Abstract Tumors with mutant KRAS, which occurs in ~95% of pancreatic cancers, ~45% of colorectal cancers, and ~30% of lung adenocarcinomas, usually have a poor response to current therapies, resulting in poor outcomes. The rationale for the new treatment approach described here for tumors with mutant KRAS arises from the critical antitumor role that we have found for the DLC1 tumor suppressor protein. DLC1 protein is frequently downregulated in a wide range of cancers. Although several mechanisms account for this downregulation, we have focused on a subset of tumor lines that express DLC1 mRNA but lack detectable DLC1 protein. Using the model system of lung adenocarcinomas with mutant KRAS that have this DLC1 phenotype, we have found that in 5 out of 5 human lung adenocarcinoma cell lines (4 with codon-12 and 1 with a codon-61 KRAS mutation), KRAS siRNAs knockdown results in readily detectable steady-state level of DLC1 protein. This phenotype of detectable steady-state DLC1 protein is also seen in these mutant KRAS lines when treated with the EZH2 inhibitor Tazemetostat. Although EZH2 is an important regulator of mRNA expression, secondary to its methylation of Lysine 27 on histone H3, the effect of EZH2 on DLC1 is mainly attributable to the EZH2 methylation of K678 on DLC1 protein in the cytoplasm, which leads to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1 protein. EZH2 inhibition prevents this degradation process, resulting in at least a 10-fold increase in DLC1 protein half-life. KRAS siRNAs knockdown phenocopies CRM1 inhibition, a major nuclear export protein, as it reduces both cytoplasmic EZH2 and DLC1 methylation. Although pharmacologic inhibition of EZH2 by Tazemetostat in the lung cancer lines stabilizes the DLC1 protein, the antitumor activity is greatly increased by the addition of the AKT inhibitor MK-2206 and/or the SRC inhibitor Saracatinib, which reverse the direct phosphorylation of DLC1 by these kinases and increase the tumor suppressor activity of DLC1. The above three-drug combination, which is well-tolerated in mice, induces potent inhibition of tumor xenograft growth that is highly dependent on DLC1 protein and is associated with induction of markers of apoptosis (Annexin V) and senescence (beta-galactosidase). Although each of these inhibitors has multiple targets, siRNAs knockdown of DLC1 abolishes ~80% of the growth inhibitory activity of the three-drug combination, strongly suggesting that DLC1 is the predominant antitumor target of the drug combination. In summary, we identify DLC1 protein is a new critical substrate for cytoplasmic EZH2; our findings suggest that EZH2 inhibitors should be considered for their potential impact on post-translational regulation in addition to their effects on gene transcription; and provide evidence that the drug combination identified here may have clinical efficacy against tumors with mutant KRAS. Citation Format: Brajendra K. Tripathi, Disha Bhargava, Xiaolan Qian, Dunrui Wang, Marian E. Durkin, Fernando J. de Miguel, Katerina Politi, Kylie J. Walters, James H. Doroshow, Douglas R. Lowy. Targeting mutant KRAS tumors by inhibiting cytoplasmic EZH2, which directly methylates and destabilizes the DLC1 tumor suppressor protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2508.