Myelodysplastic syndromes (MDS) are characterized by dysplastic ineffective hematopoiesis, cytopenias and leukemic evolution. New technological advances have allowed for improved analysis of genetic somatic defects. Among newly identified lesions, several spliceosomal factor genes were frequently found to be mutated. These included common mutations in SF3B1, ZRSR2, SRSF2 and U2AF1 as well as less prevalent mutations in PRPF8, DDX41 and others. This discovery has led to the hypothesis that alterations in the pattern of splicing of target genes plays a major role in the establishment or progression of MDS. This proposal focuses on our recent identification of inactivating mutations in the LUC7L2 gene and frequent haploinsufficiency due to deletions at 7q34 involving the LUC7L2 locus. Clinical analysis shows that deletion/low expression of LUC7L2 is associated with poor outcome. Recently published data show that LUC7L2 can reverse defective differentiation in del7q human iPS cells. Preliminary data shows that engineered human iPS cells with LUC7L2 haploinsufficiency have defective differentiation similar to del7q. LUC7L2 is thought to regulate 5' splice site choice during splicing. RNA-Seq results suggest that multiple genes have altered splicing patterns due to defects in LUC7L2. Our proposal is based on the hypothesis that mutations and/or haploinsufficiency of spliceosomal proteins such as LUC7L2 leads to specific types of missplicing of specific or distinct combinations of TSG and ultimately, that spliceosomal defects may phenocopy consequences of other molecular defects affecting specific genes or pathways. The goals of the proposal are to understand the consequences of LUC7L2 deficiency in relation to the development and progression of MDS. The effects of haploinsufficiency in primary cells and in engineered shRNA knockdown cells and LUC7L2 knock out iPS cells will be investigated in culture including effects on proliferation, differentiaion and apoptosis. We will perform splicing analysis using deep RNA sequencing to characterize splicing dysfunction and its consequences on mRNA expression to determine downstream mechanisms and target genes. To identify direct splicing targets, RNA splicing assays will be performed in vivo and in vitro with LUC7L2 knocked down and mutant knock-in cells. RNA CLIP-Seq and direct RNA binding analyses will be used to define the in vivo RNA substrates of LUC7L2 action. Finally, we will examine the roles of candidate downstream genes whose splicing and expression is altered in LUC7L2 defective cells and patient samples. Preliminary data suggests that LUC7L2 levels regulate the splicing of several downstream genes including SMAD5. Using engineered cell lines and/or patient samples, we will test the roles of the missplicing events by inducing or inhibiting alternative splice sites using antisense morpholino oligonucleotides. While in vitro testing will examine growth and differentiation, in vivo experiments will be carried out in NSG mouse xenografts containing knockdown, knock out or primary MDS cells haploinsufficient in LUC7L2.

Public Health Relevance

Recently, frequent somatic mutations in spliceosomal protein genes have been discovered in myeloid malignancies, implicating spliceosomal dysfunction as a possible novel pathway of MDS evolution. The spliceosome is responsible for processing most mRNAs in human cells. Defective processing of RNA may have various functional consequences, including mutation-specific miss-splicing and aberrant alternative splicing patterns. We propose to test the hypothesis that the oncogenic mechanisms or transforming potential of spliceosomal mutations are mediated through miss-splicing of distinct combinations of tumor suppressor genes (TSG). Thus, spliceosomal mutations may result in pathogenetic consequences similar to those produced by mutations or haploinsuffciency of TSG. Characterization of the consequences of defective spliceosomal machinery in MDS will lead to a better understanding of the pathogenesis of myeloid malignancies and point to novel therapeutic targets.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Clinical Oncology Study Section (CONC)
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El Kassar, Nahed
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Cleveland Clinic Lerner
Other Basic Sciences
Schools of Medicine
United States
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Saygin, Caner; Hirsch, Cassandra; Przychodzen, Bartlomiej et al. (2018) Mutations in DNMT3A, U2AF1, and EZH2 identify intermediate-risk acute myeloid leukemia patients with poor outcome after CR1. Blood Cancer J 8:4
Nagata, Yasunobu; Narumi, Satoshi; Guan, Yihong et al. (2018) Germline loss-of-function SAMD9 and SAMD9L alterations in adult myelodysplastic syndromes. Blood 132:2309-2313
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