Senior Scientist, Dewpoint Therapeutics
|Type||Kitchen Table Talk|
On September 15, we were beyond thrilled to welcome Broder Schmidt, from Stanford University School of Medicine, for a lively Kitchen Table Talk—finally broadcast in part from Dewpoint’s actual (and new) kitchen table!
Broder has been interested in phase transitions throughout his training. He did his PhD studies with Dirk Görlich at Max Planck Institute for Biophysical Chemistry, where he helped shape our current understanding of the nuclear pore’s selectivity and permeability. He is currently a postdoc studying functional consequences of aberrant TDP43 condensates with Rajat Rohatgi, where they have published several elegant studies including the effects of phase separation on splicing.
Broder delivered a phenomenal lecture that spanned many of the main concepts currently emerging from the condensates field: from thermodynamics and reentrant behavior to partitioning of small molecules into condensates. I’m happy to share it with you below. And make sure to check out Broder’s recent bioRxiv preprint because it shows that condensates not only can be drugged, but that we have been doing so unknowingly.
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Bede Portz (00:01):
So it’s a real thrill to introduce Broder. Having had countless conversations with him about science, I find him to be one of the deeper and more rigorous thinkers in our field. And Broder’s been interested in phase transitions throughout his training, dating back to his time as a Ph.D. candidate with Dirk Gorlich. He had an excellent paper where he reconstituted via phase separation the nuclear pore in vitro, and really elucidated rules about its selectivity and permeability, and that paper’s been cited nearly 200 times.
Bede Portz (00:36):
He then went on to Raj Rohatgi’s lab at Stanford, where he currently is, and he continued working on phase transitions there despite it not really being a phase separation lab. And there Broder had a series of papers that were really elegant on TDP43. The first was really prescient. It sort of helped to usher in this current interest in condensates with multi-layered topologies. And then the follow-up paper was really a careful dissection of the molecular grammar of TDP43’s self assembly, and how that relates to function. I note that second paper in Nature Communications also involved this ingenious splicing reporter assay that’s now been used by other groups.
Bede Portz (01:24):
More recently, he has turned his attention to cancer, where he’s studying the partitioning of chemo-therapeutic agents into condensates, and this paper really highlights some of the things that make Broder unique. So Broder reads essentially every paper, and his most recent work on cancer really is a synthesis of everything that’s happening in our field right now, from thermodynamics to reentrant behavior, to the partitioning of small molecules into condensates with specificity. I think that really reflects who Broder is as a scientist.
Bede Portz (02:01):
I finally want to add that Broder’s also a leader. He’s one of the founders of the intrinsically disordered special interest group at Stanford and the Carnegie Institute. And that organization exploded during COVID and provided an opportunity for scientists from diverse backgrounds and across the globe to present their work in an environment that was otherwise devoid of in-person meetings. I owe Broder a debt of gratitude for that organization because I gave a talk there, and that helped put me on Dewpoint’s radar, for which I’m obviously very grateful.
Bede Portz (02:39):
I want to highlight that although Broder has only been in the cancer game for a short period of time, he recently won a Forbeck scholarship for his work in that arena. And without further adieu, I’m going to turn it over to Broder.
Broder Schmidt (02:53):
Well, thank you Bede for this very kind introduction. And thank you for Dewpoint for inviting me today to discuss approaches to target disordered proteins. But I wanted to actually start out our discussions by acknowledging one of the most powerful concepts in biochemistry, and that is the idea that structure is function. The idea that the three dimensional folding of a protein is intimately linked to its function. For example, this kinase that I show here that adds phosphate groups to proteins. In fact, we now know that cells rely so heavily on correctly folded proteins that they employ a sophisticated network of chaperones and spend vast amounts of energy to ensure the proper folding of proteins…
Broder Schmidt (03:36):
However, it is now very clear that many proteins contain extended regions that do not fold into a stable tertiary structure, even in the presence of chaperones, and we call these unfolded protein domain–that you can see here in this green shade in this AlphaFold2 prediction–intrinsically disordered regions or short IDRs. And I want to emphasize that I’m not just cherry picking one example here. In fact, if you go to the DepMap database, you’ll find that roughly 15% of all genes that encode for essential proteins across hundreds of different cell lines are more than 30% disordered.
