Going round in circles: The story of extrachromosomal DNA
AI-generated image via Canva, prompt: circle of DNA double helix
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When you think about DNA, I’m willing to bet you either zoom in to imagine the double helix itself, or zoom out, picturing DNA neatly bundled up into X-shaped chromosomes. But while it is true that most of our genome - a staggering 2 metres of DNA in every cell - is packaged into these structures, that’s not the whole story.
If you’ve been paying attention to our previous episodes, you’ll also know that mitochondria also have their own little package of DNA, as do chloroplasts - the light-harvesting structures in plants.
And then there’s the weird stuff.
Our story starts in 1964, with two researchers named Yasuo Hotta and Alix Bassel, who were working at the University of Illinois to understand how DNA is organised in the chromosomes of complex organisms like mammals.
Their experiments involved looking at the DNA of boar sperm using electron microscopy, which uses electron beams rather than light rays to generate incredibly high resolution images of the contents of cells. Boar sperm might seem like an unusual choice of study material, but not only was it fairly easy to acquire (I’ll leave you to picture how), it was relatively easy to isolate DNA from the cells for the complicated preparation techniques required for electron microscopy.
Looking at the images, Hotta and Bassel noticed something strange. Nestled in amongst the regular chromosomes were circles of DNA of various sizes - the larger circles they named "double minutes" as they seemed to appear in pairs. This was the first time these structures, now known as extrachromosomal DNA, were spotted in mammalian cells.
About a year later, three scientists — David Cox, Catherine Yuncken, and Arthur Spriggs — were examining chromosomes extracted from neuroblastoma tumour cells under the microscope. This particular cancer, usually found in young children, originates in nerve cells, and can be very challenging to treat effectively. They too spotted “double minutes” lurking around the chromosomes of these cancer cells - the first time extrachromosomal DNA was spotted in human cells.
Over the years, a handful of researchers kept on investigating these mysterious little rings, but extrachromosomal DNA was largely ignored by most of the scientific community for the next five decades. Why? It was generally thought that although they were a biological curiosity, these rings probably played little to no role in cancer, or in biology in general. Most people thought they were just genetic trash. After all these tiny little dots under the microscope couldn’t be that important, could they?
During this time, the field of cytogenetics - the study of chromosomes - was growing rapidly, driven by improvements in microscopy and staining techniques. These new advancements revealed ever more detailed information about chromosomes and how they got messed up in cancer cells, which we’ve covered in more detail in our previous Genetics Unzipped episode Fusion genes and cancer cures: The story of the Philadelphia Chromosome.
Cancer became increasingly viewed as a disease of mutated genes and rearranged chromosomes, creating altered proteins that drive cancer cells to proliferate out of control. At the same time, drug developers increasingly focused on designing highly targeted therapies that would lock on to these faulty proteins and block their function, with the aim of stopping tumours in their tracks. But there was a problem. A drug would work for a bit and the cancer would shrink, but then it stopped working and the cancer came back. Or sometimes they just didn’t work at all. For all the hype about these ‘magic bullets’, and the hefty price tags, they weren’t the cure for cancer we’d all been hoping for.
The reasons for this are many fold, but one of them is extrachromosomal DNA. And the reason we now know about that is thanks in no small part to the work of one scientist, Paul Mischel. But why did these long-ignored little circles catch his eye?
Mischel, alongside many others in the cancer research field, was searching for an explanation as to why some cancers, like glioblastoma brain tumours, don’t respond to a specific type of therapy, called EGFR inhibitors. In theory, EGFR inhibitors should work brilliantly - they target mutations in the epidermal growth factor receptor gene, a gene which promotes aggressive tumour growth. However, in reality, they often fail to effectively shrink tumours in patients with cancers carrying the very mutations they specifically target. This paradox has puzzled the cancer research community for years.
Ever increasing circles
Advances in genetic sequencing technologies mean the scientists of today spend less time peering through the microscope. It was these methods that drove the completion of the Human Genome Project, and many other subsequent large-scale sequencing programmes aimed at reading the DNA from thousands of tumour samples. But these techniques specifically focus on reading DNA packaged in chromosomes, and ignore everything else. Mischel decided to go back to basics, and dusted off the microscope to take a closer look at the cells in glioblastoma tumours. Just like the scientists in 1965, he spotted those tiny dots - extrachromosomal DNA. But unlike back then, Mischel had specially stained the tumours beforehand using a technique that highlights specific genes.
Mischel stained the EGFR genes red, and the chromosomes blue. Based on the scientific consensus of just 10 years ago, what Mischel expected to see was a clump of red standing out on a strand of blue, representing an array of multiplied mutated EGFR genes within a chromosome. Instead what he saw were small red dots, sprinkled about outside the blue chromosomes. Slowly, the horrible truth began to dawn. Rather than being housed within a chromosome, these aberrant EGFR genes were located on tiny circles of extrachromosomal DNA. And that might explain why these cancers were so difficult to treat with drugs that ought to be a magic bullet.
