Survival rates for pediatric B-cell acute lymphoblastic leukemia (B-ALL) have improved dramatically, but outcomes for the 15% who relapse and for adults with B-ALL remain poor. Up to 40% of pediatric B-ALL patients require central nervous system (CNS) prophylaxis that causes significant treatment-related morbidities. p53-/- Rag-2-/- Prkdcscid;scid triple mutant (TM) mice spontaneously develop B-ALL that disseminates to the CNS. We used this model to investigate molecular mechanisms that drive CNS dissemination of leukemic B-cells. We show that CNS-disseminating B-ALLs had recurrent genomic rearrangements that replaced N-terminal Fms-like tyrosine kinase 3 (Flt3) exons with endogenous retrovirus (ERV) transcriptional control elements. ERV-Flt3 fusion genes encoded truncated FLT3 (trFLT3) proteins that induced ligand-independent STAT5 phosphorylation and proliferation of hematopoietic progenitor cells. Furthermore, trFLT3 promoted de novo development of CNS-disseminating B-ALL from hematopoietic progenitors. Thus, a novel mutational mechanism involving ERV-mediated FLT3 activation can drive the development of B-ALL characterized by high degree of CNS-invasion. Ectopic expression of trFlt3 suggested that TM B-ALLs initiate prior to B-cell commitment, since Flt3 is normally repressed by PAX5 upon B-cell commitment, co-incident with Cd19 expression. In support of this idea, we report evidence of Flt3 amplification in a rare subset of CD19- progenitors, and we show that CD19- FLT3+ cells from leukemic TM mice contain leukemia-initiating cells. Finally, we compared gene expression profiles of trFl3+ and trFl3- B-ALLs to identify potential Flt3 effectors important for CNS dissemination. TM B-ALLs uniquely expressed RANKL, a key regulator of osteoclast differentiation and normal B-cell development. FLT3 inhibition decreased RANKL expression, suggesting at least partial dependence on trFLT3 signaling. RANKL-expressing TM B-ALLs decreased trabecular bone density after adoptive transfer to normal mice, demonstrating a role for RANKL in leukemia-associated bone pathology. Importantly, a RANKL biologic antagonist inhibited CNS dissemination of TM B-ALLs in adoptive transfer experiments. Thus, my studies identified novel ligand-independent Flt3 mutations that arise prior to B-cell commitment and promote development of CNS-disseminating B-ALLs. Furthermore, I identified RANKL as a potential therapeutic target that may limit leukemia CNS dissemination and leukemia-associated bone pathology.

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

Survival rates for pediatric B-cell acute lymphoblastic leukemia (B-ALL) have improved dramatically, but outcomes for the 15% who relapse and for adults with B-ALL remain poor. Up to 40% of pediatric B-ALL patients require central nervous system (CNS) prophylaxis that causes significant treatment-related morbidities. p53-/- Rag-2-/- Prkdcscid;scid triple mutant (TM) mice spontaneously develop B-ALL that disseminates to the CNS. We used this model to investigate molecular mechanisms that drive CNS dissemination of leukemic B-cells. We show that CNS-disseminating B-ALLs had recurrent genomic rearrangements that replaced N-terminal Fms-like tyrosine kinase 3 (Flt3) exons with endogenous retrovirus (ERV) transcriptional control elements. ERV-Flt3 fusion genes encoded truncated FLT3 (trFLT3) proteins that induced ligand-independent STAT5 phosphorylation and proliferation of hematopoietic progenitor cells. Furthermore, trFLT3 promoted de novo development of CNS-disseminating B-ALL from hematopoietic progenitors. Thus, a novel mutational mechanism involving ERV-mediated FLT3 activation can drive the development of B-ALL characterized by high degree of CNS-invasion. Ectopic expression of trFlt3 suggested that TM B-ALLs initiate prior to B-cell commitment, since Flt3 is normally repressed by PAX5 upon B-cell commitment, co-incident with Cd19 expression. In support of this idea, we report evidence of Flt3 amplification in a rare subset of CD19- progenitors, and we show that CD19- FLT3+ cells from leukemic TM mice contain leukemia-initiating cells. Finally, we compared gene expression profiles of trFl3+ and trFl3- B-ALLs to identify potential Flt3 effectors important for CNS dissemination. TM B-ALLs uniquely expressed RANKL, a key regulator of osteoclast differentiation and normal B-cell development. FLT3 inhibition decreased RANKL expression, suggesting at least partial dependence on trFLT3 signaling. RANKL-expressing TM B-ALLs decreased trabecular bone density after adoptive transfer to normal mice, demonstrating a role for RANKL in leukemia-associated bone pathology. Importantly, a RANKL biologic antagonist inhibited CNS dissemination of TM B-ALLs in adoptive transfer experiments. Thus, my studies identified novel ligand-independent Flt3 mutations that arise prior to B-cell commitment and promote development of CNS-disseminating B-ALLs. Furthermore, I identified RANKL as a potential therapeutic target that may limit leukemia CNS dissemination and leukemia-associated bone pathology.

