Repurposing drugs and finding new therapeutic targets for cutaneous malignancies

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

Abstract

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer with a predilection for the elderly and the immunosuppressed. The FDA has approved the checkpoint inhibitors avelumab and pembrolizumab for the treatment of advance stage MCC, however these treatments provide durable benefit for less than 50% of MCC patients. Therefore, new treatment regimens are urgently needed. Fortunately, our laboratory has identified several compounds with the potential to be repurposed as an effective treatment for MCC. Our preliminary data suggest that disulfiram (DSF), an aldehyde dehydrogenase (ALDH) inhibitor, may be an effective treatment for MCC. In addition to inhibiting ALDH, disulfiram is known to increase the effectiveness of chemotherapeutic drugs. DSF was also shown to be more effective when complexed to copper. After immunotherapy, the standard of care for late stage MCC is chemotherapy. The most common chemotherapeutic treatment for MCC is etoposide in combination with cisplatin. The mechanism by which DSF increases the effectiveness of anticancer agents like etoposide is unknown. Therefore we will investigative the effectiveness of DSF plus copper for the treatment of MCC in the presence or absence of etoposide. Investigating the mechanism of DSF plus copper in MCC cell lines in the presence and absence of VP-16. Copper increases DSF potency in MCC leading to reduce viability. To identify novel treatments for MCC, we conducted high-throughput drug screening of approximately 4,000 small molecules in collaboration with the National Center for Advancing Translational Sciences (NCATS). We identified several drugs that showed therapeutic advantage in MCC compared to control cells based on the metric area under the dose response curve (AUC). AUC was used to identify compounds that were both potent and efficacious. The MCC cell lines analyzed were MCC-13, MCC-26, UISO, MKL1, MKL2 and WaGa, while the control cell line was a spontaneously transformed keratinocyte cell line, HaCaT. Among the MCC-selective agents was DSF, which was selected for development because it is already approved by the FDA for the treatment of alcoholism and, therefore, can be immediately repurposed for the treatment of cancer. In follow-up studies, DSF alone was more potent and efficacious in MCC cells than controls. Adding copper at physiological levels to DSF increased its potency, and preliminary studies suggest that DSF plus copper synergized with etoposide in reducing MCC viability. The addition of DSF plus copper improved the effective dose (ED50) of etoposide from the uM to the pM range in MCC cells. Taken together, these data show that DSF selectively reduces the viability of MCC relative to control cell lines in vitro and adding copper and etoposide enhances this effect. DSF and copper synergize with etoposide We sought to confirm whether DSF and copper would increase the potency and enhance the function of etoposide, an adjunct therapy for MCC. To this end, we started by investigating if DSF and copper would increase the potency of etoposide. The addition of DSF and copper notably reduced the effective dose of etoposide required to kill the MCC cell lines when compared to etoposide alone. Subsequently, we tested the effect of adding DSF and copper on the double stranded DNA break function of etoposide. As examined by immunofluorescent staining, the combined treatment of DSF, copper, and etoposide led to a robust increase in H2AX foci presentation in the nucleus of our MCC cells. These results indicate that DFS plus copper increased the potency of etoposide and enhanced its ability to induce double stranded DNA breaks. Mechanism of DSF and copper action in MCC Since, our MCC cell lines were undergoing non-apoptotic cell death, we wanted to determine if autophagy was being induced and what effect autophagy was having on our MCC cell lines. To this end, we treated MCC cells with vehicle control or DSF plus copper for 20 hours. Following treatment, we examined the expression of LC3B, which is a marker of autophagy that functions in the formation of the autophagosome. Immunofluorescent staining of LC3B showed an increase in autophagosome formation following treatment with DSF plus copper, indicating that autophagy is induced in response to this treatment in MCC. We decided to focus on whether DSF plus copper induced immunogenic cell death (ICD). To test the ICD response of DSF plus copper, we treated MCC cells with vehicle control, DSF alone, copper alone, or DSF plus copper for 20 hours and measured the translocation of the ICD marker HMGB1 from the nucleus to the cytoplasm where HMGB1 form foci. DSF plus copper led to increased foci of HMGB1 protein that was not observed in control samples. In addition to HMGB1, we also looked at the ICD marker calreticulin (CRT), which translocates from the endoplasmic reticulum (ER) to the cell membrane for recognition and activation of immune cells. We monitored CRT membrane expression via mean fluorescent intensity (MFI) in response to DSF and copper by flow cytometry and observed an increase in the membrane presentation of CRT following DSF plus copper treatment. Thus, the combined treatment of DSF and copper induced markers of ICD. In the future, we plan to investigate whether this treatment induces other ICD markers such as ATP release or heat stock protein (HSP) membrane expression. Consistent with the immunogenicity of MCC and that DSF plus copper induce ICD markers, we found that the combined treatment of DSF and copper lead to increased expression of Program Cell Death Ligand 1 (PD-L1) at the cell membrane as measured by flow cytometry. Our findings suggest that DSF plus copper may enhance the effectiveness of current immunotherapies for the treatment of MCC when dosed in combination. We plan to explore this further in the future using in vivo mouse models for Melanoma as there are currently no mouse models of MCC. We have shown that DSF selectively reduces MCC cell viability. Copper increased DSF potency and combined DSF and copper synergized with etoposide to enhance etoposide function in MCC cells. Subsequently, we determined that mice tolerate the combined treatment of DSF, copper, and etoposide. These results indicate that DSF plus copper can be repurposed for the treatment of advanced stage MCC, in combination with etoposide. In the future, we will complete efficacy studies for DSF, copper, and etoposide in mice xenografts of MCC. Additionally, we showed that DSF plus copper was cytostatic and induced non-apoptotic cell death, potentially through the activation of autophagy. As DSF only has about a 15-minute half-life in mice, in the future we will use PLGA-(DSF/copper) nanoparticles to determine the efficacy of DSF and copper in a xenograft mouse model of Merkel cell carcinoma. We also plan to investigate the role of DSF plus copper on the methylome of MCC cells and confirm our finding with ATAC-seq. We also will work with the NCATS to identify new drugs that synergize with DSF and copper for the treatment of not only Merkel cell carcinoma but acral lentiginous melanoma and squamous cell carcinoma.