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Andrea Miyahira: Hi, everyone, I'm Andrea Miyahira, here at the Prostate Cancer Foundation. Today, I'm talking with Dr. Asmaa El-Kenawi, an assistant professor at Indiana University. Her group has recently published a paper, "Elevated Methionine Flux Drives Pyroptosis Evasion in Persister Cancer Cells, in Cancer Research." Dr. El-Kenawi, thanks for joining us today.

Asmaa El-Kenawi: Thank you for having me. Thank you again, Andrea, for having me to represent my colleagues and present our recent paper about the role of methionine in driving therapeutic resistance.

As many of us know, we have a lot of effective therapies in cancer, however, this is only for a limited time, and eventually, therapeutic resistance is inevitable, and it represents a major clinical problem, since the patients who develop therapeutic resistance tend to be refractory to immunotherapy. We have a limited number of therapies that are available for them, and most of these patients will actually die from the disease.

The majority of research that has been done to identify mechanisms of therapeutic resistance focuses on the genetic aspect. However, if we look at some of the published data, such as this data from Adam Sowalsky, which he compared the transcription of residual tumor cells to the tumor before treatment, and what we found is that there are a lot of intrinsic immune pathways that are being activated, involving the inflammasome, hallmark, and also TNF alpha pathway. One of the questions that we had early on, how the cells post-treatment are sustaining intrinsic immune activation, because we see that in many cancers, including in different treatments, and how their resistance to inflammatory cell death can develop.

Before going to that, I would like to use a slide to introduce a type of cell death, which many of these cell deaths can be inflammatory or non-inflammatory. The most famous ones, which are pyroptosis and necroptosis, tend to be inflammatory cell deaths that drive immune activation in a tumor microenvironment. So, it's a good thing to have, but meanwhile, if it's sustained or chronically activated, it can derive therapeutic resistance. We started by asking the question whether the different cells remaining after treatment, have any characteristic of any major cell death. And by going through one after another marker, we found that the majority of cells left after treatment, I'm using the example here, docetaxel-treated 145, which is a prostate cancer cell, has morphological characteristics of pyroptosis. In addition, we used the caspase family, the caspase-1 activation, as another marker of pyroptosis, and we found that these residual tumor cells have an active caspase-1.

So, what's pyroptosis in detail? Pyroptosis is a highly inflammatory form of lytic programmed cell death, and the way it gets activated is by having caspase-1 truncating a protein called gasdermin. This gasdermin then translocates to the plasma membrane, creating pores, which allow the release of many inflammatory cytokines, such as IL-18, and large metabolites, such as ATP or UTP, which are major energy currencies for the cell. But if they're released outside, it would actually activate an immune response. This marker can be detected when cells are treated or the remaining cells left behind, and actually, we can detect these large ATP compounds in the supernatant, using mass spec. And we saw there's a high release of these metabolites in the supernatant. We also saw the large proteins that cannot be released unless there are pores in the plasma membrane, the LDH, outside the cell membranes treated with different treatments as well. We used here the tyrosine kinase inhibitor in the cell line, that also includes prostate cancer cells.

What is the nomenclature that we can use for these cells remaining after treatment? We prefer to use a nomenclature that, actually, the researchers derived from bacteria research, because the kinetics of cell death actually resembled the death of bacteria by antibiotics, as we see here. So, we used in our paper to define those resistant cells as persisters, which might also resemble other populations of resistant cells that have been characterized by other researchers in the field as well.

Without going through further research that has been published, we know that these cells die by pyroptosis and that pyroptosis is essential for a therapeutic response. But, that question, we were trying to answer in this paper, was can adaptation to pyroptosis play a role in therapy resistance? We did not know the answer to that question, so we started by an unbiased approach, where we wanted to differentiate between the genetic and non-genetic mechanisms of resistance. The way we did that, we modified the Nobel Luria-Delbrück experiment, which goes back to 1943. We had done the selection process in a small well, basically, a 96-well plate, to allow us to monitor the evolution of resistance over 75 days. We were able to select a group of cells that actually developed resistance so fast. We predicted that these cells would have a mutation, and the other cells that didn't develop resistance so fast, we predicted that these cells might have actually developed resistance by a non-genetic mechanism. We selected those and we expanded different cell lines from them. Then, we did a multi-omic characterization.

The first step was to prove that there were genetic versus non-genetic alterations in the cells. And what we found, indeed, was that the early resistant clone had a genetic alteration, a mutation in EGFR, as we see here, the later or descendant of the persister would not have any genetic alteration. So, this resistant mechanism was purely non-genetic and it was very interesting to see.

