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Schematic illustraiton showing DNA strand with broken strands

The study, published in the journal Cell Stem Cell, enables researchers to decode DNA sequence and organization at many different locations simultaneously in single cancer cells.

Cancers result from the accumulation of mutations within cells, but the pattern of mutations differs across cancer cells, even if they are from a single patient – tumours are not homogenous, and are instead made up of different lineages, each with its own distinct combination of mutations.

This makes treating patients difficult, and often results in the disease returning. “In order to efficiently eradicate these cancerous clones, we first need to precisely understand how key genes and pathways are corrupted by specific mutations”, says DPhil student Sven Turkalj, the study’s joint first author.

 Understanding DNA accessibility

Within the nucleus of cells, DNA is packaged around proteins, forming a complex known as chromatin. Because of this packaging, only certain parts of the DNA sequence are accessible to the cell at any one time. When a gene is switched on, the chromatin surrounding the gene tends to open, whereas genes that are switched off tend to be closed. Looking at these patterns of chromatin accessibility can tell scientists about both about the cell’s identity, and the processes that control genes being switched on or off.

In normal cells, the process of switching genes on and off is carefully controlled, but in cancer, mutations disrupt this careful control. This leads to abnormal activation or silencing of critical genes, resulting in cancers such as Acute Myeloid Leukaemia, a blood and bone marrow malignancy which is the most aggressive acute leukaemia in adults.  But there is currently no method that allows researchers to efficiently link chromatin accessibility with exactly which genes are mutated in a single cell.

Professor Paresh Vyas, the senior author on the study, said: “To better understand these processes, our lab developed a novel method capable of isolating single cells from cancerous tissues, and allowing for a simultaneous readout of their mutation status and chromatin accessibility.”

The method, known by the abbreviation GTAC (Genotyping with the Assay for Transposase-Accessible Chromatin), allows scientists to look at single cancer cells and understand how specific genes are abnormally regulated in different malignant cancer cell lineages.  

Cancer genesis

The research team used this method to study a bone marrow sample from a patient with Acute Myeloid Leukaemia. They discovered the presence of multiple genetic lineages within the leukaemia cells, even though they were from the same patient.  Using GTAC to couple this information with chromatin accessibility, they were able to track the evolution of the leukaemia cells and determine how each mutation had changed chromatin accessibility. They found that the early, pre-leukaemic mutations did not cause excessive gene de-regulation, but a later mutation in a gene called RUNX1 led to a drastic reprogramming of DNA structure, leading to the outgrowth of cancerous cells.

But the evolution of these malignant cells didn’t stop there: several cancer cells further evolved by acquiring mutations in the gene called BCOR, indicating that cells with BCOR mutations were somehow being selected because they were more likely to survive and escape the body’s normal regulation.

At first sight, these cells were similar to cancerous leukaemia cells without a BCOR mutation. However, when the research team looked at which areas of chromatin were accessible in cells with a BCOR mutation, they found abnormal activation of genes that control a cell’s ability to renew itself and differentiate into other kinds of cells. The lab further identified that in cells with the BCOR mutations, several genes that maintain leukemic growth were switched on, via aberrant accessibility of chromatin elements next to these genes.

Dr Asger Jakobsen, the study’s joint first author, said “Many leukaemia researchers and clinicians believe that anti-tumour therapies can usually kill cells that are dividing a lot, but they often fail to eradicate more dormant and silent cells that go on to birth more cancer cells.

“Our method suggests that specific combinations of mutations push malignant cells towards states resembling dangerous leukaemic ‘mother’ cells, and it identifies chromatin elements which might regulate this transition.

The research team hopes that the proof of concept with the most aggressive adult blood cancer will encourage other researchers to use this new method to understand the evolution and mechanisms behind many different kinds of cancers.

Sven Turkalj said “Most importantly, we hope that by applying GTAC to patient samples acquired over many different time points, researchers and clinicians can begin to understand how genetic and epigenetic evolution allow malignant cells to win in the Darwinian battle against cancer treatments, eventually helping develop better treatments”.

Read the full paper.