Through the Smokescreen

For many years I was lucky enough to teach in a cancer biology course for third year natural science and medical students. Quite a few of those guys would already be eyeing up research careers and, within just a few months, some might be working on the very topics that came up in lectures. Nothing went down better, therefore, than talking about a nifty new method that had given easy-to-grasp results clearly of direct relevance to cancer.

Three cheers then for Mikhail Denissenko and friends who in 1996 published the first absolutely unequivocal evidence that a chemical in cigarette smoke could directly damage a bit of DNA that provides a major protection against cancer. The compound bound directly to several guanines in the DNA sequence that encodes P53 – the protein often called ‘the guardian of the genome’ – causing mutations. A pity poor old Fritz Lickint wasn’t around for a celebratory drink – it was he, back in the 1930s, that first spotted the link between smoking and lung cancer.

This was absolutely brilliant for showing how proteins switched on genes – and how that switch could be perturbed by mutations – because, just a couple of years earlier, Yunje Cho’s group at the Memorial Sloan-Kettering Cancer Center in New York had made crystals of P53 stuck to DNA and used X-rays to reveal the structure. This showed that six sites (amino acids) in the centre of the P53 protein poked like fingers into the groove of double-stranded DNA.

x-ray-picCentral core of P53 (grey ribbon) binding to the groove in double-stranded DNA (blue). The six amino acids (residues) most commonly mutated in p53 are shown in yellow (from Cho et al., 1994).

So that was how P53 ‘talked’ to DNA to control the expression of specific genes. What could be better then, in a talk on how DNA damage can lead to cancer, than the story of a specific chemical doing nasty things to a gene that encodes perhaps the most revered of anti-cancer proteins?

The only thing baffling the students must have been the tobacco companies insisting, as they continued to do for years, that smoking was good for you.

And twenty-something years on …?

Well, it’s taken a couple of revolutions (scientific, of course!) but in that time we’ve advanced to being able to sequence genomes at a fantastic speed for next to nothing in terms of cost. In that period too more and more data have accumulated showing the pervasive influence of the weed. In particular that not only does it cause cancer in tissues directly exposed to cigarette smoke (lung, oesophagus, larynx, mouth and throat) but it also promotes cancers in places that never see inhaled smoke: kidney, bladder, liver, pancreas, stomach, cervix, colon, rectum and white blood cells (acute myeloid leukemia). However, up until now we’ve had very little idea of what, if anything, these effects have in common in terms of molecular damage.

Applying the power of modern sequencing, Ludmil Alexandrov of the Los Alamos National Lab, along with the Wellcome Trust Sanger Institute’s Michael Stratton and their colleagues have pieced together whole-genome sequences and exome sequences (those are just the DNA that encode proteins – about 1% of the total) of over 5,000 tumours. These covered 17 smoking-associated forms of cancer and permitted comparison of tobacco smokers with never-smokers.

Let’s hear it for consistent science!

The most obvious question then is do the latest results confirm the efforts of Denissenko & Co., now some 20 years old? The latest work found that smoking could increase the mutation load in the form of multiple, distinct ‘mutational signatures’, each contributing to different extents in different cancers. And indeed in lung and larynx tumours they found the guanine-to-thymine base-pair change that Denissenko et al had observed as the result of a specific chemical attaching to DNA.

For lung cancer they concluded that, all told, about 150 mutations accumulate in a given lung cell as a result of smoking a pack of cigarettes a day for a year.

Turning to tissues that are not directly exposed to smoke, things are a bit less clear. In liver and kidney cancers smokers have a bigger load of mutations than non-smokers (as in the lung). However, and somewhat surprisingly, in other smoking-associated cancer types there were no clear differences. And even odder, there was no difference in the methylation of DNA between smokers and non-smokers – that’s the chemical tags that can be added to DNA to tune the process of transforming the genetic code into proteins. Which was strange because we know that such ‘epigenetic’ changes can occur in response to external factors, e.g., diet.

What’s going on?

Not clear beyond the clear fact that tissues directly exposed to smoke accumulate cancer-driving mutations – and the longer the exposure the bigger the burden. For tissues that don’t see smoke its effect must be indirect. A possible way for this to happen would be for smoke to cause mild inflammation that in turn causes chemical signals to be released into the circulation that in turn affect how efficiently cells repair damage to their DNA.

raleighs_first_pipe_in_england-jpeg

Sir Walt showing off on his return                         to England

Whose fault it is anyway?

So tobacco-promoted cancers still retain some of their molecular mystery as well as presenting an appalling and globally growing problem. These days a popular pastime is to find someone else to blame for anything and everything – and in the case of smoking we all know who the front-runner is. But although Sir Walter Raleigh brought tobacco to Europe (in 1578), it had clearly been in use by American natives long before he turned up and, going in the opposite direction (à la Marco Polo), the Chinese had been at it since at least the early 1500s. To its credit, China had an anti-smoking movement by 1639, during the Ming Dynasty. One of their Emperors decreed that tobacco addicts be executed and the Qing Emperor Kangxi went a step further by beheading anyone who even possessed tobacco.

And paying the price

And paying the price

If you’re thinking maybe we should get a touch more Draconian in our anti-smoking measures, it’s worth pointing out that the Chinese model hasn’t worked out too well so far. China’s currently heading for three million cancer deaths annually. About 400,000 of these are from lung cancer and the smoking trends mean this figure will be 700,000 annual deaths by 2020. The global cancer map is a great way to keep up with the stats of both lung cancer and the rest – though it’s not for those of a nervous disposition!

