(P 19) INVOLVEMENT OF O-GLCNAC TRANSFERASE IN RESISTANCE TO DOXORUBICIN AND CELL DEATH IN PRECLINICAL MODELS OF LEIOMYOSARCOMA AND DESMOID TUMORS

Objective: Different histopathological subtypes of sarcoma exhibit distinct aggressive properties and clinical behavior. Desmoid type fibromatosis (DTF) originates from fibroblasts, is locally aggressive, and often recurs post-surgery but typically does not metastasize. In contrast, leiomyosarcomas (LMS) are malignant tumors that arise from smooth muscle cells and frequently metastasize to distant organs. Standard treatment for sarcoma shows unsatisfactory response rates, and emerging targeted therapies and immunotherapies yield variable outcomes. Therefore, it is highly needed to discover new therapeutic targets for sarcoma. We seek to address this challenge by studying onco-metabolism, specifically the role of hexosamine biosynthesis pathway (HBP) that provides precursors for protein glycosylation. We previously found that GFPT2, the first and rate-limiting enzyme of HBP, is associated with poor prognosis in LMS patients (PMID: 33941787), and is frequently expressed also in DTF tumors (unpublished data). In many types of cancer, aberrant glycosylation affects oncogenes, tumor suppressors, and protein kinases, contributing to tumor development and chemotherapy resistance. We are in particular interested in the type of glycosylation called O-GlcNAcylation, the modification of proteins by O-GlcNAc transferase (OGT) that occurs downstream of HBP. Studying this pathway in parallel in LMS and DTF will offer insights in mesenchymal neoplasia with varied biologic and clinical behaviors. Here, we sought to evaluate the effect of OGT inhibition alone and in combination with standard chemotherapy in preclinical models of LMS and DTF.

Methods: We used 6 LMS cell lines: 3 uterine LMS lines from ATCC (SK-LMS-1, SK-UT-1, SK-UT-1B), and 3 cell lines derived in the laboratory of Dr. Fletcher (LMS03, LMS04, LMS05). We also obtained DES23 cell line of DTF derived in the laboratory of Dr. Pollock. We characterized the baseline levels of OGT and O-GlcNAc expression in these cell lines by Western Blot. We also evaluated the baseline gene expression of the OGT gene using qRT-PCR. Next, we treated these cell lines with doxorubicin (DOX), OSMI-1 (inhibitor of OGT) and the combination of thereof for 24 hours, to study the effect of OGT inhibition alone or in combination with standard treatment. We measured the cell viability using Cell Titer Glo assay and evaluated apoptosis using Annexin V/7AAD flow cytometry-based assay.

Results: We demonstrated that the baseline OGT gene expression and protein levels were similar across all cell lines, with comparable O-GlcNAcylation levels. We found that OSMI-1 treatment efficiently decreased cell viability in all 7 cell lines except SK-LMS-1 (Figure1). SK-LMS-1 showed resistance to OSMI-induced cell death and apoptosis. Combining OSMI-1 with DOX also did not decrease cell viability in SK-LMS-1. However, treatment with OSMI-1 and DOX as a single agents and in combination increased cell death in cell lines SK-UT-1, SK-UT-1B, LMS05, and DES23 Figure1). Interestingly, OSMI-1 but not DOX decreased cell viability in LMS03 and LMS04 cell lines.

Conclusion: We demonstrated that targeting OGT decreased cell viability and could improve the efficiency of standard chemotherapy in selected cell lines. These results warrant further studies to explore therapeutic potential of targeting HBP in LMS and DTF. In the next steps, we will test cell death after treatment with an expanded panel of 4 OGT inhibitors (OSMI-1, OSMI-4, BADGP, ST045849). We will also use our newly developed 3D models of LMS and DTF to evaluate cell proliferation, invasion, hypoxia, metabolism, and cell death. Finally, we aim to combine OGT inhibition with other standard therapies for LMS and DTF, to assess potential combinatory effects on viability and drug resistance. In the next steps, we will also seek to identify O-GlcNAcylated proteins involved in tumor progression and drug resistance in LMS and DTF. These studies will further elucidate the therapeutic effect of HBP-targeting in LMS and DTF.