Mischel decided to investigate further. When the tumours were treated with EGFR inhibitors, the red dots disappeared - but once the drug was removed, the extrachromosomal DNA quickly reappeared, and those cancer promoting genes were back in action. But why did those pesky circles reappear again?
Extrachromosomal DNA contributes to something called tumour heterogeneity - this means the collection of cells within a tumour are all different to each other, expressing different genetic mutations. This can make the tumour very hard to treat, as cancer cells replicate rapidly - when you find a therapy that kills off some of the cells with a certain gene, the cancer cells with a different mutation survive, and the tumour keeps growing.
So how does extrachromosomal DNA contribute to this problem? Well, when cells replicate, each chromosome is copied and the two copies divided between the two cells that form, so each daughter cell ends up with more or less the same DNA as its parent. But genes carried on extrachromosomal DNA aren’t subject to the same rules as regular chromosomes when it comes to replication and inheritance.
Extrachromosomal DNA circles lack something called centromeres. Centromeres are structures found within each chromosome that enable each freshly replicated copy to be accurately and evenly shared out as a cell divides. Without centromeres to guide them, circles of extrachromosomal DNA are randomly scattered between the two daughter cells. So some cells might end up with loads, while others have very few. Cancer cells with lots of extrachromosomal DNA circles carrying the mutant EGFR gene get killed off by the drug, but the cancer cells with only a few - fewer than could be detected using Mischel’s microscopy technique - survive.
Once the treatment is stopped, the surviving cells quickly put extrachromosomal DNA back into production. And unlike the DNA tightly packed inside chromosomes, the genes on extrachromosomal DNA circles are easier for the cell to access - meaning they are easier to activate. The mutated EGFR gene in the circles gets switched back on, causing the cancer cells to start growing rapidly again. And, as before, as the cells replicate, some cells have loads of circles, others only have a few - and the cycle continues.
Squaring the circles
Mischel and his team first published their findings about extrachromosomal DNA carrying cancer-driving genes in 2013, reigniting interest in this long-ignored field. But maybe it shouldn’t have been such a surprise - after all, simpler organisms like bacteria have been up to these kinds of extrachromosomal shenanigans for a very long time. Plasmids are small circles of extrachromosomal DNA that get swapped between bacterial cells, often containing genes that enable the bugs to resist the effects of antibiotics. And in the same way that we can blame these plasmids for the rise of antimicrobial resistant superbugs, we can also blame extrachromosomal DNA circles in cancer cells for making them hard to treat and resistant to therapy.
As Mischel and his colleagues note in another paper from 2019, “In bacteria, small circular plasmids represent a prevalent and powerful mechanism for rapidly gaining selective advantage. We speculate that oncogene-containing circular extrachromosomal DNA in human cancers represents the conceptual equivalent, highlighting crucial gene variants and mechanisms for oncogenesis and therapeutic resistance.”
They aren’t wrong. Today we know that extrachromosomal DNA is found in at least one in seven cancers, and patients whose tumours carry cancer-driving genes on extrachromosomal DNA rather than in their regular chromosomes are less likely to survive their disease. And that’s not all. Extrachromosomal DNA circles can contain aberrant genetic control switches as well as genes themselves, driving high levels of gene activity to keep cancer cells multiplying. And they can even hop back into chromosomes, mixing things up even more. These troublesome toroids area major obstacle when it comes to curing cancer. So what do we do about them?
Despite its obvious significance in cancer biology, relatively little is known about extrachromosomal DNA, partly because it’s much harder to isolate and study than regular chromosomes. But we really need to figure out where these circles come from and how they work if we’re to come up with more effective ways to treat cancer.
For his part, Mischel and his colleagues are now working on a major project called eDyNAmiC - one of the charity Cancer Research UK’s Grand Challenges that aim to answer the really big questions in cancer research. In fact, it was Charles Swanton himself, who I mentioned right at the start, who championed the need to have a project looking at the mysteries of extrachromosomal DNA.
“We’re just beginning to see how important extrachromosomal DNA is in the evolution of tumours. It’s entirely unpredictable, entirely chaotic and very difficult to target,” he said, when Mischel’s winning team was announced. “We need to understand extrachromosomal DNA if we’re going to understand cancer drug resistance and this team will help us get to the heart of that problem much more quickly. We need to understand its biology, how it evolves, how its maintained, and ultimately how to target it.”
The eDyNAmiC project kicked of in 2022 with a huge multidisciplinary research team coming at the problem of extrachromosomal DNA from many angles - according to the CRUK news story, they are “Inspired by strategies used by jazz musicians to think and work creatively…uniting mathematical modelling to predict tumour evolution and biology studies challenging the understanding of how a cell functions, as well as viewing what’s happening in patients in real time.”
After many decades of going round in circles, it looks like time has finally come for extrachromosomal DNA to close the loop on cancer research, and hopefully lead to some new ideas for cures.
