Orientadores: Irene Lorand-Metze, Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T22:11:55Z (GMT). No. of bitstreams: 1 Mello_MarianaRezendeBandeirade_D.pdf: 5178635 bytes, checksum: 8ac6a3134a752be699d5f1d5d6d8ba71 (MD5) Previous issue date: 2011 / Resumo: A classificação atual da OMS para LMA reconhece a importância da pesquisa de anormalidades genéticas para diagnóstico e manejo adequado do paciente. A textura da cromatina dos núcleos está relacionada com fenômenos epigenéticos como a metilação. Nosso estudo teve como objetivos estudar o status de metilação dos genes CDKN2B (p15), CDKN2A (p16), CDKN1C (p57), TP73 (p73), ESR1 (ER) e ABCB1 (MDR1), verificar a freqüência das mutações nos genes FLT3 e NPM1 e analisar parâmetros da morfometria e textura da cromatina. Verificamos também a associação entre o perfil imunofenotípico, textura nuclear e características moleculares. Além disso, analisamos a relação destas variáveis com a sobrevida global. Foram estudados 106 pacientes com LMA de novo (19 com LMA M3 e 87 com outros subtipos de LMA) diagnosticados no nosso Serviço. Nas LMAs M3 foi encontrado 15,8% de pacientes com FLT3-TKD e 10,5% FLT3-ITD. Os pacientes com LMA M3 foram mais jovens, apresentaram menor número de leucócitos e plaquetas. Tiveram também maior expressão de CD45 e MPO do que os outros subtipos de LMA. Os pacientes com LMA M3 tiveram melhor sobrevida que os outros subtipos. Dentre esses pacientes as LMA M3 FLT3-ITD+ apresentaram pior sobrevida. O desvio padrão do nível de cinza foi um fator independente de prognóstico. Em relação aos casos de LMA não M3 foi encontrado 23,3% dos pacientes com presença da mutação FLT3-ITD, 8,1% FLT3- TKD e 29,1% da mutação no gene NPM1. As mutações nos genes NPM1 e FLT3 foram mais frequente em pacientes com cariótipo normal. Dos pacientes com outros subtipos de LMA, 3,4% apresentaram metilação no gene ESR1, 26,4% no gene CDKN2B, 11,5% no gene CDKN2A, 1,1% no CDKN1C e 23,0% no gene TP73. Os pacientes que apresentaram o gene CDKN2B metilado tiveram menor dosagem de hemoglobina e expressão do CD13. Os pacientes com metilação no gene TP73 apresentaram menor contagem de leucócitos, expressão de CD45, CD13, CD33 e MPO. Os pacientes com LMA sem maturação apresentaram menor R245. Na análise de sobrevida dos pacientes com LMA, excluindo M3, os casos com NPM1+ FLT3-ITD- tiveram melhor sobrevida. O R245, a entropia e a mutação FLT3-ITD foram fatores independentes de prognóstico. A frequência das mutações e de metilação encontrada foram semelhantes a outros estudos. O R245 pode ser uma variável nova para avaliação da textura da cromatina / Abstract: The WHO classification for acute myeloid leukemia (AML) has emphacized the cytogenetic and molecular aspects for diagnosis and proper management of the patients. The nuclear chromatin texture is related to epigenetic phenomena such as methylation. In our study we examined the frequency of mutations in FLT3 and NPM1 genes, the methylation status of genes CDKN2B (p15), CDKN2A (p16), CDKN1C (p57), TP73 (p73), ESR1 (ER) and ABCB1 (MDR1) and analyzed features of nuclear morphometry and chromatin texture. We studied the association between the immunophenotypic profile, nuclear texture and molecular features. In addition, we analyzed the relationship between these variables and overall survival of the patients. We studied 106 patients with de novo AML: 19 with promyelocytic leukemia (APL) and 87 with other subtypes of AML diagnosed in our Service. Patients with APL were younger, had lower peripheral leukocyte counts and platelets. They also showed a higher expression of CD45 and MPO than other subtypes of AML. Among them, 15.8% presented FLT3-TKD and 10.5% showed FLT3- ITD. CDKN2B was methylated in 21.0%, CDKN2A in 21.0% and TP73 in 10.5% of the patients. Methylation in CDKN1C, ER and MDR1 were not found. Patients with APL had better survival than other subtypes. The standard deviation of gray level was an independent factor of prognosis. Among the other cases of AML, we found the FLT3-ITD mutation in 23.3%, the FLT3-TKD mutation in 8.1%, and 29.1% of the patients had mutation of the NPM1 gene. Mutations in NPM1 and FLT3 were more frequent in patients with a normal karyotype. Among these patients, 3.4% had methylation of the ESR1 gene, 26.4% in CDKN2B, 11.5% in CDKN2A, 