Holiday Reading (4) – Can We Make Resistance Futile?

For those with a fondness for happy endings we should note that, despite the shortcomings of available drugs, the prospects for patients with a range of cancers have increased significantly over the latter part of the twentieth century. The overall 5-year survival rate for white Americans diagnosed between 1996 and 2004 with breast cancer was 91%; for prostate cancer and non-Hodgkin’s lymphoma the figures were 99% and 66%, respectively. These figures are part of a long-term trend of increasingly effective cancer treatment and there is no doubt that the advances in chemotherapy summarised in the earlier Holiday Readings are contributory factor. Nonetheless, the precise contribution of drug treatments remains controversial and impossible to disentangle quantitatively from other significant factors, notably earlier detection and improved surgical and radiotherapeutic methods.

Peering into the future there is no question that the gradual introduction of new anti-cancer drugs will continue and that those coming into use will be more specific and therefore less unpleasant to use. By developing combinations of drugs that can simultaneously poke the blancmange at several points it may be possible to confront tumor cells with a multiple challenge that even their nimbleness can’t evade, thereby eliminating the problem of drug resistance. Perhaps, therefore, in 20 years time we will have a drug cabinet sufficiently well stocked with cocktails that the major cancers can be tackled at key stages in their evolution, as defined by their genetic signature.

However, on the cautionary side we should note that in the limited number of studies thus far the effect of drug combinations on remission times has not been startling, being measured in months rather than years or decades. Having noted the durability of cancer cells we should not be surprised by this and the concern, of course, is that, however ingenious our efforts to develop drug cocktails, we may always come second to the adaptability of nature.

Equally perturbing is the fact that over 90% of cancer deaths arise from primary tumors spreading to other sites around the body. For this phenomenon, called metastasis, there are currently very few treatment options available and the magnitude of this problem is reflected in the fact that for metastatic breast cancer there has been little change in the survival rates over the past forty years.

Metastasis therefore remains one of the key cancer challenges. It’s over 125 years since the London physician Stepen Paget asked the critical question: ‘What is it that allows tumour cells to spread around the body?’ and it’s a daunting fact that only very recently have we made much progress towards an answer – and thus perhaps a way of controlling it. To the fore in this pursuit has been David Lyden and his colleagues at Weill Cornell Medical College in New York. Their most astonishing finding is that cells in the primary tumour release messengers into the circulation and these, in effect, tag what will become landing points for wandering cells. Astonishing because it means that these sites are determined before any tumour cells actually set foot outside the confines of the primary tumour. Lyden has christened this ‘bookmarking’ cancer. That is a quite remarkable finding – but, as ever in science, it merely shifts the question to ‘OK but what’s the messenger?’

A ray of sunshine

It might appear somewhat churlish, especially after all that funding, to end on a note of defeatist gloom so let’s finish with my ray of sunshine that represents a radical approach to the problem. It relies on the fact that small numbers of cells break away from tumors and pass into the circulation. In addition, tumours can release both DNA and small sacs – like little cells – that contain DNA, proteins and RNAs (nucleic acids closely related to DNA). These small, secreted vesicles are called exosomes – a special form of messengers, communicating with other cells by fusing to them. By transferring molecules between cells, exosomes may play a role in mediating the immune response and they are now recognized as key regulators of tumour growth and metastasis.

Step forward Lyden and friends once more who have just shown in a mouse model of pancreatic cancer that exosomes found their way to the liver during the tumour’s earliest stages. Exosomes are taken up by some of the liver cells and this sets off a chain of cell-to-cell signals that eventually cause the accumulation of a kind of molecular glue (fibronectin). This is the critical ingredient in a microenvironment that attracts tumour cells and promotes their growth as a metastasis (secondary growth). So you can think of exosomes as a kind of environmental educator.

Exosome Fig

Exosomes released from primary tumours can mark a niche for metastasis.

