Advances in the Development of Shape Similarity Methods and Their Application in Drug Discovery

Molecular similarity is a key concept in drug discovery. It is based on the assumption that structurally similar molecules frequently have similar properties. Assessment of similarity between small molecules has been highly effective in the discovery and development of various drugs. Especially, two-dimensional (2D) similarity approaches have been quite popular due to their simplicity, accuracy and efficiency. Recently, the focus has been shifted toward the development of methods involving the representation and comparison of three-dimensional (3D) conformation of small molecules. Among the 3D similarity methods, evaluation of shape similarity is now gaining attention for its application not only in virtual screening but also in molecular target prediction, drug repurposing and scaffold hopping. A wide range of methods have been developed to describe molecular shape and to determine the shape similarity between small molecules. The most widely used methods include atom distance-based methods, surface-based approaches such as spherical harmonics and 3D Zernike descriptors, atom-centered Gaussian overlay based representations. Several of these methods demonstrated excellent virtual screening performance not only retrospectively but also prospectively. In addition to methods assessing the similarity between small molecules, shape similarity approaches have been developed to compare shapes of protein structures and binding pockets. Additionally, shape comparisons between atomic models and 3D density maps allowed the fitting of atomic models into cryo-electron microscopy maps. This review aims to summarize the methodological advances in shape similarity assessment highlighting advantages, disadvantages and their application in drug discovery

Drugs targeting the retinoblastoma binding protein 6 (RBBP6): "the collision of computers and biochemistry"

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the Master of Science degree. 2 November 2017.Screening methodologies have identified specific targets that could serve as potential therapeutic markers in cancer drug design, and the Retinoblastoma binding protein 6 (RBBP6) which is predominately expressed in lung and breast cancers is one critical protein identified. This study seeks to understand the 3D structure of RBBP6 domains, with emphasis on cancer. Three of these domains have been studied in this project, i.e. the Domain With No Name (DWNN), RING Finger, and the p53-binding domain. The ubiquitin-like structure of the DWNN has implicated this domain as a ubiquitin-like modifier of other proteins such as p53, whilst the RING Finger domain has intrinsic E3 Ligase activity like MDM2 the prototypical negative regulator of p53. The DWNN and RING Finger domains have resolved solution NMR structures, whilst the p53-binding domain has none. Thus, the first initiative undertaken was to model the RBBP6 p53-binding domain using I-TASSER and eThread-Modeller web-severs. Our results demonstrated that this domain mainly constitutes of alpha-helices and loop structures. Structural quality validations of both I-TASSER and eThread-Modeller models were further assessed using Swiss-Model and ProSA (Protein structure analysis) web-servers. Analyses were focussed on specific statistical parameters (Anolea, DFire, QMEAN, ProCheck and the ProSA Z-score). Results from these analyses show that the first I-TASSER model is the best possible representation of the RBBP6 p53-binding domain depicting minimal deviation from native state. Furthermore, screening and docking studies were performed using Schrödinger-Maestro v10.7: Glide SP and drug-like molecules that would potentially serve as agonist or antagonist of RBBP6 were identified.MT 201

NMR as a “gold standard” method in drug design and discovery

Studying disease models at the molecular level is vital for drug development in order to improve treatment and prevent a wide range of human pathologies. Microbial infections are still a major challenge because pathogens rapidly and continually evolve developing drug resistance. Cancer cells also change genetically, and current therapeutic techniques may be (or may become) ineffective in many cases. The pathology of many neurological diseases remains an enigma, and the exact etiology and underlying mechanisms are still largely unknown. Viral infections spread and develop much more quickly than does the corresponding research needed to prevent and combat these infections; the present and most relevant outbreak of SARS-CoV-2, which originated in Wuhan, China, illustrates the critical and immediate need to improve drug design and development techniques. Modern day drug discovery is a time-consuming, expensive process. Each new drug takes in excess of 10 years to develop and costs on average more than a billion US dollars. This demonstrates the need of a complete redesign or novel strategies. Nuclear Magnetic Resonance (NMR) has played a critical role in drug discovery ever since its introduction several decades ago. In just three decades, NMR has become a “gold standard” platform technology in medical and pharmacology studies. In this review, we present the major applications of NMR spectroscopy in medical drug discovery and development. The basic concepts, theories, and applications of the most commonly used NMR techniques are presented. We also summarize the advantages and limitations of the primary NMR methods in drug development

