MLL/KMT2A amplifications and translocations are prevalent in infant, adult and therapy-induced leukemia. However, the molecular contributor(s) to these alterations are unclear. Here we demonstrate that histone H3 lysine 9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus regulates these amplifications and rearrangements. This balance is controlled by the cross-talk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B depletion increases H3K9me1/2 levels and reduces CTCF occupancy at the MLL/KMT2A locus, and in turn, promotes amplification and rearrangements. Depleting CTCF is also sufficient to generate these focal alterations. Furthermore, the chemotherapy Doxorubicin (Dox), which associates with therapy-induced leukemia and promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. KDM3B and CTCF overexpression rescues Dox-induced MLL/KMT2A alterations. G9a inhibition in human cells or mice also suppresses MLL/KMT2A events accompanying Dox treatment. Therefore, MLL/KMT2A amplifications and rearrangements are controlled by epigenetic regulators that are tractable drug targets, which has clinical implications. The data presented in this thesis were published in Cell in 2023. / Biomedical Sciences

Mon travail de thèse porte sur l’étude du rôle de l’histone acétyl-transférase MOZ et de l’histone méthyle-transférase MLL dans l’hématopoïèse. Elles contrôlent l’expression de nombreux gènes, nottament des gènes HOX, des facteurs de transcription connus pour leur rôle dans l’hématopoïèse normale et pathologique. Les deux protéines ont des gènes cibles communs tel qu'HOXA9. Ces observations nous ont conduit à rechercher une coopération fonctionnelle entre MOZ et MLL. Nous avons montré que MOZ était associée avec MLL dans les cellules souches/progénitrices humaines CD34+ afin d’activer la transcription des gènes HOXA5, HOXA7 et HOXA9. En effet, les deux protéines interagissent et sont recrutées au niveau de leur promoteur. Nous avons mis en évidence une interférence fonctionnelle entre ces deux facteurs épigénétiques, puisque MOZ est nécessaire au recrutement et à l’activité enzymatique de MLL au niveau des gènes HOXA5, HOXA7 et HOXA9 et réciproquement.Afin de caractériser le mécanisme d’action impliquant la coopération entre MOZ et MLL, nous avons recherché d’autres partenaires associés à ce duo. Nous avons identifié la Symplekin, un membre de la machinerie de polyadénylation. Nous avons mis en évidence l’interaction de la Symplekin avec MOZ et MLL dans les cellules de la lignée hématopoïétique humaine KG1. Les trois protéines sont co-recrutées sur le promoteur du gène HOXA9. Nous avons démontré le rôle ambivalent de la Symplekin. Bien qu’elle soit importante pour la polyadénylation et par conséquent pour la stabilité de l’ARN Hoxa9, la Symplekin empêche le recrutement de MOZ et de MLL au niveau du gène HOXA9, conduisant ainsi à une diminution de sa transcription. / My thesis project has consisted of the study of MOZ, and MLL. They are epigenetic regulators. MOZ and MLL activate transcription of HOX genes, which are transcription factors essential during haematopoiesis. MOZ and MLL have some target genes in common. In our study, we characterised a cooperation between MOZ and MLL in human haematopoietic stem/progenitor cells CD34+. They are both recruited onto HOX promoters. MOZ is essential for MLL recruitment, and this is reciprocal. In conclusion, we provided an example of a mechanism involving a direct cross-talk between two histone modifying enzymes.In order to dissect the mechanism of action of this complex, we decided to identify novel proteins interacting with both MOZ and MLL. A member of the RNA polyadenylation machinery has been isolated: Symplekin. We confirmed the interaction between MOZ, MLL and Symplekin in the human haematopoietic immature cell line KG1. We showed that Symplekin is co-recruited to HOXA9 promoter along with MOZ and MLL. We demonstrated the dual role of this member of the polyadenylation machinery. Indeed, besides the fact that Symplekin is important for Hoxa9 polyadenylation, thus its stability, it prevents MOZ and MLL recruitment onto HOXA9 promoter, leading to a decrease of HOXA9 transcription.Our work improved the understanding of the mechanism of action of MOZ and MLL in HOX control.

