Des etudes recentes ont mis en evidence qu’au moins 70% du genome humain est transcrit et produit... more Des etudes recentes ont mis en evidence qu’au moins 70% du genome humain est transcrit et produit une myriade d’ARN non codants. Au debut de ma these j’ai utilise des donnees de RNA-Seq sens-specifique pour identifier les transcrits divergents dans les tissus primaires de souris. J’ai utilise aussi des donnees ChIP-Seq afin d’analyser leurs caracteristiques epigenetiques. Nous avons trouve que la transcription divergente est associee de maniere significative a des genes lies a la regulation de la transcription et le developpement.Dans un deuxieme temps, je me suis interesse a l'identification et la caracterisation des lncRNA chez l'homme. J’ai applique des approches statistiques pour quantifier leur expression et identifier ceux qui sont (de)regules dans un contexte normal ou leucemique Dans un troisieme temps. Au cours de ma these, je me suis attache a etudier le mecanisme moleculaire epigenomique ainsi qu'a developper un pipeline bioinformatique permettant d'identifier les genes (codant ou non codant) associes a des profils H3K4me2/3 etendus. Ainsi, j’ai mis en evidences que ces profils etendus etaient directement dependants d'un processus transcriptionelle impliquant des nouveaux mecanismes de regulation. Cette etude a donne aussi lieu a une publication dont je suis cosignataire en premier auteur. (Zacarias, Belhocine et al. Journal of Immunology 2015). Cette nouvelle approche devrait s'averer tres utile dans d'autres modeles developpementaux et/ou pathologiques et peuvent etre utilise comme outil de prioritisation des candidats les plus relevant dans des approches plus globale.
Des etudes recentes ont mis en evidence qu’au moins 70% du genome humain est transcrit et produit... more Des etudes recentes ont mis en evidence qu’au moins 70% du genome humain est transcrit et produit une myriade d’ARN non codants. Au debut de ma these j’ai utilise des donnees de RNA-Seq sens-specifique pour identifier les transcrits divergents dans les tissus primaires de souris. J’ai utilise aussi des donnees ChIP-Seq afin d’analyser leurs caracteristiques epigenetiques. Nous avons trouve que la transcription divergente est associee de maniere significative a des genes lies a la regulation de la transcription et le developpement.Dans un deuxieme temps, je me suis interesse a l'identification et la caracterisation des lncRNA chez l'homme. J’ai applique des approches statistiques pour quantifier leur expression et identifier ceux qui sont (de)regules dans un contexte normal ou leucemique Dans un troisieme temps. Au cours de ma these, je me suis attache a etudier le mecanisme moleculaire epigenomique ainsi qu'a developper un pipeline bioinformatique permettant d'identi...
This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication... more This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precur... more T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precursors arrested at specific differentiation stages, including an ‘early-cortical’ thymic maturation arrest characterized by expression of cytoplasmic TCRβ but no surface T-cell receptor (TCR) and frequent ectopic expression of the TLX1/3 NK-like homeotic proteins (NKL). We designed a TCRα VJC PCR to identify clonal TCRα rearrangements in 32% of 127 T-ALLs, including 0/52 immature/TCRγδ lineage cases and 41/75 (55%) TCRαβ lineage cases. Amongst the latter, TCRα rearrangements were not identified in 30/54 (56%) of IMβ/pre-αβ early-cortical T-ALLs, of which the majority (21/30) expressed TLX1/3. We reasoned that the remaining T-ALLs might express other NKL proteins, so compared transcript levels of 46 NKL in T-ALL and normal thymic subpopulations. Ectopic overexpression of 10 NKL genes, of which six are unreported in T-ALL (NKX2-3, BARHL1, BARX2, EMX2, LBX2 and MSX2), was detectable in 17/104 (16%) T-ALLs. Virtually all NKL overexpressing T-ALLs were TCRα unrearranged and ectopic NKL transcript expression strongly repressed Eα activity, suggesting that ectopic NKL expression is the major determinant in early-cortical thymic T-ALL maturation arrest. This immunogenetic T-ALL subtype, defined by TCRβ VDJ but no TCRα VJ rearrangement, is associated with a favorable outcome in GRAALL-treated adult T-ALLs.
