In Vivo Reinsertion of Excised Episomes by the V(D)J Recombinase: A Potential Threat to Genomic Stability

It has long been thought that signal joints, the byproducts of V(D)J recombination, are not involved in the dynamics of the rearrangement process. Evidence has now started to accumulate that this is not the case, and that signal joints play unsuspected roles in events that might compromise genomic integrity. Here we show both ex vivo and in vivo that the episomal circles excised during the normal process of receptor gene rearrangement may be reintegrated into the genome through trans-V(D)J recombination occurring between the episomal signal joint and an immunoglobulin/T-cell receptor target. We further demonstrate that cryptic recombination sites involved in T-cell acute lymphoblastic leukemia–associated chromosomal translocations constitute hotspots of insertion. Eventually, the identification of two in vivo cases associating episomal reintegration and chromosomal translocation suggests that reintegration events are linked to genomic instability. Altogether, our data suggest that V(D)J-mediated reintegration of episomal circles, an event likely eluding classical cytogenetic screenings, might represent an additional potent source of genomic instability and lymphoid cancer

Overexpression of uridine diphospho glucuronosyltransferase 2B17 in high risk chronic lymphocytic leukemia

Uridine diphospho glucuronosyltransferase 2B17 (UGT2B17) glucuronidates androgens and xenobiotics including certain drugs. The UGT2B17 gene shows a remarkable copy number variation (CNV), which predisposes for solid tumors and influences drug response. Here, we identify a yet undescribed UGT2B17 mRNA overexpression in poor-risk chronic lymphocytic leukemia (CLL). In total, 320 CLL patients and 449 healthy donors were analyzed. High (above median) UGT2B17 expression was associated with established CLL poor prognostic factors and resulted in shorter treatment-free and overall survival (hazard ratio ([death] 2.18; 95% CI 1.18-4.01; P = .013). The prognostic impact of mRNA expression was more significant than that of UGT2B17 CNV. UGT2B17 mRNA levels in primary CLL samples directly correlated with functional glucuronidation activity toward androgens and the anticancer drug vorinostat (R > 0.9, P < .001). After treatment with fludarabine containing regimens UGT2B17 was up-regulated particularly in poor responders (P = .030). We observed an exclusive involvement of the 2B17 isoform within the UGT protein family. Gene expression profiling of a stable UGT2B17 knockdown in the CLL cell line MEC-1 demonstrated a significant involvement in key cellular processes. These findings establish a relevant role of UGT2B17 in CLL with functional consequences and potential therapeutic implications

Recombinase, chromosomal translocations and lymphoid neoplasia: targeting mistakes and repair failures.

A large number of lymphoid malignancies is characterized by specific chromosomal translocations, which are closely linked to the initial steps of pathogenesis. The hallmark of these translocations is the ectopic activation of a silent proto-oncogene through its relocation at the vicinity of an active regulatory element. Due to the unique feature of lymphoid cells to somatically rearrange and mutate receptor genes, and to the corresponding strong activity of the immune enhancers/promoters at that stage of cell development, B- and T-cell differentiation pathways represent propitious targets for chromosomal translocations and oncogene activation. Recent progress in the understanding of the V(D)J recombination process has allowed a more accurate definition of the translocation mechanisms involved, and has revealed that V(D)J-mediated translocations result both from targeting mistakes of the recombinase, and from illegitimate repair of the V(D)J recombination intermediates. Surprisingly, V(D)J-mediated translocations turn out to be restricted to two specific sub-types of lymphoid malignancies, T-cell acute lymphoblastic leukemias, and a restricted set of mature B-cell Non-Hodgkin's lymphomas

Sex-dependent association of circulating sex steroids and pituitary hormones with treatment-free survival in chronic lymphocytic leukemia patients

Chronic lymphocytic leukemia (CLL) is not considered a hormone-regulated cancer although sex is a recognized risk factor with men more frequently diagnosed and developing progressive disease. We hypothesized that variable hormonal exposure may have a sexually dimorphic influence on treatment-free survival (TFS). In 156 CLL cases, we quantitatively profiled 29 circulating steroids (progesterone, adrenal precursors, androgens, estrogens, and catechol estrogens) as well as luteinizing hormone (LH) and follicle-stimulating hormone. Median TFS was shorter for men than that for women (80.7 vs. 135.0 months, P =0.033). Circulating hormone profiles in CLL patients were significantly different from those of healthy donors. In male CLL cases, higher LH levels were associated with shorter TFS (adjusted hazard ratio (HRadj) 2.11; P =0.004). In female CLL cases, high levels of the potent androgens testosterone and dihydrotestosterone and the sum of methoxy estrogens were associated with an improved TFS with HRadj values of 0.24 (P =0.007), 0.54 (P =0.023), and 0.31 (P =0.034), respectively. Reduced TFS was observed for women with CLL exhibiting high expression of the steroid-inactivating UGT2B17 enzyme. This study is the first to establish a link between the outcome of CLL patients, sex steroids, and pituitary hormones, revealing a sex-specific hormonal imbalance associated with disease progression.(VLID)359162

