Das Neuroblastom ist eines der häufigsten Tumoren im Kindesalter. Bei Hochrisko-Neuroblastomen weisen etwa 25 % der Patienten eine MYCN-Amplifikation auf. Die Behandlung dieser Patienten bleibt eine Herausforderung. Bei genauerer Betrachtung der amplifizierten Regionen umfasst diese große genomische Bereiche, die nicht nur MYCN, sondern auch Passagiergene und genregulatorische Elemente enthalten. Um MYCN-amplifizierte Neuroblastome zu behandeln, versuchten wir festzustellen, ob Co-Amplifikationen von Passagiergenen kollaterale therapeutische Vulnerabilitäten darstellen könnten. Durch die Analyse von Kopienzahl-Daten von 238 MYCN-amplifizierten Patienten identifizierten wir das DEAD-Box-Helicase-1 (DDX1)-Gen als ein Gen, welches häufig mit MYCN auf dem gleichen genomischen Fragment amplifiziert ist. Die Analyse eines CRISPR-Cas9-Funktionsverlust- Screens aus der Cancer Dependency Map, welche über 700 humanen Krebszelllinien beinhalten, zeigt, dass das Überleben von MYCN-amplifizierten Krebszellen mit DDX1-Co-Amplifikation von der gesteigerten Aktivität des mammalian target of rapamycin complex 1 (mTORC1) abhängt. Interaktionsproteomik identifizierte Dihydrolipoyl-S-Succinyltransferase (DLST), ein Bestandteil des Tricarboxylsäure (TCA)-Zyklusenzyms α-Ketoglutarat-Dehydrogenase (α-KGDH)-Komplexes, als Interaktionspartner von DDX1 in Mitochondrien. Lebendzell- Stoffwechselanalysen legten nahe, dass diese Interaktion die TCA-Aktivität beeinträchtigen und zu einer Anhäufung von α-Ketoglutarat (α-KG) führen kann, indem sie dessen Umwandlung in Succinyl-CoA stört. Die Anhäufung von α-KG verursacht metabolischen Stress und löst Zelltod aus, der durch eine gesteigerte mTORC1-Aktivität in Krebszellen kompensiert wird. Folglich führte die Störung der mTORC1-Funktion zu Zelltod, insbesondere in Zellen mit hoher DDX1-Kopienzahl. So kann die strukturell verknüpfte Co-Amplifikation eines Passagiergens (DDX1) und eines Onkogens (MYCN) auf dem gleichen Amplicon zu kollateralen Vulnerabilitäten bei Neuroblastomen führen. / Neuroblastoma is one of the most common childhood tumors. In high-risk neuroblastoma, around 25% of patients harbor MYCN amplification. Treating neuroblastoma patients with MYCN amplification remains challenging. Taking a closer look at MYCN-amplified regions, DNA amplification encompasses large genomic regions harboring not only MYCN but also containing passenger genes and gene regulatory elements. To treat MYCN-amplified neuroblastoma, we sought to determine whether passenger co-amplifications can create collateral therapeutic vulnerabilities. By analyzing copy number data from 238 MYCN-amplified patients, we identified the DEAD-Box Helicase 1 (DDX1) gene to be frequently co-amplified with MYCN on the same genomic fragment. Analysis of CRISPR-Cas9 loss-of-function screens from the Cancer Dependency Map across over 700 human cancer cell lines revealed that the survival of MYCN-amplified cancer cells with DDX1 co-amplification depends on the enhanced activity of the mammalian target of rapamycin complex 1 (mTORC1). Interaction proteomics identified dihydrolipoamide S-succinyltransferase (DLST), a component of the tricarboxylic acid (TCA) cycle enzyme α-ketoglutarate dehydrogenase (α-KGDH) complex, as an interaction partner of DDX1 in mitochondria. Live-cell metabolomics suggested that this interaction can impair TCA activity and lead to the accumulation of α-ketoglutarate (α-KG) by interfering with its conversion to succinyl-CoA. Accumulation of α-KG, in turn, caused metabolic stress and triggered cell death, which was compensated for by enhanced mTORC1 activity in cancer cells. Consequently, disruption of mTORC1 function resulted in cell death, specifically in cells with an aberrantly high copy number of DDX1. Thus, structurally linked co-amplification of a passenger gene (DDX1) and an oncogene (MYCN) on the same amplicon can result in collateral vulnerabilities in neuroblastoma.

