(P 51) ASSESSING THE IN VIRTO EFFICACY OF DISULFIRAM AND ENZALUTAMIDE COMBINATION THERAPY IN METASTATIC OSTEOSARCOMA

Objective: Aldehyde dehydrogenases (ALDH) are overexpressed in many cancer types. Previous studies have shown that metastatic osteosarcoma (OS) tissues and cell cultures have increased expression of ALDH1A1. Furthermore, in vitro and in vivo studies using the ALDH-inhibitor Disulfiram have been promising. However, combination therapy is the clinical standard to prevent treatment resistance and reduce effective doses, reducing the risks of toxicity and associated side effects. Pathway analyses of sequencing data predicted androgen receptor (AR) to directly activate ALDH1A1 in OS lung metastases samples. Enzalutamide is an AR-inhibitor currently used to treat prostate cancer. In this study, we investigated the effects of the combination of Disulfiram and Enzalutamide in metastatic OS cell lines in vitro.

Methods: Primary OS SaOS-2 and its metastatic clone, SaOS-LM2, were treated with Disulfiram and Enzalutamide alone and in a combination matrix. Single treatment dose curves were created using the Incucyte SX1, monitoring cell viability for 7 days. For combination matrix experiments, cell viability was measured after 72h treatment and SynergyFinder 3.0 was used to evaluate the combination matrix data. Promising combination treatment regimens will be further evaluated by apoptosis analysis and clonogenic survival assays.

Results: SaOS-2 and SaOS-LM2 were sensitive to Disulfiram treatment for 7 days at all single doses tested. However, these OS cell lines were not as sensitive to single-dose Enzalutamide treatment. The combination matrix analysis revealed that treated separately. The combination had an average Bliss synergy score of –4.62 ± 4.22 (95% CI) in SaOS-2 and –3.04 ± 3.13 (95% CI) in SaOS-LM2. The highest levels of synergy were observed at 0.1 µM Disulfiram and 33 µM Enzalutamide with a score of –0.82 (± 4.22) in SaOS-2 cells. SaOS-LM2 displayed the highest level of synergy at concentrations of 1.1 µM Disulfiram and 33 µM Enzalutamide with a score of 6.09 (± 3.13). These scores indicate that Enzalutamide and Disulfiram have an overall additive effect. SynergyFinder results also revealed antagonistic effects of the combination treatment when treating SaOS-2 with doses of Enzalutamide at 11 µM and lower, an effect that was not observed in SaOS-LM2 cells.

Conclusion: Disulfiram and Enzalutamide have an additive effect when used in combination to treat OS in vitro. When used in combination, both drugs can be used at lower concentrations to achieve effective cell death. However, the effective dose of Enzalutamide remains three times greater than reported IC50s in cancers responsive to AR inhibition. Furthermore, Enzalutamide displayed antagonistic effects at these reported clinical doses when treating the primary OS cell line SaOS-2 in combination with Disulfiram. Low-dose Disulfiram has relatively low non-targeted toxicity and poses far less side effects than most chemotherapies used in the standard care of OS patients. Because treatment of metastatic OS has been promising with clinically relevant doses, we are further investigating Disulfiram with other chemotherapy agents.