The small sacs of goodies called exosomes are carried to the liver where they fuse with some cells, setting off a chain reaction that produces a sticky protein – fibronectin – a kind of glue for immune cells and tumour cells. (from Costa-Silva, B., Lyden, D. et al., Nature Cell Biology 17, 816–826, 2015).

The recent, remarkable technical advances that permit the isolation of exosomes also make it possible to fish out circulating tumour cells and tumour DNA from a mere teaspoonful of blood.

Circulating tumour cells have already been used to monitor patient responses to chemotherapy – when a treatment works the numbers drop: a gradual rise is the earliest indicator of the treatment failing. Even more exciting, this approach offers the possibility of detecting the presence of cancers years, perhaps decades, earlier than can presently be achieved. Coupling this to the capacity to sequence the DNA of the isolated cells to yield a genetic signature of the individual tumor can provide the basis for drug treatment. There are still considerable reservations attached to this approach but if it does drastically shift the stage at which we can detect tumors it may also transpire that their more naïve forms, in which fewer mutations have accumulated, are more susceptible to inhibitory drugs. If that were to be the case then even our currently rather bare, though slowly expanding, drug cabinet may turn out to be quite powerful.

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Holiday Reading (3) – Stopping the Juggernaut

The mutations that drive cancers fall into two major groups: those that reduce or eliminate the activity of affected proteins and those that have the opposite effect and render the protein abnormally active. It’s intuitively easy to see how the latter work: if a protein (or more than one) in a pathway that tells cells to proliferate becomes more efficient the process is accelerated. Less obvious is how losing an activity might have a similar effect but this comes about because the process by which one cell becomes two (called the cell cycle) is controlled by both positive and negative factors (accelerators and brakes if you will). This concept of a balancing act – signals pulling in opposite directions – is a common theme in biology. In the complex and ever changing environment of a cell the pressure to reproduce is balanced by cues that ask crucial questions. Are there sufficient nutrients available to support growth? Is the DNA undamaged, i.e. in a fit state to be replicated? If the answer to any of these questions is ‘no’ the cell cycle machinery applies the brakes, so that operations are suspended until circumstances change. The loss of negative regulators releases a critical restraint so that cells have a green light to divide even when they should not – a recipe for cancer.

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The cell cycle.

Cells are stimulated by growth factors to leave a quiescent state (G0) and enter the cell cycle – two growth phases (G1 & G2), S phase where DNA is duplicated and mitosis (M) in which the cells divide to give to identical daughter cells. Checkpoints can arrest progression if, for example, DNA is damaged. 

We’re all familiar with this kind of message tug-of-war at the level of the whole animal. We’ve eaten a cream cake and the schoolboy within is saying ‘go on, have another’ whilst the voice of wisdom is whispering ‘if you go on for long enough you’ll end up spherical.’

Because loss of key negative regulators occurs in almost all cancers it is a high priority to find ways of replacing inactivated or lost genes. Thus far, however, successful ‘gene therapy’ approaches have not been forthcoming with perhaps the exception of the emerging field of immunotherapy. The aim here is to boost the activity of the immune system of an individual – in other words to give an innate anti-cancer defense a helping hand. The immune system can affect solid cancers through what’s become known as the tumour microenvironment – the variety of cells and messengers that flock to the site of the abnormal growth. We’ve referred to these as ‘groupies’ and they include white blood cells. They’re drawn to the scene of the crime by chemical signals released by the tumour – the initial aim being to liquidate the intruder (i.e. tumour cells). However, if this fails, a two-way communication sees would-be killers converted to avid supporters that are essential for cancer development and spread.

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The tumour microenvironment. Tumour cells release chemical messengers that attract other types of cell, in particular those that mediate the immune response. If the cancer cells are not eliminated a two-way signaling system is established that helps tumour development.

There is much optimism that this will evolve into a really effective therapy but it is too early for unreserved confidence.

