Analyzing the Effectiveness of Targeting Extrachromosomal DNA in Cancer Treatment

By Natalie Zhu, USA

Abstract:

Extrachromosomal DNA (ecDNA) is a small circular piece of DNA found outside of the chromosome. It was first discovered in the 1960’s, but it was largely overlooked until recent research revealed the increased prevalence of ecDNA molecules in cancer tumors, with around 17.1% of cases containing ecDNA. Additionally, it is most commonly found in late-stage cancers, fast-growing cancers, and drug-resistant cancers (Bailey et al., 2024). By targeting ecDNA in cancer treatments, this approach would resolve the issue of cancer drug resistance during chemotherapy treatment, thereby making it more effective. This proposal seeks to determine whether treating ecDNA will increase the success rate of chemotherapy drugs on a fast-growing cancer like HER2+ breast cancer.

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Introduction:

One of the most common types of cancer treatment, chemotherapy is used world-wide as an integral method to treat various types of cancer. However, a frequent pitfall of chemotherapy is that cancer cells often develop a resistance to the drugs used during treatment, making cancer notoriously difficult to treat. For example, some data shows that up to 90% of cancer mortality is linked to drug resistance, and around 1 in every 6 deaths are caused by cancer (Bukowski et al.). Due to their rapidly dividing nature, tumor cells are able to quickly adapt to their environment, causing them to resist treatment or reform after starting treatment (National Cancer Institute, 2016). One factor that may contribute to drug resistance is the presence of ecDNA in many malignant tumors.

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ecDNA is formed when chromosomal damage causes DNA to break off and reform into circular fragments (Dong et al., 2023). Because of ecDNA’s circular structure, the DNA contained within the chromosome is less compact and therefore more easily accessible, leading to an overexpression of oncogenes. Some of these oncogene amplicons can accelerate the growth of intra-tumoral heterogeneity, where distinct cell subpopulations with different molecular profiles can originate from a single tumor (Dong et al., 2023), (Luo et al., 2023). This heightened variety can increase the chance of survival of a tumor and thus amplifies its ability to become resistant to targeted treatment. Approximately half of all human cancers contain widespread amounts of ecDNA, especially in fast-growing cancers like HER2+ breast cancer (Yang et al., 2022). In breast cancer, the oncogene HER2+ becomes amplified by ecDNA (Lu et al., 2021) , and using a specified treatment to target ecDNA first could improve the efficacy of typical chemotherapy drugs on HER2 breast cancer cells and minimize drug resistance.

Literature Review:

Although the discovery of ecDNA has been around for a while, the role of ecDNA in cancer cells and drug resistance is a fairly recent finding. Thus, the literature regarding ecDNA treatment is limited and largely speculative. However, a crucial breakthrough occurred in a study by the team eDyNAmiC. In this study, researchers found that ecDNA was particularly vulnerable to the inhibition of Checkpoint 1 (CHK1) during the cell cycle, as it tends to be over reliant on CHK1 to manage DNA damage (Tang et al., 2024). By developing a synthetic drug BBI-2779, they were able to interfere with CHK1, causing an increased number of damaged ecDNA cells to pass through the cell cycle unregulated and undergo cell death. They tested BBI-2779 in colon cancer cells both in vitro and in vivo through mice, demonstrating its ability to reduce ecDNA tumor cell count and decrease its resistance to the chemotherapy drug infigratinib. A limiting factor of this study is that it relies solely on the effects of CHK1 inhibition on colon cancer, and measures only the short term effects of this treatment. This proposal seeks to expand the research on ecDNA-targeted cancer treatment and extrapolate these findings to another cancer type, HER2 breast cancer, that is also affected by ecDNA, and to test if this method is feasible as a way to improve the effectiveness of chemotherapy treatment for other cancers in the long term.

Methods:​ 

Tumor samples will be collected surgically from patients with HER2+ breast cancer after informed consent. The tissue cells will be isolated by centrifugation and maintained in a 95% humidified, 5% CO2 environment at 37˚C. To track the amounts of ecDNA in each sample, metaphase DNA FISH (fluorescence in situ hybridization) can be used to determine defective chromosomal cells in metaphase (Wang et al., 2024). Cells are treated with 50ng/ml nocodazole to suspend them in mitosis and then treated with DAPI to stain the cells. Place the cells on sterilized slides and use DNA probes to hybridize them. Obtain images through conventional fluorescence microscopy, then wash the cells with a buffered saline solution to remove the nocodazole.

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Next, UCN-01, which has shown to be effective in inhibiting CHK1 in breast cancer (Jiang et al., 2024), will be added to the cells. Separate the cells into 6 wells of around 15,000 cells. Half of the cells will be incubated in a solution of UCN-01 for 16 h at 37˚C, then washed in a saline solution, and the other half will remain untreated as a control. A treatment of trastuzumab, commonly used to treat HER2+ breast cancer (Malwina Stanowicka-Grada and Senkus, 2023) will be applied to each well. Measure the mass of each well before treatment. Once treatment is completed, repeat the FISH method for one week, 3 weeks, and 6 months after treatment is complete to study the long-term effects of the treatment, paying attention to the regrowth of any tumor cells. Additionally, remeasure the mass of the tumor before completing the FISH method for all three measurement periods.

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For each image obtained, use the Circle_Finder or Circle_Map software to identify and score ecDNA junctions (Wang et al., 2024). Using log-rank tests, compare the survival rates of the ecDNA between the treated and untreated groups. Use an unpaired, one-tailed 2-sample t-test to compare if the change of mass of the treated group is larger than that of the control. Both tests will use an alpha of 0.05.

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Conclusion:

There is an urgent need to understand more about the mechanisms of ecDNA and its role in cancer treatment. In this proposal, we expect to find that using UCN-01 to target HER2+ breast cancer will result in a smaller survival rate of ecDNA and a greater shrinkage of tumor size, which would open the door to exploiting ecDNA weaknesses to improve cancer treatment. However, because this study utilizes cancer cells outside of a living organism, it may not be the best indicator of a viable treatment in humans. We try to mitigate this by using cells derived directly from patients instead of premade cell lines. Depending on the results of this study, further research may be needed to test the viability of treatment in living beings.

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References :

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