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The next step in personalised cancer medicine

Researchers at the Botnar Research Centre, University of Oxford have developed technology that facilitates standalone long-read Oxford Nanopore sequencing of single cells. This breakthrough technology has the potential to open new avenues within genomics and enable future discoveries to understand the causes of many human diseases.

The work, in part supported by grants from the UKRI (Innovate UK, EPSRC and MRC), results from a collaboration with researchers from the Department of Chemistry at Oxford University, ATDBio, a world leader in complex oligonucleotide chemistry, and pharmaceutical company BristolMyersSquibbs. The study has been published in this week’s issue of Nature Biotechnology.

“The application of accurate long-read single-cell sequencing will have a transformative effect on the wider single-cell sequencing community, as longer and full-length transcriptomic sequencing allows users to capture more information about the transcriptional and functional state of a cell,” says Assistant Professor Adam Cribbs, senior author of the paper and Group Leader in Systems Biology and Next Generation Sequencing Analysis at the Botnar Research Centre. “This means that we move closer to being able to better understand and diagnose diseases such as cancer”.

Single-cell genomics, the ability to examine all information contained in an individual cell, is a rapidly evolving field and is dominated by droplet-based short-read single-cell sequencing applications. In this approach, cells are encapsulated with barcoded RNA-capture microbeads into droplets within an oil emulsion. Each droplet becomes a discrete reaction vessel, associating a different barcode with each cell’s RNA and a unique molecular identifier (UMI) with each RNA transcript.  Once barcoded, RNA from all cells can be pooled and processed conventionally for next generation sequencing.  During sequencing, both the original RNA sequence and the associated barcode and UMI are determined. Key to measuring abundance of each RNA and correctly associating them with their cell of origin is accurate assignment of the UMIs and barcodes.

Long-read sequencing approaches, such as those of Oxford Nanopore Technologies, are currently revolutionising bulk sequencing approaches. “Long-read single-cell technology has the potential to interrogate not only RNA abundance, but also splice variants, structural variation and chimeric transcripts at the single-cell level. Collectively, the ability to determine these features accurately will improve diagnostics and biological understanding. However, Nanopore sequencing can be inaccurate, which hinders the critical steps of barcode and UMI assignment, making its application to single-cell sequencing challenging,” explains Dr Martin Philpott, first author of the paper and Director of the Next-Generation sequencing facility at the Botnar Research Centre.

To overcome these challenges, the team has developed a new approach called single-cell corrected long-read sequencing (scCOLOR-seq) that identifies and corrects errors in the barcode and UMI sequences, permitting standalone cDNA Nanopore sequencing of single cells. “Each mRNA molecule is tagged with a short sequence which identifies it within a certain droplet,” adds Dr Cribbs. “However, Nanopore long-read sequencing is too error prone to reliably sequence these tags, making it difficult to map the mRNA back to its specific cell. What we’ve been able to do is to develop a practical method for building redundancy into the tag, allowing inaccuracies within the sequencing to be pinpointed, and then correct them. The mRNA can then be linked back to an individual cell.”

The research was developed in collaboration with ATDBio, an Oxford/Southampton based company, created by Professor Tom Brown Sr at the Chemistry Department, University of Oxford. Dr Tom Brown Jnr, Chief Scientific Officer of ATDBio, says, “The new scCOLOR-seq method is the first of many innovations resulting from our collaboration with the Botnar Research Centre team. The collaboration has been one of our most interesting and successful, and we are pleased to see our work recognised in Nature Biotechnology. It’s a great example of how we at ATDBio can apply our expert knowledge of nucleic acid chemistry and complex oligonucleotide synthesis to difficult problems in biology and beyond, together with our corporate and academic partners”.

“This study demonstrates an incredible cross-disciplinary team effort to advance single-cell technologies and is the result of strategic investments into these technologies at our department,” adds Professor Udo Oppermann, Director of Laboratory Sciences at the Botnar Research Centre and co-senior author of the paper. “We will continue our collaborative efforts to develop innovative single-cell approaches and – as demonstrated in the paper- apply this to molecular analyses in primary and secondary bone and other haematological cancers. Our intention is to advance these technologies in personalised medicine approaches such as cancer diagnosis allowing rational clinical decision making.”