References

Denissenko, M.F. et al. ( (1996). Preferential Formation of Benzo[a]pyrene Adducts at Lung Cancer Mutational Hotspots in P53.Science 274, 430–432.

Cho, Y. et al. (1994). Crystal Structure of a p53 Tumor Suppressor-DNA Complex: Understanding Tumorigenic Mutations. Science, 265, 346-355.

Alexandrov, L.D. et al. (2016). Mutational signatures associated with tobacco smoking in human cancer. Science 354, 618-622.

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Seeing a New World

May I wish readers a Happy New Year – and indeed extend my felicitations to non-readers with the hope that they too will become followers! What a good idea! Not least because I suspect many are viewing the new year with a mixture of anxiety and despair. But I can promise there’s nothing like the sanity of science to restore you after a few minutes contemplating how we’re doing on the economic and political fronts.

Your starter for 2017

By happy chance a few weeks ago I tried to explain how it’s now possible to ‘re-write the manual of life’ – that is, to engineer our DNA, to fix broken genes if you like. This means that, in theory, it’s possible to correct errors in our genetic code that cause genetic diseases. As there are over 6,000 of these and they include Down syndrome, cystic fibrosis and Alzheimer’s disease, there’s no need to say it’s important. There are several ways of going about this but the one I described is called CRISPR and it’s had a lot of media coverage.

Right on cue

Well done then Keiichiro Suzuki, Juan Carlos Belmonte and friends from the Salk Institute in California together with colleagues from other centres in Spain, Saudi Arabia and China for their December paper describing a new CRISPR twist. They used a rat model of retinitis pigmentosa, a genetic disease that is a major cause of inherited blindness, afflicting about one and a half million people worldwide (one in 4,000 in the UK).

The CRISPR-Cas9 system is great but it works best in dividing cells (e.g., in skin and gut that are renewing all the time) and it’s particularly useful for knocking out genes rather than inserting new DNA. The latest modification allows a new gene to be inserted into a specific site in the DNA of cells that are not dividing (e.g., those of the eye or brain).

The bits of CRISPR-Cas9, which insert DNA at very precise locations within the genome, are delivered to target cells as part of an inert virus. However, the package also includes DNA that encourages the cells to use a repair process that can be turned on even in non-dividing cells. So CRISPR-Cas9 cuts the cell’s DNA at an exact sequence and the cell then repairs the double-strand breaks (by a process called non-homologous end joining (NHEJ) that glues the broken ends directly together). Give the cell a new bit of DNA (e.g., your favorite gene) and that will get patched in – bear in mind that the cell doesn’t ‘know’ what it’s doing: it just tries to fix damaged DNA with whatever’s at hand.

And the target?

Retinitis pigmentosa occurs when a chunk of a gene called Mertk is lost. After quite a lot of experiments to show that their method worked, Suzuki, Belmonte & Co made a viral carrier that included a normal Mertk gene and injected it under the retina of rats with the disease. After about 5 weeks the rats were making Mertk RNA as a result of the gene being correctly ‘knocked-in’ to eye cells. The light-detecting region of the eye, greatly reduced by the disease, was significantly restored, with associated appearance of MERTK protein.

      Diseased    Normal     Treated                         Diseased         Normal         Treated

pic

Left trio: Sections of the light-detecting layers of the eye in diseased (left), normal (centre) and diseased post-treatment rats (right). Right trio: corresponding fluorescence images showing MERTK expression (red: highlighted by white arrows); Cells labeled blue. (Suzuki et al. Nature 1–6 (2016) doi:10.1038/nature20565)

How did the rats see it?

Well, after treatment they were able to detect light and had significantly recovered their visual functions, albeit not to completely normal levels.

The usual caveats apply: the method isn’t hyper-efficient and a human treatment is still a long way off. Nevertheless, it’s a significant step.

The same group has also shown, using a way of re-programming the expression of just four genes, that it’s possible to arrest the signs of ageing. In other words, in mice this time, tinkering with these genes can increase lifespan – and yes, we have versions of these genes and in us they also control cell renewal.

So the New Year message is clear to see. If we can avoid turning the planet into a desert or blowing ourselves to smithereens the future is really rosy – and maybe even infinite!

References

Suzuki, K. et al. (2016). In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144-149.

Ocampo, A. et al. (2016). In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming. Cell 167, 1719–1733.

Cockles and Mussels, Alive, Alive-O!

And so they are across the globe, not forgetting clams, a term that can cover all bivalve molluscs – a huge number of species (over 15,000), all having a two-part, hinged shell. The body inside doesn’t have a backbone, making it soft and edible on a scale of keeping-you-alive to orgasmic, depending on the consumer – oysters and scallops are part of the family.

Bivalves are particularly common on rocky and sandy coasts where they potter happily along, generally burrowing into sediment although some of them, scallops for instance, can swim. By and large their only problem is that humans like to eat them.

Clamming up

However, it gradually emerged in the 1970s that there was another cloud hovering over some of these gastronomic delights. Their commercial importance had drawn attention to the fact that soft-shell clams living along the east coast of North America, together with mussels on the west coast and cockles in Ireland, were dying in large numbers. The cause was an unusual type of cancer in which leukemia-like cells reproduce until they turn the blood milky and the animals die, in effect, from asphyxiation. In soft-shell clams, also known as sand gapers and steamers, the disease has spread over 1,500 km from Chesapeake Bay to Prince Edward Island.

A 2009 study had shown that as the disease progresses there is a rise in the number of blood cells that have abnormally high amounts of DNA (in clams typically four times the normal number of chromosomes – i.e. they’re tetraploid). In parallel with this change the cells make increasing amounts of an enzyme called reverse transcriptase (RT).

