Funding science for a cure… Part I
by Zhenya Senyak
MPN research is funded by big pharma, universities, governments, and non-profit foundations. The MPN Research Foundation is the only organization funding basic scientific research into the causes and cures of MPNs exclusively. Since 1999, MPNRF, “founded by MPN patients for MPN patients,” has employed tough selection procedures to find new avenues of research and fund both new and veteran scientists researching the pathogenesis of myeloproliferative neoplasms.
MPNRF has invested over $8.5 million in basic science research, contributing to historic discoveries and current drug development. MPNforum Magazine wanted to look behind the laboratory door to get an idea of how that investment is paying off. In this series of articles, we’ll be reporting on some of the people and processes powering science on our behalf. And in the process, hopefully visit the birthplace of the next generation of drugs that might finally produce a cure for our blood cancers.
As patients and caregivers, most of our attention is directed to the principal investigators conducting clinical trials of MPN drugs, rock stars within the MPN community. Less attention is paid to bench scientists and theoreticians who search for MPN disease-causing mutations and prospective drug targets through experimentation, math modeling, genetic sequencing and analysis. This is the story of two such scientists.
To appreciate the complexity of the task faced by scientists and mathematicians working this terrain, some basic facts about the nature of MPNs and the molecular world in which it arises, are useful So, before opening that lab door, how about a quick review of some basic genetics applicable to our MPNs.
Consider our DNA, how filled with beauty this constellation, how cloaked in mystery. This mass of six billion base pairs is a deep and cosmic secret locked in the dark night of the cell nucleus. In each cell in our body it expresses its only message. “Behold, Me!”
Our double helix DNA is our immutable calling card, recognizable wherever it is flashed, blood drop, cheek swab, hand scrape, tissue and organ all stamped with this endlessly repeated mantra. “Behold… Me!”
In MPN disease, somewhere along that sugar-phosphate crystalline scaffolding strung with beads of linked nucleotides, is harbored the mutated instruction set that created our MPN. These new orders from within our DNA set in motion a cascade of events that produce our blood cancer cells.
With the advent of our MPN, our “Me” now includes the proliferation of a blood cell line, possibly insignificant, potentially catastrophic.
Find and cancel that order, we are cured. Find that order and interrupt it, confuse it, block it….and for a time we are relieved of MPN’s worst excesses. Search and find it. But how?
The bare bones version goes like this: Our DNA contains a long code. When decoded, proteins are made, cells created. Somewhere in that long line of code things got garbled up, mutated. As a result our blood producing system started chunking out far more of one or more blood lines than needed. By sequencing the DNA of someone with our MPN, spelling out the code letter by letter, and matching it up against a healthy human we might discover the mutation — sort of a bug in a computer program — and fix it.
The search for the single or multiple anomalies causing our MPNs takes place in an ocean of irrelevant and dynamic data, data that expands and shifts under the pressure of another complication: Mutation.
Mutations are common. They come about during the course of reproduction, through environmental insult, through chance, entropy, or inherited predisposition.
Any change in the nucleotide sequence of the genome is a mutation. Sometimes a single base pair substitution in DNA can have a deadly effect, producing an amino acid where say, a valine replaces glutamic acid. That single amino acid change caused by the mutation of a single base pair would affect the way hemoglobin molecules load into red cells and produces the damaged red blood cells of sickle cell anemia.
Sometimes a single or even multiple amino acid change has no effect on a protein’s function. (Proteins are three dimensional, resulting from specific folding of the produced polypeptides or amino acids. As long as the shape remains relatively unchanged, biological function may not be affected.)
Mutation makes the search for genetic causes of MPNs easier. We are now looking for something pathogenic, something that doesn’t appear in healthy humans without the mutation. Even though a composite human genome was been fully sequenced a decade ago, it hasn’t been fully analyzed – and may never be – and the functional analysis, the relationships between gene expression and disease states is still at the very beginning So simply discovering a mutation – say the JAK2 v617F mutation – doesn’t automatically buy us much.
