Étude des mécanismes moléculaires impliquant l'homéoprotéine MEIS1 dans le développement de leucémies myéloïdes aigües

Les leucémies myéloïdes aigües résultent d’un dérèglement du processus de l’hématopoïèse et regroupent des maladies hétérogènes qui présentent des profils cliniques et génétiques variés. La compréhension des processus cellulaires responsables de l’initiation et du maintien de ces cancers permettrait de développer des outils thérapeutiques efficaces et ciblés. Au cours des dernières années, une quantité croissante d’anomalies génétiques reliées au développement de leucémies ont été corrélées à une expression anormale des gènes HOX et de leurs cofacteurs MEIS et PBX. Des modèles expérimentaux murins ont confirmé le rôle direct joué par ces protéines dans le développement de leucémies. En effet, la protéine MEIS1 collabore avec HOXA9 dans la leucémogenèse et requiert pour ce faire trois domaines distincts. Deux de ces domaines sont conservés chez PREP1, un membre de la même classe d’homéoprotéine que MEIS1. En utilisant une approche de gain-de-fonction, j’ai confirmé l’importance du rôle joué par le domaine C-terminal de MEIS1 dans l’accélération des leucémies induites par HOXA9. J’ai également montré que l’activité de ce domaine était corrélée avec une signature transcriptionnelle associée à la prolifération cellulaire. J’ai ensuite réalisé un criblage à haut débit afin d’identifier des antagonistes de l’interaction MEIS-PBX, également essentielle à l’accélération des leucémies HOX. À cette fin, j’ai développé un essai de transfert d’énergie de résonance de bioluminescence (BRET) permettant de détecter la dimérisation MEIS-PBX dans les cellules vivantes. Plus de 115 000 composés chimiques ont été testés et suite à une confirmation par un essai orthogonal, une vingtaine de molécules ont été identifiées comme inhibiteurs potentiels. Ces composés pourront être rapidement testés sur la prolifération de cellules leucémiques primaires dans un contexte d’étude préclinique. Finalement, deux approches protéomiques complémentaires ont permis d’identifier des partenaires potentiels de MEIS1 et PREP1. La catégorisation fonctionnelle de ces candidats suggère un nouveau rôle pour ces homéoprotéines dans l’épissage de l’ARN et dans la reconnaissance de l’ADN méthylé.Acute myeloid leukemias are the result of a perturbed hematopoietic process and regroup heterogeneous diseases with distinct clinical and genetic profiles. Identifying and understanding the faulty cellular processes would allow the development of targeted and efficient therapeutic tools. Over the last 15 years, a growing number of disease-linked genetic anomalies have been correlated with abnormal expression levels of HOX genes and their cofactors MEIS and PBX. Mouse model experimentations revealed a direct role for these proteins in leukemogenesis. Indeed, the protein MEIS1 collaborates with HOXA9 in the acceleration of leukemia development. This specific function requires the presence of three different domains, two of which are highly conserved in PREP1, another member of the MEIS class of homeoproteins. Using a gain-of-function approach, I confirmed the importance of the C-terminal domain of MEIS1 in the acceleration of HOXA9-induced leukemias. I also correlated the activity of this domain with a transcriptional signature related to cell proliferation. Furthermore, I performed a high-throughput screen to identify antagonists of the MEIS-PBX interaction, also required for acceleration of HOX-induced leukemogenesis. In this regard I developed an assay that exploits bioluminescence resonance energy transfer (BRET) to monitor the MEIS-PBX dimerization in living cells. More than 115 000 compounds were tested and upon confirmation of their activity using an orthogonal assay, 20 small molecules were identified as potential inhibitors. These compounds will be rapidly tested on proliferation of primary leukemic cells in a preclinical setting. Finally two complementary proteomic approaches allowed the identification of new potential partners of MEIS1 and PREP1. The functional clustering of these candidates suggests a new role for homeoproteins in mRNA splicing and methylated DNA recognition

Expression profile of CREB knockdown in myeloid leukemia cells.

