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Andrea Miyahira: Hi. I'm Andrea Miyahira at the Prostate Cancer Foundation. With me is Dr. Christopher Barbieri of Weill Cornell Medicine, who will present a recent Nature Communications paper, "Canonical Androgen Response Element Motifs are Tumor Suppressive Regulatory Elements in the Prostate." Thanks, Dr. Barbieri for joining and sharing this.

Christopher Barbieri: Thanks. It's a pleasure to be here with you today. And I want to thank the Prostate Cancer Foundation for all their support of this project and over the years.

So pleasure to talk about this project. I'm really proud of this work from a former PCF Young Investigator who's in my lab, as well as another very talented postdoc. And really, this work focuses on trying to dissect out the cancer-associated and normal programs controlled by the androgen receptor in prostate cancer and how those change.

So in the big picture, the core of this project is really around the idea that there's more than one transcriptional program controlled by the androgen receptor. As we all know, the androgen receptor is a critical protein in prostate cancer. But in the normal prostate, it actually controls growth suppression and differentiation. And that changes in prostate cancer.

So androgen receptor programs in the normal prostate are associated with turning on genes that essentially tell prostate cells to act like prostate cells and to differentiate but actually suppress their growth. That changes in prostate cancer, where you still get activation of the differentiation-associated genes, but it actually gets hijacked to cause those prostate cancer cells to proliferate as well. So our question is going into this.

We knew that androgen receptor reprogramming, this process of taking this differentiation factor and hijacking it in cancer to make it drive cancer-associated proliferation, was a process. We knew that part of this reprogramming is associated with the androgen receptor going to new places, guided by cofactors or cooperating transcription factors like FOXA1 and HOXB13. But what we knew less about was how important the loss of function of the androgen receptor at its normal sites was, and how important that was in terms of whether that was a critical factor for the cancer actually starting to act like cancer. And we also kind of were interested in the question of whether we could potentially turn the normal program back on and take cancer cells, and really flip the switch back and promote them to differentiate and to stop growing, if we turn those programs back on.

So we knew going into this that canonical androgen receptor response elements, or AREs—which are the DNA motifs that the androgen receptor binds to in the normal prostate—were very important in the normal program. But we needed to figure out a way, essentially, to target those specifically to ask the questions, what were those doing by themselves? And so a really brilliant strategy from Mike Augello, who is a postdoc in the lab and a PCF Young Investigator, was to design these artificial transcription factors that he called modifiers of AR-containing chromatin, where the DNA-binding domain of the androgen receptor alone could sit down on these androgen receptor response elements but not interact with any of the cofactors.

And then we hooked those up to specific domains that allowed it to either activate the genes in the neighborhood or deactivate the genes in the neighborhood to see what the AREs alone controlled. And what we found from that is that the activating constructs—the ones that turned on the genes controlled by AREs—really resulted in a decrease in the cell cycle and really stopped the prostate cancer cells from growing, while strongly engaging classic androgen receptor target genes, like PSA and others. Repressing the androgen receptor response elements really didn't matter to prostate cancer cells, and they actually grew a little bit better when we did that.

That was actually the opposite of what happened in totally normal prostate organoids that were genetically normal, not cancer cells, where they didn't care at all if we activated the AREs—they were quite happy—but normal cells were disrupted and died when we repressed the AREs. So again, this helped us realize that these AREs were controlling more of the normal-like program and less of the hijacked oncogenic program. We were able to see that, when we looked at patient prognosis for these, it was very clear that the genes controlled by AREs—the higher their expression was in tumors, the better the patient's prognosis was. So the more the normal-like program persisted, the better the prognosis for these tumors.

And interestingly, that was both in primary untreated prostate cancers and in castration-resistant prostate cancer (CRPC), where, again, the more of the normal prostate character these tumors retained, the better their prognosis, and the better they responded to therapies targeting the androgen receptor. And then finally, we worked to define some of the actual switches that flip back and forth between these. We found that the epigenetic factor HDAC3, which is a histone deacetylase, was one factor that could help turn off some of this growth-suppressive normal program, and that it was one way that cancer cells—by upregulating HDAC3—were able to switch to the more oncogenic program.