UFSRAT:Ultra-fast shape recognition with atom types -The discovery of novel bioactive small molecular scaffolds for FKBP12 and 11βHSD1

MOTIVATION:Using molecular similarity to discover bioactive small molecules with novel chemical scaffolds can be computationally demanding. We describe Ultra-fast Shape Recognition with Atom Types (UFSRAT), an efficient algorithm that considers both the 3D distribution (shape) and electrostatics of atoms to score and retrieve molecules capable of making similar interactions to those of the supplied query. RESULTS:Computational optimization and pre-calculation of molecular descriptors enables a query molecule to be run against a database containing 3.8 million molecules and results returned in under 10 seconds on modest hardware. UFSRAT has been used in pipelines to identify bioactive molecules for two clinically relevant drug targets; FK506-Binding Protein 12 and 11β-hydroxysteroid dehydrogenase type 1. In the case of FK506-Binding Protein 12, UFSRAT was used as the first step in a structure-based virtual screening pipeline, yielding many actives, of which the most active shows a KD, app of 281 µM and contains a substructure present in the query compound. Success was also achieved running solely the UFSRAT technique to identify new actives for 11β-hydroxysteroid dehydrogenase type 1, for which the most active displays an IC50 of 67 nM in a cell based assay and contains a substructure radically different to the query. This demonstrates the valuable ability of the UFSRAT algorithm to perform scaffold hops. AVAILABILITY AND IMPLEMENTATION:A web-based implementation of the algorithm is freely available at http://opus.bch.ed.ac.uk/ufsrat/

Das MYCN-Onkogen als Marker für minimale Resterkrankung und therapeutisches Ziel beim Neuroblastom

