Testing of the Survivin Suppressant YM155 in a Large Panel of Drug-Resistant Neuroblastoma Cell Lines

The survivin suppressant YM155 is a drug candidate for neuroblastoma. Here, we tested YM155 in 101 neuroblastoma cell lines (19 parental cell lines, 82 drug-adapted sublines). Seventy seven (77) cell lines displayed YM155 IC$_{50}$s in the range of clinical YM155 concentrations. ABCB1 was an important determinant of YM155 resistance. The activity of the ABCB1 inhibitor zosuquidar ranged from being similar to that of the structurally different ABCB1 inhibitor verapamil to being 65-fold higher. ABCB1 sequence variations may be responsible for this, suggesting that the design of variant-specific ABCB1 inhibitors may be possible. Further, we showed that ABCC1 confers YM155 resistance. Previously, p53 depletion had resulted in decreased YM155 sensitivity. However, $TP53$-mutant cells were not generally less sensitive to YM155 than $TP53$ wild-type cells in this study. Finally, YM155 cross-resistance profiles differed between cells adapted to drugs as similar as cisplatin and carboplatin. In conclusion, the large cell line panel was necessary to reveal an unanticipated complexity of the YM155 response in neuroblastoma cell lines with acquired drug resistance. Novel findings include that ABCC1 mediates YM155 resistance and that YM155 cross-resistance profiles differ between cell lines adapted to drugs as similar as cisplatin and carboplatin

Studies on the interaction of the inhibitor of apoptosis protein Survivin with DNA-dependent protein kinase to modulate DNA double-strand break repair

During the last years, the ability of tumour cells to evade apoptosis was considered to be an important mechanism to develop resistance against tumour therapies. In this context, members of the inhibitor of apoptosis protein (IAP) family gained increasing attention. Survivin, the smallest member of the IAP family, is a functionally unique protein that is involved in a variety of molecular mechanisms and cellular networks including cell proliferation, regulation of apoptosis and metastasis formation. Furthermore, an overexpression of Survivin in the tumour tissue was correlated with tumour progression as well as a decreased survival of the patients. Besides inhibition of apoptosis and its role as a member of the chromosomal passenger complex, Survivin was also found being accumulated in the nucleus after irradiation. That accumulation was linked to a modulation of DNA double-strand break repair due to its interaction with DNA repair factors such as the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). The aim of this thesis was to gain further insight on the molecular mechanisms facilitating a Survivin-mediated regulation of DNA repair by characterising the interaction between Survivin and DNA-PKcs, a major enzyme in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway, in more detail. Docking of Survivin wild type (wt) and a X-linked IAP (XIAP) binding site deletion mutant (ΔXIAP) of Survivin to DNA-PKcs was evaluated in colorectal cancer SW480 and glioblastoma LN-229 cells via immunoprecipitation experiments. These experiments indicated that recombinant Survivin (wt) was able to co-immunoprecipitate with DNA-PKcs in both lines while the ΔXIAP mutant of Survivin did not show complexation to DNA-PKcs. In case of the Aurora-B kinase it has been reported that Survivin stimulates Aurora-B kinase activity by binding to the catalytic domain. In analogy, an interaction of Survivin with the kinase domain of DNA-PKcs (PI3K) was analysed by different methods, including GST pulldown assay, NanoLuc Binary Technology (NanoBiT®) complementation assay and flow cytometry-based Förster resonance energy transfer (FRET). All of these methods confirmed an interaction between Survivin and the PI3K domain of DNA-PKcs, indicating that Survivin is binding directly to the kinase domain but not to other domains like the HEAT1 and FATC domain. Additionally, functional analysis, such as autophosphorylation of serine 2056 of DNA-PKcs, revealed a decreased DNA-PK activity after Survivin knockdown in both SW480 and LN-229 cells. Finally, attenuation of endogenous Survivin in the ΔXIAP mutant of Survivin resulted in a decreased DNA-PK activity measured by SignaTECT kinase assay, while recombinant Survivin (wt) rescued DNA-PK activity following irradiation with 4 Gy. In conclusion these findings for the first time indicate that Survivin not only interacts with DNA-PKcs but directly binds to its kinase domain. Thus, it modulates DNA-PKcs kinase activity and as a consequence repair of radiation induced DNA double-strand breaks. These results add a further facet to the plethora of functions exerted by the nodal protein Survivin in the cellular radiation response in cancer cells

Studies on the interaction of the inhibitor of apoptosis protein Survivin with DNA-dependent protein kinase to modulate DNA double-strand break repair

