The Hippo signaling pathway plays an essential role in organ size control and tumorigenesis. Loss of Hippo signal and hyper-activation of the downstream oncogenic YAP signaling are commonly observed in various types of cancers. We previously identified STRN3-containing PP2A phosphatase as a negative regulator of MST1/2 kinases (i.e. Hippo) in gastric cancer (GC), opening the possibility of selectively targeting the PP2Aa-STRN3-MST1/2 axis to recover Hippo signaling against cancer. Here, we further discovered 1) disulfiram (DSF), an FDA-approved drug, which can similarly block the binding of STRN3 to PP2A core enzyme, and 2) CX-6258 (CX), a chemical inhibitor, that can disrupt the interaction between STRN3 and MST1/2, both allowing reactivation of Hippo activity to inhibit GC. More importantly, we found these two compounds, via a MST1/2 kinase-dependent manner, inhibit DNA repair to sensitize GC towards chemotherapy. In addition, we identified thiram (TH), a structural analog of DSF, can function similarly to inhibit cancer cell proliferation or enhance chemotherapy sensitivity. Interestingly, inclusion of copper ion enhanced such effects of DSF and TH on GC treatment. Overall, this work demonstrated that pharmacological targeting of the PP2Aa-STRN3-MST1/2 axis by drug compounds can potently recover Hippo signal for tumor treatment.Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.