Integrative analysis of RUNX1 downstream pathways and target genes.

BACKGROUND: The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. RESULTS: Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFbeta, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFbeta. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. CONCLUSION: This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications

t(15;21) translocations leading to the concurrent downregulation of RUNX1 and its transcription factor partner genes SIN3A and TCF12 in myeloid disorders.

Through a combined approach integrating RNA-Seq, SNP-array, FISH and PCR techniques, we identified two novel t(15;21) translocations leading to the inactivation of RUNX1 and its partners SIN3A and TCF12. One is a complex t(15;21)(q24;q22), with both breakpoints mapped at the nucleotide level, joining RUNX1 to SIN3A and UBL7-AS1 in a patient with myelodysplasia. The other is a recurrent t(15;21)(q21;q22), juxtaposing RUNX1 and TCF12, with an opposite transcriptional orientation, in three myeloid leukemia cases. Since our transcriptome analysis indicated a significant number of differentially expressed genes associated with both translocations, we speculate an important pathogenetic role for these alterations involving RUNX1.

Caractérisation clinique et moléculaire de nouvelles translocations chromosomiques ciblant le gène RUNX1 dans les leucémies aiguës de l’adulte

La leucémie aiguë myéloïde est une hémopathie maligne génétiquement hétérogène caractérisée par de fréquents réarrangements impliquant la bande chromosomique 21q22 et le gène RUNX1. Dans ce groupe d’anomalies, les translocations t(8;21)(q22;q22) et t(3;21)(q26;q22), associées respectivement à un pronostic favorable et défavorable, sont les mieux étudiées. Or, plus de la moitié des réarrangements ciblant RUNX1 ne sont toujours pas caractérisés au niveau clinique et moléculaire. Les principaux objectifs de cette thèse sont de caractériser quatre nouvelles translocations ciblant RUNX1 et d’étudier la dérégulation transcriptionnelle associée à ces anomalies au niveau de cibles plus spécifiques ayant un rôle dans l’auto-renouvellement ou dans la différenciation hématopoïétique. À l’aide des techniques de cytogénétique et de biologie moléculaire, deux nouveaux partenaires de RUNX1, soit CLCA2 et SV2B, ont été identifiés au sein des t(1;21)(p22.3;q22) et t(15;21)(q26.1;q22) et la récurrence des partenaires USP42 et TRPS1 a été démontrée suite à l’étude des t(7;21)(p22.1;q22) et t(8;21)(q23.3;q22). Ce travail a permis de confirmer l’existence de divers modes de dérégulation de RUNX1 dans les leucémies aiguës. L’expression présumée de protéines chimériques et/ou d’isoformes tronquées de RUNX1, un dosage aberrant des transcrits de RUNX1 et la surexpression des gènes partenaires sont des conséquences révélées par l’étude de ces fusions. Le séquençage et l’analyse des jonctions génomiques des fusions récurrentes RUNX1-USP42/USP42-RUNX1 et RUNX1-TRPS1/TRPS1-RUNX1 ont démontré la présence de signatures moléculaires caractéristiques du mode de recombinaison non-homologue de type NHEJ. En raison de la structure et de la composition différente des jonctions, l’implication de composantes distinctes du mécanisme NHEJ a été proposée. Enfin, des analyses par PCR quantitative en temps réel nous ont permis de démontrer l’existence de cibles de dérégulation partagées par les fusions récurrentes et plus rares de RUNX1. Nous avons démontré que CEBPA est moins exprimé dans la majorité des spécimens étudiés présentant une fusion de RUNX1 par rapport aux spécimens avec un caryotype normal alors que JUP, une composante effectrice de la voie Wnt, est plutôt surexprimé. Malgré l’activation transcriptionnelle de JUP dans l’ensemble de ces spécimens, certaines cibles de la voie Wnt telles que CCND1 et MYC sont différemment exprimées dans ces cellules, appuyant l’hétérogénéité décrite dans ce groupe de leucémies. Malgré l’implication de partenaires variés, nos données d’expression démontrent que les chimères et les protéines tronquées de RUNX1 partagent des cibles communes d’activation et de répression transcriptionnelle et établissent, pour la première fois, des évidences moléculaires suggérant l’existence de similitudes entre la fusion récurrente RUNX1-RUNX1T1 et quatre fusions plus rares de RUNX1. Puisque des rechutes surviennent fréquemment dans ce groupe génétique, l’inhibition de JUP pourrait être une option thérapeutique intéressante et ceci est appuyé par les bénéfices observés lors de l’inhibition de la voie Wnt dans d’autres groupes génétiques de leucémies aiguës.

