The Problem: Mesothelioma is a devastating cancer where the majority of patients will die from their disease. The last significant advance in treatment for mesothelioma was the introduction of immunotherapy, which was approved by the FDA in 2020. However, the benefits of current systemic therapeutic approaches to mesothelioma are modest and not curative. New treatments are needed. Knowledge to be Addressed: The long-term goal of this laboratory is to identify key principles of cancer biology that will allow the appropriate selection of the most effective combination therapies based on upfront molecular signatures. The objective of this application as a next step in the pursuit of this long-term goal is to validate that dihydroorotate dehydrogenase (DHODH) is a viable target to inhibit as part of a treatment strategy against mesothelioma. DHODH is a key (and rate-limiting) enzyme in de novo pyrimidine synthesis and has been demonstrated to be highly expressed in several cancers, including mesothelioma. Hypothesis: Our central hypothesis is that inhibition of DHODH will result in decreased cell proliferation in vitro and decreased tumor growth in vivo. Secondarily, we also hypothesize that 1) combination treatment with DHODH inhibition plus dinaciclib (inhibitor of CDKs 1, 2, 5, and 9 and 2) combination treatment with DHODH inhibition with immune checkpoint inhibition will result in synergistic effects on cell proliferation in vitro and tumor growth in vivo. Preliminary Studies: We have demonstrated that the DHODH inhibitor BAY2402234 potently inhibits mesothelioma cell proliferation in vitro. In addition, we have demonstrated that the combination of BAY2402334 and dinaciclib is synergistic in decreasing cell proliferation in vitro. Specific aims: 1) Validate that inhibition of DHODH inhibits mesothelioma growth and proliferation. 2) Identify the mechanisms of DHODH inhibition on related targetable pathways in mesothelioma to validate a drug combination to inform the design of a near-term clinical trial. Significance and Innovation: The proposed work is innovative because inhibition of DHODH has never been studied in mesothelioma before. Positive results in this study will validate DHODH as a target to purse in treatment strategies against mesothelioma. In addition, establishing a link between pyrimidine synthesis, several CDKs, and immune checkpoint inhibition will be novel. The proposed research will de-risk future preclinical and clinical studies and will aid in the design a clinical trial evaluating the effectiveness of DHODH inhibition in the treatment of mesothelioma.