A natureza heterogênea da Leucemia Mielóide Aguda (LMA) tornou-se um desafio para o sucesso da quimioterapia convencional com o agente Citarabina (Ara-C), especialmente em leucemias com prognóstico desfavorável, como aquelas portadoras do rearranjo MLL. Visto que as células de LMA-MLL são consideradas sensíveis ao Ara-C quando comparadas às leucemias que não apresentam o rearranjo, mas a recaída à doença é frequente, a presente tese propôs estudar a relação entre características biológicas relacionadas às bases da resistêmcia ao Ara-C em LMA-MLL. A abordagem proposta foi a seleção de subpopulações de linhagens celulares portadoras do rearranjo MLL submetidas ao tratamento em longo prazo com Ara-C, comparando-as com as linhagens não expostas à droga. As células foram caracterizadas quanto: 1) ao potencial proliferativo na presença ou ausência de Ara-C; 2) a distribuição das células no ciclo celular; 3) a distribuição de marcadores clássicos de superfície de células-tronco hematopoiéticas, CD34 e CD38; e 4) o perfil de expressão global dos RNAs transcritos. O tratamento em longo prazo selecionou células mais resistentes ao Ara-C que as células parentais. Além disso, quanto ao ciclo celular, as células selecionadas com Ara-C apresentaram apoptose reduzida (fase sub-G1), acúmulo na fase de síntese (fase S) e aumento da capacidade proliferativa após reexposição à droga (fase G2-M). Quanto à análise de marcadores de células-tronco hematopoiéticas, observou-se que após o tratamento em longo com Ara-C, uma das linhagens celulares apresentou distribuição bimodal do marcador CD38. Quando separadas por sorting em citometria de fluxo, observou-se que as subpopulações com níveis distintos de expressão de CD38, denominadas MV-4-11 CD38High e MV-4-11 CD38Low apresentaram resposta distinta ao tratamento com Ara-C. Quando avaliadas quanto ao perfil global de expressão gênica, constatou-se que MV-4-11 CD38High eram mais semelhantes às células parentais, e que MV-4-11 CD38Low formavam um grupo isolado, distinto das outras duas populações celulares. A análise de ontologia gênica (GO) evidenciou que entre as categorias mais representativas de processos biológicos estavam atividades associadas à capacidade proliferativa, ao desenvolvimento e a resposta a estímulos. As análises de agrupamentos hierárquicos mostraram que: 1) o cluster de genes do desenvolvimento HOXA estava mais expresso nas células MV-4-11 CD38Low do que em MV-4-11 CD38High, que apresentaram expressão mais elevada do cluster HOXB; 2) o gene HOX mais diferencialmente expresso foi HOXA13, associado na literatura com prognóstico desfavorável em outros tipos de câncer; 3) dos genes associados a resposta a estímulos, o único relacionado à via de metabolização do Ara-C diferencialmente expresso entre as linhagens foi NME1; 4) aqueles que participam das vias de reparo de pareamento incorreto, reparo por excisão de bases e por excisão de nucleotídeos encontraram-se mais expressos nas células MV-4-11 CD38High que em MV-4-11 CD38Low. Além disso, diversas quinases dependentes de ciclinas (CDKs) também estiveram diferencialmente expressas entre MV-4-11 CD38High e MV-4-11 CD38Low. Sugere-se por fim, que o modelo in vitro proposto neste estudo para simular a situação de resistência ao Ara-C em subpopulações de LMA-MLL, demonstrou que os mecanismos de resposta à Citarabina nesta doença, vão além de alterações na detoxificação e metabolização da droga, e parecem mais associados a vantagens proliferativas e do desenvolvimento das células leucêmicas. Estas vias devem ser exploradas como alvos potenciais na terapia combinada