The JAK2 Mutation
In 2005, four separate laboratories working from different approaches uncovered the prevalence of a single mutation nearly universal in polycythemia vera and common in about 50% of other MPNs. (Technically, this was a JAK2 point mutation at codon 617 resulting in Guanine and Thymine swapping places at nucleotide 1849 in an expressed segment of the JAK2 gene. As a result, in its final form, phenylalanine substituted for valine at codon 617, and the infamous JAK2 V617F mutation was created.)
After several false starts and failed clinical trials, it was those discoveries in 2005 that led in 2011 to acceptance of ruxolitinib as the first approved therapy for myelofibrosis
Among the discoverers of this mutation and principal authors of a key paper in Cancer Cell that year were Ross Levine and Benjamin Ebert. It is to their labs we will soon turn our attention, as they are among the recipients of MPNRF grants,. Long-time colleagues and associates, Levine and Ebert are following two separate pathways in search of the same solution.
Two main areas of MPN genetic research look for mutations in heritable and sporadic forms of the disease. Now that we know MPNs are clonal stem cell disorders, the question arises as to why? Did we inherit the disease or at least a genetic weakness to develop the disease under circumstances others wouldn’t? Or is it purely sporadic, a result of environmental misfortune, radiation, accident?
The answer may lie in our chromosomes.
Chromosomes are strange and shape shifting beasts. They emerge out of the sea of nucleic acid during times of reproduction, 23 paired chromosomes, diploid, mom and dad continuing their bond all the way into the heart of each cell of their progeny.
An exception are the germ cells, the sperm and ovum cells, the X and Y cells which are singular. In human reproduction, the union of germ cells joins two halves of haploid DNA into a new diploid being, and issues a unique calling card to the newborn child “Behold, it’s Me!”
Is our MPN stamped on this new card? Has this baby inherited familial MPN? Or is this baby carrying a set of instructions that will make it likely or nearly inevitable the there is an MPN in the future? Or is this a perfectly healthy baby who will never suffer an MPN unless some sporadic event – trauma, radiation, mutation – occurs?
Thus two main current lines of research into the nature of our mutant clone arise – familial and sporadic.
As part of its mission to fund basic genetic and molecular research in pursuit of an MPN cure, the MPN Research Foundation in 2011 made two $150,000 two year grant to a team of researchers to use whole genome sequencing to identify variant forms of genes that contribute to the development of a myeloproliferative neoplasm. One grant was award to Robert Kralovics in Vienna, the other was shared by two labs working in the familial/sporadic area.
One lab, under Ross Levine at New York City’s Sloan Kettering is focused on sporadic MPNs, the other under Ben Ebert at Harvard Medical School and Brigham and Women’s Hospital in Boston on familial MPNs.
Our first visit was with Dr. Ross Levine, one of the young stars at Memorial Sloan Kettering in New York, who has already made his mark in MPN medical history with his work on the JAK2 V617F mutation, published in 2005.
Ross Levine is a New Yorker who attended Harvard and the Johns Hopkins School of Medicine on his way to a post –doc clinical fellowship in hematology/oncology at Dana-Farber. It was there that he acquired his interest in leukemia genetics and a spot in the laboratory of Gary Gililand, the director of the Dana Farber/Harvard Cancer Center
He arrived at an historic moment in MPN research. By sequencing DNA from cancer cells and comparing those results with noncancerous cells from the same patients, he participated in the discovery that a single mutation in a signaling molecule was found in most patients. This was an early discovery of the JAK2 V617F mutation. (Here is a free complete PDF of one historic report on the finding that appeared in the New England Journal of Medicine.)
MPNforum: Who actually gets credit for being first?” we asked.
“It depends on what you mean. These studies represent large collaborative efforts. .The fact that there were so many people on our manuscript, and on the other JAK2 discovery efforts, demonstrates these are collective efforts.
