International MPN News, Science & Opinion

Robert Kralovics

CALR – The Kralovics perspective.

by Zhenya Senyak

 Robert Kralovics tells his story and describes his research in a straightforward way that leaves much unsaid.

“I’m a citizen of the Czech Republic. My training was originally in Czechoslavakia,  (Comenius University) in Bratslava. kralovics on skypeIn Slovakia in the Czech Republic.  It doesn’t exist anymore.  My professional PhD work was in genetics and microbiology, the biophysics of plant genomes.  I wanted human genomics…but this was the only open PhD course at the time.”

With excellent lab skills, even before completion of his PhD work,  Kralovics got an offer in 1995 to join the Czech hematologist/researcher Josef Prchal in Birmingham, Alabama for a one year program.  It was his introduction to myeloproliferative neoplasms and contemporary genetics research techniques.  After a year, Kralovics returned to the Czech Republic, wrapped up his PhD and rejoined Prchal to continue research into hematologic disorders.

“I eventually stayed with him seven years altogether. We moved from Birmingham to Houston Texas, to Baylor College of Medicine. I upgraded from post doc to assistant professor at Baylor.  But obviously, like many Europeans, I had a strong connection back home. In 2001,  I returned and joined the lab of Radek Skoda in Switzerland, in Basel and brought a project with me that initially started in Joe Prchal’s lab. “

Got the picture?  Of course not.  What’s missing is life in the old Soviet Union.  Kralovics was born shortly after the USSR tanks rolled into Prague to crush the Czech rebellion.  He grew up in a country that no longer exists, swallowed up by world politics following the end of WW II. He was a student activist during the 1989 protests. His opportunities as a budding scientist were seriously limited under the Communist regime.

What’s missing from this picture is the “project” and the emergence of a young hematology superstar who first entered the MPN world as a graduate student on a year’s tour of duty in America. And there’s little hint of emergence of the  scientist who first stumbled into the neighborhood of the first major genetic finding to explain the course of an MPN — loss of heterozygosity somewhere on  Chromosome 9 — and then worked relentlessly for years to pin down the precise address and function of his discovery.

After intense lab work ruling out a possible deletion, Kralovics concluded theKralovics experimental biology myeloproliferation was caused by a mutational defect in the chromosome. He and Prchal published their findings in Experimental Hematology, in March, 2002.

By the time this little noticed paper was published, helped along by homesickness and a destructive tropical storm that flooded Baylor College and interrupted his lab work, Kralovics was already back in Europe, at  Radek Skoda’s lab in Basel, Switzerland. He continued work on the Project and published an expanded view of his findings, with Prchal,  in November, 2003, Kralovics Exp HEm 2002this time in Blood. Acquired uniparental disomy of chromosome 9p is a frequent stem cell defect in polycythemia vera”

Here’s how Kralovics describes it:  “And I think  I performed one of the first genome wide screenings of some chromosomal operations using modern technology and I found some specific defect on Chromosome 9…  We published this with Joe in a small journal in 2002  and it turned out that this defect was the JAK 2 Kinase.”

Back in Europe, Kralovics found something else he desperately needed.

“In the US we have problems with patient numbers. Collection was really small in Joe’s lab but after moving to Switzerland we had not only the Swiss data but access to Italian data through Mario Cazzola  at Pavia University. We extended the patient collection to almost 300 patients.  And then we narrowed down  this specific deficit on Chromosome 9 to 15 genes  and among them was JAK2 kinase V617F wihich is how we found the mutation.”

When Kralovics published his paper on the JAK2 V617F mutation in 2005,  “A Gain-of-Function Mutation of JAK2 in Myeloproliferative Disorders”   he was fully associated with Skoda, Cazzola, Heinz Gisslinger and his European colleagues.

It was this group that would work together for the next six years to produce the answer to one of the most baffling of MPN genetic questions.  Virtually all PV patients and half of ET and MF patients are positive for the JAK2 mutation.  Discovery of the JAK2 V617F may have provided a new diagnostic tool and a drug target but what is causing MPN in those without that mutation?

“In 2006 after this success,” said Kralovics, “I applied for grants from the MPN Research Foundation and one in Vienna  from the Austrian Academy of Science, CeMM.  In collaboration with Heinz Gisslinger we expanded our tissue collection and since then we have one of the largest  biobanks in the world.

“We have over 800 patients biobanked clinically and characterized as we slowly  raised money to do the genomic analysis. So almost half  of these patients have high resolution genetics done and from this effort sequencing study became viable.

“We undertook several levels of genetic analysis… Looking at cross genomic changes , chromosomal  aberations using SNP array technology. That same technology allowed us to map constitutional genetic associations  and in 2009  we published a paper in Nature Genetics on how the JAK2 locus predisposed for mutagenesis….that came out from this type of analysis.

“By doing genetic analysis beforehand we identified  a suitable candidate for genetic sequencing. Since we already know a whole lot of their genetic data, we could cherry pick the myelofibrosis patients with no JAK2 mutation and no Thromboopoeitin receptor mutations like MPL or related chromsomal alterations.  By doing that we ended up with very clean exome data.

