Impact of RNA splicing factor mutations on circular RNA biogenesis in leukemia

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PROJECT SUMMARY/ABSTRACT Approximately 10% of new cancer diagnoses in the United States are hematological malignancies that include a spectrum of blood cancers and related disorders. In chronic lymphocytic leukemia (CLL) and myeloid neoplasms, somatic hotspot mutations frequently occur in five RNA splicing factors (SFs): SF3B1, SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA. These mutations drive aberrant splicing of mRNAs to promote leukemogenesis. Recently, a novel class of RNAs called circular RNA (circRNA) was found to be aberrantly expressed in many types of liquid tumors. Unlike mRNAs that form through normal splicing to produce linear RNAs, circRNAs are produced through backsplicing that results in RNA circularization. Because of advances to detection and annotation methods, circRNAs are now known to possess functional and clinical significances, suggesting a novel role in cancer biology. However, to date, their precise role in hematological malignancies, namely leukemia, remains undefined. The long-term goal is to investigate the functions and therapeutic potentials of circRNAs. Moreover, the role of SF mutations (SF3B1, SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA) in the aberrant expression of circRNAs remains unknown. Thus, the overall objective is to link SF mutations to aberrant circRNA expressions. To this end, I hypothesize that mutations in SF3B1 and other SFs such as SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA upregulate circRNA abundance to promote leukemogenesis. The rationale for this research is that linking SF mutations to aberrant expressions of circRNAs would define a novel regulatory axis, unlocking new therapeutic opportunities for treating leukemia. Preliminary data from CLL patient B cells and cell lines showed that mutant SF3B1 promoted aberrant expression of circRNAs in biological important molecular pathways such as protein transport and cell cycle regulation. To identify circRNAs critical for cell survival, I have validated the emergent CRISPR CasRX technology as a tool for screening circRNAs. I have also prototyped an experimental workflow for validating the functions of these circRNAs. Additionally, to further determine the role of SF mutations on circRNA biogenesis, cell lines with somatic mutations for SF3B1, SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA have been established. Finally, I have engineered cell-based and minigene-based reporter systems to investigate the mechanisms of backsplicing. Using this collection of tools, I propose the following aims: to identify and validate the functional impact of SF mutation-associated circRNA in leukemia (Aim 1) and to determine the mechanism of backsplicing (Aim 2). I expect the findings from this proposal will define a novel regulatory axis linking SF mutations and circRNAs aberrant expressions to leukemogenesis.