Ludwig Oxford and Oxford University welcome Professor Stefan Constantinescu

Oxford’s cancer community is delighted to welcome Professor Stefan Constantinescu, a physician scientist and authority on the signalling pathways and molecular mechanisms of blood cancers, especially myeloproliferative neoplasms, a collection of slow growing blood cancers that can progress to acute malignancies. He is a member of the Ludwig Institute for Cancer Research, Professor of Cell Biology at the Université catholique de Louvain, Director of Research (Honorary) at the Fonds National de la Recherche Scientifique (FRS-FNRS), Belgium and President of the Federation of European Academies of Medicine (FEAM). Constantinescu will spend 25% of his time at the Ludwig Oxford Branch and the remainder of the time at his existing Ludwig laboratory in Brussels.

Constantinescu has received many honors for his work, including membership of the Royal Academy of Medicine of Belgium and the Belgian Government prize for basic medical sciences. He is internationally known for his groundbreaking contributions to our understanding of the mutations and mechanisms that drive myeloproliferative disorders. In a fruitful collaboration with William Vainchenker, he discovered that a mutation (V617F) in a signalling enzyme named Janus kinase 2 (JAK2) occurs in most patients with polycythemia vera, in which red blood cells accumulate abnormally. Constantinescu’s subsequent work demonstrated how this mutation causes disease, leading to the development of novel therapies to treat myeloproliferative disorders and the widespread clinical use of genetic tests to detect the mutation.

Constantinescu has also identified and characterised other common mutations in the thrombopoietin receptor that cause these blood disorders. He has further demonstrated that mutated calreticulins –“chaperone” proteins that otherwise help fold other proteins appropriately—can induce myeloproliferative disorders via abnormal activation of the thrombopoietin receptor, identifying a novel oncogenic mechanism. His discoveries have helped transform the field and continue to open new avenues for the development of targeted therapies.

Constantinescu’s Ludwig Oxford lab will focus on a systematic study of signalling and epigenetic regulation during oncogenesis in chronic myeloid cancers and their progression to the severe condition, secondary acute myeloid leukaemia. Ludwig Oxford’s research programme will be enhanced by Constantinescu’s presence, and his own research programme will benefit from Ludwig Oxford’s expertise in cancer epigenetics, represented by the laboratories of Yang Shi, Chunxiao Song, Skirmantas Kriaucionis and Benjamin Schuster-Böckler.

Read more about the new Constantinescu research group here.

New method for cost-effective genome-wide DNA methylation analysis

Cytosine, one of the four DNA bases, can be chemically modified by the addition of a molecule known as a methyl group to form 5-methylcytosine. This “epigenetic” modification has long been known to regulate gene expression and plays a critical role in processes like embryonic development. Its levels and distribution are also distinct in different tissues and are significantly altered in cancers. Analysing methylation patterns of DNA shed into blood and other bodily fluids by tumours can thus reveal both the presence and the location of a cancerous growth.

In 2019, Dr Chunxiao Song (Ludwig Institute for Cancer Research, Oxford Branch, Nuffield Department of Medicine) and his team developed TET-assisted pyridine borane sequencing (TAPS) for mapping DNA methylation. The technology was spun out in 2020 to establish the biotechnology company Base Genomics, which was acquired for $410 million by Exact Sciences in October 2020. Compared to the previous gold standard for sequencing DNA methylation, TAPS is far more cost-effective and sensitive, and generates cleaner data to allow for additional genetic analysis.

Yet despite its advantages, TAPS still relies on whole-genome sequencing, which remains an expensive approach for detecting DNA methylation since just ~4% of all cytosines in the genome are methylated. Chunxiao and his team have now developed a new method that cuts costs further by sequencing only those regions of the genome that contain methylated cytosines.

Building on the TAPS method, postdocs Dr Jingfei Cheng and Dr Paulina Siejka-Zielińska made use of molecular scissors called endonucleases that recognise and cut specific DNA sites. During TAPS, methylated cytosines are chemically converted to an altered base called dihydrouracil (DHU). The researchers found an endonuclease called USER enzyme that specifically cuts at DHU. Because of the enzyme specificity, they knew that all the DNA fragments produced had methylation sites at the beginnings and ends. By then size-selecting the DNA to exclude the larger, uncut DNA, only the smaller, cut DNA fragments with methylation sites are sequenced, making this approach more cost-effective for studying DNA methylation at base-pair resolution.