That was pretty surprising as RT does what its name suggests: reverses part of the central dogma of molecular biology (DNA makes RNA makes protein) by using RNA as a template to make DNA. RT is usually carried by viruses whose hereditary material is RNA (rather than DNA – so they’re called retroviruses). As part of their life cycle they turn their genomes into DNA that inserts into the host’s genome – which gets reproduced (as RNA) to make more viruses.

But how did RT get into clams? Enter Michael Metzger and Stephen Goff from Columbia University in New York, together with Carol Reinisch and James Sherry from Environment Canada, who began to unravel the mystery.

Jumping genes

Using high throughput sequencing they showed that clam genomes contain stretches of about 5,000 bases that came about when the RNA of a virus was copied into DNA by RT (reverse transcriptase) and then inserted into the host chromosome. Normal clams have from two to ten copies of this ‘repetitive element’ that Metzger & Co dubbed Steamer. That wasn’t too surprising as we have repetitive DNA too – it makes up about half the human genome. Many of these repeated sequences can move around within the genome – they’re often called ‘jumping genes’ – and it’s easy to see how this can happen when RT uses RNA to make DNA that can then pop into new sites in the genome. And you might guess that this process could damage the host DNA in ways that might lead to disease.

A long jump?

It turned out that the diseased clams had suffered massive amplification of Steamer to the extent that they carry 150 to 300 copies of the sequence. So that’s about 30 times as many Steamer DNAs being scattered across the clam genome – but how could that cause the same disease all the way from New York to Prince Edward Island? The answer came from peering into the DNA sequences of the tumour cells: they were virtually identical to each other – but they were different to those of their hosts! Meaning? The damage that led to leukemia, caused by shoe-horning 100s of extra copies of Steamer into clam genomes, only occurred once. And the staggering implication of that finding is that the cancer spread from a single ‘founder’ clam throughout these marine-dwelling molluscs. The resemblance to the way the cancer spreads in Tasmanian devils is striking.

Fishier and fishier

Fast forward to June 2016 and the latest contribution from the Metzger group reporting four more examples of transmissible cancer in bivalves – in mussels from British Columbia, in golden carpet shell clams from the Spanish coast and two forms in cockles.

Each appears to cause the same type of leukemia previously found in clams. The disease appears to be transmitted ‘horizontally’, i.e. by living cancer cells, descended from a single common ancestor, passing directly from one animal to another. Indeed, if you transplant blood cells from infected animals into normal clams they get leukemia.

 Species hopping

All that is quite amazing but the genetic analysis came up with an even more bizarre finding. In the golden carpet shell clams DNA from cancer cells showed no match with normal DNA from this species. It was clearly derived from a different species, which turned out to be the pullet shell clam – a species that, by and large doesn’t get cancer. So they have presumably come up with a way of resisting a cancer that arose in them, whilst at the same time being able to pass live tumour cells on to another species!!clam-transfer-pic

Cancer cell transmission between different species of shellfish. Cancer cells can arise in one species (pullet shell clams) that do not themselves develop leukemia but are able to pass live cells to another species (golden carpet shell clams) that do get leukemia (Metzger et al. 2016).

We have no idea how the cancer cells survive transfer. It seems most likely that they are taken up through the siphons that molluscs use for feeding, respiration, etc. and then somehow get across the walls of the respiratory/digestive systems. In the first step they would have to survive exposure to sea water which contains a lot more salt than cells are happy in. The ‘isotonic’ saline used in drips to infuse patients contains 0.9% salt whereas seawater, with 3.5%, is ‘hypertonic’ – cells put in a hypertonic solution will shrink as water is drawn out of the cell into the surrounding solution. Presumably the cells shrivel up a bit but some at least take this in their stride and recover to reproduce in their new host. Equally obscure is how a species can protect itself from a cancer that it can pass to another species.

These amazing findings throw a different light on the care-free underwater life depicted in Disney’s The Little Mermaid, in which the popular song ‘Under the Sea’ fails to mention floating cancer.

Can this happen to us?!!

Well, not as far as we know. But the fact that the known number of cancers that can be passed from one animal to another has now risen to nine does make you wonder. However, there’s no evidence that it happens in humans in anything like the normal course of events. There are examples of person-to-person transfer, notably during organ transplantation, and there is one recent case of cancerous cells from a tapeworm colonising a human host. But these are very rare, the latter occurring in a patient with a severely weakened immune system, and there is no example of spread beyond two people.

Phew! What a relief! So now we can concentrate on following developments both in Tasmania and beneath the waves in the hope that, not only can we go on satisfying our lust for clam bakes and chowders, but that these incredible creatures will reveal secrets that will benefit mankind.

References

AboElkhair, M. et al. (2009). Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria. Diseases of Aquatic Organisms 84, 57-63.

Arriagada, G. et al. (2014). Activation of transcription and retrotransposition of a novel retroelement, Steamer, in neoplastic hemocytes of the mollusk Mya arenaria. PNAS 2014 111 (39) 14175-14180; published ahead of print September 8, 2014, doi:10.1073/pnas.1409945111.

Metzger, M.J. et al. (2015). Horizontal Transmission of Clonal Cancer Cells Causes Leukemia in Soft-Shell Clams. Cell 161, 255–263.

Metzger, M.J. et al. (2016). Widespread transmission of independent cancer lineages within multiple bivalve species. Nature 534, 705–709.

Muehlenbachs, A. et al. (2015). Malignant Transformation of Hymenolepis nana in a Human Host. N Engl J Med 2015; 373:1845-1852.

