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Janus kinases (JAKs) classically signal by activating STAT transcription factors but can also regulate gene expression by epigenetically phosphorylating histone H3 on tyrosine 41 (H3Y41-P). In diffuse large B-cell lymphomas (DLBCLs), JAK signaling is a feature of the activated B-cell (ABC) subtype and is triggered by autocrine production of IL-6 and IL-10. Whether this signaling involves STAT activation, epigenetic modification of chromatin, or both mechanisms is unknown. Here we use genetic and pharmacological inhibition to showthat JAK1 signaling sustains the survival of ABC DLBCL cells. Whereas STAT3 contributed to the survival of ABC DLBCL cell lines, forced STAT3 activity could not protect these cells from death following JAK1 inhibition, suggesting epigenetic JAK1 action. JAK1 regulated the expression of nearly 3,000 genes in ABC DLBCL cells, and the chromatin surrounding many of these genes was modified by H3Y41-P marks that were diminished by JAK1 inhibition. These JAK1 epigenetic target genes encode important regulators of ABC DLBCL proliferation and survival, including IRF4, MYD88, andMYC. A smallmolecule JAK1 inhibitor cooperatedwith the BTK inhibitor ibrutinib in reducing IRF4 levels and acted synergistically to kill ABC DLBCL cells, suggesting that this combination should be evaluated in clinical trials. [ABSTRACT FROM AUTHOR]

Burkitt's lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL in developing countries, necessitating new strategies. The normal germinal centre B cell is the presumed cell of origin for both BL and diffuse large B-cell lymphoma (DLBCL), yet gene expression analysis suggests that these malignancies may use different oncogenic pathways. BL is subdivided into a sporadic subtype that is diagnosed in developed countries, the Epstein-Barr-virus-associated endemic subtype, and an HIV-associated subtype, but it is unclear whether these subtypes use similar or divergent oncogenic mechanisms. Here we used high-throughput RNA sequencing and RNA interference screening to discover essential regulatory pathways in BL that cooperate with MYC, the defining oncogene of this cancer. In 70% of sporadic BL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival phosphatidylinositol-3-OH kinase pathway in BL, in part by augmenting tonic B-cell receptor signalling. In 38% of sporadic BL cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. These findings suggest opportunities to improve therapy for patients with BL. [ABSTRACT FROM AUTHOR]

Genomics has provided a lever to pry open lymphoid cells and examine their regulatory biology. The large body of available gene expression data has also allowed us to define the of coordinately expressed genes, termed gene expression signatures, which characterize the states of cellular physiology that reflect cellular differentiation, activation of signaling pathways, and the action of transcription factors. Gene expression signatures that reflect the action of individual transcription factors can be defined by perturbing transcription factor function using RNA interference (RNAi), small-molecule inhibition, and dominant-negative approaches. We have used this methodology to define gene expression signatures of various transcription factors controlling B-cell differentiation and activation, including BCL-6, B lymphocyte-induced maturation protein-1 (Blimp-1), X-box binding protein-1 (XBP1), nuclear factor-κB (NF-κB), and c-myc. We have also curated a wide variety of gene expression signatures from the literature and assembled these into a signature database. Statistical methods can define whether any signature in this database is differentially expressed in independent biological samples, an approach we have used to gain mechanistic insights into the origin and clinical behavior of B-cell lymphomas. We also discuss the use of genomic-scale RNAi libraries to identify genes and pathways that may serve as therapeutic targets in B-cell malignancies. [ABSTRACT FROM AUTHOR]

Background: Myc-induced lymphoblastic B-cell lymphoma (LBL) in iMycEμmice may provide a model system for the study of the mechanism by which human MYC facilitates the initiation and progression of B cell and plasma cell neoplasms in human beings. We have recently shown that gene-targeted iMycEμ mice that carry a His6-tagged mouse Myc cDNA, MycHis, just 5' of the immunoglobulin heavy-chain enhancer, Eμ, are prone to B cell and plasma cell tumors. The predominant tumor (∼50%) that arose in the iMycEμ mice on the mixed genetic background of segregating C57BL/6 and 129/SvJ alleles was LBL. The purpose of this study was to establish and characterize a cell line, designated iMycEμ-1, for the in-depth evaluation of LBL in vitro. Methods: The morphological features and the surface marker expression profile of the iMycEμ-1 cells were evaluated using cytological methods and FACS, respectively. The cytogenetic make-up of the iMycEμ-1 cells was assessed by spectral karyotyping (SKY). The expression of the inserted MycHis gene was determined using RT-PCR and qPCR. Clonotypic immunoglobulin gene arrangements were detected by Southern blotting. The global gene expression program of the iMycEμ-1 cells and the expression of 768 "pathway" genes were determined with the help of the Mouse Lymphochip© and Superarray© cDNA micro- and macroarrays, respectively. Array results were verified, in part, by RT-PCR and qPCR. Results: Consistent with their derivation from LBL, the iMycEμ-1 cells were found to be neoplastic IgMhighIgDlow lymphoblasts that expressed typical B-cell surface markers including CD40, CD54 (ICAM -1), CD80 (B7-1) and CD86 (B7-2). The iMycEμ-1 cells harbored a reciprocal T(9;11) and three non-reciprocal chromosomal translocations, overexpressed MycHis at the expense of normal Myc, and exhibited gene expression changes on Mouse Lymphochip© microarrays that were consistent with MycHis-driven B-cell neoplasia. Upon comparison to normal B cells using eight different Superarray© cDNA macroarrays, the iMycEμ-1 cells showed the highest number of changes on the NF?B array. Conclusion: The iMycEμ-1 cells may provide a uniquely useful model system to study the growth and survival requirements of Myc-driven mouse LBL in vitro. [ABSTRACT FROM AUTHOR]

A common V(D)J recombinase that recognizes a conserved recombination signal sequence (RSS) mediates the assembly of immunoglobulin (Ig) and T cell receptor (TCR) genes in B and T cell precursors. The rearrangement of particular Ig and TCR gene segments, however, is tightly regulated with respect to cell lineage and developmental stage. Using an in vitro system, we analyzed recombinase cleavage of RSSs flanking Ig and TCR gene segments in nuclei. Wefound that both the lineage-specificity and temporal ordering of gene rearrangement is reflected in the accessibility of RSSs within chromatin to in vitro cleavage. [ABSTRACT FROM AUTHOR]

Endosomal TLRs play an important role in systemic autoimmune diseases, such as systemic erythematosus lupus, in which DNAand RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune-prone mice develop more severe clinical disease, whereas TLR7-deficient and TLR7/9-double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we directly compared the functional properties of autoantigen-activated wild-type, TLR9-deficient, and TLR7-deficient B cells in an experimental system in which proliferation depends on BCR/TLR coengagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than are either wild-type or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody-producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, as well as TLR7 to promote, the clinical features of systemic erythematosus lupus. [ABSTRACT FROM AUTHOR]