FunGeST lab – an insight into liver tumors genomics

Group Leader

Actual Projects

Role of viral infection and integration in liver carcinogenesis

Collaborators: S Imbeaud (IR), C Peneau (PhD st), T La Bella (PhD st), J Zucman-Rossi (PUPH);

Involvement of pathogen agents in human carcinogenesis remain to be investigated. Our recent identification of AAV2 as the fourth virus involved in insertional mutagenesis prompt us to develop a project to evaluate consequences of HBV, HCV and AAV infections in liver tumorigenesis by integrating the analysis of viral and tumor genome alterations. Our aim is to reconstruct the natural history of the viruses (genome variation, replication, structural alterations, selection…) in the context of tumor cell selection in patients putatively treated by antiviral therapies or exposed to multiple pathogens. We will also search for additional pathogens in WGS data and collaborate with the international ICGC viruses project.

Fundings: LNCC, ANRS, Biomerieux Foundation

New molecular subtypes and cancer driver genes in pediatric liver tumors

Collaborators: G Morcrette (PhD st), E Letouzé (CR Inserm), J Pilet (PhD st), S Imbeaud (IR), G Couchy (IE, PhD st), Julien Calderaro (pathologist), JC Nault (MCUPH), J Zucman-Rossi (PUPH); in cooperation with SIOPEL,

Most of pediatric liver neoplasms are poorly analyszed at the genomic and molecular level. They include hepatocellular carcinomas (HCC), fibrolamellar carcinomas (FLC), hepatoblastomas (HB), transitional liver cell tumors (TLCT) and also benign neoplasms, hepatocellular adenomas (HCA) and focal nodular hyperplasias (FNH). We aim to perform an integrated genomic analysis of these pediatric hepatocellular tumors to better understand their mechanism of tumorigenesis. To this purpose, we have collected a series of 193 patients with pediatric frozen liver tumors part of the national HEPATOBIO tumor resource. Within this series, 48 cases/67 frozen tumor samples have been selected for whole genome sequencing (WGS) and RNA sequencing (RNAseq) within the GEPELIN project funded by France Génomique for the WGS part. Our specific aims are to (1) identify putative driver genes altered in pediatric tumors and validate them functionally (2) perform new transcriptomic classification to derive new altered pathways and diagnostic/prognostic markers, (3) characterize the mutational and chromosome rearrangement signatures to identify new mechanism of carcinogenesis (4) search for therapeutic targets. These results will be compared with the adult liver tumors data that we have accumulated in the lab.

Fundings:  LNCC, France Genomique.

Innovative genomic data integration to decipher the interaction between risk factors, endogeneous cellular processes and genomic alterations in liver carcinogenesis

Collaborators: E Letouzé , J Shinde , S Imbeaud (IR), L Meunier (PhD st), Q Bayard (M2), T Hirsch (Post-doc), J Zucman-Rossi (PUPH

In the last 5 years, we have generated large genomic data sets including 350 tumors analyzed by whole exome sequencing, 270 by RNA-seq, 250 by methylation arrays and 100 by whole genome sequencing. We will use this exceptional resource to unravel key questions in the natural history of liver cancers. (1) What mutational processes drive tumorigenesis? We have so far identified 10 point-mutation signatures associated with known (aflatoxin B1, tobacco) or unknown mutagenic processes in liver cancers. We will extend these analyses to signatures of indels and structural rearrangements, and perform a meta-analysis of all published liver cancer data sets to unravel the association of signatures with risk factors and predisposing variants. (2) What are the missing drivers in liver cancers? Analysis of coding mutations by us and others revealed tens of driver genes and pathways but 30% of tumors still have no identified driver event. In order to identify non-coding driver alterations, we will use whole genome and RNA-seq data to systematicallly screen for mutations and structural rearrangements modifying regulatory regions, chromatin context and 3’ UTR sequences and affecting mRNA expression and stability. (3) How to the genomic, epigenomic and transcriptional layers interact in tumor cells? We will develop innovative strategies to unravel the connexions between genomic alterations, DNA methylation and gene expression profiles. (4) What is the timing of genomic alterations along tumorigenesis? We and others have developed statistical approaches to time mutational signatures, copy-number alterations and driver events in the life history of a cancer using intra-tumor heterogeneity. We will conduct an ambitious project to analyze the evolution of genomic, transcriptional and micro-environment features in 25 patients with multiple samples along the treatment.

