Cancer immunotherapy has experienced remarkable advances in the last decades. Striking clinical responses have been achieved for several solid cancers, particularly cancer types with a high... Show moreCancer immunotherapy has experienced remarkable advances in the last decades. Striking clinical responses have been achieved for several solid cancers, particularly cancer types with a high mutation burden, which placed tumour-mutated antigens (neoantigens) centre stage as targets of tumour immunity and cancer immunotherapy. Neoantigens can be presented in complex with HLA molecules on the tumour cell surface, where T cells with the correct specificity can recognize the neoantigen as ‘non-self’ which will trigger killing of the tumour cell by the T cell. In theory, cancers with a low/moderate mutation burden that present neoantigens in complex with HLA class molecules could still be eligible for T cell-mediated immunotherapy. This thesis, describes the finding that neoantigen-specific T cells are present in mismatch-repair proficient (MMR-p) colorectal cancer patients, a low mutation burden cancer type. Moreover, CD39 and CD103 were found as cell surface markers that pinpoint the T cell population that contains the neoantigen-specific T cells. In addition, subsequent metastasis of a melanoma patient cohort were studied and revealed that also at advanced, late-stage disease, neoantigen-directed T cell therapy is, in theory, still applicable. Taken together, the studies reveal potential for the development of neoantigen-directed cancer immunotherapy for a broader patient population. Show less
To generate a successful novel therapy, a deep understanding of oncogenesis in combination with mechanistic understanding of anti-cancer compounds are needed. The work described in this thesis aims... Show moreTo generate a successful novel therapy, a deep understanding of oncogenesis in combination with mechanistic understanding of anti-cancer compounds are needed. The work described in this thesis aims to contribute to the knowledge on SUMO regulated oncogenesis, understanding the consequences of abolishment of SUMO signaling and exploiting the potential of SUMO E1 inhibitors. To this end, we describe SUMO as a potential biomarker for cancer aggressiveness and increase our understanding on SUMO’s role in cell cycle progression. We exploited the potential of SUMO E1 inhibition by combining with hypomethylating compound 5-Aza-2’ deoxycytidine, leading to increased cytostatic efficacy. Furthermore, we repurposed the SUMO E1 inhibitor TAK981 and hypomethylating drug 5-Aza-2’ deoxycytidine to improve engineered TCR (eTCR) T cell therapy and broaden our understanding of its immunomodulatory potential. Show less
The aim of this thesis was to develop novel treatment strategies for different types of eye melanoma utilizing zebrafish models. We first establish orthotopic and ectopic xenograft models for uveal... Show moreThe aim of this thesis was to develop novel treatment strategies for different types of eye melanoma utilizing zebrafish models. We first establish orthotopic and ectopic xenograft models for uveal and conjunctival melanoma by engraftment of the immortalized cells derived from these tumors into zebrafish embryos. Next, we expanded these models with spheroids and zebrafish patient-derived xenografts for pre-clinical, personalized screening of anti-uveal melanoma drug responses. We demonstrated that these models can be harnessed to explore the in vivo interactions of the tumor cells with blood vessels and macrophages leading to angiogenic response. We finally apply the conjunctival melanoma model to clarify the inhibitory effects of ginsenosides and correlate their structures with potential antitumoral mechanisms. Show less
Cells constitute the tissues of our body and are responsible for producing various changes in response to different situations. For instance, the repair of damaged DNA. DNA resides within the cell... Show moreCells constitute the tissues of our body and are responsible for producing various changes in response to different situations. For instance, the repair of damaged DNA. DNA resides within the cell nucleus and can be transcribed and translated into proteins, which play vital roles in numerous cellular processes. The cell relies on modifying existing proteins to carry out essential functions. These modifications can involve the conjugation of small molecules such as Ubiquitin (Ub) or Small Ubiquitin-like Modifiers (SUMOs), leading to protein degradation, conformational changes or intracellular relocation of critical proteins. The conjugation of these small molecules involves a well-orchestrated sequence of enzymatic activities performed by dedicated enzymes: E1 (activating), E2 (conjugating) and E3 (ligase). Among these, the E3 ligase enzymes hold significant importance as they confer substrate specificity.In this thesis, we have developed an advanced Mass-Spectrometry technology called TULIP2 (Targets for Ubiquitin Ligases Identified by Proteomics 2), which facilitates the identification of Ubiquitination targets for specific E3 ligases of interest. Using this technology, we have investigated the BRCA1-BARD1 E3 ligase and explore the in vivo role of the E2 UBE2D3. Furthermore, we have adapted the TULIP2 technology to create the SUMO Activated Target Traps (SATTs), enabling the identification of an E3-specific SUMO proteome. Show less
