Hi, my name is Saqib Haque, I'm a medical student at Johns Hopkins, and today I'm going to be talking about epigenetic modulation as a novel treatment strategy for Chordoma. I have no disclosures. For background, as many of you know, Chordoma is a rare aggressive cancer of the spine and skull base that's derived from undifferentiated remnants of the notochord. Management typically involves a maximal safe surgical resection as well as adjuvant radiotherapy, but there's many clinical challenges for Chordoma. First it's invasive and has a tendency to invade critical regional anatomy that makes surgery very demanding. They're highly radio-resistant, often requiring doses of radiation that are well above the tolerance thresholds of the brain and spinal cord, and they're also chemo-resistant, they're insensitive to traditional cytotoxic agents. And in terms of their biology, we really know relatively little about Chordoma aside from homework or expression of bradyuria. But there has been some recent work looking at the epigenetic landscape of Chordoma, looking at distinct patterns and deregulations in methylation of DNA and histones. To briefly touch on a few studies, this was one study that found that there were 20 genes that were differentially methylated between Chordoma and normal healthy blood, and of those, 15 were hypermethylated and 5 were hypomethylated. This is another study comparing DNA methylation patterns in recurrent versus non-recurrent Chordomas as well as some healthy controls, and they again found differences in methylation patterns across these groups, and they also showed that treatment with an inhibitor of DNA methylation in vitro was able to restore expression of genes that were previously not expressed due to hypermethylation. Moving on to histone methylation, this was a study looking at, it was profiling histone marks across Chordomas, and they found that there were actually highly conserved histone marks in different samples, and they were distinct from other cancers as well as healthy controls, and they identified some pathognomonic marks that are included here. This is another study in a slightly more translational perspective, looking at using drugs that target that histone methylation in Chordoma, and so here they used HDAC inhibitors, and they showed that HDAC inhibitors led to cell cycle arrest and cell death in vitro in Chordoma. That brings us to this concept of epigenetic reprogramming, which is a broad term that's really referring to modifications or resetting of the epigenetic landscape, these methylation patterns that I've been talking about, and this actually happens normally during development. It can also happen in pathologic processes like cancer, but interestingly recently there's been some work looking at potentially using this as a therapeutic strategy, deliberately using drugs that alter these methylation patterns for potential therapeutic effects in cancers, and so two drugs that have been used for this purpose include DZnep, which is a global inhibitor of histone methylation, and Dacitabine, which is an inhibitor of DNA methylation, and I've included a few studies here that discuss these concepts. And so we were interested in using this strategy in Chordoma, and so we wanted to investigate the potential of inhibiting histone and DNA methylation in Chordoma in vitro, both independently and in combination with each other. So we performed a number of assays that I'll walk through. The first is a cell growth or proliferation assay. This is using the UM-COR1 pliable Chordoma cell line. What we did here is we took six well plates, we plated an equal number of UM-COR1 cells in each well, and we treated them with either vehicle controls, so just water, with DZnep, Dacitabine, or the combination of DZnep, Dacitabine, and then we simply counted the number of cells that were alive at days two, four, and six following treatments, and we took representative photographs. So the y-axis is cell number from counting, x-axis is time. And so here we're seeing the growth of the control group, but following treatment with DZnep, we actually saw a notable reduction in cell growth, cell number, following treatment at multiple time points. With Dacitabine, we similarly saw a significant decrease in cell growth, although to a lesser extent than with DZnep, and with a combination of the two, we saw a significantly greater reduction in cell growth compared to either agent alone, and so we were excited about this because this was a proof of concept that using DZnep, the inhibitor of histone methylation, and Dacitabine for DNA methylation, we were actually able to reduce chordoma cell growth. We had done the same thing in a sacral chordoma cell line, UCH1, and we saw some variability with the individual drugs relative to the clio line, but the combination actually had a similar global effect with significant reduction in cell number over time. This is the same data in a bar graph format, just looking at the cell counts at the end of the experiment. This next slide is looking at a clonogenic or colony formation assay. So here we wanted to look at whether treatment with our drugs could decrease colony formation of these chordoma cells over time. So what we did was we again had six well plates, we plated a very small number of chordoma cells, so here this was 700 cells, and we treated them with our drugs of interest and simply allowed them to grow into colonies over a period of about two to three weeks. At the end of the experiment, we can stain the wells with crystal violet, which