The candidate, Dr. Rafael Bejar, presents a 5-year career development plan that seeks to characterize the broad range of mutations present in patients with myelodysplastic syndromes while establishing an academic career as a physician scientist in the field of hematology.
The specific aims of this proposal are to: (1) study a clinically annotated cohort of 439 bone marrow samples from patients with MDS to determine if the mutations they carry are associated with common clinical features of MDS and overall patient survival, (2) perform a pooled shRNA screen targeting each of the genes from the commonly deleted regions of chromosome 5q to determine which genes, when knocked down in human CD34+ hematopoietic cells, lead to clonal expansion in a stromal co-culture system, and (3) functionally characterize EZH2 mutations identified in MDS patient samples to determine their effects on hematopoietic differentiation, clonal expansion, and mature cell function. Myelodysplastic syndromes are clonal disorders of hematopoiesis that cause inefficient blood cell production, low blood counts, and risk of progression to acute leukemia. The prognosis for patients with MDS is highly variable, ranging from weeks to years. Chromosomal abnormalities present in some cases help stratify patients into risk groups, but prognosis is largely determined by clinical parameters. MDS patients are known to have acquired DNA mutations in their diseased cells, however, these genetic lesions have yet to be incorporated into prognostic scoring systems. Molecular markers are needed to better classify subtypes of MDS, define prognostic risk, and identify pathogenic mechanisms associated with the development and progression of these disorders. There is abundant evidence that genes that are somatically mutated and functional in one tumor type can play a role in other tumor types. A comprehensive analysis of known cancer genes in MDS has not been performed. In order to identify oncogenic mutations in MDS, a set of DNA primer extension/mass spectroscopic assays will be used to determine which of 439 patient samples contain any of 953 mutations in 111 known cancer genes. Tumor suppressor genes known to be mutated in MDS and other myeloid disorders will be sequenced in this cohort using the 454 quantitative next-generation sequencing platform. These highly sensitive techniques allow for detection of mutations even if samples contain a large fraction of normal cells or mutations are present in only a small subclone of diseased cells. With the help of collaborator Donna Neuberg and her team, a statistical analysis will be performed to determine if mutations are correlated with MDS phenotypes and the overall survival of patients even after known risk factors are considered. In collaboration with Dr. David Root from the RNAi Consortium at the Broad Institute, a pooled short hairpin RNA interference screen in human CD34+ hematopoietic cells will be performed. Genes from regions of chromosome 5q that are commonly deleted in patients with MDS will be targeted to determine which confer a clonal advantage when knocked down. Finally, the functional role of MDS associated EZH2 mutations will be examined in human hematopoietic assays. These include tests of clonal expansion, differentiation, histone methylation, and mature cell functions such as neutrophil activation, migration, phagocytosis, and actin polymerization. Dr. Bejar is well qualified to carry out the research outlined in this proposal. He has successfully completed a project of comparable complexity as part of his PhD thesis. His mentor, Dr. Benjamin Ebert, has experience studying the genetic basis of myelodysplastic syndromes and has published the result of short hairpin RNA interference screen that discovered RPS14 as a critical gene lost in patients with the 5q-minus syndrome subtype of MDS. Along with Dr. Ebert, Dr. Bejar has published an invited review on the genetic basis of MDS in Hematology/Oncology Clinics of North America and a review on pathogenic mechanisms in MDS in the Journal of Clinical Oncology. The preliminary work described in this proposal has been accepted for publication in the New England Journal of Medicine. Dr. Bejar has recruited a team of outstanding mentors that that in addition to Dr. Ebert, include Dr. David Williams as a co-mentor, Dr. David Steensma as a clinical mentor, and Dr. Nancy Berliner, head of the Hematology division at Brigham and Women's Hospital, as a fourth member of his advisory committee. The advisory committee will meet, at minimum, every six months to ensure a successful scientific research program. Included in the career development plan are courses on biostatistics and genomic methods at the Harvard Medical School and School of Public Health. Successful completion the specific aims and career development plan outlined in this proposal will allow the candidate to advance his academic career as an independent investigator in the field of hematology.

Public Health Relevance

The goals of this project are to identify genetic mutations associated with the development and clinical manifestations of myelodysplastic syndromes (MDS) and to model their effects on hematopoietic growth and differentiation in vitro. To accomplish these goals, we will perform: (1) a survey of mutations in known cancer genes in a large cohort of MDS patient samples with detailed clinical annotation, (2) a pooled shRNA screen to identify genes from the commonly deleted portion of chromosome 5q, that when knocked down, provide a clonal advantage to hematopoietic cells, and (3) functionally characterize the mechanisms through which mutations in the EZH2 gene contribute to the development and progression of MDS. We anticipate that the results of these studies will identify novel genetic lesions and pathogenic mechanisms that can molecularly classify subtypes of MDS, helping to predict survival of patients with these disorders.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Clinical Investigator Award (CIA) (K08)
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Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
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Bishop, Terry Rogers
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Brigham and Women's Hospital
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Bejar, Rafael; Stevenson, Kristen E; Caughey, Bennett et al. (2014) Somatic mutations predict poor outcome in patients with myelodysplastic syndrome after hematopoietic stem-cell transplantation. J Clin Oncol 32:2691-8
Schneider, Rebekka K; Ademà, Vera; Heckl, Dirk et al. (2014) Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS. Cancer Cell 26:509-20
Bejar, Rafael; Lord, Allegra; Stevenson, Kristen et al. (2014) TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients. Blood 124:2705-12
Murphy, D M; Bejar, R; Stevenson, K et al. (2013) NRAS mutations with low allele burden have independent prognostic significance for patients with lower risk myelodysplastic syndromes. Leukemia 27:2077-81