Another Peek At The Poor Little Devils

A couple of years ago (July 2014) I wrote a piece called Heir of the Dog that featured Tasmanian devils. The size of a small dog, these iconic little chaps are the largest meat-eating marsupials in the world. I’d run into them at The Lone Pine Koala Sanctuary in Brisbane where they’re keeping company with the dozy, furry tree-climbers as part of a programme to save them – the devils, that is – from extinction by cancer.

animal-fact-guide

A Tasmanian devil. Photo: Animal Fact Guide

1024px-sarcophilus_harrisii_taranna

 

 

 

 

 

 

 

imgresTheir problem comes from their inclination to bite one another, thereby directly passing on living cancer cells (causing devil facial tumour disease – DFTD). At that time the only other known example of transmissible cancer was a rare disease in dogs (canine transmissible venereal tumour – CTVT).

Genetic archaeology

DNA sequencing (i.e. whole genome analysis) had shown that the sexually transmitted dog disease probably arose thousands of years ago in a wolf or East Asian breed of dog and that the descendants of those cells are now present in infected dogs around the world.

The same approach applied to the Tasmanian devil showed that the cancer first arose in a female. Cells derived from that original tumour have subsequently spread through the Tasmanian population, the clone evolving (i.e. genetically diverging) over time. In contrast to the canine disease, DFTD is probably not more than 20 years old. Nevertheless, it spread through the wild population to the extent that the species was listed as endangered in 2008 by the International Union for Conservation of Nature.

Which is why a lot of effort is going into saving them, one approach being a number of breeding programmes in mainland Australia, with the aim of transferring uninfected animals to Tasmania.

One good turn …?

We’re all in favour of saving the little fellows, even if you probably wouldn’t want one as a pet. But, smelly and ferocious as he is, the Tasmanian devil is turning out to be remarkable in ways that suggest they might repay our efforts to keep them going. Things have moved apace down under with Greg Woods, Ruth Pye, Elizabeth Murchison, Andrew Storfer and colleagues from the Universities of Tasmania, Cambridge, Southampton and Washington State making some remarkable discoveries.

Infected animals do indeed develop the most unpleasant, large tumours that are virtually 100% fatal – to the extent that DFTD has wiped out 80% of Tasmanian devils in just 20 years. But some animals survive, even though models of the epidemiology say they shouldn’t. Andrew Storfer’s group asked how they pulled off this trick by looking for genetic changes in almost 300 devils. Quite amazingly, they found that even in a period as short as 20 years there were seven different genes that appeared to have changed (i.e. mutated) in response to selection imposed by the disease. Five of these genes encode proteins known to be associated with cancer risk or the immune system in other mammals, including humans. It seems that the mutations help their immune system to adapt so that it can recognize and destroy tumour cells.

In parallel with those studies, Greg Woods and his team now have a vaccine that looks promising early in trials – in other words a way of boosting natural immunity. We are only just beginning to find ways of giving the human immune system a helping hand – hence the burgeoning field of immunotherapy – so anything that works in another animal might give some useful pointers for us.

sick-tasTasmanian devil facial tumour disease.

This has killed 80% of the wild Australian animals in just a few decades.

Photograph: Menna Jones.

As if that wasn’t enough, a second strain of cancer has been found in a small group of male Tasmanian devils. It causes fatal facial tumours that look much the same as the first DFTD. However, it has a completely different genetic cause – so different in fact that it carries a Y chromosome, clear indication that the two forms of the disease arose by quite distinct mechanisms – which makes this marsupial the only species known to be affected by two types of transmissible of cancer.

Milk and human kindness

On top of all that some brave souls at Sydney University, Emma Peel and Menna Jones, decided in that way that scientists do, to collect some milk from the ferocious furries, just to see if it was interesting. Astonishingly the marsupial milk contained small proteins (peptides) that could kill a variety of bugs. They’re called cathelicidins and one of the things they can target is methicillin-resistant Staphylococcus aureus – MRSA – one of the dreaded ‘superbugs’ that are resistant to penicillin and other antibiotics. It’s not clear whether these peptides help to protect the devils from cancer but if that’s how turns out it might be incredibly important for us. As for their antibiotic potential, well, as it’s predicted that by 2050 superbugs will be killing one of us every three seconds you could say that opportunity beckons.

So that’s all incredibly exciting – and not just for the Tassie devils. ­ But another reason for returning to this story is that the devils have recently been joined by another example of extraordinary cancer transmission – and this one comes from the last place on the planet that you’d look for it ….

References

Murchison, E.P. et al. (2012). Genome Sequencing and Analysis of the Tasmanian Devil and Its Transmissible Cancer. Cell 148, 780–791.

Pye, R.J. et al. (2016). A second transmissible cancer in Tasmanian devils. Proceedings of the National Academy of Sciences USA, 113, 374–379.

Epstein, B. et al. (2016). Rapid evolutionary response to a transmissible cancer in Tasmanian devils. Nature Communications 7, Article number: 12684: doi:10.1038/ncomms12684.

Peel, E. et al. (2016). Cathelicidins in the Tasmanian devil (Sarcophilus harrisii). Scientific Reports 6, Article number: 35019. doi:10.1038/srep35019.

How Does DNA Do It?

Last time we left you with the mother of all molecular cliffhangers: how can it be that the simple four-letter code of DNA can carry the information to make all life? Early in that piece we’d thrown in the fact that the human genome (i.e. our DNA) is made up of three billion letters. As Watson and Crick showed 53 years ago, it’s actually two intertwined molecules, each with three thousand million letters – but it’s the number that’s important because that carries all that’s needed to make you and me.

dnaBut, if you’re like me, you have real problems grasping the meaning of numbers much above 100 – so that ‘millions’, yet alone ‘billions’, come across simply as ‘lots’– and we’re left shaking in our head in bewilderment as to how it works.