Neuroblastoma, the most common extracranial solid childhood cancer, arises from precursors of the developing sympathetic nervous system. MYCN oncogene amplification is a determinant of high risk and occurs in ~25% of neuroblastomas. Despite intensive treatment, more than half these patients succumb to their disease, implying persistence of therapy-resistant MYCN-amplified minimal residual neuroblastoma cells. This thesis proposes a comprehensive concept for the specific diagnostic detection of the MYCN amplicon and evaluates new treatment options for MYCN-amplified neuroblastoma. Disease-relevant nucleotide changes, structural gene rearrangements and copy number alterations were detected in tumor material by next-generation sequencing of a customized hybrid capture-based targeted panel. Unique MYCN amplicon breakpoints in the rearranged gene constitute a target sequence for a personalized minimal residual disease (MRD) PCR diagnostic. MYCN amplicon breakpoints in neuroblastoma cell lines and tumors were identified and recovered by individual, semi-quantitative PCR assays and Sanger sequencing. The assay was further developed for highly sensitive, real-time quantitative and droplet digital PCR detection for selected MYCN breakpoints in cell lines. MRD level detected in bone marrow aspirates collected during therapy outlined different disease courses in patients, including MRD persistence until relapse and good response to the first treatment course. Combining multi-agent chemotherapy in current high-risk protocols with indirect MYCN inhibitors provides a potential route to improve poor cure rates for MYCN-amplified neuroblastomas. Different hyperactive biological networks in MYCN-amplified neuroblastoma were tackled using small molecule inhibitors of the bromodomain and extra-terminal (BET) domain-containing protein BRD4, phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (PLK1). BET (JQ1, OTX015 and TEN-010) and kinase (alpelisib, volasertib and rigosertib) inhibitors demonstrated anti-cancer activity by diminishing viability in cell line-based drug screens at nanomolar to low micromolar concentrations. Rigosertib treatment altered PLK1 and PI3K signaling and strongly impaired the cellular ability for wound healing and colony formation. In line with in vitro observations, rigosertib reduced tumor growth in patient-derived neuroblastoma xenografts in mice. Combining OTX015 and volasertib produced synergistic anti-tumor responses in two MYCN-amplified neuroblastoma cell lines. To prevent MYCN-driven proliferation of tumor cells, further indirect MYCN targets are also being considered. This is exemplified by a substrate of PLK1, ASPM, which is elevated in MYCN-amplified primary neuroblastomas. Knockdown of ASPM, a microtubule-associated protein involved in mitotic spindle assembly, in MYCN-amplified neuroblastoma cell lines reduced viability and proliferation, accompanying a neuronal differentiation phenotype with neurite-like outgrowth, cytoskeletal changes and increased expression of differentiation markers. This study presents clinical implementable molecular diagnostics to pinpoint unique MYCN-amplified neuroblastoma cells within non-invasively accessible biopsy material, and proposes indirect small molecule-based MYCN therapies and potentially new drug targets for a personalized treatment.Das Neuroblastom, der häufigste extrakranielle solide Krebs im Kindesalter, entsteht aus Vorläuferzellen des sich entwickelnden sympathischen Nervensystems. Eine Amplifikation des MYCN-Onkogens ist ein bestimmender Faktor für ein hohes Risiko und tritt bei ~25% der Neuroblastome auf. Trotz intensiver Behandlung erliegt mehr als die Hälfte dieser Patienten ihrer Krankheit, was die Persistenz therapieresistenter, MYCN-amplifizierter minimaler Restneuroblastomzellen impliziert. Diese Arbeit stellt ein umfassendes Konzept für den spezifischen, diagnostischen Nachweis des MYCN-Amplikons vor und evaluiert neue Behandlungsoptionen für MYCN-amplifizierte Neuroblastome. Krankheitsrelevante Nukleotidveränderungen, strukturelle Genrearrangements und Kopienzahl-veränderungen wurden im Tumormaterial mit Hilfe eines maßgeschneiderten, zielgerichteten hybrid-capture-basierten Next Generation Sequencing (NGS) Assays nachgewiesen. Einzigartige MYCN-Amplikon-Bruchpunkte im rearrangierten Gen stellen eine Zielsequenz für eine personalisierte PCR-Diagnostik der minimalen Resterkrankung (MRD) dar. MYCN-Amplikon-Bruchpunkte in Neuroblastom-Zelllinien und Tumoren wurden durch individuelle, semi-quantitative PCR-Assays und Sanger Sequenzierung identifiziert und wiedererkannt. Der Assay wurde für den hochsensitiven, quantitativen Echtzeit- und digitalen Tröpfchen-PCR-Nachweis für ausgewählte MYCN-Bruchpunkte in Zelllinien weiterentwickelt. Die MRD Level, die in den während der Therapie gesammelten Knochenmarkaspiraten nachgewiesen wurden, skizzierten die verschiedenen Krankheitsverläufe bei den Patienten, einschließlich der MRD-Persistenz bis zum Rezidiv und des guten Ansprechens auf den ersten Behandlungsabschnitt. Die Kombination der Multi-Wirkstoff-Chemotherapie in den aktuellen Hochrisikoprotokollen mit indirekten MYCN-Inhibitoren stellt einen möglichen Weg dar, die schlechten Heilungsraten für MYCN-amplifizierte Neuroblastome zu verbessern. Verschiedene, hyperaktive biologische Netzwerke in MYCN-amplifizierten Neuroblastomen wurden mit niedermolekularen Inhibitoren der Bromdomäne und des extra-terminalen (BET) domänenhaltigen Proteins BRD4, der Phosphoinositid-3-Kinase (PI3K) und der polo-ähnlichen Kinase 1 (PLK1) behandelt. BET (JQ1, OTX015 und TEN-010) und Kinase-Inhibitoren (Alpelisib, Volasertib und Rigosertib) zeigten eine krebshemmende Wirkung, indem sie die Viabilität in zelllinienbasierten Wirkstoff-Screens bei nanomolaren bis niedrigen mikromolaren Konzentrationen verminderten. Die Behandlung mit Rigosertib veränderte die PLK1- und PI3K-Signalübertragung und beeinträchtigte die zelluläre Fähigkeit zur Wundheilung und Koloniebildung stark. In Übereinstimmung mit In-vitro-Beobachtungen reduzierte Rigosertib das Tumorwachstum in von Patienten stammenden Neuroblastom-Xenografts bei Mäusen. Die Kombination von OTX015 und Rigosertib erzeugte synergistische antitumorale Aktivität in zwei MYCN-amplifizierten Neuroblastom-Zelllinien. Um die MYCN-gesteuerte Proliferation von Tumorzellen zu verhindern, werden weitere indirekte MYCN-Targets in Betracht gezogen. Ein Beispiel hierfür ist ein Substrat von PLK1, ASPM, das in MYCN-amplifizierten, primären Neuroblastomen erhöht ist. Das Herunterregulieren von ASPM, einem Mikrotubuli-assoziierten Protein, das an der mitotischen Spindelanordnung beteiligt ist, führte in MYCN-amplifizierten Neuroblastom-Zelllinien zu einer verminderten Viabilität und Proliferation, was mit einem neuronalen Differenzierungsphänotyp mit neuritenartigem Auswuchs, zytoskelettalen Veränderungen und erhöhter Expression von Differenzierungsmarkern einherging. Diese Studie stellt eine klinisch umsetzbare, molekulare Diagnostik vor, um einzigartige MYCN-amplifizierte Neuroblastomzellen in nicht-invasiv zugänglichem Biopsiematerial zu detektieren, und schlägt indirekte, niedermolekular-basierende MYCN-Therapien und potenziell neue Zielmoleküle für eine personalisierte Krebsbehandlung vor