Supplementary Figure 1. Anti-CD3 stimulation of primary human T-ALLs. Supplementary Figure 2. TCR... more Supplementary Figure 1. Anti-CD3 stimulation of primary human T-ALLs. Supplementary Figure 2. TCR stimulation by in vivo administration of agonistic monoclonal antibody in a preventive setting inhibits human TCR+ T-ALL development. Supplementary Figure 3. TCR signaling is essential to the anti-leukemic effect of OKT3. Supplementary Table 1. Immunophenotypic and oncogenic characteristics of T-ALL samples.
supplementary data Figure S1. Gene expression signatures define distinct molecular groups of T-AL... more supplementary data Figure S1. Gene expression signatures define distinct molecular groups of T-ALL. Figure S2. Global flow chart of the patients Figure S3. Comparison of outcomes between LALA94 patients with central lab onco-genetic study performed (dotted line) and patients without (full line). Figure S4. Epigenetic histone marks. Figure S5. Kaplan-Meier graph for OS is shown for TAL1+ patients treated on LALA-94 vs. GRAALL-2003/2005 trials. Figure S6. TAL1 expression normalised to GAPDH by RTQ-PCR in 8 PDX treated with L-asparaginase (Rf. Figure 4H). Figure S7. (A) Correlation analysis between ASNS expression and ASNS promoter methylation ratio. (B) ASNS transcriptional expression normalized to GAPDH in T-ALL, in normal Bone Marrow (BM) (100%, 50%, 10% and 1%) and in normal Peripheral Blood Cells (PBL) (100%, 50%, 10% and 1. (C) comparison of ASNS transcriptional expression in non-sorted diagnostic sample, blast and non-blast sorted cells in two patients with a hypermethylated ASN...
Purpose:Biological explanation for discrepancies in patient-related response to chemotherapy depe... more Purpose:Biological explanation for discrepancies in patient-related response to chemotherapy depending on the underlying oncogenic events is a promising research area. TLX1- or TLX3-deregulated T-cell acute lymphoblastic leukemias (T-ALL; TLX1/3+) share an immature cortical phenotype and similar transcriptional signatures. However, their prognostic impacts differ, and inconsistent clinical outcome has been reported for TLX3. We therefore hypothesized that the overlapping transcriptional profiles of TLX1+ and TLX3+ T-ALLs would allow identification of candidate genes, which might determine their distinct clinical outcomes.Experimental Design:We compared TLX1+ and TLX3+ adult T-ALL outcome in the successive French national LALA-94 and GRAALL-2003/2005 multicentric trials and analyzed transcriptomic data to identify differentially expressed genes. Epigenetic regulation of asparagine synthetase (ASNS) and in vitro l-asparaginase sensitivity were evaluated for T-ALL cell lines and primar...
Des études récentes ont mis en évidence qu’au moins 70% du génome humain est transcrit et produit... more Des études récentes ont mis en évidence qu’au moins 70% du génome humain est transcrit et produit une myriade d’ARN non codants. Au début de ma thèse j’ai utilisé des données de RNA-Seq sens-spécifique pour identifier les transcrits divergents dans les tissus primaires de souris. J’ai utilisé aussi des données ChIP-Seq afin d’analyser leurs caractéristiques épigénétiques. Nous avons trouvé que la transcription divergente est associée de manière significative à des gènes liés à la régulation de la transcription et le développement.Dans un deuxième temps, je me suis intéressé à l'identification et la caractérisation des lncRNA chez l'homme. J’ai appliqué des approches statistiques pour quantifier leur expression et identifier ceux qui sont (dé)régulés dans un contexte normal ou leucémique Dans un troisième temps. Au cours de ma thèse, je me suis attaché à étudier le mécanisme moléculaire épigénomique ainsi qu'à développer un pipeline bioinformatique permettant d'identi...
Broad domains of H3K4 methylation have been associated with consistent expression of tissue-speci... more Broad domains of H3K4 methylation have been associated with consistent expression of tissue-specific, cell identity, and tumor suppressor genes. Here, we identified broad domain–associated genes in healthy human thymic T cell populations and a collection of T cell acute lymphoblastic leukemia (T-ALL) primary samples and cell lines. We found that broad domains are highly dynamic throughout T cell differentiation, and their varying breadth allows the distinction between normal and neoplastic cells. Although broad domains preferentially associate with cell identity and tumor suppressor genes in normal thymocytes, they flag key oncogenes in T-ALL samples. Moreover, the expression of broad domain–associated genes, both coding and noncoding, is frequently deregulated in T-ALL. Using two distinct leukemic models, we showed that the ectopic expression of T-ALL oncogenic transcription factor preferentially impacts the expression of broad domain–associated genes in preleukemic cells. Finally,...
