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This work presents the application of spiroligomers as inhibitors of protein-protein interactions. After the discovery of an acyl-transfer coupling reaction by Dr. Zachary Brown, a previous graduate student of Schafmeister group, the synthesis of highly functionalized spiroligomers that mimic the helical domain of p53 was undertaken before each molecule was tested for binding to HDM2, a natural binding partner of p53. A library of molecules was synthesized on solid support that altered the stereochemistry along the spiroligomer as well as the presented functional groups. It was determined that spiroligomers enter human liver cancer cells through passive diffusion and induces a biological response in both a dose- and time-dependent manner. The synthesis of additional spiroligomer analogues achieved low micromolar to high nanomolar range activity during screening in direct and competitive binding assays. In parallel to the project above, a series of spiroligomers that mimic the side chains of the leucine zipper region of Max were synthesized in an effort to disrupt the interaction of the protein with c-Myc. The series of compounds contained various stereocenter combinations and different functional groups as before but were made in solution before testing for inhibition. Initial binding assays resulted in low micromolar activity, however, secondary assays (ELISA and cellular assays) did not confirm the inhibitory effect of spiroligomers on the c-Myc/Max heterodimer. In summary, this work illustrates that spiroligomers are capable mimics of helical peptides and can induce a biological response. / Chemistry

Amyotrophic Lateral Sclerosis (ALS) is a non-cell-autonomous disease. In early 2000, seminal and concordant studies demonstrated that glia modulates disease progression in a mouse model expressing a mutant form of SOD1. This effect has not been validated yet in a transgenic mouse expressing TDP-43Q331K, so we selectively deleted the transgenic TDP-43 in astrocytes and observed an improvement of motor symptoms and a rescue of the cognitive impairment. We performed RNA sequencing of astrocytes purified from this mouse model, observing an increased expression of genes related to proliferation and de-differentiation at the early symptomatic stage. Additionally, we confirmed a relative increase in glial cell proliferation by Ki67 staining in vivo and by EdU staining in primary astrocytic cultures derived from this mouse model. By interrogating the genomes of 23.475 ALS patients versus 12.577 healthy controls, we revealed the enrichment of SNPs in the responsive elements of transcription factors linked to proliferation with a hub around c-myc. No differences were observed in c-Myc RNA and protein expression and stability in primary TDP-43Q331K astrocytes, while phosphorylated (active) c-Myc form was found to be increased by immunostaining. We observed this increase in several ALS models, including primary astrocytes expressing SOD1G93A, iNPC-derived astrocytes obtained from C9orf72 and sporadic ALS patients, and HEK293 cells overexpressing TDP-43. Interestingly, phosphorylated c-Myc form was found at higher levels than controls also in iPSCderived motoneurons expressing C9orf72, TDP-43M337V, SOD1I114T or derived from sporadic patients. c-Myc plays many functions in the cell, among which it enhances the release of extracellular vesicles (EVs), nanolipid structures shown to impact the non-cell autonomous degeneration in vitro. Our in vitro model showed a significant increase in the production of small EVs derived from ALS astrocytes. We observed that WT glia-derived EVs play a prosurvival effect on receiving neurons; this effect was lost when neurons were treated with gliaderived EVs from TDP-43Q331K cultures or WT cultures overexpressing c-Myc. Proteomic analysis revealed that ALS EVs and EVs derived from c-Myc-overexpressing cells were deficient in extracellular matrix proteins and integrins. Interestingly, the EVs isolated from TDP-43Q331K primary astrocytes were also less prone to enter recipient neurons. In addition, we notice that the pro-survival effect induced by WT glial EVs was lost on transgenic neurons, suggesting that receiving neurons carrying the mutation feature surface or endocytic alterations that prevent the transmission of the EV-induced prosurvival effect. To further characterize this hypothesis, we showed that HEK293 cells over-expressing TDP-43 feature impaired EV uptake and that iPSC-derived TDP-43M337V, SOD1I114T, and sporadic motoneurons feature altered endocytic pathways. All these data highlight alterations in the glial-to-neuron communication in ALS and suggest that the expression and function of integrins and extracellular matrix proteins should be further investigated in ALS, specifically focusing on rescuing neuronal endocytic pathways to achieve a functional treatment.