Identification of a new translocation that disrupts the RUNX1 gene in a patient with de novo acute myeloid leukemia

Translocation (8;21)(q22;q22)/RUNX1-RUNX1T1 is a molecular marker that is usually associated with a favorable outcome in both pediatric and adult patients with acute myeloid leukemia (AML). The present report describes the results of hematologic, cytogenetic, and fluorescence in situ hybridization analysis of a case of AML with maturation in a 23-year-old woman. Cytogenetic analysis revealed a balanced translocation involving chromosomal band 21q22, which disrupts the RUNX1 gene, and 10q22, with the following karyotype: 45,X,-X,t(10;21)(q24;q22)[cp16]/46,XX [4]. Interphase FISH showed, in 67% of the 300 interphase nuclei analyzed, three signals for RUNX1 and two RUNX1T1, but no signals corresponding to RUNX1-RUNX1T1 fusion gene. These results were corroborated by RT-PCR, which revealed negative results for the amplification of RUNX1-RUNX1T1 fusion gene. The patient was refractory to conventional and salvage chemotherapy regimens and early relapsed after unrelated donor bone marrow transplantation (BMT), dying of pneumonia, acute respiratory failure, and sepsis on day +80 after BMT, 1 year after diagnosis.

Integrative analysis of RUNX1 downstream pathways and target genes

BACKGROUND: The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. RESULTS: Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFbeta, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFbeta. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. CONCLUSION: This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.

The Runx1 transcription factor controls CSF-1-dependent and -independent growth and survival of macrophages

Gene translocations that repress the function of the Runx1 transcription factor play a critical role in the development of myeloid leukemia. In this report, we demonstrate that Runx1 precisely regulates c-fms (CSF-1 receptor) gene expression. Runx1 controlled expression by binding to multiple sites within the mouse c-fms gene, allowing interaction between promoter and downstream enhancer elements. The runx1 and c-fms genes showed an identical pattern of expression in mature macrophages. Runx1 expression was repressed in CSF-1 stimulated, proliferating bone marrow-derived macrophages (BMM) and significantly increased in quiescent, CSF-1 starved cells. The RAW264.7 and Mono-Mac-6, macrophage-like cell lines expressed low levels of Runx1 and both showed growth arrest and cell death with ectopic expression of Runx1. The EM-3 cell line, which represents an early myeloid progenitor cell line, showed growth arrest with Runx1 expression in the absence of any detectable changes in cell differentiation. These findings suggest that Runx1 regulates growth and survival of myeloid cells and provide a novel insight into the role of Runx family gene translocations in leukemogenesis.

Identification Of Target Genes Using Gene Expression Profile Of Granulocytes From Patients With Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors.

Differential gene expression analysis by suppression subtractive hybridization with correlation to the metabolic pathways involved in chronic myeloid leukemia (CML) may provide a new insight into the pathogenesis of CML. Among the overexpressed genes found in CML at diagnosis are SEPT5, RUNX1, MIER1, KPNA6 and FLT3, while PAN3, TOB1 and ITCH were decreased when compared to healthy volunteers. Some genes were identified and involved in CML for the first time, including TOB1, which showed a low expression in patients with CML during tyrosine kinase inhibitor treatment with no complete cytogenetic response. In agreement, reduced expression of TOB1 was also observed in resistant patients with CML compared to responsive patients. This might be related to the deregulation of apoptosis and the signaling pathway leading to resistance. Most of the identified genes were related to the regulation of nuclear factor κB (NF-κB), AKT, interferon and interleukin-4 (IL-4) in healthy cells. The results of this study combined with literature data show specific gene pathways that might be explored as markers to assess the evolution and prognosis of CML as well as identify new therapeutic targets.

Increased Levels of NOTCH1, NF-kappa B, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

As previously shown, higher levels of NOTCH1 and increased NF-kappa B signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow ( BM). Differences between BM and UCB cell composition also account for this finding. The CD133 marker defines a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purified human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a significantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells ( CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a significantly higher frequency ( than expected by chance) of NF-kappa B-binding sites (BS), including potentially novel NF-kappa B targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all significantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-kappa B, and other important TFs on more primitive HSC sets.