ao Ara-C. / The heterogeneity of Acute Myeloid Leukemia (AML) became a challenge for the success of the conventional chemotherapy agent Cytarabine (Ara-C), especially in leukemias with poor prognosis, as those harboring MLL rearrangement. Since AML-MLL cells are considered sensitive to Ara-C when compared with leukemias that do not carry the rearrangement, but relapse is frequent, the present dissertation proposed to study the relationship between biological characteristics related to the basis of chemoresistance to Ara-C in AML-MLL. We proposed an approach based on the selection of subpopulations of cell lines bearing MLL rearrangement submitted to the long-term treatment with Ara-C, comparing them with the cell lines that were not previously exposed to the drug. The cells were characterized according to: 1) the proliferative potential in the presence and absence of Ara-C; 2) the distribution of the cells in the cell cycle; 3) distribution of hematopoietic stem cell classic surface markers, CD34 and CD38; and, 4) global expression profile of transcribed RNAs. The long-term treatment selected cells that are more resistant to Ara-C than the cells that were not previously treated (parental cells). Besides, according to cell cycle, the cells selected by Ara-C treatment present decreased apoptosis (sub-G1 phase), accumulation in the synthesis phase (S-phase) and increase in the proliferative capability after re-exposition to the drug (G2-M phase). Regarding the hematopoietic stem cell markers, we observed that after Ara-C long-term treatment, one of the cell lines exhibited a bimodal distribution of the CD38 marker. When sorted by flow cytometry, we observed that both subpopulations with distinct levels of CD38 expression, called MV-4-11 CD38High and MV-4-11 CD38Low also showed distinct response to Ara-C. When evaluated regarding to their global gene expression profiles, we verified that MV-4-11 CD38High were more closely related to the parental cells, and MV-4-11 CD38Low made up an isolated group, distinct of the other cell populations. Gene ontology (GO) analysis revealed that among the most representative categories of biological processes, activities associated with proliferative capability, development and response to stimuli were included. The hierarchical clustering analysis showed that: 1) the cluster HOXA of genes of development was more expressed in the MV-4-11 CD38Low than in the MV-4-11 CD38High cells, that presented increased expression of HOXB cluster; 2) the most differentially expressed HOX gene was HOXA13, which according to the literature is associated with poor prognosis in other types of cancer; 3) among the genes associated with response to stimuli, the only one related to Ara-C-metabolizing pathway that was differentially expressed between the cell lines was NME1; 4) those genes that take part in the mismatch repair, base excision repair and nucleotide excision repair pathways were more expressed in the MV-4-11 CD38High than in the MV-4-11 CD38Low cells. Additionally, several cyclin-dependent kinases (CDKs) were also differentially expressed between MV-4-11 CD38High and MV-4-11 CD38Low. Finally, we suggest that the in vitro model proposed in this study to mimic the situation of chemoresistance to Ara-C in subpopulations of AML-MLL, showed that the mechanisms of Ara-C response in this disease, go beyond changes in drug detoxification and metabolization, and seem more associated to proliferative and development advantages of the leukemic cells. These pathways should be explored as potential targets to Ara-C combination therapies.

