Introdução: O Transplante Alogênico de Célula Tronco Hematopoiética (TCTH alogênico) é um procedimento de alto potencial curativo para a Leucemia Mielóide Aguda (LMA), principalmente pelo efeito “graft versus leukemia” (GVL), que leva a redução do risco de recaída. Atualmente, os pacientes com LMA de risco intermediário são submetidos ao procedimento caso possuam doador aparentado. Pacientes sem doador aparentado disponível são submetidos a tratamento de consolidação com quimioterapia, com maior chance de recaída da doença. Acredita-se que os pacientes submetidos ao TCTH tenham maiores sobrevida global e livre de doença, a despeito das altas taxas de morbimortalidade. A classificação de risco é extremamente importante para escolha terapêutica pós remissão. Assim, a realização da pesquisa de marcadores moleculares, para refinar a estratificação prognóstica, tem importância especial no grupo de risco intermediário, complementando a avaliação citogenética, e auxiliando na decisão terapêutica, sendo cada vez mais necessária, apesar de não disponível em todos os centros. Material e métodos: Foram avaliados os pacientes com LMA de risco intermediário em primeira Remissão Completa (1RC) do Serviço de Hematologia e TCTH do Hospital de Clínicas de Porto Alegre do período de 01 de abril de 1999 a 01 de outubro de 2014, com pelo menos 1 ano de seguimento após o tratamento, através de revisão de prontuários. Os dados foram dispostos no programa Excel e posteriormente exportados para o programa SPSS v. 18.0 para análise estatística. Resultados: Foram avaliados 69 pacientes, sendo 45 pacientes submetidos a consolidação com quimioterapia (“QT”) e 24 submetidos a TCTH Alogênico (“TCTH Alogênico”). A média de idade do grupo “QT” foi de 47,8 anos e do grupo “TCTH Alogênico” foi de 35,5 anos, com diferença estatisticamente significativa (P<0,001). Não houve diferença na distribuição entre o sexo. A mediana de tempo de seguimento do grupo “QT” foi de 1,1 anos (intervalo interquartil de 0,4 a 2,5 ) e no grupo “TCTH Alogênico” foi de 2,7 anos (intervalo interquartil de 0,4 a 5,5), sem diferença estatisticamente significativa na distribuição dos tempos de seguimento entre os grupos (P=0,236). A sobrevida do grupo “QT” em 12 meses foi de 52,3% e no grupo “TCTH Alogênico” foi de 62,5%. Aos 24 meses, a sobrevida do grupo “QT” foi de 31,7% e no grupo “TCTH Alogênico” foi de 58,3% e em 5 anos de 21,1% e 53,8%, respectivamente. O teste do Long-Rank aponta uma diferença estatisticamente significativa nas sobrevidas entre os grupos após 5 anos, com Hazard Ratio (HR) para óbito de 2,2 (IC 95%: 1,1-4,2), P=0,027, porém ao ajustarmos a relação pela idade esta associação perde significância estatística (HR:1,6 IC95%:1 - 1,1; P=0,246) Discussão: Os dados evidenciaram melhor sobrevida no grupo submetido à TCTH alogênico, porém o grupo submetido ao procedimento apresentava média de idade menor. No entanto, apesar da perda da significância estatística, o HR corrigido para idade permanece maior para o grupo sem TCTH, o que pode dever-se ao “n” pequeno da amostra. Identificar quais pacientes terão benefício com TCTH torna-se cada vez mais um desafio. O uso de marcadores moleculares são importantes no refinamento da estratificação de risco do grupo de risco intermediário, podendo auxiliar nessa decisão. Além disso, com o advento da possibilidade de condicionamentos não mieloablativos como alternativa aos pacientes mais velhos e com escore de comorbidades pior e a melhor terapia de suporte, talvez possamos ser menos conservadores na indicação desse procedimento, identificando assim aqueles que poderão obter melhores resultados no tratamento de uma doença tão agressiva e grave. / Background: Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is a high potentially curative procedure to Acute Myeloid Leukemia (AML), mainly by the “graft-versus-leukemia” (GVL) effect, which leads to reduced risk of relapse. Nowadays, intermediate risk AML patients are submitted to this procedure if a matched sibling donor is available. Patients without a sibling donor are submitted to consolidation with chemotherapy, with a greater chance of relapse. It is believed that patients submitted to allo-HSCT have a greater overall survival and disease-free survival, even though it presents high morbidity and mortality rates. Risk stratification is extremely important to post-remission treatment choice. Molecular markers research is especially important in intermediate risk group, complementing cytogenetic evaluation to a better prognostic stratification