The introduction of an increasing number of individualized molecular targeted therapies into clinical routine mirrors their importance in modern cancer prevention and treatment. Well-known examples for targeted agents are the monoclonal antibody trastuzumab and the selective estrogen receptor modulator tamoxifen. The identification of an unaltered gene in tumor tissue in colon cancer (KRAS) is a predictor for the patient’s response to targeted therapy with a monoclonal antibody (cetuximab). Targeted therapy for hereditary breast and ovarian cancer has become a reality with the approval of olaparib for platin-sensitive late relapsed BRCA-associated ovarian cancer in December 2014. This manuscript reviews the status quo of poly-ADP-ribose polymerase inhibitors (PARPi) in the therapy of breast and ovarian cancer as well as the struggle for carboplatin as a potential standard of care for triple-negative and, in particular, BRCA-associated breast cancer. Details of the mechanism of action with information on tumor development are provided, and an outlook for further relevant research is given. The efficacy of agents against molecular targets together with the identification of an increasing number of cancer-associated genes will open the floodgates to a new era of treatment decision-making based on molecular tumor profiles. Current clinical trials involving patients with BRCA-associated cancer explore the efficacy of the molecular targeted therapeutics platinum and PARPi.

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Zelluläre Seneszenz wird als terminaler Zellzyklusarrest definiert, der mit dem Altern und funktionellen Verlust von Geweben verknüpft ist. Eine Seneszenzreaktion wird ebenso durch Onkogene und zytotoxischen Stress verursacht. Die Ausführung des Seneszenzprogramms wird durch eine zeitlich hochdynamische Aktivität von Transkriptionsfaktoren (TF) bedingt. Interessanterweise kann die Zelllinienzugehörigkeit einer Zelle durch die Expression von linien-aberranten TF überschrieben werden. Die vorliegende Arbeit untersucht Chemotherapie-induzierte Seneszenz (TIS) in Bcl-2 überexprimierenden, deshalb vor Apoptose geschützten, murinen Eµ-Myc B-Zell Lymphomen in An- oder Abwesenheit der Seneszenz-essentiellen Histonmethyltransferase Suv39h1. Analysen auf Transkriptom- und auf Proteinebene ergeben dabei, dass in einer Seneszenz-spezifischen Weise die TF AP-1, PU.1 und C/EBPβ induziert werden, welche normalerweise für die Funktion und Entwicklung myeloischer Zelllinien bedeutend sind. Dementsprechend korreliert der Seneszenzzustand mit Transkripten, Oberflächenmarkern und einer enzymatischen Funktion der myeloischen Linie. Indem die identifizierten TFs heruntergeschaltet oder überexprimiert werden, wird ihre direkte Beteiligung an der Linienuntreue der TIS Lymphome demonstriert. TIS-Kapazität wird als für den Erfolg von Krebstherapie günstige Eigenschaft betrachtet, da sie zu einem Wachstumsblock führt. Nichtsdestotrotz können sich verweilende TIS Zellen krebsbiologisch auch nachteilig auswirken. Anhand von murinen und humanen, klinisch annotieren Transkriptomdatensätzen kann hier in beiden Spezies ein myeloisch verschobenes, Linienuntreue anzeigendes Genexpressionsprofil mit einer besseren Überlebensprognose korreliert werden. Die vorliegenden Befunde legen nahe, dass die Modulation von TF Aktivitäten in Seneszenz einen potentiellen therapeutischen Angriffspunkt darstellt, um den für den Therapieerfolg nützlichen Zweig des TIS Phänotyps zu befördern. / Cellular senescence is regarded as an irreversible cell cycle arrest associated with tissue aging and its functional decline. A senescence response is also evoked by oncogenic and cytotoxic stress. The execution of the senescence program relies on a highly dynamic sequence of transcription factor (TF) activities. Interestingly, cell lineage commitment can be overridden by the expression of lineage-aberrant TFs. This thesis examines chemotherapy-induced senescence (TIS) in Bcl-2 overexpressing, thus apoptosis-protected, murine Eμ-Myc B-cell lymphomas with or without the senescence-essential histone methyltransferase Suv39h1. Transcriptome as well as protein level analyses reveal senescence-specific induction of the TFs AP-1, PU.1 and C/EBPβ which are typically crucial for myeloid lineage commitment and function. Correspondingly, the senescent state associates with myeloid lineage transcripts, surface markers and enzymatic function, reminiscent of, but not equal to a transdifferentiation phenotype. By knocking down and overexpressing the identified TFs, we demonstrate their direct involvement in the lineage infidelity of TIS lymphomas. TIS-capacity is viewed as beneficial to cancer therapy outcome due to its block on proliferation. However, lingering TIS cells can also be detrimental due to the acquisition of latent stemness properties or tumor-protective remodeling of their microenvironment. By interrogating murine and human, clinical course-annotated transcriptome data sets, an association between a myeloid-skewed, lineage infidelity indicating gene expression profile and better tumor prognosis is established in both species. The presented findings suggest that modulation of the senescent TF activities could be therapeutically exploited to foster the cancer patient-beneficial branch of the TIS phenotype.

Error-free DNA replication is paramount to maintaining genomic integrity. Despite being highly regulated, the process of DNA replication is often challenged by various intrinsic and extrinsic sources of replication stress. Failure to maintain the DNA replication quality reduces genomic stability, cell survival and results in diseases, such as cancer. Thus, cells rely on the replication stress response that detects perturbations in DNA replication and pauses or arrests cellular replication. Similar to other intrinsic replication obstacles, oncogene expression also induces the replication stress response that acts as a barrier to cancer, thereby mystifying how cancer develops. Here, we demonstrate that oncogene expression, similar to other replication stress inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness unless counteracted by translesion synthesis (TLS). Moreover, we find that TLS subverts the replication stress response in a wide range of cancer cell lines indicating that TLS is a previously unappreciated and unique cancer vulnerability. Mechanistically, we reveal that upon replication stress, TLS restricts replication fork slowing, reversal, and fork degradation, while maintaining continuous replication. Furthermore, we demonstrate that a small molecule inhibitor targeting the TLS factor, REV1, not only disrupts DNA replication and cancer cell fitness, but also synergizes with other therapies that induce replication gaps. Thus, our study places TLS at the center of cancer cell fitness as a necessary adaptation to overcome replication stress.

Established biopharmaceuticals such as antibodies and derivatives thereof are relatively large. In cancer therapy, this creates a steep drug concentration gradient within tumors, leaving cells far from blood vessels effectively untreated. Continuous pseudo treatments should foster the development of drug resistance and might lead to eventual disease relapse. Drug concentration gradients can be operationalized as tissue penetration efficiencies, which are functions of molecular size. However, small particles are also subject to potent renal clearance, collapsing the therapeutic window beyond clinical applications. In this master’s thesis, spatial bispecificity was engineered into a single albumin binding domain (ABD). Resulting ABD derived affinity proteins (ADAPTs) are saved from urinary excretion by the grace of HSA, but in the more static microenvironment of tumors, following HSA dissociation, they are capable of tissue penetration efficiencies bestowed only upon smaller particles. To this end, phage display was used to raise ADAPTs against the cancer associated proteins human epidermal growth factor receptor 2 (HER2) and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), but also the inflammation marker C-reactive protein (CRP). Via Sanger sequencing, 9 variants were picked for protein production and characterization, among which two spatially bispecific binders were found. ADAPTs were also evaluated for aggregation tendencies, structural conformity to library design, and thermal stability. One ADAPT, binding HER2, passed all tests of initial characterizations. Deep sequencing was used to analyze selection output, from which many more binders should be screened in future experiments. / Etablerade bioläkemedel liksom antikroppar och deras derivat är relativt stora protein. Som cancerterapeutiska skapar de således branta koncentrationsgradienter utgående från tumörpenetrerande blodkärl. Detta riskerar att lämna vissa cancerceller utanför det terapeutiska fönstret. Det svaga selektionstryck som således verkar i tumörperiferin fostrar cancerceller till att utveckla resistens mot detsamma. Koncentrationsgradienten beror på proteinets vävnadspenetrarande förmåga, vilken är en funktion av proteinets storlek. Mindre proteiner borde därmed lättare ackumuleras i hela tumören och förebygga resistensutveckling. Problemet med små proteiner är deras mycket korta halveringstid i serum, en följd av relativt obehindrad filtrering ut i urinen via njurarna. I det här examensarbetet utvecklades rumsbispecifika bindare av cancerassocierade protein med hjälp av fagdisplayselektioner från ett proteinbibliotek baserat på en enda albuminbindande domän (ABD). Resulterande ABD deriverade affinitetsprotein (ADAPT) undkommer ovan nämnda filtrering tack vare sin naturligt starka interaktion med humant serumalbumin (HSA). I den mer långsamt flödande tumörmikromiljön tillåts ADAPTerna efter albumindissociation sedan utöva en bland bioläkemedel överlägsen vävnadspenetration. Tre parallella selektionsspår utfördes mot de cancerassocierade målproteinerna human epidermal growth factor receptor 2 (HER2) och carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) samt den utsöndrade inflammationsmarkören C-reaktivt protein (CRP). Via Sangersekvensering kunde flera kandidater identifieras. Bland 6 karakteriserade ADAPTer uppvisade samtliga hög HSA-affinitet, tre konstaterades interagera specifikt med sitt målprotein, och två verkade binda även rumsbispecifikt. ADAPTer utvärderades även för sin benägenhet att bilda aggregat, strukturell överensstämmelse med experimentell design, och värmestabilitet. Endast en bindare, mot HER2, klarade sig genom alla prövningar som proteinkarakteriseringen innebar utan underkänt. Även en högparallel sekvensering utav selektionsresultat utfördes, men utanför de tidsramar som tillät ytterligare karakterisering.

