Molecular and cytological features of the mouse B-cell lymphoma line iMyc(Eμ)-1

BACKGROUND: Myc-induced lymphoblastic B-cell lymphoma (LBL) in iMyc(Eμ )mice 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 iMyc(Eμ )mice that carry a His(6)-tagged mouse Myc cDNA, Myc(His), 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 iMyc(Eμ )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 iMyc(Eμ)-1, for the in-depth evaluation of LBL in vitro. METHODS: The morphological features and the surface marker expression profile of the iMyc(Eμ)-1 cells were evaluated using cytological methods and FACS, respectively. The cytogenetic make-up of the iMyc(Eμ)-1 cells was assessed by spectral karyotyping (SKY). The expression of the inserted Myc(His )gene was determined using RT-PCR and qPCR. Clonotypic immunoglobulin gene arrangements were detected by Southern blotting. The global gene expression program of the iMyc(Eμ)-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 iMyc(Eμ)-1 cells were found to be neoplastic IgM(high)IgD(low )lymphoblasts that expressed typical B-cell surface markers including CD40, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2). The iMyc(Eμ)-1 cells harbored a reciprocal T(9;11) and three non-reciprocal chromosomal translocations, over-expressed Myc(His )at the expense of normal Myc, and exhibited gene expression changes on Mouse Lymphochip(© )microarrays that were consistent with Myc(His)-driven B-cell neoplasia. Upon comparison to normal B cells using eight different Superarray(© )cDNA macroarrays, the iMyc(Eμ)-1 cells showed the highest number of changes on the NFκB array. CONCLUSION: The iMyc(Eμ)-1 cells may provide a uniquely useful model system to study the growth and survival requirements of Myc-driven mouse LBL in vitro

Distinct gene expression profiles in different B-cell compartments in human peripheral lymphoid organs

BACKGROUND: There are three major B-cell compartments in peripheral lymphoid organs: the germinal center (GC), the mantle zone (MNZ) and the marginal zone (MGZ). Unique sets of B-cells reside in these compartments, and they have specific functional roles in humoral immune response. MNZ B cells are naïve cells in a quiescent state and may participate in GC reactions upon proper stimulation. The adult splenic MGZ contains mostly memory B cells and is also known to provide a rapid response to particulate antigens. The GC B-cells proliferate rapidly and undergo selection and affinity maturation. The B-cell maturational process is accompanied by changes in the expression of cell-surface and intracellular proteins and requires signals from the specialized microenvironments. RESULTS: We performed laser microdissection of the three compartments for gene expression profiling by cDNA microarray. The transcriptional program of the GC was dominated by upregulation of genes associated with proliferation and DNA repair or recombination. The MNZ and MGZ showed increased expression of genes promoting cellular quiescence. The three compartments also revealed distinct repertoires of apoptosis-associated genes, chemokines and chemokine receptors. The MNZ and GC showed upregulation of CCL20 and CCL18 respectively. The MGZ was characterized by high expression of many chemokines genes e.g. CXCL12, CCL3, CCL14 and IFN-associated genes, consistent with its role in rapid response to infections. A stromal signature was identified including genes associated with macrophages or with synthesis of extracellular matrix and genes that influenced lymphocyte migration and survival. Differentially expressed genes that did not belong to the above categories include the well characterized BCL6 and CD10 and many others whose function is not known. CONCLUSIONS: Transcriptional profiling of B-cell compartments has identified groups of genes involved in critical molecular and cellular events that affect proliferation, survival migration, and differentiation of the cells. The gene expression study of normal B-cell compartments may additionally contribute to our understanding of the molecular abnormalities of the corresponding lymphoid tumors

Regulation of normal B-cell differentiation and malignant B-cell survival by OCT2.

The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3 Finally, by introducing mutations designed to disrupt the OCT2-OCA-B interface, we reveal a requirement for this protein-protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell-restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. DJH was supported by a Kay Kendall Leukaemia Fund Intermediate Fellowship from the UK.This is the author accepted manuscript. The final version is available from the National Academy of Sciences via http://dx.doi.org/10.1073/pnas.160055711

'Gene shaving' as a method for identifying distinct sets of genes with similar expression patterns

BACKGROUND: Large gene expression studies, such as those conducted using DNA arrays, often provide millions of different pieces of data. To address the problem of analyzing such data, we describe a statistical method, which we have called 'gene shaving'. The method identifies subsets of genes with coherent expression patterns and large variation across conditions. Gene shaving differs from hierarchical clustering and other widely used methods for analyzing gene expression studies in that genes may belong to more than one cluster, and the clustering may be supervised by an outcome measure. The technique can be 'unsupervised', that is, the genes and samples are treated as unlabeled, or partially or fully supervised by using known properties of the genes or samples to assist in finding meaningful groupings. RESULTS: We illustrate the use of the gene shaving method to analyze gene expression measurements made on samples from patients with diffuse large B-cell lymphoma. The method identifies a small cluster of genes whose expression is highly predictive of survival. CONCLUSIONS: The gene shaving method is a potentially useful tool for exploration of gene expression data and identification of interesting clusters of genes worth further investigation

XBP1, Downstream of Blimp-1, Expands the Secretory Apparatus and Other Organelles, and Increases Protein Synthesis in Plasma Cell Differentiation

AbstractThe differentiation of B cells into immunoglobulin-secreting plasma cells is controlled by two transcription factors, Blimp-1 and XBP1. By gene expression profiling, we defined a set of genes whose induction during mouse plasmacytic differentiation is dependent on Blimp-1 and/or XBP1. Blimp-1-deficient B cells failed to upregulate most plasma cell-specific genes, including xbp1. Differentiating xbp1-deficient B cells induced Blimp-1 normally but failed to upregulate genes encoding many secretory pathway components. Conversely, ectopic expression of XBP1 induced a wide spectrum of secretory pathway genes and physically expanded the endoplasmic reticulum. In addition, XBP1 increased cell size, lysosome content, mitochondrial mass and function, ribosome numbers, and total protein synthesis. Thus, XBP1 coordinates diverse changes in cellular structure and function resulting in the characteristic phenotype of professional secretory cells