allows us to visualize them with the naked eye and under the microscope. So here we're showing the UM-COR1 clival line, and on the left is the control group. Each of these dots represent chordoma colonies. And on the right, what we're seeing here, this is a photograph showing complete elimination of colony formation following treatment with 5-micromolar DZNab. At the bottom, this is showing the quantification. We did the same thing in the sacral line, UCH1, and we again saw a significant reduction in colony formation, although there were still some surviving colonies here. We were excited about this. This was an initial experiment with a higher dose of DZNab, but it was again a proof of concept that we could inhibit colony formation with these drugs. So we moved forward with the UM-COR1 cell line, here using our combination of DZNab and decitabine, and what we saw looking at the representative photographs on the left and the quantification on the right is in the control group, we're seeing really prominent colony formation. DZNab group, a significant reduction in colony formation relative to controls. Similarly with DZNabs, significant relative to controls, although to a lesser extent than with the DZNab. And then finally, with the combination therapy group, we see a significantly greater reduction relative to either DZNab or decitabine alone and relative to controls. Next we wanted to look at cell death, and here we used flow cytometry with staining for Annexin 5, which I have in a schematic here. In early stages of cell death and in later stages, Annexin 5 binds to phosphatidylserines that have translocated to the outer cell membrane. It's essentially a way that we can measure the number of cells that are undergoing cell death. And so we again took our UM-COR1 cell line. We treated them with control, DZNab, decitabine, or the combination of the two, prepared them for flow cytometry and looked at the number of cells staining positively for Annexin 5. And so on the y-axis here, the percent Annexin positive cells is representing the percent of cells that are dying. And so here we're seeing a low level of basal cell death in our control group. Following DZNab, we see a significant increase in the number of Annexin 5 positive cells or dying cells. Similarly, with decitabine, we see a significant increase, although again, to a lesser extent than DZNab, similar to what we've seen in prior assays. And then most notably with the combination of the two, we see a significantly greater increase in cell death relative to either monotherapy alone and particularly relative to controls. And then finally, given the invasive nature that I mentioned with Chordoma earlier, we wanted to look at whether treatment with our drugs could inhibit the invasive potential of these Chordoma cells. And so we used a matrigel invasion assay. The way this works is there are these transwell inserts that you can physically place into each well, the 24-well plate, and they're coated with matrigel, which is a gelatinous protein mixture that effectively resembles an extracellular matrix. And we know from cancer biology that one of the earliest steps of metastasis is invasion through that basement membrane. And so we're essentially recapitulating in vitro that process of cells invading through a basement membrane. And so what we do is we pipette cells in media without serum, which is like food for those cells. We pipette that on the top layer of the matrigel membrane. And then on the bottom layer, we have media with serum, so with food. And so the cells have this chemotactic gradient, this drive to want to invade through that matrigel membrane, again, recapitulating those early steps of invasion in cancer. And so here we did the same thing treating with our drugs of interest and looked at effects of invasion. And so what we're seeing on the y-axis is the number of invading cells per high-powered field visualized under a microscope. And we're first showing the number of cells for our control group in the representative picture on the right. Following treatment with DZNEP, we saw a notable reduction in the number of cells that were invading following DZNEP monotherapy. Following treatment with Dizibine, we did not see a significant reduction, although there did seem to be a non-significant reduction relative to controls. And with the combination of the two, we saw significantly greater reduction in cell invasion relative to either agent alone and particularly relative to the controls, and again, with representative photographs on the right side. And so we concluded that inhibiting histone and DNA methylation independently decreased chordoma growth and clonogenic potential in addition to increasing cell death. Inhibiting histone methylation decreased chordoma cell invasion, and the combination of the two strategies led to a greater reduction in chordoma cell growth, clonogenic potential and invasion, and a greater increase in cell death compared to either strategy alone. And so we concluded that epigenetic reprogramming may represent a potentially promising therapeutic strategy for chordoma that warrants further attention. I'd like to thank everybody on this slide who, thank them for their mentorship and their incredible collaboration throughout all of this work. Without them, none of this would have been possible at all. So thank you, and I've included my contact information at the bottom right for any questions. Thank you very much.

epigenetic modulation

Chordoma

treatment strategy

DNA methylation

histone methylation