Normal T-cell differentiation requires a complex regulatory network which supports a series of ma... more Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4-CD8-double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Ca...
Introduction: Traditional classification of acute lymphoblastic and myeloid leukemias (ALLs and A... more Introduction: Traditional classification of acute lymphoblastic and myeloid leukemias (ALLs and AMLs) remains heavily based on phenotypic resemblance to normal hematopoietic precursors of the respective lineages. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, which often have poor responses to treatment. This system also takes little account of modern concepts of hematopoietic identity that are mainly based on transcriptional signature identification and functional assays. Recent advances in genome-wide analytical methods developed to reconstruct landscapes of normal differentiation now provide an opportunity to re-evaluate traditional binary approaches to myeloid and lymphoid lineage assignment in leukemia. Methods: We used novel computational tools, including the recently described Iterative Clustering and Guide Gene Selection (ICGS) method to perform transcriptional analyses of a series of 125 T-ALLs and AMLs, which com...
Purpose:Biological explanation for discrepancies in patient-related response to chemotherapy depe... more Purpose:Biological explanation for discrepancies in patient-related response to chemotherapy depending on the underlying oncogenic events is a promising research area. TLX1- or TLX3-deregulated T-cell acute lymphoblastic leukemias (T-ALL; TLX1/3+) share an immature cortical phenotype and similar transcriptional signatures. However, their prognostic impacts differ, and inconsistent clinical outcome has been reported for TLX3. We therefore hypothesized that the overlapping transcriptional profiles of TLX1+ and TLX3+ T-ALLs would allow identification of candidate genes, which might determine their distinct clinical outcomes.Experimental Design:We compared TLX1+ and TLX3+ adult T-ALL outcome in the successive French national LALA-94 and GRAALL-2003/2005 multicentric trials and analyzed transcriptomic data to identify differentially expressed genes. Epigenetic regulation of asparagine synthetase (ASNS) and in vitro l-asparaginase sensitivity were evaluated for T-ALL cell lines and primar...
Des etudes recentes ont mis en evidence qu’au moins 70% du genome humain est transcrit et produit... more Des etudes recentes ont mis en evidence qu’au moins 70% du genome humain est transcrit et produit une myriade d’ARN non codants. Au debut de ma these j’ai utilise des donnees de RNA-Seq sens-specifique pour identifier les transcrits divergents dans les tissus primaires de souris. J’ai utilise aussi des donnees ChIP-Seq afin d’analyser leurs caracteristiques epigenetiques. Nous avons trouve que la transcription divergente est associee de maniere significative a des genes lies a la regulation de la transcription et le developpement.Dans un deuxieme temps, je me suis interesse a l'identification et la caracterisation des lncRNA chez l'homme. J’ai applique des approches statistiques pour quantifier leur expression et identifier ceux qui sont (de)regules dans un contexte normal ou leucemique Dans un troisieme temps. Au cours de ma these, je me suis attache a etudier le mecanisme moleculaire epigenomique ainsi qu'a developper un pipeline bioinformatique permettant d'identifier les genes (codant ou non codant) associes a des profils H3K4me2/3 etendus. Ainsi, j’ai mis en evidences que ces profils etendus etaient directement dependants d'un processus transcriptionelle impliquant des nouveaux mecanismes de regulation. Cette etude a donne aussi lieu a une publication dont je suis cosignataire en premier auteur. (Zacarias, Belhocine et al. Journal of Immunology 2015). Cette nouvelle approche devrait s'averer tres utile dans d'autres modeles developpementaux et/ou pathologiques et peuvent etre utilise comme outil de prioritisation des candidats les plus relevant dans des approches plus globale.
Des etudes recentes ont mis en evidence qu’au moins 70% du genome humain est transcrit et produit... more Des etudes recentes ont mis en evidence qu’au moins 70% du genome humain est transcrit et produit une myriade d’ARN non codants. Au debut de ma these j’ai utilise des donnees de RNA-Seq sens-specifique pour identifier les transcrits divergents dans les tissus primaires de souris. J’ai utilise aussi des donnees ChIP-Seq afin d’analyser leurs caracteristiques epigenetiques. Nous avons trouve que la transcription divergente est associee de maniere significative a des genes lies a la regulation de la transcription et le developpement.Dans un deuxieme temps, je me suis interesse a l'identification et la caracterisation des lncRNA chez l'homme. J’ai applique des approches statistiques pour quantifier leur expression et identifier ceux qui sont (de)regules dans un contexte normal ou leucemique Dans un troisieme temps. Au cours de ma these, je me suis attache a etudier le mecanisme moleculaire epigenomique ainsi qu'a developper un pipeline bioinformatique permettant d'identi...