<p dir="ltr">Although guideline-recommended long-term use of adjuvant hormone therapy (HT) is highly effective in improving breast cancer outcomes, empirical HT use is suboptimal and not well-understood. The overall objective of this study was to assess associations between patient characteristics, physician characteristics, hospital characteristics, and community characteristics and use of hormone therapy operationalized as initiation, adherence, and persistence. A retrospective analysis of the Surveillance, Epidemiology, and End Results (SEER) registry linked with Medicare claims was conducted. The full study sample included older women diagnosed with hormone receptor-positive stage I-III breast cancer from 2010 through 2014. Initiation was defined as filling at least one prescription for HT. Adherence was defined as having proportion of days covered (PDC) of 0.80 or more in the first year. Persistence was defined as having no HT discontinuation, i.e., a break of at least 90 continuous days at any point during the study period. Length of persistence was calculated as time from therapy initiation to discontinuation. All analyses were conducted using SAS 9.4. An a priori alpha level of 0.05 was used to determine significance for all the analyses. ICC values were assessed to test for clustering at physician, hospital, and zip code level. A marginal model with clustering at zip code level was used to assess associations between study predictor variables and outcome variables. Logistic Regression models were constructed to assess associations between HT initiation and adherence and study predictors while Cox Proportional Hazards models were constructed to assess associations between HT persistence and study predictor variables. Final models were restricted to the patients who had non-missing provider data (15,014 patients for initiation, 9,949 patients for adherence/persistence). 65.46[64.93, 66.00] % of all candidates initiated HT, first year adherence rate was 76.77[76.17, 77.36] % and overall persistence rate was 64.24[63.55, 64.92]. Initiation of hormone therapy was associated with age, race, marital status, dual eligibility, tumor stage and HER2 status, physician specialty, hospital ownership, and zip code median education level; adherence to hormone therapy was associated with type of hormone therapy, medication switches, HER2 status of tumor, physician specialty, teaching hospital status and zip code median education level; and persistence with hormone therapy was associated with type of hormone therapy, medication switches, physician specialty, hospital physician count and urban/rural residence.</p>

Telomerase is expressed in the majority (>85%) of tumours but not in differentiated normal tissue. This enzyme catalyses the elongation of telomeres – a process critical for continued cell proliferation. Telomerase is a potential novel target for molecularly-targeted radiotherapy (mRT), due to its nuclear localization and expression profile. The radiolabelling of telomerase inhibitors may accelerate and enhance the cytotoxicity of such molecules, as a result of irradiation of the DNA. An oligonucleotide targeting telomerase RNA (hTR), shown to inhibit enzyme activity in vitro, was selected for study. Complementary and non-targeting control oligonucleotides were conjugated to a metal chelator (DTPA) to allow radiolabelling with indium-111. The radioiodination of MST-312, BIBR-1532 and flavonoid-derived small molecule inhibitors of telomerase was also pursued. The inhibitory activity of the candidate molecules was analysed using the telomeric repeat amplification protocol (TRAP). The internalization of inhibitors was assessed by gamma-counting following cell lysis. The clonogenic assay was employed to measure the effect of modified inhibitors on cell survival. Small molecule telomerase inhibitors were modified for labelling with iodine-123, which led to a modest decrease in inhibitory potency, compared to the parent molecules. Radiolabelled small molecules exhibited poor stability and internalization into cancer cells, so were unsuitable for mRT. Modified oligonucleotides potently inhibited telomerase activity, whereas a non-targeting oligonucleotide exhibited no inhibitory activity. Indium-111 radiolabelled oligonucleotides decreased the clonogenic survival of telomerase-positive breast cancer cells but not telomerase-negative cells, in a sequence-specific manner. Accordingly, complementary radiolabelled oligonucleotides were found to induce the DNA damage marker γH2AX. Oligonucleotides localized to nuclear Cajal bodies, the sites of telomerase assembly, in a proportion of cancer cells. Telomerase inhibitors of different classes were radiolabelled with Auger electron-emitting radionuclides, and delivered to cells. Radiolabelled oligonucleotides targeting telomerase significantly reduced the clonogenicity of cancer cells in vitro. This study represents a novel approach for the mRT of telomerase-positive cancers.