Broder Schmidt (04:12):
And this brings up the question of, what is the function of these disordered domains? And work by many groups over the last 10 years or so have revealed a number of key functional categories, such as barrier formation by FG domains, interaction platforms, regulatory domains such as the Pol-II C-terminal domain, environmental sensing, or transcriptional activation.
Broder Schmidt (04:35):
And as you can appreciate from this list of very fundamental and essential functions, the dysregulation and the dysfunction of disordered proteins is linked to diseases, in particular cancer and neurodegenerative diseases as Bede alluded to in his introduction.
Broder Schmidt (04:55):
So it’s really become very clear that intrinsically disordered proteins are emerging as key regulators of cell state in health and disease. So what if we could drug disordered proteins? We should be able to control cell state and treat these devastating diseases.
Broder Schmidt (05:14):
And what I want to talk to you about today is that it turns out that oxaliplatin, a cornerstone of cancer therapy since two decades, is exactly doing that. And specifically, what we found is that oxaliplatin leverages the fact that many disordered proteins do not function in isolation, but rather function in a pack, similar to wolves. And instead of being a social behavior, in the case of disordered proteins, this is a biophysical property that is encoded in the sequence, that manifests in the phase separation of the disordered proteins into biomolecular condensates.
Broder Schmidt (05:52):
I know I’m preaching to the choir here, but I do want to point out that a few examples of where the phase separation of IDPs and biomolecular condensates have been firmly linked to function. And one of the examples would be ribosome assembly, which occurs at the nucleoli which are liquid-like condensates Bede mentioned. The nuclear pore barrier, and another example would be, RNA transport granules, for example, which are RNA-protein granules.
Broder Schmidt (06:21):
So what is this oxaliplatin and how does it work? And I want to point out right from the beginning that this is actually a collaboration. Answering this question is a collaboration with Zane Jaafar and Onn Brandman, also at Stanford Biochemistry.
Broder Schmidt (06:39):
Oxaliplatin is a third generation organoplatinum compound that is comprised of a platinum warhead, an oxalate leaving group, and a diaminocyclohexane side chain. And much like earlier generations of platinum compounds, in particular cisplatin and carboplatin, oxaliplatin is believed to target and alkylate DNA, which causes crosslinks. And these crosslinks lead then to oxidative stress, replication stress, and DNA damage, which ultimately results in cell cycle arrest and cell death.
Broder Schmidt (07:09):
However, a number of key findings in the recent past actually have questioned that this is really the mechanism of action of oxaliplatin. In 2017, in a very beautiful functional genomics approach, it was shown that oxaliplatin actually has a genetic signature that is more reminiscent of ribosome biogenesis stress rather than DNA crosslinking.
Broder Schmidt (07:33):
Earlier this year, Emily Sutton in the lab of Victoria DeRose showed that oxaliplatin inhibits ribosomal RNA synthesis. And Zane and Onn, when mapping RNA-seq reads to primary transcripts either from Pol-I (which originated in the nucleolus) or Pol-II (as a control), noticed that oxaliplatin also interferes with RNA processing, as you can see here in this additional peak of map reads to this very end of the 45S rRNA that is usually cut out in the maturation of this ribosomal RNA.
Broder Schmidt (08:11):
And also this year, it was basically shown using nanoSIMS or if you want to imaging mass spec, that oxaliplatin actually accumulates in nucleoli.
Broder Schmidt (08:19):
So what are nucleoli? Nucleoli are multilayered ribosome factories that form around ribosomal DNA in the nucleus. And it’s firmly established that each of these different layers has its own function. Now in the center, this is the fibrillar center, rRNA transcription occurs. Surrounding this fibrillar center, FC, is the dense fibrillar component, or DFC, where pre-rRNAs are processed. And then all of this is embedded in granular component, or GC, where actually ribosomal subunits assemble.
Broder Schmidt (08:53):
And as you can imagine from these different functions, these different layers also require a different set of proteins and key proteins in the FC will be the RNA polymerase I. The scaffold and the key RNA modification enzyme in the DFC is fibrillarin, and the probably best-studied scaffold of the GC is nucleophosmin. I highlight these proteins here because we can actually use them as markers to study these different phases.