Background Multiple myeloma is an incurable cancer disease that emerges from the bone marrow. Bone morphogenetic proteins (BMPs) are ligands that activates intracellular signaling pathways causing activation of transcription factors. Previous studies show that BMP treatment of myeloma cells induce apoptosis, a mechanism dependent on downregulation of c-MYC. BMPs uses different receptors on myeloma cells, but it is still unclear how the intracellular signaling pathway leading to apoptosis works. A recent whole genome CRISPR/Cas9 knockout screening suggested FOXO1 as a gene involved in the mechanism of apoptosis during BMP treatment. We therefore aimed to investigate further on how FOXO1 has an impact on BMP induced apoptosis. Methods Our hypothesis was that knockout of FOXO1 would protect the cells from apoptosis. To begin to address this issue we tested INA-6 FOXO1 knock-out cell clones that was generated before the start of the project and treated them with BMP-9 to look for effects on cell viability and protein expression. We measured cell viability using CellTiter-Glo® 2.0 Cell viability assay and expression of c-MYC and FOXO1 protein using Western blot. Results and conclusions Treatment with BMP-9 for 72 hours showed a decrease in viability of the cells, up to 98%. Protein expression of c-MYC was inhibited by BMP-9 treatment while a constant expression of FOXO1 was seen in all cells clones regardless of BMP treatment. Expression of FOXO1 in the FOXO1 knock-out cells indicates that the knock-out has not worked. More experiments are needed to clarify the role of FOXO1 in BMP-induced apoptosis.

La polykystose rénale autosomique dominante (ADPKD) est une des maladies génétiques les plus communes. ADPKD se manifeste le plus souvent au stade adulte par la présence de kystes rénaux, et bien souvent de kystes hépatiques, avec une progression très variable. ADPKD mène à une insuffisance rénale: les seuls recours sont la dialyse puis la transplantation rénale. Les mutations dispersées sur les gènes PKD1 (majoritairement; la protéine polycystine-1, PC1) et PKD2 (la protéine polycystine-2, PC2) sont responsables de l’ADPKD. Le mécanisme pathogénétique de perte de fonction (LOF) et donc d’un effet récessif cellulaire est évoqué comme causatif de l’ADPKD. LOF est en effet supporté par les modèles murins d’inactivation de gènes PKD1/PKD2, qui développent de kystes, quoique in utéro et avec une rapidité impressionnante dans les reins mais pas dans le foie. Malgré de nombreuses études in vitro, le rôle de PC1/PC2 membranaire/ciliaire reste plutôt hypothétique et contexte-dépendant. Ces études ont associé PC1/PC2 à une panoplie de voies de signalisation et ont souligné une complexité structurelle et fonctionnelle exceptionnelle, dont l’implication a été testée notamment chez les modèles de LOF. Toutefois, les observations patho-cellulaires chez l’humain dont une expression soutenue, voire augmentée, de PKD1/PC1 et l’absence de phénotypes extrarénaux particuliers remet en question l’exclusivité du mécanisme de LOF. Il était donc primordial 1) d’éclaircir le mécanisme pathogénétique, 2) de générer des outils in vivo authentiques d’ADPKD en terme d’initiation et de progression de la maladie et 3) de mieux connaitre les fonctions des PC1/PC2 indispensables pour une translation clinique adéquate. Cette thèse aborde tous ces points. Tout d’abord, nous avons démontré qu’une augmentation de PKD1 endogène sauvage, tout comme chez l’humain, est pathogénétique en générant et caractérisant en détail un modèle murin transgénique de Pkd1 (Pkd1TAG). Ce modèle reproduit non seulement les caractéristiques humaines rénales, associées aux défauts du cil primaire, mais aussi extrarénales comme les kystes hépatiques. La sévérité du phénotype corrèle avec le niveau d’expression de Pkd1 ce qui supporte fortement un modèle de dosage. Dans un deuxième temps, nous avons démontré par les études de complémentations génétiques que ces deux organes reposent sur une balance du clivage GPS de Pc1, une modification post-traductionelle typique des aGPCR, et dont l’activité et l’abondance semblent strictement contrôlées. De plus, nous avons caractérisé extensivement la biogénèse de Pc1 et de ses dérivés in vivo générés suite au clivage GPS. Nous avons identifié une toute nouvelle forme et prédominante à la membrane, la forme Pc1deN, en plus de confirmer deux fragments N- et C-terminal de Pc1 (NTF et CTF, respectivement) qui eux s’associent de manière non-covalente. Nous avons démontré de façon importante que le trafic de Pc1deN i.e., une forme NTF détachée du CTF, est toutefois dépendant de l’intégrité du fragment CTF in vivo. Par la suite, nous avons généré un premier modèle humanisant une mutation PKD1 non-sens tronquée au niveau du domaine NTF(E3043X) en la reproduisant chez une souris transgénique (Pkd1extra). Structurellement, cette mutation, qui mimique la forme Pc1deN, s’est également avérée causative de PKD. Le modèle Pkd1extra a permis entre autre de postuler l’existence d’une cross-interaction entre différentes formes de Pc1. De plus, nos deux modèles murins sont tous les deux associés à des niveaux altérés de c-Myc et Pc2, et soutiennent une implication réelle de ces derniers dans l’ADPKD tou comme une interaction fonctionnelle entre les polycystines. Finalement, nous avons démontré un chevauchement significatif entre l’ADPKD et le dommage rénal aigüe (ischémie/AKI) dont une expression augmentée de Pc1 et Pc2 mais aussi une stimulation de plusieurs facteurs