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The development of novel, tumor-selective and boron-rich compounds as potential agents for use in boron neutron capture therapy (BNCT) represents a very important field in cancer treatment by radiation therapy. Here, we report the design and synthesis of two promising compounds that combine meta-carborane, a water-soluble monosaccharide and a linking unit, namely glycine or ethylenediamine, for facile coupling with various tumor-selective biomolecules bearing a free amino or carboxylic acid group. In this work, coupling experiments with two selected biomolecules, a coumarin derivative and folic acid, were included. The task of every component in this approach was carefully chosen: the carborane moiety supplies ten boron atoms, which is a tenfold increase in boron content compared to the l-boronophenylalanine (l-BPA) presently used in BNCT; the sugar moiety compensates for the hydrophobic character of the carborane; the linking unit, depending on the chosen biomolecule, acts as the connection between the tumor-selective component and the boron-rich moiety; and the respective tumor-selective biomolecule provides the necessary selectivity. This approach makes it possible to develop a modular and feasible strategy for the synthesis of readily obtainable boron-rich agents with optimized properties for potential applications in BNCT.

The amount of boron accumulated in tumor tissue plays an important role regarding the success of the boron neutron capture therapy (BNCT). In this article, we report a modular system, combining readily available starting materials, like glycine, 1,3,5-triazine and the well-known 9-mercapto-1,7-dicarba-closo-dodecaborane(12), as well as alpha-d-galactopyranose for increased hydrophilicity, with a novel boron-rich tris-meta-carboranyl thiol.

Cancer is still one of the most common causes of death world-wide and in parallel there is a need to update the repertoire of therapies that withstand resistance of recurrent cancers. Since the introduction of antibody therapies as anti-cancer pharmaceuticals, recognized as immunotherapy in health care, it has been an increasing field in cancer therapy, as a more targeted treatment compared to chemotherapy. Despite the great success, immunotherapy rely on parenteral administration, partly due to poor tissue penetration. If the treatment is administered intravenously, specialized personnel is required, in addition to that it can be inconvenient for the patient. Also, pharmaceuticals based on antibodies often require costly production steps which yields a high-priced treatment. To approach this problem, researchers have developed small affinity domains with the aim to increase tissue penetration while keeping a high specificity to its target. Albumin Binding Domain Derived Affinity Protein (ADAPT) is an example of a small affinity domain of only 7 kDa, which is based on albumin binding domain (ABD) from the streptococcal protein G. Recently, it was shown that the ADAPTs can be further engineered to bind albumin and another relevant target protein of interest simultaneously, which suggests a tolerable half-life in patient serum, alternative administration routes and lower production costs compared to antibody treatments. Furthermore, less side effects are expected due to higher specificity compared to chemotherapy. This work presents the characterization of novel ADAPT proteins that the target the cancer relatedproteins C-C motif ligand 7 (CCL7), vascular endothelial growth factor A (VEGF-A) and carcinoembryonic antigen related cell adhesion molecule 5 (CEACAM5). The new constructs were produced recombinantly in Escherichia coli (E. coli) and purified using affinity chromatography. Moreover, the results demonstrate bispecific binding with high affinity towards serum albumin and CCL7 and CEACAM5 respectively, while the ADAPT variants targeting VEGF-A remain to be further developed. Lastly, the importance of different amino acids for structural and binding properties of one CEACAM5 binder are stated. It reveals that the target binding relies on hydrophobic interactions which also can be connected to its poor structural attributes. Accordingly, this project adds new insights about the ADAPTs which can be useful in research towards future clinical applications aimed to improve cancer treatments.

High Intensity Focussed Ultrasound (HIFU) ablation is a promising technology for the non-invasive, targeted treatment of certain types of cancer. The technique functions by subjecting tumours to a cytotoxic level of intense, localised heating, while leaving the surrounding tissue unharmed. However, a number of limitations in the available HIFU treatment monitoring methods are currently hampering the effectiveness and clinical adoption of the therapy. This work aims to develop improved metrics of HIFU-induced biological damage that are specifically suited to monitoring and controlling HIFU ablation. Firstly, an optical method that enables straightforward quantification of thermal damage in protein-embedding hydrogels is developed. Secondly, hydrogels embedded with different cell lines are used to assess the performance of common temperature-based metrics of cell death across a range of HIFU-relevant conditions. Finally, a novel, passive acoustic detector designed for the real-time monitoring of HIFU-induced tissue damage is proposed. The detector is shown to predict lesioning with over 80% accuracy in regimes that are very likely to create lesions (60 J of acoustic energy or more), with an error rate of less than 6% for exposures that are too short to cause lesioning (up to 1 s long). The proposed detector could therefore provide a low-cost means of effectively monitoring clinical HIFU treatments passively and in real time.