A different angle

To get some sort of a grip on the scale of information that genomes can carry, it might be helpful to look at DNA from the other end, so to speak. This approach started five years ago among a group who work on applying computer technology to handling biological data – i.e. how to acquire, store, analyse and interpret the tsunami of genetic information now being produced. It’s a new field called bioinformatics.

What set the bioinformatics bods thinking is a point that will have occurred to you as an internet user (and who isn’t?). How can we deal with the unimaginable amount of info we want to store? That includes everything from your holiday snaps to the tons of scientific data, including the continuing flood of genomics. If ‘millions’ leaves you boggling, how about the estimate for the global digital archive of 44 trillion gigabytes by 2020 (I think that’s 44 followed by 21 zeros). That’s a 10-fold increase from 2013.

Whatever the numbers are, they’re unimaginable but, aside from being boggled by the facts, a slight problem is that storing that amount on conventional memory sticks would use at least 10 times the amount of available silicon. So, as they say, we have a problem.

DNA to the rescue?

The boffins worked out that if you could use the storage capacity of DNA as efficiently as possible, the length you’d need to squeeze in all those trilla-bytes would correspond to about a kilogram of DNA. Put another way, the storage density of DNA is 1000 times that of flash memories. How would that work? Well, in principle it’s a simple, four-step process:

  1. Convert text to binary code (ones and zeroes)
  2. Convert binary code to triplet code (called ‘trits’: zeroes, ones and twos of a 3-digit code)
  3. Use trit code to make DNA (0, 1 or 2 translated into a base, A,T,G or C, that differs from the one just used.
  4. DNA made in overlapping fragments (to give 4 copies of each piece of code)

Plain sailing?

One of the first experiments encoded Shakespeare’s sonnets in DNA, which showed that the idea was feasible – what scientists call a ‘proof of principle’. Of course, that’s only a beginning. There are big problems to overcome, like being able to make DNA strands cheaply and quickly enough and to be able to access the data required with the ease we’re used to with hard drives and flash memories. On the flip side, DNA preserved in permafrost has been sequenced from woolly mammoths tens of thousands of years old and from horses entombed for 700,000 years, so we know that as a storage medium it’s rather more durable than anything currently in use.

For the record

The key point here is that, at the moment, DNA appears to be the only option if we are not to grind to a halt on the information storage front. Regardless of solving the problems involved, that alone gives a new perspective to the coding power of those four little bases, A, C, G and T.

References

Extance, A. (2016). How DNA could store all the world’s data. Nature 537, 22–24.

Goldman, N. et al. (2013). Nature 494, 77-80.

Orlando, L. et al. (2013). Nature 499, 74-78.

Long-live the Revolutions!!

There’s a general view that most folk don’t know much about science and, because almost day by day, science plays a more prominent role in our lives, that’s considered to be a Bad Thing. Us scientists are therefore always being told to get off our backsides and spread the word – and I try to do my bit in Betrayed by Nature, in Secret of Life (a new book shortly to be published) and in these follow-up blogs.

We may be making some progress – and, I have to admit, television has probably done more than me – though I am available (t.v. & movie head honchos please note). As one piece of evidence you could cite the way ‘DNA’ has become part of the universal lexicon, albeit often nonsensically. As evidence I call Sony Corp. Chief Executive Kazuo Hirai, as reported in The Wall Street Journal: “I’ve said this from day one. Some things at Sony are literally written into our DNA …”

Well, of course, that’s gibberish Kazuo old bean – but we know what you mean. Or do we? Most probably couldn’t tell you what the acronym stands for – but that doesn’t matter if they can explain that it’s the stuff (a ‘molecule’ would be better still!) that carries the information of inheritance and, as such, is responsible for all life. Go to the top of the class those who add that the code is in the form of chemicals called bases and there are just four of them (A, C, G & T). Something that simple doesn’t seem enough for all life but the secret is lies in the vast lengths of DNA involved. The human genome, for example, is made up of three billion letters.

A little bit of what is now history …

In the mid-1980s a number of scientists from around the world began to talk about the possibility of working out the sequence of letters that make up human DNA and thus identifying and mapping all the genes encoded by the human genome. From this emerged The Human Genome Project, a massive international collaboration, conceived in 1984 and completed in 2003. I quite often refer to this achievement as the ‘Greatest Revolution’ – meaning the biggest technical advance in the history of biology.

As that fantastic enterprise steadily advanced to its triumphant conclusion, it was accompanied by a series of mini-revolutions in technology that sky-rocketed the speed of sequencing and slashed the cost – the combined effect being an increase the efficiency of the whole process of more than 100 million-fold.

Brings us to the present …

These quite astonishing developments have continued since 2003 such that by 2009 it was possible to sequence 12 individuals in one study. By August 2016 groups from all over the world, coming together under the banner of The Exome Aggregation Consortium (ExAC), have raised the stakes 5,000-fold by sequencing no fewer than 60,706 individuals.

The name of the outfit tells you that there’s what you might think of as a very small swizz here: they didn’t sequence all the DNA, just the regions that code for proteins (exomes) – only about 1% of the three billion letters. But what highlights the power of current methods is not only the huge number of individuals sequenced but the depth of coverage – that is, the number of times each base (letter) in each individual exome was sequenced. In effect, it’s doing the same experiment so many times that errors are eliminated. Thus even genetic variants in just one person can be picked out.

seq-pic

Sequence variants between individuals. For most proteins the stretches of genomic DNA that encode their sequence  are split into regions called exons. All the expressed genes in a genome make up the exomeBy repeated sequencing The Exome Aggregation Consortium have shown that genetic variants in even one person can be reliably identified. Variants from the normal sequence found in four people are shown in red, bold letters.