Urological Cancer 2021

Cancer of the urological sphere is a disease continuously increasing in numbers in the statistics of tumor malignancies in Western countries. Although this fact is mainly due to the contemporary increase of life expectancy of the people in these geographic areas, many other factors do contribute as well to this growth. Urological cancer is a complex and varied disease of different organs and mainly affects the male population. In fact, kidney, prostate, and bladder cancer are regularly included in the top-ten list of the most frequent neoplasms in males in most statistics. The female population, however, has also increasingly found itself affected by renal and bladder cancer in the last decade. Considering these altogether, urological cancer is a problem of major concern in developed societies. This Topic Issue of Cancers intends to shed some light into the complexity of this field and will consider all useful and appropriate contributions that scientists and clinicians may provide to improve urological cancer knowledge for patients’ benefit. The precise identification of the molecular routes involved, the diagnostic pathological criteria in the grey zones, the dilemma of T1G3 management, and the possible treatment options between superficial, nonmuscle-invasive and muscle-invasive diseases will be particularly welcomed in this Issue

Functionally Relevant Macromolecular Interactions of Disordered Proteins

Disordered proteins are relatively recent newcomers in protein science. They were first described in detail by Wright and Dyson, in their J. Mol. Biol. paper in 1999. First, it was generally thought for more than a decade that disordered proteins or disordered parts of proteins have different amino acid compositions than folded proteins, and various prediction methods were developed based on this principle. These methods were suitable for distinguishing between the disordered (unstructured) and structured proteins known at that time. In addition, they could predict the site where a folded protein binds to the disordered part of a protein, shaping the latter into a well-defined 3D structure. Recently, however, evidence has emerged for a new type of disordered protein family whose members can undergo coupled folding and binding without the involvement of any folded proteins. Instead, they interact with each other, stabilizing their structure via “mutual synergistic folding” and, surprisingly, they exhibit the same residue composition as the folded protein. Increasingly more examples have been found where disordered proteins interact with non-protein macromolecules, adding to the already large variety of protein–protein interactions. There is also a very new phenomenon when proteins are involved in phase separation, which can represent a weak but functionally important macromolecular interaction. These phenomena are presented and discussed in the chapters of this book

