SRSF2 Is Essential For Hematopoiesis and Its Mutations Dysregulate Alternative RNA Splicing In MDS

Abstract Myelodysplastic syndromes (MDS) are a group of neoplasms that are ineffective in generating multiple lineages of myeloid cells and have various risks to progress to acute myeloid leukemia. Recent genome-wide sequencing studies reveal that mutations in genes of splicing factors are commonly associated with MDS. However, the importance of these splicing factors in hematopoiesis has been unclear and the causal effect of their mutations on MDS development remains to be determined. One of these newly identified genes is SRSF2, and its mutations have been linked to poor survival among MDS patients. Interestingly, most of SRSF2 mutations occur at proline 95 and the majority of these mutations change this proline to histidine (P95H). Given that SRSF2 is a well-characterized splicing factor involved in both constitutive and regulated splicing, we hypothesize that SRSF2 plays an important role in normal hematopoiesis and the SRSF2 mutations induce specific changes in alternative splicing that favor disease progression. We first examined the role of SRSF2 in hematopoiesis by generating Srsf2 null mutation in mouse blood cells via crossing conditional Srsf2 knockout mice (Srsf2f/f) with blood cell-specific Cre transgenic mice (Vav-Cre). The mutant mice produced significantly fewer definitive blood cells (10% of wild type controls), exhibited increased apoptosis in the remaining blood cells, and died during embryonic development. Importantly, we detected no hematopoietic stem/progenitor cells (lineage-/cKit+) in E14 fetal livers of Vav-Cre/Srsf2f/f mice. These results indicate that SRSF2 is essential for hematopoiesis during embryonic development. We next examined the role of SRSF2 in adult hematopoiesis by injecting polyIC into mice that carry a polyIC inducible Cre expression unit. Unexpectedly, after multiple polyIC treatments, the Srsf2f/f mice stayed alive during several months of observation. Time course genotyping analyses of polyIC treated mice revealed an increased rate of incomplete Srsf2 deletion in peripheral blood cells. These observations suggest that Srsf2 ablation did not cause immediate cell lethality in differentiated blood cells, but the gene is indispensable for the function of blood stem/progenitor cells. Since mutations of splicing factors are generally heterozygous in MDS patients, we also examined mice with Srsf2+/- blood cells. No obvious defect of hematopoiesis was observed under normal conditions or in response to stress with 5-FU treatment and sublethal irradiation. To gain molecular insight into the splicing activity of MDS-associated mutant forms of SRSF2, we performed large-scale alternative splicing surveys by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) previously developed in our lab, which offers a robust and cost-effective platform for splicing profiling. Compared to vector transduction controls, we found that overexpression of both wild type and P95H SRSF2 induced many, but distinct changes in alternative splicing in lineage-negative bone marrow cells, and importantly, we noted several changes in genes with known roles in hematopoietic malignancies that were uniquely induced by the mutant SRSF2. To further link the mutations to altered splicing in MDS patients, we also applied RASL-seq to a large number of MDS patient samples with or without mutations in SRSF2 or other splicing regulators. The data revealed a specific set of alternative splicing events that are commonly linked to MDS with splicing factor mutations. These findings strongly suggest that many of these mutations in splicing regulators are gain-of-function mutations that are causal to MDS. In conclusion, we report that SRSF2 plays an essential role in hematopoietic stem/progenitor cells and that the MDS-associated mutations in SRSF2 have a dominant effect on RNA alternative splicing. These findings provide functional information and molecular basis of SRSF2 and its MDS-related mutations in hematopoiesis and related clinical disorders. Disclosures: No relevant conflicts of interest to declare

Runx1 exon 6-related alternative splicing isoforms differentially regulate hematopoiesis in mice.

