Intérêt de la cytogénétique dans les syndromes lymphoprolifératifs chroniques

Chronic B-cell lymphoid proliferations are characterised by the presence of multiple recurrent chromosomal aberrations. A certain number of these chromosomal anomalies have been found to represent disease-specific tumour markers : the t(11;14) in mantle cell lymphoma, the t(14;18) in follicular lymphoma and the t(11;18) in lowgrade MALT lymphoma, for instance. These tumour-specific genetic markers are not only useful in patient diagnosis and follow-up, but can also have great prognostic significance. Recent prospective studies in chronic lymphocytic leukaemias have shown that in multivariate analysis, 17p and 11q deletions are the two strongest independant predictors of survival followed by age and RAI stage.The occurrence of common genetic changes in different categories of chronic lymphoid disorders is suggestive of common disease mechanisms in these apparently distinct disease entities. Therefore, it is to be expected that further multidisciplinary studies on these lymphomas would identify other potentially disease- or progression-specific chromosomal aberrations useful for accurate classification.Finally, cytogenetic studies coupled with FISH mapping have already proved instrumental in identifying hot spots of rearrangement in human cancer which in turn have served as chromosomal guides for the cloning of new genes important in oncogenesis or tumour progression.     

Chromosome stability, in the absence of apoptosis, is critical for suppression of tumorigenesis in Trp53 mutant mice

The p53 protein integrates multiple upstream signals and functions as a tumor suppressor by activating distinct downstream genes. At the cellular level, p53 induces apoptosis, cell cycle arrest and senescence. A rare mutant form of p53 with the amino acid substitution R175P, found in human tumors, is completely defective in initiating apoptosis but still induces cell cycle arrest. To decipher the functional importance of these pathways in spontaneous tumorigenesis, we used homologous recombination to generate mice with mutant p53-R172P (the mouse equivalent of R175P in humans). Mice inheriting two copies of this mutation (Trp53(515C/515C)) escape the early onset of thymic lymphomas that characterize Trp53-null mice. At 7 months of age, 90% of Trp53-null mice had died, but 85% of Trp53(515C/515C) mice were alive and tumor-free, indicating that p53-dependent apoptosis was not required for suppression of early onset of spontaneous tumors. The lymphomas and sarcomas that eventually developed in Trp53(515C/515C) mice retained a diploid chromosome number, in sharp contrast to aneuploidy observed in tumors and cells from Trp53-null mice. The ability of mutant p53-R172P to induce a partial cell cycle arrest and retain chromosome stability are crucial for suppression of early onset tumorigenesis.     

A common polymorphism acts as an intragenic modifier of mutant p53 behaviour

The p73 protein, a homologue of the tumour-suppressor protein p53, can activate p53-responsive promoters and induce apoptosis in p53-deficient cells. Here we report that some tumour-derived p53 mutants can bind to and inactivate p73. The binding of such mutants is influenced by whether TP53 (encoding p53) codon 72, by virtue of a common polymorphism in the human population, encodes Arg or Pro. The ability of mutant p53 to bind p73, neutralize p73-induced apoptosis and transform cells in cooperation with EJ-Ras was enhanced when codon 72 encoded Arg. We found that the Arg-containing allele was preferentially mutated and retained in squamous cell tumours arising in Arg/Pro germline heterozygotes. Thus, inactivation of p53 family members may contribute to the biological properties of a subset of p53 mutants, and a polymorphic residue within p53 affects mutant behaviour.     

Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity

Expression of oncogenic Ras in primary human cells activates p53, thereby protecting cells from transformation. We show that in Ras-expressing IMR-90 cells, p53 is phosphorylated at Ser33 and Ser46 by the p38 mitogen-activated protein kinase (MAPK). Activity of p38 MAPK is regulated by the p53-inducible phosphatase PPM1D, creating a potential feedback loop. Expression of oncogenic Ras suppresses PPM1D mRNA induction, leaving p53 phosphorylated at Ser33 and Ser46 and in an active state. Retrovirus-mediated overexpression of PPM1D reduced p53 phosphorylation at these sites, abrogated Ras-induced apoptosis and partially rescued cells from cell-cycle arrest. Inactivation of p38 MAPK (the product of Mapk14) in vivo by gene targeting or by PPM1D overexpression expedited tumor formation after injection of mouse embryo fibroblasts (MEFs) expressing E1A+Ras into nude mice. The gene encoding PPM1D (PPM1D, at 17q22/q23) is amplified in human breast-tumor cell lines and in approximately 11% of primary breast tumors, most of which harbor wildtype p53. These findings suggest that inactivation of the p38 MAPK through PPM1D overexpression resulting from PPM1D amplification contributes to the development of human cancers by suppressing p53 activation.     