The team has named the new technique endonuclease enrichment TAPS (eeTAPS), and details on the method can be found in their publication in Nucleic Acids Research.

Registration open for Cancer Early Detection and Epigenetics Symposium

Join us and our co-hosts for this free virtual event on 28-29th April 2021 to hear the latest developments from international leaders in these fields

Improving immunotherapy through epigenetics

Immunotherapy has shown remarkable efficacy against a range of cancers. One approach, termed immune checkpoint blockade therapy, blocks an inhibitory immune receptor called PD-1 to take the brakes off the immune system and allow it to kill cancer cells. However, despite this success, anti-PD-1 therapy is ineffective in the majority of cancer patients.

Research is underway to discover strategies that can overcome tumour resistance to immunotherapy. A promising avenue for further investigation is the manipulation of epigenetic regulators. Epigenetic regulators influence the expression of genes without changing the underlying DNA sequence. They can dampen the response of the immune system and their inhibition has been shown to enhance the response to anti-PD-1 treatment. However, because epigenetic regulators are involved in several aspects of the anti-tumour immune response, inhibiting them can result in potentially opposing effects, with the result of little or no overall benefit.

In this paper published in the journal Cancer Discovery, Professor Yang Shi and his laboratory explore the opposing effects of inhibiting one such epigenetic regulator, LSD1. Using mouse and tumour cell models, they show that when LSD1 is repressed, there is a greater immune cell infiltration into the tumour but this is counteracted by the increased production of a cell regulatory protein called TGF-β that suppresses the ability of these infiltrating immune cells to kill cancer cells.

To tackle these conflicting effects, the team experimentally depleted both LSD1 and TGF-β during anti-PD-1 therapy and demonstrated a significant increase in immune cell infiltration, cytotoxicity and cancer cell killing. This combination treatment led to eradication of these previously resistant tumours and long-lasting protection from tumour re-challenge, making it a promising future strategy for increasing the efficacy of this important class of cancer treatment.

Developing a system to simultaneously detect genetic and epigenetic information

Many diseases are associated with changes to the DNA sequence, most notably cancer. Also altered in disease is the way that the DNA is decorated with chemical modifications such as methylation (epigenetic modifications). Being able to extract genetic and epigenetic information using DNA sequencing has revolutionised biomedical research and has led to new ways to diagnose diseases. A particular interest currently is in using genetic and epigenetic characteristics of tumour DNA circulating in the blood or other bodily fluids as a strategy for detecting cancer earlier. However, despite the potential utility of combining genetic and epigenetic information to enhance disease detection, no methods currently exist that can efficiently simultaneously extract this information from the same DNA sequencing data.

Up until now, DNA methylation has predominantly been detected using methods that rely on a process called bisulphite conversion. Bisulphite is a harsh chemical that damages DNA, resulting in decreased sensitivity and a high error rate in the sequencing data. Because it is not known whether any changes in the DNA compared to a reference genome are introduced by bisulphite or real mutations, it is very challenging to simultaneously detect methylation and mutation data using these methods.

Recently, a new bisulphite-free method for detecting DNA methylation called TET-assisted pyridine borane sequencing (TAPS) has been developed by Ludwig Oxford’s Dr Chunxiao Song and Dr Benjamin Schuster-Böckler. This method is both cheaper than bisulphite sequencing and importantly produces data of higher quality, similar to that of standard DNA sequencing.

In this project, funded by an MRC Methodology Research Grant, Dr Benjamin Schuster-Böckler will collaborate with Professor Gerton Lunter (Visiting Professor, Radcliffe Department of Medicine) to develop algorithms that simultaneously detect mutations and DNA methylation from TAPS data.  Experimental data will be provided in collaboration with Ludwig Oxford’s Dr Chunxiao Song and Professor Xin Lu, and Professor Ellie Barnes (Nuffield Department of Medicine). Test data will be used to train machine-learning algorithms to optimise the accuracy of the sequencing method and to establish the best possible experimental parameters for this technique.

