Dans la leucémie aigue myéloïde (LAM), la cytogénétique et les anomalies moléculaires sont fortement associées au pronostic : au sein des LAM à caryotype normal, les duplications de FLT3 (FLT3-ITD) ont un mauvais pronostic alors que les mutations de NPM1 et de CEBPA sont associées à un pronostic favorable. Récemment, les mutations du gène TET2 ont été identifiées dans les pathologies myéloïdes malignes. De plus, les approches de séquençage à haut débit ont permis de mettre en évidence la présence dans la LAM de mutations récurrentes du gène IDH1 dont les altérations avait été préalablement reportées dans le gliome au même titre qu’IDH2. Nous avons donc évalué la fréquence et la signification pronostique des altérations de TET2, IDH1 et IDH2 dans la LAM de novo. Les altérations génomiques et les variations de séquence du gène TET2 ont été étudiées dans une cohorte de 147 patients atteints de LAM de novo, tous traités par chimiothérapie intensive. Les mutations d’IDH1 et IDH2 (IDH1m et IDH2m) ont été étudiées dans une cohorte plus étendue réunissant 520 patients adultes inclus dans les protocoles thérapeutiques ALFA-9801 et ALFA9802. Dans la LAM de novo, les mutations ponctuelles de TET2 ont été retrouvées chez 17% des patients étudiés, soit une fréquence non significativement différente de celle retrouvée dans les syndromes myélodysplasiques ou dans les néoplasmes myéloprolifératifs. En revanche, les délétions et les pertes d’hétérozygotie sans perte de copie impliquant le gène TET2 sont vraiment rares dans la LAM. La fréquence des mutations de TET2 ne semble pas significativement différente chez les patients ayant atteints la rémission complète comparée à celle des patients en échec thérapeutiques. Les mutations de TET2 sont associées aux mutations de NPM1 (p=.032) mais ne semblent avoir d’impact sur la survie globale (OS) ou la survie sans maladie (DFS), ni dans la cohorte globale, ni dans le sous-groupe des LAM à caryotype normal.. La prévalence des mutations de IDH1 et IDH2 s’élève respectivement à 9.6% et 3% des LAM étudiées. Ces mutations sont principalement retrouvées dans le sous-groupe des LAM à caryotype normal. Dans ce sous groupe, les mutations d’IDH1 sont associées avec celles de NPM1 (p=.008) mais mutuellement exclusives avec celles atteignant CEBPA (p=.03). La fréquence de FLT3-ITD est similaire chez les patients avec et sans mutation d’IDH1. En revanche, aucune mutation de NPM1 ou de CEBPA et aucune duplication de FLT3 n’a été détectée chez les patients portant une mutation d’IDH2. Dans les LAM à caryotype normal, les mutations d’IDH1 ont été détectées chez 19% des patients avec le génotype favorable défini par la présence d’une mutation de NPM1 ou de CEBPA en l’absence de duplication de FLT3. Au sein de ce sous-groupe, la présence de mutations d’IDH1 est associé à un risque plus élevé de rechute (5y-RR: 71% pour IDH1m vs 38%, p=.01). Dans les LAM à caryotype normal, le génotype favorable pourrait ainsi etre redéfini par la présence d’une mutation de NPM1 ou de CEBPA sans duplication de FLT3 ni mutation de IDH1. Dans la cohorte étudiée, les patients caractérisés par ce génotype ont en effet un risque de rechute inférieur (5y-RR: 39% vs 76%, p<.0001) et une survie globale plus longue (5y-OS: 68% vs 29%, p<.0001). Les mutations d’IDH2 ont quant à elles un pronostic fortement péjoratif. Au sein des LAM à caryotype normal, les patients avec mutation de IDH2 ont un risque de rechute plus élevé (5y-RR: 100% pour IDH2m vs 60%, p=.01) et une survie globale plus courte (5y-OS: 