“Science has always been, in part, about individualism and innovation and giving credit to people who make discoveries, but the reality of this sort of science, where there are large genome sequencing efforts, there are often many people each contributing to effect a much greater good, a larger effort than could be done in separate, individual efforts.”
And yet, Ross concedes credit is an important issue in medical research.
“These studies are expensive, we always have to be cobbling together support. The Foundation was a great help. We also turn to STARR, NIH, LLS and other foundations. This is a tough funding climate and we are all competing for funds.”
The work with Gilliland confirmed Ross in his path as a scientific researcher into blood cancers working in clinical medicine, specializing in the leukemias. “I’m a physician/scientists. About 80% of my time is spent on research and 20% with my chronic or acute leukemia patients.” That research time is spent in the Levine Laboratory at Sloan Kettering where much of the work on the MPN Research Foundation funded grant is done.
Although the grant was made to identify variant forms of genes that contribute to MPD pathogenesis, the work has been expanded and redefined over the past year. Ross’s work focuses on sporadic MPNs, diseases that arise somatically as a result of mutation.
MPNforum: What is your grant about?
“The aims of our grant are to do two important things, First, by applying these new sequencing technologies can we uncover some substantive new insights into what causes MPNs by really cataloging inherited and acquired alterations in the genome that contribute to MPNs?
“In our lab we are studying a cohort of patients who have sporadic, non-inherited, MPNs, and understand what’s wrong in their cancer cells that is not wrong in their normal cells. That can tell us what all the somatic mutations are which are acquired in MPN cells which are not heritable.
“And secondly, Ben and Ann Mullaly’s group is putting effort into collecting families with multiple affected members to see if maybe we could understand what might be the allele that predisposed the MPN in the rare cases where it’s inherited.
“These genomics studies represent our first aim, and we are taking similar approaches to answering slightly different parts of the question of why patients develop MPNs, None of these efforts are going to be exhaustive but the intent is to really understand the MPN genome I think that’s just the first step
“The second step is to functionalize (our findings). What we learned from these sequencing studies this last year is that the genomic data does not by itself tell us what is important, but rather we must study the specific genetic variants in the lab to figure out which ones contribute to MPN development. So Ben and I work really hard to use both cell culture and mouse modeling to really discern what these alleles do and our intent will be to show whether specific genetic alterations are biologically relevant either to the development of MPN, to the response to specific treatments or some aspect of MPN biology we don’t know yet.”
MPNforum: How is it going?
“Great. We’re hoping to compete our first set of analyses in the next few months to give us a sense of what all the potential candidates are and we’re hoping to then be in a position the first quarter of this year to begin functional studies.
Again, it’s a large effort. It takes many people in both of our labs working together. It’s going to take all of us working together to combine our data from different grants in different groups and we’re eager to do that as well.”
Searching for a mutation or series of mutations coding a gene that results in myeloproliferative neoplasms requires narrowing the search, reducing the oceans of data encoded in the dynamically unfolding human genome.
Our total complement of genes is our genome. Some 20,000 or so of our genes — long segments of DNA residing on chromosomes — express sequences, creating proteins or regulating functions. Protein coding sequences — exons — only account for perhaps 2% of the genome, the rest made up of intros, (intercepting segments of genes that don’t code for a protein and are spliced out) and DNA between genes, intergenic material, which is essentially dark matter whose function is unknown. It is believed that some of these DNA segments are regulatory and about 80% of the bases in the genome can be expressed.
Given this overly rich stew of data, and the expense — Steve Jobs is reported to have had his whole genome sequenced for $100,000 — plus staggering data load and time — Levine and Ebert focus on the exome, the expressed part of the genome formed of the exons.