The obstacle of reversing mutations that eliminate the function of a gene has led to the current position in which practically all anti-cancer agents in use are inhibitors, that is, they block the activity of a protein (or proteins) resulting in the arrest of cell proliferation – which may ultimately lead to cell death. Almost all these drugs are not specific for tumour cells: they hit some component of the cell replication machinery and will block division in any cell they reach – which is why so many give rise to the side-effects notoriously associated with cancer chemotherapy. For example, the taxanes – widely used in this context – stick to protein cables to prevent them from pulling duplicated DNA strands apart so that cells, in effect, become frozen in final stages of division. Other classes of agent target different aspects of the cell cycle.

It is somewhat surprising that non-tumour specific agents work as well as they do but their obvious shortcomings have provided a major incentive for the development of ‘specific’ drugs – meaning ones that hit only tumour cells and leave normal tissue alone. Several of these have come into use over the past 15 years and more are in various stages of clinical trials. They’re specific because they knock out the activity of mutant proteins that are made only in tumour cells. Notable examples are Zelboraf® manufactured by Roche (hits the mutated form of a cell messenger – called BRAF – that drives a high proportion of malignant melanomas) and Gleevec® (Novartis AG: blocks a hybrid protein – BCR-ABL – that is usually formed in a type of leukemia).

These ‘targeted therapies’ are designed to not so much to poke the blancmange as to zap it by knocking out the activity of critical mutant proteins that are the product of cancer evolution. And they have produced spectacular remissions. However, in common with all other anti-cancer drugs, they suffer from the shortcoming that, almost inevitably, tumours develop resistance to their effects and the disease re-surfaces. The most remarkable and distressing aspect of drug resistance is that it commonly occurs on a timescale of months.

And being outwitted

Tumour cells use two tactics to neutralize anything thrown at them before it can neutralize them. One is to treat the agent as garbage and activate proteins in the cell membrane that pump it out. That’s pretty smart but what’s really staggering is the flexibility cells show in adapting their signal pathways to counter the effect of a drug blocking a specific target. Just about any feat of molecular gymnastics that you can imagine has been shown to occur, ranging from switching to other pathways in the signalling network to short-circuit the block, to evolving secondary mutations in the target mutant protein so that the drug can no longer stick to it. Launching specific drugs at cells may give them a mighty poke in a particularly tender spot, and indeed many cells may die as a result, but almost inevitably some survive. The blancmange shakes itself, comes up with a counter and gets down to business again. This quite extraordinary resilience of tumour cells derives from the infinite adaptability of the genome and we might do well to reflect that in trying to come up with anti-cancer drugs we are taking on an adversary that has overcome the challenges involved in generating the umpteen million species to have emerged during the earth’s lifetime.

Not the least disheartening aspect of this scenario is that when tumours recur after an initial drug treatment they are often more efficient at propagating themselves, i.e. more aggressive, than their precursors.

Mission Impossible?

We make great play in these pages of the wonders of the genetic revolution. So we should. The technology is simply breathtaking, and the amount of data we can gather is so incomprehensibly vast the latest generation of computers is straining at the seams to record it all and, of course, it unveils the vision of a new world. No field has felt the impact more than cancer biology which now holds the promise that, shortly after being found, tumors will be sequenced: on the basis of identified ‘driver’ mutations appropriate drug cocktails will be devised to prevent remission after the initial treatment and these can even be tested in mouse ‘avatars’ to confirm their effectiveness against the patient’s own tumor cells. Finally, even if recurrence sets in at a later date, the same procedure can be repeated and a new drug combo used to target any evolution undergone by the cancer. The era of ‘personalized medicine’ has arrived.

Every Silver Lining …

But there are a few murky clouds drifting across this sky blue portrait of triumph.