and, although it is still not available in all health centers, it is more and more necessary. Materials and Methods: We evaluated intermediate risk AML patients in first Complete Remission (CR1) at the Hematology Service and Bone Marrow Transplantation from Hospital de Clínicas de Porto Alegre from April 1st 1999 to October 1st 2014, and which had, at least, a one year follow-up after treatment, by conducting a medical record review. Data was inserted in Microsoft Excel 2010 spreadsheets and after exported to SPSS v. 18.0 to statistical analysis. Results: Among the 69 patients analyzed, 45 were submitted to consolidation with chemotherapy (Intermediate risk AML – non allo-HSCT) and 24 of then submitted to allo-HSCT (Intermediate risk AML – allo-HSCT). The average age of Intermediate risk AML – non allo-HSCT was 47.8 years old and Intermediate risk AML – allo-HSCT was 35.5 years old, with statistically significance difference (P<0,001). There was no difference regard sex of patients. The median follow-up in the Intermediate risk AML – non allo-HSCT was 1.1 years (interquartile rage of 0.4 to 2.5) and in the Intermediate risk AML – allo-HSCT was 2.7 years (interquartile rage of 0.4 to 5.5), with no statistically significance difference in follow-up time distribution between groups (P=0.236). Intermediate risk AML – non allo-HSCT survival in 12 months was 52.3% and in the Intermediate risk AML – allo-HSCT was 62.5%. In 24 months, Intermediate risk AML – non allo-HSCT survival was 31.7% and in Intermediate risk AML – allo HSCT survival was 58.3% and in 5 years it was 21.1% and 53.8% respectively. Long- Rank test indicates a statistically significant difference in survival between groups after 5 years, with hazard ratio (HR) for death of 2.2 (IC95% 1.1 – 4.2), P=0.027, but when we adjust the relation to age, this association loses statistical significance (HR:1.6 95%CI: 1 – 1.1; P=0.246). Discussion: Data showed a better survival rate to the group submitted to allo-HSCT, but the group presented a lower average age. However, despite de loss of statistical significance, Hazard Ratio (HR), adjusted to age remains higher to the non allo-HSCT group. It can be explained by the small number of the sample. Identifying which patients will benefit from allo-HSCT becomes increasingly challenging. The use of molecular markers are important in the refinement of risk stratification in intermediate risk group, assisting in the decision. Moreover, with the advent of the possibility of nonmyeloablative conditioning as an alternative to older patients and with worst rates of comorbidity, and the better supporting therapy, we may be less conservative in indicating this procedure, identifying the patients who may obtain better results during treatment of such aggressive and serious disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Internal tandem duplications in FMS-like receptor tyrosine kinase (FLT3-ITDs) are seen in approximately 25% of all acute myeloid leukemia (AML) patients. FLT3-ITDs induce FLT3 ligand (FL)-independent cellular hyperproliferation, promiscuous and aberrant activation of STAT5, and confer a poor prognosis in patients; however, the molecular mechanisms contributing to FLT3-ITD-induced malignancy remain largely unknown. The protein tyrosine phosphatase, Shp2, is important for normal hematopoiesis as well as hematopoietic stem cell (HSC) differentiation, engraftment, and self-renewal. Furthermore, FLT3-ITD- or constitutive active STAT5-expressing CD34+ cells demonstrate enhanced hematopoietic stem cell self-renewal. Together with the previous findings that Shp2 is critical for normal hematopoiesis, that dysregulated Shp2 function contributes to myeloid malignancies, and that Shp2 has been shown to interact with WT-FLT3 tyrosine 599, which is commonly duplicated in FLT3-ITDs, a positive role for Shp2 in FLT3-ITD-induced signaling and leukemogenesis is implied. I demonstrated that Shp2 is constitutively associated with the reported FLT3-ITDs, N51-FLT3 and N73-FLT3, compared to WT-FLT3; therefore, I hypothesized that increased Shp2 recruitment to N51-FLT3 or N73-FLT3 contributes to hyperproliferation and hyperactivation of STAT5. I also hypothesized that Shp2 cooperates with STAT5 to activate STAT5 transcriptional targets contributing to the up-regulation of pro-leukemic proteins. Finally, I hypothesized that reduction of Shp2 would result in diminished N51-FLT3-induced hyperproliferation and activation of STAT5 in vitro, and prevent FLT3-ITD-induced malignancy in vivo. I found that