Nonhuman primate adenoviruses have formed a valuable alternative for the use of human adenoviruses in vaccine development and gene therapy strategies by virtue of the low seroprevalence of... Show moreNonhuman primate adenoviruses have formed a valuable alternative for the use of human adenoviruses in vaccine development and gene therapy strategies by virtue of the low seroprevalence of neutralizing immunity in the human population. The more recent use of several human adenoviruses as oncolytic agents has exhibited excellent safety profiles and firm evidence of clinical efficacy. This raises the question whether nonhuman primate adenoviruses could also be employed for viral oncolysis in human patients. The research in this thesis provides a rational and data-supported decision on the use of nonhuman primate adenoviruses as a base for the development of new oncolytic derivatives with limited neutralizing immunity in the human population. Moreover, the development of a potent new gorilla-derived oncolytic adenovirus named GoraVir shows the feasibility of the approach. Hopefully, this research provides some reassurance regarding the future use of replication-competent nonhuman primate adenovirus vectors as therapeutic agents. Show less
This thesis aims to improve patient outcomes after major oncological gastrointestinal surgery, by identifying targets for optimizing perioperative care. On the one hand, identifying (adjustable)... Show moreThis thesis aims to improve patient outcomes after major oncological gastrointestinal surgery, by identifying targets for optimizing perioperative care. On the one hand, identifying (adjustable) prognostic factors for postoperative complications and then using them in prediction models, and gaining insight into the quality of life after surgery. Improving preoperative risk assessment and subsequent personalization of postoperative care can reduce the rate of postoperative complications and mortality. In addition, risk prediction can support clinical knowledge in making treatment decisions. Modifiable prognostic factors are particularly important to identify as they may be optimized prior to surgery. Modifiable prognostic factors can then be used in prehabilitation programs to improve a patient's physical condition and thus improve patient outcomes. In addition, information about treatment consequences and treatment-related health problems is important for treatment decision-making for patients and healthcare professionals. Especially, if several treatment options are available, knowledge about (long-term) consequences of the treatment options is important for shared decision-making. This knowledge can also contribute to optimizing postoperative care and rehabilitation programs. In conclusion, the leads described in this thesis for improving postoperative care can be used to improve short- and long-term patient outcomes after complex oncological gastrointestinal surgery. Show less
The use of existing medications for diseases they were not originally developed for is called drug repositioning. A popular drug repositioning method to find new drugs against specific cancer types... Show moreThe use of existing medications for diseases they were not originally developed for is called drug repositioning. A popular drug repositioning method to find new drugs against specific cancer types is to search for drugs which are expected to bring back the gene expression activity of cancer cells to that of healthy cells (‘normalization’). One of the main research goals of this thesis was to investigate of this method could also be used on the gene expression profiles of individual tumors, enabling personalization of drug repositioning candidates for each patient. We initially had some success with this approach but this eventually lead to a systematic validation of the underlying principle using almost 10,000 tumor samples across 18 different tumor types. Unfortunately, the predictive power of the method was found to be much lower than previously reported and the part that remained could be nullified by correcting the gene expression profiles of the drugs for the downstream effects of reduced cell division. These results indicate that the current use of the method does not result in drug repositioning candidates specific for a tumor type but is only able to select generally cell-toxic drugs. Show less
Type I interferons (IFN-I) are pleiotropic cytokines that were originally identified for their antiviral properties and are now recognized for playing key roles in the defense against a range of... Show moreType I interferons (IFN-I) are pleiotropic cytokines that were originally identified for their antiviral properties and are now recognized for playing key roles in the defense against a range of other microorganisms as well as cancer. Their production should be well-controlled to be of benefit to the host, as excessive or chronic IFN-I expression leads to adverse effects such as immunosuppression or the induction of severe immunopathology.The studies presented in this thesis are aimed at uncovering mechanisms that regulate the production of IFN-I. The obtained knowledge on the involved molecular processes, may aid the development of targeted therapies that enhance or intercept IFN-I responses for maximum host protection while minimizing damage. Show less