The expression of Delta NTP73, TATP73 and TP53 genes in acute myeloid leukaemia is associated with recurrent cytogenetic abnormalities and in vitro susceptibility to cytarabine cytotoxicity

TP73 encodes for two proteins: full-length TAp73 and Delta Np73, which have little transcriptional activity and exert dominant-negative function towards TP53 and TAp73. We compared TATP73 and Delta NTP73 expression in acute myeloid leukaemia (AML) samples and normal CD34(+) progenitors. Both forms were more highly expressed in leukaemic cells. Amongst AML blasts, TATP73 was more expressed in AML harbouring the recurrent genetic abnormalities (RGA): PML-RARA, RUNX1-RUNX1T1 and CBFB-MYH11, whereas higher Delta NTP73 expression was detected in non-RGA cases. TP53 expression did not vary according to Delta NTP73/TATP73 expression ratio. Leukaemic cells with higher Delta NTP73/TATP73 ratios were significantly more resistant to cytarabine-induced apoptosis.

The role of complex chromosome translocations in leukemia

Resumo A tumorigénese é um processo de transformação celular que se desenrola tipicamente em várias etapas. Os diferentes níveis de evolução tumoral resultam da acumulação sucessiva de mutações genéticas numa célula normal que lhe conferem uma vantagem selectiva no respectivo meio tecidular. As mutações podem manifestar-se sob a forma de alterações nucleotídicas pontuais ao nível da sequência de DNA, levando a uma desregulação da função proteíca ou à formação de proteínas não-funcionais, ou através de alterações cromossómicas numéricas ou estruturais. Na leucemia, por exemplo, os genes híbridos que resultam de translocações cromossómicas desempenham um importante papel no processo tumorigénico. Estes genes são transcritos sob a forma de um RNA mensageiro de fusão, o qual é traduzido numa proteína híbrida com função oncogénica. Frequentemente, os subtipos de doença leucémica estão associados com translocações cromossómicas que envolvem 2 pontos de quebra recorrentes e específicos. É disto exemplo a leucemia mielóide crónica, em que uma translocação recíproca entre os cromossomas 9 e 22 conduz à formação de um gene de fusão BCR-ABL1. Em diferentes subtipos de doença, existe também uma pequena proporção de casos que apresenta translocações cromossómicas complexas, que envolvem um ou mais pontos de quebra adicionais em outras localizações genómicas além das que estão implicadas na formação dos genes de fusão. Por vezes, os pontos de quebra estão também associados a delecções extensas de material genético que se pensa terem uma função importante na tumorigénese. No entanto, o papel destas regiões genómicas no desenvolvimento tumoral não tem sido um motivo recorrente de estudo. Neste contexto, o objectivo desta dissertação foi o de determinar o potencial papel tumorigénico de alterações génicas adicionais ocorridas nos pontos de quebra de translocações cromossómicas complexas. Para a prossecução do objectivo proposto, foram estudados 5 rearranjos cromossómicos distintos associados com diferentes tipos de doença hematológica maligna, nomeadamente a leucemia linfoblástica aguda de células B (2 casos), leucemia mielóide aguda, neoplasma mieloproliferativo e síndrome mielodisplásico/neoplasma ieloproliferativo, não classificável. O mapeamento dos pontos de quebra foi efectuado utilizando a hibridação fluorescente in situ e diferentes metodologias de biologia molecular, tendo como base a informação inicial da análise citogenética. Em casos seleccionados, o papel dos novos genes candidatos foi avaliado in vitro utilizando modelos de linhas celulares, nomeadamente no que respeita às funções de controlo da proliferação celular e de regulação transcricional. De entre os 5 casos estudados, quatro deles evidenciaram translocações complexas envolvendo 3 cromossomas, nomeadamente t(12;21;5)(p13;q22;q13), t(12;6;15)(p13;p24~25;q22), t(9;11;19)(p22;q23;p13) e t(X;20;16)(p11;q13;q23). No caso remanescente, foi observada uma translocação dicêntrica dic(9;12)(p11;p11) acompanhada de delecções extensas em ambos os pontos de quebra. Nos casos com t(12;21;5) e t(9;11;19) as translocações estavam associadas com a presença de genes de fusão recorrentes, nomeadamente TV6(12p13)-RUNX1(21q22) e TLL(11q23)-MLLT3(9p22), indicando que se tratavam de rearranjos complexos das translocações t(12;21) e t(9;11) associadas com a leucemia linfoblástica aguda de células B e a leucemia mielóide aguda, respectivamente. O papel dos pontos de quebra adicionais foi estudado em detalhe no caso com t(9;11;19). Através da metodologia de long distance inverse-polymerase chain reaction, foram identificados os pontos de quebra na sequência de DNA dos 3 cromossomas envolvidos na translocação. Além dos pontos de quebra nos genes MLL e MLLT3, foi observado que o local de quebra no cromossoma 19 interrompeu a sequência de um novo gene, designado CCDC94,conduzindo à sua haplo-insuficiência nas células com t(9;11;19). Através de ensaios de reverse transcription-polymerase chain reaction