We then went ahead and we did a transcriptomic analysis, and actually, as a validation of our previous hypothesis and the data from Adam Sowalsky, we found that the activation of the intrinsic immune pathway was evident in these transcriptomic analyses, as we see here, mainly the interferon alpha, beta, was the top activated pathway. This also includes a range of inflammasome activation, and the pathway that was responsible for sensing dangerous signals in persisters, mainly the cGAS-STING, suggesting which one of the pathways was driving pyroptosis as well, suggesting that somehow these cells sustain inflammatory activation, but meanwhile, remain viable long enough to access proliferative programs afterwards.

To make the story short, we went through the paper dissecting the mechanism, and what we found was that the cells under pyroptosis tend to actually activate a metabolism program to sustain the plasma membrane integrity long enough. These cells, because of this metabolism change, would actually undergo epigenetic reprogramming, leading to perforation. How we did that, I'm using just an example of some of the data of the paper. Metabolic characterization of the persister cells shows that the most significant change was in the methionine and taurine pathway. The methionine tended to actually be utilized by cells producing taurine, so we wondered if this methionine flux would actually be utilized by persisters.

We developed an assay using isotope labels to define the process of flowing in or flowing out of different metabolites. It depended on adding a carbon-labeled metabolite to the cells and then asking whether what is the abundance of this metabolite over time, either secreted by cells or the metabolite we already added in. And what we found, again, as an example here, was that methionine was the top metabolite being used by cells. So, where is that methionine going? Well, we found that this methionine, by being converted to taurine, was actually sustaining the plasma membrane, meanwhile, it affected the epigenetic landscape of cells. Here is a genome-wide DNA hypermethylation of these persisters. As we see here, there is a very high level of methylation in these cells, and we go through these in details in the paper as well.

In summary, the idea of the paper is that we were trying to prove that there is a possibility of metabolism-dependent epigenetic reprogramming, which is independent from any genetic mutation, that can cause evasion of cell death; we used pyroptosis in this paper. With that, I would like to thank my colleagues, my collaborators, and the Moffitt Cancer Center, and my new institution, Indiana University, since I'm starting to have a lot of collaborations for this project. I'm happy to take any questions.

Andrea Miyahira: Thank you, Dr. El-Kenawi, for sharing that. Have you compared the biology or phenotypes of these persisters with either cancer stem cells or with the PACs, the polyploid cancer cells, that were described by Sarah Amend, Ken Pienta, and team?

Asmaa El-Kenawi: Yes. One of the things that we discussed in the discussion of this paper is how similar the persisters are to the PACs, including the big nucleus, so we wouldn't characterize that. But we haven't published that particularly in the paper. We also saw that, morphologically, they had the same phenotype, including in live imaging. We also saw on the transcription level that there were a lot of the PACs markers being activated or sustained in the persister, including markers for cancer stem cells as well.

Andrea Miyahira: Thanks. Do you know how these persisters might be therapeutically targeted? Are we thinking of immunotherapy, or epigenetic-targeting agents, or metabolism-targeting agents in synergy?

Asmaa El-Kenawi: This is what we are actually working on at the moment, trying to identify a therapy that can block emergent self-resistance, or at least delay it. If you think about it, these cells can be there, but the problem is that they develop a proliferation program, and we want to have them as quiescent as possible, not starting proliferation. So, what we are thinking is, probably, maybe, by identifying the mechanism of methionine uptake, or blocking these mechanisms, we could actually prevent them from advancing or adopting a more epigenetic landscape to start the proliferation, or maybe by using epigenetic-targeting drugs, which we show in the paper, were effective in treating these persisters.

Andrea Miyahira: Thanks. I think a lot of your paper was in other types of cancer, but have you investigated these mechanisms of the persisters in prostate cancer cells?

Asmaa El-Kenawi: We did use a range of prostate cancer cells, especially G145, which can be treated with chemotherapy, because it's an example of an AR-negative cell line. We also have unpublished data related to the paper, where we used furcation as well, and we saw the same morphological change. We also treated different prostate cancer cells with a tyrosine kinase inhibitor as an additional way to prove that it's a general phenomenon. It's not unique to a certain therapy. But what we're trying to do right now is, we're doing the mouse work, more advanced techniques, to prove that these persisters left behind after treatment in a mouse actually sustain the same transcription profile of the persister that we see in vivo. So, we're using a prostate cancer model for that.

Andrea Miyahira: Thank you. And what are your next steps?

Asmaa El-Kenawi: Our next steps are to define how methionine is being consumed by these persistents and how to block that. We're using a metabolomics approach as well. We also wanted to utilize one of the metabolism-related biomarkers to detect whether these persisters have emerged from the quiescent state, so that would act as a biomarker of resistance or monitoring the recurrence as well.

Andrea Miyahira: Okay. Well, thank you so much, Dr. El-Kenawi, for coming and sharing this with us today.

Asmaa El-Kenawi: Thank you for having me, Andrea. Good to see you.