The presence of abnormal nucleoli has been used as a marker for aggressive cancers for over 100 years. The nucleolus is the location for ribosomal RNA (rRNA) biogenesis, which requires approximately 80% of the energy generated by cancer cells. The first step of rRNA biogenesis is the transcription of pre-rRNA, which involves the binding of TIF-IA/RNA Pol I to the UBF/SL1 complex at the rDNA promoter. Many oncogenic mutations, such as Myc amplification and AKT activation, have been associated with the promotion of pre- rRNA synthesis. However, as pre-rRNA synthesis is a housekeeping function, direct targeting of pre-rRNA synthesis for cancer therapy has been controversial. Lung cancer is the leading cause of cancer death in the United States, with the majority of patients having non- small cell lung cancer (NSCLC). The 3rd most commonly mutated gene in NSCLC adenocarcinoma is the serine/threonine kinase LKB1 (STK11), and ~25,000 patients have mutations in LKB1. We recently discovered that LKB1 protein promotes TIF-IA-mediated pre-rRNA synthesis when intracellular UTP is down-regulated, and LKB1-mutant cells lacking such regulation are prone to apoptosis. Based upon these preliminary and published data, we hypothesize that LKB1 inactivation in NSCLC represents a unique acquired tumor vulnerability that can be targeted because cells deficient in LKB1 have lost the ability to promote TIF-IA mediated transcription. The objectives of this proposal are i) to define the molecular mechanism underlying the regulation of TIF-IA mediated pre-rRNA transcription by LKB1, and ii) to determine whether pharmacologic inhibition of de novo uridine monophosphate (UMP) synthesis can specifically suppress LKB1-mutant lung tumors in vivo. These objectives will be met by performing preclinical mouse trials in a unique and clinically- relevant genetically-engineered mouse model (GEMM) and patient derived xenograft (PDX) models to examine inhibition of de novo UMP synthesis by leflunomide as a strategy for inhibiting LKB1-mutant tumors. Since leflunomide is a commonly used clinical agent, we propose that the data from this study will provide a rational foundation to develop a future clinical trial with this inhibitor specifically targeting patients with LKB1 loss-of-function mutations.