Les thérapies de différenciation sont une alternative prometteuse aux drogues génotoxiques utilisées en chimiothérapie pour le traitement de nombreux cancers. En particulier, l’acide tout-trans rétinoïque (ATRA) est utilisé avec succès pour traiter la leucémie aiguë promyélocytaire, un sous-type des leucémies aiguës myéloïdes (LAM). Malheureusement, son efficacité clinique est limitée dans les autres sous-types des LAM. Cela est en particulier du à une répression épigénétique des gènes de réponse à l’ATRA. Les SUMO constituent une famille de modificateurs post-traductionnels apparentés à l’ubiquitine dont la conjugaison sur de nombreuses protéines, appelée sumoylation, est impliquée dans la régulation de nombreux processus cellulaires, dont la transcription. Dans ce contexte, l’objective de ma thèse a été de comprendre le rôle de la sumoylation dans la réponse des LAM aux thérapies de différenciation. Nous avons pu montrer que la sumoylation réprime la différenciation induite par ATRA dans plusieurs lignées cellulaires, des cellules primaires de patients y compris celles résistantes à la chimiothérapie. L’inhibition de la sumoylation par les inhibiteurs pharmacologiques ou la surexpression des désumoylases augmente de façon remarquable la différenciation par ATRA et, à l’inverse l’augmentation de la sumoylation suite à une surexpression de SUMO ou son enzyme de conjugaison Ubc9 réduit fortement l’efficacité d’ATRA. L’ATRA synergise avec l’inhibition de la sumoylation pour limiter la prolifération des cellules de LAM in vitro et in vivo. D’un point de vue mécanistique, l’inhibition de la sumoylation favorise la différenciation des cellules de LAM en facilitant l’expression des gènes responsables de la différenciation myéloïde. Ainsi, cibler la sumoylation constitue une approche prometteuse pour sensibiliser la LAM aux thérapies de différenciation. / Differentiation therapies are a promising alternative to genotoxic-based chemotherapies in the treatment of many cancers. In particular, All-trans-retinoic acid (ATRA) is successfully used for Acute Promyelocytic Leukemias, a subtype of Acute Myeloid Leukemias. However, its clinical efficiency is very limited in the other AML subtypes, in particular because of epigenetic repression of ATRA-responsive genes. SUMOs are a family of post-translational modifiers related to ubiquitin and their conjugation, sumoylation, to their substrate proteins regulate many processes including gene transcription. The aim of my thesis was to understand the role of sumoylation in AML responses to treatments. I showed that sumoylation represses ATRA-induced differentiation in many AML cell lines and primary patient samples, including those resistant to chemotherapies. Inhibition of sumoylation with pharmacological inhibitors or overexpression of desumoylases markedly increased their differentiation by ATRA and increasing sumoylation by overexpression of SUMO or its conjugating enzyme Ubc9 strongly reduce ATRA efficiency. Inhibition of sumoylation synergize with ATRA to arrest AML cells proliferation both in vitro and in vivo. Mechanistically, inhibition of sumoylation primes AML cells for differentiation by facilitating the expression of master genes of the myeloid differentiation. Targeting the SUMO pathway thus constitute a promising approach to sensitize AML to differentiation therapies.

Defective chromatin remodeling proteins are associated with both germline and acquired human disease. PHF6 is encoded by an X-linked gene that is predominantly expressed in the brain and thymus. Structurally, PHF6 contains nuclear and nucleolar localization sequences as well as two ZaP domains, which bind dsDNA. Germline mutations in PHF6 are the cause of BFLS, an XLID, while somatic PHF6 mutations have been identified in T-ALL, AML, and CML. Indeed, screening of a pediatric cohort of nine T-ALL patients revealed a novel H329Q mutation. In a further clinical analysis, T-ALL onset occurred in a 9-year old male BFLS patient with an R342X mutation, suggesting that BFLS might be a cancer predisposition syndrome. To better understand its protein function, recombinant PHF6 was co-immunoprecipitated for a mass spectrometry based proteomic screen. Notably, PHF6 co-purified with multiple constituents of the NuRD complex, an important transcriptional regulator during embryogenesis and lineage commitment with particularly well characterized responsibilities during lymphogenesis. PHF6-NuRD localization was restricted to the nucleoplasm, however PHF6 also co-purified with several ribosomal and splicing proteins. When examined further, PHF6 was found to be recruited to the nucleolus by an RNA-mediated interaction and co-localized within the subnucleolar FC and DFC compartments. ChIP-qPCR revealed that PHF6 binds to transcribed regions of rDNA, resulting in the repression of rRNA. These data thus present a model of PHF6 acting as a tumour suppressor by mediating both nucleoplasmic and nucleolar transcriptional events.

