Abstract 7110: Characterizing pY240-PTEN:Ki-67 interactions and development of a PPI disrupting peptide

Abstract Glioblastoma (GBM) is the most aggressive and common malignant primary brain tumor. Standard of care for GBM patients involves chemotherapy and ionizing radiation which induce DNA damage to kill tumor cells; however resistance to these treatment modalities commonly develop through various mechanisms. One novel mechanism of radioresistance is through enhanced homologous recombination (HR) DNA damage response (DDR) mediated by nuclear-localized tyrosine-phosphorylated PTEN (pY240-PTEN) recruited to chromatin through interaction with the PP1 Binding Domain (PP1BD) of Ki-67. PTEN is known to play an important tumor-suppressive role and is found to be mutated in approximately 40% of GBMs. A Y240F-PTEN knock-in mouse model has shown that loss of Y240 phosphorylation results in IR sensitivity. Ki-67 dependent, pY240-PTEN facilitated DDR may be regulated by currently uncharacterized interactions within the Ki-67 PP1BD and determination of these interactions may enable development of complex-disrupting peptides capable of radiosensitizing glioma cells. To this end, I have identified residues in the Ki-67 PP1BD that are essential for pY240-PTEN interaction via streptavidin pulldown of N-terminal biotinylated peptides synthesized with alanine mutations at all residues spanning the Ki-67 PP1BD. Orthogonally, glioma cell lines have been engineered that stably express Ki-67 minigene constructs harboring mutation of the identified PP1BD residues. The effects of these mutations on PTEN:Ki-67 interaction, as well as DDR, HR, chromatin accessibility, and colony formation efficiency are currently being interrogated. Competitor peptides have been designed based on candidate Ki-67 residues and evaluated for their ability to bind PTEN. Further optimization of these peptides has led to candidates that are being assessed for stability and potential to radiosensitize glioma cells. In addition, regulation of pY240-PTEN:Ki-67 interaction by posttranslational modification of the Ki-67 PP1BD by aurora B kinase and cyclin dependent kinase 1 has been investigated through pulldown of biotinylated peptides harboring phosphorylated candidate residues. Lastly, Ki-67 has been knocked out in U87 glioma cells, as well as TS528 glioma stem like cells, and the resulting effect on IR sensitivity and DDR is being investigated in the absence, or presence of PTEN. Overall, this project has begun to characterize the regulation and critical molecular interactions between pY240-PTEN and Ki-67, elucidating potential strategies that could disrupt these interactions to enhance the efficacy of radiotherapy. Citation Format: Brandon Jones, Sejal Patel, Frank Furnari. Characterizing pY240-PTEN:Ki-67 interactions and development of a PPI disrupting peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7110.