genetic disruption of Ptpn11, the gene encoding Shp2, or pharmacologic inhibition of Shp2 with the novel Shp2 inhibitor, II-B08, resulted in significantly reduced FLT3-ITD-induced hematopoietic cell hyperproliferation and STAT5 hyperphosphorylation. I also demonstrated a novel role of Shp2 in the nucleus of FLT3-ITD-expressing hematopoietic cells where Shp2 and STAT5 co-localized at the promoter region of STAT5-transcriptional target and pro-survival protein, Bcl-XL. Furthermore, using a Shp2flox/flox;Mx1Cre+ mouse model, I demonstrated that reduced Shp2 expression in hematopoietic cells resulted in an increased latency to and reduced severity of FLT3-ITD-induced malignancy. Collectively, these findings demonstrate that Shp2 plays an integral role in FLT3-ITD-induced malignancy and suggest that targeting Shp2 may be a future therapeutic option for treating FLT3-ITD-positive AML patients.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

Acute Myeloid Leukemia (AML), the most common form of acute leukemia in adults, is only curable in approximately 30% of all cases. Despite prognostic risk stratification using sub-typing and cytogenetic analysis to direct therapy, the mortality and relapse rate remains high. AML patients with normal karyotypes are defined as intermediate risk and are the most challenging to treat. Somatic mutations may be the key in refining prognostic stratification and providing useful therapeutic targets. The FMS-like tyrosine kinase 3 (FLT3) and Nucleophosmin (NPM1) genes have common mutated forms that are associated with overall survival and response to therapy. We assessed mutations in the FLT3 and NPM1 genes and their levels of expression in twenty eight AML patients in the presence and absence of HIV and their response to induction therapy. Furthermore, we used a novel technique, High Resolution Melting (HRM) Analysis to detect FLT3 Internal Tandem Duplications (ITD) and NPM1 exon 12 mutations. Five of the patients in this study were HIV positive, three of whom did not survive post-induction therapy. Of the AML patients, 17.9% were positive for the NPM1 mutation and 21% had mutated FLT3. Interestingly, the presence of the FLT3 and NPM1 mutations were coupled with an increase in expression levels of FLT3 and NPM1 from presentation to post-induction respectively and the loss of these mutations were coupled with a decrease in levels of expression from presentation to post-induction. However, an increase/decrease from presentation to post-induction did not necessarily denote the presence/absence of a mutation. Therefore, while mutational status of genes may generally confer mRNA levels, our results showed that there existed no definitive trend between mRNA levels of NPM1 and FLT3 expression and mutational status. We found that the HRM method was definitive for the simpler NPM1 mutation however detection of the FLT3-ITD mutation was challenging. There isn’t a clear distinction between mutated and non-mutated FLT3 due to the formation of hetero-duplexes during analysis, making detection highly subjective and error-prone. Sequencing allowed confirmation of mutated FLT3 and non-mutated FLT3 which were not in all instances in concordance with HRM analysis. The prognostic value in terms of overall survival of NPM1 and FLT3 mutations in this study is indefinite. Furthermore, the analysis of the HIV positive AML patients revealed no clear correlation between NPM1 and FLT3 levels of mRNA expression and mutational status. Also, the small number of HIV positive AML patients did not allow for conclusions to be made regarding HIV status and survival when affected with AML. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Background: Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/ relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. Methods: Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo((R)), ALDH activity by ALDELUOR(TM), and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo(TM) Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing. Results: Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright "stem-like" populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the beta 5 proteasome subunit. BTZ-resistance conferred increased resistance toAra-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+ induced apoptosis and proteasome inhibition independent of CT-like activity inhibition. Conclusions: We provide evidence that DSF/Cu2+ overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+ as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL.

