Zachary Klaassen: Hi, my name is Zach Klaassen, urologic oncologist at the Georgia Cancer Center in Augusta, Georgia. We're on UroToday at ASCO 2025, in Chicago. Delighted to be joined by medical oncologist Dr. Bishoy Faltas at Weill Cornell Medicine. Thanks so much for joining us, Bishoy.

Bishoy M. Faltas: Thank you, Zach. It's a pleasure to be here.

Zachary Klaassen:  Just some amazing data you presented at ASCO looking at the CLONEVO study. And really we're looking at other targets to investigate. And so just tell us a little bit about the background of how this whole phase II trial came about.

Bishoy M. Faltas: So CLONEVO is a window of opportunity study of abemaciclib which is a CDK4/6 inhibitor. It's an oral CDK4/6 inhibitor that's approved for patients with form of metastatic breast cancer. And it's a CDK4/6 inhibitor, as I mentioned, and we started getting interested in that early during my postdoctoral work. And we started identifying those cyclin D1 amplification events.

And so those are high copy number of amplification events of cyclin D1 that are also associated with deletions of the 9p21 locus that includes CDKN2A, CDKN2B So p16 is CDKN2A and MTAP and then wild type RB. So that molecular subtype is a subtype that we recently published on in our paper in Nature. The highlight of that publication, though, was that a lot of these cyclin D1 amplifications occur within extrachromosomal DNA, and extrachromosomal DNA is actually fascinating structures.

These are circular structures of DNA that live and persist outside of the chromosomes, and they get segregated into cancer cells asymmetrically because they lack centromeres. And they harbor very high copy number of cyclin D1 because they don't play by the rules of normal chromosomes. So they have these very high copy numbers of cyclin D1. So we predicted that very high copy number of cyclin D1 is going to activate CDK4 because it binds to CDK4 and CDK6, which are these cyclin dependent kinases, to form active complexes that essentially control-- this is one of the main control points for the cell cycle.

So by using abemaciclib, we can block that whole cascade of events and stop the cell from dividing. So that was the rationale for why we chose this specific drug to target this specific pathway in these patients.

Zachary Klaassen: And with a high copy number, I mean it's an ideal target, isn't it?

Bishoy M. Faltas: Exactly.

Zachary Klaassen: So tell us a little about the design, maybe the intervention. How many patients for this clinical trial?

Bishoy M. Faltas: Yeah, so this was a window of opportunity study. So for cisplatin ineligible patients and as you know the standard of care for patients with muscle invasive bladder cancer is neoadjuvant chemotherapy now with immunotherapy followed by radical cystectomy. At that time, it was just chemotherapy. But as you know also about half of our patients are ineligible to get cisplatin due to a variety of reasons, kidney function, other aspects that preclude them from getting chemotherapy. So that's what we designed. That's the target population for this study.

And we, initially, of course had hopes that we would see activity with the drug. But we didn't want to delay their cystectomy. So we designed this as a window of opportunity study because otherwise those patients would just go straight to cystectomy or have a clinical trial. But we didn't want to delay their cystectomy because that's associated with negative outcomes. So we designed this to have to be again, hence the phrase window of opportunity. So that's our window.

And that's four to eight weeks of abemaciclib. Most patients actually ended up having four weeks. And this is an oral medication that's taken twice a day and stopped 24 hours before the cystectomy.

Zachary Klaassen: I see.

Bishoy M. Faltas: And it's very safe in terms of not associated with any negative surgical outcomes or healing or anything like that. And we know that from the breast cancer literature.

Zachary Klaassen: Excellent. And just tell us about some of the great molecular data you presented, some early efficacy data as well.

Bishoy M. Faltas: Yeah, so some of the efficacy data that we found was very promising. Only with four weeks of abemaciclib, we see 31% pathologic downstaging. We had three patients--

Zachary Klaassen: Four weeks, isn't it?

Bishoy M. Faltas: That's right, yes. And we had three patients who had path CRs. And I know that my urology colleagues would ask me, well, maybe it's just the TURBT. And maybe it was the TURBT. But we actually looked at using circulating tumor DNA to check because we had every patient enrolled after having a TURBT.

So we had a ctDNA time point at the day of enrollment and then on the day of starting abemaciclib and then two days after starting, two weeks after starting.

Zachary Klaassen: So you were thinking ahead. The surgeon was going to take all the credit. You guys said, well, we'll make sure we take a look at that.