Fundings: Cancer environment, HTE HETCOLI, France Genomique, Canceropole

Genetic predisposition to benign and malignant liver tumors

E Letouzé (CR Inserm), P Nahon (PUPH), E Trepo (Post-doc), J Yang (Ph st), JC Nault (MCUPH), Zucman-Rossi (PUPH)

Candidate gene studies have uncovered a limited number of variants reproducibly linked to hepatocellular carcinoma (HCC). The HECAM project aims to identify newgermline variants predisposing to HCC in the French population and to test their ability to modify patient care in clinical practice. Therefore, a genome-wide association study will be performed in a multicenter cohort of chronic liver disease patients with (n = 2,066) and without (n = 2,666) HCC using the Illumina Infinium Global Screening Array including ~ 660,000 markers). Validation of the top variants will be performed in two French replication cohorts of cirrhotic patients included in HCC surveillance programs (n = 2,249). The performance for HCC risk stratification will be finally assessed through integration of genetic information into specific algorithm-based prediction models. An original integrative approach including somatic information already generated will be used for prioritizing variants impacting liver carcinogenesis. Finally, genotype-phenotype associations will be performed to build prognostic models. We also plan to analyze benign liver tumors to search for genetic predisposition to the development of hepatocellular adenoma in a cohort of 500 patients with in parallel an epidemiological study.

Fundings: HECAM, ANRS, AFEF

 

Team

Biologists

Jessica ZUCMAN-ROSSI

MD, PhD

PUPHex University Paris 5

Gabrielle COUCHY

PhD student (EPHE)

IE Inserm

Barkha GUPTA

MD PhD

Post Doc HECAM

Stefano CARUSO

PhD

Post Doc Labex

Théo HIRSCH

PhD

Post Doc Cancéropole

Tiziana LA BELLA

PhD student

University Paris 7

Camille PENEAU

PhD student

Inserm

Jie YANG

MD, PhD Student

Chinese grant

Benedicte NOBLET

-

IE Inserm

Guillaume MORCRETTE

MD, PhD Student

-

Alix DEMORY

MD, Master Student

Olivier MSIKA

MD, Master Student

Clinicians

Fundings

 

 

 

 

 

Latest Publications

APC germline hepatoblastomas demonstrate cisplatin-induced intratumor tertiary lymphoid structures. Morcrette G, Hirsch TZ, Badour E, Pilet J, Caruso S, Calderaro J, Martin Y, Imbeaud S, Letouzé E, Rebouissou S, Branchereau S, Taque S, Chardot C, Guettier C, Scoazec JY, Fabre M, Brugières L, Zucman-Rossi J. Oncoimmunology. 2019 Mar 28;8(6):e1583547. doi: 10.1080/2162402X.2019.1583547. eCollection 2019.

Genomic Medicine and Implications for Hepatocellular Carcinoma Prevention and Therapy. Dhanasekaran R, Nault JC, Roberts LR, Zucman-Rossi J. Gastroenterology. 2019 Jan;156(2):492-509. doi: 10.1053/j.gastro.2018.11.001. Epub 2018 Nov 4. Review.

Systemic AA Amyloidosis Caused by Inflammatory Hepatocellular Adenoma. Calderaro J, Letouzé E, Bayard Q, Boulai A, Renault V, Deleuze JF, Bestard O, Franco D, Zafrani ES, Nault JC, Moutschen M, Zucman-Rossi J. N Engl J Med. 2018 Sep 20;379(12):1178-1180. doi: 10.1056/NEJMc1805673.

