CRISPRi, transcriptomic and proteomic profiling in peripheral nervous system cell models reveal how NF1 loss rewires Ras signaling, alters MEK inhibitor response, and nominates KRAS as a direct neurofibromin effector and therapeutic target.
0:03Oh, oh, oh. Whoa. Hmm. Welcome to Base by Base, the papercast that brings genomics to you, wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. So for 20 years, oncology has wrestled with a paradox.
0:33How do you drug a ghost? Yeah, it's a well, it's a huge challenge. Right, because, I mean, we've gotten really good at stopping rogue hyperactive proteins. If a mutated gene produces an overactive enzyme, like a gain of function on copagene.
0:50We just design a small molecule to shut it down. Exactly. You just block the active site and turn it off. But what happens when the cancer isn't caused by a rogue protein doing too much, but by a crucial protein, just, well, just vanishing.
1:02The biological brake pedal has been completely deleted from the genome. And you obviously can't design a drug to bind to something that no longer exists in the cell. Right. It's like a car speeding down a mountain.
1:13Fixing a stuck accelerator is straightforward. But if the brakes completely vanish, how do you stop the car without wrecking the engine? You really have to get creative. Exactly. So how could this change the way we treat some of the most stubborn treatment resistant nervous system tumors?
1:27Today, we celebrate the work of Harish Vasudevan, Frank McCormick, and their incredible research teams at UCSF and the NCIRS Initiative, who have advanced our understanding of neurofibermen function in the peripheral nervous system.
1:42Hi, everyone. And yeah, it's great to be here with you to dide into this. This really is the defining challenge of tumor suppressor biology. It really is. And to grasp what this team accomplished in our deep dive today, we need to look at the specific clinical context, right?
1:57We're talking about neurofibermatosis type one or NF1. Right, it's a congenital cancer predisposition syndrome. It's driven entirely by mutations in the NF1 gene. And people with this condition are uniquely vulnerable to tumors in their peripheral nervous system.
2:12Yeah, specifically plexiform neurofibroma. Which originate in the nerve sheets, right? And they can undergo malignant transformation into some pretty aggressive sarcomas. They absolutely can. And this entire cascade stems from the loss of just a single protein encoded by that gene neural fibromen.
2:27Okay, let's unpack this. What exactly does neurofibermen do in a healthy cell? Well, it functions as a RAS GAP. Yeah, GTP's activating protein. So if you're familiar with cellular signaling, you know, RAS is one of the most critical growth switches in biology.
2:43It's a huge driver of cell division. Exactly. When RAS is bound to a molecule called GTP, it's turned on, just constantly firing growth signals deep into the cell. So where does neurofibermen come in? Neural fiberman's job is to insert this highly specialized structural domain into RAS, which accelerates the hydrolysis of that GTP into GDP?
3:03Oh, I see Yeah, it beads it up by roughly a 100,000 fold. It rapidly switches raws to off. It's the essential negative regulator. Precisely. So when the NF1 gene is mutated and neuro fibromen is lost. That hydraulysis just doesn't happen.
3:17Rass remains permanently bound to GTP. It sits at the top of the signaling cascade, locked in the on position. Right. Continually activating the downstream RAF MER key pathway. Essentially screams at the cell to keep dividing.
3:31Okay, but the current clinical standard of care for these tumors is to use MEK inhibitors, right? Like Salimatinib. But if you look at the architecture of the pathway, If the problem starts all the way up at RAS, why are we blocking MAK?
3:46isn't that like trying to dam a flooding river miles downstream from the broken reservoir? That is a perfect analogy. And yeah, it is biologically inefficient. We targeted MEK simply because we had the chemical tools to bind to it, not because it was the optimal intervention point.
4:02Right, it's just a downstream mitigation strategy. Exactly. And in the context of NF1, that strategy runs into a severe bottleneck regarding the therapeutic window. How so? Well, in these patients, the tumors themselves have lost both functional copies of the NF1 gene.
4:18their NF1 mole. Okay. But the surrounding healthy tissue, the nerves, the cardiovascular system, they still retain one functional copy. They operate in a heterosygus plus minus state. Oh, I see. Which means if you flood the patient system with a heavy dose of a downstream MEK inhibitor, you weren't just slowing down the tumor.
4:36Right. You are heavily suppressing a vital growth pathway in every single healthy cell in the patient's body that relies on that remaining half dose of neurofiberment. Wow. So you run into severe toxicity.
4:49You do, and you run into inevitable. resistance. Right. So because downstream damming isn't perfect, The researchers needed to map the entire river system, right? Like upstream and downstream. Exactly.
5:01To bypass those limitations. They couldn't just guess at upstream interventions. They had to systematically chart the entire biochemical network. And they used immortalized peripheral nerve cells for this, right?
5:12IPNs. Yeah, as their baseline model. But rather than using standard CRISPR to completely knock out genes, which often just triggers immediate cell death and, well, ruins the experiment, they deployed CRISPR interference, or CRISPR.
