LAUSR

GB is the most common, most aggressive primary brain tumor with a deleterious prognosis. Novel approaches to improve therapy are urgently needed. ASPP2 is a tumor suppressor, directly interacting with p53 to induce apoptosis. We recently identified a dominant-negative isoform of ASPP2, named ASPP2kappa(k), lacking the important p53 binding sites. We now show that ASPP2k is highly expressed and functionally active in high grade glioma and that silencing of ASPP2k reactivates induction of apoptosis via p53. Freshly resected glioma (n=76) were assessed for ASPP2k expression. Isogenic, p53wt cell models, stably suppressing or overexpressing ASPP2k were established in A-172 and U-251 cells and ex vivo from GB samples and the role of ASPP2k in induction of apoptosis, cellular proliferation, migration and invasion was assessed. A proteome array allowed to study pathways involved in ASPP2k signaling. An ASPP2k knock-down, NOD/SCID xenotransplant mouse model was established to study the role of ASPP2k in tumor engraftment and progression. Frequent overexpression of ASPP2k in glioma was confirmed in virtually all patients analyzed. Expression patterns varied widely (up to 40-fold compared to tumor-free tissue). Expression of ASPP2k thereby correlates with tumor biology - with highest levels found in aggressive grade IV GB. We further demonstrate that expression of ASPP2k results in impaired induction of apoptosis in all GB models - and knock down of ASPP2k renders cells more susceptible towards temozolamide (TMZ), γ-irradiation (avg. 25% TMZ, 40% irradiation) or combinational approaches compared to controls. Vice versa, overexpression of ASPP2κ further attenuates induction of apoptosis. Even more, ASPP2k directly affects other cellular functions in GB, rendering cells to a more aggressive phenotype with increased proliferation (approx. 25%), migration (approx. 65%) and invasion rates compared to the knock down strains. In line, silencing of ASPP2k resulted in strong upregulation of crucial p53 phosphorylation sites (S15/S46), as well as cell cycle regulators (p21, p27 and pRAD) in a proteome array, arguing for re-activation of crucial p53-mediated pathways in the ASPP2k silenced cells. Results were confirmed by western immunoblotting and FACS analyses. Ongoing xenotransplant mouse models confirm that silencing of ASPP2k in GB models attenuates tumor engraftment, development and progression in vivo. In summary, we here show that ASPP2k is highly expressed in GB and directly contributes to aggressiveness of the disease as well as resistance towards therapy. Silencing of ASPP2k thereby re-activates crucial p53 pathways, resulting in re-activation of p53-mediated apoptosis pathways as well as re-installation of cell cycle checkpoints. Future studies evaluating ASPP2k as a prognostic/predictive model and potential target for therapy are warranted. Citation Format: Marlon Hafner, Alessia Ruiba, Leonie Kampa, Vincent Kälin, Marian Neidert, Konrad Buchauer, Guido Henke, Ludwig Plasswilm, Marcus M. Schittenhelmn, Kerstin M. Kampa-Schittenhelm. Inhibition of oncogenic ASPP2kappa(k), a dominant-negative isoform of the tumorsuppressor ASPP2, results in reactivation of p53 in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2535.