Myelodysplastic syndrome (MDS) is a grouping of disease phenotypes characterized as hematological pre-malignancies. MDS treatment focuses on preventing the further transformation of diseased cells into acute myeloid leukemia (AML). A recent unique finding of myeloid pre-malignancies and malignancies is the mutation of components of the spliceosome, the nuclear complex that catalyzes pre-mRNA splicing during gene expression. Whole exome sequencing of MDS patients has identified novel mutations of the gene DDX41 in the germline and somatically. The germline mutation results in a frameshift in the protein, most likely leading to a nonfunctional polypeptide, while the somatic mutation is missense. Patients present primarily as compound heterozygotes, and these mutations confer a more advanced stage of disease and a decreased patient survival outcome. The gene DDX41 encodes a member of the DEAD-box RNA helicase family. Although proteomics has identified DDX41 as a component of the spliceosome catalytic core, no information is available on the exact role the protein plays in splicing or on its interaction partners in the spliceosome. We hypothesize that DDX41 is a bona fide tumor suppressor gene (TSG) and that mutations of it result in the aberrant splicing of other TSGs, resulting in myeloid tumorigenesis. In this project, we will investigate the roles of DDX41 in the spliceosome as well as in myeloid neoplasia. We will determine the RNA interaction sequences of wild type and mutant DDX41 in vivo using a form of crosslinking-immunoprecipitation to purify RNAs bound to DDX41 followed by high-throughput sequencing to analyze these RNAs. We will investigate the effects of DDX41 mutations on the biochemical steps of splicing using DDX41-deficient nuclear extract created from mammalian cells in an in vitro system. We will analyze alterations in splicing as well as cellular phenotype conferred by DDX41 mutations in mammalian cells using in vivo splicing assays as well as RNA- seq. Since MDS patients with DDX41 mutations respond very well to lenalidomide treatment, we will study the impact of this drug on DDX41 both in vivo and in vitro to determine a potential mechanism of action. Successful completion of this application will result in new information on the role a spliceosome core component plays in gene expression as well as disease. Furthermore, the opportunities provided by this fellowship application will enhance the training of a young scientist in the process of collaborative translational research.

Public Health Relevance

Advances in hematology patient sequencing have led to the discovery of mutations in genes encoding components of the spliceosome, the nuclear complex responsible for the splicing of precursor messenger RNA. The goals of this application are to analyze the biochemical role of the protein DDX41 in the spliceosome and how mutations of DDX41 promote malignant transformation of certain blood cells. The knowledge gained from this application may prove beneficial in the generation of new treatment options for patients with these mutations.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31CA192885-03
Application #
9332094
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mcneil Ford, Nicole
Project Start
2015-09-01
Project End
2018-08-31
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195