5:24Which acts like a precise dimmer switch. Yes, exactly. They used a deactivated cast 9 enzyme fused to a repressor domain, which lets them bind to a target gene and just precisely dial down his transcription.
5:36So they use this volume knob to selectively repress NF1 and then upstream inputs to Roz to watch how the cellular network reacted. Right. In real time. And the reaction mapping is where the methodology gets truly rigorous.
5:50They used parallel multi-omic profiling, specifically RNA sequencing and foster proteomics. But wait, why do we need all these different maps? I mean, if RNA sec tells us what the cell is building, why do we need the phosphor proteomics?
6:06Oh, that's a great question. Because RNA is just the blueprint, right? It's the transcriptomic inventory. Okay, so it shows the roads and infrastructure the cell has built. Exactly. But in mutant tumors, the real chaos is happening through post-translational modifications.
6:19That's why the phosphorportiomix is the critical layer. It acts like live GPS traffic data. Ah, so you need to know not just what parts are in the factory, but which machines are actually plugged in and running.
6:30Precisely. It shows us exactly where the phosphoration events are happening, mapping out which signaling proteins are actively driving the disease. Okay, so now that we have our molecular blueprints and flashlights, what do they actually find in the dark, let's talk about the salimituna resistance first.
6:45Well, when they turned down NF1 expression, Ross went into overdrive, as expected. But that live traffic data show that the cell doesn't just passively accept a downstream blockade when you apply a MEK inhibitor.
6:58It initiates compensatory rewiring, right? Aggressive rewiring. NF1 loss actually rewires the cell's feedback loops. The proteomic data demonstrated that the cell dynamically shifts its dependence towards specific cell cycle kindnesses.
7:11Specifically CBK1 and CDK2, right? Yeah. The cell basically builds a bypass highway around the MEK blockade, directly accelerating the engines of cellular division. Wow. So that makes the cells fundamentally less sensitive to the MEK Inhibitor Solimentum.
7:27Exactly. The downstream intervention point is simply too far removed from the source of the biological error. Okay, so that downstream failure immediately forces the intervention strategy upstream. Let's look at the upstream battles.
7:38They looked at SHP 2 and SOS one, right? Yes So SOS one is a logical 1st target. Its primary job is the exact opposite of neurofibermen. It actively pulls GDP out of ROS and replaces it with fresh GDP, turning RAS on.
7:51So if the tumor lacks the neuro fiber and brake, it makes sense to try and remove the SOS one accelerator. It does. Yet when they pharmacologically inhibited SOS one, the tumor cells barely register the change.
8:04Wait, nothing happened? Proliferation just continued? Checked, unchecked. It just kept going. Why? I mean, if you're severing the primary supply line, why didn't the pathway stall? It comes down to evolutionary redundancy.
8:15Tumors are supreme escape artists. SOS one shares highly conserved catalytic domains with a nearly identical protein called SOS 2. Ah, so the moment SOS one is taken offline, SOS 2 just seamlessly steps in.
8:28Immediately. It slots into the exact same protein complexes and continues exchanging GTP onto RAS. It's basically a cellular game of whack-a-mole. That is wild. But targeting SHP2, the protein encoded by the PTPN 11 gene, that yielded a drastically different outcome, right?
8:43Radically different. When they used Crispree to repress PTPN 11, they observed a physiological mirror image of the NF1 loss. The exact inverse. Instead of massive proliferation, the cells dramatically slowed down, promoted cell differentiation, and they actually shifted back toward a non-malignant phenotype.
9:02Why does that happen with SHP2, but not SOS one? Because SHP2 acts as a massive signal integration scaffold. By removing that scaffold, you effectively starve the hyperactive ROS pool of its continuous activation signals.
9:16And doing that strips away the humor's ability to maintain those bypass highways we discussed. The cells became highly sensitive to Simu Matina began. They did. It completely restored the vulnerability.
9:26Okay, here's where it gets really interesting because while combining upstream and downstream inhibition is powerful, they still wanted to strike at the direct source. Right, which brings us to apex proximity label.
9:38Yes. So they use this to map the immediate physical environment of neurofibermen. Can you explain how this molecular tagging system works? It's molecular engineering at its finest. The fused and engineered enzyme directly to neurofibermen.
9:52When you supply the right chemicals, it generates these short-lived radicals that have a half-life of less than a millisecond. So they can only travel a microscopic distance before they bind to whatever is nearby.
10:02Exactly, roughly 20 nanometers. You're effectively creating a localized chemical explosion that permanently tags any protein sitting in the immediate spatial vicinity of neurofibermen. And then they just run those tag proteins through mass spec to see exactly who neuro fiberman was hanging out with.
10:19Right. And this spatial mapping addressed a massive historical assumption. You see, Roz isn't just a single protein. It's a family of isoforms. H rays, N race, and Kreyas. And for decades, we assumed neurofibermen was a universal break for all of them, right?