0% pour IDH2m vs 46%, p=.005). Ainsi, l’étude des altérations concernant les gènes TET2, IDH1 et IDH2 a apporté des éléments supplémentaires de caractérisation des sous types de LAM. La détection des altérations de TET2 a permis d’évaluer leur fréquence dans la LAM mais n’a pas mis en évidence de sous groupe possédant une signification clinique ou pronostique particulière. / In acute myeloid leukemia (AML), both cytogenetic and molecular abnormalities are strongly associated with prognosis. In particular, in cytogenetically normal AML (CN-AML), FLT3-ITD (internal tandem duplication) carries adverse prognostic factor whereas NPM1 or CEBPA mutations are associated with favorable outcome. Recently, mutations of the ten eleven translocation 2 gene (TET2) have been reported in myeloid neoplasms. Further more, whole-genome sequencing identified recurrent mutations of IDH1 and IDH2 previously reported to be involved in gliomas. We evaluated the frequency and prognostic value of TET2, IDH1 and IDH2 alterations in de novo AML. Genomic alteration and sequence variation of TET2 gene were investigated in a cohort of 147 patients with de novo AML all treated with intensive chemotherapy. IDH1 and IDH2 mutations (IDH1m and IDH2m) were investigated on a wider cohort of 520 adults with AML homogeneously treated in the French ALFA-9801 and -9802 trials In de novo AML, point mutations of TET2 were found in 17% of studied patients which appear to be as frequent as in myelodysplastic syndrome or myeloproliferative neoplasms whereas TET2 deletion or copy neutral loss of heterozygosity are rare. The incidence of TET2 mutations were associated with NPM1 mutations (NPM1m) (p=0.032) and appeared similar in patients who achieved or not CR with intensive chemotherapy, but the presence of TET2 mutations did not affect OS and DFS in the overall population and in CN-AML patients. The prevalence of IDH1m and IDH2m was respectively 9.6% and 3.0% in the studied cohort, mostly associated with normal cytogenetics. In patients with CN-AML, IDH1m were associated with NPM1m (p=.008) but exclusive of CEBPAm (p=.03). The rate of FLT3-ITD was similar in IDH1m and IDH1wt patients. In contrary, no other mutations were detected in IDH2m patients. In CN-AML patients, IDH1m were found in 19% of favorable genotype ((NPM1m or CEBPAm) without FLT3-ITD) and were associated with a higher risk of relapse (5y-RR: 71% for IDH1m vs 38%, p=.01) and a trend to shorter overall survival (5y-OS: 45% for IDH1m vs 64%, p=.10). Favorable genotype in CN-AML could thus be defined by the association of NPM1m or CEBPAm with neither FLT3-ITD nor IDH1m. Patients with this favourable profile (N=62) had a lower RR (5y-RR: 39% vs 76%, p<.0001) and a better OS (5y-OS: 68% vs 29%, p<.0001). In IDH2m CN-AML patients (N=7), we observed a higher risk of induction failure, a higher RR (5y-RR: 100% for IDH2m vs 60%, p=.01) and a shorter OS (5y-OS: 0% for IDH2m vs 46%, p=.005). The investigation of alterations concerning TET2, IDH1 and IDH2 genes brought new elements of characterization of AML subtypes. Detection of TET2 alterations permit to obtain an evaluation of their frequency in AML but did not highlight a subset of cases with clinical or prognostic significance. However, investigation of IDH1m and IDH2m showed that these mutations are associated with a poor prognosis in AML contrarily to what is reported in gliomas. Screening of IDH1m could help to identify high risk patients within the subset of CN-AML with a favorable genotype.

No description available.