We took the exome issue to Dr. Benjamin Ebert, professor in the Harvard Medical School, associate of the Broad Institute and a clinical hematologist at Brigham and Women’s Hospital in Boston.The specialty of his lab is to combine genomic analyses of patient samples with functional screens to identify critical genes for hematologic malignancies, differentiation and HSC renewal. His lab team includes Ann Mullally and Luke Poveromo, in collaboration with a wider group of investigators.
Compared to sequencing entire genomes “it is much more cost effective” says Ebert, “to work on the exome. We save money and can do a larger number of subjects. And when you find a mutation in a coding sequence you can more readily postulate whether or not it changes the function of a protein.
” Most of the genome is filled with polymorphisms between genes and we have much less understanding of how to work those out, or even whether they are functionally relevant or not. So while you can get a lot more information with whole genome sequencing, we don’t know how to use a lot of that information right now in any case.”
“There are clearly sequences in (the intergenic regions) that are important for the regulation of genes, how much they’re turned on or off, and therefore could alter the phenotype of the disease a little bit. But it seems less likely that they are going to have a powerful influence on whether or not you’re going to get a disease. Nevertheless there are clearly polymorphisms, genetic variants that can influence some complex disease but are unlikely to cause a genetic disease.
“Probably 100% of MPNs have somatic mutations. We already know about a lot of the mutations. We don’t know much about the familial forms. The major drivers of familial (inherited) MPNs are completely unknown.”
MPNforum: Isn’t it a question of inheriting a predisposition, a haplotype?
“It’s possible a collection of genetic variations, a haplotype, can be transmitted through the germline. But it’s basically a difference of how powerful the predisposition is. Some genetic variants or haplotypes may cause a slightly increased risk of acquiring an MPN than someone without that variant or haplotype. Then there are other variants that make it extremely likely, perhaps almost assured, that someone would get an MPN eventually. It’s just a question of how powerful the genetic predisposition is”
“In general, in this type of research, there are common variants with a small effect and rare variants with a powerful effect. What we’re looking for now are the rare variants with a powerful effect. The JAK2 haplotype falls into the category of a more common variant with a weaker effect.”
” The identification of novel genetic variants that predispose patients to developing MPNs would help explain the biology of MPNs in those patients and we hope it will give us information on how to target MPNs and also enable us to predict who inherits those alleles and maybe help us screen for them.
MPNforum: Getting back to the JAK2 mutation, how much do we now know of its function?
“We know quite a lot about parts of its function. We know it definitely potentiates signaling from various cytokine receptors including the EPO receptor and that’s a major part of the disease phenotype. You get increased erythropoietin signaling and that’s why red blood cell counts are high …but I think there are other functions we haven’t fully elucidated…”
MPNforum: How is it in all the JAK2 clinical trials the drug being tested was effective regardless of JAK2 status?
“That’s a profound issue. There are many potential explanations. One possibility is that some patiens who don’t have the JAK2 mutation have another mutation we haven’t found yet that has the same consequences on JAK signaling.
MPNforum: How is your familial MPN project going?
“We have completed sequencing of 20 samples so far. We have narrowed down to a list of possible genes that are mutated in those samples and in order to figure out which of the genes is the right one we need to look at additional samples. We are now looking at larger sets of samles to validate the causative genetic variants.
” we are focused particularly on Jewish MPN patients.
Ashkenazi Jews have a higher incidence of MPNs, indicating a particular genetic predisposition in this population. Using recently developed DNA sequencing technologies we have identified a number of candidate genes that may contribute to the genetic predisposition to MPN in Jewish individuals.
As a starting point it seems like the first place we might make a strong discovery.
(Ed. If you are an Ashkenazi Jewish MPN patient and have at least one living first-degree relative (i.e., parent, child, or sibling) who also has MPN (Polycythemia Vera, Essential Thrombocythemia or Myelofibrosis) and are potentially interested in participating in this study, please contact Dr. Ann Mullally at firstname.lastname@example.org in the laboratory of Dr. Benjamin Ebert (Brigham and Women’s Hospital, Harvard Medical School, Boston) for further details. )
© 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.