Hi, I'm Riccardo Serra, postdoc fellow at the Interior Lab at Johns Hopkins, and today I'm going to present disulfiram and copper combination as a way to target MPL4 cancer stem cells and extend survival in medulloblastoma. I have no disclosures, and I first would like to give a brief introduction to the SHH-driven and group 3 tumors. These are two common subgroups of medulloblastoma. On the left, the SHH, they are usually fairly benign, but there's a subpopulation characterized by TP53 mutation, which presents in about 30% of the cases, there's typically poor prognosis. And on the other end, group 3 tumors are usually very aggressive. They're characterized by a MYC amplification and PVT1 fusion with frequent chromosomal rearrangement. Another interesting point about group 3 is that most of the lines used for in vitro and in vivo experiments are derived from this medulloblastoma group. There have been several chemotherapeutics in the last years that have been developed and tested both preclinically and clinically, but there is still a fairly strong need for new strategies to address these groups. We then turn to disulfiram and copper, which is a new combination, a repurposed combination of drugs that have been used for several decades in different pathologies, especially disulfiram for chronic alcoholism. And you can see a few papers that have been published in the last five to six years. On top, you can see a paper from Nature explaining the mechanism of action of disulfiram and copper in tumor cells, and then two other papers on the role of disulfiram and copper in brain tumors, and a few clinical trials that have been carried out over the last five to six years. These are our goals. Basically testing this new combination in high-risk medulloblastoma lines, try to assess in vitro their cytotoxicity and the mechanism of action at the molecular level, and then also we try to test in vivo the efficacy in prolonging average survival in two xenograft models, and also we assess ex vivo the markers of stem cellness as well as DNA damage and MPL4 that are usually targeted by disulfiram and copper. These are our results. We first look at cytotoxicity with a CCK8 assay in an SHH driven with TP53 mutation line, which is UW228, and we saw that this combination is already very effective at nanomolar concentrations, and then we turn to all the other lines, which are mainly from group 3, but there's also a wild type SHH driven line, ONS76, and we noticed that the IC50 is usually between 100 and 150 nanomolar already. We then looked at an XN5 and an XN5PI positive cells, so single and double positive, meaning apoptotic and necrotic cells, and we showed that disulfiram and copper is very toxic at 24 and 48 hours after treatment in all these lines. And then we investigated the effect of disulfiram and copper on stem-like cells in group 3 cell lines in vitro. We investigated this by running flow cytometry and staining these cells for two common markers of stemness, which are CD133 and nestin, and you can see here a significant decrease in combination therapy after 12 hours of treatment with 150 nanomolar of disulfiram and copper. We also tried to confirm this with western blotting, and we showed that aldehyde dehydrogenase, which is another common marker of stem cellness and one of the main targets of disulfiram, is reduced after treatment both in vitro, on the left in two different cell lines, and in vivo on the right. And then we investigated the mechanisms of action at the molecular level, and we showed increased fraction of cleaved PARP and also increased expression of AAEF after treatment. You can see these two groups here on top, and then also altered cleavage of NRF1 into some inactive fractions, and we confirmed that with another assay later on, and I'll show you the results. And also disulfiram and copper was able to induce DNA damage, and we also saw an increase with cellular aggregation of NPL4, which is the main target in cancer cells of disulfiram and copper. As I was saying before, DNA damage is also present after treatment with disulfiram and copper. You can see it here also with inhibition of the DNA repair protein CHK1, and also an altered cleavage pattern of the NRF1, both with disulfiram and copper, and then we confirmed this with a p97 inhibitor, which is part of the p97 cascade. And we saw that when you block the p97 cascade, you get a similar profile of cleavage of NRF1. So that's probably the effect of disulfiram and copper in cancer cells. We then turned to an immunofluorescent assay to look at the aggregation of NPL4 after treatment, and we noticed an increased aggregation in the cell nucleus, and also an higher expression of the marker of DNA damage, H2AX, and an increased expression and nuclear translocation from the mitochondria of AIF here on the right after treatment. We then moved in vivo, and we first assessed the safety of our combination with three different dosages of disulfiram, 50, 150, and 250 milligrams per kilogram of weight, and a fixed dosage of copper of 2 milligrams per kilogram. And we also had a control group, and we noticed no change in weight between the four different groups after 22 days of treatment. We then studied the efficacy of disulfiram and copper in two xenograft models of moduloblastoma, D425 med and D341, and we noticed an increased survival after treatment with all disulfiram and copper for 21 days. And then we also wanted to confirm the in vivo results by running Wesson blotting on the tumor mass, and we noticed an increase of AIF after treatment, as well as NPL4 blockage and cell aggregation, and DNA damage in all the tumors that we tested. And then we ran an immunohistochemistry analysis on control tumors and disulfiram and copper treated tumors, and we noticed a decrease in Ki-67 expression at the nuclear level after treatment, and an increase in CLIV-Caspase 3, which is a marker of apoptosis after treatment. We also noticed a small increase in NPL4, the main target of disulfiram and copper, and no change in NuN and GFAP expression among the different groups, meaning that there was no damage probably to neurons and astrocytes after treatment. So in summary, in vitro we achieved cell apoptosis and death after combination treatment with higher expression of Annexin 5 and Annexin 5PI, as well as decreased expression of markers of stem cellness and higher PARP and AIF expression post-treatment. We also noticed NPL4 aggregation with different methods and p97 pathway inhibition. In vivo we were able to achieve higher survival with the combination of disulfiram and copper, as well as lower cell proliferation and higher apoptosis with IHC. So there's obviously some limitations to our study, definitely the use of xenograft model is one, and also the fact that we would like to use more combination therapies in the future both in vitro and in vivo. So in conclusion, again we were able to achieve cytotoxicity in vivo in two different groups of medulloblastoma, and also to show an anti-stem cell effect of this combination. The inhibition of NPL4 was shown both in vitro and in vivo, and we were also able to achieve longer survival in vivo. No cytotoxicity was observed in these animals as well. So in the future we're planning to assess more combinations and also test them in genetically engineered mouse models of medulloblastoma. With this I would like to thank our collaborators in the lab, all the postdocs, and my mentors Dr. Bram and Tyler. And I want to thank you for your attention.

Riccardo Serra

disulfiram

copper

MPL4 cancer stem cells

medulloblastoma