“We detected the CALR mutation in six of the six patients we sequenced. Because our preliminary work was very focused, our analysis did not need many patients.”

The practical side of discovery and therapy 

Despite laying reasonable claim to both major genetic discoveries currently powering MPN research, Robert Kralovics is no academic bench scientist. He has a deeply practical perspective on funding research and bringing the results to benefit patients. And the patent holders.  

Along the way his aggressive application for genetic patents has raised some eyebrows.

In sharing credit with collaborators,Kralovics we have to note our connection with the MPN Research Foundation. I started my group in 2006. Soon after I applied for the first grant from what was then called the MPD Research Foundation because obviously research money was not very available in Europe at the time, or for that matter in the US.  Basically, we got two consecutive funding grants  from the MPNRF.  They invested almost half a million dollars in our work…altogether I think over $400 thousand dollars into this project.

“Eventually, it was this money that made the difference. Most of the money raised by the Foundation comes from patients and that’s  why I always give the biggest credit to the MPN patients themselves and others who raised this research money.”

And why patents…?

“There’s some misunderstanding about our filing for patents. It’s not the gene we are patenting but the way the technology is used to define the mutation.  This is different.  Now, the patents we are filing are much wider. Not only the diagnosis  but also several  ideas of therapy and some means to vaccinate patients against MPNs.

“Our patent has 88 claims and will be public in September this year because there’s a 12 months pending review phase for both European and US patents.

“Patenting protection of intellectual property is not only legal and moral but there’s a fundamental practical problem.  If you don’t have patent protection of ideas you can’t raise money to pursue  a project, to take it all the way to clinical use.  Commercial entities develop a drug or diagnostic test. It would be hard to fund this kind of effort from public resources.  So patent restrictions are bad in one sense but essential for commercial development. This is the reason we filed these patents, in order not to find ourselves unable to take these findings to market. For that, we need an industry partner.”

Why can’t the CALR mutation exist alongside the JAK2 mutation?

“There could be several explanations. One is simple genetics.  If you max out one pathway a mutation that makes use of the same pathway has no effect, so there’s no benefit. 100% maxium growth.  So if you have JAK2 already in place and then you have CALR, the cell can probably not proliferate any faster and there would be no selective advantage to the double mutant.  We don’t say that some cells may not have double mutantgenesis targets but what we’re saying is it doesn’t have a clonal advantage to grow out.”

Why would the CALR exon 9 mutation create proliferation in the megakaryocyte line?

In the endoplastic reticulum, calreticulum is a scaffold protein, it helps proper folding of proteins into the final three dimensional structure. The mutated protein ends in a different way than the wild type.  So this novel mutant somehow preserves the interaction with the thrombopoietin receptor — we don’t know whether it just locks it in during interaction with the receptor. We don’t understand how or why only the thrombopoietin receptor has this specific reaction.

So once the interaction is established the mutant calreticiulin acts like thrombopoietin, irreversibly locks to dimers of the thrombopoietin receptor and that alone is triggering JAK signaling down the line. This probably happens  all the way because the receptor trafficked from the ER to the cellular surface in the vesicles.  Inside the vesicle is where the extracellular domain of the protein is and also the calreticulum binding the receptor. And once it travels to the surface it probably continues signaling and this action may not be reversible.  We’re trying to raise antibodies to see if we can compete it out.

What’s the impact on the MPN phenotype of the CALR mutation? 

The JAK2 mutation influences more than a single receptor, all the other cytokines involved should make the JAK2 positive impact much broader.  In the CALR cases, the influence is on a single receptor so that’s why you only have a more exaggerated platelet phenotype. The JAK positive  mutation creates hyperactive signaling so there’s more red cells, more white blood cells.  In CALR positive cases,   the white blood cells are lower and the platelet counts are higher. That’s one reason the Italian group concludes CALR patients have a better thrombotic profile because of those lower WBCs.   We have to wait for a proper prospective study, something like Tony Green’s PT-1 study, a large set of ET patients.  All the data we have is retrospective.    It is clear to us these CALR patients behave differently and they have a better prognosis.  I think the explanation is they have only a single cytokine receptor aberration where the JAK psotive has a broader phenotypic impact because more receptors are involved.


Take me back to the Contents

© MPNforum.com, 2014. Unauthorized use and/or duplication of this material without express and written permission is prohibited. Excerpts and links may be used, provided that full and clear credit is given to MPNforum.com with appropriate and specific direction to the original content.


 

Comments on: "Robert Kralovics" (2)

  1. Henriett Bodnár said:

    I would also add to the list Prof Gisslinger from Wienna!! I do hope we can benefit once from this new discovery!

  2. Bonnie Kaye Evans said:

    Thank God for medical geniuses such as Green and Kralovics. Imagine being able to save lives with new discoveries. Our MPN Foundation funded the project. Great to see our donations making a difference in the MPN world.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: