Unlocking Prostate Cancer Therapeutics: The Potential of Minor Spliceosome Inhibition - Mark Rubin

June 11, 2023

In this conversation, Andrea Miyahira is joined by a research team including Anke Augspach, Rahul Kanadia, and Mark Rubin. They discuss their paper, "Minor Intron Splicing is Critical for Survival of Lethal Prostate Cancer". The paper dissects the role of minor introns, which constitute less than 0.5% of all introns, in gene expression and their involvement in prostate cancer onset and progression. The conversation uncovers the strategy they've developed using siRNA against U6atac to inhibit the minor spliceosome, showing promise in reducing prostate cancer cell survival without impacting normal cells. The discussion ends with Rubin outlining their translational plans, which involve designing a drug delivery strategy and identifying potential applications in other cancers. Their pioneering work points to minor spliceosomes as a potential third-generation target for prostate cancer treatment.


Mark Rubin, MD, University of Bern, Bern, Switzerland

Anke Augspach, PhD, University of Bern, Bern, Switzerland

Rahul Kanadia, PhD, University of Connecticut, Storrs, CT

Andrea K Miyahira, PhD, Director of Global Research & Scientific Communications, The Prostate Cancer Foundation

Read the Full Video Transcript

Andrea Miyahira: Hi, everyone. Thank you for joining us today. I'm Dr. Andrea Miyahira at the Prostate Cancer Foundation. Today I have a wonderful team joining me today. Perhaps you can all introduce yourselves.

Anke Augspach: Yes. Hi, I am Dr. Anke Augspach. I am a postdoc in Mark Rubin's group here in Switzerland in Bern.

Rahul Kanadia: Hi, I'm Rahul Kanadia. I am a professor in Physiology and Neurobiology Department at the University of Connecticut.

Mark Rubin: And hi, I'm Mark Rubin. I'm the director of the Bern Center for Precision Medicine.

Andrea Miyahira: This team recently published the paper, Minor Intron Splicing is Critical for Survival of Lethal Prostate Cancer in Molecular Cell. Thank you all for joining me today to discuss this paper.

Rahul Kanadia: I'm going to start with discussing the Augspach et al., Molecular Cell paper entitled, Minor Intron Splicing is Critical for Survival of Lethal Prostate Cancer. To begin, what I'm showing you is an anthropomorphic view of a eukaryotic gene, where exons are boxes and introns are lines. But in vivo, the system must recognize exon-intron boundaries, and that is achieved through consensus sequences, shown here in yellow, where this is the 5' splice site branch point sequence and 3' splice site. Of course, consensus sequences come in many flavors and there is a divergent sequence that I'm showing you here that diverges from GTAG to ATAT, and these introns have distinct branch point sequence as well.

So the question one can ask is, how did these sequences become the sequences that mark the exon-intron boundary? And for that we have to go back to the manner in which splicing has evolved and it occurs. For 99.5% of the introns, we call major introns, the 5' splice site is actually recognized by the base pairing of U1 snRNA to the 5' splice site, U2 snRNA with the branch point, and U4, U6, and U5 together help with proteins assistance splicing, and this machinery is therefore referred to as the major spliceosome.

However, the divergent sequence that I showed were less than 0.5% of the introns, we used the word minor introns to describe them, have analogous snRNAs, so U11 for U1, U12 for U2, U4atac, U6atac, and it actually uses U5 of the major spliceosome, and this machinery is termed as the minor spliceosome. And what is interesting is how these minor introns are found. Minor intron-containing genes, or MIGS, actually consists mostly of major introns, where only one of the intron is a minor intron. So the proper expression of these MIGS requires a coordinated action of major and minor spliceosomes to produce a fully functional mRNA.

The question then one could ask is, how did these genes acquire a minor intron and why are they kept? And so the way to answer that question is simply to say what do they do as a function? If you do a GO term enrichment analysis of these 714 MAGs, you find MAP kinase activity, voltage-gated ion channel activity, but one of the highest number of MIGs that are enriched in cell cycle. Not only that, they execute all aspects of the different stages of cell cycle.

Here is where Anke really bridged the two fields and the two labs, because she wanted to ask, can we exploit the minor spliceosome to combat disease? And so, one of the first questions we had to ask is, are MIGS even involved in prostate cancer onset and/or progression? And so what we are looking at here is MIG expression does, in fact, sort by the different stages of PCa from GTEx of healthy tissue, primary PCA data from TCGA, and Stand Up to Cancer. So if MIGs are actually showing this progression, you would imagine that the minor spliceosome components themselves also might track because the MIGs are tracking with progression.

Here are the snRNAs I described and we're going to focus on U6atac snRNA because it shows a very dramatic increase, because normally it is under rapid turnover kinetics. So in primary PCa we don't see enough of it by in situ hybridization, but in primary PCa with metastatic potential you begin to see cells increase U6atac expression, whereas in metastatic PCa, it is really highly expressed. Therefore, together one can predict that the minor spliceosome activity itself would be increasing with prostate cancer progression. And for that, Anke designed a clever strategy where you have luciferase interrupted, either by a major intron or a minor intron, and then you transfect in different cells and organoids across PCa progression, pre PCa CRPC-Adeno and CRPC-NE, and ask, does the luciferase activity read out the activity of the splicing? And/ in fact, major splicing does not seem to show any change across progression, but minor intron splicing reported by luciferase actually does go up across progression of PCa.

