Uveal melanoma (UM) is a rare ocular tumor. Up to 50% of the patients develop distant metastases predominantly targeting the liver. The median survival after diagnosis of patients with hepatic... Show moreUveal melanoma (UM) is a rare ocular tumor. Up to 50% of the patients develop distant metastases predominantly targeting the liver. The median survival after diagnosis of patients with hepatic metastases is approximately 4-6 months and hardly increased in the past decades due to lack of novel effective therapeutic options. Within the scope of this thesis we investigated the signaling landscape of metastatic UM and searched for novel avenues of therapy. In Chapter 2 we demonstrate that combinations of the multitarget drug Trabectedin with either the CK2/Clk double-inhibitor Silmitasertib or with the c-MET/TAM receptor inhibitors show synergistic growth inhibitory effects and induce apoptosis of UM cells in vitro. Chapter 3 describes the application of a CRISPR-Cas9 synthetic lethality screen for identification of molecular targets whose inhibition synergistically enhances the effect of the mTOR inhibitor everolimus in UM cells. In Chapter 4 we show that the combination of genetic depletion YAP1/TAZ together with Mcl-1 inhibition resulted in a synergistic inhibitory effect on the viability of UM cell lines. In Chapter 5 we analyzed the phospho-proteome of two UM metastatic cell lines and a primary tumor cell line from the same individual, and studied the role of MARK3 in YAP1/TAZ signaling. Show less
The past two decades have seen the growing development and consequent vast application of next-generation genome editing tools in fundamental and applied research. Nowadays GE based on RNA-guided... Show moreThe past two decades have seen the growing development and consequent vast application of next-generation genome editing tools in fundamental and applied research. Nowadays GE based on RNA-guided nucleases (e.g., engineered CRISPR-Cas9 nucleases) are the most common tools for targeted genetic modification. Nevertheless, these technologies are in need of increased efficiency and accuracy, especially looking forward to translation into diverse clinical applications. The work presented in this thesis focuses on improving the efficiency and accuracy of genome editing, particularly in cells with high therapeutic potential, such as induced pluripotent stem cells (iPSCs), by investigating the feasibility of using adenoviral vectors to test novel genome editing approaches and by exploring the possible applications of a scarless strategy. Show less
Sluimer, L.M.; Bullock, E.; Rätze, M.A.K.; Enserink, L.; Overbeeke, C.; Hornsveld, M.; ... ; Tavares, S. 2023
High expression of the non-receptor tyrosine kinase FER is an independent prognostic factor that correlates with poor survival in breast cancer patients. To investigate whether the kinase activity... Show moreHigh expression of the non-receptor tyrosine kinase FER is an independent prognostic factor that correlates with poor survival in breast cancer patients. To investigate whether the kinase activity of FER is essential for its oncogenic properties, we developed an ATP analogue-sensitive knock-in allele (FERASKI). Specific FER kinase inhibition in MDA-MB-231 cells reduces migration and invasion, as well as metastasis when xenografted into a mouse model of breast cancer. Using the FERASKI system, we identified Ski family transcriptional corepressor 1 (SKOR1) as a direct FER kinase substrate. SKOR1 loss phenocopies FER inhibition, leading to impaired proliferation, migration and invasion, and inhibition of breast cancer growth and metastasis formation in mice. We show that SKOR1 Y234, a candidate FER phosphorylation site, is essential for FER-dependent tumor progression. Finally, our work suggests that the SKOR1 Y234 residue promotes Smad2/3 signaling through SKOR1 binding to Smad3. Our study thus identifies SKOR1 as a mediator of FER-dependent progression of high-risk breast cancers. Show less
Gaykema, L.H.; Nieuwland, R.Y. van; Dekkers, M.C.; Essen, M.F. van; Heidt, S.; Zaldumbide, A.; ... ; Kooten, C. van 2023
End stage renal disease is an increasing problem worldwide driven by aging of the population and increased prevalence of metabolic disorders and cardiovascular disease. Currently, kidney... Show moreEnd stage renal disease is an increasing problem worldwide driven by aging of the population and increased prevalence of metabolic disorders and cardiovascular disease. Currently, kidney transplantation is the only curative option, but donor organ shortages greatly limit its application. Regenerative medicine has the potential to solve the shortage by using stem cells to grow the desired tissues, like kidney tissue. Immune rejection poses a great threat towards the implementation of stem cell derived tissues and various strategies have been explored to limit the immune response towards these tissues. However, these studies are limited by targeting mainly T cell mediated immune rejection while the rejection process also involves innate and humoral immunity. In this study we investigate whether inhibition of the complement system in human induced pluripotent stem cells (iPSC) could provide protection from such immune injury. To this end we created knock-in iPSC lines of the membrane bound complement inhibitor CD55 to create a transplant-specific protection towards complement activation. CD55 inhibits the central driver of the complement cascade, C3 convertase, and we show that overexpression is able to decrease complement activation on both iPSCs as well as differentiated kidney organoids upon stimulation with anti-HLA antibodies to mimic the mechanism of humoral rejection. Show less