Chromodomain Helicase DNA-Binding Protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double stranded break (DSB) repair. Here, we show that depletion of CHD4 in Acute Myeloid Leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ATM pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic-agent induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34+ hematopoetic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor formatting behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.

Un des mécanismes d’évasion des cellules tumorales au système immunitaire fait intervenir la famille des molécules de type B7. Ces molécules de costimulation ont aussi un rôle dans les mécanismes de tolérance immunitaire. La molécule B7-H1 (PD-L1 ou CD274) inhibe directement les lymphocytes T cytotoxiques (CTL) et est fortement exprimée à la surface de nombreux types de cellules tumorales. Les mécanismes de régulation de son expression ne sont pas bien connus. Il existe une augmentation d’expression de cette molécule après stimulation par l’IFN&#947; et récemment les ligands des Toll-Like-Receptor (TLR) 2, 4 et 9 ont été impliqués dans sa régulation au niveau du myélome multiple. L’implication possible des TLR dans l’expression de B7-H1 suggère un rôle possible de pathogènes dans l’échappement des tumeurs au système immunitaireIl n’existe aucune donnée dans la littérature sur l’expression des TLR au niveau des cellules blastiques de patients atteints de leucémies aigues myéloblastique (LAM). Nous avons dans un premier temps étudié l’expression des TLR dans les LAM et l’inductibilité de l’expression de la molécule B7-H1 par les ligands des TLR en cytométrie en flux. Nos résultats montrent que les TLR sont exprimés dans les LAM, avec une grande variabilité suivant les patients. On observe une augmentation significative de l’expression de B7-H1 après stimulation par le LPS (ligand TLR4) alors qu’elle n’est pas significative pour les autres ligands (PGN et ODN). Comme dans les tumeurs solides et le myélome multiple, l’expression de B7-H1 est augmentée par l’IFN &#947;. Des tests de lyse CTL sont en cours afin de confirmer le rôle fonctionnel de cette expression de B7-H1 via les TLR dans les LAM.Une autre partie de l’étude a été réalisée sur deux lignées murines leucémiques : DA1-3B et WEHI-3B, afin de disposer de modèles expérimentaux d’expression de B7-H1. On retrouve sur ces deux modèles l’augmentation d’expression de B7-H1 après stimulation par l’IFN &#947; et les ligands des TLR. L’utilisation de différents inhibiteurs chimiques des voies de signalisation suggère le rôle des voies MEK/ERK et de la voie JAK/STAT dans l’expression de cette molécule. La voie PI3kinase/Akt semble au contraire jouer un rôle inhibiteur. Le travail se poursuit avec la transfection de transdominants négatifs des différentes voies et de mutants constitutivement actifs. L’objectif à terme est de tester des stratégies d’immunothérapies des LAM par blocage pharmacologique de l’expression de B7-H1. / B7-H1 (PD-L1) is a B7-related protein that inhibits T-cell responses. B7-H1 participates in the immunoescape of cancer cells and is also involved in the long-term persistence of leukemic cells in a mouse model. B7-H1 can be constitutively expressed by cancer cells but is also induced by various stimuli. We therefore examined the constitutive and inducible expression of B7-H1 and the consequences of expression in human acute myeloid leukemia (AML). We analyzed B7-H1 expression in a cohort of 79 patients with AML. Blast cells were also studied after incubation with interferon-gamma or TLR ligands. Functionality was evaluated by cytotoxic T-cell activity against blast cells. Expression of B7-H1 at diagnosis was high in 18% of patients. Expression of toll-like receptors (TLR) 2, 