Argininosuccinate synthase 1 and periportal gene expression in sonic hedgehog hepatocellular adenomas. Nault JC, Couchy G, Caruso S, Meunier L, Caruana L, Letouzé E, Rebouissou S, Paradis V, Calderaro J, Zucman-Rossi J. Hepatology. 2018 Sep;68(3):964-976. doi: 10.1002/hep.29884. Epub 2018 Jun 6

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Mesothelioma – the 2nd expertise of FunGeST

Group Leader

Actual Projects

D Jean, MC Jaurand, F Le Pimpec-Barthes, C Meiller, W Blum, L Quetel. Fundings: LNCC IdF and CIT, Chancellerie des Universités de Paris-Legs Poix

Malignant pleural mesothelioma (MPM) is a rare tumor, with a poor prognosis mainly due to the lack of efficient treatment. It is therefore important to develop new therapies that take into account the heterogeneity of MPM at the molecular level. We have recently defined a molecular classification of MPMs that defines two main groups C1 and C2. A subgroup of C2, with a double mutation in the Hippo, NF2 and LATS2 pathway genes, was identified. The C2 group and the NF2 / LATS2 double mutant subgroup contain MPMs from patients with a very poor prognosis. We  focus on four major aims:

(1) Refine the molecular classification of malignant pleural mesothelioma and transfer it to clinic: Integrated multi-omic analysis using our large collection of tumors will allow to identify new tumor subgroups. Identification of specific biomarkers and development of prediction tools should facilitate the implementation of this classification to clinic.

(2) Determine the mechanisms of mesothelial carcinogenesis: Functional analysis using our cell lines biobank will lead to define the contribution of specific gene alterations and signal pathways to carcinogenesis.

(3) Develop therapeutic strategy: High-content screening and validation using in vitro and in vivo models will be used to study the correlation between anti-tumor compounds sensibility and the molecular phenotype and will constitute a first step toward precision medicine for mesothelioma.

(4) Characterize intra-tumor heterogeneity: Molecular analysis will be performed to explore heterogeneity of tumor samples at different anatomical sites and presence of cancer stem cells.

 

Past Works

We performed a genetic and transcriptomic characterization of malignant pleural mesothelioma (MPM) using our tumor biobanks. We identified the first recurrent oncogenic mutation in the TERT promoter and showed this alteration was frequent in MPM of the sarcomatoid subtype (1). Based on transcriptomic data, we defined a robust molecular classification consisting of two groups (C1 and C2) with different molecular profiles, gene alterations, histology subtypes, and survival outcomes. One of the major interests was to separate the epithelioid MPM, the most frequent histologic subtype, according to their survival prognosis (2). Recently, by coupling transcriptomic and genetic analysis, we identified a new specific MPM molecular subgroup (C2^LN) characterized by a co-occurring mutation in LATS2 and NF2 tumor suppressor genes. We identified a specific biomarker and highlighted a high sensitivity to mTOR/PI3K/AKT (PF-04691502) inhibitor treatment for this subgroup (3).

Tallet et al, Oncogene, 2014; de Reynies et al… Jean, Clinical Cancer Research, 2014; Tranchant et al, Clinical Cancer Research, 2017 

Fundings

 

 

 

 

 

Team

Didier JEAN

PhD - CR1

Inserm

Marie-Claude JAURAND

PhD

DR1 Inserm - Emeritat

Clément MEILLER

-

IE Inserm

Françoise LE PIMPEC BARTHES

MD

PUPH1 University Paris 5

Arthur DUBOIS

M2 Student

 

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Computational Cancer Genome Analysis

Team Leader

Projects

In the Computational Cancer Genome Analysis group, we analyze different genomic datasets (whole-exome and whole-genome sequencing, RNA-seq, DNA methylation data), and we develop innovative computational tools to better understand the origin and the molecular diversity of tumors. We explore several research axes, as described below.

 

Identification of genes driving hepatocellular carcinogenesis

Tumor cells harbor numerous molecular alterations (mutations, chromosome gains and losses, translocations) that can alter the function or the activity of target genes. We develop computational approaches to integrate these alterations and identify genes recurrently altered, which are likely to play a key role in oncogenesis. By analyzing the exome sequences of 250 hepatocellular carcinomas, we recently identified 161 putative driver genes belonging to 11 major cellular pathways (Schulze, Imbeaud, Letouzé et al., Nat Genet 2015). We now analyze whole-genome sequencing data to identify non-coding mutations likely to affect the regulatory sequences of target genes, like the activating mutations of TERT promoter (Nault et al., Nat Commun 2013).