A natureza heterogênea da Leucemia Mielóide Aguda (LMA) tornou-se um desafio para o sucesso da quimioterapia convencional com o agente Citarabina (Ara-C), especialmente em leucemias com prognóstico desfavorável, como aquelas portadoras do rearranjo MLL. Visto que as células de LMA-MLL são consideradas sensíveis ao Ara-C quando comparadas às leucemias que não apresentam o rearranjo, mas a recaída à doença é frequente, a presente tese propôs estudar a relação entre características biológicas relacionadas às bases da resistêmcia ao Ara-C em LMA-MLL. A abordagem proposta foi a seleção de subpopulações de linhagens celulares portadoras do rearranjo MLL submetidas ao tratamento em longo prazo com Ara-C, comparando-as com as linhagens não expostas à droga. As células foram caracterizadas quanto: 1) ao potencial proliferativo na presença ou ausência de Ara-C; 2) a distribuição das células no ciclo celular; 3) a distribuição de marcadores clássicos de superfície de células-tronco hematopoiéticas, CD34 e CD38; e 4) o perfil de expressão global dos RNAs transcritos. O tratamento em longo prazo selecionou células mais resistentes ao Ara-C que as células parentais. Além disso, quanto ao ciclo celular, as células selecionadas com Ara-C apresentaram apoptose reduzida (fase sub-G1), acúmulo na fase de síntese (fase S) e aumento da capacidade proliferativa após reexposição à droga (fase G2-M). Quanto à análise de marcadores de células-tronco hematopoiéticas, observou-se que após o tratamento em longo com Ara-C, uma das linhagens celulares apresentou distribuição bimodal do marcador CD38. Quando separadas por sorting em citometria de fluxo, observou-se que as subpopulações com níveis distintos de expressão de CD38, denominadas MV-4-11 CD38High e MV-4-11 CD38Low apresentaram resposta distinta ao tratamento com Ara-C. Quando avaliadas quanto ao perfil global de expressão gênica, constatou-se que MV-4-11 CD38High eram mais semelhantes às células parentais, e que MV-4-11 CD38Low formavam um grupo isolado, distinto das outras duas populações celulares. A análise de ontologia gênica (GO) evidenciou que entre as categorias mais representativas de processos biológicos estavam atividades associadas à capacidade proliferativa, ao desenvolvimento e a resposta a estímulos. As análises de agrupamentos hierárquicos mostraram que: 1) o cluster de genes do desenvolvimento HOXA estava mais expresso nas células MV-4-11 CD38Low do que em MV-4-11 CD38High, que apresentaram expressão mais elevada do cluster HOXB; 2) o gene HOX mais diferencialmente expresso foi HOXA13, associado na literatura com prognóstico desfavorável em outros tipos de câncer; 3) dos genes associados a resposta a estímulos, o único relacionado à via de metabolização do Ara-C diferencialmente expresso entre as linhagens foi NME1; 4) aqueles que participam das vias de reparo de pareamento incorreto, reparo por excisão de bases e por excisão de nucleotídeos encontraram-se mais expressos nas células MV-4-11 CD38High que em MV-4-11 CD38Low. Além disso, diversas quinases dependentes de ciclinas (CDKs) também estiveram diferencialmente expressas entre MV-4-11 CD38High e MV-4-11 CD38Low. Sugere-se por fim, que o modelo in vitro proposto neste estudo para simular a situação de resistência ao Ara-C em subpopulações de LMA-MLL, demonstrou que os mecanismos de resposta à Citarabina nesta doença, vão além de alterações na detoxificação e metabolização da droga, e parecem mais associados a vantagens proliferativas e do desenvolvimento das células leucêmicas. Estas vias devem ser exploradas como alvos potenciais na terapia combinada ao Ara-C. / The heterogeneity of Acute Myeloid Leukemia (AML) became a challenge for the success of the conventional chemotherapy agent Cytarabine (Ara-C), especially in leukemias with poor prognosis, as those harboring MLL rearrangement. Since AML-MLL cells are considered sensitive to Ara-C when compared with leukemias that do not carry the rearrangement, but relapse is frequent, the present dissertation proposed to study the relationship between biological characteristics related to the basis of chemoresistance to Ara-C in AML-MLL. We proposed an approach based on the selection of subpopulations of cell lines bearing MLL rearrangement submitted to the long-term treatment with Ara-C, comparing them with the cell lines that were not previously exposed to the drug. The cells were characterized according to: 1) the proliferative potential in the presence and absence of Ara-C; 2) the distribution of the cells in the cell cycle; 3) distribution of hematopoietic stem cell classic surface markers, CD34 and CD38; and, 4) global expression profile of transcribed RNAs. The long-term treatment selected cells that are more resistant to Ara-C than the cells that were not previously treated (parental cells). Besides, according to cell cycle, the cells selected by Ara-C treatment present decreased apoptosis (sub-G1 phase), accumulation in the synthesis phase (S-phase) and increase in the proliferative capability after re-exposition to the drug (G2-M phase). Regarding the