The resulting method will greatly increase the utility of the TAPS technique and will make it possible to routinely query a patient’s genetic background, while simultaneously measuring their epigenetic state. This will lead to a much broader understanding of the role of epigenetics in disease and would raise the possibility of using combined genetic and epigenetic information from sequencing data to aid earlier detection of cancer.

Image attribution: Darryl Leja, National Human Genome Research Institute (NHGRI) from Bethesda, MD, USA, CC BY 2.0 https://creativecommons.org/licenses/by/2.0, via Wikimedia Commons

Understanding how inherited and acquired mutations interact to affect cancer

There are two types of genetic variation that affect cancer. So-called somatic variation results from changes (mutations) in a person’s DNA that are acquired during their lifetime in individual parts of the body. These mutations only occur in some cells in the body and are often the result of damage with age or by carcinogens such as sunlight, smoking and some infections. By contrast, germline variation is inherited and so occurs in every cell in the body since birth. An example of germline variation is inheriting a mutation in the BRCA1 gene, which is associated with increased risk of breast cancer in families with these mutations.

Many research studies have investigated the separate effects of somatic and germline variation on cancer risk, progression and response to therapy. However, these studies generate an incomplete picture. For example, designing bespoke therapies to target cancer cells containing specific somatic mutations has had variable success, perhaps in part due to differing underlying germline variation between individuals. To make further progress, we need to learn more about whether germline and somatic variants interact to affect cancer. This is the question that Dr Ping Zhang asked as a post-doc in Dr Gareth Bond’s lab when it was at the Oxford Branch of the Ludwig Institute for Cancer Research.

In this paper published recently in the journal Cancer Research, the Ludwig Oxford researchers worked with colleagues at several other institutions to investigate the interplay between germline and somatic variants affecting the activity of the p53 tumour suppressor protein. p53 is a key protector against the development of cancers and somatic mutations in the gene coding for p53 are found in over half of all human cancers. Perturbation of p53 activity also influences cancer progression and drug response.

In this study, the team discovered evidence that germline cancer-risk p53 pathway mutations cooperate with somatic p53 gene mutations to alter cancer risk, progression and response to therapy, and can be used to identify novel, more effective therapies. With this increased understanding, this work has the potential to guide further discovery of future anti-cancer drug targets and novel combination therapies for enhancing precision medicine.

This work was funded by the CRUK Oxford Centre Development Fund along with the Ludwig Institute for Cancer Research, and the Nuffield Department of Medicine.

New sequencing methods for distinguishing DNA modifications

Chemical modifications made to the DNA base cytosine play an important role in the regulation of gene expression across the genome. Cytosine can be chemically modified in four ways, with 5-methylcytosine (5mC) being the most common. Demethylation of 5mC by the TET family of enzymes results in the stable intermediates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). From what has been discovered so far, these modifications appear to have distinct functions. For example, 5mC is associated with repressed regions of the genome whereas 5hmC is present in active ones. However, to study these modifications further, robust sequencing methods are needed that can detect each of these four modifications specifically.

The traditional gold standard method for detecting DNA methylation is bisulphite sequencing. However, this relies on a harsh chemical treatment that degrades most of the DNA sample and is an indirect detection method, which decreases sequencing quality. Recently, a bisulphite-free method called TAPS has been developed by Ludwig Oxford’s Song lab, which has the advantage of preserving more of the DNA, increasing sensitivity, and directly detecting modified cytosines for improved DNA sequencing quality.

Despite its advantages, TAPS cannot distinguish between the different types of cytosine modifications. Other methods already exist that can do so but these use subtraction, for example, measuring 5mC and subtracting this signal from a combined measure of 5mC and 5hmC to obtain 5hmC levels. In addition to the disadvantages of using bisulphite and/or indirect detection strategies, these subtraction methods also need higher sequencing depths and generate very noisy data that can be difficult to interpret. New subtraction-free methods are therefore needed to specifically, directly and sensitively detect these four cytosine modifications in the genome.

