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Rather than waiting for a single family to accumulate $30,000 - $60,000 in a fund, this fund combines the gifts of several individuals or families to create a research grant. Gifts to this fund are combined with gifts from other donors; once the fund reaches $60,000, a research grant award is made. Distinct funds exist for
Bone marrow failure syndromes are not only serious and debilitating diseases but they can also progress toward leukemia. Identifying the specific factors contributing into this malignant transition is extremely important. Our group focus in past few years has been the role of the immune system in bone marrow failure and we showed the importance of specific subtypes of immune cells in both pathogenesis and malignant transformation of these diseases. The AAMDSIF research grant will allow us to focus on differences in immune changes in aplastic anemia and paroxysmal nocturnal hemoglobinuria (PNH) and will enable us to utilize the state of the art technologies (such as multidimensional mass cytometry and high throughput DNA sequencing) to further understand the role of immune system in these diseases as well as identifying new important biological markers for diagnosis and classification of aplastic anemia and PNH.
The aim of this grant is to identify the specific immune dysregulation in paroxysmal nocturnal haemoglobinuria (PNH) and aplastic anaemia (AA)/PNH, and also correlate the interplay between the PNH cells and specific T-cell subsets called NKT-cells in a sequential longitudinal study. We will also identify ways that different therapies modulate the immune response and utilise it for the therapeutics of marrow failures. Cutting edge technologies such as mass-cytometry (CyTOF) and high precision DNA sequencing (deep sequencing) are being used to address these questions.
Progress so far Patient recruitment:
We have identified 89 patients with PNH through the King’s College London (KCL) tissue bank which are consented to participate in research projects (10 patients were recruited since last year). Among these patients, we have access to peripheral blood mononuclear cells (PBMCs) of 26 patients at time of diagnosis and following therapy with C5 inhibitor (Eculizumab). PBMCs from these patients are stored as viable cells and being used for this study. We have also recruited 16 AA patients with a small to moderate PNH clone (with no cases of hemolytic PNH) and 8 age matched healthy volunteers (HDs) as control groups thus far.
Identifying the specific immune signature(s) for PNH and AA/PNH
The first aim of this research was to identify a specific signature which identifies AA/PNH patients and could potentially predict their response to immunosuppressive therapy (IST). We have used CyTOF technology and multiple bioinformatics packages to identify potential immune signature(s), which predicts response to immunosuppression in AA/PNH. This enabled us to distinguish two distinct subpopulations of human T-cells (known as T regulatory cells (Tregs)), in AA and HDs and to demonstrate clear differences in AA that predicted response to treatment. The results from this study are under revision for publication and the AAMDSIF has been acknowledged in this work. Up to now, blood samples from 5 PNH patients have been stained and analysed pre and post Eculizumab by the same method (10 samples) and the preliminary data suggest that type of Treg cells could also predict response to therapy in PNH. We have successfully optimized the CyTOF panel for identification of another important immune cells in PNH (CD8+ T-cell). We were able to identify the classic CD8+ T-cell subpopulations as well as novel clusters of cells in PNH patients. We will continue to analyse more samples by CyTOF to confirm the persistence and biological relevance of the identified clusters in PNH, AA/PNH.
Plans for the second year
We would like to continue this work by accomplishing the following tasks:
1. Finalise the CyTOF run for all collected samples from PNH patients pre and post Eculizumab and finalise the data analysis.
2. Once the CyTOF data analysis has been completed, and the set of markers which accurately define the identified cells in PNH, these markers will be used to isolate these cells and perform the additional molecular studies as outlined in our application.
The ultimate goal of the second part is to identify NKT-cells within the expanded population of cells and see whether these cells can distinguish PNH from AA/PNH and how they would affect the response to Eculizumab.
We do not yet fully understand the process of disease progression in MDS. We know certain genes are involved because mutations in those genes have been found in MDS patients. We need to understand which genes are important early in the disease and which ones act later. Our goal is to identify the genes that act early in MDS, so that ultimately, our research will lead to new treatment options that are more specific and effective.