<p>Glioma development and progression are driven by complex genetic alterations, including point mutations and gain or loss of genomic copy number, as well as epigenetic aberrations, including DNA methylation and histone modifications. However, the molecular mechanisms underlying the causes and effects of these alterations are poorly understood, and improved treatments are greatly needed. Here, we report a comprehensive evaluation of the recurrent genomic alterations in gliomas and further dissect the molecular effects of the most frequently-occurring genomic events. First, we performed a multifaceted genomic analysis to identify genes targeted by copy number alteration in glioblastoma, the most aggressive malignant glioma. We identify EGFR negative regulator, <italic>ERRFI1</italic>, as a glioblastoma-targeted gene within the minimal region of deletion in 1p36.23. Furthermore, we demonstrate that Aurora-A kinase substrate, <italic>TACC3</italic>, displays gain of copy number on 4p16.3 and is overexpressed in a grade-specific pattern. Next, using a gene targeting approach, we knocked-in a single copy of the most frequently observed point mutation in gliomas, <italic>IDH1<super>R132H/WT</super></italic>, into a human cancer cell line. We show that heterozygous expression of the <italic>IDH1<super>R132H</super></italic> allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Together, these data provide insight on genetic and epigenetic alterations which drive human gliomas.</p> / Dissertation

IntroductionAcute myeloid leukemia (AML) is caused by a wide range of genetic aberrations, includingmutations within the genes that encode the enzymes isocitrate dehydrogenase 1 and 2(IDH1/2). Drugs that target mutant IDH1/2 are now available, which makes assessment of themutational status of IDH1/2 important in clinical diagnostics of AML. A promising method todetect these mutations is the droplet digital polymerase chain reaction (ddPCR), which showsadvantages of a high sensitivity and a simple workflow.AimTo evaluate ddPCR as method of choice to detect hotspot mutations in IDH1 (codon R132)and IDH2 (codon R140 and R172) in patients with AML.MethodsFifteen AML patients known to be positive for IDH1/2 diagnosed by a previously performednext generation sequencing (NGS) were selected for evaluation of ddPCR. Diagnosticsamples were tested for 14 patients, whereas follow-up samples were tested for one patient.ddPCR was performed using QX200™ Droplet Digital PCR system and data were presentedas fractional abundance of mutant allele.ResultsThe amount of mutant IDH1/2 in samples reported by ddPCR correlated well with the resultsfrom NGS when using probes that target their specific mutation. The detection limit formutant allele in the background of wild type IDH1/2 was 0,5% for IDH2 p.R140Q and 0.1%for IDH1 p.R132C/H.ConclusionddPCR that target specific mutations shows a great potential in measuring minimal residualdisease during follow-up. However, its use in screening for mutant IDH1/2 at the time ofdiagnosis is limited and alternative approaches should be considered.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19891号 / 医博第4140号 / 新制||医||1016(附属図書館) / 32968 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 山田 泰広, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le processus conduisant à la prolifération et différenciation des cellules souches hématopoïétiques (CSH) en cellules de toutes les lignées sanguines s’appelle l’hématopoïèse. Bien que l'engagement des CSH soit régi par les cytokines, les facteurs de transcription, les modificateurs épigénétiques et la niche des CSH, notre groupe a constaté que leur engagement vers la lignée érythroïde dépendait aussi du métabolisme de la glutamine. La glutaminolyse contribue à la biosynthèse des nucléotides de novo ainsi qu’à la production de l'alpha-kétoglutarate (αKG), intermédiaire métabolique du cycle TCA (Oburoglu et al. 2014). Il est cependant important de noter que la différenciation érythroïde est un processus unique, où chaque cellule fille est structurellement et fonctionnellement différente de sa cellule mère. Chaque division définit un stade de différenciation précis avec un dernier cycle de division produisant un réticulocyte énucléé. Ainsi, nous avons émis l'hypothèse que les réseaux métaboliques mobilisés dans les progéniteurs érythroïdes changent en fonction du stade de différenciation et que ces réseaux régulent la transition des progéniteurs d'un stade à l'autre.Au cours de ma thèse, j’ai caractérisé les états métaboliques associés aux différents stades de différenciation des progéniteurs érythroïdes. Nous avons ainsi montré qu'aux stades précoces de différenciation