Haolong Li: Thank you, Andrea. It's a great honor here at UroToday and discuss our recent work with Andrea that this work titled Genome-scale CRISPR screening identify PTGES3 as a druggable AR modulator. This is a big group effort from back when I was training at UCSF from my late mentor, Dr. Felix Feng, and my co-mentor, Luke Gilbert. And then a big shout-out for all the collaborators, as well as the reviewer for the help, suggestion. And then most importantly, this work was funded by Prostate Cancer Foundation. And then the work started by the center question that we're interested in the center target of androgen receptor, AR. We first want to know the fundamental AR biology will regulates AR. To do this, we first create a endogenous AR reporter in prostate cancer cell line. And we systematically use this genome-scale CRISPRi screening using this AR reporter. And then in this screening, we identify PTGES3 as a novel AR regulator. And we did quite a bit mechanism works that we found PTGES3 regulates AR protein levels and function. And it also represents a potential therapeutic target in the AR treatment-directed therapy treatment-resistant prostate cancer. And then to just give a little bit of background on AR androgen receptor. It has been known for a long time, and then work from our group as well as many others found that androgen receptor has this gene amplification as well as enhancer amplification and sometime both of them have amplified.
There's also hypermethylation in the promoter or the enhancer regions. This all result in high level of transcript of AR as well as result in high protein levels of AR. These excessive overexpressed AR can be stimulate even under the really low hormone levels that drive the prostate cancer cell to survive and the proliferation. One of the two big question at a time that really caught our attention is compared to normal prostate, why is the cancer cells can sustain such a high level of AR protein? And the other question we were wondering is can we unbiasly systematically characterize every single gene's impact on AR to understanding the AR biology? If we can do so, can we rank them and then potentially know what's the prioritized target for next-generation AR-targeted therapy? We've started our journey by first really created a reporter that truly faithfully reflects AR gene expression. Our approach here is a little bit new idea and then a little bit creative idea here by using these split fluorescence protein. We have these type of small tag of the fluorescence protein 11 and then the larger counterpart.
Neither of them have a fluorescence, but only when combined it will light up and reflects AR gene expression. We first CRISPR knock in the neon green 11 part into the N-terminus of AR and then have large part expression there. And then we have through quite a bit effort that we finally had this AR reporter established in prostate cancer cell line, which faithfully reflects the AR expression as well as the localization and then the cells behave exactly the same as the parental cell line. Little bit of visual to look at it. There's a baseline of AR expressed in the nuclear. It can stimulate by androgen, can quashed by anti-androgen. And if you put AR degrader, the protein completely disappeared. We also went on to genotypically and phenotypically characterize these cell lines to truly with the test these AR reporter in the prostate cancer cell line. With these AR reporting in hand, we use the state-of-the-art genome-scale CRISPR screening here. The way it works is we have a large pool of guide RNA that targeting every single coding gene in the genome. We infect the cells with really low MOI, ensuring in this pool of cells, one cell only gets one perturbation.
Collectively, they simultaneously reflect perturbations of the entire genome. We then put all these pool of cells through flow cytometer, sort them based on the AR expression, reflects by the green fluorescence with the bottom 25 and then top 25. And then we sequence the guide RNA in these bottom and the top populations and then use these to infer the regulators of AR. To look at the result in these volcano plot, on this side is a gene that when you knock down them, reduce AR expression and the vice versa on the other end. If we focus on this zone in this screening that we identify knockdown AR, reduce AR expression, which tells us the positive control works. We also uncover some of the canonical regulators of AR that previously known GRHL2, HOXB13, and GATA2. In the top five hits that we found this gene called prostaglandin E synthase 3, PTGES3, as the top novel AR regulator that previously have less knowledge on the regulations of AR protein levels.