hematopoietic stem cell markers, we observed that after Ara-C long-term treatment, one of the cell lines exhibited a bimodal distribution of the CD38 marker. When sorted by flow cytometry, we observed that both subpopulations with distinct levels of CD38 expression, called MV-4-11 CD38High and MV-4-11 CD38Low also showed distinct response to Ara-C. When evaluated regarding to their global gene expression profiles, we verified that MV-4-11 CD38High were more closely related to the parental cells, and MV-4-11 CD38Low made up an isolated group, distinct of the other cell populations. Gene ontology (GO) analysis revealed that among the most representative categories of biological processes, activities associated with proliferative capability, development and response to stimuli were included. The hierarchical clustering analysis showed that: 1) the cluster HOXA of genes of development was more expressed in the MV-4-11 CD38Low than in the MV-4-11 CD38High cells, that presented increased expression of HOXB cluster; 2) the most differentially expressed HOX gene was HOXA13, which according to the literature is associated with poor prognosis in other types of cancer; 3) among the genes associated with response to stimuli, the only one related to Ara-C-metabolizing pathway that was differentially expressed between the cell lines was NME1; 4) those genes that take part in the mismatch repair, base excision repair and nucleotide excision repair pathways were more expressed in the MV-4-11 CD38High than in the MV-4-11 CD38Low cells. Additionally, several cyclin-dependent kinases (CDKs) were also differentially expressed between MV-4-11 CD38High and MV-4-11 CD38Low. Finally, we suggest that the in vitro model proposed in this study to mimic the situation of chemoresistance to Ara-C in subpopulations of AML-MLL, showed that the mechanisms of Ara-C response in this disease, go beyond changes in drug detoxification and metabolization, and seem more associated to proliferative and development advantages of the leukemic cells. These pathways should be explored as potential targets to Ara-C combination therapies.

Acute myeloid leukemia (AML) is the second most common type of leukemia and accounts for 80% of adult acute leukemia cases and is characterized by the accumulation of poorly or undifferentiated myeloid blast cells. Standard treatment includes chemotherapy, which if unsuccessful, is followed by more rigorous chemotherapy as well as stem cell transplantation. Considering most patients are over the age of 45, these more rigorous therapies are not always possible, and as such, new therapies must be developed. Furthermore, AML patients harboring a chromosomal rearrangement involving Multiple Lineage Leukemia (MLL) that results in the expression of an MLL fusion protein exhibit far worse prognoses than patients without. In recent years, Danio rerio (zebrafish) has emerged as a powerful model organism for investigating human blood malignancies due to the conservation of hematopoiesis between humans and zebrafish. The first objective of this study was to develop a transient transgenic AML model in zebrafish, and the second objective was to determine if co-treatment with two medications currently in human trials for AML, Venetoclax and Flavopiridol, would be more effective than using either drug individually. In order to develop a transient transgenic AML model, we first developed a DNA construct encoding a known mixed lineage leukemia (MLL) fusion protein associated with human AML, MLL-ENL, driven by the zebrafish lysozyme C (lyz) promoter, which drives myeloid specific expression in zebrafish. We then microinjected single-cell zebrafish embryos with DNA encoding lyz driven MLL-ENL along with transposase mRNA to facilitate the genomic integration of MLL-ENL. Injected embryos were first tested for MLL-ENL expression, and subsequently tested for AML phenotypic characteristics, via whole mount in-situ hybridization (WISH) at 72 hours post fertilization (hpf). First, WISH analysis utilizing a human MLL riboprobe verified MLL-ENL expression in injected embryos, and WISH analysis utilizing the same MLL riboprobe revealed an expansion and clustering of MLL positive cells in injected embryos, characteristic of an AML phenotype. Embryos injected with MLL-ENL DNA were then treated with either DMSO (vehicle), 200 nanomolar (nM) Venetoclax, 200 nM Flavopiridol, or 200 nM Venetoclax and 200 nM Flavopiridol from 24 hpf to 72 hpf. MLL WISH analysis of injected and treated embryos revealed a reduction in MLL positive cells in both Venetoclax treated embryos and Flavopiridol treated embryos, and an even greater reduction in MLL positive cells in embryos treated with both Venetoclax and Flavopiridol, compared to controls. Although further analysis is required to be confident, these data suggest that we successfully developed an AML transient transgenic model in zebrafish. Furthermore, these data suggest that Venetoclax and Flavopiridol co-treatment could yield better outcomes for AML patients than treatment with either drug individually.