This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication... more This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precur... more T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precursors arrested at specific differentiation stages, including an ‘early-cortical’ thymic maturation arrest characterized by expression of cytoplasmic TCRβ but no surface T-cell receptor (TCR) and frequent ectopic expression of the TLX1/3 NK-like homeotic proteins (NKL). We designed a TCRα VJC PCR to identify clonal TCRα rearrangements in 32% of 127 T-ALLs, including 0/52 immature/TCRγδ lineage cases and 41/75 (55%) TCRαβ lineage cases. Amongst the latter, TCRα rearrangements were not identified in 30/54 (56%) of IMβ/pre-αβ early-cortical T-ALLs, of which the majority (21/30) expressed TLX1/3. We reasoned that the remaining T-ALLs might express other NKL proteins, so compared transcript levels of 46 NKL in T-ALL and normal thymic subpopulations. Ectopic overexpression of 10 NKL genes, of which six are unreported in T-ALL (NKX2-3, BARHL1, BARX2, EMX2, LBX2 and MSX2), was detectable in 17/104 (16%) T-ALLs. Virtually all NKL overexpressing T-ALLs were TCRα unrearranged and ectopic NKL transcript expression strongly repressed Eα activity, suggesting that ectopic NKL expression is the major determinant in early-cortical thymic T-ALL maturation arrest. This immunogenetic T-ALL subtype, defined by TCRβ VDJ but no TCRα VJ rearrangement, is associated with a favorable outcome in GRAALL-treated adult T-ALLs.
Supplementary Figure 1. Anti-CD3 stimulation of primary human T-ALLs. Supplementary Figure 2. TCR... more Supplementary Figure 1. Anti-CD3 stimulation of primary human T-ALLs. Supplementary Figure 2. TCR stimulation by in vivo administration of agonistic monoclonal antibody in a preventive setting inhibits human TCR+ T-ALL development. Supplementary Figure 3. TCR signaling is essential to the anti-leukemic effect of OKT3. Supplementary Table 1. Immunophenotypic and oncogenic characteristics of T-ALL samples.
supplementary data Figure S1. Gene expression signatures define distinct molecular groups of T-AL... more supplementary data Figure S1. Gene expression signatures define distinct molecular groups of T-ALL. Figure S2. Global flow chart of the patients Figure S3. Comparison of outcomes between LALA94 patients with central lab onco-genetic study performed (dotted line) and patients without (full line). Figure S4. Epigenetic histone marks. Figure S5. Kaplan-Meier graph for OS is shown for TAL1+ patients treated on LALA-94 vs. GRAALL-2003/2005 trials. Figure S6. TAL1 expression normalised to GAPDH by RTQ-PCR in 8 PDX treated with L-asparaginase (Rf. Figure 4H). Figure S7. (A) Correlation analysis between ASNS expression and ASNS promoter methylation ratio. (B) ASNS transcriptional expression normalized to GAPDH in T-ALL, in normal Bone Marrow (BM) (100%, 50%, 10% and 1%) and in normal Peripheral Blood Cells (PBL) (100%, 50%, 10% and 1. (C) comparison of ASNS transcriptional expression in non-sorted diagnostic sample, blast and non-blast sorted cells in two patients with a hypermethylated ASN...
Purpose:Biological explanation for discrepancies in patient-related response to chemotherapy depe... more Purpose:Biological explanation for discrepancies in patient-related response to chemotherapy depending on the underlying oncogenic events is a promising research area. TLX1- or TLX3-deregulated T-cell acute lymphoblastic leukemias (T-ALL; TLX1/3+) share an immature cortical phenotype and similar transcriptional signatures. However, their prognostic impacts differ, and inconsistent clinical outcome has been reported for TLX3. We therefore hypothesized that the overlapping transcriptional profiles of TLX1+ and TLX3+ T-ALLs would allow identification of candidate genes, which might determine their distinct clinical outcomes.Experimental Design:We compared TLX1+ and TLX3+ adult T-ALL outcome in the successive French national LALA-94 and GRAALL-2003/2005 multicentric trials and analyzed transcriptomic data to identify differentially expressed genes. Epigenetic regulation of asparagine synthetase (ASNS) and in vitro l-asparaginase sensitivity were evaluated for T-ALL cell lines and primar...
