Analysis of Gene Expression Changes upon Topobexin Treatment and TOP2B-knockout in hiPSC derived cardiomyocytes

This study demonstrates that the TOP2B-selective inhibitor topobexin effectively phenocopies the effects of TOP2B gene knockout in hiPSC-derived cardiomyocytes, establishing it as a viable alternative to genetic deletion for investigating TOP2B function and screening cardioprotectants against anthracycline-induced cardiotoxicity.

The Gist

The Big Picture: Why This Matters

Imagine your heart is a high-performance engine. For this engine to run smoothly, it needs a specific mechanic named TOP2B. This mechanic is a protein that helps untangle the DNA "wires" inside your heart cells so they can read their instructions and function correctly.

Scientists have long suspected that when this mechanic is missing or blocked by certain chemotherapy drugs (like doxorubicin), the heart gets damaged. However, studying this in humans is hard because we can't just cut out the mechanic in a living person to see what happens.

This study used a clever workaround: Human Induced Pluripotent Stem Cells (hiPSCs). Think of these as "blank slate" cells that can turn into anything. The researchers turned them into heart cells (cardiomyocytes) in a lab dish. They created two groups:

1. The "Normal" Group: Heart cells with the TOP2B mechanic.
2. The "Missing Mechanic" Group: Heart cells where they used a genetic "scissors" (CRISPR) to delete the TOP2B gene entirely.

The Experiment: What Did They Do?

1. Growing the Heart Cells
The researchers grew both groups of cells until they started beating like tiny hearts.

• The Result: Both groups worked! They both became beating heart cells. However, the "Missing Mechanic" group was a little slower to get started, taking about 1.5 days longer to start beating than the normal group. It's like a car that still starts, but the engine takes a few extra seconds to warm up.

2. The "Chemical Scissors" Test
Deleting a gene is permanent and takes a long time. The researchers wanted to know: Can we just use a drug to temporarily "turn off" the mechanic and get the same result?

They treated the normal heart cells with two different drugs:

• Drug A (Dexrazoxane/ICRF-187): An old drug that blocks all types of DNA untangling mechanics.
• Drug B (Topobexin): A brand-new, super-specific drug that only blocks the TOP2B mechanic.

3. The "DNA Library" Check (RNA Sequencing)
To see what was happening inside the cells, they took a snapshot of the "instruction manuals" (RNA) being read by the cells. This is like checking the library logs to see which books the cells are reading.

The Surprising Findings

The "Missing Mechanic" vs. The "Drug"

• The Genetic Knockout: When they deleted the TOP2B gene, the cells changed their behavior in a specific way. They stopped reading some books and started reading others.
• The Old Drug (Drug A): Surprisingly, this drug barely changed anything. The cells acted almost exactly the same as the normal ones.
• The New Drug (Topobexin): This was the big win. When they used Topobexin, the normal cells changed their behavior almost exactly like the "Missing Mechanic" cells did.

The Analogy:
Imagine a factory (the cell) with a specific supervisor (TOP2B).

• Scenario 1: You fire the supervisor (Gene Knockout). The factory changes its workflow.
• Scenario 2: You use a generic tool that locks every door in the factory (Old Drug). The factory barely notices because the doors weren't the problem.
• Scenario 3: You use a specific tool that locks only the supervisor's office (Topobexin). The factory changes its workflow in the exact same way as if the supervisor had been fired.

Why Is This a Big Deal?

1. A Faster Way to Study Heart Health
Usually, to study what happens when a gene is missing, scientists have to spend months or years creating "knockout" animals or cells. This study shows that Topobexin acts as a "shortcut." You can just add the drug to normal cells, and it mimics the genetic deletion instantly. It's like using a "simulation mode" in a video game instead of rebuilding the whole engine.

2. Protecting the Heart from Chemotherapy
Many cancer drugs (anthracyclines) kill cancer cells by breaking DNA, but they accidentally hurt the heart because the heart relies heavily on TOP2B.

• This research confirms that TOP2B is the culprit behind this heart damage.
• More importantly, it suggests that Topobexin could be used as a "shield." If you give cancer patients Topobexin alongside their chemotherapy, it might block TOP2B in the heart cells, protecting them from the damage, while the cancer cells (which rely on a different mechanic, TOP2A) are still destroyed.

The Bottom Line

The researchers proved that:

1. Heart cells can survive and beat even without the TOP2B gene (though they are a bit slower).
2. A new drug called Topobexin is a perfect "stand-in" for deleting that gene.
3. This opens the door to developing new, safer cancer treatments that protect the heart without needing complex genetic engineering.

In short, they found a "magic key" (Topobexin) that can temporarily turn off a specific heart protein, mimicking a genetic deletion, which could lead to better ways to save hearts during cancer treatment.

Technical Summary

1. Problem Statement

The role of DNA topoisomerase II beta (TOP2B) in cardiomyocyte differentiation and function is not fully understood, despite its established link to anthracycline-induced cardiotoxicity (a major side effect of chemotherapy). While previous studies using embryonic fibroblasts or conditional murine knockouts confirmed TOP2B as the driver of this toxicity, human-specific mechanisms remain under-characterized.

• Limitations of existing models: Animal models and immortalized cell lines often fail to replicate human-specific gene regulation and electrophysiology.
• Limitations of current inhibition methods: Previous attempts to mitigate cardiotoxicity using siRNA knockdown are difficult to translate clinically due to delivery challenges. Small molecule inhibitors like dexrazoxane (ICRF-187) are non-selective (targeting both TOP2A and TOP2B), making it difficult to isolate the specific effects of TOP2B inhibition.
• Research Gap: There is a need for a human-specific, genetically tractable model to study TOP2B function in cardiomyocytes and to validate whether a selective TOP2B inhibitor can mimic the protective effects of genetic knockout.