érythroïde, avant la différenciation terminale, les progéniteurs hématopoïétiques présentent une activité métabolique accrue avec un niveau de phosphorylation oxydative (OXPHOS) plus élevé. Ces données sont en corrélation avec l'augmentation de la génération de l’αKG à ces stades de différenciation. De plus, nous avons constaté une augmentation de l’OXPHOS de ces progéniteurs en présence d’αKG exogène. Cependant, la différenciation terminale des précurseurs érythroïdes, caractérisée par la perte de la masse mitochondriale et de leur potentiel membranaire, est associée à une diminution du niveau d'OXPHOS. Ainsi, l'administration exogène d’αKG, a fortement atténué la différenciation érythroïde terminale et l'énucléation, sans affecter la différenciation des pro-érythroblastes. Inversement, un antagoniste de l’αKG (diméthyloxalylglycine, DMOG) n'a pas altéré la différenciation terminale ou l'énucléation, malgré l'abrogation de l'OXPHOS dans les érythroblastes.Ces données suggèrent que la production d’αKG et sa contribution à l’OXPHOS perturbent l'énucléation des globules rouges. C'est pourquoi, dans le but de réduire les niveaux intracellulaires d’αKG, nous avons inhibé l’expression de l'isocitrate déshydrogénase I (IDH1), enzyme cytosolique catalysant la conversion de l'isocitrate en αKG. Cependant, comme IDH1 peut catalyser les réactions dans les deux sens, la diminution de son expression pourrait également augmenter les niveaux d’αKG. En effet, nous avons constaté que le knockdown d'IDH1 entraînait une forte atténuation de la différenciation terminale et de l'énucléation des précurseurs érythroïdes. Cet effet est probablement dû à un déséquilibre de la disponibilité des substrats ; ainsi l’administration ectopique de l’αKG ainsi que du citrate renforce l’altération de la différenciation terminale des précurseurs érythroïdes IDH1-/- ainsi que leur énucléation. Cette étude identifie donc un rôle crucial pour le métabolite αKG dans la régulation de la fonction mitochondriale et de l’OXPHOS, processus qui sont une condition sine qua non pour la différenciation des précurseurs érythroïdes au stade proérythroblaste. Nous montrons en outre que la suppression d’OXPHOS et la catalyse d’intermédiaires du TCA, substrats d’IDH1, sont requis pour les phases terminales de la différenciation érythroïde et l'énucléation.En conclusion, les résultats obtenus au cours de ma thèse mettent en évidence la nature dynamique des réseaux métaboliques qui régulent la progression des précurseurs érythroïdes tout au long des différents stades de la différenciation érythroïde. / Hematopoiesis is the process whereby hematopoietic stem cells (HSCs) proliferate and differentiate to all blood cell lineages. While HSC commitment is known to be regulated by cytokines, transcription factors, epigenetic modifiers and the HSC niche, our group found that specification of HSCs to the red cell lineage is dependent on glutamine metabolism. Glutaminolysis contributes to de novo nucleotide biosynthesis and to the generation of the alpha-ketoglutarate (αKG) TCA cycle metabolite (Oburoglu et al. 2014). Importantly though, erythroid differentiation is a unique process as each daughter cell is structurally and functionally different from its parent cell. Each division defines a stage of differentiation with the final division cycle resulting in the production of an enucleated reticulocyte which further matures to a biconcave erythrocyte. Thus, we hypothesized that progenitor metabolic networks change as a function of the erythroid differentiation stage and moreover, that they regulate the transition of progenitors from one stage of differentiation to the next.During my PhD, I assessed the metabolic alterations that occur as a function of the erythroid differentiation stage. We showed that at early stages of human red cell development, prior to terminal differentiation, hematopoietic progenitors exhibited an increased metabolic activity with a significantly higher level of oxidative phosphorylation (OXPHOS). This correlated with the increased generation of αKG and indeed, we found that ectopic αKG directly augmented OXPHOS in these progenitors. However, the terminal differentiation of erythroid precursors, characterized by the loss of mitochondrial mass and membrane potential, was associated with a decreased level of OXPHOS. Notably, ectopic αKG, which did not alter pro-erythroblast erythroid differentiation, severely attenuated terminal differentiation and enucleation. Conversely, an αKG antagonist (dimethyloxalyl glycine, DMOG) did not negatively impact on terminal differentiation or enucleation despite abrogating OXPHOS in erythroblasts.These data suggested that the production of