Abstract Although cancer usually arises from the combined effects of oncogene activation and tumor suppressor gene inactivation, most targeted cancer treatments are focused on the inhibition of oncoproteins, with less consideration given to the possible concomitant effects on tumor suppressors. Here we describe a critical role of the DLC1 tumor suppressor in the growth regulation of cancer and have identified two kinases that directly phosphorylate DLC1 and attenuate its tumor suppressor activity, leading to the hypothesis that reactivation of DLC1 by the relevant kinase inhibitors should be evaluated as a candidate biomarker of clinical response. We determined that the SRC and AKT kinases directly phosphorylate and attenuate the Rho-GAP and tumor suppressor activities of DLC1 by distinct, cooperative mechanisms and that inhibition of the SRC and/or AKT kinases has strong antitumor activity in two DLC1-positive tumor models that have high SRC and AKT activities, the MMTV-PyMT breast cancer model and tumor xenografts from a NSCLC line. In both models, treatment with the kinase inhibitors results in cooperative DLC1 reactivation, which was monitored by the reactivation of the Rho-GAP and tumor suppressor activities of DLC1. Reactivation of DLC1 makes a critical contribution to the antitumor activity of the AKT or the SRC inhibitors in both models, and the combined treatment with inhibitors of both kinases has even greater Rho-GAP and antitumor activities, which is correlated with greater reductions in RhoA-GTP and its downstream signaling. Furthermore, the combined treatment with AKT and SRC inhibitors induced cellular senescence and apoptosis to a greater degree than single agent treatment, as measured by beta-galactosidase and annexin V expression after short-term tumor treatment. Their antitumor activity is much weaker in isogenic tumors that are DLC1-negative or express DLC1 mutants that are not activatable by the AKT and the SRC inhibitors. A limitation of this therapeutic approach is that it is likely to benefit only those tumors that express moderate to high levels of DLC1 protein. However, several types of solid tumors express DLC1 mRNA but have lost DLC1 protein expression through the epigenetic destabilization of the protein. We hypothesize that it could be beneficial to treat this class of solid tumors with a combination of drugs that can stabilize the DLC1 protein and concurrently inhibit the AKT and SRC activities. Our results indicate the feasibility of this approach, as treatment with proteasome inhibitors in several tumor cell lines that lack detectable DLC1 protein but express its mRNA increased the steady state level of DLC1 protein, which enabled SRC and AKT inhibitors to more potently inhibit tumor cell growth and reduce RhoA-GTP and its signaling. Thus, reactivation of the DLC1 tumor suppressor can be a key therapeutic target, in contrast to the usual situation where the focus is principally on inhibiting pro-oncogenic factors. Citation Format: Brajendra K. Tripathi, Meghan F. Anderman, James H. Doroshow, Douglas R. Lowy. Targeting the DLC1 Rho-GAP tumor suppressor protein to treat solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4513.