Andrea Miyahira: Hello, everybody. I am Andrea Miyahira on the Prostate Most cancers Basis, and thanks for becoming a member of me at the moment. With me is Dr. Abhijit Parolia, an assistant professor on the College of Michigan. He’ll current his group’s newest paper, “NSD2 is a Requisite Subunit of the AR/FOXA1 neo-Enhanceosome in Selling Prostate Tumorigenesis.” This has been launched as a pre-print in Bioarchives and is underneath evaluation. Thanks a lot for taking the time to be with us at the moment, Dr. Parolia.
Abhijit Parolia: Nice. Thanks, Andrea, for that sort introduction in addition to the invitation to come back and share this new work from our group. It actually facilities on finding out the mechanistic particulars of AR’s practical duality in regular prostate epithelium versus the cancerous or malignant variant of prostate most cancers cells.
Let me start by introducing you to the traditional prostate. It’s a walnut-shaped organ that sits on the base of the bladder and microscopically contains glandular buildings, predominantly made up of two kinds of epithelial cells: the outer basal epithelium and the inward lumen-facing epithelial cells referred to as luminal cells, which distinctively specific the androgen receptor protein.
Now, androgen receptor protein exercise in these luminal cells is completely vital. That terminal differentiation, shifting from these luminal progenitors, proven in crimson, to this terminal state, proven in blue, is the place AR actually drives their correct maturation right into a non-dividing state and reasonably promotes their secretory luminal capabilities in producing that prostatic fluid. Now, the androgen receptor primarily does so by binding to its ligand, testosterone or the first male hormone. As soon as internalized, it will get transformed into DHT and binds the AR, which triggers AR dimerization. Now, this homodimer translocates into the nucleus and binds to the DNA in a sequence-specific method. These are primarily referred to as androgen response components and comprise palindromic sequences containing two half AR websites oriented in an inverted conformation. Now, like most transcription components, the androgen receptor would not work in isolation. It reasonably works in live performance with a bunch of epigenetic and chromatin transforming proteins that regulate its activating or repressive transcriptional duties. So within the regular epithelium, AR actually drives a non-dividing secretory luminal cell state.
In distinction, and really very intriguingly, in most cancers that originates from the prostate epithelium, AR is predominantly expressed in virtually 90-95% of that main illness. These two seminal research again within the Nineteen Forties realized that in the event you take away androgen or outcompete it by estrogen administration, you set off dramatic atrophy of the malignant prostate epithelium. And ever since, androgen deprivation therapies following radiation or prostatectomy of the first illness have been the mainstay of remedy within the medical administration of the illness. You see these profound cyclical patterns of illness remission with both ADT within the type of castration or second-line therapies like enzalutamide or abiraterone that instantly bind to AR within the ligand pocket and outcompete DHT’s exercise and, due to this fact, repress AR operate.
So at the least within the malignant context, in distinction to the traditional epithelium, it seems that oncogenic AR actually drives the hyperproliferative, invasive, and survival properties of the malignant prostate epithelium. This brings me to the first thesis of this challenge: how can we clarify, mechanistically, the profound antitumor impact that we see in prostate most cancers cells upon inhibiting this terminal differentiation issue?
One of many main implications, due to this fact, that the broader subject has additionally began believing and interrogating, is that AR exercise will get dramatically reprogrammed seemingly from its terminal differentiation axes within the regular prostate epithelium to extra of an oncogenic exercise as soon as the prostate epithelium is remodeled. Increasing on these concepts, the seminal papers from the Pomerantz, Freedman, and Zwart laboratories counsel that somatically altered or cancer-specific epigenetic or chromatin transforming components may seemingly be concerned or implicated in reprogramming AR as soon as this transformation happens.
On this state, we hypothesize that there are two modes or types of the AR protein: one within the regular epithelium that drives a nonproliferative state, and the second, an oncogenic variant that sustains survival and metastatic and invasive properties of the malignant prostate epithelium.
To determine these subunits or components that may very well be implicated, now we have adopted varied orthogonal approaches. One distinctive strategy now we have taken is synthesizing these reporter cell strains, which we discuss with because the endogenous AR reporters. Endogenous is vital as a result of we hope to seize epigenetic regulation, on this case, of the androgen receptor protein. So we went into the KLK locus in LNCaP epithelial cells or prostate most cancers cells, and we engineered the KLK3 locus, KLK3 being a cognate AR goal, to include proper downstream of the KLK promoter an mCherry assemble fused by a P2A endopeptidase sequence to the KLK3 gene.