1.1% in the CDKN1C and 23.0% in TP73. Patients with methylated CDKN2B showed lower hemoglobin levels and a lower expression of CD13. Patients with methylation in the TP73 gene had lower peripheral leukocyte counts and lower expression of CD45, CD13, CD33 and MPO. Patients with AML without maturation had lower R245. In the survival analysis of patients with AML, except APL, the cases with NPM1+ FLT3 ITD- had better survival. The R245, entropy and FLT3-ITD mutation were independent predictors of prognosis. The frequency of mutations and methylation found were similar with other studies. The R245 can be a new variable for evaluation of chromatin texture / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Dans les leucémies aiguës myéloïdes (LAM), l’activation anormale de nombreuses voies de signalisation intracellulaires favorise la croissance et la survie des cellules tumorales. L’amélioration des connaissances biologiques de ces pathologies hétérogènes, dont le pronostic est réservé, devrait permettre le développement de thérapies ciblées. La kinase oncogénique mTOR est présente au sein de deux complexes, parmi lesquels mTORC1, activé constitutivement dans la majorité des blastes primaires de patients porteurs de LAM, qui contrôle la synthèse protéique, et mTORC2 activé constitutivement dans 50% des LAM. Les inhibiteurs allostériques de mTORC1 (la rapamycine et ses dérivés) n’inhibent pas la phosphorylation du répresseur traductionnel 4E-BP1, ne diminuent pas la traduction et induisent peu d’apoptose in vitro dans les LAM. Utilisés en monothérapie, leur effet est décevant. De plus ces inhibiteurs n’agissent pas sur le complexe mTORC2. J’ai étudié l’effet d’un inhibiteur catalytique de mTOR, l’AZD8055, actif sur les deux complexes. In vitro, l’AZD8055 inhibe efficacement la signalisation en aval de mTORC1 et de mTORC2, dont les sites de phosphorylation de 4E-BP1 résistants à la rapamycine, ainsi que la synthèse protéique. Il diminue la prolifération, bloque le cycle cellulaire en phase G0G1 et induit une apoptose caspase-dépendante dans les blastes primaires de LAM. Il diminue également la clonogénicité des progéniteurs leucémiques, sans affecter celle des cellules CD34+ normales. Dans un modèle murin de xéno-transplantation, l’AZD8055 inhibe la croissance tumorale et améliore la survie des souris traitées. Je me suis également intéressée à la régulation de l’activité de mTORC1 par les acides aminés. Dans les cellules de mammifères, l’activation de mTORC1 nécessite la présence de glutamine et de leucine qui agissent en coopération via deux transporteurs membranaires, SLC1A5 et SLC7A5/SLC3A2. J’ai montré que la privation en glutamine, obtenue par l’activité glutaminase de la drogue L-asparaginase ou par l’utilisation de milieux de culture spécifiques dépourvus sélectivement en acides aminés, inhibe l’activation de mTORC1 et induit de l’apoptose dans diverses lignées leucémiques et dans les blastes primaires de LAM. La L-asparaginase inhibe la synthèse protéique et ses effets fonctionnels sont liés à son activité glutaminase. J’ai pu également constater une augmentation de l’expression protéique de la glutamine synthase induite par la Lasparaginase, dont l’inhibition majore l’apoptose induite par la L-asparaginase dans certaines lignées leucémiques. J’ai également étudié l’effet de l’inhibition spécifique par un shARN inductible du transporteur SLC1A5, qui permet l’import de glutamine. L’inhibition de SLC1A5 bloque la réactivation de mTORC1 par l’association leucine/glutamine après privation et induit de l’apoptose dans la lignée leucémique MOLM14. Cette inhibition diminue la croissance tumorale dans un modèle de xénogreffe / Acute myeloid leukaemias (AML) are heterogeneous diseases associated with poor prognosis. In AML, aberrant activation of many signaling pathways enhances proliferation and survival of leukemic blast cells. Understanding the mechanisms underlying survival of tumoral cells should allow the development of targeted therapies. The oncogenic kinase mTOR belongs to two distinct multimeric complexes. MTORC1 that controls protein translation, is constitutively activated in most of primary blast cells at AML diagnosis, while mTORC2 is constitutively activated in about half of AML samples. In AML, some phosphorylation events of the