%0 Generic
%A Rösch, Lisa
%C Heidelberg
%D 2023
%F heidok:32850
%R 10.11588/heidok.00032850
%T ERBB and P-glycoprotein inhibitors break resistance in relapsed neuroblastoma through P-glycoprotein
%U https://archiv.ub.uni-heidelberg.de/volltextserver/32850/
%X Neuroblastoma is a pediatric tumor of the sympathetic nervous system that most commonly affects infants and young children. It is a disease with very variable outcomes, which range from spontaneous regression to aggressive disease. Particularly problematic are high-risk cases after relapse, with only 10% of patients surviving for longer than five years and chemotherapy resistance making treatment difficult. In this thesis, I examined differences between primary and relapsed patient gene expression data and between chemotherapy-sensitive and chemotherapy-resistant neuroblastoma cell lines to find a vulnerability that can be targeted to overcome chemotherapy resistance and lead to cell death. I further investigated which type of cell death was induced.  My analysis of gene expression data from primary and relapsed neuroblastoma patients suggested that the ERBB family of receptor tyrosine kinases, particularly ERBB4, plays a role in relapsed high-risk neuroblastomas. The ERBB family is crucial in development and is well known for its link to cancer. Furthermore, I functionally examined the resistance breaking effect of 15 clinically relevant drugs on a neuroblastoma cell line that was treated to be resistant to the standard-of-care chemotherapeutic vincristine (VCR). Tariquidar, an inhibitor of P-glycoprotein (P-gp), and afatinib, a FDA approved inhibitor of the ERBB family, were the two most efficient drugs in breaking resistance in this screen. P-gp/ABCB1 is a transmembrane transporter that very efficiently eliminates drugs and other xenobiotics from the cell and has long been recognized for its contribution to chemotherapy resistance.  Analysing gene expression datasets of more than 50 different neuroblastoma cell lines (primary and relapsed) and more than 160 neuroblastoma patient samples from the pediatric precision medicine platform INFORM (Individualized Therapy For Relapsed Malignancies in Childhood) confirmed a crucial role of P-gp in neuroblastoma resistance at relapse. The ERBB family appeared to play a minor part. I mechanistically investigated chemotherapy resistance through four pairs of vincristine- sensitive/resistant neuroblastoma sublines - each pair with the same genetic background - and two additional high-risk neuroblastoma cell lines. I effectively overcame resistance by the addition of the pan-ERBB family inhibitors afatinib and lapatinib, as well as the P-gp inhibitors tariquidar and verapamil, to VCR treatment, which synergistically reduced viability. Functional analyses of the ERBB downstream pathways, as well as ERBB4 knock-down, showed the resistance breaking effect of afatinib to be unrelated to ERBB signaling and suggested an off-target effect on P-gp. ABCB1 knock-down and analysis of transporter function confirmed that resistance was mediated through P-gp.  My analysis of programmed cell death, in particular apoptosis and ferroptosis, in neuroblastoma cell lines showed that apoptosis was consistently induced, while ferroptosis contributed to cell death in the IMR-32 cell line. The combination treatments of VCR with afatinib or tariquidar in VCR-sensitive and VCR-resistant cell lines led to cell death by apoptosis.  In summary, this study showed that P-gp is an important player in chemotherapy resistance in high-risk, relapsed neuroblastoma. ERBB4, although upregulated in individual cases or cell lines, did not contribute to resistance, and the sensitizing effect of the ERBB family inhibitor afatinib was due to an off-target effect on P-gp. I further showed that both apoptosis and ferroptosis are induced in neuroblastoma cell lines, with apoptosis being the dominant mechanism of cell death when chemotherapy resistance was broken.