The development of targeted therapy has contributed tremendously to the treatment of patients with cancer. The use of highly specific affinity proteins to target cancer cells has become a standard in treatment strategies for several different cancers. In light of this, many cancer cell markers are investigated for their potential use in diagnostics and therapy. One such marker is the human epidermal growth factor receptor 3, HER3. It has been established as an important contributor to many cancer types. The function of HER3 is to relay cell growth signals from outside of the cell to the inside. Interfering with- and inhibit- ing the function of HER3 has emerged as an interesting strategy for cancer therapeutics. The studies presented in this thesis aim to target HER3 with small, engineered affinity domain proteins for therapeutic purposes. Monomeric affibody molecules have previously been engineered to bind and inhibit HER3 in vitro. Due to the relatively low expression of HER3, an increase in valency appears promising to strengthen the therapeutic potential. Affibody molecules targeting the receptor were thus linked to form bivalent and bispecific constructs and evaluated both in vitro and in vivo. In the first study of this thesis affibody molecules specific for HER3 and HER2 were fused to an albumin binding domain to form bivalent and bispecific construct. The constructs inhibited ligand-induced receptor phos- phorylation of both HER2 and HER3 more efficiently than monomeric affibody molecules. A second approach to enhance the potential of affibody molecules in tumor targeting is described in the second study, where monomeric HER3-binding affibody molecules were engineered to increase their affinity for HER3. The resulting variants showed a 20-fold in- creased affinity and higher capacity to inhibit cancer cell growth. Combining the findings of the first two studies, the third study describes the evaluation of a HER3-targeting bivalent affibody construct for potential application as a therapeutic. Here, the bivalent construct inhibited cancer cell growth in vitro and was found to slow down tumor growth in mice, while being well tolerated and showing no visible toxicity. The fourth study built upon these findings and compares a very similar bivalent construct to the clinically-investigated HER3-specific monoclonal antibody seribantumab. The affibody construct showed very comparable efficacy with the antibody in terms of decreasing tumor growth rate and ex- tending mouse survival. Collectively, these works describe for the first time the use of alternative affinity protein constructs with therapeutic potential targeting HER3. / <p>QC 20170330</p>

Cancer involves the dysregulated proliferation and growth of cells throughout the body. C-terminal binding proteins (CtBP) 1 and 2 are transcriptional co-regulators upregulated in several cancers, including breast, colorectal, and ovarian tumors. CtBPs drive oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes. CtBPs encode an intrinsic dehydrogenase activity, utilizing intracellular NADH concentrations and the substrate 4-methylthio-2-oxobutyric acid (MTOB), to regulate the recruitment of transcriptional regulatory complexes. High levels of MTOB inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. While encouraging, a good therapeutic would utilize >100-fold lower concentrations. Therefore, we endeavored to design better CtBP-specific therapeutics. The best of these drugs, 3-Cl and 4-Cl HIPP, exhibit nanomolar enzymatic inhibition and micromolar cytotoxicity and showed that CtBP enzymatic function is subject to allosteric interactions. Additionally, the function of the substrate-binding domain has yet to be examined in context of CtBP’s oncogenic activity. To this end, we created several point mutations in the CtBP substrate-binding pocket and determined key residues for CtBP’s enzymatic activity. We found that a conserved tryptophan in the catalytic domain is imperative for function and unique to CtBPs among dehydrogenases. Knowledge of this and other residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Early work interrogated the importance of these residues in cell migration. Taken together, this work addresses the utility of the CtBP substrate-binding domain as a target for cancer therapeutics.

Cancers represent a group of unprecedented heterogeneous diseases and currently available anti-cancer therapies provide highly variable efficacy with unsatisfactory cure rates. A wide range of proteomic technologies are being used in quest for newer approaches which could significantly contribute to the discovery and development of selective and specific cancer biomarkers for monitoring the disease state and anti-cancer therapy success. Taking into consideration the above aspects, this research was undertaken to study cancer cell proteomes and their changes after anti-cancer treatment with specific focus on: (a) response to conventional anthracycline/anthracenedione drugs with respect to their different clinical efficacy and (b) identification of novel targets for therapy in cancer cells resistant to biological drugs such as inhibitors of (b1) cyclin-dependent kinases and (b2) Aurora kinases. This study identified several interesting key aspects related to the effects of daunorubicin, doxorubicin and mitoxantrone. With the main focus on early time intervals when the influence of apoptosis is minimised, changes common for all three drugs belonging mainly to metabolic and cellular processes were observed. More importantly, significant changes in proteins involved in the generation of precursor...

