EZH1/2 function mostly within canonical PRC2 and exhibit proliferation-dependent redundancy that shapes mutational signatures in cancer

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Identification of genetics events involved in malignant transformation in neurofibromatosis type 1

La neurofibromatose de type 1 (NF1) est un syndrome de prédisposition tumorale causée par une mutation perte-de-fonction du gène suppresseur de tumeurs NF1. Près de la moitié des patients atteints de NF1 développent un type de tumeurs bénignes des gaines des nerfs périphériques appelés neurofibromes plexiformes. Ces tumeurs sont majoritairement constituées de cellules de Schwann présentant une inactivation somatique du deuxième allèle NF1. Les neurofibromes plexiformes peuvent se transformer en tumeurs malignes dénommées MPNST (Malignant Peripheral Nerve Sheath Tumor) qui sont des sarcomes extrêmement agressifs, résistants aux thérapies actuelles et représentant la première cause de mortalité des patients NF1. A ce jour, les acteurs à l’origine de cette transformation maligne ne sont pas clairement établis. Leur identification représente donc un enjeu majeur pour une prise en charge appropriée des patients et le développement de nouvelles molécules thérapeutiques. Dans ce contexte, le travail mené au cours de ma thèse a eu pour objectifs la recherche et la caractérisation de nouvelles voies de signalisations impliquées dans la tumorigenèse NF1. D’une part, une approche orientée par les travaux antérieurs au laboratoire a permis de montrer l’implication de la voie WNT dans la tumorigenèse NF1. D’autre part, une approche génomique plus large a conduit à la mise en évidence de l’inactivation du répresseur transcriptionnel PRC2 (Polycomb Repressive Complex 2) dans près de la moitié des MPNST. La génération de modèles cellulaires in vitro a facilité l’exploration des gènes surexprimés lors de la perte de fonction du PRC2. Elle a également permis d’entreprendre un crible lentiCRISPR pan-génomique à la recherche des gènes essentiels à la survie des cellules tumorales mutées pour le PRC2.Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by loss-offunction mutations in the NF1 tumor suppressor gene. Almost half of NF1 patients develop a specific type of benign peripheral nerve sheath tumor called plexiform neurofibromas. These tumors are mainly composed of Schwann cells in which the second NF1 allele is inactivated. Plexiform neurofibromas can give rise to malignant tumors called MPNST that are extremely aggressive sarcomas, resistant to therapy and which represents the first cause of early demise of NF1 patients. The molecular mechanisms underlying this malignant transformation remain enigmatic. Their identification is crucial for appropriate management of NF1 patients and development of new therapies. The goal of my PhD was to identify and characterize new signaling pathways involved in NF1 tumorigenesis. On the one hand, we highlighted the involvement of WNT pathway in NF1 tumorigenesis. On the other hand, a larger genomic approach led to the identification of the transcriptional repressor PRC2 (Polycomb Repressive Complex 2) inactivation in almost half of MPNST. We have generated various cell models, which facilitated the exploration of genes aberrantly expressed consequently to PRC2 loss-offunction. These models also allowed performing a pan-genomic lentiCRISPR screen searching for essential genes for PRC2-mutated tumor cells survival

Identification des évènements génétiques impliqués dans la transformation maligne de la neurofibromatose de type 1

Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by loss-offunction mutations in the NF1 tumor suppressor gene. Almost half of NF1 patients develop a specific type of benign peripheral nerve sheath tumor called plexiform neurofibromas. These tumors are mainly composed of Schwann cells in which the second NF1 allele is inactivated. Plexiform neurofibromas can give rise to malignant tumors called MPNST that are extremely aggressive sarcomas, resistant to therapy and which represents the first cause of early demise of NF1 patients. The molecular mechanisms underlying this malignant transformation remain enigmatic. Their identification is crucial for appropriate management of NF1 patients and development of new therapies. The goal of my PhD was to identify and characterize new signaling pathways involved in NF1 tumorigenesis. On the one hand, we highlighted the involvement of WNT pathway in NF1 tumorigenesis. On the other hand, a larger genomic approach led to the identification of the transcriptional repressor PRC2 (Polycomb Repressive Complex 2) inactivation in almost half of MPNST. We have generated various cell models, which facilitated the exploration of genes aberrantly expressed consequently to PRC2 loss-offunction. These models also allowed performing a pan-genomic lentiCRISPR screen searching for essential genes for PRC2-mutated tumor cells survival.La neurofibromatose de type 1 (NF1) est un syndrome de prédisposition tumorale causée par une mutation perte-de-fonction du gène suppresseur de tumeurs NF1. Près de la moitié des patients atteints de NF1 développent un type de tumeurs bénignes des gaines des nerfs périphériques appelés neurofibromes plexiformes. Ces tumeurs sont majoritairement constituées de cellules de Schwann présentant une inactivation somatique du deuxième allèle NF1. Les neurofibromes plexiformes peuvent se transformer en tumeurs malignes dénommées MPNST (Malignant Peripheral Nerve Sheath Tumor) qui sont des sarcomes extrêmement agressifs, résistants aux thérapies actuelles et représentant la première cause de mortalité des patients NF1. A ce jour, les acteurs à l’origine de cette transformation maligne ne sont pas clairement établis. Leur identification représente donc un enjeu majeur pour une prise en charge appropriée des patients et le développement de nouvelles molécules thérapeutiques. Dans ce contexte, le travail mené au cours de ma thèse a eu pour objectifs la recherche et la caractérisation de nouvelles voies de signalisations impliquées dans la tumorigenèse NF1. D’une part, une approche orientée par les travaux antérieurs au laboratoire a permis de montrer l’implication de la voie WNT dans la tumorigenèse NF1. D’autre part, une approche génomique plus large a conduit à la mise en évidence de l’inactivation du répresseur transcriptionnel PRC2 (Polycomb Repressive Complex 2) dans près de la moitié des MPNST. La génération de modèles cellulaires in vitro a facilité l’exploration des gènes surexprimés lors de la perte de fonction du PRC2. Elle a également permis d’entreprendre un crible lentiCRISPR pan-génomique à la recherche des gènes essentiels à la survie des cellules tumorales mutées pour le PRC2

Mutations in SETD2 cause a novel overgrowth condition

International audienceBACKGROUND: Overgrowth conditions are a heterogeneous group of disorders characterised by increased growth and variable features, including macrocephaly, distinctive facial appearance and various degrees of learning difficulties and intellectual disability. Among them, Sotos and Weaver syndromes are clinically well defined and due to heterozygous mutations in NSD1 and EZH2, respectively. NSD1 and EZH2 are both histone-modifying enzymes. These two epigenetic writers catalyse two specific post-translational modifications of histones: methylation of histone 3 lysine 36 (H3K36) and lysine 27 (H3K27). We postulated that mutations in writers of these two chromatin marks could cause overgrowth conditions, resembling Sotos or Weaver syndromes, in patients with no NSD1 or EZH2 abnormalities. METHODS: We analysed the coding sequences of 14 H3K27 methylation-related genes and eight H3K36 methylation-related genes using a targeted next-generation sequencing approach in three Sotos, 11 'Sotos-like' and two Weaver syndrome patients. RESULTS: We identified two heterozygous mutations in the SETD2 gene in two patients with 'Sotos-like' syndrome: one missense p.Leu1815Trp de novo mutation in a boy and one nonsense p.Gln274* mutation in an adopted girl. SETD2 is non-redundantly responsible for H3K36 trimethylation. The two probands shared similar clinical features, including postnatal overgrowth, macrocephaly, obesity, speech delay and advanced carpal ossification. CONCLUSIONS: Our results illustrate the power of targeted next-generation sequencing to identify rare disease-causing variants. We provide a compelling argument for Sotos and Sotos-like syndromes as epigenetic diseases caused by loss-of-function mutations of epigenetic writers of the H3K36 histone mark