4, and 9 was detected in one-third of AML samples. Expression of TLR2 and TLR4 ligands or IFN-&#61543; induced by B7-H1 was found to protect AML cells from CTL-mediated lysis. Spontaneous B7-H1 expression was also found to be enhanced at relapse in some patients. MEK inhibitors including UO126 and AZD6244 reduced B7-H1 expression and restored CTL-mediated lysis of blast cells. In AML, B7-H1 expression by blasts represents a possible immune escape mechanism. The inducibility of B7-H1 expression by IFN-&#61543; or TLR ligands suggests that various stimuli, either produced during the immune response against leukemia cells or released by infectious microorganisms, could protect leukemic cells from T-cells. The efficacy of MEK inhibitors against B7-H1-mediated inhibition of CTLs suggests a possible cancer immunotherapy strategy using targeted drugs.

Les leucémies aiguës myéloïdes (LAM) sont des hémopathies malignes caractérisées par une accumulation dans la moelle et le sang de progéniteurs hématopoïétiques bloqués dans un stade différenciation. La mutation FLT3-ITD, qui entraîne une activation constitutive du récepteur à activité tyrosine kinase FLT3, est retrouvée dans 20-25% des LAM et est associée à un mauvais pronostique. De nombreux inhibiteurs de FLT3 ont été développés et certains sont testés en clinique mais des études mettent en évidence l'apparition de résistance. Une meilleure compréhension des mécanismes oncogéniques de FLT3-ITD est donc nécessaire afin d'améliorer le traitement des LAM. Mes travaux de thèse ont été centrés sur l'analyse du processus autophagique qui correspond à l'un des mécanismes de résistance décrits dans les cellules cancéreuses en réponse aux traitements. Au cours de cette étude, nous avons constaté que l'expression de FLT3-ITD augmente l'autophagie basale des cellules de LAM, et que l'inhibition du récepteur réduit cette autophagie dans des échantillons primaires de LAM et dans des lignées cellulaires. Nous avons pu montrer que l'autophagie est requise pour la prolifération et la survie in vitro et in vivo des cellules de LAM et que sont ciblage permet de surmonté la résistance aux inhibiteurs de FLT3. De plus, nous avons identifié la protéine ATF4 comme un acteur essentiel au processus d'autophagie en aval de FLT3-ITD. Ces résultats suggèrent que le ciblage de l'autophagie ou d'ATF4 chez les patients exprimant les mutations de FLT3 peut représenter une stratégie thérapeutique prometteuse et innovatrice dans les LAM. / Acute myeloid leukemias (AMLs) are a family of hematological malignancies characterized by an accumulation in the marrow and blood of hematopoietic progenitors blocked in their differentiation process. The FLT3-ITD mutation is found in 20-25% of AMLs and is associated with a poor prognosis. Different FLT3 inhibitors have been developed and some of them are clinically tested but resistance to treatment has been observed in many patients. A better understanding of AML biology is necessary in order to improve the treatment of AMLs. My thesis project focused on the analysis of the autophagic process, which is one of the mechanisms described in the resistance of cancer cells. In this study, we found that the FLT3-ITD expression increases basal autophagy in AML cells, and that the receptor inhibition reduced this autophagy in primary patient samples and cell lines. We show that autophagy is required for proliferation and survival in vitro and in vivo of leukemic cells lines and inhibition of autophagy overcomes resistance to FLT3 inhibitors. In addition, we identified the ATF4 protein as a key actor of the autophagy process induced by the FLT3-ITD mutation. These results suggest that targeting autophagy or ATF4 may represent a promising and innovative therapeutic strategy for FLT3 mutated AMLs.

