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The adaptive B cell response is driven by the expansion, somatic hypermutation, and selection of B cell clonal lineages. A high number of clonal lineages in a B cell population indicates a highly diverse repertoire, while clonal size distribution and sequence diversity reflect antigen selective pressure. Identifying clonal lineages is fundamental to many repertoire studies, including repertoire comparisons, clonal tracking, and statistical analysis. Several methods have been developed to group sequences from high-throughput B cell repertoire data. Current methods use clustering algorithms to group clonally-related sequences based on their similarities or distances. Such approaches create groups by optimizing a single objective that typically minimizes intra-clonal distances. However, optimizing several objective functions can be advantageous and boost the algorithm convergence rate. Here we propose MobiLLe, a new method based on multi-objective clustering. Our approach requires V(D)J annotations to obtain the initial groups and iteratively applies two objective functions that optimize cohesion and separation within clonal lineages simultaneously. We show that our method greatly improves clonal lineage grouping on simulated benchmarks with varied mutation rates compared to other tools. When applied to experimental repertoires generated from high-throughput sequencing, its clustering results are comparable to the most performing tools and can reproduce the results of previous publications. The method based on multi-objective clustering can accurately identify clonally-related antibody sequences and presents the lowest running time among state-of-art tools. All these features constitute an attractive option for repertoire analysis, particularly in the clinical context. MobiLLe can potentially help unravel the mechanisms involved in developing and evolving B cell malignancies.

In this work, we used comparative transcriptomics to identify regulatory outliers (ROs) in the human pathogen Candida glabrata. ROs are genes that have very different expression patterns compared to their orthologs in other species. From comparative transcriptome analyses of the response of eight yeast species to toxic doses of selenite, a pleiotropic stress inducer, we identified 38 ROs in C. glabrata. Using transcriptome analyses of C. glabrata response to five different stresses, we pointed out five ROs which were more particularly responsive to iron starvation, a process which is very important for C. glabrata virulence. Global chromatin Immunoprecipitation and gene profiling analyses showed that four of these genes are actually new targets of the iron starvation responsive Aft2 transcription factor in C. glabrata. Two of them (HBS1 and DOM34b) are required for C. glabrata optimal growth in iron limited conditions. In S. cerevisiae, the orthologs of these two genes are involved in ribosome rescue by the NO GO decay (NGD) pathway. Hence, our results suggest a specific contribution of NGD co-factors to the C. glabrata adaptation to iron starvation.

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.

The oncogenic process leading to nasopharyngeal carcinoma (NPC) requires the combination of genetic and epigenetic alterations, latent infection by the Epstein-Barr virus and local inflammation. A transcriptome analysis of NPC xenografts identified the gene encoding the cellular inhibitor of apoptosis protein 2 (c-IAP2) among the top five most intensely expressed. Consistently, the very high levels of the c-IAP2 protein were detected in 11 of 13 NPC biopsies. RMT 5265, a structural analog of second mitochondria-derived activator of caspase (SMAC), induced the rapid degradation of c-IAP2 in nasopharyngeal epithelial cells, whether malignant or not, but blocked clonal cell growth in NPC cells only. In short-term experiments, RMT 5265 induced apoptosis in a fraction of NPC cells, and this apoptosis was dramatically enhanced when RMT 5265 was combined with Toll-like receptor 3 (TLR3) stimulation. By contrast, the cooperative effect with tumor necrosis factor α was only marginal. The apoptosis induced by the combination of RMT 5265 and TLR3 stimulation was mediated by caspase-8 and associated with a decrease in the cellular content of the long isoform of FLICE-like inhibitory protein. Similar caspase-8 activation was obtained when siRNA knockdown of c-IAP2 was combined with TLR3 stimulation. In conclusion, c-IAP2 has a specific protective function in NPC cells challenged by TLR3 agonists. This protective function is probably important to make NPC cells tolerant to their own production of small viral RNAs, which are potential agonists of TLR3. Our data will help to design a rational use of IAP inhibitors in NPC patients.

Gorlin's or nevoid basal cell carcinoma syndrome (NBCCS) causes predisposition to basal cell carcinoma (BCC), the commonest cancer in adult human. Mutations in the tumor suppressor gene PTCH1 are responsible for this autosomal dominant syndrome. In NBCCS patients, as in the general population, ultraviolet exposure is a major risk factor for BCC development. However these patients also develop BCCs in sun-protected areas of the skin, suggesting the existence of other mechanisms for BCC predisposition in NBCCS patients. As increasing evidence supports the idea that the stroma influences carcinoma development, we hypothesized that NBCCS fibroblasts could facilitate BCC occurence of the patients. WT (n = 3) and NBCCS fibroblasts bearing either nonsense (n = 3) or missense (n = 3) PTCH1 mutations were cultured in dermal equivalents made of a collagen matrix and their transcriptomes were compared by whole genome microarray analyses. Strikingly, NBCCS fibroblasts over-expressed mRNAs encoding pro-tumoral factors such as Matrix Metalloproteinases 1 and 3 and tenascin C. They also over-expressed mRNA of pro-proliferative diffusible factors such as fibroblast growth factor 7 and the stromal cell-derived factor 1 alpha, known for its expression in carcinoma associated fibroblasts. These data indicate that the PTCH1(+/-) genotype of healthy NBCCS fibroblasts results in phenotypic traits highly reminiscent of those of BCC associated fibroblasts, a clue to the yet mysterious proneness to non photo-exposed BCCs in NBCCS patients.

Background:RET/PTC1 rearrangement is the most common genetic alteration identified to date in papillary thyroid carcinomas (PTC) and represents an interesting target for small interfering RNA (siRNA) strategies because it is present only in the tumor cells and not in the normal cells. Our aims were (i) to target the RET/PTC1oncogene by siRNAs, (ii) to assess the knockdown effects on cell growth and cell cycle regulation, and (iii) to identify genes affected by the RET/PTC1silencing.Methods:Three efficient siRNAs previously designed in our laboratory in a model of murine PTC (RP-1 cells) were used to knockdown RET/PTC1in the TPC-1 cells. By reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (Q-RT-PCR) they were found unable to silence RET/PTC1. After sequencing, we redesigned an siRNA against RET/PTC1(siRNARET/PTC1) and compared it for its efficiency and specificity with an siRNA against RET(siRNARET) in the TPC-1 cells, in human cell lines that expressed RET(MCF-7 and BT-474 cells), and in the murine RP-1 cells. The effects on cell cycle growth (MTT tests), cell cycle (flow cytometry), and apoptosis (TUNEL method) were studied. Genes affected by the RET/PTC1knockdown were identified by microarray analysis followed by Q-RT-PCR validation.Results:A mutation was found by sequencing within the H4part of the RET/PTC1 junction leading to a 297T→G substitution. The redesigned siRNARET/PTC1 inhibits about 85% of the oncogene expression in the human TCP-1 cells. The specificity of the siRNARET/PTC1 was confirmed by the absence of a silencing effect on the human breast MCF-7 and BT-474 cells without RET/PTC1and the murine RP-1 with 297G→T mutation. The downregulation of RET/PTC1modified the cell cycle and induced an apoptotic response. Microarray analysis revealed an inhibition of E2F2transcription factor known to be involved in the cell cycle regulation.Conclusions:This study shows the impact of a point mutation within a junction oncogene on the siRNA design. In the case of a therapeutic approach by siRNA, the junction oncogene must be systematically sequenced. The E2F2gene regulation would have a biological significance and seems to be directly mediated by RET/PTC1. [ABSTRACT FROM AUTHOR]