translational repressor 4E-BP1, are resistant to allosteric inhibitors of mTORC1 including rapamycin and its analogs. These first generation inhibitors of mTORC1 have only few effects on AML and do not induce significant apoptosis in vitro. I have tested a second generation mTOR kinase inhibitor active on both mTORC1 and mTORC2 complexes. In vitro, AZD8055 blocked mTORC1 and mTORC2 signaling, including 4E-BP1 rapamycin resistant phosphorylation events and protein synthesis. This compound decreased AML blast cells proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependant apoptosis in leukemic cells but not in normal immature CD34+ cells. Finally, AZD8055 reduced tumor growth and improved survival in xenografted mouse model. In the second part of this work, I have studied the regulation of mTORC1 by amino acids in AML. In mammalian cells, activation of mTORC1 requires the presence of glutamine and leucine acting together via two membrane transporters, SLC1A5 and SLC7A5/SLC3A2. I showed that glutamine deprivation, obtained by L-asparaginase glutaminase activity or specific alpha-MEM use, inhibited mTORC1 and induced apoptosis in AML cell lines and primary AML blasts. L-asparaginase also inhibited protein synthesis and I have observed a correlation between the functional effects of L-asparaginase and its glutaminase activity. L-asparaginase induced an up-regulation of glutamine synthase (GS) protein and shRNA-induced GS inhibition increased L-asparaginase-dependant apoptosis in the MV4-11 AML cell line. I have also studied the effects of SLC1A5 inhibition with an inducible shRNA expressed in MOLM14 cells. Inhibition of this high afffinity transporter for glutamine blocked mTORC1 stimulation by leucine and glutamine after deprivation and induced apoptosis in MOLM-14 cell line. SLC1A5 inhibition reduced tumor growth and improved survival in transplanted mice

Les protéines du groupe Polycomb (PcG) sont des facteurs épigénétiques dont le rôle est de maintenir la répression de leurs gènes cibles au niveau de la chromatine via la modification des protéines histones. EZH2 est une protéine clé dans les mécanismes de régulation puisqu’elle catalyse la mise en place de la marque répressive H3K27me3. Dans le cadre de ma thèse, je me suis intéressée au modèle des leucémies aiguës myéloïdes (LAM) dans lesquelles, contrairement à d’autres pathologies myéloïdes, des mutations affectant EZH2 ou des membres Polycomb ne sont retrouvées que très rarement (˂1%). Des études ont montré que dans ce type de leucémies, de nombreux gènes cibles d’EZH2 sont dérégulés bien que son activité répressive soit toujours présente, mettant en évidence d’éventuels défauts de recrutement de cette protéine. Parmi les facteurs de transcription susceptibles de réguler l’association des protéines PcG à la chromatine, se trouve PLZF qui est un candidat intéressant. En effet, le laboratoire a mis en évidence une interaction entre PLZF et la protéine Polycomb BMI-1 et a montré que la distribution génomique de PLZF concorde avec celle de certains composants des Polycomb. L’objectif de mon travail de thèse a donc été de déterminer dans quelle mesure PLZF intervient dans le recrutement ou l’activité d’EZH2. / Polycomb group (PcG) proteins are epigenetic factors which play a major role in maintaining epigenetic silencing via histone modifications at the chromatin level. EZH2 is a key regulator that catalyzes the trimethylation of H3K27, which is a repressive mark. During my PhD, I was interested in the acute myeloid leukemia (AML) model in which, unlike other myeloid malignancies, EZH2 or other PcG protein mutations are very rare (˂1%). Studies have shown that in this type of leukemia, many of EZH2 target genes are deregulated although its repressive activity is still present highlighting possible EZH2 recruitment defects. Among the transcription factors that regulate the association of PcG proteins to chromatin, the transcription factor PLZF is an interesting candidate. Indeed, the laboratory has demonstrated an interaction between PLZF and the Polycomb protein BMI -1 and showed that the genomic distribution of PLZF is consistent with that of some Polycomb components. The aim of my thesis was therefore to determine in which extent PLZF is involved in the recruitment or activity of EZH2.