It turns out that there are about 7.5 million variants and they pop up remarkably often – at one in every eight sites (bases). About half only occur once (which illustrates why DNA fingerprinting, aka DNA profiling, is so sensitive). As Jay Shendure put it, this gives us a “glimpse of the bottom of the well of genetic variation in humans.”

One of the major results of this study is that, by filtering out common variants from those associated with specific diseases, it will help to pin down the causes of Mendelian diseases (i.e. genetic disorders caused by change or alteration in a single gene, e.g., cystic fibrosis, haemophilia, sickle-cell anaemia, phenylketonuria). It’s clear that, over the next ten years, tens of millions of human genomes will be sequenced which will reveal the underlying causes of the thousands of genetic disorders.

The prize … and the puzzle

The technology is breathtaking, the amount of information being accumulated beyond comprehension. Needless to say, private enterprise has leapt on the bandwagon and you can now get your genome sequenced by, for example, 23andMe who offer “a personalised DNA service providing information and tools for individuals to learn about and explore their DNA. Find out if you are at risk for passing on an inherited condition, who you’re related to etc.” All for a mere $199!!

But you could say that the endpoint – the reason for grappling with DNA in the first place – is easy to see: eventually we will be able to define the molecular drivers of all genetic diseases and from that will follow ever improving methods of treatment and prevention.

Nevertheless, in that wonderful world I suspect we will still find ourselves brought up short by the underlying question: how one earth does DNA manage to carry the information necessary for all life?

For those who like to ponder such things, in the next piece we’ll try to help by looking at DNA from a different angle.

References

Ng, SB. et al. (2009). Nature 461, 272-276.

Lek, M. et al. (2016). Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291.

Re-writing the Manual of Life

A little while ago we talked about a fantastic triumph by a team at Great Ormond Street Hospital (Gosh! Wonderful GOSH) in using a form of immunotherapy to save a little girl. What they did was to take the T cells from a sample of her blood and use gene editing – molecular cutting and pasting – to remove some genes and add others before growing more of the cells and then putting them back into the patient.

Gene editing – genetic engineering that removes or inserts sections of DNA – uses engineered nucleases, enzymes that snip DNA but do so in a controlled way by homing in on a specific site (i.e. a defined sequence of As, Cs, Gs and Ts).

We mentioned that there are four main ways of doing this kind of engineering – the GOSH group used ‘transcription activator-like effectors’ (TALEs). However, the method that has made the biggest headlines is called CRISPR/Cas, and it has been very much in the news because a legal battle is underway to determine who did what in its development and who, therefore, will be first in line for a Nobel Prize.

Fortunately we can ignore such base pursuits and look instead at where this technology might be taking us.

What is CRISPR/Cas?

CRISPRs (pronounced crispers) are bits of DNA that contain short repetitions of base sequence, each next to a ‘spacer’ sequence. The spacers have accumulated in bacteria as a defence mechanism – they’re part of the bacterial immune system – and they’re identical to sequences found in viruses that infect microbes. In other words, the cunning bugs pick up bits of dangerous viruses to make a rogues gallery so they can recognize and attack those viruses next time they pop in.

Close to CRISPR sit genes encoding Cas proteins (enzymes that cut DNA, so they’re ‘nucleases’). When the CRISPR-spacer DNA is read by the machinery of the cell to make RNA, the spacer regions stick to Cas proteins and the whole complex, including the viral sequences, can roam the cell seeking a virus with genetic material that matches the CRISPR RNA. The CRISPR RNA sticks to the virus and Cas chops its DNA – end of virus. So Cas, by binding to CRISPR RNA, becomes an RNA-guided DNA cutter.

crispr-pic

CRISPR-CAS: Bug defence against invaders. Viruses can attack bacteria just as they can human cells. Over time bugs have evolved a cunning defence strategy: they insert short bits of viral DNA into their own genome (above). These contain repeated sequences of bases and each is followed by short segments of ‘spacer DNA’ (above). This happens next to DNA that encodes Cas proteins so that both are ‘read’ to make RNA (transcription). Cas proteins bind to spacer RNA, leaving the adjacent viral RNA free to attach to any complementary viral DNA it encounters. The Cas enzyme is thus guided to DNA that it can cleave. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.

Why is CRISPR/Cas in the headlines?

We saw in Gosh! Wonderful GOSH how the Great Ormond Street Hospital team tinkered with DNA and in Self Help – Part 2 we summarized another way of doing this using viruses (notably a disabled form of the human immunodeficiency virus) to carry novel genes into cells.

A further arm of immunotherapy attempts to reverse an effect called checkpoint blockade whereby the immune system response to tumours is damped down – e.g. by using antibodies that target a protein called PD-1 (Self Help – Part 1).

Now comes news of a Chinese trial which will be the first time cells modified using CRISPR–Cas9 gene editing have been injected into people. The chap in charge is Lu You from Sichuan University’s West China Hospital in Chengdu and the plan is to take T cells from the blood patients with metastatic non-small cell lung cancer for whom chemotherapy, radiation therapy and other treatments have failed.

The target will be the PD-1 gene, the idea being that, if you want to stop PD-1 doing its stuff, far better than mucking about with antibodies is to just knock out its gene: no gene no protein! What could possibly go wrong?