  1. The first is that, as we’ve seen in Family Tree of Breast Cancer and Molecular Mosaics, cancers are an incredible mixture – that is, the mutation signature varies depending on the region sampled in primary tumors and is different for individual metastases. This means that a ‘signature’ at best represents a dominant hand of mutations and, worse still, it’s continuously evolving.
  2. The second problem is that, although there are several hundred ‘anti-cancer’ drugs that have been approved for use by the FDA against specific types or stages of cancer, fewer than half a dozen are ‘specific’ – meaning that they hit only tumor cells and leave normal tissue alone. The ‘few’ work because they knock out the activity of mutant proteins that are made only in tumor cells. Notable examples are vemurafinib/Zelboraf (hits the mutated form of BRAF that drives a high proportion of malignant melanomas) and imatinib/Gleevec (blocks the BCR-ABL protein that is formed in most chronic myelogenous leukemias) – and these ‘targetted therapies’ have produced spectacular remissions. Other agents that have attracted much media attention include Herceptin (trastuzumab), a monoclonal antibody that sticks to a protein present in large amounts on the surface of some types of breast cancer cell. This type of agent is highly specific for the protein it targets (i.e. it doesn’t interact with anything else) but it isn’t specific for cancer cells per se. They work because cells heavily loaded with the target get a relatively big hit – a kind of tall poppy syndrome.
  3. Virtually all other chemo agents work on the same principle: in essence they affect every cell they manage to reach and any anti-cancer effect is due to tumor cells being a bit more susceptible. Which is why, of course, the efficacy of any drug combo is to a considerable extent a matter of luck and side effects are such a common problem.
  4. Unquestionably more anti-cancer drugs will be developed, those that do come on line will be more specific and therefore less unpleasant to use, so it may well be that in 20 years time we will have a drug cabinet that is sufficiently well stocked to tackle the major cancers at key stages in their evolution. Which is all well and good but, regardless of how they work and what is meant by ‘specificity’, the biggest problem of all will remain. Resistance – the capacity of tumor cells to neutralize anything that is used with the idea of neutralizing them. They do this by two main routes (1) pumping out the drug and (2) adapting to reduce drug efficacy. The obvious counter is simply to throw more of the drug at them but, in the end, side-effects impose a limit. What this means is that even when drugs have initially startling effects, as do vemurafinib and imatinib, patients eventually become refractive and tumors recur.

MAPK

Cell signalling: cells receive many signals from messengers that attach to receptor proteins spanning the outer membrane. Activated receptors turn on relays of proteins (RAS, A, B, C, D) that talk to the nucleus, switching on genes that drive proliferation. RAS proteins are a focus for many incoming signals and they also set off several relay chains that converge on the nucleus. They work at the cell membrane to which they are escorted from where they’re made by a protein called PDEdelta. A new drug, deltarasin, blocks the escort’s action so that RAS cannot find its way to work and cell growth is arrested.

A Different Line of Attack

In view of that rather gloomy assessment should we try an alternative approach? The personalized scenario involves drug combos tailored to the individual cancer at a given stage of development. But if that seems unlikely to provide a solution remotely near to the ideal, is there another way of selecting targets? Time to try ‘impersonalized medicine’ perhaps?

This notion comes from the thought that what we’re trying to do is block signals that release the brakes on cell proliferation. Many distinct signal pathways impact on the machinery that drives this process, themselves driven by different types of external signal, but it would seem obvious that somewhere along the line these must converge on one or two key regulators – master controllers if you like of cell multiplication. Indeed they do and one of these foci is a protein called RAS (there are three close relatives in the RAS family). RAS is a major junction in cell signalling: many messages from the outside world eventually converge on RAS and lots of pathways radiate from it. When a cell launches itself into the division cycle it does so as an integrated response to these signals.

RAS is mutated to a hyperactive form in about 20% of human cancers (turning on cell growth) so obviously it would be good to have a drug that can hit RAS and an enormous amount of effort has gone into coming up with one. Unfortunately a variety of clever strategies aimed directly at RAS proteins simply haven’t worked. Enter Gunther Zimmermann and his team.