La biomédecine et la biophotonique sont des champs de recherches en plein expansion qui grandissent à vive allure, constituant un secteur entier d'activités novatrices. Ce secteur, vraiment interdisciplinaire, comprend le développement de nouveaux nanomatériaux, de sources lumineuses et l'élaboration de nouveaux concepts, de dispositifs/équipements pour quantifier la conversion de photons et leurs interactions. L'importance décisive du diagnostic précoce et du traitement individuel des patients exige des thérapies soigneusement ciblées et la capacité de provoquer sélectivement la mort cellulaire des cellules malades. Malgré les progrès spectaculaires réalisés en utilisant les points quantiques ou des molécules biologiques organiques pour l'imagerie biologique et la libération ciblée de médicaments, plusieurs problèmes restent à résoudre : obtenir une sélectivité accrue pour une accumulation spécifique dans les tumeurs et une amélioration de l'efficacité des traitements. D'autres problèmes incluent la cytotoxicité et la génotoxicité, l'élimination lente et la stabilité chimique imparfaite. Des espérances nouvelles sont portées par de nouvelles classes de matériaux inorganiques comme les nanoparticules à base de silicium ou à base de carbone, qui pourraient faire preuves de caractéristiques de stabilité plus prometteuses tant pour le diagnostic médical que pour la thérapie. Pour cette raison, la découverte de nouveaux agents de marquage et de transport de médicaments représente un champ important de la recherche avec un potentiel de croissance renforcé / Biomedicine and biophotonics related businesses are currently growing at a breathtaking pace, thereby comprising one of the fastest growing sectors of innovative economy. This sector is truly interdisciplinary, including, very prominently, the development of novel nanomaterials, light sources, or novel device/equipment concepts to carry out photon conversion or interaction. The great importance of disease diagnosis at a very early stage and of the individual treatment of patients requires a carefully targeted therapy and the ability to induce cell death selectively in diseased cells. Despite the tremendous progress achieved by using quantum dots or organic molecules for bio-imaging and drug delivery, some problems still remain to be solved: increased selectivity for tumor accumulation, and enhancement of treatment efficiency. Other potential problems include cyto- and genotoxicity, slow clearance and low chemical stability. Significant expectations are now related to novel classes of inorganic materials, such as silicon-based or carbon-based nanoparticles, which could exhibit more stable and promising characteristics for both medical diagnostics and therapy. For this reason, new labeling and drug delivery agents for medical application is an important field of research with strongly-growing potential.The 5 types of group IV nanoparticles had been synthesized by various methods. First one is the porous silicon, produced by the electrochemical etching of bulk silicon wafer. That well-known technique gives the material with remarkably bright photoluminescence and the complicated porous structure. The porous silicon particles are the agglomerates of the small silicon crystallites with 3nm size. Second type is 20 nm crystalline silicon particles, produced by the laser ablation of the bulk silicon in water. Those particles have lack of PL under UV excitation, but they can luminesce under 2photon excitation conditions. 3rd type of the particles is the 8 nm nanodiamonds

Gold nanoparticle has emerged as an attractive platform for drug delivery applications by complementing the existing drug delivery carriers. Currently, only a few gold nanoparticle-based anticancer drug delivery systems have been reported, compared to the polymer-based delivery systems. Additionally, there is still a lack of understanding for the behavior and fate of the gold-drug conjugate in the body that further attention is required. The overall goal of this thesis is to investigate the in vivo behavior of colloidal gold nanoparticle and its therapeutic efficacy in an animal model, especially in a drug delivery application. To achieve this goal, we investigated the feasibility of using colloidal gold nanoparticle as an anticancer agent delivery vehicle for treatment of cancer. Then, long-term clearance, toxicity, and biodistribution of colloidal gold nanoparticle were studied to further aid in understanding of using colloidal gold nanoparticle as a drug delivery platform. In particular, two representative sizes of gold nanoparticles, 5nm and 60nm, were investigated for the size effect on the therapeutic efficacy, toxicity, biodistribution, and clearance in cancer nanotherapy. We believe that nanoparticle size plays a critical role in not only delivering the drug to the target site but also determining the in vivo behavior such as biodistribution and clearance. By choosing an appropriate size scale for the system, we successfully used the small-sized gold nanoparticles for drug delivery applications, which also displayed no apparent toxicity with desirable clearance from the biological system. This work is significant by providing an insight on a potential ideal candidate for drug delivery carrier for cancer nanotherapy.

Histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated a wealth of biological effects, including anti-proliferative, anti-inflammatory, anti-parasitic, and cognition-enhancing activities. The recent FDA approvals of the inhibitors SAHA and FK-228 have validated HDACi clinical use in cutaneous T cell lymphoma, while numerous clinical trials are currently ongoing using HDACi against a variety of disease states. While the future of the HDAC field looks increasingly promising, there are lingering issues hindering broader use. Recent data point to dysregulation of specific HDAC isoforms in many disease states. However, most current HDACi are pan-inhibitors, lacking the specificity to target individual isoforms. Adding to this, there are currently 18 identified HDAC isoforms, and most lack a defined crystal structure, further complicating the task of designing isoform-specific inhibitors. Most importantly, HDACi have demonstrated a lack of efficacy against solid tumors in the clinic, a major obstacle to broader use in cancer therapy. Several of these issues could more fully be addressed through specific targeting of HDACi, and could bring HDACi into wider and more efficacious pharmaceutical use. Targeting the specific tissue or organelle where HDAC dysregulation occurs could confer greater efficacy in vivo. To this end, we have created four classes of compounds: (1) aryltriazolyl HDACi that potently inhibit HDAC activity and prostate cancer cell growth, (2) dual-targeted inhibitors of Topoisomerase II and HDAC and (3) dual-targeted inhibitors of Topoisomerase I and HDAC, both of which have potent inhibition against both target enzymes as well as cancer cell lines, and finally (4) macrocyclic HDACi that potently inhibit the growth of lung cancer cell lines and preferentially target lung tissue in vivo.

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