Submitted by Andre Moraes Queiroz (andre.moraesqueiroz@ufpe.br) on 2015-04-14T13:40:30Z No. of bitstreams: 2 Dissertação Aleide Lima.pdf: 1775977 bytes, checksum: 02788df7796db55323f52a980da5d08a (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-14T13:40:30Z (GMT). No. of bitstreams: 2 Dissertação Aleide Lima.pdf: 1775977 bytes, checksum: 02788df7796db55323f52a980da5d08a (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013 / Marcadores moleculares, como mutações nos genes FLT3 e NPM1, são ferramentas úteis para a avaliação prognóstica de pacientes com leucemia mieloide aguda (LMA) e, até o momento, não tinham sido estudadas em pacientes com LMA no Estado de Pernambuco. Dessa forma, esse trabalho teve como objetivo caracterizar pacientes adultos com LMA diagnosticados na Fundação HEMOPE de acordo com achados clínico-laboratoriais e as mutações nos genes FLT3 e NPM1. Foram incluídos 115 pacientes com LMA de novo (15 com leucemia promielocítica aguda (LPA) e 100 com outros subtipos LMA). As frequências das mutações FLT3/ITD, FLT3/TKD e NPM1 nos pacientes não-LPA foram de 22%, 2% e 24%, respectivamente. Nos pacientes com LPA, a frequência foi de 40% e 6,7% para as mutações FLT3/ITD e NPM1, respectivamente, não sendo diagnosticado nenhum caso com mutação FLT3/TKD. As mutações FLT3/ITD e no NPM1 foram relacionadas com alta contagem de leucócitos (p=0,021; p=0,012) e mutações no NPM1 foram mais frequentes em pacientes com mais de 60 anos (p=0,01) e no grupo de cariótipo normal (p=0,008). Não foi observada diferença nas sobrevidas global (SG) e livre de doença (SLD) e nas taxas de remissão completa (TRC) e de recaída (TR) entre os grupos com e sem mutação FLT3/ITD e no NPM1 quando avaliados os pacientes não-LPA; entretanto, pacientes LPA com mutação FLT3/ITD apresentaram menores TRC, SG e SLD. Os resultados comprovam o valor preditivo da mutação FLT3/ITD para um curso clínico desfavorável na LPA do adulto, enquanto que, para os pacientes não-LPA, as mutações FLT3/ITD e no NPM1, aparentemente, não apresentaram o mesmo impacto prognóstico.

