Neural G0:a quiescent-like state found in neuroepithelial-derived cells and glioma

Single‐cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA‐seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent‐like state in neuroepithelial‐derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non‐dividing neural progenitors. Putative glioblastoma stem‐like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down‐regulation of quiescence‐associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent‐like state found in neuroepithelial‐derived cells and gliomas

A Meta-Analysis and Genome-Wide Association Study of Platelet Count and Mean Platelet Volume in African Americans

Several genetic variants associated with platelet count and mean platelet volume (MPV) were recently reported in people of European ancestry. In this meta-analysis of 7 genome-wide association studies (GWAS) enrolling African Americans, our aim was to identify novel genetic variants associated with platelet count and MPV. For all cohorts, GWAS analysis was performed using additive models after adjusting for age, sex, and population stratification. For both platelet phenotypes, meta-analyses were conducted using inverse-variance weighted fixed-effect models. Platelet aggregation assays in whole blood were performed in the participants of the GeneSTAR cohort. Genetic variants in ten independent regions were associated with platelet count (N = 16,388) with p<5×10−8 of which 5 have not been associated with platelet count in previous GWAS. The novel genetic variants associated with platelet count were in the following regions (the most significant SNP, closest gene, and p-value): 6p22 (rs12526480, LRRC16A, p = 9.1×10−9), 7q11 (rs13236689, CD36, p = 2.8×10−9), 10q21 (rs7896518, JMJD1C, p = 2.3×10−12), 11q13 (rs477895, BAD, p = 4.9×10−8), and 20q13 (rs151361, SLMO2, p = 9.4×10−9). Three of these loci (10q21, 11q13, and 20q13) were replicated in European Americans (N = 14,909) and one (11q13) in Hispanic Americans (N = 3,462). For MPV (N = 4,531), genetic variants in 3 regions were significant at p<5×10−8, two of which were also associated with platelet count. Previously reported regions that were also significant in this study were 6p21, 6q23, 7q22, 12q24, and 19p13 for platelet count and 7q22, 17q11, and 19p13 for MPV. The most significant SNP in 1 region was also associated with ADP-induced maximal platelet aggregation in whole blood (12q24). Thus through a meta-analysis of GWAS enrolling African Americans, we have identified 5 novel regions associated with platelet count of which 3 were replicated in other ethnic groups. In addition, we also found one region associated with platelet aggregation that may play a potential role in atherothrombosis

Anomalies moléculaires du gène PAX5 dans les leucémies aiguës lymphoblastiques de la lignée B