A Leucemia Mieloide Aguda (LMA) é uma neoplasia originada a partir da expansão clonal de blastos da linhagem mieloide em medula óssea, sangue periférico e outros tecidos. Entre as mutações mais frequentemente detectadas nas LMAs, se encontra a mutação do tipo duplicação interna em tandem (FLT3-ITD) que é detectada em aproximadamente 25% dos pacientes adultos. Esta mutação no receptor de tirosina quinase FLT3 é uma inserção de 3 a 400 pares de base na região juxtamembrana do receptor, a qual é responsável pelo controle da atividade enzimática dos domínios tirosina quinase. Quando esta mutação se encontra presente, a região juxtamembrana perde a capacidade de controlar a ativação dos domínios tirosina quinase e o receptor fica constitutivamente ativo conferindo uma vantagem proliferativa ao clone leucêmico. Esta mutação é considerada de mal prognóstico e já foram desenvolvidos inibidores de tirosina quinase específicos para o receptor FLT3 (FLT3 TKI). Porém, os resultados dos primeiros ensaios clínicos não apresentaram a efetividade esperada e continua a busca de novas combinações de drogas que contribuam a aumentar a efetividade destes inibidores. É por isso que este trabalho teve por objetivo testar a combinação de FLT3 TKIs com o ácido trans-retinoico (ATRA) já aplicado no tratamento da Leucemia Promielocítica Aguda (LPA) com PML-RARA. A combinação de FLT3 TKIs com ATRA induziu a morte celular programada de forma precoce tanto na linhagem de LMA MV4-11 como na MOLM-13. Esta morte celular observada foi inibida com pré- tratamento com inibidor de caspases QVD. O tratamento combinado in vivo em camundongos Nod Scid Gamma (NSG) transplantados com células MOLM-13, aumentou a sobrevida dos animais e diminuiu a percentagem de células CD45 humanas em medula óssea, baço e sangue periférico. No seu conjunto, nossos dados sugerem que o ATRA aumenta o efeito citotóxico dos FLT3-TKIs. Este achado poderá ser relevante para o tratamento de pacientes com LMA portadores de mutaçãoes ITD no gene FLT3 / Acute Myeloid Leukemia (LMA) is hematological disease that arises from the clonal expansion of a myeloid blast in bone marrow, peripheral blood and other tissues. Among LMAs mutations most frequently detected, 25% of adult patients carry the FLT3-ITD mutation. This mutation is a pair base insertion of 3 to 400 in the juxtamembrane domain of the receptor and leads to the constitutive activation of the kinase domains. It gives the leukemic clone a proliferative advantage and it is associated with a bag prognosis. FLT3 tyrosine kinase inhibitors (FLT3 TKI) were already developed in order to improved patients\' treatment. However, results from the first clinical trials were not as promising as expected. Therefore, there is still room for testing new drug combinations that could improve FLT3 TKIs efficacy. The main objective of this work was to test FLT3 TKIs in combination with all-trans retinoic acid (ATRA) already used in Acute Promyelocytic Leukemia (APL) with PMLRARA treatment. The combination of FLT3-TKIs and ATRA induced early programmed cell death in both the MV4-11 and MOLM-13 LMA lines. This early cell death was inhibited with QVD caspase inhibitor pre-treatment. In vivo combined treatment in Nod Scid Gamma (NSG) mice transplanted with MOLM-13 cells, increased animals survival and decreased the percentage of human CD45 cells in bone marrow, spleen and peripheral blood. Taken together, our data suggest that ATRA increases the cytotoxic effect of FLT3- TKIs. This finding may be relevant for the treatment of patients with AML with ITD mutations in the FLT3 gene