Epithelial-mesenchymal plasticity (EMP) and tumor cell migration play an important role in cancer progression, and an improved understanding of the mechanisms underlying these concepts is essential... Show moreEpithelial-mesenchymal plasticity (EMP) and tumor cell migration play an important role in cancer progression, and an improved understanding of the mechanisms underlying these concepts is essential for developing new targeted approaches. In this thesis, we studied these mechanisms using mathematical and computational approaches.First, we summarized and reviewed previous computational approaches that have been used to decipher EMP regulation. We then created mathematical models to explore (1) how different regulatory networks can explain epithelial-mesenchymal transition (EMT) in different cell contexts, and (2) how EMP and immune regulation can interact to cause tumor immunoevasion.Next, we studied the role of cell density in migration characteristics of triple-negative breast cancer cell lines by using a combined experimental and computational approach. We show how clustering and pseudopodial dynamics, potentially influenced by EMT-related factors, can alter the migratory behavior of these cell lines.Jointly, our work has shown that computational modeling can be used to test hypotheses based on experimental data, and generate testable hypotheses, making it a valuable addition to wet-lab experiments. Importantly, we identified mechanisms related to potential therapeutic targets, hopefully leading to improved targeted therapies and reduced cancer mortality. Show less
Immune checkpoint therapies that aim to (re)activate the immune response against cancer cells have shown promising results in a variety of tumor types. Yet, a large fraction of cancer patients does... Show moreImmune checkpoint therapies that aim to (re)activate the immune response against cancer cells have shown promising results in a variety of tumor types. Yet, a large fraction of cancer patients does not benefit from these therapies. While the presence of a substantial number of immune cells, and in particular T cells, in the tumor is generally related with a better clinical response to checkpoint therapies, the T cells in the tumor are diverse in their capacity to eliminate the tumor. In order to improve treatment outcome of cancer patients, we require a better understanding of the roles of different T cells in the response (and resistance) to immune checkpoint therapy. The development of single cell profiling technologies has provided us with a powerful tool to analyze the state and functionality of individual cells. In this thesis, I have used single cell profiling methods in combination with innovative experimental technologies to unravel the diversity of T cells in human tumors and define the changes in the profiles of T cells that occur in response to treatment with immune checkpoint therapy to dissect which T cells are important for therapy response. Show less
All cellular organisms contain genomic DNA which provides the instructions for their correct development and functioning. Damage to this DNA may interfere with critical cellular processes such as... Show moreAll cellular organisms contain genomic DNA which provides the instructions for their correct development and functioning. Damage to this DNA may interfere with critical cellular processes such as transcription and replication and has the potential to drive mutagenesis. In turn, this may underlie inherited disorders and accelerate progression of diseases such as cancer and neurodegenerative disorders. The protection of cells and organisms against these devastating effects of DNA damage relies on the DNA damage response (DDR), which comprises a complex network of signaling and repair pathways that coordinate the sensing, signaling and repair of DNA lesions while accommodating suitable adjustments in for instance chromatin structure and cell cycle progression. Not only does the DDR dictate the appropriate repair pathway for several types of DNA damage, including DNA double-strand breaks (DSB), it also modulates replication fork surveillance mechanisms in response to DNA replication stress (RS). While many core proteins have been studied in detail, the full repertoire of factors involved in these pathways remains unknown. Clearly, extending our knowledge on regulators of the DDR will contribute to our understanding of the development, and possibly the treatment, of the numerous disorders that are associated with defects in the DDR. The research described in this thesis has successfully identified and characterized novel factors in DSB repair and the RS response. Show less
The spindle-assembly checkpoint (SAC) is a safety mechanism which secures accurate chromosome segregation during mitosis. BUB1, a serine/threonine kinase, is one of the proteins involved in this... Show moreThe spindle-assembly checkpoint (SAC) is a safety mechanism which secures accurate chromosome segregation during mitosis. BUB1, a serine/threonine kinase, is one of the proteins involved in this checkpoint and its inhibition is thought to have therapeutic potential for the treatment of cancer. Although the exact role of BUB1 in the SAC remains controversial, inhibition of its kinase function has previously been shown to reduce tumor size in mouse xenograft models when combined with paclitaxel. The research described in this thesis aimed to develop novel BUB1 kinase inhibitors for which high-throughput screening was used as starting point for drug discovery. Medicinal chemistry efforts were performed to improve potency after which the obtained inhibitors were further evaluated in cellular assays. In addition, the development of a cellular BUB1 target engagement assay is described. Hit optimization led to the discovery of two lead compounds with good physicochemical properties, subnanomolar affinity for BUB1, good cellular BUB1 target engagement, acceptable selectivity over other kinases and a favorable in vitro ADME profile. Show less
