Project Details
Projekt
Long-read sequencing of SF3B1 mutated and wildtype chronic lymphocytic leukemia and myelodysplastic syndrome to identify mechanisms of genomic instability and therapy resistance and their impact on prognosis.
Subject Area
Hematology, Oncology
Term
from 2019 to 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 418085361
Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the Western world. One of the most frequently mutated genes in CLL is the splicing factor SF3B1, which is associated with an adverse outcome in CLL. Mutations in SF3B1 have been shown to induce splicing alterations and are associated with alterations of the DNA damage response, which in turn influences chromosomal stability. Interestingly, SF3B1 is also recurrently mutated in myelodysplastic syndrome (MDS), a disease of the hematopoietic stem cell. However, in contrast to CLL, SF3B1 mutations in MDS are associated with a favorable prognosis. The overall goal of this study is to analyze the effect of SF3B1 mutations on RNA splicing, repeat expansion and genomic stability in CLL. To answer these questions, we will employ long-read sequencing using single molecule real time sequencing (SMRT) by PacBio. We will perform Isoform sequencing of RNA (Iso-seq) of CLL-samples mutated and wildtype for SF3B1 and of healthy B-cells. Bioinformatics analyses will enable an improved detection of splicing isoforms in SF3B1 mutated samples in conjunction with our analyses with short-read sequencing in the CRU286 project (Aim 1). In addition, we plan to perform Iso-seq of MDS samples mutated and wildtype for SF3B1 and healthy hematopoietic stem cells and compare the isoforms affected by mutated SF3B1 mutations in MDS and CLL. This will enable us to uncover differences between SF3B1 mutated CLL and MDS cases which might be responsible for the differences in prognosis (Aim 2).Furthermore, besides Iso-seq we plan to perform long-read sequencing of genomic DNA to identify structural variations. Our analyses of the CRU286 short-read RNA-seq data have shown that repetitive RNA is significantly de-regulated in CLL in general, but also specifically in SF3B1 mutated cases. This, together with the connection between SF3B1 and the cellular stress response, renders alterations on a genomic level very likely which will be possible to identify with long-read sequencing. Taken together, the proposed project will ideally complement our functional analyses and (epi)genomic data sets from the CRU286 project. It will help to decipher the functional consequences of SF3B1 mutations in CLL and MDS and thereby may give insight in mechanisms of tumor progression.
DFG Programme
Research Grants