RUNX1 is an important transcription factor for hematopoiesis. There are multiple alternatively spliced isoforms of RUNX1. The best known isoforms are RUNX1a from use of exon 7A and RUNX1b and c from use of exon 7B. RUNX1a has unique functions due to its lack of C-terminal regions common to RUNX1b and c. Here, we report that the ortholog of human RUNX1a was only found in primates. Furthermore, we characterized 3 Runx1 isoforms generated by exon 6 alternative splicing. Runx1bEx6(-) (Runx1b without exon 6) and a unique mouse Runx1bEx6e showed higher colony-forming activity than the full-length Runx1b (Runx1bEx6(+)). They also facilitated the transactivation of Runx1bEx6(+). To gain insight into in vivo functions, we analyzed a knock-in (KI) mouse model that lacks isoforms Runx1b/cEx6(-) and Runx1bEx6e. KI mice had significantly fewer lineage-Sca1(+)c-Kit(+) cells, short-term hematopoietic stem cells (HSCs) and multipotent progenitors than controls. In vivo competitive repopulation assays demonstrated a sevenfold difference of functional HSCs between wild-type and KI mice. Together, our results show that Runx1 isoforms involving exon 6 support high self-renewal capacity in vitro, and their loss results in reduction of the HSC pool in vivo, which underscore the importance of fine-tuning RNA splicing in hematopoiesis

SRSF2 Is Essential For Hematopoiesis and Its Mutations Dysregulate Alternative RNA Splicing In MDS

Abstract Myelodysplastic syndromes (MDS) are a group of neoplasms that are ineffective in generating multiple lineages of myeloid cells and have various risks to progress to acute myeloid leukemia. Recent genome-wide sequencing studies reveal that mutations in genes of splicing factors are commonly associated with MDS. However, the importance of these splicing factors in hematopoiesis has been unclear and the causal effect of their mutations on MDS development remains to be determined. One of these newly identified genes is SRSF2, and its mutations have been linked to poor survival among MDS patients. Interestingly, most of SRSF2 mutations occur at proline 95 and the majority of these mutations change this proline to histidine (P95H). Given that SRSF2 is a well-characterized splicing factor involved in both constitutive and regulated splicing, we hypothesize that SRSF2 plays an important role in normal hematopoiesis and the SRSF2 mutations induce specific changes in alternative splicing that favor disease progression. We first examined the role of SRSF2 in hematopoiesis by generating Srsf2 null mutation in mouse blood cells via crossing conditional Srsf2 knockout mice (Srsf2f/f) with blood cell-specific Cre transgenic mice (Vav-Cre). The mutant mice produced significantly fewer definitive blood cells (10% of wild type controls), exhibited increased apoptosis in the remaining blood cells, and died during embryonic development. Importantly, we detected no hematopoietic stem/progenitor cells (lineage-/cKit+) in E14 fetal livers of Vav-Cre/Srsf2f/f mice. These results indicate that SRSF2 is essential for hematopoiesis during embryonic development. We next examined the role of SRSF2 in adult hematopoiesis by injecting polyIC into mice that carry a polyIC inducible Cre expression unit. Unexpectedly, after multiple polyIC treatments, the Srsf2f/f mice stayed alive during several months of observation. Time course genotyping analyses of polyIC treated mice revealed an increased rate of incomplete Srsf2 deletion in peripheral blood cells. These observations suggest that Srsf2 ablation did not cause immediate cell lethality in differentiated blood cells, but the gene is indispensable for the function of blood stem/progenitor cells. Since mutations of splicing factors are generally heterozygous in MDS patients, we also examined mice with Srsf2+/- blood cells. No obvious defect of hematopoiesis was observed under normal conditions or in response to stress with 5-FU treatment and sublethal irradiation. To gain molecular insight into the splicing activity of MDS-associated mutant forms of SRSF2, we performed large-scale alternative splicing surveys by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) previously developed in our lab, which offers a robust and cost-effective platform for splicing profiling. Compared to vector transduction controls, we found that overexpression of both wild type and P95H SRSF2 induced many, but distinct changes in alternative splicing in lineage-negative bone marrow cells, and importantly, we noted several changes in genes with known roles in hematopoietic malignancies that were uniquely induced by the mutant SRSF2. To further link the mutations to altered splicing in MDS patients, we also applied RASL-seq to a large number of MDS patient samples with or without mutations in SRSF2 or other splicing regulators. The data revealed a specific set of alternative splicing events that are commonly linked to MDS with splicing factor mutations. These findings strongly suggest that many of these mutations in splicing regulators are gain-of-function mutations that are causal to MDS. In conclusion, we report that SRSF2 plays an essential role in hematopoietic stem/progenitor cells and that the MDS-associated mutations in SRSF2 have a dominant effect on RNA alternative splicing. These findings provide functional information and molecular basis of SRSF2 and its MDS-related mutations in hematopoiesis and related clinical disorders. Disclosures: No relevant conflicts of interest to declare