Novel Treatment Strategies for Glioblastoma : Therapeutic Potential of Phenolic Derivatives and Orphan G-protein Coupled Receptor Ligand

Glioblastoma (GBM) is a prevalent brain tumor with a high mortality rate worldwide. Although many efforts have been made to explore potential therapeutic strategies, the treatment for GBM remains obscure. Phenolic compounds have received considerable attention in cancer biology owing to their therapeutic applications. Indeed, phenolic compounds with alkylaminophenol core have been approved by the U.S. Food and Drug Administration to treat several diseases. The present study aims at exploring the anti-tumor activity of three different alkylaminophenols, namely 2-((3,4-dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP), 2- ((1,2,3,4-tetrahydroquinolin-1-yl)(4-methoxyphenyl)methyl)phenol (THMPP), and N-(2-hydroxy-5-nitrophenyl(4'-methylphenyl)methyl)indoline (HNPMI) against GBM cell growth and proliferation. Our results reveal that THTMP has potent inhibitory activity against GBM cells and could target GBM cancer stem cells (GSCs) via arresting the cell cycle at the G1/S phase and inducing reactive oxygen species- mediated apoptosis. Furthermore, THTMP could target GSCs by modulating epidermal growth factor receptor (EGFR) and GSC signaling pathways. In addition, the G-protein coupled receptor 17 (GPR17) targeted signaling pathway has also grasped attention in the treatment of GBM. Our preliminary study has revealed that GPR17 interaction with its ligand, 2-[[5-(3-morpholin-4-ylsulfonylphenyl)-4-[4- (trifluoromethoxy) phenyl]-1,2,4-triazol-3-yl] sulfanyl]-N-(4-propan-2ylphenyl) acetamide (namely, T0510.3657 or T0), could potentially regulate the intracellular signaling communication of GBM. We have identified that T0 downregulates the concentration of adenosine 3',5'-cyclic monophosphate (cAMP) through activating GPR17 signaling. Here, we have characterized the effect of T0 and the underlying molecular mechanism in inducing GBM cell death. Towards combinatorial drug development, the lead phenolic compound and the GPR17 ligand were used to investigate the anti-cancer effect against GBM. The results show that THTMP has a higher synergistic effect when combined with T0 than the temozolomide (TMZ) in inducing GBM cell death. Furthermore, this study reveals that combining THTMP with T0 would increase the inhibitory effect against mesenchymal GBM cells compared to a single THTMP/T0/TMZ treatment. In addition, the combination THTMP+T0 could decrease the migration, invasion, and colony formation ability of glioblastoma cells. The combination also has the ability to arrest the cell cycle at the S phase as well as to induce ROS-, caspase- and mitogen-activated protein kinase (MAPK)-mediated apoptosis. The activation of intrinsic apoptosis is found to be regulated by XIAP, p53, cIAP-1, cIAP-2, HSP27, cytochrome c, cleaved caspases-3, and Bcl-2. The combinatorial drug treatment shows the promising anti-tumor property in the GBM xenograft model since it can reduce tumor volume. Our findings imply the coordinated administration of THTMP and T0 as a potential therapy that can be used for GBM treatment

Hypoxia-Induced Radioresistance in Human Non-Small Cell Lung Carcinoma Cell Lines

