Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis

SUMMARY Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ~11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently-mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in MDS patients.

We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of patients with de novo myelodysplastic syndromes (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis.

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MLL rearrangements are translocation mutations that cause both acute lymphoblastic leukemia and acute myeloid leukemia (AML). These translocations can occur as sole clonal driver mutations in infant leukemias, suggesting that fetal or neonatal hematopoietic progenitors may be exquisitely sensitive to transformation by MLL fusion proteins. To test this possibility, we used transgenic mice to induce one translocation product, MLL-ENL, during fetal, neonatal, juvenile and adult stages of life. When MLL-ENL was induced in fetal or neonatal mice, almost all died of AML. In contrast, when MLL-ENL was induced in adult mice, most survived for >1 year despite sustained transgene expression. AML initiation was most efficient when MLL-ENL was induced in neonates, and even transient suppression of MLL-ENL in neonates could prevent AML in most mice. MLL-ENL target genes were induced more efficiently in neonatal progenitors than in adult progenitors, consistent with the distinct AML initiation efficiencies. Interestingly, transplantation stress mitigated the developmental barrier to leukemogenesis. Since fetal/neonatal progenitors were highly competent to initiate MLL-ENL–driven AML, we tested whether Lin28b, a fetal master regulator, could accelerate leukemogenesis. Surprisingly, Lin28b suppressed AML initiation rather than accelerating it. This may explain why MLL rearrangements often occur before birth in human infant leukemia patients, but transformation usually does not occur until after birth, when Lin28b levels decline. Our findings show that the efficiency of MLL-ENL–driven AML initiation changes through the course of pre- and postnatal development, and developmental programs can be manipulated to impede transformation.