During the last years, the ability of tumour cells to evade apoptosis was considered to be an important mechanism to develop resistance against tumour therapies. In this context, members of the inhibitor of apoptosis protein (IAP) family gained increasing attention. Survivin, the smallest member of the IAP family, is a functionally unique protein that is involved in a variety of molecular mechanisms and cellular networks including cell proliferation, regulation of apoptosis and metastasis formation. Furthermore, an overexpression of Survivin in the tumour tissue was correlated with tumour progression as well as a decreased survival of the patients. Besides inhibition of apoptosis and its role as a member of the chromosomal passenger complex, Survivin was also found being accumulated in the nucleus after irradiation. That accumulation was linked to a modulation of DNA double-strand break repair due to its interaction with DNA repair factors such as the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). The aim of this thesis was to gain further insight on the molecular mechanisms facilitating a Survivin-mediated regulation of DNA repair by characterising the interaction between Survivin and DNA-PKcs, a major enzyme in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway, in more detail. Docking of Survivin wild type (wt) and a X-linked IAP (XIAP) binding site deletion mutant (ΔXIAP) of Survivin to DNA-PKcs was evaluated in colorectal cancer SW480 and glioblastoma LN-229 cells via immunoprecipitation experiments. These experiments indicated that recombinant Survivin (wt) was able to co-immunoprecipitate with DNA-PKcs in both lines while the ΔXIAP mutant of Survivin did not show complexation to DNA-PKcs. In case of the Aurora-B kinase it has been reported that Survivin stimulates Aurora-B kinase activity by binding to the catalytic domain. In analogy, an interaction of Survivin with the kinase domain of DNA-PKcs (PI3K) was analysed by different methods, including GST pulldown assay, NanoLuc Binary Technology (NanoBiT®) complementation assay and flow cytometry-based Förster resonance energy transfer (FRET). All of these methods confirmed an interaction between Survivin and the PI3K domain of DNA-PKcs, indicating that Survivin is binding directly to the kinase domain but not to other domains like the HEAT1 and FATC domain. Additionally, functional analysis, such as autophosphorylation of serine 2056 of DNA-PKcs, revealed a decreased DNA-PK activity after Survivin knockdown in both SW480 and LN-229 cells. Finally, attenuation of endogenous Survivin in the ΔXIAP mutant of Survivin resulted in a decreased DNA-PK activity measured by SignaTECT kinase assay, while recombinant Survivin (wt) rescued DNA-PK activity following irradiation with 4 Gy. In conclusion these findings for the first time indicate that Survivin not only interacts with DNA-PKcs but directly binds to its kinase domain. Thus, it modulates DNA-PKcs kinase activity and as a consequence repair of radiation induced DNA double-strand breaks. These results add a further facet to the plethora of functions exerted by the nodal protein Survivin in the cellular radiation response in cancer cells

Ionizing Radiation Induces Morphological Changes and Immunological Modulation of Jurkat Cells

Impairment or stimulation of the immune system by ionizing radiation (IR) impacts on immune surveillance of tumor cells and non-malignant cells and can either foster therapy response or side effects/toxicities of radiation therapy. For a better understanding of the mechanisms by which IR modulates T-cell activation and alters functional properties of these immune cells, we exposed human immortalized Jurkat cells and peripheral blood lymphocytes (PBL) to X-ray doses between 0.1 and 5 Gy. This resulted in cellular responses, which are typically observed also in naïve T-lymphocytes in response of T-cell receptor immune stimulation or mitogens. These responses include oscillations of cytosolic Ca2+, an upregulation of CD25 surface expression, interleukin-2 and interferon-γ synthesis, elevated expression of Ca2+ sensitive K+ channels and an increase in cell diameter. The latter was sensitive to inhibition by the immunosuppressant cyclosporine A, Ca2+ buffer BAPTA-AM, and the CDK1-inhibitor RO3306, indicating the involvement of Ca2+-dependent immune activation and radiation-induced cell cycle arrest. Furthermore, on a functional level, Jurkat and PBL cell adhesion to endothelial cells was increased upon radiation exposure and was highly dependent on an upregulation of integrin beta-1 expression and clustering. In conclusion, we here report that IR impacts on immune activation and functional properties of T-lymphocytes that may have implications in both toxic effects and treatment response to combined radiation and immune therapy in cancer patients

image_1_Ionizing Radiation Induces Morphological Changes and Immunological Modulation of Jurkat Cells.tiff

<p>Impairment or stimulation of the immune system by ionizing radiation (IR) impacts on immune surveillance of tumor cells and non-malignant cells and can either foster therapy response or side effects/toxicities of radiation therapy. For a better understanding of the mechanisms by which IR modulates T-cell activation and alters functional properties of these immune cells, we exposed human immortalized Jurkat cells and peripheral blood lymphocytes (PBL) to X-ray doses between 0.1 and 5 Gy. This resulted in cellular responses, which are typically observed also in naïve T-lymphocytes in response of T-cell receptor immune stimulation or mitogens. These responses include oscillations of cytosolic Ca²⁺, an upregulation of CD25 surface expression, interleukin-2 and interferon-γ synthesis, elevated expression of Ca²⁺ sensitive K⁺ channels and an increase in cell diameter. The latter was sensitive to inhibition by the immunosuppressant cyclosporine A, Ca²⁺ buffer BAPTA-AM, and the CDK1-inhibitor RO3306, indicating the involvement of Ca²⁺-dependent immune activation and radiation-induced cell cycle arrest. Furthermore, on a functional level, Jurkat and PBL cell adhesion to endothelial cells was increased upon radiation exposure and was highly dependent on an upregulation of integrin beta-1 expression and clustering. In conclusion, we here report that IR impacts on immune activation and functional properties of T-lymphocytes that may have implications in both toxic effects and treatment response to combined radiation and immune therapy in cancer patients.</p