Acquired Molecular Defects in Spliceosome Machinery: Novel Pathogenetic Pathways in Myeloid Leukemogenesis

Abstract 271

In MDS and other myeloid malignancies, deletions of chromosomal material can lead to decreased gene expression. Inactivating mutations may have similar consequences leading to decreased or absent gene function. Through these events dysfunction of tumor suppressor genes (TSGs) constitutes the key pathogenic mechanism in clonal evolution. In recent years, a large number of new somatic mutations affecting a variety of TSGs have been identified.

In the search for new mutational events associated with MDS subtypes or phenotypes, we have applied whole exome sequencing to a large group of MDS patients. In a systematic analysis of somatic events screened for recurrence and potential functional significance, we noted several somatic mutations affecting genes involved in the splicing machinery. The somatic SF3B1 mutation, initially found in a patient with refractory anemia with ring sideroblasts (RARS) and thrombocytosis (T), was subsequently detected in 22/32 patients with RARS/RARST, but not in 58 patients with other forms of MDS and MDS/myeloproliferative neoplasms (MPN) and <15% ring sideroblasts. However, in another index case, we have identified a mutation in another member of the spliceosome gene family: U2 snRNP auxiliary factor (U2AF)1 (chromosome 21q22.3), encoding the splicing factor SR family protein which closely interacts with SF3B1 and plays a critical role in recognition of the pre-mRNA branch sites. Subsequent targeted screening detected additional mutations in a total of 23 of 247 adult patients with secondary and primary acute myeloid leukemia (AML) (9%), advanced forms of MDS (11%), and lower-risk MDS (6.5%). U2AF1 mutations were most frequently observed in chronic myelomonocytic leukemia (CMML) (17%) and unlike SF3B1 mutations (with which they were mutually exclusive) were found only in 2/36 RARS/RARSt patients (6%). Similar to SF3B1, no mutations were found in juvenile myelomonocytic leukemia (JMML) and pediatric AML (N=112). Based on the chromosomal location of U2AF1 we next examined trisomy 21 and UPD21q. Homozygous mutations were found in 1 case, and, 2 copies of the mutation were detected in trisomy 21. In addition, we identified a 7-Mbp deletion encompassing U2AF1, resulting in reduced copy number of wild type. In serial samples collected at initial presentation of MDS in patients who subsequently progressed to AML, U2AF1 was identified from the very beginning of the clonal process. In total, we have identified 4 different missense mutations, located in 3 residues (S34, R156 and Q157) in 2 zinc finger domains, the most common of which was predicted to affect amino acid S34 and Q157 (in 10 and 11 patients, respectively). U2AF1 and other spliceosome proteins are abundantly expressed in CD34 cells: analyses of MDS samples showed that expression of U2AF1 was decreased (<2 fold) in 7/55, 8/80 and 12/48 patients with RA, RA with excess blasts, and RARS. Finally, after adjustment for other confounding factor mutations in U2AF1, we determined that these lesions were associated with decreased overall survival (p=.08) in the CMML cohort. Of note is that mutations of DNMT3A and ASXL1 genes frequently coincide with U2AF1 mutations, which are often present in patients with normal cytogenetics.

Discovery of other mutations in spliceosomal genes LUC7L2 (7q34) and PRPF8 (17p13.3) in a similar phenotype further substantiated the notion that spliceosomal machinery TSGs are frequently targeted by mutations. LUC7L2 is associated with the U1 snRNP spliceosomal subunit and involved in recognition of splice donor site. PRPF8 is a component of the U5/U4/U6 tri-snRNP and participates in both U2 and U12 splicing. Interestingly, loss of heterozygosity of these 2 spliceosome associated gene regions were also seen in cases with U2AF1 mutations. Moreover, a LUC7L2 nonsense mutation was detected in a secondary AML with U2AF1 mutation. For the further evaluation of functional significance of U2AF1 mutations, splice status was examined by RT-PCR based analysis. In mutant cases, pre-mRNA of several genes, including P53 and PRPF8 were not completely spliced and normally spliced fragments were decreased compared with wild type cases or healthy donors. Unspliced P53 genes were wild type.

Our findings suggest that different components of spliceosome dysregulation might have a synergetic effect on clonal evolution in myeloid malignancies, and that dysfunction of RNA splicing is a common pathway in leukemogenesis.