Saturday, April 20, 2024
4/20/2024
Project Summary Glioblastoma (GBM) is the most aggressive and common malignant primary brain tumor1. Standard of care for GBM patients involves chemotherapy and ionizing radiation (IR) which induce DNA damage to kill tumor cells; however resistance to these treatment modalities commonly develop through various mechanisms2–4. One novel mechanism of radioresistance identified in the Furnari lab 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-675. PTEN (Phosphatase and Tensin Homolog) is known to play an important tumor-suppressive role and is found to be mutated in approximately 40% of GBMs6. While PTEN cytosolic lipid phosphatase activity inhibition of PI3K signaling has been well established7,8, the function of nuclear PTEN remains less clear. A Y240F-PTEN knock-in mouse model has shown that loss of Y240 phosphorylation results in IR sensitivity5. Preliminary experiments also indicate PTEN:Ki-67 interaction can be disrupted by overexpressed PP1¿, which binds to the RVxF small linear interacting motif (SLiM) located in the Ki-67 PP1BD, and through use of a competitor peptide based on the Ki- 67-Repoman SLiM (KiR-SLiM)5,9. I hypothesize that Ki-67 dependent, pY240-PTEN facilitated DDR is regulated by currently uncharacterized interactions within SLiMs of the Ki-67 PP1BD and that characterization of these interactions will enable development of complex-disrupting peptides capable of radiosensitizing glioma cells. I will firstly identify residues in the Ki-67 KiR-SLiM motif that are essential for pY240-PTEN interaction. This will be accomplished via streptavidin pulldown of N-terminal biotinylated peptides designed based on the KiR-SLiM. Orthogonally, Ki-67 minigene constructs harboring mutation of the identified PP1BD residues will be utilized in GBM models to examine their effects on PTEN:Ki-67 interaction, as well as DDR, HR, chromatin accessibility, and colony formation efficiency. Competitor peptides will be designed based on candidate Ki-67 residues and evaluated for their ability to disrupt PTEN:Ki-67 interaction and radiosensitize glioma cells. Secondly, regulation of pY240-PTEN:Ki-67 interaction by posttranslational modification of Ki-67 SLiMs by aurora B kinase and cyclin dependent kinase 1 will be investigated through site directed mutagenesis and pharmacological inhibition. Discoveries regarding SLiM regulation will be incorporated into competitor peptides to optimize specific disruption of the pY240-PTEN:Ki-67 complex. Lastly, mutations determined to have the greatest impact on pY240-PTEN:Ki-67 interactions will be CRISPR edited into oncogenic mouse astrocytes with WT versus knock-in Y240F PTEN and the effects of these mutations will be investigated in vitro and in vivo5. Overall, this project aims to characterize the regulation and critical molecular interactions between pY240-PTEN and Ki-67, culminating in strategies that will disrupt these interactions to enhance the efficacy of radiotherapy.
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