Abstract 74: Exploring the expression of cancer-intrinsic PD1 in osteosarcoma cells: A comprehensive proteomic analysis of its functional landscape

Abstract Cancer treatment was revolutionized by immune checkpoint inhibitors (ICIs) targeting the PD1/PDL1 axis - a brake to the immune system. Despite remarkable success, some patients do not respond to therapy or rapidly deteriorate due to an unclear mechanism. As reported for several types of tumors, PD1 receptor is not solely expressed on immune cells but also on cancer cells. Depending on the tumor type, it may act either as a suppressor or a tumor promoter. The limited response to ICIs was also reported in osteosarcoma (OSA), the most common type of bone cancer, mostly affecting children and young adults. With no significant improvement in therapy over the past 30 years, there is an urgent need for therapeutic advancement. This study aimed to determine if PD1 protein is expressed in OSA and whether it has a functional role, potentially affecting the response to ICIs. Strikingly, our results revealed both surface and intracellular PD1 expression in U2OS osteosarcoma cells, confirmed by Western Blot, flow cytometry and immunofluorescence analyses. siRNA-mediated PDCD1 gene silencing significantly increased cell migration and viability in scratch and viability assays. LC-MS-based global proteomics showed that PDCD1 knockdown markedly affects U2OS cells’ proteomic landscape compared to the control. To dissect PD1 receptor downstream signaling effects, we subjected the identified proteins to GO and GSEA large-scale data interpretation tools. The results strongly suggested that PDCD1 silencing leads to the enrichment of proteins involved in cell growth, migration and motility, corresponding to the cellular effects we initially observed. Importantly, the analysis clearly indicated positive regulation of MAPK cascade in cells with perturbed PD1 expression, aligning with PD1 mediated MAPK regulation in T cells. To reveal the mechanism underlying PD1 signaling, we focused on proteins with significantly changed expression in response to the knockdown. Using the STRING database, we created their interacting network, revealing that numerous proteins are closely related, and the majority of them are phosphoproteins, indicating significance in cellular signaling. Remarkably, many of the STRING-analyzed proteins belong to intersecting pathways, such as AKT1S1 and LAMTOR proteins, the critical components for mTORC1 signaling, or selected members of the Src family kinases along with their negative regulator CSK. In summary, we report previously undiscovered functional expression of PD1 in OSA cells, providing insights into the landscape of PD1 downstream signaling. Our studies indicate the role of PD1 in regulating cell growth, proliferation, and motility, suggesting osteosarcoma-expressed PD1 as a tumor suppressor. Further studies are urgently needed to investigate its therapeutic significance and explore if we could make ICIs more effective to treat osteosarcoma. Citation Format: Katarzyna Joanna Dziubek, Jakub Faktor, Ted Hupp, Maciej Parys, Sachin Kote. Exploring the expression of cancer-intrinsic PD1 in osteosarcoma cells: A comprehensive proteomic analysis of its functional landscape 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 (6Suppl): Abstract nr 74.