Abstract 403: Characterization of PROTACs for targeting tissue transglutaminase 2 (TG2) in cancer

Abstract Introduction: Tissue transglutaminase (TG2) is a multifunctional enzyme involved in the interaction between fibronectin and integrins, as well as in GTP hydrolysis. TG2 is upregulated and active in several cancers, including ovarian cancer (OC). Due to its multifunctional nature and involvement in various processes that promote cancer progression, targeting TG2 therapeutically has proven challenging. Here, we explore the development of PROteolysis TArgeting Chimeras (PROTACs) against TG2 as a strategy to induce protein degradation and target TG2 in cancer. Materials and Methods: OVCAR5 or SKOV3 OC cells expressing TG2 were treated with different concentrations of PROTACs for 6 or 24 hours. TG2 expression was measured via Western blot (WB) and TG2 activity was assessed by using an enzymatic activity assay. PROTACs that induced TG2 degradation were further validated through reverse phase protein array (RPPA) analysis, adhesion assays, migration assays, and in-vivo omentum adhesion assays. Results: Over 50 compounds were screened. WB analysis showed significant (p < 0.05) degradation of TG2 using three PROTACs: P74, P404, and P405, in both SKOV3 and OVCAR5 cells at 24 hours of treatment, without decreased cell viability (p < 0.05). Degradation was stable at 48 hours. Migration and adhesion assays revealed that PROTACs reduced both adhesion and migration capabilities of the cells at 24 hours (p < 0.005). Additionally, PROTACs reduced the enzymatic activity of TG2, demonstrating the ability to disrupt other TG2 functions (p < 0.001). MG132 treatment prevented PROTAC induced degradation of TG2 showing that the degradation is proteasome dependent. (p < 0.05). Reverse phase proteomic analysis (RPPA) of P74-treated cells identified 136 differentially expressed proteins, including a reduction in TG2 and an increase in VHL expression. The most affected pathways in PROTAC-treated cells included cell adhesion, the PI3K/AKT pathway, and others (FDR < 0.05, fold change < 0.5). PROTACs P74 and P405 also reduced in vivo adhesion of OVCAR5 cells to the omentum in an intraperitoneal omental colonization assay (p < 0.05). Conclusion: Treatment of OC cells with PROTACs P74, P404, and P405 significantly reduced TG2 expression levels, disrupted migration and adhesion, and impaired TG2 enzymatic activity. Furthermore, PROTACs reduced cell adhesion in-vivo, highlighting their potential as therapeutic agents Citation Format: Andres Felipe Valdivia, Purav Vagadia, Natalia Masnica, Joshua Li Zhu, Vanessa Hernandez, Ana Maria Isac, Gary Schiltz, Daniela Matei. Characterization of PROTACs for targeting tissue transglutaminase 2 (TG2) in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 403.