Background: TGM (Transglutaminase) family enzymes are Ca2+ dependent and engaged in specific posttranslational modifications by cross-linking extracellular matrix (ECM) proteins. ECM protein complex is more stable and resistant to enzymatic degradation. These enzymes modify proteins by cross-linking epsilon-(gamma-glutamyl), lysine, or (gamma-glutamyl) polyamine bonds. TGM2 (Transglutaminase 2) is a ubiquitous enzyme, and its expression is identified in the cytoplasm, plasma membrane, and the nucleus of various cells. The membrane-bound form of TGM2 binds GTP and functions as a G protein. TGM2 regulates cell survival, proliferation, migration, and modulation through ECM organization. We explored the role of TGM2 in Glioblastoma cells and its role in modulating the tumor microenvironment, cell proliferation, and survival. In addition to TGM2's role in GBM tumors, its expression has been linked to resistance to certain therapies in glioblastoma was investigated. Targeted modulation of macrophages with TGM2 inhibitors impacts the immune response against GBM, potentially enhancing the efficacy. Understanding the intricate interplay between TGM2, and GBM-associated macrophages is a key component of the tumor microenvironment (TME) and crucial for the development of targeted TGM2 inhibitors.

Materials and Methods: Human Transglutaminase 2 (hTGM2) activity of inhibitors was assayed using the fluorescent transamination assay incorporating dansylcadaverine into glutamine-donor substrate N, N-dimethyl casein, and DCC. Cellular efficacies of selected TGM2 inhibitors evaluated in U138 and U87 glioblastoma cells in CellTiter-Glo Luminescent cell viability assay.

Results: We have identified a series of novel small molecule TGM2 inhibitors based on our initial lead fragment hits with an IC50s 6-29 uM from our in-house MolecuLern fragment library. Fragments2Lead (F2L) optimizations strategies, synthesis, and SAR studies provided a lead SLX-9029 and SLX-9031 inhibitors with IC50s of 1.2, 1.5 uM in inhibiting TGM2 activity in our established cell-free fluorescent transamination assay. The initial TMG2 inhibitor leads further screening for TGM2-expressed U138 and U87 glioblastoma cells demonstrating promising cellular efficacies as singles agents. Additional GBM cell profiling, combination studies along with safety secondary pharmacology, cellular toxicities, and PK results will be discussed.

Conclusion: In summary, we identified promising lead TGM2 inhibitors that show activity in both cell-free and cell-based TGM2-expressed GMB cells. The TGM family members, selectivity, additional GBM cellular profiling, ADME, cellular toxicity, and in vivo PK studies are underway, and these results will be presented.

Citation Format: Hariprasad Vankayalapati, Chenyu Lin, Zhaoang li, Kyle Medley, David J. Bearss. Identification of a novel and selective Transglutaminase 2 (TGM2) inhibitors modulate tumor microenvironment in Glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB444.