Broder Schmidt (09:24):
As I alluded to early on, beautiful work from Cliff Brangwynne and Tony Hyman has really established that nucleoli overall behave like liquids. And we can see this here in these beautiful DIC images where a nucleolus that come into proximity fuse over time just as the raindrops on your windshield will do.
Broder Schmidt (09:48):
Following this observation, Marina Feric in Cliff Brangwynne’s lab has established that actually these different sub-layers have their own material properties that slightly vary. So the granular component is very viscous, a viscous liquid, whereas the DFC and possibly also the fibrillar center have an elastic component so they behave a little bit more gelly, if you will.
Broder Schmidt (10:13):
Then in a series of papers over the past years, spearheaded by the labs of Richard Kriwacki and Cliff Brangwynne, it was reestablished that these different material properties in the phases and the different solubility of ribosomal components as they mature in these different layers are actually the basis of the vectorial assembly of ribosomal subunits.
Broder Schmidt (10:36):
So given this link of oxaliplatin to nucleoli and the link of phase separation and multi-phase organization of the nucleoli to function, we wondered how this reactive molecule, oxaliplatin would affect phase separation of the nucleoli? To this end we were very fortunate that Manuel Leonetti at the Chan Zuckerberg Institute shared with us a cell line where the endogenously labeled nucleophosmin and fibrillarin with fluorescent proteins. And we combined this with a FISH staining against this 5′ ETS, and this 5′ terminal region in the 45S rRNA as a marker of rRNA. So then we can have a look at these different phases.
Broder Schmidt (11:21):
When we treated the cells with cisplatin, a molecule that has established to cause DNA crosslinks, we didn’t observe any effect on nucleoli. However, when we added oxaliplatin to the cells, we observed a number of striking effects. First of all, you can see that the size of the nucleoli dramatically reduced. We can also appreciate that the nucleoli are much more spherical. And if you look closely at the different markers, all of a sudden you observe that this nucleoli ultrastructure is altered and disrupted by oxaliplatin. We can actually see how nucleophosmin concentrates together, rounds up. We can see that fibrillarin has the most strong signal here at those points where the nucleophosmin signal is the lowest. And then this rRNA seems to accumulate at the interface of these two layers. And you can appreciate this very nicely here in this 3D reconstruction where you again have the rRNA sandwiched between this fibrillarin cap and the nucleophosmin.
Broder Schmidt (12:30):
This was really indicative to us of major changes in the phase properties of the nucleolus, especially this rounding-up effect suggests that there were also massive changes in the biophysical properties such as the surface tension. And indeed, when we estimated this, we found that changes in surface tension are at least one order of magnitude compared to the untreated or the cisplatin treated case.
Broder Schmidt (12:53):
What is interesting is that we not only observed that oxaliplatin caused the disintegration of the nucleolus ultrastructure at large. When we actually looked at multiple markers here in the granular component, in particular nucleophosmin and SURF6, which have been previously shown to actually interact and form the scaffold of the granular component. This was work from Diana Mitrea in Richard Kriwacki’s lab, when she was there. We noticed that oxaliplatin actually, I’m sorry, I’m skipping ahead, also demixes these two, this granular component.
Broder Schmidt (13:34):
You can actually see this here, nucleophosmin again, becomes more spherical and we see that the SURF6 signal actually localizes to the outer surface, the outer rim of this nucleophosmin phase. And inspired by the work from Diana and Richard, what this really suggested to us is that there was a switch from these heterotypic nucleophosmin and SURF6 interaction to more homotypic interactions. And to follow this up, we decided maybe we can use oxaliplatin to carefully map phase diagrams of this behavior.
Broder Schmidt (14:10):
This was really inspired by the idea that if we just take a standard two-dimensional phase diagram, right? What we typically plot is an interaction parameter, that we typically express as a function of temperature or salt concentration against protein concentration. So basically when we start out here, this one phase regime of the phase diagram, increase the concentration to go to this point two, we will have phase separation. And if we then, for example, change the temperature, increase the temperature, we can dissolve these phases.
Broder Schmidt (14:40):
So we were wondering, perhaps oxaliplatin could be a modulator of this interaction parameter and so we decided to plot this against the concentrations of nucleophosmin. These concentrations we can easily measure from our microscopy images, right? The concentration is essentially proportional to the fluorescence intensity so we quantify the signal here in the nucleoplasmin, the light phase, and we quantify the signal in the nucleoli and our dense phase and we plotted it here in our plot.
