Feature: Rush of blood

Dr Benjamin Kile almost became a lawyer. Yet had he gone ahead and pursued that particular career path, he would have missed out on receiving one of this country’s highest scientific accolades, and we would have missed out on him solving a 100-year-old mystery of a blood platelet’s brief existence and revealing how a known cancer gene plays an essential role in blood cell formation.

After completing a science/law degree and almost becoming a lawyer, Kile threw in his lot with the sciences via an Honours degree working on knockout mice and cell division, and was won over by the world of molecular biology.

However, it was his subsequent PhD with Professors Doug Hilton and Warren Alexander at the Walter and Eliza Hall Institute of Medical Research (WEHI) that introduced him to blood cells and cancer.

Less than a decade after completing his PhD, Kile’s work was recognised with him being named the Science Minister’s Life Scientist of the Year in 2010. Not one to rest on his laurels, Kile has continued on his quest to unravel the life and times of the humble platelet and its family members on several fronts, with his discoveries making a demonstrable difference to drug development.

Dr Emma JosefssonIn particular, together with WEHI colleague Dr Emma Josefsson, he has unravelled another long-standing conundrum in the field: how the platelet’s ‘parent’ cell, the megakaryocyte, brings about its own demise.

After his PhD, Kile took up a postdoc with Monica Justice at Baylor College of Medicine in Houston, one of the major centres of the genome revolution, with arrays of robots pumping out vast amounts of sequence data.

Kile became the blood guy on a huge screen being conducted to find genes regulating just about every biological process in the mouse. “There really was a renaissance in genetics screens going on around that time, and I just learnt everything I could,” he says.

In 2004, Kile brought his Baylor tricks back to WEHI, where he initially set up a simple screen to identify factors regulating platelet production. “Of course, like all good genetic screens, it did exactly the opposite,” says Kile.

“Instead of finding out how a mouse with thrombocytopenia (fewer circulating platelets than normal) might be making less platelets, we stumbled across something that in fact regulates when and how a platelet is destroyed. That was really the Eureka moment… and everything else has gone from there.”

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The platelet story

To put the recent megakaryocyte work with Josefsson in context, we first need to recap on Kile’s prize-winning platelet research. Platelets are tiny anucleate cells in our circulation that are essential for blood clotting, rapidly marshalling their forces at the first sign of vessel damage to form a plug and stop the bleeding. They are also increasingly implicated as important players in many other functions including immunity and the spread of cancer.

Since one of the pioneers of platelet research, William Duke, reported in 1910 that platelets work hard and die young, with them circulating for only around 10 days in humans, people have speculated about the mechanisms regulating platelet survival and lifespan.

The prevailing theory for many years was that unavoidable and progressive external damage to platelets in the blood eventually sends a ‘find and destroy’ message to the body’s immune scavenger cells.

Healthy bone marrow (top) contains many cell types including platelet-producing megakaryocytes (brown). The pro-survival protein Bcl-xL is important for keeping megakaryocytes alive. After chemotherapy (below) many cell types in the bone marrow, including megakaryocytes, are killed through a process requiring the pro-death proteins Bax and Bak. Platelet numbers can be depleted in the blood by chemotherapy because of its toxicity for both platelets and megakaryocytes. (Image: Walter and Eliza Hall Institute)However, together with his WEHI colleague Professor David Huang, Kile showed that something completely unexpected was going on to ensure the platelet’s early exit from the stage. “We showed that platelets have an internal life span ‘timer,’ governed by opposing forces.”

The team’s mouse screen initially turned up a pro-survival protein called Bcl-xL, whose job it was to keep circulating platelets alive. They then discovered that the apoptotic death activator, Bak, was also at work trying to kill the platelets, if not for the restraining actions of Bcl-xL.

“So each platelet has this little molecular egg timer on board, functioning in equilibrium, until eventually the pro-survival protein runs out. This sets off the classical apoptosis pathway, and that cell gets cleared from the circulation.”

This was a landmark finding for Kile’s group, with their 2007 Cell paper gaining international recognition in the fields of both haematology and apoptosis. “Even though it was a surprise, the mechanism actually made a lot of sense,” says Kile.

Because of Kile’s background in haematopoiesis and how platelets are formed, his mind then turned towards the mother of all platelets, the megakaryocyte. “These are the other fascinating component in this whole system. They are massive progenitor cells in the bone marrow with a very complicated cytoplasm that reorganises and then fragments to shed thousands of platelets into the circulation.

“So, just how does the parent cell make all these progeny that carry their own built-in death machine, and does the megakaryocyte have this egg timer mechanism going on as well?”

