Increased utrophin expression in healthy and DMD patient derived myoblasts in response to ERK1/2 and EZH2 inhibitor treatment

This study demonstrates that inhibiting ERK1/2 and EZH2 increases utrophin expression in human myoblasts, with a unique sustained effect in Duchenne muscular dystrophy (DMD) patient-derived cells that also reverses altered myogenic transcription factor profiles, suggesting a promising therapeutic strategy to compensate for dystrophin loss and restore muscle regeneration.

The Gist

The Big Picture: A Broken Bridge and a Backup Plan

Imagine your muscles are like a busy suspension bridge. To keep the bridge from falling apart under the weight of traffic (movement), it needs strong cables called dystrophin.

In people with Duchenne Muscular Dystrophy (DMD), the instructions to build these cables are broken. Without dystrophin, the bridge is fragile. Every time the muscles move, the "road surface" (the cell membrane) gets ripped and torn. Over time, the muscle tissue gets damaged, scarred, and eventually fails.

The Problem: We can't easily fix the broken instructions (the gene mutation) yet.
The Proposed Solution: Nature has a "backup cable" called utrophin. In babies, this backup cable is used to build the bridge. But as we grow up, the body switches to the main cable (dystrophin) and turns off the backup (utrophin).

The scientists in this paper asked: What if we could trick the adult muscle cells into turning the backup cable (utrophin) back on? Could that save the bridge?

The Experiment: Finding the "On" Switch

The researchers knew that two specific "switches" in the cell's control room might be keeping the utrophin backup turned off. These switches are called ERK1/2 and EZH2.

Think of these switches like a dimmer switch on a light that is currently set to "Off" (or very low). The researchers wanted to see if they could use special tools (drugs) to flip those switches and turn the light (utrophin) back on.

They tested two drugs:

1. LY3214996: A tool to turn off the ERK1/2 switch.
2. GSK503: A tool to turn off the EZH2 switch.

What They Did (The Lab Work)

They took muscle cells from two groups:

1. Healthy people: People with working "main cables."
2. DMD patients: People with broken "main cables."

They treated these cells with the drugs for 24 hours and watched what happened.

The Results: Good News and a Surprise

1. The Drugs Worked (The Light Turned On)
When they treated the cells, the "backup cable" (utrophin) started appearing in huge amounts. This happened in both healthy cells and DMD patient cells. It was like flipping a switch and seeing the backup lights flood the room.

2. The "Memory" Effect (The Big Surprise)
This is the most exciting part.

• In Healthy Cells: When the researchers stopped giving the drugs, the utrophin levels went back down. It was like turning off the light switch; the light went out.
• In DMD Cells: When they stopped the drugs, the utrophin levels stayed high. Even though the "switch" was removed, the cells remembered to keep the backup cable running.

Analogy: Imagine you are teaching a student to study.

• Healthy cells are like a student who only studies while the teacher is in the room. When the teacher leaves, the student stops.
• DMD cells are like a student who, once they realize they need to study to survive, keeps studying even after the teacher leaves the room. The broken "main cable" (dystrophin) seems to make the cells desperate enough to keep the backup system running permanently once triggered.

3. Fixing the "Construction Crew"
Muscle cells need to grow and repair themselves. The researchers found that in DMD cells, the "construction crew" (the proteins that tell cells to grow and divide) was confused and chaotic.

• The DMD cells were growing too fast (like a construction crew running around without a plan).
• The drugs helped calm them down and get them back on track, making them behave more like healthy, organized workers.

Why This Matters

This paper suggests a potential new treatment for DMD that doesn't require fixing the broken gene directly. Instead, it uses drugs to:

1. Turn on the backup cable (utrophin) to protect the muscle from damage.
2. Fix the construction crew so the muscle can repair itself properly.
3. Create a lasting effect specifically in DMD patients, where the treatment "sticks" even after the drugs are gone.

The Bottom Line

The scientists found a way to use drugs to wake up a natural backup system in muscle cells. While this is still early research (done in a lab dish, not yet in people), it offers a glimmer of hope. It suggests that we might be able to treat DMD by helping the body's own "spare parts" take over the job of the broken parts, potentially slowing down or stopping the disease.

In short: They found a way to flip a switch that turns on a life-saving backup system, and in sick cells, that system seems to stay on even after the switch is removed.

Technical Summary

1. Problem Statement

Duchenne Muscular Dystrophy (DMD) is a fatal, progressive X-linked muscle-wasting disorder caused by loss-of-function mutations in the dystrophin (DMD) gene. The absence of dystrophin destabilizes the dystrophin-associated protein complex (DAPC), leading to sarcolemma fragility, muscle degeneration, and eventual respiratory or cardiac failure.

The Therapeutic Gap:

• Utrophin as a Substitute: Utrophin (UTRN) is an embryonic homologue of dystrophin. Upregulating utrophin in adult muscle fibers has been proposed as a therapeutic strategy to compensate for the lack of dystrophin.
• Previous Failures: Small molecule candidates like Ezutromid failed in clinical trials.
• Model Discrepancies: Mouse models (e.g., mdx mice) often show milder phenotypes than human DMD patients due to more effective compensatory regeneration, making preclinical translation difficult.
• Regulatory Complexity: Utrophin expression is tightly regulated by multiple promoters, enhancers, and post-transcriptional mechanisms, making it difficult to target with single agents.

Research Question: Can the inhibition of ERK1/2 and EZH2, previously identified in mouse models, effectively upregulate utrophin in human myoblasts (both healthy and DMD-derived) and restore the altered regenerative capacity of DMD cells?