La myélofibrose primitive (MFP) est un néoplasme myéloprolifératif (NMP) chronique BCR-ABL1-négatif associant une dérégulation de l’hématopoïèse (myéloprolifération, dysmégacaryopoïèse et migration des cellules souches et progéniteurs hématopoïétiques (CSH/PH)) à une altération du stroma médullaire et splénique (fibrose ostéomyélosclérose, néoangiogenèse). Le mégacaryocyte (MK) est un acteur majeur de sa pathogenèse, via la production de cytokines et facteurs fibrosants, dans un contexte inflammatoire. Plusieurs arguments suggèrent que les mutations JAK2V617F et MPL515L/K qui caractérisent les NMP ne sont pas les événements initiaux de la MFP car elles ne sont retrouvées que chez la moitié des patients. L’objectif de mon travail a été de rechercher si d’autres anomalies, géniques ou non, pouvaient expliquer la pathogenèse de la MFP. Pour cela, parallèlement à une démarche génomique (transcriptome et CGH array), nous avons développé une approche de biologie cellulaire ciblée sur le rôle du stroma hématopoïétique. Bien que n’ayant pas identifié d’autres anomalies génomiques que celles décrites dans la littérature et en particulier, la délétion 13q, les approches génomiques que nous avons développées nous ont permis de préciser les bornes de cette délétion dans les PH CD34+ et les polynucléaires des patients. Cette délétion (région chromosomique minimale 13q14-13q21) est située à 2 mégabases (télomérique) du cluster FLT où est localisé le gène FLT3. Plusieurs arguments nous ont ensuite conduits à rechercher si le couple Flt3-ligand/Flt3 était impliqué dans la dérégulation de l’hématopoïèse et plus particulièrement dans la dysmégacaryopoïèse observée chez les patients. Parmi ceux-ci, citons : 1) l’existence d’une modulation d’expression de gènes inclus dans la zone de délétion 13q et dans le cluster FLT, dont le gène FLT3 et 2) le fait que Flt3, un récepteur clé de la régulation de l’hématopoïèse primitive, soit souvent impliqué dans la pathogenèse d’hémopathies malignes et que son ligand, Flt3-ligand, soit majoritairement produit par le stroma hématopoïétique. Notre étude montre une dérégulation de Flt3 et des MAPKs p38 dans les PH CD34+ et les MK des patients atteints de MFP et ceci, quelque soit leur statut mutationnel Jak2. Elle démontre également que la persistance de la stimulation de l’axe Flt3/p38 en réponse à une production accrue de Flt3 ligand, participe à la dysmégacaryopoïèse qui caractérise la maladie. En effet, nous avons mis en évidence : 1) une augmentation du taux sérique de Flt3 ligand et de son expression par les cellules du stroma médullaire et splénique ainsi que par les PH des patients atteints de MFP, 2) une surexpression spécifique de son récepteur Flt3 et de sa phosphorylation dans les CSH/PH CD34+ et les progéniteurs mégacaryocytaires (MK), qui persistent au cours de la différenciation MK, quelque soit le statut mutationnel de Jak2 des patients, 3) une activation de Flt3 dans les progéniteurs MK en réponse au Flt3 ligand conduisant à la phosphorylation en cascade de la voie de signalisation des MAPKs p38 et à l’expression de ses gènes cibles tels que AP-1, p53, NFATc4, ATF2, IL-8, 4) une restauration de la mégacaryopoïèse et une inhibition de la migration (Flt3-ligand)-dépendante des progéniteurs MK des patients après inhibition de Flt3 ou de p38.Nos résultats confirment l’importance d’une altération des MAPKs dans une dérégulation de l’hématopoïèse et soulignent le rôle d’une activation persistante de la voie p38, via le couple Flt3-ligand/Flt3, dans la dysmégacaryopoïèse qui caractérise la myélofibrose primitive. Ils suggèrent également que cette dérégulation participe au processus inflammatoire à l’origine de la réaction stromale et « lit » d’une transformation leucémique potentielle. Ce dialogue altéré entre les cellules hématopoïétiques pathologiques (Bad seeds), en particulier mégacaryocytaires et les cellules stromales (Bad soil), conforte notre concept « Bad seeds in Bad soil ». / The primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (NMP) BCR-ABL1-negative associating a dysregulation of hematopoiesis (myeloproliferation, dysmegacaryopoiesis and egress of hematopoietic stem and progenitor cells (HSC / PH)) from an altered bone marrow stroma (osteosclerosis, fibrosis, angiogenesis) to the spleen. The megakaryocyte (MK) is a major player in its pathogenesis through the production of cytokines and fibrotic factors in an inflammatory context. Several arguments suggest that mutations JAK2V617F and MPL515L / K which characterize the NMP are not the initial events of the PMF since they are found only in half of patients. The aim of my work was to investigate whether other abnormalities, genetic or otherwise, could explain the pathogenesis of the PMF. For this, a process parallel to genomics (transcriptome and CGH array), we developed a cell biology approach focused on the role of hematopoietic stroma.Although we have not identified other genomic abnormalities as those described in the literature and in particular, deletion 13q, by genomic approaches we have clarified the limits of this deletion in the PH CD34+ and