The MPNRF science grants: Behind the Lab Door
On the pathway to a major break-through in PV treatment
By Zhenya Senyak
Not many of us really want to know the details of the science behind drug discovery. The mind boggles at concepts like BCR-ABL and JAK2 mutations. Conceptually, the Philadelphia Chromosome is more remote than the Marianas Trench at the bottom of the Pacific Oceanr.
But whether we understand the workings of science or not, the labs where it is practiced are where the real action is in our myeloproliferative world. Before a miracle drug hits the market, before clinical trials, there is only the bright blade of science interposed between us and our blood cancer.
Ever since an important signaling mutation, common to many with myeloproliferative neoplasms, was found five years ago, research has accelerated. With discovery of that JAK2 mutation (LINK), there has been an international explosion of research and publishing in the MPN arena, mostly focused on inhibiting the action of the JAK2 enzyme and its deadly effect on blood production.
One scientist is taking a different approach. And his big idea may soon advance us another giant step toward understanding and curing MPNs. And that cure is a common drug that might already be in our own medicine cabinet or the corner pharmacy.
What happened in Shenyang
Built on the plains of the Liao River, bordered by the forests of the Changai Mountains, the northeastern Chinese city of Shenyang has rarely been a peaceful place.
For half the 20th Century, Shenyang was at the center of war and occupation. The Japanese invaded in force to create the
Manchuko State out of this portion of what was then Manchuria. After WWII, Shenyang became a civil war battleground as first Soviet troops and then Chinese Nationalists occupied the city until driven out by the Chinese Communist forces in 1948. Today, this heavily industrialized Northeastern Chinese city — hot and humid in the summer, and very cold in winter — is home to over 8 million people. It is also home to the PRC’s first medical school, China Medical University (中国医科大学/中國醫科大學) It is in this city that Shaoguang Li grew up.
Shortly after graduating from China Medical School, Shaoguang Li, then a medical doctor, left to study in the United States. His primary interest was research with emphasis on leukemia In the US, he studied at Tulane University in New Orleans earning his PhD degree for Cell and Molecular Biology. He went on to complete his post doctoral degree at Harvard Medical School, obtained an appointment as Associate Professor at the University of Massachusetts and established his own lab. In the course of his work with Chronic Myeloid Leukemia (CML) he began to study a related blood disease, polycythemia vera. This work produced a flash of insight.
He had a big idea he wanted to pursue. And to do it, he needed funding.
What happened in Chicago
For its 2010 grant program, the MPN Research Foundation sent out a Request for Proposals to individual researchers and medical research institutions. Sometimes these RFPs, as in the MF Concept Grant (LINK),are sharply focused. For that year, the Foundation cast a wide net trying to find both experienced and new investigators working on innovative research with high promise of productive results.
The competition was fierce, fueled both by MPN research stimulated by the JAK2 discoveries and the onset of the Great Recession of 2008, The prize: Nearly $1 million: Three $150,000/year two-year grants in Established Investigator Awards as well as $75,000 Awards to New Investigators. Only about 10% of all applicants would come away with anything.
Project coordinator Barbara Van Husen, president of the Foundation, would end up forwarding over 50 proposals to Dr. Andrew Schafer,Weill Cornell Medical School and head of the MPNRF Scientific Advisory Board. Dr. Schafer parceled out the proposals to teams of two reviewers each, to score each proposal individually.
After the scored proposals arrived back to the Foundation’s North Michigan Avenue HQ in Chicago, they were copied and shipped off to each of the 19 international scientific and academic advisors who would soon gather around the big conference table in Chicago to help determine what future direction MPN science might take.
Members of the preliminary review team presented their case, their reasons for assigning scores. During the ensuing discussions – sometimes lively – each adviser would assign his or her own score to a proposal. At the end, after tabulation and ranking of research proposals, there were further discussions. At stake: Funding science that may well alter the shape of basic MPN research for years. Finally, the first awards are announced. .
A $150,000 per year, two-year grant was awarded to Shaoguang Li.
The Lab at the University of Massachusetts
In Worcester, Mass., on the outer ring of the Boston Metropolitan Area, is the Aaron Lazare Medical Research Building, the University of Massachusetts’ ten story curved glass high tech research facility. It is here that Shaoguang Li would take his grant to test his big idea,
The big idea
From his work with Leukemia Stem Cells (LSCs) in Chronic Myeloid Leukemia, Shaoguang knew that, among mouse populations, the ALOX5 gene was necessary to induce CML. Remove the ALOX5 and the LSCs were disrupted, essentially terminating the disease. He saw that there were essential similarilties between CML and the myeloproliferative neoplasms, particularly polycythemia vera.
Once he started looking at the MPNs and researching the JAK2 V617F mutation, it was obvious that the JAK2 mutation could not be the only cause of MPNs – since MPNs existed in people who were negative for the mutation. There must be something downstream of the cause related to the mutated JAK2 pathway, some recipient in the cell that’s actually critical to the onset of the disease.
Part of that process seems clear: Phosphate groups act as on/off switchs on proteins. Mutant JAK2 ,hopelessly auto-phosphorylated endlessly signals for more blood production and the STAT protein picks up the message, is itself phosphyrolated and translocates into the cell nucleus to trigger the transcription of genes that results in translation into messages, proteins, and in the case of MPNs, trouble. And while pretty much everyone with PV has the JAK2 mutation, not even half those who are JAK2 positive have PV. What’s going on.