Broder Schmidt (15:18):
Obviously, the concentration of oxaliplatin we know because we added it to the cells. What’s interesting when we titrated in increasing amounts of oxaliplatin, we first of all observed that the light phase concentration didn’t change much. Or in other words, the critical concentration for phase separation didn’t change much. What we noticed is that the dense phase concentration changed quite a bit. If we compare this here to the untreated case, we see that there’s an increased driving force for nucleophosmin phase separation.
Broder Schmidt (15:46):
Surprisingly, we observe exactly the opposite for SURF6 where if you compare this to the untreated case, we observe that there’s a decreased driving force for phase separation. The dense force phase concentration is clearly reduced.
Broder Schmidt (16:03):
Now, increased and decreased driving force, right? This is a qualitative term. It turns out we can actually quantify this, and I want to point out that this is inspired, really, by the beautiful work from Josh Riback when he was a postdoc in Cliff Brangwynne’s lab. And he essentially showed that the free energy, the free transfer energy of, let’s say, a nucleophosmin from the light phase into the nucleoli is a function of the partitioning coefficient, or the ratio of the dense to light phase concentration. Which means there’s nothing else than essentially the width of the binodal, here, in our phase diagram.
Broder Schmidt (16:37):
This now allows us to essentially quantify this change in driving force compared to the untreated case, right? So this is essentially a delta delta G that we calculate here. And we can see in the case of nucleophosmin, if the delta G for the untreated case is smaller than the delta G in the treated case, the delta delta G will be negative, so there will be a greater driving force.
Broder Schmidt (17:05):
What this analysis now allows us is to build a super phase diagram where we now can compare in one plot the behavior of two different proteins, in this case, nucleophosmin and SURF6. Before I show you the data, I kind of want to walk you through this somewhat more complex plot. We have, essentially three quadrants in this plot, only two of which really matter for our analysis. This first quadrant here, both the delta delta G for nucleophosmin and SURF6 will be positive, meaning that both their phase separation will be energetically more unfavorable.
Broder Schmidt (17:41):
In this second quadrant, we have a negative delta delta G for nucleophosmin but a positive delta delta G for SURF6, meaning that the phase separation of nucleophosmin in this regime will be energetically more favorable, whereas the phase separation of SURF6 will be energetically less favorable. What I’m about to plot here is various drugs at various concentrations and the concentrations will be color coded. I’m not only showing cisplatin and oxaliplatin, the two clinically used anti-cancer drugs we’ve already compared before. I’m also adding a third drug here, which is actinomycin D, which is a selective RNA polymerase inhibitor, which is actually not clinically used because it’s so toxic that there’s no therapeutic window.
Broder Schmidt (18:31):
So when you add clinical concentrations of cisplatin nothing happens to the nucleoli as we have seen in the images. But as you increase the concentration of cisplatin, we start to observe this demixing effect. If we have very high concentrations of cisplatin, the extent of this demixing is actually very similar to what we observed with clinical and low concentrations of oxaliplatin.
Broder Schmidt (18:54):
As you can see here, what is interesting, as we titrate in more oxaliplatin, we actually have this inflection point and the delta delta G for nucleophosmin transfer from the light phase to the dense phase becomes less negative. And if we add actinomycin D, we can actually push this entire system into this regime where both the delta delta G for nucleophosmin and SURF6 are positive, indicative of the complete dissolution of nucleoli.
Broder Schmidt (19:28):
What is really cool about it is now we essentially have a map where we can compare drug concentrations to effects on nucleoli. We have an idea in what regime we would expect to have a therapeutic window that is similar to oxaliplatin. What was also very cool to see when Zane actually treated purified fibrillarin and nucleophosmin with oxaliplatin and cisplatin, he observed the same type of dose dependence. You can see that oxaliplatin readily modifies fibrillarin as visualized here on this protein gel, this is read out with antibody staining against fibrillarin or nucleophosmin. We can see here this shit on the gel, indicative of the crosslinks. We can see oxaliplatin readily does this at clinical concentration, and the amount of this modification increases with increasing concentration of oxaliplatin whereas cisplatin is much weaker in this effect. And from his analysis it also appears that fibrillarin is more heavily modified compared to nucleophosmin.