Now, this enables us to make use of mCherry as a cognate AR goal gene. We deal with these cells with a targeted epigenetic information RNA library, incubate them for about 5 to 7 days, deal with them with DHT, after which FACS type them into mCherry excessive and low populations. We then have a look at the information RNA sequences, and in the event you rank these enrichment profiles from the low mCherry cells to the excessive mCherry cells, you discover that, amidst different recognized co-activators of AR like BRD4 and TRIM24, NSD2 information RNAs are enriched within the mCherry-low inhabitants. This means that NSD2 is definitely a co-activator of AR exercise in prostate most cancers cells.
Now, what’s NSD2? It is really a K36 dimethyltransferase. It’s primarily concerned within the activation of gene expression, and that is thought to occur by antagonism of the PRC2 or EZH2 repressive duties that deposit the K36 trimethyl mark on the histones of the nuclear zones. It belongs to this NSD2 household or NSD gene household, the place NSD2 really notably templates two splice isoforms. So it is the identical gene producing two splice isoforms, with the lengthy kind having the SET area, which is the catalytically lively variant.
Now, intriguingly, and this, once more, builds on a few of the main work from the first reviews from Cory Abate-Shen group, the place they’ve really gone in and instructed that NSD2 reveals this improve upon transformation. However what we have executed on this paper is we have linked a singular cohort of prostatectomy specimens, and right here in a patient-matched setting, you’ll be able to really respect that the adjoining or the traditional tissue proximal to the cancerous lesion has little or no expression of NSD2, however in the identical affected person, the malignant lesions purchase NSD2 expression. So, actually, NSD2 is expressed upon malignant transformation. And right here you’ll be able to see a abstract from about 4 impartial sufferers. We have executed this in a TMA cohort as properly utilizing IF, which is a really delicate strategy the place you’ll be able to see AR is expressed within the benign epithelium in addition to the malignant types of prostate most cancers, however the NSD2 protein is sort of undetectable within the regular epithelial cells and is just expressed when these cells are fashioned both within the main prostate most cancers or the castration-resistant variant of the metastatic type of this illness.
By way of practical validation, we went forward and took these parental strains, and in the event you knock down each the lengthy or the quick isoform, you’ll be able to really see a downregulation of KLK3, which validates a CRISPR display. However extra importantly, on the proper, I really present you some fashions that now we have CRISPR-engineered, and you’ll respect both lack of each the lengthy and the quick isoform or the lengthy isoform alone, which is, once more, the catalytically lively variant. You see a marked loss within the exercise of AR goal genes or the expression of AR goal genes.
Now, notably, what’s actually spectacular to notice is that the AR protein ranges themselves don’t change, however its potential to activate or drive the expression of its cognate targets is dramatically attenuated within the absence of the catalytic exercise of NSD2. And even shifting away from these choose targets in a gene expression transcriptomic assay in RNA-seq, you’ll be able to see a wider AR-activated gene set to be dramatically blunted in its expression upon the lack of the NSD2 protein, which parallels a loss within the proliferative signature and a few of the different oncogenic hallmarks that I’ll share in subsequent slides.
However extra impressively, provided that we really see no change within the androgen receptor protein itself, we wished to see if there are any modifications within the binding of AR to the chromatin. And right here we had been in for a shock. We really discovered that within the wild-type NSD2 cells, AR sure a large cistrome, however whenever you take away NSD2, regardless of no change within the ranges of the AR protein, we noticed this dramatic downsizing of the androgen receptor cistrome, with virtually 65% of the websites being completely cleaned in cells that lack the exercise of the NSD2 protein. Now, that is really true even whenever you have a look at the warmth map. There’s completely no binding of those components whereas AR continues to bind simply fantastic on the impartial websites. And that is what actually led us to now divide AR binding components into NSD2-dependent and impartial compartments.
While you now interrogate FOXA1 binding, it stays simply intact. NSD2 would not regulate the binding of FOXA1, however within the absence of AR loading, you really see a dramatic attenuation of the activation mark within the type of K27 acetylation, actually suggesting that within the absence of NSD2, the dependent components are decommissioned as enhancer websites in these NSD2-null prostate most cancers cells.
Now, in the event you have a look at the motifs that basically underlie these components, we actually wished to differentiate what’s the distinctive function of dependent versus impartial websites. So whenever you interrogate simply the impartial components, you’ll be able to really see the recurrence of those full palindromic AREs that I launched to you earlier, the place you see the palindromic sequence. However this sequence wasn’t evident after we appeared on the dependent cistrome. The highest-ranking motif was, actually, the FOXA1 AR half-motif, the place the FOXA1 DNA sequence is juxtaposed to just one half of the AR sequence or the AR motif. Now, that is actually intriguing and actually means that NSD2 is maybe important in AR binding at these chimeric AR half-sites, and that is maybe extra evident in these read-density plots the place you’ll be able to respect that the NSD2-independent web site stays unaltered whereas in cis-proximity and chromosome 10, you see an entire elimination of AR binding at this web site. And in the event you interrogate the motifs, you really see the NSD2-dependent web site has the FOXA1 AR half-element whereas the NSD2-independent web site contains these full palindromic AREs.
So is there any relevance relating to these motifs being detected in main prostate most cancers sufferers? To essentially handle this query, we went again to Mark Pomerantz’s research from Matthew Freedman’s lab the place we downloaded the first AR cistromes generated from affected person tumor biopsies. We outlined regular CRPC and first particular AR binding websites, and whenever you now interrogate what motifs are particularly enriched within the main most cancers versus the traditional epithelium or mCRPC versus the traditional epithelium, you notice that the FOXA1 AR half-element or the AR FOX, so that is the inverted orientation, are particularly and markedly enriched within the most cancers epithelium.
One other notable function is that the complete palindromic AREs, if something, confirmed slight depletion upon development to a malignant state. In order that’s actually thrilling and means that the cancerous type of AR binding actually occurs at these chimeric sequences versus the complete palindromic motifs.
With the lack of NSD2, due to this fact, you see this marked attenuation in proliferation, you see a loss within the invasion or migration potential, in addition to a loss within the potential to kind colonies in addition to graft in mice, all of this being hallmark cancerous phenotypes. So what we actually consider is going on is that NSD2-deficient cells are resembling extra a standard epithelium versus a cancerous state.
Now, all this was properly and good, however we have all the time had a translational focus in our group, so our apparent query was, can it’s therapeutically focused in superior castration-resistant prostate cancers? And within the curiosity of time, whereas I don’t share the whole thing of how we labored on the compensation and so forth and so forth, we realized that NSD1 plus 2-targeting gave the impression to be essentially the most optimum approach of inducing cytotoxicity. And to essentially have the ability to make pharmacological brokers that might permit us to set off twin degradation of each of those isoforms, we really leveraged this warhead that was printed in 2021 by a bunch and tried to, with our chemist collaborators, convert them into these highly effective PROTACs that will bind to those proteins of curiosity and induce their proteasomal degradation, thereby sort of degrading these and affecting a therapeutic impact.
We synthesized virtually upwards of 150 odd derivatives and arrived at this explicit twin compound, which we name LLC0150. It primarily engages each NSD2 and 1, we consider, by way of the WWP1 area, which is discovered within the excessive N-terminal half, and you’ll respect in these Westerns, really triggering a substantial lack of each NSD1 in addition to 2 with out altering NSD3 expression. And within the cancerous cells, whenever you deal with the VCaP cells, on this case, with LLC0150, you induce a marked loss within the expression of AR MYC in addition to AR goal genes whereas inducing c-PARP accumulation, which means that these cells are experiencing some kind of apoptotic cell demise or stress.
We have now gone on to profile this twin compound extra comprehensively in a bigger panel of about 122 regular and cancerous human-derived cell strains. Right here we discover that whereas the NSD2-mutant types of most cancers—largely heme malignancies the place NSD2 is rearranged or mutated in an activating style in virtually 15-20% of the circumstances—are, as anticipated, essentially the most delicate. However that is instantly adopted by AR-positive types of the prostate epithelium, proven in crimson, and, to our shock, even AR-positive breast tumors, which is absolutely reassuring and distinguishes fairly dramatically from the aspect of toxicity seen in regular cells, proven in grey, and even AR-negative types of the tumor, proven in blue.
So with this, we’re fairly enthusiastic about reaching the conclusion that there are two modes wherein AR interacts with the chromatin. The predominant mode happens on the palindromic full AREs, maybe within the regular epithelium, the place AR is ready to occupy these websites even independently of NSD2 operate. However upon transformation, with the achieve or the ectopic achieve, reasonably, of NSD2, AR features entry to a bigger cistrome comprising these FOX AR half-motifs that we consider represent the AR neo-enhanceosome, or reasonably its cancer-specific complicated, which amplifies or wires its oncogenic exercise, driving proliferation, metastases, and different most cancers hallmark phenotypes.
With that, I am completely happy to introduce you to the workforce. Arul, my main mentor, is a vital participant, in addition to Irfan, with whom we collaborated at UPenn, who has executed a variety of the interactome work that I didn’t current as a result of time constraints. In my lab, primarily two people, each of them graduate college students, Sanjana and Yihan, have considerably contributed to this work. So I am completely happy to take any questions you might have, Andrea.
Andrea Miyahira: Thanks a lot for sharing this actually thrilling research. What causes NSD2 upregulation in prostate most cancers, and do you see any genomic alterations?