Then when we went on, actually did a lot of mechanics studies on how this PTGES3 regulates AR. To summarize our finding that based on the literature in the cytosol of the prostate cancer cells, PTGES3 has this dual function can serve as a chaperone protein, as well as the enzyme function that potentially regulates AR. We went out and did a lot of mechanism study on these. We also found that PTGES3 in prostate cancer cells has this nuclear localization that potentially help the active AR that to regulate as AR function serve as somewhat as a coactivator of AR function as well. And we also has this collaboration with Dr. Elizabeth Wasmuth, which is a Prostate Cancer Young Investigator. We combine her structured biology knowledge as well as our FoldAI to predict potentially how this works. We actually successfully show that PTGES3 interacts with AR-dimer binded to ARE. We also has in vitro cell assays to demonstrate this activity as well. With all these knowledge in hand, we're really curious whether PTGES3 will be a potential therapeutic target for prostate cancer. In this cohort of patients, when we look at ADT-treated patients, that high PTGES3 patients have a worse outcomes compared to the low PTGES3 population showing this has some therapeutic potential. The other thing we do is we systematically knock down PTGES3 in these ARV7 expressed 22R1 cells that shows reduced expression of reduced growth of the cells. And then this is also true in the enzalutamide-resistant MR49F models as well. This is also true in AR gene amplification, also ARV7 positive cell lines. And then we also further validate knocking down PTGES3 in vivo also repressed tumor model growth.
This shows the potential that PTGES3 could be a good therapeutic target for prostate cancer. Just quickly to summarize what we found in this work, we developed this live-cell endogenous AR reporter using a genome-scale CRISPRi screening and identified genes that requires for AR protein maintenance, so provide a fundamental AR biology. Prostaglandin E synthase 3, PTGES3, emerge as a top and unexpected regulators of AR, a little bit different than the canonical traditional AR co-factors or chaperones. PTGES3 loss reduce AR protein levels, and then reduce AR-driven transcription, and then can impair the survival of AR-driven therapy-resistant prostate cancer model. PTGES3 can directly bind to AR, and it is required for AR function at a target gene in the nucleus. High PTGES3 expression is associated with resistant to AR-directed therapies and then poor clinical outcomes. And then we didn't show in this work, but with the support of the Prostate Cancer Foundation, we actually had a early proof-of-concept, covalent PTGES3 inhibitor that can phenocopy the genetic AR suppression and support PTGES3 can be a therapeutic target. A little bit of take-home message for this work is PTGES3 is novel regulators of AR stability transcription activity in prostate cancer. Targeting PTGES3 represents a new strategy that suppress AR signaling that is distinguished from the current AR-directed therapies.
And then PTGES3 inhibition has the potential to overcome some of the common mechanisms of the resistance in AR-driven disease. And then I just want to use this time quickly thank the effort from my lab, and then from my co-mentor Luke Gilbert's lab, and then my collaborators at Fred Hutch, and then especially my peer, Prostate Cancer Young Investigator, Dr. Elizabeth Wasmuth. And then of course my PhD mentor, Dr. Felix Feng, and then Dr. Kevan Shokat and then everyone in the Feng Lab and our wonderful collaborator, and then the support from Prostate Cancer Foundation. Thank you.
Andrea Miyahira: Well, thank you so much for sharing this interesting study. What impacts does PTGES3 have on AR variants like ARV7?
Haolong Li: Yeah, that is actually a great, great question. We're interested as well. The V7 represents this resistant mechanisms in prostate cancer. We actually had some preliminary data that showing knocking down PTGES3 reduced V7 expression. This was done, I believe it's in 22R1 and then VCaP cells. And then the specific how this works, we have two hypotheses. One is through reducing the full-length AR, somewhat similar to other people observe as that as well, or there's a new work that we are thinking to further explore how specifically that these PTGES3 impact V7, this last variant expression.
Andrea Miyahira: Thank you. Do you think that PTGES3 can impact interactions with or efficacy of direct AR inhibitors such as enzalutamide or the AR-targeting PROTACs?