Inhibiting RAS Signalling

RAS proteins do their signaling attached to the inside of the outer membrane of the cell – but they’re made in the interior and to get to their place of work they are escorted to the membrane by a protein called PDEδ (a phosphodiesterase). To upset this cosy arrangement, the Dortmund group developed small molecule, deltarasin, that sticks tightly to the escort which, in response, changes shape just enough to prevent it being able to hold hands  with RAS. The result is that the key signaller (KRAS in fact) is no longer distributed to the membrane. This prevents it working and impairs the growth of KRAS-mutant pancreatic tumour cells.

The great attraction of this approach is that it’s indirect – so the hope is that cells won’t realize that RAS is wandering aimlessly around doing nothing and therefore not simply overwhelm the drug by making more mutant RAS. It remains to be seen how many off-target effects this drug has but for the moment an exciting new idea holds the promise of hitting cancers where it hurts them most – in a key node essential for unregulated cell growth.

References

Baker, N.M. and Der, C.J. (2013). Cancer: Drug for an ‘undruggable’ protein. Nature 497, 577–578.

Zimmermann, G., Papke, B., Ismail, S., Vartak, N., Chandra, A., Hoffmann, M., Hahn, S.A., Triola, G., Wittinghofer, A., Bastiaens, P.I.H. and Waldmann, H. (2013). Small molecule inhibition of the KRAS–PDEδ interaction impairs oncogenic KRAS signaling. Nature 497, 638–642.

Signs of Resistance

In Beware of Greeks … we noted that in one sort of leukemia at least, tumour cells have come up with an extraordinary way of escaping from the bone marrow where they start life into the circulation where they cause trouble – by releasing pieces of their own DNA that then break down the retaining barrier.

Keeping track of tumors

Curious behaviour though it may be, there’s nothing new about the idea of cells shedding bits of their genetic code – that was first shown to happen over 60 years ago. What is novel is the evidence that not only does this happen in a variety of cancer cells but that modern methods enable those fragments to be isolated from just a teaspoonful of blood: the sequence of the DNA can then be determined – which gives the mutational signature of the original tumour. A remarkable development has now shown that repeating these steps over a period of time can reveal the response of secondary tumours (metastases) to drug treatment (chemotherapy).

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One great advantage of this blood sampling method is that it is as near as makes no difference ‘non-invasive’. That is, it uses only a (small) blood sample and there’s no need for painful excavations to dig out tumour samples. The study, largely funded by Cancer Research UK, looked at three major cancers (breast, ovarian and lung) and identified specific mutations caused by drugs over a period of one to two years. For good measure they also took tumour samples to show that the mutation patterns found in circulating DNA did indeed represent what had gone on in the tumour itself. In other words, they had established what scientists like to call ‘proof of principle’ – i.e. we can do it!

There’s another more subtle advantage of this approach in that it gets round a problem we described in Molecular Mosaics: tumours are a mixture and the mutational signature differs depending on which bit you sample and sequence. The cell-free DNA fragments collected from blood are a gemisch – an averaged signature if you like – that may therefore give a better picture of the target for drug cocktails at any given time during tumour evolution.

Why is this so important?

There are two main reasons why it’s difficult to exaggerate the potential important of this step. The first is that metastasis accounts for over 90% of cancer deaths, the second that the fiendish ingenuity with which tumours negate chemotherapy, i.e. develop drug resistance, is one of the biggest challenges to successful treatment. So, the sooner changes that enable tumours to become insensitive to drugs can be detected the better in terms of adjusting the treatment regime. Even more exciting, however, is that notion that the DNA shed by cancers into the circulation may permit detection years or even decades earlier than is possible with any of the current methods (e.g., mammography) – with screening being carried put routinely from blood samples. Being even more optimistic, very early stage tumours may be particularly susceptible to appropriate drug combos, so that we might look forward to the day when chemotherapy replaces surgery as the first line of treatment for most cancers.

Reference

Murtaza, M. et al., (2013). Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497, 108–112.