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Johns Hopkins’ Robert Georgantas On microRNAs and Hematopoiesis

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NAME: Robert Georgantas
 
POSITION: Research associate, Johns Hopkins University
 
BACKGROUND:
Postdoc, Johns Hopkins University — 2001-2005;
PhD, immunology/molecular biology, Johns Hopkins University — 2001;
BS, immunology, University of Illinois — 2001;
Immunologist and molecular biologist, Johns Hopkins University — 1994-2001;
Cell and molecular biologist, Argonne National Laboratory — 1992-1994;
Chemist, Utopia Instruments Co. — 1990-1992
 
At Johns Hopkins University, Robert Georgantas runs a lab within the research group of Curt Civin, who was recently awarded a three-year grant from the National Institutes of Health to investigate the role of microRNAs in erythroid development.
 
Last week, RNAi News spoke with Georgantas, whose lab is overseeing the NIH-funded work, to discuss the research and its implications.
 

 
Let’s start with a little background on the lab and the overall focus there.
 
Curt [Civin] is very famous for having discovered the hematopoietic stem cell about twenty years ago; he actually came up with an antibody that allowed it to be purified. Since then, his lab has been focused on the mechanisms that lead to differentiation of that stem cell to its different progenitor cells.
 
My focus, when I originally came to his lab as a postdoc, was trying to figure out the genetics of that [process] — basically look at the gene-expression patterns, look at the epigenetics, and figure out how those played into hematopoietic differentiation.
 
The way that we ended up in the microRNA field is kind of an interesting story. We were competing with two other groups — one from Stanford [University] and one from Princeton [University] — to do microRNA profiling of the hematopoietic stem cell, determine exactly which genes are expressed at different stages of hematopoiesis, and use that information to figure out which genes actually drove differentiation.
 
The funny thing that came out of our [2004] study [in Cancer Research]… was that, looking at the mRNA profiles, we couldn’t really predict what the hematopoietic stem cell was doing. The interesting thing we noticed that the other two groups didn’t pick up on was that pretty much every gene that had been shown to be involved in hematopoiesis — things like the GATA factors, as well as a number of other important transcriptions factors — were equally expressed in [both] the hematopoietic stem cell [and] in progenitor cells.
 
Being in Curt’s lab and being involved in surface markers for purifying the hematopoietic stem cell, we had five new candidate surface markers that came out of the study. The interesting thing from those that we noticed right away was that when we looked at the mRNA expression, these genes were expressed really highly. But when we [tried] to find the protein on the surface of the cell, they weren’t actually expressed.
 
That was basically our first clue that instead of having some type of normal genetic mechanism where you have DNA turning into RNA turning into protein going in to control differentiation, we noticed right away that there was a block at the RNA-to-protein step, or what we call a translational block.
 
We came up with this theory we called the prime stem cell model [wherein] the stem cell [was] already expressing all of the genes that it needed to become any of its differentiated progeny, but that there had to be some translational mechanism that was blocking the mRNAs from turning into proteins, and that was what actually causing control of differentiation for hematopoiesis.
 
Curt has a peer who was at Thomas Jefferson [University] at the time named Carlo Croce. [Prior to the Cancer Research publication,] we went to a leukemia symposia at which Carlo and Curt and I were giving talks. Carlo gave his talk about a new phenomenon called microRNAs that he was look at as being involved in cancer.
 
Carlo’s lab had actually made the first microRNA microarray, and it was pretty much the only reagent available at the time for measuring microRNAs other than Northern blots. So [after the symposia] we collaborated with him to repeat the studies we had done looking at mRNA expression in the different cell subsets, but this time we did it for microRNAs.
 
You can look at expression data and say, ‘This gene is expressed, this gene is not expressed.’ This is nice information but it doesn’t really tell you anything. I went and made a huge database and [developed] a set of software tools that would let us take the microRNA data and overlay it on top of the mRNA data.
 
