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. We found that complexing DSF with copper increased its potency against MCC cells. Treatment with DSF plus copper was cytostatic, induced autophagy, and caused non-apoptotic cell death in MCC cells. Interestingly it also increased the expression of immunogenic cell death markers and increased Program Cell Death Ligand 1 (PD-L1) expression at the membrane. We also observed that DSF plus copper synergized with the topoisomerase II inhibitor etoposide to reduce MCC cell viability by enhancing DNA damage as evidenced by gamma H2A.X foci in the nucleus. 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. We found that the combined treatment of DSF and copper lead to increased expression of 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. DSF-Copper (Cu) nanoparticles (NP) show similar in vitro efficacy to DSF plus copper. Despite dramatic in vitro responses in MCC cell lines, mouse xenograft tumor responses to DSF plus copper have been limited. To improve the in vivo first pass metabolism and half-life of DSF in the bloodstream, we developed Metal-Organic Framework (MOF) and Polylactic-co-glycolic acid (PLGA) NP formulations of DSF-Cu. The MOF NP surround DSF with a cage of copper and Methylimidazole. This NP is taken up by tumor cells via endocytosis and the MOF is broken down in the lysosome releasing DSF and Cu. The PLGA NP are a polymer-based NP, where the polymer surrounds a complexed form of DSF and Cu. This NP is more hydrophobic and is degraded by the hydrolysis of the ester linkages or through erosion in aqueous environments. We determined the in vitro activity of two NP formulations by performing cell viability assays after treatment with increasing doses of the two NP formulation and 50 nM DSF plus 2.4 M Cu in MCC13, MCC26, MKL-1 and WaGa cell lines. In MKL-1 cells both NP formulations showed similar efficacy to 50 nM DSF plus 2.4 M Cu. The DSF-equivalent effective concentration 50 (EC50) in MKL-1 cells was 460 nM for the MOF NP and 26.4 nM for the PLGA NP. Similar results were obtained in MCC13, MCC26 and WaGa cell lines. Thus, DSF-Cu NP show in vitro activity in MCC cells. 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. Acral Lentiginous Melanoma: ALM is a rare form of melanoma that occurs on the skin of the palms, soles, ventral digits, and nail units. No FDA approved treatments exist for ALM. At an incidence rate of 2 per million persons per years, ALM represents less than 5% of all cutaneous melanomas, but causes the majority of skin cancer related deaths in minorities. Although ALM is less common in individuals of European descent, it is the most commonly diagnosed form of melanoma in individuals of African, Hispanic, or Asian descent. Furthermore, the 5-year survival rate is worse for individuals of African descent (66.9%) compared Hispanic (72%), Asian (76.6%) and non-Hispanic Whites (84%). DSF plus Cu reduces acral lentiginous melanoma cell viability Leveraging our expertise in repurposing DSF, we treated six ALM cell lines with 50 nM DSF and 1 M Cu. In MB2204 cells, DSF plus Cu reduced cell viability with an average EC50 of 75 nM when compared to DSF alone. This result was consistent for the 5 additional ALM cell lines tested. This finding suggests that DSF plus Cu might also be an effective treatment of ALM. In the future, we will conduct a high-throughput drug screen in collaboration with NCATS to find additional novel treatments for ALM and measure gene expression dysregulation via RNA sequencing.