Abstract 345: Altered ALDH1A1 expression and cellular copper levels between low and highly metastatic osteosarcoma provides a case for novel repurposing of disulfiram
Authors: Mandell Jonathan B. , Douglas Nerone , Ukani Vrutika , Anderson Carolyn , Beumer Jan , Watters Rebecca , Weiss Kurt 
Published: September 2020

Osteosarcoma (OS) is the most common primary bone cancer and disproportionately affects pediatric and adolescent patients. Survival rates for OS patients with metastatic disease is only 15-30% and has not improved in several decades. Novel treatments targeting specifically metastatic markers of OS are needed. ALDH1A1 is a known cancer stem marker and highly expressed in metastatic cancer cells. Disulfiram (Antabuse®) is an FDA-approved anti-alcoholism drug which acts as an ALDH inhibitor and has been widely studied for repurposing as an effective therapy in multiple types of cancer. Disulfiram cytotoxicity in cancer cells has been shown to be copper-dependent due to its ability to function as a bivalent metal ion chelator of copper (Cu). Cu concentrations of OS patient tumors, human OS cell lines, and OS patient plasma were determined using a Perkin Elmer AAnalyst 600 atomic absorption spectrophotometer. qPCR using custom primers for ALDH1A1 was performed on human OS cell lines and OS patient tumors differing in metastatic potential. OS cell cytotoxicity assays were performed with disulfiram and copper chloride after 24 hours of treatment. Balb/C mice were challenged intratibial with Luciferase+ K7M2, and treated with disulfiram and copper gluconate by oral gavage. Primary tumor growth and pulmonary metastases were monitored using the IVIS XR live imaging system. Metastatic OS patient tumors displayed significantly increased ALDH1A1 expression compared to non-metastatic OS patient tumors (n=8, P=0.02). Metastatic OS tumors displayed decreased Cu levels compared to non-metastatic OS tumors (n=18, P=0.05). Conversely, serum Cu levels from OS patients with metastases demonstrated increased blood Cu levels compared with non-metastatic patients (n=24, P=0.01). qPCR displayed ALDH1A1 expression was significantly increased in highly metastatic human OS cell lines LM2 and LM7 compared to low metastatic SaOS-2 (n=3, P=0.002). Intracellular Cu was inversely proportional to metastatic phenotype in human OS cell lines SaOS-2&gt;LM2&gt;LM7. Copper chloride displayed clear potentiation of disulfiram resulting in a strong cytotoxic effect in both SaOS-2 and LM7. Doxorubicin IC50 for SaOS-2 and LM7 was 1.2 µM and 2.5 µM respectively, while disulfiram IC50 with a 50 nM copper chloride supplement for SaOS-2 and LM7 was 1.2 µM and 0.6 µM respectively. In our preclinical mouse model, after primary tumor resection, differences in recurrent growth at amputation site were seen between treatment groups. Disulfiram and copper gluconate combination treated mice displayed 13% (1/8) recurrence rates at the primary tumor amputation site, while untreated animals displayed 60% (9/15) recurrence rates. This study demonstrates that both ALDH1A1 and Cu bioavailability are altered between low and highly metastatic OS. Since ALDH1A1 is a direct target and Cu is a potentiator of disulfiram, this supports the potential of using this repurposed drug in combination with surgical and chemotherapy interventions for metastatic OS.

Citation Format: Jonathan B. Mandell, Nerone Douglas, Vrutika Ukani, Carolyn Anderson, Jan Beumer, Rebecca Watters, Kurt Weiss. Altered ALDH1A1 expression and cellular copper levels between low and highly metastatic osteosarcoma provides a case for novel repurposing of disulfiram [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 345.