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Since the late 1990s, the predominant thinking in the field was that to make platelets, megakaryocytes must deliberately undergo a specialised form of apoptosis in which caspases are activated to dismantle the cells and release the platelet offspring. And, according to Kile, there was lots of circumstantial evidence around to support that theory.

For example, mice carrying mutations that impair apoptosis have fewer circulating platelets and megakaryocytes with enhanced survival mechanisms or inhibited caspases are not so good at making platelets.

However, there was a paradox in this theory about megakaryocytes, which became more evident following Kile’s discoveries about the survival mechanisms in platelets. “If megakaryocytes need to go through apoptosis to produce platelets, but platelets have their apoptosis program shut down until it is activated to terminate their life in the circulation, then how do you trigger apoptosis in the parent cell while keeping it turned-off in the progeny?”

Furthermore, the idea of megakaryocytes committing suicide in order to make platelets was inconsistent with the thrombocytopenia observed in response to cellular stresses such as chemotherapy, which appear to kill megakaryocytes via apoptosis, thereby reducing the number of platelets in circulation.

Ripe for discovery

To Kile, this seemed ripe for study, and he knew that together with WEHI colleague and platelet expert, Dr Emma Josefsson, they had the tools and expertise to answer the question.

“We had observed that megakaryocytes express all the bits and pieces needed for the same mechanism we identified in platelets, and the literature supported the idea that these things could be activated, but no one had really had a proper look at it. In the end we did what we do best: delete pieces of the apoptotic pathway genetically and see how the cells respond.”

What Kile and Josefsson found overturned the theory that megakaryocytes must undergo classical apoptosis for platelet production. Instead, these parent cells seem programmed to restrain apoptosis long enough to survive and progress safely through the platelet formation and shedding process.

“We showed that deleting some of the pro-survival proteins in megakaryocytes induces cell death, but doesn’t stimulate platelet production at all. In fact, it does the complete opposite and destroys the megakaryocyte, along with platelet production,” says Kile.

“Equally, deleting the pro-death proteins and effectively knocking out the apoptotic machinery protects the megakaryocyte from insults like chemotherapy, ameliorating the acute loss of platelets.” This work is also helping to pinpoint the pathways chemotherapies might trigger in megakaryocytes and their precursors, which so far have remained elusive.

Kile and Josefsson published some of this work last year in The Journal of Experimental Medicine and this year in Blood. According to Kile, the results are “slowly dismantling this idea in peoples’ minds that megakaryocytes actively undergo apoptosis to produce platelets.

We now think that megakaryocytes function more like their progeny, in that they have this particular pro/anti-death machinery to keep in balance if they are to survive and stay in the business of making platelets.

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“One of the exciting translational implications that has emerged from all of this work relates to how the apoptotic pathway is being exploited in cancer therapy,” says Kile. “Particularly, with new targeted molecules called BH3 mimetics.

“Since the essential role of Bcl-2 family proteins in controlling apoptosis was uncovered in the 1980s, the holy grail has always been to exploit these cell death programs to kill cancer cells, and those drugs are now actually in the clinic,” he says.

The first of these is a compound called ABT-263, which is marketed by Abbott Laboratories and Genentech as Navitoclax. It is designed to specifically inhibit pro-survival proteins, including Bcl-xL, in cancer cells, thereby triggering their apoptotic death. “As it happens, the clinical trials of this drug were just about to start at the time we were figuring out the link between Bcl-xL and platelet lifespan.

“So, we treated mice with a compound structurally related to Navitoclax, and lo and behold, their platelets were wiped out. This fitted neatly with our genetic studies, demonstrating that if you inhibit Bcl-xL, you kill platelets and cause thrombocytopenia, which is often a dose-limiting toxicological side effect of standard chemotherapies.”

This work provided a proof of mechanism for both Kile’s team and the drug companies. It indicated that Navitoclax would likely cause thrombocytopenia in patients – which it did – and that this was an on-target effect of the drug. Subsequently, the clinicians developing Navitoclax have largely managed to work around the platelet effect by altering the dosage schedule etc, and the drug has now progressed into phase II trials.

For Kile, it was gratifying to see such an immediate translational effect of his research. As a basic molecular biologist, with a desire to understand how things work, Kile knows that sometimes one must pluck up the courage to say: “I think it is important to understand the basics of the system, and I am not entirely sure how this is going to translate.”

In his case, it certainly turned out well. Asking some seemingly esoteric questions about megakaryocytes and platelets has proven terrifically informative in terms of biochemical pathways and physiological processes. But it has also allowed the team at WEHI new insights into the major side effect of a new cancer drug, such that actions could be taken to alleviate the adverse effect in patients early in the clinical trial process.