A Diverse Repertoire of Human Immunoglobulin Variable Genes in a Chicken B Cell Line is Generated by Both Gene Conversion and Somatic Hypermutation

Abstract Immunoglobulin (Ig) gene somatic hypermutation (SHM) is a prognostic factor in small B-cell lymphomas, as demonstrated by studies reporting that hypermutated chronic lymphocytic leukemia (CLL) and splenic marginal zone lymphoma (SMZL) cases show a better prognosis, while hypermutated mantle cell lymphoma (MCL) cases display specific clinicopathological features (leukemic course, increased survival). However both the mechanisms and markers of SHM are poorly characterized, with the partial exception of the role of the genes ZAP70 and AID. With the purpose of identifying SHM surrogate markers in small B-cell lymphomas, we analyzed IgVH mutational status and expression profiles of 93 small B-cell lymphoma patient samples including SMZL (24 cases), MCL (33 cases) and CLL (36 cases). Patients were classified into two groups: high SHM (>5% mutations) and low SHM (<5% mutations). T-test analysis with 100,000 permutations was performed and 39 genes were identified whose expression is significantly different (p<0.005, FDR<0.05) between cases with high and low SHM burdens. To dissect the molecular mechanisms of Ig somatic hypermutation and validate the observed findings, SHM was induced in a model system and compared to results in patient samples. The BL2 cell line was used, and SHM was induced by treating the cells with IL4 and CD40. After 24 hours, active SHM was confirmed by western blot using an anti-AID monoclonal antibody on treated and untreated cells. These same cells were analyzed using microarrays, to identify the genes that were induced or downregulated during the SHM activation. A total of 29 genes (18 upregulated and 11 downregulated) were identified which are significantly differentially expressed in the cell line model during induction of SHM (expression change >0.4 Log2 scale) and are also differentially expressed between cases with high and low SHM (p<0.001, FDR<0.1). In both analyses, the upregulated genes are implicated in transcription, DNA repair and replication and chromosome maintenance, correlating well with previous hypotheses indicating that active transcription is necessary for SHM. Based on these observations, a group of 10 key genes, implicated in DNA repair, replication and transcription, were selected and protein expression was analyzed in a set of tissue microrrays containing 150 paraffin embedded small B-cell lymphoma cases, for which clinical data is available and SHM status is known. These proteins may provide a surrogate marker for prognosis and/or analysis of IgVH SHM in patient samples using paraffin-embedded tissue samples.

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Spliceosome Mediated RNA Trans-Splicing for Targeting Kappa+ B-Cell Neoplasms

Blood ◽

10.1182/blood.v124.21.3633.3633 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3633-3633

Author(s):

Lukas Weiss ◽

Christina Gruber ◽

Ulrich Koller ◽

Josefina Piñón-Hofbauer ◽

Stefan Hainzl ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Line ◽

Light Chain ◽

Somatic Hypermutation ◽

Kappa Light Chain ◽

Specific Therapy ◽

Trans Splicing ◽

Anti Cd20 ◽

Light Chain Locus

Abstract Introduction B-cells express either kappa or lambda light chains but not both. B-cell directed therapies such as anti-CD20 antibodies deplete healthy and malignant CD20+ B-cells but spare CD20- plasma and myeloma cells. Furthermore anti-CD20 antibody mediated B-cell depletion can lead to severe hypoimmunoglobulinemia and thereby predispose for infections. Light-chain specific targeting would allow a more specific therapy of lymphomas and myeloma, with collateral damage limited to only half of healthy B-cells. Due to the heterogeneity of immunoglobulines, lymphoma specific therapy so far required customized solutions for every single patient. Despite having 40 variable and 5 joining regions, the kappa light chain has only 1 constant region (IGKC). Therefore the 5’ IGKC intron is conserved in kappa light chain pre-mRNA, regardless of any recombination or somatic hypermutation in the VJ-region. Spliceosome-mediated RNA trans-splicing allows endogenous pre-mRNA to be converted into a new gene product via exon replacement. This can be achieved by the introduction of an RNA trans-splicing molecule (RTMRTM) that binds to endogenous target RNA and induces trans-splicing between the target gene and the RTM. RTMs contain a binding domain (BD), which defines the target specificity, splicing elements for efficient trans-splicing, and the desired coding sequence. This proof-of-principle study was aimed at demonstrating the feasibility of light-chain specific targeting through spliceosome-mediated RNA trans-splicing. Methods This study was approved by the local ethics committee of the provincial government of Salzburg. Potential RTMs for trans-splicing were identified using a fluorescence screening procedure as previously described (Gruber C et al. Mol Cancer Ther. 2011 Feb;10(2):233-41.). Primary lymphoma and myeloma samples were collected during routine diagnostic testing. Total RNA isolation was performed with RNeasy® isolation kit (QIAGEN) and reverse transcription of RNA using the i-script cDNASynthesis Kit (Bio-Rad). Quantitative real-time polymerase chain reaction was performed using GoTaq® qPCR Master Mix (Promega). For western blotting, cell lysates were separated using 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis. After transferring the proteins onto a nitrocellulose membrane, immune detection of specific proteins was performed using anti-Green Fluorescent Protein (GFP) antibodies and enhanced chemiluminescence detection. Results BD screening identified RTM 55 as the most efficient trans-splicing molecule and was therefore used for further experiments. Since the light chain locus undergoes substantial physiologic genetic changes in the process of VJ rearrangement and somatic hypermutation, we analysed whether these changes extend to the target sequence of the BD of RTM 55. Sequencing of the complete 5’ IGKC intron in primary kappa+ CLL (N=4), DLBCL (N=3) and myeloma cells (N=3) only showed occasional point mutations. In the following, effective endogenous trans-splicing was confirmed in 2 different kappa+ cell lines, the DLBCL cell line SUDHL4 and the CLL cell line MEC2. We could detect successful trans-splicing on the mRNA level and sequencing of PCR-products confirmed accurate trans-splicing with the last base of the J-region adjoining the first base of the GFP gene. Stable retroviral transfection of SUDHL4 cells also allowed the successful detection of the kappa light-chain-GFP fusion protein by western blotting. Surprisingly, the lambda+ myeloma cell line U266 as well as FACS-sorted healthy lambda+ B-cells showed high expression levels of kappa light chain mRNA. This leaky mRNA expression of the non-dominant light chain could not be seen in kappa+ cells, which may be explained by the programmed sequential activation of the kappa and lambda loci during B-cell development. Conclusions We could show that the kappa light chain can be specifically targeted by spliceosome-mediated RNA trans-splicing. The possibility to reprogram the light chain pre-mRNA offers numerous possible applications, such as suicide gene therapy for lymphoma and myeloma. The unexpectedly high expression of kappa mRNA in lambda+ cells but not vice-versa, raises the question whether the lambda light chain locus would offer an even better, since more specific target Disclosures No relevant conflicts of interest to declare.

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