?B deregulation during lymphomagenesis. <br/

To respond or not to respond to CD40 agonism: That is the prediction

In the context of the clinical development of a CD40-stimulatory monoclonal antibody for the treatment of B-cell cancers, a tumor mRNA-based gene signature reflecting CD40 signaling pathway activation status was identified that could be used to predict responding and non-responding patients with diffuse large B cell lymphoma (DLBCL)

LMP1 signaling can replace CD40 signaling in B cells in vivo and has unique features of inducing class-switch recombination to IgG1.

The Epstein-Barr virus (EBV) protein LMP1 is considered to be a functional homologue of the CD40 receptor. However, in contrast to the latter, LMP1 is a constitutively active signaling molecule. To compare B cell-specific LMP1 and CD40 signaling in an unambiguous manner, we generated transgenic mice conditionally expressing a CD40/LMP1 fusion protein, which retained the LMP1 cytoplasmic tail but has lost the constitutive activity of LMP1 and needs to be activated by the CD40 ligand. We show that LMP1 signaling can completely substitute CD40 signaling in B cells, leading to normal B-cell development, activation, and immune responses including class-switch recombination, germinal center formation, and somatic hypermutation. In addition, the LMP1-signaling domain has a unique property in that it can induce class-switch recombination to IgG1 independent of cytokines. Thus, our data indicate that LMP1 has evolved to imitate T-helper cell function allowing activation, proliferation, and differentiation of EBV-infected B cells independent of T cells

Studying Copy Number Variations in Cell-Free DNA: The Example of AR in Prostate Cancer

Serum and plasma cell-free DNA (cfDNA) has been shown as an informative noninvasive source of biomarkers for different diseases, including cancer. Starting from the hypothesis that the gain of androgen receptor (AR) gene is a frequent aberration in advanced prostate cancer patients, we analyzed it in cfDNA as a potential predictive biomarker of specific treatments. Here we report a general protocol that may be considered to analyze gene copy number variations in serum or plasma fluids

Reproducing indolent B-cell lymphoma transformation with T-cell immunosuppression in LMP1/CD40-expressing mice

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B cell expansion and lymphomagenesis induced by chronic CD40 signaling is strictly dependent on CD19.

CD40, a member of the Tumor necrosis factor receptor family, is expressed on all mature B-cells and on most B-cell lymphomas. Recently, we have shown that constitutive activation of CD40-signaling in B-cells induced by a fusion protein consisting of the transmembrane part of the Epstein-Barr viral Latent Membrane Protein 1 (LMP1) and the cytoplasmic part of CD40 (LMP1/CD40) drives B-cell lymphoma development in transgenic mice. Since LMP1/CD40-expressing B-cells showed an upregulation of CD19, we investigated CD19 function in CD40-driven B-cell expansion and lymphomagenesis. Here, we demonstrate that ablation of CD19 in LMP1/CD40 transgenic mice resulted in a severe loss and reduced life span of mature B-cells and completely abrogated development of B-cell lymphoma. CD19 is localized to lipid rafts and constitutively activated by the LMP1/CD40 fusion protein in B-cells. We provide evidence that the improved survival and malignant transformation of LMP1/CD40-expressing B-cells is dependent on activation of the MAPK Erk that is mediated through CD19 in a PI3K-dependent manner. Our data suggest that constitutively active CD40 is dependent on CD19 to transmit survival- and proliferation-signals. Moreover, we detected a similarly functioning prosurvival pathway involving phosphorylated CD19 and PI3K-dependent Erk-phosphorylation in human Diffuse Large B Cell Lymphoma cell lines. Our data provides evidence that CD19 plays an important role in transmitting survival and proliferation signals downstream of CD40 and therefore might be an interesting therapeutic target for the treatment of lymphoma undergoing chronic CD40-signaling