Antitumor effect of a new pyruvate kinase M2 (PKM2) inhibitor Acyclovir in experimental esophageal adenocarcinoma

Background: Esophageal adenocarcinoma (EAC) is one of the most aggressive human cancers with poor prognosis even with modern combination therapies due to high resistance to chemotherapy. Therefore, new therapeutic approaches are urgently needed. Pyruvate kinase M2 (PKM2) is not only a key enzyme regulating cancer glycolysis but also translocate into the nucleus to regulate transcription. Although PKM2 is overexpressed in various tumor tissues including EAC, its functional role in esophageal EAC chemotherapy remains unexplored. The objective of this study was to determine whether acyclovir can act as an inhibitor of PKM2 and EAC tumor growth. Methods: Virtual in silico screening using molecular docking was conducted to screen an FDA-approved chemical library for identification of potential PKM2 inhibitors. HER-2 overexpressing OE19, LPR-OE19 and OE33 EAC cell lines were used. The lapatinib-resistant OE19 (LPR-OE19) cell line was generated from parent OE19 cells through intermittent exposure to increasing concentrations of lapatinib for over six months. Acyclovir and nanoparticle albumin-bound paclitaxel (nab-paclitaxel), alone or in combination, were tested for effects on cell proliferation, lactate production, apoptosis, signaling pathways and tumor growth. Antiproliferative activities were measured by WST-1 assay. Western blotting was performed to evaluate expression of PKM2, apoptotic markers and cell signaling proteins. In-vivo antitumor efficacy was measured in a patient-derived xenograft (PDX) model of human EAC. Results: Molecular docking identified acyclovir as a potential PKM2 inhibitor that showed lowest docking and glide scores, indicating a strong binding interaction with PKM2. Acyclovir inhibited both PKM2 expression and lactate production. It inhibited cell proliferation in 2D, 3D and organoid cultures of EAC in a dose dependent manner. Interestingly, addition of nab-paclitaxel enhanced the antiproliferative effect of acyclovir. Acyclovir increased expression of proapoptotic proteins and decreased expression of phospho AKT in EAC cells. In a subcutaneous PDX model, acyclovir induced a tumor regression compared to control as monotherapy, and acyclovir in combination with nab-paclitaxel showed a significant enhancement effect of tumor regression. The net change in tumor size in the control, acyclovir, nab-paclitaxel, and combination groups was 551.19 ± 99.69 mm 3 , 347.33 ± 71.41 mm 3 , 221.55 ± 43.73 mm 3 , and 159.37 ± 39.29 mm 3 . Reduction in PDX tumor growth corroborated decreased tumor cell proliferation results. Conclusions: These data suggest that acyclovir, acting as a PKM2 inhibitor, in combination with nab-paclitaxel should be further investigated as a potential therapeutic strategy for HER2-positive EAC patients and could be a novel treatment strategy for EAC.