Acute myeloid leukemia (AML) is a highly heterogeneous disorder that results from a block in the differentiation of hematopoietic progenitor cells along with uncontrolled proliferation. In approximately 60% of cases, specific recurrent chromosomal aberrations can be identified by modern cytogenetic techniques, and is an important indicator to classify patients into three prognostic categories: favorable, intermediate, and poor risk. Currently, favorable risk patients are usually treated with chemotherapy while poor risk patients receive allogeneic stem cell transplantation. However, the largest subgroup of AML patients (approximately 40%) has no identifiable cytogenetic abnormalities and is classified as intermediate risk. In this special subgroup of patients, a number of studies have demonstrated the relationship between different translocations involving the mixed lineage leukemia (MLL) gene and patient prognosis. The heterogeneity of MLL-rearranged AML is reflected by the identification of more than 70 different fusion partners of this gene and the panel is continuously increasing. The aim of this study is to develop a sensitive molecular profiling test for relevant risk stratification that can help in the decision of treatment and/or follow-up strategy.

Acute myeloid leukemia (AML) is a clonal neoplastic disorder organized as a cellular hierarchy, with the self-renewing leukemia stem cell (LSC) at the apex. Recurrent mutations in transcription factors (TF) and epigenetic regulators suggest that AML is driven by aberrant transcriptional circuits, but these circuits have not been fully defined in an LSC model. To study transcriptional mechanisms relevant to leukemogenesis in vivo, we generated a murine serial transplantation model of MLL-AF9-driven, myelomonocytic leukemia with genetically- and phenotypically-defined LSCs. Using this model, we pursued two related lines of investigation. First, we performed an in vivo RNA interference (RNAi) screen to identify transcription factors required for LSC function. This screen highlighted the circadian rhythm TFs, Clock and Bmal1, as genes essential for the survival of murine leukemia cells, and we validated this finding with CRISPR/Cas-based genome editing and knockdown studies in AML cell lines. Utilizing luciferase reporter mice to track expression of the circadian target gene Per2, we demonstrated that both leukemic and normal hematopoietic cells have the capacity for oscillating, circadian-dependent gene expression. Importantly, using murine knockout models, we found that normal hematopoietic stem and progenitor cells (HSPC), in contrast to leukemia cells, do not depend on Bmal1. We further demonstrated that selective depletion of LSCs following circadian perturbation is mediated through enhanced myeloid differentiation. ChIP-Seq studies revealed that the circadian rhythm network is integrally connected to the LSC self-renewal circuitry and highlighted putative Clock/Bmal1 targets in leukemia, providing a mechanistic basis for our findings. Second, we performed a functional and genomic characterization of our MLL-AF9 serial transplantation model to explore mechanisms of disease evolution and clonal selection in AML. Limiting dilution studies demonstrated that serial transplantation results in a reduction in disease latency, dramatic enrichment of leukemia-initiating cells (LIC), and reconfiguration of the LSC hierarchy. While mutations in known AML-associated genes were not linked to disease progression, RNA-sequencing (RNA-Seq) demonstrated that the increase in LIC frequency in serially transplanted leukemias is driven by changes in cell cycle and differentiation. In aggregate, these studies offer insights into the biological mechanisms regulating LSC self-renewal and disease evolution in AML.

No description available.