verificámos que o gene CCDC94 é expresso ubiquitariamente em tecidos humanos normais. A análise informática da sequência prevista da proteína CCDC94 indicou uma elevada identidade de aminoácidos com a proteína cwf16, envolvida na regulação do ciclo celular da levedura Schizosaccharomyces pombe. Através da clonagem do DNA complementar de CCDC94 em vectores de expressão, e após a transfecção destes em culturas de linhas celulares in vitro, observámos que este gene codifica uma proteína de localização exclusivamente nuclear. A expressão ectópica da proteína CCDC94 diminuiu a progressão do ciclo celular e a proliferação das células em cultura. Inversamente, a supressão do transcrito do gene CCDC94 através de interferência de RNA conduziu a um aumento significativo da proliferação celular, confirmando que CCDC94 regula negativamente a proliferação e a progressão do ciclo celular. Estes resultados mostram que os pontos de quebra adicionais, presentes em translocações cromossómicas complexas em leucemia, podem resultar na haplo-insuficiência de genes controladores dos mecanismos proliferativos, cooperando desta forma com a acção das proteínas de fusão para proporcionar ao clone leucémico uma proliferação celular descontrolada. Nos restantes 3 casos estudados não foram identificados genes de fusão. Ao invés, todos aqueles apresentaram delecções de extensão variável associadas com os pontos de quebra cromossómicos. No caso com t(12;6;15), identificámos uma delecção de 1.2 megabases de DNA na banda 12p13 que resultou na eliminação de 9 genes incluindo ETV6 e CDKN1B. O gene ETV6 codifica um factor de transcrição que é essencial para a formação das diferentes linhagens hematopoiéticas na medula óssea, enquanto CDKN1B é traduzido numa proteína responsável por bloquear a entrada das células na fase G1 do ciclo celular e,consequentemente, por travar a proliferação celular. Neste contexto, os resultados obtidos indicam que a perda simultânea de ETV6 e de CDKN1B, através de uma translocação cromossómica complexa, constituiu uma acção cooperativa na leucemogénese. A mesma noção pode aplicar-se ao caso com dic(9;12), no qual pelo menos 2 genes que codificam para factores de transcrição importantes na linhagem hematopoiética, PAX5 no cromossoma 9 e ETV6 no cromossoma 12, estavam deleccionados como resultado do rearranjo cromossómico. Dado que o factor de transcrição PAX5 regula negativamente a expressão do gene FLT3, que desempenha uma função pró-proliferativa, é expectável que a haplo-insuficiência de PAX5 no caso com dic(9;12) terá tido como consequência uma elevação dos níveis de expressão de FLT3, contribuindo deste modo para uma proliferação celular aumentada. A t(X;20;16) foi identificada num doente com trombocitémia essencial (TE), uma doença que está intimamente relacionada com alterações de vias intracelulares reguladas por citocinas. Neste caso, através da utilização de um array genómico, identificámos a presença de pequenas delecções associadas com os pontos de quebra nos cromossomas 16 e 20. No cromossoma 16 apenas um gene, MAF, estava deleccionado, enquanto no cromossoma 20 a delecção tinha abrangido 3 genes. Dos genes deleccionados, dois deles, NFATC2 (20q13) e MAF (16q23), codificam proteínas que operam como reguladores transcricionais de citocinas hematopoiéticas. Dado que NFATC2 se localiza numa região que constitui um alvo frequente de delecções em neoplasmas ieloproliferativos, incluindo a trombocitémia essencial,efectuámos um estudo detalhado do papel deste gene na proliferação megacariocítica e na regulação da expressão de uma citocina hematopoiética (GM-CSF), implicada na maturação das diferentes linhagens mielóides. Utilizando um modelo de linha celular de trombocitémia essencial, verificámos que a supressão do transcrito do gene NFATC2 in vitro, por interferência de RNA, estava associada com um aumento da proliferação celular. Em concordância, o bloqueio da activação da proteína NFATC2 através de um inibidor específico da sua interacção com a calcineurina, conduziu a um aumento da proliferação celular in vitro. Utilizando a PCR quantitativa em tempo real, detectou-se um aumento da produção do RNA de GM-CSF em ambos os ensaios celulares, indicando que o factor de transcrição NFATC2 pode regular negativamente a expressão de GM-CSF em células de trombocitémia essencial. No geral, estes resultados mostram que a redução dos níveis fisiológicos do transcrito NFATC2, ou a redução da respectiva actividade proteica, estão relacionados com a proliferação de megacariocitos através do aumento da produção de GM-CSF. De acordo com estes resultados, verificámos que as células dos doentes com TE apresentam níveis mais baixos do transcrito NFATC2 do que a população normal. Dado que o factor de transcrição MAF desempenha igualmente um papel como regular transcricional de citocinas, é plausível que a haplo-insuficiência dos genes NFATC2 e MAF, resultante do rearranjo cromossómico complexo t(X;20;16), teve um efeito cooperativo importante na