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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Self Help – Part 2

In the second type of cancer immunotherapy a sample of a patient’s T lymphocytes is grown in the lab. This permits either expansion of the number of cells that recognize the tumour or genetic engineering to modify the cells so they express receptors on their surface that target them to the tumour cell surface. Infusion of these manipulated cells into the patient enhances tumour cell killing. We’re now in the realms of ‘personalized medicine’.

A little more of a good thing

The first of these methods picks up a weakness in the patient’s immune system whereby it makes lymphocytes that kill tumour cells but can’t make enough – their protective effect is overwhelmed by the growing cancer. By taking small pieces of surgically removed tumours and growing them in the lab, it’s possible to select those T cells that have killing capacity. These are expanded over a few weeks to make enough cells to keep on growing when they’re infused back into the patient. The upshot is a hefty boost for the natural anti-tumour defence system. The pioneer of this method, called adoptive cell therapy, is Steven Rosenberg (National Cancer Institute, Bethesda) and it has been particularly effective for melanomas. Responses are substantially improved by treatment with drugs that reduce the white cell count before samples are taken for T cell selection – probably because the system responds by making growth factors to restore the balance and these drive the expansion of the infused cells.

A wonderful benefit of this method is its efficacy against metastases – i.e. tumour growths that have spread from the primary site – perhaps not surprising as it’s what Rosenberg calls a “living” treatment, in other words it just gives a helping hand to what nature is already trying to do.

93. Fig. 1Selecting naturally occurring T cells with anti-tumour activity

Tumour fragments are grown in the laboratory: lymphocytes that kill tumour cells are selected and expanded in culture.  About 6 weeks growth yields enough cells to infuse into the patient.

Gene therapy

A more sophisticated approach to boosting innate immunity is to introduce new genes into the genetic material (the genome) of T cells to target them to tumour cells with greater efficiency. An ordinary blood sample suffices as a starting point from which T cells are isolated. One way of getting them to take up novel genes uses viruses – essentially just genetic material wrapped in an envelope. The virus is ‘disabled’ so that it has none of its original disease-causing capacity but retains infectivity – it sticks to cells. ‘Disabling’ means taking just enough of the original genome to make the virus – a viral skeleton – and then inserting your favourite gene, so the engineered form is just a handy vehicle for carrying genes. No need to panic, therefore, if you see a press headline of the “HIV cures cancer” variety: it just means that the human immunodeficiency virus – well and truly disabled – has been used as the gene carrier.

93. Fig. 2

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.

 This method of re-directing T cells to a desired target was pioneered by Gideon Gross and colleagues at The Weizmann Institute of Science in Israel in the late 1980s and it has led to sensational recent results in treating chronic lymphocytic leukemia (CLL), albeit in just a few patients so far. To the fore have been Renier Brentjens and his group from the Memorial Sloan-Kettering Cancer Center, New York. The genetic modification they used made the patient’s T cells express an artificial receptor on their surface (called a chimeric antigen receptor). This T cell receptor was designed to stick specifically to a protein known to be displayed on the surface of CLL cells. The result was that the T cells, originally unable to ‘see’ the leukemic cells, now homed in on them with high efficiency. Astonishingly, and wonderfully, the modified cells divide in the patient so that, in effect, their immune system has been permanently super-charged.

A critical part of the strategy is that CLL cells carry a known molecular target but the absence of such defined markers for most cancers is currently a severe limitation. On the bright side, however, this type of gene therapy has now been attempted in at least three different centres and, despite inevitable minor differences in method, it clearly works.

One of the leading figures in gene therapy is Carl June of the University of Pennsylvania. Some of his colleagues have made a brilliant video explaining how it works whilst June himself has described in wonderfully humble fashion what it means to work in this field.

References

Rosenberg, S.A. and Restifo, N.P. (2015). Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348, 62-68.

Gross, G., et al. (1989). Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptorswith antibody-type specificity. Proc. Natl. Acad. Sci. U.S.A. 86, 10024–10028.

Brentjens, R.J., et al. (2013). CD19-Targeted T Cells Rapidly Induce Molecular Remissions in Adults with Chemotherapy-Refractory Acute Lymphoblastic Leukemia. Sci Transl Med., 5, 177ra38. DOI:10.1126/scitranslmed.3005930.

Kalos, M., et al. (2011). T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci. Transl. Med. 3, 95ra73.

Kochenderfer, J.N., et al. (2012). B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor–transduced T cells. Blood 119, 2709–2720.