BackgroundThe cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution.MethodsTo understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA.ResultsBy combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA.ConclusionWe have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle

Targeting Chromatin Complexes in Myeloid Malignancies and Beyond: From Basic Mechanisms to Clinical Innovation

The aberrant function of chromatin regulatory networks (epigenetics) is a hallmark of cancer promoting oncogenic gene expression. A growing body of evidence suggests that the disruption of specific chromatin-associated protein complexes has therapeutic potential in malignant conditions, particularly those that are driven by aberrant chromatin modifiers. Of note, a number of enzymatic inhibitors that block the catalytic function of histone modifying enzymes have been established and entered clinical trials. Unfortunately, many of these molecules do not have potent single-agent activity. One potential explanation for this phenomenon is the fact that those drugs do not profoundly disrupt the integrity of the aberrant network of multiprotein complexes on chromatin. Recent advances in drug development have led to the establishment of novel inhibitors of protein–protein interactions as well as targeted protein degraders that may provide inroads to longstanding effort to physically disrupt oncogenic multiprotein complexes on chromatin. In this review, we summarize some of the current concepts on the role epigenetic modifiers in malignant chromatin states with a specific focus on myeloid malignancies and recent advances in early-phase clinical trials

Targeting protein–protein interactions in hematologic malignancies: still a challenge or a great opportunity for future therapies?

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110054/1/imr12244.pd

Proteomics of Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is characterized by specific cytogenetic aberrations that are strong determinants of prognostic outcome and therapeutic response. Because the pathological outcome of AML patients with cytogentic abnormalities differs considerably we hypothesized that their proteome may also differ specifically in their expression pattern, protein interaction pathways and posttranslational modifications. We performed this study using 42 AML patients diagnosed for various cytogenetic abnormalities based on two-dimensional gel electrophoresis and MALDI TOF Tandem MS (MS/MS) analysis. We could identify significant differences in the proteome and posttranslational modifications of peptides, later confirmed by other methods, between cytogenetic groups. The interactome analysis based on computational bioinformatics reveals a major regulating networks, MAPK8 and MYC for complex aberrant karyotype, TP53 for t(8;21), TP53- MYC- PRKAC for 11q23, JUN and MYC for Inv(16). We could show in our validation and characterisation experiments that survivin is a novel target of t(8;21) leukemia and AML1-ETO directly regulates its expression to induce the differentiation block that could be overcome by silencing its expression. Further, we analysed 42 MS spectra representative of hnRNPH1, Calreticulin and hnRNPA2/B1 in a peak explorer which reveals a cytogenetic specific posttranslational modification of β-O-linked N-acetyl glucosamine (O-GlcNAc) of hnRNPH1 in AML patients with 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation and methylation of hnRNPA2/B1 in patients with translocations of t(8;21) and inv(16). This report may lead to a new thinking about the AML pathogenesis as differences at PTM level could be used to distinguish different subtypes of AML besides for testing the therapeutic significance. Further, we characterised the biological role of survivin identified specifically from t(8;21) patients. We could show that AML1-ETO induces the expression of survivin both in a cell line model and in primary human hematopoietic precursors. AML1-ETO activates the basal transcription of the survivin promoter and binds to the only AML1 core enhancer binding sequence, TGTGGT, in survivin promotor. Repression of AML1-ETO mediated induction of survivin expression by a specific short hairpin RNA restores C/EBPα protein and its basal transcriptional activity on its own promotor. This restoration differentiates AML1-ETO positive leukemic cells to terminal granulocytic differentiation and growth arrest. These observations indicate that the antiapoptotic survivin protein, which holds a great therapeutic promise, is a critical mediator of AML1-ETO induced defective granulopoiesis. Thus, proving that AML1-ETO induces inhibition of granulocytic differentiation by activating survivin expressio

Dissecting BMI1 Protein-Protein Interactions Through Chemical Biology.