The goal of our research is to gain a better understanding of disease progression in myelodysplastic syndrome (MDS) so more effective therapies can be identified. We are investigating the role of genes that are mutated early in disease progression. These early mutations are thought to have a role in driving the disease and could pre-dispose the cell to further mutations resulting in more severe disease. We are studying mutations in two groups of genes that are commonly mutated in MDS, regulators of epigenetic modifications and regulators of splicing. Both of these processes are tightly regulated in normal cells and alterations to them could affect many different genes. We want to understand how these two processes interact in MDS, and how they could trigger downstream mutations. Our research involves using zebrafish with a mutation in a key splicing factor, sf3b1, to look at what happens to epigenetic modifications when you lack Sf3b1. Zebrafish are useful for this type of study as they have a short generation time, their embryos are transparent and easy to manipulate. Additionally zebrafish have equivalents of almost all human genes.
We have found that mutations in sf3b1 lead to a decrease in the levels a key epigenetic gene, tet2. The tet2 gene produces an enzyme that modifies DNA by converting a methyl (5mC) residue to a hydroxymethyl residue (5hmC). We found a reduction in the overall levels of 5hmC in our sf3b1 mutant zebrafish. The levels of 5hmC are known to be altered in MDS and numerous other cancers. Our data provides evidence of a link between splicing and epigenetic modifiers. We hope to further our understanding of how these processes interact and determine how this interaction could affect other genes in the context of MDS. This research will provide valuable insight into the processes that are altered early in MDS and will facilitate the development of more effective therapies.
Myelodysplastic syndromes are a group of diseases that occur when blood cells acquire multiple mutations or genetic changes that alter cellular function and behavior. These mutations can occur as a normal byproduct of aging or can result from exposure to certain mutagenic agents. All mature blood cells are produced by hematopoietic stemcells (HSCs) via the formation of intermediate cells known as progenitors that will eventually give rise to all mature blood cell lineages. When mutations occur early in this process numerous downstream cells inherit these mutations and can then acquire further mutations resulting in a loss of the ability to make functional mature blood populations. The loss of normal functions in these cells is reflected in the low blood cell counts observed in MDS patients. Current therapies are often ineffective at removing mutated HSCs and early progenitors that can restart the MDS process resulting in relapse.
The goal of our research is to understand the earliest mutations and changes that occur in HSCs and their progenitors, so we can develop more effective therapies that efficiently target the majority of mutated cells. We are using zebrafish to study how mutations in two groups of factors thought to be drivers of disease (splicing factors and epigenetic modifiers) interact in MDS and how they could pre-dispose cells to further mutations. Our zebrafish have a mutation in a key splicing factor, sf3b1 (splicing factor 3b, subunit 1), which is one of the most commonly mutated genes found in MDS patient blood cells. These mutants have defects in early hematopoiesis, reinforcing the importance of splicing factors in blood cell development. Zebrafish are useful for this type of study due to their short generation time, ease of manipulation and they also have equivalents of almost all human genes.
In addition to splicing factors, we are also interested in epigenetic modifiers, specifically TET2, the most commonly mutated epigenetic modifier in MDS. TET2 encodes an enzyme that is important in DNA methylation, which is part of a global mechanism of regulating when genes are turned on or off. We found reduced levels of the tet2 gene in sf3b1 mutant zebrafish. We also found that tet2 and sf3b1 interact during early blood development in zebrafish. Additionally, we used a powerful technique to identify all the genes that were mis-regulated in sf3b1 mutants. From this analysis, we identified a number of epigenetic modifiers that are mis-regulated in response to loss of sf3b1 and we will be investigating how these genes alter the behavior and function of blood cells. Finally, we are performing a small molecule screen to look for compounds that correct the blood defect we see in sf3b1 mutants. This screen will be valuable in finding drugs that could represent novel therapeutic agents for MDS treatment.
