Bon Ham Yip

Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells.

The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34+ cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicing/processing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1–BCLAF1–SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34+ cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.

Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1 -mutant myelodysplastic syndromes

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3′ splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3′ splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts

Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.

The U2AF1S34F mutation induces lineage-specific splicing alterations in myelodysplastic syndromes

Mutations of the splicing factor–encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor–associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be used therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity and support the development of therapies targeting splicing abnormalities.

Erratum: Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells (Leukemia (2015) 29 (1092-1103) 10.1038/leu.2014.331)

Correction to: Leukemia (2015) 29, 1092–1103; doi:10.1038/leu.2014.331; published online 23 December 2014. Since the publication of the above article the authors would like to add the following to the Acknowledgements section: AP and JB acknowledge support by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme.

Effects of L -leucine in 5q- syndrome and other RPS14-deficient erythroblasts

family, or, as in our case, reveal a (potential) founding mutation for FPD/AML. Vigilance in identifying a RUNX1 mutation in patients suspected of FPD/AML is required as allogenic SCT is often the only potentially curative treatment. Given clinical heterogeneity of the disorder, candidate sibling donors should then be investigated for being carrier of the abnormality. Furthermore, a de novo 21q RUNX1 translocation, when present in the germ line of a carrier could be transmitted to the next generation, and could result in FPD/AML, but also in chromosomal unbalanced offspring depending on which derivative chromosomes are segregating.

Application of genome editing technologies to the study and treatment of hematological disease.

Genome editing technologies have advanced significantly over the past few years, providing a fast and effective tool to precisely manipulate the genome at specific locations. The three commonly used genome editing technologies are Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas9 (CRISPR/Cas9) system. ZFNs and TALENs consist of endonucleases fused to a DNA-binding domain, while the CRISPR/Cas9 system uses guide RNAs to target the bacterial Cas9 endonuclease to the desired genomic location. The double-strand breaks made by these endonucleases are repaired in the cells either by non-homologous end joining, resulting in the introduction of insertions/deletions, or, if a repair template is provided, by homology directed repair. The ZFNs, TALENs and CRISPR/Cas9 systems take advantage of these repair mechanisms for targeted genome modification and have been successfully used to manipulate the genome in human cells. These genome editing tools can be used to investigate gene function, to discover new therapeutic targets, and to develop disease models. Moreover, these genome editing technologies have great potential in gene therapy. Here, we review the latest advances in the application of genome editing technology to the study and treatment of hematological disorders.

Impact of Splicing Factor Mutations on Pre-mRNA Splicing in the Myelodysplastic Syndromes

Splicing is an essential cellular process which is carried out by the spliceosome in order to remove the introns and join the exons present in pre-mRNA transcripts. A variety of spliceosomal mutations have been recently identified in the myelodysplastic syndromes (MDS), a heterogeneous group of hematopoietic stem cell malignancies, revealing a new leukemogenic pathway involving spliceosomal dysfunction. Splicing factor genes are the most frequently mutated genes found in MDS, with mutations occurring in more than half of all patients. The high mutation frequency in different components of the spliceosome in MDS indicates that aberrant splicing may be a common consequence of these mutations in this disorder. RNA sequencing studies using MDS patient bone marrow cells and different mouse models have identified several downstream targets of the splicing factor mutations. Aberrant splicing of these target genes may contribute to MDS pathogenesis, however functional studies are required in order to fully determine the effects of the aberrant isoforms on disease phenotype. Splicing inhibitors are currently being developed and may be used as therapeutic agents to target aberrant pre-mRNA splicing in MDS and other cancers with splicing factor mutations. The mouse models expressing splicing factor mutations may prove particularly valuable for pre-clinical testing of these drugs.

Activation of the mTOR signaling pathway by L -leucine in 5q- syndrome and other RPS14-deficient erythroblasts

Activation of the mTOR signaling pathway by L -leucine in 5q- syndrome and other RPS14-deficient erythroblasts

L-leucine increases translation of RPS14 and LARP1 in erythroblasts from del(5q) myelodysplastic syndrome patients

Deletion of the long arm of chromosome 5 [del(5q)] is the most common cytogenetic abnormality found in the myelodysplastic syndromes (MDS).[1][1] Patients with the 5q-syndrome have macrocytic anemia and the del(5q) as the sole karyotypic abnormality.[1][1] Haploinsufficiency of the ribosomal protein