BMI1 has emerged as a key oncogenic factor in many cancers, associated with unregulated cellular proliferation, tumor metastasis and cancer-initiating cell self-renewal. BMI1 is best characterized as a component of the canonical vertebrate polycomb repression complex 1 (PRC1) which negatively regulate transcription of hundreds of genes through ubiquitination of histone H2A. Previous work suggested that BMI1 has multiple protein binding partners within the PRC1 complex and we were motivated by the prospects to target these protein-protein interactions (PPIs) with small molecule inhibitors. This dissertation describes a multi-pronged campaign to: 1) characterize BMI1 PPIs at the molecular level and 2) develop novel chemical tools to explore BMI1 function in both normal and cancer biology. Using X-ray crystallography and solution NMR approaches we solved the 3D structure of BMI1 in complex with its PRC1 binding partner protein PHC2. Supporting biochemical and biophysical characterization of the BMI1 PPI domain demonstrated a novel mode of self-association of this domain. Mutagenic disruption of both BMI1-PHC2 and BMI1-BMI1 interactions blocks cellular proliferation demonstrating that multiple PPIs are critical for BMI1 function. To identify small molecule inhibitors of BMI1 we designed two biochemical assays to quantify the BMI1-PHC2 interaction and these assays were used as a platform for high-throughput screening. Through this screen we identified three classes of small molecule inhibitors that bind directly to BMI1 to disrupt the BMI1-PHC2 interaction, representing three different strategies for BMI1 inhibitor development. As a complementary approach to inhibit BMI1 we developed a specific inhibitor of Ring1B/BMI1- mediated H2A ubiquitination with potent inhibitory activity both in vitro and in cells. Mechanistic characterization demonstrates that Ring1B/BMI1 inhibitors induce significant protein conformational change and the inhibitor-bound conformation is incompatible with nucleosome binding by Ring1B. These molecules represent the first direct-binding inhibitors of Ring1B/BMI1 and have a novel mechanism of action to block direct protein-nucleosome interaction. Overall, this work contributes to the understanding of BMI1 function through characterization of its multiple PPIs and demonstrates that these interactions can be inhibited by small molecules representing novel strategies to target this protein for development of new chemical tools or potential therapeutics for cancer.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113363/1/flvgray_1.pd

WNT-DEPENDENT REGENERATIVE FUNCTION IS INDUCED IN LEUKEMIA-INITIATING AC133BRIGHT CELLS

The Cancer Stem Cell model supported the notion that leukemia was initiated and maintained in vivo by a small fraction of leukemia-initiating cells (LICs). Previous studies have suggested the involvement of Wnt signaling pathway in Acute Myeloid Leukemia (AML) by the ability to sustain the development of LICs. A novel hematopoietic stem and progenitor cell marker, monoclonal antibody AC133, recognizes the CD34bright CD38- subset of human acute myeloid leukemia cells, suggesting that it may be an early marker for the LICs. During the first part of my phD program we previously evaluated the ability of leukemic AC133+ fraction, to perform engraftment following to xenotransplantation in immunodeficient mouse model Rag2-/-\u3b3c-/-. The results showed that the surface marker AC133 is able to enrich for the cell fraction that contains the LICs. In consideration of our previously reported data, derived from the expression profiling analysis performed in normal (n=10) and leukemic (n=33) human long-term reconstituting AC133+ cells, we revealed that the ligand-dependent Wnt signaling is induced in AML through a diffuse expression and release of WNT10B, a hematopoietic stem cells regenerative-associated molecule. In situ detection performed on bone marrow biopsies of AML patients, showed the activation of the Wnt pathway, through the concomitant presence of the ligand WNT10B and of the active dephosphorylated \u3b2-catenin form, suggesting an autocrine / paracrine-type ligand-dependent activation mechanism. In consideration of the link between hematopoietic regeneration and developmental signaling, we transplanted primary AC133+ AML A46 cells into developing zebrafish. This biosensor model revealed the formation of ectopic structures by activation of dorsal organizer markers that act downstream of the Wnt pathway. These results suggested that the misappropriating Wnt associated functions can promote pathological stem cell-like regeneration responsiveness. The analyses performed in situ retained information on the cellular localization, enabling determination of the activity status of individual cells and allowing the tumor environment view. Taking this issue into consideration, during the second part of my phD program, I set up the application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The mRNA in situ detection technique is based on padlock probes ligation and target priming rolling circle amplification allowing the single nucleotide resolution in heterogenous tissues. The mRNA in situ detection performed on bone marrow biopsies derived from AML patients, showed a diffuse localization pattern of WNT10B molecule in the tissue. Conversely, only the AC133bright cell population shows the Wnt signaling activation signature represented by the cytoplasmatic accumulation and nuclear translocation of the active form of \u3b2-catenin. In spite of this, we previously evidenced that the regenerative function of WNT signaling pathway is defined by the up-regulation of WNT10B, WNT10A, WNT2B and WNT6 loci, we identified the WNT10B as a major locus associated with the regenerative function and over-expressed by all AML patients. By the molecular evaluation of the WNT10B transcript, we isolated an aberrant splicing variant (WNT10BIVS1), that identify Non Core-Binding Factor Leukemia (NCBFL) class and whose potential role is discussed. Moreover, we demonstrate that the function of "leukemia stem cell", present in the cell population enriched for the marker AC133bright, is strictly related to regenerative function associated with WNT signaling, defining the key role of WNT10B ligand as a specific molecular marker for leuchemogenesis. This thesis defines the new suitable approaches to characterize the leukemia-initiating cells (LICs) and suggest the role of WNT10B as a new suitable target for AML