Lung cancer accounts for 25 % of cancer-related deaths. Non-small cell lung carcinoma (NSCLC) constitutes 85 % of all lung cancers. Radiotherapy is used in treatment of over half of lung cancer patients. Tumor hypoxia is associated with treatment resistance particularly in the context of radiotherapy. Targeting tumor hypoxia to increase radiotherapy efficacy has met limited success clinically with no measurable mortality benefit. High linear energy transfer (LET) carbon ions are being used increasingly in cancer clinical trials and have the theoretical advantage of being less sensitive to the influence of oxygen. Studying DNA damage response (DDR) to low- (X-rays) and high-LET ionizing radiation under hypoxia may help identify molecular processes that can be potentially targeted therapeutically to overcome hypoxia-induced radioresistance. Additionally, tumor cells often experience reversible hypoxia due to tumor shrinkage secondary to treatment, neo-angiogenesis as well as intermittent vasospasm of feeding vessels. Thus, impact of reoxygenation on radioresistance also warrants greater understanding. The Nuclear Factor Kappa B (NF-kB) pathway is associated with cellular inflammatory response to stressors like ionizing radiation and hypoxia and has been associated with enhanced cell survival. However, the role of NF-kB pathway in hypoxia-induced radioresistance remains elusive. Therefore, the radioresistance of NSCLC cells was evaluated under continuous hypoxia and following reoxygenation by performing clonogenic assays following irradiation with the objective of correlating hypoxia induced radioresistance in NSCLC cells to DDR in terms of DNA double strand break (DSB) induction, DSB repair, cell cycle progression as well as activation of pro-survival NF-kB pathway. A549 (p53-wt) and H358 (p53-null) NSCLC cell lines were incubated after seeding for 48 h under hypoxia (0.1 % and 1 % O2) and normoxia (20 % O2) and irradiated using X-rays (200 KeV) and carbon ions (on target energy 25.7 MeV/nucleon). Following irradiation, hypoxic cells were either allowed to reoxygenate (transient hypoxia) or kept hypoxic (continuous hypoxia) till end of experiments. Radioresistance was evaluated in normoxic, continuously hypoxic and transiently hypoxic cells using Puck’s colony forming ability (CFA) assay. DDR four hours following irradiation was assessed at transcriptional level in terms of cell cycle modulation, DNA DSB repair and activation of pro-survival NF-kB pathway by carrying out RNA sequencing and differential expression analysis of relevant genes in continuously and transiently hypoxic cells in comparison to normoxic controls. Cell cycle progression was determined by flow cytometry of cells after staining their nuclei with 4’,6’-diamidino-2-phenylindole (DAPI). DSB induction and repair was assessed using gamma H2AX immunofluorescence microscopy and NF-kB pathway activation was evaluated by p65 (NF-kB subunit) nuclear translocation using p65 immunofluorescence microscopy, and by measuring production of NF-kB target proteins, interleukin (IL) 6 and IL-8. CFA assays revealed that hypoxic cells were more radioresistant compared to normoxic controls when they were given 24 h to repair (late plating) following both X-rays and carbon ions exposure. Radioresistance was higher at 0.1 % O2 compared 1 % O2. Continuously hypoxic cells were more radiosensitive compared to normoxic controls when they were immediately re-seeded for growth of colonies following irradiation (immediate plating). This radiosensitivity was reversed if hypoxic cells were reoxygenated following irradiation (transient hypoxia). Carbon ions had a greater relative biological effectiveness (RBE) in killing hypoxic cells compared to X-rays. Cell cycle progression under hypoxia following both X-rays and carbon ions exposure in A549 and H358 cell lines was slowed as indicated by a greater proportion of cells in G1 phase and smaller population of cells in G2 phase compared to normoxic controls. Phase redistribution was similar at 0.1 % and 1 % O2. Differential expression analysis of cell cycle genes revealed weak transcriptional regulation in both cell lines indicating importance of post-translational cell cycle regulation in response to irradiation under hypoxia. Reoxygenation did not affect cell cycle phase distribution in first 24 h. DSB induction assessment based on H2AX foci count 1 h after irradiation was lower under continuous and transient hypoxia in case of X-rays exposure but not in case of carbon ions exposure. No residual DSBs were observed at a dose of 2 Gy X-rays but DSBs induced by carbon ions took longer to resolve compared to those caused by X-rays. Differential expression analysis of DSB repair genes was unremarkable but that for NF-B target genes showed overexpression of several pro-survival and pro-proliferation genes under hypoxia. The gene expression signature of both cell lines was unique with minimal overlap. Gene expression signature also varied following X-rays and carbon ions exposure. In case of H358 cells, reoxygenation resulted in transcriptional regulation of several genes not regulated under continuous hypoxia. IL-6 (following carbon ions exposure) and IL-8 (following both X-rays and carbon ions exposure) were upregulated in A549 cells while in H358 cells, only IL-8 was upregulated upon reoxygenation. Nuclear translocation of cytosolic p65 was found to occur earlier (at 2 h vs. 6 h) in A549 and H358 cells under continuous hypoxia (1 % O2) following both X-rays and carbon ion exposure compared to normoxia. Reoxygenation had a minimal effect on p65 nuclear translocation in A549 cells. In H358 cells, p65 nuclear localization increased in response to reoxygenation but was not affected by irradiation. Both IL-6 and IL-8 secretion by A549 cells was amplified under hypoxia regardless of reoxygenation and irradiation resulted in its further increase. IL-8 secretion by H358 cells was increased under both continuous and transient hypoxia but irradiation further increased its production only under continuous hypoxia. Hypoxia-induced radioresistance in continuously and transiently hypoxic A549 cells following X-rays and carbon ion exposure was found to be associated with cell cycle phase redistribution toward the radioresistant G1 phase, lesser DSB induction, earlier NF-B activation, greater NF-kB target gene expression and higher NF-B target protein synthesis and secretion. The same was true in case of continuously hypoxic H358 cells following X-rays exposure. However, transiently hypoxic H358 cells behaved differently: reoxygenation increased basal p65 nuclear translocation and IL-8 secretion but irradiation did not lead to further increase in p65 nuclear intensity and IL-8 secretion. Hypoxia-induced radioresistance effects faster growing cells (A549) more than less rapidly dividing cells (H358). Reoxygenation does not alter effects of hypoxia on cell survival, DNA damage and cell cycle but it does affect both cell lines differently in terms of NF-kB pathway activation and transcription of its target genes. While this work does not establish a causal relationship between NF-kB activation and radioresistance seen in hypoxic NSCLC cells, the association does show promise for further investigation into NF-kB as a potential molecular target for therapy in NSCLC. Although NF-kB activation is seen following high LET radiation exposure as well, hypoxic cells are more radiosensitive to carbon ions compared to low LET X-rays. Moreover, IL-6 and IL-8 secretion may serve as potential prognostic indicators of radioresistance