So together, the idea, therefore, is, can we now use a strategy to inhibit the minor spliceosome? Anke designed a surprisingly effective strategy on using siRNA against U6atac to inhibit the minor spliceosome. One of the first things we did was take RNA-Seq analysis to study whether or not minor introns are affected. And simply what we do is we look for reads that show transcripts with retained introns and divide by the sum of retained plus successfully spliced, and then we run this across all of the minor intron-containing genes. And we see elevated minor intron retention in LNCaP, C4-2, 22Rv1, and the organoid PM154, as seen by this box plot. So then the question is, what does this mean downstream?

When we combine RNA-Seq data with mass spec data and actually find the transcripts that show minor in trans splicing defect in the corresponding protein down regulation, you actually find that majority of those are enriching for cell cycle and mitotic progression, which we confirmed through FACS analysis, where you can see this is in C4-2 and PM154. We see a significant reduction of cells in S phase in both C4-2 and PM154. And the question, therefore, is, if cell cycle is altered in these cells, one would predict their survival would also be compromised. And in fact, that is what we see.

What you're looking at at the bottom panel, C4-2 cells that are either treated with siScrambled, siU6atac, or siRNPC3, a component of the minor spliceosome, you see there is significant death occurring in C4-4 cells. What is fascinating is that when we do the exact same experiment in normal mouse prostate cells and fibroblasts, they do not experience this high cell death. This suggests that maybe this is something we can test and compare to the existing approved drugs and see if the siU6atac least compares well with them.

We first tried in LNCaP, so what you're looking at are these different colors, and across time, confluence of the cells. siU6atac is the orange and what you're seeing is scrambled at the top, gray or the black, but what you find is in LNCaP, siU6atac is just the same as the approved drugs of enzalutamide or siEZH2 plus enzalutamide.

Where things got interesting is where we look for LNCaP resistant to enzalutamide. At this juncture, siU6atac outperforms the others. And in fact, in C4-2 which are beginning to transition, they actually do also, siU6atac outperforms siEZH2 or enzalutamide plus siEZH2. So taken together, one of the things we'd like to propose is that minor spliceosome is potentially a third-generation target for prostate cancer. These are the main stars. Anke is the start.

Andrea Miyahira: Thank you so much for sharing that. What functions do the minor spliceosome and siU6atac perform in normal adult cells? And what toxicities might you expect from targeting siU6atac?

Rahul Kanadia: I'll take that question. The minor spliceosome is ubiquitous. It is required for expression of, give or take, 714 genes. One of the things, like I showed, the fibroblasts are resistant. It seems that cancers are especially vulnerable to minor spliceosome and siU6atac. We'll have to find the right, or as mark and I like to call, the Goldilocks spot, where we take out siU6atac just enough where cancer is susceptible but not the normal tissue. And so that is the next phase of the evolution of this project.

Andrea Miyahira: Thank you. New treatments such as anti-AR therapies alter minor intron splicing in prostate cancer?

Anke Augspach: Good question. We do indeed see a strong and significant increase in minor spliceosome activity when we do a long-term treatment with ARs, such as enzalutamide or abiraterone, and that goes along with the fact that overexpression of U6atac triggers resistance towards these treatments.

Andrea Miyahira: Thanks. I guess a follow-up question then, does this mechanism play a role in lineage plasticity or progression to prostate cancer with neuroendocrin features?

Mark Rubin: Yeah, I'll take that question. That's obviously a question that our lab has been thinking about for many years now. And this story I think is quite exciting, because what we see is that one theory about lineage plasticity is that the tumor cells hijack normal developmental machinery. In this example we see that the machinery used for neural development, which has to be blocked normally because normal epithelial cells should have turned it off, and what we see is that in the transition of advanced cancers to then resistant tumors, we often see that machinery is hijacked and we see neural differentiation. What we're seeing with U6atac is it has the ability to regulate this machinery, such that it enhances the differentiation of this neural machinery that's usually turned off. And that's through gene called REST that we've spoken about before and you've probably heard about this before in other presentations.

Andrea Miyahira: Yeah, very interesting. Does your team have any translational plans for this study?

Mark Rubin: Yeah, I'll take that question. It's something we're extremely excited about. You saw the data related to therapy and how U6atac is very effective in a castration-resistant tumor setting, so one of our first challenges is, how can we develop a drug delivery strategy, either for the si, or can we develop small molecules against the minor spliceosome? I think one of the key concepts that we really need to bring up is that, and as we've learned from our Rahul, is that we're not a question of turning off minor splicing, because that would be deleterious for this for all the normal cells, it's really thinking as a rheostat and we want to turn it down to enough that affects the cancer cells, but not the normal cells. And so that's a challenge we have and we have, I think, a good game plan for that and that's something we're very excited about. So that's one aspect.

The other thing is, say we're talking about prostate cancer, but we also think this is extremely important in other cancers as well. In the paper we show some preliminary data and we think that our targeting strategy will also require a biomarker strategy, which I think will require collaboration with a number of teams throughout the world to try to address some of the other cancers that we think might be important and might be targetable by modulating U6atac or the minor spliceosome.

Andrea Miyahira: Okay. Well, thank you all for joining me today. I think this paper is super interesting and important. I'm excited to see this new potential therapeutic target and biomarker and what you guys will do in the future. Thank you.

Mark Rubin: Thank you.

Rahul Kanadia: Thank you.

Anke Augspach: Thank you.