Akut myeloisk leukemi (AML) är en invasiv sjukdom som kräver en stor omställning för patienten. Sjuksköterskan har därmed en betydelsefull roll för att kunna bidra till professionell omvårdnad. Syftet med studien var att beskriva patientens upplevelse av att leva med sjukdomen AML. Studien var en litteraturstudie där tio resultatartiklar inkluderades, fem kvalitativa och fem kvantitativa. Innehållsanalys användes som analysmetod, vilket resulterade i tre kategorier: upplevelsen av begränsningar i livet, behovet av stöd och upplevelsen av välbefinnande. I resultatet framkom det att patienter med AML upplever begränsningar i vardagen och vanligtvis är symtomen anledningen till det. Stödet, både professionellt och socialt, har stor betydelse för patienterna. Livskvaliteten hos patienterna kan påverkas både positivt och negativt under sjukdomsförloppet. För att omvårdnaden och bemötandet av patienter med sjukdomen skall förbättras krävs det mer forskning kring ämnet omvårdnad och hur patienternas upplevelse av sjukdomen ser ut. / Acute myeloid leukemia (AML) is an invasive disease that requires a major transformation for the patient. The nurse therefore has an important role in contributing to professional nursing. The purpose of the study was to describe the patients experience of living with the disease AML. The study was a literature study where ten result articles were included, five qualitative and five quantitative. Content analysis was used as an analytical method, which resulted in three categories: experience of life limitations, the need for support and the experience of well-being. It appears that patients with AML feel limited in everyday life, usually the symptoms are the cause of the restrictions. Support, both professionally and socially, is of great importance to patients with AML. The quality of life of patients can be affected both positively and negatively during the disease. In order to improve nursing and treatment of the patients, more research is needed on the subject of nursing and how the patients experience the disease.

INTRODUCTION: Older patients with AML (> 60 years) often receive intensive EOL care including hospitalizations and chemotherapy close to death. Intensive EOL care has been shown to increase emotional and financial burdens for patients and families, while often not aligning with patients’ preferences. However, factors associated with the intensity of EOL care in this population are unknown. OBJECTIVES: There is a need to better understand the factors associated with intense EOL care, in hopes of providing more informed, high-quality EOL care in line with patient preferences and decreasing burdens associated with unnecessary healthcare. We aim to describe the associations between the intensity of EOL care, patient demographics, and baseline psychological distress in older patients with AML. METHODS: We conducted a secondary analysis of two supportive care studies including 168 deceased older patients with AML. We assessed patients’ demographics, quality of life (QOL) [Functional Assessment Cancer Therapy-Leukemia], and anxiety and depression symptoms [Hospital Anxiety and Depression Scale (HADS); Patient Health Questionnaire (PHQ-9)] at the time of diagnosis. We used multivariate logistic regression models to examine the association among demographic factors, patient-reported outcomes, and the following EOL care outcomes abstracted from the electronic health record: 1) hospitalizations in the last 7 days of life; 2) receipt of chemotherapy in the last 30 days of life; and 3) hospice utilization. RESULTS: The median age of the cohort was 69 (range 20-100), and the majority were males (63.7% 107/168). Overall, 66.7% (110/165) of patients were hospitalized in the last 7 days of life, 51.8% (71/137) received chemotherapy in the last 30 days of life, and 40.7% (70/168) utilized hospice services. In multivariate models, higher education (OR = 1.54, SE=0.24, P=0.006), and elevated depression symptoms [PHQ-9: OR=1.09, SE=0.04, P=0.028] at the time of diagnosis were associated with higher odds of being hospitalized in the last 7 days of life. In contrast, higher QOL at diagnosis [OR=0.98, SE=0.01, P=0.009] was associated with lower odds of being hospitalized in the last 7 days of life. Depression symptoms at the time of diagnosis as measured by the HADS was the only factor associated with the receipt of chemotherapy in the last 30 days of life [HADS-Depression: OR=1.10, SE=0.05, P=0.042]. Patients factors were not associated with hospice utilization. CONCLUSIONS: Older patients with AML who are more educated and report elevated depression symptoms and lower QOL at the time of diagnosis were more likely to receive intensive EOL care. These findings identify a population at the time of diagnosis of AML who are at higher risk for hospitalizations and chemotherapy use at the EOL and who may benefit from targeted supportive care interventions.

No description available.

Acute myeloid leukemia (AML) is a very heterogeneous disease associated with cytogenetic aberrations and genetic mutations. Many of these changes have been revealed and their detection became usual part of the diagnostic process today. However, changes of expression profiles of small, noncoding RNAs, so called microRNAs (miRNAs), are less known and not used for diagnostics yet. These RNAs, 19-24 nucleotides long, take part in the regulation of expression of different genes through complementary base pairing to the 3'non- translated region (3'UTR) of the target messenger RNA (mRNA). They can influence key processes of the cell, like differentiation, proliferation or apoptosis. The changes in expression of different miRNAs are known from different types of cancers. In solid tumors, they are usually detected from bioptic samples; but also plasma samples are now in the center of attention as so called liquid biopsies providing the information about molecular genetic events in the organism. Many studies have revealed deregulated miRNAs in the bone marrow, full blood or isolated progenitor cells (CD34+) of AML patients, only four of them have analyzed plasma samples. We focused on the plasma samples and we targeted on such miRNAs, which levels differ at AML diagnosis and after the chemotherapy. Out of...

Treatment of acute myeloid leukemia, an aggressive hematopoietic malignancy of myeloid progenitors, has remained rather stagnant over the course of several decades. Infusions of cytarabine and anthracycline antibiotics have dominated the landscape of AML therapy, with minor changes to dosing schedule occasionally making slight adjustments to efficacy or tolerability. Improvements in prognosis have been bittersweet, with most progress seen in younger populations less likely to get the disease, and already more likely to achieve remission and to meet survival milestones. Much of this progress is attributed to other factors, such as improved supportive care and availability of hematopoietic stem cell and platelet transfusion. In most patients, occupying the 60-and-above demographic, improvements in survival have not been significant. In turn, the population impact of AML has changed little over time. While accounting for about one-third of total leukemia cases and one percent of total cancer cases, AML accounts for about one half of total leukemia deaths and two percent of total cancer deaths. Most advances straying away from standard treatment have been in important pathways that could be impactful in subsets of the overall AML patient population. Tyrosine kinases are implicated in numerous cancers including AML, with activity-enhancing mutations conferring growth advantages to malignant cells. About one-third of AML patients have mutations in one such kinase, FLT3, and may benefit from inhibitors to tyrosine kinases overall and from FLT3- specific agents. Mutations in isocitrate dehydrogenases highlight another subpopulation, about one-fifth of AML patients, who might benefit from emerging agents that inhibit these pathways from creating a leukemia-favoring environment in the bone marrow. Other pathways similarly implicated in numerous cancers including AML are being targeted with new agents that can benefit some AML patients, such as Hedgehog signaling and apoptotic regulation. Still, breakthroughs are needed that can help most AML patients, particularly in the cases of relapsed leukemia that occurs in most patients within a year or two after remission is achieved. CD33 is among a few molecular targets for AML, though it is just as ubiquitously expressed on healthy myeloid cells. Antibody-drug conjugates like Mylotarg have made progress in this approach, though hematopoietic toxicities have made treatment difficult in older populations. Clever techniques such as ablation of CD33 from healthy myeloid progenitors may be supportive in CD33-based approaches, and immunotherapy involving CD33-targeting is a rapidly growing research focus. This dissertation describes a new type of bispecific antibody that binds CD33 on AML and CD3 on cytotoxic T cells in a proof-of-concept study. Various formats for bifunctional molecules have