Cooperation between RUNX1-ETO9a and Novel Transcriptional Partner KLF6 in Upregulation of <i>Alox5</i> in Acute Myeloid Leukemia

<div><p>Fusion protein RUNX1-ETO (AML1-ETO, RUNX1-RUNX1T1) is expressed as the result of the 8q22;21q22 translocation [t(8;21)], which is one of the most common chromosomal abnormalities found in acute myeloid leukemia. RUNX1-ETO is thought to promote leukemia development through the aberrant regulation of RUNX1 (AML1) target genes. Repression of these genes occurs via the recruitment of the corepressors N-COR and SMRT due to their interaction with ETO. Mechanisms of RUNX1-ETO target gene upregulation remain less well understood. Here we show that RUNX1-ETO9a, the leukemogenic alternatively spliced transcript expressed from t(8;21), upregulates target gene <i>Alox5</i>, which is a gene critically required for the promotion of chronic myeloid leukemia development by BCR-ABL. Loss of <i>Alox5</i> expression reduces activity of RUNX1-ETO9a, MLL-AF9 and PML-RARα <i>in vitro</i>. However, <i>Alox5</i> is not essential for the induction of leukemia by RUNX1-ETO9a <i>in vivo</i>. Finally, we demonstrate that the upregulation of <i>Alox5</i> by RUNX1-ETO9a occurs via the C₂H₂ zinc finger transcription factor KLF6, a protein required for early hematopoiesis and yolk sac development. Furthermore, <i>KLF6</i> is specifically upregulated by RUNX1-ETO in human leukemia cells. This identifies KLF6 as a novel mediator of t(8;21) target gene regulation, providing a new mechanism for RUNX1-ETO transcriptional control.</p></div

<i>Alox5</i> involvement in hematopoietic cell self-renewal.

<p>(A) <i>Alox5</i> required for long-term self-renewal of hematopoietic cells by RE9a. Colony numbers from wildtype or <i>Alox5</i>-/- bone marrow cells transduced with control (MIP) or RE9a retrovirus and serially replated in methylcellulose. Data shown are averages with standard deviations of a representative dataset. Four independent assays were performed. (B) Typical colony images after 9^th replating from (A) taken using Nikon Eclipse TS100 microscope with 2×/0.06 objective lens and Nikon DS Camera Control Unit DS-U2 system. (C) Flow cytometric analysis of replated cells from (A). Cells from 3^rd, 6^th and 9^th replatings were stained for myeloid lineage markers Gr-1 and CD11b. Representative data from four independent assays shown. (D) and (E) Lack of <i>Alox5</i> decreases colony formation potential of hematopoietic cells transduced with MLL-AF9 and PML-RARα. Wildtype or <i>Alox5</i>-/- bone marrow cells were transduced with MIP and MLL-AF9 (D) or PML-RARα (E) retrovirus and serially replated in methylcellusose. Data shown are averages and standard deviations of representative datasets. Three independent assays were performed.</p

Loss of <i>Alox5</i> does not block RE9a leukemia induction <i>in vivo</i>.

<p>(A) Survival of mice receiving wildtype or <i>Alox5</i>-/- fetal liver cells transduced by control (MigR1) or RE9a retrovirus. Number of mice in each cohort shown at right. WT median survival: 30.71 weeks; <i>Alox5</i>-/- median survival: 29.43 weeks; <i>p</i> = 0.39. (B) Presence of hematopoietic blast cells in tissues of mice transplanted with RE9a-transduced wildtype or <i>Alox5</i>-/- cells. Peripheral blood smears and cytocentrifugation of bone marrow and spleen cells were stained with Wright-Giemsa solutions. (C) Immunophenotype of myeloid progenitor cells in wildtype and <i>Alox5</i>-/- leukemias. Distribution of EGFP⁺Lin⁻Sca-1⁻c-Kit⁺ leukemic cells harvested from spleen shown based on expression of CD34 and Fcγ receptors II/III (FcγRII/III). At least 4 mice analyzed per genotype, with representative distributions shown.</p