Due to the generous support from the Aplastic Anemia & MDS International Foundation, we took a valuable step forward in understanding how early mutations contribute to the disease process in MDS. The data generated by this work will continue to be utilized to better understand how mutations in splicing and epigenetic genes contribute to blood development and how their mis-regulation can result in MDS.
Bone marrow failure is a serious condition that occurs when the bone marrow stops making enough healthy blood cells. A risk for bone marrow failure is genetic instability, including exacerbated shortening of telomeres (repetitive DNA sequences that cap chromosome ends). Using different genetic and biochemical approaches, this proposal will use cells derived from bone marrow failure patients that have telomere attrition as a platform for the development of clinical therapies against this disease. These experiments will increase our knowledge on stem cell function and regulation in bone marrow failure syndromes.
Telomeres represent the extremities of our chromosomes and are composed of long stretches of repetitive DNA sequences that are bound to several proteins, which are required to maintain its structure. It has been observed in humans that telomeres become progressively shorter with age. This shortening has been linked to the fact that every time a cell divides, it is unable to replicate the very end of our DNA molecules, represented by telomeres. Therefore, telomeres get progressively shorter with continuous cellular division throughout the human lifetime. When a cell reaches a critical telomere length, after several rounds of division, it becomes unable to divide and dies. Therefore it is not surprising that telomere shortening correlates with loss of tissue function, and has been associated with degenerative aging in humans.
The correct function of our tissues and organs is dependent on adult stem cells. When these cells divide they are able to maintain their own state, in a process termed self-renewal, and also generate the cells that perform the specific function in any given tissue. For instance, hematopoietic stem cells are blood-forming stem cells that are found in the bone marrow and therefore must be able to grow for the entire life of an individual, giving rise to 1 trillion blood cells every day. Therefore the maintenance of telomeres above critical length is vital for hematopoietic stem cells and the circulatory system. In fact, these cells have telomerase, a dedicated protein complex that elongates telomeres and maintains their stability. The consequences of not having efficient telomere maintenance are catastrophic for the circulatory system, since hematopoietic stem cells will become unable to maintain their self-renewal to generate blood cells. Several mutations in telomerase have been identified in patients suffering from dyskeratosis congenita and aplastic anemia, two severe forms of bone marrow failure. These patients have extremely short telomeres and are also at an elevated risk for developing cancer and other systemic tissue dysfunction.
Research regarding dyskeratosis congenita and aplastic anemia has been hampered by a lack of adequate models. To circumvent this issue, we are using human pluripotent stem cells harboring disease-associated mutations as a platform to understand the cellular and molecular mechanisms behind bone marrow failure caused by telomere shortening. Recently we developed the technology to differentiate these stem cells in a controlled, quantitative fashion, to become any particular blood cell type, such as red blood cells, present in the circulatory system. This allows us, for the first time, to reproduce the clinical effect of this disease, in a tissue culture dish, and therefore precisely understand the disease progression in dyskeratosis congenita and aplastic anemia. Our goal is to significantly increase the knowledge on the mechanisms leading to bone marrow failure in patients with mutations in telomerase. For that, we are thankful for the Aplastic Anemia & MDS International Foundation, whose generous support is essential to help our group delineate novel therapies against this devastating disease.
Myelodysplasia is a bone marrow failure syndrome with a tendency to progress to leukemia. A characteristic finding in blood cell precursors of some individuals with myelodysplasia is the ringed sideroblast, a cell that accumulates large amounts of toxic iron in mitochondria. Recently, the presence of these abnormal mitochondria has been correlated with mutations of the splice factor SF3B1. We plan to investigate the mitochondria of these cells with perturbed SF3B1, aiming to gain insight into mitochondrial causes of myelodysplasia. This may point to new therapies.
