Disulfiram/Copper Induces Immunogenic Cell Death and Enhances CD47 Blockade in Hepatocellular Carcinoma

SIMPLE SUMMARY: The combination of disulfiram and copper is a novel anti-cancer drug under clinical development for the treatment of several tumor types including hepatocellular carcinoma. In this study, we investigated the potential of disulfiram and copper to induce immunogenic cell death and whether it could enhance the efficacy of immune checkpoint blockade. Our results showed that treatment with disulfiram and copper induced the release of damage-associated molecular patterns, such as calreticulin, ATP, and high mobility group box 1; thus, eliciting the maturation and activation of dendritic cells. The treatment with disulfiram and copper further enhanced the efficacy of CD47 blockade. Mechanically, disulfiram and copper promoted the nuclear accumulation and aggregation of nuclear protein localization protein 4 to inhibit the ubiquitin-proteasome system, thus, inducing endoplasmic reticulum stress. Taken together, the present findings suggest the potential clinical applications of disulfiram and copper in hepatocellular carcinoma. ABSTRACT: Some chemotherapeutic agents have been found to enhance antitumor immunity by inducing immunogenic cell death (ICD). The combination of disulfiram (DSF) and copper (Cu) has demonstrated anti-tumor effects in a range of malignancies including hepatocellular carcinoma (HCC). However, the potential of DSF/Cu as an ICD inducer and whether it can enhance the efficacy of the immune checkpoint blockade in HCC remains unknown. Here, we showed that DSF/Cu-treated HCC cells exhibited characteristics of ICD in vitro, such as calreticulin (CRT) exposure, ATP secretion, and high mobility group box 1 (HMGB1) release. DSF/Cu-treated HCC cells elicited significant immune memory in a vaccination assay. DSF/Cu treatment promoted dendritic cell activation and maturation. The combination of DSF/Cu and CD47 blockade further facilitated DC maturation and subsequently enhanced CD8(+) T cell cytotoxicity. Mechanically, DSF/Cu promoted the nuclear accumulation and aggregation of nuclear protein localization protein 4 (NPL4) to inhibit the ubiquitin-proteasome system; thus, inducing endoplasmic reticulum (ER) stress. The inhibition of NPL4 induced ICD-associated damage-associated molecular patterns. Collectively, our findings demonstrated that DSF/Cu-induced ICD-mediated immune activation in HCC enhanced the efficacy of CD47 blockade.