Les lymphocytes B sont la clé de voute de cellules de l'immunité adaptative humorale. La mise en place de la lignée B dépend de l'expression coordonnée de facteurs de transcription. Parmi ceux-ci, PAX5 a un rôle prépondérant en activant des gènes spécifiques de la lignée B et en réprimant des gènes nécessaires à l'engagement vers les autres lignages hématopoïétiques. Son expression est finement régulée durant la lymphopoïèse B jusqu'à son extinction dans les cellules matures permettant la différenciation terminale en cellules plasmocytaires productrices d'anticorps. La délétion homozygote de Pax5 dans les modèles murins conduit à une trans ou une dédifférenciation des cellules B dans plusieurs autres lignées hématopoïétiques. Les mutations de facteurs de transcription clés sont fréquemment détectées dans les hémopathies malignes. Dans ce contexte, PAX5 a été décrit comme la cible préférentielle de mutations somatiques dans les LAL-B de l'enfant dans 38.9% de cas. Nous avons réalisé une étude des mutations de PAX5 dans les LAL-B de l'adulte, inclus dans le protocole clinique GRAALL03 (Group of Research in Adult Acute Lymphoblastic Leukemia) et avons montré que PAX5 était altéré dans 34% des cas. De plus les délétions de PAX5 sont associées à la présence de BCR-ABL1 alors que les mutations ponctuelles sont toujours isolées. Ceci laisse penser que ces deux types d'altérations de PAX5 ne sont pas équivalents, la mutation ponctuelle pourrait être un événement princeps de la leucémogenèse alors que la délétion de PAX5 ne serait qu'une conséquence de l'instabilité génétique d'une cellule dans un processus leucémique. Nous avons également réalisé une recherche de translocations impliquant PAX5 dans une cohorte du GFCH (Groupe Francophone de Cytogénétique Hématologique) de 153 patients LAL-B possédant un réarrangement du 9p. Enfin nous avons entrepris la caractérisation fonctionnelle des mutants de PAX5 en utilisant le système de différenciation ex vivo décrit par Rolink et montré que les différents types de mutations n'ont pas le même impact sur la pathogenèse B.B cell lymphocytes are essential for humoral adaptive immunity. The expression of transcription factors drives the commitment of cells in B lineage. Among those factors, PAX5 fulfils a dual role by repressing B lineage ‘inappropriate’ genes and simultaneously activating B lineage–specific genes. PAX5 is expressed at similar levels throughout B cell development from the pro-B to the mature B cell stage and is subsequently repressed during terminal plasma cell differentiation. When Pax5–/– pro-B cells are stimulated with other lineage­appropriate cytokines, they are able to differentiate into functional macrophages, granulocytes, dendritic cells, osteoclasts and natural killer cells. Transcription factors mutations are frequently detected in hematopoietic cancers. The PAX5 gene was the prevalent target of somatic mutation, being altered in 38.9% of cases. We screened PAX5 mutations in a unique cohort of adult BCP-ALL treated according to the protocols of the GRAALL03 (Group of Research on Adult Acute Lymphoblastic Leukemia) and we reported that PAX5 is mutated in 34% of cases. Moreover, PAX5 complete loss and PAX5 point mutations differ. Indeed, PAX5 complete loss is significantly associated with BCR-ABL1 fusion gene and seems to be a secondary event consequence of genetic instability, whereas PAX5 point mutation might be the initial event in leukemogenesis. Using classic cytogenetic techniques and a newly developed molecular strategy, we investigated 9p abnormalities, focusing especially on the PAX5 locus, on 153 childhood and adult B-ALL include in the Groupe Francophone de Cytogénétique Hématologique (GFCH). Finally we undertook the functional characterization of PAX5 mutants using the ex vivo differentiation system described by Rolink and showed that the various kind of muations not have the same impact on the B cell pathogenesis

CRISPR/Cas9-Based Approaches for Investigating Mechanisms of Oncogene Activation and Tumor Suppression

Cancer is largely a disease of the genome. Cancer development is thought to involve the gradual acquisition of mutations that can activate oncogenes and/or inactivate tumor suppressor genes, resulting in a series of genetic changes that stimulate growth, attenuate cell death, destroy checkpoint controls, promote further genetic instability, and enable metastasis. In the first part of my thesis, I focused on deciphering how activation of oncogenes by structural copy number alterations (SCNAs) that relocate enhancers in close proximity to oncogenes can be achieved, rather than activation by mutation or amplification. This mechanism was recently described as enhancer hijacking (EHJ). I contributed to the understanding of the EHJ mechanism at two genomic loci, IGF2, a known oncogenic locus in colorectal cancer and IRS4, a gene identified as a top pan-cancer EHJ candidate. To achieve this, I recapitulated the rearrangements associated with EHJ in colorectal and lung cell lines using the CRISPR/Cas9 genomic engineering system and tested for IGF2 and IRS4 overexpression, respectively. The rearrangements were successfully reconstructed; however an increase in gene expression was not achieved, suggesting a more complex mechanism of activation or context-dependency than initially anticipated. Investigation of the tumor promoting role of IRS4 was supported using mouse xenografts, where constitutive overexpression of IRS4 leads to formation of larger tumors in comparison to control tumors. In the second part of this thesis I emphasized on the identification of genes, which, when disrupted, lead to sustained cell growth and can be potential tumor suppressors. To achieve this, I employed systematic screens on cells with different genetic backgrounds using a combination of CRISPR/Cas9-based whole genome knockout libraries and the powerful anchorage independent growth assay. I was able to verify known tumor suppressor genes, which include components of the Hippo and mTOR pathways, as well as to identify novel candidates including FRYL and AHR. Furthermore, a growth screen under non-selecting conditions was performed and identified numerous candidates found in the initial anchorage independent growth screen, which further supports the growth promoting roles of the candidate genes. In conclusion, in my study I identified potential tumor suppressors that lead untransformed cells to enhanced as well as anchorage independent growth

