University thesis:
Dissertation, Universität Freiburg, 2021
Footnote:
Description:
Abstract: Myelodysplastic syndromes (MDS) represent a heterogenous group of clonal stem cell disorders with the potential of malignant transformation to acute myeloid leukemia. The disease is caused by recurrent genetic lesions affecting different pathways of the myeloid precursor cell, including splicing factors (SF). Recurrent mutations in the splicing factor SRSF2 occur frequently in myeloid neoplasms and portend a poor prognosis. To determine how mutations in SRSF2 alter its function in vivo, hematopoietic cell lines with inducible expression of Flag-tagged SRSF2WT and SRSF2P95H were generated. HITS-CLIP (“High-Throughput Sequencing after UV-Cross-Linking and RNA Immuno-Precipitation) and RNA deep sequencing (RNA-Seq) were performed to correlate RNA binding and splice events. Exon-specific primers were designed around putative molecular targets of both differential binding and splicing. The identified genes of interest were functionally characterized via cloning and knockdown experiments.<br><br>In the present work, we observe that the majority of differential binding events do not translate into alternative splicing of exons with SRSF2P95H binding sites. Rather, alternative splice alterations appear to be dominated by indirect effects. SRSF2P95H targets are enriched in RNA processing and splicing genes, including several members of the hnRNP and SR families of proteins, suggesting a “splicing-cascade” phenotype wherein mutation of a single SF leads to widespread modifications in multiple RNA processing and splicing proteins. We demonstrate that multiple SF, most notably of the hnRNP protein family, are recurrently mis-spliced in diverse SRSF2MUT cell systems and SRSF2MUT primary patient samples. Furthermore, we find that SRSF2 mutations result in the retention of a poison intron of the tumor suppressor and splicing factor DDX41. Interestingly, the burden of SF mis-splicing in SRSF2MUT patients correlates with increased splicing of anti- apoptotic splice isoforms, including the usage of a cryptic 5’ splice site of CASP8. By differentiating Srsf2P95H/WT EML cells down the myeloid lineage, we illustrate that the splice outcomes of the identified target genes are dynamic in nature and influenced by differentiation. We show that the effects of the SRSF2 mutation on hematopoiesis and lineage skewing are mirrored by the knockdown of hnRNP target genes, such as HNRNPA2/B1 and HNRNPH1. The obtained technical skills and understanding of myelopoiesis was then transferred to the University of Freiburg Medical Center, where a model was developed to study oncogenic fusion protein variants of AML1/ETO discovered in t(8;21)/t(9;22)-overlap leukemia.<br><br>In summary, changes of in vivo consensus motif binding preference underline the highly context- specific nature of SRSF2MUT-mRNA interactions. Our data suggests a model, whereby subtle, but broad disruption of splicing sets off a cascade of gene regulatory events that together affect hematopoiesis and drive cancer. These findings provide the mechanistic rationale for studying the generation of neoepitopes in SRSF2 mutant disease and ascertaining the role of immune therapy in exploiting this potential Achilles heel