Broder Schmidt (20:34):
What was really cool to see is that this really suggested to us that oxaliplatin my not only modify nucleic acids on RNAs in this case, but also proteins, and in particular disordered proteins like fibrillarin.
Broder Schmidt (20:48):
Well, but what about actinomycin-D. Unlike oxaliplatin, actinomycin-D is not a reactive molecule but as you’ll recall from this plot, it has a very strong effect on nucleoli. What we found very helpful to discern the difference between these two graphs was actually not only a titration series but also a temporal analysis of the effect. Particularly what we did, we looked at nucleolar function and used as a proxy this 5′ ETS signal, so the rRNA signal in our FISH. We looked at demixing simply by measuring the changes in the eccentricity of nucleophosmin. Essentially asking for it to go from an elongated shape to a more rounded shape.
Broder Schmidt (21:33):
What we observed is when we treated the cells with actinomycin-D, there was an immediate effect on nucleolar function. On RNA transcription and processing. However, the effect on morphology, the demixing effect by actinomycin-D was actually delayed by at least half an hour. And in the case of oxaliplatin, we observed that the function and the demixing effect actually happened around the same time.
Broder Schmidt (22:01):
So what we think is going on here is that oxaliplatin first causes the nucleolar demixing, which then leads to the dysfunction of nucleoli. Whereas, actinomycin-D first inhibits the function and then the lack of rRNAs will cause the nucleolar demixing.
Broder Schmidt (22:21):
The model that we proposed it that actinomycin-D passively defuses into nucleoli where it then specifically inhibits polymerase I which immediately kills cells. So in a way, you can think of actinomycin-D as a kill switch. If you add it, boom, RNA transcription stops, cells die. Because this is so effective there is essentially no therapeutic window because cancer cells and non-cancer cells will just die right away.
Broder Schmidt (22:49):
Now in the case of oxaliplatin, we believe that the drug enriches in nucleoli due to matching physicochemical properties and this is in line with the observation from the nanoSIMS that oxaliplatin does enrich nucleoli. And it’s also very much in line with beautiful work from Isaac Klein and Rick Young that showed that this is also the case for a number of other chemotherapy drugs.
Broder Schmidt (23:16):
This enrichment, then, allows oxaliplatin to chemically modify nucleolar scaffold components, especially fibrillarin and rRNA, which then causes the nucleolar demixing, cell cycle arrest, and more importantly, a more gradual decline in ribosome assembly. So oxaliplatin acts more like a dimmer that causes a slow cell death. And what will be very interesting in future work to see whether oxaliplatin exploits the increased means of cancer cells and ribosomes to sustain rapid growth or, perhaps also and, whether it exploits difference in nucleolar morphology, and nucleolar biophysical properties that are already present in cancer cells and it just exacerbates that effect.
Broder Schmidt (24:07):
What this really suggests is that, in my opinion a new paradigm for drug development. What I mean by this is if we look at the conventional approach to develop drugs, they must have strong focus on targeting specific molecules with high affinity binders. This is known as the ‘lock and key’ model. And what our work and the work of Isaac and others suggests is there is now another way to develop drugs and this is by targeting the biophysics of condensates. I call this the ‘seek and disrupt the pack’ model because you’re not directly targeting the function but the biophysics. The idea is you can expand the therapeutic window of many drugs and you actually can drug essential cellular functions such as transcription and translation.
Broder Schmidt (24:56):
In the last few minutes of my talk, I actually want to take on a more critical stance of this model and ask the question of how broadly applicable it is? We actually know that many disease-linked IDPs stray from the pack, so to speak. So the question really is, can you drug the physics of lone wolf IDPs, as I kind of called this a little bit eccentrically in my title.
Broder Schmidt (25:20):
The short answer is that it turns out it’s still very tricky. But what do I mean by all of this? And to explain this to you, I want to give you an example, and this example is the RNA binding protein TDP43, which is a very relevant example. TDP43 is predominantly localized in the nucleus and is actively transported into the nucleus. There’s also been some reports of some passive diffusion out of the nucleus. So, there’s some low level of shuttling. TDP43 is tightly linked to a number of neurodegenerative diseases, in particular amyotrophic lateral sclerosis, frontal temporal dementia, and the dementia late.