αKG and its subsequent contribution to oxidative phosphorylation perturb red cell enucleation. We therefore downregulated isocitrate dehydrogenase I (IDH1), the cytosolic enzyme that catalyzes the conversion of isocitrate to αKG, by an shRNA approach in an attempt to decrease αKG levels. However, because IDH1 can catalyze both the forward and reverse reactions, its downregulation could also increase αKG levels. Indeed, we found that IDH1 knockdown resulted in a severe attenuation of terminal erythroid differentiation and enucleation. This effect was likely due to an imbalance in substrate availability––both ectopic αKG as well as citrate further decreased polychromatic to orthochromatic erythroblast differentiation and the subsequent enucleation of IDH1-knockdown erythroid precursors. Thus, the present study identifies a crucial role for the αKG metabolite in regulating mitochondrial function and oxidative phosphorylation, processes that are a sine qua non for erythroid precursors at the pro-erythroblast stage. We further show that terminal erythroid differentiation and enucleation requires OXPHOS suppression and the IDH1-mediated enzymatic catalysis of its TCA substrates.To conclude, the results generated during my PhD highlight the dynamic nature of the metabolic networks that regulate the progression of erythroid precursors through the distinct stages of erythroid differentiation.

Depuis que Parsons et col. ont découvert en 2008 que le gène de l’isocitrate déhydrogénase 1 (IDH1) est fréquemment muté dans les glioblastomes (12%), de nombreuses équipes ont étudié la prévalence et les caractéristiques des mutations des gènes IDH1 et 2 dans les gliomes.Les mutations du gène IDH1 sont observées dans environ 40% des gliomes. La mutation d’IDH1 la plus fréquentes dans les gliomes (>90% des cas) est la mutation R132H. La fréquence des mutations IDH1 et 2 est inversement corrélée au grade des gliomes (grade II ~80%, III ~50%, and IV ~10%). Les mutations IDH1/2 ont une valeur diagnostique ainsi que pronostique (associées à une meilleure survie). Pendant ce travail de thèse nous avons dans une première partie analysé la distribution de ces mutations IDH1/2 dans les différents gliomes, leur association avec d’autres altérations génétiques, ainsi que leur valeur diagnostique et pronostique dans une cohorte de 1332 patients atteints de gliomes. Nous confirmons sur cette très grande cohorte les données de la littérature et affinons la valeur pronostique des mutations IDH1/2. Dans une seconde partie, nous avons mis en évidence dans les gliomes un polymorphisme (SNP) du gène IDH1 (SNP rs 11554137; C (cytosine) substituted by T (thymin)) précédemment observé dans les leucémies myéloïdes aigues. Ce SNP, codon 105, est localisé dans le même exon que le codon 132 fréquemment muté, et nous avons montré qu’il est associé à une moins bonne survie des patients atteints de gliomes. Les mutations du codon 132 causent une baisse de l’activité enzymatique normale d’IDH1/2 qui est remplacé par le gain d’une nouvelle. Les protéines IDH1/2 mutés, au lieu de produire de l’alpha-cétoglutarate de façon NADP dépendante, réduisent de façon NADPH dépendante l’alpha-cétoglutarate en 2-hydroxyglutarate (2HG). Une forte concentration de 2HG et une baisse de la quantité de NADPH peuvent sensibiliser les tumeurs au stress oxidatif et donc potentialiser l’effet de la radiothérapie, ce qui pourrait expliquer la meilleure survie de ces patients. Nous avons donc dans une troisième partie étudié in vitro l’impact de la mutation IDH1R132H sur la survie après radiothérapie de cellules tumorales exprimant de façon stable ce gène muté. Les résultats obtenus montrent que dans certaines conditions ces cellules pourraient être plus radiosensibles que les mêmes cellules exprimant le gène IDH1 non-muté.Dans ce travail de thèse, nous avons donc étudié le gène IDH1 dans les gliomes de patients et tenté par une approche fonctionnelle in vitro d’évaluer l’impact de la mutation IDH1R132H sur la radiosensibilité des cellules tumorales. / Since Parsons et al. (2008) found the frequent mutations of IDH1 (12%) in GBMs, various reports have studied the prevalence and characteristic of IDH1 and IDH2 mutations.The mutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in nearly 40% of gliomas. The frequency of IDH1 mutations are inversely connected with grade II (~80%), III (~50%), and IV (~ 10%) gliomas. Importantly, the status of IDH1 mutations is associated with a better outcome and demonstrated a diagnostic value. We analyzed also these mutations in distribution, association with tumor-derived other genetic alterations and the diagnostic and prognostic value in a cohort of 1332 glioma