patogénese da trombocitémia essencial através da alteração do padrão normal de expressão das citocinas hematopoiéticas. Em síntese, efectuámos nesta dissertação um estudo citogenético de 4 translocações cromossómicas complexas incluindo t(12;21;5), t(12;6;15), t(9;11;19) e t(X;20;16), e de uma translocação dicêntrica dic(9;12), associadas com diferentes neoplasmas hematológicos. Em casos seleccionados efectuámos também um estudo molecular detalhado das regiões dos pontos de quebra. Esta análise permitiu-nos identificar 2 genes, CCDC94 no cromossoma 19 e NFATC2 no cromossoma 20, cuja haplo-insuficiência pode promover o aumento da proliferação celular das células leucémicas. A partir destes estudos podem ser retiradas 2 noções principais: (i) Os pontos de quebra adicionais, que ocorrem em translocações complexas associadas com a formação de genes de fusão, podem ter como consequência a desregulação de genes controladores da proliferação celular (e.g., CCDC94); (ii) As translocações complexas caracterizadas pela ausência de genes de fusão recorrentes poderão estar preferencialmente associadas com a presença de delecções, envolvendo um ou mais genes, nos pontos de quebra; nestas situações, serão necessários pelo menos 2 genes com funções celulares semelhantes (e.g., NFATC2 e MAF) ou complementares (e.g., ETV6 e CDKN1B) para, quando deleccionados, promoverem de forma cooperativa a leucemogénese. Nestes termos, o modelo de alterações genéticas sequenciais que caracteriza o desenvolvimento do cancro pode ser substituído por um modelo em que vários genes-alvo são simultaneamente desregulados pela formação de uma translocação cromossómica complexa, evitando deste modo a necessidade de ocorrência de alterações genéticas subsequentes.----------------------ABSTRACT: Tumourigenesis is a multistep process which results from the accumulation of successive genetic mutations in a normal cell. In leukemia for instance, recurrent translocations play a part in this process by generating fusion genes which lead to the production of hybrid proteins with an oncogenic role. However, a minor subset of chromosomal translocations referred to as complex or variant involves extra breakpoints at variable genome locations in addition to those implicated in the formation of fusion genes. We aimed to describe in this work the role, if any, of genes located at extra breakpoint locations or which are affected by breakpoint-adjacent deletions through the study of 5 leukemia patients.Two of the patients presented with TV6(12p13)-RUNX1(21q22) and MLL(11q23)- MLLT3(9p22) fusion genes as a result of a t(12;21;5) and a t(9;11;19), respectively. Detailed molecular characterization of the extra breakpoint at chromosome 19 in the latter case revealed that a novel ubiquitously expressed gene, CCDC94, with a potential role in cell cycle regulation, was disrupted by the breakpoint. We demonstrated using in vitro cellular assays that this gene codifies for a nuclear protein which negatively regulates cell cycle progression. These data shows that extra breakpoint locations of complex translocations may result in haplo-insufficiency of critical proliferation genes, thereby cooperating with the generation of hybrid proteins to provide unrestrained cell proliferation. In the other 3 patients there were reakpoint-associated deletions which precluded the formation of putative fusion genes. In a case with a t(12;6;15) we characterized a deletion at 12p13 which eliminated ETV6 and 8 other genes including CDKN1B. These findings indicate that concomitant loss of ETV6 and CDKN1B, which encodes a cyclin-dependent kinase inhibitor responsible for blocking entry of cells into the G1 phase of the cell cycle, acted cooperatively to promote leukemogenic proliferation. The same notion applied to a case with a dic(9;12) in which 2 genes encoding hematopoietic transcription factors - ETV6 and PAX5 (9p13)- were deleted as a result of breakpoint-adjacent deletions. Similarly, we found that 2 transcription factor genes involved in the regulation of cytokine expression, NFATC2 (20q13) and MAF (16q23), were involved in deletions contiguous to the breakpoints in a patient with a t(X;20;16). In vitro suppression of NFATC2 mRNA or inhibiton of NFATC2 protein activity enhanced cell proliferation as a result of an increase in the production of a myeloid-lineage stimulating hematopoietic cytokine, GM-CSF. These results suggest that haplo-insufficiency of NFATC2 and MAF genes had a cooperative effect in inducing cell proliferation as a result of a disregulation of cytokine production. Two main conclusions may be drawn from our studies: (i) In complex translocations associated with the production of fusion genes, additional breakpoints may cooperate in tumourigenesis by targeting genes that control cell proliferation; (ii) In complex translocations associated with small breakpoint-adjacent deletions, at least 2 genes with similar or complementary functions need to be deregulated to promote tumourigenesis.