2. Methodology

The study utilized Human Induced Pluripotent Stem Cells (hiPSCs) to create a comparative model system involving genetic deletion and pharmacological inhibition.

• Cell Line Generation:
  • CRISPR-Cas9 Knockout: A specific clone (18D) of hiPSCs was generated with a genomic deletion of TOP2B (BKO) by targeting Exon 1.
  • Validation: Knockout was confirmed via PCR genotyping, DNA sequencing, and Western blotting (showing absence of TOP2B protein).
• Differentiation:
  • Both Wild-Type (WT) and BKO hiPSCs were differentiated into cardiomyocytes (CMs) using a monolayer protocol involving CHIR99021 (GSK3 inhibitor) and Activin A.
  • Spontaneous contractility (beating sheets) was monitored.
• Pharmacological Treatment:
  • Differentiated WT CMs were treated with:
    1. Topobexin: A novel, selective catalytic inhibitor of TOP2B (10 µM, 3 hours).
    2. Dexrazoxane (ICRF-187): A non-selective TOP2 inhibitor (100 µM, 3 hours).
• Transcriptomic Analysis:
  • RNA was extracted from four conditions: Undifferentiated WT, Undifferentiated BKO, Differentiated WT CM, and Differentiated BKO CM.
  • Additional RNA was extracted from WT CMs treated with Topobexin or ICRF-187.
  • Sequencing: RNA-seq was performed (Illumina NovaSeq 6000) with four biological replicates per condition.
  • Bioinformatics: Analysis included Principal Component Analysis (PCA), differential expression analysis (DESeq2), and Gene Set Enrichment Analysis (GSEA).

3. Key Contributions

• Generation of a Human TOP2B-null Model: Successfully created and differentiated hiPSC-derived cardiomyocytes lacking TOP2B, demonstrating that TOP2B is not essential for the formation of beating cardiomyocytes but affects differentiation kinetics.
• Validation of Topobexin: Provided the first transcriptomic evidence that Topobexin, a selective TOP2B inhibitor, largely phenocopies the genetic knockout of TOP2B in human cardiomyocytes.
• Comparative Transcriptomics: Established a comprehensive dataset comparing genetic inactivation vs. pharmacological inhibition, distinguishing between direct TOP2B targets and downstream regulatory effects.
• Alternative to Knockout: Demonstrated that Topobexin can serve as a rapid, reversible alternative to CRISPR-Cas9 knockout for studying TOP2B function in various cell lines.

4. Key Results

• Differentiation Kinetics:
  • Both WT and BKO cells differentiated into beating cardiomyocytes.
  • Delay: BKO cells took approximately 1.5 days longer to reach the beating stage (mean 10.5 days) compared to WT (mean 9 days), suggesting TOP2B plays a role in the timing of differentiation.
• Transcriptional Changes upon Differentiation:
  • Differentiation resulted in the upregulation of cardiac markers (e.g., ACTN2, MYL7, TNNT2) and downregulation of pluripotency markers (e.g., NANOG, DPPA4) in both genotypes.
  • The transcriptional shift from stem cell to CM was the dominant factor in the data (PC1), while the difference between WT and BKO CMs was a secondary but distinct factor (PC2).
• Genetic vs. Pharmacological Inhibition:
  • BKO vs. WT: 530 genes were downregulated and 760 upregulated in BKO CMs compared to WT.
  • Topobexin vs. WT: Topobexin treatment caused significant changes (912 down, 601 up), whereas ICRF-187 had negligible effects on the transcriptome (only 8 genes altered).
  • Correlation: There was a strong correlation (Pearson $r = 0.88$) in the direction and magnitude of gene expression changes between the BKO cells and Topobexin-treated cells. Approximately 50% of the genes altered in the knockout were also altered by Topobexin.
• Long Gene Analysis:
  • Consistent with previous findings in neuroblastoma cells, downregulated transcripts in BKO and Topobexin-treated cells were slightly enriched in the upper quartile of gene lengths, supporting the hypothesis that TOP2B facilitates the transcription of long genes.
• Pathway Enrichment:
  • Gene Set Enrichment Analysis (GSEA) revealed that the most significantly affected biological processes in both BKO and Topobexin-treated cells were related to cellular and tissue development and differentiation.

5. Significance

• Mechanistic Insight: The study confirms that while TOP2B is not strictly required for cardiomyocyte survival or beating, its absence or inhibition significantly alters the transcriptional landscape, particularly regarding developmental pathways and long-gene transcription.
• Therapeutic Implications: The finding that Topobexin phenocopies the genetic knockout validates its use as a tool to study anthracycline cardiotoxicity. Since Topobexin is a small molecule, it offers a clinically viable strategy to protect the heart during chemotherapy without the need for complex gene editing.
• Model Utility: The hiPSC-derived TOP2B-null model provides a robust, human-specific platform for screening cardioprotectants and studying cardiac development.
• Methodological Advancement: The paper establishes that selective catalytic inhibition (Topobexin) is a superior and faster alternative to genetic knockout for investigating TOP2B function in diverse cell types, bypassing the time and technical hurdles of CRISPR editing.

Data Availability: The RNA-seq data is publicly available in the Gene Expression Omnibus (GEO) under accession number GSE262148.