So the take-home messages here are that we've been able to dissect out and define really distinct AR transcriptional programs, both in a normal-like growth-suppressive one and an oncogenic one, that have distinct regulatory elements and distinct control of them. We can show that if we do reactivate the normal-like program by activating AREs, it can result in tumor suppression in vitro and in vivo. And we've seen that the transcriptional signatures associated with these are strongly prognostic in both hormone-naive and in castration-resistant prostate cancer. And so we think we can use some of these things as readouts going forward to help define these transcriptional programs more cleanly and really understand the mechanisms by which they get hijacked in prostate cancer, and by which we might be able to turn them back on for therapeutic reasons. Thank you.

Andrea Miyahira: Thank you so much for sharing this. So, are there approaches for promoting canonical activity in prostate cancer? For instance, BAT therapy with super physiologic testosterone? I don't know, maybe adding an HDAC inhibitor?

Christopher Barbieri: Yeah, exactly. I think those are exactly the type of approaches we're thinking of. So super physiologic testosterone or bipolar androgen therapy is a very interesting approach.

We do see, when we look at some of the studies on super physiologic testosterone done by Pete Nelson and other people, a lot of the same programs are engaged by super physiologic testosterone, as we see controlled by these androgen response elements. So it may be a question of, sometimes at least, are we just pushing the androgen receptor hard enough with higher levels of testosterone to get it to go to the places it used to go, and forcing it there? And then bipolar androgen therapy—again, the same thing with those approaches—whether that's a means to keep these normal-like programs at least engaged somewhat during the patient's journey and really improve the ability to target those axes. I think it's a great question.

Andrea Miyahira: Thanks. And what do you think the evolutionary purpose is for having these HOXB13, FOXA1, AREs? Do they have any roles in development or other cellular programs?

Christopher Barbieri: I think it's likely there's developmental reasons for having them because, as you said, clearly, they are important to prostate cells. The cancers find ways to rely on them and become addicted to them to grow better. What we don't know is what context they're important in.

I mean, a lot of these developmental programs—they're important maybe for a very specific time window, for a very specific set of cells, basically. And the cancer cells rewire that and take advantage of those developmental programs to drive their proliferation better and survive. So I think understanding the biology and the signaling of when those things are important and when they're important may allow us to really understand how to target that reprogramming effect in prostate cancer better.

Andrea Miyahira: Thanks. And does the role of the canonical versus the oncogenic ARE activity change during disease progression to CRPC?

Christopher Barbieri: Yeah. So we definitely see that the genes controlled by canonical AREs remain relevant in CRPC. We worked with some of the Stand Up to Cancer dream teams and Felix Feng's group to look at the data in CRPC patients.

And we can clearly see that patients that are expressing more of these classic normal-like androgen receptor target genes have better prognosis in CRPC. And more than that, if you look at the luminal character of those tumors, they are more luminal—they're more classic adenocarcinoma CRPC. So again, it tells you something about the biology: that they may be more anchored to their prostate identity compared to other CRPCs which shift away from that, essentially.

Andrea Miyahira: And have you compared these ARE signatures with other biomarkers of aggressive disease?

Christopher Barbieri: Yeah. I mean, it's very variable. Like I said, they do show prognostic and predictive biomarker information about ARE-targeting therapies.

But I think it tells you more about the biology of the tumor and how prostate-like it is and how much like a luminal adenocarcinoma it is, which itself has its own prognostic implications. So I think these things are telling us about biology. I'm not sure if they're going to be as good as biomarkers that were developed purely for response reasons to answer those questions, though, basically.

Andrea Miyahira: And what are your next steps? And do you have any translational plans?

Christopher Barbieri: Yeah. So I think that the most important thing we've been able to get out of these is we now have a clearer picture of these programs, and we can use them as readouts basically.

So we've done a lot and are continuing to do a lot of, number one, functional genomic screens to really understand: OK, what are some of the key cofactors, cooperating transcription factors, epigenetic modifiers that can push things in one direction or the other between these programs? And from that, we may be able to get therapeutic targets out of those real targetable nodes. Hand in hand with that, we're also trying to do more small molecule screens to say: OK, are there actual drugs, either new ones or ones we already have in hand, that can push these programs the way we would like them to go and potentially engage a more growth-suppressive transcription from the androgen receptor?

Andrea Miyahira: OK, well, thanks so much for sharing this study with us.

Christopher Barbieri: My pleasure. Thanks for having me.