 

 

Mutational signatures

Somatic mutations that drive cancer progression are the consequence of spontaneous enzymatic conversions, replication errors, or mutagenic exposures like tobacco or UV light. These mutational processes leave imprints on the tumor genome that can be identified as mutational signatures, caracterized by specific types of mutations or mutations occurring in specific genomic contexts. For instance, tobacco carcinogenes induce mostly C>A mutations, whereas defects in DNA mismatch repair genes lead to an enriched frequency of C>T mutations in NCG trinucleotide context. By analyzing a large series of liver tumors by whole-exome sequencing, we have identified 2 new mutational signatures caracteristic of liver tumors (Schulze, Imbeaud, Letouzé et al., Nat Genet 2015). One of these signatures, caracterized by frequent C>A mutations at GCC trinucleotides, could be related to exposure to aflatoxin B1, a toxin produced by a mushroom in warm and wet countries of Africa and Asia. We now analyze whole-genome sequencing data from a new series of tumors, associated with diverse risk factors, to identify new signatures and distinguish mutational process operative in the early and late steps of oncogenesis. In collaboration with Pr Pierre Laurent-Puig’s team (UMR-S 1147), we also analyze the mutational signatures of other tumor types, like lung and colorectal cancer.

 

Clonal evolution of liver tumors

Tumors develop through the expansion of cell clones having acquired genomic alterations that confer them a proliferative advantage over surrounding cells. Several clones may coexist within a single tumor, including a dominant clone and one or more minor subclones. Understanding tumor heterogeneity is essential as subclones may harbor specific genetic defects conferring resistance to treatment. Besides, reconstructing the clonal architecture of a tumor allows to distinguish early from late genetic alterations, and to better understand the role of each driver gene. We currently analyze whole-genome sequencing data to reconstruct the clonal architecture of 50 liver tumors. In particular, we analyze cases of adenomas having progressed to carcinomas (Pilati et al., Cancer Cell 2014), to identify the molecular events triggering malignant transformation.

 

 

Epigenetic signatures of oncogenic processes

Epigenetic profiles (DNA methylation, histone modifications, chromatin conformation) are highly rearranged in tumor cells. Besides, numerous epigenetic regulators, implicated in DNA (de)methylation or chromatin remodeling, are frequently mutated, in particular in liver cancers. However, the mechanistic link between these molecular alterations and the epigenetic profiles of tumors remains poorly understood. By analyzing the methylome of a large series of liver tumors previously characterized by whole-exome sequencing, we wish to identify specific DNA methylation signatures. We will then correlate these signatures with clinical (exposure to risk factors) and molecular annotations (mutations in epigenetic regulators) to identify the cause of each pattern. This project aims at understanding how altered epigenetic profiles are established, and how the affect the transcriptome of tumor cells. In collaboration with INSERM UMR970 team (Dr Judith Favier), we also study the hypermethylator phenotype induced by succinate dehydrogenase mutations in paraganglioma (Letouzé et al., Cancer Cell 2013).

 

 

Insertional mutagenesis by AAV and HBV virus

Virus, including hepatitis B virus (HBV) and hepatitis C, are major causes of hepatocellular carcinomas (HCC) worldwide. HBV is a well-known oncogenic DNA virus in liver tumors that induces insertional mutagenesis, chromosome instability and expression of oncogenic viral proteins. Currently only 2 other DNA viruses (merkel polyomavirus and human papilloma virus) are known to induce oncogenic insertional mutagenesis in human. Recently we obtained very innovative results showing that genomic approaches can help to identify new risk factors of HCC and modified our understanding of the disease. We showed that the adeno-associated virus type 2 (AAV2) is involved in HCC development on normal liver due to insertional mutagenesis in cancer genes (1). Currently, AAV is the 8th virus known associated to human cancer. The first identification of recurrent oncogenic AAV2 insertions in liver tumors leads us to develop a research project aiming to identify viral sequences and possibly new AAV integration sites in different types of liver tumors. As recombinant AAV is used as a vector for gene therapy in human clinical trials, a fine understanding of the mechanisms of carcinogenesis of the wild type virus is mandatory to assess the potential risk of cancer development after gene therapy. The objective is to quantify the contribution of AAV infection to the development of liver tumors in patients with liver disease related to various etiologies or without liver disease. We aim to identify new risk factors of hepatocellular carcinoma by analyzing tumor genomes and to validate them in epidemio-molecular studies through a comprehensive analysis of the tumor, host and viral genomes.