In this paper published in Nature Communications, Dr Yibin Liu from Dr Chunxiao Song’s lab (Ludwig Oxford) and Dr Zhiyuan Hu from Professor Ahmed Ahmed’s lab (Weatherall Institute of Molecular Medicine and Nuffield Department of Women’s and Reproductive Health, University of Oxford) have developed a suite of TAPS-related whole genome sequencing methods for specifically detecting 5mC, 5hmC, 5fC and 5caC. They have named these TAPSβ (for 5mC), chemical-assisted pyridine borane sequencing (CAPS; for 5hmC), pyridine borane sequencing (PS; for 5caC and 5fC) and pyridine borane sequencing for 5caC (PS-c; for 5caC).

With these new methods, the research community is now armed to tackle more of the questions about the distinct and important functions of cytosine modifications in the genome and how their distribution is altered in diseases such as in cancer.

Potential of DNA-based blood tests for detecting pancreatic cancer earlier

Pancreatic cancer is sadly a disease with very poor outcomes and only 7.3% of people survive this cancer for 5 years or longer in England (Cancer Research UK). The majority of patients with pancreatic cancer are diagnosed too late for potentially curable treatment to be applied and so there is an urgent need to detect pancreatic cancers earlier with the aim of improving outcomes from this disease.

One strategy for earlier detection is to screen people before they experience any symptoms using a minimally invasive test such as a blood test to look for indicators of pancreatic cancer. Published in the journal Cancers, Dr Shivan Sivakumar (Department of Oncology and Oxford University Hospitals NHS Trust) and colleagues Dr Jedrzej Jaworski (University of Oxford) and Dr Robert Morgan (University of Manchester and Christie NHS Foundation Trust) review the potential of cancer DNA in the blood as an effective and reliable indicator of pancreatic cancer.

DNA from cancer cells can be distinguished from DNA from healthy tissue using either genetic or epigenetic methods (or a combination of both). In the genetic method, cancer can be detected by looking at the DNA sequence, with the presence of cancer-associated DNA sequence changes called mutations or altered fragmentation patterns indicating cancer. In the epigenetic method, chemical modifications to the DNA called methylation are measured that have been shown to change in cancer.

In this review, the authors discuss the potential for DNA-based blood tests for pancreatic cancer earlier detection, the challenges that still need to be overcome and the future perspectives.

Read the full review article on the Cancers journal website.

Pancreatic cancer blood test research in Oxford

In Oxford, we have a couple of research projects underway to study both the genetic and epigenetic methods for detecting pancreatic cancer-derived DNA in the blood.

Dr Siim Pauklin (Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences) is working to identify a pancreatic cancer-specific DNA signature. In the long-term, it is hoped that this can be used as the basis of a simple blood test to detect the presence of pancreatic cancer earlier. This project is funded by the Pancreatic Cancer UK Research Innovation Fund. Read more about Siim’s project here.

Dr Chunxiao Song (Ludwig Institute for Cancer Research) is collaborating with Dr Shivan Sivakumar to apply his TAPS technology to pancreatic cancer. TAPS is a new, more sensitive method for detecting methylation on DNA, which gives it an advantage over other detection methods for measuring the very small levels of circulating tumour DNA in the blood. The team are working to identify patterns of DNA methylation that are specific for pancreatic cancer with the aim of developing this into a diagnostic test. Read more about Chunxiao’s and Shivan’s project on the CRUK Oxford Centre website.

Research projects to detect pancreatic cancer in the blood through non-DNA markers are also in progress in Oxford.

Oxford Cancer alumni’s biotech success

Scenic Biotech was founded in March 2017 as a spin-out of the University of Oxford and the Netherlands Cancer Institute. The company is based on the Cell-seq technology developed by co-founders Sebastian Nijman and Thijn Brummelkamp in their academic labs.

Cell-seq is a large-scale genetic screening platform that allows the identification of genetic modifiers – or disease suppressors – that act to decrease the severity of a disease. These disease-specific genetic modifiers are difficult to identify by more traditional population genetics approaches, especially in the case of rare genetic diseases. By mapping all the genetic modifiers that can influence the severity of a particular disease, Cell-seq unveils a new class of potential drug targets that can be taken forward for drug development.

In a deal worth $375m, Scenic Biotech has recently entered into a strategic collaboration with Genentech, a member of the Roche Group. This will enable discovery, development and commercialisation of novel therapeutics that target genetic modifiers.