patients.A synonymous single nucleotide polymorphism [SNP rs 11554137; C (cytosine) substituted by T (thymin)] has been studied in gliomas patients. The SNP rs 11554137 (in codon 105) are located in the same exon with the IDH1 R132 mutations (in codon 132). And gliomas patients with SNP rs 11554137: C>T had a poorer outcome than patients without SNP rs 11554137. This was observed a similarly adverse effect in survival in patients with AML. Mutations in codon 132 can cause a decrease of IDH1/2 activity and also gain a new enzyme function for the NADPH dependent reduction of alpha-ketoglutarate to 2-hydroxyglutarate. High 2HG and low NADPH levels might sensitize tumors to oxidative stress, potentiating response to radiotherapy, and may account for the prolonged survival of patients harboring the mutations. So we studied further the alterations of function in IDH1R132H mutant cells in vitro. Based on the decrease of defence and the increase of impairing factors in tumor cells, we found that the tumors harbouring IDH1 mutations may have an elevated radiosensitivity. In the present study, we described the impact of IDH1 mutations in gliomas and search for new perspectives for the treatment strategy.

<p>Gliomas are tumors of the central nervous system for which improvements in treatment are critically needed. Mutations in IDH1 and IDH2, which encode the cytosolic and mitochondrial NADP+-dependent isocitrate dehydrogenases, respectively, are frequent in gliomas. Here, we summarize recent literature concerning gliomas, the normal cellular functions of IDH1/2, the epidemiology of IDH1/2 mutations, and the understanding of the function of IDH1/2 mutations in cancer. We then show in vitro using liquid chromatography-mass spectrometry that a function of many IDH1/2 mutations is to produce 2-hydroxyglutarate. Next, we use a mass spectrometry based platform to characterize metabolic changes in a glioma cell line expressing IDH1/2 mutants and show that the IDH mutants are associated with lowered N-acetylated amino acids both in this cell line model and in primary tumor tissue. Finally, we develop and characterize a Drosophila melanogaster (fruit fly) model of IDH1/2-mutated cancer by expressing the mutated Drosophila homolog of IDH1 in fly tissues using the UAS-Gal4 binary expression system. These results delineate downstream molecular players that likely play a role in IDH1/2-mutated cancer and provide a model organism for interrogation of genetic networks that interact with IDH1/2 mutation. These findings refine our understanding of glioma pathogenesis and may inform the design of new glioma therapies.</p> / Dissertation

The aim of the study was to identify molecular markers that can determine progression of low grade glioma. This was done using various approaches such as IDH1 and IDH2 mutation analysis, MGMT methylation analysis, copy number analysis using array comparative genomic hybridisation and identification of differentially expressed miRNAs using miRNA microarray analysis. IDH1 mutation was present at a frequency of 71% in low grade glioma and was identified as an independent marker for improved OS in a multivariate analysis, which confirms the previous findings in low grade glioma studies. IDH1 mutation was associated with MGMT promoter methylation when partially methylated tumours were grouped with methylated tumours. Grade II and grade III tumour comparison analysis revealed 14 novel significant miRNAs with differential expression. A miRNA signature was shown for histological subtypes, oligoastrocytoma and anaplastic oligoastrocytoma, following the miRNA expression analysis in grade II and grade III tumors based on histology. Oligoastrocytoma presented a more similar profile to oligodendroglioma, but anaplastic oligoastrocytoma was more similar to anaplastic astrocytoma. Five novel miRNAs were identified in grade III tumours, when comparing IDH1 mutant and IDH1 wild type tumours. Analysis of paired samples of primary/recurrent tumours revealed that additional genomic changes may promote tumour progression. For each of the pair, the two samples were genomically different and in each case, the reccurent tumours had more copy number aberrations than the corresponding primary tumours. Cell cultures derived from the tumour biopsies were not representative of the low grade glioma in vivo, which was evident from the differences identified in the miRNA expression and copy number changes in the paired samples. IDH1 mutation present in tumour biopsies was not maintained in their respective cell cultures. These findings give an insight into the molecular mechanisms involved in the tumourigenesis of low grade glioma and also tumour progression.