Recent Developments in Cancer Systems Biology

This ebook includes original research articles and reviews to update readers on the state of the art systems approach to not only discover novel diagnostic and prognostic biomarkers for several cancer types, but also evaluate methodologies to map out important genomic signatures. In addition, therapeutic targets and drug repurposing have been emphasized for a variety of cancer types. In particular, new and established researchers who desire to learn about cancer systems biology and why it is possibly the leading front to a personalized medicine approach will enjoy reading this book

Remodelling of the nuclear envelope during KSHV lytic infection

Kaposi’s sarcoma associated herpesvirus (KSHV) is a human tumour virus and key aetiological agent for several malignancies including Kaposi’s sarcoma. KSHV exhibits a biphasic life cycle split between a persistent latent period with minimal gene expression and a lytic period with an expression cascade that culminates in the release of nascent virions. Crucially, the lytic phase has been shown to be important for tumorigenesis and the spread of Kaposi’s sarcoma. The Nuclear Pore Complex (NPC) is a protein mega-complex that regulates nucleocytoplasmic transport. It is formed by multiple copies of individual nucleoporins that combine into a sophisticated protein gateway. The regulation of nuclear access makes it a target for viruses that subvert the NPC in order to hijack the cell for viral replication. Whilst herpesvirus can induce changes at the NPC, little is known about KSHV NPC remodelling. This study presents an investigation of how KSHV targets the NPC during its lytic infection highlighting the targeting a specific nucleoporin, Nup98, and an attempt at broader interactomic analysis using proximity dependent biotin identification. Nup98 is specifically downregulated early during lytic infection by the E3 ubiquitin ligase activity of viral protein RTA. This appears to be related to the repression of expression at viral ORF50 promoters when Nup98 is overexpressed in the nucleoplasm. This study also highlights how depletion of Nup98 is detrimental to the virus, leading to failed virion egress. In summary, this project highlights how KSHV specifically targets one population of Nup98 but requires NPC-bound Nup98 to sequester a cellular mRNA for CHMP7 protein to ensure virion egress. It also provides the first attempt at using interactomic techniques to create a comprehensive, semi-quantitative profile of changes to the NPC during KSHV lytic infection that can pave the way for further interaction studies and the development of targeted antivirals for KSHV

Structure, Activity, and Function of Protein Methyltransferases

This collection of review articles describes the structure, function and mechanism of individual protein methyltransferase enzymes including protein lysine methyltransferases, protein arginine methyltransferases, and also the less abundant protein histidine methyltransferases and protein N-terminal end methyltransferases. The topics covered in the individual reviews include structural aspects (domain architecture, homologs and paralogs, and structure), biochemical properties (mechanism, sequence specificity, product specificity, regulation, and histone and non-histone substrates), cellular features (subcellular localization, expression patterns, cellular roles and function, biological effects of substrate protein methylation, connection to cell signaling pathways, and connection to chromatin regulation) and their role in diseases. This review book is a useful resource for scientists working on protein methylation and protein methyltransferases and those interested in joining this emerging research field