G protein-coupled receptors (GPCRs), one of the largest families of membrane proteins, are responsive to a diverse set of physiological endogenous ligands including hormones and neurotransmitters.... Show moreG protein-coupled receptors (GPCRs), one of the largest families of membrane proteins, are responsive to a diverse set of physiological endogenous ligands including hormones and neurotransmitters. Due to the various GPCR ligand binding domains present on GPCRs and their sensitivities to a diverse array of ligands, these proteins have shown to be very ‘druggable’ as they are the main target for an estimated 30% of approved drugs. A growing body of evidence shows a prominent role of GPCRs in all phases of cancer with a mutation frequency of approximately 20% in all cancers. Mutations occurring in GPCRs can severely alter their normal function and may ultimately convert their physiological and pathological roles. One particular class of rhodopsin-like GPCRs included in this thesis are the adenosine receptors (ARs). Due to the accumulation of adenosine in the tumor microenvironment, all four subtypes of ARs might be targets for the development of novel approaches for the treatment of cancer. For each of the four subtypes, a number of somatic mutations have been identified in patient isolates. In this thesis, we examined them on receptor activation and ligand binding using reference adenosine receptor ligands, and determined the impact mutations have on these pharmacological readouts. Show less
T cell exhaustion is a state of T cell hypofunction arising during persistent viral infections and cancer. Recent advances in the field of immunology uncover the roles of cytokines in regulating T... Show moreT cell exhaustion is a state of T cell hypofunction arising during persistent viral infections and cancer. Recent advances in the field of immunology uncover the roles of cytokines in regulating T cell responses. Using LCMV Clone-13 as a model of persistent viral infection, this thesis investigates the roles of IL-27 and IFN-I in regulating T cells during infection. In addition, the thesis explores the potential of JAK inhibitor in rescuing T cell exhaustion during persistent viral infection and cancer. Show less
Death in all types of melanomas is generally caused by metastasis. Uveal melanoma (UM) is the most common intraocular melanoma, there are currently no (patient-derived) animal models that... Show moreDeath in all types of melanomas is generally caused by metastasis. Uveal melanoma (UM) is the most common intraocular melanoma, there are currently no (patient-derived) animal models that faithfully recapitulate metastatic dissemination of UM. Here we generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma. In doing so we can recapitulate the etiology of cancer in its totality. Subsequently, we developed a patient-derived zebrafish xenograft (zf-PDX) model, using spheroid cultures generated from metastatic and primary UM tissues. Harnessing this versatile model, we reveal high sensitivity of circulating UM cells to ferroptosis induction in vivo by Erastin and RSL3, implicating ferroptosis as a new potential therapy in metastatic UM.Increased melanin levels in cutaneous melanoma are associated with decreased patient survival. Melanin levels in primary uveal melanoma patient cells positively correlate with their metastatic potential in zebrafish. Modulation of melanin levels of pan-melanoma cells results in enhanced/reduced metastatic potential upon increased or decreased melanin levels, respectively. Melanin depletion sensitizes melanoma cells to ferroptosis inducers in zebrafish leading to a decreased metastatic burden. Collectively, our data identify melanin biosynthetic enzymes as potential future target to treat melanoma and show that melanin protects metastasizing melanoma cells from ferroptosis. Show less
Despite the interest and success in immunotherapy, our current understanding of how the immune activation balance – on the macro level – and the expression of receptors and their ligands – on the... Show moreDespite the interest and success in immunotherapy, our current understanding of how the immune activation balance – on the macro level – and the expression of receptors and their ligands – on the micro level – shape health and disease is still limited. By identifying novel regulatory networks that dictate immune cell activity in physiologically relevant settings, well-designed pooled genetic screens could offer a relevant approach to address this knowledge gap. Genetic screens have become a powerful tool to identifygenes that affect the response and phenotype of a cell in an unbiased manner.In this thesis, I aimed to identify regulators of immune cell activation in the context of cancer through a combination of in vitro and in vivo genetic screens and genetically modified mouse models. Specifically, this was achieved by both 1) using genetic screening technologies to identify novel cancer cell-expressed inhibitory ligands or regulatorsof immune-modulating ligands in an unbiased manner, and 2) investigating the effect of these regulators on the cellular components and signaling pathways that constitute the local tumor microenvironment and potentially dictate anti-tumor efficacy. Show less