Spatial proximity of translocation-prone gene loci in human lymphomas

Cancer cells frequently have disease-specific chromosome rearrangements. It is poorly understood why translocations between chromosomes recur at specific breakpoints in the genome. Here we provide evidence that higher-order spatial genome organization is a contributing factor in the formation of recurrent translocations. We show that MYC, BCL and immunoglobulin loci, which are recurrently translocated in various B-cell lymphomas, are preferentially positioned in close spatial proximity relative to each other in normal B cells. Loci in spatial proximity are non-randomly positioned towards the nuclear interior in normal B cells. This locus proximity is the consequence of higher-order genome structure rather than a property of individual genes. Our results suggest that the formation of specific translocations in human lymphomas, and perhaps other tissues, is determined in part by higher-order spatial organization of the genome.     

Genetic variation in DLG5 is associated with inflammatory bowel disease

Crohn disease and ulcerative colitis are two subphenotypes of inflammatory bowel disease (IBD), a complex disorder resulting from gene-environment interaction. We refined our previously defined linkage region for IBD on chromosome 10q23 and used positional cloning to identify genetic variants in DLG5 associated with IBD. DLG5 encodes a scaffolding protein involved in the maintenance of epithelial integrity. We identified two distinct haplotypes with a replicable distortion in transmission (P = 0.000023 and P = 0.004 for association with IBD, P = 0.00012 and P = 0.04 for association with Crohn disease). One of the risk-associated DLG5 haplotypes is distinguished from the common haplotype by a nonsynonymous single-nucleotide polymorphism 113G-->A, resulting in the amino acid substitution R30Q in the DUF622 domain of DLG5. This mutation probably impedes scaffolding of DLG5. We stratified the study sample according to the presence of risk-associated CARD15 variants to study potential gene-gene interaction. We found a significant difference in association of the 113A DLG5 variant with Crohn disease in affected individuals carrying the risk-associated CARD15 alleles versus those carrying non-risk-associated CARD15 alleles. This is suggestive of a complex pattern of gene-gene interaction between DLG5 and CARD15, reflecting the complex nature of polygenic diseases. Further functional studies will evaluate the biological significance of DLG5 variants.     

Birc2 (cIap1) regulates endothelial cell integrity and blood vessel homeostasis

Integrity of the blood vessel wall is essential for vascular homeostasis and organ function. A dynamic balance between endothelial cell survival and apoptosis contributes to this integrity during vascular development and pathological angiogenesis. The genetic and molecular mechanisms regulating these processes in vivo are still largely unknown. Here, we show that Birc2 (also known as cIap1) is essential for maintaining endothelial cell survival and blood vessel homeostasis during vascular development. Using a forward-genetic approach, we identified a zebrafish null mutant for birc2, which shows severe hemorrhage and vascular regression due to endothelial cell integrity defects and apoptosis. Using genetic and molecular approaches, we show that Birc2 positively regulates the formation of the TNF receptor complex I in endothelial cells, thereby promoting NF-kappaB activation and maintaining vessel integrity and stabilization. In the absence of Birc2, a caspase-8-dependent apoptotic program takes place that leads to vessel regression. Our findings identify Birc2 and TNF signaling components as critical regulators of vascular integrity and endothelial cell survival, thereby providing an additional target pathway for the control of angiogenesis and blood vessel homeostasis during embryogenesis, regeneration and tumorigenesis.     

Germline gain-of-function mutations in SOS1 cause Noonan syndrome

Noonan syndrome, the most common single-gene cause of congenital heart disease, is characterized by short stature, characteristic facies, learning problems and leukemia predisposition. Gain-of-function mutations in PTPN11, encoding the tyrosine phosphatase SHP2, cause approximately 50% of Noonan syndrome cases. SHP2 is required for RAS-ERK MAP kinase (MAPK) cascade activation, and Noonan syndrome mutants enhance ERK activation ex vivo and in mice. KRAS mutations account for <5% of cases of Noonan syndrome, but the gene(s) responsible for the remainder are unknown. We identified missense mutations in SOS1, which encodes an essential RAS guanine nucleotide-exchange factor (RAS-GEF), in approximately 20% of cases of Noonan syndrome without PTPN11 mutation. The prevalence of specific cardiac defects differs in SOS1 mutation-associated Noonan syndrome. Noonan syndrome-associated SOS1 mutations are hypermorphs encoding products that enhance RAS and ERK activation. Our results identify SOS1 mutants as a major cause of Noonan syndrome, representing the first example of activating GEF mutations associated with human disease and providing new insights into RAS-GEF regulation.     