Noninvasive Prenatal Screening for Trisomy 21 in Patients with a Vanishing Twin

A vanishing twin (VT) occurs in up to 30% of early diagnosed twin pregnancies and is associated with an increased risk of fetal aneuploidy. Here, we describe our experience in a large VT population of 847 patients that underwent noninvasive prenatal testing (NIPT) for common fetal trisomies over a three-year period. All patients underwent an ultrasound examination prior to NIPT. Two comparison populations were included, namely, the singleton (n = 105,560) and the viable multiple gestation pregnancy samples (n = 9691) collected over the same period. All NIPT samples in the VT population received a result, of which 14 were high-risk for trisomy 21 (1.6%), nine for trisomy 18 (1.1%), and six for trisomy 13 (0.7%). Diagnostic testing confirmed the presence of trisomy 21 in 6/12 samples, giving a positive predictive value of 50%. One trisomy 18 case and no trisomy 13 cases were confirmed. The time between fetal demise and NIPT sampling did not appear to affect the number of true- or false-positive cases. In conclusion, NIPT is an effective screening method for trisomy 21 in the surviving fetus(es) in VT pregnancies. For trisomies 18 and 13, a positive NIPT should be interpreted carefully and ultrasound monitoring is preferrable over invasive diagnostic testing

PRC2 loss amplifies Ras-driven transcription and confers sensitivity to BRD4-based therapies

The polycomb repressive complex 2 (PRC2) exerts oncogenic effects in many tumour types. However, loss-of-function mutations in PRC2 components occur in a subset of haematopoietic malignancies, suggesting that this complex plays a dichotomous and poorly understood role in cancer. Here we provide genomic, cellular, and mouse modelling data demonstrating that the polycomb group gene SUZ12 functions as tumour suppressor in PNS tumours, high-grade gliomas and melanomas by cooperating with mutations in NF1. NF1 encodes a Ras GTPase-activating protein (RasGAP) and its loss drives cancer by activating Ras. We show that SUZ12 loss potentiates the effects of NF1 mutations by amplifying Ras-driven transcription through effects on chromatin. Importantly, however, SUZ12 inactivation also triggers an epigenetic switch that sensitizes these cancers to bromodomain inhibitors. Collectively, these studies not only reveal an unexpected connection between the PRC2 complex, NF1 and Ras, but also identify a promising epigenetic-based therapeutic strategy that may be exploited for a variety of cancers.status: publishe

MicroRNAome profiling in benign and malignant neurofibromatosis type 1-associated nerve sheath tumors: evidences of PTEN pathway alterations in early NF1 tumorigenesis

International audienceBackground: Neurofibromatosis type 1 (NF1) is a common dominant tumor predisposition syndrome affecting 1 in 3,500 individuals. The hallmarks of NF1 are the development of peripheral nerve sheath tumors either benign (dermal and plexiform neurofibromas) or malignant (MPNSTs). Results: To comprehensively characterize the role of microRNAs in NF1 tumorigenesis, we analyzed 377 miRNAs expression in a large panel of dermal and plexiform neurofibromas, and MPNSTs. The most significantly upregulated miRNA in plexiform neurofibromas was miR-486-3p that targets the major tumor suppressor gene, PTEN. We confirmed PTEN downregulation at mRNA level. In plexiform neurofibromas, we also report aberrant expression of four miRNAs involved in the RAS-MAPK pathway (miR-370, miR-143, miR-181a, and miR-145). In MPNSTs, significant deregulated miRNAs were involved in PTEN repression (miR-301a, miR-19a, and miR-106b), RAS-MAPK pathway regulation (Let-7b, miR-195, and miR-10b), mesenchymal transition (miR-200c, let-7b, miR-135a, miR-135b, and miR-9), HOX genes expression (miR-210, miR-196b, miR-10a, miR-10b, and miR-9), and cell cycle progression (miR-195, let-7b, miR-20a, miR-210, miR-129-3p, miR-449a, and miR-106b). Conclusion: We confirmed the implication of PTEN in genesis of plexiform neurofibromas and MPNSTs in NF1. Markedly deregulated miRNAs might have potential diagnostic or prognostic value and could represent novel strategies for effective pharmacological therapies of NF1 tumors

Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study.

OBJECTIVE:NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. METHOD:After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. RESULTS:The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample's trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. CONCLUSION:Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women