[To do so], we made a bunch of algorithms that predicted which microRNAs would control which mRNAs, as well as use some data that was already available on the internet, [and] put that all in one huge software tool so that we could go through and predict exactly what each microRNA would do in hematopoiesis. We had one of those eureka moments where as soon as we made all of the predictions we said, ‘These have got to be it!’ … All of a sudden, everything lined up in the right spot where this microRNA was expressed in this cell subtype and it would control these transcription factors and stop differentiation.
 
We’ve actually gone through and functionally tested three of the microRNAs we made predictions for and they actually do control hematopoiesis. And there have been another four publication from outside our group that have confirmed our predictions, [which was published in the Proceedings of the National Academy of Sciences].
 
Is this tool available somewhere for other researchers?
 
This is just in our lab at the moment. We are actually trying to find a software company to develop it into a tool. The problem is, the way it is written at the moment, it has to be custom-written for each dataset. All the algorithms and tools that you overlay on top of the dataset are portable, but each time you do a new dataset you have to make a new baseline database.
 
So three microRNAs stood out from that work?
 
The first [of the] three was micro-155, which … blocks myelopoiesis and erythropoiesis. We also have a publication in press showing that microRNA-16 blocks erythropoiesis. Right now, we’re working on microRNA-128a, which we think blocks all hematopoiesis, including lymphopoiesis.
 
In regards to the NIH grant, is that supporting this work?
 
Yes. It is supporting us looking specifically at erythropoiesis. There are four or five different microRNAs that we think control different stages of erythropoiesis, and that grant supports that work.
 
We also have a Maryland Stem Cell grant to look at really early steps of hematopoiesis, as well as some other [funding awards].
 
Long term, what are the implications of microRNA control of these differentiation processes?
 
We’re working with [an undisclosed] big drug company right now to turn some of these into [miRNA-targeting] drugs. What we foresee is that any translation work that comes out of this … [will] have a lot of utility in regenerative medicine.
 
One of the things that Irv Weissman’s group at Stanford [University School of Medicine] has found is that as people age, their hematopoietic system becomes weaker. That kind of holds up with what we see when geriatric patients become anemic and their immune system starts to falter.
 
What we’re really hoping is that we can take advantage of some of these microRNAs to do something similar to what we can do with erythropoietin where we can basically give a small-molecule drug that will stimulate specific parts of the hematopoietic system. We foresee being able to drive the hematopoietic system to make more lymphocytes, make more platelets … for people with bleeding disorders, [or] make the next erythropoietin — something that will drive red blood cell development.
 
Another area we’re really actively looking at is … working specifically to use these findings to expand the hematopoietic stem cell. One of the huge problems in our field, as well as in bone-marrow transplantation, is that the hematopoietic stem cell, as soon as you pull it out of the body, starts to differentiate. [This makes it] almost impossible to culture it in a dish for more than three days.
 
One of the things we think we’ll be able to do, and we have some early data for this, is to express something like microRNA-155 or microRNA-128a to stop the cell from differentiating so you can culture it.
 
Now if you do a bone-marrow transplant, you need a donor. What we’re hoping to do in a few years is pull out a few hematopoietic stem cells, type those as normal, then expand them for a given patient and transplant those back in.
 
The third thing that’s been [ongoing] is that, because we have microRNA data for the normal hematopoietic system, we’ve started comparing that data to abnormal hematopoiesis — things like leukemias and lymphomas.
 
What’s really different [about] our work … is that we do differential expression so we can say, ‘This [miRNA] is expressed in normal [tissue] and expressed in leukemia cells, so it’s probably not important.’
 
We now have two [miRNAs identified] in the lab that are expressed in normal hematopoiesis but are not expressed in any cancer that we’ve looked at so far. And I have a postdoc looking at what those two microRNAs do in leukemia and lymphoma. What we’ve found is that they are the most potent tumor suppressors that have ever been found. If we replace these microRNAs in a cancer cell, it commits apoptosis and dies because [at least one of the miRNAs] hits every single drug-resistance and apoptosis pathway that is known.
When it is expressed in the cancer cell, it turns down the drug resistance proteins and up-regulates apoptosis.

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