Well, wonderful though CRISPR is, it doesn’t always hit the right target but in this trial the cells can be tested to make sure it’s the PD-1 gene that’s been zonked – so that shouldn’t be a problem. However, it’s a blockbuster in that all the multiplied T cells put back into the patient will be active – i.e. will have lost the PD-1 brake. Whilst that may be good for zonking tumours, goodness knows what it might do elsewhere.

The initial trial is on a small scale – just 10 people. If there are problems one possibility is to try to take the T cells from the site of the tumour, which might select those specifically targeting the tumour – not straightforward as lung cancers are difficult to get at.

Anyone for a DNA upgrade?

It’s hard to say where all this is leading. However, as Chinese scientists have already made the first CRISPR-edited human embryos and the first CRISPR-edited monkeys, the only safe bet is that China will be to the fore.

 

The Shocking Effect of Boiled Bugs

There’s never a dull moment in science – well, not many – and at the moment no field is fizzing more than immunotherapy. Just the other day in Outsourcing the Immune Response we talked about the astonishing finding that cells from healthy people could be used to boost the immune response – a variant on the idea of taking from patients cells that attack cancers, growing them in the lab and using genetic engineering to increase potency (generally called adoptive cell therapy).

A general prod

Just when you thought that was as smart as it could get, along comes Angus Dalgleish and chums from various centres in the UK and Spain with yet another way to give the immune system a shock. They used microorganisms (i.e. bugs) as a tweaker. The idea is that bacteria (that have been heat-killed) are injected, they interact with the host’s immune system and, by altering the proteins expressed on immune cells (macrophages, natural killer cells and T cells) can boost the immune response. That in turn can act to kill tumour cells. It’s a general ‘immunomodulatory’ effect. Dalgleish describes it as “rather like depth-charging the immune system which has been sent to sleep”. Well, giving it a prod at least.

bugs-pic

Inactivating bugs (bacteria) and waking up the immune system.

And a promising effect

The Anglo-Spanish effort used IMM-101 (a heat-killed suspension of a bacterium called Mycobacterium obuense) injected under the skin, which has no significant side-effects. The trial was carried out in patients with advanced pancreatic cancer, a disease with dismal prognosis, and IMM-101 immunotherapy was combined with the standard chemotherapy drug (gemcitabine). IMM-101increased survival from a median of 4.4 months to 7 months with some patients living for more than a year and one for nearly three years.

Although the trial numbers are small as yet, this is a very exciting advance because it looks as though immunotherapy may be able to control one of the most serious of cancers in which its incidence nearly matches its mortality.

References

Dalgleish, A. et al. (2016). Randomised, open-label, phase II study of gemcitabine with and without IMM-101 for advanced pancreatic cancer. British Journal of Cancer doi: 10.1038/bjc.2016.271.

 

Outsourcing the Immune Response

We’re very trendy in these pages, for no other reason than that the idea is to keep up to date with exciting events in cancer biology. Accordingly, we have recently talked quite a lot about the emerging field of cancer immunotherapy – the notion that our in-built immune system will try to kill cancer cells as they emerge, because it ‘sees’ them as being to some extent ‘foreign’, but that when tumours make their presence known it has not been able to do the job completely. The idea of immunotherapy is to give our in-house system a helping hand and we’ve seen some of the approaches in Self Help – Part 2 and Gosh! Wonderful GOSH.

The immune see-saw

Our immune system walks a tight-rope: on the one hand it should attack and eliminate any ‘foreign’ cells it sees (so that we aren’t killed by infections) but, on the other, if it’s too efficient it will start destroying out own cells (which is what happens in auto-immune diseases such as Graves disease (overactive thyroid gland) and rheumatoid arthritis.

Like much of our biology, then, it’s a tug-of-war: to kill or to ignore? And, like the cell cycle that determines whether a cell should grow and divide to make two cells, it’s controlled by the balance between ‘accelerators’ and ‘brakes’. The main targets for anti-tumour immune activity are mutated proteins that appear on the surface of cancer cells – called neo-antigens (see The Shape of Things to Come?)

The aim of immunotherapy then is to boost tumour responses by disabling the ‘brakes’. And it’s had some startling successes with patients going into long-term remission. So the basic idea works but there’s a problem: generally immunotherapy doesn’t work and, so far, in only about one in ten of patients have there been significant effects.

Sub-contracting to soup-up detection

Until now it’s seemed that only a very small fraction of expressed neo-antigens (less than 1%) can turn on an immune response in cancer patients. In an exciting new take on this problem, a team of researchers from the universities of Oslo and Copenhagen have asked: “if someone’s immune cells aren’t up to recognizing and fighting their tumours (i.e. ‘seeing’ neo-antigens), could someone else’s help?” It turns out that many more than 1 in 100 neo-antigens are able to cause an immune response. Even more exciting (and surprising), immune cells (T cells) from healthy donors can react to these neo-antigens and, in vitro at least (i.e. in cells grown in the laboratory), can kill tumour cells.

118. pic

Genetic modification of blood lymphocytes

T cells are isolated from a blood sample and novel genes inserted into their DNA. The engineered T cells are expanded and then infused into the patient. In the latest development T cells from healthy donors are screened for reactivity against neo-antigens expressed in a patient’s melanoma. T cell receptors that  recognise neo-antigens are sequenced and then transferred to the patient’s T cells.

How does that work?

T cells (lymphocytes) circulating in the blood act, in effect, as scouts, scanning the surface of all cells, including cancer cells, for the presence of any protein fragments on their surface that should not be there. The first contact with such foreign protein fragments switches on a process called priming that ultimately enables T cells to kill the aberrant cells (see Invisible Army Rouses Home Guard).