Abhijit Parolia: Proper, that is a really fascinating query. Really, that is the very first thing we examined in our genomic datasets. We see no amplifications of the NSD2 gene. In heme malignancies, there are literally activating hotspot mutations within the SET area. We do not see these in prostate most cancers. There’s one group—there is a JCI paper, I overlook the lead investigator—they projected that AKT phosphorylates NSD2, inflicting stabilization, however that’s nonetheless, I feel, an open query and really a lot of curiosity to our group by way of what actually results in aberrant or this overseas expression of NSD2 within the cells. I can inform you it appears to be extra by way of protein modifications reasonably than mRNA at this level.
Andrea Miyahira: Okay, thanks. And have you ever evaluated what fraction of prostate cancers, together with throughout totally different levels, are pushed by NSD2?
Abhijit Parolia: That is very intriguing. There are two causes for this. I feel, previous to this, there have been a few publications targeted on NSD2. So we’re not the primary to focus on this protein, however they targeted on its expression within the neuroendocrine or the AR-negative type of the tumor. Objectively, in the event you rank its expression in all types of regular or malignant prostate tissue, neuroendocrine prostate most cancers has the best expression of NSD2. It virtually mirrors what occurs to EZH2 expression within the prostate epithelium. So there may be definitely proof that NSD2 performs a job within the neuroendocrine tumor, which isn’t one thing we’re interrogating actively, however I do know Michael Shen’s group is engaged on it.
By way of our understanding, virtually each type of the traditional or main tumors that specific AR appear to additionally specific NSD2. So I might speculate that each one types, AR-positive in addition to AR-negative, specific NSD2. Nonetheless, its operate might evolve from supporting and unlocking the oncogenic exercise of AR within the main transformation to antagonizing, maybe, EZH2 within the neuroendocrine type of the tumor, the place EZH2 additionally takes on a bigger function by way of its hyper expression.
Andrea Miyahira: Okay, thanks. What are NSD1 and NSD2’s capabilities in regular tissues? Do they work together with AR in regular contexts?
Abhijit Parolia: NSD2 may be very simple; it isn’t even expressed within the epithelium within the regular tissue that now we have checked out thus far. We’re nonetheless making an attempt to make use of extra antibodies to validate that with IF, which is seemingly essentially the most delicate methodology. We’re additionally making an attempt to do it on the RNA degree simply to make sure by way of the manufacturing of the lively transcript. So by way of NSD2, I do not speculate that to be a standard operate. For NSD1, we’re within the means of validating a few of these antibodies for IHC. At this level, I do not know if it is expressed within the regular epithelium. However I can inform you that it is regarded as the first NSD2 paralog that competes with EZH2 and its repressive capabilities. So I would not be shocked if it has some function within the regular epithelium in that capability.
Andrea Miyahira: Okay, thanks. And what most cancers options are activated by way of the NSD2-dependent chimeric AR-binding websites?
Abhijit Parolia: Really, the very first thing we thought after we arrived at this protein was, “Oh, if we now inhibit it, we’re going to see acute cytotoxicity.” And we do not. We’ve got by no means really seen cytotoxic results from NSD2 inhibition alone, and that is what led us to essentially making an attempt to grasp if that was paralog compensation, maybe. And that is what actually made us arrive at this twin PROTAC of 1+2.
However one factor may be very, very sure. NSD2 loss alone definitely strips prostate most cancers cells of the first malignant phenotype. So that they develop dramatically slower, they don’t invade in any respect, there’s completely no potential emigrate and invade, and this could be one thing that NSD2 wires throughout epithelial tumors, it seems, they usually definitely don’t graft in mice. So all of that makes us consider that maybe cardinal hallmark options of a malignant cell are being wired by way of NSD2’s redistribution of AR, however survival just isn’t considered one of them. So it’s a must to hit NSD1 plus NSD2 to induce cytotoxicity.
Andrea Miyahira: Okay, thanks. What are your translational plans for the NSD1/2 PROTAC? And do you foresee any damaging penalties to NSD1/2 blockade?
Abhijit Parolia: Proper. Actually, at this level, we solely have a twin compound. LLC0150 has little or no in vivo bioavailability, which we try to enhance, and solubility as properly. So we have been a bit hampered in our potential to check its tolerability in mice by way of security. One other situation is that this variant would not bind mouse orthologs, so having the ability to carry out these research in mice has been a little bit of a problem. That stated, we’re actively working with our medicinal chemist collaborators. We’re doing a variety of SAR research to enhance on this compound and maybe arrive at a extra drug-like variant that we are able to inhibit within the in vivo context to deal with these questions.
Andrea Miyahira: Okay. Properly, thanks a lot for sharing this, and good luck with the remainder of your research.
Abhijit Parolia: Thanks a lot, Andrea. I actually respect the invitation. Thanks.
