Abstract 4831: DSF-Cu complex sensitizes patient-derived unmethylated MGMT expressing brain tumor cells to temozolomide

Abstract

Abstract Introduction: O6 methylguanine DNA methyltransferase (MGMT) repairs the DNA damage caused by alkylating agents [temozolomide (TMZ)] leading to chemoresistence. Members of the aldehyde dehydrogenase (ALDH) family of isoenzymes serve as markers of cancer stem cells and contribute to chemotherapy resistance. Disulfiram (DSF) inhibits MGMT through ubiquitin mediated degradation; it is also a specific inhibitor of ALDH. We therefore, hypothesized that DSF through MGMT and ALDH inhibition decreases stemness and sensitizes GBM cells to TMZ. Methods: Normal astrocytes as well as established MGMT expressing GBM cell lines (LN18, T98G, U138, U118) and MGMT expressing (unmethylated) patient derived glioblastoma cells (ANII 7730 and ANII 7754) were treated with DSF/Cu +/- TMZ in various doses and combinations. We have also evaluated MGMT, ALDH levels and Sox2 expression. Cell viability, apoptotic assay and caspase 3/7 assay were used to evaluate inhibitory effect of various treatment combinations. Results: DSF and TMZ have a minimal effect on normal astrocyte growth. DSF alone inhibited MGMT in established MGMT expressing glioblastoma cells (5 to 10 µM). DSF alone was unable to inhibit MGMT in patient derived brain tumor cells (up to 20µM). Addition of copper (</=1µM) significantly inhibited MGMT (~ 90%) in patient derived GBM cells which correlated with significant growth inhibition even when DSF was used at low concentrations (</=1µM). This suggests that addition of copper to DSF may reduce any potential, dose dependent DSF related neurotoxicity. Temozolomide alone did not inhibit MGMT in established cell lines nor patient derived brain tumor cell lines (up to 1000µM). DSF+Cu further sensitized patient derived GBM cells to TMZ and significantly inhibited GBM cell growth (~ 95%) and did not inhibit normal astrocyte growth. Further DSF+Cu and DSF+Cu+TMZ combinations caused significant apoptotic cell death and significantly increased caspase 3/7 in patient derived unmethylated GBM cells. Similarly, combination of DSF+Cu and TMZ+DSF+Cu caused significant drop in ALDH activity in patient derived brain tumor cells compared to untreated controls and single agents. DSF induced ALDH and MGMT inhibition correlated with decrease in SOX2. MGMT expression canceled by CRISPR/Cas9 led to significant decrease in SOX2 expression in primary cell cultures. Conclusions: Our findings suggest that DSF/Cu treatment, a dual MGMT and ALDH inhibitor, suppresses stemness. Furthermore, our results confirm that combination of TMZ and DSF/Cu significantly inhibited glioblastoma cell growth compared to TMZ alone and untreated controls. Citation Format: George C. Bobustuc, Amin B. Kassam, Richard A. Rovin, Deborah Donohoe, Dmitry Bosenko, Santhi D. Konduri. DSF-Cu complex sensitizes patient-derived unmethylated MGMT expressing brain tumor cells to temozolomide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4831.