been created and used clinically, including antibody-drug conjugates and bispecific antibodies that simultaneously engage antigens on two different types of cells. Those like the one described here, bispecific T-cell engagers, have typically taken the form of single-chain fusion proteins containing the variable regions binding to both antigens of interest. Other bispecific antibodies have imitated naturally-occurring immunoglobulin structures, boasting superior pharmacokinetics while facing steep obstacles in large-scale production. The single-chain fusions, easier to produce, can face difficulties in full engagement, with loss of function sometimes seen in fusion partners at the C-terminus. We propose a new format, believed to present two antigen-binding domains in N-terminal positions on a two-chain heterodimeric structure. Capitalizing on an elegantly designed system of hydrophobic cores and hydrogen-bonding networks generating an orthogonal heterodimer, we added an immunoglobulin hinge region to secure a permanently-bound heterodimer, and attached domains binding to CD3 and CD33. We hypothesized that this design, ensured to present its antibody components at N-termini, could bind two antigens at a distance appropriate for facilitating T cell cytotoxicity to AML. After expressing and purifying these proteins in mammalian cells, we demonstrated their ability to persist as a bispecific heterodimer. We showed in vitro that our bispecific heterodimers could bind both CD3+ and CD33+ cells, and that they bolstered T cell cytotoxicity to AML cell lines in a dose-dependent manner. Monomeric components bound only CD3+ or CD33+ cells depending on antibody variable domain present, and had no effect on T cell cytotoxicity. In a mouse model of minimal residual disease, T cells alone did not have a significant effect on the growth of AML, nor did they have an effect on overall survival. T cells with bispecific heterodimer greatly extended survival, and mice of this treatment group were free of leukemia. These findings suggest that this format for bispecific proteins allows for robust simultaneous engagement with both antigens of interest in a manner conducive to T cell cytotoxicity against AML. We believe this presents a compelling modular system for bispecific antibodies, where CD3- and CD33-binding domains can be readily swapped with domains binding to other cancer- or immune cell-specific antigens, and can be further developed into a trispecific system engaging other immune cells or extending half-life with anti-albumin or Fc domains.

La mitochondrie est restée longtemps cantonnée au rôle de centrale énergétique cellulaire. On sait désormais qu'elle est aussi la principale source d'espèces réactives oxygénées, impliquées dans le stress oxydant et la signalisation inter- cellulaire. Le dérèglement de l'activité mitochondriale est ainsi susceptible d'être la cause de l'apparition et de la progression de maladies associées au vieillissement, comme le cancer et les maladies neurodégénératives. Dans le cadre de la leucémie aiguë myéloïde, des études menées par l'équipe dirigée par Jean-Emmanuel Sarry du Centre de Recherche en Cancérologie de Toulouse, ont montré qu'il est possible de sensibiliser les cellules jusqu'alors résistantes à la chimiothérapie, en ciblant préalablement la fonction mitochondriale. C'est dans ce contexte que s'inscrit le sujet de cette de thèse, portant sur la conception et la réalisation de micro-capteurs électrochimiques dédiées à l'analyse du métabolisme mitochondrial, à l'échelle de la mitochondrie isolée. La fabrication des microsystèmes s'est déroulée dans la salle blanche du Laboratoire d'Analyse et d'Architecture des Systèmes de Toulouse, sous l'encadrement des chercheurs Jérôme Launay et Pierre Temple-Boyer, spécialisés dans la conception de capteurs pour la détection d'espèces en phase liquide. Finalement, un système complet assurant le couplage à la microscopie et à la gestion des fluides a été fabriqué, validé, et breveté. Les résultats obtenus nous permettent d'envisager l'analyse à l'échelle de la mitochondrie unique par une approche parallélisée, ce qui n'a encore jamais été réalisé. / The role of mitochondria have been