Abstract 2995: Inhibition of pyrimidine synthesis and the cell cycle in mesothelioma
Authors: George Scaria, Marian Kratzke, Stephen Porter, Betsy Kren, Mark A. Klein
Journal: Cancer Research (2025)

Introduction: Novel approaches to the treatment of advanced mesothelioma are needed, as mesothelioma is considered incurable in the vast majority of cases. Targeting 1) pyrimidine biosynthesis via inhibition of dihydroorotate dehydrogenase (DHODH) and 2) DHODH in combination with inhibition of select cyclin-dependent kinases (CDKs) are potential approaches for treating mesothelioma, as the cell cycle is altered most mesothelioma cells. Methods: We conducted cell proliferation assays in mesothelioma cell lines to determine the IC50 for the DHODH inhibitor BAY2402234 as compared with the older generation DHODH inhibitor brequinar. BAY2402334, brequinar, or control were incubated with mesothelioma cells for 72 hours and cell viability was determined using a Cell Counting Kit-8 spectrophotometric assay. Experiments were conducted with and without uridine at 100 mM to determine the effect of “uridine rescue” on the IC50 values for each DHODH inhibitor. We also determined mRNA expression via RNA-sequencing (RNA-Seq) and protein expression via immunoblotting of key mRNA transcripts and corresponding proteins. We then conducted cell proliferation assays to determine if the combination of BAY2402234 plus dinaciclib (inhibitor of CDKs 1, 2, 5, and 9) could be additive or synergistic. Results: BAY2402234 exhibited a significant degree of potency against 4 mesothelioma cell lines with IC50 values ranging from 55 picomolar (pM) to 4.63 nanomolar (nM). Uridine rescue resulted in a significant shift in the fitted curves (suggesting decreased efficacy of BAY2402234 due to uridine supplementation), which supports the notion that DHODH is a major mechanism of BAY2402234 as anticipated. Overall, BAY2402234 was between 59 to 1,811 times more potent than brequinar against mesothelioma cells. After exposure to BAY2402334 or control for 48 hours, H2373 cells were evaluated via RNA-Seq and immunoblotting. Of note, PD-L1 mRNA increased approximately 7-fold, and PD-L1 protein expression increased 1.5-2-fold. In addition, mRNA expression of CDKN1A (p21 protein), Cyclin D1, Cyclin A1, and CDK1 increased. Cytokine pathway interactions also increased. For several concentration combinations, the combination index was &amp;lt; 1, suggesting a synergistic effect of that combination of drugs on decreasing cell proliferation. Conclusions: BAY2402234 is an extremely potent inhibitor of cell growth in multiple mesothelioma cell lines and 59-1,881 times more potent than brequinar; 2) BAY2402234 and dinaciclib act synergistically, suggesting an interaction between pyrimidine biosynthesis and one or more of the following CDKs: CDK1, CDK2, CDK5, and/or CDK9; and 3) BAY2402234 results in increased PD-L1 expression and increased cytokine pathway mRNA expression, suggesting the potential for enhancement of immune checkpoint blockade. Citation Format: George Scaria, Marian Kratzke, Stephen Porter, Betsy Kren, Mark A. Klein. Inhibition of pyrimidine synthesis and the cell cycle in mesothelioma [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 2995.