Caractérisation cytogénétique et moléculaire des translocations chromosomiques dans la phase blastique de la leucémie myéloïde chronique

La leucémie myéloïde chronique (LMC) est un modèle d’évolution tumorale dans les cancers humains. Le processus d’évolution de la LMC de la phase chronique (PC) à la phase blastique (PB) est caractérisé par un arrêt de différenciation et l’acquisition de la capacité d’autorenouvellement incontrôlé d’une cellule souche ou d’un progéniteur hématopoïétique. La LMC en PB est associée à la présence d’anomalies génétiques additionnelles à la fusion BCR-ABL1 qui résulte de la translocation chromosomique t(9;22). Contrairement aux patients en PC, les patients en PB de la LMC n’obtiennent pas une réponse moléculaire complète à long terme avec 1’Imatinib mesylate, un inhibiteur de la tyrosine kinase (ITK) BCR-ABL1. De plus, les ITKs de deuxième et troisième générations sont moins efficaces en PB de la LMC lorsque les cellules leucémiques ont acquis une résistance au traitement indépendante des mutations de BCR-ABL1. Les mécanismes moléculaires des voies de signalisation impliquées dans la progression de la LMC en PB ne sont pas entièrement élucidés. Le but de notre travail est de caractériser de nouvelles anomalies génétiques dans la PB de la LMC. Nous avons identifié en cytogénétique, quatre nouvelles translocations chromosomiques : t(1;21)(p36;q22), t(7;17)(p15;q22), t(8;17)(q11;q22) et t(2;12)(q31;p13) dans les cellules leucémiques de patients en PB de la LMC résistants au traitement. En utilisant des techniques d'hybridation in situ en fluorescence, de RT-PCR et de séquençage, nous avons délimité les régions à investiguer au niveau des points de cassure et identifié un réarrangement de plusieurs gènes codant pour des facteurs de transcription importants lors de l’hématopoïèse tels que RUNX1, ETV6, PRDM16 et HOXA. L’altération de ces gènes pourrait expliquer l’arrêt de différenciation et/ou l’acquisition de la capacité d’autorenouvellement caractéristiques de la LMC en PB. Nous avons identifié les fusions RUNX1-PRDM16, MSI2-HOXA, MSI2-SOX17 et ETV6-HOXD11, respectivement associées aux translocations chromosomiques t(1;21), t(7;17), t(8;17) et t(2;12). Ces fusions génèrent différents transcrits alternatifs qui maintiennent et altèrent le cadre ouvert de lecture. L’analyse des séquences des transcrits chimériques identifiés dans ce projet, incluant RUNX1-PRDM16, MSI2-HOXA9, MSI2-HOXA10, MSI2-HOXA11 et ETV6-HOXD11, nous a permis de prédire les domaines fonctionnels potentiellement présents au niveau des protéines chimériques prédites. Les transcrits de fusion qui respectent le cadre ouvert de lecture peuvent générer des domaines fonctionnels des deux partenaires. C’est le cas des deux transcrits identifiés pour la fusion RUNX1-PRDM16 où le domaine de liaison à l’ADN RHD (Runt homology domain) de RUNX1 est fusionné avec la quasi-totalité des domaines de PRDM16. Les transcrits de fusion qui ne respectent pas le cadre ouvert de lecture donnent des formes tronquées des transcrits RUNX1, MSI2 et ETV6. La juxtaposition des régions promotrices de ces derniers en 5’ de leurs partenaires entraîne l’activation de la forme courte oncogénique de PRDM16 dans la t(1;21) ou de différents gènes HOXA/D dans les t(7;17) et t(2;12), ainsi que l’expression aberrante d’un nouveau transcrit alternatif de SOX17 dans la t(8;17). Notre étude nous a permis d’identifier de nouveaux gènes de fusion et/ou une activation de gènes qui pourraient coopérer avec la fusion BCR-ABL1 dans la progression de la LMC et être impliqués dans la résistance au traitement de la LMC en phase avancée. La caractérisation des événements génétiques associés à la transformation blastique de la LMC est essentielle pour l’investigation des voies moléculaires impliquées dans cette phase de la maladie. Investiguer la résistance au traitement de ces patients pourrait aussi contribuer à identifier de nouvelles cibles thérapeutiques dans cette leucémie.