cystogéniques tel que la tubérine, la β-caténine et l’oncogène c-Myc. Nos études ont donc apporté des évidences cruciales sur la contribution du gène dosage dans l’ADPKD. Nous avons développé deux modèles murins qui serviront d’outil pour l’analyse de la pathologie humaine ainsi que pour la validation préclinique ADPKD. L’identification d’une nouvelle forme de Pc1 ajoute un niveau de complexité supplémentaire expliquant en partie une capacité de régulation de plusieurs voies de signalisation par Pc1. Nos résultats nous amènent à proposer de nouvelles approches thérapeutiques: d’une part, le ciblage de CTF i.e., de style chaperonne, et d’autre part le ciblage de modulateurs intracellulaires (c-Myc, Pc2, Hif1α). Ensemble, nos travaux sont d’une importance primordiale du point de vue informatif et pratique pour un avancement vers une thérapie contre l’ADPKD. Le partage de voies communes entre AKI et ADPKD ouvre la voie aux approches thérapeutiques parallèles pour un traitement assurément beaucoup plus rapide. / Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic diseases. ADPKD is manifested by the presence of renal cysts detected most often in the adult stage, and frequently liver cysts, with highly variable progression. ADPKD leads to kidney failure with the only recourse of dialysis and eventual kidney transplantation. Mutations dispersed throughout the PKD1 gene (major player, the polycystin-1 protein, PC1) and the PKD2 gene (polycystin-2 protein, PC2) are responsible for ADPKD. The loss of function (LOF) pathogenetic mechanism, and therefore a cellular recessive effect, has been suggested as causative of ADPKD. LOF is indeed supported by the PKD1/PKD2 gene inactivation mouse models, which develop cysts, although in utero with impressive speed in the kidney but not in the liver. Despite many in vitro studies, the membrane/ciliary role of PC1/PC2 remains rather hypothetical and context-dependent. These studies have associated PC1/PC2 to a variety of signaling pathways and underlined exceptional structural and functional complexity, whose involvement has been tested especially in LOF models. However, pathocellular observations in humans with sustained and even increased expression of PKD1/PC1, and the absence of particular human extrarenal phenotypes questions the exclusivity of the LOF mechanism. It was therefore essential 1) to clarify the pathogenetic mechanism, 2) to generate in vivo tools authentic of ADPKD in terms of initiation and progression of the disease and 3) to better understand the essential functions of PC1/PC2 for an adequate clinical translation. This thesis addresses all of these issues. First, we demonstrated that an increase in endogenous PKD1, just like in humans, is pathogenetic by generating and characterizing in detail a transgenic mouse model of Pkd1 (Pkd1TAG). This model not only reproduces the renal human characteristics associated with defects of the primary cilium, but also the extrarenal, namely, liver cysts. The severity of the phenotype correlates with the expression level of Pkd1, which strongly supports a dosage model. Secondly, we have demonstrated with genetic complementation studies that these two organs rely on a balance of Pc1 GPS cleavage, a typical post-translational modification of aGPCR, whose activity and abundance seem strictly controlled. Furthermore, we have extensively characterized Pc1 biogenesis and its derivatives in vivo generated upon GPS cleavage. We have identified a new form, predominantly on the membrane, the Pc1deN form, in addition to confirming the two N- and C-terminal Pc1 fragments (NTF and CTF, respectively), which associate non-covalently. Importantly, we have demonstrated that traffic of Pc1deN i.e., the NTF form detached from the CTF, is still dependant on the integrity of the CTF fragment. Next, we generated a first model humanizing a PKD1 nonsense truncated mutation at the level of the NTF(E3043X) domain by reproducing it in a transgenic mouse (Pkd1extra). Structurally, this mutation, which mimics Pc1deN, has also been shown to be causative of PKD. The Pkd1extra model allowed the proposition of the existence of a cross-interaction between different forms of Pc1. In addition, our two mouse models are both associated with altered levels of c-Myc and Pc2, which is supportive of their involvement in ADPKD and a functional interaction between the polycystins. Finally, we have shown a significant overlap between ADPKD and acute renal injury (ischemia/AKI) namely increased expression of Pc1 and Pc2 but also stimulation of several cystogenic factors such as tuberin, β-catenin and the oncogene c-Myc. Our studies have therefore given crucial evidence to the contribution of PKD1 gene dosage mechanism in ADPKD. We have developed two mouse models, which can serve as a tool for the analysis of human pathology as well as for preclinical validation of ADPKD. The identification of a new form of Pc1 adds an additional level of complexity in part explaining the regulation capacity of Pc1 on several signaling pathways. Our findings lead us to propose new therapeutic approaches: firstly, targeting the CTF i.e., chaperone style, and also targeting intracellular modulators (c-Myc, Pc2, Hif1α). Together, our work is of paramount importance in an informative point of view and practical perspective for progress towards a therapy for treating ADPKD. The sharing of common pathways between AKI and ADPKD paves the way for parallel therapeutic approaches for assured much faster treatment.

