Through large-scale DNA sequencing, we recently identified the core spliceosome factor, SF3B1, as a novel cancer gene in chronic lymphocytic leukemia (CLL). SF3B1 functions in the catalytic core of the U2 small nuclear ribonucleoprotein (U2 snRNP), an essential RNA-protein complex involved in pre-mRNA splicing. Several features of this new cancer gene make it a high priority for investigation. First, SF3B1 is mutated at a high frequency in CLL, with all mutations localizing to a discrete gene region, and with half recurrent at K700E. Second, mutation in SF3B1 is significantly associated with del(11q), a cytogenetic abnormality associated with aggressive disease, and yet, is an independent predictive marker of poor prognosis. Third, CLL samples with SF3B1 mutation demonstrate altered pre-mRNA splicing in known spliceosome target genes compared to samples with wildtype SF3B1. Finally, SF3B1-K700E was recently identified as a recurring mutation in myelodysplasia. Mutations in SF3B1 and modulation of RNA splicing are likely to represent a novel oncogenic process across hematologic malignancies and preferentially in aggressive forms of CLL. Our hypothesis is that mutated SF3B1 generates mis-spliced pre-mRNAs, encoding proteins that promote leukemogenesis. Our strategy for functionally linking SF3B1 mutation and CLL pathogenesis is to first define the effects of SF3B1 mutation on survival and proliferation of B cells, and on other pathways that we recently identified as critical to CLL (inflammation, Notch1 and Wnt signaling, DNA damage/repair) (Aim 1). Second, we will identify the mechanism by which these changes in cellular function are mediated (Aim 2). Since SF3B1 is essential for RNA splicing, we will examine the effects of SF3B1 mutation on critical protein-protein and protein-RNA interactions within the spliceosome, and use RNA sequencing to globally identify the novel splice variants generated by SF3B1 mutation. Understanding of the critical cellular processes affected by SF3B1 mutation (per Aim 1) will prioritize the candidate splice variants to be functionally validated as crucial to promoting CLL. Because SF3B1 mutation appears to function in a cell lineage specific context, we will carry out all studies in B cell lines or primary normal B cells;into which mutated or wildtype SF3B1 is introduced using novel biomolecule delivery or infection methods that our group has pioneered. Additionally, we will validate our findings using CLL samples naturally harboring SF3B1 mutations. Third, to definitively establish the role of SF3B1 mutation in driving CLL, we are generating a transgenic conditional knock-in SF3B1-K700E mouse (Aim 3). We will examine these mice for in vivo evidence of lymphohematopoietic accumulation of clonal B cells when SF3B1-K700E expression is restricted to CD19+ B cells alone or in combination with other CLL-generating genetic alterations. The proposed studies are anticipated to provide critical insights into a novel splicing mechanism underlying CLL (with implications for other blood cancers) that are expected to contribute to improved strategies to treat this incurable disease, especially given the development of spliceosome inhibitors.

Public Health Relevance

Through large-scale DNA sequencing of patient samples, we recently discovered a new cancer gene -- SF3B1 -- which controls how specific segments of RNA are joined together (called 'RNA splicing') to make a full messenger RNA which is then used to make proteins. We found that SF3B1 is defective in an aggressive form of the most common adult B cell leukemia, chronic lymphocytic leukemia (CLL). We now propose to study how SF3B1 contributes to the development of leukemia, for example, by finding out which mRNAs are spliced incorrectly in CLL patients with defective SF3B1. By understanding how RNA splicing is defective in CLL, we hope to deepen our knowledge of how splicing contributes to the generation of this and other cancers, and to suggest new therapeutic targets for this currently incurable cancer.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL116452-01
Application #
8417317
Study Section
Molecular and Cellular Hematology (MCH)
Program Officer
Di Fronzo, Nancy L
Project Start
2013-02-01
Project End
2018-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
1
Fiscal Year
2013
Total Cost
$413,759
Indirect Cost
$175,759
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
076580745
City
Boston
State
MA
Country
United States
Zip Code
02215
Gruber, Michaela; Wu, Catherine J (2014) Evolving understanding of the CLL genome. Semin Hematol 51:177-87
Landau, D A; Carter, S L; Getz, G et al. (2014) Clonal evolution in hematological malignancies and therapeutic implications. Leukemia 28:34-43
Wan, Youzhong; Wu, Catherine J (2013) SF3B1 mutations in chronic lymphocytic leukemia. Blood 121:4627-34
Landau, Dan A; Wu, Catherine J (2013) Chronic lymphocytic leukemia: molecular heterogeneity revealed by high-throughput genomics. Genome Med 5:47