Broder Schmidt (25:59):
Really, the clinical hallmark of these diseases is that TDP43 forms these cytoplasmic aggregates in this disease. Now, in its physiological form, TDP43 has been shown to associate with a number of different condensates, including paraspeckles, TDP43 foci, nuclear stress bodies in the nucleus, and RNA traffic granules and stress granules in the cytoplasm. I think what is important to highlight here is that in none of these cases, there’s really very firm evidence suggesting that TDP43 is a scaffold of any of these condensates to a similar degree, as for example, fibrillarin is for the DFC.
Broder Schmidt (26:42):
This brings up a number of key questions that actually Bede very nicely highlighted in his recent review. That is, in what phase does TDP43 function? Does it have different functions in different phases? How does this vary between cell types? And then more from a drug development perspective, is TDP43 phase separation obligatory? Meaning, if we would target one condensate, can we live with the consequences this may have on another TDP43-containing condensate?
Broder Schmidt (27:15):
I think these are big questions that will take a few years to fully unravel. And I think one of the major barriers to finding good answers to these questions is the need of new quantitative tools to study the function of disordered proteins. And, as Bede said, we’ve actually spent quite some time on developing such tools for TDP43 and I just wanted to give you a quick taste of it as time is limited.
Broder Schmidt (27:43):
So TDP43, one of the best understood functions of this RNA-binding protein is the skipping of cryptic exons. TDP43 binds to defined sites of RNAs. This binding will then signal to the cell, to the splicing machinery, that the exon here has to be skipped. And we thought, we can use this function, and if we just take such a TDP43-dependent exon and put it into a fusion between a GFP and an mCherry we could essentially build a fluorescent reporter for this splicing event.
Broder Schmidt (28:21):
So the idea is that in the presence of functional TDP43, this exon gets skipped and we get both a GFP and an mCherry signal. However, if we have non-functional TDP43, we would only get a GFP signal. This now allows us to read out the splicing event in a quantitative and facile manner on a signal cell level using flow cytometry.
Broder Schmidt (28:44):
You can see this here, we transfected this reporter into wild type cells and then transfected it into TDP43 knock-out cells, and can see here this difference in the flow cytometry tool. What this tool now allows is high-throughput studies to study the effect of sequence, secondary structure, phase separation, or even genetic interactions on the function of TDP43. Yeah, again, I will not go into much more detail about this. If you have questions about this, please reach out or refer to our papers.
Broder Schmidt (29:21):
With this I actually want to thank you for joining my talk, and I want to thank you. I want to thank both the Brandman and the Rohatgi labs for providing an exciting environment to do these studies, and particularly Onn, Zane, and Raj who have been really a fun team to work with on the oxaliplatin project. We had help from Scott Dixon and Jason Rodencal at Stanford with some of the cell viability assays that I didn’t go into. I mentioned Manuel Leonetti from the Chan Zuckerberg Initiative, who contributed the cell lines.
Broder Schmidt (29:55):
I also want to acknowledge Ariana Barreau and Carly Weber-Levine, two very talented undergrads that worked with me on the TDP43 function and the phase separation project. I want to thank the funding agencies. And I do want to also thank IDPSIG and friends for really carrying me through this pandemic. And with this, I’m happy to take your questions and hope that we have a lively discussion.
Bede Portz (30:31):
That was really marvelous Broder, and I think the transfer-free energy work is really an incredible advance because it parametrizes what a therapeutic index looks like, with a biophysical grounding, and I think that’s pretty neat. So it really motivated me to think about your Cell Reports paper where you have these vacuolated TDP43 condensates and those are large and readily visualizable and as you articulated, that’s a key drug target in the context of neurodegenerative disease. So have you thought about combining the advances from your recent work with that reporter and looking for modulators of the phase behavior of TDP43 using your vacuolated reporter?
Broder Schmidt (31:24):
Yeah, that’s a great question and the short answer is yes, I have thought about it. But, I don’t have any data. I haven’t done it yet. And yeah, so this other report system that we were mentioning that it essentially is, just to get everybody up to speed, this is essentially a trick to essentially overemphasize the contributions of the disordered domain to TDP43 phase separation. And the advantage of it really is that you form gigantic droplets in the nucleus that are easily screenable. So I think, yes, there’s a lot of potential in using such an image-based approach to do essentially a similar study as we did for oxaliplatin in the nucleolus, for TDP43.