Expression levels of blood microRNAs as biomarker of cognitive decline due to Alzheimer's disease

Studies investigating differential miRNAs expression levels in patients with Alzheimer’s disease (AD) abounded in the last decades and catalysed the interest towards miRNAs as novel non-invasive biomarkers of AD. Chapter 1 provides an overview of AD’s pathogenesis, discusses the evolution of the disease’s definition, and introduces miRNAs. In Chapter 2, a systematic review and a P-value based meta-analysis of 107 studies investigate miRNA expression levels in AD patients. This leads to a prioritisation of 25, 32 and 5 dysregulated miRNAs at study-wide significance in the brain, the blood and the cerebrospinal fluid (CSF) of AD patients, respectively. A pathway enrichment analysis for the top dysregulated miRNAs in the brain confirms their role in regulating biological functions implicated in AD. In Chapter 3, expression levels of the 32 dysregulated miRNAs in the blood and 6 top dysregulated miRNAs in the brain of AD patients, are assessed using real-time quantitative polymerase chain reaction in the blood of cognitively healthy individuals from the CHARIOT-PRO cohort. Low performers on the total Repeatable Battery for the Assessment of Neuropsychological Status scale show downregulation of six miRNAs (hsa-miR-128-3p, hsa-miR-144-5p, hsa-miR-146a-5p, hsa-miR-26a-5p, hsa-miR-29c-3p and hsa-miR-363-3p). Pathway enrichment analysis highlights involvement in pathways initiating early pathogenetic changes in AD. Finally, in chapter 4, whole-genome sequencing data from the Alzheimer’s Disease Neuroimaging Initiative is used to perform an association analysis between polymorphisms within the six miRNAs’ genes and CSF biomarkers of neurodegeneration. A functional annotation of significant variants highlights expression quantitative trait loci, location in enhancer regions and alterations in the binding sites of transcription factors regulating neuronal function. The association of variants located within the same miRNA gene with different markers of neurodegeneration reveals a positive correlation between members of the amyloid cascade and microglial activation in the CSF. The final chapter highlights the clinical relevance of these findings and discusses future perspectives.Open Acces

Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer

ABSTRACT: BACKGROUND: Structural rearrangements form a major class of somatic variation in cancer genomes. Local chromosome shattering, termed chromothripsis, is a mechanism proposed to be the cause of clustered chromosomal rearrangements and was recently described to occur in a small percentage of tumors. The significance of these clusters for tumor development or metastatic spread is largely unclear. RESULTS: We used genome-wide long mate-pair sequencing and SNP array profiling to reveal that chromothripsis is a widespread phenomenon in primary colorectal cancer and metastases. We find large and small chromothripsis events in nearly every colorectal tumor sample and show that several breakpoints of chromothripsis clusters and isolated rearrangements affect cancer genes, including NOTCH2, EXO1 and MLL3. We complemented the structural variation studies by sequencing the coding regions of a cancer exome in all colorectal tumor samples and found somatic mutations in 24 genes, including APC, KRAS, SMAD4 and PIK3CA. A pairwise comparison of somatic variations in primary and metastatic samples indicated that many chromothripsis clusters, isolated rearrangements and point mutations are exclusively present in either the primary tumor or the metastasis and may affect cancer genes in a lesion-specific manner. CONCLUSIONS: We conclude that chromothripsis is a prevalent mechanism driving structural rearrangements in colorectal cancer and show that a complex interplay between point mutations, simple copy number changes and chromothripsis events drive colorectal tumor development and metastasis.

Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

Funding Wellcome (WT097452MA) Constantine Alifrangis Wellcome Trust (106555/Z/14/Z) Neelam Kumar Cancer Research UK (A17341) Henning Walczak Cancer Research UK Ultan McDermott Wellcome (WT107963AIA) Samuel M Janes The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Peer reviewedPublisher PD