Myelodysplastic syndromes (MDS) are premalignant hematopoietic stem cell disorders characterized by peripheral blood cytopenias, ineffective hematopoiesis, and an increased proneness to acquire acute leukemia. Few drugs can stabilize the disease, and typically only transiently. No new drugs have been approved by the FDA and although there are therapeutics currently under development, they are unlikely to be approved. Recent results from sequencing studies showed that over half of MDS patients have splicing factor (SF) mutations, the 3 most commonly mutated being: splicing factor 3B, subunit 1 (SF3B1), serine/arginine- rich splicing factor 2 (SRSF2) and U2 small nuclear RNA auxiliary factor 1 (U2AF1). SF mutations occur early in the course of the disease, which along with their prevalence in MDS strongly suggests that SF mutations are instrumental in the pathogenesis of the condition and may hold the key to new therapeutic opportunities, but the downstream mechanisms are currently unknown. All mutations are heterozygous and specific to certain hotspots, which suggests that the mutations result in a functional alteration of the SFs rather than just functional impairment. The Papapetrou laboratory has pioneered the use of pluripotent stem cells (iPSC) to model MDS and has provided proof-of-principle of the model system being appropriate for studying the genetic mechanisms of MDS. They have derived iPSC lines with the SRSF2 P95L mutation and validated that MDS phenotypes can be observed upon hematopoietic differentiation. We used an enhanced version of the CLIP- seq method (eCLIP) to characterize in preliminary experiments the interaction between mutant SRSF2 and RNA. In this project we will investigate the role of these SF mutations and seek to identify downstream effects of each mutation. This will help us gain new insight into the pathogenesis of MDS and identify mechanisms and pathways that may constitute promising therapeutic targets, and the iPSC lines that will be generated can provide a new platform for drug screening. During this fellowship I will learn the details of the many ground- breaking methods used in the Papapetrou lab to use iPSCs for disease modeling. With the help of my co- sponsor I will build on the skills in genetic engineering I will learn from the Papapetrou lab and also become immersed with the methods of CRISPR-screening. The cutting-edge techniques used in the proposed aims will also generate a lot of data for which more intricate analysis methods can be developed to further challenge my problem solving skills as a bioinformatician in new ways.
Myelodysplastic syndromes (MDS) are common premalignant hematopoietic stem cell disorders for which no new drugs have been approved in recent years. Splicing factor (SF) mutations have recently been discovered as the most frequent class of mutations in MDS. In this research proposal we will use induced pluripotent stem cells with MDS-specific SF mutations to characterize downstream effects of the mutations in order to gain new insights into the pathogenesis of MDS and identify new therapeutic targets.