Cannabinoid Receptors Are Overexpressed in CLL but of Limited Potential for Therapeutic Exploitation

<div><p>The cannabinoid receptors 1 and 2 (CNR1&2) are overexpressed in a variety of malignant diseases and cannabinoids can have noteworthy impact on tumor cell viability and tumor growth. Patients diagnosed with chronic lymphocytic leukemia (CLL) present with very heterogeneous disease characteristics translating into highly differential risk properties. To meet the urgent need for refinement in risk stratification at diagnosis and the search for novel therapies we studied CNR expression and response to cannabinoid treatment in CLL. Expression levels of CNR1&2 were determined in 107 CLL patients by real-time PCR and analyzed with regard to prognostic markers and survival. Cell viability of primary CLL cells was determined in suspension and co-culture after incubation in increasing cannabinoid concentrations under normal and reduced serum conditions and in combination with fludarabine. Impact of cannabinoids on migration of CLL cells towards CXCL12 was determined in transwell plates. We found CNR1&2 to be overexpressed in CLL compared to healthy B-cells. Discriminating between high and low expressing subgroups, only high CNR1 expression was associated with two established high risk markers and conferred significantly shorter overall and treatment free survival. Viability of CLL primary cells was reduced in a dose dependent fashion upon incubation with cannabinoids, however, healthy cells were similarly affected. Under serum reduced conditions, no significant differences were observed within suspension and co-culture, respectively, however, the feeder layer contributed significantly to the survival of CLL cells compared to suspension culture conditions. No significant differences were observed when treating CLL cells with cannabinoids in combination with fludarabine. Interestingly, biologic activity of cannabinoids was independent of both CNR1&2 expression. Finally, we did not observe an inhibition of CXCL12-induced migration by cannabinoids. In contrast to other tumor entities, our data suggest a limited usability of cannabinoids for CLL therapy. Nonetheless, we could define CNR1 mRNA expression as novel prognostic marker.</p></div

“Candidatus Thiobios zoothamnicoli,” an Ectosymbiotic Bacterium Covering the Giant Marine Ciliate Zoothamnium niveum

Zoothamnium niveum is a giant, colonial marine ciliate from sulfide-rich habitats obligatorily covered with chemoautotrophic, sulfide-oxidizing bacteria which appear as coccoid rods and rods with a series of intermediate shapes. Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization showed that the ectosymbiont of Z. niveum belongs to only one pleomorphic phylotype. The Z. niveum ectosymbiont is only moderately related to previously identified groups of thiotrophic symbionts within the Gammaproteobacteria, and shows highest 16S rRNA sequence similarity with the free-living sulfur-oxidizing bacterial strain ODIII6 from shallow-water hydrothermal vents of the Mediterranean Sea (94.5%) and an endosymbiont from a deep-sea hydrothermal vent gastropod of the Indian Ocean Ridge (93.1%). A replacement of this specific ectosymbiont by a variety of other bacteria was observed only for senescent basal parts of the host colonies. The taxonomic status “Candidatus Thiobios zoothamnicoli” is proposed for the ectosymbiont of Z. niveum based on its ultrastructure, its 16S rRNA gene, the intergenic spacer region, and its partial 23S rRNA gene sequence

Comparison of patient characteristics between CNR1 high and low mRNA expressing groups.

<p>Comparison of patient characteristics between CNR1 high and low mRNA expressing groups.</p

High CNR1 mRNA expression (≥ 1.52) confers significantly shorter survival in CLL patients (n = 107).

<p>(A) High expressing patients had a mean overall survival (OS) of 153 months compared to 277 months in low expressing patients (p = 0.001). (B) The mean treatment free survival (TFS) was 75 months in the CNR1 high group vs. 150 months in the CNR1 low group (p<0.0001).</p

Cytotoxic impact of cannabinoids on primary cells from healthy individuals.

<p>PBMC from 3 healthy donors were incubated in triplicates in suspension culture in increasing concentrations of compounds. Viability was determined after 48h, mean values and standard deviations are shown. (A) (R)-(+)-methanandamide. (B) (-)-cannabidiol. (C) ACEA. (D) JWH133. (E) AM251. (F) AM630. Note different scale on x-axis in A and D, note different scale on y-axis in D.</p

Cytotoxic impact of cannabinoids on CLL primary cells.

<p>PBMC from CLL patients were incubated in triplicates both in suspension culture and in co-culture with M2-10B4 mouse fibroblast cells in increasing concentrations of compounds. Viability was determined after 48h, mean values and standard deviations are shown. (A) (R)-(+)-methanandamide (N = 10). (B) (-)-cannabidiol (N = 18). (C) ACEA (N = 16). (D) JWH133 (N = 16). (E) AM251 (N = 16). (F) AM630 (N = 16). For ACEA, JWH133, and AM251, the 50% reduction in viability required for IC₅₀ calculation could not be reached in co-culture. Note different scale on x-axis in A and D.</p