Since it appears that the mutated JAK2 enzyme might be signalling some cascade of events that do in fact cause PV, Shaoguang Li suspected the gene Alox5 was implicated in the pathway and would be an effective target gene to control PV.. But where in the pathway would he find it?
Stopping you from driving to D.C.
“This is a brand new idea.,” said Shaoguang Li. ” Over the years, researchers have targeted the reason for disease, say the BCR-ABL function…We learned however that the BCR ABL or JAK2 or whatever, cannot induce disease without using key pathways in the cell.“
“So we began to identify the key pathways downstream ..target the downstream pathway, alone or in combination with traditonal targeting of [[mutations] like BCR-ABLE and JAK2 Let me give you an example. Where do you live?
“OK, let’s say you don’t fly. You drive from North Carolina to Washington,D.C. So your car is critical for you to get from one place to another, However, what road are you going to take, this highway that highway? For me to stop you from going from N.C. to D.C. I can make sure you can’t. I can break your car, give you a flat tire,no gas, do something on your car.
“But say I don’t know where your car is. I don’t know if you have people to protect your car… However, if I can find a way to block the key pathway to go from N.C. to D.C, like a key bridge to cross, and I block the bridge you cannot get to D.C. In order for you to achieve your objective, you have to have a car and your car has to have an available pathway.”
Seen in this light, — the Mapquest approach to genomic targeting — the Big Idea seems a little inevitable. But before Professor Li could blow up any pathway bridges he would have to first find the bridge that lies on the JAK2 polycythemia vera pathway. And to do that he needed mice.
How do you give a mouse PV?
Shaoguang’s plan was straightforward. He already had already seen that the Alox5 gene was essential to the development of a similar disease, CML and he knew the mutant JAK2 protein activated several molecules that could be signaling the typical blood proliferation associated with PV, such as STAT5 and ERK.
What he didn’t know was whether Alox5 was upstream, between JAK2 and those molecules, or downstream, between the signaling molecules and the genes expressing proliferation. His lab started by using viruses to induce PV by introducing the expressed JAK mutation into the bone marrow cells of two types of mice. One is the so-called Wild Type (WT) or natural, and the other Alox5 Knock out or Alox-/- mice, mice totally without the Alox 5 gene. They would then transplant those cells into Wild Type mice.
. Four months later, his lab separated proteins from the spleen cells of these mice and analyzed them, by detecting phosphorylation levels, to see which proteins had been activated. If Alox5 is upstream, these proteins would not be activated, if downstream they would. And if these signalling proteins had no relationship to Alox5 their state would be unchanged.
PV therapy – another part of the project
Whether active upstream or downstream, Professor Li was pretty sure from his earlier work on CML and PV, that the Alox5 gene was involved in the PV pathway.
Shaoguan Li was principal author of a paper in Nature Genetics, in 2009, “Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia” (LINK) in which he noted treatment of CML mice with an Alox5 inhibitors prolonged survival. There, he attributed the result to the targeting and inhibition of cancer stem cells. “In the absence of Alox5, BCR-ABL failed to induce chromic myeloid leukemia in mice.”
Under his MPNRF grant he now wanted to further test the mechanism and effectiveness of an Alox5 inhibitor in controlling PV over the long-term.
The inhibitor? Zileuton, sold in your local pharmacy as Zyflo, a commonly used drug to prevent asthma attacks. It works because it’s a leukotriene inhibitor. Leukotrienes are those substances your body releases to close down parts of your airway when you breathe in substances to which you’re allergic.
What is Alox5 and why should we care?
Alox5 is a protein-coding gene, the name an abbreviation for arachidonate 5-lipoxygenase. The gene encodes a protein expressed in bone marrow cells and converts arachidonic acid to leukotriennes. These leukotriennes mediate many inflammatory and allergic conditions. Lipoxygenases are part of the LOX family we met earlier (LINK) in the article on reversing fibrosis at Katya Ravid’s lab. For more information see www,genecards.org (ALOX5 Gene).
Is this work pioneering a new direction in PV and MPN research?
For 2000 years, from before Hippocrates on through today, a front line treatment for disease has been phlebotomy. Bleeding a patient has varied in intent – from purging bad blood and humors to balancing blood — and in technique, from blood sticks and leeches to hypodermic needles and venesection supplies. But whether accomplished by physicians, barbers or the blood letting leeches of the 19th Century, the therapeutic use of the technique today is fairly limited to the reduction of blood volumes in polycythemia or the iron overload of hemachromatosis.
(The barber pole is a holdover from those times, the red standing for blood, the white the tourniquet and the pole the blood stick.)
Then the development of drug therapies, most notably the use of hydroxyurea and an escalating choice of drugs aimed at the effects of the disease, interferons, primarily, has lessened the requirement for frequent phlebotomies. And, since nearly all PV patients carry the JAK2 genetic mutation, it’s anticipated that fairly soon one or more of the JAK inhibitors will be available to PV patients.
Advances in genomics, molecular biology and biotechnology permitting closer targeting of disease mechanisms will undoubtedly uncover new means to inhibit the ravages of PV.
And now, scientific research focused on the signalling pathway that leads from upstream mutation to ultimate proliferation promises a new direction, about as far from the barbershops and leeches as blood treatment can get.
© Zhenya Senyak and MPNforum.com, 2012. Unauthorized use and/or duplication of this material without express and written permission from this blog’s author and/or owner is strictly prohibited. Excerpts and links may be used, provided that full and clear credit is given to Zhenya Senyak and MPNforum.com with appropriate and specific direction to the original content.