polymorphonuclear patients. This deletion (chromosomal region 13q14-13q21 minimum) is located 2 megabases (telomeric) of the cluster where is located the FLT gene FLT3. Several arguments have then led to inquire whether the couple was involved in Flt3-ligand/Flt3 deregulation of hematopoiesis, especially in the dysmegakaryopoiesis observed in patients. Among these are: 1) the existence of an expression modulation of genes included in the area of deletion 13q and FLT in the cluster, as gene FLT3 and 2) the fact that Flt3, a key receptor the regulation of primitive hematopoiesis, is often implicated in the pathogenesis of hematologic malignancies and its ligand, Flt3-ligand, was predominantly produced by the hematopoietic stroma.Our study shows dysregulation of Flt3 and p38 MAPKs in CD34+ and PH MK from patients with PMF and this, whatever their Jak2 mutation status. It also shows that persistent stimulation of the axis Flt3/p38 in response to increased production of Flt3 ligand, participates in the dysmegacaryopoiesis that characterizes the disease. Indeed, we have highlighted: 1) an increase in serum Flt3 ligand and its expression by stromal cells and bone marrow and spleen by PH patients with PMF, 2) a specific overexpression of its receptor Flt3 and its phosphorylation in HSC / PH CD34+ and megakaryocytic progenitors (MK), which persist during the MK differentiation, regardless of the mutational status of Jak2 patients, 3) activation of Flt3 in MK progenitors by the Flt3 ligand leads to phosphorylation cascade signaling pathway, p38 MAPK and expression of its target genes such as AP-1, p53, NFATc4, ATF2, IL-8, 4) a restoration of megakaryopoiesis and inhibition of migration (Flt3-ligand)-dependent patients after of MK progenitors by Flt3 or p38 inhibitors.Our results confirm the importance of an alteration of MAPKs in a deregulation of hematopoiesis and highlight the role of a persistent activation of the p38 pathway, via the couple Flt3-ligand/Flt3 in the dysmegakaryopoiesis that characterizes idiopathic myelofibrosis. They also suggest that this dysregulation contributes to the inflammatory process at the origin of the stromal reaction and "bed" of a leukemic transformation potential. The dialogue among impaired hematopoietic cell disease (Bad Seeds), especially the stromal cells and megakaryocyte (Bad Soil), reinforces our concept of "Bad Seeds in Bad Soil". This work could help improve the dialogue with therapeutic approaches targeting the axis Flt3-ligand/Flt3 mediated by activation of p38 which, by reducing the inflammatory process, re-establish a link between the "seed" and the "Soil".

Sammanfattning Bakgrund: Akut myeloisk leukemi (AML) är en av de vanligaste typerna av leukemi, dock är denna cancer sällsynt. AML kan drabba personer i alla åldrar men förekommer vanligast hos vuxna personer som är över 40 år. Idag befinner sig ett antal olika FLT3-hämmare i olika faser i kliniska studier. Kliniska prövningar av FLT3-hämmare för AML visar inte särskilt lovande resultat, detta på grund av framväxande läkemedelsresistans mot FLT3-hämmare. FLT3-ITD och FLT3-TKD är de vanligaste mutationerna som förekommer hos AML-patienter. Läkemedlet gilteritinib är speciellt bland FLT3-hämmare eftersom det hämmar både TKD- och ITD-mutationer. Syfte: Syftet med denna litteraturstudie var att undersöka tolerabiliteten av gilteritinib samt undersöka om gilteritinib har god effekt hos refraktär/recidiv AML-patienter med FLT3-mutation. Metod: Databasen Pubmed användes för att hitta veteskapliga artiklar som ska analyseras. För att hitta artiklarna användes sökorden "(gilteritinib) AND (relapsed/refractory)". Resultat: Generellt tolererade det flesta patienter gilteritinib tämligen bra. Frekvensen av fullständig remission/fullständig remission med partiell hematologisk återhämtning var högre med gilteritinib jämfört med salvage kemoterapi samt gilteritinib har överlevnadsfördelar jämfört med salvage kemoterapi. Patienter som uppnådde fullständig remission/fullständig remission med partiell hematologisk återhämtning hade längre totalt överlevnad och hade högre grad av FLT3-ITD-clearance jämfört med patienter som inte uppnådde fullständig remission/fullständig remission med partiell hematologisk återhämtning Slutsats: Det flesta patienter från studierna tolererade gilteritnib ganska bra. Gilteritinib har stort terapeutisk fönster samt har överlevnadsfördelar jämfört med salvage kemoterapi. Begräsningar med studierna var att det inte var så många studiedeltagare. Detta ger mindre pålitliga resultat men det är inte så enkelt att genomföra stora studier med många patienter eftersom AML är en sällsynt cancer. / ABSTRACT Background: Acute myeloid leukaemia (AML) is one of the most common types of leukaemia, even if this cancer is relatively rare. AML can affect people of all ages but is most common in adults over 40 years old. Today, a number of FLT3 inhibitors are in various stages of clinical trials. Clinical trials of FLT3 inhibitors for AML are not showing very promising results, due to emerging drug resistance to FLT3 inhibitors. FLT3-ITD and FLT3-TKD are the