Impact of cellular localization of Survivin on DNA damage response, DNA repair, replication, cytotoxicity and clastogenicity upon exposure to topoisomerase I inhibitors

• Applicant: Professorin Maja Tomicic-Christmann, Ph.D.
• Subject Area: Toxicology, Laboratory Medicine
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 462114959

Survivin (also known as BIRC5), the smallest inhibitor of apoptosis (IAP) is an evolutionary conserved eukaryotic protein that can inhibit cell death (apoptosis) and is essential for cell division (mitosis). It can passively diffuse into the nucleus but is actively exported from the nucleus. Survivin is physiologically expressed during development and in actively proliferating cells, but is highly upregulated in most, if not all cancers. Consequently, Survivin has received a significant attention as a potential oncotherapeutic target. In the past twenty years a lot of information on its structure, domains, key interacting partners and signaling, transcriptional regulation of its gene (BIRC5), post-translational modifications by phosphorylation or acetylation, or on its role in migration and angiogenesis has been collected. The aim of this application is to investigate whether Survivin expression or distinct cellular localization (nucleus vs. cytoplasm) influences the sensitivity ofcolorectal cancer (CRC) cells to topoisomerase I (TOP1) inhibitors and which mechanisms are behind it. Also, we aim to examine what function Survivin has in the DNA damage response (DDR), DNA repair, apoptosis, autophagy, senescence, as well as in replication and clastogenicity. For this purpose we have generated different Survivin-GFP expressing clones, which on one hand express the fusion protein predominantly in the cytoplasm and on the other primarily in the nucleus, and have verified the nuclear translocation and accumulation of exogenous and endogenous Survivin upon irinotecan exposure. In cooperation with Synthego, we are also planning to use CRISPR-Cas9 technology to produce double knock-in cell clones that accumulate endogenous Survivin in the nucleus. Also, we experienced huge differences in clonogenic survival (colony-forming assay) upon irinotecan, with high survival rates in the clones expressing cytosolic Survivin vs. those with the mutated export sequence (accumulation of Survivin in the nucleus). In order to elucidate the underlying mechanisms, on the one hand, nuclear interaction partners of Survivin should be identified by means of mass spectrometry-based proteome / interactome analysis in order to gain knowledge for a potential generation of therapeutically usable inhibitors that would inhibit the protein-protein interactions of Survivin. In addition, we want to investigate whether Survivin and XIAP-expressing CRC cell clones can be sensitized to TOP1-inhibiting compounds by IAP inhibitors and inhibitors of DDR and DNA repair.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Markus Christmann