In the midst of running a very busy lab (“three hundred thousand fish”), treating patients (“I see pediatric hematology patients and adult oncology patients”), creating two companies (Fate Therapeutics and Scholar Rock), and playing the shofar, Dr Leonard Zon found time to talk to JEM. Dr Zon’s fascination in college with hormonal regulation of blood cell development led to over three decades of groundbreaking work, from identifying GATA-1 as a key regulator of erythrocyte development, to his pioneering role in making zebrafish one of the dominant models in hematopoiesis research.
Dr Zon has recently published in JEM on how the nucleoside adenosine promotes hematopoietic development, which together with two other papers in the same issue (one of which he also co-authored) help understand how blood flow in the developing embryo regulates the emergence of hematopoietic stem cells.
A natural storyteller, Dr Zon gave us enough material for more than one post. Enjoy part I.
Illustration by Madalena Parreira.
How did you get involved in basic research?
LZ I went to Jefferson Medical College and I thought I wanted to be a community pediatrician. I had done research all during college, but I didn’t know that I wanted to do research. Then I went to a lecture by Allan Erslev. Now, Allan Esrlev was the person who discovered erythropoietin. He did the seminal experiment, where he took anemic rabbit sera and shot it into a bunny that was normal, and that bunny developed polycythemia. I was so blown away that hormones could actually control blood cells, and blood cell development, that I thought this is what I want to go into. So I set up a long career of examining cell specific signals and cell fate decisions. That has really captivated my thoughts for 30, 35 years.
Then I came to Harvard to do my clinical training and during my fellowship we had the opportunity to work in a basic science lab, and so I joined Stu Orkin’s laboratory. It was really there that I felt like I got to a place that I really enjoyed, and just loved the intellectual debates and curiosity that went on. So it really kind of hit its stride when I was a fellow. At that point, I had cloned this transcription factor called GATA1, which is required for red blood cell development. It binds to every red blood cell gene and activates it. So that was an exciting time, and that let me ultimately set up my own lab.
Tell us a bit about your recent work on zebrafish hematopoiesis in JEM.
LZ This was a really nice paper, and I’m very happy that we were able to publish it with all the other papers. It really is a very good group of papers together, that I think complement each other. We had found that adenosine worked in a screen looking for engraftment in the caudal hematopoietic territory, which is the fetal liver equivalent (in zebrafish, ed. note). This chemical had also come up in another screen and so it seemed something that was very biologically active, and we really pursued it. A number of adenosine analogs increased the engraftment of the stem cells in this “fetal liver.”
We saw that in the developing aorta, the adenosine analogs really regulated hematopoietic stem cell and progenitor formation. We could use an antagonist to the adenosine pathway and also show that this would block the production of the stem cells. We were able to figure out that adenosine worked through a particular receptor, the A2B receptor. Then we showed that what happens is that you get more budding events if you give a receptor agonist. We could do this with live imaging using confocal and actually watch these things happen. We could also use an antisense approach, or morpholino, approach to the receptor. When you knockdown the receptor, the budding events happened, but then the cells started to die, and they would kind of burst. It meant that the pathway was not only sufficient to induce stem cells, but was also required, at a certain level. Together with George Daley’s lab, we were able to demonstrate that in mouse AGM cultures, as well as embryonic stem cell cultures, activation of this pathway promoted hematopoietic development. That was nice, that the process was conserved.
We went on to show a little about the mechanism. There’s a chemokine, CXCL8, also known as IL8, and what we realized is that adenosine increased the levels of IL8. We found a mutant for IL8, and it had defects in the stem cells in the aorta, particularly through these budding events. The activation of the pathway seems to go through cyclic AMP/PKA. Basically, you have this activation of the receptor, you increase cyclic AMP in the endothelial cell, which leads to the production of growth factors, which would include IL8. This has an impact on the budding event, particularly getting this emerging hematopoietic stem cell, or progenitor cell.
That story was very complimentary to other stories that you published*, the first one being blood flow, and how blood flow initiates this process. George and I had both had papers on that. But we’re able to demonstrate that one of the major activities of the blood flow was actually prostaglandin, and that could also activate a cyclic AMP pathway, and be able to stimulate the production of the blood stem cells. Also, George had been working on CREB as a target gene, particularly as it regulated some of the BMP responsiveness in this region. We were able to look at that and show that this pathway probably also involves the activation of CREB. The stories together really form a very nice unit and show that there are these activators that are triggered by a variety of environmental events including the blood flow, and you have factors like adenosine and prostaglandins that stimulate the initiation of this stem cell budding.
*Ed. note David Traver penned an open-access summary of the three papers.
Another fascinating story is your work on PGE2, where you went from zebrafish to mouse, to clinical trials.
LZ We’re in phase II trials right now. We’re in the midst of testing fifty patients. This was a fantastic story, and I have to give a lot of credit to Trista North and Wolfram Goessling, who were doing the work in the laboratory. They ended up doing a chemical screen to look for inducers of stem cell number in the developing aorta, which is where all vertebrates form their blood stem cells. We found 35 chemicals that can increase blood stem cells. This prostaglandin was definitely the big winner. There was a huge increase in the number of stem cells in the aorta. So we went ahead to see if it was therapeutically useful. We used the mouse competitive transplant assay as a method to see if it would work. We treated marrow with prostaglandin and then competed against untreated marrow that was genetically marked. What we saw was a fourfold increase in the number of stem cells that engrafted if the stem cells had been treated just for two hours with prostaglandin.
Then we took human cord blood. Cord blood is very interesting because the cord blood stem cell transplants work really well, but there are very few stem cells per cord. The current standard of care is to give two cord blood’s worth of stem cells. If just one cord is given, you don’t have an absolute engraftment, so doubling the dose of stem cells gives you a pretty good significant rate of engraftment. The problem with this is that if you put two immune systems in a third person then they start fighting each other. One of the cord bloods will win, in terms of an engraftment, over time. Most people think it would be nicer if you could treat with one cord blood and have it amplified in some way. There are also thoughts about bringing a lot of cord blood that’s been stored that has an inadequate dose of stem cells. If you cold goose those cords up with some drugs it might be useful to actually transplant those—which might be better matched to a patient.
So, we treated cord blood with prostaglandin, transplanted it into immune-deficient mice, and we saw more mice had human blood, and they were more chimeric. Then we decided to do a clinical trial, which was very exciting. I definitely think that in my life, it is the most interesting thing we’ve done. Some of the experiments that we had to do to get through the FDA were extremely boring. At one point we had to change our buffers, because we were using mouse buffers, and we needed to switch to human buffers. I didn’t think that was a big deal. They told me we had to redo all of our preclinical studies in the new buffer.
Thousands of dollars later, the result was basically the same, but that allowed us to go to the FDA and get approval. Then we were able to do this trial, and in the trial we treated 12 patients who had leukemia. They were getting two cord bloods as standard of care, and we basically repeated the mouse experiment in the human by treating one of the cord bloods with the prostaglandin. What we saw was in 10 out of 12, the treated cord was the one that engrafted, and also the treated cord had early engraftment with neutrophils—early recovery, I should say, with neutrophils and platelets about four and a half days earlier.
That’s been really great. We published that in the journal Blood, and then we went on to do a phase II clinical trial. I should say that I started a company as result of this, called Fate Therapeutics, and they’re actually running the phase II trial. I don’t have as much to do with it as I did at the initiation of the first trial. But it is to treat six patients and see how it works. So it is an interesting time, going to phase II trials now for pediatric patients, for inborn errors of metabolism. There are actually three trials that are going on.