Haolong Li: That is a great question. Actually, this is some of the work we're really interested in how PTGES3 in foundational AR biology, but we are also interested whether it's related to treatment resistance. We have some preliminary data that in the work in the paper, also now included in the paper that we show knocking down PTGES3 can reduce the enzalutamide-resistant model that enzalutamide-resistant 22R1 and then MR49F cell growth. We also has a little bit of data that's showing in some of the, it can enhance the treatment response for the PROTACs. And then over on the contra end, over-expression PTGES3 reverse that can protect the AR proteins from get degraded by the PROTACs. Again, a lot of work need to do to illustrate how this works and how does having more PTGES3 protect AR protein from degradation.
Andrea Miyahira: Okay, thank you. And have you determined whether PTGES3 is mutated in prostate cancer or if levels change during disease progression or the acquisition of resistance to AR-targeted therapy?
Haolong Li: Yeah, this is a really, really good question. In terms of mutation, it's pretty interesting. Again, this is a super interesting protein that actually has relatively small, 23 kDa. So the mutation actually does not happen very frequently for PTGES3. There are two mutation that we describe, which is a functional mutation, but doesn't necessarily mean they happens in prostate cancer. One is related to their chaperone function, one is the enzyme function. More or less, this is from a fundamental biology study, less of this was observed in patient or in human so most of the mutation does not happen very frequent in prostate cancer. In terms of the expression, I would say that most of the cancer patient or cancer cells have a higher expression of PTGES3, specifically in the hormone-driven ones in prostate cancer and breast cancer. And then we are currently want to getting into explore these paired pre- and post-enza/abi patient samples to understand how the expression of PTGES3 change that after the treatment, and then so to know that will be really important on how do we anchor the treatment or therapeutic window for PTGES3.
Andrea Miyahira: Okay. And based on this data, it sounds like you think PTGES3 does have potential as a therapeutic target, but what context do you think and what side effects might you anticipate? And do you know if it has a role in non-AR pathways or non-prostate cells that may be impacted by targeting this?
Haolong Li: Yeah, that's a really great question in terms of thinking this as a therapeutic target. Now from our data, we found that PTGES3 is relatively, or I would say pretty specific to AR-positive prostate cancer cell lines and is not that essential or not essential at all for the non-AR-positive prostate cancer cell line PC3 or DU 145. Now with that being said, PTGES3 were known that this is back long time ago in the early 2010s that the fundamental work on PTGES3 on glucocorticoid receptor. There's a crystal structure of PTGES3 also helping with glucocorticoid receptor as well. We foresee there's something that will cross-function with glucocorticoid receptor. And then this is not necessarily a bad thing for prostate cancer treatment because as a previous work that proposed that glucocorticoid receptor actually could be a alternative way for the enzalutamide-resistant or treatment-resistant mechanism. So having targeting PTGES3 may actually targeting both receptors, so that is something that we currently explore.
Andrea Miyahira: Okay, thanks. And finally, what are your next steps in these studies and your biggest questions that you plan to investigate next about PTGES3 biology or any translational work?
Haolong Li: It's still really fascinating to us when we anchor on this protein because it is one of the protein that identified very early in the biology knowledge side, but still so much to know about this small protein and then the function of its chaperone protein and then the enzyme function that we did not have discussed quite very thoroughly just only in this work for the sake of focusing on the AR signaling. And then also the nuclear function, how specific and how does that work and can we get a structure on how this all goes into what regions of PTGES3 interacts with AR? We have some predictions, but we need more data, actually crystal structure or other type of biochemistry data showing that.
Why that is important is it help us to understanding these fundamental AR biology, how AR interacts with PTGES3 to design. We have, again, with the support of the Prostate Cancer Foundation, we have a proof-of-concept compound that this is a huge collaboration with Feng lab, with Dr. Kevan Shokat lab who developed the first KRAS G12C inhibitor. We actually used a similar method to develop this covalent PTGES3 inhibitor. That will be the next wave of further improve these proof-of-concept inhibitor, and how do we actually translate these into more preclinical drugs and then actually convert the knowledge into benefit clinic.
Andrea Miyahira: Okay. Well, thank you so much, Dr. Li, for sharing this with us, and good luck with your next studies.
Haolong Li: Thank you.