Myelodysplasia is a bone marrow failure syndrome with a tendancy to progress to leukemia. Recently mutations in the splice factor SF3B1 were linked to myelodysplasia and sideroblastic anemia. We used a drug meayamycin B (made to Dr K. Koide) to inhibit SF3B1 activity and to mimic the disease in bone marrow precursor cells. We have found that there are changes in iron metabolism that are unique to these precursor cells and that may be important for causing the disease.
Blood cells develop in the bone marrow and are released into the blood. Myelodysplasia refers to a group of disorders characterized by ineffective blood cell formation and a tendency to progress to leukemia, or cancer of the blood. Treatments of myelodysplasia are indirect and unsatisfactory, typically directed at mitigating the secondary effects of the disease and in reducing complications. With the advent of low cost DNA sequencing, scientists have been able to correlate changes in DNA of bone marrow cells with particular types of myelodysplasia. In particular, a high incidence of mutations in a gene, SF3B1, has been correlated with subtypes of myelodysplasia with ringed sideroblasts. Ringed sideroblasts are red blood cell precursors in the bone marrow that fail to develop normally because of iron trapped in the mitochondria. The goal of our project was to mutate or inhibit SF3B1 in immortal cell lines and in cells derived from healthy bone marrow to see if we could generate ringed sideroblasts. If inhibition of SF3B1 led to the formation of ringed sideroblasts, then we would biochemically characterize these cells and provide information that might lead to new therapeutic approaches.
Our initial experiments used an immortal (cancer) cell line (K562) that was derived from a cell with the potential to form red blood cells. These cells are difficult to manipulate genetically, so we employed a chemical inhibitor of SF3B1 called meayamycin. This was a chemical developed by our collaborator Kazunori Koide (University of Pittsburgh). Meayamycin did alter cellular iron metabolism, but not in the way that we hypothesized. The drug treated cells behaved in a way that would limit iron accumulation. Indeed when we measured iron uptake in cells that had been treated with meaymycin, we observed that cells accumulated less iron, not more. This was surprising because this drug had been reported (Visconte et al. 2012, Blood 120: 3173-3186) to trigger the formation of ringed sideroblasts in healthy bone marrow (blood precursor) cells. We attempted to repeat this published observation, but were unsuccessful. Instead the drug proved to be toxic with prolonged exposure and killed most of the bone marrow cells. We presented these results at the American Society for Hematology meeting in December 2014, at which another investigator informed us that he had seen the same results as us and did not see the formation of ringed sideroblasts. We decided to try a molecular genetic approach to inhibit SF3B1 called siRNA.
For the siRNA studies we had to use a different cell line (HeLa) that would more readily take up DNA to permit genetic manipulation. We used gene biomarkers to assess the effects of the siRNA. The siRNA technique was successful in that the level of SF3B1 in the cell was decreased by five-fold. We also observed changes in a gene called ABCB7, depletion of which had previously been shown to cause iron accumulation in mitochondria. Other changes, particularly those concerning genes involved in iron metabolism were the opposite to what we had observed with meaymycin and the K562 cell line. We tested the effect of meayamycin on the HeLa cells and observed the same results that we had obtained with the siRNA approach, indicating that the downstream effects of inhibiting SF3B1 are dependent on cell type.
In summary using two different methods of SF3B1 inhibition and different cell types we have shown that less SF3B1 does lead to altered cellular iron metabolism. However, the changes were complex and dependent on the cell type. Our research suggests that simple loss of function of SF3B1 in bone marrow cells does not cause formation of ringed sideroblasts. However the type of mutations in SF3B1 that have been correlated with the formation of ringed sideroblasts may be "gain of function" mutations. Recently a technique called CRISPR has been developed and we are currently using this to see if we can test this possibility.