Broder Schmidt (32:14):
But what I wanted to highlight is it’s a bit more tricky if you go after things like TDP43 because there’s essentially much more unknown about the function of the protein per se, which is why I think functional reporters are invaluable also in this approach.
Bede Portz (32:32):
Thanks Broder. Alex Holehouse has a question.
Alex Holehouse (32:36):
Hello, Broder, beautiful talk. I’m out in the corridor, my lab is watching it on our big screen, so I escaped to try and avoid the echo, but let me know if you get feedback and I can move further away. So this is really beautiful, and one thing I was wondering with the oxaliplatin inhibition. Oxaliplatin will inhibit polymerase, but do you have a sense of the specificity from Pol-I versus Pol-II, versus Pol-III, because you can sort of imagine that there may be an effect of inhibiting Pol-II, for example, either in the release of lots of now, partially synthesized mRNA transcripts, or the inhibition of using nucleotides in the nucleus, and I’m wondering if you’ve thought at all about that, and what that contribution might look like.
Broder Schmidt (33:16):
Right. So, I do want to point out just for clarification. So actinomycin-D is the drug that-
Alex Holehouse (33:22):
Sorry, sorry. Actinomycin-D, yes.
Broder Schmidt (33:23):
Targets polymerase, and yes, thank you for pointing out, actually. It can inhibit multiple polymerases, like RNA Pol-I, Pol-II. And essentially this is concentration-dependent. At lower concentrations you get somewhat a specificity for RNA Pol-I. I do think that the extreme toxicity of this drug is related to the fact that essentially you can shut off not only rRNA transcription, you can shut off all transcription with it. So I think I would kind of turn around your question, and I would say that the really neat effect of oxaliplatin is that because it targets the nucleolus, it actually does allow you to inhibit only the function of nucleoli, in this case, Pol-I related transcripts. So I think this is part of the reason why oxaliplatin actually does have a therapeutic window, in contrast to actinomycin-D. I hope that kind of trick of mind answers your question.
Alex Holehouse (34:34):
Yeah, I guess one thing I was thinking, is I think alpha-amanitin is specific to I and III, but not II, and that might be an alternative to sort of decouple the impacts of broad mRNA transcription. But yeah, beautiful. That’s great.
Broder Schmidt (34:49):
So I think that in that respect, if you go to Josh Riback’s paper, he has essentially shown [inaudible 00:34:59]. Also if you look at Rich’s papers, right. So the rRNA plays a critical role for phase separation, like it does in other systems, like in RNA granules. So taking away the rRNA will also alter the nucleolar properties, the phase separation properties. So that’s why I think there is a secondary effect if you inhibit this polymerase, then you will have a consequence on the nucleolar function. And what I didn’t show, and I maybe should have put this in, right, is if you look at viability of cells in response to different drugs, you can also see that actinomycin-D has a very strong effect early on, where cisplatin and oxaliplatin have a much more delayed effect.
Bede Portz (35:54):
So there’s a very provocative question from Nic Fawzi in the chat.
Nicolas Fawzi (35:58):
Sorry, I’m here. I was wondering, Broder, excellent talk. I was wondering if IDP-modifying drugs are going to be covalent modifiers? I think you showed that the proteins were covalently modified in the gel. That was kind of the first question. And then I was wondering are ribosomal RNAs in this case modified as well? What is the mechanism of action? Maybe you said it, maybe I just missed it.
Broder Schmidt (36:39):
Yeah, I don’t think this is a provocative question at all, it’s actually a great question. So I do think, I do have a very special interest, I guess, in drugs that do covalently modify proteins and also rRNAs in the context of drugging condensates because I think in a way, sometimes I like to think about oxaliplatin and those platinum modifications as aberrant or drug-induced post-translation modifications that essentially alter the valency of the IDRs in the rRNAs.
Broder Schmidt (37:17):
And then, to your second question, actually, whether rRNAs are modified. I think actually we do have some data on this. And essentially, what this mapping of the reads to the different transcripts does, we can actually use this also to map with a nucleotide resolution where you have a platinum crosslink, because this kind of protocol actually involves a circularization of the RNA so you can sequence right up to where you have this crosslink. So the fact that we see this peak here is actually indicative of the fact that oxaliplatin does modify this rRNA. There are also other evidence for this in the literature.