 

 

Transcriptome deregulation in liver tumors

Transcriptome profiling of liver tumors using DNA microarrays allowed us to identify homogeneous molecular groups of tumors, associated with distinct molecular alterations and risk factors (Boyault et al., Hepatology 2007). With the advent of high-throughput sequencing, we can now study the transcriptome by directly sequencing the RNAs extracted from a tumor sample (RNA-seq technique). These data give access to an unprecedented wealth of information. In addition to gene expression levels, RNA-seq data give access to the structure and sequence of transcripts, and allow the discovery of new genes. We have generated in the lab the RNA-seq profiles of a large series of liver tumors. We now develop innovative bioinformatic approaches to (1) refine the molecular classfication of liver tumors, (2) analyze the deregulation of non-coding RNAs, (3) detect alternative splicing and allele specific expression in tumors, and (4) identify the mutations that are actually expressed (more likely to play an oncogenic role) and RNA editing events.

 

 

Development of an annotation database

The development of genetics and new in-depth and high throughput technologies results in massive data production. Moreover, the multiplicity of programs and genomic annotations through “genome browsers” has created a “jungle” of terminology which requires the establishment and use of a unified vocabulary. In this context, we design and develop a database for structuring, annotation and exploitation of this mass of data generated by genomics programs. This project will, in collaboration with the EBCI Company, the implementation of an efficient IT infrastructure to meet our mixed data model, including clinic, histological and molecular datasets, to lead (1) the optimization of the architecture of our data model, (2) the management of the annotation including updates, and (3) optimizing a Web server to allow biologists to efficiently query data.

 

Team

Publications

APC germline hepatoblastomas demonstrate cisplatin-induced intratumor tertiary lymphoid structures. Morcrette G, Hirsch TZ, Badour E, Pilet J, Caruso S, Calderaro J, Martin Y, Imbeaud S, Letouzé E, Rebouissou S, Branchereau S, Taque S, Chardot C, Guettier C, Scoazec JY, Fabre M, Brugières L, Zucman-Rossi J. Oncoimmunology. 2019 Mar 28;8(6):e1583547. doi: 10.1080/2162402X.2019.1583547. eCollection 2019.

Analysis of Liver Cancer Cell Lines Identifies Agents With Likely Efficacy Against Hepatocellular Carcinoma and Markers of Response. Caruso S, Calatayud AL, Pilet J, La Bella T, Rekik S, Imbeaud S, Letouzé E, Meunier L, Bayard Q, Rohr-Udilova N, Péneau C, Grasl-Kraupp B, de Koning L, Ouine B, Bioulac-Sage P, Couchy G, Calderaro J, Nault JC, Zucman-Rossi J, Rebouissou S. Gastroenterology. 2019 May 4. pii: S0016-5085(19)36771-X. doi: 10.1053/j.gastro.2019.05.001.

A 17-Beta-Hydroxysteroid Dehydrogenase 13 Variant Protects From Hepatocellular Carcinoma Development in Alcoholic Liver Disease. Yang J, Trépo E, Nahon P, Cao Q, Moreno C, Letouzé E, Imbeaud S, Bayard Q, Gustot T, Deviere J, Bioulac-Sage P, Calderaro J, Ganne-Carrié N, Laurent A, Blanc JF, Guyot E, Sutton A, Ziol M, Zucman-Rossi J, Nault JC. Hepatology. 2019 Mar 25. doi: 10.1002/hep.30623.

Argininosuccinate synthase 1 and periportal gene expression in sonic hedgehog hepatocellular adenomas. Nault JC, Couchy G, Caruso S, Meunier L, Caruana L, Letouzé E, Rebouissou S, Paradis V, Calderaro J, Zucman-Rossi J. Hepatology. 2018 Sep;68(3):964-976. doi: 10.1002/hep.29884. Epub 2018 Jun 6.

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