The goal of this thesis was to investigate the role of circRNAs in B-cell lymphoma and breast cancer. We focused on the characterization of their expression patterns, functional mechanisms, and regulatory roles. In the first part, we conducted RNA sequencing to characterize the circRNA landscape of three main B-cell lymphoma subtypes and normal B-cells. This was followed by more in-depth studies of the roles of circPVT1 and circZDHHC11 in B-cell lymphoma. CircPVT1 and the linear PVT1 transcripts showed opposite differential expression patterns and both supported cell growth. In contrast to other cancers, we showed that the effect on growth was independent of their ability to bind miRNAs. CircZDHHC11 was strongly enriched in the AGO2-IP fraction upon miR-150 overexpression and supported the growth of B-cell lymphoma. However, its growth-supporting effect was independent of its ability to bind miR-150. In the second part, we investigated the role of circ-NOL10 in breast cancer. Circ-NOL10 was selected based on its most pronounced downregulation in triple-negative breast cancer (TNBC) compared to control tissue. Upon binding of MTDH and CASC3, circ-NOL10 levels were strongly reduced, resulting in the release of miR-149-5p, miR-330-3p, and miR-452-5p. These miRNAs subsequently targeted PDCD4 and this promoted progression of breast cancer. Overall, our study extends current knowledge of circRNAs and highlights specific functions of selected circRNAs in B-cell lymphoma and breast cancer.

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.

The study of this thesis is related to the previously established MYC/miR-150/MYB/ZDHHC11 network regulating Burkitt lymphoma (BL) growth. Our first aim was to study the role of this network in Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL). We found a similar role of three of the network components, while miR-150 overexpression showed no or only very mild effects on HL and DLBCL growth. Therefore, we considered miR-150 as a BL-specific component. Our second aim was to study the specific roles of the protein coding and circular ZDHHC11 gene products. ZDHHC11 belongs to a family of 24 ZDHHC proteins that are characterized by a DHHC motif which mediates palmitoylation. Loss of palmitoylation induced apoptosis in three B-cell lymphoma subtypes indicating the overall importance of protein palmitoylation for B-cell lymphoma. Based on the observed expression patterns we pinpointed seven DHHC family members that might be relevant in B-cell lymphoma. Further studies to elucidate the role of the ZDHHC11 protein were hampered due to difficulties in detecting the protein but provided some evidence that this protein is not a critical factor in regulating BL growth. Lastly, we showed that circular ZDHHC11 RNA transcripts do play a critical role in growth of B cell lymphoma, but this effect is independent of binding miR-150, as deletion of the miR-150 binding site region did not alter the observed phenotype. In summary, we extended current knowledge on the role of the MYC/miR-150/MYB/ZDHHC11 network and addressed the function of ZDHHC11 transcripts and protein in B-cell lymphoma.

Classic Hodgkin lymphoma (cHL) is a cancer of the immune system common in adolescents and young adults. The chance to develop cHL is strongly influenced by genetically defined human leukocyte antigen (HLA) types. The main function of HLA is to present antigenic peptides and it is a critical player in the normal immune system. In this thesis we focused on the mechanisms of HLA-related associations with cHL. First, we showed that part of the cHL-associated HLA alleles are associated with loss of HLA expression on tumour cells. Second, we identified novel ERAP – HLA interactions that were specific for cHL patients and not observed in controls. Together these data show that antigen presentation is a critical component in defining the susceptibility to develop cHL. Lastly, we identified an association between cHL and killer cell immunoglobulin-like receptors (KIRs). These KIRs interact with HLA class I molecules expressed on target cells and play an important role in clearance of virus-infected or tumour cells. We found that KIR haplotype B protects against the development of a specific cHL subgroup (EBV+ nodular sclerosis). In addition, we observed presence of KIR2DL2 – HLA-C1 and KIR2DS2 – HLA-C1 at significantly lower carrier frequencies in this cHL subgroup compared to controls. Overall, our studies showed that multiple HLA associated molecules influence susceptibility to develop cHL. The results of this thesis further support the critical role of HLA and antigen presentation in cHL susceptibility and extends on the biological mechanisms underlying the development of cHL.

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.