Progress in understanding the etiology and effective treatment of MDS is currently hampered by the scarcity of tools to study this disease. Our goal is to harness cutting-edge human pluripotent stem cell and genetic engineering technologies to establish new models of an MDS subset characterized by loss of chromosome 7 material and use them as a novel platform to identify genes on chromosome 7 that are critical for this disease. These models should provide a powerful resource to the MDS community to investigate the cell biology, molecular pathogenesis and genetic basis of MDS, identify new therapeutic targets and perform drug screens.
Read more about this study.
Myelodysplastic syndrome (MDS) is the most common form of bone marrow failure in adults. Although a relatively common disease, we currently understand little about its etiology and we lack good models for its study. My laboratory is using a recent breakthrough technology which enables us to take cells from any individual and convert them into pluripotent stem cells, called induced pluripotent stem cells (iPSCs). These cells still contain all the genetic information of the individual they were derived from, including allpossible genetic abnormalities that may have caused or predisposed to disease. By using iPSCs derived from patient cells, we can develop new models of human disease. My laboratory was the first to generate iPSCs from patients with MDS and develop a “disease-in-a-dish” model of this syndrome.
In the project funded by the AA&MDS IF research grant we conducted the first studies to characterize the potential of blood formation of these cells and to compare it with this of normal cells derived from the same patients. These data demonstrate that our model indeed mimics hallmark characteristics of the disease and help us establish assays that we are now using to ask important questions about the genetic causes of MDS. This work will also provide us with a platform that we can use more generally in the future to study chromosomal defects implicated in myelodysplastic syndromes.
Induced pluripotent stem cells (iPSCs) -- adult cells reprogrammed back to an embryonic stem cell-like state--may better model the genetic contributions to each patient's particular disease. In a process called cellular reprogramming, researchers at Icahn School of Medicine at Mount Sinai have taken mature blood cells from patients with myelodysplastic syndrome (MDS) and reprogrammed them back into iPSCs to study the genetic origins of this rare blood cancer. The results appear in an upcoming issue of Nature Biotechnology.
In MDS, genetic mutations in the bone marrow stem cell cause the number and quality of blood-forming cells to decline irreversibly, further impairing blood production. Patients with MDS can develop severe anemia and in some cases leukemia also known as AML. But which genetic mutations are the critical ones causing this disease?
In this study, researchers took cells from patients with blood cancer MDS and turned them into stem cells to study the deletions of human chromosome 7 often associated with this disease.
"With this approach, we were able to pinpoint a region on chromosome 7 that is critical and were able to identify candidate genes residing there that may cause this disease," said lead researcher Eirini Papapetrou, MD, PhD, Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai.
Chromosomal deletions are difficult to study with existing tools because they contain a large number of genes, making it hard to pinpoint the critical ones causing cancer. Chromosome 7 deletion is a characteristic cellular abnormality in MDS and is well-recognized for decades as a marker of unfavorable prognosis. However, the role of this deletion in the development of the disease remained unclear going into this study.
Understanding the role of specific chromosomal deletions in cancers requires determining if a deletion has observable consequences as well as identifying which specific genetic elements are critically lost. Researchers used cellular reprogramming and genome engineering to dissect the loss of chromosome 7. The methods used in this study for engineering deletions can enable studies of the consequences of alterations in genes in human cells.
"Genetic engineering of human stem cells has not been used for disease-associated genomic deletions," said Dr. Papapetrou. "This work sheds new light on how blood cancer develops and also provides a new approach that can be used to study chromosomal deletions associated with a variety of human cancers, neurological and developmental diseases."
Reprogramming MDS cells could provide a powerful tool to dissect the architecture and evolution of this disease and to link the genetic make-up of MDS cells to characteristics and traits of these cells. Further dissecting the MDS stem cells at the molecular level could provide insights into the origins and development of MDS and other blood cancers. Moreover, this work could provide a platform to test and discover new treatments for these diseases.