most common mutations found in AML-patients. The drug gilteritnib is special amoung FLT3 inhibitors because it inhibits both TKD and ITD mutations. Aim: The aim of this literature review was to investigaste the tolerability and to investigate whether gilteritinib has good efficacy in relapsed/refractory AML-patients with FLT3-mutation. Method: The Pubmed database was used to identify scientific articles to analysis. To find the articles, the keyword "(gilteritinib) AND (relapsed/refractory)" were used. Results: In general, most patients tolerated gilteritinib quite well. The frequency of complete remission/complete remission with partial hematologic recovery was higher with gilteritinib compared to salvage chemotherapy. Patients who achieved complete remission/complete remission with partial hematologic recovery had longer overall survival and had higher levels of FLT3-ITD clearance compared to patients who did not achieve complete remission/complete remission with partial hematologic recovery. Conclusion: Most patients from the studies tolerated gilteritinib fairy well. Gilteritinib has large therapeutic window as well and has survival advantages over salvage chemotherapy. Limitations of the studies were that there were not many study participants. This makes for less reliable result, but it is not so easy to conduct large studies with many patients because AML is a rare cancer.

In recent years, there has been a major movement in the pharmaceutical industry towards the development of molecules that selectivity inhibit a previously-validated specific target. This is referred to as target-based drug discovery. It was hoped that adopting this approach would usher in a new golden age of drug discovery. However, this has not been the case, with issues arising such as the target’s mechanism of action being poorly understood, with it not playing the expected role in the disease progression, or feedback resistance mechanisms causing the target to lose its role in the disease. In contrast to this, in the past 20 years it has been argued that developing drugs in a target-agnostic way and screening them against an expressed phenotype i.e. phenotypic drug discovery, has been more successful, despite fewer programs being run in the manner. The AXL kinase is a receptor tyrosine kinase (RTK) and a member of the TAM family, along with MER and TYRO3. AXL has long been associated with numerous types of cancer. Having been first discovered in 1991 in acute myeloid leukaemia (AML), it has gone on to be more associated with advanced solid tumours such as brain, breast, and lung, with the trend being that increased AXL correlates with a poorer prognosis for the patient. Upon the activation of AXL by the vitamin K ligand GAS6, a series of downstream pathways are activated that go on to encourage cell survival, proliferation, and migration. In addition to this, AXL has been shown to be involved in crosstalk with other kinase pathways, resulting in AXL expression being associated with chemoresistance and survival mechanisms. Despite the promising outlook for AXL inhibitors, to date only one selective AXL inhibitor, BGB324 (formally R428) has entered clinical trials, with selective AXL inhibitors being difficult to develop due to a lack of a crystal structure or a reliable homology model. To address the aforementioned issues that target-based approaches can suffer from, and due to AXL lacking a crystal structure, the work in this thesis utilised a pragmatic drug design method that started from ligands/existing scaffolds known to inhibit the target from the literature (publications, clinical trials and patents). A series of small libraries were prepared and then tested against a selected phenotype e.g. cell viability, in at least two cell types: one that expressed the target (e.g. AXL) and one that did not. Hits were optimised for potency against the desired phenotype. The compounds then went through target deconvolution (kinase screening) to confirm the target of the inhibitors. Employing this approach, we initially synthesised two small libraries of potential AXL inhibitors. The potency of these compounds was tested using cell-based phenotypic assays, by evaluating cell viability in both native and chemo-resistant breast cancer cells. These libraries were optimised through focused combinatorial synthesis and phenotypic screening, to yield a small collection of antiproliferative hits. These hits were then profiled against a panel of twelve select kinases. The first library, while giving some important structural information, did not inhibit the kinases screened in a meaningful manner. However, the second library gave several potent compounds, inhibiting AXL, FLT3, and RET, with one compound being selective for AXL. The leads from this series were optimised further, through SAR studies, gaining important structural information in order to improve potency and selectivity of the compounds. The flexibility of the phenotypic cell-based approach allowed the pursuit of FLT3 inhibitors, resulting in the synthesis of one of the most potent FLT3 inhibitors synthesised to date.