Bede Portz (38:11):
So there’s a great question in the chat from Saskia Hutten, apologies if I got that name incorrect.
Saskia Hutten (38:18):
Oh, that’s fine, I’m flexible. Hi Broder.
Broder Schmidt (38:21):
Saskia Hutten (38:22):
I was wondering, because you can purify nucleoli from cells to high purity and they stay intact, and you can put them on EM grid and look at them under the microscope. Have you tried to add oxaliplatin to those and see whether they also change the structure or what happens, actually, to those?
Broder Schmidt (38:41):
Yeah, that’s a great question. We thought about it. We’ve gotten EM grids and we haven’t gotten around to doing this. I think-
Saskia Hutten (38:54):
[crosstalk 00:38:54] But if you witness the three-colored cell line that would maybe be by fluorescence microscopy already [inaudible 00:39:00]…
Broder Schmidt (39:03):
You mean just purify the nucleoli from those cell lines? Yeah, that’s a good idea, I think that should be for us, much more readily doable than EM. But yeah, I think, with the advent of cryo-EM, I was obviously kind of fascinated, wondering how that would look like. So hopefully something we’ll get around to doing. We do have, so far, on the image work, only the direct readout of modification of the major components in different, both in a soluble form as well as in a phase separated form.
Saskia Hutten (39:39):
Bede Portz (39:45):
So there’s a question in the chat that is pretty interesting. So do you think that, and this is something I’ve thought about, and I’m very much interested in your comments. Do you think it’s going to be possible to dissolve aggregates with IDR-targeting drugs or do you think the best shot is preventing further aggregation?
Broder Schmidt (40:08):
Yeah, well, I think that the kind of disaggregation approach, I think I’m most excited about essentially, the approaches that the Shorter lab pioneered, to essentially try to disintegrate the aggregates and refold or solubilize the proteins again. And I think it would be kind of cool using some of the functional readouts like, maybe this splicing reporter, to see whether you can actually restore function this way. I’m somewhat hesitant, I think, with modifying drugs, like oxaliplatin because you will have then, maybe a more soluble disordered protein again, but it will be modified, so it’ll be important to see how this effects the function.
Broder Schmidt (41:05):
But for that, really, I think I would say, I’m kind of more excited about kind of using the capabilities of the cells to do that in some way or the other. I guess you guys would be the ones that have more knowledge in this, but from a drug development approach, it might be easier to go after prevention of aggregation if that is at all possible. I think it’s a challenge to design drugs that target aggregates but don’t target condensates.
Diana Mitrea (41:38):
That was a beautiful talk, Broder, this is Diana. I was curious, it was fascinating to see that oxaliplatin was driving the opposite directions, the driving force for phase separation for NPM1 and SURF6. So that sort of suggested to me that there’s very strong RNA component, RNA effect component . So I was wondering, have you looked at the ribosomal proteins and see what happens to them when you treat with oxaliplatin, and have you looked at the reversibility effect? Once you wash off the compound, do the nucleoli go back and what’s the timescale of that?
Broder Schmidt (42:30):
I like your second question, that’s a great idea. We haven’t done that. In response to your first question, we know from essentially ribosome footprinting experiments that the ribosomal proteins, the translation of them gets upregulated. We also know that, from just conventional RNA-seq, that a lot of the small nucleolar RNAs, so not the rRNA that’s eventually ending up in the ribosome, but those also get upregulated. So I think, factoring in this additional complexity and how these molecules that also supposedly go to the nucleolus and suppose you also contribute in the scaffolding and contribute to the chemical environment, contribute to the material properties, I think that’ll be interesting to see how those factor in. Yeah. So there’s definitely more to come.
Bede Portz (43:36):
Yep. Wonderful, Broder, thank you very much for your time and engaging discussion afterwards. Really appreciate it. Thank you everyone for attending.
Broder Schmidt (43:47):
Thank you. That was fun.
Jill Bouchard (43:51):
With that we’ll close it down and thanks everybody for coming. Join us in October, three weeks away. Thanks again, Broder. Awesome talk.
Broder Schmidt (44:01):