Establishing a Robust In Vitro Embryonic Stem Cell Differentiation Assay to Monitor the Hematopoietic Potential of DELES Clones

Afin d’effectuer des études fonctionnelles sur le génome de la souris, notre laboratoire a généré une bibliothèque de clones de cellules souches embryonnaires (ESC) présentant des suppressions chromosomiques chevauchantes aléatoires – la bibliothèque DELES. Cette bibliothèque contient des délétions couvrant environ 25% du génome murin. Dans le laboratoire, nous comptons identifier de nouveaux déterminants du destin des cellules hématopoïétiques en utilisant cet outil. Un crible primaire utilisant la benzidine pour démontrer la présence d'hémoglobine dans des corps embryoïdes (EBS) a permis d’identifier plusieurs clones délétés présentant un phénotype hématopoïétique anormal. Comme cet essai ne vérifie que la présence d'hémoglobine, le but de mon projet est d'établir un essai in vitro de différenciation des ESC permettant de mesurer le potentiel hématopoïétique de clones DELES. Mon hypothèse est que l’essai de différenciation hématopoïétique publié par le Dr Keller peut être importé dans notre laboratoire et utilisé pour étudier l'engagement hématopoïétique des clones DELES. À l’aide d’essais de RT-QPCR et de FACS, j’ai pu contrôler la cinétique de différenciation hématopoïétique en suivant l’expression des gènes hématopoïétiques et des marqueurs de surface comme CD41, c-kit, RUNX1, GATA2, CD45, β-globine 1 et TER-119. Cet essai sera utilisé pour valider le potentiel hématopoïétique des clones DELES candidats identifiés dans le crible principal. Mon projet secondaire vise à utiliser la même stratégie rétro-virale a base de Cre-loxP utilisée pour générer la bibliothèque DELES pour générer une bibliothèque de cellules KBM-7 contenant des suppressions chromosomiques chevauchantes. Mon but ici est de tester si la lignée cellulaire leuémique humaine presque haploïde KBM-7 peut être exploitée en utilisant l'approche DELES pour créer cette bibliothèque. La bibliothèque de clones KBM-7 servira à définir les activités moléculaires de drogues anti-leucémiques potentielless que nous avons identifiées dans le laboratoire parce qu’elles inhibent la croissance cellulaire dans plusieurs échantillons de leucémie myéloïde aiguë dérivés de patients. Elle me permettra également d'identifier les voies de signalisation moléculaires qui, lorsque génétiquement perturbées, peuvent conférer une résistance à ces drogues.