Loss of genetic material from chromosome 7 (del7q) is a frequent karyotypic abnormality in MDS that is associated with poor prognosis and increased risk of progression to leukemia. Although this abnormality has been recognized since decades, we still do not understand the mechanism and the specific genes implicated. One reason for this is that chromosomal deletions are much harder to study than gene mutations because a large number of genes are included in the deletion and significant differences in the organization of chromosomes between species hamper their modeling in the mouse. To overcome these obstacles, we took advantage of cutting-edge stem cell and chromosome engineering technologies to model this deletion in human pluripotent stem cells. Specifically, we derived induced pluripotent stem cells (iPSCs) with del(7q) from the bone marrow of MDS patients. In parallel, we engineered chr7q deletions of various lengths in normal iPSCs through chromosome engineering technologies. Thus we were able to show that the culprit is reduced gene dosage (haploinsufficiency) and to pinpoint candidate genes on chr7q that may be critical to the pathogenesis of MDS.
In this study we established the first (and currently the only existing) iPSC lines from patients with MDS and developed the first iPSC model of MDS. This model opens a new research avenue for the study of MDS. It should find broader use in basic research into the pathogenesis of MDS, as well as in translational studies to guide clinical decision-making. These iPSC lines can also provide a new platform for drug screens to discover new compounds or repurpose existing ones, as well as in personalized medicine approaches. This work also established for the first time technologies for engineering large-scale chromosomal deletions in human cells that can be applied to the study of other disease-associated chromosomal deletions.
The genes responsible for MDS initiation are largely unknown. Recently, a group of genes that are important for splicing together RNA in cells were found to be mutated in up to 57% of MDS patients. How these mutations contribute to MDS initiation is unknown. We will determine whether a mutation in one of these genes (U2AF1) affects blood cell formation in mice and alters the splicing of RNA in bone marrow cells from mice and MDS patients.
Myelodysplastic syndromes (MDS) are blood cell diseases associated with life threatening infections and bleeding and can evolve to leukemia. Approximately 20,000 adults will be diagnosed with MDS each year and the associated mortality is high. We know that gene mutations occur in bone marrow cells from patients with MDS that are acquired sometime during their lifetime, but it remains largely unknown which gene mutations are responsible for MDS initiation. Recently, a group of genes that are important for splicing together RNA in cells were found to be mutated in up to half of MDS patients. How these mutations contribute to MDS initiation is unknown. We are studying a mutation in one of these genes (U2AF1) to determine how it alters blood cell formation and development. We observed that certain types of blood cells are affected more than others in young mice expressing the mutant gene and immature blood cells are increased. We are continuing to study these mice as they get older and will try to understand why blood cell development is abnormal. A better understanding of how U2AF1 mutations contribute to MDS development may uncover approaches that could be used to eliminate cells carrying these mutations in patients with MDS.
Myelodysplastic syndromes (MDS) are blood cell diseases associated with life threatening infections and bleeding and can evolve to leukemia. Approximately 20,000 adults will be diagnosed with MDS each year and the associated mortality is high. We know that gene mutations occur in bone marrow cells from patients with MDS that are acquired sometime during their lifetime, but it remains largely unknown which gene mutations are responsible for MDS initiation. Recently, a group of genes that are important for splicing together RNA in cells were found to be mutated in up to half of MDS patients. How these mutations contribute to MDS initiation is unknown. We are studying a mutation in one of these genes (U2AF1) to determine how it alters blood cell formation and development in mice. We observed that mice expressing the mutant U2AF1 gene have low white blood cell counts, similar to that seen in MDS patients. Mutant mouse expressing bone marrow cells also have changes in splicing of their RNA that are identical to bone marrow cells from MDS patients. Future studies will focus on how we could be use this mouse model to develop approaches to target cells carrying these mutations in patients with MDS.
Matthew Walter, M.D. of Washington University in St. Louis, a 2005 AA&MDSIF grantee, credited his grant from AA&MDSIF as the start of his research into the genomics of MDS. Dr. Walter and his colleagues recently published the results of their significant work in the New England Journal of Medicine. Watch his interview on the AA&MDSIF YouTube channel.