10:34We did, but the apex tag caught Carrie S redhanded. In these peripheral nerve cells, HRAs and NRAS were virtually absent from neurofibrum in's neighborhood. It exclusively associated with CaryS. Wait, exclusively?
10:49Yes. Carrie S was the most highly interconnected signaling note. That spatial exclusivity fundamentally changes our understanding of the disease mechanism. It isn't a general failure of ROS regulation.
10:59It's an acute localized failure of KRS regulation. Exactly. And this happens because these raw isoforms undergo different lipid modifications that dictate exactly which microdomains they inhabit on the cell membrane.
11:12And neurofibermen clearly traffics to those exact same microdomains as KRS. So what's the solution? If KRS is the main culprit, can we target it? We can now. Historically, KRS was the ultimate undruggable target, but modern pharmacology cracked it.
11:28The researchers introduced BI 2865, a non-covalent PanCaryS inhibitor. And when they applied this direct KRIS inhibitor to the NF1 mutant cell? The biochemical collapse of the tumor network was profound, it robustly suppressed the rogue ERK activation.
11:45Did it bypass the redundancy of the SOS proteins? Completely. And furthermore, it completely silenced those compensatory CDK1 and CDK2 cell cycle engines that drive resistance. Wow. So we've caught KRS redhanded, but how does this translate from a Petri dish to a patient?
12:00If we connect this to the bigger picture, the clinical implication is massive, directly targeting CRS provides a much cleaner therapeutic window than downstream MEK inhibition. Because you're striking the exact molecular no that was left unchecked by the missing neuro fiberman.
12:15Exactly. You aren't creating collateral damage miles downstream, and you aren't relying on complex feedback mechanisms. You are shutting off the specific engine driving the pathology. But wait, are there blind spots here?
12:28There always are. Any steady utilizing in vitro cell models carries inherent biological blind spots. For instance, a flat layer of immortalized cells lacks the three-dimensional mechanical stress of a human nervous system.
12:42Right. It doesn't have the complex tumor micro environment. Exactly. In human plexiform neurofibromas, the tumor cells constantly communicate with fiber blasts, macrophages, and the immune system, which can provide survival signals that rescue tumors from targeted therapies.
12:56And what about resistance to the KRAS inhibition itself? That's the other big unknown. By locking down KRSO effectively, you exert massive evolutionary pressure on the tumor to survive. We don't know if the tumor might eventually mutate and use HRAS or NRAS as a backup generator.
13:14Ah. So the spatial segregation we see now might just shift under pharmacological pressure. Right. Which suggests future treatments might require dynamic strategies, maybe cycling between direct KRS inhibitors and broad upstream scaffold inhibitors like SHP2.
13:29But even with those challenges, the underlying methodology here redefines the landscape. To sum it up, the loss of the NF1 tumor suppressor in peripheral nerves doesn't just accelerate growth, right? No, it drives cancer by specifically hyperactivating KRES and rewiring cellular feedback loops.
13:46And by systematically mapping these exact pathways, researchers discovered that direct KRAS inhibition can short circuit this runaway growth, offering a powerful new therapeutic alternative to existing downstream MEK inhibitors.
14:01Exactly. It fundamentally bypasses the resistance and toxicity limitations we've struggled with for years. So what does this all mean for you? What does this mean for other cancers driven by missing tumor suppressors?
14:11Could using proximity mapping to find their exact molecular neighbors unlock completely new, unexpected drug targets and diseases we thought were undruggable? It's something fascinating to think about.
14:21This episode was based on an open access article under the CCBY 4.0 license. You can find a direct link to the paper and the license in our episode description. If you enjoyed this, follow or subscribe in your podcast app and leave a 5 star rating.
14:36If you'd like to support our work, use the donation link in the description. Now, stay with us for an original track, created especially for this episode, and inspired by the article you've just heard about.
14:46Thanks for listening and join us next time as we explore more science base by base. In the lab with the bright screen glow, Signalizing where it shouldn't go, when the F1 slips, the warning's loud, Rust runs hot like a thunder cloud.
15:23Can ace wheels stop has spin too fast. Air on fire, the brakes don't last. Cells keep time with our new pace, but by tracing the switch in the baseline space. Dub the eyes down from the red light low. Put that curtain where the wild tides flow if the old road bends and the mimic cake signs fake.
15:42We'll find a new door in the choices we made up the rice down. Let it slow, let it free. Dub the rise down. Let it slow, let it breathe. Map some messages, RNA, streams, protein, sparks, and phosphore, dreams, close up, signal says, look right here.
16:02Crass in the shadows, pulled in near. Some door shut, some pathways fake. One block slips another willowake, SHP 2 pulls broader, holds the line, wire SOS swaps mask in the meantime, so we aim for the cool.
16:17Steady hands, clear sound. Keep the rhythm. Keep the noise pinned down, down, down, down, down. Dub the ass down, turn the red light. Pressure to the good cells, glow and resistance talks. We listen, we learn, but the beach stays brave, and the pages still turn, dub their eyes down, now, our cool cycles release.
16:42And night shifts into a cautious piece.