<div>Cancer treatment was revolutionized when imatinib, a protein kinase inhibitor, was approved for the treatment of chronic myeloid leukemia (CML) in 2001. Imatinib increased the five-year survival rate of CML to over 90%. Imatinib is successful because it can selectively target BCR-ABL1, the mutant kinase driver to CML. Since then, there have been over 50 small molecules approved by the FDA to treat various cancers, which are driven by aberrantly expressed or mutated protein kinases. These kinase inhibitors (KIs) target kinases such as KIT (to treat gastrointestinal stromal tumors), EGFR (to treat lung cancer), and MEK (to treat melanoma) and often inhibit the kinases by binding to the ATP binding site or an allosteric site. Despite the successes seen with these approved KIs, responses are often transient. Most patients relapse after only a few months of responding to first and second generation KIs due to mutations to the kinases and/or upregulation of compensatory pathways. For cancer-driver kinases such as BCR-ABL1 or FLT3-ITD, which are oncogenic due to a fusion protein (BCR fused to ABL1) or internal tandem duplication within the juxtamembrane domain (FLT3), relapse can also occur due to the emergence of additional mutations in the kinase domain (a phenomenon termed secondary mutation). For example, secondary mutation (T315I or E255K) in BCR-ABL1 leads to imatinib resistance whereas secondary mutation (F691L or D835Y/V/H) in FLT3-ITD leads to resistance to many first- and second-generation FLT3 inhibitors. Gilteritinib, a recently approved FLT3 inhibitor drug for acute myeloid leukemia (AML) is not effective against AML harboring FLT3-ITD (F691L). Thus, there is a clinical need for novel chemotypes that inhibit drug-resistant kinases, especially those containing secondary mutations.</div><div>Many of the FDA-approved kinase inhibitors contain common heterocycles such as quinolines, indazoles, quinazolines, quinazolinones, quinoxalines, and oxindole. The majority of KIs also contain groups that enhance aqueous solubility, such as morpholine and piperazine rings. Additionally, many KIs contain halogen substituents (mainly Cl and F). This thesis introduces novel kinase inhibitors, which contain the less explored isoquinolines, naphthyridines, and pyrido[3,4-b]pyrazine cores. These newly introduced kinase inhibitors potently inhibit drug-resistant ABL1 and FLT3 kinases, harboring secondary mutations, and have the potential for clinical translation against relapsed leukemia.</div>

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.

Die Akute Myeloische Leukämie (AML) ist eine sowohl zytogenetisch als auch molekulargenetisch äußerst heterogene Erkrankung, die durch die klonale Proliferation myeloider Vorläuferzellen sowie eine Ausreifungsblockade charakterisiert ist. Trotz des in den letzten Jahren zugenommenen Wissens über die Biologie dieser Erkrankung und Weiterentwicklung von Therapiemethoden bleibt das Gesamtüberleben der Patienten mit AML überwiegend schlecht. Damit für mehr Patienten eine Heilung der AML möglich werden kann, sind ein tieferes Verständnis über die funktionellen Zusammenhänge in der Leukämogenese, eine bessere Risikostratifizierung und neue Therapieoptionen erforderlich. Diese Habilitationsschrift fasst Publikationen zusammen, die neue molekulare Biomarker und deren klinischen Einfluss in der AML untersucht haben. Der Fokus liegt auf der Erfassung molekularbiologischer Veränderungen bei Diagnose einer AML oder im Krankheitsverlauf, die die Risikostratifizierung der Patienten verbessern kann. Außerdem gestattet die Arbeit Einblicke in die mit diesen molekularen Markern verbundene Biologie der AML, sowie mögliche neue Therapieoptionen. Der erste bis dritte Abschnitt der Arbeit fokussiert sich auf Genmutationen und Genexpressionen in der AML. Es wird dargelegt, wie das Vorhandensein bestimmter Fusionstranskripte (hier CBFB-MYH11), rekurrenter Mutationen allein (hier im DNMT3A Gen) sowie als Teil genetischer Risikoklassifikationssysteme (hier die genetischen Risikogruppen des European LeukemiaNet) und die abberrante Expression AML-assoziierter Gene (hier BAALC, ERG und MN1) Beiträge zur Prognoseabschätzung der AML bieten. Darüber hinaus hat sich in den letzten Jahren die entscheidende Rolle von MicroRNAs in der Pathophysiologie der AML herausgestellt. Heute weiß man, dass für die Leukämieentstehung und die Aggressivität der Erkankung schon die Dysregulation einer einzelnen microRNA entscheidend sein kann. Im vierten Abschnitt wird auf den progostischen Einfluss der Expressionslevel zweier MicroRNAs – miR-181a und miR-29b – und deren klinische und biologische Konsequenzen eingegangen. Außerdem wird dargestellt, wie verschiedene therapeutische Interventionen zu günstigen Änderungen des Expressionsniveaus dieser beiden neuen Biomarker und so zu potentiell neuen Therapiestrategien in der AML führen können. Weiterhin wächst die Erkenntnis, dass in der AML so gennannte Leukämie-initiierende Zellen für Therapieresistenz und Rezidive verantwortlich zu sein scheinen. Der letzte Abschnitt dieser Habilitationsschrift fokussiert sich auf das CD34+/CD38- Zellkompartiment, welches einen Großteil der Leukämie-initiiernden Zellen enthält. Es wird gezeigt, dass die Bestimmung der Größe des CD34+/CD38- Zellkompartiments bei Diagnose geeignet ist, AML Patienten mit einem erhöhten Rezidivrisiko nach allogener Stammzelltransplantation zu identifizieren. Zusammenfassend zeigt die Arbeit verschiedene Ansätze, wie neue molekulare Biomarker zu einer besseren Risikostratifizierung und einem tieferen Verständnis der AML zugrunde liegenden Biologie führen können. Des Weiteren beschreibt sie Möglichkeiten der therapeutischen Intervention und weist insgesamt auf die klinischen Implikationen dieser neuen Biomarker hin.