restricted to oxidative phosphorylation for a long time. Now it is clear that they are also the main sources of reactive oxygen species, implied in oxidative stress and cell-to-cell signaling. Thus, mitochondrial malfunction is potentially the cause of the appearance and the progression of diseases linked to ageing like cancers and neurodegenerative troubles. In the frame of acute myeloid leukemia, studies governed by Jean-Emmanuel Sarry of the Cancer Research Center of Toulouse, showed that it is possible to improve the efficacy of current chemotherapies by targeting mitochondria's function. In this context, the objective of the thesis presented here consist in the design and the manufacturing of electrochemical micro-sensors, dedicated to the analysis of the metabolic activity of isolated mitochondria. The manufacturing occurred in the clean room facilities of the Laboratory for Analysis and Architecture of Systems of Toulouse under the supervision of Jérôme Launay and Pierre Temple-Boyer, researchers specialized in the development of solutions aiming the detection of species diluted in solution. Finally, a complete system ensuring the coupling with microscopy and fluidics have been realized, validated, and patented. The results obtained allow us to consider the analysis at the scale of the single mitochondrion with a parallelized approach, thing that have never been made.

Indiana University-Purdue University Indianapolis (IUPUI) / FMS-like tyrosine receptor kinase-internal tandem duplications (FLT3-ITDs) are the most frequent deleterious mutations found in acute myeloid leukemia (AML) and portend a poor prognosis. Currently, AML patients typically achieve disease remission, yet undergo high rates of disease relapse, implying a residual post-treatment reservoir of resistant malignancy-initiating cells. This begs for new therapeutic approaches to be discovered, and suggests that targeting multiple cellular compartments is needed for improved therapeutic approaches. We have shown that the protein tyrosine phosphatase, Shp2, associates physically FLT3-ITD at tyrosine 599 (Y599) and positively regulates aberrant STAT5 activation and leukemogenesis. We also demonstrated that genetic disruption of Ptpn11, the gene encoding Shp2, increased malignancy specific survival of animals transplanted with FLT3-ITD-transduced cells, suggesting that Shp2 may regulate the function of the malignancy-initiating cell. Taken together, I hypothesized that inhibiting Shp2 can target both FLT3-ITD+ AML tumor cells as well as FLT3-ITD-expressing hematopoietic stem cells. To study this hypothesis, I employed two validation models including genetic inhibition of Shp2 interaction with FLT3-ITD in 32D cells or genetic disruption of Shp2 in FLT3-ITD-expressing HSCs. Using FLT3-ITD-expressing 32D cells as an AML tumor model, I found that mutating the Shp2 binding site on FLT3-ITD (Y599) reduced proliferation in vitro and increased latency to leukemia onset in vivo. Further, pharmacologic inhibition of Shp2 preferentially reduced proliferation of FLT3-ITD+ primary AML samples compared to FLT3-ITD- samples, and cooperated with inhibition of the lipid kinase, phospho-inositol-3-kinase (PI3K), and of the tyrosine kinase, Syk, to reduce proliferation of both FLT3-ITD+ and FLT3-ITD- AML samples. To evaluate the stem cell compartment, I crossed a murine locus-specific knock-in of FLT3-ITD with Shp2flox/flox; Mx1-Cre mice to generate FLT3-ITD; Shp2+/- mice and found that Shp2 heterozygosity dramatically inhibits hematopoietic stem cell engraftment in competitive transplant assays. Further, I found that lineage negative cells from FLT3-ITD; Shp2+/- mice demonstrated increased senescence compared to control mice, suggesting that Shp2 may regulate senescence in FLT3-ITD-expressing hematopoietic stem cells. Together, these findings indicate a cooperative relationship between the tyrosine phosphatase, Shp2, and the kinases PI3K and Syk in AML tumor cells, and indicate that Shp2 plays a positive role in the stem cell compartment to promote stem cell function of the malignancy-initiating cell in AML. Therefore, targeting Shp2 may hold therapeutic benefit for patients with FLT3-ITD+ AML.