Into the blue...Using mouse models to uncover genes driving tumorigenesis and therapy resistance in human breast cancer

To improve cancer treatments, personalized medicine approaches have aimed to identify exactly which mutations are driving tumor development in a given patient and specifically target these mutations using precision therapies. However, one of the main challenges of this approach is identifying which mutations are true drivers, as tumors typically contain many additional passenger mutations that do not actually contribute to tumor development. Besides this, many patients often relapse after prolonged treatment due to the emergence of acquired resistance, limiting the clinical effectiveness of targeted treatments. In this thesis, we focussed on using genetically engineered mouse models to identify candidate cancer genes and therapy resistance mechanisms in two different breast cancers: invasive lobular carcinoma (ILC) and triple-negative breast cancer (TNBC). For ILC, we used transposon-based insertional mutagenesis (TIM) to uncover several novel cancer genes driving ILC development. Besides this, we also developed a novel computational approach (IM-Fusion) for improving the discovery of cancer genes from TIM screens and explored mechanisms of resistance in Fgfr2-driven ILC. For TNBC, we used CRISPR-based iterative mouse modeling combined with comparative oncogenomics to identify novel drivers of BRCA1-deficient TNBC. Finally, using combined in-vivo/in-vitro screens, we identified Parg as a driver of treatment resistance in BRCA2-deficient TNBC. Divisions of Molecular Pathology and Molecular Carcinogenesis, Netherlands Cancer InstituteToxicolog