Abstract C128: PT-112, a novel immunogenic cell death inducer, causes ribosomal biogenesis inhibition and organelle stress in cancer cells

Abstract

Abstract PT-112 is a novel immunogenic small molecule under Phase 2 development in metastatic castration-resistant prostate cancer and thoracic malignancies. PT-112 has been shown to release damage-associated molecular patterns, essential immune signaling components of immunogenic cell death (ICD), and to elicit anticancer immune responses. In addition, PT-112 causes mitochondrial and endoplasmic reticulum (ER) stress in vitro. Here, we sought to elucidate early molecular effects of PT-112 that culminate in cancer cell death. We assessed nucleolar protein phenotypes in human prostate cancer cells treated with PT-112 using antibody labeling, followed by confocal microscopy. We also conducted nascent RNA sequencing in human cancer cells (non-small cell lung, prostate, and renal cell carcinoma) following 1- and 6-hour PT-112 treatment. We then performed an analysis using Enrichr across multiple databases to identify biological pathways linked to clusters of differentially expressed genes (p ≤ 0.001) in PT-112-treated cells. Pathways affected by PT-112 (p ≤ 0.05) were sorted by statistical significance. In prostate cancer cells, PT-112 caused changes in nucleolar protein labeling. The most pronounced effect, observed after 8 or more hours of treatment, was rapid NPM1 relocation from nucleoli to nucleoplasm, a hallmark of nucleolar stress and disruption of ribosomal biogenesis (RiBi). PT-112 also induced gene expression changes in the treated cancer cell lines, resulting in statistically significant repression of pathways related to RiBi, ribosomal RNA (rRNA) processing and translation. Notably, a pattern across these pathways was the reduced expression of many ribosomal genes, consistent with RiBi inhibition. Additional analysis identified oncogenic pathways such as Myc and NF-κB significantly inhibited by PT-112. The degree of pathway suppression increased with treatment time. These results demonstrate that PT-112 rapidly causes nucleolar stress and RiBi inhibition, suggesting these are central to PT-112-induced cancer cell death. This mechanism could also explain the selective killing of cancer cells by PT-112, given that aberrant nucleolar size and number as well as ribosomal activity in cancer cells make them more sensitive to these drug effects. The broad consequences of impaired ribosome production and downstream protein translation may explain PT-112’s anticancer and immunogenic effects. Additionally, these disruptions have been reported to cause an increase in free ribosomal proteins and changes in nucleolar protein localization, which have been shown to induce ER stress and shuttle pro-apoptotic proteins to the mitochondria. These additional consequences of RiBi inhibition and nucleolar stress may contribute to or drive previously observed PT-112-induced cancer organelle stress and ICD. While further validation of these findings is needed, this work deepens our understanding of PT-112’s mechanism of action. Present findings, together with demonstrated clinical activity of PT-112, position it as a promising small molecule cancer immunotherapy. Citation Format: Christina Y. Yim, Maria T. Congenie, Hannah L. Johnson, Maureen G. Mancini, Fabio Stossi, Michael A. Mancini, Joey Azofeifa, Matthew R. Price, Johan Baeck, Tyler D. Ames. PT-112, a novel immunogenic cell death inducer, causes ribosomal biogenesis inhibition and organelle stress in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C128.