<p>Despite decades worth of research, glioblastoma remains one of the most lethal cancers. The identification of <italic>IDH1</italic> as a major cancer gene in glioblastoma provides an exceptional opportunity for improving our understanding, diagnostics, and treatment of this disease. In addition to mutations in <italic>IDH1</italic>, recent studies from our laboratory have characterized the genetic landscape of gliomas and have shown the cooperation between IDH1 mutations and other oncogenic alterations such at TP53 mutations. Normally, IDH1 functions in the oxidative decarboxylation of isocitrate to &alpha;&ndash;ketoglutarate, however the mutant form confers neomorphic enzymatic activity by producing 2&ndash;hydroxyglutarate, an oncometabolite responsible for aberrant methylation in IDH1&ndash;mutated tumors, among other mutant <italic>IDH1</italic>&ndash;mediated phenotypes. To determine the role of mutant IDH1 <italic>in vivo</italic>, we generated a conditional knock&ndash;in mouse model. This genetically faithful system is both biologically and clinically relevant and will promote the understanding of mutant IDH1&ndash;mediated tumorigenesis while offering a route for therapeutic targeting.</p><p>We observed that broad expression of mutant IDH1 throughout the brain leads to hydrocephalus in 80% of animals. In assessing the earliest effects of mutant IDH1 on the brain, we determined mutant IDH1 confers a decrease in the proliferative cells of the subventricular zone of the lateral ventricle, the area which houses the neural stem cells in embryonic and adult animals. Additionally, a perturbation to the normal neural stem cell niche was observed in these animals. Combined, this data suggests that mutant IDH1 may be affecting the signaling pathways involved in differentiation in this population of cells. <italic>In vivo</italic> and <italic>in vitro</italic> studies will further elucidate mutant IDH1's effects on the differentiation patterns of neural stem cells expressing mutant IDH1.</p><p>To express mutant IDH1 in a more restricted manner and harness spatiotemporal control, we crossed mutant animals to a Nestin&ndash;CreER<super>T2</super> strain of mouse that permits expression of floxed alleles upon treatment with tamoxifen. Animals were sacrificed at the onset of symptoms or at 1&ndash;year of age. We observed the development of both low&ndash; and high&ndash;grade gliomas in approximately 15&ndash;percent of E18.5 tamoxifen&ndash;treated animals. All tumors were found in a TP53&ndash;deleted background with mutant IDH1 being detected in only those tumors with the mutant allele. Lastly, to decrease the latency and increase the penetrance of tumor formation, an orthotopic intracranial injection model was generated to allow for visualization of tumor formation and development, as well as investigation of therapeutic modalities. The models generated and the knowledge gained from these studies will offer an understanding of the biological effects of the most common mutations found in the astrocytic subset of gliomas, bringing us strides closer to determining mechanisms and therapeutic targets for <italic>IDH1</italic>&ndash;mutated cancers.</p> / Dissertation