We did not find mutation in coding areas of genes for components of TGFbeta1 signaling pathway but we detected decreased or undetectable expression of these analysed genes.The decreased expression is probably caused by epigenetic changes, so by hypermethylation and deacetylation of promoter regionsof these genes.Antiproliferative and apoptotic effect of TGF1 was analysed in AML cell lines (ML1, ML2, CTV1 and Kasumi1). ML2 cells rezistence to inhibition of DNA synthesis by TGFβ1 is not caused by mutations of genes for components of TGFβ1 signaling pathway. We found that increased SnoN (Ski-like novel gene) expression on the level of coresponding mRNA and protein is probably accountable for this rezistence. Kasumi1 and M2 cells were sensitive to induction of apoptózis caused by TGFβ1 treatment but in less extent than by proteazome inhibitor bortezomib. The difference of AML cells of different lines answers shows a great heterogeneity AML in AML patients. Prognostic factors analysis in AML with normal karyotype confirmed that CEBPA (CCAAT/enhancer binding protein alpha) mutations predict favourable prognosis but the elevated EVI1 ("Ecotropic Virus Integration Site 1") and ERG ("ETS-related gene") expression are connected with unfavourable prognosis. EVI1 is a negative marker for MDS as well. We did not confirm...

Acute myeloid leukaemia (AML) is highly heterogeneous haematological malignancy, which represents a challenge in the understanding of the disease. Relapse in AML is common, andmany relapsed patients respond poorly to conventional treatment, leading to a low survivalrate. Investigating the mutational landscape connected to relapse AML is therefore of highinterest in order to improve clinical outcome. Recurrent in-frame internal tandem duplications(ITDs) in exon 13 of the UBTF gene have previously been discovered in paediatric relapseAML, correlating to one of the DNA binding domains of the transcription factor UBTF.UBTF is involved in recruitment of RNA polymerase I and activation of the transcription ofribosomal RNA. As a result, it is an important factor in ribosome biogenesis. In this study, wehave investigated the effect of UBTF-ITDs on RNA synthesis and UBTF localization. I haveshown that ITDs lead to disturbed localization of UBTF to the nucleus, which coupled withthe previous finding that patients are heterozygous for the ITDs, indicates haploinsufficiency.This could have potential implications in AML drug resistance. We have further generated anin vitro model for investigating the effect of UBTF haploinsufficiency, which could lead toidentification of vulnerabilities to be targeted in future drug treatments.

京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第23123号 / 人健博第85号 / 新制||人健||6(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 藤井 康友, 教授 岡 昌吾, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19368号 / 医博第4045号 / 新制||医||1011(附属図書館) / 32382 / 新制||医||1011 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 吉村 長久, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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