5 Abstract Neuroblastoma is one of the most common cancer diseases diagnosed in children. This rapidly growing solid tumor is usually formed by hypoxic areas which arise as a consequence of inefficient and disorganized neovascularization. The cells stressed by hypoxia triggers transcription of many genes necessary for their survival, and conversely stop the production of proteins which are not necessarily needed for the survival in these severe conditions. The adaptation of cells to hypoxic conditions may appear due to the epigenetic regulation of metabolism associated with chromatin remodeling which involves the DNA methylation and also the posttranslational modifications of histones. Among the most important of these, there is the acetylation of lysine residues of histones associated with the DNA strands loosening, facilitated binding of transcription factors and the activation of gene expression. Thus, the first part of this study is concerned with changes in the acetylation of histones H3 and H4 of human neuroblastoma cell lines UKF-NB-3, UKF-NB-4, SH-SY5Y and SK-N-AS, cultured in parallel under standard culture conditions and in the absence of oxygen (hypoxia, 1% O2) for 24 hours, which are studied by Western blot analysis. Thereupon, the activity of histone deacetylases and histonacetyltransferases,...

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma. Patients who fail conventional therapy (~50%) have a poor prognosis and few treatment options. It is essential to understand the underlying biological processes, the progression of the disease, and utilize this information to develop new therapeutics. DLBCL patients with high C-MYC expression have a poor prognosis and new therapeutics for these patients are needed. This thesis describes work testing the hypothesis that JQ1, which can indirectly inhibit C-MYC in some tumors, can be used as an effective treatment for DLBCL. Some tumors have an unknown mechanism causing high C-MYC expression, leading me to investigate the underlying mechanisms. YY1 is a transcriptional regulator of c- Myc and has been implicated in DLBCL and as a potential regulator of the germinal center (GC) reaction. DLBCL arises from GC cells or post-GC cells. I tested the hypothesis that YY1 regulates the GC reaction. SMURF2 is an E3-ubiquitin ligase for YY1 and a tumor suppressor for DLBCL. I was interested in examining the mechanism underlying the suppression of DLBCL by SMURF2 leading to the hypothesis that SMURF2 regulates the GC. This thesis shows JQ1 leads to cell death and cellular senescence in human DLBCL cells. I conclude that BRD4 inhibition by JQ1 or derivatives could provide a new therapeutic avenue for DLBCL patients. I also show loss of YY1 perturbs the GC by decreasing the dark zone and increasing apoptosis. Finally I show modulation of SMURF2 does not affect the GC, suggesting SMURF2 utilizes a different mechanism to act as a tumor suppressor and may not modulate YY1 in the context of the GC.

Overall, this study enhances our understanding of the role of c-Myc activation in chromosome 11 repositioning in mouse PreB v-abl/myc cells and a possible interaction between telomeres, TRF2, and lamin A/C underlying this phenomenon. Additionally, the importance of human 17q25.3 is confirmed as a potential region involved in NSCLC tumorigenesis. A utilizationof the 3D telomeric organization profiles is demonstrated a tendency to categorize NSCLC patients into different prognostic subgroups, underscoring a potential future value of its clinical application. / February 2017