What the Scandinavian group did was to screen healthy individuals for tissue compatibility with a group of cancer patients. They then identified a set of 57 neo-antigens from three melanoma patients and showed that 11 of the 57 could stimulate responses in T cells from the healthy donors (T cells from the patients only reacted to two neo-antigens). Indeed the neo-antigen-specific T cells from healthy donors could kill melanoma cells carrying the corresponding mutated protein.

What can possibly go wrong?

The obvious question is, of course, how come cells from healthy folk have a broader reactivity to neo-antigens than do the cells of melanoma patients? The answer isn’t clear but presumably either cancers can make T cell priming inefficient or T cells become tolerant to tumours (i.e. they see them as ‘self’ rather than ‘non-self’).

And the future?

The more critical question is whether the problem can be short-circuited and Erlend Strønen and friends set about this by showing that T cell receptors in donor cells that recognize neo-antigens can be sequenced and expressed in the T cells of patients. This offers the possibility of a further type of adoptive cell transfer immunotherapy to the one we described in Gosh! Wonderful GOSH.

https://cancerforall.wordpress.com/2015/11/19/gosh-wonderful-gosh/

As one of the authors, Ton Schumacher, put it “Our findings show that the immune response in cancer patients can be strengthened; there is more on the cancer cells that makes them foreign that we can exploit. One way we consider doing this is finding the right donor T cells to match these neo-antigens. The receptor that is used by these donor T-cells can then be used to genetically modify the patient’s own T cells so these will be able to detect the cancer cells.”

And Johanna Olweus commented that “Our study shows that the principle of outsourcing cancer immunity to a donor is sound. However, more work needs to be done before patients can benefit from this discovery. Thus, we need to find ways to enhance the throughput. We are currently exploring high-throughput methods to identify the neo-antigens that the T cells can “see” on the cancer and isolate the responding cells. But the results showing that we can obtain cancer-specific immunity from the blood of healthy individuals are already very promising.”

References

Strønen, M. Toebes, S. Kelderman, M. M. van Buuren, W. Yang, N. van Rooij, M. Donia, M.-L. Boschen, F. Lund-Johansen, J. Olweus, T. N. Schumacher. Targeting of cancer neoantigens with donor-derived T cell receptor repertoires. Science, 2016.

“Fighting cancer with the help of someone else’s immune cells.” ScienceDaily. ScienceDaily, 19 May 2016.

In the beginning … 

You may have noticed that the American actress Angelina Jolie, who is now employed as a  Special Envoy  for the  United Nations High Commissioner for Refugees, has re-surfaced in the pages of the science media. She first hit the nerdy headlines by announcing in The New York Times that she had had a preventive double mastectomy (in 2013) and a preventive oophorectomy (in 2015).

We described the molecular biology that prompted her actions in A Taxing Inheritance. The essential facts were that she had a family history of breast and ovarian cancer: genetic testing revealed that she carried a mutation in the BRCA1 gene giving her a 87% risk of breast cancer and a 50% chance of getting ovarian cancer.

A star returns

BRCA1 and breast cancer are back in the news as a result of a paper by Jane Visvader, Geoffrey Lindeman and colleagues in Melbourne that asked a very simple question: which type of cell is driven to proliferate abnormally and give rise to a tumour by mutant BRCA1 protein? That is, pre-cancerous breast tissue contains a mixture of cell types: does cancer develop from one in particular –  and, if you blocked proliferation of that type of cell, could you prevent tumours forming?

Simple question but their paper summarises about 10 years of work to come up with a clear answer.

And the villain is …

The mature mammary gland is made up of lots of small sacs (alveoli) lined with cells that produce milk – called luminal cells. Groups of alveoli are known as lobules, linked by ducts that carry milk to the nipple. Most breast cancers start in the lobular or duct cells.

Breast fig copy

Left: Normal breast lobule showing alveoli lined with milk-producing luminal cells connected to duct leading to the nipple. Right: Normal milk sac, non-invasive cancer, invasive cancer.

Things are complicated by there being more than one type of progenitor cell but the Melbourne group were able to show that, in mice carrying mutated BRCA1, one subtype stood out in terms of its cancerous potential. These cells carried a protein on their surface called RANK (which is member of the tumour necrosis factor family). They had gross defects in their DNA repair systems (so they can’t fix genetic damage) and they’re highly proliferative. Luminal progenitors that don’t express RANK behave normally.

Slide1 copy

Scheme representing normal and abnormal cell development. The basic idea is that different types of cells evolve from a common ancestor. The Australian work identified one type of luminal progenitor cell that carries a protein called RANK on its surface (pink cell) as being a prime source of tumours. RANK+ cells have defective DNA repair systems so they accumulate mutations (red cells) more rapidly than normal cells, a feature of tumour cells.

In mice with mutant BRCA1 a monoclonal antibody (denosumab) that blocks RANK signalling markedly slowed tumour development. In a small pilot study blockade of RANK inhibited cell proliferation in breast tissue from human BRCA1-mutation carriers.

Next?

How effective blocking the activation of RANK signalling will be in preventing breast cancer is anyone’s guess but the idea behind the work of the Australian group cannot be faulted. Being able to prevent the ‘starter’ cells from launching themselves on the pathway to cancer driven by mutation in BRCA1 would mean that women in Angelina Jolie’s position would not have to contemplate the drastic course of surgery. The question is: will the preliminary mouse results lead to something that works in humans and, moreover, does so with high efficiency. As ever in cancer, watch this space – but don’t hold your breath!

References

Nolan, E. et al. (2016). RANK ligand as a potential target for breast cancer prevention in BRCA1-mutation carriers.