Being confronted with cancer is a life-changing event, with major impact to well-being, quality of life and relationships. Cancer treatments and outcomes have improved, but possess the potential to... Show moreBeing confronted with cancer is a life-changing event, with major impact to well-being, quality of life and relationships. Cancer treatments and outcomes have improved, but possess the potential to impair reproductive and sexual function. International guidelines highlight the importance of timely discussion of sexual function and fertility deterioration resulting from treatments. In spite of these developments, practice behaviour and attitudes of health professionals have been reported to vary.The first part of this thesis demonstrates barriers among oncology professionals in the Netherlands in discussing sexual function during daily practice. In general, consensus exists regarding responsibility for addressing sexual dysfunction. Despite, the implementation of discussing sexual function as a standard of care is not carried out structural.Part two describes practice routines concerning the counselling on impaired fertility and fertility preservation for cancer patients of reproductive age. Medical oncologists and oncology nurses both reported discussing the impact of treatment on fertility. However, it was not performed in all cases and depending on factors like educational level, experience, type of hospital, prognosis and chances of fertility recovery.Furthermore, testicular cancer patients were evaluated on specific items concerning the discussion, referral and process of semen cryopreservation. Long term reproductive concerns were identified among survivors. Show less
This thesis aims to gain a better understanding of NER, to elucidate new molecular mechanisms and proteins that orchestrate how DNA repair is carried out on genomic DNA that is tightly packed in... Show moreThis thesis aims to gain a better understanding of NER, to elucidate new molecular mechanisms and proteins that orchestrate how DNA repair is carried out on genomic DNA that is tightly packed in chromatin inside the living cell. It is important to obtain a better clinical picture of how inherited defects in DNA repair genes shapes phenotypes in patients with DNA repair-deficiency disorders. Show less
Tumor heterogeneity favors tumor tissue to survive and resist drugs, leading to the failure of chemotherapeutic agents to induce a therapeutic response. In addition, the absorption mechanisms,... Show moreTumor heterogeneity favors tumor tissue to survive and resist drugs, leading to the failure of chemotherapeutic agents to induce a therapeutic response. In addition, the absorption mechanisms, metabolism and excretion of chemotherapeutic drugs, which are commonly used for cancer patients and the lack of specific targeting of these drugs can cause adverse effects on treated patients. Thus, the general objective of this thesis is to investigate the biological activity of targeted poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) as a drug delivery system (DDS) for carvedilol (CVDL) or oxaliplatin (OXA), in vitro and in vivo, to treat colorectal cancer (CRC). DDSs were formulated to achieve this goal. In chapters 2, 3 and 4, our studies were discussed in detail on the formulations and characterizations of NPs as DDSs with ideal characteristics to increase the therapeutic range of drugs at the tumor site. As well as the biological evaluation of these DDS when its anti-inflammatory activity (Chapter 2) and its antitumor activity in vitro (Chapters 2, 3 and 4) and in vivo (Chapters 3 and 4). Taken together, all the DDSs studied in this thesis were able to improve the chemotherapeutic efficiency of the drugs studied in Chapters 2, 3 and 4. Show less
Upon infection or vaccination, naïve CD8+ T cells clonally expand and differentiate into antigen-specific effector cell populations, which turn into phenotypically diverse memory CD8+ T cell... Show moreUpon infection or vaccination, naïve CD8+ T cells clonally expand and differentiate into antigen-specific effector cell populations, which turn into phenotypically diverse memory CD8+ T cell subsets that differ in their cytokine polyfunctionality, cytolytic capacity and homing properties. The circulating memory CD8+ T cell pool can be largely classified in two major subsets: effector memory T (TEM) and central memory T (TCM) cells. Contrary to the classical view that lymphocytes continuously recirculate, tissue-resident memory T (TRM) cells have been discovered as cells with the unique ability to reside in tissues with limited recirculation through the blood. Antigen-specific CD8+ TRM cells are induced upon antigen encounter and localize to many different tissues, including barrier tissues, where they play a crucial role in protection against infectious and malignant disease. Within these major circulating and resident T cell subsets a variety of phenotypes and functions exists. However, the development of the heterogeneous populations of memory T cells is not fully understood. In this thesis we dissect the development and heterogeneity of antigen-specific circulating and tissue-resident CD8+ T cells upon vaccination and infection, study their protective capacity in infectious and malignant disease and use this information to improve vaccination and immunotherapeutic strategies. Show less