Life extension through neurofibromin mitochondrial regulation and antioxidant therapy for neurofibromatosis-1 in Drosophila melanogaster

We investigated the pathophysiology of neurofibromatosis-1 (NF1) in Drosophila melanogaster by inactivation or overexpression of the NF1 gene. NF1 gene mutants had shortened life spans and increased vulnerability to heat and oxidative stress in association with reduced mitochondrial respiration and elevated reactive oxygen species (ROS) production. Flies overexpressing NF1 had increased life spans, improved reproductive fitness, increased resistance to oxidative and heat stress in association with increased mitochondrial respiration and a 60% reduction in ROS production. These phenotypic effects proved to be modulated by the adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A pathway, not the Ras/Raf pathway. Treatment of wild-type D. melanogaster with cAMP analogs increased their life span, and treatment of NF1 mutants with metalloporphyrin catalytic antioxidant compounds restored their life span. Thus, neurofibromin regulates longevity and stress resistance through cAMP regulation of mitochondrial respiration and ROS production, and NF1 may be treatable using catalytic antioxidants.     

Telomerase-deficient mice with short telomeres are resistant to skin tumorigenesis

Inhibition of telomerase is proposed to limit the growth of cancer cells by triggering telomere shortening and cell death. Telomere maintenance by telomerase is sufficient, in some cell types, to allow immortal growth. Telomerase has been shown to cooperate with oncogenes in transforming cultured primary human cells into neoplastic cells, suggesting that telomerase activation contributes to malignant transformation. Moreover, telomerase inhibition in human tumour cell lines using dominant-negative versions of TERT leads to telomere shortening and cell death. These findings have led to the proposition that telomerase inhibition may result in cessation of tumour growth. The absence of telomerase from most normal cells supports the potential efficacy of anti-telomerase drugs for tumour therapy, as its inhibition is unlikely to have toxic effects. Mice deficient for Terc RNA (encoding telomerase) lack telomerase activity, and constitute a model for evaluating the role of telomerase and telomeres in tumourigenesis. Late-generation Terc-/- mice show defects in proliferative tissues and a moderate increase in the incidence of spontaneous tumours in highly proliferative cell types (lymphomas, teratocarcinomas). The appearance of these tumours is thought to be a consequence of chromosomal instability in these mice. These observations have challenged the expected effectiveness of anti-telomerase-based cancer therapies. Different cell types may nonetheless vary in their sensitivity to the chromosomal instability produced by telomere loss or to the activation of telomere-rescue mechanisms. Here we show that late-generation Terc-/- mice, which have short telomeres and are telomerase-deficient, are resistant to tumour development in multi-stage skin carcinogenesis. Our results predict that an anti-telomerase-based tumour therapy may be effective in epithelial tumours.     

Tissue-specific and reversible RNA interference in transgenic mice

Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.     

Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis

We present a genome-wide association study of ileal Crohn disease and two independent replication studies that identify several new regions of association to Crohn disease. Specifically, in addition to the previously established CARD15 and IL23R associations, we identified strong and significantly replicated associations (combined P < 10(-10)) with an intergenic region on 10q21.1 and a coding variant in ATG16L1, the latter of which was also recently reported by another group. We also report strong associations with independent replication to variation in the genomic regions encoding PHOX2B, NCF4 and a predicted gene on 16q24.1 (FAM92B). Finally, we demonstrate that ATG16L1 is expressed in intestinal epithelial cell lines and that functional knockdown of this gene abrogates autophagy of Salmonella typhimurium. Together, these findings suggest that autophagy and host cell responses to intracellular microbes are involved in the pathogenesis of Crohn disease.     

A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1

We performed a genome-wide association study of 19,779 nonsynonymous SNPs in 735 individuals with Crohn disease and 368 controls. A total of 7,159 of these SNPs were informative. We followed up on all 72 SNPs with P 0.4), these data suggest that the underlying biological process may be specific to Crohn disease.     

A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion

The identification of subtype-specific translocations has revolutionized the diagnostics of sarcoma and has provided new insight into oncogenesis. We used RNA-seq to investigate samples from individuals diagnosed with small round cell tumors of bone, possibly Ewing sarcoma, but which lacked the canonical EWSR1-ETS translocation. A new fusion was observed between BCOR (encoding the BCL6 co-repressor) and CCNB3 (encoding the testis-specific cyclin B3) on the X chromosome. RNA-seq results were confirmed by RT-PCR and through cloning of the tumor-specific genomic translocation breakpoints. In total, 24 BCOR-CCNB3-positive tumors were identified among a series of 594 sarcoma cases. Gene profiling experiments indicated that BCOR-CCNB3-positive cases are biologically distinct from other sarcomas, particularly Ewing sarcoma. Finally, we show that CCNB3 immunohistochemistry is a powerful diagnostic marker for this subgroup of sarcoma and that overexpression of BCOR-CCNB3 or of truncated CCNB3 activates S phase in NIH3T3 cells. Thus, the intrachromosomal X-chromosome fusion described here represents a new subtype of bone sarcoma caused by a newly identified gene fusion mechanism.