Parasporal proteins from Bacillus thuringiensis (Bt) are well studied in various fields of environmental biotechnology and recently in medical biology. Correspondingly, this dissertation presents the use of site-directed mutagenesis technologies to obtain mutant cry and parasporin proteins with different effects against vector-borne diseases and human cancer cell lines. The use of in silico modeling offers a possible explanation of how the altered residues are involved in changing their structure and interacting with cell membrane receptors. The proteins from this work can be used as targets for new studies that will provide more information about the mechanism of action of Bt proteins, and open the door to the employment of these biomolecules in combating diseases.

Unfortunately, lymphoma (Hodgkin’s and Non-Hodgkin’s lymphoma) is still an incurable disease for many people and new and smart treatment techniques are needed to improve the prognosis of these patients.
The goal of the research described in this dissertation was to find pre-existing medications used in other cancers, using a technique that allows to test the dependence on anti-apoptotic proteins (proteins important for the survival of cancer cells). This technique is called BH3 profiling.
By applying this technique, it has been found that tamoxifen, an affordable, anti-hormone drug that has been used in breast cancer for more than 40 years, can kill lymph node cancer cells in laboratory experiments and in animal experimental models. If tamoxifen is combined with a drug that blocks anti-apoptotic proteins, the anticancer effect is enhanced. This has led to a clinical trial in patients with untreatable aggressive lymphoma.
In addition to these important findings and applications, this thesis describes the efficacy of combinations of different drugs in lymphoma and whether these agents alter the dependence on these anti-apoptotic agents.
This project was carried out through a KWF grant.

Hodgkin lymphoma (HL) is a common malignancy in young adults. It is characterized by a minority of tumor cells surrounded by an abundant inflammatory infiltrate, which mainly consists of CD4+ T cells. The current treatment strategy has a high cure-rate, but often results in long-term treatment related adverse events. Therefore, new treatment options such as immune checkpoint blockade (ICB) are being explored. In this project we characterized CD4+ T cells and their interactions with tumor cells, with a specific focus on the role of CD4+ T cells in ICB therapy. We discovered that CD4+ T cells are weakly activated by T cell receptor (TCR)-HLA class II and CD2-CD58 interactions with the tumor cells. These T cells that are in close proximity to the tumor cells have been activated before (antigen experienced) and are diverse (polyclonal). In addition, they specifically express transcription factors TOX and TOX2 that are associated with T cell exhaustion and can be induced by chronic or repeated TCR stimulation. This suggests that Hodgkin tumor cells actively inhibit cells in their surrounding to promote tumor cell survival. We developed a long-term co-culture model and found that blocking PD-1 with nivolumab results in pronounced CD4+ T cell activation and proliferation, confirming the potential of this treatment in HL. In addition, we identified soluble PD-L1 as a promising biomarker in HL patients. In summary, our studies provide novel insights into the function of CD4+ T cells in HL biology and treatment responses. This may help improve immunotherapy in HL patients.

Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive type of non-Hodgkin lymphoma. The current treatment for the disease currently cures only 60% of the patients, which means new therapies are needed to improve patient survival. In this project, we performed a large gene expression analysis in 1800 DLBCL patient samples to find new targets to improve the current treatment. Here we found high expression of the WEE1 protein, which is involved in cell cycle regulation and repair of DNA damage. Inhibition of the WEE1 protein in DLBCL cell lines induced disruption of the normal cell cycle and high levels of DNA damage, eventually causing cell death. In addition, WEE1 inhibition showed to enhance the currently used therapies for the treatment of DLBCL, which include rituximab (anti-CD20), radiation, CHOP (first line chemotherapy) and cytarabine (second line chemotherapy). Based on these findings, we also investigated the effect of WEE1 inhibition in combination with so-called “anti-apoptotic inhibitors”, which prevent cells from protecting themselves against cell death. These inhibitors are currently being tested in clinical trials for different types of non-Hodgkin lymphoma and leukemia’s, including DLBCL . We found that WEE1 inhibition worked very well together with the anti-apoptotic inhibitors, and that combination therapy significantly enhanced cell death in DLBCL. In total, our results demonstrate that inhibition of WEE1 is very successful in DLBCL, and would likely improve the current treatment available for DLBCL.

In classical Hodgkin lymphoma (cHL) there is an urgent need for biomarkers to determine prognosis or treatment response. In this thesis we summarized current knowledge on circulating biomarkers in cHL and studied a selection of these markers as treatment response or prognostic biomarkers. In the first part of this thesis we studied the application of Thymus and Activation Regulated Chemokine (TARC) as a biomarker for treatment response and compared TARC with sGal-1, sCD163 and sCD30 and interim FDG-PET imaging. We found that TARC at diagnosis correlates with metabolic tumor volume and that serial TARC measurements during and after treatment accurately reflect treatment response. In comparison to sGal-1, sCD163 and sCD30, TARC dynamics most accurately reflected treatment response. In addition, interim TARC had a higher positive predictive value for final treatment response compared to interim FDG-PET imaging. All together, we concluded that TARC is a cheap, non-invasive and highly accurate biomarker to determine treatment response in cHL patients.
In the second part of this thesis, we focused on microRNAs (miRNAs) as circulating biomarkers. We summarized the role of miRNAs in cHL and concluded that deregulation of miRNAs is a frequent event and an important factor in the pathobiology of cHL. Next, we summarized pre-analytical, analytical and post-analytical challenges in circulating miRNAs studies in general. Finally, we performed miRNA microarray profiling of serum of patients with cHL. Using this approach, we were unable to find circulating miRNAs with prognostic value in classical Hodgkin lymphoma.