2. Methodology

The study utilized a combination of primary human cultures, immortalized cell lines, and pharmacological interventions.

• Cell Sources:
  • Healthy Controls: Primary myoblasts from 6 healthy young male volunteers (aged 20–32) and an immortalized healthy line (AB1190).
  • DMD Patients: Primary myoblasts from 16 DMD patients (aged 1–12, with various exon deletions) and two immortalized DMD lines (AB1098, AB1071).
• Pharmacological Agents:
  • LY3214996 (LY32): An ERK1/2 inhibitor.
  • GSK503: An EZH2 (histone methyltransferase) inhibitor.
  • Ruxolitinib (Ruxo): A JAK1/2 inhibitor (used as a comparative control for differentiation effects).
• Experimental Design:
  • Short-term Treatment: Proliferating myoblasts treated for 24 hours with inhibitors alone or in combination.
  • Pre-treatment/Differentiation Assay: Cells treated for 24 hours, followed by drug removal and induction of differentiation (low serum media) for up to 96 hours to assess if the utrophin upregulation is sustained in myotubes.
• Analytical Techniques:
  • RT-qPCR: Quantification of mRNA levels for UTRN, MYOD1, MYOG, and MK167 (Ki67), normalized to TBP.
  • Western Blot: Protein quantification of Utrophin and Dystrophin (normalized to Vinculin).
  • Immunofluorescence: Staining for Ki67 (proliferation), Myogenin, and MHC (differentiation) to assess nuclear intensity and cell morphology.
  • Genotyping: PCR analysis of DMD exons to confirm mutation status in patient lines.

3. Key Contributions

1. Validation in Human Models: The study successfully translates findings from a mouse bioluminescent screen to human primary and immortalized myoblasts, confirming that ERK1/2 and EZH2 inhibition upregulates utrophin in human cells.
2. Differential Response Discovery: A critical finding is the divergent response between healthy and DMD-derived cells regarding the persistence of utrophin upregulation.
   • In healthy human myoblasts, utrophin upregulation is transient and lost upon drug withdrawal and differentiation.
   • In DMD myoblasts, utrophin upregulation is sustained even after drug removal and differentiation into myotubes.
3. Restoration of Myogenic Phenotype: The study demonstrates that DMD myoblasts exhibit a distinct "proliferative/differentiation imbalance" (low MYOD1/MYOG, high Ki67) and that dual inhibition of ERK1/2 and EZH2 reverses this signature, restoring a more normal myogenic profile.

4. Results

A. Utrophin Upregulation in Proliferating Cells

• Treatment with LY32 and GSK503 (individually or combined) induced a dose-dependent increase in UTRN mRNA in both healthy and DMD myoblasts.
• The combination of both inhibitors yielded the highest upregulation (often >2-fold in DMD samples).
• Protein levels of utrophin increased correspondingly in treated cells.

B. Impact on Myogenic Markers and Proliferation

• Healthy Cells: ERK1/2 inhibition caused premature expression of differentiation markers (MYOD1, MYOG) and reduced proliferation (Ki67), but cells successfully differentiated into myotubes upon drug removal.
• DMD Cells: Basal levels of MYOD1 and MYOG were significantly lower, and Ki67 was higher compared to healthy controls, indicating a defect in the regenerative cycle.
• Treatment Effect: In DMD cells, LY32 (especially combined with GSK503) restored MYOD1 and MYOG expression to near-normal levels and significantly reduced Ki67 proliferation markers.

C. The "Sustained" Upregulation Phenomenon

• Healthy Cells: When pre-treated and then differentiated, the upregulation of utrophin was lost; myotubes returned to baseline utrophin levels.
• DMD Cells: In contrast, DMD-derived myotubes retained the elevated utrophin levels 96 hours after drug removal.
• Mechanism Hypothesis: The authors suggest this persistence is linked to the higher basal levels of utrophin in DMD cells (a compensatory mechanism) and potentially distinct chromatin landscapes at the UTRN locus in dystrophic cells that "lock in" the upregulated state once induced.

D. JAK1/2 Inhibition Comparison

• Treatment with Ruxolitinib (JAK1/2 inhibitor) also increased MYOD1 and UTRN in DMD cells, supporting the link between premature differentiation and utrophin upregulation, though the sustained effect of ERK/EZH2 inhibition was unique to the specific drug combination in DMD contexts.

5. Significance and Conclusions

Therapeutic Potential:
The study proposes a dual-inhibition strategy (ERK1/2 + EZH2) as a viable therapeutic avenue for DMD. This approach offers a "two-pronged" benefit:

1. Compensation: It increases utrophin expression to compensate for the missing dystrophin protein in muscle fibers.
2. Regeneration: It corrects the intrinsic defects in DMD satellite cells/myoblasts (restoring MYOD1 and reducing hyper-proliferation), potentially improving the muscle's ability to repair itself.

Clinical Implications:

• The finding that utrophin upregulation is sustained specifically in DMD cells (but not healthy cells) following a short-term treatment window is highly encouraging. It suggests that a transient drug administration could lead to long-term therapeutic benefits in patients, potentially reducing the need for chronic dosing.
• The study highlights the limitations of relying solely on mouse models, as the sustained effect was not observed in healthy human cells (which mimic the mouse wild-type response) but was specific to the human DMD pathology.

Future Directions:
The authors call for further investigation into the chromatin landscape of the UTRN locus in DMD vs. healthy cells to understand the mechanistic basis of the sustained upregulation. They advocate for iterative use of mouse and human models to refine these compounds for eventual clinical trials.