Des études récentes ont mis en évidence qu’au moins 70% du génome humain est transcrit et produit... more Des études récentes ont mis en évidence qu’au moins 70% du génome humain est transcrit et produit une myriade d’ARN non codants. Au début de ma thèse j’ai utilisé des données de RNA-Seq sens-spécifique pour identifier les transcrits divergents dans les tissus primaires de souris. J’ai utilisé aussi des données ChIP-Seq afin d’analyser leurs caractéristiques épigénétiques. Nous avons trouvé que la transcription divergente est associée de manière significative à des gènes liés à la régulation de la transcription et le développement.Dans un deuxième temps, je me suis intéressé à l'identification et la caractérisation des lncRNA chez l'homme. J’ai appliqué des approches statistiques pour quantifier leur expression et identifier ceux qui sont (dé)régulés dans un contexte normal ou leucémique Dans un troisième temps. Au cours de ma thèse, je me suis attaché à étudier le mécanisme moléculaire épigénomique ainsi qu'à développer un pipeline bioinformatique permettant d'identi...
Broad domains of H3K4 methylation have been associated with consistent expression of tissue-speci... more Broad domains of H3K4 methylation have been associated with consistent expression of tissue-specific, cell identity, and tumor suppressor genes. Here, we identified broad domain–associated genes in healthy human thymic T cell populations and a collection of T cell acute lymphoblastic leukemia (T-ALL) primary samples and cell lines. We found that broad domains are highly dynamic throughout T cell differentiation, and their varying breadth allows the distinction between normal and neoplastic cells. Although broad domains preferentially associate with cell identity and tumor suppressor genes in normal thymocytes, they flag key oncogenes in T-ALL samples. Moreover, the expression of broad domain–associated genes, both coding and noncoding, is frequently deregulated in T-ALL. Using two distinct leukemic models, we showed that the ectopic expression of T-ALL oncogenic transcription factor preferentially impacts the expression of broad domain–associated genes in preleukemic cells. Finally,...
Normal T-cell differentiation requires a complex regulatory network which supports a series of ma... more Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4-CD8-double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Ca...
Introduction: Traditional classification of acute lymphoblastic and myeloid leukemias (ALLs and A... more Introduction: Traditional classification of acute lymphoblastic and myeloid leukemias (ALLs and AMLs) remains heavily based on phenotypic resemblance to normal hematopoietic precursors of the respective lineages. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, which often have poor responses to treatment. This system also takes little account of modern concepts of hematopoietic identity that are mainly based on transcriptional signature identification and functional assays. Recent advances in genome-wide analytical methods developed to reconstruct landscapes of normal differentiation now provide an opportunity to re-evaluate traditional binary approaches to myeloid and lymphoid lineage assignment in leukemia. Methods: We used novel computational tools, including the recently described Iterative Clustering and Guide Gene Selection (ICGS) method to perform transcriptional analyses of a series of 125 T-ALLs and AMLs, which com...
Purpose:Biological explanation for discrepancies in patient-related response to chemotherapy depe... more Purpose:Biological explanation for discrepancies in patient-related response to chemotherapy depending on the underlying oncogenic events is a promising research area. TLX1- or TLX3-deregulated T-cell acute lymphoblastic leukemias (T-ALL; TLX1/3+) share an immature cortical phenotype and similar transcriptional signatures. However, their prognostic impacts differ, and inconsistent clinical outcome has been reported for TLX3. We therefore hypothesized that the overlapping transcriptional profiles of TLX1+ and TLX3+ T-ALLs would allow identification of candidate genes, which might determine their distinct clinical outcomes.Experimental Design:We compared TLX1+ and TLX3+ adult T-ALL outcome in the successive French national LALA-94 and GRAALL-2003/2005 multicentric trials and analyzed transcriptomic data to identify differentially expressed genes. Epigenetic regulation of asparagine synthetase (ASNS) and in vitro l-asparaginase sensitivity were evaluated for T-ALL cell lines and primar...
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Papers by Mohamed Belhocine