Nous avons étudié la régulation et les fonctions de Cell division cycle 25A (CDC25A), phosphatase impliquée dans l'activation des cyclin-dependent kinases durant le cycle cellulaire, dans les leucémies aiguës myéloïdes portant la mutation de la tyrosine kinase Fms-Like Tyrosine kinase 3 (FLT3) FLT3-ITD, de mauvais pronostic. Nous montrons que CDC25A est une cible précoce en aval de FLT3-ITD, contrairement aux autres protéines du cycle cellulaire, par un mécanisme impliquant STAT5. L'inhibition de CDC25A était à l'origine d'une inhibition de la prolifération et d'une réinduction de la différenciation des cellules primaires ou de lignées FLT3-ITD, in vitro et in vivo, en faisant une cible prometteuse dans les LAM avec la mutation FLT3-ITD. Enfin, nous avons évalué la valeur pronostique de l'expression de CDC25A au niveau ARN messager et protéique sur une série de patients traités au CHU de Toulouse par chimiothérapie intensive. / We studied the regulation and functions of Cell division cycle 25A (CDC25A), a phosphatase involved in the activation of the cyclin-dependent kinases during the cell cycle, in acute myeloid leukemia baring the mutation of the tyrosine kinase Fms-Like Tyrosine kinase 3 (FLT3) FLT3-ITD, which confers adverse prognosis. We show that CDC25A is an early target downstream of FLT3-ITD, contrarily to other cell cycle proteins, by a mechanism involving STAT5. CDC25A inhibition gives rise to inhibition of proliferation and reinduction of differentiation in FLT3-ITD primary cells and FLT3-ITD cell lines, in vitro and in vivo. CDC25A appears as a promising target in FLT3-ITD acute myeloid leukemia. We finally evaluated the prognostic value of CDC25A expression, at the mRNA and proteic level in a series of patients treated by intensive chemotherapy at Toulouse University Hospital.

Les mutations FLT3-ITD et KITD816V sont fréquemment retrouvées dans les leucémies aiguës myéloïdes où elles sont associées à un pronostic défavorable. Ces deux récepteurs à activité tyrosine kinase (RTK) mutés sont des acteurs clés de la leucémogenèse régulant la prolifération, la survie et la différenciation cellulaire. L'objectif de ce travail de thèse a été d'étudier la régulation de l'expression protéique de FLT3 en réponse aux inhibiteurs du protéasome, le rôle de l'autophagie dans les LAM KITD816V et l'impact du 2-deoxy-D-glucose sur la localisation intracellulaire des récepteurs. Les travaux réalisés ont démontré trois manières originales de cibler des cellules portant les oncogènes FLT3-ITD ou KIT en jouant sur leur dégradation, leur localisation intracellulaire et l'autophagie. / FLT3-ITD and KITD816V mutations are recurrently found in acute myeloid leukemia, where they are associated with a poor prognosis. These two Tyrosine Kinase Receptors (TKR) are involved in leukemogenesis, regulating proliferation, survival and cell differentiation. The aim of this thesis was to study the regulation of FLT3 protein expression in response to proteasome inhibitors, the role of autophagy in KITD816V-driven AML and the impact of 2-deoxy-D-glucose (2-DG) on the intracellular localization of TKRs. Our studies investigated three original ways to target cells bearing FLT3-ITD or oncogenic KIT mutations playing on their degradation, intracellular localization and autophagy.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jakub Poráč Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Effect of CDK and FLT3 inhibitors on activity of ABC efflux transporters in vitro, relation to multidrug resistance P-gp and BCRP are transmembrane proteins that form part of a large family of ABC transporters. These are ATP-driven transporters, which main task is to eliminate exogenous and endogenous substances and their metabolites from cells of both, healthy and tumour tissues. This activity is often associated with the expulsion of administered therapeutics and multiple drug resistance (MDR) in tumour cells. A promising therapy of cancer represents a newer class of drugs target the tyrosine kinase (TK), and cyclin-dependent kinases (CDK), which are cell enzymes responsible for the processes of proliferation, apoptosis and differentiation. Cyclin- dependent kinase inhibitors (CDKI) are used in the treatment of breast cancer, but at the same time they form a new group of drugs with the potential for use in hematological malignancies. In the treatment of AML, a new successful approach is TK inhibitors (TKI), which target the mutated FLT3 receptor, specifically the recently approved drugs midostaurin and...