<p>Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %.</p><p>In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML.</p><p>The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved.</p><p>Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS.</p><p>Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL).</p><p>Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.</p>

Le gène MLL (Mixed-Lineage Leukemia), un homologue du gène trithorax de la Drosophile, localisé à la bande chromosomique 11q23, est fréquemment réarrangé dans plusieurs types de leucémies, essentiellement suite à des translocations chromosomiques. Dans les différentes translocations chromosomiques, la partie N-terminale de MLL est fusionnée avec les séquences d’un gène partenaire. Malgré le grand nombre de partenaires de fusion rapportés, peu de fusions MLL ont été bien caractérisées sur le plan moléculaire. De plus, l’impact pronostique de plusieurs fusions moins fréquentes n’est pas bien établi. L’objectif de mon projet est de caractériser plusieurs translocations MLL qui ont été détectées dans 39 spécimens leucémiques collectés par la Banque de cellules leucémiques du Québec (www.bclq.gouv.qc.ca), et d’établir une corrélation entre les résultats de la cytogénétique et différents paramètres biologiques et cliniques des leucémies respectives. L’identification des gènes partenaires de fusion (GPF) dans notre série (30 échantillons étudiés), a révélé la fusion de MLL à un gène partenaire très récurrent dans 26 leucémies: MLLT3(AF9), AFF1(AF4), MLLT4(AF6), MLLT1(ENL), ELL; à un GPF modérément commun dans 1 leucémie : MLLT6(AF17); et à un partenaire rare de MLL dans 3 leucémies : GAS7 et AF15/CASC5 (2 cas). Nous avons poursuivi notre travail avec la caractérisation des points de cassure de deux fusions, soit MLL-ELL associée à un syndrome myéloprolifératif (une association rare), et MLL-GAS7 (une fusion rare de MLL), associée à une leucémie aiguë myéloïde. L’analyse des transcrits de fusion par RT-PCR et séquençage a révélé respectivement la fusion de l’exon 9 de MLL à l’exon 2 de ELL et des exons 7 ou 8 de MLL (deux transcrits) à l’exon 2 de GAS7. Ce travail permettra d’effectuer des études fonctionnelles et des projets de recherche translationnelle en utilisant ces spécimens de leucémies avec différents réarrangements de MLL, bien caractérisés sur le plan clinique et moléculaire. / The MLL (Mixed-Lineage Leukemia) gene, a human homolog of the Drosophila trithorax gene, located at chromosomal band 11q23, is frequently rearranged in several types of leukemia, mostly by chromosomal translocations. In different chromosomal translocations, the N-terminal part of MLL is fused to sequences of the partner gene. Despite the large number of fusion partners that have been reported, several gene fusions remain poorly characterized at the molecular level. Moreover, the prognostic impact of less frequent fusions is not well established. The aim of my project is to characterize different MLL fusions detected in 39 leukemic samples, collected by the Quebec Leukemia Cell Bank (www.bclq.gouv.qc.ca) and to correlate cytogenetics with the clinical and biological features of the corresponding leukemia. Identification of fusion partner genes in our series (30 samples studied), revealed fusion of MLL to one of the most frequent partners in 26 leukemias: MLLT3(AF9), AFF1(AF4), MLLT4(AF6), MLLT1(ENL), ELL; to a moderately common MLL fusion partner in 1 leukemia: MLLT6(AF17); and to a rare partner in 3 leukemias: GAS7 and AF15/CASC5 (2 cases). We have characterized the breakpoints of two fusions, MLL-ELL in a myeloproliferative syndrome (a rare association) and MLL-GAS7 (a rare MLL fusion) associated with acute myeloid leukemia. Fusion transcripts analysis by RT-PCR and sequencing revealed respectively, a fusion of MLL exon 9 to ELL exon 2 and of MLL exon 7 or exon 8 (two transcripts) to GAS7 exon 2. This study is essential to perform functional studies and translational research projects using these well characterized leukemic specimens with different MLL rearrangements.