Project Summary Dissecting the Mechanism of SETDB1 and its K867 Monoubiquitination in Lung Cancer Progression Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality worldwide and associated with high mutation burdens. During the last decade, biomarker-driven targeted therapies have achieved clear clinical benefits in patients with several oncogenic driver mutations such as EGFR, BRAF and ALK. However, the treatments for tumors with the most prevalent KRAS mutations remain challenging despite of recent breakthrough in targeting KRAS-G12C mutation. In KRAS mutant NSCLC, the constitutively activated downstream signaling ultimately leads to transcription reprogramming to sustain tumor growth and progression. This process is orchestrated by a cohort of crucial transcription factors, whose temporal and spatial expression is associated with distinct epigenetic changes. Thus, dissecting epigenetic mechanisms in this process could open new avenues for targeting strategies. SETDB1 is a principal methyltransferase catalyzing histone H3K9 methylation, a major repressive epigenetic modification. The focal amplification and upregulation of SETDB1 have been observed in a wide range of cancers including NSCLC, suggesting it has pro-oncogenic function. However, the mechanisms of action of SETDB1 in NSCLC remain elusive. In preliminary study, we uncovered that SETDB1 upregulation in lung adenocarcinoma (ADC) correlates with poorer prognosis and significantly co-occurs with KRAS gene alternations. In NSCLC cells harboring oncogenic RAS, SETDB1 loss attenuates migration and invasion, invadopodia formation and ECM degradation. These phenotypic changes correlate with reactivation of transcription factors FOXA2 and CDX2 which function as key regulatory nodes to impede metastatic progression of KRAS mutant lung ADC. More importantly, Setdb1 loss in tumor cells derived from metastatic KrasG12D;p53fl/fl lung ADC abolished their spontaneous ability to metastasize from the subcutaneous tumor to the lungs. These findings argue for a novel concept that SETDB1-mediated epigenetic mechanisms are critical for oncogenic KRAS-induced transcription reprogramming during lung ADC progression. Moreover, we have demonstrated that SETDB1 is monoubiquitinated at lysine-867 by the UBE2E family of E2 enzymes independent of E3s and this monoubiquitination is essential for SETDB1-mediated H3K9 methylation and ERV silencing. Accordingly, one objective of this proposal is to delineate SETDB1's pro-metastatic activity in KRAS mutant lung ADC using various genetically engineered mouse models and xenograft models. Another objective is to dissect the mechanisms of lysine-867 monoubiquitination in SETDB1's pro-metastatic activity in vivo and in vitro. Our central hypothesis is that SETDB1 plays important roles in KRAS mutant lung ADC progression through epigenetic mechanisms that require its monoubiquitination dependent enzymatic activity. This project is signi...