Bishoy M. Faltas: Exactly, I got to be one step ahead of you. No, but we work together with our surgical colleagues and the TURBTs and the importance of a good TURBT cannot be overstated. But really the point was to try to really understand what's the contribution of the TURBT versus the drug because in all these neoadjuvant trials, you get a path CR or a downstaging. And it's very hard to know what that really means.

The point I'm trying to make is that's a very nice use case for serial or longitudinal circulating tumor DNA to try to get at that. And in our case, we see that most of the effect is really caused by abemaciclib, because the ctDNA actually slightly goes up in some cases after the TURBT. And then as soon as we start abemaciclib, it really goes down.

We did look at cyclin D1 amplifications by whole exome sequencing. We performed whole exome sequencing of all the pre and post tissues, pre and post-abemaciclib tissues in these samples. And we also the ctDNA, I would like to point out here, was whole exome sequencing ctDNA. So we sequence all 20,000 genes. And that was done by Caris Life Sciences, and we're very grateful for their support of that study who supported all these correlatives.

And we essentially found that there was a positive correlation between pathologic response in downstaging and cyclin D1 amplification. But you also always want to know if this is translating that high copy number genomic copy number is translating to actually high protein expression. So we performed a Hyperion imaging mass cytometry, and Hyperion imaging mass cytometry is a spatial technique to look at more than many proteins, in this case more than 40 proteins and phosphoproteins. And we were able to look to see that cyclin D1 amplification in more than 630,000 cells was associated with downstaging.

We also looked at RB phosphorylation. So RB is downstream of CDK4 and CDK6. So inhibition of CDK4 and CDK6, which is the target of this drug, would result in a decrease in RB phosphorylation. So we clearly saw on target activity as evidenced by a significant decrease in RB phosphorylation and Ki-67 which is a marker of cell proliferation.

Zachary Klaassen: So great great data phase II study 20 patients a lot to jump off of and to see where this goes next. What's next for CLONEVO abemaciclib? Where are you guys taking this next direction?

Bishoy M. Faltas: Yeah. So one of the interesting findings that we found was that in the patients who were given abemaciclib, we see a downregulation not only of these cell cycle dependent pathways or proteins, but also we see downregulation of some of the DNA repair genes. And some of these are cell cycle dependent. So specifically homologous recombination DNA repair of double-strand breaks. And we saw significant downregulation of two proteins-- one called RAD51 and another one called TOPBP1, which is a scaffold that recruits additional proteins that participate in double-strand break repair.

So we tested in the laboratory. So we actually established patient-derived organoids for some of these patients in the trial and other patients that we had and we tested that sequence. Could we give a drug that induces double-strand breaks and then followed by abemaciclib to prevent the cell from repairing these double-strand breaks. And that's essentially what we did. So we did that with EV (Enfortumab Vedotin). And EV has an MMAE payload. So that's a microtubule inhibitor. So you wouldn't necessarily expect it to cause double-strand break. But in this case, we see very clearly with EV an increase in gamma H2AX, which is a marker of double-strand break.

And then we see that the sequence of EV followed by abemaciclib is a very effective sequence. So that's where we're exploring next is different sequences where we can give either chemotherapy or different antibody drug conjugates is what we're really interested in followed by abemaciclib or other CDK4/6 inhibitors. And we think we have a lot of biological rationale, as I explained to you, to believe that would be a particularly effective combination.

Zachary Klaassen: Would you see those sequences, especially with EV, would that be in the muscle invasive neoadjuvant, kind of similar space you've been working in, do you think?

Bishoy M. Faltas: So we're thinking different spaces. We are thinking in the post EV pembro setting with different antibody drug conjugates. I think that's a huge unmet need because we don't really know what to do post EV pembro in the metastatic setting. But also as EV pembro probably moves in the first line setting within the neoadjuvant setting, that could also be another angle.

We are interested in this EV abemaciclib. So without pembro, that's another angle that can probably be tested in the neoadjuvant setting. So there are a lot of different ways to do this, but we're probably going to focus on these two settings.

Zachary Klaassen: Fantastic. Just great work great discussion as always. Anything we haven't hit on from the CLONEVO presentation you want to discuss? Any take-home messages?

Bishoy M. Faltas: I think you have covered it very well. We are now wrapping up this data. There's a lot of very interesting data that we're exploring in the spatial data sets. I only showed you we're just scratching the top here but there are so much very interesting data on the effect of abemaciclib in the tumor microenvironment that we're exploring in this very rich spatial data set. So stay tuned for the manuscript.

Zachary Klaassen: We'll do. And we'll have you back on to discuss it again for sure.

Bishoy M. Faltas: Thank you so much, Zach. Appreciate it.