Increased Levels of NOTCH1, NF-kappa B, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vivo consequences of AML1-ETO fusion protein expression for hematopoiesis

The t(8;21) (q22;q22) translocation fusing the ETO (also known as MTG8) gene on human chromosome 8 with the AML1 (also called Runx1 or CBFα) gene on chromosome 21 is one of the most common genetic aberrations found in acute myeloid leukemia (AML). This chromosomal translocation occurs in 12 % of de novo AML cases and in up to 40 % of the AML-M2 subtype of the French-American-British classification. To date, the in vivo function of aberrant AML1-ETO fusion protein expression has been investigated by several groups. However, in these studies, controversial results were reported and some key issues remain unknown. Importantly, the consequences of aberrant AML1-ETO expression for self-renewing hematopoietic stem cells (HSCs), multipotent hematopoietic progenitors (MPPs) and lineage-restricted precursors are not known. rn The aim of this thesis was to develop a novel experimental AML1-ETO in vivo model that (i) overcomes the current lack of insight into the pre-leukemic condition of t(8;21)-associated AML, (ii) clarifies the in vivo consequences of AML1-ETO for HSCs, MPPs, progenitors and more mature blood cells and (iii) generates an improved mouse model suitable for mirroring the human condition. For this purpose, a conditional tet on/off mouse model expressing the AML1-ETO fusion protein from the ROSA26 (R26) locus was generated. rn Aberrant AML1-ETO activation in compound ROSA26/tetOAML1-ETO (R26/AE) mice caused high rates of mortality, an overall disruption of hematopoietic organs and a profound alteration of hematopoiesis. However, since the generalized activity of the R26 locus did not recapitulate the leukemic condition found in human patients, it was important to restrict AML1-ETO expression to blood cell lineages. Therefore, bone marrow cells from non-induced R26/AE mice were adoptively transplanted into sublethal irradiated RAG2-/- recipient mice. First signs of phenotypical differences between AML1-ETO-expressing and control mice were observed after eight to nine months of transgene induction. AML1-ETO-expressing mice showed profound changes in hematopoietic organs accompanied by manifest extramedullary hematopoiesis. In addition, a block in early erythropoiesis, B- and T-cell maturation was observed and granulopoiesis was significantly enhanced. Most interestingly, conditional activation of AML1-ETO in chimeric mice did not increase HSCs, MPPs, common lymphoid precursors (CLPs), common myeloid progenitors (CMPs) and megakaryocyte-erythrocyte progenitors (MEPs) but promoted the selective amplification of granulocyte-macrophage progenitors (GMPs). rn The results of this thesis provide clear experimental evidence how aberrant AML1-ETO modulates the developmental properties of normal hematopoiesis and establishes for the first time that AML1-ETO does not increase HSCs, MPPs and common lineage-restricted progenitor pools but specifically amplifies GMPs. The here presented mouse model not only clarifies the role of aberrant AML1-ETO for shaping hematopoietic development but in addition has strong implications for future therapeutic strategies and will be an excellent pre-clinical tool for developing and testing new approaches to treat and eventually cure AML.rn

Transcriptional dysregulation during myeloid transformation in AML

The current paradigm on leukemogenesis indicates that leukemias are propagated by leukemic stem cells. The genomic events and pathways involved in the transformation of hematopoietic precursors into leukemic stem cells are increasingly understood. This concept is based on genomic mutations or functional dysregulation of transcription factors in malignant cells of patients with acute myeloid leukemia (AML). Loss of the CCAAT/enhancer binding protein-alpha (CEBPA) function in myeloid cells in vitro and in vivo leads to a differentiation block, similar to that observed in blasts from AML patients. CEBPA alterations in specific subgroups of AML comprise genomic mutations leading to dominant-negative mutant proteins, transcriptional suppression by leukemic fusion proteins, translational inhibition by activated RNA-binding proteins, and functional inhibition by phosphorylation or increased proteasomal-dependent degradation. The PU.1 gene can be mutated or its expression or function can be blocked by leukemogenic fusion proteins in AML. Point mutations in the RUNX1/AML1 gene are also observed in specific subtypes of AML, in addition to RUNX1 being the most frequent target for chromosomal translocation in AML. These data are persuasive evidence that impaired function of particular transcription factors contributes directly to the development of human AML, and restoring their function represents a promising target for novel therapeutic strategies in AML.