AA&MDSIF Research Grant Program
Our Researchers' Reports
Oxidative Stress in the Etiology of Myelodysplasia
Dr. Kay Macleod, University of Chicago
Erwin Umbach Research Study (2007-2009)
During the second year of our work funded by the AA&MDSIF, we have built on our findings from year one in which we identified a novel mechanism to explain how blood cells lacking the function of the RB tumor suppressor accumulate increased levels of reactive oxygen species and DNA damage that results in defective blood cell function. We show that pRB interacts with a DNA-damage sensing protein called Parp-1 and that loss of pRB is associated with abnormally high levels of Parp-1 activity that feeds back to induce reactive oxygen species and more DNA damage, thus producing a detrimental positive feedback loop. Chemical inhibition of Parp-1 both prevented accumulation of reactive oxygen species and DNA damage. Importantly, this also promoted normal maturation of RB deficient blood cells in vitro. Based on these novel findings, we examined the effect of treating a mouse model of bone marrow failure and anemia with chemical agents that inhibit the abnormal activity of Parp-1 and showed that this reduced levels of oxidative stress and delayed the onset of anemia in this mouse model of disease. We are currently preparing this work for publication. We will also be presenting this data in an oral presentation at the American Society of Hematology Annual Meeting in December 2009 during the Scientific Program Session on "Apoptosis: On matters of life and death".
Differential Inhibition of Normal Stem Cells in PNH 
Jaroslaw P. Maciejewski, Cleveland Clinic Foundation Taussig Cancer Center
Pursuing New Hope Pernick Family Research Study (2005-2008)
We were extremely fortunate to have received generous support for studies as to the basic disease mechanism in PNH; how do PNH stem cells take over the blood cell production in bone marrow of patients. Using most sensitive technique, DNA microchips, we tested whether we can detect additional genetic defects and mutations which would render PNH stem cell more viable and faster growing. We did not confirm this possibility and consequently, have conducted a further search for factors which predispose immune system to more likely attack healthy stem cells and spare PNH stem cells, a situation which likely leads to their growth advantage and PNH expansion in patients. These studies resulted in identification of several genetic variants in genes regulating immune system in PNH patients. Based on these finding we are currently developing therapeutic strategies which would enable us to reverse the conditions in the marrow so that remaining healthy stem cells can outcompete disease PNH cells and thereby reverse the disease process. During our investigations, we have also serendipitously identified a pathway involving PNH proteins that may explain the propensity to blood clots in PNH.
PNH as an orphan disease is very difficult to study because the funding sources are very limited and in match with other common and well publicized diseases research applications are not competitive. The grant we have received allowed us to devote time to systematic investigations in PNH. Thank to the funding obtained we were able to investigate the mechanisms leading to selective outgrowth of PNH stem cell. Using high density DNA chips we have demonstrated that there are no genomic differences between the normal and PNH clone derived from the same patient, a finding that suggest mechanisms of clonal selection do not operate in from PNH stem cell but are rather. Through high density genotyping we have identified a number of genetic polymorphic variants which could explain propensity for PNH evolution in individual patients. Thank to the funding provided with the help of AA&MDS Int Foundation and incredible gifts of the donors, discovery of these variants allowed us to prepare larger studies involving whole genome scanning technology for which are currently await funding from federal sources. If successful the impact of initial funding will be amplified.
Microarray Profiling of Micro RNA in 5q-Syndrome
Dr. Lubomir Sokol, H. Lee Moffitt Cancer Center and Research Institute
AA&MDSIF New Investigator Award
Harold Spielberg Research Study (2007-2009)
miRNAs are small non-coding RNAs that post-trascriptionally regulate the expression of many important genes in normal and cancer cells including tumor suppressor genes and oncogenes. The Aplastic Anemia/MDS Foundation Grant supported our initial research in this new field. We studied a role of miRNAs in the pathobiology of MDS. We found that miRNA expression is significantly dysregulated in MDS vs. normal bone marrow cells suggesting that miRNAs could serve as novel biomarkers. We have also discovered a unique miRNA signature that predicted the prognosis in MDS patients. In our future research we would like to investigate the role of miRNA target molecules in the pathogenesis of MDS and use this knowledge in the development of novel personalized therapies for patients with MDS.
Telomere Maintenance in Patients with Aplastic Anemia 
Dr. Hinh Ly, Emory University
Holly Cataldo Memorial Research Study (2006-2008)
AA&MDSIF Established Investigator Award
Scientific Report (pdf)
We have recently discovered various mutations in a protein called TIN2 in patients with acquired aplastic anemia. TIN2 is a protein that is known to help maintain the integrity of the human genome. We examined samples collected from 142 AA patients and 289 healthy controls and found several TIN2 mutations in blood cells collected from AA patients. Using various assays, we showed that blood cells with TIN2 mutations grew slower than cells collected from healthy individuals, consistent with the fact that cells from patients turned over quicker than those of healthy individuals. By using various standard laboratory tests, we showed that several mechanisms might be involved in mediating premature cellular aging in patients with TIN2 mutations.
As a complementary approach, we defined the regulatory elements that control TIN2 protein expression under normal physiological condition. Results obtained from these studies offered important insight into how TIN2 mutations can lead to genome instability and to the limited marrow stem-cell reserve and renewal capacity in patients with various forms of bone-marrow failure syndrome (BMFS). To our knowledge, this is the first comprehensive functional analysis of all known mutations in the TIN2 protein in patients with AA or BMFS. This study may offer a new cellular target for the development of novel therapies against these debilitating diseases. Two separate manuscripts are in preparation to report these novel findings. We have also recently reported findings of a related study in a reputable journal (BLOOD), where we cited financial support by the AA&MDSIF (Holly Cataldo Memorial Research Study).
GENOMIC STABILITY IN MDS
Dr. Christine L. O'Keefe, Cleveland Clinic
Lindsay Minelli Research Study (2006-2008)
AA&MDSIF Young Investigator Award
Scientific Report (pdf)
Myelodysplastic syndrome is a disorder that affects the body's ability to properly make blood cells. In about half of the people with this disease, the chromosomes in the blood cells and bone marrow stem cells (the cells responsible for blood production) have been damaged. These defects are characteristic of myelodysplastic syndrome, and they can have an impact on the treatment and the progression of the disease. A standard technique, called metaphase chromosome analysis or karyotyping, is routinely used to detect these changes. However, karyotyping is not very sensitive and can only identify very large changes. It is possible that smaller chromosomal changes exist in the blood cells of patients with myelodysplastic syndrome. These so-called cryptic changes may also have an impact on diagnosis and treatment, but our standard clinical tests cannot detect them.
Recently, a new technique called single nucleotide polymorphism (SNP) arrays have been developed that can detect much smaller physical changes on chromosomes. Gene microarrays are similar to computer chips and allow us to quickly examine all the chromosomes of an individual and will greatly increase the number of patients we can test. Our laboratory has a very large collection of DNA from patients with myelodysplastic syndrome and other related diseases. Also, we have established a specialized clinical program for these diseases, therefore we will have access to enough patients to perform our study properly. We have identified small chromosomal changes in patients with myelodysplastic syndrome and other related diseases that affect the blood and bone marrow. We also identified a novel chromosomal change, uniparental disomy, and found it was common in myelodysplastic syndrome. We showed that these changes could affect the outcome of the disease, similar to the large changes previously identified by metaphase karyotyping. Finally, we studied another type of chromosomal change, copy number variants, and their association with myelodysplastic syndrome. Our investigations show that SNP arrays are a useful tool for studying blood and bone marrow diseases, and that they might directly affect diagnosis and treatment.
